アンメット・メディカル・ニーズに応える
創薬基盤研究の推進および臨床応用への展開

平成 27 年度～平成 31 年度
私立大学戦略的研究基盤形成支援事業
研究成果報告書

令和２年５月学校法人名東北医科薬科大学

大名

東北医科薬科大学

研究組織名

大学院薬学研究科

研究代表者

丹野

学

孝一

（東北医科薬科大学大学院薬学研究科 教授）

― 目 次 ―1. はしがき -------------------------------------------------------------------------------------- 11

2. 研究成果報告書概要 ----------------------------------------------------------------------- 33

3. 研究発表状況（主要論文）
研究テーマ 1
新規がん分子標的治療薬の創製 ---------------------------------------------- ？
55
研究テーマ 2
老年性神経系疾患（老人性認知症、高齢者うつ病）改善薬の開発 ----295？
研究テーマ 3
難治性疼痛に対する緩和医療法の確立 ------------------------------------ 419？

平成 27 年度～平成 31 年度「私立大学戦略的研究基盤形成支援事業」（創薬研究）研究成果報告書発刊にあたって
研究代表者

丹野 孝一

アンメット・メディカル・ニーズ（いまだ有効な治療薬/治療法のない医療ニーズ）に応える治療薬/治療法の開発は最も社会的要請の強い研究課題の一つとなっています。本学ではハイテク・リサーチ・センター整備事業「生体内分子標的理論にもとづく創薬とその臨床治療への応用（平成 17〜21 年度）」および私立大学戦略的研究基盤形成支援事業「癌および加齢性疾患の制御と QOL 向上を目指す創薬（平成 22〜26 年度）」を実施し、創薬基礎研究に関する顕著な成果を上げてきました。本プロジェクト「アンメット・メディカル・ニーズに応える創薬基盤研究の推進および臨床応用への展開」では、これまで培ってきた知識・経験・技術をもとに、アンメット・メディカル・ニーズに応える治療薬の開発を目指した創薬基盤研究を推進するとともに、東北地方における創薬研
究の拠点としての役割を果たすことを目的に企画しました。
本プロジェクトは、①制がん化合物やホルモン受容体制御化合物の分子設計、合成法の開発と機能解析、②化学物質の毒性評価および③神経精神疾患、難治
性疼痛や気管支喘息の発症メカニズムの解明などにおいて独創的で先駆的な成果を上げている 7 つの研究室（創薬化学、分子薬化学、医薬合成化学、環境衛
生学、薬理学、機能形態学、病態生理学）で組織されています。本プロジェク
トでは、これまで本学が醸成してきた基礎薬学研究を 3 つのテーマ、すなわち、「1. 新規がん分子標的治療薬の創製」、「2. 老年性神経系疾患（老人性認知症、高齢者うつ病）改善薬の開発」および「3. 難治性疼痛に対する緩和医療法の確立」に集結し、研究の進展を図ってきました。
本プロジェクトにおいて①抗腫瘍性天然物とそれらの類縁化合物ならびに新規抗腫瘍性ヌクレオシドの合成、②各種 P450 活性の評価スクリーニング系の樹立と応用、③新規選択的エストロゲン受容体モジュレーターの合成ならびに抗うつ作用・認知障害改善作用とそれらの作用機序の解明、④難治性疼痛治療薬のターゲットとなる特異的受容体の発見、および⑤µオピオイド受容体遺伝子の一塩基変異が気管支喘息の増悪因子となる可能性を明らかにするなど、それぞれの研究テーマにおいて優れた研究成果を上げることができました。本研究プ
ロジェクトの研究成果がアンメット・メディカル・ニーズに応える治療薬の開発に繋がることを期待しております。

—1—

—2—

（様式 2）
法人番号プロジェクト番号

041004
S1511001L

平成 27 年度～平成 31 年度「私立大学戦略的研究基盤形成支援事業」
研究成果報告書概要
１ 学校法人名

東北医科薬科大学

２ 大学名

３ 研究組織名

東北医科薬科大学大学院薬学研究科

東北医科薬科大学

４ プロジェクト所在地

宮城県仙台市青葉区小松島 4-4-1

５ 研究プロジェクト名

アンメット・メディカル・ニーズに応える創薬基盤研究の推進および臨床応用への展開６ 研究観点研究拠点を形成する研究７ 研究代表者
研究代表者名

所属部局名

職名

丹野 孝一

大学院薬学研究科教授

８ プロジェクト参加研究者数
９ 該当審査区分

７３ 名

理工・情報

生物・医歯

人文・社会

１０ 研究プロジェクトに参加する主な研究者
研究者名

所属・職名

丹野 孝一

薬理学教室・教授

吉村 祐一

分子薬化学教室・教授

永田 清

環境衛生学教室・教授

遠藤 泰之

創薬化学教室・教授

溝口 広一

機能形態学教室・教授

髙橋 知子

病態生理学教室・教授

大野 勲

医学教育推進センター・教授

プロジェクトでの研究課題

プロジェクトでの役割

老年性精神系疾患の発症メカニ
ズムの解明と治療薬の開発
抗腫瘍性ヌクレオシド誘導体の設計と合成
毒性発現および薬物相互作用の評価とスクリーニング系の構築
エストロゲン受容体制御にもとづく神経系疾患改善薬および新規抗がん活性化合物の探索
難治性疼痛の発症メカニズムの解明と特異的鎮痛薬の開発
オピオイド受容体遺伝子多型の解析および個別化医療への応用
オピオイド受容体遺伝子多型の解析および個別化医療への応用

老年性精神疾患改善薬の開発(研究代表者)—3—

新規ヌクレオシド系抗腫瘍薬の探索
毒性および薬物相互作用の評価
老年性精神疾患改善薬およびがん分子標的薬の開発
難治性疼痛治療薬の創製
難治性疼痛緩和療法の確立
難治性疼痛緩和療法の確立

（様式 2）
法人番号プロジェクト番号

041004
S1511001L＜研究者の変更状況（研究代表者を含む）＞
旧
ﾌﾟﾛｼﾞｪｸﾄでの研究課題
研究テーマ１・新規がん分子標的治療薬の創製」抗腫瘍性天然物
および類縁化合物の合成と構造
活性相関研究

所属・職名

研究者氏名

大学院薬学研究科・
医薬合成化学教室・ 加藤 正
教授

プロジェクトでの役割
研究代表者（変更の時期：平成 29 年 1 月 6 日）

新変更前の所属・職名
大学院薬学研究科・
環境衛生学教室・教授変更（就任）後の所属・職名
大学院薬学研究科・
環境衛生学教室・教授

研究者氏名

プロジェクトでの役割

永田 清

研究代表者

所属・職名

研究者氏名

プロジェクトでの役割

大学院薬学研究科・
環境衛生学教室・
教授

永田 清

研究代表者

変更（就任）後の所属・職名

研究者氏名

プロジェクトでの役割

創薬研究センター・教授

加藤 正

研究代表者

研究者氏名

プロジェクトでの役割

櫻田 忍

難治性疼痛治療薬の創製

研究者氏名

プロジェクトでの役割

溝口 広一

難治性疼痛治療薬の創製

旧ﾌﾟﾛｼﾞｪｸﾄでの研究課題
研究テーマ１・新規がん分子標的治療薬の創製」抗腫瘍性天然物および類縁化合物の合成と構造活性相関研究（変更の時期：平成 29 年 4 月 1 日）

新
変更前の所属・職名
大学院薬学研究科・医薬
合成化学教室・教授

旧ﾌﾟﾛｼﾞｪｸﾄでの研究課題
所属・職名
難治性疼痛の発症メカニ
大学院薬学研究科・ズムの解明と特異的鎮痛
機能形態学教室・特任教授
薬の開発（変更の時期：平成 29 年 4 月 1 日）

新
変更前の所属・職名変更（就任）後の所属・職名
大学院薬学研究科・機能 大学院薬学研究科・形態学教室・ 准教授
機能形態学教室・教授—4—

（様式 2）041004
S1511001L

法人番号
プロジェクト番号

旧
ﾌﾟﾛｼﾞｪｸﾄでの研究課題
所属・職名
研究者氏名
研究テーマ１・新規がん分子標的治療薬の創製」抗腫瘍性天然物 大学院薬学研究科・医薬
加藤 正および類縁化合物の合成と構造 合成化学教室・教授
活性相関研究

プロジェクトでの役割
研究代表者（変更の時期：平成 31 年 4 月 1 日）

新
変更前の所属・職名
大学院薬学研究科
薬理学教室・教授

変更（就任）後の所属・職名
大学院薬学研究科
薬理学教室・教授

研究者氏名

プロジェクトでの役割

丹野 孝一

研究代表者

１１ 研究の概要（※ 項目全体を１０枚以内で作成）（１）研究プロジェクトの目的・意義及び計画の概要
アンメット・メディカル・ニーズ（いまだ有効な治療薬/治療法のない医療ニーズ）に応える治療薬/治療法の開発は、最も社会的要請の強い研究課題の一つである。本学では、ハイテク・リサーチ・センター整備事
業として「生体内分子標的理論にもとづく創薬とその臨床治療への応用（平成 17〜21 年度）」、および私立大学戦略的研究基盤形成支援事業として「癌および加齢性疾患の制御と QOL 向上を目指す創薬（平成 22〜26 年度）」を実施し、創薬基礎研究に関する優れた成果をあげてきた。本プロジェクトでは、これまで蓄積してきた基礎研究成果を集約し、アンメット・メディカル・ニーズの高い疾患領域、すなわち、①がん、②
老年性神経系疾患（老人性認知症、高齢者うつ病）および③難治性疼痛に対する創薬研究の推進を図り、東北地方における創薬研究拠点としての役割を果たすことを目的として企画した。本研究プロジェクトの目
指す最終ゴールは、非臨床・臨床試験に進める有望な候補化合物を創出し、アカデミア発/日本発の画期的新薬として臨床応用研究（実用化研究）へと結び付けることである。具体的には以下の 3 つの研究テーマを実施する。

「研究テーマ １. 新規がん分子標的治療薬の創製」(1) 抗腫瘍性天然物および類縁化合物の合成と構造活性相関研究(2) 新規抗腫瘍性ヌクレオシド誘導体の設計と合成(3) 薬効/毒性発現および薬物相互作用の評価とそのスクリーニング系の構築「研究テーマ ２. 老年性神経系疾患(老人性認知症、高齢者うつ病)改善薬の開発」(1) エストロゲン受容体制御にもとづく老年性神経系疾患改善薬の設計と合成(2) 精神疾患モデル動物を用いた老年性神経系疾患改善薬の評価および作用メカニズムの解明「研究テーマ ３. 難治性疼痛に対する緩和医療法の確立」(1) 難治性疼痛の発症メカニズムの解明および特異的疼痛治療薬の創製(2) μオピオイド受容体遺伝子多型の解析および個別化医療への応用
本研究プロジェクトで計画している疾患ターゲットやメカニズムの解明、疾患に対するタンパクなどに作用する新規化合物の探索・同定、疾患モデル動物の作製、薬効/副作用の予測法の樹立、シード化合物からリード化合物さらには開発候補化合物への展開などの創薬方法論の開発は、学術的にも実践的にも大きな波及効果が期待できる。

（２）研究組織—5—

（様式 2）
法人番号プロジェクト番号

041004
S1511001L（２）研究組織
本研究組織は東北医科薬科大学大学院薬学研究科の 7 教室 30 名の教員によって構成されており、新規化合物を設計・合成する有機化学・創薬化学系研究室と機能解析や動物試験を行う生化学・薬理系研究室を本プロジェクトの 3 つの研究テーマに組織化できるように配置している。研究代表者は全体の統括、調整を行うとともに、各研究室間の連携を促進するため本プロジェクトの運営会議、成果公表の取り纏め、
成果発表会（創薬研究センターシンポジウム）の企画を行い、プロジェクトの充実に努めている。それぞれの研究テーマ組織は以下の通りである。
「研究テーマ１. 新規がん分子標的治療薬の創製」
吉村教授を責任者とし、医薬合成化学教室、分子薬化学教室および環境衛生学教室の 3 教室で組織し、教員 13 名、ポスドク 2 名、大学院博士課程学生 2 名および修士課程学生 18 名で研究を行っている。
「研究テーマ２. 老年性神経系疾患(老人性認知症、高齢者うつ病)改善薬の開発」
遠藤教授を責任者とし、創薬化学教室および薬理学教室の 2 教室で組織し、教員 7 名、大学院博士課
程学生 5 名および修士課程学生 7 名で研究を行っている。
「研究テーマ３. 難治性疼痛に対する緩和医療法の確立」
溝口教授を責任者とし、機能形態学教室および病態生理学教室の 2 教室で組織し、教員 10 名、大学院博士課程学生 4 名および修士課程学生 5 名で研究を行っている。
研究者以外に技術系職員 2 名と事務系職員 1 名が担当するなど、大学が積極的に支援を行っている。

（３）研究施設・設備等
総研究施設面積は 2108m2 であり、7 つの研究室が参画し、教員 30 名、ポスドク 2 名、大学院博士課
程学生 11 名および修士課程学生 30 名の総勢 73 名の研究者が、本プロジェクトを推進している。
本プロジェクトで設置した以下の研究設備(装置)は、多くの研究者に有効に活用されている(括弧内は平成 27〜31 年度の 5 年間の稼働時間を表す)。
・リアルタイム細胞アナライザー (1950 時間)
・超高感度等温滴定型カロリーメーター (93 時間)
・正立型共焦点レーザー顕微鏡システム (1580 時間)
・擦過行動リアルタイム定量化システム (434 時間)・全自動磁気細胞分離装置 (438 時間)（４）研究成果の概要 ※下記、１３及び１４に対応する成果には下線及び＊を付すこと。
研究テーマごとに研究成果の概要を記載する。

「研究テーマ１．新規がん分子標的治療薬の創製」「抗腫瘍性天然物および類縁化合物の合成と構造活性相関研究」では、医薬合成化学教室で創製した候補化合物「FK-A11」[ヒストン脱アセチル化酵素（HDAC）/ホスファチジルイノシトール 3-キナーゼ（PI3K）
二重阻害薬]の臨床応用に向けた大量合成法の確立、HDAC 阻害薬のリード化合物の創製、新しい分子
標的抗がん薬のシード天然物であるナキジノール B（脳腫瘍細胞増殖抑制活性物質）、リファガール[PI3K 阻害活性物質]、および TAN-1813 [ファルネシル基転移酵素（FTase）阻害活性物質] の合成法の確
立と構造活性相関の解明による新規リード化合物/候補化合物の創製を目的に研究を遂行した。その結果、①「ＦＫ-A11」の工業的製造法を確立するとともに (特許*2)、新規合成法(改良法)の開発に成功し(従来法：全 24 工程、総収率 3.5％； 改良法：全 15 工程、総収率 10％)、工業的製造法への道を拓いた(特許*2)。②スピルコスタチンおよび FK228 の類縁体合成を行い、アイソフォーム選択的 HDAC 阻害薬の創出に成功した(論文*31)。③脳腫瘍の抗がん剤シードであるナキジノールＢの全合成を世界に先駆けて達成した(論文*11)。④リファガール類縁体の合成と活性評価を行った結果、リファガールの生合成前駆体と考えられるシホノディクチアールＢが優れたキナーゼ阻害活性を示すことを見出した(特許*1、論文*33)。⑤抗がん活性を有するマクロラクトン天然物 マンデラリド A およびイソマンデラリド A の全合成を達成した(論文*34)。⑥抗がん活性を有する 9-デオキシキセニアラクトール C の合成研究では、ラクトール環を有する 9 員環部の立体選択的な構築法を開発した(学会*1, 12, 30, 35)。
—6—

（様式 2）
法人番号プロジェクト番号

041004
S1511001L「新規抗腫瘍性ヌクレオシド誘導体の設計と合成」では、新規骨格を有するヌクレオシド系分子標的抗がん薬の創製を目指し、そのデザイン、合成、および生物活性評価を行った。始めに、含硫黄誘導体を疑似糖部とする 4’-チオヌクレオシド誘導体の探索を行った。Vorbrüggen 法を用い、各種核酸塩基を導入した
3’-デオキシ-4’-チオヌクレオシド誘導体の合成を達成した(学会*96)。また、Pummerer 型チオグリコシル化反応を用い、4’-置換 4’-チオリボヌクレオシド誘導体の合成も達成した(学会*18)。さらに、ジヒドロチオ
ピラン環ヌクレオシド誘導体の構造活性相関研究のため新たな合成法の開発に取り組んだ(学会*32)。
これまでに蓄積された種々の修飾ヌクレオシド合成法を核酸医薬の開発へと展開させることを検討した。はじめに、2’-5’連結型環状ジヌクレオチドの合成を検討した(学会*44, 76)。また、前述の 4'-置換 4'チオリボヌクレオシド誘導体の合成における合成中間体を用いた糖部立体構造を固定化したヌクレオシド
誘導体の合成を行った。
4'位に置換基をもつスタブジン誘導体の構造活性相関研究とその鏡像異性体間における薬理活性への影響を解明することを目指し検討を行った。当研究室によって開発された酸化的グリコシル化反応を利用することで、4’-置換スタブジン誘導体の合成を達成した(学会*64, 97)。この合成における中間体を基質とするリパーゼを用いた光学分割を検討し、高い光学純度で両鏡像異性体を得ることに成功した(学会*16)。
得られた光学活性なヌクレオシド誘導体の絶対構造を決定し、リパーゼを用いた光学分割における立体選択性について新たな知見を得た(学会*7)。
生体分子を基本骨格とする有機分子触媒のデザインと合成を行い，不斉反応に応用可能な新規有機
分子触媒の開発を目指し検討を行った。2'位にアミノ基をもつウリジン誘導体の合成を行い、得られた各誘
導体を不斉有機触媒として用いた Diels-Alder 反応への適応を試みた(論文*1)。ペプチド構造をもつ有機分子触媒を設計、合成し、不斉反応への適応について検討した(学会*65, 73)。
立体選択的なイミノ糖誘導体の合成を検討すると共に、得られた化合物の生物活性を評価した。グルコ
シダーゼ阻害活性を持つ化合物の立体選択的合成法を確立し、本方法を用いた構造活性相関研究により、これまでに無い強い活性を持つ化合物を見いだすことに成功した(論文*7、学会*67, 94)。
新たな合成法開発の一環として、各種新反応の開拓を行った。超原子価ヨウ素とジフェニルジセレニドを用いた環化反応を検討し、分子内エーテル環化反応の開発(学会*72)、及び含窒素複素環化合物の合成へと展開させた(学会*8, 17, 20, 33)。パラジウム触媒による位置選択的炭素炭素結合形成反応を開発した(論文*26、学会*34, 43, 74, 95)。ルテニウム触媒を用いた閉環メタセシスによる種々の複素環化合物の合成を行った(論文*14、学会*6, 19, 66)。
「薬効/毒性発現および薬物相互作用の評価とそのスクリーニング系の構築」では、薬効あるいは副作
用に最も大きく寄与する要因は､投与薬物の血中濃度であり、その濃度には薬物代謝酵素 P450（CYP）活性が大きく関わっているため、薬物、食品成分を含む化学物質による薬効、毒性あるいは薬物相互作用発症を予測するために CYP 活性変動を正確に評価可能な複数のスクリーニング系を樹立した。まず、CYP
活性阻害は、アデノウイルスを用いて､複数の CYP を同時に発現させ、カクテル基質と LC/MS/MS を用いることで、一度に 5 種の CYP 活性を調べることができる評価系を樹立した（論文*29, 48、学会*77, 79, 99,105）。また、薬物動態関連遺伝子の解析を行い、新規誘導機序を明らかとし（論文*28, 39、学会*50, 54,
80, 82, 84）、特に薬物相互作用で問題となっている CYP3A4 と CYP1A2 については、これらの遺伝子とレポーター遺伝子の組み替えを行い､レポーター活性を測定することで簡便に薬物代謝酵素誘導の評価可
能なシステムの構築を行った（論文*40, 47、学会*100）。これらの評価系を用いて薬物のみならず健康食品の網羅的解析を行い、健康食品の約 20〜30％に活性阻害や誘導を引き起こす可能性があることを明らかとした（論文*41, 49、学会*26, 49、平成 30 年度および平成 31 年度創薬研究センターシンポジウム）。また、CYP3A4 を強く誘導した健康食品については、CYP3A4 と酵素誘導に関与する核内受容体をヒト化したマウスを用いて、実際に in vivo で CYP3A4 が誘導され、薬物の効果が低下するかについて調べ､誘導の強かったフォルスコリンはヒトで薬物相互作用を引き起こす可能性を明らかにした（学会*23）。さらに､薬物、
食品成分を含む化学物質による毒性発現は培養細胞に CYP を発現させ、リアルタイム細胞アナライザー
によって評価し､健康食品製品の中には細胞毒性をしたものが幾つか見出された。一方、医薬品としても用いられているビタミンＡによる催奇形性の発症が問題となっている。ビタミンＡの活性体のレチノイン酸を—7—

（様式 2）
法人番号プロジェクト番号

041004
S1511001L

代謝する CYP26 は、レチノイン酸の濃度が上昇すると誘導されてくる。この CYP26 遺伝子を用いて、組織内のごく微量のレチノイン酸濃度を測定可能な in vitro スクリーニング系の樹立に成功し（学会*45, 46, 51,
83, 101）、これを用いてアゾール系の農薬が催奇形性を発症するか予測した。一方で、Human Induced
Pluripotent Stem Cells（hiPSCs）細胞から肝細胞への分化は、現在まである程度の薬物代謝酵素活性を得ることに成功したが、成熟肝細胞が示す薬物代謝酵素活性を示すまでには至っていない。当研究室を含め、現在までに報告されている分化肝細胞における肝薬物代謝酵素群の発現比率は胎児肝細胞に類似している。特に、代表的な P450 である CYP3A においては、胎児型 P450 と呼ばれる CYP3A7 の発現量は高く、成人型 P450 である CYP3A4 の発現量が低い。これらから、当研究室で分化させた hiPSCs 由来肝細胞は胎児肝様細胞にまでは分化していると考えている。そして、肝臓が成熟する過程で起きる胎児型
CYP3A7 から成人型 CYP3A4 への発現量の入れ替わり(スイッチ)の分子機序を解明することが、hiPSCsの成熟肝細胞への分化に重要な役割を担う因子の探索の足がかりになると予想し、この機序の解明を試みた。現在のところ、肝臓特異的発現に関わっている転写因子の中で HNF3βが CYP3A7 発現を強く抑制していることを見出した（学会*24）。

「研究テーマ２．老年性神経系疾患(老人性認知症、高齢者うつ病)改善薬の開発」「エストロゲン受容体制御にもとづく老年性神経系疾患改善薬の設計と合成」では、新しい疎水性構造を用いた核内受容体制御を基盤として方向性を絞って研究を展開した。エストロゲン受容体βが神経系作用に関与する可能性を考え、αβサブタイプ選択性を一つの課題とした。分子設計・合成により、高いβ選択
的及びα選択的化合物を見出し、受容体構造との関係性を明らかにした（論文*56, 77, 96、学会*157）。
エストロゲン受容体のアゴニスト/アンタゴニスト制御をもう一つの課題とし、ジフェニルメタン型化合物の系
統合成により、疎水性構造の変化による制御法への重要な情報を見出した (論文*94)。また、新しい疎水性構造を用いた新規細胞増殖阻害薬の創製への展開も行い、がん細胞パネルスクリーニングを含む細胞増殖試験で有効な抗がん化合物を見出した（論文*55, 75, 76, 82）。薬理学教室との共同研究では、我々が開発した新規選択的エストロゲン受容体モジュレーター (SERM) である「BE360」の抗うつ、抗認知症効果を確立した（論文*84）。
「精神疾患モデル動物を用いた老年性神経系疾患改善薬の評価および作用メカニズムの解明」では、
創薬化学教室で開発した「BE360」がうつ病や認知症のモデル動物として用いられている嗅球摘出マウスのうつ様行動および認知障害の両方を改善することを見出した。「BE360」の改善メカニズムは海馬の転写因子 CREB のリン酸化を介した脳由来神経栄養因子（BDNF）増加により、海馬歯状回での神経細胞増殖促進が関与していることを明らかにした（論文*84, 学会*203）。なお、AMP 活性化プロテインキナーゼ活性化薬（論文*61）やアルツハイマー型認知症治療薬であるメマンチン（論文*71）も「BE360」と同様なメカニズ
ムで抗うつ作用を発現することを見出している。さらに、女性の更年期障害である閉経後のうつ病に対する「BE360」の効果を検討するため、メスマウスの卵巣摘出およびストレスを負荷した閉経後うつ病モデル動物を作製した。女性の更年期障害モデルのうつ様行動に対しても「BE360」が改善作用を示し、そのメカニズムとしては、上記の作用メカニズムに加え海馬において神経保護因子である Bcl-2 を増加し神経保護作用を示した（学会*148, 163, 201）。このモデル動物における子宮重量の低下は、エストラジオールまたはタモキシフェン投与によって増加するのに対して「BE360」は、子宮重量に影響を与えなかった。以上のことから、「BE360」は、生殖器に影響のない高齢期に観察されるうつ病および認知障害といった精神疾患の治療薬に成り得る可能性を示唆した（学会*148, 163, 201）。また、更年期障害は、女性のみならず男性においても認知されるようになってきた。しかしながら、男性における更年期の気分変調を検討するための動物モ
デルはほとんど見当たらなかった。上記で示した女性の更年期障害モデルを参考にし、精巣摘出マウスにストレスを負荷し、行動解析を行なった。この男性更年期障害モデル動物において衝動性様の行動を示すことを明らかにした（平成 31 年度創薬研究センターシンポジウム）。今後このモデルを用いて更なる検討を行うことにより更年期の男性に認められる衝動性に関連する病態の解明および治療薬の開発に繋がることが示唆された。
統合失調症は閉経後にも小さな発症のピークがあり、エストロゲンレベルの低下と相関している。我々—8—

（様式 2）
法人番号プロジェクト番号

041004
S1511001Lは、「BE360」の統合失調症に対する作用を検討するためモデル動物の作製を試みている（論文*54, 62）。
神経新生抑制薬（メチルアゾキシメタノール）を胎生期に投与することにより簡便な神経発達障害仮説に基づいた統合失調症モデルマウス作製に成功した（論文*62、学会*120, 130, 141, 178）。
高齢者における咀嚼や口腔機能低下と脳の機能または糖代謝系との関連性を解明するため、長期間粉末食でマウスを飼育し、その影響を検討した。その結果、粉末食飼育マウスは多動や低不安行動を発現し、その異常行動発現にはカテコールアミン系、GABA 系および前頭皮質のドパミン D4 受容体の変化が関与する可能性を示した（論文*65, 92、学会*121, 135, 156, 214）。さらに、粉末食飼育マウスでは、低不安行動の他、血清コルチコステロン濃度の増加、海馬グルココルチコイド受容体発現量の減少が認められ、これらの変化は、粉末食から固形食に切り替えて飼育することで改善することが判明した。従って、咀嚼や口腔機能の低下に起因する脳機能や糖代謝系の変化には、視床下部－下垂体―副腎皮質系が関与している可能性も示した（学会*173）。
潰瘍性大腸炎は 20-30 歳までの若い年齢層に多く発症する疾患と考えられてきたが、近年では 40 歳代から 60 歳代の発症例も増えてきている。症状として血便を伴う下痢や腹痛などの消化器症状の他に抑う
つや不安などの精神症状も多く現れてくるが、そのメカニズムについては不明である。このような背景の下、我々は消化性大腸炎に伴ううつ病モデルマウスの作製に成功し、そのメカニズムとして大腸の炎症性サイトカイン産生増加を介した海馬歯状回の神経新生の抑制に起因することを明らかにした（論文*60）。

「研究テーマ３．難治性疼痛に対する緩和医療法の確立」「難治性疼痛の発症メカニズムの解明および特異的疼痛治療薬の創製」では、dermorphin（μオピオイ
ド受容体アゴニスト）誘導体の「amidino-TAPA」をプロトタイプ化合物として、各種難治性疼痛（神経損傷性
神経障害性疼痛、炎症性疼痛、神経絞扼性がん性疼痛、多発性硬化症疼痛）に有効かつ、依存性のない
新規鎮痛薬を開発すると共に、難治性疼痛治療のターゲットとなるμオピオイド受容体スプライスバリアン
トの特定を目的に研究を遂行した。その結果、各種難治性疼痛に有効な新規鎮痛薬として、「amidino-TAPA」およびその誘導体「ADAMB」を見出した（学会*288, 289）。また、既存の麻薬性鎮痛薬である oxycodone と methadone も、特定の難治性疼痛に対して有効であることを発見した（学会*225, 228,
237, 238, 239, 286, 290, 301, 314）。これら難治性疼痛に有効な鎮痛薬は、共通した特定の薬理作用を持つことを発見し、難治性疼痛治療薬のターゲットとなる特異的受容体を見出した（学会*288, 289）。本研究成果は、難治性疼痛に対する特異的治療薬の開発において、従来とは大きく異なる新たな方向性を示した。
「μオピオイド受容体遺伝子多型の解析および個別化医療への応用」では、μオピオイド受容体遺伝子
多型と疼痛治療薬の免疫応答に及ぼす影響の関連性を解析するとともに、得られた解析結果から、疼痛治療薬によるアレルギー疾患や感染症の発症・悪化の感受性増大リスクを軽減するための情報基盤を確立し、個別化療法への応用に向けた新規疼痛治療薬の創製を目的に研究を遂行した。その結果、μオピ
オイド受容体を起点とする神経/内分泌/免疫ネットワークを介した気管支喘息の発症(論文*112, 114)および増悪機序が明らかとなり(論文*105, 117, 123, 124, 130, 133, 134)、性が神経/内分泌/免疫ネットワーク
を介した免疫応答制御に深く関与していることが示唆された(論文*110, 132)。特に、CD103＋ 樹状細胞やCD8＋ T 細胞は末梢免疫応答の修飾における責任細胞の一つであることが明らかとなった (論文*111,
115)。さらに、μオピオイド受容体遺伝子（OPRM1）の一塩基変異（A118G SNP, rs1799971)は、本邦の気管支喘息患者の 64％に認められ、遺伝子型 GG は気管支喘息の増悪因子となる可能性が示された(学会*254, 260)。日常生活環境において遺伝子型 GG は、リガンドの存在下で末梢組織に発現するμオピオイド
受容体の機能変化を誘導し、Th2 細胞分化を促進させる可能性が明らかとなった(学会*251)。受容体遺伝
子多型や性、生活習慣に関するこれらの結果は、μオピオイド受容体を標的とする難治性疼痛制御において、安全性と個別化療法の確立のために重要な基盤情報となり得ることを示した。

＜優れた成果が上がった点＞「研究テーマ１．新規がん分子標的治療薬の創製」
天然物をシードとする創薬研究では、「ＦＫ-A11」(HDAC/PI3K 二重阻害薬)の新規合成法(改良法)の開—9—

（様式 2）
法人番号プロジェクト番号

041004
S1511001L発に成功し(従来法：全 24 工程、総収率 3.5％； 改良法：全 15 工程、総収率 10％)、工業的製造法への道を拓いた(特許*2)。また、リファガール類縁体の合成と活性評価を行った結果、リファガールの生合成前駆体と考えられるシホノディクチアールＢが優れたキナーゼ阻害活性を示すことを見出した(特許*1, 論文*33)。
ヌクレオシド誘導体の設計と合成研究では、①核酸塩基としてチミン、シトシンを導入した 3’-デオキシ-4’-チオヌクレオシド誘導体の合成を達成した。さらに、核酸塩基としてウラシル、チミン、シトシンを導入した 4’-置換 4’-チオリボヌクレオシド誘導体の合成も達成した。本チオヌクレオシドに関して、糖部の立体構
造を固定化した誘導体の合成法を確立することが出来た。②抗 HIV 活性が認められなかったものの、4’フルオロメチルスタブジンの合成を達成した。さらに 4'位に置換基を持つスタブジン誘導体の光学分割に成功した。また、その絶対構造を決定し、リパーゼを用いた光学分割における立体選択性について新たな知見を得ることが出来た。③グルコシダーゼ阻害活性を持つ化合物の立体選択的合成法を確立することが出来た。また、本方法によりこれまでに無い強い活性を持つ化合物を見いだすことが出来た。④遷移金属触媒を用いた位置選択的炭素炭素結合形成反応を開発することが出来た。
薬効/毒性発現および薬物相互作用の評価とそのスクリーニング系の構築では、①正確な薬物相互作
用評価を可能するために、薬物動態関連遺伝子誘導の新規分子機構を 2 種明らかとした。また、これを基に薬物代謝酵素活性誘導および阻害評価系の構築に成功した。②薬物代謝酵素活性誘導および阻害評
価系を用いて薬物のみならず健康食品の網羅的解析を行い、健康食品の約 20〜30％に活性阻害や誘導を引き起こす可能性があることを明らかとした。③レチノイン酸の低濃度（数 nM）検出可能な手法を開発
し、化学物質によるレチノイン酸濃度変動による催奇形性発症予測可能な in vitro スクリーニング系を樹立した。④hiPSCs 細胞から肝細胞への分化は、胎児肝様細胞にまで達し、ある程度の薬物代謝酵素活性を得ることに成功した。また、肝臓特異的発現に関わっている転写因子の中で HNF3が CYP3A7 発現を強く抑制していることを見出した。

「研究テーマ２．老年性神経系疾患(老人性認知症、高齢者うつ病)改善薬の開発」
エストロゲン受容体制御にもとづく老年性神経系疾患改善薬の設計と合成では、①エストロゲン受容体
β選択性及びα選択性が高い化合物を見出し、受容体構造との関係性を明らかにした（論文*56, 77, 96、
学会*157）。②エストロゲン受容体のアゴニスト/アンタゴニスト制御をもう一つの課題とし、ジフェニルメタ
ン型化合物の系統合成により、疎水性構造の変化による制御法への重要な情報を見出した (論文*94)。
③新しい疎水性構造を用いた新規細胞増殖阻害薬の創製への展開も行い、がん細胞パネルスクリーニン
グを含む細胞増殖試験で有効な抗がん化合物を見出した（論文*55, 75, 76, 82）。④薬理学教室との共同研究では、我々が開発した新規選択的エストロゲン受容体モジュレーター (SERM) である BE360 の抗うつ、抗認知症効果を確立した（論文*84）。
精神疾患モデル動物を用いた老年性神経系疾患改善薬の評価および作用メカニズムの解明では、①「BE360」選択的エストロゲン受容体モジュレーターが精神疾患モデル動物（嗅球摘出マウス、卵巣摘出+
ストレス負荷マウス）において海馬歯状回の神経細胞の増殖を促進し、抗うつ作用および認知障害改善作用を示すことを明らかにした（論文*84、学会*148, 163, 201, 203）。さらに、②ホルモン補充療法により乳がんや子宮体がんが認められていたが「BE360」は生殖器に影響を及ぼさないことも見出した（学会*148,
163, 201）。また、③従来詳細に検討されていなかった男性の更年期障害モデル動物の作製（平成 31 年度
創薬研究センターシンポジウム）および神経発達障害仮説に基づいた簡便な統合失調症モデル動物の作
製（論文*62、学会*120, 130, 141, 178）にも成功した。

「研究テーマ３．難治性疼痛に対する緩和医療法の確立」
難治性疼痛の発症メカニズムの解明および特異的疼痛治療薬の創製では、①神経損傷性神経障害性
疼痛、炎症性疼痛、多発性硬化症疼痛、神経絞扼性がん性疼痛などの各種難治性疼痛に有効な新規鎮痛薬として、「amidino-TAPA」およびその誘導体「ADAMB」を見出した（学会*288, 289）。また、②既存の麻薬性鎮痛薬である oxycodone と methadone も、特定の難治性疼痛に対して有効であることを発見した（学会*225, 228, 237, 238, 239, 286, 290, 301, 314）。③これら難治性疼痛に有効な鎮痛薬は、共通した特定の— 10 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L

薬理作用を持つことを発見し、難治性疼痛治療薬のターゲットとなる特異的受容体を見出した（学会*288,
289）。
μオピオイド受容体遺伝子多型の解析および個別化医療への応用では、①μオピオイド受容体を起点とする神経/内分泌/免疫ネットワークを介した気管支喘息の発症(論文*112, 114)および増悪機序が明らかとなり(論文*105, 117, 123, 124, 130, 133, 134)、性が神経/内分泌/免疫ネットワークを介した免疫応答制御に深く関与していることが示唆された(論文*110, 132)。特に、CD103＋ 樹状細胞や CD8＋ T 細胞は末
梢免疫応答の修飾における責任細胞の一つであることが明らかとなった (論文*111, 115)。さらに、②μオ
ピオイド受容体遺伝子（OPRM1）の一塩基変異（A118G SNP, rs1799971)は、本邦の気管支喘息患者の64％に認められ、遺伝子型 GG は気管支喘息の増悪因子となる可能性が示された(学会*254, 260)。③日
常生活環境において遺伝子型 GG は、リガンドの存在下で末梢組織に発現するμオピオイド受容体の機能変化を誘導し、Th2 細胞分化を促進させる可能性が明らかとなった(学会*251)。

＜課題となった点＞「研究テーマ１．新規がん分子標的治療薬の創製」「FK-A11」の改良合成法は、工程数の削減および総収率の向上に繋がったものの、光学分割法を用いているため、望まない立体化学を有する副生成物が得られてしまう。そのため、副生成物を有効に利用する方法や副生成物を生じない不斉反応への展開が必要であるが、まだその目的を達成していない。一方、抗がん活性を有する 9 員環キセニアジテルペノイドである 9-デオキシキセニアラクトール C の合成研究
では、ラクトール環上への側鎖導入が困難であり大きな課題であったが、現在合成経路を見直し、カルボニル基を足掛かりとした新しい合成法を検討しており、今後の全合成および類縁化合物の系統的な合成への展開が期待される。さらに、スクアレン合成酵素阻害剤であるビサボスクアール A の合成研究では、
望む立体配置を有する四環性骨格の構築法および側鎖導入法が課題であるが、現在、光学活性体を用いて検討中である。
4’-置換 4’-チオヌクレオシド誘導体の合成において、縮環反応とアルドール反応を組み合わせたワン
ポット反応の再現性に問題があったが、塩基の当量をコントロールすることでその問題を解決した。糖部の立体構造を固定化した誘導体の合成に際し、位置選択的な保護基の導入が非常に困難であったが、塩基部を利用した選択的保護を試みたところ、予想外にも望みの化合物が一段階で合成出来た。4'位に置換基を持つスタブジン誘導体の光学分割において、当初立体選択性が非常に低いものであった。中間体の構造を最適化することにより、高い光学純度で両鏡像異性体を得ることが出来た。
CYP3A4 を強く誘導した健康食品については、CYP3A4 と酵素誘導に関与する核内受容体をヒト化したマウスを用いて、酵素誘導により薬物相互作用を実際に引き起こすか検討し、フォルスコリンはヒトで薬物相互作用を引き起こすことが示唆されたが、野生型マウスでは薬物相互作用が認められたが、必ずしもヒト化マウスの in vivo で CYP3A4 誘導を示さず、CYP3A4 以外のマウス CYP の関与が予測され、今後､新たな評価系の構築の検討が課題となった。
iPS 細胞から成熟肝細胞への分化は、まだ成功の報告例はない。我々は胎児型発現と成熟型発現を示す肝臓が成熟する過程で起きる胎児型 CYP3A7 から成人型 CYP3A4 への発現量の入れ替わり(スイッチ)の分子機序を解明することが、hiPSCs の成熟肝細胞への分化に重要と考えている。そのために､現在、CYP3A7 発現を強く抑制する HNF3βの役割の解明を行っている。

「研究テーマ２．老年性神経系疾患(老人性認知症、高齢者うつ病)改善薬の開発」
新規疎水性構造カルボランの反応性に関する知見が少なく、環への求電子試薬の導入反応の収率や再現性に問題があったが、溶媒や反応濃度などの条件検討により、その改善を行い、合成法を確立した。
受容体αβサブタイプのリガンド結合領域はアミノ酸 2 個の違いしかなく、その選択性の一般化は困難であったが、分子動力学の専門家との共同研究により、選択性の条件の検討が進展している（論文*56, 67,
94, 96、学会*108）。
「BE360」の抗うつ作用および認知障害改善作用は、エストロゲン受容体に結合して発現していることを見出した。しかしながら、エストロゲン受容体のどちらのサブタイプ（α，β）を介しているか選択的なアンタ— 11 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L

ゴニストが少なく同定できていない。さらに、エストロゲン受容体は膜や核に存在することが知られているが「BE360」が膜および／または核に結合して作用を発現しているか不明である為、「BE360」に蛍光プローブ
標識等を結合した化合物を合成し局在を評価する必要がある。また、簡便な統合失調症モデルマウス作
製に関する論文化に時間を費やし「BE360」の有効性について評価ができていないため今後検討する。

「研究テーマ３．難治性疼痛に対する緩和医療法の確立」
本研究課題において、従来から使用している各種難治性疼痛（神経損傷性神経障害性疼痛、炎症性疼
痛、多発性硬化症疼痛、神経絞扼性がん性疼痛）の実験動物モデルの他に、新たな難治性疼痛モデルとして脳腫瘍誘発疼痛の実験動物モデルの確立を試みた。しかし、有用かつ汎用性の高い実験動物モデルを確立することは出来なかった。
疼痛治療薬によるアレルギー疾患の発症・悪化の感受性増大リスクを軽減するための基盤研究において、IL-4 存在下で活性化した T 細胞に発現するμオピオイド受容体における遺伝子一塩基多型が、免疫応答制御に特に重要な役割を果たしている可能性が示された(学会*251)。このことは、我々が目的とする
難治性疼痛新規治療薬の有効性と安全性の評価において、薬物の分布および動態に加えて患者の免疫状態との相互作用を解析する必要性を示唆するものである。さらに、μオピオイド受容体の遺伝子多型による Th2 型免疫応答の亢進は、環境要因である性によって影響されることが明らかとなり、遺伝的背景と環境要因の相互作用から新規疼痛治療薬の有効性と安全性を評価する必要がある。

＜自己評価の実施結果と対応状況＞
毎年 6 月に学内において「創薬研究センターシンポジウム」を開催し、各研究室からの成果報告を行い、進捗状況と研究方針について確認と議論を行っている。また、本シンポジウムでは、学外から関連分野の第一線で活躍されている研究者（3 名）を講演者として招聘しており、その先生方からも貴重な助言を頂いている。有効な研究費運用の維持に関しては創薬研究センター運営委員会で各年度の予算執行について討議している。平成 29 年度終了時（中間報告書作成時）における研究成果を研究費配分に反映させる予定であったが、計画遂行がいずれも順調であったため従来の配分を維持した。なお、本研究プロジェクト終了時に本学自己評価委員会により詳細な審査に基づき厳正な評価が実施される予定である。

＜外部（第三者）評価の実施結果と対応状況＞
本プロジェクトを終了するにあたり、2 名の外部評価者に「計画の達成度」、「得られた研究成果」、「研究教育拠点形成」および「プロジェクト全体の評価（総合評価）」に関しての評価およびコメントを依頼した。その結果、いずれの項目とも高い評価を頂いた。また、平成 28 年度に設置された医学部との緊密な連携体制を強化することで地域医療ならびにアンメット・メディカル・ニーズに応える先導的研究拠点としての役割を担えるように発展して欲しいとのコメントを頂いた。この点に関しては本プロジェクトの研究成果を基盤に、これまで以上に医薬連携体制を強化し、本学が地域医療ならびに創薬研究の拠点になれるよう邁進していきたい。

＜研究期間終了後の展望＞
今後も本研究プロジェクトに参画した研究室が連携を取りながら、がん、老年性神経系疾患および難治性疼痛に対する新規医薬品のシードとなる化合物の創製を目指して研究を継続していきたいと考えている。それぞれの研究テーマについての今後の研究方針を以下に記述する。

「研究テーマ１．新規がん分子標的治療薬の創製」
抗がん活性を有する 9-デオキシキセニアラクトール C の合成研究に関しては、側鎖導入法を検討した後、全合成およびその類縁体を合成し、構造活性相関研究に発展させたいと考えている。さらに、本合成法は、同様な基本骨格を有するクリスタキセニシン A およびその縮環体であるプルミスクレリン A の全合成研究に応用したいと考えている。一方、マンデラリド A の全合成研究で用いた銀触媒による光学活性なフラン環およびピラン環の新規合成法は、ピペリジンやピロリジンなどの含窒素複素環の合成に応用し、— 12 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L

それらを含む天然物群の系統的な合成に活用したいと考えている。また、FTase 阻害薬「TAN-1813」の合成研究においては、モデル化合物での検討結果より、二つの重要中間体をカップリングさせるために必要な新知見を得ている。この方法論を用いて「TAN-1813」の完全化学合成を世界に先駆けて達成する。
各種グリコシル化によるチオヌクレオシド誘導体の合成を展開させると共に、新たなグリコシル化反応の開発へと展開させていく。種々の 4’-置換ヌクレオシド誘導体の合成を行うと共に、光学活性なヌクレオシド
誘導体を合成し、生物活性評価を行う。エナンチオマー間での活性の違いを評価することで、生物活性における立体化学の一般性を確立出来るものと考えられる。一方、チオヌクレオシド誘導体の構造活性相関
研究が、高い抗腫瘍活性、もしくは抗 HIV 活性を有する誘導体の創製に繋がることが期待される。特に、糖部立体構造を固定化した誘導体の合成が可能になったことから、選択的な薬理活性を発現させうる核酸医薬の発展に貢献できるに違いない。また、今回開発した炭素炭素結合形成反応は更なる結合形成反応へと展開が可能であり、種々の化合物合成へと応用できると考えられる。
肝障害発症には薬物代謝酵素活性阻害および誘導が強く関わっていることから、本研究で構築した薬物代謝酵素活性誘導および阻害評価系の有効性が証明されたので、これらの系を活用し、薬物肝障害発症リスク評価系を新たに構築する。CYP3A4 を誘導および活性阻害を強く示すことが判明した健康食品
は、ヒト化マウスを用いて、ヒトにおける薬物相互作用の予測を行って行く必要がある。また、iPS 細胞の成熟肝細胞分化の研究については、分化薬物代謝活性が CYP 発現の違いの原因を解明し、新たな分化の手法の開発を目指す。現在共同研究者等はヒト小腸上皮細胞と同等な薬物代謝活性を示す分化に成功しており、その分化手法を改良することで成熟肝細胞と同等な薬物代謝活性を有する分化を目指す。

「研究テーマ２．老年性神経系疾患(老人性認知症、高齢者うつ病)改善薬の開発」
本研究で見出したエストロゲン関連化合物の in vivo での構造活性相関により、神経薬理学的知見と、分子論的作用機構の間を埋める結果が得られることを期待している。また、本研究で見出した新しい細胞増殖阻害化合物は新しい作用機構をもつ抗がん薬の端緒となる可能性がある。
神経系への作用が期待されているエストロゲン受容体βに選択的に作用する化合物が設計および合成されている。したがって、「BE360」の抗うつ作用および認知障害改善作用メカニズムにおけるエストロゲン
受容体サブタイプの同定後、精神疾患モデル動物を用いて選択性のある化合物の有効性について検討する。さらに、簡便な統合失調症モデル動物を用いて「BE360」の効果およびその機序を行動薬理学的並びに分子生物学的手法により解明する。

「研究テーマ３．難治性疼痛に対する緩和医療法の確立」「Amidino-TAPA」や methadone の構造を基に、類似構造を持ちかつそれぞれの特許に抵触しない新規化合物を、現在合成中である。合成した新規化合物の各種難治性疼痛に対する有効性を検討し、またメカ
ニズムを解明する予定である。また、「amidino-TAPA」は精神依存性を示さないこと（論文*106、学会*271,
274, 304, 315）、methadone も著しく精神依存性が弱い麻薬性鎮痛薬であることから、新規化合物の精神依存性の有無を検討し、新規化合物を依存性の無い難治性疼痛の特異的治療薬として開発する予定である。
μオピオイド受容体遺伝子（OPRM1）の一塩基変異（A118G SNP, rs1799971)と同様の多型を有するマウス（Oprm1 SNP A112G）を用いて、内因性μオピオイド受容体アゴニストによる末梢免疫応答の変化をさらに多面的に解析する。特に Th1 型免疫応答が主体となる感染免疫応答や、Th17 型免疫応答が主体となる一部の喘息病態や創病変の動物モデルを用いて、神経/内分泌/免疫ネットワークの観点から感染症や創傷の発症と悪化の感受性増大リスクを評価する。加えて、μオピオイド受容体一塩基多型と新規疼痛治療薬の作用（疼痛緩和効果、リガンドとの親和性、細胞内シグナル伝達の差異等）を解明し、臨床試験へと発展させる。

＜研究成果の副次的効果＞「研究テーマ１．新規がん分子標的治療薬の創製」
抗がん活性を有するマンデラリド A の全合成では、合成終盤のマクロラクトン化反応において、想定し— 13 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001Lていない水酸基の位置で環化したイソマンデラリド A が得られた。本化合物の抗がん活性を評価したところ、ターゲット天然物であるマンデラリド A よりも強い生物活性を有していることが判明した(論文*34)。さらに、マンデラリド A の構造中に含まれる光学活性なピラン環およびフラン環の構築法では、金触媒を用いる方法が一般的であったが、これを銀触媒に代替えする方法論を開発し、合成コストの削減に成功した(論文*35)。また、「FK-A11」の工業的製造法の開発により(特許*2)、臨床応用に向けた動物実験や非臨床試
験などの研究が推進されることが期待できる。また、リファガールおよび関連化合物の新たな生物活性を見出せたことより(特許*1)、これら天然物をシードした創薬研究を発展させることができる。
各種グリコシル化反応により様々なヌクレオシド誘導体の合成法を確立することが出来た。今後、これらのグリコシル化を適応させることで、望みの官能基を有する種々の誘導体が合成可能となることが期待さ
れ、抗腫瘍活性、もしくは抗 HIV 活性をもつ新たな化合物の創製に貢献できるものと考えられる。4’-置換
4’-チオヌクレオシド誘導体の合成において、糖部の立体構造を容易に固定化可能となったことから、核酸
医薬の構成素子となる新たなヌクレオシドユニットの供給が容易となり、アンチセンス法に有用な人工核酸の開発に寄与できるものと考えられる。一方、選択的なグルコシダーゼ阻害活性を持つ化合物の立体選択的合成法を確立することが出来た。未だ多くの課題を残しているものの、将来的には糖尿病治療における薬物選択肢の幅を広げることに貢献できるものと考えられる。
CYP3A4 の肝臓および腸管における誘導評価を行った 2 種の in vitro スクリーニング系から得られたデータを比較したところ、CYP3A4 には従来から考えられていたものとは異なる分子機序を見出した。その一つに CYP3A4 は核内受容体である PXR に結合することで誘導されることが知られていたが、本研究の過程でプロテインキナーゼの活性化により誘導されることを明らかにした。また、hiPSCs の成熟肝細胞への分化に重要な役割を担う因子の探索を行ってきた課程で、肝臓が成熟する過程で起きる胎児型 CYP3A7
から成人型 CYP3A4 への発現量の入れ替わり(スイッチ)の分子機序に肝特異的転写因子が関与していることを見出した。特にその転写因子の一つである HNF3βは CYP3A7 の発現を強く抑制するが、その発現は胎児肝臓で低く､成熟に伴って上昇することが分かっている。従って､胎児型 CYP3A7 から成人型 CYP3A4への発現スイッチは、この転写因子の成熟に伴う上昇によって CYP3A7 発現が抑制されていることが示唆された。

「研究テーマ２．老年性神経系疾患(老人性認知症、高齢者うつ病)改善薬の開発」
本研究での構造の基礎となる新規疎水性構造の医薬分子設計への応用の方法論は、第 16 回国際ホウ素化学会議での招待講演、日本薬学会の会報での解説など、普及に努めている（図書*2、論文*50、学会*158）。また、新規疎水性骨格としてのホウ素クラスターの計算科学的解析が、本プロジェクトを通じて生体分子とのドッキング研究を通じて発展した（論文*56, 67, 94, 96、学会*108）。本研究の新規リガンドが生物学的ツールとして生物科学研究に用いられてきている（論文*81）。特に、本研究との関連性もある神経細胞分化の研究にも用いられ、進展している（論文*95）。
候補化合物「BE360」はうつ病、認知症、骨粗鬆症のモデル動物に対して有効であった。さらに、この化合物を評価する際用いた精神疾患モデル動物については、生体機能と創薬シンポジウム 2016 および第16 回生物化学若手研究者セミナー（いずれも招待講演）で発表（学会*178, 203）し、臨床を反映したモデルであることが評価され産学連携に発展し精神疾患に対する治療薬・サプリメント開発および病態解明に繋げることができた。この「BE360」については、老化に伴い生じる気分障害、記憶力低下および寝たきりにつながる骨粗鬆症による骨折といった高齢者の QOL 低下に福音をもたらす可能性を示した。

「研究テーマ３．難治性疼痛に対する緩和医療法の確立」
難治性疼痛治療薬のターゲットとなりうる特異的受容体に関して、用途特許の申請が可能か検討中である。また、新規化合物に難治性疼痛に対する有効性を見出した場合、ならびに新規化合物に精神依存性が無いことを見出した場合は、それぞれ「難治性疼痛治療薬」ならびに「依存性の無い鎮痛薬」として、特許申請を行う予定である。
OPRM1A118G SNP, rs1799971 はアジア人において多い遺伝子多型であり受容体機能を変調させる多型であることから、オピオイドネットワークを介した免疫応答制御の解析において用いた Oprm1 SNP— 14 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L

A112G マウスは、μオピオイド受容体を標的とする種々の新規化合物の有効性評価において有用なモデ
ル動物の作製に寄与できる可能性が明らかとなった。

１２ キーワード（当該研究内容をよく表していると思われるものを８項目以内で記載してください。）（１） 創薬（４） 毒性発現予測（７） 難治性疼痛治療薬（２） 分子標的抗がん薬（５） エストロゲン受容体（８） 受容体遺伝子多型（３） ヌクレオシド誘導体（６） 老年性精神疾患治療薬

１３ 研究発表の状況（研究論文等公表状況。印刷中も含む。）
上記、１１(４)に記載した研究成果に対応するものには＊を、本報告書に全文を掲載した主要論文には文献番号に二重線を付した。
＜雑誌論文＞「研究テーマ１．新規がん分子標的治療薬の創製」*1. Synthesis of 2'-Aminouridine Derivatives as an Organocatalyst for Diels-Alder Reaction,
Wakamatsu, H.; Itoh, M.; Natori, Y.; Yoshimura, Y. Nucleosides Nucleotides Nucleic Acids, in press(査読有).
2. UDP-Glucuronosyltransferase (UGT) -Mediated Attenuations of Cytochrome P450 3A4 Activity:
UGT Isoform-Dependent Mechanism of Suppression, Miyauchi, Y.; Tanaka, Y.; Nagata, K.; Yamazoe,
Y.; Mackenzie, P.I.; Yamada, H.; Ishii, Y. British J. Pharmcol. 2020, 177, 1077-1089 (査読有).
3. A Novel Approach to Oxazole-Containing Diterpenoid Synthesis from Plant Roots: Salviamines E
and F, Narita, K.; Fujisaki, N.; Sakuma, Y.; Katoh, T. Org. Biomol. Chem. 2019, 17, 655-663 (査読有).
4. A Concise Approach for Producing Optically Pure Carboxylic Acid Segments for the Synthesis of
Bicyclic Depsipeptide Histone Deacetylase Inhibitors, Narita, K.; Sayar, N.; Saijo, K.; Ishioka, C.;
Katoh, T. Synthesis, 2019, 51, 1408-1418 (査読有).
5. Facile Total Synthesis of Thailandepsins D–F: Novel Bicyclic Depsipeptide Histone Deacetylase
Inhibitors Isolated from a Microorganism, Sayar, N.; Narita, K.; Katoh, T. Synthesis, 2019, 51,
1419-1426 (査読有).
6. In Vitro and In Vivo Antitumor Activity and the Mechanism of Siphonodictyal B in Human Colon
Cancer Cells, Chikamatsu, S.; Saijo, K.; Imai, H.; Narita, K.; Kawamura, Y.; Katoh, T.; Ishioka, C. Cancer
Medicine, 2019, 8, 5662-5672 (査読有).
*7. Catalytic Asymmetric Synthesis of Stereoisomers of 1-C-n-butyl-LABs for the SAR Study of
α-Glucosidase Inhibition, Natori, Y.; Sakuma, T.; Watanabe, H.; Wakamatsu, H.; Kato, A.; Adachi, I.;
Takahata, T.; Yoshimura, Y. Tetrahedron 2019, 75(20), 2866-2876 (査読有).
8. Conjugate Vaccine Produces Long-Lasting Attenuation of Fentanyl vs. Food Choice and Blocks
Expression of Opioid Withdrawal-Induced Increases in Fentanyl Choice in Rats, Townsend, A.; Blake,
S.; Faunce, K. E.; Hwang, C. S.; Natori, Y.; Zhou, B.; Bremer, P. T.; Janda, K. D.; Bank, M. L.
Neuropsychopharmacology 2019, 44, 1681-1689 (査読有).
9. A Chemically Contiguous Hapten Approach for a Heroin–Fentanyl Vaccine, Natori, Y.; Hwang, C. S.;
Lin, L.; Smith, L. C.; Zhou, B.; Janda K. D. Beilstein J. Org. Chem. 2019, 15, 1020-1031 (査読有).
10. Phosphorylation-Deficient Mutant of Retinoid X Receptor α at Thr167 Alters Fasting Response andEnergy Metabolism in Mice, Sueyoshi, T.; Sakuma, T.; Shindo, S.; Fashe, M.; Kanayama, T.; Ray, M.;
Moore, R.; Negishi, M. A. Lab. Invest. 2019, 99, 1470-1483 (査読有).
*11. A Novel Approach to Sesquiterpenoid Benzoxazole Synthesis from Mmarine Sponges: Nakijinols A, B
and E–G, Takeda, Y.; Nakai, K.; Narita, K.; Katoh, T. Org. Biomol. Chem., 2018, 16, 3639-3647 (査読有).
12. Total Syntheses of Liphagal: A Potent and Selective Phosphoinositide 3-Kinase α (PI3Kα) Inhibitor
from the Marine Sponge Aka Coralliphaga, Narita, K.; Katoh, T. Heterocycles, 2018, 96, 3-41 (査読有).
— 15 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L

13. Improved Admixture Vaccine of Fentanyl and Heroin Hapten Immunoconjugates: Antinociceptive
Evaluation of Fentanyl-Contaminated Heroin, Hwang, C. S.; Smith, L. C.; Natori, Y.; Ellis, B.; Zhou, B.;
Janda, K. D. ACS Omega 2018, 3, 11537-11543 (査読有).
*14. Synthesis of Various Heterocycles Having Dienamide Moiety by Ring–Closing Metathesis of Ene–ynamides, Wakamatsu, H.; Sasaki, Y.; Kawahata, M.; Yamaguchi, K.; Yoshimura, Y. Synthesis 2018,50(17), 3467-3486 (査読有).
15. Glycosylation Reactions Mediated by Hypervalent Iodine: Application to the Synthesis of
Nucleosides and Carbohydrates, Yoshimura, Y.; Wakamatsu, H.; Natori, Y.; Saito, Y.; Minakawa, N.
Beilstein J. Org. Chem. 2018, 14, 1595-1618 (査読有).
16. Synthesis of 4'-Thionucleosides as Antitumor and Antiviral Agents, Yoshimura, Y.; Saito, Y.; Natori,
Y.; Wakamatsu, H. Chem. Pharm. Bull. 2018, 66, 139–146 (査読有).
17. Efficient Syntheses of Cocaine Vaccines and Their in Vivo Evaluation, Kimishima, A.; Olson, M. E.;
Natori, Y.; Janda, K. D. ACS Med. Chem. Lett. 2018, 9, 411-416 (査読有).
18. Efficacious Vaccine against Heroin Contaminated with Fentanyl, Hwang, C. S.; Smith, L. C.; Natori, Y.;
Ellis, B.; Zhou, B.; Janda, K. D. ACS Chem. Neurosci. 2018, 9, 1269-1275 (査読有).
19. Practical Synthesis of 4'-Thioribonucleosides from L-Arabinose via Novel Reductive
Ring-Contraction Reaction and Pummerer-Type Thioglycosylation, Wakamatsu, H.; Nitta, K.; Shoji,N.; Natori, Y.; Saito, Y.; Yoshimura, Y. Curr. Protoc. Nucleic Acid Chem. 2018, 71, 1.43.1–1.43.12 (査読有).
20. Sulfotransferase 4A1 Increases its Expression in Mouse Neurons as they Mature, Hashiguchi, T.;
Shindo, S.; Chen, S. H.; Hong, J. S.; Negishi, M. Drug Metab Dispos. 2018, 46, 860-864 (査読有).
21. Synthetic Study toward Total Synthesis of Marine Diterpenoid Cristaxenicin A: Synthesis of
δ-Unsaturated Lactone Bearing α-Bulky Substituent, Nakai K.; Sayar, N.; Yoshimura Y.; Watanabe,
K. Journal of Tohoku Medical and Pharmaceutical University, 2017, 64, 57-63.
22. Unified Synthesis of the Marine Sesquiterpene Quinones (+)-Smenoqualone, (–)-Ilimaquinone,(+)-Smenospongine, and (+)-Isospongiaquinone, Katoh, T.; Atsumi, S.; Saito, R.; Narita, K.; Katoh, T.
Eur. J. Org. Chem., 2017, 3837–3849 (査読有).
23. Antitumor Activity and Pharmacologic Characterization of the Depsipeptide Analog as a Novel
Histone Deacetylase/Phosphatidylinositol 3-Kinase Dual Inhibitor, Saijo, K.; Imai, H.; Chikamatsu, S.;
Narita, K.; Katoh, T.; Ishioka C. Cancer Sci., 2017, 108, 1469–1475 (査読有).
24. Total Synthesis of Marine Sesquiterpene Quinones (+)-Cyclospongiaquinone-1 and(–)-Dehydrocyclospongiaquinone-1 with a Tetracyclic Benzo[a]xanthene Skeleton, Takeda, T.;
Narita, K.; Katoh, T. Eur. J. Org. Chem., 2017, 901–907 (査読有).
25. Development of a Glycosylation Reaction: A Key to Accessing Structurally Unique Nucleosides,
Yoshimura, Y. Heterocycles 2017, 94, 1625-1651 (査読有).
*26. Palladium-Catalyzed Regioselective Hydroarylation of Ynamides with Aryl Iodides: Easy Synthesis of
Various Substituted Enamides Containing Stilbene Derivatives, Wakamatsu, H.; Yanagisawa, R.;
Kimura, S.; Osawa, N.; Natori, Y.; Yoshimura, Y. Synlett 2017, 28, 2135-2138 (査読有).
27. Activated Brain-Derived Neurotrophic Factor/TrkB Signaling in Rat Dorsal and Ventral Hippocampi
Following 10-Day Electroconvulsive Seizure Treatment, Enomoto. S.; Shimizu, K.;, Nibuya, M.;,
Suzuki, E.; Nagata, K.; Kondo, T. Neurosci. Lett., 2017, 660, 45-50 (査読有).
*28. CYP3A4 Induction Mechanism of Polycyclic Aromatic Hydrocarbons Differs from that of Rifampicin
in PXR Binding Element, Aratsu, Y.; Odagiri, R.; Shoji, R.; Watanabe, K.; Kumagai, T.; Shindo, S.;
Sasaki, T.; Nagata, K. Fundam. Toxicol. Sci., 2017, 4, 229–239 (査読有).
*29. Effect of Health Foods on Cytochrome P450-Mediated Drug Metabolism, Sasaki, T.; Sato,Y.; Kumagai, T.; Yoshinari, K.; Nagata, K. J. Pharm. Health Care Sci., 2017,3, 14 (査読有).
30. Total Synthesis of the Depsipeptide FR901375 and Preliminary Evaluation of Its Biological Activity,
Narita, K.; Katoh, Y.; Ojima, K.; Dan, S.; Yamori, T.; Ito, A.; Yoshida, M.; Katoh, T. Eur. J. Org. Chem.,
2016, 5667–5677 (査読有).
*31. Synthesis and Biological Evaluation of Novel FK228 Analogues as Potential Isoform Selective HDAC
Inhibitors, Narita, K.; Matsuhara, K.: Itoh, J.; Akiyama, Y.; Dan, S.; Yamori, T.; Ito, A.; Yoshida, M.;— 16 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L

Katoh, T. Eur. J. Med. Chem., 2016, 121, 592–609 (査読有).
32. Low-Dose Spiruchostatin-B, a Potent Histone Deacetylase Inhibitor Enhances Radiation-Induced
Apoptosis in Human Lymphoma U937 Cells via Modulation of Redox Signaling, Rehman, M. U.;
Jawaid, P.; Zhao, Q. L.; Li, P.; Narita, K.; Katoh, T.; Shimizu, T.; Kondo, T. Free Radic. Res., 2016, 50,
596–610 (査読有).
*33. Enantioselective Total Synthesis of (−)-Siphonodictyal B and (+)-8-epi-Siphonodictyal B with
Phosphatidylinositol 3-Kinase Inhibitory Activity, Kikuchi, T.; Narita, K.; Saijo, K.; Ishioka, C.; Katoh,
T. Eur. J. Org. Chem., 2016, 5659–5666 (査読有).
*34. Enantioselective Total Synthesis of Mandelalide A and Isomandelalide A: Discovery of a Cytotoxic
Ring-Expanded Isomer, Veerasamy, N.; Ghosh, A.; Li, J.; Watanabe, K.; Serrill, D. J.; Ishmael, E. J.;
McPhail, L. K.; Carter, G. R. J. Am. Chem. Soc., 2016, 138, 770–773 (査読有).
*35. Stereoselective, Ag-Catalyzed Cyclizations To Access Polysubstituted Pyran Ring Systems:
Syntesis of C1-C12 Subunit of Madeirolide A, Watanabe, K.; Li, J.; Veerasamy, N.; Ghosh, A.; Carter,
G. R. Org. Lett., 2016, 18, 1744–1747 (査読有).
36. Total Synthesis of Thailandepsin B, a Potent HDAC Inhibitor Isolated from a Microorganism, Narita,
K.; Katoh T. Chem. Pharm. Bull., 2016, 64, 913–917 (査読有).
37. Docking Study and Biological Evaluation of Pyrrolidine-based Iminosugars as Pharmacological
Chaperones for Gaucher Disease, Kato, A.; Nakagome, I.; Sato, K.; Yamamoto, A.; Adachi, I.; Nash, R
J.; Fleet, G. W. J.; Natori, Y.; Watanabe, Y.; Imahori, I.; Yoshimura, Y.; Takahata, H.; Hirono, S. Org.
Biomol. Chem. 2016, 14, 1039-1048 (査読有).
38. Development of Stereoselective Synthesis of Biologically Active Nitrogen-heterocyclic Compounds:
Applications for Syntheses of Natural Product and Organocatalyst, Natori, Y.; Imahori, T.; Yoshimura,
Y. J. Synth. Org. Chem. Jpn. 2016, 74, 335-349 (査読有).
*39. Activation of p38 Mitogen-Activated Protein Kinase by Clotrimazole Induces Multidrug
Resistance-Associated Protein 3 Activation through a Novel Transcriptional Element, Sasaki, T.;
Inami, K.; Numata, Y.; Funakoshi, K.; Yoshida, M.; Kumagai, T.; Kanno, S.; Matsui, S.; Toriyabe, T.;
Yamazoe, Y.; Yoshinari, K.; Nagata, K. J. Pharmacol. Exp. Ther., 2016, 359, 102–109 (査読有).
*40. レポーター遺伝子導入培養細胞を用いた健康食品による CYP1A1/1A2 誘導の網羅的評価、熊谷健佐々木崇光、永田 清、医療薬学, 2016, 42, 701–708 (査読有).
*41. Indirubin, a Component of Ban-Lan-Gen, Activates CYP3A4 Gene Transcription through the Human
Pregnane X Receptor, Kumagai, T.; Aratsu, Y.; Sugawara, R.; Sasaki, T.; Miyairi, S.; Nagata, K. Drug
Metab. Pharmacokinet., 2016, 31, 139–145 (査読有).
42. Directed Synthesis of 2-Spiroindolines from Indole Derivatives by Formic Acid-Induced
N-Acyliminium Ion-Conjugated Diene Spirocyclization, Abe, H.; Miyagawa, N.; Hasegawa, S.;
Kobayashi, T.; Aoyagi, S.; Kibayashi, C.; Katoh, T.; Ito, H. Tetrahedron Lett., 2015, 56, 921–924 (査読有).
43. Biochemical, Biological and Structural Properties of Romidepsin (FK228) and Its Analogs as Novel
HDAC/PI3K Dual Inhibitors, Saijo, K.; Imamura, J.; Narita, K.; Oda, A.; Shimodaira, H.; Katoh, T.;
Ishioka, C. Cancer Sci., 2015, 106, 208–215 (査読有).
44. Catalytic Asymmetric Synthesis of (-)-E-δ-Viniferin via an Intramolecular C–H Insertion of
Diaryldiazomethane using Rh2(S-TFPTTL)4, Natori, Y.; Ito, M.; Anada, M.; Nambu, H.; Hashimoto,
S.Tetrahedron Lett. 2015, 56, 4324-4327 (査読有).
45. Construction of an Isonucleoside on a 2,6-Dioxobicyclo[3.2.0]-heptane Skeleton, Yoshimura, Y.;
Kobayashi, S.; Kaneko, H.; Suzuki, T.; Imamichi, T. Molecules 2015, 20, 4623-4634 (査読有).
46. Suppression of Cytochrome P450 3A4 Function by UDP-Glucuronosyltransferase 2B7 through a
Protein-Protein Interaction: Vooperative Roles of the Cytosolic Carboxyl-Terminal Domain and the
Luminal Anchoring Region, Miyauchi, Y.; Nagata, K.; Yamazoe, Y.; Mackenzie, PI.; Yamada, H.; Ishii, Y.
Mol. Pharmacol., 2015, 88, 800–812 (査読有).
*47. Simultaneous Evaluation of Human CYP3A4 and ABCB1 Induction by Reporter Assay in LS174T
Cells, Stably Expressing their Reporter Genes, Inami, K.; Sasaki, T.; Kumagai, T.; Nagata, K. Biopharm.
Drug Dispos., 2015, 36, 139–147, 2015 (査読有).
— 17 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L*48. Development of a Highly Reproducible System to Evaluate Inhibition of Cytochrome P450 3A4
Activity by Natural Medicines, Sato, Y.; Sasaki, T.; Takahashi, S.; Kumagai, T.; Nagata, K.
J. Pharm. Pharm. Sci., 2015, 18, 316–327 (査読有).
*49. 培養細胞を用いた健康食品と医薬品との薬物相互作用の網羅的調査 熊谷 健、佐々木崇光、佐藤 裕、髙橋昌悟、細川正清、頭金正博、松永民秀、大森 栄、永田 清、東京都病院薬剤師会雑誌（THPA）, 2015, 64, 14−22 (査読無).

「研究テーマ２．老年性神経系疾患(老人性認知症、高齢者うつ病)改善薬の開発」*50. ホウ素クラスターの形状と疎水性を利用した創薬 遠藤泰之、ファルマシア, 2020, 56, (査読有)51. Liver Hydrolysate Improves Depressive-Like Behavior in Olfactory Bulbectomized Mice:
Involvement of Hippocampal Neurogenesis through the AMPK/BDNF/CREB Pathway, Nakagawasai,O.; Yamada, K.; Odaira, T.; Takahashi, K.; Nemoto, W.; Sakuma, W.; Wakou, M.; Lin, J.R.; Tan-No, K. J.
Pharmacol. Sci., in press (査読有).
52. Downregulation of Spinal Angiotensin Converting Enzyme 2 is Involved in Neuropathic Pain
Associated with Type 2 Diabetes Mellitus in Mice, Yamagata, R.; Nemoto, W.; Nakagawasai, O.;
Takahashi, K.; Tan-No, K. Biochem. Pharmacol., 2020, 174, 113825 (査読有).
53. Effect of Spinal Angiotensin-Converting Enzyme 2 Activation on the Formalin-Induced Nociceptive
Response in Mice, Nemoto, W.; Yamagata, R.; Nakagawasai, O.; Nakagawa, K.; Hung, W.Y.; Fujita, M.;
Tadano, T.; Tan-No, K. Eur. J. Pharmacol., 2020, 872, 172950 (査読有).
*54. Role of Prefrontal Cortical 5-HT2A Receptors and Serotonin Transporter in the Behavioral Deficits in
Post-Pubertal Rats Following Neonatal Lesion of the Ventral Hippocampus, Mitazaki, S.;
Nakagawasai, O.; Onogi, H.; Watanabe, K.; Takahashi, K.; Tan-No, K.; Quirion, R.; Srivastava, L.K.;
Tadano, T. Behav. Brain Res., 2020, 377, 112226 (査読有).
*55. Anti-Cancer Activity of m-Carborane-Containing Trimethoxyphenyl Derivatives through Tubulin
Polymerization Inhibition, Kaise, A.; Endo, Y.; Ohta, K. Bioorg. Med. Chem., 2019, 27, 1139-1144 (査読有).
*56. ER Subtype Selectivity of m-Carborane Containing Phenols: C-Alkyl Groups on the m-Carborane
Cage Enhance ERα Selectivity, Ohta, K.; Ogawa, T.; Kato, K.; Oda, A.; Endo, Y. Bioorg. Med. Chem.
Lett., 2019, 29, 2290-2293 (査読有).
57. Design and Synthesis of Novel Breast Cancer Therapeutic Drug Candidates Based upon the
Hydrophobic Feedback Approach of Antiestrogens, Ohta, K.; Kaise, A.; Taguchi, F.; Aoto, S.; Ogawa,
T.; Endo, Y. Molecules, 2019, 24, 3966 (査読有)58. Benzolactam-Related Compounds Promote Apoptosis of HIV-Infected Human Cells via Protein
Kinase C–Induced HIV Latency Reversal, Matsuda, K.; Kobayakawa, T.; Tsuchiya, K.; Hattori, S.;
Nomura, W.; Gatanaga, H.; Yoshimura, K.; Oka, S.; Endo, Y.; Tamamura, H.; Mitsuya, H.; Maeda, K. J.
Biol. Chem., 2019, 294, 116-129 (査読有).
59. An Automated Microliter-Scale High-Throughput Screening System (MSHTS) for Real-Time
Monitoring of Protein Aggregation Using Quantum-Dot Nanoprobes, Sasaki, R.; Tainaka, R.; Ando, Y.;
Hashi, Y.; Deepak, H. V.; Suga, Y.; Murai, Y.; Anetai, M.; Monde, K.; Ohta, K.; Ito, I.; Kikuchi, H.; Oshima,Y.; Endo, Y.; Nakao, H.; Sakono, M.; Uwai, K.; Tokuraku, K. Scientific Reports, 2019, 9, 1-9 (査読有).
*60. Effect of Enterococcus Faecalis 2001 on Colitis and Depressive-Like Behavior in Dextran Sulfate
Sodium-Treated Mice: Involvement of the Brain-Gut Axis, Takahashi, K.; Nakagawasai, O.; Nemoto,W.; Odaira, T.; Sakuma, W.; Onogi, H.; Nishijima, H.; Furihata, R.; Nemoto, Y.; Iwasa, H.; Tan-No, K.;
Tadano, T. J. Neuroinflammation, 2019, 16, 201 (査読有).
*61. Mechanisms Underpinning AMP-Activated Protein Kinase-Related Effects on Behavior and
Hippocampal Neurogenesis in an Animal Model of Depression, Odaira, T.; Nakagawasai, O.;
Takahashi, K.; Nemoto, W.; Sakuma, W.; Lin, J.L.; Tan-No, K. Neuropharmacology, 2019, 150, 121-133(査読有).
*62. Prenatal Treatment with Methylazoxymethanol Acetate as a Neurodevelopmental Disruption Modelof Schizophrenia in Mice, Takahashi, K.; Nakagawasai, O.; Sakuma, W.; Nemoto, W.; Odaira, T.; Lin,
J.L.; Onogi, H.; Srivastava, L.K.; Tan-No, K. Neuropharmacology, 2019, 150, 1-14 (査読有).
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63. Anti-Hypersensitive Effect of Angiotensin (1-7) on Streptozotocin-Induced Diabetic Neuropathic
Pain in Mice, Ogata, Y.; Nemoto, W.; Yamagata, R.; Nakagawasai, O.; Shimoyama, S.; Furukawa, T.;
Ueno, S.; Tan-No, K. Eur. J. Pain, 2019, 23, 739-749 (査読有).
64. Etidronate Attenuates Tactile Allodynia by Spinal ATP Release Inhibition in Mice with Partial Sciatic
Nerve Ligation, Yamagata, R.; Nemoto, W.; Nakagawasai, O.; Hung, W.Y.; Shima, K.; Endo, Y.; Tan-No,
K. Naunyn Schmiedebergs Arch. Pharmacol., 2019, 392, 349-357 (査読有).
*65. Involvement of Catecholaminergic and GABAAergic Mediations in the Anxiety-Related Behavior inLong-Term Powdered Diet-Fed Mice, Yaoita, F.; Tsuchiya, M.; Arai, Y.; Tadano, T.; Tan-No, K.
Neurochem. Int., 2019, 124, 1-9 (査読有).
66. Synthesis, Structure-Activity Relationships, and Mechanistic Studies of 5-Arylazo-Tropolone
Derivatives as Novel Xanthine Oxidase (XO) Inhibitors, Sato, D.; Kisen, T.; Kumagai, M.; Ohta, K.
Bioorg. Med. Chem., 2018, 26, 536-542 (査読有).
*67. Novel Androgen Receptor Full Antagonists: Design, Synthesis, and a Docking Study of a Glycerol andAminoglycerol Derivatives that Contain p-Carborane Cages, Kaise, A.; Ohta, K.; Fujii, S.; Oda, A.;
Goto, T.; Endo, Y. Bioorg. Med. Chem., 2018, 26, 3805-3811(査読有).
68. Behavioral and Molecular Pharmacological Study of the Role of Angiotensin II in Spinal Pain
Transmission, Nemoto, W., Yakugaku Zasshi, 2018, 138, 1235-1240 (査読有).
69. Effect of Repeated Oral Administration of Chondroitin Sulfate on Neuropathic Pain Induced byPartial Sciatic Nerve Ligation in Mice, Nemoto, W.; Yamada, K.; Nakagawasai, O.; Ogata, Y.; Chiba, M.;
Yamagata, R.; Sakurai, H.; Tan-No, K. J. Pharmacol. Sci., 2018, 137, 403-406 (査読有).
70. Antidepressant-Like Effect of Aripiprazole via 5-HT1A, D1, and D2 Receptors in the Prefrontal Cortex
of Olfactory Bulbectomized Mice, Takahashi, K.; Nakagawasai, O.; Nemoto, W.; Odaira, T.; Sakuma, W.;
Tan-No, K. J. Pharmacol. Sci., 2018, 137, 241-247 (査読有).
*71. Memantine Ameliorates Depressive-Like Behaviors by Regulating Hippocampal Cell Proliferation and
Neuroprotection in Olfactory Bulbectomized Mice, Takahashi, K.; Nakagawasai, O.; Nemoto, W.;
Kadota, S.; Isono, J.; Odaira, T.; Sakuma, W.; Arai, Y.; Tadano, T.; Tan-No, K. Neuropharmacology,
2018, 137, 141-155 (査読有).
72. Kappa Opioid Receptor Agonist Administration in Olfactory Bulbectomized Mice Restores Cognitive
Impairment through Cholinergic Neuron Activation, Takahashi, K.; Nakagawasai, O.; Sugawara, M.;
Sato, A.; Nemoto, W.; Tadano, T.; Tan-No, K. Biol. Pharm. Bull., 2018, 41, 957-960 (査読有).
73. Neutrophils Provide a Favorable IL-1-Mediated Immunometabolic Niche that Primes GLUT4
Translocation and Performance in Skeletal Muscles, Tsuchiya, M.; Sekiai, S.; Hatakeyama, H.; Koide,
M.; Chaweewannakorn, C.; Yaoita, F.; Tan-No, K.; Sasaki, K.; Watanabe, M.; Sugawara, S.; Endo, Y.;
Itoi, E.; Hagiwara, Y.; Kanzaki, M. Cell Rep., 2018, 23, 2354-2364 (査読有).
74. Involvement of Peripheral Alpha2A Adrenoceptor in the Acceleration of Gastrointestinal Transit andAbdominal Visceral Pain Induced by Intermittent Deprivation of REM Sleep, Yaoita, F.; Muto, M.;
Murakami, H.; Endo, S.; Kozawa, M.; Tsuchiya, M.; Tadano, T.; Tan-No, K. Physiol. Behav., 2018, 186,
52–61 (査読有).
*75. Design and Synthesis of p-Carborane-Containing Sulfamates as Multitarget Anti-Breast Cancer
Agents, Kaise, A.; Ohta, K.; Shirata, C.; Endo, Y. Bioorg. Med. Chem., 2017, 25, 6417–6426 (査読有).
*76. Novel p-Carborane-Containing Multitarget Anticancer Agents Inspired by the Metabolism of
17β-Estradiol, Kaise, A.; Ohta, K.; Endo, Y. Bioorg. Med. Chem., 2017, 25, 6371–6378 (査読有).
*77. Design and Synthesis of Iodocarborane-Containing Ligands with High Affinity and Selectivity toward
ERβ, Ohta, K.; Ogawa, T.; Endo, Y. Bioorg. Med. Chem. Lett., 2017, 27, 4030–4033 (査読有).
78. Structure Activity Relations of Rosmarinic acid Derivatives for the Amyloid β Aggregation Inhibition
and Antioxidant Properties, Taguchi, R.; Hatayama, K.; Takahashi, T.; Hayashi, T.; Sato, Y.; Sato, D.;
Ohta, K.; Nakano, H.; Seki, C.; Endo, Y.; Tokuraku, K.; Uwai, K. Eur. J. Med. Chem., 2017, 138,
1066–1075 (査読有).
79. Time-Dependent Role of Prefrontal Cortex and Hippocampus on Cognitive Improvement by
Aripiprazole in Olfactory Bulbectomized Mice, Takahashi, K.; Nakagawasai, O.; Nemoto, W.; Odaira, T.;
Arai, Y.; Hisamitsu, T.; Tan-No, K. Eur. Neuropsychopharmacol., 2017, 27, 1000–1010 (査読有).
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80. Inhibitory Effect of Angiotensin (1-7) on Angiotensin III-Induced Nociceptive Behaviour in Mice,
Nemoto, W.; Yamagata, R.; Ogata, Y.; Nakagawasai, O.; Tadano, T.; Tan-No, K. Neuropeptides, 2017,65, 71–76 (査読有).
*81. BA321, a Novel Carborane Analog that Binds to Androgen and Estrogen Receptors, Acts as a New
Selective Androgen Receptor Modulator of Bone in Male Mice, Watanabe, K.; Hirata, M.; Tominari, T.;
Matsumoto, C.: Endo, Y.; Murphy, G.; Nagase, H.; Inada, M.; Miyaura, C. Biochem. Biophys. Res.
Commun., 2016, 478, 279–285 (査読有).
*82. Design, Synthesis, and Anti-Proliferative Activity of 1-(4-Methoxyphenyl)-12-Hydroxymethylp-Carborane Derivatives, Kaise, A.; Ohta, K.; Endo, Y. Eur. J. Med. Chem., 2016, 122, 257–263 (査読有).
83. Symmetric 4,4'-(Piperidin-4-ylidenemethylene)bisphenol Derivatives as Novel Tunable Estrogen
Receptor (ER) Modulators, Sato, M.; Ohta, K.; Kaise, A.; Aoto, S.; Endo, Y. Bioorg. Med. Chem. 2016,
24, 1089–1094 (査読有).
*84. BE360, a New Selective Estrogen Receptor Modulator, Produces Antidepressant and Antidementia
Effects through the Enhancement of Hippocampal Cell Proliferation in Olfactory Bulbectomized
Mice, Nakagawasai, O.; Nemoto, W.; Onogi, H.; Moriya, T.; Lin, J-R. Odaira, T.; Yaoita, F.; Ogawa, T.;
Ohta, K.; Endo, Y.; Tan-No, K. Behav. Brain Res., 2016, 297, 315–322 (査読有).
85. Alterations in Behavioral Responses to Dopamine Agonists in Olfactory Bulbectomized Mice:
Relationship to Changes in the Striatal Dopaminergic System, Takahashi, K.; Nakagawasai, O.;
Nemoto, W.; Nakajima, T.; Arai, Y.; Hisamitsu, T.; Tan-No, K. Psychopharmacology, 2016, 233,
1311–1322 (査読有).
86. The Bisphosphonates Clodronate and Etidronate Exert Analgesic Effects by Acting on Glutamateand/or ATP-Related Pain Transmission Pathways, Shima, K.; Nemoto, W.; Tsuchiya, M.; Tan-No, K.;
Takano-Yamamoto, T.; Sugawara, S.; Endo, Y. Biol. Pharm. Bull., 2016, 39, 770–777 (査読有).
87. ERK5 Induces Ankrd1 for Catecholamine Biosynthesis and Homeostasis in Adrenal Medullary Cells.,
Obara, Y.; Nagasawa, R.; Nemoto, W.; Pellegrino, MJ.; Takahashi, M.; Habecker, BA.; Stork, PJS.;
Ichiyanagi, O.; Ito, H.; Tomita, Y.; Ishii, K.; Nakahata, N. Cell. Signal., 2016, 28, 177-189 (査読有).
88. Alterations in Behavioral Responses to Dopamine Agonists in Olfactory Bulbectomized Mice:
Relationship to Changes in the Striatal Dopaminergic System, Takahashi, K.; Nakagawasai, O.;
Nemoto, W.; Nakajima, T.; Arai, Y.; Hisamitsu, T.; Tan-No, K. Psychopharmacology, 2016, 233,
1311–1322 (査読有).
89. Involvement of Spinal Angiotensin II System in Streptozotocin-Induced Diabetic Neuropathic Pain inMice, Ogata, Y.; Nemoto, W.; Nakagawasai, O.; Yamagata, R.; Tadano, T.; Tan-No, K. Mol. Pharmacol.,2016, 90, 205–213 (査読有).
90. Chondroitin Sulfate Attenuates Formalin-Induced Persistent Tactile Allodynia, Nemoto, W.; Yamada,K.; Ogata, Y.; Nakagawasai, O.; Onodera, K.; Sakurai, H.; Tan-No, K. J. Pharmacol. Sci., 2016, 131,
275–278 (査読有).
91. Effects of Methylphenidate on the Impairment of Spontaneous Alternation Behavior in Mice
Intermittently Deprived of REM Sleep, Niijima-Yaoita, F.; Nagasawa, Y.; Tsuchiya, M.; Arai, Y.;
Tadano, T.; Tan-No, K. Neurochem. Int., 2016, 100, 128–137 (査読有).
*92. 睡眠や食習慣の乱れに起因した精神神経障害動物モデルの作製、八百板富紀枝 YAKUGAKUZASSHI, 2016, 136, 895–904 (査読有).
93. 断続的なレム断眠負荷による病態動物モデルの作製、八百板富紀枝 BIO Clinica, 2016, 31,
993–995 (査読無).
*94. Estrogenic Activity of Bis(4-hydroxyphenyl)methanes with Cyclic Hydrophobic Structure, Kojima, K.;
Ogawa, T.; Kitao, S.; Sato, M.; Oda, A.; Ohta, K.; Endo, Y. Bioorg. Med. Chem., 2015, 23, 6900–6911(査読有).
*95. Activation of RXR/PPARγ Underlies Neuroprotection by Bexarotene in Ischemic Stroke, Certo, M.;
Endo, Y.; Ohta, K.; Sakurada, S.; Bagetta, G.; Amantea, D. Pharmacol. Res., 2015, 102, 298–307 (査読有).
*96. Design and Synthesis of Carborane-Containing Estrogen Receptor-Beta (ERβ)-Selective Ligands,— 20 —

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Ohta, K.; Ogawa, T.; Oda, A.; Kaise, A.; Endo, Y. Bioorg. Med. Chem. Lett, 2015, 25, 4174–4178 (査読有).
*97. Synthesis and Biological Evaluation of Novel m-Carborane-Containing Estrogen Receptor Partial
Agonists as SERM Candidates, Ohta, K.; Ogawa, T.; Kaise, A.; Endo, Y. Bioorg. Med. Chem. Lett, 2015,
25, 3213–3216 (査読有).
98. Structure–Activity Relationship Study of Diphenylamine-Based Estrogen Receptor (ER)
Antagonists, Ohta, K.; Chiba, Y.; Kaise, A.; Endo, Y. Bioorg. Med. Chem., 2015, 23, 861–867 (査読有).
99. Involvement of p38 MAPK Activation Mediated through AT1 Receptors on Spinal Astrocytes and
Neurons in Angiotensin II- and III-Induced Nociceptive Behavior in Mice, Nemoto, W.; Ogata, Y.;
Nakagawasai, O.; Yaoita, F.; Tadano, T.; Tan-No, K. Neuropharmacology, 2015, 99, 221–231 (査読有).
100. Liver Hydrolysate Attenuates the Sickness Behavior Induced by Concanavalin A in Mice,
Nakagawasai, O.; Yamada, K.; Nemoto, W.; Sato, S.; Ogata, Y.; Miya, K.; Sakurai, H.; Tan-No, K. J.
Pharmacol. Sci., 2015, 127, 489–492 (査読有).
101. The Intrathecal Administration of Losartan, an AT1 Receptor Antagonist, Produces an
Antinociceptive Effect through the Inhibiton of p38 MAPK Phosphorylation in the Mouse FormalinTest, Nemoto, W.; Ogata, Y.; Nakagawasai, O.; Yaoita, F.; Tadano, T.; Tan-No, K. Neurosci. Lett., 2015,
585, 17–22 (査読有).

「研究テーマ３．難治性疼痛に対する緩和医療法の確立」
102. Anxiolytic-like Effects of Bergamot Essential Oil are Insensitive to Flumazenil in Rats, Rombolà, L.;
Scuteri, D.; Adornetto, A.; Straface, M.; Sakurada, T.; Sakurada, S.; Mizoguchi, H.; Corasaniti, M.T.;
Bagetta, G.; Tonin, P.; Morrone: L.A. Evid. Based Complement. Alternat. Med., 2019, Volume 2019,
Article ID 2156873 1-6 (査読有).
103. Neuropharmacology of the Neuropsychiatric Symptoms of Dementia and Role of Pain: Essential Oilof Bergamot as a Novel Therapeutic Approach, Scuteri, D.; Rombolà, L.; Morrone, L.A.; Bagetta, G.;
Sakurada, S.; Sakurada, T.; Tonin, P.; Corasaniti, M.T. Int. J. Mol. Sci., 2019, 20, E3327 1-13 (査読有).
104. Neuropharmacological Properties of the Essential Oil of Bergamot for the Clinical Management ofPain-related BPSDs, Scuteri, D.; Rombola, L.; Tridico, L.; Mizoguchi, H.; Watanabe, C.; Sakurada, T.;
Sakurada, S.; Corasaniti, M.T.; Bagetta, G.; Morrone, L.A. Curr. Med. Chem., 2019, 26, 3764-3774 (査読有).
*105. 脳と喘息病態～精神的ストレスの受容と気管支喘息の増悪を繋ぐ脳から肺への情報伝達～、宮坂智充、川上佳織、奥山-土橋 佳織、河野資、高橋知子、大野勲、『呼吸』e レポート, 2019, 3, 1-9（査読無）*106. μオピオイド受容体の多様性, 溝口広一 東北医科薬科大学研究誌, 2018, 65, 1-6 (査読有).
107. Possible Involvement of the Peripheral µ-Opioid System in Antinociception Induced by Bergamot
Essential Oil to Allodynia after Peripheral Nerve Injury, Komatsu, T.; Katsuyama, S.; Uezono, Y.;
Sakurada, C.; Tsuzuki, M.; Hamamura, K.; Bagetta, G.; Sakurada, S.; Sakurada, T. Neurosci. Lett.,
2018, 686, 127-132 (査読有).
108. Antinociceptive Effect of Inhalation of the Essential Oil of Bergamot in Mice, Scuteri, D.; Crudo, M.;
Rombolà, L.; Watanabe, C.; Mizoguchi, H.; Sakurada, S.; Sakurada, T.; Greco, R.; Corasaniti, M.T.;
Morrone, L.A.; Bagetta, G. Fitoterapia, 2018, 129, 20-24 (査読有).
109. ETosis-Derived DNA Trap Production in Middle Ear Effusion is a Common Feature of Eosinophilic
Otitis Media, Ohta, N.; Ueki, S.; Konno, Y.; Hirokawa, M.; Kubota, T.; Tomioka-Matsutani, S.; Suzuki, T.;
Ishida, Y.; Kawano, T.; Miyasaka, T.; Takahashi, T.; Suzuki, T.; Ohno, I.; Kakehata, S.; Fujieda, S.
Allergol. Int., 2018, 67, 414-416 （査読有）.
*110. 女性における喘息重症化メカニズムの解明に向けた基礎研究：樹状細胞の性差、宮坂智充、増田
千愛、川上佳織、高橋知子、大野勲、アレルギーの臨床, 2018, 38, 51(351)-55(355) (査読有).
*111. 成人女性における喘息重症化病態を基礎研究から考える：性特異的 Th2 型免疫応答を担う
CD103+樹状細胞の役割、宮坂智充、増田千愛、川上佳織、高橋知子、大野勲、アレルギーの臨床,2018, 38, 55(563)-59(567) (査読有).
*112. Increased Susceptibility to Allergic Asthma with the Impairment of Respiratory Tolerance Caused by— 21 —

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Psychological Stress, Kawano, T.; Ouchi, R.; Ishigaki, T.; Masuda, C.; Miyasaka, T.; Ohkawara, Y.; Ohta,
N.; Takayanagi, M.; Takahashi, T.; Ohno, I. Int. Arch. Allergy Immunol., 2018, 177, 1-15 （査読有）.
113. The expression of 11 beta hydroxysteroid dehydrogenase in severe allergic rhinitis, Ohta, N.;
Noguchi, N.; Takahashi, T.; Suzuki, T.; Kakuta, R.; Suzuki, Y.; Awataguchi, T.; Suzuki, T.; Takahashi, Y.;
Shoji, F.; Wada, K.; Kawano, T.; Ohno, I.; Kusano, Y.; Miyasaka, T.; Osafune, H.; Matsutani, S.;
Yaginuma, Y.; Ishida, Y.; Saito, Y.; Yamazaki, M.; Ikeda, R. Otolaryngol. Pol. 2018, 73, 18-22 （査読有）.
*114. Maternal Separation as Early-Life Stress Causes Enhanced Allergic Airway Responses by Inhibiting
Respiratory Tolerance in Mice, Ouchi, R.; Kawano, T.; Yoshida, H.; Ishii, M.; Miyasaka, T.; Ohkawara,
Y.; Takayanagi, M.; Takahashi, T.; Ohno, I. Tohoku. J. Exp. Med, 2018, 246, 155-165 （査読有）.
*115. Sex-Based Differences in CD103+ Dendritic Cells Promote Female-Predominant Th2 Cytokine
Production During Allergic Asthma, Masuda, C.; Miyasaka, T.; Kawakami, K.; Inokuchi, J.; Kawano, T.;
Dobashi-Okuyama K.; Takahashi, T.; Takayanagi, M.; Ohno, I. Clin. Exp. Allergy., 2018, 48, 379-393(査読有).
116. Combined Treatment with D-Allose, Docetaxel and Radiation Inhibits the Tumor Growth in an In Vivo
Model of Head and Neck Cancer, Hoshikawa, H.; Kamitori, K.; Indo, K.; Mori, T.; Kamata, M.; Takahashi,T.; Tokuda, M. Oncol. Lett., 2018, 15, 3422–3428 (査読有).
*117. The Interplay between Neuroendocrine Activity and Psychological Stress-Induced Exacerbation ofAllergic Asthma, Miyasaka, T.; Dobashi-Okuyama, K.; Takahashi, T.; Takayanagi, M.; Ohno, I. Allergol.
Int., 2018, 67, 32–42 (査読有).
118. Aromatherapy and Aromatic Plants for the Treatment of Behavioural and Psychological Symptomsof Dementia in Patients with Alzheimer's Disease: Clinical Evidence and Possible Mechanisms,
Scuteri, D.; Morrone, L.A.; Rombolà, L.; Avato, P.R.; Bilia, A.R.; Corasaniti, M.T.; Sakurada, S.;
Sakurada, T.; Bagetta, G. Evid. Based Complement. Alternat. Med., 2017, Volume 2017, Article ID
9416305 1-8 (査読有).
119. Antinociceptive Effect of Tebanicline for Various Noxious Stimuli-Induced Behaviours in Mice,
Hayashi, T.; Katsuyama, S.; Orito, T.; Suzuki, T.; Sakurada, S. Neurosci. Lett., 2017, 638, 46–50 (査読有).
120. The Involvement of Spinal Release of Histamine on Nociceptive Behaviors Induced by Intrathecally
Administered Spermine, Mizoguchi, H.; Watanabe, C.; Hayashi, T.; Iwata, Y.; Watanabe, H.; Katsuyama,S.; Hamamura, K.; Sakurada, T.; Ohtsu, H.; Yanai, K.; Sakurada, S. Eur. J. Pharmacol., 2017, 800, 9-15
(査読有).
121. Bergamot Essential Oil Attenuates Anxiety-Like Behaviour in Rats, Rombolà, L.; Tridico, L.; Scuteri,
D.; Sakurada, T.; Sakurada, S.; Mizoguchi, H.; Avato, P.; Corasaniti, M.T.; Bagetta, G.; Morrone, L. A.
Molecules, 2017, 22, E614: 1–11 (査読有).
122. Opioids Resistance in Chronic Pain Management, Morrone, L.A.; Scuteri, D.; Rombolà, L.; Mizoguchi,H.; Bagetta, G. Curr. Neuropharmacol., 2017, 15, 444–456 (査読有).
*123. Neuropsychiatry Phenotype in Asthma: Psychological Stress-Induced Alterations of the
Neuroendocrine-Immune System in Allergic Airway Inflammation, Ohno, I. Allergol. Int., 2017, 66,
S2–S8 (査読有).
*124. The Interrelationship between Asthma and Brain Activities: Psychological Stress-Related Asthama
as a New Asthama Phenotype, Ohno, I. Arerugi., 2017, 66, 153–160 (査読有).
125. Intrathecal Morphine-3-Glucuronide-Induced Nociceptive Behavior via δ2 Opioid Receptors in the
Spinal Cord, Komatsu, T.; Katsuyama, S.; Nagase, H.; Mizoguchi, H.; Sakurada, C.; Tsuzuki, M.;
Sakurada, S.; Sakurada. T. Pharmacol. Biochem. Behav., 2016, 140, 68–74 (査読有).
126. Involvement of Spinal Glutamate in Nociceptive Behavior Induced by Intrathecal Administration of
Hemokinin-1 in Mice, Watanabe, C.; Mizoguchi, H.; Bagetta, G.; Sakurada, S. Neurosci. Lett., 2016,
617, 236–239 (査読有).
127. ラベンダーオイルの末梢局所皮下投与によるパクリタキセル誘発性末梢神経障害性疼痛抑制効果の検討、勝山 壮、音羽 亮、武井佐和子、櫻田 忍、倉本敬二、日本未病システム学会雑誌, 2016,
22, 47-53 (査読有).
128. Analgesic Effects of 1st Generation Anti-histamines in Mice, Takahashi, M.; Shima, K.; Tsuchiya, M.;— 22 —

（様式 2）
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Hagiwara, Y.; Mizoguchi, H.; Sakurada, S.; Sugawara, S.; Fujita, T.; Tadano,T.; Watanabe, M.; Fukumoto,S.; Endo, Y. Biol. Pharm. Bull., 2016, 39, 620–624 (査読有).
129. Rational Basis for the Use of Bergamot Essential Oil in Complementary Medicine to Treat Chronic
Pain, Rombolà, L.; Amantea, D.; Russo, R.; Adornetto, A.; Berliocchi, L.; Tridico, L.; Corasaniti, M.T.;
Sakurada, S.; Sakurada, T.; Bagetta, G.; Morrone, L.A. Mini Rev. Med. Chem., 2016, 16, 721–728 (査読有).
*130. The Involvement of Central Nervous System Histamine Receptors in Psychological Stress-Induced
Exacerbation of Allergic Airway Inflammation in Mice, Miyasaka, T.; Okuyama-Dobashi, K.; Masuda,
C.; Iwami, S.; Sato, M.; Mizoguchi, H.; Kawano, T.; Ohkawara, Y.; Sakurada, S.; Takayanagi, M.; Ohno, I.
Allergol. Int., 2016, 65, S38–S44 (査読有).
131. Human CD4− CD8− Invariant Natural Killer T Cells Promote IgG Secretion from B Cells Stimulatedby Cross-Linking of Their Antigen Receptors, Miyasaka, T., Watanabe, Y., Akahori, Y., Miyamura, N.,
Ishii, K., Kinjo, Y., Miyazaki, Y., Liu, T.-Y., Uemura, Y. and Kawakami, K. World Journal of Vaccines.,
2016, 6, 34–41 (査読有).
*132. CD8+ T Cells Mediate Female-Dominant IL-4 Production and Airway Inflammation in Allergic
Asthma, Ito, C.; Okuyama-Dobashi, K.; Miyasaka, T.; Masuda, C.; Sato, M.; Kawano, T.; Ohkawara, Y.;
Kikuchi, T.: Takayanagi, M.; Ohno, I. PLoS One., 2015, 10, e0140808 (査読有)*133. ストレスによる喘息増悪の病態、宮坂智充、大野 勲、喘息, 2015, 28, 133–139 (査読有).
*134. ストレス喘息； asthma nervosa から新たな喘息フェノタイプへ、大野 勲、宮坂智充、山内広平、中村 豊、アレルギーの臨床, 2015, 35, 62–65 (査読有).

＜図書＞
1. 中分子創薬に資するペプチド・核酸・糖鎖の合成技術、「新規グリコシル化反応の開発–Pummerer型チオグリコシル化反応の開発と展開–」、若松秀章、名取良浩、斎藤有香子、吉村祐一、シーエム
シー出版、2018, 2, (p.181-188). (ISBN: 978-4-7813-1320-7)*2. Boron-Based Compounds: Potential and Emerging Applications in Medicine, ed.by Vinas, C.;
Hey-Hawkins, E. Chapter 1. Carboranes as Hydrophobic Pharmacophores: Applications for Designof Nuclear Receptor Ligands. p.3-19, Endo, Y., Wiley, July 2018. ISBN: 978-1-119-27555-8
2017
3. 基礎有機化学問題集 第２版、渡邉一弘、加藤正、他（共著）、廣川書店、2
4. トワイクロス先生の緩和ケア処方薬 第２版、溝口広一（武田文和、鈴木 勉 監訳）、医学書院、
2017、36 (p.581-593, p.595-617).
5. 疾患薬理学、溝口広一（成田 年 監修）、Neo Medical、2
2016、13 (p. 19-31).
6. Aromatherapy: Basic Mechanisms and Evidence Based Clinical Use, Watanabe, C.; Mizoguchi, H.;
Sakurada, S. (Edited by Bagetta, G.; Cosentino, M.; Sakurada, T.), CRC Press, 2015, 18 (p.251-268).

＜学会発表＞「研究テーマ１．新規がん分子標的治療薬の創製」*1. 抗がん活性を有する 9-デオキシキセニアラクトール C の合成研究、佐藤廣河、成田紘一、吉村
祐一、渡邉一弘、日本薬学会第 140 年会、京都、2020 年 3 月.
2. 抗原虫活性を有するクリスタキセニシン A の全合成研究、分銅和香、中居啓陽、Sayar Noel、佐藤廣河、成田紘一、吉村祐一、渡邉一弘、日本薬学会第 140 年会、京都、2020 年 3 月.
3. 抗炎症作用を有するメロテルペン誘導体クリソゲンエステルの合成研究、佐々木里菜、渡邉一弘、
佐藤廣河、成田紘一、吉村祐一、日本薬学会第 140 年会、京都、2020 年 3 月.
4. スクアレン合成酵素阻害剤ビサボスクアール A の合成研究、吉田昌太郎、成田紘一、佐藤廣河、
渡邉一弘、吉村祐一、日本薬学会第 140 年会、京都、2020 年 3 月.
5. アルキルヒドロキノン天然物ビオラセイド A および C の簡便合成、成田紘一、木村龍平、佐藤廣河、
渡邉一弘、吉村祐一、日本薬学会第 140 年会、京都、2020 年 3 月.
*6. エンイナミドを基質とする閉環メタセシスの新たなアプローチと複素環化合物合成への応用、若松
秀章、小野賛、髙橋将典、吉村祐一、日本薬学会第 140 年会、京都、2020 年 3 月.

*7. 4’-置換ヌクレオシド誘導体の光学分割、遠藤柚季乃、若松秀章、伊藤恭平、斎藤有香子、名取良— 23 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L

浩、吉村祐一、日本薬学会第 140 年会、京都、2020 年 3 月.

*8. 超原子価ヨウ素とジフェニルジセレニドを用いた分子間反応による含窒素複素環化合物の合成法の開発、鈴木郁、名取良浩、萬浩太、若松秀章、斎藤有香子、吉村祐一、日本薬学会第 140 年会、
京都、2020 年 3 月.
9. 4’-置換 4’-チオリボシルチミン誘導体の合成、前田璃音、若松秀章、庄子希望、名取良浩、吉村祐一、日本薬学会第 140 年会、京都、2020 年 3 月.
10. GM3 セラミド部の類似体である長鎖脂肪酸の合成研究、名取良浩、若松秀章、伊藤瑛將、袰地陽太、金野美里、斎藤有香子、井ノ口仁一、吉村祐一、日本薬学会第 140 年会、京都、2020 年 3 月.
11. スクアレン合成酵素阻害剤 ビザボスクアール A の合成研究、吉田昌太郎、成田紘一、渡邉一
弘、吉村祐一、日本薬学会第 139 年会，千葉，2019 年 3 月.
*12. 抗がん活性を有する 9–デオキシキセニアラクトール C の合成研究、佐々木里菜、佐藤廣河、成田紘一、渡邉一弘、吉村祐一、創薬懇話会 2019 in 秋保、仙台、2019 年 6 月.
13. スクアレン合成酵素阻害剤 ビザボスクアール A の合成研究、吉田昌太郎，成田紘一、佐藤廣
河、渡邉一弘、吉村祐一、第 58 回日本薬学会東北支部大会、仙台、2019 年 10 月.
14. アルキルヒドロキノン天然物 ビオラセイド A の簡便合成、成田紘一、木村龍平、佐藤廣河、渡邉
一弘、吉村祐一、第 58 回日本薬学会東北支部大会、仙台、2019 年 10 月.
15. フェンタニルが混入したヘロインに対するワクチンの開発研究、名取良浩、Candy S. Hwang、Lauren
C. Smith、Beverly Ellis、Bin Zhou、Kim D. Janda、第 58 回日本薬学会東北支部大会、仙台、2019 年
10 月.
*16. 4'-置換ヌクレオシド誘導体の光学分割、遠藤柚季乃、若松秀章、伊藤恭平、斎藤有香子、名取良浩、吉村祐一、第 58 回日本薬学会東北支部大会、仙台、2019 年 10 月.
*17. 超原子価ヨウ素とジフェニルジセレニドを用いた含窒素複素環化合物の合成法の開発、鈴木郁、名取良浩、萬浩太、若松秀章、斎藤有香子、吉村祐一、創薬懇話会 2019 in 秋保、仙台、2019 年 6 月.
*18. 縮環/アルドールタンデム反応を利用した 4'-置換 4'-チオヌクレオシドの合成、前田璃音、若松秀章、庄子希望、名取良浩、吉村祐一、日本薬学会第 139 年会、千葉、2019 年 3 月.
*19. エンイナミドの閉環メタセシスによる様々な複素環化合物の合成、若松秀章、佐々木好美、川幡正
俊、山口健太郎、吉村祐一、日本薬学会第 139 年会、千葉、2019 年 3 月.
*20. 超原子価ヨウ素とジフェニルジセレニドを用いた分子内環化反応による含窒素複素環化合物の合成法の開発、名取良浩、鈴木郁、萬浩太、須玉夏海、若松秀章、斎藤有香子、吉村祐一、日本薬学会第 139 年会、千葉、2019 年 3 月.
21. CYP26A1 レポーター遺伝子を用いたレチノイン酸の低濃度測定法の開発、永田 清、第 1 回 内外
環境応答研究会、富山、2019 年 10 月.
22. 非リン酸化 ERα（S216A）導入マウスを用いたエストロゲン受容体の新規機能の解析、進藤佐和子、
第 1 回 内外環境応答研究会、富山、2019 年 10 月.
*23. 健康食品による薬物相互作用解析、熊谷 健、令和元年 内外環境応答・代謝酵素研究会、仙台、
2019 年 9 月.
*24. 肝成熟に伴う CYP3A7 の発現低下に HNF3β が関与する? 白井晃太朗 令和元年 内外環境応答・代謝酵素研究会、仙台、2019 年 9 月.
25. UDP-グルクロン酸転移酵素（UGT）によるシトクロム P450 3A4 活性の抑制：UGT 分子種依存的な抑制機序とラット肝臓を用いた in vivo 解析、宮内 優、永田 清、山添 康、Peter MACKENZIE、田中嘉孝、石井祐次、第 46 回日本毒性学会学術年会、徳島、2019 年 6 月.
*26. プロポリス含有健康食品による CYP 誘導評価と薬物相互作用、熊谷 健、星 利香、長田大輝、渡邉夢実、進藤佐和子、永田 清、第 46 回日本毒性学会学術年会、徳島、2019 年 6 月.
27. ベンゾオキサゾール骨格を有するセスキテルペン ナキジノール A 及び B の合成、武田由貴、加藤武、熱海 秀、成田紘一、加藤 正、日本薬学会第 138 年会、金沢、2018 年 3 月.
28. 海洋天然物 Cristaxenicin A および Plumisclerin A の合成研究、Noel Sayar、中井啓陽、吉村祐一、
渡邉一弘、日本薬学会第 138 年会、金沢、2018 年 3 月.
29. 海洋由来ジテルペン系天然物 Cristaxenicin A の合成研究、中井啓陽、Noel Sayar、吉村祐一、渡邉一弘、日本薬学会第 138 年会、金沢、2018 年 3 月.
— 24 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L*30. 海洋性ジテルペノイド 9-デオキシキセニアラクトール C の合成研究、渡邉一弘、高橋央宜、吉村祐一、日本薬学会第 138 年会、金沢、2018 年 3 月.
31. フェンタニルで汚染されたヘロインに対するワクチンの開発研究、名取良浩、Candy S. Hwang、Lauren C. Smith、Beverly Ellis、Bin Zhou、Kim D. Janda、第 36 回メディシナルケミストリーシンポジ
ウム、京都、2018 年 11 月.
*32. ジヒドロチオピラン環ヌクレオシド誘導体の合成研究、斎藤有香子、山崎佳子、高橋江里佳、名取
良浩、若松秀章、吉村祐一、第 57 回日本薬学会東北支部大会、仙台、2018 年 10 月.
*33. 超原子価ヨウ素とジフェニルジセレニドを用いたピペリジン環構築法の開発、名取良浩、萬浩太、鈴木郁、若松秀章、斎藤有香子、吉村祐一、第 57 回日本薬学会東北支部大会、仙台、2018 年 10 月.
*34. パラジウム触媒を用いたイナミドの位置選択的炭素炭素結合形成反応、若松秀章、池内麻衣、吉村祐一、日本薬学会第 138 年会、金沢、2018 年 3 月.
*35. 抗がん活性を有する 9-デオキシキセニアラクトール C の合成研究: キセニアラクトール骨格の構築、渡邉一弘、高橋央宜、吉村祐一、加藤 正、第 47 回複素環化学討論会、高知、2017 年 10 月.
36. べンゾオキサゾール環を有するセスキテルペン ナキジノール B の合成研究、武田由貴、加藤武、熱海 秀、成田紘一、渡邉一弘、吉村祐一、加藤 正、第 56 回日本薬学会東北支部大会、青森、2017 年 10 月.
37. 海洋由来ジテルペノイド系天然物 Cristaxenicin A の合成研究、中井啓陽、Noel Sayar、吉村祐一、
渡邉一弘、第 56 回日本薬学会東北支部大会、青森、2017 年 10 月.
38. 多標的キナーゼ阻害薬としてのテルペノイド類化合物の同定とその特徴、近松園子、西條 憲、今井 源、成田紘一、加藤 正、石岡千加史、第 76 回日本癌学会学術総会、横浜、2017 年 9 月.
39. 多標的キナーゼ阻害薬としてのテルペノイド類化合物の同定、近松園子、西條 憲、成田紘一、
加藤 正、石岡千加史、第 21 回日本がん分子標的治療学会学術集会、福岡、2017 年 6 月.
40. 5-ヒドロキシナフトオキサゾール天然物 サルビアミン E および F の全合成、成田紘一、藤崎成美、
佐久間裕太、加藤 正、日本薬学会第 137 年会、仙台、2017 年 3 月.
41. カンナビノイド受容体アゴニスト活性を有するフェルギネン B の全合成、加藤友哉、成田紘一、加藤正、日本薬学会第 137 年会、仙台、2017 年 3 月.
42. セスキテルペンベンゾオキサゾール骨格を有するナキジノール A の合成研究、武田由貴、加藤武、熱海 秀、成田紘一、加藤 正、日本薬学会第 137 年会、仙台、2017 年 3 月.
*43. イナミドを基質としたパラジウム触媒による炭素炭素結合形成反応、若松秀章、石井彩香、菊池洋
平、髙橋彩乃、柳澤里佳、木村 翔、大澤直央、名取良浩、吉村祐一、第 64 回有機金属化学討論
会、仙台、2017 年 9 月.
*44. 2'-5'結合を有する環状ジヌクレオチドの合成研究、若生有未、名取良浩、若松秀章、吉村祐一、日本薬学会第 137 年会、仙台、2017 年 3 月.
*45. レチノイン酸類似化合物同時測定系の構築、鈴木裕之、森 謙太、進藤佐和子、熊谷 健、永田清、中村 仁、第 57 回日本薬学会東北支部大会、仙台、2017 年 10 月.
*46. アゾール構造を含む農薬によるヒト CYP 代謝活性阻害、手賀 要、鈴木裕之、進藤佐和子、熊谷健、永田 清、第 57 回日本薬学会東北支部大会、仙台、2017 年 10 月.
47. PAHs によるシトクロム P450 遺伝子発現変動のメカニズムの解析、及川和憲、渡辺孝樹、進藤佐和
子、熊谷 健、永田 清、第 57 回日本薬学会東北支部大会、仙台、2017 年 10 月.
48. UGT1A 分子種発現の各種細胞間比較と誘導剤による影響、緒方龍也、森本麻友、進藤佐和子、熊谷 健、永田 清、第 57 回日本薬学会東北支部大会、仙台、2017 年 10 月.
*49. CYP 遺伝子発現作用に及ぼす健康食品の影響、渡邉夢実、熊谷 健、佐々木崇光、永田 清、フォーラム 2017 衛生薬学・環境トキシコロジー、仙台、2017 年 9 月.
*50. CYP3A4 遺伝子の新規転写活性化について、渡辺孝樹、小田桐玲生、荒津祐輔、佐々木崇光、進藤佐和子、熊谷 健、永田 清、フォーラム 2017 衛生薬学・環境トキシコロジー、仙台、2017 年 9 月.
*51. レチノイン酸の低濃度測定可能な CYP26A1 遺伝子レポーターアッセイ系の構築、森 謙太、鈴木裕之、塩谷安奈里、佐々木崇光、進藤佐和子、熊谷 健、永田 清、フォーラム 2017 衛生薬学・環境ト
キシコロジー、仙台、2017 年 9 月.
52. 薬と健康食品との相互作用、永田 清、フォーラム 2017 衛生薬学・環境トキシコロジー、仙台、2017
年 9 月.
、平成 29 年度内外環境応答・代謝酵素研究会、福岡、
53. PXR が関わるおかしな P450 誘導、永田 清、— 25 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L

2017 年 9 月.
*54. 同一 PXR 結合配列を介した異なる CYP3A4 誘導機序について、荒津祐輔、小田桐玲生、渡辺孝
樹、佐々木崇光、熊谷 健、永田 清、第 56 回日本薬学会東北支部大会、青森、2017 年 10 月.
55. HDAC/PI3K 二重阻害剤デプシペプチド類化合物のヒト軟部肉腫細胞に対する抗腫瘍効果の評
価、西條 憲、成田紘一、加藤 正、石岡千加史、第 75 回日本癌学会学術総会、横浜、2016 年 10月.
56. 海洋天然物シクロスポンジアキノン-1 及び類縁物質の全合成、武田由貴、成田紘一、加藤 正、
第 55 回日本薬学会東北支部大会、郡山、2016 年 9 月.
57. ナフトオキサゾール骨格を有するサルビアミン F の全合成、成田紘一、藤崎成美、佐久間裕太、
加藤 正、第 55 回日本薬学会東北支部大会、郡山、2016 年 9 月.
58. ヒト軟部肉腫細胞に対する HDAC/PI3K 二重阻害剤としてのデプシペプチド類化合物の抗腫瘍効果の検討、西條 憲、成田紘一、加藤 正、石岡千加史、第 20 回日本がん分子標的治療学会、別府、
2016 年 5 月.
59. Total Synthesis of Bicyclic Depsipeptide Natural Products and Evaluation of Their Biological
Activity, Koichi Narita, Tadashi Katoh, French-Japanese Symposium on Medicinal and Fine
Chemistry (FJS 2016), Tokyo, Japan, May, 2016.
60. ヒストン脱アセチル化酵素(HDAC)阻害剤 FK228 類縁体合成、成田紘一、松原圭介、加藤 正、日本薬学会第 136 年会、横浜、2016 年 3 月.
61. 大環状デプシペプチド類天然物 FR-901375 の全合成、加藤友哉、成田紘一、加藤 正、日本薬学会第 136 年会、横浜、2016 年 3 月.
62. 生合成仮説を基軸とした(+)-リファガールの全合成、渡辺拓実、神島尭明、菊地拓也、成田紘一、
加藤 正、日本薬学会第 136 年会、横浜、2016 年 3 月.
63. 抗腫瘍活性を有するシクロスポンジアキノン-1 の全合成、武田由貴、成田紘一、加藤 正、日本薬学会第 136 年会、横浜、2016 年 3 月.
*64. 4'-フルオロメチルスタブジンの合成研究、伊藤恭平、名取良浩、菅野裕也、齋藤華子、若松秀章、
斎藤有香子、吉村祐一、第 34 回メディシナルケミストリーシンポジウム、つくば、2016 年 11 月-12 月.
*65. ペプチド構造を有する有機分子触媒を用いた不斉反応の開発、阿部美保、若松秀章、名取良浩、
吉村祐一、第 42 回反応と合成の進歩シンポジウム、静岡、2016 年 11 月.
*66. エンイナミドを基質とする閉環メタセシスの新たなアプローチ、若松秀章、髙橋将典、諏訪純一、小椋玲奈、吉村祐一、第 46 回複素環化学討論会、金沢、2016 年 9 月.
*67. イミノ糖を含む複素環化合物の合成とその生物活性評価、名取良浩、第 15 回化学系若手研究者
セミナー、仙台、2016 年 10 月.
68. 2-デオキシ-2-フルオロフコースの合成研究、名取良浩、草野宏光、顧 建国、吉村祐一、第 55 回日
本薬学会東北支部大会、郡山、2016 年 9 月.
69. イリジウム触媒を用いた含窒素複素環の構築法の開発、名取良浩、菊地俊輔、長岡杏花、曽雌歩
実、吉村祐一、第 55 回日本薬学会東北支部大会、郡山、2016 年 9 月.
70. 極長鎖 GM3 分子種による慢性炎症惹起メカニズム、狩野裕考、郷 慎司、新田昇大、ヴェイロン ル
ーカス、カッタニオ アンナ、レティッザ マリレナ、名取良浩、吉村祐一、安藤弘宗、石田秀治、樺山
一哉、下山敦史、深瀬浩一、チャンパ マリア、マウリ ラウラ、プリネッティ アレッサンドロ、ソニーノ
サンドロ、鈴木明身、井ノ口仁一、第 89 回 日本生化学会、仙台、2016 年 9 月.
71. 内因性リガンドとしての極長鎖 GM3 ガングリオシドによる慢性炎症惹起機構、狩野裕考、郷 慎司、
新田昇大、ヴェイロン ルーカス、カッタニオ アンナ、レティッザ マリレナ、名取良浩、吉村祐一、安藤弘宗、石田秀治、樺山一哉、下山敦史、深瀬浩一、チャンパ マリア、マウリ ラウラ、プリネッティ
アレッサンドロ、ソニーノ サンドロ、鈴木明身、井ノ口仁一、第 58 回日本脂質生化学会、秋田、2016
年 6 月.
*72. 超原子価ヨウ素とジフェニルジセレニドを用いた分子内エーテル環化反応、須玉夏海、斎藤有香
子、名取良浩、吉村祐一、日本薬学会第 136 年会、横浜、2016 年 3 月.
*73. 二元機能型トリペプチド有機分子触媒を用いた不斉ディールスアルダー反応、阿部美保、若松秀章、吉村祐一、日本薬学会第 136 年会、横浜、2016 年 3 月.
*74. イナミドを基質とした Mizoroki-Heck 型反応、若松秀章、柳澤里佳、木村 翔、大澤直央、名取良浩、吉村祐一、日本薬学会第 136 年会、横浜、2016 年 3 月.
— 26 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L

75. ピロリジン型イミノ糖を基盤とした新規 GCase 高親和性リガンドのデザインとファーマコロジカルシャ
ペロン効果について、加藤 敦、山本亜里紗、友原啓介、足立伊佐雄、渡邊靖香、名取良浩、吉村祐一、中込 泉、広野修一、日本薬学会第 136 年会、横浜、2016 年 3 月.
*76. 2'-5'連結型環状ジヌクレオチドの合成研究、若生有未、名取良浩、若松秀章、吉村祐一、日本薬学会第 136 年会、横浜、2016 年 3 月.
*77. ヒト肝 P450 発現模倣細胞（Ad-P450 細胞）を利用した薬物代謝評価、佐々木 崇光、佐藤 裕、熊谷 健、吉成 浩一、永田 清、第 23 回 HAB 研究機構 学術年会、つくば、2016 年 5 月.
78. ACTIVATION OF P38 MAPK BY CLOTRIMAZOLE ENHANCES THE TRANSCTIPTION OF HUMAN
MRP3 THROUGH A NOVEL TRANSCRIPTIONAL ELEMENT, Takamitsu Sasaki, Keita Inami, Takeshi
Kumagai, Kouichi Yoshinari, Kiyoshi Nagata, 11TH International ISSX meeting, Busan, Korea, 2016,
June.
*79. 健康食品による薬物代謝 P450 活性阻害の網羅的評価、、永田 清、佐藤 裕、熊谷 健、佐々木崇
光、フォーラム 2016 衛生薬学・環境トキシコロジー、東京、2016 年 9 月．
*80. 多環芳香族炭化水素による CYP2D6 発現誘導機構の解析、稲見敬太、佐々木崇光、熊谷 健、吉成浩一、永田 清、H28 度内外環境応答・代謝酵素研究会、静岡、2016 年 9 月.
*81. CYP26A1 レポーター遺伝子発現ウイルスを用いた低濃度下におけるレチノイン酸合成・代謝酵素活性阻害評価系の検討、塩谷安奈里、渡邊美智子、渡辺郁実、角田駿哉、若生俊也、佐々木崇光、熊谷 健、永田 清、H28 度内外環境応答・代謝酵素研究会、静岡、2016 年 9 月.
*82. CYP3A4 遺伝子の基本的転写活性化に関わる新規シスエレメントの探索、小田桐玲生、佐々木崇
光、熊谷 健、永田 清、H28 度内外環境応答・代謝酵素研究会、静岡、2016 年 9 月.
*83. CYP26A1 レポーター遺伝子発現ウイルスを用いたレチノイン酸の誘導評価、塩谷安奈里、渡邊美智子、渡辺郁実、角田駿哉、若生俊也、佐々木崇光、熊谷 健、永田 清、第 55 回日本薬学会東北支部大会、福島、2016 年 9 月.
*84. CYP3A4 遺伝子転写活性化に関わる新規シスエレメントの探索、小田桐玲生、佐々木崇光、熊谷健、永田 清、第 55 回日本薬学会東北支部大会、福島、2016 年 9 月.
85. デプシペプチド類天然物の全合成および生物活性評価、成田紘一、加藤友哉、加藤 正、第 33 回メディシナルケミストリーシンポジウム、千葉、2015 年、11 月.
86. 選択的 PI3Kα 阻害活性物質(+)-リファガールの全合成、菊地拓也、神島尭明、成田紘一、加藤正、第 41 回反応と合成の進歩シンポジウム、大阪、2015 年 10 月.
87. 有機化学教育(講義、実習、研究)の活性化に向けての本学の取組み―東北薬科大学を例にして―、加藤 正、第 10 回有機化学系教科担当教員会議（薬学教育協議会主催）、大阪、2015 年 10 月.
88. デプシペプチド類縁体の PI3K 阻害剤としての特性に関する検討、西條 憲、成田紘一、下平秀樹、
加藤 正、石岡千加史、第 74 回日本癌学会学術総会、名古屋、2015 年 10 月.
89. 生合成仮説を基軸とした(+)-リファガールの全合成研究、渡辺拓実、神島尭明、菊地拓也、成田紘
一、加藤 正、第 54 回日本薬学会東北支部大会、矢巾町、2015 年 9 月.
90. 大環状デプシペプチド類天然物 FR-901375 の全合成、加藤友哉、成田紘一、加藤 正、第 54 回日
本薬学会東北支部大会、矢巾町、2015 年 9 月.
91. 抗がん活性天然物マンデラリド A の全合成に向けて： Ag(I) 触媒による cis-2,5-DHF および
3,6-DHP の合成、渡邉一弘、日本薬学会東北支部 第１３回化学系若手研究者セミナー、2015 年 9月.
92. デプシペプチド類縁体の PI3K 阻害活性についての検討、李 仁、西條 憲、下平秀樹、成田紘一、
加藤 正、石岡千加史、第 19 回日本がん分子標的治療学会、松山、2015 年 6 月.
93. PTP1B 阻害活性を有するダイシダバロン A, B および C の全合成、成田紘一、福井友理恵、佐藤江里、安達剛史、加藤 正、第 26 回万有仙台シンポジウム、仙台、2015 年 6 月.
*94. 1-C-n-ブチル-L-イミノフラノース誘導体の触媒的不斉合成と酵素阻害活性評価、佐久間俊嘉、名取良浩、中川進平、加藤 敦、足立伊佐雄、吉村祐一、第 33 回メディシナルケミストリーシンポジウ
ム、千葉、2015 年 11 月.
*95. イナミドを基質とした Mizoroki-Heck 型反応とスチルベン誘導体の合成、若松秀章、大澤直央、柳澤里佳、木村 翔、名取良浩、吉村祐一、第 41 回反応と合成の進歩シンポジウム、大阪、2015 年 10月.
*96. 4'位に硫黄が置換した 3'-デオキシヌクレオシドの合成研究、安達桃子、伊藤文、名取良浩、若松秀— 27 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L章、吉村祐一、第 54 回日本薬学会東北支部大会、盛岡、2015 年 9 月.
*97. スタブジン誘導体の合成研究、伊藤恭平、菅野裕也、齋藤華子、若松秀章、名取良浩、斎藤有香
子、吉村祐一、第 54 回日本薬学会東北支部大会、盛岡、2015 年 9 月.
98. サイクリン依存性キナーゼ 1（Cdk1）が CYP2D6 及び CYP3A4 遺伝子発現に及ぼす影響の解析、稲見敬太、佐々木崇光、中島菜月、小野 緑、熊谷 健、永田 清、H27 度内外環境応答・代謝酵素研
究会、旭川、2015 年 7 月.
*99. 薬物代謝酵素発現細胞及び誘導評価細胞を用いた薬物性肝障害研究について、佐々木崇光、熊谷 健、永田 清、第 42 回日本毒性学会学術年会、金沢、2015 年 6 月.
*100. レポーター遺伝子導入細胞株を用いた健康食品による CYP1A1/1A2 誘導の網羅的評価、永田清、佐々木崇光、熊谷 健、フォーラム 2015 衛生薬学・環境トキシコロジー、神戸、2015 年 9 月.
*101. レチノイン酸代謝酵素 CYP26 の活性阻害評価系構築、新井 悠、佐々木崇光、稲見敬太、佐藤裕、熊谷 健、吉成浩一、永田 清、第 54 回日本薬学会東北支部大会、岩手、2015 年 9 月.
102. Profiles of Immune Cells and Reproduction of Immune-Mediated Drug-Induced Injury, Yasuyuki
Fujisaka, Yasuteru Kondo, Takamitsu Sasaki, Takayuki Kogure, Jun Inoue, Yu Nakagome, Yuuta
Wakui, Tomoaki Iwata, Tatsuki Morosawa, Teruyuki Umetu, Hiroyuki Suzuki, Hiroaki Yamaguchi,
Nariyasu Mano, Kiyoshi Nagata, Tooru Shimosegawa, American College of Gastroenterology 2015
annual scientific meeting, Honolulu, Hawaii, 2015, October.
103. Profiles of Ｉmmune in Ｐeripheral Ｂlood and Ｒeproduction of Ｉmmune-Ｍediated Ｄrug-Ｉnduced
Ｉnjury Ｉn Ｖitro, Yasuyuki Fujisaka, Yasuteru Kondo, Takayuki Kogure, Jun Inoue, Yu Nakagome,
Tomoaki Iwata, Tatsuki Morosawa, Teruyuki Umetu, Hiroyuki Suzuki, Hiroaki Yamaguchi, Takamitsu
Sasaki, Nariyasu Mano, Kiyoshi Nagata, Tooru Shimosegawa, 第 19 回日本肝臓学会、東京、2015 年
10 月.
104. シトクロム P450 3A4 と UDP-グルクロン酸転移酵素 1A7 の生細胞内でのタンパク質相互作用：機能的相互作用および蛍光共鳴移動（FRET）解析、江越菜月、木下亨佑、古葉弘樹、生城真一、永田清、山添 康、Peter I Mackenzie、山田英之、石井祐次、日本薬物動態学会第 30 回年会、東京、2015 年 11 月.
*105. ヒト肝細胞における P450 活性を模倣した細胞評価系の構築、佐藤 裕、佐々木崇光、新井 悠、熊谷 健、永田 清、日本薬物動態学会第 30 回年会、東京、2015 年 11 月.

「研究テーマ２．老年性神経系疾患(老人性認知症、高齢者うつ病)改善薬の開発」
106. キサンチンオキシダーゼ阻害作用を有するトロポロン誘導体の探索、太田公規、佐藤 輔、皆瀨麻子、遠藤 泰之、日本薬学会第 140 年会、京都、2020 年 3 月.
107. 抗腫瘍活性を示す m-カルボラン含有トリメトキシフェニル誘導体、皆瀨麻子、太田公規、遠藤泰之、日本薬学会第 140 年会、京都、2020 年 3 月.
*108. アンドロゲン受容体とカルボラン誘導体との複合体の分子動力学シミュレーション、金井 涼、加藤紘一、仲吉朝希、太田公規、遠藤泰之、栗本英治、小田彰史、日本薬学会第 140 年会、京都、2020
年 3 月.
109. 潰瘍性大腸炎に起因するうつ様症状に対する肝臓水解物の効果、小平貴代、中川西修、山田耕太
郎、佐久間若菜、髙橋浩平、根本 亙、石澤大輔、高橋成弥、穂積智香子、櫻井英知、丹野孝一、
日本薬学会第 140 年会、京都、2020 年 3 月.
110. 酵素分解サバペプチドの抗疲労メカニズム、中川西修、佐久間若菜、根本 亙、松本 聡、只野武、丹野孝一、日本薬学会第 140 年会、京都、2020 年 3 月.
111. 断続的断眠誘発性衝動性様行動における海馬α2A 受容体の関与、八百板富紀枝、名村幸大、柴田 楓、菅原 彩、土谷昌広、只野 武、丹野孝一、第 93 回日本薬理学会年会、横浜、2020 年 3 月.
112. 嗅球摘出マウスの海馬 AMPK の役割、小平貴代、中川西修、根本 亙、髙橋浩平、佐久間若菜、丹野孝一、第 93 回日本薬理学会年会、横浜、2020 年 3 月.
113. 球状ホウ素クラスターを基盤とする新規エストロゲン受容体リガンドの設計と合成、皆瀨麻子、大山
美優、吉見友弘、太田公規、遠藤泰之、第 37 回メディシナルケミストリーシンポジウム、東京、2019
年 11 月.
114. キサンチンオキシダーゼ阻害作用を示すトロポロン誘導体の探索、太田公規、佐藤大輔、皆瀨麻子、遠藤泰之、第 37 回メディシナルケミストリーシンポジウム、東京、2019 年 11 月.
— 28 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L

115. 新規 5-アリールアゾトロポロン誘導体の細胞増殖抑制活性と活性メカニズム、皆瀨麻子、氣仙拓也、畠山瑞季、太田公規、佐藤大輔、今野茉歩、遠藤泰之、日本薬学会第 139 年会、千葉、2019 年
3 月.
116. Liver Hydrolysate Improves Depressive-Like Behavior in Olfactory Bulbectomized Mice: Involvementof Hippocampal Neurogenesis Through AMPK/BDNF/CREB Pathway, Kotaro Yamada, Osamu
Nakagawasai, Takayo Odaira, Kohei Takahashi, Wataru Nemoto, Wakana Sakuma, Jia-Rong Lin,
Hidetomo Sakurai, Koichi Tan-No, ICoFF/ISNFF, 京都, 2019 年 12 月.
117. レプチン欠損マウスの接触性痛覚過敏に対するアンジオテンシン (1-7) の抑制効果、山縣涼太、
根本 亙、中川西修、丹野孝一、第 58 回日本薬学会東北支部大会、仙台、2019 年 10 月.
118. 長期粉末食飼育誘発性低不安行動におけるヒスタミン神経系の関与、八百板富紀枝、長谷山咲子、宮澤将之、野田芙優、土谷昌広、丹野孝一、第 58 回日本薬学会東北支部大会、仙台、2019 年
10 月.
119. Captopril の海馬 Angiotensin(1-7)/Mas 受容体系の活性化を介した抗うつ効果、小平貴代、中川西修、小野涼太郎、根本 亙、髙橋浩平、佐久間若菜、丹野孝一、第 58 回日本薬学会東北支部大会、仙台、2019 年 10 月.
*120. Schizophrenia-Like Symptoms in the Offspring of Methylazoxymethanol-Treated Mice, Kohei
Takahashi, Osamu Nakagawasai, Wakana Sakuma, Wataru Nemoto, Takayo Odaira, Jia-Rong Lin,
Hiroshi Onogi, Lalit K. Srivastava, Minoru Tsuji, Hiroshi Takeda, Koichi Tan-No, 6th Congress of Asian
College of Neuropsychopharmacology，福岡，2019 年 10 月.
*121. Involvement of Catecholaminergic and GABAAergic Mediations in the Anxiety-Related Behavior
Induced by Long-Term Powdered Food Feeding, Fukie Yaoita, Masahiro Tsuchiya, Yuichiro Arai,
Takeshi Tadano, Koichi Tan-No, 6th Congress of Asian College of Neuropsychopharmacology，福岡，2019 年 10 月.
122. 長期粉末食飼育マウスにおける結腸機能と免疫細胞の関連性、八百板富紀枝、宮澤将之、土谷昌広、土谷 忍、神埼 展、只野 武、丹野孝一、第 70 回日本薬理学会北部会、札幌、2019 年 8 月.
123. レプチン欠損マウスの神経障害性疼痛に対するアンジオテンシン (1-7) の効果、山縣涼太、根本亙、中川西修、丹野孝一、第 70 回日本薬理学会北部会、札幌、2019 年 9 月.
124. 絞扼性神経損傷マウスにおける diminazene aceturate の抗痛覚過敏作用、洪 琬貽、根本 亙、中川西修、丹野孝一、第 70 回日本薬理学会北部会、札幌、2019 年 9 月.
125. Angiotensin 変換酵素阻害薬 Captopril の抗うつ作用は Angiotensin (1-7)/Mas 受容体を介する、小平貴代、中川西修、小野涼太郎、根本亙、髙橋浩平、佐久間若菜、荒井裕一郎、只野武、丹野孝
一、第 23 回活性アミンに関するワークショップ、東京、2019 年 8 月.
126. 2 型糖尿病マウスの神経障害性疼痛における脊髄アンジオテンシン系の関与、根本 亙、山縣涼太、丹野孝一、生体機能と創薬シンポジウム 2019、東京、2019 年 8 月.
127. 糖尿病性神経障害性疼痛における脊髄アンジオテンシン系の関与の解明―新たな治療戦略を目指して―、丹野孝一、小潟佳輝、山縣涼太、根本 亙、生体機能と創薬シンポジウム 2019、東京、2019 年 8 月.
128. p-Hydroxyamphetamine Causes Prepulse Inhibition Disruptions in Mice: Contribution of
Catecholamine and Serotonin Neurotransmission, Hiroshi Onogi, Osamu Nakagawasai, Wataru
Nemoto, Satoru Mitazaki, Koichi Tan-No, Takeshi Tadano, 2019 International Society for
Neurochemistry Meeting，Montreal，2019 年 8 月.
129. Antinociceptive Effect of Diminazene Aceturate, an Angiotensin-Converting Enzyme 2 Activator, in
the Mouse Formalin Test, Wataru Nemoto, Ryota Yamagata, Osamu Nakagawasai, Shota Michimata,Koharu Nakagawa, Koichi Tan-No, 2019 International Society for Neurochemistry Meeting，Montreal，2019 年 8 月.
*130. Behavioral, Cytoarchitectural, and Neurochemical Changes in the Offspring of Methylazoxymethanol
Treated in Mice, Osamu Nakagawasai, Kohei Takahashi, Wakana Sakuma, Wataru Nemoto, Takayo
Odaira, Lin Jia-Rong, Hiroshi Onogi, Lalit K Srivastava, Koichi Tan-No, 2019 International Society for
Neurochemistry Meeting，Montreal，2019 年 8 月.
131. AMPK 活性化は嗅球摘出マウスにおいて海馬 PKCζ/NF-κB/BDNF/CREB を介して抗うつ効果を示す、小平貴代、中川西修、髙橋浩平、根本 亙、佐久間若菜、丹野孝一、日本薬学会第 139 年— 29 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L会，千葉，2019 年 3 月.
132. デキストラン硫酸ナトリウム誘発性潰瘍性大腸炎モデルマウスのうつ様症状に対する肝臓水解物の効果、中川西修、山田耕太郎、小平貴代、佐久間若菜、髙橋浩平、根本 亙、石澤大輔、大沼健
太郎、高橋成弥、大久保未佑、西村優希、穂積智香子、櫻井英知、只野 武、丹野孝一、日本薬学会第 139 年会、千葉、2019 年 3 月.
133. 海馬 AMPK シグナリングの活性化は嗅球摘出マウスのうつ様行動を改善する、小平貴代、中川西修、根本亙、髙橋浩平、佐久間若菜、丹野孝一、第 92 回日本薬理学会年会、大阪、2019 年 3 月.
134. 坐骨神経部分結紮マウスにおけるエチドロン酸の抗痛覚過敏作用について、根本 亙、山縣涼太、
中川西修、洪 琬貽、島 和弘、遠藤康男、丹野孝一、第 92 回日本薬理学会年会、大阪、2019 年 3月.
*135. 長期粉末食飼育誘発性不安関連行動におけるノルアドレナリンおよびドパミン神経系の、八百板富紀枝、土谷昌広、荒井裕一朗、只野 武、丹野孝一、第 92 回日本薬理学会年会、大阪、2019 年 3月.
136. マルチターゲット型抗腫瘍活性を示す新規カルボラン誘導体の創製、皆瀨麻子、太田公規、白田智
七美、遠藤泰之、第 36 回メディシナルケミストリーシンポジウム、京都、2018 年 11 月.
137. ホウ素クラスターの医薬活性化合物における基礎骨格としての可能性、皆瀨麻子、氣仙拓也、佐藤大輔、太田公規、遠藤泰之、第 17 回化学系若手研究者セミナー、仙台、2018 年 9 月.
138. ホウ素クラスター誘導体の抗腫瘍活性とそのメカニズムについて、皆瀨麻子、白田智七美、太田公規、遠藤泰之、日本薬学会第 138 年会、金沢、2018 年 3 月.
139. 過敏性腸症候群モデルマウスにおけるエキセナチドの効果、八百板富紀枝、川浪啓豪、今泉宏
紀、丹野孝一、第 57 回日本薬学会東北支部大会、仙台、2018 年 10 月.
140. 断眠ストレス負荷誘発性異常行動におけるヒスタミン神経系の関与、今泉宏紀、八百板富紀枝、川浪啓豪、長谷山咲子、丹野孝一、第 57 回日本薬学会東北支部大会、仙台、2018 年 10 月.
*141. 胎生期 Methylazoxymethanol 投与マウスの統合失調症モデル動物としての妥当性、髙橋浩平、中川西修、小野木弘志、根本 亙、小平貴代、佐久間若菜、丹野孝一、第 57 回日本薬学会東北支部
大会、仙台、2018 年 10 月.
142. Angiotensin (1-7) Prevents Streptozotocin-Induced Diabetic Neuropathic Pain in Mice, Wataru
Nemoto, Yoshiki Ogata, Ryota Yamagata, Osamu Nakagawasai, Koichi Tan-No, 31th European
College of Neuropsychopharmacology Congress，Barcelona，2018 年 10 月.
143. Memantine Produces Antidepressant Effect Through the Enhancement of Hippocampal
Neurogenesis in Olfactory Bulbectomized Mice, Osamu Nakagawasai, Kohei Takahashi, Wataru
Nemoto, Takayo Odaira, Wakana Sakuma, Yuichiro Arai, Takeshi Tadano, Koichi Tan-No, 31th
European College of Neuropsychopharmacology Congress，Barcelona，2018 年 10 月.
144. 断続的断眠ストレス負荷誘発性低不安行動におけるヒスタミン神経系の関与、八百板富紀枝、今泉 宏紀、川浪啓豪、土谷昌広、只野 武、丹野孝一、第 69 回日本薬理学会北部会、富山、2018 年
9 月.
145. 2 型糖尿病モデルマウスにおけるアンジオテンシン（1-7）の抗痛覚過敏作用、山縣涼太、根本 亙、
中川西修、丹野孝一、第 69 回日本薬理学会北部会、富山、2018 年 9 月.
146. メマンチンの海馬ドパミン神経系を介した抗うつ効果、髙橋浩平、中川西修、根本 亙、小平貴代、
佐久間若菜、荒井裕一朗、只野 武、丹野孝一、第 22 回活性アミンに関するワークショップ、徳島、
2018 年 7 月.
147. Hippocampal AMPK Activation Suppresses Depressive-Like Behavior in Olfactory Bulbectomized
Mice, Takayo Odaira, Osamu Nakagawasai, Wataru Nemoto, Kohei Takahashi, Wakana Sakuma,
Ryotaro Ono, Koichi Tan-No, 18th World Congress of Basic and Clinical Pharmacology，京都，2018
年 7 月.
*148. Antidepressant Effect of BE360, a New Selective Estrogen Receptor Modulator, and Its Mechanism
in Ovariectomized Mice, Wakana Sakuma, Osamu Nakagawasai, Wataru Nemoto, Takayo Odaira,Takumi Ogawa, Kiminori Ohta, Yasuyuki Endo, Koichi Tan-No, 18th World Congress of Basic andClinical Pharmacology，京都，2018 年 7 月.
149. Liver Hydrolysate Produces Antidepressant and Antidementia Effects in Olfactory Bulbectomized
Mice, Osamu Nakagawasai, Kotaro Yamada, Takayo Odaira, Wakana Sakuma, Wataru Nemoto,— 30 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L

Hidetomo Sakurai, Koichi Tan-No, 18th World Congress of Basic and Clinical Pharmacology，京都，
2018 年 7 月.
150. Involvement of Peripheral Alpha2A Adrenoceptor in the Acceleration of Gastrointestinal Transit andAbdominal Pain induced by Intermittent Sleep Deprivation, Fukie Yaoita, Keigo Kawanami, Hiroki
Imaizumi, Takeshi Tadano, Koichi Tan-No, 18th World Congress of Basic and Clinical Pharmacology，
京都，2018 年 7 月.
151. Anti-Allodynic Effect of Angiotensin (1-7) on Streptozotocin-Induced Diabetic Neuropathic Pain,
Ryota Yamagata, Wataru Nemoto, Yoshiki Ogata, Osamu Nakagawasai, Koichi Tan-No, 18th World
Congress of Basic and Clinical Pharmacology，京都，2018 年 7 月.
152. Inhibitory Effect of Repeated Oral Administration of Chondroitin Sulfate on the Formalin-Induced
Tactile Allodynia in Mice, Wataru Nemoto, Kotaro Yamada, Osamu Nakagawasai, Yoshiki Ogata,
Hidetomo Sakurai, Koichi Tan-No, 18th World Congress of Basic and Clinical Pharmacology，京都，
2018 年 7 月.
153. 身体疲労モデルマウスにおける酵素分解サバペプチドの効果、中川西修、佐久間若菜、石澤大輔、根本 亙、松本 聡、只野 武、丹野 孝一、日本薬学会第 138 年会、金沢、2018 年 3 月.
154. 神経障害性疼痛に対するコンドロイチン硫酸の抑制効果、根本 亙、山田耕太郎、中川西修、小潟佳輝、千葉聖斗、山縣涼太、櫻井英知、丹野孝一、日本薬学会第 138 年会、金沢、2018 年 3 月.
155. 嗅球摘出マウスにおけるメマンチンの抗うつ効果とその作用機序、髙橋浩平、中川西修、根本 亙、
小平貴代、佐久間若菜、丹野孝一、日本薬学会第 138 年会、金沢、2018 年 3 月.
*156. 長期粉末食飼育がマウスの情動発達に及ぼす影響について、八百板富紀枝、今泉宏紀、川浪啓
豪、土谷昌広、只野 武、丹野孝一、日本薬学会第 138 年会、金沢、2018 年 3 月.
*157. Structural Ｄifferences of the Ｌigand Ｂinding Ｐockets between Estrogen Receptor-Alpha andBeta, Koichi Kato, Kenichiro Fujii, Tomoki Nakayoshi, Yurie Watanabe, Shuichi Fukuyoshi, Kiminori
Ohta, Yasuyuki Endo, Noriyuki Yamaotsu, Shuichi Hirono, Eiji Kurimoto, Akifumi Oda, XXIX IUPAP
Conference on Computational Physics, CCP2017 , Paris, 2017 年 7 月.
*158. Hydrophobicity and Electronic Effects of Carborane for Application in Medicinal Drug Design (invitedlecture), Yasuyuki Endo, Keisuke Yamamoto, Kiminori Ohta, Hiroto Yamazaki, Asako Kaise, 16th
International Meeting on Boron Chemistry (IMEBORON XVI) , Hong Kong, 2017 年 7 月.
159. カルボラン含有非ステロイド型 2-methoxyestradiol 誘導体の抗腫瘍活性、皆瀨麻子、太田公規、遠藤泰之、日本ケミカルバイオロジー学会第 12 回年会、札幌、2017 年 6 月.
160. フェニルアゾトロポロン誘導体のキサンチンオキシダーゼ阻害活性とそのメカニズム解析、佐藤 大輔、氣仙拓也、太田公規、遠藤泰之、日本薬学会第 137 年会、仙台、2017 年 3 月.
161. Wieland-Miescher ケトン誘導体の加溶媒分解条件下における協奏的骨格転位反応、猪股浩平、遠藤泰之、日本薬学会第 137 年会，仙台，2017 年 3 月.
162. 新規 SERD 化合物の探索：カルボラン含有 ER リガンドによる ER ダウンレギュレーション、皆瀨麻子、吉見友弘、太田公規、遠藤泰之、日本薬学会第 137 年会、仙台、2017 年 3 月.
*163. 卵巣摘出マウスのうつ様行動に対する新規選択的エストロゲン受容体モジュレーターBE360 の効
果、佐久間若菜、中川西修、根本 亙、小平貴代、小川卓巳、太田公規、遠藤泰之、只野 武、丹野
孝一、第 21 回活性アミンに関するワークショップ，京都，2017 年 8 月.
164. Structural Ｄifferences of the Ｌigand Ｂinding Ｐockets between Estrogen Receptor-Alpha andBeta, Koichi Kato, Kenichiro Fujii, Tomoki Nakayoshi, Yurie Watanabe, Shuichi Fukuyoshi, Kiminori
Ohta, Yasuyuki Endo, Noriyuki Yamaotsu, Shuichi Hirono, Eiji Kurimoto, Akifumi Oda, XXIX IUPAP
Conference on Computational Physics, CCP2017 , Paris, 2017 年 7 月.
165. Hydrophobicity and Electronic Effects of Carborane for Application in Medicinal Drug Design (invitedlecture), Yasuyuki Endo, Keisuke Yamamoto, Kiminori Ohta, Hiroto Yamazaki, Asako Kaise, 16th
International Meeting on Boron Chemistry (IMEBORON XVI) , Hong Kong, 2017 年 7 月.
166. カルボラン含有非ステロイド型 2-methoxyestradiol 誘導体の抗腫瘍活性、皆瀨麻子、太田公規、遠藤泰之、日本ケミカルバイオロジー学会第 12 回年会、札幌、2017 年 6 月.
167. フェニルアゾトロポロン誘導体のキサンチンオキシダーゼ阻害活性とそのメカニズム解析、佐藤 大輔、氣仙拓也、太田公規、遠藤泰之、日本薬学会第 137 年会、仙台、2017 年 3 月.
168. Wieland-Miescher ケトン誘導体の加溶媒分解条件下における協奏的骨格転位反応、猪股浩平、遠— 31 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L

藤泰之、日本薬学会第 137 年会、仙台、2017 年 3 月.
169. 新規 SERD 化合物の探索：カルボラン含有 ER リガンドによる ER ダウンレギュレーション、皆瀨麻子、
吉見友弘、太田公規、遠藤泰之、日本薬学会第 137 年会、仙台、2017 年 3 月.
170. 脊髄疼痛伝達機構における Angiotensin II の役割に関する行動および分子薬理学的研究、根本亙、第 39 回東北薬学セミナー、仙台、2017 年 12 月.
171. 断続的レム断眠ストレス負荷誘発性衝動性様症状における海馬ノルアドレナリン神経系の関与、八百板富紀枝、川浪啓豪、今泉宏紀、柴田 楓、菅原 彩、只野 武、丹野孝一、第 56 回日本薬学会
東北支部大会、青森、2017 年 10 月.
172. 嗅球摘出マウスにおけるメマンチンの海馬 CREB-BDNF 系を介する抗うつ効果、髙橋浩平、中川西修、根本 亙、小平貴代、丹野孝一、第 68 回日本薬理学会北部会、山形、2017 年 9 月.
*173. 長期粉末食飼育誘発性低不安行動におけるグルココルチコイド受容体およびノルアドレナリン神経系の関与、八百板富紀枝、小澤美芙由、遠藤汐里、土谷昌広、荒井裕一朗、只野 武、丹野孝一、
第 68 回日本薬理学会北部会、山形、2017 年 9 月.
174. ストレプトゾトシン誘発性 1 型糖尿病マウスにおけるアンジオテンシン (1-7) の抗アロディニア作
用、山縣涼太、根本 亙、小潟佳輝、中川西修、丹野孝一、第 68 回日本薬理学会北部会、山形、
2017 年 9 月.
175. Activation of AMP-Activated Protein Kinase Induces Anti-Depressant Like Effect in Olfactory
Bulbectomized Mice, Takayo Odaira, Osamu Nakagawasai, Wataru Nemoto, Kohei Takahashi, Wakana
Sakuma, Ryotaro Ono, Koichi Tan-No, 第 60 回日本神経化学会、仙台、2017 年 9 月.
176. 海馬 AMPK 活性化による抗うつ作用メカニズムについて、小平貴代、中川西修、根本 亙、髙橋浩
平、佐久間若菜、小野涼太郎、丹野孝一、生体機能と創薬シンポジウム 2017、京都、2017 年 8 月.
177. 1 型糖尿病モデルマウスにおける神経障害性疼痛に対するアンジオテンシン (1-7) の抑制効果、山縣涼太、根本 亙、小潟佳輝、中川西修、丹野孝一、生体機能と創薬シンポジウム 2017、京都、2017 年 8 月.
*178. 神経発達障害仮説に基づいた統合失調症モデル動物作製の試み、中川西修、第 16 回生物化学若手研究者セミナー、仙台、2017 年 7 月.
179. 嗅球摘出マウスのうつ様行動に対するメマンチンの効果、髙橋浩平、中川西修、根本 亙、小平貴代、丹野孝一、日本薬学会第 137 年会、仙台、2017 年 3 月.
180. 断続的レム断眠負荷誘発性交替率低下に対するメチルフェニデートの効果、八百板富紀枝、永澤佑佳、土谷昌広、荒井裕一朗、只野 武、丹野孝一、日本薬学会第 137 年会、仙台、2017 年 3 月.
181. 嗅球摘出マウスの精神機能障害に対する肝臓水解物の効果：海馬 AMP 活性化プロテインキナーゼの関与について、中川西修、山田耕太郎、小平貴代、佐久間若菜、根本 亙、櫻井英知、丹野孝
一、日本薬学会第 137 年会、仙台、2017 年 3 月.
182. AMPK 活性化剤である AICAR は抗うつ作用をもたらす、小平貴代、中川西修、根本 亙、佐久間若菜、丹野孝一、第 90 回日本薬理学会年会、長崎、2017 年 3 月.
183. 嗅球摘出マウスの認知機能障害に対するアリピプラゾールの効果、髙橋浩平、中川西修、根本亙、小平貴代、荒井裕一朗、久光 正、丹野孝一、第 90 回日本薬理学会年会、長崎、2017 年 3 月.
184. アンジオテンシン (1-7) は糖尿病性神経障害性疼痛を抑制する、小潟佳輝、根本 亙、中川西修、
丹野孝一、第 90 回日本薬理学会年会、長崎、2017 年 3 月.
185. アンジオテンシン III 誘発性侵害刺激行動に対するアンジオテンシン (1-7) の抑制作用について、
根本 亙、中川西修、丹野孝一、第 90 回日本薬理学会年会、長崎、2017 年 3 月.
186. 嗅球摘出マウスのうつ様行動に対する Angiotensin 変換酵素阻害薬 Captopril の効果、小野涼太
郎、中川西修、小平貴代、佐久間若菜、根本 亙、丹野孝一、第 26 回神経行動薬理若手研究者の集い、福岡、2017 年 3 月.
187. フェニルアゾトロポロン誘導体のキサンチンオキシダーゼ阻害活性、佐藤大輔、氣仙拓也、太田公規、遠藤泰之、第34回メディシナルケミストリーシンポジウム、つくば、2016年11月.
188. メカニズムに着目したカフェイン酸誘導体のアミロイドβ凝集阻害活性評価、田口莉帆、高橋倫人、
畑山晃輝、太田公規、橋友理香、関 千草、中野博人、遠藤泰之、徳樂清孝、上井幸司、第34回メ
ディシナルケミストリーシンポジウム、つくば、2016年11月.
189. ホウ素クラスターを中心骨格とする新規選択的エストロゲン受容体抑制薬（SERD）の開発、皆瀨麻子、吉見友弘、太田公規、遠藤泰之、第34回メディシナルケミストリーシンポジウム、つくば、2016年— 32 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L

11月.
190. ランダムスクリーニングから見出されたトロポロン誘導体の多様な生物活性、太田公規、佐藤友紀、皆瀨麻子、遠藤泰之、第55回日本薬学会東北支部大会、郡山、2016年9月.
191. キサンチンオキシダーゼ阻害活性を示すフェニルアゾトロポロン誘導体の探索、佐藤大輔、氣仙拓也、太田公規、遠藤泰之、第55回日本薬学会東北支部大会、郡山、2016年9月.
192. 新規 XO 阻害剤の定量的構造活性相関と結合様式の解析、太田公規、佐藤 匠、石井康博、遠藤泰之、日本薬学会第 136 年会、横浜、2016 年 3 月.
193. カルボラン含有グリセロール誘導体の抗アンドロゲン活性、皆瀨麻子、太田公規、小田彰史、遠藤泰之、日本薬学会第 136 年会、横浜、2016 年 3 月.
194. ７員環含有 Wieland-Miescher ケトン誘導体における新規骨格転位反応、猪股浩平、遠藤泰之、日本薬学会第136年会、横浜、2016年3月.
195. Long-Term Feeding on Powdered Food Causes Hyperglycemia and Signs of Systemic Illness in Mice,
Hiroyuki Yoneda, Masahiro Tsuchiya, Fukie Niijima-Yaoita, Shinobu Tsuchiya, Yoshihiro Hagiwara,
Keiichi Sasaki, Shunji Sugawara, Yasuo Endo, Koichi Tan-No, Makoto Watanabe, International
Conference on Food for Health in Niigata 2016, 新潟、2016 年 11 月.
196. ニューロン新生マーカー・BrdU の脳内分布における拡散型ヌクレオシドトランスポーター1 の役割の解明、木村準也、吉田寛伸、竹生田淳、Bastos Gilmara、鈴木登紀子、吉川雄朗、根本 亙、中川西修、丹野孝一、谷内一彦、平沢典保、守屋孝洋、第 67 回日本薬理学会北部会、札幌、2016 年 9 月.
197. 断続的断眠ストレス負荷誘発性交替行動障害における海馬誘導型一酸化窒素合成酵素の関与、八百板富紀枝、永澤佑佳、土谷昌広、荒井裕一朗、只野 武、丹野孝一、第 67 回日本薬理学会北部会、札幌、2016 年 9 月.
198. 嗅球摘出マウスにおける線条体 Dopamine 受容体機能変化とそれに及ぼす抗うつ薬の影響、髙橋
浩平、中川西修、根本 亙、菊地恵理、佐久間若菜、小平貴代、丹野孝一、第 67 回日本薬理学会
北部会、札幌、2016 年 9 月.
199. 過敏性腸症候群モデルの消化器症状におけるアドレナリンα2 受容体サブタイプの関与、八百板富紀枝、村上裕樹、柴田 楓、菅原 彩、只野 武、丹野孝一、第 55 回日本薬学会東北支部大会、郡山、2016 年 9 月.
200. 1 型糖尿病モデルマウスの機械的アロディニアにおける脊髄内アンジオテンシン II 系の関与、山縣涼太、根本 亙、小潟佳輝、中川西修、八百板富紀枝、丹野孝一、生体機能と創薬シンポジウム
2016、仙台、2016 年 8 月.
*201. 更年期障害モデルマウスのうつ状態に対する新規選択的エストロゲン受容体モジュレ－ターBE360の効果、佐久間若菜、中川西修、根本 亙、小潟佳輝、八百板富紀枝、小川卓巳、太田公規、遠藤泰之、丹野孝一、生体機能と創薬シンポジウム 2016、仙台、2016 年 8 月.
202. 脊髄疼痛伝達機構におけるアンジオテンシン II の役割、根本 亙、小潟佳輝、丹野孝一、生体機能と創薬シンポジウム 2016、仙台、2016 年 8 月.
*203. 精神疾患モデル動物作製とその発現機序の解明、中川西修、生体機能と創薬シンポジウム 2016、
仙台、2016 年 8 月.
204. スカブロニン G-メチルエステルの認知障害改善作用とその機序について、小平貴代、中川西修、林嘉蓉、根本 亙、佐久間若菜、小原祐太郎、小早川優、中田雅久、丹野孝一、次世代を担う創薬・医療薬理シンポジウム 2016、仙台、2016 年 8 月.
205. うつ病モデルマウスの脳内ドパミン神経機能変化について、中川西修、髙橋浩平、根本 亙、菊地恵理、佐久間若菜、八百板富紀枝、荒井裕一朗、只野 武、丹野孝一、第 20 回活性アミンに関する
ワークショップ、筑波、2016 年 8 月.
206. カルボラン含有2-methoxyestradiol誘導体の細胞増殖抑制作用とメカニズム解析、皆瀨麻子、太田
公規、遠藤泰之、第33回メディシナルケミストリーシンポジウム、千葉、2015年11月.
207. 新規チオフェン含有エストロゲン受容体制御化合物の開発、青砥沙也加、田口 史、皆瀨麻子、太田公規、遠藤泰之、第33回メディシナルケミストリーシンポジウム、千葉、2015年11月.
208. トリアジン構造を有する新規キサンチンオキシダーゼ阻害剤の創製、太田公規、佐藤 匠、石井康
博、遠藤泰之、第33回メディシナルケミストリーシンポジウム、千葉、2015年11月.
209. 新規カルボラン含有医薬化合物創製への挑戦、皆瀨麻子、太田公規、遠藤泰之、第54回日本薬学会東北支部大会、盛岡、2015年9月.
— 33 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L

210. バナナ型構造を持つエストロゲン受容体リガンドにおけるアゴニスト－アンタゴニストバランスの制
御、太田公規、皆瀨麻子、遠藤泰之、日本ケミカルバイオロジー学会 第10回年会、仙台、2015年6月.
211. エストロゲン代謝物を基にデザインした球状ホウ素クラスター含有新規抗腫瘍性化合物の開発とメカニズム解析、皆瀨麻子、太田公規、遠藤泰之、日本ケミカルバイオロジー学会 第10回年会、仙台、2015 年6月.
212. ホルマリン誘発性アロディニアに対するコンドロイチン硫酸の効果、根本 亙、山田耕太郎、中川西修、小野寺甲仁、小潟佳輝、櫻井英知、丹野孝一、日本薬学会第 136 年会、横浜、2016 年 3 月.
213. アンジオテンシン II が関わる病態とその治療薬の薬理、丹野孝一、第 28 回東北薬科大学生涯教育
講演会、仙台、2015 年 10 月.
*214. 長期粉末食飼育誘発性マウス異常行動に対するメチルフェニデートの効果、藤巻玲香、八百板富紀枝、佐々木貴史、土谷昌広、荒井裕一朗、根本 亙、中川西修、只野 武、丹野孝一、第 54 回日
本薬学会東北支部大会、盛岡、2015 年 9 月.
215. 過敏性腸症候群モデルの消化器症状における腸管α2c アドレナリン受容体の関与、武藤正通、八百板富紀枝、村上裕樹、根本 亙、中川西修、只野 武、丹野孝一、第 54 回日本薬学会東北支部
大会、盛岡、2015 年 9 月.
216. 断続的断眠ストレス負荷誘発性情動行動障害に対する ADHD 治療薬の効果、八百板富紀枝、永澤佑佳、土谷昌広、荒井裕一朗、根本 亙、中川西修、只野 武、丹野孝一、第 66 回日本薬理学会北部会、富山、2015 年 9 月.
217. 脊髄疼痛伝達機構におけるアンジオテンシン III の役割、根本 亙、小潟佳輝、中川西修、八百板富紀枝、只野 武、丹野孝一、第 66 回日本薬理学会北部会、富山、2015 年 9 月.
218. 有痛性糖尿病性ニューロパチーにおける脊髄アンジオテンシン系の関与、小潟佳輝、根本 亙、中川西修、八百板富紀枝、只野 武、丹野孝一、第 66 回日本薬理学会北部会、富山、2015 年 9 月.
219. 有痛性糖尿病性神経障害における脊髄アンジオテンシン II の関与、小潟佳輝、根本 亙、中川西
修、八百板富紀枝、只野 武、丹野孝一、生体機能と創薬シンポジウム 2015、船橋、2015 年 8 月.
220. スカブロニン G メチルエステルは認知障害やうつ様症状を海馬の神経新生促進作用により改善する、林嘉蓉、中川西修、根本 亙、小原祐太郎、八百板富紀枝、荒井裕一朗、只野 武、小早川優、
中田雅久、丹野孝一、第 19 回活性アミンに関するワークショップ、いわき、2015 年 8 月.
221. The Effects of Liver Hydrolysate on Physical Fatigue and Sickness Behavior, Kotaro Yamada, Osamu
Nakagawasai, Wataru Nemoto, Hidetomo Sakurai, Koichi Tan-No, 12th Asian Congress of Nutrition,
横浜、2015 年 5 月.

「研究テーマ３．難治性疼痛に対する緩和医療法の確立」
222. Mechanism of Bilateral Pain under the Inflammatory State、渡辺千寿子、善積克、櫻田忍、溝口広一、第 93 回日本薬理学会年会、横浜、2020 年 3 月.
223. Inhibitory Effect of Gabapentin for Interstitial Cystitis/Bladder Pain Syndrome in Rats、善積克、渡辺千寿子、溝口広一、第 93 回日本薬理学会年会、横浜、2020 年 3 月.
224. モルヒネ非感受性μオピオイド受容体スプライスバリアント選択的作動薬を志向したモルヒナン誘導体の合成、丹羽淳一、本城綾香、米澤佑、平山重人、唐木文霞、伊藤謙之介、溝口広一、藤井秀
明、第 11 回北里化学シンポジウム、東京、2019 年 12 月.
*225. モルヒネ抵抗性炎症性疼痛の制御、溝口広一、第 11 回仙台痛みフォーラム、仙台、2019 年 12 月.
226. The Development of Bilateral Pain under the Inflammatory State, Chizuko Watanabe, Masaru
Yoshizumi, Takafumi Hayashi, Shinobu Sakurada, Hirokazu Mizoguchi, Novel Pain Therapeutics: From
Basic Research to Clinical Translation and Rehabilitation, Rende (Cosenza), Italy, October 2019.
227. Possible Involvement of Histamine on Nociceptive Behaviors Induced by Intrathecally Administered
CCK-8, Shinobu Sakurada, Takafumi Hayashi, Chizuko Watanabe, Sou Katsuyama, Tsuneyoshi
Suzuki, Tsukasa Sakurada, Novel Pain Therapeutics: From Basic Research to Clinical Translation andRehabilitation, Rende (Cosenza), Italy, October 2019.
*228. Management of Morphine-Resistant Inflammatory Pain, Hirokazu Mizoguchi, Chizuko Watanabe,
Masaru Yoshizumi, Yuta Aoki, Asami Komiyama, Shinobu Sakurada, Novel Pain Therapeutics: From
Basic Research to Clinical Translation and Rehabilitation, Rende (Cosenza), Italy, October 2019.
— 34 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L

229. LPS 誘発性間質性膀胱炎モデルラットに対するガバペンチンの効果、善積克、渡辺千寿子、溝口広一、第 58 回日本薬学会東北支部大会、仙台、2019 年 10 月.
230. 炎症性疼痛下における対側性疼痛発現機構の解明、渡辺千寿子、善積克、櫻田忍、溝口広一、第58 回日本薬学会東北支部大会、仙台、2019 年 10 月.
231. I 型糖尿病モデルマウスにおけるアロディニアの発現機序の解明、林貴史、勝山壮、渡辺千寿子、
櫻田司、鈴木常義、櫻田忍、第 70 回日本薬理学会北部会、札幌、2019 年 9 月.
232. 間質性膀胱炎モデルラットに対するガバペンチンの薬効評価、善積克、渡辺千寿子、溝口広一、第70 回日本薬理学会北部会、札幌、2019 年 9 月.
233. 間質性膀胱炎モデルラットにおけるガバペンチンの有効性、善積克、渡辺千寿子、溝口広一、第 39
回鎮痛薬・オピオイドペプチドシンポジウム、東京、2019 年 8 月.
234. 非天然型モルフィナン骨格を有する MRGPRX2 リガンドの創出、飯尾啓太、沓村憲樹、南雲康行、斉藤毅、山本直司、岩間美佐都、溝口広一、長瀬博、第 39 回鎮痛薬・オピオイドペプチドシンポジウ
ム、東京、2019 年 8 月.
235. 難治性痒みに有効な新規低分子ペプチドの創成、石田宗一郎、櫻田忍、溝口広一、関千草、上井幸司、中野博人、日本化学会北海道支部 2019 年夏季研究発表会、苫小牧、2019 年 7 月.
236. 抗掻痒作用をもつ新規低分子ペプチドの創成、石田宗一郎、櫻田忍、溝口広一、関千草、上井幸
司、中野博人、日本化学会北海道支部 2019 年夏季研究発表会、苫小牧、2019 年 7 月.
*237. 炎症性疼痛制御における methadone の特異的作用、渡辺千寿子、込山麻美、善積克、櫻田忍、溝口広一、第 13 回日本緩和医療薬学会、千葉、2019 年 6 月.
*238. 炎症性疼痛下における morphine の鎮痛作用減弱機構、溝口広一、青木祐太、渡辺千寿子、善積克、櫻田忍、第 13 回日本緩和医療薬学会、千葉、2019 年 6 月.
*239. 神経障害性疼痛に対する methadone の脊髄鎮痛作用、善積克、角貴祐、渡辺千寿子、櫻田忍、溝口広一、第 13 回日本緩和医療薬学会、千葉、2019 年 6 月.
240. Development of Agonists Toward Morphine-Insensitive µ Opioid Receptor Variants, Hideaki Fujii,
Ayaka Honjo, Yu Yonezawa, Ayano Miyota, Shigeto Hirayama, Fumika Karaki, Kennosuke Itoh,Hirokazu Mizoguchi, 20th Tetrahedron Symposium, Bangkok, Thailand, June 2019.
241. Design and Synthesis of Novel MRGPRX2 Ligands with Unnatural Morphinan Skeleton, Keita Ito,
Noriki Kutsumura, Yasuyuki Nagumo, Tsuyoshi Saitoh, Naoshi Yamamoto, Misato Iwama, Hirokazu
Mizoguchi, Hiroshi Nagase, The 27th French-Japanese Symposium on Medicinal and Fine Chemistry,
Fukuoka, May 2019.
242. Gabapentin Differently Modulates Pre-Synaptic GABA Release in the Spinal Dorsal Horn and Locus
Coeruleus of Rat after Nerve Injury, Masaru Yoshizumi, Ken-ichiro Hayashida, James C. Eisenach,
Chizuko Watanabe, Hirokazu Mizoguchi, 7th International Congress on Neuropathic Pain, London, UK,
May 2019.
243. Mirror image pain 発現機構における NMDA 受容体の関与、渡辺千寿子、善積克、川瀬涼音、櫻田忍、溝口広一、第 92 回日本薬理学会年会、大阪、2019 年 3 月.
244. 培養アストロサイトからのグルタミン酸誘発性グルタミン酸遊離におけるガバペンチンの促進効果、
善積克、林田健一郎、James C. Eisenach、河谷正仁、渡辺千寿子、溝口広一、第 92 回日本薬理学会年会、大阪、2019 年 3 月.
245. Sex-based difference in CD86 expression on CD103+ dendritic cells promotes female-predominantTh2 cytokine production during allergic asthma. Tomomitsu Miyasaka, Chiaki Masuda, Kaori
Kawakami, Jin-ichi Inokuchi, Tasuku Kawano, Kaori Dobashi-Okuyama, Motoaki Takayanagi, Tomoko
Takahashi, Isao Ohno. 17th International Congress of Immunology, Beijing, 2019, October.
246. Involvement of airway epithelial cells in dendritic cell activation leading to female-predominant Th2
cytokine production in allergic asthma. Kaori Kawakami, Tomomitsu Miyasaka, Chiaki Masuda,
Tasuku Kawano, Tomoko Takahashi, Motoaki Takayanagi, Isao Ohno. 17th International Congress of
Immunology, Beijing, 2019, October.
247. 真菌オリゴ DNA による気管支喘息の制御とその免疫機序、鈴木柊伍、平田大実、宮坂智充、佐藤光、笠松純、石井恵子、川上佳織、高橋知子、大野勲、川上和義、アレルギー・好酸球研究会
2019、東京、2019 年 10 月.
248. 女性優位な Th2 サイトカイン産生を担う樹状細胞の活性化に関与する気道上皮細胞の役割、宮坂— 35 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L

智充、川上佳織、増田千愛、河野資、高橋知子、高柳元明、大野勲、アレルギー・好酸球研究会
2019、東京、2019 年 10 月.
249. 気管支喘息増悪における好酸球数の増加に関与するμオピオイド受容体遺伝子一塩基多型、川上佳織、宮坂智充、中村豊、宮田敏、佐藤美希、目時弘仁、曽良一郎、山内広平、河野資、下川宏
明、高柳元明、高橋知子、大野勲、アレルギー・好酸球研究会 2019、東京、2019 年 10 月.
250. 喘息モデルマウスに対する気管支肺胞上皮幹細胞を用いた治療効果についての検討、石井聖人、
河野資、滝口迪瑠、高橋稜、木村諒、宮坂智充、松尾平、大野勲、高橋知子、第 58 回日本薬学会
東北支部大会、仙台、2019 年 10 月.
*251. オピオイド受容体遺伝子一塩基多型が喘息病態に及ぼす影響の解析、原田真裕美、宮坂智充、川上佳織、中村豊、宮田敏、佐藤美希、目時弘仁、曽良一郎、山内広平、河野資、下川宏明、高柳元
明、高橋知子、大野勲、第 58 回日本薬学会東北支部大会、仙台、2019 年 10 月.
252. 好中球・好酸球混合型喘息における精神的ストレスの関与～精神的ストレスによる免疫寛容の抑制と Th17 細胞の分化誘導～、河野資、石井聖人、宮坂智充、大河原雄一、高柳元明、高橋知子、大野勲、第 53 回東北アレルギー懇話会、仙台、2019 年 7 月.
253. 真菌より見出したオリゴ DNA による Th2 免疫抑制を介した気管支喘息の制御、鈴木柊伍、平田大実、宮坂智充、佐藤光、笠松純、石井恵子、川上佳織、高橋知子、大野勲、川上和義、第 68 回日本アレルギー学会学術大会、東京、2019 年 6 月.
*254. 気管支喘息増悪におけるヒトμオピオイド受容体遺伝子一塩基多型の関与、川上佳織、宮坂智充、中村豊、宮田敏、佐藤美希、目時弘仁、曽良一郎、山内広平、河野資、下川宏明、高柳元明、高橋知子、大野勲、第 68 回日本アレルギー学会学術大会、東京、2019 年 6 月.
255. 精神的ストレスが免疫寛容に及ぼす影響と喘息発症、河野資、大内竜介、石井聖人、宮坂智充、
大河原雄一、高柳元明、高橋知子、大野勲、第 68 回日本アレルギー学会学術大会、東京、2019 年
6 月.
256. 喘息気道炎症における樹状細胞の活性化に関与する気道上皮細胞の性差、川上佳織、宮坂智充、増田千愛、立崎史歩、中村豊、河野資、山内広平、高柳元明、高橋知子、大野勲、第 59 回日本
呼吸器学会学術講演会、東京、2019 年 4 月.
257. 気管支喘息における樹状細胞の活性化の性差に関与する気道上皮細胞の役割、川上佳織、宮坂智充、増田千愛、河野資、高柳元明、高橋知子、大野勲、日本薬学会第 139 年会、千葉、2019 年 3月.
258. 気管支喘息モデルマウスにおける気管支肺胞上皮幹細胞(Bronchioalveolar stem cells (BASCs))の治療効果の検討、石井聖人、河野資、滝口迪瑠、柏原若奈、平泉花菜、鈴木涼平、佐藤美菜香、宮坂智充、松尾平、高柳元明、大野勲、高橋知子、日本薬学会第 139 年会、千葉、2019 年 3 月.
259. 気管支喘息における樹状細胞活性化の性差に関与する気道上皮細胞の役割、宮坂智充、川上佳織、増田千愛、鹿野さくら、河野資、高柳元明、高橋知子、大野勲、第 1 回日本アレルギー学会東北地方会、仙台、2019 年 2 月.
*260. 気管支喘息の増悪におけるオピオイド受容体遺伝子一塩基多型の関与、川上佳織、宮坂智充、中村豊、宮田敏、佐藤美希、目時弘仁、鹿野さくら、曽良一郎、山内広平、河野資、下川宏明、高柳元
明、高橋知子、大野勲、第 1 回日本アレルギー学会東北地方会、仙台、2019 年 2 月.
261. 気管支肺胞上皮幹細胞 (Bronchioalveolar stem cells (BASCs)) の気管支喘息モデルマウスに対する効果の検討、石井聖人、河野資、滝口迪瑠、柏原若奈、平泉花菜、鈴木涼平、佐藤美菜香、宮坂智充、松尾平、高柳元明、大野勲、高橋知子、第 1 回日本アレルギー学会東北地方会、仙台、
2019 年 2 月.
262. 幼少期ストレスによる免疫寛容の抑制を介した喘息発症モデル、大内竜介、河野資、宮坂智充、大河原雄一、高柳元明、高橋知子、大野勲、第 1 回日本アレルギー学会東北地方会、仙台、2019 年 2月.
263. New Tripeptide for Antipruritic Agent, Soichiro Ishida、Shinobu Sakurada, Hirokazu Mizoguchi,
Chigusa Seki, Koji Uwai, Yuko Okuyama, Hiroto Nakano, 10th International Peptide Symposium,
Kyoto, December 2018.
264. 鎮痛作用を有する新規かご型オピオイドペプチド化合物の合成研究、岸里涼太、櫻田忍、溝口広一、林貴史、関千草、上井幸司、奥山祐子、中野博人、第 36 回メディシナルケミストリーシンポジウ
ム、京都、2018 年 11 月.
— 36 —

（様式 2）
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041004
S1511001L

265. モルヒネ非感受性μオピオイド受容体スプライスバリアント選択的作動薬を志向したモルヒナン誘導体の合成、本城綾香、米澤佑、御代田彩乃、平山重人、唐木文霞、伊藤謙之介、溝口広一、藤井秀明、第 36 回メディシナルケミストリーシンポジウム、京都、2018 年 11 月.
266. モルヒネ非感受性μオピオイド受容体選択的作動薬を志向したモルヒナン誘導体の合成、本城綾香、御代田彩乃、平山重人、唐木文霞、伊藤謙之介、溝口広一、藤井秀明、第 44 回反応と合成の進歩シンポジウム、熊本、2018 年 11 月.
267. 神経障害性疼痛における抗てんかん薬バルプロ酸の鎮痛機構、善積克、林田健一郎、渡辺千寿
子、河谷正仁、James C. Eisenach、溝口広一、第 57 回日本薬学会東北支部大会、仙台、2018 年
10 月.
268. Mirror image pain 発現機構の解明、川瀬涼音、渡辺千寿子、大野澤歩美、善積克、櫻田忍、溝口広一、第 57 回日本薬学会東北支部大会、仙台、2018 年 10 月.
269. μ受容体作動薬の末梢性抗掻痒作用、岩間美佐都、竹田詩織、福原直也、渡辺千寿子、善積克、
櫻田忍、溝口広一、第 57 回日本薬学会東北支部大会、仙台、2018 年 10 月.
270. Involvement of the Spinal Release of Histamine on Nociceptive Behaviors Induced by Spermine.
Hirokazu Mizoguchi, Chizuko Watanabe, Takafumi Hayashi, Yoko Namioka, Hiroyuki Watanabe, Toru
Orito, Masaru Yoshizumi, Kenji Onodera, Shinobu Sakurada, World Histamine Symposium 2018,
Kobe, July 2018.
*271. Identification of the µ-Opioid Receptor Splice Variants Involved in the Lack of Rewarding Effect for
Amidino-TAPA, Hirokazu Mizoguchi, Maya Yoshioka, Chizuko Watanabe, Akira Otowa, Shinobu
Sakurada, The 18th World Congress of Basic and Clinical Pharmacology, Kyoto, July 2018.
272. The Dvelopment of Mirror Image Pain in a Mouse Model of Inflammatory Pain, Chizuko Watanabe,
Takafumi Sato, Akira Otowa, Tsukasa Sakurada, Shinobu Sakurada, Hirokazu Mizoguchi, The 18th
World Congress of Basic and Clinical Pharmacology, Kyoto, July 2018.
273. 天然由来成分の抗かゆみ活性、芦部文一朗、松見繁、丸山徹也、溝口広一、櫻田忍、日本薬学会
第 138 年会、金沢、2018 年 3 月.
*274. 依存性のない新規鎮痛薬の探索、溝口広一、第 2 回仙台痛みフォーラム、仙台、2018 年 2 月.
275. クリプトコックスから見出したオリゴ DNA による気管支喘息の発症抑制効果、宮坂智充、川上和義、石井恵子、奥山（土橋）香織、川上佳織、増田千愛、河野資、高柳元明、高橋知子、大野勲、第2 回東北医真菌研究会、仙台、2018 年 12 月.
276. 気管支肺胞上皮幹細胞を用いた急性好酸球性気道炎症の治療効果の検討、石井聖人、河野資、
柏原若奈、平泉花菜、鈴木涼平、佐藤美菜香、宮坂智充、松尾平、高柳元明、大野勲、高橋知子、第 57 回日本薬学会東北支部大会、仙台、2018 年 10 月.
277. 喘息病態の性差に関与する樹状細胞サブセットの解析、鹿野さくら、宮坂智充、増田千愛、川上佳織、河野資、井ノ口仁一、高柳元明、高橋知子、大野勲、第 57 回日本薬学会東北支部大会、仙台、
2018 年 10 月.
278. 新規 Toll-like receptor 9 アゴニストによる喘息気道炎症抑制効果の検討、宮坂智充、川上和義、石井恵子、奥山香織、佐藤光、川上佳織、増田千愛、佐藤佑樹、塩田哲也、野村俊樹、渡邉祐里絵、
宮原杏奈、松本郁美、菅野恵美、丹野寛大、河野資、高柳元明、高橋知子、大野勲、アレルギー・
好酸球研究会 2018、東京、2018 年 9 月.
279. 免疫寛容誘導に対する精神的ストレスの影響と喘息発症、河野資、大内竜介、宮坂智充、大河原雄一、高柳元明、高橋知子、大野勲、アレルギー・好酸球研究会 2018、東京、2018 年 9 月.
280. 女性喘息患者における Th2 型免疫応答の亢進に関与する Estradiol の役割、川上佳織、宮坂智充、
増田千愛、河野資、高橋知子、高柳元明、大野勲、第 67 回日本アレルギー学会学術大会、千葉、
2018 年 6 月.
281. 幼少期ストレスによる喘息発症モデル―幼少期ストレスと免疫寛容の抑制―、大内竜介、河野資、
宮坂智充、大河原雄一、高柳元明、高橋知子、大野勲、第 67 回日本アレルギー学会学術大会、千葉、2018 年 6 月.
282. 女性ホルモン依存的 IL-5 産生の亢進に関与する CD103+樹状細胞の役割、宮坂智充、増田千愛、
川上佳織、河野資、井ノ口仁一、高柳元明、高橋知子、大野勲、第 58 回日本呼吸器学会学術講演
会、大阪、2018 年 4 月.
283. 幼少期ストレスによる喘息発症モデル-幼少期ストレスによる免疫寛容の抑制-、河野資、大内竜— 37 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L介、宮坂智充、大河原雄一、高柳元明、高橋知子、大野勲、第 58 回日本呼吸器学会学術講演会、
大阪、2018 年 4 月.
284. 気管支喘息重症化の性差に関与する気道上皮細胞の Estradiol 依存的機能変化、川上佳織、宮坂智充、増田千愛、河野資、高柳元明、高橋知子、大野勲、日本薬学会第 138 年会、金沢、2018 年 3月.
285. 喘息病態における女性優位な Th2 型免疫応答を誘導する CD103+ 樹状細胞の機能亢進に関与す
る 17β-estradiol の役割、増田千愛、宮坂智充、川上佳織、河野資、高柳元明、高橋知子、大野勲、日本薬学会第 138 年会、金沢、2018 年 3 月.
*286. 炎症性疼痛制御における methadone の特異的作用、込山麻美、渡辺千寿子、音羽 亮、櫻田 忍、
溝口広一、第 68 回日本薬理学会北部会、山形、2017 年 9 月.
287. 難治性掻痒症治療薬 nalfurafine の末梢性抗掻痒作用、音羽 亮、渡辺千寿子、長瀬 博、櫻田忍、溝口広一、第 68 回日本薬理学会北部会、山形、2017 年 9 月.
*288. Effectiveness of amidino-TAPA against morphine-resistant neuropathic pain、溝口広一、渡辺千寿
子、音羽 亮、櫻田 忍、第 60 回日本神経化学会大会、仙台、2017 年 9 月.
*289. Amidino-TAPA の神経障害性疼痛に対する有効性、溝口広一、渡辺千寿子、音羽 亮、櫻田 忍、第4 回包括的緩和医療科学学術研究会・第 5 回 Tokyo 疼痛緩和次世代研究会合同研究会、東京、2017 年 8 月.
*290. 炎症性疼痛下における各種麻薬性鎮痛薬における鎮痛効果とその耐性形成能、込山麻美、溝口広一、渡辺千寿子、音羽 亮、櫻田 忍、第 90 回日本薬理学会年会、長崎、2017 年 3 月.
291. Possible involvement of CD103+ dendritic cells in female-predominant Th2 priming in asthma.
MIYASAKA Tomomitsu, MASUDA Chiaki, KAWAKAMI Kaori, INOKUCHI Jin-ichi, KAWANO Tasuku,
TAKAYANAGI Motoaki, TAKAHASHI Tomoko, OHNO Isao、第 46 回日本免疫学会学術集会、仙台、
2017 年 12 月.
292. Influence of sex-related difference in dendritic cells on female-predominant Th2 cytokine productionin asthma. Tomomitsu Miyasaka, Chiaki Masuda, Kaori Kawakami, Tasuku Kawano, Jin-ichi Inokuchi,
Tomoko Takahashi, Motoaki Takayanagi, Isao Ohno. The 27th Congress of Interasma Japan / North
Asia, Kumamoto, 2017, October.
293. Early life stress increased the risk of adult-onset asthma through the inhibition of the developmentof respiratory tolerance in a murine model. Tasuku Kawano, Ryusuke Ouchi, Tomomitsu Miyasaka,
Yuichi Ohkawara, Motoaki Takayanagi, Tomoko Takahashi, Isao Ohno. The 27th Congress ofInterasma Japan / North Asia, Kumamoto, 2017, October.
294. Involvement of sex-related functions of dendritic cells in female-predominant allergic inflammation inasthma. Chiaki Masuda, Tomomitsu Miyasaka, Kaori Dobashi-Okuyama, Tasuku Kawano, Inokuchi
Jin-ichi, Tomoko Takahashi, Motoaki Takayanagi, Isao Ohno. The 11th International Conference on
Allergy, Asthma & Clinical Immunology, Edinburgh, 2017, September.
295. Male-dominant suppressive activity of CD8+ T cells on CD4+ T cells: assessing female-dominant
allergic airway inflammation、Tomomitsu Miyasaka, Chihiro Ito, Kaori Dobashi-Okuyama, Chiaki
Masuda, Toshiaki Kikuchi, Tasuku Kawano, Tomoko Takahashi, Motoaki Takayanagi, Isao Ohno. The
11th International Conference on Allergy, Asthma & Clinical Immunology, Edinburgh, 2017,
September.
296. Sex-related differences in allergic asthma: possible involvement of CD103+ dendritic cells inenhanced Th2 cytokine production in females. Tomomitsu Miyasaka, Chiaki Masuda, Kaori Kawakami,
Jin-ichi Inokuchi, Tasuku Kawano, Motoaki Takayanagi, Tomoko Takahashi, Isao Ohno. The 8th
Congress of the International Society for Gender Medicine, Sendai, 2017, September.
297. The increased susceptibility to adult-onset asthma through the inhibition of the development of
respiratory tolerance by early life stress. Tasuku Kawano, Ryusuke Ouchi, Tomomitsu Miyasaka,
Yuichi Ohkawara, Tomoko Takahashi, Motoaki Takayanagi, Isao Ohno. The 11th International
Conference on Allergy, Asthma & Clinical Immunology, Edinburgh, 2017, September.
298. 精神的ストレスによる喘息発症モデル-精神的ストレスによる免疫寛容の抑制-、河野 資、大内竜介、宮坂智充、大河原雄一、高柳元明、高橋知子、大野 勲、第 66 回日本アレルギー学会学術大会、東京、2017 年 6 月.
— 38 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L

299. 肥満という表現型は必ずしも喘息増悪と関係しない―マウス喘息モデルを用いた検討―、大河原雄一、宮坂智充、河野 資、高橋知子、大野 勲、第 66 回日本アレルギー学会学術大会、東京、 2017年 6 月.
300. 幼少期ストレスによる成人喘息発症モデル-幼少期ストレスによる免疫寛容の抑制-、河野 資、大内竜介、宮坂智充、大河原雄一、高柳元明、高橋知子、大野 勲、第 57 回呼吸器学会学術講演会、
東京、2017 年 4 月.
*301. 炎症性疼痛下における各種麻薬性鎮痛薬の鎮痛効果とその耐性形成能、込山麻美、溝口広一、
渡辺千寿子、音羽 亮、櫻田 忍、第 67 回日本薬理学会北部会、札幌、2016 年 9 月.
302. Nalfurafine の末梢性抗掻痒作用、音羽 亮、溝口広一、渡辺千寿子、長瀬 博、櫻田 忍、第 89
日本薬理学会年会、横浜、2016 年 3 月.
303. Hemokinin-1 誘発性疼痛関連行動の発現機構、渡辺千寿子、溝口広一、音羽 亮、櫻田 忍、第 89回日本薬理学会年会、横浜、2016 年 3 月．
*304. Amidino-TAPA の非依存性に関わるμオピオイド受容体スプライスバリアントの探索、溝口広一、渡辺千寿子、音羽 亮、吉岡麻也、櫻田 忍、第 89 回日本薬理学会年会、横浜、2016 年 3 月．
305. Early life stress increased the risk of adultonset asthma through the inhibition of the development of
respiratory tolerance in murine model. OUCHI Ryusuke, KAWANO Tasuku, MIYASAKA Tomomitsu,
OHKAWARA Yuichi, TAKAYANAGI Motoaki, TAKAHASHI Tomoko, OHNO Isao, 第 45 回日本免疫学会学術集会、宜野湾市、2016 年 12 月.
306. A novel Toll-like receptor 9 agonist derived from Cryptococcus neoformans attenuates asthmatic
airway responses. MIYASAKA Tomomitsu, KAWAKAMI Kazuyoshi, ISHII Keiko,
DOBASHI-OKUYAMA Kaori, NOMURA Toshiki, MASUDA Chiaki, WATANABE Yurie, MIYAHARA
Anna, MATSUMOTO Ikumi, TAKAYANAGI Motoaki, TAKAHASHI Tomoko, OHNO Isao. 第 45 回日本
免疫学会学術集会、宜野湾市、2016 年 12 月.
307. Sex-related differences in the interaction of CD4+ T cells with CD8+ T cells lead tofemale-dominant allergic airway inflammation. Tomomitsu Miyasaka, Chihiro Ito, Kaori
Dobashi-Okuyama, Chiaki Masuda, Tasuku Kawano, Yuichi Ohkawara, Toshiaki Kikuchi, Motoaki
Takayanagi, Isao Ohno. International Congress of Immunology, Melbourne, 2016, August.
308. 喘息増悪における µ オピオイド受容体遺伝子多型と臨床病態との相関、大野 勲, 宮坂智充, 中村
豊、宮田 敏、佐藤美希、高柳元明, 下川宏明、山内広平、第 65 回日本アレルギー学会学術大会
東京、2016 年 6 月.
309. 精神的ストレスによる免疫寛容の抑制と喘息発症の感受性増大の機序、河野 資、大内竜介、吉田仁美、宮坂智充、大河原雄一、高柳元明, 大野 勲. 第 65 回日本アレルギー学会学術大会、 東京
2016 年 6 月.
310. 幼少期ストレスによる免疫寛容の抑制と喘息発症リスクの増大、大内竜介、河野 資、吉田仁美、
新田典秀、宮坂智充、大河原雄一、高柳元明、大野 勲、第 56 回呼吸器学会学術講演会、京都、2016 年 4 月.
311. 重症喘息における精神的ストレスの関与～精神的ストレスによる免疫寛容の抑制と Th17 細胞の分化誘導～、河野 資、下田奈々、加羽香澄、大内竜介、新田典秀、宮坂智充、大河原雄一、高柳元
明、高橋知子、大野 勲、第 55 回日本薬学会東北支部会、郡山、2016 年 9 月.
312. ラベンダーオイルの末梢局所投与によるパクリタキセル誘発性末梢神経障害性疼痛抑制効果の検討、勝山 壮、音羽 亮、倉本敬二、櫻田 忍、第 22 回日本未病システム学会学術総会、札幌、2015
年 10 月.
313. 当院における入院患者を対象としたアロマトリートメント（精油成分）による疼痛緩和作用の検討＜第一報＞、真井健吾、椎崎正秀、前間邦生、楠直子、池末いづみ、桑波田日香里、勝山 壮、小松
生明、櫻田 司、櫻田 忍、第 9 回日本緩和医療薬学会年会、横浜、2015 年 10 月.
*314. 多発性硬化症疼痛に対するオピオイド性鎮痛薬の効果、溝口広一、渡辺千寿子、音羽 亮、長瀬博、櫻田 忍、第 9 回日本緩和医療薬学会年会、横浜、2015 年 10 月.
*315. Amidino-TAPA の非依存性に関わるμオピオイド受容体スプライスバリアントの探索、溝口広一、渡辺千寿子、音羽 亮、吉岡麻也、櫻田 忍、第 45 回日本神経精神薬理学会・第 37 回日本生物学的精神医学会合同年会、東京、2015 年 9 月.
316. 難治性掻痒症治療薬 nalfurafine の末梢性抗掻痒作用、音羽 亮、小笠原蓉子、新井田隆宏、溝口— 39 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L

広一、渡辺千寿子、勝山 壮、長瀬 博、櫻田 忍、第 66 回日本薬理学会北部会、富山、2015 年 9月.
317. Psychological stress increases susceptibility to the development of asthma through inhibiting
respiratory tolerance. KAWANO Tasuku, ISHIGAKI Takahiro, Nitta Norihide, MIYASAKA Tomomitsu,
OHKAWARA Yuichi, TAKAYANAGI Motoaki, OHNO Isao. 第 44 回日本免疫学会総会、札幌、2015
年 11 月.
318. 気管支喘息におけるμオピオイド受容体一塩基多型と臨床病態との相関、大野 勲、宮坂智充、中村 豊、宮田 敏、佐藤美希、森 拓夢、川島祐里佳、高柳元明、下川宏明、山内広平、第 101 回日
本呼吸器学会東北地方会、福島、2015 年 10 月.
319. 気管支喘息における µ オピオイド受容体一塩基多型と臨床病態との相関、大野 勲、宮坂智充、中村 豊、宮田 敏、佐藤美希、森 拓夢、川島祐里佳、高柳元明、川島宏明、山内広平、第 101 回日
本呼吸器学会東北地方会、福島市、2015 年 10 月.
320. 幼少期ストレスによる免疫寛容の抑制を介した喘息発症の機序、新田典秀、河野 資、宮坂智充、
大河原雄一、高柳元明、大野 勲、第 54 回日本薬学会東北支部大会、矢巾、2015 年 9 月.
321. 精神的ストレスによる免疫寛容の抑制と喘息発症の感受性増大の機序—ストレス誘導性グルココル
チコイドの役割—、河野 資、石垣貴裕、新田典秀、吉田仁美、宮坂智充、大河原雄一、高柳元明、
大野 勲、第 42 回日本神経内分泌学会、仙台、2015 年 9 月.
322. 肥満による気管支喘息増悪と内臓脂肪組織の免疫代謝調節機構の変化、大河原雄一、岩田敏
信、宮坂智充、河野 資、石垣貴裕、伊藤ちひろ、佐藤美希、増田千愛、高柳元明、大野 勲、第 64回日本アレルギー学会学術大会、東京、2015 年 5 月.
323. 精神的ストレスによる免疫寛容の抑制と喘息発症の感受性増大の機序、河野 資、石垣貴裕、新田典秀、宮坂智充、大河原雄一、高柳元明、大野 勲、第 64 回日本アレルギー学会学術大会、東京、2015 年 5 月.
324. 精神的ストレスによる免疫寛容の抑制と喘息発症の感受性の増大、河野 資、石垣貴裕、宮坂智充、大河原雄一、菊地利明、高柳元明、大野 勲、第 55 回呼吸器学会学術講演会、東京、2015 年 4月.

＜研究成果の公開状況＞（上記以外）
シンポジウム・学会等の実施状況、インターネットでの公開状況等＜既に実施しているもの＞《平成３１年度 創薬研究センターシンポジウム》開催日：平成 31 年 6 月 22 日(土)研究成果発表（口頭７題）
1. 麻薬類に対するワクチンの開発研究（名取 良浩、Candy S. Hwang、君嶋 敦、Lauren C. Smith、Margaret E.Olson、Beverly Ellis、
Bin Zhou、吉村 祐一、Kim D. Janda）
2. 抗原虫活性を有するクリスタキセニシン A の全合成研究（分銅 和香、中井 啓陽、Sayar Noel 、成田 紘一、吉村 祐一、渡邉 一弘）
3. 新規三次元疎水性構造単位を用いた医薬分子設計のコンセプトとその実証(遠藤 泰之、太田 公規、皆瀬 麻子)
4. プロポリス含有健康食品による薬物相互作用解析(熊谷 健、星 利香、長田 大輝、渡邉 夢実、進藤 佐和子、永田 清)
5. 間質性膀胱炎モデルラットの確立とガバペンチンの有効性(善積 克、渡辺 千寿子、溝口 広一)
6. 好中球・好酸球混合型喘息における精神的ストレスの関与～精神的ストレスによる免疫寛容の抑制とＴ細胞分化誘導～(河野 資、石井 聖人、萩庭 幹太、山口 祐弐、佐藤 伸輔、宮坂 智充、大河原 雄一、高柳 元明、高橋 知子、大野 勲)
7. 脊髄 Angiotensin 系の不均衡が糖尿病性神経障害性疼痛に寄与する(山縣 涼太、根本 亙、小潟 佳輝、中川西 修、八百板 富紀枝、丹野 孝一)
研究成果発表（ポスター７題）
1. 4’置換ヌクレオシド誘導体の合成と光学分割の検討— 40 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L(遠藤 柚季乃、若松 秀章、伊藤 恭平、斎藤 有香子、名取 良浩、吉村 祐一)
2. スクアレン合成酵素阻害剤 ビサボスクアール A の合成研究（吉田 昌太郎、成田 紘一、渡邉 一弘、吉村 祐一）
3. m-カルボラン含有トリメトキシフェニル誘導体の抗腫瘍活性（皆瀨 麻子、太田 公規、遠藤 泰之）
4. リン酸化エストロゲン受容体の免疫細胞における役割の探索（進藤 佐和子、熊谷 健、永田 清、根岸 正彦）
5. 炎症性疼痛下における TRPV1 チャネルの機能的変化（渡辺 千寿子、佐藤 駿文、善積 克、櫻田 忍、溝口 広一）
6. 気管支喘息の増悪におけるμ オピオイド受容体の一塩基多型の関与（川上 佳織、宮坂 智充、菊池 かなみ、千丸 正尊、原田 真裕美、竹内 圭亮、杉本 大幸、
佐藤 美希、河野 資、曽良 一郎、中村 豊、目時 弘仁、高柳 元明、高橋 知子、大野 勲）
7. 加齢性男性性腺機能低下症候群動物モデルの作製(佐久間 若菜、中川西 修、根本 亙、小平 貴代、八百板 富紀枝、丹野 孝一)
特別講演３題
1. オレキシン受容体選択的リガンドの設計・合成とその薬理作用(筑波大学睡眠研究機構・長瀬 博 特命教授)
2. 末梢性神経障害性疼痛治療薬としてのニコチン受容体作動薬の可能性（和歌山県立医科大学医学部・岸岡 史郎 特別顧問）
3. 薬物作用を本質的に紐解くための技術革新とその応用（星薬科大学 薬理学研究室・成田 年 主任教授）《平成３０年度 創薬研究センターシンポジウム》開催日：平成 30 年 6 月 23 日(土)研究成果発表（口頭７題）
1. 4’-置換 4’-チオリボヌクレオシド誘導体の合成研究(前田璃音、若松秀章、庄子希望、名取良浩、吉村祐一)
2. ベンゾオキサゾール骨格を有するセスキテルペン ナキジノール類の合成（武田由貴、中井啓陽、成田紘一、加藤 正）
3. 化学酵素的手法による Swaminathan-Narayanan ケトン型キラル合成素子の創製(猪股 浩平、成田 沙奈美、庄子 佳穂、三浦 亜里沙、野田 貴子、遠藤 泰之)
4. Mirror image pain発現機構におけるNMDA受容体の関与(渡辺千寿子、田代 隆、大野澤歩美、音羽 亮、善積 克、櫻田 忍、溝口広一)
5. 嗅球摘出マウスにおけるAMP-Activated Protein Kinase (AMPK) の活性化を介した抗うつ効果(小平 貴代、中川西 修、根本 亙、髙橋 浩平、佐久間 若菜、八百板 富紀枝、丹野 孝一)
6. 生体内におけるリン酸化エストロゲン受容体の脳内における新規機能の探索(進藤 佐和子、Hao Hu、熊谷 健、永田 清、根岸 正彦)
7. 気管支喘息における臨床病態の増悪に関与するμオピオイド受容体の一塩基多型(宮坂智充、佐藤美希、中村豊、宮田敏、目時弘仁、川上佳織、河野資、下川宏明 、高柳元明、
山内広平、高橋知子、大野勲)
研究成果発表（ポスター７題）
1. 2-デオキシ−2−フルオロフコース誘導体の合成研究(名取良浩、草野宏光、相原瞳、斎藤有香子、若松秀章、吉村祐一)
2. クリスタキセニシンAの全合成に向けたOxy-Cope環化前駆体の合成研究(渡邉 一弘、中井 啓陽、Sayar Noel 、成田 紘一、吉村 祐一)
3. 新規フェニルアゾトロポロン誘導体の細胞増殖抑制活性とメカニズム解析(皆瀨麻子、氣仙拓也、佐藤大輔、太田公規、遠藤泰之)
4. 神経障害性疼痛モデルラットにおけるバルプロ酸の鎮痛機構－脊髄グルタミン酸放出の調節－(善積克、林田健一郎、James C. Eisenach、渡辺千寿子、溝口広一)
5. 断続的レム断眠ストレス負荷誘発性衝動性様症状における海馬アドレナリンα2A受容体の関与(八百板富紀枝、川浪啓豪、今泉宏紀、根本亙、中川西修、丹野孝一)
6. CYP遺伝子発現誘導を引き起こす健康食品の検討(熊谷 健、渡邉夢実、進藤佐和子、永田 清)
7. 急性好酸球性気道炎症に対する気管支肺胞上皮幹細胞の抗炎症効果の検討— 41 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L(石井聖人、河野資、柏原若奈、平泉花菜、鈴木涼平、佐藤美菜香、宮坂智充、高柳元明、大野勲、
高橋知子）
特別講演３題
1. アジア圏海洋生物由来生物活性物質を創薬シードとする化学的研究(明治薬科大学薬学部・薬化学・齋藤 直樹 教授)
2.ペプチドミメティックを基盤とした中分子創薬研究（東京医科歯科大学・生体材料工学研究所・玉村 啓和 教授）
3. 環状デプシペプチド天然物の全合成、類縁体合成、活性評価、および三次元構造解析（東北大学大学院薬学研究科・反応制御化学・土井 隆行 教授）《平成２９年度 創薬研究センターシンポジウム》開催日：平成 29 年 6 月 17 日(土)研究成果発表（口頭７題）
1. トロポロン骨格を有する新規キサンチンオキシダーゼ阻害剤の探索とそのメカニズム解析（佐藤大輔、氣仙拓也、太田公規、遠藤泰之）
2. エンイナミドの閉環メタセシスによる複素環化合物の合成（若松秀章、髙橋将典、小椋玲奈、諏訪純一、吉村祐一）
3. カンナビノイド受容体に親和性を有するフェルギネン B の全合成
（成田紘一、加藤友哉、吉村祐一、加藤 正）
4. 免疫細胞におけるリン酸化エストロゲン受容体の機能的役割の解析 （進藤佐和子、根岸正彦）
5. 脊髄 Angiotensin 系は糖尿病性神経障害性疼痛の治療標的になり得るか？
（根本 亙、小潟佳輝、山縣涼太、中川西修、八百板富紀枝、丹野孝一）
6. 多発性硬化症疼痛に対する麻薬性鎮痛薬の効果 （溝口広一、渡辺千寿子、音羽 亮、櫻田 忍）
7. 幼少期ストレスよる喘息発症モデル～幼少期ストレスによる免疫寛容の抑制～（大内竜介、河野 資、宮坂智充、大河原雄一、高柳元明、高橋知子、大野 勲）
研究成果発表（ポスター８題）
1. カルボラン含有新規選択的エストロゲン受容体抑制薬（SERD）化合物の探索（皆瀨麻子、吉見友弘、太田公規、遠藤泰之）
2. 3’-デオキシ-4’-チオヌクレオシド誘導体の合成（安達桃子、若松秀章、伊藤 文、斎藤有香子、名取良浩、吉村祐一）
3. キセニアラクトール骨格構築法の開発 （渡邉一弘、高橋央宜、吉村祐一、加藤 正）
4. 健康食品による CYP 遺伝子発現作用の in vivo 評価 （熊谷 健、佐々木崇光、永田 清）
5. 更年期うつ病モデルマウスにおける新規選択的エストロゲン受容体モジュレーター BE360 の抗うつ効果 （佐久間若菜、中川西修、根本 亙、小平貴代、八百板富紀枝、小川卓巳、太田公規、遠藤泰之、 丹野孝一）
6. 糖尿病性神経障害性疼痛に対するアンジオテンシン (1-7) の抑制効果（山縣涼太、根本 亙、小潟佳輝、中川西修、八百板富紀枝、丹野孝一）
7. 炎症性疼痛下における麻薬性鎮痛薬の鎮痛効果ならびに鎮痛耐性形成（込山麻美、渡辺千寿子、音羽 亮、櫻田 忍、溝口広一）
8. 精神的ストレスによるアレルギー性気道炎症の増悪における中枢性ヒスタミン受容体の関与（岩見俊哉、宮坂智充、増田千愛、河野 資、川上佳織、高柳元明、高橋知子、大野 勲）
特別講演３題
1. オートファジー欠損マウスからわかったこと （新潟大学大学院医歯学総合研究科・小松雅明 教授）
2. サイアタンジテルペノイドの不斉全合成と生物活性 （早稲田大学理工学術院・中田雅久 教授）
3. 痛みメモリーを消去するLPAシグナル制御創薬研究（長崎大学大学院医歯薬学総合研究科・植田弘師 教授）《平成２８年度 創薬研究センターシンポジウム》開催日：平成 28 年 6 月 18 日(土)研究成果発表（口頭７題）
1. 気管支喘息における Th2 サイトカイン産生の性差に関与する樹状細胞の役割（増田千愛、宮坂智充、岩見俊哉、河野 資、大河原雄一、井ノ口仁一、高柳元明、高橋知子、大野勲）
2. 神経障害性疼痛に対する麻薬性鎮痛薬ならびに新規鎮痛薬の効果— 42 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L（溝口広一、渡辺千寿子、音羽 亮、櫻田 忍）
3. 嗅球摘出マウスにおける脳内ドパミン神経機能の変化に対する三環系抗うつ薬 Imipramine の影響（髙橋浩平、中川西修、根本 亙、菊地恵理、佐久間若菜、八百板富紀枝、丹野孝一）
4. 健康食品の CYP 活性に対する網羅的評価 （熊谷 健、佐藤 裕、佐々木崇光、永田 清）
5. 3’-デオキシ-4’-チオヌクレオシドの立体選択的合成（安達桃子、伊藤 文、名取良浩、若松秀章、吉村祐一）
6. 変異 AR に対するカルボラン含有グリセロール誘導体の活性評価（皆瀨麻子、阿部孝俊、太田公規、小田彰史、遠藤泰之）
7. ジスルフィド結合を有する大環状デプシペプチド FR901375 の全合成 （加藤友哉、成田紘一、加藤正）
研究成果発表（ポスター８題）
1. 幼少期ストレスによる喘息発症リスクの増大―ストレスによる免疫寛容の抑制― （大内竜介、河野資、新田典秀、宮坂智充、大河原雄一、高柳元明、高橋知子、大野勲）
2. 炎症性疼痛下における各種麻薬性鎮痛薬の鎮痛効果とその耐性形成能 （込山麻美、溝口広一、渡辺千寿子、音羽 亮、櫻田 忍）
3. 長期粉末食飼育誘発性マウス異常行動に対する ADHD 治療薬の効果 （八百板富紀枝、柴田 楓、
菅原 彩、土谷昌広、荒井裕一朗、根本 亙、中川西修、只野 武、丹野孝一）
4. レチノイン酸レセプター、RAR/RXR を介した CYP26A1 遺伝子発現誘導評価系の構築 （塩谷安奈里、
渡邊美智子、角田駿哉、若生俊也、佐々木崇光、熊谷 健、永田 清）
5. CYP3A4 遺伝子転写活性化の新規分子機構の解明 （小田桐玲生、佐々木崇光、熊谷 健、永田 清）
6. スタブジン誘導体の合成研究 （伊藤恭平、菅野裕也、齋藤華子、若松秀章、名取良浩、斎藤有香子、吉村祐一）
7. 高尿酸血症改善へ向けた新規 XO 阻害剤の創製 （太田公規、石井康博、佐藤 匠、遠藤泰之）
8. 生合成仮説を基軸とした(+)-リファガールの全合成 （渡辺拓実、神島尭明、菊地拓也、成田紘一、加藤 正）
特別講演（３題）
1. 二重活性化型触媒の創製を基盤とする新規分子骨格構築反応の開発（大阪大学産業科学研究所・笹井宏明 教授）
2. 新しい天然物を獲る―休眠遺伝子を活用する天然物の創出―（東北大学大学院薬学研究科・大島吉輝 教授）
3. 核酸系医薬におけるフッ素置換基の役割 （北海道大学大学院薬学研究院・松田 彰 教授）《共催講演会等》
1. セミナー（平成31年3月4日） 井上 誠 准教授（University of Illinois at Urbana-Champaign）
状況に応じた免疫システム変調と中枢神経機能変調-多発性硬化症研究をもとに
2. 特別講演会（平成29年1月13日） 今道友純 主任研究員（Frederick National Laboratory for Cancer
Research）
IL-27のHIV抑制機構とDNA刺激によるIFN-λ1の産生
3. 特別講演会（平成28年11月7日） 藤井秀明 教授（北里大学薬学部）
δオピオイド受容体逆作動薬における窒素置換基に関する構造活性相関研究
4. 特別講演会（平成28年10月21日） 渡邉廣行 研究員（Uppsala University）
脊髄オピオイド系が外傷性脳損傷後の姿勢非対称性に関与する可能性
5. 特別講演会（平成28年10月18日） 濱村賢吾 助教（第一薬科大学）“生体リズム”および“血清中エクソソーム”に着目した神経障害性疼痛の病態解明
6. 特別講演会（平成27年10月21日） Prof. David R. Williams （Indiana University）《インターネットでの公開状況》
本研究プロジェクトの目的・研究計画概要（http://www.tohoku-mpu.ac.jp/about/a20/）および平成27-29 年度の「研究進捗状況報告書の概要」（http://www.tohoku-mpu.ac.jp/wp/wp-content/uploads/2019/05/b7cbd27efd694b40bd87cdb185aa7dd7.
pdf）を公開している。

＜これから実施する予定のもの＞
本研究プロジェクトの「研究成果報告書」を公開する予定である。
— 43 —

（様式 2）
法人番号プロジェクト番号

041004
S1511001L

１４ その他の研究成果等《特許出願》*1. 発明の名称：リファガールの類縁体、及びリファガール又はその類縁体を含む多標的キナーゼ阻害剤

発明者：石岡千加史、西條 憲、加藤 正、成田紘一 (出願人: 国立大学法人 東北大学)
特許出願番号：特願 2017-091712 (出願日 平成 29 年 5 月 2 日)
特許公開番号：現時点では未公開*2. 発明の名称：デプシペプチド類化合物の製造中間体及びその製造方法(Method for Producing
Depsipeptide Compounds and Synthetic Intermediates Therefor)
発明者：石岡千加史、西條 憲、加藤 正、成田紘一 (出願人: 国立大学法人 東北大学)
国際特許出願番号：PCT/JP2017/001140 (出願日 2017 年 01 月 13 日)
国際特許公開番号：WO2017/122822 (公開日 2017 年 7 月 20 日).

《企業からの依頼講演》
3. 抗ウイルス剤探索を目的としたヌクレオシド誘導体合成法の開発、吉村祐一、富士フィルム富山事業所社内ゼミ、富山、2017 年 8 月 22 日．

１５ 「選定時」及び「中間評価時」に付された留意事項及び対応＜「選定時」に付された留意事項＞
アゴニスト/アンタゴニストバランスなどの問題は、分子動力学専門家と共同で効率的な解決を模索して欲しい。

＜「選定時」に付された留意事項への対応＞
分子動力学の専門家である小田彰史教授(名城大学薬学部)との共同研究を開始し、エストロゲン受容体β選択性やアゴニスト/アンタゴニスト制御に関して、設計・合成した化合物と受容体のドッキングシミュ
レーションを含む結果を本研究期間中に論文および学会発表するなど(論文*56, 67, 94, 96, 97、学会*164)、協力体制を作り、効率的に研究を推進している。

＜「中間評価時」に付された留意事項＞
該当なし＜「中間評価時」に付された留意事項への対応＞
留意事項としての指摘はなかったが、評価コメントの中で①若手研究員・大学院生のテニュアトラック制度導入や②AMED（日本医療研究開発機構）の積極的な活用と連携を視野に入れた研究の展開に期待が示されていた。テニュアトラック制度の導入に関しては本研究プロジェクトのプログラム中に取り入れることができなかったが、今後全学上げて取り組むべき課題であると認識している。また、本研究プロジェクトで非常に優れた化合物を見出したので、今後は AMED 等と連携しながらアンメット・メディカル・ニーズに応えられる医薬品の創出を目指していきたい。

— 44 —

（様式2）
法人番号プロジェクト番号
１６ 施設・装置・設備・研究費の支出状況（実績概要）
内
年度・区分 支出額 法 人
平 施 設
成
２ 装 置
７ 設 備
年
度 研究費
平 施 設
成
２ 装 置
８ 設 備
年
度 研究費
平 施 設
成
２ 装 置
９ 設 備
年
度 研究費
平 施 設
成
３ 装 置
０ 設 備
年
度 研究費

（千円）
訳

共同研
受託
究機関
寄付金 その他（
研究等
負担

負 担

私 学
助 成

81,206

27,071

54,13521,00011,1799,821

15,9188,881

19,041

10,00011,7509,250

041004
S1511001L

）

0
0

0
0
0
24,79900
0
29,04100
021,000

平 施 設
成
３ 装 置
１ 設 備
年
度 研究費

21,000

16,106

4,894

施 設

0

0

0

0

0

0

0

装 置

0

0

0

0

0

0

0

設 備

81,206

27,071

54,135

0

0

0

0

研究費

116,840

73,99442,84600

0

0

198,046

101,065

96,981

0

0

0

0

総
額
総

計

0
0
0

— 45 —備 考（様式2）
法人番号041004

１７ 施設・装置・設備の整備状況 （私学助成を受けたものはすべて記載してください。）《施 設》 （私学助成を受けていないものも含め、使用している施設をすべて記載してください。）
施 設 の 名 称

整備年度

研究施設面積H182,108㎡

創薬研究センター

研 究 室 等 数 使用者数

7教室

事業経費

補助金額

（千円）
補助主体

73名

※ 私学助成による補助事業として行った新増築により、整備前と比較して増加した面積㎡《装置・設備》 （私学助成を受けていないものは、主なもののみを記載してください。）
装置・設備の名称

型

整備年度

番

台

数

稼働時間数

事業経費

（千円）
補助金額

補助主体

（研究設備）
リアルタイム細胞アナライザー

H27

超高感度等温適定型

H27

カロリーメーター

xCELLigence
RTCA SPシステム

1式

1950

Affinity ITC Auto

1式

93

LV オートサンプラーシステム

正立型共焦点レーザー
顕微鏡システム

H27

Ni-E,C2+

1式

1580

擦過行動リアルタイム
定量化システムH27

SCLABA-Realシステム

1式

434

全自動磁気細胞分離装置

H27130-092-545

1式

438

１８ 研究費の支出状況【テーマ1】
年度
平成
小 科 目
消 耗 品 費光 熱 水 費
通信運搬費
印刷製本費
旅費交通費
報酬・委託料
（
賃借料
）
（
修繕費
）
計
人件費支出
（兼務職員）
教育研究経費支出
計
教育研究用機器備品

図

ｈ
ｈ
ｈ
ｈ
ｈ

13,413

8,942 私学助成

32,821

21,880 私学助成

ｈ
ｈ
ｈ
ｈ
ｈ

20,552

13,701 私学助成

8,916

5,943 私学助成

ｈ
ｈ

5,504

3,669 私学助成

（千円）
27

年度

積 算 内 訳主 な 使 途金 額
主 な 内 容
教
育
研
究
経
費
支
出
5,518 薬品・実験材料・その他
5,518 試薬・研究試料・実験用品・文房具・OA用品
009 郵便・運搬
9 サンプル等配送 等
35 論文等印刷
35 論文印刷、文献複写
777 学会・研究発表
777 学会参加、研究打合せ
261 委託・支払報酬
261 分析・検査、英文校正、講演料等
5 設備賃借費
5 容器使用料
106 設備修繕
106 機器修理、メンテナンス
6,711
6,711
ア
ル
バ
イ
ト
関
係
支
出

支 出 額

0
設 備 関 係 支 出（１個又は１組の価格が５００万円未満のもの）
2,289 機械器具
2,289 有機熔媒精製装置 他

書
計

2,289
研

究

ス

タ

ッ

フ

2,289
関

リサーチ・アシスタント

ポスト・ドクター
研究支援推進経費

計00— 46 —係支出（様式2）
法人番号041004【テーマ1】
年

（千円）
度

小 科 目

年度

積 算 内 訳主 な 使 途金 額
主 な 内 容
教
育
研
究
経
費
支
出
6,077 薬品・実験材料・その他
6,077 試薬・研究試料・実験用品・文房具・OA用品
004 郵便・運搬4 サンプル等配送 等
120 論文等印刷
120 論文印刷・文献複写
380 学会・研究発表
380 学会参加・研究打合せ
463 委託・支払報酬
463 分析・検査、英文校正、講演料等
5 設備賃借費
5 容器使用料
570 設備修繕
570 機器修理、メンテナンス
7,619
7,619
ア
ル
バ
イ
ト
関
係
支
出

支 出 額

消 耗 品 費光 熱 水 費
通信運搬費
印刷製本費
旅費交通費
報酬・委託料（ 賃借料 ）
（ 修繕費 ）
計
人件費支出
（兼務職員）
教育研究経費支出
計00
設 備 関 係 支 出（１個又は１組の価格が５００万円未満のもの）
1,381 スターラー付低温恒温槽 他
1,381 機械器具

教育研究用機器備品

図

28

平成

書
1,381
研

計

究

ス

タ

ッ

フ

1,381
関

係

支

出

リサーチ・アシスタント

ポスト・ドクター
研究支援推進経費

計3,799 研究補助

3,799 学内１人

3,799

3,799

【テーマ1】
年

（千円）
度

小 科 目
消 耗 品 費光 熱 水 費
通信運搬費
印刷製本費
旅費交通費
報酬・委託料（ 賃借料 ）
（ 修繕費 ）
計
人件費支出
（兼務職員）
教育研究経費支出
計
教育研究用機器備品

図

29

平成

年度

積 算 内 訳主 な 使 途金 額
主 な 内 容
教
育
研
究
経
費
支
出
6,718 薬品・実験材料・その他
6,718 試薬・研究試料・実験用品・文房具・OA用品
003 郵便・運搬
3 サンプル等配送 等
112 論文等印刷
112 論文印刷・文献複写
619 学会参加・研究打合せ
619 学会・研究発表
209 委託209 分析・検査、英文校正
1 設備賃借費
1 容器使用料
21 設備修繕
21 機器修理、メンテナンス
7,683
7,683
ア
ル
バ
イ
ト
関
係
支
出

支 出 額

0
0
設 備 関 係 支 出（１個又は１組の価格が５００万円未満のもの）
1,317 機械器具
1,317 薬用冷蔵ショーケース 他

書
計

1,317
研

究

ス

タ

ッ

フ

1,317
関

係

リサーチ・アシスタント

ポスト・ドクター
研究支援推進経費

計

8,041 研究補助

8,041 学内２人

8,041

8,041

— 47 —支出（様式2）
法人番号041004【テーマ1】
年

（千円）
度

小 科 目

年度

積 算 内 訳主 な 使 途金 額
主 な 内 容
教
育
研
究
経
費
支
出
6,880 薬品・実験材料・その他
6,880 試薬・研究試料・実験用品・文房具・OA用品
002 郵便・運搬
2 サンプル等配送 等
0091 学会・研究発表
91 学会参加・研究打合せ
71 委託
71 分析・検査、英文校正
4 設備賃借費
4 容器使用料
528 設備修繕
528 機器修理、メンテナンス
7,576
7,576
ア
ル
バ
イ
ト
関
係
支
出

支 出 額

消 耗 品 費光 熱 水 費
通信運搬費
印刷製本費
旅費交通費
報酬・委託料（ 賃借料 ）
（ 修繕費 ）
計
人件費支出
（兼務職員）
教育研究経費支出
計00
設 備 関 係 支 出（１個又は１組の価格が５００万円未満のもの）
1,424 機械器具
1,424 低温循環水槽クールマンパル 他

教育研究用機器備品

図

30

平成

書
1,424
研

計

究

ス

タ

ッ

フ

1,424
関

係

支

出

リサーチ・アシスタント

ポスト・ドクター
研究支援推進経費計

00【テーマ1】
年

（千円）
度

小 科 目
消 耗 品 費光 熱 水 費
通信運搬費
印刷製本費
旅費交通費
報酬・委託料（ 賃借料 ）
（ 修繕費 ）
計
人件費支出
（兼務職員）
教育研究経費支出
計
教育研究用機器備品

図書
計

リサーチ・アシスタント

ポスト・ドクター
研究支援推進経費

計

31

平成

年度

積 算 内 訳主 な 使 途金 額
主 な 内 容
教
育
研
究
経
費
支
出
8,801 試薬・研究試料・実験用品・文房具・OA用品
8,802 薬品・実験材料・その他
00
3 郵便・運搬
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18 分析・検査、英文校正
3 設備賃借費
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— 48 —

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65 論文等印刷
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196 委託・支払報酬
196 分析・検査、英文校正、講演料等
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59 論文等印刷
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56 論文等印刷
56 論文印刷・文献複写
185 学会・研究発表
185 学会参加・研究打合せ
59 委託
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30 設備賃借費
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355 学会参加・研究打合せ
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177 学会・研究発表
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計00— 53 —係支出

— 54 —

研究テーマ１
新規がん分子標的治療薬の創製— 55 —

— 56 —

Received: 20 January 2018

Revised: 19 September 2019

Accepted: 28 September 2019

DOI: 10.1111/bph.14900

RESEARCH PAPER

UDP-Glucuronosyltransferase (UGT)-mediated attenuations ofcytochrome P450 3A4 activity: UGT isoform-dependent
mechanism of suppression
Yuu Miyauchi1,2 |Yoshitaka Tanaka2Peter I. Mackenzie5||

Kiyoshi Nagata3 |Hideyuki Yamada1† |Yasushi Yamazoe4 |Yuji Ishii1

1

Laboratory of Molecular Life Sciences,
Graduate School of Pharmaceutical Sciences,
Kyushu University, Fukuoka, Japan

2

Division of Pharmaceutical Cell Biology,
Graduate School of Pharmaceutical Sciences,
Kyushu University, Fukuoka, Japan3

Department of Environmental and Health
Science, School of Pharmaceutical Sciences,
Tohoku Medical and Pharmaceutical
University, Sendai, Japan

4

Background and Purpose: Cytochrome P450 (CYP, P450) 3A4 is involved in themetabolism of 50% of drugs and its catalytic activity in vivo is not explained only
by hepatic expression levels. We previously demonstrated that UDP-glucuronosyltransferase (UGT) 2B7 suppressed CYP3A4 activity through an interaction.
In the present study, we target UGT1A9 as another candidate modulator of
CYP3A4.
Experimental Approach: We prepared co-expressed enzymes using the baculovirus-

Food Safety Commission, Cabinet Office,
Government of Japan, Tokyo, Japan

insect cell expression system and compared CYP3A4 activity in the presence and5absence of UGT1A9. Wistar rats were treated with dexamethasone and liver micro-

Department of Clinical Pharmacology,
Flinders Medical Centre and Flinders
University, Adelaide, SA, Australia
Correspondence
Yuji Ishii, Laboratory of Molecular Life
Sciences, Graduate School of Pharmaceutical
Sciences, Kyushu University, 3-1-1 Maidashi,
Higashi-ku, Fukuoka 812-8582, Japan.
Email: ishii@phar.kyushu-u.ac.jp
Funding information
Japan Society for the Promotion of Science,
Grant/Award Number: 25293039; Ministry of
Education, Culture, Sports, Science andTechnology, Grant/Award Number: 19590147;
Japan Research Foundation for ClinicalPharmacology

somes were used to elucidate the role of CYP3A–UGT1A interactions.
Key Results: UGT1A9 and UGT2B7 interacted with and suppressed CYP3A4. Kinetic
analyses showed that both of the UGTs significantly reduced Vmax of CYP3A4 activity. In addition, C-terminal truncated mutants of UGT1A9 and UGT2B7 still retained
the suppressive capacity. Dexamethasone treatment induced hepatic CYP3As andUGT1As at different magnitudes. Turnover of CYP3A was enhanced about twofoldby this treatment.
Conclusion and Implications: The changes of kinetic parameters suggested thatUGT1A9 suppressed CYP3A4 activity with almost the same mechanism as UGT2B7.
The luminal domain of UGTs contains the suppressive interaction site(s), whereas the
C-terminal domain may contribute to modulating suppression in a UGT isoformspecific manner. CYP3A–UGT1A interaction seemed to be disturbed by dexamethasone treatment and the suppression was partially cancelled. CYP3A4–UGT
interactions would help to better understand the causes of inter/intra-individual differences in CYP3A4 activity.

Abbreviations: CNX, calnexin; CPR, NADPH-cytochrome P450 reductase; CYP and P450, cytochrome P450; DDI, drug–drug interactions; HLM, human liver microsomes; SNP, single-nucleotide
polymorphism; UGT, UDP-glucuronosyltransferase.
†

Deceased

Br J Pharmacol. 2020;1–13.

wileyonlinelibrary.com/journal/bph— 57 —© 2019 The British Pharmacological Society

1

2

1

MIYAUCHI ET AL.

|

I N T RO DU CT I O NWhat is already known

Cytochrome P450 (CYP, P450) 3A4 is involved in the metabolism of• Two drug-metabolizing enzymes, cytochrome P450 (CYP)

30–50% of drugs clinically used (Thummel & Wilkinson, 1998;

and UDP-glucuronosyltransferase (UGT) form complexes.

Zanger & Schwab, 2013). Thus, it is very important to understand the• UGT2B7 interacts with and suppresses CYP3A4 activity

causes of inter-individual differences of CYP3A4 activities, especiallyby inhibiting substrate binding.

as there is a large discrepancy between the difference in its expression level and in CYP3A4-mediated drug clearance. Hepatic expres-

What does this study add

sion levels of CYP3A4 differ up to 40-fold, but the variance in• Another UGT isoform, UGT1A9 also suppresses CYP3A4

CYP3A4-mediated drug clearance is only as much as 10-fold (Lamba,

in a UGT isoform-dependent fashion.

Lin, Schuetz, & Thummel, 2002; Shimada, Yamazaki, Mimura, Inui, &• Dexamethasone affected and partially abrogated theGuengerich, 1994). In other P450 isoforms, single-nucleotide poly-

CYP3A–UGT1A interactions in rat liver.

morphisms (SNPs) clearly explain discrepancies between expression
levels and clearance; CYP2C9*2 and CYP2D*18 show lower catalytic

What is the clinical significanceactivities because the SNPs attenuate their enzymatic functions. In• CYP–UGT interactions contribute to our understanding

contrast, however, CYP3A4 SNPs which can affect activity withoutof the causes of drug–drug interactions.

changing expression levels are very rare. Thus, SNPs cannot account• These interactions also result in the large differences infor this discrepancy in the case of CYP3A4 (Johansson & Ingelman-

hepatic enzyme level between individuals.

Sundberg, 2011; Zanger & Schwab, 2013).
To address this issue, we focused on protein–protein interactions
between CYP3A4 and UDP-glucuronosyltransferase (UGT) as a posttranslational mode of regulation of CYP3A4 activity. Interactions ofP450 with NADPH-cytochrome P450 reductase (CPR) and cytochrome b5 were well examined (Hildebrandt & Estabrook, 1971; Lu,
Junk, & Coon, 1969). P450–P450 and UGT–UGT oligomerization

modulate CYP3A4 function remain unclear. To address this issue, inwere also reported and protein interactions were shown to control

the present study, we focused on UGT1A9, another major human iso-their activities (Backes & Kelley, 2003; Ishii, Takeda, & Yamada, 2010).

form and performed a comparative study with UGT2B7.

However, although P450 and UGT both localize to the endoplasmic
reticulum membrane, they had been thought to work separately
because of differences in their membrane topologies. P450 faces the2|

METHODS

cytosol whereas the majority of the UGT protein is oriented to thelumen. With UGT, only the C-terminal region is predicted to form atransmembrane helix followed by a cytosolic tail, the region composed

2.1 | Construction of recombinant baculovirus andmammalian expression vectorsof about 20 residues extruding to the cytosol (Black, 1992;
Radominska-Pandya, Czernik, Little, Battaglia, & Mackenzie, 1999).

Bac-to-Bac® Baculovirus Expression System (Life Technologies, Carls-

Despite such a large topological difference, however, it is reasonablebad, CA) was used to prepare recombinant baculovirus. The recombi-that P450 and UGT interact with each other because some chemicals

nant baculoviruses coding CYP3A4, human P450 reductase (CPR),

are hydroxylated by P450 and sequentially glucuronidated by UGT. To

UGT2B7 wild-type (WT) and its truncated mutant lacking transmem-

validate our hypothesis, we have detected P450–UGT complexes bybrane followed by cytoplasmic domain (ΔTM or Δ493-529) were pre-several biochemical methods and reported that P450 could modulate

pared as described previously (Miyauchi et al., 2015). The open

UGT function (Ishii et al., 2007; Ishii et al., 2014; Nakamura et al.,reading frame of UGT1A9 was amplified using pIRES-UGT1A9

2016; Takeda et al., 2005; Takeda et al., 2009; Taura et al., 2000).

(Uchaipichat et al., 2004) as a template, KOD-Plus-Neo DNA Polymer-

Other groups also supported our reports (Fremont, Wang, & King,ase (Toyobo Life Science, Osaka, Japan), and the following primers:

2005; Fujiwara & Itoh, 2014; Rouleau, Audet-Delage, Desjardins,sense, 50 -CGGGATCCGTGATGGCTTGCACAGGGTG-30 (primer A);

Girard-Bock, & Guillemette, 2017). In our last study, we confirmed theanti-sense, 50 -CGGGATCCCACTTCTCAATGGGTCTTGG-30 (under-

reverse effect; UGT2B7 suppressed CYP3A4 activity by inhibiting

line, BamHI site). Thermal cycling condition was as follows: initialsubstrate binding to the P450. In addition, we also determined interac-denaturation, 94 C, 2 min; 40 cycling step, 98 C, 10 s; 52 C, 30 s;tion sites of UGT2B7 with CYP3A4. Hydrophobic regions in the68 C, 1 min; hold, 4 C. The PCR products were digested with BamHI

C-terminal region and luminal membrane anchoring region were nec-

and inserted into pFastBac1 cleaved with the same enzyme. To gener-

essary for CYP3A4–UGT2B7 interaction. These two sites work coop-

ate the truncated mutant of UGT1A9 (Δ488-530) which lacks theeratively in the suppression (Miyauchi et al., 2015).

transmembrane and cytoplasmic domains, we performed PCR with

Although we reported that UGT2B7 could suppress CYP3A4, theprimer A as sense primer and the following anti-sense one: 50 -mechanism of the suppression and whether other UGT isoforms also

CGGGATCCGTGATGGCTTGCACAGGGTG-30 (underline, BamHI site).

— 58 —

3

MIYAUCHI ET AL.

The recombinant pFastBac1 vectors were transfected into the competent Escherichia coli DH10Bac™ strain (Life Technology). After positive

2.3 | Culture of COS-1 cells and transfection of
mammalian expression vector

clones were selected by blue white selection, recombinant bacmids,
baculoviral DNAs for producing recombinant virus, were prepared

COS-1 cells (RRID:CVCL_0223) were cultured in DMEM containing

according to the user's manual. In addition, we subcloned UGT1A9high glucose (Wako Pure Chemical Industries, Osaka, Japan) supplied

cDNA into the pcDNA3.1/hygro (−) vector for experiments with

with 10% FBS. Cells were seeded to 35-mm dishes and transfectedmammalian cells. An HA-tag was added to its C-terminus to improvewith 2-μg pcDNA3.1 vectors by Lipofectamine 3000 (Life Technolo-

detection in pull-down assays. The introduction of an HA-tag was per-gies). Cells were harvested with lysis buffer, 20-mM Tris-HCl (pH 7.4)

formed by two rounds of PCR. A forward part of the HA-tag wascontaining 150-mM NaCl, 1% Triton X-100, 10% glycerol, and soni-

introduced in the first round, and the latter part followed by a restric-

cated to prepare whole cell lysate. When we use the lysates for pull-tion site was introduced in the second round using pairs of primers

down assay, those were sequentially centrifuged, and supernatants

below: the first round sense, primer A; the first round anti-sense,

were collected.

50 -ATCTGGAACATCGTATGGGTAATGGGTCTTGGATTTGTGGG-30(bold, forward part of HA-tag); the second round sense, primer A; thesecondround

anti-sense,

50 -CGGGATCCTCAAGCGTAATCTGG

2.4|

Pull-down assay

AACATCGTATGGGTAAT-3 (underline, BamHI site; bold, coding HA0tag). In the second round, PCR products of the first round were used

Whole cell lysate (600 μg) was mixed with 15 μl of magnetic agaroseas the template. PCR was carried out as described above.

beads conjugated with Ni-nitrilotriacetic acid (Qiagen, Hilden, Ger-

Δ488-530-HA construct was generated by QuickChange site-directedmany) equilibrated with lysis buffer. To avoid non-specific binding, wemutagenesis with pcDNA3.1_WT-HA as a template using the follow-

added another 200-mM sodium chloride, 40-mM imidazole, 0.05%

ing primers: sense, 50 -CAGTACCATTCCTTGGACTACCCATACGATG

Emulgen 911 and 0.2% BSA to the mixture. Protein binding to the

TTCCA-30 ; anti-sense, 50 -TGGAACATCGTATGGGTAGTCCAAGGAAT

beads was conducted at 4 C for 4 hr. The beads were washed three

GGTACTG-30 . The primers were designed using Agilent QuikChange

times with 20-mM Tris-HCl (pH 7.4 at 4 C) containing 200-mM

Primer(https://www.genomics.agilent.com/

sodium chloride, 40-mM imidazole, 20% glycerol and 0.05% EmulgenprimerDesignProgram.jsp). Hexa-histidine conjugated CYP3A4 has

DesignProgram911. Then binding proteins were eluted from the beads with 20 μl ofbeen cloned into bacmid for Ni2+ pull-down assay (Miyauchi et al.,10-mM sodium phosphate (pH 7.4) containing 300-mM sodium chlo-2015). We amplified the cDNA of CYP3A4-6 × His with the following

ride, 250-mM imidazole, 20% glycerol and 0.05% Emulgen 911. Sam-primers: sense, 5 -ATAAGAATGCGGCCGCGTGATGGCTCTCATCCC

ples were stored until analysis by immunoblotting.

0

AGA-30 ;anti-sense,

50 -CCGCTCGAGTCAGTGATGGTGATGGTGATGGGCTCCACTTAC-30 (underline, NotI and XhoI site, respectively;bold, coding hexa-histidine). All of the sequences of the constructs

2.5|

Immunoblotting®

were confirmed by an ABI 3130xl Genetic analyser, using a BigDye
Terminator v3.1 Cycle Sequencing Kit (Life Technologies).

The immuno-related procedures used comply with the recommendations made by the British Journal of Pharmacology (Alexander et al.,2018). Proteins were separated by SDS-PAGE and electroblotted onto

2.2 | Culture of insect cells and preparation ofhomogenatesa PVDF membrane (Immobilon-P; Millipore, Bedford, MA; Cat#
IPVH00010). The blotted membrane was washed with TBS-T, 20-mM
Tris-HCl (pH 7.5) containing 150-mM NaCl, and 0.1% Tween 20 and

All the cellular experiments were carried out and analysed by a sin-blocked with 2% skim milk in TBS-T at room temperature for 30 min.

gle investigator, so no blinding was undertaken. Sf9 (RRID:

For detection of targets, the following primary antibodies and detec-

CVCL_0549) insect cells were cultured according to our previous

tion kit were utilized: rabbit polyclonal anti-UGT1A antibody (H-300,

study (Miyauchi et al., 2015). We prepared cell homogenates from

Cat# sc-25847, RRID:AB_2241481), mouse monoclonal anti-CPR

infected cells for the CYP3A4 assay. For preparation of cell

antibody (F-10, Cat# sc-25270, RRID:AB_627391; Santa Cruz Bio-6homogenates, 2.0 × 10 cells were seeded in 35-mm dish 24 hr

technology, Dallas, TX), goat polyclonal anti-CYP3A2 serum (Daiichi

prior to viral infection. Following medium exchange, baculoviruses

Pure Chemicals, Tokyo, Japan; Cat# 877), rabbit polyclonal anti-

were transfected. Twenty-four hours after the infection, hemin-

UGT2B7

BSA was supplied to the cells (final concentration of 1 μgml−1 of(Proteintech, Rosemont, IL; respective Cat# 16661-1-AP andhemin). Forty-eight hours after hemin supply, cells were harvested

10494-1-AP, respective RRID:AB_2214249 and AB_2263076), rabbit

in homogenization buffer, 10-mM Tris-HCl (pH 7.5) containing

polyclonal anti-calnexin (CNX) antibody (GeneTex, Irvine, CA; Cat#

0.25-M sucrose, 10% glycerol, and 1× protease inhibitor cocktail

GTX112886, RRID:AB_1949822), rabbit polyclonal anti-6-His anti-(Nacalai Tesque, Kyoto, Japan), and homogenates were preparedbody (Bethyl Laboratories, Montgomery, TX; Cat# A190-114A, RRID:

by sonication for 30 s with a Ultrasonic Cleaner UT-51N (SHARP,

AB_67321), rabbit polyclonal anti-hemagglutinin (HA) antibody

Osaka, Japan).

(Sigma-Aldrich, St. Louis, MO; Cat# H6908, RRID:AB_260070), and— 59 —

antibody,

rabbitpolyclonalanti-GAPDH

antibody

4

MIYAUCHI ET AL.

WB-MAB-3A Human CYP3A Western Blotting Kit (Corning Gentest,

5 in each group, 150–164 g of body weight) were purchased from

Woburm, MA; Cat# 458254, RRID:AB_712943). The primary anti-

CLEA Japan (Tokyo, Japan), randomly divided into two groups, andbodies were diluted 2,000-fold with TBS-T containing 2% skim milk.

maintained for 1 week. They were treated with dexamethasone

The membranes were incubated with the diluted primary antibody at

21-phosphate intraperitoneally at a dose of 80 mgkg−1 in saline every

4 C overnight. The diluted primary antibodies were collected, stored

day for 4 days. Control rats were injected with the same amount of

at 4 C and reused up to 10 times. Immunochemical detection was

saline. The day following the last treatment, livers were removed andperformed with HRP-conjugated secondary antibody, HRP-rabbit

perfused with ice-cold saline and microsomes were prepared individu-

anti-goat IgG (MP Biomedicals, Santa Ana, CA; Cat# 59301), HRP-

ally from the liver homogenates by differential centrifugation as

donkey anti-rabbit IgG, or HRP-sheep anti-mouse IgG (GE Healthcare,

described elsewhere (Oguri et al., 1996). Collected microsomes wereLittle Chalfont, UK; respective Cat# NA934V and NA931V). After theresuspended in 0.25-M sucrose, snap-frozen in liquid nitrogen andfirst antibody treatment, the membranes were extensively washedstored at −80 C prior to use. To determine relative protein levels ofwith TBS-T and incubated with the secondary antibody diluted

CYP3A, UGT1A, and CPR in the rat liver microsomes by immunoblot-

10,000-fold with TBS-T containing 2% skim milk at room temperatureting, CNX was selected as a loading control and each band was stan-for 1 hr. Then the membranes were washed extensively again.

dardized to the corresponding CNX band. CNX is an ER marker

EzWestLumi plus (ATTO, Tokyo, Japan) was utilized as a substrate ofprotein and a previous report suggested that dexamethasone treat-

HRP, and the signals were visualized and quantified with a ChemiDoc

ment does not affect CNX protein level (Fujii et al., 2006). Intensities

MP System (Bio-Rad, Hercules, CA). For detection of another target

of CYP3A were quite different between control and dexamethasone-on the same membrane, antibodies were stripped after detection. All

treated groups. Thus, we utilized 10- and 1-μg microsomes of control

the stripping processes were carried out at room temperature. Usedand dexamethasone-treated rats, respectively, in order to detect themembrane was incubated with stripping buffer, 25-mM glycine-HCl

CYP3A bands at the same range. In kinetic analyses, assays were con-(pH 2.0) containing 1% sodium dodecylsulphate for 30 min. After that,

ducted with 20-μg microsomes and luciferin-PFBE as enzyme source

the membrane was neutralized by washing three times for 5 min inand substrate, respectively, and CYP3A activity was calculated with

TBS-T, reblocked with TBS-T containing 2% skim milk for 30 min andrelative expression levels of CYP3A explained above in each sample.

used in an incubation with another primary antibody.

Animal treatment/sample preparation and the later analysis were conducted in a blinded manner.

2.6|

Assay of CYP3A4 activity
2.8|

Other methods

CYP3A4 activity was measured with P450-Glo CYP3A4 Assay System(Luciferin-PFBE: Promega, Madison, WI). CYP3A4 activity was deter-

Protein concentrations of rat liver microsomes were determined bymined by measuring chemiluminescence according to the manufac-

the method of Lowry, Rosebrough, Farr, and Randall (1951), and thoseturer's protocol with insect cell homogenates (20 μg in each well) asof lysates prepared from COS-1 cells and homogenates from insect

enzyme source. NADPH was used instead of regenerating systemcells were by Protein Assay CBB Solution (Nacalai Tesque) with BSA

because its oxidized form (NADP+) was reported to inhibit UGT func-

as a standard.

tion (Nishimura et al., 2007). In kinetic experiments, the substrate concentration varied from 6.25 to 150 μM. Data were fitted to the

2.9Michaelis–Menten model defined by the equation below:|

Data and statistical analysis

V = Vmax × S=ðKM + SÞ,Results are presented as the means ± SD of measurements obtained

where V is the reaction rate, S is the substrate concentration, Vmax is

Statistical analysis was undertaken only for studies where each groupthe maximum enzyme velocity and KM is the Michaelis constant,size was at least N = 5 with GraphPad Prism 5.04 software (GraphPad

which is equal to the concentration of substrate for half-maximal

software, La Jolla, CA; RRID:SCR_002798). Statistical significance invelocity.

comparison among the three groups was determined by ANOVA

2.7 | Preparation of microsomes fromdexamethasone-treated rats

cant and there was no significant variance in homogeneity. The statis-

from six batches of cells or five rats in each group per experiment.

followed by Dunnett's test only when the former F value was signifitical significance between the two groups was determined byStudent's t test (unpaired, two tailed), and we also confirmed that
Animal experiments in this study were approved by the Institutional

there was no significant variance in homogeneity between the two

Animal Care and Experiment Committee of Kyushu University (appli-groups. A P value less than 0.05 was considered statistically signifi-

cation approval number: A19-084). Animal studies are reported incant. Protein expression levels of rat liver CYP3A, UGT1A, and CPR

compliance with the ARRIVE guidelines (Kilkenny, Browne, Cuthill,

relative to CNX were further normalized to each mean value of con-Emerson, & Altman, 2010) and with the recommendations made bytrols to adjust for unwanted sources of variation and to show the clear

the British Journal of Pharmacology. Male Wistar rats (5 weeks old, N =effects of dexamethasone on their protein levels. The data and— 60 —

5

MIYAUCHI ET AL.

statistical analysis comply with the recommendations on experimental

immunoblotting. With every enzyme, specific bands were observed

design and analysis in pharmacology (Curtis et al., 2018).

with the same MW as those in positive controls, HLM, and/or theirstandards, and insect GAPDH was detected as loading control(Figure 1). In UGT detection, HLM showed broad bands and different

2.10

|

Materials

migrations from those of the recombinant ones. The primary antibody
we used can react with several human UGT isoforms, which resulted

Synthetic oligonucleotides were purchased from Life Technologies.

in the difference. As the results indicated the suitability of the expres-Restriction enzymes, bacterial alkaline phosphatase, and T4 DNA

sion system, we next prepared homogenates from cells that co-ligase were from Takara Bio (Shiga, Japan). Human liver microsomes

expressed the drug-metabolizing enzymes of interest.

(HLM) 50 donors pooled were from Corning® Gentest. NADPH wasfrom Oriental Yeast (Tokyo, Japan). All other reagents were of the

3.2 | Suppression of CYP3A4 activity by UGT1A9and UGT2B7

highest grade commercially available.

2.11

|

Nomenclature of targets and ligands
We estimated the effect of each UGT on CYP3A4 activity using Sf9

Key protein targets and ligands in this article are hyperlinked tohomogenates as enzyme sources. The effect of UGTs on CYP3A4

corresponding entries in http://www.guidetopharmacology.org, thefunction was examined by comparing CYP3A4 activity between

commontoP450/CPR doubly expressing homogenates and P450/CPR/UGT ter-

PHARMACOLOGY (Harding et al., 2018), and are permanently

nary expressing ones (none, UGT1A9, and UGT2B7 in Figure 2,

archived in the Concise Guide to PHARMACOLOGY 2019/20

respectively). It is well known that P450 activity highly depends on(Alexander et al., 2019).

CPR (Lu et al., 1969; Miwa & Lu, 1984). To avoid misestimating theportal

for

data

from

the

IUPHAR/BPS

Guide

effect of UGTs, we quantified the enzymes by immunoblotting

3|

RESULTS(Figure S1). Although expression levels of CYP3A4 and CPR weremarkedly different among the three groups, their P450/CPR ratios

3.1 | Confirmation of CYP3A4, UGTs and CRP
expression in insect microsomes

were comparable (Table S1). Co-expression of UGT1A9/2B7 significantly suppressed CYP3A4 activity (Figure 2a), which was consistent
with our last study reporting UGT2B7-mediated suppression of

The drug-metabolizing enzymes were transiently expressed in insect

CYP3A4 (Miyauchi et al., 2015). Further, we performed kinetic analy-

cells and their presence was confirmed in prepared homogenates by

sis to characterize UGT isoform-dependent differences in the

F I G U R E 1 Immunoblotting to confirm the expression of the recombinant enzymes, CYP3A4, CPR, UGT1A9, and UGT2B7. Antibodies against
human-CYP3A, human-CPR, human-UGT1A, human-UGT2B7, and human-GAPDH were used as primary antibodies. HLM and recombinant
enzymes were positive controls. The protein amounts loaded were as follows: HLM, 10 μg; Supersomes (as a CYP3A4 standard), 5 pmol of
CYP3A4; purified CPR, 100 ng; and the Sf9 homogenates (as denoted), 20 μg, respectively. Mock represents homogenates prepared from Sf9
cells infected with control virus— 61 —

6

MIYAUCHI ET AL.

F I G U R E 2 Comparison of CYP3A4 activity in the absence and presence of UGT1A9/2B7. Sf9 cells were seeded in 35-mm dishes andindependently infected with recombinant baculoviruses. CYP3A4 activity was measured with Sf9 homogenates (20-μg protein) as enzyme source
and luciferin-PFBE (50 μM) as a substrate. The mean ± SD of CYP3A4 activities are shown (N = 6). The result of quantitative immunoblotting isshown in Figure S1, and protein levels of expressed enzymes are summarized in Table S1 (a). Kinetic analysis to estimate the effect of
coexpression of UGT1A9 and UGT2B7 on CYP3A4 activity. Using Sf9 homogenates as enzyme source, CYP3A4 activity was compared between
CYP3A4/CPR homogenates (none, open circle) and CYP3A4/CPR/UGT homogenates (UGT1A9, grey triangle; UGT2B7, closed square). Data
were fitted to the Michaelis–Menten model. Each plot represents the mean ± SD of six different samples (b). Calculated Vmax and KM values werecompared. The kinetic parameters were determined in each sample, to obtain six sets of Vmax and KM values in each group. The mean ± SD of six
sets of parameters are shown, and the values are listed in Table 1 (c). Statistical significance was determined by ANOVA followed by Dunnett's
test only when the former F value was significant and there was no significant variance in homogeneity (vs. none)

suppression of CYP3A4 activity. We used the same homogenates in

the parameters are listed in Table 1. Both of the UGTs significantly

kinetic analysis as an enzyme source. The data were fitted to the

reduced Vmax of CYP3A4 activity, but only UGT2B7 decreased KM

Michaelis–Menten model (Figure 2b). We conducted curve fitting in(Figure 2c and Table 1).

every sample to obtain six sets of Vmax and KM in each group. The calculated parameters are presented in Figure 2c, and the mean ± SD of

T A B L E 2 Carboxyl-terminal sequences of deletion mutants of
UGT1A9 and UGT2B7T A B L E 1 Effect of coexpression of UGT1A9 and UGT2B7 on
CYP3A4 activity
Kinetic parameter
Construct

Vmax (nmolhr−1nmol P450−1)

KM (μM)None

3.94 ± 0.77

269.2 ± 71.37

UGT1A92.02 ± 0.81*

211.4 ± 94.23

UGT2B7

1.58 ± 0.44*

146.2 ± 61.61*

Note. The mean ± SD of calculated parameters in Figure 2c (N = 6 in eachgroup) are listed.
Abbreviations: P450, cytochrome P450; UGT,
UDP-glucuronosyltransferase.
*
P < .05 versus none.

ProteinSequenceUGT1A9

D487VIGFLLAVVLTVAFITFK505CCAYGYRKCLGKKGRVKKAHKSKTH530

UGT1A9 ΔTM

D487

UGT2B7

D493VIGFLLVCVATVIFIVTK510CCLFCFWKF
ARKAKKGKND529

UGT2B7 ΔTM

D493

Note. Truncated mutants of UGT1A9 and UGT2B7 lacking the C-terminal
region were constructed. The postulated transmembrane regions of UGT
are underlined (Mackenzie, 1986; Radominska-Pandya et al., 1999) andproceed the cytosolic tail. The subscript numbers represent the location of
the residues of each UGT counted from the N-terminal methionine of themature form.
Abbreviation: UGT, UDP-glucuronosyltransferase.

— 62 —

7

MIYAUCHI ET AL.

3.3 | Role of carboxyl-terminal region of UGT1A9in the suppression of CYP3A4 activity

CYP3A4 activity (Miyauchi et al., 2015). However, it remains unclear
which domain is more critical for the suppression caused by UGT1A9(Figure 2). To address this issue, we generated a UGT1A9 truncatedWe previously indicated that both the C-terminal region and luminal

mutant that lacks the C-terminal region, containing the transmem-

membrane binding domains were necessary for UGT2B7 to suppress

brane helix and following cytosolic tail (ΔTM, Table 2). First, we

F I G U R E 3 Pull-down assay to detect protein–protein interactions between CYP3A4 and UGT1A9 WT/ΔTM. CYP3A4-6 × His andUGT1A9-HA were transiently expressed in COS-1 cells, and lysates were prepared. Pull-down assay was performed by mixing Ni-nitrilotriacetic
acid magnetic beads and 600 μg of lysate. Bound proteins were eluted by buffer containing a high level of imidazole. Pull-down samples and20-μg lysates (3% input) were analysed by immunoblotting

F I G U R E 4 Effect of C-terminal truncated mutants of UGT1A9 and UGT2B7 on CYP3A4 activity. CYP3A4 activity was measured with Sf9
homogenates (20-μg protein) as enzyme source and luciferin-PFBE (50 μM) as a substrate. The mean ± SD of CYP3A4 activities are shown (N = 6).
The result of quantitative immunoblotting is shown in Figure S2, and protein levels of expressed enzymes are summarized in Table S2 (a). Kinetic
analysis showing the effect of UGT truncated mutants. The homogenates mentioned above were used as enzyme sources, and CYP3A4 activity
was compared between CYP3A4/CPR homogenates (none, open circle) and CYP3A4/CPR/UGT homogenates (UGT1A9 ΔTM, grey triangle;
UGT2B7 ΔTM, closed square). Data were fitted to the Michaelis–Menten model. Each plot represents the mean ± SD of the six samples (b).
Calculated Vmax and KM values were compared. The kinetic parameters were determined in each sample, so we obtained six pairs of Vmax and KM
values in each group. The mean ± SD of six calculated parameters are shown, and the values are listed in Table 3 (c). Statistical significance wasdetermined by ANOVA followed by Dunnett's test only when the former F value was significant and there was no significant variance inhomogeneity (vs. none)— 63 —

8

MIYAUCHI ET AL.

examined the role of this region in the interaction with CYP3A4 bydetection. We have already ascertained that the epitope tag did not

pull-down assay. To mimic physiological conditions, we used mamma-

inhibit P450–UGT interactions (Nakamura et al., 2016). Both WT andlian cells (COS-1) instead of insect cells in this assay. An HA-tag was

ΔTM UGT1A9 forms were co-precipitated with CYP3A4-6 × His

conjugated to the C-terminus of UGT1A9 to improve sensitivity of(Figure 3), which suggested that UGT1A9 could interact with CYP3A4

T A B L E 3 Effect of C-terminal deletion mutants (ΔTM) of
UGT1A9 and UGT2B7 on CYP3A4 activity

T A B L E 4 Effect of dexamethasone treatment on hepatic CYP3A
activity in Wistar rat

Kinetic parameter−1

Kinetic parameter−1

Construct

Vmax (nmolhr nmol P450 )KM (μM)

None

1.71 ± 0.30

57.9 ± 9.3

Treatment

Vmax (pmolmin−1 relative
CYP3A−1

KM (μM)UGT1A90.35 ± 0.12*

96.7 ± 18.2*

Control

0.167 ± 0.040

81.2 ± 17.7

UGT2B70.70 ± 0.16*

103.8 ± 17.4*

Dexamethasone

0.244 ± 0.059*

47.7 ± 6.5*

Note. The mean ± SD of calculated parameters in Figure 4c (N = 6 in eachgroup) are listed.
Abbreviations: P450, cytochrome P450; UGT,
UDP-glucuronosyltransferase.
*
P < .05 versus none.

Note. Assays were conducted with rat liver microsomes and luciferin
derivative as enzyme sources and substrate, respectively. The mean ± SD
value of the calculated parameters shown in Figure 5c (N = 5 in eachgroup) are listed.
*
P < .05 versus control.

F I G U R E 5 Effect of dexamethasone treatment on CYP3A–UGT1A interaction in rat liver. Induction ranges of CYP3As, UGT1As, and CPR
were determined by immunoblotting. Wistar rats (male, 5 weeks old, N = 5 in each group) were treated with dexamethasone in saline at a dose of
80 mgkg−1 in a day for 4 days. The control group was treated with the same amount of saline. One day after the last treatment, liver microsomes
were prepared and analysed individually. Dex represents dexamethasone-treated rats. Calnexin (CNX), an ER marker protein, was also detected asa loading control. Relative protein levels (control mean = 1.0) are shown as the mean ± SD. The amount of microsomes utilized in the
immunoblotting was 10 μg except for detection of CYP3As in dexamethasone-treated rats (1 μg) (a). CYP3A turnovers are shown as the mean ±SD (N = 5) for each substrate concentration. Kinetic analysis with individual rat liver microsomes. Data were fitted to the Michaelis–Menten
model. Values of the y axis represent turnover of CYP3A, an activity related to CYP3A levels as determined by immunoblotting shown in
Figure 5a, not an activity per microsomal protein (b). Five sets of Vmax and KM for each group were calculated. These are shown as the mean ± SD.
Statistical significance was determined by Student's t test (unpaired, two tailed) (c)— 64 —

9

MIYAUCHI ET AL.

FIGURE 6

Postulated mechanism of UGT-mediated suppression of CYP3A4 activity

without the C-terminal region. We then determined whether the

Figure 5a. CNX, an ER marker protein, was used as a loading control,mutant could suppress CYP3A4 activity to the same extent as

and signals of CYP3A, UGT1A, and CPR were standardized to that ofUGT1A9 WT. We prepared coexpressed homogenates using the

CNX to calculate relative expression levels (Figure 5b). Fold induc-baculovirus-insect cell system, containing unconjugated CYP3A4 andtions, the value of expression levels after dexamethasone treatment

UGT1A9 or UGT2B7 ΔTM. Both of the truncated mutants still func-

divided by those of control, are also presented. First, CYP3A levels

tioned as a suppressor of CYP3A4 activity (Figure 4a; and the results

were compared with anti-CYP3A2 antibody which can cross-reactof quantitative immunoblotting are shown in Figure S2 and summa-

with CYP3A1. Almost the same levels of CYP3As were detected in

rized in Table S2). Further, kinetic analysis indicated that they signifi-

10 μg of microsomes from control rats and 1 μg of those from

cantly reduced Vmax of CYP3A4 activity as shown for their WT

dexamethasone-treated rats, suggesting that the treatment induces

counterparts (Figure 4b,c and Table 3). In contrast, the effects of the

CYP3As at about 10-fold (in detail, 8.7-fold). UGT1As were also

ΔTM mutants on KM of CYP3A4 activity were far different from thoseinduced by dexamethasone, but induction rates were lower thanobserved in their WT: Both of the ΔTM mutants significantly

those of CYP3A, 4.6-fold, while the treatment had little effect on

increased KM (Figure 2c and Table 1). Therefore, it is suggested that

CPR, 2.7-fold (Figure 5a). The result of kinetic analysis is shown in

the C-terminal region of UGT1A9 and UGT2B7 has a unique modify-

Figure 5b, calculated parameters are shown in Figure 5c, and theing role in the suppression of CYP3A4 activity. Nevertheless, the lumi-

values are listed in Table 4. Dexamethasone treatment significantly

nal domain of both UGT1A9 and UGT2B7 is crucial to modulating

increased Vmax and reduced KM of CYP3A activity.

CYP3A4 function.

4
3.4 | Dexamethasone treatment cancelled UGT1Adependent suppression of CYP3A in rat liver|

DI SCU SSION AND CO NCLUSIO NSThis study demonstrates that UGT1A9, like UGT2B7, alters CYP3A4
activity. We have reported UGT2B7-mediated suppression of

For further evaluation of the role of CYP3A–UGT1A interactions, we

CYP3A4, so this is the second study supporting our hypothesis that

focused on dexamethasone, a strong inducer of hepatic CYP3A1,

P450 and UGT work cooperatively through protein–protein interac-

CYP3A2, UGT1A1, and 1A6 in rat (De Martin et al., 2014; Ghosal,

tions. Both UGT1A9 and UGT2B7 suppressed CYP3A4 activity

Sadrieh, Reik, Levin, & Thomas, 1996; Jemnitz, Veres, Monostory, &(Figure 2a), mainly by a significant reduction in Vmax. There were also

Vereczkey, 2000). The UGT-mediated suppression of CYP3As should

effects on KM which were slightly different between the UGT

be affected if dexamethasone treatment induces CYP3As andisoforms (Tables 1 and 3). UGT2B7 markedly decreased the KM ofUGT1As to different extents. To confirm that, we calculated CYP3A

CYP3A4 activity, which was not observed in our last study (Miyauchi

turnover (activity per relative CYP3A levels, but not the activity per

et al., 2015). In that study, microsomes were utilized as enzyme source

microsomal protein), with the luciferin derivative as a substrate. We

while homogenates were used in this study, so the presence of thedetermined the impact of dexamethasone treatment on hepatic

other cellular components may have affected the results. In our previ-

CYP3A–UGT1A interactions by using liver microsomes (N = 5 in eachous study, we tried to determine the step(s) in the P450 catalytic cycle

group). The results of quantitative immunoblotting are shown inat which UGT2B7 suppresses P450 function. UGT2B7 down-— 65 —

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MIYAUCHI ET AL.

regulated CYP3A4 function not only at the substrate oxidation step

substrate to P450 can be altered by the depth of P450 in the mem-but also during NADPH consumption, H2O2 generation and substratebrane (Berka, Hendrychova, Anzenbacher, & Otyepka, 2011; Berka,oxidation driven by cumene hydroperoxide (Shunt pathway). Based

Paloncyova, Anzenbacher, & Otyepka, 2013). Another group also

on these data, we suspected that UGT2B7 suppresses substrate bind-reported that the lipid content of the membrane could change theing to P450, the start point of catalytic cycle, and proved that by com-degree of P450 insertion, thereby affecting P450 activity, especiallyparing substrate-binding difference spectra in the presence andwith CYP3A4 (Ahn, Guengerich, & Yun, 1998; Kim, Ahn, & Yun,absence of the UGT (Miyauchi et al., 2015). The present study rev-2003). Thus, the change of P450 location in the membrane could elicit

ealed that UGT1A9 also suppressed CYP3A4 and the suppressive

suppression of CYP3A4 activity.

effect was equal or more significant than UGT2B7 (Figures 2 and 4).

A key question is whether the CYP3A4–UGT interaction occurs

Given that, it is reasonable to consider that UGT1A9, like UGT2B7,

and regulates CYP3A4 under physiological conditions. We have con-inhibits substrate binding to P450. However, we cannot judge whichfirmedUGT is a better suppressor of CYP3A4 because their expression levels

CYP1A1-conjugated affinity chromatography (Taura et al., 2004),

were quantified by immunoblotting with different primary antibodies.

GST-overlay assay (Ishii et al., 2014; Takeda et al., 2005), chemical

Further, it is difficult to determine the percentage of UGT interacting

crosslinking (Takeda et al., 2009) and co-immunoprecipitation/pull-with CYP3A4 in our co-expression system. Both UGTs may contribute

down assay (Ishii et al., 2007; Miyauchi et al., 2015; Nakamura

theinteraction

withseveralbiochemical

methods:

to the reduction of toxic H2O2 by inhibiting substrate binding to

et al., 2016). In addition, other groups also detected P450–UGTCYP3A4 in order to reduce oxidative stress elicited by the P450 underinteractions by MS using human tissue samples (Fujiwara & Itoh,physiological conditions.

2014; Rouleau et al., 2017). We have demonstrated that rat

The C-terminal region of UGT is believed to be involved in UGTCYP3As form a metabolosome with several UGT isoforms evidenced

localization (Iyanagi et al., 1986; Meech & Mackenzie, 1997). In addi-

by co-immunoprecipitation using anti-CYP3A2 antibody (Ishii et al.,tion to its role as a targeting signal, this region was also reported to be2007). Further, UGTs in the CYP3A–UGT complex of the co-important for maintaining catalytic function in UGT1A6 (Ouzzine,

immunoprecipitate are also catalytically active (Ishii et al., 2007). It

Magdalou, Burchell, & Fournel-Gigleux, 1999). In the case of UGT1A9,

is conceivable that there is physiological significance for the meta-

however, the hexa-His-tag fused C-terminal truncated mutant could

bolosome, CYP3A–UGT complex. In the present study, we suggest

be purified in the baculovirus-insect cell system, with reduced butthat dexamethasone treatment could partially cancel UGT1A-

measurable activity (Kurkela, Morsky, Hirvonen, Kostiainen, & Finel,

mediated CYP3A suppression by higher inductions of CYP3A com-2004). Thus, the role of this region in glucuronidation activity seems

pared to UGTs. We have reported that rat P450 interacts with andsomehow different among UGT isoforms in addition to suppression ofregulates UGT as well as human P450, and CYP3A4 regulates activi-

CYP3A4 activity, as observed in the effects on KM (Tables 1 and 3).

ties of human UGT1A1 and UGT1A6 through protein–protein inter-

Currently, splice variants of UGT1A and 2B7 have been reported inaction (Ishii et al., 2007; Ishii et al., 2014; Nakamura et al., 2016).

several human tissues. These variants have shorter C-terminal regions

Given the results, it is reasonable to postulate that rat UGT1A1 andthan WT as they lack the transmembrane and cytoplasmic domains.

1A6 suppress CYP3A, like human UGT1A9. Dexamethasone treat-

Although the variants themselves exhibit no marked activity, they play

ment up-regulated CYP3A turnover and significantly increased Vmax

a role as suppressors of WT UGT function through protein–protein

and reduced KM of CYP3A activity, suggesting that a portion ofinteraction (Bellemare, Rouleau, Harvey, & Guillemette, 2010;

CYP3A showed higher catalytic activity in livers of dexamethasone-

Guillemette, Levesque, & Rouleau, 2014; Menard et al., 2011). Our

treated rats (Figure 5 and Table 4). This result suggested that dexa-

results with truncated mutants of UGTs raise the possibility that themethasone treatment increased the amount of CYP3A that is not

UGT variants also interact with and suppress CYP3A4 under physio-

complexed to UGT1A, resulting in the increase of CYP3A affinity tological conditions.

the substrate and higher catalytic activity. This implies that CYP3A–

It has been proposed that UGT has another membrane-

UGT interactions regulate P450 activity under physiological condi-

associating region in its luminal domain (Laakkonen & Finel, 2010;

tions and this regulation is partly ablated when the molecular ratios

Meech & Mackenzie, 1998; Ouzzine et al., 1999). In P450, similarly,of P450 and UGT are markedly altered by chemical-elicited induc-

the FG loop is also predicted to be associated with membranetions. Mismatch between CYP3A4 protein level and its catalytic(Brown & Black, 1989; Szczesna-Skorupa, Ahn, Chen, Doray, &activity in HLM also supported the presence of the interaction

Kemper, 1995). Given this, it is reasonable to predict that P450 andin vivo. Wandel and his colleagues (1998) utilized several batches of

UGT interact with each other through the membrane. This study indi-

HLM and examined the relationship between CYP3A4 protein levels

cated that UGT1A9 also interacted with and suppressed CYP3A4 inand activity using midazolam, one representative CYP3A substrate

the absence of its C-terminal region (Figures 3 and 4), which led toand reported that this relationship was weak (r2 = .31). In that

our current model of UGT-mediated suppression of CYP3A4

study, HLM from patient #13 had a high CYP3A4 content but failed(Figure 6). The main interaction site of UGT is the luminal membrane

to show a corresponding high catalytic activity. The hepatic expres-binding domain and this domain may interact with the FG loop ofsion level of UGTs in this patient might be higher than that in other

CYP3A4. Interaction with UGT could drag CYP3A4 more deeply into

patients, although those remain to be established. Further, Kummer,

the ER membrane. From a recent computer simulation, influx of

Hammann, Haschke, and Krahenbuhl (2016) reported interesting— 66 —

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MIYAUCHI ET AL.

clinical data about the care of Crigler–Najjar syndrome type II

DECLARATION OF T RANSPARENCY AND SCI ENTIFIC

patients. Phenobarbital is utilized to up-regulate expression of

RIGOURUGT1A1 in these patients. However, they demonstrated that

This declaration acknowledges that this paper adheres to the princi-

St. John's Wort, a strong inducer of CYP3A4 but not UGT1A1, alsoples for transparent reporting and scientific rigour of preclinicalreduced total serum bilirubin and worked as an alternative drug toresearch as stated in the BJP guidelines for Design & Analysis,

phenobarbital. We previously showed that CYP3A4 increased

Immunoblotting

UGT1A1 activity through protein–protein interaction, an effect that

Experimentation, and as recommended by funding agencies, pub-would well explain the clinical report (Ishii et al., 2014). Hence,lishers, and other organizations engaged with supporting research.

andImmunochemistry,

andAnimalin vivo evidence supports the existence of P450–UGT interactions
MEETINGS IN WH ICH A PART OF DATA WERE

under physiological conditions.
Some drugs like phenobarbital and rifampicin can induce P450s

PR ES ENT E D

through activation of nuclear receptors and cause drug–drug interac-18th Microsomes and Drug Oxidation (MDO)–12th European

tions (DDI), an important issue for clinical therapeutics (Fuhr, 2000;

Regional Meeting of International Society for the Study of Xenobi-

Harmsen, Meijerman, Beijnen, & Schellens, 2007). Induction of hepatic

otics (ISSX; 2012), 28th Japanese Society of the Study of Xenobi-

expression levels of P450 has been the main focus as a trigger of suchotics (JSSX; 2013), 134th Annual meeting of Japanese Society of

DDI, but drugs could also affect P450–UGT complexes as shown with

Pharmaceutical Sciences (2014), 19th North American Regional

dexamethasone, thereby altering P450 activity (Figure 5). To date, theMeeting of ISSX–29th JSSX (2014; Miyauchi et al.), and 20th

effect on P450–UGT interactions is indistinguishable from that of

MDO (2014; Ishii et al.).

P450 induction in altering P450 activity. Thus, we have to establish
methods to quantify P450–UGT complexation and comprehensively

OR CIDassess the effects on P450 activity in order to minimize the risk

Yuji Ishii

https://orcid.org/0000-0002-2069-0219

of DDI.
In conclusion, we demonstrate for the first time that UGT1A9,
a major UGT isoform, can suppress CYP3A4 activity throughprotein–protein interaction. Further, we conclude that the molecularmechanism

underlying

UGT1A9-mediatedsuppressionof

CYP3A4 activity is very similar to that of UGT2B7. However, there
are 22 UGT isoforms in human (Rowland, Miners, & Mackenzie,2013), and their effects on CYP3A4 may be different. Thus, further
studies are necessary to estimate their effects comprehensively.
CYP3A4–UGT interactions are one aspect of the post-translational
regulation of CYP3A4 activity, which may lead to a better understanding of the causes of the large inter-individual differences inP450 activities.
ACKNOWLEDGEMEN TS
The authors thank Mr. Ken-ichi Yamada for constructing theUGT1A9-HA in pcDNA3.1/hygro (−) vector. The authors also thankthe Research Support Center, Graduate School of Medical Sciences,
Kyushu University, for technical support. This study was supported inpart by the Japan Research Foundation for Clinical Pharmacology; theGrant-in-Aid for Scientific Research (B)(25293039) from Japan Society
for the Promotion of Science; and the Grant-in-Aid for Scientific
Research (C)(19590147) from the Ministry of Education, Culture,
Sports, Science and Technology to Y.I.
CONF LICT OF IN TE RE ST
The authors declare no conflict of interest.
AUTHOR CONTRIBUTIONS
All the authors participated in the research design. Y.M. and
Y.I. performed the experiments and analysed the data. Y.M., Y.T.,
P.I.M., and Y.I. wrote the manuscript.

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glucuronosyltransferase 2B7: Evidence for protein-protein associationand possible involvement of CYP3A4 J-helix in the interaction. Molecular Pharmacology, 75(4), 956–964. https://doi.org/10.1124/mol.108.
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Taura, K., Naito, E., Ishii, Y., Mori, M. A., Oguri, K., & Yamada, H. (2004).
Cytochrome P450 1A1 (CYP1A1) inhibitor α-naphthoflavone interferes with UDP-glucuronosyltransferase (UGT) activity in intact but
not in permeabilized hepatic microsomes from 3-methylcholanthrenetreated rats: Possible involvement of UGT-P450 interactions. Biological & Pharmaceutical Bulletin, 27(1), 56–60.
Taura, K. I., Yamada, H., Hagino, Y., Ishii, Y., Mori, M. A., & Oguri, K.
(2000). Interaction between cytochrome P450 and other drugmetabolizing enzymes: Evidence for an association of CYP1A1 withmicrosomal epoxide hydrolase and UDP-glucuronosyltransferase. Biochemical and Biophysical Research Communications, 273(3),1048–1052. https://doi.org/10.1006/bbrc.2000.3076
Thummel, K. E., & Wilkinson, G. R. (1998). In vitro and in vivo drug interactions involving human CYP3A. Annual Review of Pharmacology and Toxicology, 38, 389–430. https://doi.org/10.1146/annurev.pharmtox.38.
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Galetin, A., Houston, J. B., & Miners, J. O. (2004). Human UDPglucuronosyltransferases: Isoform selectivity and kinetics of
4-methylumbelliferone and 1-naphthol glucuronidation, effects oforganic solvents, and inhibition by diclofenac and probenecid. Drug
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SUPPORTING INF ORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of this article.

— 69 —How to cite this article: Miyauchi Y, Tanaka Y, Nagata K, et al.
UDP-Glucuronosyltransferase (UGT)-mediated attenuations ofcytochrome P450 3A4 activity: UGT isoform-dependent
mechanism of suppression. Br J Pharmacol. 2020;1–13.
https://doi.org/10.1111/bph.14900

Received: 28 December 2018
DOI: 10.1002/cam4.2409|

Revised: 23 June 2019|

Accepted: 25 June 2019

ORIGINAL RESEARCHIn Vitro and in Vivo antitumor activity and the mechanism of
siphonodictyal B in human colon cancer cells
Sonoko Chikamatsu1

Yoshifumi Kawamura1| Ken Saijo1,2| Tadashi Katoh4

1Department of Clinical Oncology, Instituteof Development, Aging and Cancer, Tohoku
University, Sendai, Japan2

Department of Medical Oncology, Tohoku
University Hospital, Sendai, Japan

3

Laboratory of Synthetic and Medicinal
Chemistry, Faculty of Pharmaceutical
Sciences, Tohoku Medical andPharmaceutical University, Sendai, Japan

4

Faculty of Pharmaceutical
Sciences, Research Center for Drug
Discovery, Tohoku Medical andPharmaceutical University, Sendai, Japan
Correspondence
Chikashi Ishioka, Department of ClinicalOncology, Institute of Development, Aging
and Cancer, Tohoku University, 4‐1, Seiryo‐
machi, Aoba‐ku, Sendai, Japan.
Email: chikashi@tohoku.ac.jp| Hiroo Imai1,2 | Koichi Narita3 || Chikashi Ishioka1,2

Abstract
Liphagal, isolated from the marine sponge Aka coralliphaga, exhibits phosphatidylinositol 3‐kinase alpha (PI3Kα) inhibitory activity and cytotoxic effects in humancancer cells. Siphonodictyal B, the biogenetic precursor of liphagal, also has PI3K
inhibitory activity. However, its cytotoxic or antitumor activities have not been evaluated. In this study, we demonstrated that siphonodictyal B inhibits several kinases
such as CDK4/6, CDK7, and PIM2 in addition to PI3K in vitro and that siphonodictyal B exhibits more potent cytotoxic effects than liphagal against human coloncancer cell lines. Furthermore, treatment with siphonodictyal B resulted in increased
PARP cleavage, a larger sub‐G1 fraction, and a larger annexin V‐positive cell population, all of which are indicative of apoptosis induction. As a mechanism of apoptosis
induction, we found that siphonodictyal B activates the p38 MAPK pathway, leading
the upregulation of proapoptotic factors. Moreover, siphonodictyal B increased ROS
levels, thus promoting p38 MAPK pathway activation. NAC, an ROS scavenger, almost completely reversed both the cytotoxic and p38 MAPK pathway‐activating effects of siphonodictyal B. These results indicate that the p38 MAPK pathway might
be involved downstream of ROS signaling as part of the mechanism of siphonodictyal B‐induced apoptosis. Finally, siphonodictyal B displayed antitumor effects in ahuman colon cancer xenograft mouse model and increased p38 phosphorylation intumor tissue. These results suggest that siphonodictyal B could serve as the basis of
a novel anticancer drug.
KEYWORDS
liphagal, p38 MAPK pathway, reactive oxygen species, siphonodictyal B, terpenoid1|

IN T RO D U C T IO N

Compounds isolated from natural products have attracted attention, as some possess unique chemical structures and biological activities that can lead to the development of novel
anticancer drugs.1-3 Terpenoids are the most widespreadgroup of natural products with a chemically characteristic
structure of five‐carbon isoprene units.1,2 Liphagal and siphonodictyal B are meroterpenoids that were isolated fromthe marine sponge Aka coralliphaga4,5 (Figure 1). Generally,
it is difficult to obtain sufficient samples of natural products
for biological assays or developing pharmaceutical productsThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original
work is properly cited.
© 2019 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.
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Cancer Medicine. 2019;8:5662–5672.

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because of their scarcity. Meanwhile, the total synthesis ofsuch natural compounds has been pursued with great enthusiasm. Concerning liphagal and siphonodictyal B, efficientand successful total synthetic methods have been reported.5
In the synthetic process, siphonodictyal B corresponds to thebiogenetic precursor of liphagal.5
Furthermore, both liphagal and siphonodictyal B havebeen reported to exert inhibitory activity against phosphatidylinositol 3‐kinase (PI3K),4,6 a lipid kinase that converts
phosphatidylinositol 4,5‐bisphosphate to phosphatidylinositol 3,4,5‐triphosphate (PIP3). PIP3 in turn activates protein
kinase B (AKT) and downstream molecules, leading to thepromotion of cell growth, proliferation, and survival.7 In
many types of cancer cells, the PI3K‐AKT pathway is frequently activated through gain‐of‐function mutations in
PIK3CA, which encodes p110α, the catalytic subunit of
PI3K.8 Therefore, PI3K is considered a potential drug target for cancer therapy.9,10 Although liphagal was reported to
exhibit cytotoxic effects in human colon cancer cells,4 theeffects of siphonodictyal B on human cancer cell have neverbeen evaluated. In the present study, we illustrated that siphonodictyal B induced apoptosis more potently than liphagal in
human colon cancer cells. Then, we focused on siphonodictyal B and analyzed the mechanism by which siphonodictyal
B induces apoptosis. Furthermore, we confirmed the in vivo
antitumor activity of siphonodictyal B in a human colon cancer cell xenograft mouse model.

2
2.1

|

M ATE R IA L S A N D ME T HODS|

Reagents

Liphagal and siphonodictyal B were synthesized chemically and provided by Tadashi Katoh at Tohoku Medical
and Pharmaceutical University (Sendai, Japan). GDC0941
was purchased from Abcam (Cambridge, UK). Palbociclib
was purchased from ChemoScene (Monmouth Junction, NJ,
USA). SB203580 was purchased from Cayman Chemical
Company (Ann Arbor, MI, USA). N‐acetyl‐l‐cysteine(NAC) was purchased from Sigma‐Aldrich (St. Louis, MO,
USA).

2.2

|

F I G U R E 1 Chemical structure and kinome inhibition plot.
A, Chemical structure of liphagal and siphonodictyal B. B, Plot ofkinome inhibition by siphonodictyal B. The inhibition of 313 protein
kinases by siphonodictyal B at 10 μmol/L was evaluated using theoff‐chip mobility shift assay or immobilized metal ion affinity‐based
fluorescence polarization assay. The strengths of the inhibitory
activities against kinases are illustrated by the sizes of the circles.
PI3K was not included in the tree because of its lipid kinase features.
Therefore, the circle was written in a separate frame

2.3

Cell lines and cell culture

The human colon cancer cell lines HCT 116 and SW480 werepurchased from American Type Culture Collection (ATCC,
Manassas, VA, USA). HT‐29 was a gift from Dr John M.
Mariandason at Ludwig Institute for Cancer Research, Austin
Hospital (Heidelberg, VIC, Australia). Early passage aliquots
were frozen. Cells used in this study were replenished fromthe frozen stocks. All cell lines were cultured in RPMI‐1640
medium supplemented 10% fetal bovine serum at 37°C in an
atmosphere of 5%CO2.

|

Enzyme assay in cell‐free system

Cellular kinase activities were evaluated using nonradioisotopic methods such as the off‐chip mobility shift assay or
IMAP (Carna Biosciences, Kobe, Japan).11 A kinase inhibition profiling panel was produced on the basis of the kinase
inhibition rates. In off‐chip mobility shift assay, compound
solution was prepared in assay buffer (20 mmol/L HEPES,
0.01% Triton X‐100, 2 mmol/L DTT, pH 7.5) and incubated in a 384‐well plate at room temperature. The reaction
was stopped by adding Termination Buffer (QuickScout
Screening Assist MSA; Carna Biosciences, Kobe, Japan).

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CDK7/Cyclin H/
MAT1

PIM2

PIK3CA/PIK3R1a

6.78

1.27

5.31

4.12

69.1

13.7

6.99

2.62

KinaseCDK4/Cyclin D3

CDK6/
Cyclin D3

Liphagal

19.9

Siphonodictyal
B

32.9

TABLE 1

Evaluation of kinases
inhibitory activities of liphagal or
siphonodictyal B

Kinase inhibitory activities were measured by the off‐chip mobility shift assay. IC50 (μmol/L) value wasshown.
aKikuchi T, Narita K, Saijo K, Ishioka C, Katoh T. Enantioselective Total Synthesis of (−)‐Siphonodictyal
B and (+)‐8‐epi‐Siphonodictyal B with Phosphatidylinositol 3‐Kinase α (PI3Kα) Inhibitory Activity.
2016;2016(34):5659‐66.

The substrate peptide and the phosphorylated peptide in thereaction solution were separated and quantified by LabChip
system (Perkin Elmer, MA, USA). In IMAP assay, compound solution was prepared in assay buffer (20 mmol/L
HEPES, 0.01% Tween‐20, 2 mmol/L DTT, pH 7.4) andincubated in a 384‐well black plate at room temperature.
IMAP binding reagent (IMAP Screening Express kit;
Molecular Devices, CA, USA) was added and incubatedfor 30 minutes. The kinase reaction was evaluated at thefluorescence polarization.

2.4

|

2.5

Cells were seeded in 96‐well plates at 4000 cells/well andincubated for 24 hours, then treated with each concentration of siphonodictyal B. In experiments with SB203580
or NAC, cells were further preincubated for 24 or 1 hour
before application of siphonodictyal B, respectively.
Cells were incubated with Cell Counting Kit‐8 (Dojindo
Molecular Technologies, Kumamoto, Japan) for 1.5 hours.
Then, the absorbance of each well was measured using
SpectraMaxM2e (Molecular Devices, CA, USA). The ratios of surviving cells to control cells treated with 0.1%
DMSO were calculated.

Cell cycle analysis|

Western blot analysis

A total of 1 × 106 HCT 116 cells were seeded and incubatedfor 24 hours. Cells were treated with each concentration of
siphonodictyal B for 24 hours. Cells were harvested, washedin PBS, and fixed with 70% ethanol overnight at 4°C. After
that, cells were incubated with RNase and propidium iodide (PI). The measurement was performed using FC‐500(Beckman Coulter, CA, USA).

2.6

Cell proliferation assay|

HCT 116 cells were seeded in six‐well plates at
2 × 105 cells/well and incubated for 24 houra. Cells
were treated with each concentration of siphonidictyal B,
GDC0941, palbociclib, or a combination of GDC0941 andpalbociclib for 48 hours. Cells were harvested and lysedusing RIPA buffer. The proteins were separated on an
SDS‐PAGE and transferred to PVDF membranes. Then,the membranes were blocked for 1 hour and incubatedwith primary antibodies overnight at 4°C. The membranes
were washed and incubated with secondary antibodies
for 1 hour at room temperature. Next, the resultant bands
were detected using an Odyssey Infrared Imaging systemF I G U R E 2 Effects by siphonodictyal B on cell proliferation, the cell cycle, and apoptosis. A, The antiproliferative effects of liphagal andsiphonodictyal B. HCT 116 cells were treated with liphagal or siphonodictyal B at a concentration of 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 μmol/Lfor 24 h. The ratios of surviving cells to control cells treated with 0.1% DMSO were calculated. Data are the means ± SD of three independent
experiments performed triplicate. B, Flow cytometric analysis of the cell cycle. The G1, S, G2/M, and sub‐G1 phase populations are shown.
HCT 116 cells were treated with siphonodictyal B at a concentration of 0.1, 0.5, or 1.0 μmol/L for 24 h. Siphonodictyal B induced G1 arrestand increased the sub‐G1 population. C, Western blot analysis of cells treated with 2 μmol/L GDC0941, 10 μmol/L palbociclib, a combinationof 2 μmol/L GDC0941 and 10 μmol/L palbociclib (GDC + palbociclib), or siphonodictyal B (0.1, 0.5, or 1 μmol/L) for 48 h. Cleaved PARP,
phospho‐AKT, phospho‐RB, phospho‐p38, and Bim were analyzed. D, Western blot analysis of phospho‐p38 expression in cells treated withsiphonodictyal B with or without SB203580 for 48 h. E, Antiproliferative effects of siphonodictyal B with or without SB203580 for 48 h. The
ratios of surviving cells to control cells were calculated. Data are the means ± SD of three independent experiments performed triplicate. *P < 0.05
compared with cells treated with siphonodictyal B alone. F, Flow cytometric analyses of apoptosis using annexin V and PI double staining in HCT
116 cells. HCT 116 cells were treated with siphonodictyal B with or without SB203580 for 48 h. The percentages of apoptotic cells are shown. G,
The bar charts representing the proportion of apoptotic cells expressed as the fold increase vs that for untreated cells. Data are the means ± SD ofthree independent experiments. *P < 0.05 compared with cells treated with siphonodictyal B alone— 72 —

| 5665

CHIKAMATSU eT Al(LI‐COR, NB, USA). Antibodies against phospho‐RB(Ser780), AKT, phospho‐AKT (Ser437), PARP, phospho‐
p38 MAPK (Thr180/Tyr182), p38 MAPK, and Bim werepurchased from Cell Signaling Technology (Danvers, MA,
USA). Antibodies against β‐actin and α‐tubulin were purchased from Sigma‐Aldrich.

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F I G U R E 3 Elevation of intracellular ROS levels by siphonodictyal B. A, Flow cytometry of intracellular ROS levels using DCFDA staining
in HCT 116 cells. HCT 116 cells were treated with siphonodictyal B with or without NAC for 2 h. B, Antiproliferative effects of siphonodictyal
B with or without NAC for 24 h. The ratios of surviving cells to control cells were calculated. Data are the means ± SD of three independent
experiments performed triplicate. *P < 0.05 compared with cells treated with siphonodictyal B alone. C, Flow cytometry of apoptosis using
Annexin V and PI double staining in HCT 116 cells. HCT 116 cells were treated with siphonodictyal B with or without NAC for 24 h. The
percentages of apoptotic cells are shown. D, The bar charts express the proportion of apoptotic cells as fold increases vs the number of cells treatedwith 0.1% DMSO. Data are the means ± SD of three independent experiments. *P < 0.05 compared with cells treated with siphonodictyal B alone.
E, Western blotting of phospho‐p38 in cells treated with siphonodictyal B with or without NAC for 24 h

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2.7|

siRNA transfection

2.12

HCT 116 cells were seeded in 24‐well plates at 2.5 × 104 cells/
well and incubated for 24 hours. Cells were transfected with
6 pmol/well siRNA against p38 or negative control siRNAusing Lipofectamine 2000 Transfection Reagent (Thermo
Fisher Scientific, Waltham, MA, USA). All siRNAs werepurchased from Thermo Fisher Scientific.

2.8|

Annexin V‐PI double staining

2.9 | Reactive oxygen species(ROS) detection
HCT 116 cells were treated with each drug for 2 hours andstained using a DCFDA/H2DCFDA—Cellular Reactive
Oxygen Species Detection Assay Kit (Abcam). The measurement was performed using FC‐500.

2.10

|

Xenograft mouse modelAll animal experiments were performed according to
the Regulations for Animal Experiments and Related
Activities at Tohoku University. Six‐week‐old female
Balb/c nude mice were purchased from Charles River
Laboratories (Wilmington, MA, USA). HCT 116 cells(5 × 106) were implanted subcutaneously into the rightflanks of nude mice. After tumor formation was confirmed,
mice were assigned to treatment with 20 mg/kg siphonodictyal B or vehicle (four mice per group) administeredintraperitoneally every 3 days. Siphonodictyal B was
dissolved in saline with 2.5% CremophorEL® and 2.5%
ethanol. Tumor volumes were calculated on the basis oftumor mass using the formula length × width × width/2(mm3). The body weights of mice were also measured
every 3 days.

2.11

|

Immunohistochemistry

Tumor tissues were collected from mice, fixed in 10% formalin, and embedded in paraffin. Tumor sections on slides were
dewaxed, rehydrated, and incubated with a specific antibody
against phospho‐p38.

Statistical analysis

All data were presented as the mean ± standard deviation. Statistical analysis was performed using JMP Pro
12 software (SAS, Cary, NC, USA). Unless otherwise
noted, the significance of differences was examined using
Student's t tests. P < 0.05 indicated a statistically significant difference.

3

HCT 116 cells were seeded in six‐well plates at
2 × 105 cells/well and incubated for 24 hours. In experiments with SB203580 or NAC, cells were further preincubated for 24 or 1 hour before application of siphonodictyal
B, respectively. Cells were washed with PBS and stainedusing an Annexin V‐FITC Apoptosis Detection Kit(Nacalai Tesque, Kyoto, Japan). The measurement was
performed using FC‐500.

||

RESULTS

3.1 | Evaluation of the kinase inhibitory
activities of liphagal and siphonodictyal B incell‐free system
Although liphagal and siphonodictyal B were reported
to have PI3K (p110α/p85α) inhibitory activity with IC50
values of 4.1 and 2.6 μmol/L, respectively,6 their inhibitory activities against other kinases have not been evaluated. Therefore, we evaluated the inhibitory activities ofliphagal and siphonodictyal B against 313 kinases usinga kinase panel assay. As shown in Figure S1 and Figure
1B, liphagal and siphonodictyal B had inhibitory activities against several kinases. In particular, liphagal andsiphonodictyal B strongly inhibited activities against cyclin‐dependent kinase 4 (CDK4), CDK6, CDK7, and proto‐
oncogene proviral integration site for murine leukemia
virus‐2 (PIM‐2). The IC50 values of liphagal and siphonodictyal B against CDK4, CDK6, CDK7, and PIM2 areshown in Table 1.

3.2 | Cytotoxic effects of liphagal andsiphonodictyal B against human colon cancer
cell lines
We performed cell proliferation assays to evaluate the cytotoxic effects of liphagal and siphonodictyal B on humancolon cancer cell lines. Siphonodictyal B exhibited more potent cytotoxic effects than liphagal in HCT 116 cells (Figure2A). Similar results were also shown in HT‐29 and SW480
cells (Figure S2). Based on these results, we focused on
siphonodictyal B in subsequent experiments.

3.3 | The effects of siphonodictyal B on thecell cycle in HCT 116 cells
We performed FACS analyses to evaluate the effects ofsiphonodictyal B on the cell cycle. As shown in Figure 2B,
both the G1 and sub‐G1 populations were slightly increased
by treatment with 0.5 μmol/L siphonodictyal B. Moreover,the sub‐G1 phase was clearly increased by treatment with
1.0 μmol/L siphonodictyal B. These results revealed that— 75 —

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siphonodictyal B induces apoptosis at higher concentrationsin HCT 116 cells.

3.4 | Siphonodictyal B induces apoptosis in
HCT 116 via the induction of Bim
We performed Western blotting to investigate the molecular mechanism of apoptosis induced by siphonodictyal B.
As shown in Figure 2C, treatment with 0.5 or 1.0 μmol/Lof siphonodictyal B increased the levels of the apoptosis‐
promoting protein Bim concentration dependently in HCT
116 cells. Treatment with 1.0 μmol/L siphonodictyal B also
induced PARP cleavage. These results suggested that apoptosis induced by siphonodictyal B is attributable to the
induction of Bim. Although siphonodictyal B has PI3K and
CDK4/6 inhibitory activity, it did not inhibit AKT phosphorylation at Ser473 or RB phosphorylation at Ser780 in
HCT 116 cells. By contrast, the PI3K inhibitor GDC0941
inhibited AKT phosphorylation. The CDK4 inhibitor palbociclib inhibited RB phosphorylation. Moreover, thecombination of GDC0941 and palbociclib increased PARP
cleavage. From these results, it was assumed that inhibitory
activities against PI3K and CDK4/6 did not contribute toapoptosis induced by siphonodictyal B.

3.5 | Siphonodictyal B induces apoptosis
through p38 phosphorylation
It was previously reported that apoptosis is induced throughactivation of the p38 MAPK pathway in HCT 116 cells.12,13
Therefore, we performed Western blotting to evaluate theeffects of siphonodictyal B on the p38 MAPK pathway. As
shown in Figure 2C, treatment with 1.0 μmol/L siphonodictyal
B for 48 hour clearly induced p38 phosphorylation. Additionof the p38‐specific inhibitor SB203580 to siphonodictyal B
resulted in reduced p38 phosphorylation (Figure 2D). In thecell proliferation assay, the addition of SB203580 partiallycanceled the cytotoxic effect of siphonodictyal B (Figure2E). Likewise, in the annexin V‐PI double staining assay, thepopulation of apoptotic cells induced by siphonodictyal B wasattenuated by the addition of SB203580 (Figure 2F,G).
Moreover, the contribution of the p38 pathway was further validated via an RNAi approach. The protein level ofphosphorylated p38 was significantly lower in cells treatedwith p38 siRNA (si#1 and si#2) than in cells treated with
control RNA (siScr) (Figure S2A). Depletion of p38 partiallyreduced the cytotoxic effect of siphonodictyal B (Figure
S2B). As expected, the annexin V‐PI double staining assay
revealed that the induction of apoptosis by siphonodictyal
B was lower in cells treated with p38 siRNA than in cells
treated with control siRNA (Figure S2C,D). From these results, it was considered that in HCT 116 cells, siphonodictyal
B induced apoptosis partially through the induction of p38
phosphorylation.

3.6 | Siphonodictyal B induced apoptosis in
HCT 116 cells by increasing ROS production
It had been reported that several terpenoids increased ROS levels and subsequently induced apoptosis in cancer cells.14-17 We
performed 2′,7′‐dichlorofluorescin diacetate (DCFDA) staining and FACS analysis to evaluate whether apoptosis induction
by siphonodictyal B is related to ROS production. As shownin Figure 3A, ROS levels were higher in cells treated withsiphonodictyal B than in those treated with DMSO. However,increased ROS production induced by siphonodictyal B wascompletely canceled by the addition of NAC, a scavenger of
ROS (Figure 3A). The cytotoxic effects induced by siphonodictyal B alone were almost completely rescued by the additionof NAC (Figure 3B). Furthermore, the population of apoptotic
cells induced by siphonodictyal B was greatly reduced by theaddition of NAC (Figure 3C,3). These results indicated that
siphonodictyal B induced apoptosis through the induction of
ROS production in HCT 116 cells. The upregulation of phosphorylated p38 by siphonodictyal B was reduced by NAC(Figure 3E). These results suggested that siphonodictyal B induced apoptosis through the increase of ROS production andsubsequent activation of the p38 MAPK pathway in HCT 116
cells.

3.7 | Evaluation of the in vivo antitumor
activity of siphonodictyal B
Antitumor efficacy was evaluated using HCT 116 xenograft mouse models. Mice were divided in two groups (control group: solvent was intraperitoneally administered every
3 days and siphonodictyal B group: 20 mg/kg of siphonodictyal B was intraperitoneally administered every 3 days). As

F I G U R E 4 Antitumor activity of siphonodictyal B in vivo. (A‐C) HCT116 cells were implanted subcutaneously into the right flanks of mice.
Mice were treated with siphonodictyal B (20 mg/kg) or vehicle via intraperitoneal injection every 3 d. Four mice were used for each treatmentgroup. The tumor volumes in mice treated with vehicle or siphonodictyal B. The siphonodictyal B injection times are shown by black arrows. Data
are the means ± SD from four mice. *P < 0.05 compared with the control group. Tumor weights were measured and represented by the boxplot.
Data are the medians ± SD from four mice. *P < 0.05 compared with the control group. Body weights of mice were measured every 3 days. The
relative ratios to the initial weights are shown. The siphonodictyal B injection times are shown by black arrows. Data are the means ± SD from four
mice. Activation of the p38 MAPK pathway induced by siphonodictyal B in vivo. The in vivo biologic activity of siphonodictyal B was evaluated
via immunohistochemistry using an antibody against phospho‐p38— 76 —

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shown in Figure 4A,B, the tumor volume and weight wereboth significantly smaller in the siphonodictyal B group thanin the control group. Body weights of mice in the siphonodictyal B group were similar to those in control mice (Figure 4C).
To confirm the induction of p38 MAPK pathway activation by siphonodictyal B in tumor tissue, tumors were isolated
from mice after all treatments. The status of phosphorylated
p38 in tumor tissue was immunohistochemically determinedusing the specific antibody. As shown in Figure 4D, tumor
tissue in the siphonodictyal B group was strongly stained by
the phosphorylated p38 antibody compared to the findings in
the control group. This result verified the pharmacodynamic
effects of siphonodictyal B in vivo, which is consistent with
p38 MAPK pathway activation in vitro.

4|

D IS C U SS ION

In the present study, we demonstrated that the terpenoid
siphonodictyal B might induce apoptosis in colon cancer
cells via ROS‐p38 MAPK pathway activation in vitro andinhibited tumor growth in vivo.
We observed that siphonodictyal B had kinase inhibitory
activity against CDK4 and PI3K using the off‐chip mobility shift assay. However, siphonodictyal B did not reduce theexpression levels of phosphorylated AKT and phosphorylated RB. It is known that mTORC1 and S6K regulate thenegative feedback loop that attenuates insulin/IGF receptor
signaling. mTORC1/S6K inhibitors suppress this negative
feedback loop, thus upregulating the PI3K/AKT and ERK
pathways.18 Similarly, as this example, cell signaling pathways complicatedly interfere with each other. We speculated
that the inhibitory effects of siphonodictyal B on individual
kinases were masked because the multitarget effects of thecompound might influence multiple signaling pathways thatinterfere with each other, although we did not pursue thisissue in this study.
Both liphagal and siphonodictyal B induced cell growth
inhibitory activity in a concentration‐dependent manner.
Although it was already known that liphagal inhibited cellproliferation,4 the effect of siphonodictyal B on cell proliferation had not been previously studied as far as we know.
We elucidated that siphonodictyal B exhibits more potent cell
growth inhibitory activity than liphagal.
5‐FU, irinotecan, and oxaliplatin, cetuximab (antiepidermal growth factor receptor antibody), and aflibercept(vascular endothelial growth factor antagonist) are key
drugs for colon cancer chemotherapy.19-22 Previous studies
reported that mutation of RAS, BRAF, TP53, and PIK3CA
is involved in resistance to these drugs.23-26 HCT 116(KRAS G13D mutant, BRAF wild type, TP53 wild type,and PIK3CA H1047R mutant), HT‐29 (KRAS wild type,
BRAF V600E mutant, TP53 R237H mutant, and PIK3CA

P499T mutant), and SW480 (KRAS G12V mutant, BRAF
wild type, TP53 R237H mutant, and PIK3CA wild type)cells were used to evaluate the anticell proliferation activity of siphonodictyal B, which was similar in the three celllines (Figure S2).These results suggest that anticell proliferation effect of siphonodictyal B is independent of thesegenes, it is expected that siphonodictyal B might overcome
these resistances.
In normal cells, apoptosis is strictly regulated to maintain a healthy balance of survival and death.27 Apoptosis
occurs via activation of the caspase family through intrinsic
and extrinsic apoptotic pathways. The intrinsic pathway controls caspase family activation through the expression of theproapoptotic (eg, Bim, BAK, BAX) and antiapoptotic proteins (eg, BCL‐2, MCL‐1, XIAP).28 In this study, we foundthat siphonodictyal B induced apoptosis via upregulation ofBim.
Terpenoids such as echnocystic acid, dehydrocostus lactone, and furanodiene have the ability to induce apoptosis
through p38 MAPK pathway activation.29 We found that
downregulation of the p38 MAPK pathway at least partiallyattenuated siphonodictyal B‐induced apoptosis in HCT 116
cells. The result showed that siphonodictyal B induces apoptosis at least in part through p38 MAPK pathway activation.
Previous research found that the p38 MAPK pathway
was activated by various extracellular or endogenous cellular stimuli. ROS represent a major trigger of the p38 MAPK
pathway.30 Moreover, it is known that moderate levels of
ROS play an important role in healthy cell proliferation anddifferentiation.31 However, excessive ROS levels reaching
the toxic threshold result in irreversible damage to intracellular molecules, such as lipids, proteins, and nucleic acids.32
Therefore, the antioxidant defense system tightly controls
ROS generation in normal cells. Conversely, ROS production
is upregulated in cancer cells, and elevated ROS levels areassociated with abnormal cell proliferation. However, excessive levels of ROS also damage cancer cells. Therefore, if
there is a drug that modulates a balance of ROS generationand elimination, it may be a novel anticancer drug with a few
side effects that is less toxic to normal cells.33 In the present
study, siphonodictyal B markedly promoted ROS generationand induced apoptosis.
As shown in Figure 4B,E, NAC almost completely reversed both cell growth inhibition and p38 phosphorylationinduced by siphonodictyal B. However, when the p38 MAPK
pathway was downregulated via p38 inhibition or depletion,
the cytotoxic and apoptosis‐inducing effects of siphonodictyal B were partially attenuated. These results also suggestthat the p38 MAPK pathway is involved downstream of ROS
signaling as part of the mechanism of siphonodictyal B‐induced apoptosis in HCT 116 cells.
Also, siphonodictyal B significantly suppressed tumor
growth in vivo. We preliminarily investigated the antitumor— 78 —

| 5671

CHIKAMATSU eT Al

effects of siphonodictyal Bintraperitoneally administrated at
a dosage of 20 mg/kg. The antitumor effect is expected to
be further enhanced by optimizing the administration route,frequency, and dosage in the future. We hope that siphonodictyal B might become a promising seed of novel anticancer
drug.
CONFLICT OF INTEREST
The authors have no conflicts to disclose in relation to thiswork.
DATA AVAILABILIT Y STATEMENT
The data that support the findings of this study are available
from the corresponding author upon reasonable request.
ORCID
https://orcid.
Sonoko Chikamatsuorg/0000-0002-5322-0723
Ken Saijo

https://orcid.org/0000-0003-0179-3789

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SUPPORTING INFORMATION
Additional supporting information may be found online in
the Supporting Information section at the end of the article.

— 80 —How to cite this article: Chikamatsu S, Saijo K, Imai
H, et al. In Vitro and in Vivo antitumor activity and themechanism of siphonodictyal B in human colon cancer
cells. Cancer Med. 2019;8:5662–5672. https://doi.
org/10.1002/cam4.2409

Tetrahedron 75 (2019) 2866e2876

Contents lists available at ScienceDirect

Tetrahedron
journal homepage: www.elsevier.com/locate/tet

Catalytic asymmetric synthesis of stereoisomers of 1-C-n-butyl-LABs
for the SAR study of a-glucosidase inhibition
Yoshihiro Natori a, *, Toshihiro Sakuma a, Haruka Watanabe a, Hideaki Wakamatsu a,
Atsushi Kato b, Isao Adachi b, Hiroki Takahata c, 1, Yuichi Yoshimura a, **abcFaculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Sendai, 981-8558, Japan
Department of Hospital Pharmacy, University of Toyama, Toyama, 930-0194, Japan
Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University, Sendai, 981-8558, Japana r t i c l e i n f o

a b s t r a c t

Article history:
Received 7 March 2019
Received in revised form
27 March 2019
Accepted 1 April 2019
Available online 5 April 2019

We achieved synthesis of seven stereoisomers of 1-C-n-butyl-L-iminofuranose derivatives using catalytic
asymmetric alkylation and Negishi coupling as key reactions. The synthetic strategy based on these key
reactions was quite useful, since both a- and b-iminofuranoses could be obtained by switching thechirality of the ligand employed for the AAA reaction. The common intermediates for a- and b-isomers
were subjected to further manipulations to install a diol unit at C2 and C3 to give the desired stereoisomers of L-iminofuranose derivatives. We achieved the preparation of all stereoisomers of 1-C-n-butylL-iminofuranose derivatives, with the exception of b-lyxo type iminofuranose. It is noteworthy that asynthetic route for many stereoisomers of iminofuranose derivatives was developed. Unfortunately, noneof the L-iminofuranoses obtained showed inhibitory activity against the a-glycosidases examinedde.g.,maltase, sucrase, and isomaltase.
© 2019 Published by Elsevier Ltd.

Keywords:
Stereoselective synthesis
Iminosugar
a-Glucosidase inhibitor

1. Introduction
Diabetes mellitus is a chronic metabolic disease caused by
dysfunction of insulin, and leads to hyperglycemia. It is categorized
into two major types, types 1 and 2, as well as several minor types.
Patients with type 1 diabetes lack production of insulin due toautoimmune pancreatic b-cell destruction. Thus, injection of insulin is indispensable for their treatment. On the other hand, inadequate insulin secretion and tolerance toward insulin give rise totype 2 diabetes, which is the most common form of diabetes.
Indeed, 90% of individuals with diabetes have the type 2 form [1].
WHO reported that diabetes caused 1.5 million deaths in the worldin 2012 and 422 million people had diabetes in 2014 [2].
The cause of type 2 diabetes is complicated, but is thought toinvolve both genetic factors and lifestyle-related factors such as
obesity, physical activity, diet and stress. As a consequence of diabetes, many organs are damaged, increasing the risk of heart* Corresponding author.
** Corresponding author.
E-mail addresses: natori-y@tohoku-mpu.ac.jp (Y. Natori), yoshimura@tohokumpu.ac.jp (Y. Yoshimura).
1
Deceased on 1st March, 2014.

disease and stroke, neuropathy in the feet, diabetic retinopathy andkidney failure. Therefore, as the number of patients with diabetes
rises, there is a concomitant rise in global health care costs. The
initial management of type 2 diabetes consists of exercise and dietary changes to control the blood glucose levels [3]. In patients
whose blood glucose levels are not lowered by these approaches,
antidiabetic drugs are often used. Antidiabetic drugs are classiﬁed
into several mechanistic classes, i.e., sulfonylureas, biguanides,
meglitinides, thiazolidinediones, DPP IV inhibitors, SGLT2 inhibitors, a-glucosidase inhibitors, and bile acid sequestrants. aGlucosidase inhibitors, which are oral antidiabetic agents, block thehydrolysis of disaccharides (e.g., maltose and sucrose) and suppress
postprandial hyperglycemia [4]. As depicted in Fig. 1, three aglucosidase inhibitors (acarbose, voglibose, and miglitol) have been
approved and used in clinical treatment [5]. Miglitol is an iminosugar derivative that can act as a transition state analogue whilebinding to an active site of a-glucosidase (Fig. 1) [6].
For some time now, we have been focused on the design andsynthesis of novel iminosugar derivatives with potential glycosidase inhibitory actions [7]. Recently, we discovered a new inhibitor,
a-1-C-butyl-1,4-dideoxy-1,4-imino-L-arabinitol (a-1-C-butyl-LAB,1a), which strongly inhibits several a-glucosidases. The structureactivity relationship (SAR) investigations of LAB were mainly

https://doi.org/10.1016/j.tet.2019.04.003
0040-4020/© 2019 Published by Elsevier Ltd.

— 81 —

Y. Natori et al. / Tetrahedron 75 (2019) 2866e2876

2867

Fig. 1. Structures of approved a-glucosidase inhibitors.

performed on C1 substituents, and revealed that a-glucosidases areable to recognize alkyl substituents at the C1 position. Further
support was provided by a docking simulation between an enzyme
and 1-C-butyl-LAB, which led us to synthesize 1-C-(aryl)butyl-LAB
derivatives that exhibited potent inhibitory activities toward aglucosidases, including maltase, isomaltase and sucrose. On theother hand, introduction of a second alkyl group into either the C3
or N-position of 1a strongly diminished their inhibitory activities.
In addition, a D-enantiomer of 1a, a-1-C-butyl-1,4-dideoxy-1,4imino-D-arabinitol (a-1-C-butyl-DAB), showed only moderate to
weak inhibitory activities toward the same enzymes (Fig. 2).
These results prompted us to further investigate the SAR of LABs,
focusing on the effects of the stereochemistry of hydroxyl groups
on a pyrrolidine ring. Herein we describe our synthesis of the stereoisomers of 1a (1-C-n-butyl-L-iminofuranoses 1), which proceeds with an asymmetric allylic alkylation reaction as a key step.
This synthesis should make an important contribution to the SAR
study of iminosugars as potential a-glycosidase inhibitors.
2. Results and discussion
Our original synthesis of a-1-C-butyl-LAB 1a was achieved bystarting from a chiral oxazolidinone 2. We converted 2 to the key
intermediate 6 by means of palladium-catalyzed asymmetric allylic
alkylations (AAA) [8,9] and Negishi cross-coupling [10] (Scheme 1).
In the former AAA reaction, an (S,S)-naphthyl Trost ligand was used
to control a newly formed chiral center and obtain (R)-2-aminohomoallylalchol derivative 4. After ring closing metathesis andiodination of the primary hydroxyl group, the latter Negishi
coupling of 5 allowed us to synthesize many iminosugar derivatives

Fig. 2. Structures of 1-C-n-butyl-LAB, -DAB and -iminofuranoses 1.

— 82 —

Scheme 1. Preparation of chiral intermediate 6 for synthesizing 1-C-n-butyl-a-L-iminofuranose derivatives.

having side chains of various lengths substituted at the C1 position.
By tracing the previous method, we prepared the same key intermediate 6 from which the synthesis of 2- and 3-stereoisomers of a1-C-butyl-LAB 1 was started.
Among the 2- and 3-stereoisomers, iminofuranoses having lyxoand ribo-conﬁgurations were obtainable by simple cis-dihydroxylation of 6 with osmium tetroxide. In addition, selective formationof lyxo- and ribo-iminofuranoses would be achieved by restricting
the approach of the oxidative agent. We previously reported that
epoxidation of 6 proceeded selectively through the b-side, since thea-side was shielded by a bulky n-butyl group [7,11]. Thus, the cisdihydroxylation of 6 by treatment with a catalytic amount of
K2OsO4 and N-methylmorpholine N-oxide (NMO) selectively gave
an iminofuranose derivative 7 in good yield. NOE experiments of 7
clearly showed that it had a lyxo-conﬁguration as expected (Fig. 1Sin the Supplementary Data). Cleavage of the oxazolidinone ringunder basic conditions afforded a-1-C-butyl-lyxo-iminofuranose(1b) in 97% yield.
On the other hand, to synthesize the ribo-iminofuranose derivative, it is necessary to block the b-side and induce a-sideoxidation [11]. A simple molecular modeling consideration
revealed that the rigid structure of 7 ﬁxed by a fused oxazolidinone ring forced the bulky butyl group to occupy a pseudo-axial
position. The resulting disposition of the substituent acted toshield the a-side. Therefore, cleavage of the oxazolidinone ringand introduction of a bulky protecting group at the C5-primaryhydroxyl group might efﬁciently switch the stereoselectivity ofoxidation from the b-to a-side. Following this strategy, an oxazolidinone ring of 6 was hydrolyzed by treatment with aqueous
NaOH to give a dihydropyrrole derivative 8. The primary alcohol of
8 was protected by a bulky TBDPS group and the remaining iminogroup was acetylated to give an N-acetyldihydropyrrole 9 quantitatively. As we expected, cis-dihydroxylation of 9 under theconditions used for 6 selectively produced a ribo-iminofuranose
derivative 10, deprotection of which under acidic conditions gavean HCl salt of a-1-C-butyl-ribo-iminofuranose (1c), with anexcellent yield, as a sole product (Scheme 2). The selectivity of
dihydroxylation was affected by an N-protecting group: introduction of an N-nosyl group, instead of acetyl, resulted in a slightdecrease of selectivity (ribo: lyxo ¼ 9 : 1; Scheme 1S in the Supplementary Data). The stereochemistry was conﬁrmed by NOE
experiments of major dihydroxylated product of nosyl derivative,
which was deprotected to afford the same iminosugar as in 1c

2868

Y. Natori et al. / Tetrahedron 75 (2019) 2866e2876

Scheme 2. Syntheses of 1-C-n-butyl-a-L-iminofuranoses 1b and 1c.

(Fig. 2S and Scheme 2S in the Supplementary Data).
The fact that epoxidation of 6 and the subsequent oxirane ringcleavage gave only the arabino-iminofuranose derivative, as reported previously [7], implied the difﬁculty of synthesizing the
xylo-derivative by this synthetic route. As an alternative approach,
we intended to synthesize the xylo-derivative from lyxo-iminofuranose 1b by epimerizing C2-carbon after selective protection of the
C3- and C5-hydroxyl groups.
After N-acetylation of 1b by successive acylation and selective
deacylation steps, the resulting product was treated with 2,2dimethoxypropane (2,2-DMP) in the presence of 10camphorsulfonic acid (CSA) to give a ketal 11 with a yield of 58%
in 3 steps. Although many attempts were made to invert the stereochemistry at C2 of the ketal 11 by SN2 reactions, including the
Mitsunobu reaction, none of the reactions gave a desired producthaving a xylo-conﬁguration (data not shown). Next, we tried toobtain the xylo-iminofuranose derivative by using oxidation and ahydride reduction sequence for epimerization at the C2 position.
The oxidation of 11 was performed by treatment with CrO3-pyridine-Ac2O in dichloromethane to give a ketone 12 in 52% yield.
Among the hydride reductions of ketone 12 examined with various
hydride sources, the reaction with LiBH4 showed the best result(Table 1S in the Supplementary Data). Treatment of 12 with LiBH4
in EtOH at room temperature gave an inseparable mixture of lyxoand xylo-iminofuranose derivatives 11 and 13 (11 : 13 ¼ 3 : 2) in 66%
yield. To separate the diastereomeric mixture, secondary hydroxyl
groups of 11 and 13 were once protected by a p-anisoyl group andthe resulting mixture was separated by a silica gel column to give panisoates 14a and 14b in 43% and 29% yield, respectively. Finally, allprotecting groups of 14b were deprotected at once by treatment
with reﬂuxing aqueous HCl to give a-1-C-butyl-xylo-iminofuranose(1d) as an HCl salt (Scheme 3).
Having completed the synthesis of a-1-C-butyl-iminofuranoses,
we proceeded to synthesize a series of b-C-n-butyl derivatives. A
common intermediate for b-C-n-butyl-iminofuranoses was prepared along with that for the a-C-n-butyl derivatives mentioned
above. A chiral oxazolidinone 2 was subjected to an AAA reaction
using an (R,R)-naphthyl Trost ligand in place of the (S,S)-ligand,giving the (S)-2-amino-homoallylalchol derivative 15a and its

epimer 15b, after protection of primary hydroxyl groups with a
TBDPS, in 61% and 6% yield, respectively. RCM reaction of 15a in thepresence of Grubbs' 1st catalyst, followed by desilylation, gave adihydropyrrole 17. Iodination of 17 with I2 and triphenylphosphine
gave 18, which was subjected to the Negishi cross-coupling reaction with n-propyl zinc iodide to give the common intermediate 19
for synthesizing b-C-n-butyl-iminofuranoses (Scheme 4).
cis-Dihydroxylation of 19 by treatment with K2OsO4 and NMO asin the case of 6 and 9 gave a diol 20 as a sole product because ofsteric hindrance around the C1 and C4 substituents. Hydrolysis of
20 under acidic conditions cleaved an oxazolidinone ring to give 1C-n-butyl-b-L-ribo-iminofuranose (1g) as an HCl salt (Scheme 5).
Epoxidation of 19, on the other hand, by a dioxirane derivative
generated from triﬂuoroacetone and Oxone® gave a mixture ofdiastereomers (12 : 1) in 88% yield. The structure of the major
product was expected to be 21a. It is considered that the epoxidation from the a-side [11] proceeded with the same stereoselectivity as the dihydroxylation from 19 to 20.
As described above, a bulky butyl group at C1 of 6 strongly
shielded an a-side of the C2 position of iminosugar to limit thereactionde.g., to limit epoxidation and dihydroxylationdonly fromthe b-side [11] or to restrict cleavage of epoxide only from the C3side. This steric effect no longer existed in the intermediate 21asince a butyl group occupied a b-position, resulting in a loss ofreaction bias around the C2 position. Thus, cleavage of the epoxidering of 21a was expected to occur on either the C2 or C3 side. Thissuggested that both xylo- and arabino-derivatives could be obtained from one intermediate in the case of the b-isomer. Indeed,treatment of 21a under acidic conditions efﬁciently cleaved its
epoxide ring to give the desired xylo- and arabino-iminosugar derivatives 22a,b as an inseparable mixture. To separate the mixtureof diastereomers, we intended to use selective acetal formation
between the C4 and C5 hydroxyl groups of the xylo-iminosugar,
which could not be allowed for the arabino-derivative. The oxazolidinone ring of 22a,b was opened by NaOH under reﬂuxing conditions and the resulting products were further converted to Nacetyl derivatives 23a,b in 2 steps. Treatment of 23a,b with 2,2dimethoxypropane (2,2-DMP) and CSA resulted in the formationof acetal 24 and triol 23b, which were easily separated by silica gelcolumn chromatography, in 54% and 29% yield, respectively.
Deprotection of 24 by treatment with 6 mol/L HCl under reﬂux
conditions gave 1-C-n-butyl-b-L-xylo-iminofuranose (1h) as a HCl
salt. Similarly, 1-C-n-butyl-b-L-arabino-iminofuranose (1e) was
obtained from 25 quantitatively. The NOE experiments of 1h clearly
showed that it had a xylo conﬁguration (Fig. 3S in the Supplementary Data). Since 1e was obtained from the common epoxide
21a, which produced 1h, the results also suggested that 1e had an
arabino conﬁguration.
To achieve synthesis of the 1-C-n-butyl-b-L-lyxo-iminofuranose(1f), we tried to epimerize stereochemistry at C2 of acetal 24 by the
Mitsunobu reaction. However, the reaction to obtain p-nitrobenzoate 25 did not proceed under the Mitsunobu conditions (pnitrobenzoic acid, triphenylphosphine and DEAD or Bis(2methoxyethyl) azodicarboxylate [12] in THF). As mentioned
above, in the case of the xylo-iminofuranose derivative, the oxidation and hydride reduction sequence gave a desired epimerized
product. However, the method failed to yield either lyxo-iminofuranose derivative (Scheme 3S in the Supplementary Data).
With the seven stereoisomers of L-iminofuranoses (1a-e, 1g, h)
in hand, we next evaluated the inhibitory activities of the stereoisomers against rat intestinal a-glycosidases including maltase,sucrase, and isomaltase [13]. The results are summarized in Table 1.
Among the tested compounds, the original a-arabino derivative 1a
was only active against all a-glycosidases. Its 20 -epimer a-lyxoiminofuranose 1b showed moderate inhibitory activity against— 83 —

Y. Natori et al. / Tetrahedron 75 (2019) 2866e2876

2869

Scheme 3. Synthesis of 1-C-n-butyl-a-L-iminofuranose 2d.

sucrase and weak activity against maltase. The other stereoisomers
did not show any inhibition toward a-glycosidases. Our previous
docking study demonstrated that a long aliphatic substituent at C1
of 1a was required to ﬁt a hydrophobic pocket existing in a catalytic
domain of rat maltase.
The fact that the obtained b-isomers (1e, g, h) exhibited
diminished inhibitory activities in comparison with that of 1a wasin accordance with the results of the docking simulations. Together
with the ﬁnding that D-iminofuranoses did not inhibit activity toward a-glycosidases, the results also revealed that the speciﬁcplacement of all three hydroxyl groups on iminofuranose was
highly important for their binding to the enzyme. On the other
hand, the result that the lyxo derivatives showed weak inhibition
suggests that a SAR study on C2 substitution of iminosugar mightalso yield new inhibitors, since the reduction of inhibitory activity
by this substitution was less than those for the C3 and C4
substituents.
3. Conclusion
We achieved the synthesis of seven stereoisomers of 1-C-nbutyl-L-iminofuranose derivatives by using AAA and Negishi
coupling as key reactions. The synthetic strategy based on these key
reactions is quite attractive because both a- and b-iminofuranoses
can be obtained by changing a chiral ligand of the palladiumcatalyst for the AAA reaction. The common intermediates for a- and— 84 —

b-isomers obtained were converted to the desired iminofuranose
derivatives by stepwise introduction of diol units. Although a b-Llyxo type iminofuranose having all cis-substituted groups could not
be synthesized, it is worth noting that a synthetic route for many
stereoisomers of iminofuranose derivatives was developed. Unfortunately, none of the L-iminofuranoses except the L-arabino derivative showed inhibitory activity against a-glycosidases. Attempts
to synthesize new iminosugar derivatives based on this strategy are
ongoing in our laboratories, and the results, including further SAR
studies of iminosugars, will be reported in the future.
4. Experimental section
4.1. General experimental procedures
All reactions were run under an atmosphere of argon unlessotherwise indicated. Infrared (IR) spectra were recorded on a
Perkin-Elmer 1600 series FT-IR spectrometer. Mass spectra wererecorded on a JEOL JMN-DX 303/JMA-DA 5000 spectrometer. Microanalyses were performed on a Perkin-Elmer CHN 2400
elemental analyzer. Optical rotations were measured with a JASCO
DIP-360 or JASCO P-1020 digital polarimeter at the sodium D line
(589 nm) with the solvent and concentration indicated.
Proton nuclear magnetic resonance (1H NMR) spectra wererecorded on a JEOL JNM-EX 270 (270 MHz), JEOL JNM-AL 400(400 MHz), Varian Unity-500 (500 MHz), or JNM-LA (600 MHz)

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Y. Natori et al. / Tetrahedron 75 (2019) 2866e2876

were removed from the mixture by azeotropic distillations withtoluene (2 � 5 mL) under reduced pressure and the residue was
puriﬁed by silica gel column chromatography (hexane/EtOAc ¼ 3/1)
to give diol 7 (398 mg, 97%) as brown oil; [a]26
D ¼ þ25.7 (c 0.10,
CH3OH). 1H NMR (400 MHz, CD3OD) d: 0.94 (t, J ¼ 7.2 Hz, 3H),
1.34e1.57 (m, 5H), 1.67e1.76 (m, 1H), 3.41e3.46 (m, 1H), 3.84 (t,J ¼ 3.4 Hz, 1H), 3.97 (dd, J ¼ 3.4, 7.2 Hz, 1H), 4.02e4.05 (m, 1H), 4.40
(t, J ¼ 8.7 Hz, 1H), 4.49 (dd, J ¼ 3.4, 8.7 Hz, 1H). 13C NMR (100 MHz,
CD3OD) d: 14.40, 23.67, 29.68, 34.95, 62.28, 63.76, 64.46, 73.10,
81.29, 164.99. IR (NaCl) cm�1: 3445, 2932, 1731. EI-MS (m/z): 215(Mþ). HRMS Calcd for C10H17NO4: 215.1158; Found: 215.1149.
4.2.2. a-1-C-Butyl-L-lyxo-iminofuranose (1b)NaOH (761 mg, 19.0 mmol) was added to a solution of diol 7(394 mg, 1.83 mmol) in EtOH/H2O (2:1, 27 mL), and the mixture wasreﬂuxed for 5 h. The reaction mixture was cooled to room temperature and concentrated. The residue was puriﬁed by silica gelcolumn (Et2O/CH3OH/28% NH3 aq. ¼ 80 : 20: 1) to yield 1b (339 mg,
198%) as a white solid; [a]26
D ¼ �42.0 (c 0.50, CH3OH). H NMR(400 MHz, CD3OD) d: 0.94 (t, J ¼ 7.0 Hz, 3H), 1.29e1.49 (m, 5H),
1.66e1.70 (m, 1H), 3.01e3.06 (m, 1H), 3.25e3.30 (m, 1H), 3.65e3.71(m, 2H), 3.78 (dd, J ¼ 6.3, 11.2 Hz, 1H), 4.07 (t, J ¼ 4.4 Hz, 1H). 13C
NMR (100 MHz, CD3OD) d: 14.34, 23.86, 30.20, 34.79, 61.72, 61.89,
62.04, 73.43, 79.08. IR (KBr) cm�1: 3438, 3264, 2955, 2871. EI-MS(m/z): 189 (Mþ). HRMS Calcd for C9H19NO3: 189.1365; Found:189.1370.

Scheme 4. Preparation of chiral intermediate 19 for synthesizing 1-C-n-butyl-b-Liminofuranose derivatives.

spectrometer using tetramethylsilane as the internal standard.
Chemical shifts are reported relative to the internal standard (tetramethylsilane, dH 0.00; CDCl3, dH 7.26; CD3OD, dH 3.31; D2O, dH
4.79). The following abbreviations are used: s ¼ singlet,d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet, br ¼ broad.
Carbon-13 nuclear magnetic resonance (13C NMR) spectra wererecorded using a JEOL JNM-AL 400 (100 MHz) or Varian Unity-500(125 MHz). The following internal references were used: CDCl3 (d77.0) or CD3OD (d 49.0). Acetone was used as an internal standard in
13
C NMR in D2O. Column chromatography was carried out on silica
gel 60N (40e50 mm) for ﬂash chromatography purchased from
Kanto Chemical. Dichloromethane (CH2Cl2), tetrahydrofuran (THF),
and N,N-dimethylacetamide (DMA) were dehydrated grade andpurchased from Kanto Chemical.
4.2. Synthesis
Compounds 2, 4, 5, 6 were prepared according to the reported
procedures [7,8].
4.2.1. (5S,6S,7R,7aS)-5-Butyl-6,7-dihydroxytetrahydropyrrolo[1,2c]oxazol-3(1H)-one (7)4-Metylmorpholine N-oxide (1.76 mL, 8.42 mmol) and potassium osmate dihydrate (97.3 mg, 0.38 mmol) were added to a solution of compound 6 (347 mg, 1.91 mmol) in acetone/H2O (2:1,
54 mL). After the mixture was stirred at room temperature for 5 h,
the solvents were removed under reduced pressure. Traces of water

4.2.3. {(2R,5S)-5-Butyl-2,5-dihydro-1H-pyrrol-2-yl}methanol (8)NaOH (830 mg, 20.7 mmol) was added to a solution of compound 6 (394 mg, 1.83 mmol) in EtOH/H2O (2:1, 24 mL), and the
mixture was reﬂuxed for 1 h. The reaction mixture was cooled toroom temperature and concentrated. The residue was passed
through a short pad of silica gel (eluting with CH3OH/28% NH3
aq. ¼ 100 : 1). The ﬁltrate was then concentrated, and the residue
was puriﬁed by silica gel chromatography (CH2Cl2/CH3OH/28% NH3
aq. ¼ 90 : 9: 1) to provide alcohol 8 (275 mg, 89%) as a colorless oil.
1[a]23
D ¼ þ366 (c 0.81, CH3OH). H NMR (400 MHz, CD3OD) d: 0.93 (t,J ¼ 7.0 Hz, 3H), 1.31e1.44 (m, 4H), 1.45e1.57 (m, 2H), 3.49 (d,J ¼ 5.8 Hz, 2H), 3.93e3.98 (m, 1H), 4.00e4.05 (m, 1H), 5.79e5.81(m, 1H), 5.87e5.90 (m, 1H). 13C NMR (100 MHz, CD3OD) d: 14.37,
23.87, 29.77, 37.31, 65.95, 66.04, 67.82, 129.80, 134.66. IR (NaCl)
cm�1: 3306, 2926, 1643. EI-MS (m/z): 155 (Mþ); HRMS Calcd for
C9H17NO: 155.1310; Found: 155.1308.
4.2.4. 1-[(2S,5R)-2-Butyl-5-[{(tert-butyldiphenylsilyl)oxy}methyl]2,5-dihydro-1H-pyrrol-1-yl]ethanone (9)tert-Butylchlorodiphenylsilane (251 mL, 0.97 mmol) was added
to a solution of alcohol 8 (100 mg, 0.64 mmol) and imidazole(87.7 mg, 1.29 mmol) in CH2Cl2 (3.0 mL) at 0 � C. The reaction
mixture was stirred at room temperature for 1 h. The reaction was
quenched with sat. NaHCO3 aq. (5 mL), and the whole mixture was
extracted with CH2Cl2 (5 mL � 2). The combined organic layers
were washed with sat. NaCl aq. (10 mL) and dried over anhydrous
Na2SO4i. Filtration and evaporation in vacuo furnished the crude
product, which was puriﬁed by silica gel chromatography (hexane:
ethylacetate ¼ 20:1 /10:1) to provide TBDPS ether (214 mg, 84%)1
as a pale yellow oil; [a]23
D ¼ þ151 (c 0.98, CHCl3); H NMR (400 MHz,CDCl3) d: 0.90 (t, J ¼ 6.8 Hz, 3H), 1.05 (s, 9H), 1.21e1.50 (m, 6H), 3.59
(d, J ¼ 6.1 Hz, 2H), 3.93 (dd, J ¼ 6.1, 10.9 Hz, 1H), 4.10e4.14 (m, 1H),
5.75 (td, J ¼ 1.8, 3.9 Hz, 1H), 5.81 (td, J ¼ 1.8, 3.9 Hz, 1H), 7.35e7.44(m, 6H), 7.65 (dt, J ¼ 2.1, 7.9 Hz, 4H); 13C NMR (100 MHz, CDCl3) d:
14.07, 19.25, 22.84, 26.87, 28.68, 37.15, 64.67, 66.59, 67.35, 127.63,
129.13, 129.61, 133.70, 133.94, 135.58, 135.61; IR (NaCl) cm�1: 3070,
2998, 2957, 2929, 2857; EI-MS (m/z): 393 (Mþ); HRMS Calcd for
C25H35NOSi: 393.2488; Found: 393.2506.

— 85 —

Y. Natori et al. / Tetrahedron 75 (2019) 2866e2876

2871

Scheme 5. Synthesis of 1-C-n-butyl-b-L-iminofuranose derivatives 1e, g, h.

Table 1
IC50 (mM) values of 1-C-n-butyl-L-iminofuranoses for rat intestinal a-glycosidases.
Comp

1a
1b
1c
1d
1e
1g
1h

Conﬁg

a-arabino
a-lyxoa-riboa-xylob-arabino
b-ribob-xyloa-Glycosidases
MaltaseSucraseIsomaltase

0.13
350.9
NI
NI
NI
NI
NI

0.032
22.5
NI
NI
423
NI
331

4.7
NI
NI
NI
NI
NI
NI

NI: no inhibition (less than 50% inhibition at 1000 mM).

NEt3 (49.3 mL, 0.35 mmol), DMAP (2.7 mg, 0.02 mmol) and acetic
anhydride (25.0 mL, 0.27 mmol) were added to a solution of theobtained TBDPS ether in THF (4.0 mL). After stirring at room temperature for 3 h, the reaction was quenched with sat. NH4Cl aq.
(10 mL). The whole mixture was extracted with ethylacetate(10 mL � 2), and the combined organic layers were washed withsat. NaCl aq. (10 mL) and dried over anhydrous Na2SO4. Filtrationand evaporation in vacuo furnished the crude product, which was
puriﬁed by silica gel column chromatography (hexane/
EtOAc ¼ 20:1 /10:1) to provide compound 9 (88.4 mg, 91%) as a1colorless oil; [a]27
D ¼ þ174 (c 0.57, CHCl3). H NMR (400 MHz, CDCl3,
ca 1.4 : 1.0 mixture of amide rotamers) (major) d: 0.85e0.91 (m,3H), 1.02 (s, 9H), 1.17e1.26 (m, 4H), 1.68e1.72 (m, 1H), 1.88e1.92 (m,1H), 2.08 (s, 3H), 3.98 (dd, J ¼ 2.3, 9.8 Hz, 1H), 4.07e4.13 (m, 1H),
4.58e4.63 (m, 1H), 4.75e4.79 (m, 1H), 5.77 (td, J ¼ 2.3, 4.3 Hz, 1H),
5.81e5.88 (m, 1H), 7.34e7.46 (m, 6H), 7.60e7.65 (m, 4H); (minor) d:
0.85e0.91 (m, 3H), 1.04 (s, 9H), 1.17e1.26 (m, 5H), 1.68e1.72 (m,— 86 —

1H), 1.82 (s, 3H), 3.57 (dd, J ¼ 6.6, 10.1 Hz, 1H), 3.79 (dd, J ¼ 3.4,
10.1 Hz, 1H), 4.49e4.54 (m, 1H), 4.71e4.75 (m, 1H), 5.81e5.88 (m,2H), 7.34e7.46 (m, 6H), 7.60e7.65 (m, 4H). 13C NMR (100 MHz,CDCl3) d: 13.97, 14.07, 19.10, 19.25, 22.31, 22.59, 22.65, 22.66, 25.74,
26.27, 26.72, 26.75, 30.92, 34.71, 62.19, 65.56, 65.63, 66.09, 66.72,
66.85, 127.16, 127.53, 127.75, 127.77, 128.52, 129.52, 129.86, 132.87,
133.06, 133.68, 135.48, 135.52, 168.93, 169.28. IR (NaCl) cm�1: 3071,
3049, 3014, 2998, 2957, 2930, 2858, 1651, 1630, 1589. EI-MS (m/z):
435 (Mþ); HRMS Calcd for C27H37NO2Si: 435.2594; Found:435.2586.
4.2.5. 1-((2S,3R,4S,5S)-2-Butyl-5-[{(tert-butyldiphenylsilyl)oxy}
methyl]-3,4-dihydroxypyrrolidin-1-yl)ethanone (10)4-metylmorpholine N-oxide (357 mL, 1.7 mmol) and potassium
osmate dihydrate (19.8 mg, 0.08 mmol) were added to a solution ofalkene 9 in acetone/H2O (1.6:1, 4.7 mL). After stirring at room
temperature for 20 h, the solvents were removed under reducedpressure. Traces of water were removed from the mixture byazeotropic distillations with toluene (5 mL � 2) under reducedpressure. The residue was puriﬁed by silica gel column chromatography (hexane/ethylacetate ¼ 20/1) to give diol 10 (182 mg, 83%)1
as a yellow amorphous solid; [a]26
D ¼ þ20.3 (c 1.74, CHCl3). H NMR(400 MHz, CDCl3, mixture of amide rotamers) d: 0.85e0.94 (m, 3H),
1.03e1.07 (m, 9H), 1.32e1.56 (m, 5H), 1.79 (brs, 1H), 2.04 and 2.08 (s,3H), 2.92e3.08 (m, 1H), 3.40e3.84 (m, 2H), 3.88e4.21 (m, 2H),
4.29e4.37 (m, 1H), 4.51e4.63 (m, 1H), 7.34e7.49 (m, 6H), 7.54e7.67(m, 4H). 13C NMR (100 MHz, CDCl3, mixture of amide rotamers) d:
13.89, 13.97, 19.20, 21.95, 22.03, 22.47, 22.89, 26.68, 26.80, 26.88,
28.75, 29.96, 30.17, 32.15, 34.30, 59.03, 59.06, 61.23, 61.71, 65.08,
66.21, 70.31, 70.40, 71.59, 74.36, 74.54, 127.72, 127.75, 127.86, 127.95,

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Y. Natori et al. / Tetrahedron 75 (2019) 2866e2876

128.10, 128.21, 129.80, 130.09, 130.21, 131.52, 131.78, 132.99, 133.11,
135.45, 135.49, 135.51, 135.71, 170.39, 170.54. IR (KBr) cm�1: 3375,
3072, 3052, 2958, 2932, 2859, 1615, 1464, 1428. EI-MS (m/z): 469(Mþ). HRMS Calcd for C27H39NO4Si: 469.2648; Found: 469.2648.
4.2.6. a-1-C-Butyl-L-ribo-iminofuranose (1c) hydrochloride
A solution of 10 (34.6 mg, 0.07 mmol) in 6 mol/L HCl (1.0 mL)
was stirred at 100 � C for 12 h. The whole mixture was transferred
into a separate funnel and washed with CH2Cl2 (3 mL � 2). The
water layer was evaporated to obtain imino sugar 1c hydrochloride(18.1 mg, quant.) as a white solid; mp > 143 � C (decomp.).
1[a]21
D ¼ �30.9 (c 0.71, CH3OH). H NMR (400 MHz, CD3OD) d: 0.96 (t,J ¼ 7.0 Hz,3H), 1.35e1.48 (m, 4H), 1.68e1.72 (m, 1H), 1.85e1.95 (m,1H), 3.44e3.50 (m, 2H), 3.77 (dd, J ¼ 5.6, 12.0 Hz, 1H), 3.89 (dd,J ¼ 3.0, 12.0 Hz, 1H), 4.08 (t, J ¼ 3.0 Hz, 1H), 4.16e4.19 (m, 1H). 13C
NMR (100 MHz, CD3OD) d: 14.11, 23.55, 27.45, 29.13, 59.70, 63.23,
63.81, 71.98, 73.30. IR (KBr) cm�1: 3363, 2945, 2537, 1618. EI-MS (m/z): 189 (Mþ). HRMS Calcd for C9H19NO3: 189.1365; Found: 189.1358.
4.2.7. 1-{(4aS,6S,7S,7aR)-6-Butyl-7-hydroxy-2,2dimethyltetrahydro-[1,3]dioxino[5,4-b]pyrrol-5(6H)-yl}ethanone(11)
NEt3 (710 mL, 5.06 mmol), DMAP (61.8 mg, 0.51 mmol) and acetic
anhydride (478 mL, 5.06 mmol) were added to a solution of compound 1b (319 mg, 1.69 mmol) in THF (20 mL). After stirring at room
temperature for 4 h, the mixture was evaporated. The residue wassolved in NEt3/CH3OH (NEt3: CH3OH ¼ 1 : 4, 20 mL). After stirring atroom temperature for 48 h, the mixture was evaporated to afford
the residue, which was puriﬁed by silica gel column chromatography (Et2O: CH3OH ¼ 10 : 1 / 5 : 1) to provide the N-Ac-triol
1(291 mg, 75%) as a colorless oil; [a]27
D ¼ þ19.4 (c 4.57, CH3OH). H
NMR (400 MHz, CD3OD, ca 1.5:1.0 mixture of amide rotamers) d:
0.91e0.97 (m, 3H), 1.14e1.61 (m, 6H), 2.08 and 2.14 (s, 3H),
3.64e3.68 (m, 1H), 3.79e3.95 (m, 2H), 4.03e4.17 (m, 2H),
4.48e4.53 (m, 1H). 13C NMR (100 MHz, CD3OD, mixture of amide
rotamers) d: 14.33, 14.34, 21.94, 23.00, 23.39, 23.49, 29.76, 29.82,
32.14, 35.00, 49.85, 57.30, 58.91, 61.94, 62.56, 67.04, 67.92, 70.57,
71.43, 74.46, 75.22, 172.87, 173.29. IR (NaCl) cm�1: 3270, 2933, 1614.
EI-MS (m/z): 231 (Mþ). HRMS Calcd for C11H21NO4: 231.1471;
Found: 231.1480.
Activated MS3Å (molecular sieve 3 Å) (120 mg) was added to asolution of the obtained triol (289 mg, 0.27 mmol) in CH2Cl2 (3 mL).
2,2-dimethoxy propane (306 mL, 2.50 mmol) and CSA (10camphorsulfonic acid) (87.0 mg, 0.38 mmol) were added to thesuspension. After stirring at room temperature for 12 h, the reaction mixture was ﬁltered with a pad of celite eluting with EtOAc.
The solvents were evaporated to furnished the residue, which was
puriﬁed by silica gel column chromatography (hexane: ethyl acetate ¼ 4 : 1 / 0 : 1) to provide compound 11 (140 mg, 67%) as a1colorless oil; [a]25
D ¼ þ5.47 (c 0.79, CH3OH). H NMR (400 MHz,CDCl3, mixture of amide rotamers) d: 0.87e0.95 (m, 3H), 1.25e1.59(m, 6H), 1.39 (s, 3H), 1.42 (s, 3H), 2.07 (s, 3H), 3.11 (d, J ¼ 2.9 Hz, 1H),
3.61e3.63 (m, 1H), 3.73e3.88 (m, 1H), 3.98e4.09 (m, 1H), 4.15e4.24(m, 1H), 4.29e4.38 (m, 1H), 4.38e4.50 (m, 1H). 13C NMR (100 MHz,CDCl3) d: 14.05, 22.69, 22.75, 24.45, 26.22, 27.80, 32.41, 55.58, 60.72,
65.31, 69.11, 74.29, 99.90, 170.65. IR (NaCl) cm�1: 3428, 2987, 2934,
2873, 1737, 1634. EI-MS (m/z): 271 (Mþ). HRMS Calcd for
C14H25NO4: 271.1784; Found: 271.1776.
4.2.8. (4aS,6S,7aR)-5-Acetyl-6-butyl-2,2-dimethyltetrahydro-[1,3]dioxino[5,4-b]pyrrol-7-(7aH)-one (12)
CrO3 (68.9 mg, 0.689 mmol) was added to a solution of pyridine(111 mL, 1.38 mmol) and acetic anhydride (65.l mL, 0.689 mmol) in
CH2Cl2 (5.0 mL), then the mixture was cooled in an ice bath. A solution of compound 11 (62.3 mg, 0.23 mmol) in CH2Cl2 (1.0 mL) was

added to the CrO3-pyridine-acetic anhydride complex at 0 � C. After
stirring at room temperature for 6 h, the reaction was quenchedwith 2-propanol (53 mL, 0.69 mmol). After stirring at room temperature for 2 h, the mixture was ﬁltered with a pad of celite eluting
with EtOAc. The solvents were evaporated to furnished the residue,
which was puriﬁed by silica gel column chromatography (hexane:
ethyl acetate ¼ 5 : 1 / 2 : 1) to provide ketone 12 (32.4 mg, 52%) as1
a colorless oil; [a]26
D ¼ þ26.9 (c 1.31, CHCl3). H NMR (400 MHz,CDCl3, mixture of amide rotamers) d: 0.84e0.95 (m, 3H), 0.96e1.54(m, 4H), 1.37 (s, 3H), 1.40 (s, 3H), 1.74e2.09 (m, 2H), 2.11 and 2.13 (s,3H), 3.63e3.73 (m, 1H), 4.10e4.17 (m, 1H), 4.24e4.33 (m, 1H),
4.37e4.42 (m, 1H), 4.45e4.50 (m, 1H). 13C NMR (100 MHz, CDCl3,
mixture of amide rotamers) d: 13.67, 13.77, 22.03, 22.35, 22.40,
22.59, 25.50, 25.55, 26.20, 26.35, 30.10, 33.10, 53.00, 54.57, 60.73,
61.61, 61.73, 62.65, 70.39, 72.51, 100.82, 101.26, 170.03, 170.41,
209.26, 210.06. IR (NaCl) cm�1: 3428, 2924, 2859, 1770, 1634. EI-MS(m/z): 269 (Mþ). HRMS Calcd for C14H23NO4: 269.1627; Found:269.1629.
4.2.9. 1-{(4aS,6S,7S,7aR)-6-Butyl-7-hydroxy-2,2dimethyltetrahydro-[1,3]dioxino[5,4-b]pyrrol-5(6H)-yl}ethanone(11), 1-{(4aS,6S,7R,7aR)-6-Butyl-7-hydroxy-2,2dimethyltetrahydro-[1,3]dioxino[5,4-b]pyrrol-5(6H)-yl}ethanone(13)
LiBH4 (15.3 mg, 0.70 mmol) was added to a solution of ketone 12(37.9 mg 0.141 mmol) in EtOH (2.0 mL) at 0 � C. After stirring at room
temperature for 12 h, the reaction was quenched by addition of sat.
NH4Cl aq. (5.0 mL). The whole mixture was extracted with CH2Cl2(5 mL � 2) and the combined organic layers were washed with sat.
NaCl aq. (5 mL). The organic layer was dried over anhydrous
Na2SO4. Filtration and evaporation in vacuo furnished the crude
product, which was puriﬁed by silica gel column chromatography(hexane/EtOAc ¼ 5 : 1 /1 : 1) to provide an inseparable mixture of
11 and 13 (11 : 13 ¼ 3 : 2, 25.2 mg, 66%). The diastereomer ratio wasdetermined by HPLC analysis [Inertsil® CN-3 column (30 : 1 hexane/iPrOH; 1.0 mL/min): 11 tR 3.89 min; 13 tR 6.57 min];1[a]20
D ¼ þ19.4 (c 2.32, CHCl3). H NMR (400 MHz, CDCl3, mixture ofamide rotamers) (mixture of 11 and 13) d: 0.88e0.94 (m, 3H),
1.25e1.58 (m, 12H), 2.03 and 2.07 (s, 3H), 2.76 (brs, 0.3H), 3.11 (d,J ¼ 2.9 Hz, 0.5H), 3.61e3.80 (m, 2H), 3.91e4.19 (m, 3H), 4.36e4.50(m, 2H). 13C NMR (100 MHz, CDCl3) d: 13.80, 13.88, 13.92, 13.97,
22.40, 22.49, 22.62, 22.90, 23.22, 23.36, 23.48, 24.17, 24.39, 24.46,
25.82, 26.00, 26.29, 27.13, 27.51, 28.01, 28.54, 29.47, 29.59, 31.78,
32.14, 55.15, 55.36, 56.12, 57.12, 59.90, 60.40, 60.60, 60.77, 61.37,
61.59, 63.41, 65.03, 65.72, 68.88, 69.88, 73.10, 74.00, 74.41, 74.79,
75.77, 99.15, 99.65, 99.79, 169.57, 170.53, 170.69. IR (NaCl) cm�1:
3391, 2933,2873,1622, 1416. EI-MS (m/z): 271 (Mþ). HRMS Calcd for
C14H25NO4: 271.1784; Found: 271.1773.
4.2.10. (4aS,6S,7S,7aR)-5-Acetyl-6-butyl-2,2-dimethylhexahydro[1,3]dioxino[5,4- b]pyrrol-7-yl 4-methoxybenzoate (14a),(4aS,6S,7R,7aR)-5-Acetyl-6-butyl-2,2-dimethylhexahydro-[1,3]dioxino[5,4-b]pyrrol-7-yl 4-methoxybenzoate (14b)
A mixture of 11 and 13 (11 : 13 ¼ 3 : 2, 27.0 mg, 0.10 mmol) was
dissolved in CH2Cl2 (1.0 mL) and the solution was cooled in an ice
bath. NEt3 (42.1 mL, 0.30 mmol), DMAP (2.4 mg, 0.02 mmol) and pmethoxybenzoyl chloride (20.3 mL, 0.15 mmol) were added to asolution at 0 � C. After stirring at room temperature for 52 h, thereaction was quenched by addition of sat. NaHCO3 aq. (3.0 mL). The
whole mixture was extracted with CH2Cl2 (5 mL � 2) and the
combined organic layers were washed with sat. NaCl aq. (5 mL). The
organic layer was dried over anhydrous Na2SO4. Filtration andevaporation in vacuo furnished the crude product, which was puriﬁed by silica gel column chromatography (hexane/EtOAc ¼ 10 : 1)
to afford the mixture of 14a and 14b. The mixture of 14a and 14b— 87 —

Y. Natori et al. / Tetrahedron 75 (2019) 2866e2876

was puriﬁed by preparative TLC (TLC 20 � 20 cm2, Et2O/hexane ¼ 4: 1) to afford ester 14a (16.2 mg, 43%) as a colorless oil and ester 14b1(11.0 mg, 29%) as a colorless oil. 14a: [a]25
D ¼ �13.6 (c 0.90, CHCl3). H
NMR (400 MHz, CDCl3, mixture of amide rotamers) d: 0.87e0.93(m, 3H), 1.20 (s, 3H), 1.24e1.66 (m, 6H), 1.34 (s, 3H), 2.04 and 2.10 (s,3H), 3.73e3.80 (m, 1H), 3.87 (s, 3H), 3.99e4.05 (m, 1H), 4.08e4.16(m, 1H), 4.28e4.50 (m, 1H), 4.61e4.73 (m, 1H), 5.40e5.46 (m, 1H),
6.92e6.95 (m, 2H), 7.98e8.02 (m, 2H). 13C NMR (100 MHz, CDCl3,
mixture of amide rotamers) d: 13.88, 13.95, 22.54, 22.56, 22.61,
23.10, 24.13, 24.61, 26.23, 26.25, 27.42, 30.29, 32.94, 55.46, 55.72,
56.10, 60.35, 61.45, 61.54, 62.75, 68.32, 69.22, 74.55, 75.74, 99.46,
99.61, 113.70, 113.73, 122.07, 131.71, 131.75, 163.63, 165.59, 170.15. IR(NaCl) cm�1: 2935, 1715, 1645, 1606, 1513. EI-MS (m/z): 405 (Mþ).
HRMS Calcd for C22H31NO3: 405.2151; Found: 405.2160.
1
14b: [a]25
D ¼ þ8.96 (c 0.81, CHCl3). H NMR (400 MHz, CDCl3,
mixture of amide rotamers) d: 0.77e0.82 (m, 3H), 1.14e1.32 (m, 4H),
1.35 (s, 3H), 1.39 (s, 3H), 1.50e1.56 (m, 1H), 1.63e1.71 (m, 1H), 2.06
and 2.12 (s, 3H), 3.64e3.83 (m, 1H), 3.88 (s, 3H), 4.04e4.21 (m, 1H),
4.29e4.45 (m, 3H), 5.41e5.48 (m, 1H), 6.93e6.97 (d, J ¼ 8.8 Hz, 2H),
7.97e8.02 (m, 2H). 13C NMR (100 MHz, CDCl3, mixture of amide
rotamers) d: 13.78, 13.88, 22.78, 22.84, 23.13, 23.26, 23.30, 26.20,
26.57, 28.03, 28.08, 28.29, 29.68, 29.83, 55.21, 55.49, 58.88, 59.93,
60.86, 61.49, 72.42, 72.48, 75.63, 77.40, 99.51, 99.89, 113.83, 121.49,
123.75, 131.76, 131.84, 163.79, 163.88, 165.19, 165.47, 170.08, 170.67.
IR (NaCl) cm�1: 3473, 2934, 2873, 1715, 1651, 1606, 1581, 1512. EIMS (m/z): 405 (Mþ). HRMS Calcd for C22H31NO6: 405.2151;
Found: 405.2151.

2873

3H), 0.05 (s, 3H), 0.86 (s, 9H), 3.65 (dd, J ¼ 4.8, 9.2 Hz, 1H), 3.88e4.12(m, 3H), 4.37e4.41 (m, 2H), 5.19e5.23 (m, 2H), 5.27e5.35 (m, 2H),
5.72e5.79 (m, 1H), 5.96e6.05 (m, 1H). 13C NMR (100 MHz, CDCl3) d:
5.56, 18.05, 25.73, 58.61, 60.99, 62.89, 67.26, 118.53, 120.41, 132.75,
135.94, 157.16. IR (NaCl) cm�1: 3499, 3080, 2934, 1753, 1405.
EI-MS (m/z): 297 (Mþ). HRMS Calcd for C15H27NO3Si: 297.1760;
Found: 297.1749.
4.2.13. (5S,7aR)-5-[{(tert-Butyldimethylsilyl)oxy}methyl]-1,7adihydropyrrolo[1,2- c]oxazol-3(5H)-one (16)A solution of diene 15a (4.76 g, 16.0 mmol) in CH2Cl2 (115 mL)
was degassed with argon for 30 min and then heated to 40 � C. Four
portions of Grubbs 1st catalyst (each 71 mg, 0.863 mmol) wereadded after 0 h, 10.5 h, 24 h and 65 h. The reaction was stirred an
additional 7 h for a total reaction time of 72 h, then cooled to room
temperature and concentrated. The residue was puriﬁed by silica
gel column chromatography (hexane: ethylacetate ¼ 9 : 1 /1 : 1)
to yield compound 16 (3.84 g, 89%) as a white gum; [a]26
D ¼ þ3.20 (c0.95, CH2Cl2). 1H NMR (400 MHz, CDCl3) d: 0.04 (s, 3H), 0.05 (s, 3H),
0.86 (s, 9H), 3.83 (dd, J ¼ 1.7, 10.4 Hz, 1H), 4.01 (dd, J ¼ 7.8, 10.4, Hz,
1H), 4.30e4.33 (m, 1H), 4.36 (dd, J ¼ 4.1, 10.4 Hz, 1H), 4.47 (t,J ¼ 7.8 Hz, 1H), 4.95e4.98 (m, 1H), 5.82 (m, 2H). 13C NMR (100 MHz,CDCl3) d: 5.74, 18.25, 25.73, 58.43, 66.07, 67.08, 69.63, 126.47, 133.55,
158.94. IR (NaCl) cm�1: 2956, 1758. EI-MS (m/z): 269 (Mþ). HRMS
Calcd for C13H23NO3Si: 269.1447; Found: 269.1439.

4.2.11. a-1-C-1-Butyl-L-xylo-iminofuranose (1d) hydrochloride
A solution of 14b (12.0 mg, 0.03 mmol) in 6 mol/L HCl (1.0 mL)
was stirred at 100 � C for 12 h. The whole mixture was transferred
into a separate funnel and washed with CH2Cl2 (3 mL � 3). The
water layer was evaporated to obtain 1d hydrochloride (5.0 mg,69%) as a white solid; mp > 142 � C (decomp.). [a]21
D ¼ þ4.74 (c 0.49,
CH3OH). 1H NMR (400 MHz, CD3OD) d: 0.97 (t, J ¼ 7.0 Hz,3H),
1.36e1.47 (m, 4H), 1.71e1.77 (m, 1H), 1.79e1.88 (m, 1H), 3.70 (td,J ¼ 2.9, 7.5 Hz, 1H), 3.79e3.94 (m, 3H), 4.04e4.05 (m, 1H), 4.18
(J ¼ 1.4 Hz, 1H). 13C NMR (100 MHz, CD3OD) d: 14.17, 23.63, 27.00,
29.71, 59.41, 63.39, 64.76, 76.16, 76.41. IR (KBr) cm�1: 3392, 2961,
2497, 1601. EI-MS (m/z): 189 (Mþ). HRMS Calcd for C9H19NO3:
189.1365; Found: 189.1361.

4.2.14. (5S,7aR)-5-(Hydroxymethyl)-1,7a-dihydropyrrolo[1,2-c]oxazol-3(5H)-one (17)A solution of tetra-n-butylammonium ﬂuoride (1.0 M solution in
THF, 25 mL, 25 mmol) was added to a solution of compound 16 in
THF (53 mL) and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated and puriﬁed by silica gel column chromatography (hexane: ethylacetate ¼ 1: 1 / 0 : 1) to yield alcohol 17 (1.31 g, 73%) as a white solid; mp1
97e99 � C. [a]26
D ¼ þ3.35 (c 1.20, CH2Cl2); H NMR (400 MHz, CDCl3)
d: 3.83e3.89 (m, 1H), 4.02 (dd, J ¼ 3.4, 10.6 Hz, 1H), 4.10e4.18 (m,2H), 4.43 (brs, 1H), 4.60 (t, J ¼ 8.7 Hz, 1H), 4.97e4.98 (m, 1H), 5.82
(d, J ¼ 5.8 Hz, 1H), 5.96 (d, J ¼ 5.8 Hz, 1H). 13C NMR (100 MHz, CDCl3)
d: 61.96, 66.48, 68.10, 69.44, 129.14, 132.13, 160.21. IR (NaCl) cm�1:
3393, 1723. EI-MS (m/z): 155 (Mþ). HRMS Calcd for C7H9NO3:
155.0582; Found: 155.0580.

4.2.12. (R)-3-[(S)-1-{(tert-Butyldimethylsilyl)oxy}but-3-en-2-yl]-4vinyloxazolidin-2-one (15a)Butadiene monoxide (3.15 mL, 39.1 mmol) was added to a solution of oxazolidinone 2 (2.95 g, 26.1 mmol), (R,R)-Naphthl Trost
ligand(619 mg,
3.0 mol%),
Tris(dibenzylideneaceton)
dipalladium(0)-chloroform adduct (270 mg, 1.0 mol%) and DBU(390 mL, 10 mol%) in CH2Cl2. After stirring at room temperature for
72 h, the mixture was evaporated to afford the residue, which was
puriﬁed by silica gel column chromatography (hexane: ethylacetate ¼ 2 : 1) to provide the alcohol (4.62 g) as a colorless oil. The
alcohol was dissolved in CH2Cl2 (84 mL) and the solution wascooled to 0 � C. Imidazole (3.44 g, 50.5 mmol) and tert-butyldimethylsilyl chloride (4.57 g, 30.3 mmol) were added to the solution.
After stirring at room temperature for 1 h, the reaction was
quenched by addition of H2O (30 mL). The whole mixture was
extracted with EtOAc (100 mL � 2) and the combined organic layers
were washed with sat. NaCl aq. (50 mL). The organic layer was driedover anhydrous Na2SO4. Filtration and evaporation in vacuo furnished the crude product, which was puriﬁed by twice silica gelcolumn chromatography (1st column; hexane: ethylacetate ¼ 20 :1 /1 : 1, 2nd column; CH2Cl2: hexane ¼ 1 : 1 / CH2Cl2:
CH3OH ¼ 5 : 1) to give 15a (2.94 g, 63%) as a colorless oil;1[a]23
D ¼ þ3.94 (c 0.93, CH2Cl2). H NMR (400 MHz, CDCl3) d: 0.04 (s,4.2.15. (5S,7aR)-5-(Iodomethyl)-1,7a-dihydropyrrolo[1,2-c]oxazol3(5H)-one (18)
Alcohol 17 (1.25 g, 8.06 mmol), imidazole (1.37 g, 20.2 mmol)
and triphenyl phosphine (5.29 g, 20.6 mmol) were dissolved in
CH2Cl2 (37 mL) and the solution was cooled to 0 � C. Iodine chips(4.09 g, 16.1 mmol) were added to a solution at 0 � C, and then thereaction was stirred for 10 min. The reaction was quenched byaddition of sodium thiosulfate aq. (1.0 M, 10 mL), changing thebrown reaction mixture to a colorless one. The whole mixture was
extracted with CH2Cl2 (30 mL � 3) and the combined organic layers
were dried over anhydrous Na2SO4. Filtration and evaporation in
vacuo furnished the crude product, which was puriﬁed by silica gelcolumn chromatography (hexane: ethylacetate ¼ 4 : 1) to give 18(1.75 g 82%) as a pale yellow solid; mp 80e82 � C. [a]26
D ¼ þ3.31 (c1.09, CHCl3). 1H NMR (400 MHz, CDCl3) d: 3.73 (dd, J ¼ 2.7, 10.7 Hz,
1H), 4.09 (dd, J ¼ 4.8, 10.7 Hz, 1H), 4.26e4.30 (m, 2H), 4.55 (t,J ¼ 8.0 Hz, 1H), 5.01e5.03 (m, 1H), 5.80 (d, J ¼ 6.3 Hz, 1H), 6.00 (d,J ¼ 6.3 Hz, 1H). 13C NMR (100 MHz, CDCl3) d: 7.64, 62.63, 66.93,
69.10, 127.40, 135.33, 157.37. IR (KBr) cm�1: 1732. EI-MS (m/z): 264(Mþ). HRMS Calcd for C7H8INO2: 264.9600; Found: 264.9603; Anal.
Calcd for C7H8INO2: C, 31.72; H, 3.04; N, 5.28. Found: C, 31.72; H,
3.01; N, 5.22.

— 88 —

2874

Y. Natori et al. / Tetrahedron 75 (2019) 2866e2876

4.2.16. (5R,7aR)-5-Butyl-1,7a-dihydropyrrolo[1,2-c]oxazol-3(5H)one (19)Preparation of a solution of n-propylzinc iodide in N,Ndimethylacetamide; Iodine chips (173 mg, 0.68 mmol) were added
to a suspension of dried zinc powder (1.34 g, 20.5 mmol) in N,Ndimethylacetamide (13 mL). The mixture was stirred for 30 min atroom temperature, changing the brown suspension to a colorlessone. 1-Iodopropane (1.34 mL, 13.7 mmol) was added to the suspension. After stirring for 3 h at room temperature, the suspension
was used as a solution of n-propylzinc iodide in N,Ndimethylacetamide.
Negishi Coupling; Yellow bis(1,5-cyclooctadiene)nickel(0)(132 mg, 0.480 mmol, 16 mol%) and (R,R)-2,6-bis(4-isopropyl-2oxazolin-2-yl)pyridine (291 mg, 0.966 mmol, 32 mol%) wereadded to N,N-dimethylacetamide (19 mL), and the resulting
mixture was stirred for 30 min at room temperature. The resulting
deep-blue solution was added to a mixture of compound 18(800 mg, 3.02 mmol) and pre-prepared n-propylzinc iodide solution (13 mL). After stirring for 20 h at room temperature, the reaction was quenched by addition of iodine chips (400 mg, 3.15 mmol).
After additional stirring for 10 min, the dark-brown mixture was
puriﬁed by silica gel column chromatography (hexane: ethylacetate ¼ 4 : 1) to give compound 19 (279 mg, 51%) as a colorless1oil; [a]23
D ¼ �1.91 (c 0.93, CHCl3). H NMR (400 MHz, CDCl3) d: 0.91
(t, J ¼ 7.2 Hz, 3H), 1.24e1.40 (m, 4H), 1.79e1.83 (m, 1H), 2.27e2.31(m, 1H), 3.98 (dd, J ¼ 7.7, 9.7 Hz, 1H), 4.27e4.30 (m, 1H), 4.49 (t,J ¼ 7.7 Hz, 1H), 4.93e5.00 (m, 1H), 5.84 (dt, J ¼ 1.9, 6.3 Hz, 1H), 5.94
(dt, J ¼ 1.9, 6.3 Hz, 1H). 13C NMR (100 MHz, CDCl3) d: 13.95, 22.60,
27.59, 29.07, 64.29, 66.76, 69.16, 126.22, 135.43, 157.82. IR (NaCl)
cm�1: 2927, 2861, 1748. EI-MS (m/z): 181 (Mþ). HRMS Calcd for
C10H15NO2: 181.1103; Found: 181.1099.
4.2.17. (5R,6R,7S,7aS)-5-Butyl-6,7-dihydroxytetrahydropyrrolo[1,2c]oxazol-3(1H)-one (20)4-Metylmorpholine N-oxide (151 mL, 0.73 mmol) and potassium
osmate dihydrate (8.4 mg, 0.03 mmol) were added to a solution ofcompound 19 in acetone/H2O (2 : 1, 3 mL). After stirring at room
temperature for 47 h, the solvents were removed under reducedpressure. Traces of water were removed from the mixture byazeotropic distillations with toluene (1 mL � 2) under reducedpressure. The residue was puriﬁed by silica gel column chromatography (hexane: ethylacetate ¼ 4 : 1 /1 : 1) to give diol 20
1(24.0 mg, 67%) as a brown oil; [a]22
D ¼ �0.42 (c 1.18, CHCl3). H NMR(400 MHz, CDCl3) d: 0.93 (t, J ¼ 7.0 Hz, 3H), 1.32e1.54 (m, 4H),
1.83e1.90 (m, 1H), 2.24e2.33 (m, 1H), 3.20e3.25 (m, 1H), 3.98e4.03(m, 1H), 4.17 (t, J ¼ 3.3 Hz, 1H), 4.23 (t, J ¼ 5.7 Hz, 1H), 4.28 (t,J ¼ 8.7 Hz, 1H), 4.50 (dd, J ¼ 5.7, 8.7 Hz, 1H). 13C NMR (100 MHz,CDCl3) d: 13.99, 22.71, 24.29, 29.73, 60.72, 62.38, 63.26, 70.57, 73.15,
160.29. IR (NaCl) cm�1: 3371, 2930, 2872, 1731.EI-MS (m/z): 215(Mþ). HRMS Calcd for C10H17NO4: 215.1158; Found: 215.1158.
4.2.18. b-1-C-Butyl-L-ribo-iminofuranose (1g) hydrochloride
A solution of 20 (15.0 mg, 0.07 mmol) in 6 mol/L HCl (2.0 mL)
was stirred at 100 � C for 12 h. The whole mixture was transferred
into a separate funnel and washed with CH2Cl2 (2 mL � 3). The
water layer was evaporated to obtain iminosugar 1g hydrochloride
1(13.4 mg, 88%) as a colorless oil; [a]24
D ¼ �0.65 (c 0.73, CH3OH). H
NMR (400 MHz, D2O) d: 0.88 (t, J ¼ 6.8 Hz, 3H), 1.33e1.41 (m, 4H),
1.71e1.75 (m, 1H), 1.81e1.85 (m, 1H), 3.52 (brs, 1H), 3.75e3.80 (m,1H), 3.81e3.87 (m, 1H), 3.93 (dd, J ¼ 3.9, 11.8 Hz, 1H), 4.32 (m, 1H),
4.52 (dd, J ¼ 3.9, 7.0 Hz, 1H). 13C NMR (100 MHz, D2O) d: 13.65,
22.36, 26.19, 28.22, 58.73, 61.44, 61.49, 70.63, 71.01. IR (NaCl) cm�1:
3351, 2930, 1572. EI-MS (m/z): 189 (Mþþ1). HRMS Calcd for
C9H20NO3: 190.1443; Found: 190.1439.

4.2.19. (1aS,1bS,6R,6aR)-6-Butyltetrahydrooxireno[20 ,30 :3,4]pyrrolo[1,2-c]oxazol-4(1aH)-one (21a), (1aR,1bS,6R,6aS)-6Butyltetrahydrooxireno[20 ,30 :3,4]pyrrolo[1,2-c]oxazol-4(1aH)-one(21b). To a vial with 19 (180 mg, 0.910 mmol) were added CH3CN(7 mL) and 4 � 10�4 M aqueous solution of ethylenediaminetetraacetic acid (4.5 mL). The solution was cooled to 0 � C, and 1,1,1- triﬂuoroacetone (814 mL, 9.12 mmol) was added. A mixture of solid
Oxone® (2.80 g, 4.56 mmol) and NaHCO3 (574 mg, 6.84 mmol) was
added in four portions over 2 h. The reaction was stirred for 2 h at
0 � C, then diluted with 5 mL of H2O, and extracted with CH2Cl2(3 � 20 mL). The organic layers were combined and dried overanhydrous Na2SO4. Filtration and evaporation in vacuo furnishedthe crude product, which was puriﬁed by silica gel column chromatography (Et2O) to provide epoxide 21a (145 mg, 81%) as acolorless oil and epoxide 21b (14.0 mg, 8%) as a colorless oil.
1
21a: [a]26
D ¼ �0.06 (c 0.64, CHCl3). H NMR (400 MHz, CDCl3) d:0.95 (t, J ¼ 7.0 Hz, 3H), 1.38e1.54 (m, 4H), 1.72e1.82 (m, 1H),
2.50e2.59 (m, 1H), 3.52 (dd, J ¼ 4.8, 9.7 Hz, 1H), 3.56 (d, J ¼ 2.9 Hz,
1H), 3.68 (d, J ¼ 2.9 Hz, 1H), 4.14e4.18 (m, 1H), 4.31 (dd, J ¼ 6.8,
8.7 Hz, 1H), 4.41 (t, J ¼ 8.7 Hz, 1H). 13C NMR (100 MHz, CDCl3) d:
14.01, 22.70, 25.26, 29.51, 53.89, 58.77, 59.89, 60.10, 63.30, 158.14. IR(NaCl) cm�1: 3494, 2959, 2872, 1747. EI-MS (m/z): 197 (Mþ). HRMS
Calcd for C10H15NO3: 197.1052; Found: 197.1043.
1
21b: [a]21
D ¼ þ6.41 (c 0.50, CHCl3). H NMR (400 MHz, CDCl3) d:
0.94 (t, J ¼ 9.3 Hz, 3H), 1.32e1.50 (m, 4H), 1.73e1.82 (m, 1H),
2.28e2.36 (m, 1H), 3.64 (d, J ¼ 2.9 Hz, 1H), 3.70 (dd, J ¼ 2.9, 8.8 Hz,
1H), 3.80 (d, J ¼ 2.9 Hz, 1H), 4.03 (dd, J ¼ 8.3, 11.2 Hz, 1H), 4.35 (dd,J ¼ 8.3, 11.2 Hz, 1H), 4.49 (t, J ¼ 8.3 Hz, 1H). 13C NMR (100 MHz,CDCl3) d: 13.92, 22.65, 26.61, 27.59, 57.45, 59.40, 61.40, 64.56, 65.46,
157.58. IR (NaCl) cm�1: 2926, 1747. EI-MS (m/z): 197 (Mþ). HRMS
Calcd for C10H15NO3: 197.1052; Found: 197.1046.
4.2.20. (5R,6R,7R,7aS)-5-Butyl-6,7-dihydroxytetrahydropyrrolo[1,2-c]oxazol-3(1H)-one (22a), (5R,6S,7S,7aS)-5-Butyl-6,7dihydroxytetrahydropyrrolo[1,2-c]oxazol-3(1H)-one (22b)
Triﬂuoroacetic acid (416 mL, 5.44 mmol) was added to a solutionof the epoxide 21a (91.0 mg, 0.46 mmol) in THF/H2O (3/2, 9.5 mL).
After being stirred at 80 � C for 72 h, the reaction mixture was cooled
to room temperature and concentrated. The product was puriﬁed
by silica gel column chromatography (Et2O only / Et2O:
CH3OH ¼ 20 : 1) to yield an inseparable mixture of 22a and 22b(22a: 22b ¼ 2.5 : 1.0, 82.0 mg, 83%) as a colorless oil; [a]20
D ¼ þ5.47(c 1.02, CH3OH). 1H NMR (400 MHz, CD3OD, mixture of 22a and22b) d: 0.93e0.97 (m, 3H), 1.29e1.63 (m, 5H), 1.80e1.89 (m, 1H),
2.18e2.26 (m, 1H), 2.44e2.52 (m, 0.7H), 3.23 (ddd, J ¼ 1.5, 4.4,
10.2 Hz, 0.7H), 3.45e3.49 (m, 0.3H), 3.57 (brs, 0.3H), 3.79 (brs,0.7H), 3.87e3.91 (m, 1H), 4.06 (brs, 0.7H), 4.13e4.18 (m, 0.3H), 4.39(s, 2H), 4.56 t, J ¼ 9.3 Hz, 0.3H). 13C NMR (100 MHz, CD3OD, mixtureof 22a and 22b) d: 14.32, 23.70, 23.82, 24.94, 28.68, 30.13, 30.28,
30.91, 63.69, 64.36, 64.91, 66.77, 68.31, 68.81, 75.15, 79.91, 81.48,
84.69, 159.54, 162.41. IR (NaCl) cm�1: 3382, 2925, 1715. EI-MS (m/z):
215 (Mþ). HRMS Calcd for C10H17NO4: 215.1158; Found: 215.1158.
4.2.21. b-1-C-Butyl-L-xylo-iminofuranose (1h), b-1-C-Butyl-Larabino-iminofuranose (1e)A mixture of 22a and 22b (22a: 22b ¼ 2.5 : 1.0, 43.0 mg,
0.200 mmol) was dissolved in EtOH/H2O (2:1, 3 mL) and NaOH
(761 mg, 19.0 mmol) was added to the solution. The mixture wasreﬂuxed for 1 h, then cooled to room temperature and concentrated. The residue was puriﬁed by a silica gel column (Et2O:
CH3OH: 28% NH3 aq. ¼ 80 : 20: 1 / Et2O: CH3OH: 28% NH3 aq. ¼ 0 :100: 2) to yield an inseparable mixture of 1h and 1e (1h: 1e ¼ 2.3 :1
1, 36.0 mg, 97%) as a colorless oil; [a]25
D ¼ þ0.79 (c 3.26, CH3OH); H
NMR (400 MHz, CD3OD, mixture of 1h and 1e) d: 0.95 (t, J ¼ 7.0 Hz,— 89 —

Y. Natori et al. / Tetrahedron 75 (2019) 2866e2876

3H), 1.33e1.49 (m, 4H), 1.55e1.74 (m, 2H), 2.81e2.86 (m, 0.7H),
3.00e3.04 (m, 0.3H), 3.13e3.18 (m, 0.3H), 3.21e3.26 (m, 0.7H),
3.63e3.86 (m, 3H), 3.98e4.00 (m, 1H); 13C NMR (100 MHz, CD3OD,
mixture of 1h and 1e) d: 14.28, 23.66, 23.87, 28.10, 30.14, 30.19,
34.26, 60.60, 62.70, 63.35, 63.78, 67.08, 69.31, 78.24, 78.83, 80.12,
83.34. IR (NaCl) cm�1: 3320, 2934, 1682. EI-MS (m/z): 189 (Mþ).
HRMS Calcd for C9H19NO3: 189.1365; Found: 189.1368.
4.2.22. 1-{(2R,3R,4R,5S)-2-Butyl-3,4-dihydroxy-5-(hydroxymethyl)pyrrolidin-1-yl}ethanone (23a), 1-{(2R,3S,4S,5S)-2-Butyl-3,4dihydroxy-5-(hydroxymethyl)pyrrolidin-1-yl}ethanone (23b)
A mixture of 1h and 1e (136 mg, 0.720 mmol) was dissolved in
THF (5 mL), and then NEt3 (220 mL, 1.58 mmol), DMAP (8.77 mg,
0.07 mmol) and acetic anhydride (122 mL, 1.45 mmol) were added
to the solution. After stirring at room temperature for 4 h, themixture was evaporated. The residue was solved in NEt3/CH3OH(NEt3: CH3OH ¼ 1 : 4, 10 mL). After stirring at room temperature for
48 h, the mixture was evaporated to afford the residue, which was
puriﬁed by silica gel column chromatography (Et2O: CH3OH ¼ 10 :1 / 5 : 1) to provide an inseparable mixture of 23a and 23b
1(145 mg, 87%) as a colorless oil; [a]23
D ¼ þ14.4 (c 0.26, CH3OH). H
NMR (400 MHz, CD3OD, mixture of amide rotamers) (mixture of
23a and 23b) d: 0.90e0.97 (m, 3H), 1.36e2.05 (m, 6H), 2.09e2.13(m, 3H), 3.59e4.20 (m, 6H). 13C NMR (100 MHz, CD3OD, mixture ofamide rotamers) (mixture of 23a and 23b) d: 14.37, 14.43, 22.12,
22.24, 23.66, 23.84, 24.01, 24.04, 28.18, 28.89, 29.70, 29.80, 30.27,
31.17, 33.72, 34.36, 62.00, 62.14, 62.23, 62.86, 63.71, 63.77, 63.83,
66.34, 68.75, 69.38, 77.23, 77.78, 78.06, 79.72, 81.23, 173.29, 174.11.
IR (NaCl) cm�1: 3343, 2925, 1731, 1600. EI-MS (m/z): 231 (Mþ).
HRMS Calcd for C11H21NO4: 231.1471; Found: 231.1468.
4.2.23. 1-{(4aS,6R,7R,7aR)-6-Butyl-7-hydroxy-2,2dimethyltetrahydro-[1,3]dioxino[5,4-b]pyrrol-5(6H)-yl}ethanone(24), 1-{(2R,3S,4S,5S)-2-Butyl-3,4-dihydroxy-5-(hydroxymethyl)pyrrolidin-1-yl}ethanone (23b)Activated MS3Å (120 mg) was added to a solution of triols 23aand 23b (62.8 mg, 0.270 mmol) in CH2Cl2 (6 mL). 2,2-dimethoxy
propane (306 mL, 2.50 mmol) and CSA (18.9 mg, 0.08 mmol) wereadded to the suspension. After stirring at room temperature for
12 h, the reaction mixture was ﬁltered with a pad of celite eluting
with CH3OH. The solvents were evaporated to furnish the residue,
which was puriﬁed by silica gel column chromatography (CH2Cl2:
CH3OH ¼ 20 : 1 /10 : 1) to provide compound 24 (27.7 mg, 38%) asa colorless oil and 23b (9.5 mg, 15%) as a colorless oil.
1
24: [a]26
D ¼ þ0.85 (c 2.03, CHCl3). H NMR (400 MHz, CDCl3, ca
2.0 : 1.0 mixture of amide rotamers) (major) d: 0.90e0.95 (m, 3H),
1.33e1.45 (m, 10H), 1.63e1.69 (m, 1H), 1.90 (brs, 1H), 2.02 and 2.07
(s, 3H), 3.40 (brs, 1H), 3.48e3.78 (m, 2H), 4.00e4.29 (m, 4H). 13C
NMR (100 MHz, CDCl3, mixture of amide rotamers) d: 13.92, 21.51,
21.76, 22.57, 22.62, 24.48, 25.94, 28.89, 28.91, 31.52, 32.74, 55.69,
57.97, 60.95, 61.62, 67.03, 68.80, 75.51, 76.05, 76.88, 98.73, 99.83,
170.13, 170.92. IR (NaCl) cm�1: 3362, 2933, 1728, 1621. EI-MS (m/z):
271 (Mþ). HRMS Calcd for C14H25NO4: 271.1784; Found: 271.1786.
123b: [a]25
D ¼ þ1.65 (c 1.81, CH3OH). H NMR (400 MHz, CD3OD,
mixture of amide rotamers) d: 0.90e0.96 (m, 3H), 1.29e1.66 (m,5H), 1.83 (brs, 1H), 2.10 and 2.12 (s, 3H), 3.63e3.83 (m, 3H),
3.87e4.13 (m, 3H). 13C NMR (100 MHz, CD3OD, mixture of amide
rotamers) d: 14.35, 14.45, 21.98, 22.47, 24.00, 24.03, 29.04, 29.80,
30.27, 31.17, 62.23, 62.26, 62.87, 63.18, 66.36, 69.38, 76.55, 76.74,
77.23, 79.03, 173.88, 173.91. IR (NaCl) cm�1: 3334, 2958, 1614. EI-MS(m/z): 231 (Mþ). HRMS Calcd for C11H21NO4: 231.1471; Found:
231.1476.
4.2.24. b-1-C-Butyl-L-xylo-iminofuranose (1h) hydrochloride
A solution of 24 (15.0 mg, 0.07 mmol) in 6 mol/L HCl (2.0 mL)— 90 —

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was stirred at 100 � C for 12 h. The whole mixture was transferred
into a separate funnel and washed with CH2Cl2 (2 mL � 3). The
water layer was evaporated to obtain 1h·HCl (24.2 mg, quant) as a1brown oil; [a]23
D ¼ þ1.93 (c 0.97, CH3OH). H NMR (400 MHz, D2O) d:
0.88 (t, J ¼ 6.8 Hz, 3H), 1.32e1.42 (m, 4H), 1.78e1.88 (m, 2H), 3.40
(brs, 1H), 3.81 (t, J ¼ 4.1 Hz, 1H), 3.87e3.92 (m, 1H), 3.98 (dd, J ¼ 4.1,
11.8 Hz, 1H), 4.05 (d, J ¼ 1.7 Hz, 1H), 4.23 (d, J ¼ 1.7 Hz, 1H). 13C NMR(100 MHz, D2O) d: 13.62, 22.17, 28.54, 31.28, 57.65, 63.66, 66.67,
75.43, 79.79. IR (NaCl) cm�1: 3365, 2928, 1585. EI-MS (m/z): 189(Mþ). HRMS Calcd for C9H19NO3: 189.1365; Found: 189.1370.
4.2.25. b-1-C-Butyl-L-arabino-iminofuranose (1e) hydrochloride
A solution of 23b (15.0 mg, 0.07 mmol) in 6 mol/L HCl (2.0 mL)
was stirred at 100 � C for 12 h. The whole mixture was transferred
into a separate funnel and washed with CH2Cl2 (2 mL � 3). The
water layer was evaporated to obtain 1e·HCl (5.0 mg, 79%) as a1brown oil. [a]23
D ¼ þ2.88 (c 0.77, CH3OH). H NMR (400 MHz, D2O) d:
0.89 (t, J ¼ 6.5 Hz, 3H), 1.38 (brs, 4H), 1.74e1.84 (m, 2H), 3.56 (brs,
1H), 3.73 (t, J ¼ 6.5 Hz, 1H), 3.80 (t, J ¼ 11.3 Hz, 1H), 3.94 (dd, J ¼ 4.8,
11.3 Hz, 1H), 4.07 (brs, 1H), 4.16 (brs, 1H). 13C NMR (100 MHz, D2O)
d: 13.66, 22.44, 25.29, 28.39, 60.35, 63.51, 68.15, 75.82, 76.73. IR(NaCl) cm�1: 3401, 3307, 3051, 2954, 1562. EI-MS (m/z): 189 (Mþ).
HRMS Calcd for C9H19NO3: 189.1365; Found: 189.1366.
4.3. Biological evaluation
Brush border membranes were prepared from the rat small
intestine according to our previous method and were assayed at pH
5.8 for rat intestinal maltase, sucrase, and isomaltase using theappropriate disaccharides as substrates [13]. For rat intestinal aglucosidase activities, the reaction mixture contained 25 mM substrate and the appropriate amount of enzyme, and the incubations
were performed for 10e30 min at 37 � C. The reaction was stopped
by heating at 100 � C for 3 min. After centrifugation (600 g; 10 min),the resulting reaction mixture was subjected to a Glucose CII-test
Wako (Wako Pure Chemical Industries, Osaka, Japan). The absorbance at 505 nm was measured to determine the amount of Dglucose released.
Acknowledgements
This work was supported by JSPS KAKENHI Grant Number
18K06553. This work was supported by a grant from the Strategic
Research Foundation Grant-aided Project for Private Universities
from the Ministry of Education, Culture, Sport, Science, and Technology, Japan (MEXT).
Appendix A. Supplementary data
Supplementary data to this article can be found online athttps://doi.org/10.1016/j.tet.2019.04.003.
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[2] World Health Organization. Global Report on Diabetes. Available online:
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182e197.
[4] U. Ghani, Eur. J. Med. Chem. 103 (2015) 133e162.
[5] (a) P. Compain, O.R. Martin, IminosugarsdFrom Synthesis to Therapeutic
Applications, John Wiley & Sons, Ltd., West Sussex, England, 2007;(b) N. Asano, K. Oseki, E. Tomioka, H. Kizu, K. Matsui, Carbohydr. Res. 259(1994) 243e255;(c) R.J. Nash, A. Kato, C.-Y. Yu, G.W. Fleet, J. Future Med. Chem. 3 (2011)
1513e1521;(d) G. Horne, F.X. Wilson, Prog. Med. Chem. 50 (2011) 135e176.

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391e396;(b) H. Bischoff, Eur. J. Clin. Invest. 24 (1994) 3e10;(c) N. Kuboyama, I. Hayashi, T. Yamaguchi, Folia Pharmacol. Jpn. 127 (2006)
223e232.
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Y. Natori, Y. Watanabe, T. Imahori, Y. Yoshimura, H. Takahata, S. Hirono, Org.
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I. Adachi, A. Kato, H. Takahata, Bioorg. Med. Chem. Lett 24 (2014) 3298e3301;(d) A. Kato, E. Hayashi, S. Miyauchi, I. Adachi, T. Imahori, Y. Natori,
Y. Yoshimura, R.J. Nash, H. Shimaoka, I. Nakagome, J. Koseki, S. Hirono,
H. Takahara, J. Med. Chem. 55 (2012) 10347e10362;(e) Y. Natori, S. Kikuchi, Y. Yoshimura, A. Kato, I. Adachi, H. Takahata,

Heterocycles 86 (2012) 1401e1417;(f) Y. Natori, T. Imahori, K. Murakami, Y. Yoshimura, S. Nakagawa, A. Kato,
I. Adachi, H. Takahata, Bioorg. Med. Chem. Lett 21 (2011) 738e741.
[8] B.M. Trost, D.B. Horne, M.J. Woltering, Chem. Eur J. 12 (2006) 6607e6620.
[9] R.L. Grange, E.A. Clizbe, P.A. Evans, Synthesis 48 (2016) 2911e2968.
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Ed. 46 (2007) 8790e8795.
[11] In this manuscript, the words “a-side” and “b-side” were used according to
the deﬁnition of carbohydrate nomenclature. The same side as the hydroxymethyl group or the equivalent was “b-side”, and the opposite side was the“a-side”[12] T. Sugimura, K. Hagiya, Chem. Lett. 36 (2007) 566e567.
[13] M. Kessler, O. Acuto, C. Storelli, H. Murer, M. Müller, G. Semenza, Biochim.
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A novel approach to sesquiterpenoid benzoxazole
synthesis from marine sponges: nakijinols A, B andE–G†
Yuki Takeda, Keiyo Nakai, Koichi Narita and Tadashi Katoh*

Nakijinols A, B and analogues E through G, which are structurally unique and biologically signiﬁcant sesquiterpenoid benzoxazoles, can be eﬃciently obtained in a highly uniﬁed manner from the sesquiterpeReceived 26th March 2018,
Accepted 21st April 2018
DOI: 10.1039/c8ob00721g
rsc.li/obc

noid quinone, smenospongine. The starting material is accessible from the (+)-5-methyl Wieland–
Miescher ketone. The synthetic method features strategic construction of the requisite dihydroxylated
benzoxazole substructure via the ring closure of the N-(2-hydroxyphenyl)-formamide or -acetamide
moiety. The synthesis of nakijinols is reported here for the ﬁrst time. The absolute conﬁgurations of nakijinols A and E were also established.

Introduction
In 1995, Kobayashi et al.1 reported the isolation and structural
elucidation of a novel sesquiterpenoid benzoxazole,2 nakijinol
A (1, Fig. 1) from an Okinawan marine sponge belonging to
the Spongiidae family. Even though the constitutional structure and relative stereochemistry of 1 have been both determined via extensive spectroscopic studies and 2D NMR experiments, its absolute configuration remains to be assigned.1 It is
well established that this marine natural product has a transfused decalin ring connected to a dihydroxylated benzoxazole
ring through a methylene linkage; the decalin unit contains
four contiguous asymmetric carbon centres (C-5, C-8, C-9 andC-10) and an endo-olefinic double bond (C-3–C-4 linkage).1
Remarkably, 1 was the first example of naturally occurring
sesquiterpenoids that contained a benzoxazole ring. However,the biological activity of 1 has not been established.1 Nakijinol
B (2) was originally isolated by Motti et al.3 in 2011 from themarine sponge Dactylospongia elegans, and its structure was
assigned as the C-4 exo-olefinic isomer of 1. Nakijinol B has
shown anti-proliferative activity against several human cancer
cell lines in the micromolar range.3 In 2014, nakijinol C (3),wherein the dihydroxybenzoxazole unit of 1 has been replaced
by a methyl trihydroxybenzoate unit, was isolated by
Kobayashi et al.4 from the same sponge in which 1 had been
detected. It has been reported that 3 exhibits antimicrobial

Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University,
4-4-1 Komatsushima, Aoba-ku, Sendai, 981-8558, Japan.
E-mail: katoh@tohoku-mpu.ac.jp† Electronic supplementary information (ESI) available: Copies of 1H and 13C
NMR spectra for all new compounds. See DOI: 10.1039/c8ob00721g

Fig. 1 Structures of nakijinols A–C (1–3), E–G (5–7), 5-epi-nakijinol
D (4), smenospongine (8), ilimaquinone (9) and O-methylilimaquinone(10).

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Organic & Biomolecular Chemistry

activity against several types of bacteria and fungi.4 In thesame year, 5-epi-nakijinol D (4)5 was isolated by Proksch et al.6
from the sponge Dactylospongia metachromia. The structure of
4 contains a unique 2,6-dimethylbenzodioxazole unit.6
Nakijinol E (5), which incorporates a methyl group at C-22
on the benzoxazole ring, was obtained from Smenospongia
sponges by Hamann et al.7 in 2015. It has been shown that 5
has the potential to exert antiproliferative activity via the inhibition of β-catenin expression.7 More recently, Lin et al.8
reported the isolation and characterisation of novel nakijinols
F (6) and G (7), along with known nakijinols 2 and 5, from adiﬀerent species of the marine sponge, namely Hyrtios sp. The
absolute configurations of nakijinols B, F and G have been
assigned as depicted in structures 2, 6 and 7, respectively. Thiswas done using a combination of single-crystal X-ray diﬀraction and electronic circular dichroism analyses.8 Nakijinol G
has been reported to exhibit protein tyrosine phosphatase 1B
inhibitory activity at a low micromolar level.8 To the best of
our knowledge, these are the only members of the nakijinol
family that have been isolated so far.

Results and discussion
Synthetic plan for 1, 2 and 5–7
Retrosynthetic analysis of nakijinols A (1), B (2) and E–G (5–7)
is outlined in Scheme 1. The key element of this plan was thestrategic construction of the doubly hydroxy-substituted benzoxazole ring system. Thus, we envisioned that the requisite
benzoxazole moiety could be established through ring closureof the appropriately functionalised form- and acet-imidoyl
chloride intermediates 11 and 12 via intramolecular substitution (i.e. a nucleophilic addition–elimination sequence) of
the chlorine atom by the phenolic hydroxy group in the ortho
position (i.e. 11 → 2; 12 → 7). Therefore, intermediates 11 and12 could be obtained via imidoyl chloride formation of thecorresponding N-phenyl-formamide 13 and -acetamide 14,
respectively, both of which were accessible from smenospongine (8) via formylation or acetylation of the amino group, followed by reduction of the quinone moiety. Starting material 8
was prepared from an enantiomerically pure sample of 15(>99% ee) according to the method previously reported 9a (27%overall yield in 15 steps). The cyclized product 2 (i.e. nakijinol B)
was converted into nakijinol A (1) via isomerization at the C-4
olefinic double bond. In addition, site-selective O-methylationof the C-18 hydroxy group in 2 provided nakijinol F (6).
Similarly, nakijinol E (5), which possessed the C-22 methylgroup, was derived from the other cyclized product 7 (i.e. nakijinol G) by site-selective O-methylation of the C-18 hydroxygroup.
There are several reports regarding the synthesis of benzoxazoles from the corresponding N-acylaminophenols.10
However, to the best of our knowledge, the method for synthesizing dihydroxy-substituted benzoxazoles (e.g. 2 and 7) fromaminohydroxyquinone derivatives (e.g. 8) was previously
unheard of.11 Thus, this approach posed a challenge from a

Scheme 1

Plan for the synthesis of nakijinols A (1), B (2) and E–G (5–7).

synthesis viewpoint. Furthermore, another hurdle was choosing mild reaction conditions that would have no impact on the
acid-labile exo-olefin functionality at C-4, especially during thesynthesis of 2 and 7 from 8.
Synthesis of Nakijinol B (2)Nakijinol B (2) was made as shown in Scheme 2. The synthesis
commenced with formylation of the amino group in smenospongine (8),9a which aﬀorded the corresponding formamide
16 in 73% yield. There were initial attempts to directly convert
16 into the N-(trihydroxyphenyl)formamide 13, a hypotheticalsubstrate for the key benzoxazole ring formation (Scheme 1),
through reduction of the quinone moiety under several conditions [(i) Na2S2O4, MeCN/H2O, MeOH/H2O, or EtOAc/H2O,
0 °C to room temperature;12 (ii) Zn, AcOH, room tempera-

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in structure 11 (Scheme 1). Interestingly, a mixture of 19 and
20 could be converted to the sole product 20 in quantitative
yields when this mixture was exposed to triethylamine in
MeCN at room temperature.16 This indicated that the C-18
acetate 20 was thermodynamically more stable than the C-17
acetate 19. The structure of 20 was assigned based on analysisof the heteronuclear multiple bond correlation (HMBC) spectrum of the C-17 O-methylated product 21 (see ESI†), which
had been derived from 20. To advance the synthesis, deprotection of the O-acetyl group in a mixture of 19 and 20 was
conducted using 2.5% aqueous ammonia in MeOH at 0 °C toaﬀord the first target compound, nakijinol B (2), in 92% yield.
The spectroscopic properties of 2 (IR, MS and both 1H and 13C
NMR spectroscopy) were identical to those of natural 2.3,8 The
optical rotation of synthetic 2 was in agreement with that ofnatural 2.
Synthesis of nakijinols A (1) and F (6)Scheme 2 Synthesis of nakijinols B (2). (a) HCO2H, Ac2O, 60 °C, 2 h;then 8, room temp., 2 h, 73%; (b) Ac2O, DMAP, THF, 0 °C to room temp.,
81%; (c) Na2S2O4, MeCM/H2O (1 : 1), room temp., 30 min; (d) PPh3, CCl4,
MeCN, 50 °C, 2 h, 98% (2 steps, 19/20 ca.1 : 3); (e) NH3 (2.5% aq.),
MeOH, 0 °C, 10 min, 92%. Ac = acetyl, DMAP = 4-(dimethylamino)
pyridine.

ture;13 (iii) NaBH4, MeOH or EtOH, 0 °C to temperature14]. Allof these experiments were unsuccessful and resulted indecomposition of the products. This was likely due to the presence of the sensitive hydroxy group at C-17 in the substrate 16.
Given this, it was decided that masking the reactive hydroxygroup as an acetate would be the best way to improve ourresults. Acetylation of the hydroxy group in 16 provided thecorresponding acetate 17 in 81% yield. Subsequent reductionof the quinone moiety in 17 with Na2S2O4 15 in MeCN/H2O(1 : 1) at room temperature furnished the requisite hydroquinone 18. However, since the product was unstable andeasily susceptible to oxidation in air, it was used immediately
for subsequent reactions without further purification. The
crucial benzoxazole ring formation was eﬃciently achieved bytreating the crude product 18 with triphenylphosphine (PPh3)
and carbon tetrachloride (CCl4)10e in MeCN at 50 °C for 2 h.
The benzoxazole products 19 and 20 (the O-acetyl-migration
product of 19) were obtained as an inseparable mixture in 98%
yield over two steps. Using 400 MHz 1H NMR spectroscopic
analysis, the ratio of products 19 and 20 was estimated to beapproximately 1 : 3. We believe that this ring-closing reaction
proceeded via the reactive imidoyl chloride species as depicted

After obtaining the first target compound, nakijinol B (2), we
next approached the synthesis of the second and third target
compounds, nakijinols A (1) and F (6), as shown in Scheme 3.
Acid-induced isomerization of the C-4 exo-olefin moiety in 2
was eﬃciently achieved through treatment with p-toluenesulfonic acid in CH2Cl2 at room temperature to provide nakijinol
A (1) in good yield (86%). It is worth noting that the sensitive
dihydroxylated benzoxazole moiety present in 2 remained
intact during acid-induced olefin isomerization. On the other
hand, site-selective O-methylation of the C-18 hydroxy group in
2 was successfully achieved using Me2SO4 in the presence of
Cs2CO3 17 in acetone at room temperature. This produced nakijinol F (6) in 82% yield. Note that no formation of the C-17
O-methylated product was observed in this reaction, probably
owing to steric crowding around the C-17 hydroxy group in
substrate 2. The spectroscopic properties of 1 and 6 were iden-

Scheme 3 Synthesis of nakijinols A (1) and F (6). (a) pTsOH·H2O,
CH2Cl2, room temp., 1 h, 86%; (b) Me2SO4, Cs2CO3, acetone, room
temp., 30 min, 82%. pTsOH = p-toluenesulfonic acid.

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Organic & Biomolecular Chemistrytical to those obtained for natural 1 1 and 6,8 respectively. The
optical rotations of synthetic 1 and 6 were consistent withthose reported for natural 1 and 6, respectively. The synthesisof 1 also led to the confirmation of its absolute configuration.

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Synthesis of nakijinols E (5) and G (7)Having successfully developed a novel method for constructing
the key dihydroxybenzoxazole ring system, we next focused onsynthesizing the final two target compounds, nakijinols E (5)and G (7). Both of these compounds have a methyl group at
C-22 on the benzoxazole ring, as shown in Scheme 4. Startingfrom 8, the synthesis was performed in a manner similar tothat described for nakijinols B (2) (Scheme 2) and F (6)(Scheme 3). Twofold acetylation of the amino and the hydroxygroups in 8 aﬀorded the corresponding N,O-diacetylatedquinone 22 in 80% yield. Subsequent reduction of thequinone moiety in 22 using Na2S2O4 provided hydroquinone
23, which was immediately utilized without further purification due to its unstable nature. The crucial benzoxazole ringformation of 23 proceeded smoothly and cleanly under the
aforementioned conditions (cf. 18 → 19 + 20 in Scheme 2),leading to the formation of the requisite benzoxazole products
24 and 25 (ca. 2 : 3)18 as an inseparable mixture in excellent

yield (98%). Removal of the O-acetyl group from a mixture of
24 and 25 furnished the fourth target compound, nakijinol
G (7), in a quantitative yield of 99%. Finally, site-selective
O-methylation of the C-18 hydroxy group in 7 provided thefinal target compound, nakijinol E (5), in 89% yield. The spectroscopic properties of 7 and 5 were identical to those ofnatural 7 8 and 5.7,8 Optical rotation values for synthetic 7 and5 matched those reported for natural 7 and 5, respectively. The
synthesis of 5 reported here led to confirmation of its absoluteconfiguration.

Conclusions
We have achieved the highly eﬃcient synthesis of nakijinols
A (1), B (2) and E–G (5–7) for the first time starting fromsmenospongine (8) (1: 45.8% overall yield over six steps; 2:
53.3% overall yield over five steps; 5: 69.0% overall yield over
five steps; 6: 43.7% overall yield over six steps and 7: 77.6%
overall yield over four steps). The key step of the synthesis was
oxazole-ring formation via ring closure of the N-(2-hydroxyphenyl)-formamide or -acetamide moiety. This was undertaken by
PPh3/CCl4 in MeCN at 50 °C and provided the requisite dihydroxylated benzoxazole ring substructure in excellent yield(98%) [cf. 18 → 19 + 20 in Scheme 2; 23 → 24 + 25 in
Scheme 4]. Importantly, this general and flexible synthesis canpotentially produce additional analogues of nakijinols inenantiomerically pure forms (e.g. analogues possessing
various alkyl substituents at C-22 on the benzoxazole ring) inorder to explore their structure–activity relationships. Further
investigations to identify the mode of action of nakijinols
using the synthetic samples are currently in progress.

Experimental section
General experimental procedures

Scheme 4 Synthesis of nakijinols E (5) and G (7). (a) Ac2O, DMAP, THF,
60 °C, 2 h, 80%; (b) Na2S2O4, MeCM/H2O (5 : 1), room temp., 10 min;(c) PPh3, CCl4, MeCN, 50 °C, 2 h, 98% (2 steps, 24/25 ca. 2 : 3); (d) NH3(2.5% aq.), MeOH, 0 °C, 10 min, 99%; (e) Me2SO4, Cs2CO3, acetone,
room temp., 45 min, 89%.

All reactions involving air- and moisture-sensitive reagentswere carried out using oven dried glassware and standard
syringe-septum cap techniques. Routine monitorings of reaction were carried out using glass-supported Merck silica gel 60
F254 TLC plates. Flash column chromatography was performed on Kanto Chemical Silica Gel 60N (spherical, neutral
40–50 nm) with the solvents indicated. All solvents andreagents were used as supplied with following exceptions.
Tetrahydrofuran (THF) was freshly distilled from Na metal/benzophenone under argon. CH2Cl2 was distilled from
calcium hydride under argon. Measurements of opticalrotations were performed with an Anton Paar MCP-100 automatic digital polarimeter. 1H and 13C NMR spectroscopic data
were recorded with JEOL AL-400 (400 MHz) and JEOL AL-600(600 MHz) spectrometers. Chemical shifts were expressed in
ppm using Me4Si (δ = 0) as an internal standard. Infrared (IR)
spectral measurements were carried out with a JASCO FT/
IR-4100 spectrometer. Low- and High-resolution mass (HRMS)

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spectra were measured on a JEOL JMS-700 high-resolution
mass spectrometer.
N-{4-Hydroxy-3,6-dioxo-5-{[(1R,2S,4aS,8aS)-1,2,4a-trimethyl5-methylenedecahydronaphthalen-1-yl]methyl}cyclohexa-1,4dienyl}formamide (16). Acetic anhydride (5.50 mL, 5.8 mmol)
was added dropwise to a stirred solution of formic acid
(2.50 mL, 6.1 mmol) at 0 °C under argon, and the reaction
mixture was stirred at 60 °C for 2 h. A solution of mixed acidanhydride (AcOCHO) prepared above was added dropwise to a
stirred powder of smenospongine (8)9a (200 mg, 0.58 mmol) at
room temperature. After 2 h, the reaction was quenched withwater (20 mL) at 0 °C, and the resulting mixture was extractedwith EtOAc (3 × 30 mL). The combined extracts were washedwith brine (2 × 30 mL), then dried with Na2SO4. The mixture
was concentrated in vacuo, and the residue was purified bycolumn chromatography (hexane/EtOAc, 3 : 1) to give 16(158 mg, 73%) as an orange solid. Recrystallization fromhexane/EtOAc (2 : 1) gave an analytical sample of 16 as orange
1
prisms. M.p. 235.0–236.5 °C. [α]25
D = + 6.0 (c = 0.10, CHCl3). H
NMR (400 MHz, CDCl3): δ = 0.74 (dd, J = 11.7, 2.0 Hz, 1H), 0.85
(s, 3H), 0.96 (d, J = 6.3 Hz, 3H), 1.04 (s, 3H), 1.10–1.17 (m, 2H),
1.33–1.42 (m, 3H), 1.47 (dd, J = 12.1, 3.4 Hz, 1H), 1.50–1.54 (m,1H), 1.84–1.89 (m, 1H), 2.05–2.09 (m, 2H), 2.33 (td, J = 13.8,
5.0 Hz, 1H), 2.45 (d, J = 13.7 Hz, 1H), 2.55 (d, J = 13.7 Hz, 1H),
4.44 (s, 1H), 4.45 (s, 1H), 7.50 (brs, 1H), 7.64 (s, 1H), 8.53 (br s,
1H), 8.63 (s, 1H) ppm. 13C NMR (100 MHz, CDCl3): δ = 17.3 (q),17.9 (q), 20.5 (q), 23.3 (t), 28.0 (t), 29.7 (t), 32.6 (t), 32.9 (t), 36.6(t), 38.2 (d), 40.5 (s), 43.4 (s), 50.3 (d), 102.8 (t), 115.2 (d), 116.5(s), 131.9 (s), 154.2 (s), 159.5 (s), 160.0 (d), 160.1 (s), 182.7 (s)ppm. IR (neat): ν = 3853, 3734, 3648, 3291, 2924, 2359, 1717,
1636, 1540, 1489, 1373, 1193, 891, 772, 668 cm−1. HRMS (EI):
calcd for C22H29NO4 [M]+ 371.2097; found 371.2084.
4-Formamido-3,6-dioxo-2-{[(1R,2S,4aS,8aS)-1,2,4a-trimethyl5-methylenedecahydronaphthalen-1-yl]methyl}cyclohexa-1,4dienyl acetate (17). Acetic anhydride (42.9 μL, 0.45 mmol) and4-(dimethylamino)pyridine (3.10 mg, 25 μmol) were added to a
stirred solution of 16 (46.9 mg, 0.13 mmol) in THF (4.0 mL) at
0 °C under argon, and the mixture was stirred at room temperature for 1 h. The reaction was quenched with saturated
aqueous NaHCO3 (5 mL) at 0 °C, and the resulting mixture was
extracted with EtOAc (2 × 20 mL). The combined extracts were
washed with brine (2 × 10 mL), then dried with Na2SO4. The
mixture was concentrated in vacuo, and the residue was purified by column chromatography (hexane/EtOAc, 4 : 1) to give17 (42.2 mg, 81%) as a yellow amorphous powder. [α]25
D = + 4.2
(c = 1.00, CHCl3). 1H NMR (400 MHz, CDCl3): δ = 0.70 (d, J =11.2 Hz, 1H), 0.85 (s, 3H), 0.92 (d, J = 6.3 Hz, 3H), 1.05 (s, 3H),
1.20–1.26 (m, 1H), 1.35–1.38 (m, 2H), 1.40 (m, 1H), 1.44 (m,1H), 1.48–1.55 (m, 2H), 1.86–1.93 (m, 2H), 2.05–2.10 (m, 1H),
2.25–2.34 (m, 1H), 2.34 (s, 3H), 2.41 (d, J = 13.2 Hz, 1H), 2.55
(d, J = 13.2 Hz, 1H), 4.46 (s, 1H), 4.48 (s, 1H), 7.56 (s, 1H), 8.30
(br s, 1H), 8.60 (s, 1H) ppm. 13C NMR (100 MHz, CDCl3): δ =17.1 (q), 18.1 (q), 20.5 (q), 20.5 (q), 23.5 (t), 27.8 (t), 28.6 (t),32.8 (t), 33.7 (t), 36.4 (t), 40.5 (s), 44.0 (s), 50.9 (s), 103.4 (t),114.3 (d), 133.0 (s), 137.5 (s), 152.0 (s), 159.3 (d), 159.4 (s),167.7 (s), 179.8 (s), 183.0 (s) ppm. FTIR (neat) ν = 3734, 3648,

2929, 2357, 1780, 1715, 1667, 1612, 1507, 1312, 1219, 1150,
884, 772, 670, 638 cm−1. HRMS (EI): calcd for C24H31NO5 [M]+413.2202; found 413.2189.
Mixture of 5-hydroxy-7-{[(1R,2S,4aS,8aS)-1,2,4a-trimethyl-5methylenedecahydronaphthalen-1-yl]methyl}benzo[d]oxazol-6yl acetate (19) and 6-hydroxy-7-{[(1R,2S,4aS,8aS)-1,2,4a-trimethyl-5-methylenedecahydronaphthalen-1-yl]methyl}benzo[d]oxazol-5-yl acetate (20). A solution of sodium hydrosulfite(846 mg, 4.9 mmol) in water (6.0 mL) was added dropwise to a
stirred solution of 17 (50.0 mg, 0.12 mmol) in acetonitrile(6.0 mL) at room temperature. After 30 min, the reaction
mixture was poured into water (20 mL), and the resulting
mixture was extracted with EtOAc (3 × 20 mL). The combined
extracts were washed with brine (2 × 15 mL), then dried with
Na2SO4. Concentration in vacuo gave 4-formamido-3,6-dihydroxy-2-{[(1R,2S,4aS,8aS)-1,2,4a-trimethyl-5-methylenedecahydronaphthalen-1-yl]methyl}phenyl acetate (18) (60.7 mg). This
product was used for the next reaction without further
purification.
Triphenylphosphine (255 mg, 0.97 mmol) and carbon tetrachloride (47.3 μL, 0.49 mmol) was added to a stirred solutionof the crude product 18 (60.7 mg) in acetonitrile (6.0 mL) at
room temperature, and the reaction mixture was further
stirred at 50 °C for 2 h. After cooling to room temperature, thereaction mixture was poured into 5% aqueous NaHCO3(20 mL), and the resulting mixture was extracted with EtOAc(3 × 20 mL). The combined extracts were washed with brine(2 × 20 mL), then dried with Na2SO4. The mixture was concentrated in vacuo, and the residue was purified by column chromatography (hexane/EtOAc, 2 : 1) to give an inseparable mixtureof 19 and 20 (ca. 1 : 3) (47.2 mg, 98%, 2 steps). 1H NMR(400 MHz, CDCl3): δ = 0.87 (dd, J = 12.2, 2.4 Hz, 1/4H), 0.91
(dd, J = 12.2, 2.4 Hz, 3/4H), 0.92 (s, 3/4H), 0.94 (s, 9/4H), 0.99
(d, J = 5.4 Hz, 3/4H), 1.03 (d, J = 5.9 Hz, 9/4H), 1.06 (s, 3/4H),
1.06 (s, 9/4H), 1.19–1.28 (m, 2/4H), 1.19–1.28 (m, 4/6H),
1.40–1.60 (m, 5/4H), 1.40–1.60 (m, 15/4H), 1.89–1.92 (m, 1/4H),
1.89–1.92 (m, 3/4H), 2.20–2.23 (m, 1/4H), 2.20–2.23 (m, 3/4H),
2.30–2.36 (m, 1/4H), 2.30–2.36 (m, 3/4H), 2.38 (s, 3/4H), 2.39(s, 9/4H), 2.74 (d, J = 13.2 Hz, 1/4H), 2.80 (d, J = 13.2 Hz, 1/4H),
2.87 (d, J = 14.1 Hz, 3/4H), 2.96 (d, J = 14.1 Hz, 3/4H), 4.35
(s, 1/4H), 4.35 (s, 3/4H), 4.39 (s, 1/4H), 4.39 (s, 3/4H), 5.53 (s,3/4H), 6.27 (br s, 1/4H), 7.23 (s, 1/4H), 7.98 (s, 3/4H), 8.02 (s,1/4H) ppm. 13C NMR (100 MHz, CDCl3): δ = 17.2 (q, 1/4C), 17.3
(q, 3/4C), 18.2 (q, 3/4C), 18.5 (q, 1/4C), 20.4 (q, 1/4C), 20.5 (q,3/4C), 20.8 (q, 1/4C), 21.1 (q, 3/4C), 23.3 (t, 3/4C), 23.5 (t, 1/4C),
28.0 (t, 1/4C), 28.1 (t, 3/4C), 28.7 (t, 1C), 32.8 (t, 1/4C), 32.9 (t,3/4C), 35.0 (t, 3/4C), 35.9 (t, 1/4C), 36.3 (t, 1C), 37.5 (d, 1/4C),
37.6 (d, 3/4C), 40.4 (s, 1C), 43.0 (s, 1/4C), 43.1 (s, 3/4C), 49.7 (d,3/4C), 49.9 (d, 1/4C), 102.8 (t, 3/4C), 103.0 (t, 1/4C), 105.4 (d,1/4C), 111.2 (d, 3/4C), 112.0 (s, 3/4C), 117.8 (s, 1/4C), 131.8 (s,3/4C), 136.6 (s, 3/4C), 136.8 (s, 1/4C), 137.9 (s, 1/4C), 144.9(s, 1/4C), 145.4 (s, 3/4C), 145.6 (s, 1/4C), 151.9 (s, 3/4C), 152.8(s, 1/4C), 159.6 (s, 1/4C), 159.9 (s, 3/4C), 168.9 (s, 3/4C), 169.1(s, 1/4C) ppm.
Conversion of a mixture of 19 and 20 to single compound
20. Triethylamine (0.13 mL, 0.92 mmol) was added to a stirred

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solution of a mixture of 19 and 20 (ca. 1 : 3, 18.7 mg) in acetonitrile (1.0 mL) at room temperature. After 5 min, the reaction
mixture was concentrated in vacuo to give a single product of
20 (18.7 mg, 100%) as a pale yellow amorphous solid; [α]25D =−46.4 (c = 0.11, CHCl3). 1H NMR (400 MHz, CDCl3): δ =0.88–0.93 (m, 1H), 0.94 (s, 3H), 1.04 (d, J = 4.8 Hz, 3H), 1.06 (s,3H), 1.19–1.26 (m, 2H), 1.39–1.42 (m, 3H), 1.44–1.48 (m, 1H),
1.57 (td, J = 11.6, 4.0 Hz, 1H), 1.89–1.93 (m, 1H), 2.05 (dd, J =13.5, 3.9 Hz, 1H), 2.22 (br d, J = 13.0, 1H), 2.34 (td, J = 14.5, 4.7
Hz, 1H), 2.87 (d, J = 14.0 Hz, 1H), 2.96 (d, J = 14.0 Hz, 1H), 4.35
(s, 1H), 4.40 (s, 1H), 7.46 (s, 1H), 7.98 (s, 1H) ppm. 13C NMR(100 MHz, CDCl3): δ = 17.3 (q), 18.2 (q), 20.5 (q), 21.1 (q), 23.3(t), 28.1 (t), 28.7 (t), 32.9 (t), 35.0 (t), 36.3 (t), 37.6 (d), 40.4 (s),43.1 (s), 49.7 (d), 102.8 (t), 111.2 (d), 112.0 (s), 131.8 (s), 136.6(s), 145.4 (s), 148.7 (s), 151.9 (d), 159.9 (s), 168.9 (s) ppm. FTIR
(neat): ν = 3648, 3566, 2925, 2359, 1771, 1635, 1521, 1457,
1219, 772, 674 cm−1. HRMS (EI): calcd for C24H31NO4 [M]+397.2253; found 397.2236.
6-Methoxy-7-{[(1R,2S,4aS,8aS)-1,2,4a-trimethyl-5-methylenedecahydronaphthalen-1-yl]methyl}benzo[d]oxazol-5-yl acetate (21).
Cesium carbonate (7.89 mg, 24 μmol) and dimethyl sulfate(9.53 μL, 0.10 mmol) were added to a stirred solution of 20(8.0 mg, 20 μmol) in acetone (2.0 mL) at room temperature.
After 1 h, the reaction was quenched with saturated aqueous
NH4Cl (10 mL), and the resulting mixture was extracted with
CHCl3 (3 × 10 mL). The combined extracts were washed withbrine (2 × 10 mL), then dried with Na2SO4. The mixture wasconcentrated in vacuo, and the residue was purified by column
chromatography (hexane/EtOAc, 3 : 1) to give 21 (5.8 mg, 70%)
as a colourless viscous liquid. [α]25
D = −27.3 (c = 0.11, CH3OH).
1
H NMR (400 MHz, CDCl3): δ = 0.88 (dd, J = 11.7, 2.0 Hz, 1H),
0.93 (s, 3H), 0.97 (d, J = 4.9 Hz, 1H), 1.05 (s, 3H), 1.21 (m, 3H),
1.34–1.46 (m, 4H), 1.47–1.58 (m, 2H), 1.85–1.91 (m, 1H), 2.05
(m, 2H), 2.28–2.33 (m, 1H), 2.36 (s, 3H), 2.84 (d, J = 13.7 Hz,
1H), 2.90 (d, J = 13.7 Hz, 1H), 3.73 (s, 3H), 4.35 (s, 1H), 4.39 (s,1H), 7.39 (s, 1H), 8.03 (s, 1H) ppm. 13C NMR (100 MHz,CDCl3): δ = 17.3 (q), 18.2 (q), 20.5 (q), 20.9 (q), 23.4 (t), 28.0 (t),28.5 (t), 32.9 (t), 35.3 (t), 36.3 (d), 37.6 (s), 40.4 (s), 43.2 (d),49.7 (t), 61.1 (t), 102.9 (t), 112.6 (d), 118.4 (s), 134.8 (s),141.9 (s), 148.3 (s), 150.0 (d), 159.8 (s), 169.1 (s) ppm. FTIR
(neat) ν = 2923, 2359, 1771, 1540, 1507, 1457, 1220, 894, 772,
694, 683, 635, 617, 606 cm−1. HRMS (EI): calcd for C25H33NO4[M]+ 411.2410; found 411.2407.
Nakijinol B (2)2.5% Aqueous ammonia (40.0 μL, 58 μmol) was added to a
stirred solution of a mixture of 19 and 20 (ca. 1 : 3) (18.7 mg,
47 μmol) in MeOH (3.0 mL) at 0 °C. After 10 min, the reaction
was quenched with saturated aqueous NH4Cl (15 mL) at 0 °C,
and the resulting mixture was extracted with EtOAc(3 × 20 mL). The combined extracts were washed with brine(2 × 15 mL), then dried with Na2SO4. The mixture was concentrated in vacuo, and the residue was purified by column chromatography (hexane/EtOAc, 1 : 1) to give 2 (15.3 mg, 92%) as apale pink solid. Recrystallization from MeOH gave an analytical sample of 2 as colourless prisms. M.p. 259.5–260.5 °C {lit.8825
M.p. 213–218 °C}. [α]25
D = −29.3 (c = 0.08, MeOH) {ref. [α]D =3
25−27.3 (c = 0.11, MeOH); ref. [α]D = −6.7 (c = 0.075, MeOH)}.
The 1H and 13C NMR, IR, and MS spectra (see below) are identical to those of natural nakijinol B (2).3,8 1H NMR (400 MHz,
CD3OD): δ = 0.93–0.96 (m, 1H), 0.95 (s, 3H), 1.07 (s, 3H), 1.08
(d, J = 5.9 Hz, 3H), 1.28 (m, 1H), 1.38–1.43 (m, 3H), 1.45–1.47(m, 2H), 1.55 (m, 1H), 1.86–1.91 (m, 1H), 2.01–2.05 (m, 1H),
2.31–2.35 (m, 1H), 2.35–2.39 (m, 1H), 2.86 (d, J = 14.1 Hz, 1H),
2.94(d, J = 14.1 Hz, 1H), 4.32 (s, 1H), 4.35 (s, 1H), 6.97 (s, 1H),
8.20 (s, 1H) ppm. 1H NMR (400 MHz, DMSO-d6): δ = 0.83 (1H,
dd, J = 11.7, 2.0 Hz), 0.87 (3H, s), 0.99 (3H, s), 1.00 (3H, d, J =4.9 Hz), 1.05–1.10 (1H, m), 1.13–1.24 (1H, m), 1.34–1.38 (4H,m), 1.45 (1H, m), 1.81 (1H, m), 1.98 (1H, dd, J = 13.3, 3.7 Hz),2.21 (1H, d, J = 13.3 Hz), 2.27 (1H, td, J = 13.7, 5.4 Hz), 2.74
(1H, d, J = 14.1), 2.81 (1H, d, J = 14.1), 4.28 (1H, s), 4.34 (1H, s),
6.94 (1H, s), 8.36 (1H, s) ppm. 13C NMR (100 MHz, CD3OD):
δ = 17.9 (q), 18.9 (q), 21.0 (q), 24.4 (t), 29.2 (t), 29.9 (t), 34.0 (t),35.5 (t), 37.8 (t), 38.7 (d), 41.6 (s), 44.0 (s), 50.8 (d), 102.2 (d),103.1 (t), 110.8 (s), 131.3 (s), 144.6 (s), 146.3 (s,) 146.4 (s), 152.8(d), 161.4 (s) ppm. 13C NMR (100 MHz, DMSO-d6): δ = 17.2 (q),18.2 (q), 20.1 (q), 22.7 (t), 27.5 (t), 27.9 (t), 32.3 (t), 34.4 (t), 36.1(t), 37.0 (d), 39.8 (s), 42.2 (s), 48.9 (d), 102.2 (d), 102.8 (t), 109.2(s), 130.2 (s), 143.1 (s), 144.2 (s), 144.4 (s), 151.7 (d), 159.3 (s)ppm. FTIR (neat): ν = 3679, 2972, 2865, 2844, 1456, 1321, 1055,
1033, 1013 cm−1. HRMS (EI): calcd for C22H29NO3 [M]+355.2147; found 355.2155.

Nakijinol A (1)p-Toluenesufonic acid monohydrate (17.2 mg, 0.10 mmol) was
added to a stirred solution of 2 (18.0 mg, 51 μmol) in CH2Cl2(3.0 mL) at room temperature. After 1 h, the reaction was
quenched with saturated aqueous NaHCO3 (5 mL) at 0 °C, andthe reaction mixture was extracted with EtOAc (3 × 10 mL). The
combined extracts were washed with brine (2 × 10 mL), thendried with Na2SO4. The mixture was concentrated in vacuo,
and the residue was purified by column chromatography(hexane/EtOAc, 1 : 1) to give 1 (15.4 mg, 86%) as a colorlesssolid. Recrystallization from MeOH gave an analytical sample
of 1 as colourless prisms. M.p. 245.5–247.0 °C. [α]20
D = −29.3(c = 0.15, MeOH) {lit.1 [α]20
=
−172
(c
=
0.03,
MeOH)}.
The 1HD
13
and C NMR, IR, and MS spectra (see below) are identical tothose of natural nakijinol A (1).1 1H NMR (600 MHz, CDCl3):
δ = 0.95 (s, 3H), 0.94–0.96 (m, 1H), 1.03 (s, 3H), 1.06 (d, J = 6.6
Hz, 3H), 1.17 (dd, J = 12.3, 1.6 Hz, 1H), 1.38–1.41 (m, 2H),
1.45–1.50 (m, 1H), 1.51 (br s, 3H), 1.58–1.62 (m, 2H), 1.93–1.97(m, 1H), 1.97–2.07 (m, 1H), 2.17–2.21 (m, 1H), 2.89 (d, J =14.3 Hz, 1H), 3.01 (d, J = 14.3 Hz, 1H), 5.09 (br s, 1H), 5.65
(br s, 1H), 5.73 (s, 1H), 7.14 (s, 1H), 7.95 (s, 1H) ppm. 1H NMR(600 MHz, CD3OD): δ = 0.89–0.94 (1H, m), 0.95 (3H, s), 1.03
(3H, s), 1.06 (3H, d, J = 6.2 Hz), 1.17 (1H, m), 1.36–1.40 (2H,m), 1.47 (3H, brs), 1.51–1.56 (1H, m), 1.58–1.61 (2H, m), 1.98
(2H, m), 2.24–2.27 (2H, m), 2.91 (1H, d, J = 13.9 Hz), 3.01 (1H,d, J = 13.9 Hz), 5.06 (1H, br s), 6.97 (1H, s), 8.22 (1H, s) ppm.
13
C NMR (150 MHz, CDCl3): δ = 17.3 (q), 18.0 (q), 18.2 (q), 20.0(t), 20.2 (q), 27.0 (t), 28.0 (t), 34.7 (t), 35.7 (t), 37.4 (d), 38.4 (s),42.8 (s), 47.1 (d), 103.4 (d), 110.4 (s), 120.6 (d), 131.3 (s), 141.03644 | Org. Biomol. Chem., 2018, 16, 3639–3647

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(s), 143.4 (s), 144.1 (s), 145.8 (s), 151.3 (d) ppm. 13C NMR(150 MHz, CD3OD): δ = 17.9 (q), 18.4 (q), 18.6 (q), 20.7 (q), 21.1(q), 28.0 (t), 29.3 (t), 35.6 (t), 37.3 (d), 38.8 (d), 39.6 (s), 43.8 (s),49.6 (d), 102.6 (d), 111.1 (s), 121.7 (d), 131.4 (s), 145.0 (s), 146.3(s), 146.3 (s), 144.8 (s), 152.8 (d) ppm. FTIR (neat): ν = 3679,
2972, 2865, 2844, 1456, 1321, 1055, 1033, 1013 cm−1. HRMS
(EI): calcd for C22H29NO3 [M]+ 355.2147; found 355.2143.
Nakijinol F (6)Cesium carbonate (14.9 mg, 46 μmol) and dimethyl sulfate(4.34 μL, 46 μmol) were added to a stirred solution of 2
(14.8 mg, 42 μmol) in acetone (2.0 mL) at room temperature.
After 30 min, the reaction was quenched with saturated
aqueous NH4Cl (10 mL) at 0 °C, and the reaction mixture was
extracted with EtOAc (3 × 20 mL). The combined extracts were
washed with brine (2 × 10 mL), then dried with Na2SO4. The
mixture was concentrated in vacuo, and the residue was purified by column chromatography (hexane/EtOAc, 4 : 1) to give 6(12.6 mg, 82%) as a colourlesss solid. Recrystallization fromhexane/EtOAc (1 : 1) gave an analytical sample of 6 as colourlesss plates. M.p. 202.5–204.0 °C. [α]25
D = −28.4 (c = 0.13,
MeOH) {ref.8 [α]25
=
−16.2
(c
=
0.11,
MeOH)}.
The 1H and 13C
D
NMR, IR, and MS spectra (see below) are identical to those ofnatural nakijinol F (6).8 1H NMR (400 MHz, CDCl3): δ = 0.94
(dd, J = 11.7, 2.0 Hz, 1H), 0.94 (s, 3H), 1.06 (d, J = 6.0 Hz, 3H),
1.06 (s, 3H), 1.19–1.24 (m, 1H), 1.24–1.28 (m, 1H), 1.37–1.43(m, 2H), 1.37–1.43 (m, 2H), 1.43–1.47 (m, 1H), 1.46–1.58 (m,1H), 1.90–1.96 (m, 1H), 2.03–2.06 (m, 1H), 2.26–2.30 (m, 1H),
2.30–2.35 (m, 1H), 2.85 (d, J = 13.6 Hz, 1H), 2.95 (d, J =13.6 Hz, 1H), 3.95 (s, 3H), 4.33 (s, 1H,), 4.37 (s, 1H), 5.99 (s,1H), 7.11 (s, 1H), 7.92 (s, 1H) ppm. 13C NMR (100 MHz,CDCl3): δ = 17.4 (q), 18.2 (q), 20.5 (q), 23.3 (t), 28.1 (t), 28.7 (t),33.0 (t), 34.6 (t), 36.4 (t), 37.5 (d), 40.4 (s), 43.0 (s), 49.5 (d),56.5 (q), 99.3 (d), 102.5 (t), 109.5 (s), 131.1 (s), 144.4 (s), 145.0(s), 145.7 (s), 151.9 (d), 160.3 (s) ppm. FTIR (neat): ν = 2928,
2359, 1623, 1523, 1460, 1426, 1362, 1323, 1244, 1219, 1095,
941, 827, 772, 678, 668, 609 cm−1. HRMS (EI): calcd for
C23H31NO3 [M]+ 369.2304; found 369.2292.
4-Acetamido-3,6-dioxo-2-{[(1R,2S,4aS,8aS)-1,2,4a-trimethyl-5methylenedecahydronaphthalen-1-yl]methyl}cyclohexa-1,4-dienyl
acetate (22). 4-(Dimethylamino)pyridine (7.30 mg, 60 μmol)
were added to a stirred solution of 8 (102.9 mg, 0.3 mmol) in
acetic anhydride (2.00 mL, 21 mmol) at room temperature,and the mixture was stirred at 60 °C for 2 h. After cooling toroom temperature, the mixture was concentrated in vacuo, andthe residue was purified by column chromatography (hexane/
EtOAc, 4 : 1) to give 22 (102.4 mg, 80%) as an orange amor1
phous powder. [α]25
D = + 8.00 (c = 0.10, CHCl3). H NMR(400 MHz, CDCl3): δ = 0.72 (d, J = 10.8 Hz, 1H), 0.85 (s, 3H),
0.92 (d, J = 6.4 Hz, 1H), 1.05 (s, 3H), 1.15–1.26 (m, 2H),
1.35–1.52 (m, H), 1.80–1.90 (m, 2H), 2.08–2.10 (m, 1H), 2.25 (s,3H), 2.34 (s, 3H), 2.42 (d, J = 13.2 Hz, 1H), 2.52 (d, J = 13.2 Hz,
1H), 4.47 (s, 1H), 4.48 (s, 1H), 7.55 (s, 1H), 8.12 (s, 1H) ppm.
13
C NMR (100 MHz, CDCl3): δ = 17.1 (q), 18.2 (q), 20.5 (q, 2C),
23.6 (q), 25.0 (s), 27.9 (q), 28.7 (t), 32.8 (t), 33.8 (t), 36.4 (t), 38.3(t), 40.6 (d), 43.9 (d), 50.9 (t), 103.4 (d), 112.9 (s), 132.8 (s),138.7 (s), 152.1 (s), 159.3 (s), 167.8 (s), 169.1 (s), 180.0 (s), 183.5(s) ppm. FTIR (neat): ν = 3445, 2926, 2359, 1773, 1663, 1498,
1370, 1312, 1147, 1016 cm−1. HRMS (EI): calcd for C25H33NO5[M]+ 427.2359; found 427.2378.
Mixture of 5-hydroxy-2-methyl-7-{[(1R,2S,4aS,8aS)-1,2,4a-trimethyl-5-methylenedecahydronaphthalen-1-yl]methyl}benzo[d]oxazol-6-yl acetate (24) and 6-hydroxy-2-methyl-7-{[(1R,2S,4aS,8aS)1,2,4a-trimethyl-5-methylenedecahydronaphthalen-1-yl]methyl}benzo[d]oxazol-5-yl acetate (25). A solution of sodium hydrosulfite (348 mg, 2.0 mmol) in water (1.0 mL) was added dropwise
to a stirred solution of 22 (21.4 mg, 50 μmol) in acetonitrile(5.0 mL) at room temperature. After 5 min, the reaction
mixture was poured into water (20 mL), and the resulting
mixture was extracted with EtOAc (3 × 20 mL). The combined
extracts were washed with brine (2 × 15 mL), then dried with
Na2SO4. Concentration in vacuo gave 4-acetamido-3,6-dihydroxy-2-{[(1R,2S,4aS,8aS)-1,2,4a-trimethyl-5-methylenedecahydronaphthalen-1-yl]methyl}phenyl acetate (23) (30.2 mg). This
product was used for the next reaction without further
purification.
Triphenylphosphine (52.4 mg, 0.20 mmol) and carbon
tetrachloride (9.70 μL, 0.10 mmol) was added to a stirred solution of the crude product 23 (30.2 mg) in acetonitrile (2 mL) at
room temperature, and the reaction mixture was further
stirred at 65 °C for 1.5 h. After cooling to room temperature,
the reaction mixture was poured into 5% aqueous NaHCO3(20 mL), and the resulting mixture was extracted with EtOAc(3 × 20 mL). The combined extracts were washed with brine(2 × 10 mL), then dried with Na2SO4. The mixture was concentrated in vacuo, and the residue was purified by column chromatography (hexane/EtOAc, 1 : 1) to give an inseparable mixtureof 24 and 25 (ca. 2 : 3) (20.2 mg, 98%, 2 steps). 1H NMR(400 MHz, CDCl3): δ = 0.87 (dd, J = 11.7, 2.0 Hz, 2/5H),
0.89–0.93 (m, 3/5H), 0.91 (s, 6/5H), 0.93 (s, 9/5H), 1.02 (d, J =6.8 Hz, 6/5H), 1.03 (d, J = 6.8 Hz, 9/5H), 1.06 (s, 6/5H), 1.06 (s,9/5H), 1.21–1.58 (m, 14/5H), 1.21–1.58 (m, 21/5H), 1.89 (m,2/5H), 1.89 (m, 3/5H), 2.02–2.13 (m, 4/5H), 2.02–2.13 (m,6/5H), 2.27 (s, 6/5H), 2.34 (s, 9/5H), 2.24–2.34 (m, 2/5H),
2.24–2.34 (m, 3/5H), 2.55 (s, 6/5H), 2.56 (s, 9/5H), 2.68 (d, J =14.1 Hz, 2/5H), 2.77 (d, J = 14.1 Hz, 2/5H), 2.83 (d, J = 14.1 Hz,
3/5H), 2.91 (d, J = 14.1 Hz, 3/5H), 4.35 (s, 2/5H), 4.35 (s, 3/5H),
4.39 (s, 2/5H), 4.39 (s, 3/5H), 6.14 (br s, 2/5H), 7.11 (s, 2/5H),
7.27 (s, 3/5H) ppm. 13C NMR (100 MHz, CDCl3): δ = 14.4 (q,1C), 17.3 (q, 1C), 18.3 (q, 3/5C), 18.5 (q, 2/5C), 20.4 (q, 2/5C),
20.5 (q, 3/5C), 20.7 (q, 2/5C), 21.0 (q, 3/5C), 23.3 (t, 3/5C), 23.4
(t, 2/5C), 27.9 (t, 2/5C), 28.0 (t, 3/5C), 28.7 (t, 2/5C), 28.8 (t,3/5C), 32.8 (t, 2/5C), 32.9 (t, 3/5C), 34.8 (t, 3/5C), 35.5 (t, 2/5C),
36.2 (t, 2/5C, 2/5C), 36.3 (t, 3/5C), 37.1 (d, 2/5C), 37.4 (d, 3/5C),
40.4 (s, 1C), 42.8 (s, 3/5C), 43.0 (s, 2/5C), 49.5 (d, 1C), 102.8(t, 3/5C), 103.0 (t, 2/5C), 104.1 (d, 2/5C), 110.2 (d, 3/5C), 111.7(s, 3/5C), 116.9 (s, 2/5C), 132.8 (s, 3/5C), 136.2 (s, 3/5C), 137.2(s, 2/5C), 137.7 (s, 2/5C), 144.8 (s, 3/5C), 145.3 (s, 2/5C), 145.8(s, 2/5C), 159.8 (s, 2/5C), 149.6 (s, 3/5C), 159.5 (s, 3/5C), 163.2(s, 3/5C), 164.1 (s, 2/5C), 169.1 (s, 3/5C), 169.4 (s, 2/5C) ppm.
HRMS (EI): calcd for C25H33NO4 [M]+ 411.2410; found411.2407.

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Nakijinol G (7)2.5% Aqueous ammonia (0.10 mL, 0.15 mmol) was added to a
stirred solution of a mixture of 24 and 25 (ca. 2 : 3) (41.1 mg,
0.10 mmol) in MeOH (5.0 mL) at room temperature. After
10 min, the reaction was quenched with saturated aqueous
NH4Cl (20 mL) at 0 °C, and the resulting mixture was extractedwith CHCl3 (3 × 20 mL). The combined extracts were washedwith brine (2 × 15 mL), then dried with Na2SO4. The mixture
was concentrated in vacuo, and the residue was purified bycolumn chromatography (hexane/EtOAc, 1 : 1) to give 7(36.4 mg, 99%) as a colourless solid. Recrystallization from
CHCl3 gave an analytical sample of 7 as colourless prisms.
825
M.p. 266.0–266.5 °C. [α]25
D = −42.0 (c = 0.10, MeOH) {ref. [α]D =1
13−18.4 (c = 0.10, MeOH)}. The H and C NMR, IR, and MS
spectra (see below) are identical to those of natural nakijinol G(7).8 1H NMR (400 MHz, DMSO-d6): δ = 0.82 (d, J = 10.1 Hz,
1H), 0.87 (s, 3H), 1.08–1.12 (m, 2H), 1.01 (s, 3H), 1.02 (d, J =4.9 Hz, 3H), 1.14–1.17 (m, 2H), 1.30–1.38 (m, 5H), 1.40–1.44(m, 1H), 1.79–1.88 (m, 1H), 1.98–2.00 (m, 1H), 2.22–2.26 (m,1H), 2.25–2.28 (m, 1H), 2.45 (s, 3H), 2.71 (d, J = 14.4 Hz, 1H),
2.78 (d, J = 14.4 Hz, 1H), 4.29 (s, 1H), 4.35 (s, 1H), 6.83 (s, 1H)
ppm. 13C NMR (100 MHz, DMSO-d6): δ = 14.0 (q), 17.4 (q), 18.3(q), 20.2 (q), 22.8 (t), 27.5 (t), 28.1 (t), 32.4 (t), 34.2 (t), 36.2 (t),36.7 (d), 39.5 (s), 42.2 (d), 48.7 (d), 101.9 (d), 102.8 (d), 108.8(s), 131.5 (s), 142.5 (s), 143.4 (s), 145.1 (s), 159.3 (s), 160.8 (s)ppm. FTIR (neat): ν = 2922, 1731, 1456, 1382, 1321, 1054, 1032,
1011, 975, 880, 741, 690, 651, 639, 624 cm−1. HRMS (EI): calcd
for C23H31NO3 [M]+ 369.2304; found 369.2292.
Nakijinol E (5)Cesium carbonate (11.7 mg, 36 μmol) and dimethyl sulfate(3.10 μL, 33 μmol) were added to a stirred solution of 7
(11.1 mg, 30 μmol) in acetone (2.0 mL) at room temperature.
After 10 min, the reaction was quenched with saturated
aqueous NH4Cl (10 mL) at 0 °C, and the reaction mixture was
extracted with EtOAc (3 × 20 mL). The combined extracts were
washed with brine (2 × 10 mL), then dried with Na2SO4. The
mixture was concentrated in vacuo, and the residue was purified by column chromatography (CH2Cl2/EtOAc, 9 : 1) to give 5(10.2 mg, 89%) as a colourless amorphous solid. [α]25
D = −23.6(c = 0.11, MeOH) {ref.7 [α]25
=
−29.0
(c
=
0.10,
MeOH)}.
The 1HD
13
and C NMR, IR, and MS spectra (see below) are identical tothose of natural nakijinol E (5).7 1H NMR (400 MHz, CDCl3):
δ = 0.93 (dd, J = 11.9, 2.3 Hz, 1H), 0.93 (s, 3H), 1.06 (s, 3H),
1.07 (d, J = 5.9 Hz, 3H), 1.15–1.23 (m, 1H), 1.21–1.26 (m, 3H),
1.30–1.38 (m, 1H), 1.38–1.46 (m, 3H), 1.50–1.53 (m, 1H),
1.86–1.90 (m, 1H), 2.00–2.03 (m, 1H), 2.26–2.38 (m, 1H),
2.28–2.38 (m, 1H), 2.55 (s, 3H), 2.82 (d, J = 14.2 Hz, 1H), 2.91
(d, J = 14.2 Hz, 1H), 3.92 (s, 3H), 4.34 (s, 1H), 4.38 (s, 1H), 5.91
(s, 1H), 6.98 (s, 1H) ppm. 13C NMR (100 MHz, CDCl3): δ = 14.5(q), 17.5 (q), 18.3 (q), 20.5 (q), 23.2 (t), 28.0 (t), 28.7 (t), 33.0 (t),34.4 (t), 36.3 (t), 37.1 (d), 40.4 (s), 42.8 (s), 49.1 (d), 56.4 (q),98.7 (d), 102.4 (t), 109.0 (s), 132.3 (s), 143.4 (s), 144.3 (s), 146.3(s), 160.3 (s), 161.8 (s) ppm. FTIR (neat): ν = 3527, 3083, 2925,
2857, 1729, 1633, 1584, 1459, 1431, 1382, 1324, 1281, 1242,

1213, 1192, 1168, 1130, 1070, 1050, 1007, 991, 941, 889, 829,
756 cm−1. HRMS (EI): calcd for C24H33NO3 [M]+ 383.2460;
found 383.2462.

Conﬂicts of interest
There are no conflicts to declare.

Acknowledgements
This study was financially supported by a JSPS KAKENHI(Grant Number JP15K07865) and a Grant-in-Aid for theStrategic Research Foundation Program at Private Universities(Grant Number S15110010L) from MEXT.

Notes and references
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5 The epi-nomenclature was conveniently used for the newcompound 4 to indicate its cis-fused decalin ring moiety,even though the corresponding trans-fused isomer (i.e.
nakijinol D) has not been reported yet. See: ref. 6.
6 G. Daletos, N. J. de Voogd, W. E. G. Müller, V. Wray, W. Lin,D. Feger, M. Kubbutat, A. H. Aly and P. Proksch, J. Nat.
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15 The reducing reagent Na2S2O4 has been used most widely
for the conversion of sesquiterpenoid quinones to thecorresponding hydroquinones. See: ref. 11e–g.
16 This operation made it possible to carry out the full assignments for the 1H and 13C NMR spectra of the benzoxazole
product (i.e. 20) (see ESI†).
17 The use of K2CO3 instead of Cs2CO3 resulted in a lower
yield of product 6 (∼70%).
18 Interestingly, being diﬀerent from the case of a mixture of
19 and 20 (cf. Scheme 2), exposure of a mixture of 24 and
25 to triethylamine in MeCN could not produce 25 as asingle product, but only resulted in a change of the composition ratio from appropriately 2 : 3 to 1 : 3.

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— 101 —— 102 —— 103 —— 104 —— 105 —— 106 —— 107 —— 108 —— 109 —— 110 —— 111 —— 112 —— 113 —— 114 —— 115 —— 116 —— 117 —— 118 —— 119 —— 120 —— 121 —Glycosylation reactions mediated by hypervalent iodine:application to the synthesis of nucleosides and carbohydrates
Yuichi Yoshimura*1, Hideaki Wakamatsu1, Yoshihiro Natori1, Yukako Saito1
and Noriaki Minakawa2

ReviewOpen Access

Address:
1Faculty of Pharmaceutical Sciences, Tohoku Medical andPharmaceutical University, Komatsushima 4-4-1, Aoba-ku, Sendai,
981-8558, Japan and 2Graduate School of Pharmaceutical Science,
Tokushima University, Shomachi 1-78-1, Tokushima, 770-8505,
Japan

Beilstein J. Org. Chem. 2018, 14, 1595–1618.
doi:10.3762/bjoc.14.137

Email:Yuichi Yoshimura* - yoshimura@tohoku-mpu.ac.jp

This article is part of the Thematic Series "Hypervalent iodine chemistry inorganic synthesis".

* Corresponding author

Guest Editor: T. Wirth

Keywords:
glycosylation; hypervalent iodine; Lewis acid; nucleoside;oligosaccharide© 2018 Yoshimura et al.; licensee Beilstein-Institut.
License and terms: see end of document.

Received: 02 February 2018
Accepted: 15 June 2018
Published: 28 June 2018

Abstract

To synthesize nucleoside and oligosaccharide derivatives, we often use a glycosylation reaction to form a glycoside bond. Coupling reactions between a nucleobase and a sugar donor in the former case, and the reaction between an acceptor and a sugar donorof in the latter are carried out in the presence of an appropriate activator. As an activator of the glycosylation, a combination of a
Lewis acid catalyst and a hypervalent iodine was developed for synthesizing 4’-thionucleosides, which could be applied for the synthesis of 4’-selenonucleosides as well. The extension of hypervalent iodine-mediated glycosylation allowed us to couple a nucleobase with cyclic allylsilanes and glycal derivatives to yield carbocyclic nucleosides and 2’,3’-unsaturated nucleosides, respectively.
In addition, the combination of hypervalent iodine and Lewis acid could be used for the glycosylation of glycals and thioglycosides
to produce disaccharides. In this paper, we review the use of hypervalent iodine-mediated glycosylation reactions for the synthesisof nucleosides and oligosaccharide derivatives.

Introduction

Nucleic acids and oligosaccharides are both mandatory polymers for the maintenance of life and cell growth. The former
exists in nuclei and codes genetic information, which is transformed into proteins through a transcription process known as
the “central dogma” (i.e., DNA makes RNA makes proteins).

The latter make up the cell walls of microorganisms and also
play a role in transmitting information on the cell surface,
whose interactions with proteins are a starting point for signaltransduction into cells [1]. Since both types of polymers are
essential for cell viability, their biological synthesis, including
1595— 122 —

Beilstein J. Org. Chem. 2018, 14, 1595–1618.

the synthesis of their monomer units, e.g., nucleotides, is highly
regulated. Damage to these vital molecules often results incongenital disease with ultimately fatal consequences [2,3]. Accordingly, the study of polymers and their biosynthesis is quite
important, and informs the development of new drugs fordiseases including cancers and infectious diseases caused byviruses [4-7]. Indeed, many drugs related to nucleic acids andoligosaccharides have been developed and used in clinicalfields. Synthetic chemists have contributed to the studies bysupplying biological tools for the analyses of these polymers,
as well as by synthesizing effective drug candidates for the
diseases mentioned above [4,8-14].
To synthesize nucleoside and oligosaccharide derivatives,
glycosylation reactions are often used to form a glycoside bond.
In the case of nucleoside synthesis, a coupling reaction between a persilylated nucleobase and a sugar donor is typicallyused [15-17]. On the other hand, the reaction between an
acceptor and sugar donor is carried out in the presence of an
appropriate activator for oligosaccharide synthesis [18,19]. In
both cases, a Lewis acid is generally used as an activator forsugar donors. Our previous review focused on the developmentof glycosylation reactions and their application to the synthesisof nucleoside derivatives [17]. In this review, we showed our
glycosylation reactions under oxidative conditions. These werequite useful and the conceptually similar reactions were widelyused for synthesizing nucleoside derivatives. Recently, a combination of a Lewis acid catalyst and hypervalent iodine was developed for synthesizing 4’-thionucleosides, which was basedon a Pummerer-type reaction coupled with oxidation. The
concept of the oxidative glycosylation reaction was successfully applied to the synthesis of other nucleoside derivatives, including 4’-selenonucleosides and carbocyclic nucleosides. The
hypervalent iodine-mediated glycosylation has also been used
for oligosaccharide synthesis employing glycals and thioglycosides as sugar donors. In this review, we survey the synthesis of
nucleoside and disaccharide derivatives under oxidative conditions mostly based on the hypervalent iodine-mediated glycosylation reactions.

ReviewSynthesis of 4’-thionucleosides
Over the last decade, we have steadily pursued the identification of novel antitumor and antiviral nucleoside derivatives[17,20-22]. Matsuda and co-workers reported a 2’-substituted
cytidine derivative, DMDC (1), with potent antitumor activity[23,24]. In other reports, Walker [25] and Secrist [26] independently described the potent antiherpesvirus activity of 2’-deoxy4’-thionucleoside 2, in which sulfur was introduced in place ofthe sugar ring oxygen of 2’-deoxynucleoside. The results for
2’-substituted nucleosides and 2’-deoxy-4’-thionucleosides
strongly suggested that 2’-substituted 4’-thionucleosides would
be promising candidates for novel antitumor agents. Thus, we
designed a novel 2’-substituted 4’-thiocytidine, 4’-thioDMDC(3), as our target molecule for potential antitumor agents[27,28] (Figure 1).
At the time we started our project, there had been no reports
regarding the synthesis of even 2-substituted 4-thiosugar derivatives. We thus developed the first synthetic route accessing the
4-thiosugar derivative by way of bicyclic intermediate 8 fromdiacetoneglucose (5). Construction of the bicyclic ring of 8 wasachieved by consecutive inter-/intramolecular SN2 reactions of
the dimesylate derivative 7 obtained by manipulations of 5.
After acetal hydrolysis and the subsequent hydride reduction,
4-thioarabinose derivative 9 was obtained in good yield. Introduction of a TBDPS group at the primary hydroxy group of 9,oxidation and Wittig reaction, followed by deprotection of the
benzyl group, gave allyl alcohol 11.
The most popular method to form a glycosyl bond between thesugar moiety and the base of a nucleoside is a Vorbrüggen reaction [15,16], in which a silylated base and sugar donor, e.g.,1-acetoxy sugar, are condensed by a Lewis acid catalyst. It was
clear that this reaction could also be used in the synthesis of4’-thionucleosides as well as normal “4’-oxy” nucleosides.
However, for reasons which will be described later, we decided
to develop an alternative method to build the glycosyl bond of
4’-thionucleosides by using a direct coupling of a 4-thiosugar

Figure 1: Design of potential antineoplastic nucleosides.

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sulfoxide and a silylated base under sila-Pummerer conditions[29,30]. We found that treatment of 12, obtained by oxidationof 11, with excess persilylated N4-acetylcytosine in the presence of TMSOTf as a Lewis acid gave an inseparable mixtureof α- and β-anomers of 4’-thioDMDC derivatives 15 in goodyield. Based on the study of the sila-Pummerer reaction by Kita,
it was plausible that the reaction proceeded via the formation ofsulfenium ion 14 which was formed by β-elimination of silylated sulfoxide 13. The 4’-thioDMDC derivative 15 was deprotected and the resulting anomeric mixture was separated tofurnish 4’-thioDMDC (3) and its α-anomer [27,28] (Scheme 1).
After we reported the synthesis of 4’-thioDMDC using a
Pummerer-type glycosylation reaction, Minakawa and Matsuda
applied the reaction to the syntheses of 4’-thioribonucleosides.
Applying the synthetic scheme of 2’-deoxy-4’-thionucleoside
by Walker to a ribo derivative, 2-dimethoxybenzoate 20 was
prepared from tribenzylated ribose 16. Introduction of adimethoxybenzoyl (DMBz) group at the 2-position and diastereoselective formation of sulfoxide 20, favored in Pummerertype glycosylation reactions and cases where the approach of
the nucleophile is restricted, were the key strategies for theirsynthesis of 4’-thioribonucleosides. Under optimized conditions, the desired 4’-thiouridine derivative 21 was the sole product and it was obtained in excellent yield (Scheme 2). Using themethod developed, they succeeded in preparing all four kinds of4’-thioribonucleosides [31].

We also synthesized 4’-thioribonucleosides constructing theskeleton of the 4-thioribose via a ring-contraction reaction
under reductive conditions [32] from 2-mesylate 23, which was
obtained from 22. As shown in Scheme 3, the reaction firststarted to form an episulfonium ion 24 triggered by intramolecular SN2 reaction at the 5-position by sulfur atom. Secondary,ring contraction from thiopyranose to thiofuranose occurred to
produce 5-aldehyde 26. Finally, hydride reduction of 26 gavethe 4-thiofuranose derivative 27. The Pummerer-type glycosylation reaction of 5-O-silylated sulfoxide 28, by treating with 2,4bis(trimethylsilyl)uracil (29) and excess diisopropylethylamine(DIPEA) in the presence of TMSOTf, gave 4’-thiouridine
derivative 30 in a good yield. The reaction stereoselectively
proceeded and resulted the predominant formation of the
β-anomer due to steric hindrance of the 2,3-di-O-isopropylidene group.
Before our reports regarding the Pummerer-type glycosylation,
the synthesis of 4’-thionucleosides was based on the known
chemistry: typically, a 1-acetoxy-4-thiosugar or its synthetic
equivalent was obtained from natural sugars and subjected to
the Vorbrüggen reaction as in the case of 2’-deoxy-4’-thionucleosides [25,26]. When synthesizing 4’-thionucleosides by theway of a sulfide derivative 31, the known chemistry should lead
us to use a classical Pummerer reaction to produce 1-acetoxy
derivative 33 after converting 31 to the corresponding sulfoxide 32. Even though this scheme should be promising

Scheme 1: Synthesis of 4’-thioDMDC.

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Scheme 2: Synthesis of 4’-thioribonucleosides by Minakawa and Matsuda.

Scheme 3: Synthesis of 4’-thioribonucleosides by Yoshimura.

enough, we intended to introduce an additional synthetic idea
based on the fact that both of the reaction intermediate of the
Vorbrüggen reaction [15,16] of 33 and the sila-Pummerer reaction developed by Kita [29,30] involving sulfoxide 32 would be
the same sulfenium ion 34. This new glycosylation reaction was
unique and attractive since it was capable of skipping a step. In
other words, the reaction could directly access sulfenium ion 34
from sulfoxide 32. Thus we developed the Pummerer-type
glycosylation as mentioned above. From these results it can be
deduced that the expected sulfenium ion had formed and thatthe concept of the Pummerer-type glycosylation was actuallyeffective for the formation of the glycosyl bond of 4’-thionucleosides. After we had reported our synthesis of 4’-thioDMDC,
the method was widely adopted for the synthesis of 4’-thionucleoside derivatives by other groups and became a standard approach for the glycosylation [33-37]. On the other hand, theconversion from the sulfide to 4’-thionucleoside using thePummerer-type glycosylation included an oxidation step. If theoxidation of sulfide 31 and the Pummerer-type glycosylation of
the sulfoxide 32 could be performed in the same flask, the reaction could bypass two of the reaction steps and would directly
produce 4’-thionucleoside 35 from 31. Indeed, the utilization of
hypervalent iodine would have enabled this short-cut reaction(Figure 2).
Hypervalent iodine reagents have been widely used in organic
synthesis [38]. Although originally used as oxidative agents,
their use has spread to coupling reactions, including those forthe formation of C–C bonds [39-43]. In the case of C–N bondformation, introduction of an azido group using PhI=O andTMSN3 was reported by Kita and co-workers [44]. Their paper
prompted Nishizono et al. to study the glycosylation reaction
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Figure 2: Concept of the Pummerer-type glycosylation and hypervalent iodine-mediated glycosylation.

for 4’-thionucleosides using hypervalent iodine reagents. As a4-thiosugar donor, 2-p-methoxybenzoate derivative 36 was prepared following Matsuda’s method as shown in Scheme 2, andthen was subjected to the Pummerer-type glycosylation mediated by hypervalent iodine. Treatment of 36 with bis(trifluoroacetoxy)iodobenzene (PIFA) and uracil in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) and triethylamine gave a 5:1 mixture of 4’-thiouridine derivative 37 in 55%
yield. The reaction of 36 with iodosylbenzene (PhI=O)
proceeded stereoselectively and gave only the β-anomer of 37
in 53% yield [45] (Scheme 4).
The mechanism of hypervalent iodine-mediated glycosylation
can be expressed as shown in Figure 3. The activated hypervalent iodine reagents in the presence of TMSOTf reacted a sulfur
atom of 36 to give 38, in which elimination of iodobenzene
and HX might subsequently occur to generate a sulfenium ion
40 (path a). The nucleophilic attack of the silylated base to
the sulfenium ion 40 favored approaching from the β-face togive only the β-anomer 37 as in the case of Minakawa andMatsuda’s synthesis described above.

Nishizono considered that the difference between the stereoselectivities of the coupling reactions in methods A and B wascaused by the existence of another reaction path of the sulfonium salt (38 or 39). In path b, the 4-thiosugar 41 was generated and reacted with a nucleobase, giving a mixture of α- andβ-anomers since the reaction might occur by the simple SN2reaction. Thus, the reaction proceeded through both paths a andb in method A, but path a was predominant in the reaction of
method B [45] (Figure 3).
Nishizono et al. applied the hypervalent iodine-mediated glycosylation to purine 4’-thionucleosides [46]. However, the reaction of 36 with 6-chloropurine resulted in the formation of aregioisomer reacting at the 4-position without any formation of
the desired purine 4’-thionucleoside. The result should relate to
the acidity of the α hydrogen adjacent to a sulfur atom, which
affects the regioselectivity of the reaction. To study the effects
of a protecting group on the reaction, the regioselectivity of thereaction was examined using 42 and 43, which were obtained
from 27. When the 5-hydroxy group was protected with abenzoyl group, the coupling reaction of 42 occurred at the 4-po-

Scheme 4: Oxidative glycosylation of 4-thioribose mediated by hypervalent iodine.

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Figure 3: Speculated mechanism of oxidative glycosylation mediated by hypervalent iodine.

sition, as in the case mentioned above, to give 46 in 44% yieldalong with the desired product and its N7 isomer. In contrast,
switching the protecting group of 27 at the 5-position to TBS
resulted in the exclusive formation of 45 reacted at the 1-position (28%) along with the N7 stereoisomer (10%). These results
support the above-mentioned hypothesis. Finally, 4'-thioadenosine (49) was synthesized by treating 45 with TFA followed bymethanolic ammonia [46] (Scheme 5).

The same group attempted to apply the oxidative coupling reaction to the synthesis of thietane nucleosides [47]. The substrateof the coupling reaction was prepared as shown in Scheme 6
starting from benzyloxyacetaldehyde (50). When a hypervalentiodine reagent was used for glycosylation with a diastereomericmixture of sulfide 53, the reaction stereoselectively gave thering-expanded nucleoside 54 in 30% yield, but did not give thedesired thietane nucleoside at all (Scheme 6).

Scheme 5: Synthesis of purine 4’-thioribonucleosides using hypervalent iodine-mediated glycosylation.

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Scheme 6: Unexpected glycosylation of a thietanose derivative.

Considered that the ring-expansion occurred in the absence ofthe hypervalent iodine reagent, the Nishizono and co-workers
speculated that the reaction mechanism was as shown in
Scheme 7. First, the Lewis acid catalyzed the intramolecular
SN2 reaction of sulfur to form the epi-sulfonium ion 55, which
proceeded only from the cis-isomer due to the steric requirement. The subsequent nucleophilic attack leaving the benzoate
anion resulted in the formation of a ring-expanded product 56,
which became a substrate of the hypervalent iodine-mediatedglycosylation. As a result, 4’-thiofurano nucleoside 54 wasstereoselectively obtained with the assistance of the neighboring benzoyl group as in 58.
The desired thietanonucleosides 62 and 63 with an anomerichydroxymethyl group were synthesized by the Pummerer-type
glycosylation reaction of trans-cyclobutane sulfoxide 59. The
authors concluded that the stereochemistry of the sulfoxide andthe nature of the protecting groups had no significant effect on
the yield of the Pummerer-type glycosylation [47] (Scheme 8).

Pummerer-type glycosylation, which was developed by ourgroup, improved the synthesis of 4’-thionucleosides. It greatly
contributed to search new biological active nucleoside derivatives. The use of hypervalent iodine reagents helped to further
improve their synthesis by saving reaction steps to improve synthetic efficiency.

Synthesis of 4’-selenonucleosides
The unique biological activity of 4’-thionucleosides triggered
the synthesis of their chalcogen isosters, 4’-selenonucleosides,
the activity of which were reported. The first synthesis of
4’-selenonucleosides was reported by Jeong and co-workers in2008 [48,49].
As in the case of the 4’-thioribonucleoside described in
Scheme 3, Jeong et al. chose a 2,3-di-O-isopropylidene-protected intermediate as a donor of glycosylation, which was synthesized based on their method developed for 4’-thionucleosides. Starting from compound 64, which was obtained from

Scheme 7: Speculated mechanism of the ring expansion of a thietanose derivative.

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Scheme 8: Synthesis of thietanonucleosides using the Pummerer-type glycosylation.

D-gulonic γ-lactone, dimesylate 66 was prepared. The consecutive inter-/intramolecular SN2 reactions of 66 by selenide anion
gave a 4-seleno sugar 67 in an excellent yield. After converting
67 to the corresponding selenoxide, the resulting 68 was immediately treated with uracil or N4-benzoylcytosine under the same
conditions for Pummerer-type glycosylation to give the desired
4’-selenouridine and 4’-selenocytidine derivatives in moderate
yields. Deprotection of the nucleoside derivatives afforded
4’-selenouridine and 4’-selenocytidine, respectively [48](Scheme 9). In the year in which the first synthesis of
4’-selenonucleoside was reported, Jayakanthan et al. used thesame strategy to synthesize 4’-selenonucleosides, including
4’-selenoadenosine [50].
After successful application of the Pummerer-type glycosylation to the synthesis of 4’-selenonucleosides, Jeong’s group reported various 4’-selenonucleoside derivatives by using thesame method [51-58]. Minakawa and his group attempted tosynthesize 4’-selenonucleosides based on their method de-scribed in Scheme 2 [59]. However, the Pummerer-type glycosylation of selenoxide 74 obtained from 73 gave the desired
4’-selenonucleoside in low yield along with the formation ofdiselenide 76 and deoxygenated 73 (Scheme 10). One of thereasons for the unsatisfactory result was the instability of
selenoxide 74. Jeong et al. faced the same problem andsuppressed decomposition by the immediate reaction after
synthesizing the corresponding selenoxide [48].
To overcome these problems, Minakawa decided to use hypervalent iodine for the glycosylation reaction [59] as in Nishizono’s synthesis of 4’-thionucleosides [45]. First, they optimized the reaction conditions by examining the reaction of 73
with uracil in the presence of hypervalent iodine reagents. Noneof the desired pyrimidine nucleoside 75 was formed when thereaction was performed by treatment with iodosylbenzene,
TMSOTf and triethylamine in the presence of the silylated
uracil (Table 1, entry 1). Instead of trimethylamine, 2,6-lutidine
was employed to give 75 in 48% yield together with selenoxide

Scheme 9: First synthesis of 4’-selenonucleosides.

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Scheme 10: The Pummerer-type glycosylation of 4-selenoxide 74.

74 (20%) and starting 73 (8%) (Table 1, entry 2). The use of
more reactive hypervalent iodine agents (PIFA and diacetoxyiodobenzene) did not improve the chemical yield of 75 (Table 1, entries 3 and 4). When 73 was treated with iodosylbenzene, TMSOTf, 2,6-lutidine and the silylated uracil in dichloroethane at 50 °C, the reaction gave 75 in 64% yield while suppressing the formation of 74 (Table 1, entry 5).
Minakawa’s group attempted to apply the aforementioned reaction to the synthesis of purine derivatives [60]. Based on thereports by Jeong et al., who synthesized 4’-selenoadenosineusing the Vorbrüggen reaction [53], they conceived that thehypervalent iodine-mediated reaction of “disarmed” sugar
donor 73 bearing an electron-withdrawing group at the 2-position would not readily yield the desired purine derivative.
Therefore, they decided to use “armed” seleno sugar 67 as adonor for the hypervalent iodine-mediated glycosylation reaction as in Jeong’s synthesis.

The reaction of 67 was performed by treating with silylated
6-chloropurine, iodosylbenzene, TMSOTf and 2,6-lutidine indichloroethane at 85 °C for 2.5 h to give the desired N9-isomer
78 in 39% yield along with the formation of the N7-isomer 77(31%) and the α-isomer (8%, N7/N9 mixture). On the other
hand, consumption of 67 required longer times and subsequent
isomerization to 78 was insufficient at 50 °C, giving 78 in 31%
yield with the predominant formation of 77 (40%). The separated N7 isomer 77 was successfully isomerized to the desired N9
isomer 78 in 53% yield upon treatment with TMSOTf in toluene at 90 °C. Under similar conditions, the hypervalent iodinemediated glycosylation reaction of 67 in the presence of 2,6dichloropurine was conducted. The coupling reaction proceeded
to give an inseparable mixture of N7-isomer 80 and N9-isomer
81 in 64% yield (80:81 = 1:1). To isomerize the undesired
N7-isomer to the desired product as in the case of 2,6-dichloropurine, the subsequent treatment of the resulting mixture withTMSOTf in toluene at 90 °C gave rise to exclusive formation of

Table 1: The Pummerer-like glycosylation reaction mediated by hypervalent iodine.

Conditions

Yield (%)

Entry

Hypervalent iodine (1.2 equiv)

Base (8 equiv) Solvent

Temp (°C)

Time (h)

75

74

73

1
2
3
4
5

PhIO
PhIO
PhI(OCOCF3)2
PhI(OAc)2
PhIO

Et3N
2,6-lutidine
2,6-lutidine
2,6-lutidine
2,6-lutidine

0
rt
rt
rt
50

4.5
17
3
5
1.5

0
48
38
25
64

0
20
0
0
0

33
8
40
20
13

CH2Cl2CH2Cl2CH2Cl2CH2Cl2
ClCH2CH2Cl

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the desired N9-isomer 81 in 62% yield. Finally, 81 was converted to the desired guanosine derivative 82 [60] (Scheme 11).
As in the case of 4’-thionucleosides, the use of hypervalentiodine greatly improved the glycosylation reaction with 4-seleosugars by skipping the preparation of unstable selenoxide derivatives.

Synthesis of carbocyclic nucleosides
As described above, in the hypervalent iodine-mediated glycosylation, a thiosugar donor 83 was oxidized to a cationic intermediate 84 with the assistance of a Lewis acid (TMSOTf) and abase and the subsequent nucleophilic attack of silylated base to

84 gave the desired nucleoside 85. The success of the hypervalent iodine-mediated glycosylation led us to apply the reaction
to the synthesis of carbocyclic nucleosides. In addition, we werealso encouraged by the study of Ochiai, who developed theFriedel–Crafts reaction via umpolung of allylsilanes using
hypervalent-iodine reagents [61] and the pioneering work on
C–N bond formation using hypervalent iodine by Kita [62].
Thus, we envisioned the use of allylsilanes as a pseudosugar
donor for the synthesis of carbocyclic nucleosides. We expected to couple a cyclic allylsilane 86, which could act as a
pseudosugar donor for carbocyclic nucleosides 88, with a persilylated nucleobase by using a combination of hypervalentiodine and an appropriate Lewis acid (Figure 4).

Scheme 11: Synthesis of purine 4’-selenonucleosides using hypervalent iodine-mediated glycosylation.

Figure 4: Concept of the oxidative coupling reaction applicable to the synthesis of carbocyclic nucleosides.

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As shown in Scheme 12, an oxidative coupling reaction was examined using a model reaction [63]. Cycloalkenylsilanes 89a,b
and 90a,b were prepared by hydrosilylation of cyclopentadieneand cyclohexadiene. Using TMSOTf as a Lewis acid, the hypervalent iodine-mediated coupling reaction of 89a,b and 90a,bwith silylated uracil 29 was examined and the results are
summarized in Table 2. Our first attempt to couple triethoxysilanes 89a,b with 29 in the presence of diacetoxyiodobenzene
gave cycloalkenyluracil 91a and 91b in 45% and 49% yields respectively (Table 2, entries 1 and 2). On the other hand, the useof trialkylsilanes 90a and 90b successfully improved the chemical yield of 91a and 91b (Table 2, entries 3 and 4). In contrast,
the reactions using PIFA, iodosylbenzene, and [hydroxyl(tosyloxy)iodo]benzene (PhI(OH)OTs) resulted in a decrease of thereaction yield (Table 2, entries 5–7).

trimethylsilylbutadiene 92 and dimethyl fumarate (93), whichgave cyclohexene diester 94 (1:1 mixture of diastereomers)[64]. Reduction and subsequent separation by silica gel column
chromatography gave diols 95a and 95b, the hydroxy groups ofwhich were protected to give di-TBDPS derivatives 96a and96b. The resulting cyclohexenylsilanes 96a and 96b were subjected to the oxidative coupling reaction with 2,4-bis(trimethylsilyl)uracil (29) using diacetoxyiodobenzene, respectively, andthe results are shown in Table 3. The reaction of 96a gave an
inseparable mixture containing 4 stereoisomers of 97a–d with aratio of 6:10:2:1.5, which was determined based on the analysis
of its 1H NMR spectrum. The reaction of 96b also gave a similar result. In both reactions, the formation of cyclohexadiene 98
was observed. These results strongly supported that the reaction proceeded through the carbocation intermediate, as expected and depicted in Figure 4, since 98 was considered to beformed by E1 elimination of the allyl cation intermediate. The
fact that 96a and 96b showed different reactivities could be explained by the steric interaction between the substituents on thecyclohexene ring and the nucleobase approaching. Compounds
97a–d were converted to the corresponding cytosine analogues[63]. During the course of conversion, all the stereoisomers
were separated. Among them, only the cytosine derivative 99
showed weak anti-HIV activity (Scheme 13 and Table 3).
Table 3: Summary of the oxidative coupling reactions of 96a and 96b.

Scheme 12: Oxidative coupling reaction mediated by hypervalentiodine.

comp

Table 2: Summary of the oxidative coupling reaction using hypervalent iodine.

entry

comp

I(III)

Time (h)yield (%)

1
2
3
4
5
6
7

89a
89b
90a
90b
90b
90b
90b

PhI(OAc)2
PhI(OAc)2
PhI(OAc)2
PhI(OAc)2
PhI(O2CCF3)2
PhIO
PhI(OH)OTs

15
15
1
1
1
1
1

91a: 45
91b: 49
91a: 65
91b: 65
91b: 55
91b: 57
91b: 29

96a
96b

time

1h
24 h

yield (%)

ratio

97a–d 98

recov. 97a:97b:97c:97d

60
50

0
20

18
11

6:10:2.0:1.5
3:10:2.5:0.5

An oxidative coupling reaction for synthesizing carbocyclic
nucleosides mediated by hypervalent iodine was developed.
Since the Friedel–Crafts type reaction involved carbocationintermediate, the reaction always gave a mixture of products.
Unfortunately, the reaction was not efficient. However, it isworthy that the oxidative coupling reaction contains a novel
type of C–N bond formation and would help to synthesize newcarbocyclic nucleosides.

To prove the usefulness of the oxidative coupling reaction
mediated by hypervalent iodine, the reaction was applied to thesynthesis of a carbocyclic nucleoside derivative designed as apotential anti-HIV agent.
As a target, cyclohexenylcytosine 99 was designed and wasplanned to synthesize using the oxidative coupling reaction. To
prepare the substrate of the coupling reaction, cyclohexenylsilane 96 was synthesized using the Diels–Alder reaction of

Synthesis of dihydropyranonucleosides
The success of the oxidative coupling reaction for constructing
a carbocyclic nucleoside skeleton led us to develop a glycosylation reaction applicable to glycal derivatives. Since an electronrich enol ether unit of glycal could react with oxidative agents,
it was expected to form a cationic intermediate as in the case ofallylsilanes described above. A direct coupling of glycals with
nucleobases is challenging, since it is formally a C–N bond1605— 132 —

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Scheme 13: Synthesis of cyclohexenyl nucleosides using an oxidative coupling reaction.

forming reaction with cleaving of the inactive C–H bond at theγ-position. Actually, the C–N bond-forming reactions using
hypervalent iodine agents have attracted much attention [62,6568]. In the case of the hypervalent iodine-catalyzed coupling
reaction with allylsilanes (Figure 5), the reaction involves thefollowing 2 steps: 1) the generation of allyl cation 87 by the oxidation of an allylsilane 86 with PhI(OAc)2 and TMSOTf, and2) the subsequent nucleophilic attack of the persilylated base to
87 as shown in Figure 5. Therefore, we expected that subjecting

Figure 5: Concept of the oxidative coupling reaction of glycal derivatives.

the electron-rich glycal 100 to the hypervalent iodine-mediatedreaction described above would generate an oxocarbenium ion
101 to serve as an intermediate, giving a nucleoside 102.
First, we attempted model reactions of the oxidative coupling toenol ether using a TMSOTf/PhI(OAc)2 system. After several
attempts, we found that the reaction of 3,4-dihydro-2H-pyran(DHP, 103) with PhI(OAc)2 and TMSOTf, starting at −40 °C
and then gradually raised to room temperature, gave a dihydropyranyluracil derivative 104 in 31% yield [69]. We also
found that when Cu(OTf)2 was used as a catalyst in place of
TMSOTf, the reaction gave 104 in 24% yield (Scheme 14).
We speculated that the mechanism of the oxidative coupling
reaction was as shown in Scheme 15. DHP (103) was reactedwith PhI(OAc)2 to produce an acetoxyiodobenzene derivative
105 with the assistance of TMSOTf. With respect to the pathway from the intermediate 105 to the N1-substituted uracil 104,
there were two plausible routes. In path a, a nucleophilic attackof 2,4-bis(trimethylsilyl)uracil (29) occurs prior to an elimination. In path b, on the other hand, an allylic carbocation 110
formed from 108 reacts with 29. From the result that the reaction of 2,3-dihydrofuran gave side products generated from an
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Scheme 14: Oxidative coupling reaction of silylated uracil and DHP using hypervalent iodine.

intermediate resembling 107 (data not shown), it was strongly
suggested that the oxidative coupling reaction preferred path arather than path b (Scheme 15).
Because further optimization of the oxidative coupling reaction
was not successful, we decided to examine the effect of addinga co-catalyst. The speculated reaction mechanism depicted in
Scheme 15 suggested that the instability of the intermediates
105 and 106 might have caused the low yield of the oxidative
coupling. Based on this idea, we intended to use (PhSe)2 as a
co-catalyst, since it might prevent the formation of unstable 105
and 106 and yield 102 in one step (Figure 6).

We examined the effect of (PhSe)2 as an additive by the reaction of various glycals and their chemical equivalents [69] andthe results are summarized in Table 4. The reaction of 103 and29 was performed by treatment with PhI(OAc)2 and (PhSe)2 in
the presence of catalytic amounts of TMSOTf to selectively
yield a trans-isomer of 1-(3-phenylselanyltetrahydropyran-2yl)uracil (116) in 73% yield (Table 4, entry 1). Although thisresult was unexpected, it was important, since the reaction
appeared to be applicable to access various nucleoside derivatives, including 2’-deoxynucleosides. More importantly, we
could avoid the use of unstable reagents such as PhSeBr. In
other words, the reaction using hypervalent iodine and stable

Scheme 15: Proposed mechanism of the oxidative coupling reaction mediated by hypervalent iodine.

Figure 6: Synthesis of 2’,3’-unsaturated nucleosides using hypervalent iodine and a co-catalyst.

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(PhSe)2 in the presence of a Lewis acid would be expected toyield the same products as the reaction using PhSeBr. The reaction with dihydrofuran (112) furnished 1-(3-phenylselanyltetrahydrofuran-2-yl)uracil (117) in 31% yield (Table 4, entry 2).
The reaction of 113 with 29 at −5 °C afforded 118 in 69% yield(Table 4, entry 3). The reaction of 114 gave an anomeric mixture of 119 in 80% yield with the predominant formation of the
β-nucleoside (Table 4, entry 4). In contrast, the oxidative glycosylation reaction of D-glucal 115 gave a 1:1 mixture of α-120and β-120 in 64% yield (Table 4, entry 5). From these data, theoxidative coupling reaction mediated by hypervalent iodine ofglycal derivatives can clearly be regarded as a new glycosylation reaction that is applicable to the synthesis of 2’-deoxy- and2’,3’-dideoxydidehydronucleosides, some of which are known
to have anti-HIV activity (Table 4).
To reveal the scope of this reaction, we designed a new dihydropyranonucleoside as a potential anti-HIV agent andattempted to synthesize it by using the oxidative coupling reaction [70]. First, the PMB-protected epoxide 121 was converted
to diene 122. The dihydropyran ring of 123 was constructed by
RCM of 122 catalyzed by a Grubbs 1st generation catalyst. The

Table 4: Summary of the oxidative coupling reaction of bis(trimethylsilyl)uracil 29 with enol ethers using the TMSOTf/PhI(OAc)2/(PhSe)2 system.

entry

enol ether

product

1

yield (%)

73
103
116

2

31
112
117

3

69

113
118

80
(α:β=1:2)

4
114

1608

— 135 —

Beilstein J. Org. Chem. 2018, 14, 1595–1618.

Table 4: Summary of the oxidative coupling reaction of bis(trimethylsilyl)uracil 29 with enol ethers using the TMSOTf/PhI(OAc)2/(PhSe)2 system.
(continued)64(α:β=1:1)

5
115

isomerization of the double bond in 123 by treatment with aWilkinson catalyst under basic conditions afforded glycal 124(Scheme 16).
The hypervalent iodine-mediated glycosylation of 2,4-bis(trimethylsilyl)uracil (29) with glycal 124 gave an inseparable mixture of α- and β-anomers 125 (α:β = 1:2) in 51% yield as we expected. Compound 125 was then oxidized by treatment with
mCPBA, followed by elimination of the resulting selenoxide togive 126. After the separation of anomers, the major β-anomer
was converted into a cytosine derivative 127 [70]. However,
127 did not show any activity against HIV whereas its 5’-thiocounterpart did show anti-HIV activity (Scheme 16).
The reaction mediated by hypervalent iodine provides an alternative method for constructing glycosidic bonds of nucleosidederivatives by using a glycal as sugar donor. Its usefulness wasproved by applying the reaction to synthesize new nucleosidederivatives as mentioned above.

Synthesis of acyclic nucleosides
It is known that the oxidative C–C bond cleavage of glycols,
epoxides, and olefins takes place by the action of hypervalentiodine [38,71,72]. For example, Havare and Plattner reported
the oxidative cleavage of α-aryl aldehydes using iodosylbenzene to give chain-shortened carbonyl compounds and formaldehyde [71]. In the field of carbohydrate chemistry, similar
deformylation by action of hypervalent iodine has also been
demonstrated: the β-fragmentation reaction of an anomericalkoxy radical of carbohydrates was mediated by a hypervalentiodine reagent [73]. The reaction results in the formation ofcarbohydrates with a reduction of one carbon. From the viewpoint of the synthetic method, the reaction would be useful for
dehomologation of aldoses and preparation of chiral synthons

Scheme 16: Synthesis of dihydropyranonucleoside.

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Beilstein J. Org. Chem. 2018, 14, 1595–1618.

deriving from sugars. The reaction procedure involves theinitial formation of an alkoxy anomeric radical by a hypervalent iodine reagent in the presence of iodine, which triggers the
β-fragmentation of the C1–C2 bond. As a result, a C2 radical isgenerated and is further oxidized to a carbocation that is reactedwith nucleophilic agents to give the desired products.
Boto et al. applied the reaction to the one-pot synthesis ofacyclic nucleosides that belong to an important class of nucleosides with antiviral activity [74]. First, they tried to synthesize
acyclic nucleosides in a stepwise manner. The substrates 128
and 129 for the fragmentation reaction were synthesized fromribose in a few steps by the conventional method. The oxidative scission of 128 and 129 was carried out by treatment withdiacetoxyiodobenzene and iodine under irradiation with visible
light to give acetoxy acetals 130 and 131 in good yields withhigh stereoselectivities. As shown in Scheme 17, the reaction
was expected to proceed via the formation of anomeric alkoxyl
radicals, which underwent fragmentation to produce radical
132. The radical 132 could be trapped with iodine, giving iodide
133. The oxycarbenium ion 134 generated by the extrusion ofiodide from 133 reacted with the acetoxy ion to furnish the resulting acetate derivatives. The acetates 130 and 131 were thentreated with silylated thymine or N 4-benzoylcytosine in thepresence of a Lewis acid to give the desired acyclic nucleosides
135 and 136 in excellent yields. The results revealed that thenucleophilic attack of the nucleobase selectively occurred fromthe less hindered side of the oxycarbenium ion intermediates,
giving 1’,2’-trans isomers as major products (Scheme 17).

Based on the conditions for the stepwise fragmentation andglycosylation procedure, Boto et al. explored the one-pot
version of the reaction [74]. When the β-fragmentation, the firststep of oxidative glycosylation, was carried out in CH2Cl2 andthen the Lewis acid and the silylated base were added, theacyclic nucleosides were obtained in low yields. Boto andco-workers overcame this problem by replacing the solvent
before glycosylation. After the fragmentation reaction was
finished, the solvent (CH2Cl2) was removed and replaced withacetonitrile. The resulting mixture was treated with TMSOTf
and the silylated base. Under the optimized conditions, the reactions of ribose derivative 128, mannose derivative 137, andrhamnose derivative 138 gave the desired acyclic nucleosides
in excellent yields as shown in Scheme 18. It is worth notingthat the overall yields for the one-pot process are comparable
or superior to those obtained with the two-step procedure(Scheme 18).

Synthesis of disaccharides
Classically, carbohydrates have been considered primarily anenergy source for life – as in the cases of glucose, fructose andtheir oligosaccharides, e.g., starch. However, more recently it
has been revealed that oligosaccharides and glycoconjugates
also play important roles in various biological processes, asmentioned earlier. As a result, the increasing significance ofoligosaccharides in biological events has led to a strong demand
for synthetic routes towards oligosaccharides, which would also
contribute to the identification and development of drug candidates. For example, cancer immunotherapy based on vaccines

Scheme 17: Synthesis of acetoxyacetals using hypervalent iodine and addition of silylated base.

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Beilstein J. Org. Chem. 2018, 14, 1595–1618.

Scheme 18: One-pot fragmentation-nucleophilic additions mediated by hypervalent iodine.

derived from carbohydrate antigen–adjuvant combinations hasreceived much attention in recent years [75-77]. However, thedifficulties associated with the isolation of tumor-associatedcarbohydrate antigens from natural sources have impeded extensive research. Thus, the most promising approach to the
supply of these antigens is to develop a suitable method fortheir chemical synthesis.
To date, various glycosylation reactions capable of constructing oligosaccharides with high stereoselectivities have been reported [18,19]. Thioglycosides are often used as a sugar donor
in these reactions due to their stability under various conditions

and specific activation with thiophilic agents. For example, oneof the typical conditions used for the construction of oligosaccharides is the combination of Lewis acids and iodine or its
chemical equivalents. Fukase and co-workers reported a glycosylation reaction with thioglycoside using hypervalent iodine
reagents in the 1990s [78,79]. The outline and postulated mechanism of the reaction are shown in Figure 7. The reaction of
iodosylbenzene and electrophiles, e.g., triflic anhydride or
Lewis acids, should generate a potent thiophile 143 that reacts
with thioglycoside 144 to form an oxocarbenium ion 145. The
resulting oxocarbenium ion 145 should in turn react with asugar acceptor to give the glycosylated product 147 (Figure 7).

Figure 7: The reaction of thioglycoside with hypervalent iodine in the presence of Lewis acids.

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Beilstein J. Org. Chem. 2018, 14, 1595–1618.

By this reaction, Fukase et al. reported the glycosylation of
methyl thioglycoside 148 as a sugar donor to give disaccharides 150 and 152 in high chemical yields as depicted in
Scheme 19. As mentioned above, not only triflic anhydride, butvarious Lewis acids (TMSOTf, Sn(OTf) 2, Yb(OTf) 3) and a
Brønsted acid (TfOH) were proven useful as activators, bywhich the reaction finished in a short time and gave the products with high stereoselectivity [79].
Recently, the reaction was revisited by Kajimoto et al., whosought a glycosylation reaction that could be applied todisarmed thioglycosides using hypervalent iodine reagents[80,81]. One of the reactions they examined was the glycosylation reaction of methyl 2-phthalimidothioglucopyranoside
153 with methyl tribenzylglucopyranoside 149 by PIFA in thepresence of various acid catalysts. The results showed that thereaction with PIFA and TfOH afforded the best result, giving
disaccharide 154 in 77% yield. On the other hand, the useof bis[cyclohexyl]trifluoromethanesulfonylborane [(cycloHex)2BOTf] and methanesulfonic acid resulted in a poor yield.
The synthesis of disaccharides under the optimized conditions
was performed using “odorless” thioglycoside 155 and 149 as
the donor and the acceptor [81]. Even with the combination of“disarmed” 155 and “armed” 149, the reaction gave rise to thedesired disaccharide 157 in 87% yield. The same reaction of thecorresponding 3-epimer 156 proceeded smoothly to give thedisaccharide 158 in good yield (Scheme 20).
Randolph and Danishefsky reported a glycal assembly strategy
to the synthesis of a branched oligosaccharide [82]. Bennett andco-workers reported that phenyl(trifluoroethyl)iodonium triflimide was a stable promoter for glycosylation reactions using
thioglycoside donors [83]. Since the reactions often were unselective in the absence of C2 acetate-directing groups, Bennett et
al. investigated the compatibility of the above-mentioned reaction in nitrile solvents documented to have a β-directing effect,with the aim of developing a glycosylation that can be selectively achieved in the absence of directing groups. After preliminary screens, they found that the reaction in the presence ofphenyl(trifluoroethyl)iodonium triflimide 160 and the nonnucleophilic base 2,4,6-tri-tert-butylpyrimidine (TTBP) at 0 °C
with the solvent combination of 2:1 CH2Cl2/pivalonitrile provided the optimal reaction outcome. However, they also encountered a problem: the reduced solubility of substrate in the solvent system resulted in lower yields. They therefore examined
mixed nitrile solvents again, and eventually found that a quaternary solvent mixture composed of 6:1:1:1 CH2Cl2/acetonitrile/isobutyronitrile/pivalonitrile greatly improved both the chemical yields and stereoselectivity, as shown in Scheme 21. The
results suggested that both the solvent system and iodonium salt
promoter are required for selectivity.
Even though glycals have a π-electron-rich enol ether unit,
reports regarding transformations involving glycal oxidation
as well as installation of heteroatom substituents at the C2
position were limited. In 2001, Gin’s group reported theC2-acycloxyglycosylation procedure based on hypervalentiodine chemistry [84]. In this reaction, the use of a combinationof hypervalent iodine and Lewis acid was key, as in the reactions described above. In this procedure, a solution of the glycal

Scheme 19: Synthesis of disaccharides employing thioglycosides under an oxidative coupling reaction mediated by hypervalent iodine.

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Beilstein J. Org. Chem. 2018, 14, 1595–1618.

Scheme 20: Synthesis of disaccharides using disarmed thioglycosides by hypervalent iodine-mediated glycosylation.

Scheme 21: Glycosylation using aryl(trifluoroethyl)iodium triflimide.

donor and a (diacyloxyiodo)benzene reagent was first treatedwith BF3·OEt2. Then, the glycosyl acceptor (R”OH) and a catalytic amount of TfOH were added to the mixture, giving the
1,2-trans disubstituted C2-acyloxylglycoside. A plausible
mechanism of the reaction is shown in Figure 8. The first stepof the reaction between glycal 161 and (diacyloxyiodo)benzene
formed the glycosyl ester intermediate 162 bearing a phenyl
iodonium(III) functionality at C2, which was transformed to adiacyloxylated product 163. As evidence in support of thismechanism, they reported that 163 was indeed isolated when
the reaction was finished at the first step. In the second step, theresulting diacyloxylated product 163 could effectively glycosylate the appropriate acceptor by the action of TfOH to give theC2-acyloxyglycoside 164 with good selectivity at the anomeric
position as a consequence of participation by the neighboring
C2 acyloxy group (Figure 8).

Figure 8: Expected mechanism of hypervalent iodine-mediated glycosylation with glycals.

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Beilstein J. Org. Chem. 2018, 14, 1595–1618.

They prepared C2-acyloxy glycosides 165, 166, 169, 170 and
172 using hypervalent iodine-mediated coupling reactions withglycals, and the results are shown in Scheme 22. Either (diacetoxyiodo)benzene or (dibenzoyloxyiodo)benzene could serve as
an efficient oxidant, and the reactions utilizing them gave theproducts installing either the acetate or benzoate functionality,
respectively, at the C2-position. Both glucal 115 and galactal
167 were amenable to the oxidative glycosylation reaction tostereoselectively give C2-acyloxylated β-glycosides in goodyields [84] (Scheme 22).
Hotha and co-workers utilized the reaction of glycals withhypervalent iodine reagents for the stereoselective synthesis of
C2 deoxyglycosides and amino acid glycoconjugates [85]. In
their work, they also utilized an important chemical attribute ofcetylammonium bromide (CTAB) – namely, CTAB forms surfactant-assembled lipophilic nanoreactors stable in organic sol-

vents, which could be used for regioselective functionalizationof indenes. Therefore, they investigated the regioselective iodination of glycals by using CTAB and hypervalent iodine
reagents for the synthesis of 2-deoxy-2-iodoacetates. In the preliminary experiments, the reaction between per-O-acetylglucal(177) and PhI(OAc) 2 in CTAB and KI gave trans-2-iodo
α-acetate and its corresponding bromo acetate in a 94:5 ratio.
The latter was expected to be formed by halide counter ion
exchange between CTAB and KI. Since the reaction occurred as
expected, it was applied to the synthesis of amino acid conjugates. Acetyl groups of the (diacetoxyiodo)benzene were
exchanged with N- and O-protected amino acids by slow evaporation of a mixture of PhI(OAc)2 and amino acid 173 and 174 inchlorobenzene to give PhI(OCOR)2 compounds 175 and 176.
The formation of iodo ester glycosides 178 and 179 from 175
and 176 was achieved in very good yields under the conditions
shown in Scheme 23.

Scheme 22: Synthesis of oligosaccharides by hypervalent iodine-mediated glycosylation with glycals.

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Beilstein J. Org. Chem. 2018, 14, 1595–1618.

Scheme 23: Synthesis of 2-deoxy amino acid glycosides.

Notably, the resulting iodo ester glycosides 178 and 179 wereconsidered to have self-assembled structures versatile for thesynthesis of serenylated and threonylated glycosides by intramolecular glycosylation. In addition, the access to 2-deoxyglycosides should be easily achievable by subsequent radical
deiodination of the products. After several experiments, treatment with a catalytic amount of TMSOTf was found to be suitable for the intramolecular glycosylation, giving the corresponding acid, which was easily converted to the corresponding methyl ester 180 under EDCI/DMAP/MeOH conditions[85]. Similarly, the reaction of the threonine derivative 179
afforded 181 in good yield. Radical deiodination of 180 and 181
using Bu3SnH and AIBN successfully gave 2-deoxy-β-glycosides 182 and 183, which were difficult to synthesize from thecorresponding 2-deoxy sugar derivative in a stereoselective
manner (Scheme 23).
As mentioned above, the iodo ester glycosides were considered
to have self-assembled structures suitable for intramolecularglycosylation. As depicted in Figure 9, treatment of 184 withTMSOTf first cleaved the silyl ether to form 185, which wascorrectly positioned to undergo intramolecular glycosidation.

As a result, the Lewis acid could also facilitate the departure of
the anomeric ester and the resulting 185 gave rise to the intramolecular nucleophilic attack to furnish the corresponding acid186.
Glycals and thioglycosides were often used as sugar donors forthe glycosylation of oligosaccharides. It is interesting that thehypervalent iodine-mediated oxidative reactions with theses derivatives provide a different method to build glycosidic bonds.
Diversity in glycoside bond forming reactions would contributeto improve the oligosaccharide synthesis.

Conclusion

The Pummerer-type glycosylation includes oxidation ofa sulfide to the corresponding sulfoxide followed by the
TMSOTf-mediated coupling reaction. The reaction utilizing
hypervalent iodine reagents could bypass one step of thePummerer-type glycosylation and directly give 4’-thionucleosides from the corresponding sulfide derivative. The reaction
could be efficiently applied to the synthesis of 4’-selenonucleosides as well as 4’-thionucleosides. Based on the concept ofhypervalent iodine-mediated glycosylation, a reaction applic-

Figure 9: Rationale for the intramolecular migration of the amino acid unit.

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Beilstein J. Org. Chem. 2018, 14, 1595–1618.

able to the synthesis of carbocyclic nucleosides and a coupling
reaction between nucleobase and glycal derivatives were developed. The latter reaction was employed to synthesize dihydropyranonucleosides. Oxidative scission is a characteristic
reaction mediated by hypervalent iodine reagents and is typically used for dehomologation of sugars. A one-pot glycosylation
using this reaction was also developed for the synthesis ofacyclic nucleoside derivatives. In addition to nucleoside synthesis, hypervalent iodine-mediated glycosylation could also be
applied to the synthesis of oligosaccharides and glycoconjugates when thioglycosides and glycals were used as sugar
donors. There is no doubt that the use of hypervalent iodine
reagents greatly improved the efficiency of the synthesis of
nucleosides and oligosaccharides. The results of these syntheses demonstrate the power of glycoside bond-forming reactions,and should assist in the future identification or synthesis of biologically active nucleoside and glycoconjugate derivatives.

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Our work described in this review article was supported in partby a Grant-in-Aid for Scientific Research from JSPS and by agrant from the Strategic Research Foundation Grant-aided Project for Private Universities from the Ministry of Education, Culture, Sport, Science, and Technology, Japan (MEXT).

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Fundamental Toxicological Sciences (Fundam. Toxicol. Sci.)
Vol.4, No.5, 229-239, 2017

Fundamental Toxicological Sciences
URL : http://www.fundtoxicolsci.org/index_e.html

Original Article

CYP3A4 induction mechanism of polycyclic aromatichydrocarbons differs from that of rifampicin in PXR binding
element
Yusuke Aratsu1,2, Reo Odagiri1, Rie Shoji1, Kouki Watanabe1, Takeshi Kumagai1,
Sawako Shindo1, Takamitsu Sasaki1,3 and Kiyoshi Nagata1
Department of Environmental Health Science, Faculty of Pharmaceutical Sciences, Tohoku Medical andPharmaceutical University, 4-4-1 Komatsushima, Aoba-ku Sendai, Miyagi 981-8558, Japan
2Drug Metabolism and Pharmacokinetics Research Laboratories, Central Pharmaceutical Research Institute,Japan tobacco Inc.,1-1 Murasaki-cho Takatsuki, Osaka 569-1125, Japan
3Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizoka,
52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
1(Received September 22, 2017; Accepted September 27, 2017)

ABSTRACT — CYP3A4 is an important drug-metabolizing enzyme induced by various compounds
causing drug-drug interactions. However, the molecular mechanism of CYP3A4 induction is not completely understood. CYP3A4 induction is caused by pregnane X receptor (PXR) through binding to somePXR binding elements. These elements comprise an everted repeat separated by six nucleotides in the
promoter region and distal nuclear receptor binding element 1 (dNR-1) as well as the essential distal
nuclear receptor binding element for CYP3A4 induction (eNR3A4) in the enhancer region of the CYP3A4
gene. Recently, we found that polycyclic aromatic hydrocarbons including anthracene induce CYP3A4 in
HepG2 cells with a different induction profile from that of rifampicin (RF), a typical PXR ligand. Whena CYP3A4 reporter plasmid in which the eNR3A4 DNA fragment binds directly to the CYP3A4 promoter (-362 bases) was evaluated in a reporter assay, dibenz[a,h]anthracene (DBA) induced reporter activity,
while RF did not. To be induced reporter activity by RF, more 14 nucleotides 5′ upstream of the eNR3A4(rifampicin eNR3A4: reNR3A4) DNA fragment were required. However, eNR3A4 and reNR3A4 did not
respond to recombinant PXR without dNR-1. These results suggest that eNR3A4 and reNR3A4 are necessary for CYP3A4 induction by DBA and RF, respectively, and that dNR-1 is indispensable for fullinduction through PXR.
Key words: CYP3A4 induction, Pregnane X receptor, Rifampicin, Polycyclic aromatic hydrocarbons,
Dibenz[a,h]anthracene, PXR binding element
INTRODUCTIONCYP3A4 is one of the most important drug-metabolizing enzymes involved in the metabolism of more than
50% of marketed drugs (Wienkers and Heath, 2005).
Numerous studies have examined drug-drug interac-tions (DDIs) mediated by CYP3A4. DDIs may cause
serious problems from a clinical perspective. Particularly, CYP3A4 induction decreases concomitant drug exposure and efficacy in the human body. Therefore, the U.S.
Food and Drug Administration (FDA, 2012), European Medicines Agency (EMA, 2012), and Pharmaceuti-

Correspondence: Kiyoshi Nagata (E-mail: nagataki@tohoku-mpu.ac.jp)
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Y. Aratsu et al.

cals and Medical Devices Agency (PMDA, 2014) havereleased guidance for DDI where required to be includedin the package insert and examined through clinical trials.
Many pharmaceutical companies have attempted to estimate the risk of DDI on the clinical side and develop lowrisk compounds by estimating inducibility using in vitro tools such as hepatocytes. However, it is very difficult
to precisely evaluate the induction potency of individual compounds because of their complicate mechanisms
compared to CYP inhibition.
A number of studies have demonstrated that CYP3A4
inducers activate transcription of CYP3A4 mediated by
nuclear receptors such as pregnane X receptor (PXR,
NR1I2) (Bertilsson et al., 1998; Blumberg et al., 1998;
Lehmann et al., 1998), constitutive androstane receptor(NR1I3) (Goodwin et al., 2002), and vitamin D receptor (NR1I1) (Drocourt et al., 2002). PXR is a main regulator of drug-metabolizing enzymes and transporters(Synold et al., 2001; Geick et al., 2001). Human PXR is
activated by binding to various drugs such as rifampicin(RF), phenytoin, and carbamazepine (Kamiguchi, 2010),
transferred into the nucleus, and activates CYP3A4 transcription by binding to the regulatory region of the geneas a heterodimer with retinoid X receptor (Wan et al.,2000).
New activation pathways of CYP3A4 transcription
have also been reported. For example cyclin-dependent
kinase 2 negatively regulates PXR activity through phosphorylation of Ser350 (Lin et al., 2008). Other reports indicated that promoter transactivation on CYP3A4 and UDPglucuronosyltransferase 1A1 were reduced by mutationof Ser350 (Elias et al., 2014; Sugatani et al., 2010).
Several nucleotide sequences of the PXR binding element have been identified in CYP3A4, but are not fully understood. Some PXR binding elements are composed of an everted repeat separated by six nucleotides(ER-6) in the promoter region and distal nuclear receptor binding element1 (dNR-1) (Blumberg et al., 1998;
Lehmann et al., 1998; Goodwin et al., 1999), as well as
the essential distal nuclear receptor binding element for
CYP3A4 induction (eNR3A4) (Toriyabe et al., 2009) inthe enhancer region of CYP3A4. These elements have acluster of TGA(A/C)CT-like sequences recognized by the
nuclear receptor family of transcription factors (Goodwin
et al., 1999).
We recently reported that poly aromatic hydrocarbons (PAHs) and indirubin, which were thought to be arylhydrocarbon receptor (AhR) ligands, induced CYP3A4
in HepG2 cells. Their induction is mediated through
PXR, but not through AhR (Kumagai et al., 2012;
Kumagai et al., 2016). In addition, the molecular mech-anism of CYP3A4 induction by PAHs and indirubin differ from those by RF and clotrimazole (CTZ), typical CYP3A4 inducers, in the responsible element of the
CYP3A4 gene. In this study, the roles of PXR binding element in eNR3A4 and dNR-1 were investigated to clarify
the CYP3A4 induction mechanism.
MATeRIAlS AND MeThODS
Materials
RF, 3-methylcholanthrene, benzo[e]pyrene, and 1,2benzanthracene were purchased from Sigma-Aldrich(St. Louis, MO, USA). The other PAHs listed Fig. 1. wereobtained from Tokyo Chemical Industry (Tokyo, Japan).
Dimethyl sulfoxide (DMSO) was purchased from Nacalai Tesque (Kyoto, Japan). CTZ and 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) were purchased from Wako
Pure Chemical Industries (Osaka, Japan).
Cell culture
Human hepatoma cell line HepG2 cells were obtained
from the RIKEN BRC (Tukuba, Japan) through Cell
Resource Center for Biomedical Research (Tohoku
University). The HepG2-derived cell line clone 3-1-20
stably expressing the CYP3A4-luciferase reporter gene
was established as reported previously (Noracharttiyapot
et al., 2006). These cells were cultured in Dulbecco’s
modified Eagle’s medium (Wako Pure Chemical Industries) supplemented with non-essential amino acids(Invitrogen, Carlsbad, CA, USA), 10% heat-inactivatedfetal bovine serum (Biowest, Nuaillé, France), and antibiotic-antimycotic (Invitrogen).
Reporter gene constructs and expression
vectors
pCYP3A4-362, pCYP3A4-362-7.7k, pCYP3A4-362m-7.7k, pCYP3A4-362-7.7km, and pCYP3A4-362m-7.
7km and pCYP3A4-362-7.7∆Bsite were constructed as
described previously (Takada et al., 2004). pCYP3A4362-7.7k∆eNR3A4 was reported previously (Toriyabe et al., 2009). pCYP3A4-362+dNR-1, pCYP3A4362+eNR3A4, pCYP3A4-362+rifampicin eNR3A4(reNR3A4), and pCYP3A4-362+14αβ were preparedby inserting the dNR1, eNR3A4, reNR3A4, and 14αβ
DNA fragments, which were isolated by PCR with primers (Table 1) and digested with MluI and BglII, into the
MluI and BglII sites of pCYP3A4-362. pCYP3A4-362+eNR3A4+dNR-1 and pCYP3A4-362+reNR3A4+dNR-1
were made from pCYP3A4-362+eNR3A4 andpCYP3A4-362+reNR3A4 by inserting the dNR-1 DNA
fragment digested with Acc65I and MulI into those ofVol. 4 No. 5— 187 —

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Different CYP3A4 induction mechanism between rifampicin and PAHs

Table 1. Primers used for construction of reporter gene plasmids
dNR1
eNR3A4
reNR3A4
14αβ

FW
RV
FW
RV
FW
RV
FW
RV

Primer sequence
gcgggtaccATCTCAGCTGAATGAACTTGCTGAC
gcgacgcgtAGGAAAGCAGAGGGTCAGCAAGTTC
gcgacgcgtATTAAACCTTGTCCTGTGTTGACCCCAGGT
gcgagatctTGAAAAGAATATGATAGCTTGT
gcgacgcgtCAGTGATTATTAAACCTTGTC
gcgagatctTGAAAAGAATATGATAGCTTGT
gcgacgcgtCAGTGATTATTAAACCTTGTC
gcgagatctTTCACCTGGGGTCAACACAGG

the Acc65I and MulI sites. pCYP3A4-362-7.7k∆mα,pCYP3A4-362-7.7k∆mβ, pCYP3A4-362-7.7k∆mγ,
pCYP3A4-362-7.7k∆mδ, and pCYP3A4-362-7.7k∆mεwere prepared from reporter plasmids mutated in theα, β, γ, δ, and ε sites as previously reported (Toriyabe
et al., 2009) by digestion with XhoI, treatment with S1
nuclease, and self-ligation. Point-mutated reporter plasmids were constructed with the QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA)using pCYP3A4-362-7.7k as a template.
luciferase assay for reporter plasmid
HepG2 cells were seeded into a 48-well tissue culture plate (BD Biosciences, Franklin Lakes, NJ, USA) at
3.0 × 104 cells per well the day before transfection. Eachreporter plasmid and pGL4.82 (Promega, Madison, WI,USA) were cotransfected using Targefect F-1 (Targeting
Systems, El Cajon, CA, USA) according to the manufacturer’s protocols. Transfection efficiency was normalized
by Renilla luciferase activity of pGL4.82. After transfection, HepG2 cells were cultured in the presence of various compounds dissolved in DMSO for 48 hr. Control
cells were treated with vehicle (0.1% DMSO). After 48 hr
incubation, the cells were washed with PBS and suspended in passive lysis buffer (Promega). Luciferase activities
were evaluated using the Dual-Luciferase assay system
and GloMaxTM 96 Microplate Luminometer (Promega)according to the manufacturer’s protocols. Fold-induction
values were calculated from the luminescence normalized
as the ratio of control samples.
luciferase assay for PAhs
3-1-20 cells were seeded into 96-well tissue cultured
plate at 1.0 × 10 4 cells per well the day before treatment with various compounds dissolved in DMSO. After
48 hr incubation, the cells were washed with PBS and suspended in passive lysis buffer. Luciferase activities were
evaluated using the Luciferase assay system (Promega)with GloMaxTM 96 Microplate Luminometer. The luminescence of each sample was normalized to the pro-tein concentration using the Protein Assay Kit (Bio-Rad,
Hercules, CA, USA).
Construction of adenovirus expression vector
and infection
The human PXR-expressing adenovirus (AdhPXR)
was constructed as described previously (Matsubara etal., 2007). The β-galactosidase-expressing adenovirus(AdCont; AxCALacZ), used as a control adenovirus, wasprovided by Dr. Izumi Saito (Tokyo University, Tokyo,
Japan) (Miyake, 1996). The titer of the adenoviruses, the
50% titer culture infectious dose (TCID50), was determined as reported previously (Matsubara et al., 2007).
Multiplicity of infection was calculated as the TCID 50
based on the number of cells. One day before transfection, cells were seeded in 48-well plates. The adenovirus
infection protocol was described previously (Kumagai etal., 2012).
Statistical analysis
Data are presented as the mean ± standard deviation(S.D.) and were evaluated by paired Student’s t-test.
A P value < 0.05 was considered significant.
ReSUlTSeffect of PAhs on CYP3A4 reporter activity in
3-1-20 cells
First, CYP3A4 reporter activity by PAHs was investigated by using HepG2-derived cell line clone, 3-1-20
stably expressing the CYP3A4-luciferase reporter gene.
Many PAHs increased CYP3A4 reporter activity. Amongthem, dibenz[a,h]anthracene (DBA) and dibenz[a, c]
anthracene strongly increased reporter activity (20-30fold), while benzo[e]pyrene, pyrene, naphthalene showedno activation, and dibenz[de,kl]anthracene showed minimal activation of CYP3A4 transcription (Fig. 2).

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Y. Aratsu et al.

Fig. 1.

Structure of RF, CTZ and PAHs.

Fig. 2.

Effect of PAHs on CYP3A4 reporter activity in 3-1-20 cells. Clone 3-1-20 cells were seeded at 1 × 104 cells in a 96-welltissue culture plate and pre-incubated for 24 hr before treatment with various compounds. The cells were treated with RF(10 μM), CTZ (10 μM) and various PAHs (1 μM) for 48 hr and reporter activity was measured by the luciferase assay. Reporter activities are expressed as the fold to that in the vehicle-treated cells. Data are shown as the mean ± S.D. from threedifferent samples. *P < 0.05, **P < 0.005, differences from vehicle-treated cells.

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Different CYP3A4 induction mechanism between rifampicin and PAHs

effect of mutation in preR-6 and dNR-1 onreporter activity
Next, transient transfection assays were conducted byusing CYP3A4 luciferase reporter plasmids containing
three elements (dNR-1, eNR3A4, and prER-6), whichbind to PXR (pCYP3A4-362-7.7k). The role of these elements was investigated using reporter plasmids containing mutations in dNR-1 and/or prER-6 (pCYP3A4-3627.7km, pCYP3A4-362m-7.7k, pCYP3A4-362m-7.7km).
Reporter activity was compared among the reporter plasmids using PXR ligands (RF and CTZ) and AhR ligands (DBA and TCDD) (Fig. 3). All inducers increased
reporter activity (8.2-, 7.9-, 11.0-, and 9.1-fold, respectively) in cells transfected with pCYP3A4-362-7.7k.
The construct mutated in prER-6, pCYP3A4-362m-7.7k,
showed nearly the same activity as pCYP3A4-362-7.7k for
all inducers. In contrast, the constructs mutated in dNR-1,
pCYP3A4-362-7.7km, in dNR-1 and pER-6, pCYP3A4362m-7.7km showed very low responses to RF and CTZ(pCYP3A4-362-7.7km: 2.7- and 1.8-fold and pCYP3A4362m-7.7km: 1.5- and 2.3-fold, respectively). However,
DBA and TCDD showed relatively high responses compared to those of RF and CTZ in pCYP3A4-362-7.7km(10.4- and 6.2-fold) and pCYP3A4-362m-7.7km (5.0and 5.1-fold). Similar results were observed in cells treated with indirubin (data not shown).

Fig. 3.

effect of deletion in enhancer region on thereporter activity
To verify the involvement of eNR3A4 for CYP3A4
induction, transient transfection assays were conducted by using several CYP3A4 luciferase reporter plasmids with deletions in various enhancer regions(Fig. 4). RF did not increase reporter activity in cells treated with deleted plasmids in this experiment, pCYP3A4362-7.7k∆eNR3A4, pCYP3A4-362-7.7k∆Bsite,
pCYP3A4-362+dNR-1, pCYP3A4-362+eNR3A4, andpCYP3A4-362, although RF increased reporter activity incells treated with pCYP3A4-362-7.7k (17.9-fold). In contrast, DBA increased reporter activity to some extent inall plasmids used in this experiment. Of all deleted plasmids, pCYP3A4-362-7.7k∆Bsite and pCYP3A4-362+eNR3A4 showed a high reporter activity (5.4- and 16.3fold, respectively) following treatment with DBA.
effect of overexpressing PXR on the reporter
activity
It is widely known that PXR is a major transcription factor involved in CYP3A4 induction. The effect of
PXR on CYP3A4 induction by RF and DBA was investigated by overexpressing human PXR in HepG2 cells
using adenovirus (Fig. 4). The induced reporter activity (17.9-fold) by RF was dramatically increased (51.3-

Effect of mutation in CYP3A4 promoter and/or enhancer region on reporter activity induced by various compounds. HepG2
cells were seeded at 3 × 104 cells in a 48-well tissue culture plate. The cells were incubated for 24 hr before transfection.
The transfection protocol is described in the “Materials and Methods”. Closed boxes represent mutated PXR-binding elements. The cells were treated with RF (10 μM), CTZ (10 μM), DBA (10 μM), or TCDD (10 nM) for 48 hr. Luciferase
activity was normalized to Renilla luciferase activity. The values were expressed as the fold induction of the vehicle control(DMSO) group. Data are shown as the mean ± S.D. from six different samples. *P < 0.05, **P < 0.005, differences fromvehicle-treated cells.
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Y. Aratsu et al.

Fig. 4.

Effect of overexpressing PXR on CYP3A4 reporter activity in HepG2 cells by RF and DBA. HepG2 cells were seeded at
3 × 104 cells in a 48-well tissue culture plate. The cells were incubated for 24 hr before transfection. The transfection protocol is described in the “Materials and Methods”. After 24 hr of Ad-PXR (3 MOI) infection, the cells were treated with RF(10 μM) or DBA (10 μM) for 48 hr. Luciferase activity was normalized to Renilla luciferase activity. The values were expressed as the fold induction of the vehicle control (DMSO) group. Data are shown as the mean ± S.D. from three different
samples. *P < 0.05, **P < 0.005, differences from vehicle-treated cells.

Fig. 5.

Sequence of eNR3A4 and reNA3A4 of CYP3A4 and reporter constructs containing mutations and or deletions. These elements contain a cluster of TGA(A/C)CT-like sequences recognized in the nuclear receptor family of transcription factors.

fold) in cells by overexpressing PXR after transfection ofpCYP3A4-362-7.7k. In cells transfected with pCYP3A4362-7.7k∆Bsite, reporter activity also increased by overexpressing PXR. Similarly, DBA highly increased thereporter activity (6.0- and 5.4-fold, respectively) in cells
transfected with pCYP3A4-362-7.7k and pCYP3A4-

362-7.7k∆Bsite, and these activities were dramaticallyincreased (17.5- and 15.8-fold, respectively) by overexpressing PXR. In contrast, increased reporter activity bytreatment with DBA was not activated by overexpressing
PXR in cells transfected with pCYP3A4-362+eNR3A4 in
which the eNR3A4 element binds directly to the promot-

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Different CYP3A4 induction mechanism between rifampicin and PAHs

er region (-362).
Effect of 14 bases 5′-upstream of eNR3A4 on
reporter activity
Because increased reporter activity was not observedin pCYP3A4-362+eNR3A4 treated with RF, pCYP3A4362+reNR3A4 containing 14 additional bases 5′-upstreamof eNR3A4 was transfected into HepG2 cells. The results
are shown in Fig. 6. RF and DBA increased reporter activity in cells transfected with pCYP3A4-362+reNR3A4(5.1- and 7.2-fold, respectively), but these reporter activities were not significantly increased by overexpressing PXR (8.3- and 7.3-fold, respectively). Similar results
were observed in cells transfected with pCYP3A4362+14αβ, although the reporter activity was increased tosome extent without overexpressing PXR.
Role of dNR-1 for CYP3A4 induction
In the study above shown, we suggest that eNR3A4
and reNR3A4 are essential elements for CYP3A4 geneactivation by DBA and RF. However, it remained unclear
how dNR-1, eNR3A4, and reNR3A4 function in CYP3A4
induction. To verify the involvement of dNR-1, eNR3A4,
and reNR3A4, further transient transfection assays wereconducted using CYP3A4 luciferase reporter plasmids
with or without dNR-1 (Fig. 7). RF did not increase
reporter activity in cells treated with pCYP3A4-362+eNR3A4 and pCYP3A4-362+dNR-1+eNR3A4. Howev-

Fig. 6.

er, RF induced reporter activity (4.5-fold) was dramatically increased (29.7-fold) in cells overexpressing PXR after
transfection of pCYP3A4-362+dNR-1+reNR3A4. In contrast, DBA increased reporter activity in cells transfected with any reporter constructs and the induced activity
was increased by overexpressing PXR in cells transfected with pCYP3A4-362+dNR-1+eNR3A4 and pCYP3A4362+dNR-1+reNR3A4 (8.4- and 18.1-fold).
effect of deletion in eNR3A4 region on reporter
activity
Several TGA(A/C)CT-like sequences binding to nuclear receptor family members were observed in the eNR3A4
region. To verify the role of half-sites in eNR3A4 for
CYP3A4 induction, several CYP3A4 luciferase reporter plasmids with mutated and deleted half-sites wereconstructed. The results are shown in Fig. 8. RF did notincrease reporter activity in cells treated with pCYP3A4362-7.7k∆mα and pCYP3A4-362-7.7k∆mβ, even byoverexpressing PXR. However, DBA increased reporter
activity in pCYP3A4-362-7.7k∆mα and pCYP3A4-3627.7k∆mβ and the activity was not increased by overexpressing PXR. Moreover, reporter activity increased by
RF and DBA was lower in pCYP3A4-362-7.7k∆mγ andpCYP3A4-362-7.7k∆mδ than in pCYP3A4-362-7.7k,
although these activities were increased by overexpressing PXR.

Effect of DNA deletion in CYP3A4 enhancer region and overexpressing PXR on reporter activity in HepG2 cells induced
by RF and DBA. HepG2 cells were seeded at 3 × 104 cells in a 48-well tissue culture plate. The cells were incubated for
24 hr before transfection. The transfection protocol is described in the “Materials and Methods”. After 24 hr of Ad-PXR (3MOI) infection, the cells were treated with RF (10 μM) or DBA (10 μM) for 48 hr. Luciferase activity was normalized toRenilla luciferase activity. The values were expressed as the fold induction of the vehicle control (DMSO) group. Data areshown as the mean ± S.D. from three different samples. *P < 0.05, **P < 0.005, differences from vehicle-treated cells.
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Y. Aratsu et al.

Fig. 7.

Effect of DNA deletion in CYP3A4 enhancer region and overexpressing PXR on reporter activity in HepG2 cells induced
by RF and DBA. HepG2 cells were seeded at 3 × 104 cells in a 48-well tissue culture plate. The cells were incubated for
24 hr before transfection. The transfection protocol is described in the “Materials and Methods”. After 24 hr of Ad-PXR (3MOI) infection, the cells were treated with RF (10 μM) or DBA (10 μM) for 48 hr. Luciferase activity was normalized toRenilla luciferase activity. The values are expressed as the fold induction of the vehicle control (DMSO) group. Data areshown as the mean ± S.D. from six different samples. *P < 0.05, **P < 0.005, differences from vehicle-treated cells.

Fig. 8.

Effect of DNA deletion in eNR3A4 and overexpressing PXR on reporter activity in HepG2 cells induced by RF and DBA.
HepG2 cells were seeded at 3 × 104 cells in 48-well tissue culture plate. The cells were incubated for 24 hr before transfection. The transfection protocol is described in the “Materials and Methods”. After 24 hr of Ad-PXR (3 MOI) infection, thecells were treated with RF (10 μM) or DBA (10 μM) for 48 hr. Luciferase activity was normalized to Renilla luciferase
activity. The values are expressed as the fold induction of the vehicle control (DMSO) group. Data are shown as the mean ±S.D. from five different samples. *P < 0.05, **P < 0.005, differences from vehicle-treated cells.

effect of point mutation in eNR3A4 region onreporter activity
To confirm the role of the half-site in eNR3A4 in
detail, point mutations were introduced into pCYP3A4-

362-7.7k at several bases of the eNR3A4 DNA fragment
as indicated with an arrow by changing to A. Mutation
No. 3, 4, 8, and 9 showed low activity compared to thatof pCYP3A4-362-7.7k by RF and DBA.

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Different CYP3A4 induction mechanism between rifampicin and PAHs

DISCUSSION
We first evaluated CYP3A4 induction by PAHs using
3-1-20 cells stably expressing the CYP3A4-luciferase
reporter gene (Noracharttiyapot et al., 2006). The results
showed that many PAHs increased CYP3A4 reporter
activity (Fig. 2). PAHs are known as AhR ligands, andactivated AhR binds to a cluster of CACGC-like sequences (Watson and Hankinson, 1992), but a CACGC-likesequence was not identified in the promoter and enhancer region of CYP3A4 used in this experiment. As shown
previously, CYP3A4 activation by PAHs such as 3-methylchoranthrene and TCDD was increased by overexpressing PXR and decreased by PXR-siRNA (Kumagai et al.,2012). One of the PAHs, DBA, increased reporter activity
in cells transfected with pCYP3A4-362-7.7k by overexpressing PXR (Fig. 4). PAHs including anthracene in theirchemical structure likely activate the CYP3A4 reporter gene in HepG2 cells, but compounds such as benzo[e]pyrene, pyrene and dibenz[de, kl]anthracene categorized
as condensed-ring annulation types only minimally activated the CYP3A4 reporter gene. However, it is unclear
whether PXR binds to these compounds. In this study,
because of its high induction potency, we used DBA toevaluate the CYP3A4 induction mechanism of PAHs.
It has been reported that dNR-1, eNR3A4, and prER-6
are very important for CYP3A4 induction (Blumberg etal., 1998; Lehmann et al., 1998; Goodwin et al., 1999;
Toriyabe et al., 2009). In this study, we investigated therole of these PXR binding elements on CYP3A4 induction in detail by using several reporter plasmids, including
CYP3A4 enhancer region into which we introduced deletion and mutations. The results indicate strong involvement not only of dNR-1, but also of eNR3A4 in the transcriptional activation of CYP3A4 by RF and CTZ, whileeNR3A4 appeared to be important for transcriptional activation of CYP3A4 by DBA and TCDD (Fig. 3). To confirm the involvement of eNR3A4, the reporter activities of each plasmid containing deletions in the enhancer
region were measured. The results showed that eNR3A4
is more important than dNR-1 in activating CYP3A4 transcription by DBA, as demonstrated by the pCYP3A4362-7.7k∆Bsite and CYP3A4-362+eNR3A4 containing
an eNR3A4 DNA fragment (Fig. 4).
We previously reported that indirubin, a ligand of
AhR, induces CYP3A4 through PXR (Kumagai et al.,2012; Kumagai et al., 2016) and other group also reported PAHs induce CYP3A4 (Luckert et al., 2013). In addition, indirubin showed a similar profile of transcriptional activation of CYP3A4 to that of TCDD and DBA (datanot shown) in HepG2 cells. To confirm the involvementof PXR in DBA, CYP3A4 reporter activity was examined by overexpressing PXR in HepG2 cells. As a result,plasmids containing eNR3A4, pCYP3A4-362-7.7k, andpCYP3A4-362-7.7k∆Bsite showed increased reporter
activity by overexpressing PXR. pCYP3A4-362+eNR3A4
showed increased the reporter activity by DBA, but notby RF. The increased reporter activity was not enhancedby overexpressing PXR (Fig. 4). These results suggestthat CYP3A4 induction through eNR3A4 by DBA does
not depend only on PXR. There are some reports of
new activation pathways of CYP3A4 transcription. For
example, cyclin-dependent kinase2 negatively regulates
the activity of PXR through phosphorylation of Ser350(Lin et al., 2008) and cell proliferation in Huh7 cells(Sivertsson et al., 2013), but the mechanism of CYP3A4
induction by DBA remains unclear. Therefore, further
studies are needed to determine the CYP3A4 induction
mechanism by DBA.
In addition, RF did not increase reporter activity incells transfected with pCYP3A4-362+eNR3A4 (Fig. 4).
The present results suggest that eNR3A4 is not sufficientfor the CYP3A4 transcription. We previously reported
that the α and β sites in the eNR3A4 DNA fragment are
key elements for induction of CYP3A4 (Toriyabe et al.,2009). Therefore, we predicted that a specific DNA fragment is required for CYP3A4 induction other than the αand β sites. Reporter plasmids containing an extra 14-basepair DNA fragment 5′-upstream of eNR3A4 named as
reNR3A4 were constructed. In addition, we constructeda reporter plasmid, pCYP3A4-362+14αβ, with the extra
14-base pair DNA fragment and α and β sites. The resultsof transient transfection assays indicated that the 14- basepair DNA fragment is necessary for CYP3A4 induction by RF other than the PXR binding element (Fig. 5).
However, overexpressing PXR did not increase reporter
activity in either pCYP3A4-362+eNR3A4 or pCYP3A4362+reNR3A4. Therefore, to verify the role of dNR-1,
pCYP3A4-362+dNR-1+reNR3A4 and pCYP3A4-362+dNR-1+eNR3A4 were prepared. The increased reporter activity of pCYP3A4-362+dNR-1+reNR3A4 by DBA
and RF was dramatically enhanced by overexpressing
PXR, but the reporter activity of pCYP3A4-362+dNR1+eNR3A4 was enhanced by overexpressing PXR following DBA treatment. These results indicate that for
full CYP3A4 induction, dNR-1 is indispensable and the
14-base pair DNA fragment 5′-upstream of eNR3A4 isnecessary for RF other than binding elements of dNR-1,
but not for DBA.
We further verified the role of each half-site (α, β, γ,δ, ε) in eNR3A4 using CYP3A4 reporter constructs in
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Y. Aratsu et al.

Fig. 9.

Effect of DNA point mutation in γ and δ half-sites of eNR3A4 on reporter activity induced by RF and DBA. HepG2 cells
were seeded at 3 × 104 cells in a 48-well tissue culture plate. The cells were incubated for 24 hr before transfection. The
transfection protocol is described in the “Materials and Methods”. After 24 hr of Ad-PXR (3 MOI) infection, the cells weretreated with RF (10 μM) or DBA (10 μM) for 48 hr. Luciferase activity was normalized to Renilla luciferase activity. The
values are expressed as the fold-induction of the vehicle control (DMSO) group. Data are shown as the mean from two different samples.

which various DNA deletions and single mutations wereintroduced. As previously reported, α and β sites arevery important for CYP3A4 induction by RF (Fig. 8)(Toriyabe et al., 2009), but this study showed that α andβ sites are not essential for CYP3A4 induction by DBA.
Similar results were also observed by mutation of α andβ sites (data not shown). Deletion of the γ and δ sites
decreased reporter activity, which was increased by DBA
and RF, but did not disappear. These results suggest thatthe γ and δ sites are also important for CYP3A4 induction. This idea was confirmed by mutation analysis of γand δ sites as shown in Fig. 9.
In conclusion, we demonstrated that the molecularmechanism of CYP3A4 induction differs between RF
and DBA in the PXR binding element. While reNR3A4
is an important DNA fragment for CYP3A4 induction by
RF, eNR3A4 is an important DNA fragment for CYP3A4
induction by DBA. Furthermore, our results might suggest that CYP3A4 induction by DBA and RF use different
transcription factors, although we did not confirm transcription factor binding to eNR3A4 and reNR3A4. The
results of our study revealed the role of each half-sites
in eNR3A4. The α and β sites are essential for CYP3A4
induction by RF, but not by DBA; in addition to the α andβ sites, the γ and δ sites are necessary for CYP3A4 induction. Moreover, to obtain full CYP3A4 induction through
PXR by RF and DBA, dNR-1 was indispensable.
ACKNOWleDgMeNTS
This work was supported by a Health and Labor Sciences Research Grant for Research on Food Safety (No.
22230301) from the Ministry of Health, Labor and Welfare of Japan; Grant-in-Aid for Scientific Research (B)(No. 23790193); and Japanese Ministry of Education,
Culture, Sports, Science and Technology (MEXT)-Supported Program for the Strategic Research Foundation atPrivate Universities.
Conflict of interest---- The authors declare that there isno conflict of interest.
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DOI 10.1186/s40780-017-0083-x

RESEARCH ARTICLE

Open Access

Effect of health foods on cytochrome P450mediated drug metabolism
Takamitsu Sasaki1,2, Yu Sato1, Takeshi Kumagai1, Kouichi Yoshinari2 and Kiyoshi Nagata1*
Abstract
Background: Health foods have been widely sold and consumed in Japan. There has been an increase in reports ofadverse effects in association with the expanding health food market. While health food-drug interactions are a particularconcern from the viewpoint of safe and effective use of health foods, information regarding such interactions is limited
owing to the lack of established methods to assess the effects of health food products on drug metabolism. We therefore
developed cells that mimicked the activities of cytochrome P450 1A2 (CYP1A2), CYP2C9, CYP2C19, CYP2D6, and CYP3A4,
which strongly contribute to drug metabolism in human hepatocytes, and established a system to assess the inhibitory
activity of health foods toward P450-mediated metabolism.
Methods: We simultaneously infected HepG2 cells with five P450-expressing adenoviruses (Ad-CYP1A2, Ad-CYP2C9, AdCYP2C19, Ad-CYP2D6, and Ad-CYP3A4) to mimic the activity levels of these P450s in human hepatocytes, and named
them Ad-P450 cells. The activity levels of P450s in Ad-P450 cells and human hepatocytes were calculated via
simultaneous liquid chromatography/tandem mass spectrometry analysis utilizing a P450 substrate cocktail.
Results: We established Ad-P450 cells mimicking the activity levels of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in
human hepatocytes. We determined the Km values of P450 substrates and IC50 values of P450 inhibitors in Ad-P450 cells.
These values were approximately equivalent to those obtained in previous studies. We investigated the inhibitory effectsof 172 health foods that were recently in circulation in Japan on P450-mediated metabolism using Ad-P450 cells. Of the
172 health foods, five products (two products having dietary effects, one turmeric-based product, one collagen-based
product, and one propolis-containing product) simultaneously inhibited the five P450s by more than 50%. Another 29
products were also confirmed to inhibit one or more P450s.
Conclusions: We established a comprehensive assessment system to elucidate the effects of health foods on
P450-mediated metabolism and identified the inhibitory activity of 34 of 172 health foods toward the drugmetabolizing P450s. Our results may provide useful information to predict health food-drug interactions.
Keywords: Health food, Health food-drug interaction, Cytochrome P450, Inhibition

BackgroundFoods with health claims and so-called ‘health foods’
have been widely sold and consumed in Japan. Amongthese foods, Foods for Specified Health Uses (FOSHU)
have shown scientific evidence-based beneficial effects
on physiological conditions in both healthy and diseased
individuals. Although most health foods contain ingredients that promote health and improve health-related
conditions, the effectiveness of these products has not* Correspondence: nagataki@tohoku-mpu.ac.jp
1
Department of Environmental and Health Science, School of Pharmaceutical
Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1
Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, Japan
Full list of author information is available at the end of the article

been proven. Consumers therefore select products basedon advertisements, which are usually non-scientific.
However, the consumption of health foods has increased
rapidly, because they are inexpensive and readily available in comparison with FOSHU. Reporting of adverse
effects has also increased in association with the expansion of the health food market [1]. Several studies havereported that drug-induced liver injury is caused not
only by prescription drugs but also by dietary and herbal
supplements [2, 3]. Consumers have recently become
more aware of the benefits and risks of using healthfoods, through information provided by relevant regulatory agencies. However, many consumers believe that

© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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Sasaki et al. Journal of Pharmaceutical Health Care and Sciences (2017) 3:14

products derived from natural substances are harmless.
Furthermore, some consumers use multiple health foods
at the same time. Provision of information that isaccompanied by scientific evidence on the safety andefficacy of health foods is essential to maintain publichealth and decrease medical expenses.
Health food-drug interactions are a particular concern
from the viewpoint of safe and effective use of healthfoods. Common interactions are associated with the
inhibition of drug-metabolizing enzymes, particularly
cytochrome P450s (CYPs, P450s). For example, green
tea extract has been reported to inhibit CYP2C9,CYP2D6, and CYP3A4 in human liver microsomes [4].
Epigallocatechin gallate, the most abundant catechin ingreen tee, is also a potent inhibitor of CYP3A in humanliver and intestinal microsomes [5]. While ginkgolide A,
B, C, J, and bilobalide, known as constituents of Ginkgo
biloba, have shown weak or negligible inhibition of
CYP1A2, CYP2C9, and CYP3A in human liver microsomes, other constituents, such as ginkgolic acid I andII, have been reported to inhibit CYP1A2, CYP2C9, andCYP2C19 [6, 7]. Although many reports have provided
useful information on the safe and effective use of healthfoods in patients taking drugs, most of this information
is limited to the effects of the health foods main ingredients on P450-mediated metabolism. However, healthfoods that are on the market in Japan contain a numberof ingredients, and such products may contain impurities. We recently reported the effects of health foods
that are available in Japan on CYP2D6-mediated metabolism [8]. We have confirmed that a product containing
curcumin is a potent inhibitor of CYP2D6. Coleus
forskohlii extract- and collagen-based products also inhibit CYP2D6. The inhibition by forskolin and collagen
had not been previously reported. Thus, it is difficult todeduce the effects of health foods on P450-mediatedmetabolism solely from the assessment of their main ingredients. We therefore believe that the development ofa P450 inhibition screening system for complete healthfood products, rather than their main ingredients, may
lead to more appropriate use of the products.
Human hepatocytes are recommended as the mostreliable tool for the assessment of drug metabolism anddrug-drug interactions [9]. However, due to their highcost and lot-to-lot variations in drug metabolism, it isdifficult to continuously obtain human primary hepatocytes that have the same metabolic activities, and therefore these cells are unsuitable for high-throughput
testing. In this study, we have utilized hepatocellular
carcinoma cells (HepG2 cells) and P450-expressing adenoviruses to establish cells (named Ad-P450 cells) thatmimic the activities of CYP1A2, CYP2C9, CYP2C19,
CYP2D6, and CYP3A4, which strongly contribute tohuman drug metabolism in human hepatocytes [10]. We

Page 2 of 11

have established a system to assess the inhibitory effectsof health foods on P450-mediated metabolism using AdP450 cells.

Methods
Reagents

Phenacetin, acetaminophen, dextromethorphan, dextrorphan, furafyllin, and sulfaphenazole were purchased from
Sigma-Aldrich (St. Louis, MO, USA). Quinidine waspurchased from Tokyo Chemical Industry (Tokyo, Japan).
4-hydroxydiclofenac and 1-hydroxymidazolam were purchased from Becton Dickinson (Franklin Lakes, NJ, USA).
5-Hydroxyomeprazole was purchased from Toronto
Research Chemical (North York, Canada). Ketoconazole
was purchased from LKT Laboratories (St. Paul, MN,
USA). Health foods were purchased from Japanese retailmarkets. All other reagents were of the highest grade
available from Wako Pure Chemical Industries (Osaka,
Japan) and Sigma-Aldrich. Oligonucleotides were commercially synthesized by Fasmac (Atsugi, Japan).
Construction of recombinant adenovirus

The open reading frames of CYP1A2, CYP2C9, CYP2C19,
and CYP3A4 were amplified by PCR from cDNA obtained from human hepatocytes by using primers specific for CYP1A2 (forward: 5′-CACCATGGCATTGTCCCAGTCTGTTC-3′; reverse: 5′-TCAGTTGATGGAGAAGCGCAGCCG-3′), CYP2C9 (forward: 5′-CACCATGGATTCTCTTGTGGTCCTTG-3′; reverse: 5′-TCA
GACAGGAATGAAGCACAGCTGGTAG-3′), CYP2C19(forward: 5′-CACCATGGATCCTTTTGTGGTCCTTG
TG-3′; reverse: 5′-TCAGACAGGAATGAAGCACAG
CTGA-3′), and CYP3A4 (forward: 5′-CACCATGGCTCTCATCCCAGACTTGGC-3′; reverse: 5′-TCAGGCTCCACTTACGGTGCCATC-3′), respectively. Constructsof these P450-expressing adenoviruses, Ad-CYP1A2, AdCYP2C9, Ad-CYP2C19, and Ad-CYP3A4, were madeaccording to the procedure described [11]. Preparation of
Ad-CYP2D6 was previously described [8].
Human hepatocytes and cell culture

Human cryopreserved primary hepatocytes (lot HEP187265,
54-year-old Caucasian woman) were purchased from Biopredic International (Rennes, France). The hepatocytes were
thawed and cultured using the medium kit (Biopredic International) according to the manufacturer’s protocol. The cells
were seeded in type I collagen-coated 48-well plate at 8.5 ×
104 cells/well. After 12 h, the cell medium was changed with
culture medium (Biopredic International) containing P450
substrate cocktail (100 μM phenacetin, 25 μM diclofenac,
10 μM omeprazole, 10 μM dextromethorphan, and 10 μM
midazolam) and then the cells were incubated for 24 h.

— 198 —

Sasaki et al. Journal of Pharmaceutical Health Care and Sciences (2017) 3:14

Preparation of HepG2 cells mimicking the activity levelsof CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in
human hepatocytes (Ad-P450 cells)

Page 3 of 11

Effect of health foods and curcumin on P450-mediatedmetabolism

HepG2 cells were purchased from RIKEN cell Bank
(Tsukuba, Japan) and were cultured in Dulbecco’s modified Eagle’s medium (Wako Pure Chemical Industries)
supplemented with 10% fetal bovine serum (Biowest,
Miami, FL, USA), non-essential amino acids (Thermo
Fisher Scientific, Maltham, MA, USA), and antibioticantimycotic (Thermo Fisher Scientific) under 5% CO295% air at 37 °C. The cells were seeded in a 48-well tissue culture plate (Becton Dickinson) at 5.0 × 104 cells/
well. After 48 h, the cells were simultaneously infectedwith Ad-CYP1A2 at 5 multiplicity of infection (MOI),
Ad-CYP2C9 at 1 MOI, Ad-CYP2C19 at 2 MOI, AdCYP2D6 at 0.05 MOI, and Ad-CYP3A4 at 10 MOI. The
cells were cultured for 72 h and subsequently used as
Ad-P450 cells. The cells were incubated in culture
medium containing P450 substrate cocktail for 5 h.

Assessment of P450 activity and inhibition in Ad-P450
cells

Ad-P450 cells were incubated in culture medium
containing phenacetin (2.5–100 μM), diclofenac (0.5–20 μM), omeprazole (0.25–10 μM), dextromethorphan(1–50 μM), or midazolam (0.25–10 μM) for 5 h. In
P450 inhibition assessments, Ad-P450 cells were incubated in culture medium containing P450 substrate
cocktail and each typical P450 inhibitors (furafylline for
CYP1A2 [0.05–30 μM], sulfaphenazole for CYP2C9[0.01–10 μM], ticlopidine for CYP2C19 [0.3–300 μM],
quinidine for CYP2D6 [0.01–10 μM], or ketoconazole
for CYP3A4 [0.01–10 μM]) for 5 h.

Preparation of health food extracts and curcumin

Recommended daily dose of health foods (tablet, capsule, and powder) was incubated in 10 mL of 70%
ethanol for 2 h at 37 °C. The resulting solution wascentrifuged at 3500 × g for 15 min and the supernatant was used as a test solution. Liquid-type healthfoods were used as test solutions without extraction
procedure. Curcumin was dissolved in dimethyl sulfoxide (DMSO).

Ad-P450 cells were incubated in culture medium containing P450 substrate cocktail and test solution (0.5%)
or curcumin (10–300 μM) for 5 h.
Liquid chromatography/tandem mass spectrometry (LCMS/MS) analysis

Collected medium was mixed with an equal volume ofethyl acetate containing 0.1 μM nitrazepam (internal
standard). After shaking, the mixtures were centrifuged at2500 × g for 10 min and the supernatants were evaporated
to dryness at 60 °C in a block incubator. The residues weredissolved in acetonitrile containing 0.1% acetic acid andthe solutions were subjected to liquid chromatography/
tandem mass spectrometry (LC-MS/MS) analysis.
The Prominence system (Shimadzu Corporation,
Kyoto, Japan) equipped with LCMS-8040 system (Shimadzu Corporation) was used for LC-MS/MS analysis
with an electrospray ionization interface. The ionization
mode used was positive in the multiple reaction monitoring. The chromatographic separation was performedon Xterra MS C18 columns (2.1 mm × 100 mm, 5 μm)(Waters, Milford, MA, USA). The column oventemperature was maintained at 40 °C. The mobile phaseconsisted of acetonitrile containing 0.1% acetic acid andwater containing 0.1% acetic acid (85:15) with a flow rateof 200 μL/min. The LC-MS/MS conditions were shownin Table 1. Desolvation line temperature and heat block
temperature was 250 and 400 °C, respectively. Nebulizer
gas flow rate and drying gas flow rate were 3 and 15 L/
min, respectively.
Data analysis

The Km, Vmax, and IC50 values were determined using
Prism software (version 6.0; GraphPad Software Inc.,San Diego, CA, USA). The other calculations were performed using Excel (Microsoft, Seattle, MA, USA).

Results
Establishment of Ad-P450 cells

The activity levels of CYP1A2 (phenacetin O-deethylation activity), CYP2C9 (diclofenac 4′-hydroxylation activity), CYP2C19 (omeprazole 5-hydroxylation activity),CYP2D6 (dextromethorphan O-demethylation activity),

Table 1 MS/MS selected reaction monitoring transitions and collision energies for ions of metabolite and internal standard in the
cocktail assays
CYP1A2CYP2C9CYP2C19

CYP2D6

CYP3A4

Substrate

Phenacetin

Diclofenac

Omeprazole

Dextromethorphan

Midazolam

Nitrazepam

Metabolite

Acetaminophen

4′-Hydroxy-diclofenac

5-Hydroxy-omeprazole

Dextrorphan

1′-Hydroxy-midazolam-

Collision energy (eV)

19

33

13

42

23

25

Product ion (m/z)

110.05229.95213.95157.05324.05236.10— 199 —

IS

Sasaki et al. Journal of Pharmaceutical Health Care and Sciences (2017) 3:14

Activity (pmol/well/h)

1000

Ad-P450 cells
Human hepatocytes

100
10

1
0.1

CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4

Fig. 1 Activity levels of CYP1A2, CYP2C9, CYP2C19, CYP2D6, andCYP3A4 in Ad-P450 cells and human hepatocytes. Five human
P450s were expressed in HepG2 cells as described in section titled‘Materials and methods’. The cells (Ad-P450 cells) were cultured
for 72 h and then incubated in culture medium containing P450
substrate cocktail for 5 h. Human cryopreserved primary hepatocytes
seeded in type I collagen-coated 48-well plate at a density of
8.5 × 104 cells/well were incubated with culture medium containing
P450 substrate cocktail for 24 h. These media were collected andmetabolites were analyzed by LC-MS/MS. The activity levels are shownas means ± SD (n = 3)

Page 4 of 11

and CYP3A4 (midazolam 1′-hydroxylation activity) in
human hepatocytes were calculated via simultaneous
LC-MS/MS analysis utilizing the P450 substrate cocktail.
The activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6,
and CYP3A4 were 59.48, 106.71, 0.94, 0.17, and 3.86
pmol/well/h, respectively (Fig. 1). Based on these data,
we simultaneously infected HepG2 cells with AdCYP1A2 (5 MOI), Ad-CYP2C9 (1 MOI), Ad-CYP2C19(2 MOI), Ad-CYP2D6 (0.05 MOI), and Ad-CYP3A4 (10MOI) to mimic the activity levels of CYP1A2, CYP2C9,CYP2C19, CYP2D6, and CYP3A4 in human hepatocytes.
The activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6,
and CYP3A4 in the infected HepG2 cells were 31.22,
77.02, 0.66, 0.16, and 2.33 pmol/well/h, respectively(Fig. 1). Although the P450 activities in HepG2 cells
infected with five P450-expressing adenoviruses wereslightly lower in comparison with those in human hepatocytes, the ratios of the activities of each P450
were almost identical between these two cells (Fig. 1).
We thus named the established HepG2 cells mimicking the activity levels of CYP1A2, CYP2C9,CYP2C19, CYP2D6, and CYP3A4 in human hepatocytes, Ad-P450 cells.

Fig. 2 Kinetic analysis of P450-mediated metabolism in Ad-P450 cells. Ad-P450 cells were incubated in culture medium containing phenacetin(2.5–100 μM), diclofenac (0.5–20 μM), omeprazole (0.25–10 μM), dextromethorphan (1–50 μM), or midazolam (0.25–10 μM) for 5 h. These media
were collected and metabolites were analyzed by LC-MS/MS. The activity levels are shown as means ± SD (n = 3)

— 200 —

Sasaki et al. Journal of Pharmaceutical Health Care and Sciences (2017) 3:14

Page 5 of 11

Fig. 3 Effect of representative inhibitors on P450-mediated metabolism in Ad-P450 cells. Ad-P450 cells were incubated in culture medium containing P450 substrate cocktail and each typical P450 inhibitor (furafylline [0.05–30 μM], sulfaphenazole [0.01–10 μM], ticlopidine [0.3–300 μM],
quinidine [0.01–10 μM], or ketoconazole [0.01–10 μM]) for 5 h. These media were collected and metabolites were analyzed by LC-MS/MS. The activity levels are shown as means ± SD (n = 3)

are useful for the assessment of P450-mediated drug metabolism, and drug-drug or health food-drug interactions.

Kinetic analysis and inhibition assessment in Ad-P450
cells

To investigate the properties of Ad-P450 cells, we performed kinetic analyses using P450 substrates and calculated IC50 values for specific representative P450 inhibitors(CYP1A2, furafylline; CYP2C9, sulfaphenazole; CYP2C19,
ticlopidine; CYP2D6, quinidine; CYP3A4, ketoconazole) in
Ad-P450 cells (Figs. 2 and 3). The Michaelis-Menten
equation was used to calculate the Km and Vmax values
from the metabolic reaction rates of the five P450s (Table 2).
The specific P450 inhibitors used showed concentrationdependent inhibition, and the IC50 values are shown in
Table 2. These values are similar to those previously
reported [12, 13]. These results suggest that Ad-P450 cells

Effects of health foods on P450-mediated metabolism

We investigated the inhibitory effects of 172 health foods,
whose uses in Japan were confirmed in our previous survey [8], on P450-mediated metabolism in Ad-P450 cells.
The results, classified by main ingredients or expected
effects, are shown in Fig. 4 and are summarized in Table 3.
Products that inhibited any P450 by more than 50% wereconsidered to have P450 inhibitory activity.
Of the 172 products, five products [two products having
dietary effects (diet, no. 65 and 66), one turmeric-based
product (no. 125), one collagen-based product (no. 152),

Table 2 Kinetic parameters of P450-mediated drug metabolism and IC50 values of P450 inhibitors in Ad-P450 cells
EnzymeMetabolic reaction

Inhibitor

Km (μM)

Vmax (pmol/well/hr)

IC50 (μM)

CYP1A2

Phenacetin O-deethylation

Furafylline

16.32

100.300.57

CYP2C9Diclofenac 4′-hydroxylation

Sulfaphenazole

5.87

239.9

0.53

CYP2C19

Omeprazole 5-hydroxylation

Ticlopidine

2.75

4.66

1.11

CYP2D6

Dextromethorphan O-demethylation

Quinidine

17.14

1.10

0.14

CYP3A4

Midazolam 1′-hydroxylation

Ketoconazole

1.00

12.30

0.12

— 201 —

Sasaki et al. Journal of Pharmaceutical Health Care and Sciences (2017) 3:14

Page 6 of 11

Fig. 4 Effect of health foods on P450-mediated metabolism in Ad-P450 cells. Ad-P450 cells were incubated in culture medium containing P450 substrate
cocktail and test solution (0.5%) for 5 h. These media were collected and metabolites were analyzed by LC-MS/MS. The P450s activities in Ad-P450 cells
cultured in P450 substrate cocktail and 0.5% extractant (70% ethanol) for 5 h were set to 100%. The activity levels are shown as means ± SD (n = 3)

— 202 —

Sasaki et al. Journal of Pharmaceutical Health Care and Sciences (2017) 3:14

and one other product (propolis-containing product,
no. 168)] simultaneously inhibited the five P450s by
more than 50%. Two sesamin-based products (no.
135 and 136) simultaneously inhibited the four P450s
except CYP1A2. Five products [collagen-based (no.
36), diet (no. 70), turmeric-based (no. 124), St. John’swort (SJW)-based (no. 145 and 146)] simultaneously
inhibited three P450s (CYP1A2, CYP2C9/CYP2D6,
and CYP3A4). A vitamin-, an isoflavone, and a vitamin/mineral-based product (no. 15, 62, and 131)
simultaneously inhibited two P450s (CYP1A2 andCYP2C9). In addition, nineteen other products were
found to inhibit one of the five P450s. Therefore, ourresults demonstrated that 34 (19.8%) of 172 healthfoods have P450 inhibitory activities.
Effects of curcumin on P450-mediated metabolism

Of the five products that simultaneously inhibitedthe five P450s, we further examined the effects of
their ingredients in the turmeric-based products on
P450-mediated metabolism. Curcumin is a known
constituent of turmeric and a P450 inhibitor [14]. In
our system using Ad-P450 cells, curcumin inhibitedthe five P450s in a concentration-dependent manner(Fig. 5). The residual activities in Ad-P450 cells
treated with 50 μM curcumin were 58.3% for
CYP1A2, 25.9% for CYP2C9, 72.7% for CYP2C19,
71.9% for CYP2D6, and 61.9% for CYP3A4. Similar
to the results of turmeric-based products, CYP2C9
was most strongly inhibited by curcumin, although
this inhibition was observed only at a relatively highconcentration.

Discussion
In order to comprehensively elucidate health fooddrug interactions, we developed Ad-P450 cells mimicking the activity levels of CYP1A2, CYP2C9,CYP2C19, CYP2D6, and CYP3A4 in human hepatocytes and established a P450 inhibition assessment
system. Of the 172 health food products tested, fiveproducts simultaneously inhibited all the five P450s,
and another 29 products inhibited at least one of theP450s. Furthermore, the results of the inhibition of
CYP2D6 in Ad-P450 cells were consistent with thoseobtained using Ad-CYP2D6-infected cells in our previous study [8].
To develop Ad-P450 cells that mimicked the drugmetabolizing activity of human hepatocytes, the fiveP450s that most strongly contribute to drug metabolism in human livers were expressed in HepG2 cells at
levels showing the same activity as those of humanhepatocytes (Fig. 1). We determined the Km values
for typical P450 reactions and IC50 values of representative P450 inhibitors in the Ad-P450 cells. These

Page 7 of 11

values were approximately equivalent to those obtained in previous studies (Figs. 2 and 3, Table 2) [12,13]. These results show that the Ad-P450 cells areuseful tools to assess drug metabolism and healthfood-drug interactions.
Based on the data from our previous survey [8], we
investigated the effects of health foods, for which
actual use in Japan has been confirmed, on the fiveP450s. Our results showed that five products (twodiet products, one turmeric-based product, one
collagen-based product, and one propolis-containing
product) inhibited the five P450s by more than 50%(Fig. 4). The two diet products (no. 65 and 66) contained C. forskohlii extract powder (containing 10%
forskolin). However, there are few reports concerning
their P450 inhibitory activities. These results suggestthat unidentified ingredients might be involved in
P450 inhibition, since these two diet products do notinclude common ingredients other than C. forskohlii
extract.
A turmeric-based product (no. 125) also inhibitedthe five P450s, with CYP2C9 being the most inhibited. Curcumin is a polyphenolic component in turmeric that inhibits CYP1A2, CYP2B6, CYP2C9,CYP2D6, and CYP3A4, and its IC50 values are particularly low for CYP2C9 [14]. Since the product was
obtained from Curcuma longa, which is curcuminrich, and strong inhibitory activity of other turmericbased products (no. 124 and 126) with little/no C.
longa had not been observed, the inhibition of thefive P450s by the turmeric-based product was thought
to be due to curcumin. In this study, we confirmedthat curcumin inhibited the five P450s in aconcentration-dependent manner in Ad-P450 cells(Fig. 5). However, strong inhibition was found only at
relatively high concentrations. These results suggestthat P450 inhibition by the turmeric-based productcould also be associated with ingredients other thancurcumin.
Based on the product labels, the collagen-based product (no. 152) that showed inhibitory activities toward thefive P450s contained silibinin in addition to collagen, butother collagen-based products (no. 29–38 and 151) did
not. Silibinin has been reported to inhibit CYP2C9 and
CYP3A4 through mechanism-based inhibition (MBI)[15]. It was recently reported that a metabolite of rutaecarpine, a principal constituent of Evodia rutaecarpa,
strongly inhibits P450s [16], suggesting that health fooddrug interactions could be caused by P450 inhibition
through MBI.
We also revealed that two sesamin-based products (no.
135 and 136) inhibited CYP2C9, CYP2C19, CYP2D6, andCYP3A4. Sesamin is a known competitive inhibitor of
CYP1A2, CYP2C9, and CYP3A4 and the reported Ki— 203 —

Sasaki et al. Journal of Pharmaceutical Health Care and Sciences (2017) 3:14

Page 8 of 11

Table 3 Health foods with inhibitory activity and residual activity of P450s
Product classification
Vitamin

Glucosamine
CollagenNumbera
4/19

2/11
3/12

Product nameInhibited P450

Relative activity (%)b
CYP1A2CYP2C9CYP2C19

CYP2D6

CYP3A4

Vitamin-4

CYP2C9

73.0

8.6

―

62.8

64.0

Vitamin-11

CYP2C9

95.1

18.1

―

―

65.4

Vitamin-13

CYP2C9

92.4

23.3

―

88.1

65.3

Vitamin-15

CYP1A2, CYP2C9

38.2

7.3

―

62.6

72.6

Glucosamine-27

CYP1A2

45.1

83.6

92.4

65.6

56.2

Glucosamine-28

CYP1A2

36.4

70.0

70.2

50.7

53.2

Collagen-29

CYP2C9

96.1

31.8

―

81.4

71.4

Collagen-36

CYP2C9, CYP2D6, CYP3A4

61.4

27.7

69.1

46.2

42.2

Collagen-152

CYP1A2, CYP2C9, CYP2C19,
CYP2D6, CYP3A4

26.3

8.0

30.4

35.6

26.6

Mineral-39

CYP2C9

52.3

41.5

―

―

70.1

Mineral

1/14

Plant

1/6

Plant-57

CYP2C9

58.1

28.4

63.6

63.5

98.3

Isoflavone

1/10

Isoflavone-62

CYP1A2, CYP2C9

35.7

8.5

―

97.4

75.0

Diet

6/9

Diet-65

CYP1A2, CYP2C9, CYP2C19,
CYP2D6, CYP3A4

22.3

3.8

32.4

34.7

23.9

Diet-66

CYP1A2, CYP2C9, CYP2C19,
CYP2D6, CYP3A4

24.2

3.4

20.4

19.7

17.1

Diet-67

CYP2C9

76.5

44.3

―

77.6

55.1

Diet-68

CYP2C9

77.4

46.3

99.1

75.8

55.3

Diet-69

CYP2C9

94.7

48.5

―

68.2

60.0

Diet-70

CYP1A2, CYP2C9, CYP3A4

49.1

19.0

68.4

65.3

40.2

Garlic

1/10

Garlic-160

CYP1A2

45.2

77.3

―

53.4

56.3

Lutein/berry

0/11

―

―

―

―

―

―

―

Aojiru

0/10

―

―

―

―

―

―

―

Amino acid

0/6

―

―

―

―

―

―

―

Chlorella

0/7

―

―

―

―

―

―

―

Lactic acid bacterium

0/5

―

―

―

―

―

―

―

Other

1/3

Other-168

CYP1A2, CYP2C9, CYP2C19,
CYP2D6, CYP3A4

7.6

5.3

0.0

20.9

32.5

Oyster

0/3

―

―

―

―

―

―

―

Vinegar

1/6

Vinegar-169

CYP2C9

―

46.4

70.8

83.9

59.6

CoQ10

0/4

―

―

―

―

―

―

―

Turmeric

3/4

Turmeric-124

CYP1A2, CYP2C9, CYP3A4

47.2

49.4

67.6

66.4

30.2

Turmeric-125

CYP1A2, CYP2C9, CYP2C19,
CYP2D6, CYP3A4

19.2

5.6

10.2

35.4

25.1

Turmeric-126

CYP1A2

18.3

70.9

85.7

61.8

57.8

DHA

0/3

―

―

―

―

―

―

―

Vitamin/mineral

2/3

Vitamin/mineral-131

CYP1A2, CYP2C9

49.2

7.3

89.6

71.7

59.0

Vitamin/mineral-133

CYP2C9

―

28.4

―

―

71.5

Enzyme

1/1

Enzyme-134

CYP2D6

―

94.6

―

36.6

62.5

Sesamin

3/3

Sesamin-135

CYP2C9, CYP2C19, CYP2D6,
CYP3A4

56.8

12.5

25.6

31.5

31.4

Sesamin-136

CYP2C9, CYP2C19, CYP2D6,
CYP3A4

58.5

15.7

32.4

43.4

31.0

Sesamin-172

CYP2C9

83.0

49.4

62.8

57.3

51.1

Vegetable-139

CYP1A2

36.8

86.8

97.4

―

71.5

Vegetable

1/3— 204 —

Sasaki et al. Journal of Pharmaceutical Health Care and Sciences (2017) 3:14

Page 9 of 11

Table 3 Health foods with inhibitory activity and residual activity of P450s (Continued)
Royal jelly

0/3

―

―

―

―

―

―

―

Animal1/1

Animal-143

CYP2C9

88.5

24.6

―

88.6

61.5

Tea

0/1

―

―

―

―

―

―

―

SJW

2/2

SJW-145

CYP1A2, CYP2C9, CYP3A4

26.5

31.3

78.0

57.0

32.5

SJW-146

CYP1A2, CYP2C9, CYP3A4

21.4

22.3

64.3

53.8

29.9

α-Lipoic acid

0/2

―

―

―

―

―

―

―

a

Number of products with inhibitory activity/Number of products examinedb
P450s that were not inhibited by products are shown as ―values are 75, 24, and 4.2 μM, respectively [17]. Our
results support the previous study, since the inhibition of
CYP1A2 by two sesamin-based products was weaker thanthat of other P450s.
We confirmed that one isoflavone-based (no. 62) and
one vitamin-based (no. 15) product inhibited CYP1A2
and CYP2C9. The isoflavone-based product contained
isoflavones derived from soybean and red clover, such as
genistein and biochanin A, which reportedly inhibit
CYP1A2 [18, 19]. In contrast, few reports are available
on P450 inhibition by vitamins in vitamin-based
products, although lipid-soluble vitamins, such as vitamin A and vitamin D, have been reported to induce
CYPs [20, 21]. Based on the product label, the vitaminbased product (no. 15) does not contain vitamin A andvitamin D, while the amounts of water-soluble vitamins,
such as vitamins B1, B2, B6, B12, and pantothenic acid,
were higher than those in other vitamin-based products.
Although further study is needed to elucidate the P450

inhibition by vitamin-based product, the use of excessive
amounts of water-soluble vitamins should be avoided toprevent health food-drug interactions.
In this study, we established a system to assess the inhibitory effects of health foods on P450-mediated metabolism using Ad-P450 cells. In contrast to the assessmentof health food-drug interactions using human hepatocytes,
assessment using Ad-P450 cells might not provide an accurate prediction of the interaction because of the limited
number of P450s expressed in Ad-P450 cells. However,this established assessment system is easily applicable tohealth food-drug interactions testing for many healthfoods, because Ad-P450 cells are inexpensive with little tono lot-to-lot variations. In comparison with common
assessment systems of health food-drug interactions
using liver microsomes, our established system is including absorption process of chemical compounds inhealth foods into cells [11]. Moreover, this system has
a valuable advantage, where it can mimic hepatocytes

Fig. 5 Effect of curcumin on P450-mediated metabolism in Ad-P450 cells. Ad-P450 cells were incubated in culture medium containing P450 substrate
cocktail and curcumin (10–300 μM) for 5 h. These media were collected and metabolites were analyzed by LC-MS/MS. The P450 activities in
Ad-P450 cells cultured in P450 substrate cocktail and DMSO (0.5%) for 5 h were set to 100%. The activity levels are shown as means ± SD (n = 3). The
limit of quantitation is 18.90 ng/mL for acetaminophen, 39.02 ng/mL for 4′-hydroxydiclofenac, 0.24 ng/mL for 5-hydroxyomeprazole, 0.25 ng/mL for
dextrorphan, and 2.14 ng/mL for 1′-hydroxymidazolam. N.D.: not detected— 205 —

Sasaki et al. Journal of Pharmaceutical Health Care and Sciences (2017) 3:14

of P450-mediated metabolism, which showed interindividual variations by adjusting the ration of infection
amount and species of P450-expressing adenovirus[22]. Furthermore, it is of great importance to assess
the effects of health foods on the five P450s thatmost strongly contribute to drug metabolism in human livers in order to obtain beneficial and fundamental information under the current situation where
there is limited scientific evidence regarding healthfood-drug interactions.

Conclusions
We established a comprehensive assessment system toinvestigate the effects of health foods on P450-mediatedmetabolism and found that 34 of the 172 health foods
have the potential to inhibit human P450 activities. This
report is the first to investigate the P450 inhibitory effects of a large number of health foods under the same
conditions. Our results provide useful information tounderstand and predict health food-drug interactions.
Abbreviations
Ad: Adenovirus; FOSHU: Foods for Specified Health Uses; LC-MS/MS: Liquid
chromatography/tandem mass spectrometry; MBI: Mechanism-based
inhibition; MOI: Multiplicity of infection; PCR: Polymerase chain reaction
Acknowledgements
Not applicable
Funding
This work was supported by a Health and Labor Sciences Research Grant for
Research on Food Safety (No. 22230301) from the Ministry of Health, Labor,and Welfare, Japan.
Availability of data and material
Data sharing not applicable to this article as no datasets were generated oranalyzed during the current study.
Authors’ contributions
TS and KN participated in the research design. TS and YS conducted
experiments. TS, YS, and TK performed the data analysis. TS and KN
contributed new reagents or analytic tools. TS, KY, and TK wrote orcontributed to the writing of the manuscript. All authors read and approved
the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not applicable
Ethics approval and consent to participate
Not applicablePublisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.
Author details
1
Department of Environmental and Health Science, School of Pharmaceutical
Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1
Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, Japan. 2Department of
Molecular Toxicology, School of Pharmaceutical Sciences, University of
Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan.

Page 10 of 11

Received: 23 February 2017 Accepted: 19 April 2017

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European Journal of Medicinal Chemistry 121 (2016) 592e609

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistryjournal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Synthesis and biological evaluation of novel FK228 analogues aspotential isoform selective HDAC inhibitors
Koichi Narita a, Keisuke Matsuhara a, Jun Itoh a, Yui Akiyama a, Singo Dan b,
Takao Yamori b, c, Akihiro Ito d, Minoru Yoshida d, Tadashi Katoh a, *a

Laboratory of Synthetic and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1
Komatsushima, Aoba-ku, Sendai 981-8558, Japan
Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-10-6 Ariake, Koto-ku, Tokyo 135-8550,
Japanc
Pharmaceuticals and Medical Devices Agency (PMDA), 3-3-2 Kasumigaseki, Chiyoda-ku, Tokyo 100-0013, Japand
Chemical Genetics Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japanba r t i c l e i n f o

a b s t r a c t

Article history:
Received 24 February 2016
Received in revised form
14 May 2016
Accepted 16 May 2016
Available online 18 May 2016

Novel C4- and C7-modiﬁed FK228 analogues were efﬁciently synthesized in a highly convergent anduniﬁed manner. This synthesis features the amide condensation of glycine-D-cysteine-containing segments with D-valine-containing segments for the direct assembly of the corresponding seco-acids, which
are key precursors of macrolactones. The HDAC inhibition assay and cell-growth inhibition analysis of thesynthesized analogues revealed novel aspects of their structure-activity relationship. This study
demonstrated that simple modiﬁcation at the C4 and C7 side chains in FK228 is effective for improving
both HDAC inhibitory activity and isoform selectivity; moreover, potent and highly isoform-selectiveclass I HDAC1 inhibitors were identiﬁed.
© 2016 Elsevier Masson SAS. All rights reserved.

Keywords:
FK228 analogues
Histone deacetylase inhibitors
Structure-activity relationshipBicyclic depsipeptide
Total synthesis

1. Introduction
The reversible acetylation and deacetylation of histones plays animportant role for regulating gene expression by altering chromatin architecture [1]. Histone acetyltransferases (HATs) catalyse
the transfer of acetyl groups from acetyl-CoA to the ε-amino groups
on histone lysine residues, which relaxes the chromatin structureand epigenetically promotes gene transcription. Conversely, histone deacetylases (HDACs) catalyse the removal of acetyl groups
from the N-acetyl lysine residues on histones, which contracts thechromatin structure and epigenetically suppresses gene transcription. The inhibition of HDAC enzymatic activity has been demonstrated to affect transcriptional events that are involved in growtharrest, differentiation, proliferation, cell cycle regulation, protein
turnover and/or apoptosis in transformed tumour cell cultures.
Consequently, HDAC-inhibiting compounds are considered to bepotential drugs for targeted cancer therapy [2].

* Corresponding author.
E-mail address: katoh@tohoku-mpu.ac.jp (T. Katoh).

There are 18 human HDAC isoforms, which are grouped into
four major classes: class I (HDACs 1, 2, 3 and 8), class II (class IIa:
HDACs 4, 5, 7 and 9; class IIb: HDACs 6 and 10), and class IV (HDAC
11) are Zn2þ-dependent metallohydrolases, whereas class III HDACs(SirTs 1e7) are NADþ-dependent sirtuins [3]. Inhibiting class I
HDACs is considered to be an effective mechanism for anticancer
agents, whereas inhibiting class II HDACs may cause undesirable
side effects such as serious cardiac hypertrophy [4]; therefore, incancer chemotherapy, the potent and selective inhibition of class I
HDACs is highly desirable.
In 2009, the U.S. Food and Drug Administration (FDA) approved
the bicyclic depsipeptide HDAC inhibitor FK228 (romidepsin, 1,
Fig. 1) for treating cutaneous T-cell lymphoma and other peripheral
T-cell lymphomas [5]; however, FK228 is known to be associatedwith an unresolved cardiotoxicity issue [5b,6], which highlights therequirement for further identifying and developing new HDAC inhibitors with high efﬁcacy and low toxicity. FK228 was ﬁrst
discovered by researchers from Fujisawa Pharmaceutical Co. Ltd.
(now Astellas Pharma Inc.) in 1994, who isolated it from a culture
broth of Chromobacterium violaceum (No. 968) [7]. Yoshida et al.
proposed a molecular mechanism by which FK228 inhibits HDAC

http://dx.doi.org/10.1016/j.ejmech.2016.05.031
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

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593[13a,14c,15b,16,18a], we became interested in synthesizing FK228
analogues and evaluating their isoform selectivity in order to
identify the potent and class I-selective HDAC inhibitors. There areseveral studies in the literature on synthesizing FK228 analogues[20]. Note that the synthesis and evaluation of FK228 analogues,
which was reported by Ganesan et al., revealed novel aspects of
their structureeactivity relationship (SAR) [20c]. From this SAR
study, three primary critical features that were related to the HDAC
inhibitory activity of 1 were identiﬁed; i) the 16-memberedmacrocyclic scaffold is indispensable, ii) the unsaturated Dhb
moiety can be replaced with other amino acids and iii) the C4
isopropyl group in the L-valine moiety is not always necessary.
Although these results are very useful for designing novel FK228
analogues, no information on the effects of the stereochemistry in
the amino acid components was reported.
Based on the results presented by Ganesan et al., we designed eightnovel C4- and C7-modiﬁed FK228 analogues, which were denoted as
FK-A1 to FK-A8 (13aeh, Fig. 2). Note that the L-valine and Dhb moieties of 1 were replaced by simple amino acids such as glycine, D-/Lalanine, D-/L-phenylalanine and D-/L-leucine. We envisaged that
13aeh could be readily synthesized via the total synthesis we had
previously developed for bicyclic depsipeptide natural products.
In this study, we describe the synthesis of FK228 analogues
13aeh using a synthetic strategy that was developed previously in
our laboratory. Then, to investigate the effect of the C7 stereochemistry and amino acid substituent groups, these synthesizedanalogues were subjected to an HDAC inhibition assay and antiproliferative analysis.
2. Results and discussion
2.1. Chemistry
2.1.1. General synthetic strategy for the novel FK228 analogues FKA1 to FK-A8 (13aeh)
The selection of the method that was used for constructing the

Fig. 1. Structures of FK228 (romidepsin; 1), spiruchostatins AeD (2e5), FR901375 (6),burkholdacs A (thailandepsin C; 7) and B (thailandepsin A; 8), and thailandepsins E (9),B (10), D (11) and F (12). i-Pr ¼ isopropyl, s-Bu ¼ sec-butyl, i-Bu ¼ isobutyl.

[8]. In that mechanism, FK228, which itself serves as a stable prodrug, is activated by reductive cleavage of the disulﬁde bond afterincorporation into the cells, and the released sulfhydryl group in
the butenyl side chain interacts with the zinc cation located at thebinding pocket of the HDACs, resulting in a potent inhibitory effect.
The structural features of FK228 are a 16-membered bicyclic depsipeptide comprising dehydrobutyrine (Dhb), L-valine, D-cysteine,
D-valine, (3S,4E)-3-hydroxy-7-mercapto-4-heptenoic acid (Hmh)and a characteristic disulﬁde bond linkage. Since the discovery ofFK228, several structurally similar bicyclic depsipeptide HDAC inhibitors have been isolated from bacterial fermentations, including
spiruchostatins AeD (2e5) [9], FR901375 (6) [10], burkholdacs A (7)and B (8) [11] and thailandepsins AeF (7e12) [12] (thailandepsins A
and C are identical to burkholdacs B and A, respectively). The
potentially therapeutic biological properties and unique structuralfeatures of 1e12 have made them attractive targets for total synthesis, and several such syntheses of these natural products havebeen published in the literature [13e19].
During our research on the synthesis and biological evaluationof bicyclic depsipeptide HDAC inhibitors such as 1e5, 7 and 8

Fig. 2. Structures of the novel FK228 analogues FK-A1 to FK-A8 (13aeh). Bn ¼ benzyl.

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16-membered macrocyclic ring is crucial to successfully achieve thetotal synthesis of this class of natural products. In our previously
reported total syntheses of FK228 (1) [13a], spiruchostatins AeD(2e5) [14c,15b,16] and burkholdacs A (7) and B (8) [18a], we
employed two different reliable methods such as Shiina’s macrocyclization [21] and Mitsunobu macrocyclization [22]. For synthesizing 2e5, 7 and 8, we employed Shiina’s macrocyclization,
whereas for synthesizing 1, we employed Mitsunobu macrocyclization. Note that Shiina’s macrocyclization was superior to the
Mitsunobu macrocyclization in terms of chemical yield, reaction
cleanness and ease of puriﬁcation; however, in the case of 1, allattempts to conduct Shiina’s macrocyclization proved unsuccessful,
most possibly because of the sterically hindered isopropyl group in
the L-valine unit. Remarkably, using Shiina’s macrolactonization,
Ganesan et al. successfully synthesized FK228 analogues in which
the L-valine moiety was replaced by glycine [20c]; therefore, we
decided to perform the key macrolactonization using Shiina’s
method in our synthesis.
Our general synthetic plan is outlined in Scheme 1. We envisioned that the target molecules 13aeh could be synthesized by
Shiina’s macrolactonization of the corresponding seco-acids 14aeh,
followed by internal disulﬁde bond formation. The key element of
this scheme is the highly convergent assembly of 14aeh by the
direct condensation of the glycine-D-cysteine-containing segments
15aeh and the D-valine-containing segment 16. Segments 15aeh
are formed by the condensation of the D-cysteine derivative 17,amino acid 18 and glycine methyl ester hydrochloride (19). The
remaining segment 16 is obtained using our previously described
total synthesis of 1 [13a] and 5 [16].
2.1.2. Synthesis of FK-A1 (13a)First, the synthesis of segment 15a was performed from L-

Scheme 1. Retrosynthetic analysis for 13aeh. Tr ¼ triphenylmethyl, PMB ¼ 4methoxylbenzyl, Boc ¼ tert-butoxycarbonyl.

threonine (18a), as shown in Scheme 2. Protection of the aminogroup in 18a with a 9-ﬂuorenylmethoxycarbonyl (Fmoc) group,
which was followed by condensation of the resulting carbamate 20
with glycine methyl ester hydrochloride (19), provided the desired
dipeptide 21 in 88% yield over the two steps. After deprotection of
the N-Fmoc group in 21, the free amine 22 was subjected tocondensation with the D-cysteine derivative 17, which resulted in
the tripeptide 23 in 53% yield over the two steps. At this stage, thethreonine part of 23 is converted to an unsaturated Dhb moiety via
a two-step sequence that involves mesylation of the hydroxy group(94% yield) followed by elimination of the resulting mesylate 24(89% yield), thus affording the desired product 25. Deprotection of
the N-Boc group in 25 is efﬁciently achieved using BF3$OEt2, leading
to the desired segment 15a in 88% yield.
Subsequently, the synthesis of FK-A1 (13a) was accomplished byassembling the two segments 15a and 16, as shown in Scheme 3.
Treating 15a and 16 with HATU (1.3 equiv) and HOAt (1.3 equiv) in
the presence of i-Pr2NEt (2.6 equiv) in CH2Cl2/MeCN at �20 � C for
5 h afforded the desired condensation product 26a in 80% yield
without appreciable epimerization at the C21 stereogenic centre[13a]. Then, the condensation product 26a was converted to therequired seco-acid 14a with an overall yield of 87% via the successive removal of both the PMB and methyl-protecting groups. The
critical macrolactonization of 14a is successfully performed using
Shiina’s method [MNBA (1.3 equiv), DMAP (3.0 equiv), CH2Cl2(1.0 mM), rt, 12 h] [21], and the desired cyclization product 28a is
obtained in 64% yield. Finally, disulﬁde bond formation accompanied by oxidative S-Tr deprotection of 28a is efﬁciently achieved bybrief exposure to I2 in a dilute CH2Cl2/MeOH solution (0.5 mM) atroom temperature, which results in the production of the desired
FK-A1 (13a) in 97% yield.
2.1.3. Synthesis of FK-A2 to FK-A8 (13beh)
As shown in Scheme 4, using glycine (18b: R1 ¼ R2 ¼ H), D-/Lalanine (18c,d: R1 ¼ a-/b-Me, R2 ¼ H), D-/L-phenylalanine (18e,f:
R1 ¼ a-/b-Bn, R2 ¼ H) and D-/L-leucine (18g,h: R1 ¼ a-/b-i-Bu,
R2 ¼ H) as starting materials, the remaining FK228 analogues, i.e.

Scheme 2. Synthesis of segment 15a. a) FmocCl, 10% aq. Na2CO3, dioxane, rt; b) 19,
PyBOP, i-Pr2NEt, MeCN, rt, 88% (2 steps); c) Et2NH, MeCN, rt; d) 17, PyBOP, i-Pr2NEt,
MeCN, rt, 53% (2 steps); e) MsCl, Et3N, CH2Cl2, 0 � C, 94%; f) DABCO, THF, rt, 89%; g)
BF3$Et2O, CH2Cl2, rt, 88%. FmocCl ¼ 9-ﬂuorenylmethoxycarbonyl chloride,
PyBOP ¼ (benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexaﬂuorophosphate,
MsCl ¼ methanesulfonyl chloride, DABCO ¼ 1,4-diazabicyclo-[2.2.2]octane.

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K. Narita et al. / European Journal of Medicinal Chemistry 121 (2016) 592e609

Scheme 3. Synthesis of FK-A1 (13a). a) HATU, HOAt, i-Pr2NEt, CH2Cl2/MeCN, �20 � C,
80%; b) DDQ, CH2Cl2/H2O, rt, 90%; c) LiOH, THF/H2O, 0 � C, 97%; d) MNBA, DMAP,
CH2Cl2, rt, 64%; e) I2, MeOH/CH2Cl2, rt, 97%. HATU ¼ O-(7-azabenzotriazol-1-yl)hexaﬂuorophosphate,HOAt¼
1-hydroxy-7N,N,N0 ,N’-tetramethyluronium
azabenzotriazole, DDQ ¼ 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, MNBA ¼ 2methyl-6-nitrobenzoic anhydride, DMAP ¼ 4-dimethylaminopyridine.

FK-A2 to FK-A8 (13beh), were efﬁciently synthesized in a manner
similar to that described for synthesizing FK-A1 (13a).
2.2. Biological evaluation
2.2.1. HDAC inhibition assay
To determine their potency and isoform selectivity, the synthesized FK228 analogues 13aeh were tested for their HDAC
inhibitory activity against HDAC1 (class I) and HDAC6 (class IIb). In
this assay, the clinically approved FK228 (1) was used as a positive
control, and the results are summarized in Table 1. FK-A1 (13a),which lacks the C4 isopropyl group that is present in 1, exhibitedhigh HDAC1 inhibitory activity (IC50 ¼ 4.7 nM), although its potency is approximately half of that of 1 (IC50 ¼ 2.4 nM). Thisobservation is in good agreement with a previous SAR study thatwas conducted by Ganesan et al. [20c]. On the other hand, FK-A2(13b), which lacks both the C4 isopropyl and the C7 ethylidenegroups present in 1, exhibits much lower inhibition potency(IC50 ¼ 92.4 nM) compared to 1. These results indicate that the C4
substituent is not responsible for HDAC inhibitory activity, whereas
the C7 substituent is indispensable.
With respect to the effect of the R1 substituent (including the C7
stereochemistry), FK-A3 (13c), FK-A5 (13e) and FK-A7 (13g), whichwere synthesized from the corresponding D-amino acids, exhibit
excellent potency against HDAC1 in the low nanomolar range(IC50 ¼ 0.54e2.1 nM). Importantly, the potencies of 13c, 13e and 13g

Scheme 4. Synthesis of FK-A2 to FK-A8 (13beh). a) FmocCl, 10% aq. Na2CO3, dioxane,
rt; b) 18, PyBOP, i-Pr2NEt, MeCN, rt; c) Et2NH, MeCN, rt; d) 17, PyBOP, i-Pr2NEt, MeCN,
rt; e) BF3$Et2O, CH2Cl2, rt; f) 16, HATU, HOAt, i-Pr2NEt, CH2Cl2/MeCN, �20 � C; g) DDQ,
CH2Cl2/H2O, rt; h) LiOH, THF/H2O, 0 � C; i) MNBA, DMAP, CH2Cl2, rt, 64%; j) I2, MeOH/
CH2Cl2, rt.

Table 1
HDAC inhibitory activity of FK228 and analogues FK-A1eFK-A8 (13aeh).
IC50a (nM)Compoundc

FK228 (1)FK-A1 (13a)FK-A2 (13b)FK-A3 (13c)FK-A4 (13d)FK-A5 (13e)FK-A6 (13f)
FK-A7 (13g)
FK-A8 (13h)aHDAC1 (class I)b

HDAC6 (class IIb)b

SId

2.4 ± 0.16
4.7 ± 0.88
92.4 ± 30.0
2.1 ± 1.0
184 ± 69.1
0.54 ± 0.10
40.8 ± 4.9
0.96 ± 0.14
11.4 ± 4.1

109 ± 35.8859 ± 117
1316 ± 178
1008 ± 461
1022 ± 38.6
54.9 ± 14.5355 ± 828452 ± 1066
2255 ± 577

45
182
14
480
6
102
9
8804
198

The concentration that induces 50% inhibition against HDACs.
The enzyme assay was performed in the presence of 0.1 mM dithiothreitol(DTT).
c
Positive control used in this study was synthesized in our laboratory [13a].
d
Selectivity index (HDAC6 IC50/HDAC1 IC50) as the selectivity towards class I
HDAC1 over class IIb HDAC6.

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K. Narita et al. / European Journal of Medicinal Chemistry 121 (2016) 592e609

were estimated to be equal to or two-fold higher than that of 1. FKA5 shows the highest potency (IC50 ¼ 0.54 nM) among the analogues that were tested in this assay; however, FK-A4 (13d), FK-A6(13f) and FK-A8 (13h), which were synthesized from the corresponding L-amino acids, showed considerably lower inhibitory activity (IC50 ¼ 11.4e184 nM). These results suggest that the C7
stereochemistry plays an important role in conferring potency andthat the D-amino acid side chains are preferable as the R1 substituents. To the best of our knowledge, the SAR of the C7 stereochemistry in FK228 has not been previously reported. Thus, theseﬁndings provide new insight for designing novel synthetic depsipeptide HDAC inhibitors.
As we expected, 13aeh exhibit much lower HDAC6 inhibitory
activity, with IC50 values in the range of 55e8452 nM. For convenience, isoform selectivity (class I HDAC1/class IIb HDAC6) isexpressed as a selective index (SI) value (HDAC6 IC50/HDAC1 IC50).
The isoform selectivity of 13a (SI ¼ 182) is four-fold higher than thatof 1 (SI ¼ 45), suggesting that removal of the C4 substituent iseffective for increasing isoform selectivity. However, FK-A2 (13b)exhibits lower isoform selectivity (SI ¼ 14), suggesting that removalof the C7 substituent reduces isoform selectivity. Among theremaining analogues 13ceh, compounds 13c, 13e and 13g, which
possess D-amino acid side chains at the C7 position, exhibit higher
isoform selectivity (SI ¼ 102e8804) compared to compounds 13d,
13f and 13h, which possess L-amino acid side chains (SI ¼ 6e198).
Note that FK-A7 (13g, SI ¼ 8804) exhibits approximately 200-foldhigher isoform selectivity compared to 1 (SI ¼ 45), which, to thebest our knowledge, represents the highest level of class I/II
selectivity among the naturally occurring and synthetic bicyclic
depsipeptides that have been reported to date.
These results reveal that a simple transformation of the C7
substituent (an ethylidene group) of 1 into a D-amino acid side
chain is effective for improving both the HDAC inhibitory activity
and the isoform selectivity. This structural alternation may confer apreferable conformational change onto the macrocyclic depsipeptide scaffold, thereby enhancing the discrimination and interactions between the macrocycle and the HDAC1 enzyme ‘rim’
located outside the active site binding pocket [23].
2.2.2. Cell-growth inhibition assay
The growth inhibitory activity of the synthesized analogues
13aeh was simultaneously evaluated with FK228 (1) as a referencecompound, using a panel of 39 human cancer cell lines at theJapanese Foundation for Cancer Research [24]. The organs fromwhich the cell lines were derived were as follows (number of cells
in parentheses): breast (5), central nervous system (6), colon (5),lung (7), melanoma (1), ovary (5), kidney (2), stomach (6) andprostate (2). Dose-response curves were measured at 5 different
concentrations (from 10�11 to 10�7 M, from 10�10 to 10�6 M or from10�8 to 10�4 M) for each compound, and the concentration thatcaused 50% cell growth inhibition (GI50) was compared with that of
the control.
The GI50 values of the tested analogues 13aeh together with 1
are shown in Table 2. The potency order of these analogues wasestimated using the MG-MID value (mean value of GI50 over all of
the cell lines tested) to be 1 (6.2 nM) > 13e (9.5 nM) > 13c(43 nM) > 13g (56 nM) > 13a (115 nM) [ 13f (1071 nM) > 13h(1259 nM) [ 13b (14,000 nM) > 13d (20,000 nM).
These results correspond reasonably with the abilities of theanalogues to inhibit HDAC1, as discussed above. Analogues 13c, 13eand 13g, which possess D-amino acid side chains, exhibit considerably more potency (GI50 ¼ 9.5e56 nM) compared to analogues
13d, 13f and 13h, which possess L-amino acid side chains(GI50 ¼ 1071e20,000 nM). Interestingly, analogue 13g, which exhibits the highest HDAC isoform selectivity (SI ¼ 8804), exhibits thelowest sensitivity against SF-295 (brain cancer) among the 39 celllines. This result is signiﬁcantly different from the analogues 13aef
and 13h, all of which exhibit their lowest sensitivity against HCT-15(colon cancer). The change in the cell-growth inhibition pattern for
13g might be caused by its HDAC isoform selectivity. Moreover,analogue 13e, which exhibits the highest MG-MID value(GI50 ¼ 9.5 nM) among the tested analogues, exhibits two-foldhigher sensitivity to NCI-H522 (lung cancer, GI50 ¼ 0.72 nM)
compared to that of 1 (GI50 ¼ 1.8 nM). One possible explanation forthis observation is that analogue 13e, which possesses an aromaticring in the C7 side chain, has an additional molecular target (e.g. a
different type of protein kinase) [25]; therefore, further investigation is necessary. Considering both HDAC inhibitory and antiproliferative activities, 13c, 13e and 13g are promising candidates
for developing novel anticancer agents with high efﬁcacy and low
toxicity.
3. Conclusion
We synthesized eight new FK228 analogues, i.e. FK-A1 to FK-A8(13aef), in a highly convergent and uniﬁed manner. The present
preliminary biological evaluation revealed novel aspects of the SAR
for this class of depsipeptide HDAC inhibitor. In particular,
replacement of the C4 isopropyl and C7 ethylidene substituents of
FK228 by a hydrogen atom and a D-amino acid side chain, respectively (13c, 13e and 13g), was quite effective for improving HDAC1
inhibitory activity and isoform selectivity. Furthermore, incorporating an aromatic ring into the C7 side chain (13e) was demonstrated to result in an improvement of the speciﬁcity to cancer celllines. These ﬁndings are useful for designing and developing new
anticancer agents that work by the isoform-selective inhibition of
HDACs. Further investigation of the activity of the synthesizedanalogues 13c, 13e and 13g using an animal model is currently
underway in our laboratories and will be reported in due course.
4. Experimental
4.1. Chemistry
All reactions involving air- and moisture-sensitive reagentswere carried out using oven dried glassware and standard syringeseptum cap techniques. Routine monitorings of reaction were carried out using glass-supported Merck silica gel 60 F254 TLC plates.
Flash column chromatography was performed on Kanto Chemical
Silica Gel 60N (spherical, neutral 40e50 nm) with the solvents
indicated.
All solvents and reagents were used as supplied with following
exceptions. Tetrahydrofuran (THF) was freshly distilled from Na
metal/benzophenone under argon. CH2Cl2, MeCN, Et2NH, and iPr2NEt were distilled from calcium hydride under argon.
Measurements of optical rotations were performed with a
JASCO DIP-370 automatic digital polarimeter. 1H and 13C NMR
spectra were measured with a JEOL AL-400 (400 MHz) spectrometer. Chemical shifts were expressed in ppm using Me4Si (d ¼ 0) as
an internal standard. The following abbreviations are used: singlet(s), doublet (d), triplet (t), quartet (q), multiplet (m) and broad (br).
Infrared (IR) spectral measurements were carried out with a JASCO
FT/IR-4100 spectrometer. Low- and High-resolution mass (HRMS)
spectra were measured on a JEOL JMS-700 high resolution mass
spectrometer.
4.1.1. Fmoc-L-ThreGly-OMe (21)A solution of 9-ﬂuorenylmethyl chloroformate (FmocCl)(652 mg, 2.5 mmol) in dioxane (10 mL) was added dropwise to a
stirred solution of L-threonine (L-Thr) (18a) (300 mg, 2.5 mmol) in— 212 —

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K. Narita et al. / European Journal of Medicinal Chemistry 121 (2016) 592e609
Table 2Growth inhibition of FK228 analogues FK-A1 to FK-A8 (13aeh) against a panel of 39 human cancer cell lines.
GI50a (nM)
Origin of cancer

Cell line

1

13a

13b

13c

13d

13e

13f

13g

13h

Breast

HBC-4
BSY-1
HBC-5
MCF-7
MDA-MB-231
U-251
SF-268SF-295
SF-539
SNB-75
SNB-78HCC2998
KM-12
HT-29
HCT-15
HCT-116
NCI-H23
NCI-H226
NCI-H522
NCI-H460
A549
DMS273
DMS114LOX-IMVIOVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
RXF-631L
ACHN
St-4
MKN1
MKN7
MKN28
MKN45
MKN74
DU-145
PC-3

6.9
8.5
13
4.2
5.5
3.9
4.9
4.0
3.6
7.2
9.6
3.1
3.4
3.3
450c
3.1
4.6
8.9
1.8b
3.0
2.6
5.8
3.6
2.5
4.6
20
2.8
5.5
3.3
6.6
20
22
3.2
4.9
17
14
3.0
6.0
18
6.2

170
200
630
190
53
120
110
280
10b
10b
10b
32
45
42
20,000c
26
130
410
10b
56
52
230
43
25
64
270
49
54
63
230
460
1100
73
380
1400
1900
100
39
170
115

13,00018,00039,00033,0009200
17,00012,00040,000
3300
950
610b
13,00012,0002700
100,000c
2500
36,00061,000680
15,00010,00047,0005200
2900
13,00046,0003100
18,00012,00091,00043,00081,00012,00047,00095,000100,000c
16,0005400
11,00014,000

30
36
160
37
21
19
31
71
10b
10b
10b
14
20
13
6700c
10b
31
110
10b
15
15
89
17
11
22
100
14
31
19
110
270
410
36
260
440
500
35
21
70
43

17,00027,00021,00017,00016,00017,00017,00051,00015,00014,00013,00013,00015,00012,000100,000c
11,00027,00021,0001700b
43,00018,00047,00015,0004500
16,00022,00015,00031,00016,000100,000c
100,000c
62,00014,00043,00024,000100,000c
18,00015,00013,00020,000

37
36
84
62
8.7
22
5.0
21
3.3
8.7
8.6
3.2
2.8
3.0
100c
2.6
4.4
4.8
0.72b
6.2
3.4
20
4.1
2.2
17
27
6.8
4.6
2.4
7.9
24
34
2.6
35
78
64
5.9
3.6
5.7
9.5

690
1300
1900
1900
1200
1500
220
1800
860
1300
540
1100
740
570
26,000c
250
1100
1200
200
1100
880
1500
620
160b
1100
1900
1400
1400
1200
1800
11,000
2000
290
1900
2100
1800
500
590
1100
1071

70
69
270
52
39
10b
11
1100c
57
43
30
27
33
33
590
22
39
59
10b
39
23
74
36
23
40
87
31
42
23
68
200
230
21
280
270
240
44
37
69
56

1400
1500
1700
1600
1200
330
820
1500
1500
1000
1800
1300
1100
690
17,000c
340
1200
1600
200
1100
720
1400
680
180b
1500
1600
1200
1500
1500
3200
9400
2100
1100
1800
1800
2200
1500
830
1400
1259

Central nervous system (Brain)

Colon

Lung

Melanoma
Ovary

KidneyStomachProstate
MG-MIDda

Concentration that induces 50% inhibition of cell growth compared to the control.
The most sensitive cell.
c
The least sensitive cell.
dMean value of GI50 over all cell lines tested.
b

10% aqueous Na2CO3 (10 mL) at room temperature. After 2 h, thereaction mixture was diluted with H2O (130 mL), and the resulting
mixture was washed with Et2O (2 � 40 mL). Concentrated H2SO4
was slowly added to the aqueous layer until pH was 2e3, and theresulting solution was extracted with EtOAc (3 � 100 mL). The
combined extracts were washed with H2O (2 � 50 mL) and brine(2 � 50 mL), then dried over Na2SO4. Concentration of the solventin vacuo afforded Fmoc-L-Thr (20) (859 mg) as a colorless amorphous solid, which was used for the next reaction without further
puriﬁcation.
i-Pr2NEt (1.71 mL, 10 mmol) was added dropwise to a stirred
solution of the crude 20 (859 mg) and glycine methyl ester (GlyOMe) hydrochloride (19) (348 mg, 2.8 mmol) in MeCN (25 mL)
containing (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexaﬂuorophosphate (PyBOP) (1.97 g, 3.8 mmol) at room temperature
under argon. After 1 h, concentration of the solvent in vacuo
afforded a residue, which was puriﬁed by column chromatography(hexane/EtOAc 1:1) to give 21 (912 mg, 88%, 2 steps) as a colorless
1
amorphous solid. [a]27
D �29.1 (c 1.0, CHCl3); H NMR (400 MHz,CDCl3): d 1.18 (3H, d, J ¼ 6.3 Hz), 3.62 (1H, s), 3.70 (3H, s), 4.02 (2H,d, J ¼ 5.4 Hz), 4.21 (2H, q, J ¼ 6.8 Hz), 4.37e4.40 (3H, m), 5.99 (1H, d,
J ¼ 7.8 Hz), 7.15 (1H, br t, J ¼ 4.9 Hz), 7.29 (2H, t, J ¼ 7.3 Hz), 7.38 (2H,t, J ¼ 7.6 Hz), 7.58 (2H, d, J ¼ 7.3 Hz), 7.75 (2H, d, J ¼ 7.3 Hz); 13C NMR(100 MHz, CDCl3): d 18.1, 41.1, 47.1, 52.4, 58.8, 67.0, 67.2, 120.0 (2C),
125.0 (2C), 127.0 (2C), 127.71, 127.72, 141.3 (2C), 143.59, 143.64,
156.8, 170.2, 171.3; IR (neat): 3331, 3017, 2975, 2952, 1724, 1714,
1667, 1530, 1449, 1215, 1106, 758, 740 cm�1; HRMS (EI): m/z calcd
for C22H24N2O6 (Mþ) 412.1634, found 412.1628.
4.1.2. Boc-D-CyseL-ThreGly-OMe (23)
Et2NH (2.3 mL, 22 mmol) was added to a stirred solution of 21(912 mg, 2.2 mmol) in MeCN (30 mL) at room temperature. After
3 h, the reaction mixture was concentrated in vacuo to afford NH2L-ThreGly-OMe (22) (421 mg) as a colorless oil, which was used forthe next reaction without further puriﬁcation.
i-Pr2NEt (0.98 mL, 5.8 mmol) was added dropwise to a stirred
solution of the crude amine 22 (421 mg, 2.2 mmol) and N-Boc-DCys(Tr)-OH (17) (1.23 g, 2.7 mmol) in MeCN (22 mL) containing
PyBOP (1.50 g, 2.9 mmol) at room temperature under argon. After
1 h, concentration of the solvent in vacuo afforded a residue, which
was puriﬁed by column chromatography (hexane/EtOAc 1:1) togive 23 (745 mg, 53%, 2 steps) as a colorless amorphous solid. [a]28D�27.5 (c 0.10, CHCl3). 1H NMR (400 MHz, CDCl3): d 1.12 (3H, d,
J ¼ 6.3 Hz), 1.40 (9H, s), 2.59 (1H, dd, J ¼ 12.9, 4.9 Hz), 2.78 (1H, dd,— 213 —

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K. Narita et al. / European Journal of Medicinal Chemistry 121 (2016) 592e609

J ¼ 12.4, 7.8 Hz), 3.30 (1H, br s), 3.60 (1H, br d, J ¼ 4.9 Hz), 3.65e3.74(4H, m), 4.04 (1H, dd, J ¼ 17.6, 5.4 Hz), 4.23 (1H, d, J ¼ 7.8 Hz), 4.46
(1H, br s), 5.00 (1H, d, J ¼ 6.3 Hz), 6.82 (1H, d, J ¼ 7.8 Hz), 7.21e7.31(9H, m), 7.39e7.44 (7H, m); 13C NMR (100 MHz, CDCl3): d 18.5, 28.2
(3C), 33.5, 41.0, 52.3, 54.3, 57.8, 66.6, 67.3, 80.7, 127.0 (3C), 128.1(6C), 129.5 (6C), 144.2 (3C), 155.5, 170.2, 171.4 (2C); IR (neat): 3387,
3303, 3275, 2979, 2921, 2852, 1685, 1676, 1654, 1642, 1541, 1533,
1508, 1219, 1163, 1101, 877, 700 cm�1; HRMS (FAB): m/z calcd for
C34H42N3O7S (MþþH) 636.2738, found 636.2759.
4.1.3. Boc-D-Cys(Tr)-L-Thr(OMs)-Gly-OMe (24)Methanesulfonyl chloride (MsCl) (0.245 mL, 3.2 mmol) was
added slowly to a stirred solution of 23 (745 mg, 1.2 mmol) in THF(23 mL) containing Et3N (0.98 mL, 7.0 mmol) at 0 � C under argon,
and stirring was continued for 1 h at room temperature. The reaction was quenched with 1 M HCl (5 mL) at 0 � C. The resulting
mixture was extracted with CHCl3 (3 � 30 mL), and the combined
extracts were washed with brine (2 � 20 mL), then dried overMgSO4. Concentration of the solvent in vacuo afforded a residue,
which was puriﬁed by column chromatography (hexane/EtOAc 2:1)
to give 24 (785 mg, 94%) as a colorless amorphous solid. [a]27
D �21.0(c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3): d 1.35e1.44 (12H), 2.60
(1H, dd, J ¼ 13.3, 5.3 Hz), 2.84 (1H, dd, J ¼ 13.5, 7.7 Hz), 3.02 (3H, s),
3.21e3.34 (1H, m), 3.50 (1H, dd, J ¼ 17.4, 5.3 Hz), 3.68 (3H, s), 4.06
(1H, dd, J ¼ 17.4, 6.3 Hz), 4.60 (1H, d, J ¼ 8.7 Hz), 4.87 (1H, d,
J ¼ 4.4 Hz), 5.44 (1H, qd, J ¼ 6.5, 1.4 Hz), 6.38 (1H, d, J ¼ 8.7 Hz),7.24e7.34 (9H, m), 7.40e7.42 (6H, m), 7.69 (1H, br s); 13C NMR(100 MHz, CDCl3): d 18.5, 28.2 (3C), 31.5, 33.0, 37.7, 41.0, 52.2, 54.8,
56.5, 67.6, 81.2, 127.2 (3C), 128.2 (6C), 129.4 (6C), 144.0 (3C), 155.8,
168.9, 169.4, 171.2; IR (neat): 3330, 3057, 2979, 2935, 1752, 1675,
1517, 1491, 1364, 1211, 1175, 975, 912, 742, 700 cm�1; HRMS (FAB):m/z calcd for C35H42N3O9S2 (MþeH) 712.2357, found 712.2363.
4.1.4. Boc-D-Cys(Tr)eDhbeGly-OMe (25)
1,4-Diazabicyclo[2.2.2]octane (DABCO) (478 mg, 4.3 mmol) was
added to a stirred solution of 24 (761 mg, 1.1 mmol) in THF (21 mL)at room temperature under argon. After 24 h, concentration of thesolvent in vacuo afforded a residue, which was puriﬁed by column
chromatography (hexane/EtOAc 2:1) to give 25 (585 mg, 89%) as a1colorless amorphous solid. [a]29
D �3.6 (c 1.0, CHCl3); H NMR(400 MHz, CDCl3): d 1.40 (9H, s), 1.69 (3H, d, J ¼ 7.3 Hz), 2.64 (1H, dd,
J ¼ 12.7, 5.9 Hz), 2.80 (1H, dd, J ¼ 12.7, 6.8 Hz), 3.64e3.73 (4H, m),
3.87 (2H, d, J ¼ 5.4 Hz), 4.90 (1H, d, J ¼ 5.4 Hz), 6.86 (1H, q,
J ¼ 6.8 Hz), 7.12e7.22 (2H, m), 7.22e7.32 (9H, m), 7.41e7.43 (6H, m);
13
C NMR (100 MHz, CDCl3): d 13.5, 28.2 (3C), 33.2, 41.2, 52.1, 54.3,
67.4, 81.0, 127.1 (3C), 128.0, 128.2 (6C), 129.4 (6C), 134.0, 144.1 (3C),
155.9, 164,2, 169.4, 170.1; IR (neat): 3390, 3345, 3235, 3059, 3019,
2974, 2932, 1739, 1713, 1671, 1659, 1650, 1538, 1518, 1495, 1440,
1367, 1218, 1167, 1063, 910, 738, 700 cm�1; HRMS (FAB): m/z calcd
for C34H40N3O6S (MþþH) 618.2632, found 618.2628.
4.1.5. H2N-D-Cys(Tr)eDhbeGly-OMe (15a)A solution of BF3$Et2O (0.41 mL, 3.2 mmol) in CH2Cl2 (3.2 mL)
was added dropwise to a stirred solution of 25 (571 mg, 0.93 mmol)in CH2Cl2 (46 mL) at 0 � C, and stirring was continued for 5 h at room
temperature. The reaction was quenched with saturated aqueous
NaHCO3 (15 mL) at 0 � C. The resulting mixture was extracted with
CHCl3 (3 � 40 mL), and the combined extracts were washed withbrine (2 � 20 mL), then dried over MgSO4. Concentration of thesolvent in vacuo afforded a residue, which was puriﬁed by column
chromatography (CHCl3/MeOH 20:1) to give 15a (422 mg, 88%) as a1colorless amorphous solid. [a]27
D þ2.0 (c 1.0, CHCl3); H NMR(400 MHz, CDCl3): d 1.62 (2H, br s), 1.67 (3H, d, J ¼ 7.3 Hz), 2.65 (1H,
dd, J ¼ 12.2, 4.4 Hz), 2.77 (1H, dd, J ¼ 12.2, 4.4 Hz), 3.25 (1H, dd,
J ¼ 6.1, 4.4 Hz), 3.67 (3H, s), 3.79 (1H, dd, J ¼ 18.0, 5.4 Hz), 3.92 (1H,

dd, J ¼ 18.0, 5.9 Hz), 6.63 (1H, q, J ¼ 7.3 Hz), 6.97 (1H, br t, J ¼ 4.9 Hz),
7.19e7.30 (9H, m), 7.40e7.42 (6H, m), 8.53 (1H, br s); 13C NMR(100 MHz, CDCl3): d 13.5, 37.2, 41.1, 52.1, 53.9, 66.9, 126.8 (3C), 127.9(6C), 128.8, 129.4 (6C), 130.7, 144.3 (3C), 164.6, 170.2, 172.1; IR(neat): 3304, 3057, 3015, 2951, 1749, 1669, 1642, 1525, 1488, 1443,
1212, 1033, 847, 745, 700 cm�1; HRMS (FAB): m/z calcd for
C29H32N3O4S (MþþH) 518.2108, found 518.2127.
4.1.6. (5E,7S,11R,14S,17Z)-Methyl 17-ethylidene-11-isopropyl-7-(4methoxybenzyloxy)-9,12,15,18-tetraoxo-1,1,1-triphenyl-14tritylthiomethyl-2-thia-10,13,16,19-tetraazahenicos-5-en-21-oate(26a)i-Pr2NEt (0.16 mL, 0.96 mmol) was added dropwise to a stirred
solution of 15a (190 mg, 0.37 mmol) and 16 (234 mg, 0.37 mmol) in
CH2Cl2/MeCN 2:1 (8.0 mL) containing O-(7-azabenzotriazol-1-yl)hexaﬂuorophosphate(HATU)
N,N,N0 ,N0 -tetramethyluronium(182 mg, 0.48 mmol) and 1-hydroxy-7-azabenzotriazole (HOAt)(65 mg, 0.48 mmol) at �20 � C under argon. After 5 h, the reaction
mixture was diluted with CHCl3 (80 mL). The organic layer waswashed successively with 10% aqueous HCl (2 � 20 mL), saturated
aqueous NaHCO3 (2 � 20 mL) and brine (2 � 20 mL), then driedover MgSO4. Concentration of the solvent in vacuo afforded a residue, which was puriﬁed by column chromatography (hexane/
EtOAc 2:1) to give 26a (334 mg, 80%) as a colorless amorphous
1
solid. [a]29
D 0 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): d 0.71 (3H, d,
J ¼ 7.0 Hz), 0.99 (3H, d, J ¼ 6.8 Hz), 1.69 (3H, d, J ¼ 6.8 Hz), 1.91e2.27(5H, m), 2.37e2.45 (2H, m), 2.54 (1H, dd, J ¼ 12.9, 5.6 Hz), 2.93 (1H,
dd, J ¼ 12.7, 6.3 Hz), 3.64 (3H, s), 3.79 (3H, s), 3.85e4.03 (5H, m),
4.21 (1H, d, J ¼ 11.2 Hz), 4.46 (1H, d, J ¼ 10.7 Hz), 5.27 (1H, dd,
J ¼ 15.4, 8.0 Hz), 5.51 (1H, dt, J ¼ 15.1, 6.8 Hz), 6.64 (1H, d, J ¼ 6.8 Hz),
6.81e6.87 (3H, m), 7.07 (1H, t, J ¼ 5.6 Hz), 7.17e7.29 (21H, m),
7.36e7.41 (12H, m), 7.68 (1H, s); 13C NMR (100 MHz, CDCl3): d 13.5,
17.7, 19.4, 29.4, 31.25, 31.30, 32.9, 41.3, 42.3, 52.0, 53.0, 55.3, 59.7,
66.6, 67.2, 70.0, 76.4, 113.9 (2C), 126.6 (3C), 127.0 (3C), 127.9 (6C),
128.1 (6C), 128.3, 129.4 (6C), 129.5 (7C), 129.6, 129.8 (2C), 133.6,
133.7, 144.1 (3C), 144.8 (3C), 159.4, 164.2, 168.7, 170.3, 171.9, 172.3; IR(neat): 3271, 3057, 3016, 2959, 2930, 1752, 1634, 1536, 1512, 1443,
1247, 1216, 1078, 1034, 768, 744, 700 cm�1; HRMS (FAB): m/z calcd
for C68H73N4O8S2 (MþþH) 1137.4864, found 1137.4889.
4.1.7. (5E,7S,11R,14S,17Z)-Methyl 17-ethylidene-7-hydroxy-11isopropyl-9,12,15,18-tetraoxo-1,1,1-triphenyl-14-tritylthiomethyl-2thia-10,13,16,19-tetraazahenicos-5-en-21-oate (27a)2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (112 mg,
0.49 mmol) was added in small portions to a stirred solution of 26a(373 mg, 0.33 mmol) in CH2Cl2/H2O 9:1 (16 mL) at room temperature. After 3 h, the mixture was diluted with CHCl3 (60 mL), andthe organic layer was washed with saturated aqueous NaHCO3(2 � 20 mL) and brine (2 � 20 mL), then dried over MgSO4. Concentration of the solvent in vacuo afforded a residue, which was
puriﬁed by column chromatography (hexane/EtOAc 1:5) to give 27a(299 mg, 90%) as a colorless amorphous solid. [a]29
D þ0.19 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 0.93 (3H, d, J ¼ 6.8 Hz), 0.98
(3H, d, J ¼ 6.3 Hz), 1.71 (3H, d, J ¼ 7.3 Hz), 1.97e2.31 (6H, m), 2.36
(1H, dd, J ¼ 13.2, 2.0 Hz), 2.64 (1H, dd, J ¼ 12.7, 4.9 Hz), 2.75 (1H, dd,
J ¼ 12.7, 7.8 Hz), 3.61 (3H, s), 3.79 (1H, br s), 3.84 (1H, dd, J ¼ 18.0,
4.9 Hz), 4.02 (1H, dd, J ¼ 18.0, 5.4 Hz), 4.11 (1H, t, J ¼ 5.6 Hz), 4.20
(1H, td, J ¼ 7.8, 5.4 Hz), 4.29e4.37 (1H, m), 5.35 (1H, dd, J ¼ 15.1,
5.9 Hz), 5.49 (1H, dt, J ¼ 15.1, 6.3 Hz), 6.32 (1H, d, J ¼ 5.9 Hz), 6.67
(1H, q, J ¼ 7.1 Hz), 6.90 (1H, t, J ¼ 5.4 Hz), 7.13e7.29 (19H, m),
7.35e7.41 (12H, m), 7.78 (1H, br s); 13C NMR (100 MHz, CDCl3):
d 13.2, 17.8, 19.5, 29.1, 31.3 (2C), 33.1, 41.3, 43.9, 52.2, 52.5, 60.2, 66.6,
67.0, 69.6, 126.6 (3C), 126.9 (3C), 127.8 (6C), 128.0 (6C), 129.0, 129.4(6C), 129.5 (6C), 129.9, 132.5, 132.6, 144.3 (3C), 144.8 (3C), 164.7,
169.2, 170.7, 171.4, 173.1; IR (neat): 3284, 3057, 3017, 2962, 2928,— 214 —

K. Narita et al. / European Journal of Medicinal Chemistry 121 (2016) 592e609

1747, 1634, 1531, 1489, 1443, 1215, 1033, 745, 700 cm�1; HRMS
(FAB): m/z calcd for C60H65N4O7S2 (MþþH) 1017.4289, found1017.4280.
4.1.8. (5E,7S,11R,14S,17Z)-17-Ethylidene-7-hydroxy-11-isopropyl9,12,15,18-tetraoxo-1,1,1-triphenyl-14-tritylthiomethyl-2-thia10,13,16,19-tetraazahenicos-5-en-21-oic acid (14a)
LiOH$H2O (24.7 mg, 0.59 mmol) was added dropwise to a stirred
solution of 27a (299 mg, 0.94 mmol) in THF/H2O 4:1 (15 mL) at 0 � C.
After 4 h, 10% aqueous HCl was added to the mixture at 0 � C until pH
was 6. The resulting mixture was extracted with EtOAc (3 � 30 mL),and the combined extracts were washed with saturated aqueous
NaHCO3 (2 � 15 mL) and brine (2 � 15 mL), then dried over Na2SO4.
Concentration of the solvent in vacuo afforded a residue, which was
puriﬁed by column chromatography (CHCl3/MeOH 6:1) to give 14a(289 mg, 97%) as a colorless amorphous solid. [a]29
D þ5.5 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3/CD3OD 10: 1): d 0.83 (3H, d,
J ¼ 5.9 Hz), 0.85 (3H, d, J ¼ 5.4 Hz), 1.65 (3H, d, J ¼ 6.8 Hz), 1.96e2.09(3H, m), 2.12e2.22 (2H, m), 2.33 (2H, d, J ¼ 6.3 Hz), 2.49e2.71 (2H,m), 3.57e3.82 (2H, m), 4.00e4.19 (2H, m), 4.33 (1H, q, J ¼ 5.9 Hz),5.37 (1H, dd, J ¼ 15.1, 5.9 Hz), 5.50 (1H, dt, J ¼ 15.1, 6.3 Hz), 6.67 (1H,q, J ¼ 5.9 Hz), 7.18e7.28 (19H, m), 7.33e7.40 (15H, m); 13C NMR(100 MHz, CDCl3/CD3OD 10: 1): d 13.3, 17.7, 19.0, 30.0, 31.2, 31.3,
32.3, 43.1, 43.3, 53.1, 59.0, 66.4, 66.9, 68.9, 126.5 (3C), 126.8 (3C),
127.7 (6C), 127.9 (6C), 128.5, 129.3 (6C), 129.4 (6C), 131.9, 132.1,
132.6, 144.1 (3C), 144.7 (3C), 164.7, 169.8, 172.9, 176.0, 183.3; IR(neat): 3290, 3056, 3016, 2963, 2927, 1628, 1528, 1489, 1443, 1317,
1217, 1033, 744, 700 cm�1; HRMS (FAB): m/z calcd for C59H63N4O7S2(MþþH) 1003.4133, found 1003.4124.
4.1.9. (9S,12R,16S,Z)-6-Ethylidene-12-isopropyl-16-[(E)-4tritylthiobut-1-enyl]-9-tritylthiomethyl-1-oxa-4,7,10,13tetraazacyclohexadecane-2,5,8,11,14-pentaone (28a)A solution of 14a (154 mg, 0.15 mmol) in CH2Cl2 (15 mL) was
added very slowly to a stirred solution of 2-methyl-6-nitrobenzoic
anhydride (MNBA) (68.7 mg, 0.20 mmol) in CH2Cl2 (150 mL, 1.0 mM
concentration) containing 4-dimethylaminopyridine (DMAP)(56.3 mg, 0.46 mmol) at room temperature over 10 h. After 2 h, themixture was diluted with CH2Cl2 (150 mL), and the organic layer
was washed with saturated aqueous NaHCO3 (2 � 50 mL), water(2 � 20 mL) and brine (2 � 50 mL), then dried over MgSO4. Concentration of the solvent in vacuo afforded a residue, which was
puriﬁed by column chromatography (EtOAc) to give 28a (97.3 mg,
164%) as a colorless amorphous solid. [a]28
D �24.0 (c 1.0, CHCl3); H
NMR (400 MHz, CDCl3/CD3OD 10:1): d 0.86 (3H, d, J ¼ 6.8 Hz), 0.94
(3H, d, J ¼ 6.8 Hz), 1.64 (3H, d, J ¼ 6.8 Hz), 1.94e2.26 (5H, m), 2.44
(1H, dd, J ¼ 14.6, 2.0 Hz), 2.58 (1H, dd, J ¼ 14.1, 10.2 Hz), 2.80 (1H, dd,
J ¼ 13.7, 4.9 Hz), 2.86 (1H, dd, J ¼ 13.7, 8.8 Hz), 3.27e3.38 (1H, m),
3.71 (1H, dd, J ¼ 16.6, 3.9 Hz), 4.24e4.41 (2H, m), 5.37 (1H, dd,
J ¼ 15.6, 6.3 Hz), 5.54e5.62 (2H, m), 6.85 (1H, q, J ¼ 6.8 Hz),7.15e7.32 (20H, m), 7.38e7.42 (13H, m), 8.16 (1H, d, J ¼ 6.3 Hz); 13C
NMR (100 MHz, CDCl3/CD3OD 10: 1): d 13.2, 17.2, 19.3, 30.9, 31.1,
31.2, 31.8, 41.3, 41.6, 53.9, 58.2, 66.5, 67.0, 72.2, 126.5 (3C), 126.6,(3C), 127.7 (6C), 127.8 (6C), 128.0, 129.27 (6C), 129.34 (7C), 132.6,
135.3, 144.2 (3C), 144.6 (3C), 164.2, 168.9, 169.0, 170.5, 173.0; IR(neat): 3316, 3057, 3018, 2964, 2929, 1749, 1667, 1659, 1650, 1644,
1538, 1488, 1444, 1217, 1193, 1033, 768, 700 cm�1; HRMS (FAB): m/z
calcd for C59H61N4O6S2 (MþþH) 985.4027, found 985.4037.
4.1.10. FK-A1 (13a)A solution of 28a (87.7 mg, 89 mmol) in CH2Cl2/MeOH 9:1(22 mL) was added dropwise to a vigorously stirred solution of I2(226 mg, 0.89 mmol) in CH2Cl2/MeOH 9:1 (178 mL, 0.5 mM concentration) over 10 min at room temperature. After 10 min, thereaction was quenched with 0.2 M ascorbic acid/citric acid buffer599(10 mL, adjusted to pH 4.0) at room temperature. The resulting
mixture was diluted with CH2Cl2 (100 mL) and water (50 mL). The
organic layer was washed with brine (2 � 50 mL) and dried overMgSO4. Concentration of the solvent in vacuo afforded a residue,
which was puriﬁed by column chromatography (CHCl3/MeOH,
15:1) to give 13a (42.8 mg, 97%) as a colorless amorphous solid.
1[a]28
D þ54.9 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3/CD3OD 10:1):
d 1.11 (3H, d, J ¼ 6.3 Hz), 1.12 (3H, d, J ¼ 6.3 Hz), 1.76 (3H, d,
J ¼ 6.8 Hz), 2.24 (1H, dq, J ¼ 12.7, 6.8 Hz), 2.41e2.53 (1H, m),
2.66e2.78 (2H, m), 2.81 (1H, dd, J ¼ 13.2, 6.3 Hz), 2.93 (1H, ddd,
J ¼ 13.2, 8.8, 3.4 Hz), 3.13e3.25 (3H, m), 3.97 (1H, dd, J ¼ 4.9, 3.4 Hz),4.10 (1H, dd, J ¼ 18.0, 3.9 Hz), 4.26 (1H, dd, J ¼ 17.6, 4.9 Hz), 4.82 (1H,q, J ¼ 5.4 Hz), 5.72e5.91 (3H, m), 6.71 (1H, q, J ¼ 7.3 Hz), 7.03 (1H, t,
J ¼ 4.1 Hz), 7.67 (1H, d, J ¼ 6.3 Hz), 8.01 (1H, d, J ¼ 2.9 Hz), 8.55 (1H,s); 13C NMR (100 MHz, CDCl3/CD3OD 10:1): d 13.6, 18.8, 18.9, 29.2,
30.4, 35.5, 37.3, 38.0, 42.8, 56.3, 62.5, 69.7, 128.0, 129.0, 129.5, 132.6,
164.2, 166.9, 168.7, 171.6, 172.1; IR (neat): 3434, 3356, 3011, 2965,
2929, 1748, 1650, 1519, 1406, 1252, 979, 753 cm�1; HRMS (FAB): m/z
calcd for C21H31N4O6S2 (MþþH) 499.1680, found 499.1695.
4.1.11. Fmoc-GlyeGly-OMe (30b)Compound 30b was synthesized from Gly (18b) (300 mg,
4.0 mmol) in a manner similar to that described for the synthesis of
21. Puriﬁcation by column chromatography (CHCl3/MeOH 30:1)
gave 30b (1.12 g, 78%, 2 steps) as colorless amorphous solid. 1H NMR(400 MHz, CDCl3): d 3.76 (3H, s), 3.93 (2H, d, J ¼ 4.9 Hz), 4.06 (2H, d,
J ¼ 4.9 Hz), 4.23 (1H, t, J ¼ 6.8 Hz), 4.45 (2H, d, J ¼ 6.8 Hz), 5.47 (1H,
br s), 6.48 (1H, br s), 7.31 (2H, td, J ¼ 7.3, 1.0 Hz), 7.40 (2H, t,
J ¼ 7.3 Hz), 7.59 (2H, d, J ¼ 7.3 Hz), 7.77 (2H, d, J ¼ 7.3 Hz); 13C NMR(100 MHz, CDCl3): d 41.1, 44.3, 47.1, 52.4, 67.2, 120.0 (2C), 125.0 (2C),
127.0 (2C), 127.7 (2C), 141.3 (2C), 143.7 (2C), 156.6, 169.3, 170.1; IR(KBr): 3387, 3294, 3076, 2948, 1742, 1715, 1653, 1555, 1536, 1285,
1243, 1234, 1166, 756, 733 cm�1; HRMS (EI): m/z calcd for
C20H20N2O5 (Mþ) 368.1372, found 368.1364.
4.1.12. Boc-D-Cys(Tr)eGlyeGly-OMe (32b)Compound 32b was synthesized from 30b (500 mg, 1.4 mmol) ina manner similar to that described for the synthesis of 23. Puriﬁcation by column chromatography (CHCl3/MeOH 20:1) gave 32b(588 mg, 73%, 2 steps) as a colorless amorphous solid. [a]26
D �14.4 (c1.0, CHCl3). 1H NMR (400 MHz, CDCl3): d 1.40 (9H, s), 2.63 (1H, dd,
J ¼ 12.9, 5.4 Hz), 2.76 (1H, dd, J ¼ 12.7, 7.3 Hz), 3.65e3.73 (4H, m),
3.89 (2H, d, J ¼ 5.9 Hz), 3.95 (2H, t, J ¼ 5.4 Hz), 4.79 (1H, br d,
J ¼ 4.4 Hz), 6.51 (1H, br t, J ¼ 5.9 Hz), 7.00 (1H, br s), 7.22e7.32 (9H,m), 7.40e7.42 (6H, m); 13C NMR (100 MHz, CDCl3): d 28.2 (3C), 33.6,
40.8, 42.9, 52.2, 54.0, 67.3, 80.7, 127.0 (3C), 128.1 (6C), 129.5 (6C),
144.2 (3C), 155.6, 169.0, 169.9, 170.9; IR (neat): 3317, 3008, 2978,
1749, 1667, 1521, 1490, 1443, 1367, 1247, 1214, 1166, 1033, 852, 752,
700 cm�1; HRMS (FAB): m/z calcd for C32H38N3O6S (MþþH)592.2476, found 592.2475.
4.1.13. H2N-D-Cys(Tr)eGlyeGly-OMe (15b)Compound 15b was synthesized from 32b (1.46 g, 2.5 mmol) in amanner similar to that described for the synthesis of 15a. Puriﬁcation by column chromatography (CHCl3/MeOH 20:1) gave 15b(873 mg, 72%) as a colorless amorphous solid. [a]28
D �13.8 (c 1.1,
CHCl3); 1H NMR (400 MHz, CDCl3): d 1.48 (2H, br s), 2.69 (2H, d,
J ¼ 5.9 Hz), 3.16 (1H, t, J ¼ 5.9 Hz), 3.70 (3H, s), 3.80e3.99 (4H, m),
6.82 (1H, t, J ¼ 5.9 Hz), 7.20e7.31 (9H, m), 7.41e7.43 (6H, m), 7.69
(1H, t, J ¼ 5.9 Hz); 13C NMR (100 MHz, CDCl3): d 37.3, 40.8, 42.8,
52.2, 53.8, 66.9, 126.8 (3C), 128.0 (6C), 129.5 (6C), 144.4 (3C), 169.2,
169.9, 173.8; IR (neat): 3353, 3317, 3057, 2951, 1748, 1660, 1520,
1489, 1443, 1372, 1212, 1183, 1033, 984, 886, 848, 745, 700 cm�1;
HRMS (FAB): m/z calcd for C27H30N3O4S (MþþH) 492.1952, found492.1942.

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4.1.14. (7S,11R,14S,E)-Methyl 11-isopropyl-7-(4methoxybenzyloxy)-9,12,15,18-tetraoxo-1,1,1-triphenyl-14tritylthiomethyl-2-thia-10,13,16,19-tetraazahenicos-5-en-21-oate(26b)Compound 26b was synthesized from 15b (77.1 mg, 0.16 mmol)
in a manner similar to that described for the synthesis of 26a. Puriﬁcation by column chromatography (CHCl3/MeOH 30:1) gave 26b(152 mg, 87%) as a colorless amorphous solid. [a]27
D þ10.4 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 0.73 (3H, d, J ¼ 6.8 Hz), 0.82
(3H, d, J ¼ 6.8 Hz), 1.95 (1H, dq, J ¼ 13.2, 6.8 Hz), 2.06e2.16 (2H, m),
2.21e2.25 (2H, m), 2.44 (2H, d, J ¼ 5.9 Hz), 2.58 (1H, dd, J ¼ 13.2,
5.9 Hz), 2.78 (1H, dd, J ¼ 13.2, 6.8 Hz), 3.65 (3H, s), 3.75 (3H, s),
3.81e3.89 (3H, m), 3.96 (2H, dt, J ¼ 18.0, 6.3 Hz), 4.08 (1H, q,
J ¼ 6.3 Hz), 4.14 (1H, t, J ¼ 6.8 Hz), 4.21 (1H, d, J ¼ 10.7 Hz), 4.41 (1H,d, J ¼ 10.7 Hz), 5.29 (1H, dd, J ¼ 15.1, 8.3 Hz), 5.54 (1H, dt, J ¼ 15.1,
6.8 Hz), 6.81 (2H, d, J ¼ 8.3 Hz), 6.97 (1H, d, J ¼ 7.3 Hz), 7.02 (1H, t,
J ¼ 5.6 Hz), 7.15e7.30 (22H, m), 7.35e7.42 (12H, m); 13C NMR(100 MHz, CDCl3): d 17.8, 19.3, 29.9, 31.2, 31.3, 33.3, 40.8, 42.3, 42.8,
52.1, 52.6, 55.2, 59.0, 66.5, 67.0, 70.0, 76.5, 113.7 (2C), 126.5 (3C),
126.8 (3C), 127.8 (6C), 128.0 (6C), 129.4 (6C), 129.5 (6C), 129.6, 129.8(2C), 129.9, 133.2, 144.2 (3C), 144.7 (3C), 159.3, 169.1, 169.9, 170.2,
171.5, 171.6; IR (neat): 3287, 3082, 3058, 3016, 2960, 2931, 1744,
1691, 1634, 1513, 1490, 1443, 1247, 1216, 1181, 1079, 1034, 975, 822,
752, 700 cm�1; HRMS (FAB): m/z calcd for C66H71N4O8S2 (MþþH)1111.4708, found 1111.4731.
4.1.15. (7S,11R,14S,E)-Methyl 7-hydroxy-11-isopropyl-9,12,15,18tetraoxo-1,1,1-triphenyl-14- tritylthiomethyl-2-thia-10,13,16,19tetraazahenicos-5-en-21-oate (27b)Compound 27b was synthesized from 26b (108 mg, 97 mmol) ina manner similar to that described for the synthesis of 27a. Puriﬁcation by column chromatography (CHCl3/MeOH 30:1) gave 27b(95.6 mg, 99%) as a colorless amorphous solid. [a]26
D �17.0 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 0.89 (3H, d, J ¼ 6.8 Hz), 0.93
(3H, d, J ¼ 6.8 Hz), 2.03e2.08 (2H, m), 2.12e2.22 (3H, m), 2.28 (1H,
dd, J ¼ 13.7, 9.8 Hz), 2.39 (1H, dd, J ¼ 13.7, 2.9 Hz), 2.61e2.69 (2H,m), 3.60 (3H, s), 3.73 (1H, dd, J ¼ 17.1, 5.4 Hz), 3.79 (1H, dd, J ¼ 18.0,
5.4 Hz), 3.89 (1H, br s), 3.96 (1H, dd, J ¼ 18.0, 5.9 Hz), 4.03 (1H, dd,
J ¼ 16.6, 6.3 Hz), 4.18 (1H, t, J ¼ 6.3 Hz), 4.23 (1H, q, J ¼ 7.1 Hz),4.33e4.43 (1H, m), 5.38 (1H, dd, J ¼ 15.1, 5.9 Hz), 5.53 (1H, dt,
J ¼ 15.1, 6.3 Hz), 6.54 (1H, d, J ¼ 6.8 Hz), 7.14e7.28 (20H, m),7.31e7.41 (13H, m); 13C NMR (100 MHz, CDCl3): d 17.7, 19.4, 29.4,
31.2 (2C), 33.4, 40.8, 43.0, 43.7, 52.2, 52.3, 59.5, 66.5, 66.8, 69.4,
126.5 (3C), 126.8 (3C), 127.8 (6C), 127.9 (6C), 129.4 (6C), 129.5 (6C),
129.7, 132.6, 144.2 (3C), 144.7 (3C), 169.5, 170.46, 170.50, 171.3,
172.8; IR (neat): 3289, 3082, 3058, 3016, 2962, 2929, 1744, 1635,
1531, 1489, 1443, 1216, 1183, 1082, 1033, 973, 851, 752, 700 cm�1;
HRMS (FAB): m/z calcd for C58H63N4O7S2 (MþþH) 991.4133, found991.4120.
4.1.16. (7S,11R,14S,E)-7-Hydroxy-11-isopropyl-9,12,15,18-tetraoxo1,1,1-triphenyl-14-tritylthiomethyl-2-thia-10,13,16,19tetraazahenicos-5-en-21-oic acid (14b)Compound 14b was synthesized from 27b (106 mg, 0.11 mmol)
in a manner similar to that described for the synthesis of 14a. Puriﬁcation by column chromatography (CHCl3/MeOH 5:1) gave 14b(79.5 mg, 76%) as a colorless amorphous solid. [a]29
D �12.5 (c 1.0,
CHCl3). 1H NMR (400 MHz, CD3OD/CDCl3 5:1): d 0.93 (3H, d,
J ¼ 5.8 Hz), 0.94 (3H, d, J ¼ 6.3 Hz), 1.98e2.07 (2H, m), 2.09e2.21(3H, m), 2.35 (1H, dd, J ¼ 13.5, 4.3 Hz), 2.46 (1H, dd, J ¼ 13.5, 9.2 Hz),2.62 (1H, dd, J ¼ 12.6, 8.7 Hz), 2.70 (1H, dd, J ¼ 12.6, 5.8 Hz), 3.74
(1H, d, J ¼ 16.9 Hz), 3.76 (2H, s), 3.96 (1H, d, J ¼ 16.9 Hz), 4.12e4.15(2H, m), 4.36e4.40 (1H, m), 5.42 (1H, dd, J ¼ 15.0, 6.3 Hz), 5.54 (1H,
dt, J ¼ 15.0, 6.8 Hz), 7.13e7.24 (18H, m), 7.24e7.39 (12H, m); 13C
NMR (100 MHz, CD3OD/CDCl3 5:1, 40 � C): d 18.4, 19.7, 30.9, 32.2,

32.4, 34.0, 42.1, 43.4, 44.1, 54.0, 60.6, 67.6, 67.9, 70.2, 127.5 (3C),
127.7, (3C), 128.6 (6C), 128.8 (6C), 130.4 (6C), 130.46, 130.50 (6C),
134.1, 145.5 (3C), 146.0 (3C), 171.3, 172.2, 173.3, 173.5, 174.5; IR(neat): 3299, 3057, 3017, 2964, 2928, 1725, 1635, 1536, 1488, 1444,
1218, 1033, 971, 752, 700 cm�1; HRMS (FAB): m/z calcd for
C57H61N4O7S2 (MþþH) 977.3976, found 977.3962.
4.1.17. (9S,12R,16S)-12-Isopropyl-16-[(E)-4-tritylthiobut-1-enyl]-9tritylthiomethyl-1-oxa-4,7,10,13-tetraazacyclohexadecane2,5,8,11,14-pentaone (28b)Compound 28b was synthesized from 14b (72.1 mg, 74 mmol) ina manner similar to that described for the synthesis of 28a. Puriﬁcation by column chromatography (CHCl3/MeOH 30:1) gave 28b(59.9 mg, 85%) as a colorless amorphous solid. [a]29
D �24.5 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 0.79 (3H, d, J ¼ 6.8 Hz), 0.91
(3H, d, J ¼ 6.3 Hz), 1.85e2.03 (2H, m), 2.08e2.24 (3H, m), 2.42 (1H,
dd, J ¼ 13.7, 9.8 Hz), 2.47e2.54 (1H, m), 2.75 (1H, dd, J ¼ 13.2,
3.9 Hz), 2.86 (1H, dd, J ¼ 13.2, 9.8 Hz), 3.32e3.42 (1H, m), 3.42e3.55(2H, m), 4.30 (1H, dd, J ¼ 17.1, 8.3 Hz), 4.46 (1H, dd, J ¼ 16.6, 9.3 Hz),4.72 (1H, dd, J ¼ 9.3, 3.9 Hz), 5.34 (1H, dd, J ¼ 15.6, 6.3 Hz),5.49e5.64 (2H, m), 6.30 (1H, d, J ¼ 8.8 Hz), 7.12e7.28 (20H, m),
7.33e7.39 (12H, m), 8.08 (1H, d, J ¼ 5.9 Hz); 13C NMR (100 MHz,CDCl3): d 16.7, 19.6, 30.9, 31.4, 31.6, 32.4, 40.9, 42.7, 43.6, 54.8, 56.6,
66.7, 67.0, 72.4, 126.6 (3C), 126.7, (3C), 127.6, 127.8 (6C), 127.9 (6C),
129.4 (6C), 129.5 (6C), 132.5, 144.5 (3C), 144.7 (3C), 169.8, 169.9,
170.2, 170.4, 172.7; IR (neat): 3300, 3057, 3018, 2964, 2931, 1737,
1661, 1539, 1489, 1444, 1214, 1033, 974, 744, 700 cm�1; HRMS (FAB):m/z calcd for C57H59N4O6S2 (MþþH) 959.3871, found 959.3891.
4.1.18. FK-A2 (13b)Compound 13b was synthesized from 28b (59.9 mg, 62 mmol) ina manner similar to that described for the synthesis of 13a. Puriﬁcation by column chromatography (CHCl3/MeOH 10:1) gave 13b(22.0 mg, 75%) as a colorless amorphous solid. [a]24
D �39.7 (c 0.70,
CHCl3); 1H NMR (400 MHz, CDCl3): d 1.09 (3H, d, J ¼ 3.4 Hz), 1.10
(3H, d, J ¼ 2.9 Hz), 2.30 (1H, dq, J ¼ 12.1, 6.3 Hz), 2.45e2.60 (1H, m),
2.63e2.80 (2H, m), 2.88e2.99 (2H, m), 3.08 (1H, ddd, J ¼ 13.0, 9.7,
2.9 Hz), 3.18 (1H, dd, J ¼ 15.0, 4.3 Hz), 3.27 (1H, dd, J ¼ 15.5, 10.1 Hz),3.51 (1H, dd, J ¼ 16.4, 3.4 Hz), 4.04 (1H, t, J ¼ 3.9 Hz), 4.10e4.21 (2H,m), 4.44 (1H, dd, J ¼ 16.4, 8.7 Hz), 4.76e4.84 (1H, m), 5.68e5.82(2H, m), 5.82e5.95 (1H, m), 6.78 (1H, d, J ¼ 2.9 Hz), 6.95 (1H, t,
J ¼ 4.3 Hz), 7.49 (1H, d, J ¼ 6.8 Hz), 7.68 (1H, dd, J ¼ 8.2, 2.9 Hz); 13C
NMR (100 MHz, CDCl3): d 18.6, 19.4, 29.3, 31.1, 37.1, 37.5, 39.2, 42.3,
43.5, 55.9, 62.3, 69.7, 129.1, 129.5, 167.0, 168.9, 170.3, 171.2, 171.3; IR(neat): 3298, 3011, 2965, 2932, 1742, 1658, 1529, 1258, 1191, 1031,
979, 754 cm�1; HRMS (EI): m/z calcd for C19H28N4O6S2 (Mþ)
472.1450, found 472.1463.
4.1.19. Fmoc-D-AlaeGly-OMe (30c)Compound 30c was synthesized from D-alanine (D-Ala) (18c)(500 mg, 5.6 mmol) in a manner similar to that described for thesynthesis of 21. Puriﬁcation by column chromatography (hexane/
EtOAc 2:3) gave 30c (1.98 g, 92%, 2 steps) as a colorless amorphous
1
solid. [a]29
D þ13.5 (c 1.1, CHCl3); H NMR (400 MHz, CDCl3): d 1.40
(3H, d, J ¼ 6.8 Hz), 3.70 (3H, s), 4.01 (2H, d, J ¼ 4.8 Hz), 4.19 (1H, t,
J ¼ 6.8 Hz), 4.29e4.40 (1H, m), 4.39 (2H, d, J ¼ 6.8 Hz), 5.61 (1H, d,
J ¼ 7.7 Hz), 6.82 (1H, br s), 7.29 (2H, t, J ¼ 7.2 Hz), 7.38 (2H, t,
J ¼ 7.2 Hz), 7.57 (2H, dd, J ¼ 7.2, 3.6 Hz), 7.74 (2H, d, J ¼ 7.7 Hz); 13C
NMR (100 MHz, CDCl3): d 18.5, 41.2, 47.1, 50.4, 52.4, 67.1, 120.0 (2C),
125.0 (2C), 127.1 (2C), 127.7 (2C), 141.3 (2C), 143.7 (2C), 156.0, 170.1,
172.5; IR (KBr): 3323, 3293, 3067, 2982, 2951, 2940, 2890, 1731,
1720, 1693, 1648, 1541, 1449, 1329, 1301, 1266, 1254, 1105, 1089,
1051, 1023, 758, 741, 670, 645 cm�1; HRMS (EI): m/z calcd for
C21H22N2O5 (Mþ) 382.1529, found 382.1526.

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4.1.20. Boc-D-Cys(Tr)eD-AlaeGly-OMe (32c)Compound 32c was synthesized from 30c (200 mg, 0.52 mmol)
in a manner similar to that described for the synthesis of 23. Puriﬁcation by column chromatography (hexane/EtOAc 1:2) gave 32c(260 mg, 82%, 2 steps) as a colorless amorphous solid. [a]24
D þ5.3 (c1.0, CHCl3); 1H NMR (400 MHz, CDCl3): d 1.36 (3H, d, J ¼ 6.8 Hz), 1.41
(9H, s), 2.61 (1H, dd, J ¼ 12.9, 5.9 Hz), 2.70 (1H, dd, J ¼ 12.7, 6.8 Hz),3.69 (3H, s), 3.70e3.84 (2H, m), 3.96 (1H, dd, J ¼ 17.6, 5.9 Hz), 4.47
(1H, dq, J ¼ 7.4, 7.2 Hz), 4.81 (1H, br s), 6.35 (1H, br s), 6.96 (1H, br s),
7.21e7.32 (9H, m), 7.41e7.43 (6H, m); 13C NMR (100 MHz, CDCl3):
d 17.9, 28.1 (3C), 33.7, 40.8, 48.6, 52.0, 53.4, 67.0, 80.2, 126.8 (3C),
127.9 (6C), 129.3 (6C), 144.1 (3C), 155.3, 169.8, 170.2, 172.0; IR (neat):3285, 3058, 2978, 2928, 1756, 1680, 1645, 1523, 1491, 1445, 1367,
1209, 1167, 1049, 1020, 851, 751, 700 cm�1; HRMS (FAB): m/z calcd
for C33H40N3O6S (MþþH) 606,2632, found 606.2641.
4.1.21. H2N-D-Cys(Tr)eD-AlaeGly-OMe (15c)Compound 15c was synthesized from 32c (70.0 mg, 0.12 mmol)
in a manner similar to that described for the synthesis of 15a. Puriﬁcation by column chromatography (CHCl3/MeOH 20:1) gave 15c(52.0 mg, 89%) as a colorless amorphous solid. [a]25
D þ26.9 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 1.35 (3H, d, J ¼ 6.8 Hz), 1.42
(2H, br s), 2.58 (1H, dd, J ¼ 12.6, 8.2 Hz), 2.70 (1H, dd, J ¼ 12.6,
4.3 Hz), 3.00 (1H, dd, J ¼ 8.2, 4.3 Hz), 3.71 (3H, s), 3.89 (1H, dd,
J ¼ 17.9, 5.3 Hz), 3.98 (1H, dd, J ¼ 17.9, 5.3 Hz), 4.42 (1H, quint,
J ¼ 7.2 Hz), 6.87 (1H, t, J ¼ 5.3 Hz), 7.20e7.31 (9H, m), 7.39e7.45 (7H,m); 13C NMR (100 MHz, CDCl3): d 17.4, 37.1, 41.0, 48.3, 52.2, 53.9,
67.0, 126.8 (3C), 128.0 (6C), 129.5 (6C), 144.5 (3C), 170.0, 172.2, 173.4;
IR (neat): 3311, 3057, 3016, 2952, 1751, 1652, 1514, 1444, 1369, 1211,
1182, 1032, 982, 886, 848, 746, 700 cm�1; HRMS (FAB): m/z calcd for
C28H32N3O4S (MþþH) 506.2108, found 506.2106.
4.1.22. (7S,11R,14S,17R,E)-Methyl 11-isopropyl-7-(4methoxybenzyloxy)-17-methyl-9,12,15,18-tetraoxo-1,1,1-triphenyl14-tritylthiomethyl-2-thia-10,13,16,19-tetraazahenicos-5-en-21oate (26c)Compound 26c was synthesized from 15c (101 mg, 0.20 mmol)
in a manner similar to that described for the synthesis of 26a. Puriﬁcation by column chromatography (CHCl3/MeOH 30:1) gave 26c(193 mg, 86%) as a colorless amorphous solid. [a]27
D �0.39 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 0.72 (3H, d, J ¼ 6.8 Hz), 0.79
(3H, d, J ¼ 6.8 Hz), 1.33 (3H, d, J ¼ 6.8 Hz), 1.96 (1H, dq, J ¼ 13.4,
6.7 Hz), 2.05e2.15 (2H, m), 2.21e2.25 (2H, m), 2.38e2.52 (3H, m),
2.70 (1H, dd, J ¼ 13.2, 7.3 Hz), 3.65 (3H, s), 3.76 (3H, s), 3.89 (2H, d,
J ¼ 5.4 Hz), 4.00 (1H, td, J ¼ 4.4, 2.9 Hz), 4.08 (1H, q, J ¼ 6.3 Hz), 4.19
(1H, d, J ¼ 10.7 Hz), 4.30 (1H, t, J ¼ 7.1 Hz), 4.34 (1H, d, J ¼ 10.7 Hz),4.72 (1H, dq, J ¼ 7.2, 7.1 Hz), 5.28 (1H, dd, J ¼ 16.4, 7.8 Hz), 5.51 (1H,
dt, J ¼ 15.1, 6.8 Hz), 6.81 (2H, d, J ¼ 8.3 Hz), 6.91 (1H, br s), 7.14e7.28(22H, m), 7.35e7.42 (12H, m), 7.49 (1H, br s); 13C NMR (100 MHz,CDCl3): d 17.9, 18.3, 19.2, 30.6, 31.2, 31.3, 34.0, 41.0, 42.5, 48.7, 52.0,
52.1, 55.2, 58.8, 66.5, 67.0, 69.9, 76.4, 113.7 (2C), 126.6 (3C), 126.8(3C), 127.8 (6C), 128.0 (6C), 129.4 (6C), 129.5 (6C), 129.7, 129.8 (2C),
130.0, 133.1, 144.2 (3C), 144.8 (3C), 159.2, 169.4, 170.0, 171.3 (2C),
172.3; IR (neat): 3277, 3082, 3059, 3029, 3018, 2960, 2931, 1752,
1692, 1631, 1537, 1489, 1444, 1246, 1215, 1181, 1080, 1034, 976, 822,
752, 700 cm�1; HRMS (FAB): m/z calcd for C67H73N4O8S2 (MþþH)1125.4864, found 1125.4873.
4.1.23. (7S,11R,14S,17R,E)-Methyl 7-hydroxy-11-isopropyl-17methyl-9,12,15,18-tetraoxo-1,1,1-triphenyl-14-tritylthiomethyl-2thia-10,13,16,19-tetraazahenicos-5-en-21-oate (27c)Compound 27c was synthesized from 26c (193 mg, 0.17 mmol)
in a manner similar to that described for the synthesis of 27a. Puriﬁcation by column chromatography (CHCl3/MeOH 40:1) gave 27c(140 mg, 81%) as a colorless amorphous solid. [a]27
D �1.6 (c 1.0,

601

CHCl3); 1H NMR (400 MHz, CDCl3): d 0.89 (3H, d, J ¼ 7.3 Hz), 0.96
(3H, d, J ¼ 6.8 Hz), 1.40 (3H, d, J ¼ 7.3 Hz), 2.05e2.10 (2H, m),
2.20e2.28 (4H, m), 2.36 (1H, dd, J ¼ 13.7, 2.9 Hz), 2.53e2.63 (2H, m),
2.92 (1H, br s), 3.67 (3H, s), 3.81 (1H, dd, J ¼ 18.0, 5.4 Hz), 3.96 (1H,
dd, J ¼ 18.0, 5.9 Hz), 4.10e4.20 (2H, m), 4.29e4.33 (1H, m), 4.42 (1H,
quint, J ¼ 7.3 Hz), 5.36 (1H, dd, J ¼ 15.6, 6.3 Hz), 5.39 (1H, dt, J ¼ 15.6,
6.3 Hz), 6.20 (1H, d, J ¼ 6.8 Hz), 6.81 (1H, d, J ¼ 7.8 Hz), 7.01 (1H, t,
J ¼ 4.9 Hz), 7.05 (1H, d, J ¼ 7.3 Hz), 7.17e7.30 (18H, m), 7.36e7.42(12H, m); 13C NMR (100 MHz, CDCl3): d 17.9, 18.1, 19.2, 30.4, 31.30,
31.32, 33.9, 41.0, 43.5, 49.0, 52.0, 52.2, 58.8, 66.6, 66.8, 69.6, 126.6(3C), 126.9 (3C), 127.8 (6C), 128.0 (6C), 129.4 (6C), 129.5 (6C), 130.0,
132.5, 144.2 (3C), 144.8 (3C), 169.7, 170.3, 171.3, 172.2, 172.3; IR(neat): 3279, 3083, 3060, 3030, 3017, 2963, 2928, 1746, 1691, 1632,
1537, 1488, 1444, 1217, 1183, 1081, 1033, 971, 853, 751, 699 cm�1;
HRMS (FAB): m/z calcd for C59H65N4O7S2 (MþþH) 1005.4289, found1005.4321.
4.1.24. (7S,11R,14S,17R,E)-7-Hydroxy-11-isopropyl-17-methyl9,12,15,18-tetraoxo-1,1,1-triphenyl-14-tritylthiomethyl-2-thia10,13,16,19-tetraazahenicos-5-en-21-oic acid (14c)Compound 14c was synthesized from 27c (106 mg, 0.11 mmol)
in a manner similar to that described for the synthesis of 14a. Puriﬁcation by column chromatography (CHCl3/MeOH 5:1) gave 14c(134 mg, 97%) as a colorless amorphous solid. [a]29
D �15.4 (c 1.0,
CHCl3); 1H NMR (400 MHz, CD3OD/CDCl3 3:1): d 0.91 (3H, d,
J ¼ 6.8 Hz), 0.93 (3H, d, J ¼ 7.3 Hz), 1.34 (3H, d, J ¼ 6.8 Hz), 2.01e2.06(2H, m), 2.10e2.21 (3H, m), 2.36 (1H, dd, J ¼ 13.7, 4.4 Hz), 2.46 (1H,
dd, J ¼ 13.7, 8.8 Hz), 2.57 (1H, dd, J ¼ 11.7, 8.8 Hz), 2.64 (1H, dd,
J ¼ 12.2, 5.9 Hz), 3.69 (1H, d, J ¼ 17.6 Hz), 3.79 (1H, d, J ¼ 18.0 Hz),4.16 (1H, d, J ¼ 5.9 Hz), 4.18e4.26 (1H, m), 4.30e4.43 (2H, m), 5.43
(1H, dd, J ¼ 15.6, 5.9 Hz), 5.53 (1H, dt, J ¼ 15.1, 6.3 Hz), 7.14e7.24(18H, m), 7.33e7.39 (12H, m); 13C NMR (100 MHz, CD3OD/CDCl3
3:1): d 18.0, 18.3, 19.7, 30.9, 32.2, 32.3, 34.0, 42.5, 44.1, 50.2, 53.7,
60.4, 67.5, 67.8, 70.3, 127.5 (3C), 127.7, (3C), 128.6 (6C), 128.8 (6C),
130.36 (6C), 130.44 (6C), 130.5, 133.9, 145.4 (3C), 145.9 (3C), 171.4,
173.4, 173.9, 174.1, 174.2; IR (neat): 3291, 3057, 3017, 2965, 2929,
1727, 1636, 1527, 1490, 1444, 1405, 1217, 1034, 971, 754, 700 cm�1;
HRMS (FAB): m/z calcd for C58H62N4O7S2Na (MþþNa) 1013.3952,
found 1013.3991.
4.1.25. (6R,9S,12R,16S)-12-Isopropyl-6-methyl-16-[(E)-4tritylthiobut-1-enyl]-9-tritylthiomethyl-1-oxa-4,7,10,13tetraazacyclohexadecane-2,5,8,11,14-pentaone (28c)Compound 28c was synthesized from 14c (134 mg, 0.14 mmol)
in a manner similar to that described for the synthesis of 28a. Puriﬁcation by column chromatography (CHCl3/MeOH 40:1) gave 28c(108 mg, 85%) as a colorless amorphous solid. [a]27
D �2.2 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 0.85 (3H, d, J ¼ 6.8 Hz), 0.92
(3H, d, J ¼ 6.8 Hz), 1.44 (3H, d, J ¼ 7.3 Hz), 1.92e2.17 (5H, m), 2.38
(1H, dd, J ¼ 14.1, 9.8 Hz), 2.43e2.52 (1H, m), 2.64 (1H, dd, J ¼ 12.7,
5.4 Hz), 2.81 (1H, dd, J ¼ 12.2, 8.8 Hz), 3.56 (1H, q, J ¼ 6.8 Hz),3.90e4.02 (2H, m), 4.16 (1H, quint, J ¼ 7.3 Hz), 4.25 (1H, t,
J ¼ 7.1 Hz), 5.31 (1H, dd, J ¼ 15.6, 6.3 Hz), 5.49e5.61 (2H, m), 6.33
(1H, d, J ¼ 8.3 Hz), 7.16e7.30 (20H, m), 7.36e7.39 (12H, m), 7.81 (1H,
br s); 13C NMR (100 MHz, CDCl3, 40 � C): d 16.7, 18.0, 19.6, 30.9, 31.1,
31.4, 31.8, 41.7, 42.4, 50.2, 56.0, 58.4, 66.7, 67.2, 72.1, 126.6 (3C), 126.9(3C), 127.9 (6C), 128.0, 128.1 (6C), 129.4 (6C), 129.6 (6C), 132.5, 144.4(3C), 144.9 (3C), 168.9, 169.8, 170.2, 172.2, 173.0; IR (neat): 3307,
3057, 3016, 2964, 2932, 1745, 1660, 1651, 1538, 1490, 1444, 1265,
1215, 1033, 972, 750, 700 cm�1. HRMS (FAB): m/z calcd for
C58H60N4O6S2Na (MþþNa) 995.3846, found 995.3847.
4.1.26. FK-A3 (13c)Compound 13c was synthesized from 28c (108 mg, 0.11 mmol)
in a manner similar to that described for the synthesis of 13a.

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Puriﬁcation by column chromatography (CHCl3/MeOH 20:1) gave
13c (42.4 mg, 79%) as a colorless amorphous solid. [a]25
D þ19.8 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 1.09 (6H, d, J ¼ 5.9 Hz), 1.54
(3H, d, J ¼ 6.8 Hz), 2.23 (1H, dq, J ¼ 12.7, 6.8 Hz), 2.49e2.59 (1H, m),
2.66e2.77 (2H, m), 2.94e3.03 (3H, m), 3.11 (1H, ddd, J ¼ 13.2, 9.3,
3.4 Hz), 3.33 (1H, dd, J ¼ 16.1, 11.2 Hz), 3.77 (1H, q, J ¼ 6.8 Hz), 3.88
(1H, dd, J ¼ 17.6, 2.0 Hz), 4.06 (1H, dd, J ¼ 5.9, 4.4 Hz), 4.41 (1H, dd,
J ¼ 17.6, 6.3 Hz), 4.76 (1H, ddd, J ¼ 11.2, 7.3, 3.9 Hz), 5.69e5.80 (2H,m), 5.80e5.91 (1H, m), 6.64 (1H, d, J ¼ 3.9 Hz), 7.11 (1H, d,
J ¼ 3.9 Hz), 7.59 (1H, d, J ¼ 7.3 Hz), 7.73 (1H, d, J ¼ 6.3 Hz); 13C NMR(100 MHz, CDCl3): d 15.1, 19.3, 19.5, 29.2, 31.1, 34.2, 37.5, 38.1, 43.1,
51.2, 55.1, 62.0, 69.4, 129.7, 130.3, 168.0, 170.03, 170.05, 170.8, 172.4;
IR (neat): 3342, 3012, 2965, 2934, 1742, 1659, 1525, 1260, 1186, 1024,
979, 754 cm�1; HRMS (EI): m/z calcd for C20H30N4O6S2 (Mþ)
486.1607, found 486.1616.
4.1.27. Fmoc-L-AlaeGly-OMe (30d)Compound 30d was synthesized from L-alanine (L-Ala) (18d)(500 mg, 5.6 mmol) in a manner similar to that described for thesynthesis of 21. Puriﬁcation by column chromatography (hexane/
EtOAc, 2:3) gave 30d (1.99 g, 93%, 2 steps) as a colorless amorphous
1
solid. [a]29
D �13.2 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): d 1.41
(3H, d, J ¼ 6.3 Hz), 3.72 (3H, s), 4.02 (2H, d, J ¼ 4.9 Hz), 4.20 (1H, t,
J ¼ 6.8 Hz), 4.27e4.38 (1H, m), 4.40 (2H, d, J ¼ 6.3 Hz), 5.51 (1H, d,
J ¼ 6.3 Hz), 6.71 (1H, br s), 7.30 (2H, t, J ¼ 7.3 Hz), 7.39 (2H, t,
J ¼ 7.3 Hz), 7.58 (2H, dd, J ¼ 6.8, 2.4 Hz), 7.75 (2H, d, J ¼ 7.3 Hz); 13C
NMR (100 MHz, CDCl3): d 18.5, 41.2, 47.1, 50.4, 52.4, 67.1, 120.0 (2C),
125.0 (2C), 127.1 (2C), 127.7 (2C), 141.3 (2C), 143.7 (2C), 156.0, 170.0,
172.5; IR (KBr): 3322, 3293, 3067, 2983, 2951, 2941, 2890, 1731,
1720, 1693, 1648, 1541, 1449, 1329, 1301, 1266, 1254, 1105, 1089,
1051, 1023, 758, 741, 671, 645 cm�1; HRMS (EI): m/z calcd for
C21H22N2O5 (Mþ) 382.1529, found 382.1520.

4.1.30. (7S,11R,14S,17S,E)-Methyl 11-isopropyl-7-(4methoxybenzyloxy)-17-methyl-9,12,15,18-tetraoxo-1,1,1-triphenyl14-tritylthiomethyl-2-thia-10,13,16,19-tetraazahenicos-5-en-21oate (26d)Compound 26d was synthesized from 15d (119 mg, 0.24 mmol)
in a manner similar to that described for the synthesis of 26a. Puriﬁcation by column chromatography (CHCl3/MeOH 40:1) gave 26d(240 mg, 91%) as a colorless amorphous solid. [a]27
D �4.0 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 0.69 (3H, d, J ¼ 6.8 Hz), 0.79
(3H, d, J ¼ 6.8 Hz), 1.34 (3H, d, J ¼ 7.3 Hz), 2.27 (1H, dq, J ¼ 13.2,
6.6 Hz), 2.05e2.14 (2H, m), 2.21e2.25 (2H, m), 2.47 (2H, d,
J ¼ 5.4 Hz), 2.56 (1H, dd, J ¼ 12.7, 5.4 Hz), 2.78 (1H, dd, J ¼ 13.4,
7.1 Hz), 3.64 (3H, s), 3.77 (3H, s), 3.82 (1H, dd, J ¼ 18.0, 5.4 Hz),3.89e3.99 (2H, m), 4.06 (1H, q, J ¼ 6.7 Hz), 4.16 (1H, t, J ¼ 6.6 Hz),4.23 (1H, d, J ¼ 10.7 Hz), 4.41 (1H, d, J ¼ 10.7 Hz), 4.57 (1H, dq,
J ¼ 7.4, 7.2 Hz), 5.29 (1H, dd, J ¼ 15.4, 8.0 Hz), 5.55 (1H, dt, J ¼ 15.1,
6.8 Hz), 6.78 (1H, d, J ¼ 8.3 Hz), 6.82 (2H, d, J ¼ 8.8 Hz), 7.01 (1H, d,
J ¼ 7.3 Hz), 7.16e7.28 (22H, m), 7.33e7.42 (12H, m); 13C NMR(100 MHz, CDCl3): d 17.7, 17.8, 19.3, 30.3, 31.2, 31.3, 33.7, 40.9, 42.4,
48.9, 52.1, 52.5, 55.2, 58.9, 66.5, 67.0, 69.9, 76.4, 113.8 (2C), 126.6(3C), 126.8 (3C), 127.8 (6C), 128.0 (6C), 129.45 (6C), 129.48 (6C),
129.7, 129.8 (2C), 129.9, 133.1, 144.3 (3C), 144.8 (3C), 159.3, 169.5,
170.1, 171.2, 171.6, 172.4; IR (neat): 3291, 3058, 3016, 2961, 2931,
1752, 1635, 1513, 1490, 1444, 1390, 1368, 1247, 1215, 1181, 1079,
1034, 975, 847, 823, 751, 700 cm�1; HRMS (FAB): m/z calcd for
C67H73N4O8S2 (MþþH) 1125.4864, found 1125.4873.

4.1.28. Boc-D-Cys(Tr)eL-AlaeGly-OMe (32d)Compound 32d was synthesized from 30d (1.65 g, 4.3 mmol) ina manner similar to that described for the synthesis of 23. Puriﬁcation by column chromatography (hexane/EtOAc 1:2) gave 32d(2.24 g, 86%, 2 steps) as a colorless amorphous solid. [a]25
D �31.4 (c1.0, CHCl3); 1H NMR (400 MHz, CDCl3): d 1.36 (3H, d, J ¼ 6.8 Hz), 1.40
(9H, s), 2.55 (1H, dd, J ¼ 12.9, 5.4 Hz), 2.75 (1H, dd, J ¼ 12.0, 6.8 Hz),3.66e3.73 (4H, m), 3.82 (1H, dd, J ¼ 18.0, 5.4 Hz), 4.00 (1H, dd,
J ¼ 18.0, 5.4 Hz), 4.48 (1H, dq, J ¼ 7.2, 7.1 Hz), 4.90 (1H, br s), 6.39
(1H, br s), 7.04 (1H, br s), 7.04e7.31 (9H, m), 7.39e7.41 (6H, m); 13C
NMR (100 MHz, CDCl3): d 17.9, 28.0 (3C), 33.7, 40.8, 48.5, 51.9, 53.6,
66.8, 80.0, 126.7 (3C), 127.8 (6C), 129.3 (6C), 144.1 (3C), 155.2, 169.9,
170.2, 172.2; IR (neat): 3299, 3058, 3006, 2978, 2932, 1752, 1652,
1518, 1491, 1445, 1367, 1213, 1166, 1049, 1021, 854, 751, 700 cm�1;
HRMS (FAB): m/z calcd for C33H40N3O6S (MþþH) 606,2632, found;606.2625.

4.1.31. (7S,11R,14S,17S,E)-Methyl 7-hydroxy-11-isopropyl-17methyl-9,12,15,18-tetraoxo-1,1,1-triphenyl-14-tritylthiomethyl-2thia-10,13,16,19-tetraazahenicos-5-en-21-oate (27d)Compound 27d was synthesized from 26d (240 mg, 0.21 mmol)
in a manner similar to that described for the synthesis of 27a. Puriﬁcation by column chromatography (CHCl3/MeOH 50:1) gave 27d(213 mg, 99%) as a colorless amorphous solid. [a]27
D �37.8 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 0.95 (3H, d, J ¼ 6.8 Hz), 1.00
(3H, d, J ¼ 6.8 Hz), 1.42 (3H, d, J ¼ 7.3 Hz), 2.03e2.08 (2H, m),
2.18e2.37 (4H, m), 2.42 (1H, dd, J ¼ 13.2, 2.0 Hz), 2.64 (2H, d,
J ¼ 6.8 Hz), 3.54 (3H, s), 3.61 (1H, dd, J ¼ 18.0, 4.4 Hz), 4.00 (1H, br s),
4.08e4.10 (2H, m), 4.33e4.45 (3H, m), 5.38 (1H, dd, J ¼ 15.6, 5.9 Hz),5.59 (1H, dt, J ¼ 15.6, 6.3 Hz), 6.01 (1H, d, J ¼ 5.8 Hz), 6.96 (1H, t,
J ¼ 5.6 Hz), 7.02 (1H, d, J ¼ 6.8 Hz), 7.16e7.30 (19H, m), 7.33e7.42(12H, m); 13C NMR (100 MHz, CDCl3): d 16.1, 17.4, 19.5, 29.0, 31.1,
31.3, 33.6, 40.7, 43.7, 48.8, 51.9, 52.1, 59.9, 66.5, 66.7, 69.3, 126.5 (3C),
126.6 (3C), 127.7 (6C), 127.8 (6C), 129.38 (6C), 129.42 (6C), 129.7,
132.7, 144.3 (3C), 144.7 (3C), 170.3, 170.7, 170.8, 172.9, 173.0; IR(neat): 3291, 3082, 3058, 3017, 2964, 2930, 1745, 1633, 1531, 1490,
1444, 1391, 1370, 1216, 1183, 1081, 1033, 972, 850, 751, 700 cm�1;
HRMS (FAB): m/z calcd for C59H65N4O7S2 (MþþH) 1005.4289, found1005.4293.

4.1.29. H2N-D-Cys(Tr)eL-AlaeGly-OMe (15d)Compound 15d was synthesized from 32d (1.83 g, 3.0 mmol) ina manner similar to that described for the synthesis of 15a. Puriﬁcation by column chromatography (CHCl3/MeOH 15:1) gave 15d(1.43 g, 94%) as a colorless amorphous solid. [a]25
D �64.5 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 1.34 (3H, d, J ¼ 6.8 Hz), 1.47
(2H, br s), 2.64 (2H, d, J ¼ 4.9 Hz), 3.05 (1H, t, J ¼ 5.6 Hz), 3.64 (3H, s),
3.78 (1H, dd, J ¼ 17.6, 5.4 Hz), 3.90 (1H, dd, J ¼ 17.6, 5.9 Hz), 4.49
(1H, dq, J ¼ 7.2, 7.1 Hz), 7.18e7.28 (10H, m), 7.39e7.41 (6H, m), 7.58
(1H, d, J ¼ 7.8 Hz); 13C NMR (100 MHz, CDCl3): d 17.6, 37.3, 40.7, 48.2,
51.9, 53.8, 66.7, 126.7 (3C), 127.8 (6C), 129.3 (6C), 144.3 (3C), 169.8,
172.3, 173.0; IR (neat): 3308, 3057, 3016, 2951, 1750, 1654, 1509,
1444, 1369, 1211, 1183, 1032, 981, 886, 848, 745, 700 cm�1; HRMS
(FAB): m/z calcd for C28H32N3O4S (MþþH) 506.2108, found506.2109.

4.1.32. (7S,11R,14S,17S,E)-7-Hydroxy-11-isopropyl-17-methyl9,12,15,18-tetraoxo-1,1,1-triphenyl-14- tritylthiomethyl-2-thia10,13,16,19-tetraazahenicos-5-en-21-oic acid (14d)Compound 14d was synthesized from 27d (213 mg, 0.21 mmol)
in a manner similar to that described for the synthesis of 14a. Puriﬁcation by column chromatography (CHCl3/MeOH 5:1) gave 14d(209 mg, 99%) as a colorless amorphous solid. [a]30
D �16.9 (c 1.0,
CHCl3); 1H NMR (400 MHz, CD3OD/CDCl3 3:1): d 0.94 (3H, d,
J ¼ 5.4 Hz), 0.95 (3H, d, J ¼ 6.3 Hz), 1.37 (3H, d, J ¼ 6.8 Hz), 2.01e2.06(2H, m), 2.09e2.21 (3H, m), 2.36 (1H, dd, J ¼ 13.7, 4.4 Hz), 2.48 (1H,
dd, J ¼ 13.7, 9.6 Hz), 2.57 (1H, dd, J ¼ 12.2, 8.8 Hz), 2.64 (1H, dd,
J ¼ 12.7, 5.9 Hz), 3.79 (2H, s), 4.08 (1H, d, J ¼ 5.4 Hz), 4.13 (1H, dd,
J ¼ 8.3, 6.3 Hz), 4.29 (1H, q, J ¼ 6.8 Hz), 4.39e4.43 (1H, m), 5.43 (1H,
dd, J ¼ 15.1, 6.3 Hz), 5.55 (1H, dt, J ¼ 15.1, 6.3 Hz), 7.15e7.26 (18H, m),
7.35e7.39 (12H, m); 13C NMR (100 MHz, CD3OD/CDCl3 3:1, 45 � C):— 218 —

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d 17.6, 18.2, 19.6, 30.5, 32.0, 32.2, 33.8, 41.7, 43.9, 50.0, 53.7, 60.4,
67.4, 67.7, 69.8, 127.3 (3C), 127.5, (3C), 128.4 (6C), 128.6 (6C), 130.2(6C), 130.26, 130.27 (6C), 133.7, 145.3 (3C), 145.7 (3C), 171.3, 172.5,
173.1, 174.2, 174.3; IR (neat): 3297, 3057, 3018, 2964, 2928, 1725,
1638, 1531, 1490, 1444, 1217, 1033, 970, 750, 700 cm�1; HRMS (FAB):m/z calcd for C58H63N4O7S2 (MþþH) 991.4133, found 991.4153.
4.1.33. (6S,9S,12R,16S)-12-Isopropyl-6-methyl-16-[(E)-4tritylthiobut-1-enyl]-9-tritylthiomethyl-1-oxa-4,7,10,13tetraazacyclohexadecane-2,5,8,11,14-pentaone (28d)Compound 28d was synthesized from 14d (104 mg, 0.11 mmol)
im a manner similar to that described for the synthesis of 28a.
Puriﬁcation by column chromatography (CHCl3/MeOH 40:1) gave
28d (87.6 mg, 86%) as a colorless amorphous solid. [a]27
D �16.3 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 0.85 (3H, d, J ¼ 6.3 Hz), 0.95
(3H, d, J ¼ 6.3 Hz), 1.20 (3H, d, J ¼ 7.2 Hz), 1.88e2.18 (5H, m),
2.42e2.57 (2H, m), 2.75 (1H, dd, J ¼ 12.6, 3.4 Hz), 2.91 (1H, dd,
J ¼ 12.6, 9.7 Hz), 3.49 (1H, dd, J ¼ 16.4, 3.4 Hz), 3.56e3.64 (1H, m),
4.29 (1H, dd, J ¼ 16.4, 8.2 Hz), 4.34e4.51 (2H, m), 5.37 (1H, dd,
J ¼ 15.5, 6.3 Hz), 5.47e5.62 (2H, m), 6.17 (1H, br d, J ¼ 5.3 Hz),7.13e7.27 (20H, m), 7.33e7.38 (12H, m), 7.67 (1H, br s); 13C NMR(100 MHz, CDCl3, 40 � C): d 17.2, 17.6, 19.8, 31.1, 31.4, 32.1, 32.2, 42.1,
42.5, 48.5, 54.6, 57.7, 66.7, 66.9, 72.0, 126.6 (3C), 126.8 (3C), 127.8(6C), 127.9 (6C), 128.0, 129.5 (6C), 129.6 (6C), 132.8, 144.6 (3C), 144.8(3C), 169.0, 169.2, 169.8, 172.3, 172.8; IR (neat): 3289, 3058, 3016,
2965, 2933, 1734, 1654, 1540, 1489, 1444, 1216, 1195, 1033, 980, 751,
700 cm�1; HRMS (FAB): m/z calcd for C58H60N4O6S2Na (MþþNa)995.3846, found 995.3822.
4.1.34. FK-A4 (13d)Compound 13d was synthesized from 28d (87.0 mg, 93 mmol) ina manner similar to that described for the synthesis of 13a. Puriﬁcation by column chromatography (CHCl3/MeOH 20:1) gave 13d(40.0 mg, 89%) as a colorless amorphous solid. [a]27
D �30.4 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 1.09 (3H, d, J ¼ 4.4 Hz), 1.10
(3H, d, J ¼ 3.9 Hz), 1.42 (3H, d, J ¼ 7.3 Hz), 2.30 (1H, dq, J ¼ 12.2,
6.8 Hz), 2.46e2.57 (1H, m), 2.62e2.76 (2H, m), 2.89e2.96 (2H, m),
3.08 (1H, ddd, J ¼ 13.2, 8.8, 2.9 Hz), 3.14 (1H, dd, J ¼ 15.1, 3.9 Hz),3.27 (1H, dd, J ¼ 15.1, 10.2 Hz), 4.06 (1H, t, J ¼ 4.4 Hz), 4.11 (1H, dd,
J ¼ 17.6, 5.4 Hz), 4.20 (1H, dd, J ¼ 17.6, 5.4 Hz), 4.54 (1H, q,
J ¼ 7.3 Hz), 4.80 (1H, ddd, J ¼ 10.2, 6.8, 3.9 Hz), 5.70e5.81 (2H, m),
5.83e5.94 (1H, m), 6.48 (1H, d, J ¼ 2.9 Hz), 6.90 (1H, t, J ¼ 4.9 Hz),7.43 (1H, d, J ¼ 6.8 Hz), 7.48 (1H, d, J ¼ 8.3 Hz); 13C NMR (100 MHz,CDCl3): d 17.2, 18.7, 19.4, 29.3, 31.3, 37.5, 37.6, 39.5, 42.3, 49.5, 56.0,
62.2, 69.5, 128.9, 129.7, 167.3, 169.6, 171.0, 171.1171.9; IR (neat): 3297,
3009, 2966, 2933, 1742, 1655, 1524, 1253, 1190, 1136, 1024, 979,
754 cm�1; HRMS (EI): m/z calcd for C20H30N4O6S2 (Mþ) 486.1607,
found 486.1601.
4.1.35. Fmoc-D-PheeGly-OMe (30e)Compound 30e was synthesized from D-phenylalanine (D-Phe)(18e) (500 mg, 3.0 mmol) in a manner similar to that described forthe synthesis of 21. Puriﬁcation by column chromatography (CHCl3/
MeOH 30:1) gave 30e (971 mg, 70%, 2 steps) as a colorless amor1
phous solid. [a]25
D þ11.1 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3):
d 3.07e3.11 (2H, m), 3.68 (3H, s), 3.89e4.02 (2H, m), 4.15 (1H, t,
J ¼ 6.8 Hz), 4.26e4.30 (1H, m), 4.37e4.41 (1H, m), 4.52 (1H, d,
J ¼ 5.9 Hz), 5.55 (1H, br d, J ¼ 6.8 Hz), 6.57 (1H, br s), 7.19e7.29 (7H,m), 7.38 (2H, t, J ¼ 7.3 Hz), 7.50 (2H, t, J ¼ 7.3 Hz), 7.74 (2H, d,
J ¼ 7.3 Hz); 13C NMR (100 MHz, CDCl3): d 38.4, 41.1, 46.3, 52.2, 55.9,
67.0, 119.9 (2C), 125.0, 126.9 (2C), 127.0 (2C), 127.6 (2C), 128.6 (2C),
129.2 (2C), 136.3, 141.2 (2C), 143.6 (2C), 156.0, 169.8, 171.3; IR (neat):
3301, 3064, 3029, 2952, 1748, 1660, 1538, 1449, 1259, 1213, 1036,
758, 740, 700 cm�1; HRMS (FAB): m/z calcd for C27H27N2O5 (MþþH)459.1920, found 459.1924.

603

4.1.36. Boc-D-Cys(Tr)eD-PheeGly-OMe (32e)Compound 32e was synthesized from 30e (775 mg, 1.7 mmol) ina manner similar to that described for the synthesis of 23. Puriﬁcation by column chromatography (hexane/EtOAc 1:1) gave 32e(1.07 g, 92%, 2 steps) as a colorless amorphous solid. [a]24
D þ15.4 (c1.0, CHCl3); 1H NMR (400 MHz, CDCl3): d 1.34 (9H, s), 2.52e2.62(2H, m), 3.08 (1H, dd, J ¼ 14.2, 6.3Hz), 3.15 (1H, dd, J ¼ 13.9, 6.8 Hz),3.60 (1H, dd, J ¼ 11.7, 5.9 Hz), 3.65 (3H, s), 3.71 (1H, dd, J ¼ 17.8,
5.9 Hz), 3.93 (1H, dd, J ¼ 18.1, 5.9 Hz), 4.64 (1H, d, J ¼ 5.4 Hz), 4.73
(1H, dd, J ¼ 14.9, 6.3Hz), 6.32 (1H, d, J ¼ 8.3 Hz), 7.00 (1H, br s),
7.13e7.29 (14H, m), 7.37e7.40 (6H, m); 13C NMR (100 MHz, CDCl3):
d 28.1 (3C), 33.2, 37.0, 41.0, 52.1, 53.4, 54.2, 67.4, 80.6, 126.9, 127.0(3C), 128.1 (6C), 128.6 (2C), 129.3 (2C), 129.4 (6C), 136.1, 144.1 (3C),
155.7, 169.6, 170.2, 170.8; IR (neat): 3277, 3060, 3030, 2979, 2928,
1740, 1698, 1675, 1645, 1531, 1443, 1392, 1366, 1279, 1173, 1032, 746,
700 cm�1; HRMS (FAB): m/z calcd for C39H44N3O6S (MþþH)682.2951, found 682.2957.
4.1.37. H2N-D-Cys(Tr)eD-PheeGly-OMe (15e)Compound 15e was synthesized from 32e (1.14 g, 1.7 mmol) in amanner similar to that described for the synthesis of 15a. Puriﬁcation by column chromatography (CHCl3/MeOH 40:1) gave 15e(837 mg, 86%) as a colorless amorphous solid. [a]25
D þ11.5 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 1.26 (2H, br s), 2.39 (1H, dd,
J ¼ 12.9, 8.3 Hz), 2.59 (1H, dd, J ¼ 13.2, 4.4 Hz), 2.85 (1H, dd, J ¼ 8.3,
4.4 Hz), 3.01 (1H, dd, J ¼ 13.9, 7.3 Hz), 3.11 (1H, dd, J ¼ 13.9, 6.8 Hz),3.67 (3H, s), 3.80 (1H, dd, J ¼ 15.3, 5.4 Hz), 3.94 (1H, dd, J ¼ 18.1,
5.4 Hz), 4.64 (1H, dd, J ¼ 15.1, 7.8 Hz), 6.80 (1H, t, J ¼ 5.4 Hz),7.13e7.28 (14H, m), 7.39e7.45 (7H, m); 13C NMR (100 MHz, CDCl3):
d 36.9, 37.4, 41.0, 52.2, 53.6, 53.9, 66.9, 126.8 (4C), 127.9 (6C), 128.5(2C), 129.2 (2C), 129.5 (6C), 136.4, 144.4 (3C), 170.0, 171.0, 173.5; IR(neat): 3308, 3059, 3029, 2926, 1748, 1652, 1539, 1517, 1443, 1372,
1208, 1082, 1033, 744, 700, 667 cm�1; HRMS (FAB): m/z calcd for
C34H36N3O4S (MþþH) 582.2427, found 582.2421.
4.1.38. (7S,11R,14S,17R,E)-Methyl 17-benzyl-11-isopropyl-7-(4methoxybenzyloxy)-9,12,15,18-tetraoxo-1,1,1-triphenyl-14tritylthiomethyl-2-thia-10,13,16,19-tetraazahenicos-5-en-21-oate(26e)Compound 26e was synthesized from 15e (200 mg, 0.34 mmol)
in a manner similar to that described for the synthesis of 26a. Puriﬁcation by column chromatography (CHCl3/MeOH 40:1) gave 26e(409 mg, 99%) as a colorless amorphous solid. [a]25
D �3.4 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 0.65 (3H, d, J ¼ 6.8 Hz), 0.78
(3H, d, J ¼ 6.8 Hz), 1.89e1.98 (1H, m), 2.07e2.16 (2H, m), 2.19e2.27(2H, m), 2.32e2.48 (3H, m), 2.70 (1H, dd, J ¼ 12.7, 6.3 Hz), 3.08 (1H,
dd, J ¼ 14.1, 9.3 Hz), 3.26 (1H, dd, J ¼ 14.4, 5.4 Hz), 3.66 (3H, s),
3.74e3.80 (4H, m), 3.87 (1H, dd, J ¼ 5.9, 12.4 Hz), 3.95e4.05 (3H, m),
4.17 (1H, d, J ¼ 10.7 Hz), 4.43 (1H, d, J ¼ 10.7 Hz), 4.80 (1H, dt, J ¼ 8.7,
4.9 Hz), 5.29 (1H, dd, J ¼ 15.4, 7.8 Hz), 5.51 (1H, dt, J ¼ 15.1, 6.4 Hz)
6.52 (1H, br d, J ¼ 5.9 Hz), 6.79e6.82 (3H, m), 7.11e7.32 (33H, m),7.41e7.43 (6H, m); 13C NMR (100 MHz, CDCl3): d 17.7, 19.4, 29.7, 31.2,
31.3, 33.1, 36.9, 41.0, 42.3, 52.0, 52.4, 54.0, 55.2, 59.5, 66.6, 67.1, 70.0,
76.4, 113.8 (2C), 126.5, 126.6 (3C), 126.9 (3C), 127.7 (6C), 127.8 (6C),
128.1 (2C), 128.3, 129.2 (2C), 129.4 (6C), 129.5 (6C), 129.6, 129.9 (2C),
133.6, 137.1, 144.0 (3C), 144.8 (3C), 159.4, 169.6, 169.8, 171.1, 171.6,
171.9; IR (neat): 3280, 3059, 3030, 2959, 2929, 1743, 1635, 1514,
1443, 1247, 1216, 1079, 1034, 976, 745, 699 cm�1; HRMS (FAB): m/z
calcd for C73H77N4O8S2 (MþþH) 1201.5183, found 1201.5183.
4.1.39. (7S,11R,14S,17R,E)-Methyl 17-benzyl-7-hydroxy-11isopropyl-9,12,15,18-tetraoxo-1,1,1-triphenyl-14-tritylthiomethyl-2thia-10,13,16,19-tetraazahenicos-5-en-21-oate (27e)Compound 27e was synthesized from 26e (409 mg, 0.34 mmol)
in a manner similar to that described for the synthesis of 27a.

— 219 —

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Puriﬁcation by column chromatography (CHCl3/MeOH 50:1) gave
27e (331 mg, 90%) as a colorless amorphous solid. [a]25
D þ3.2 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 0.85 (3H, d, J ¼ 7.3 Hz), 0.92
(3H, d, J ¼ 7.3 Hz), 2.04e2.35 (7H, m), 2.43 (1H, dd, J ¼ 12.7, 5.4 Hz),2.49 (1H, dd, J ¼ 12.7, 7.3 Hz), 2.70 (1H, br s), 3.10 (1H, dd, J ¼ 14.6,
9.3 Hz), 3.29 (1H, dd, J ¼ 14.6, 4.7 Hz), 3.66 (3H, s), 3.76 (1H, dd,
J ¼ 18.1, 5.4 Hz), 3.96e4.02 (2H, m), 4.14e4.16 (2H, m), 4.77 (1H, dt,
J ¼ 8.8, 4.9 Hz), 5.31 (1H, dd, J ¼ 15.4, 6.3 Hz), 5.42 (1H, dt, J ¼ 15.1,
6.6 Hz), 6.29 (1H, br d, J ¼ 6.8 Hz), 6.87 (1H, br d, J ¼ 7.8 Hz), 6.98
(1H, br d, J ¼ 6.8 Hz), 7.09e7.32 (30H, m), 7.39e7.44 (6H, m); 13C
NMR (100 MHz, CDCl3): 17.6, 19.5, 29.4, 31.27, 31.31, 33.0, 33.6, 41.1,
43.8, 52.1, 52.9, 53.9, 59.7, 66.7, 66.9, 69.6, 126.5, 126.7 (3C), 127.0(3C), 127.9 (6C), 128.1 (6C), 128.4 (2C), 129.0 (2C), 129.3 (6C), 129.6(6C), 130.4, 132.3, 137.2, 144.0 (3C), 144.8 (3C), 169.9, 170.0, 171.1,
171.7, 172.4; IR (neat): 3275, 3060, 3030, 2962, 2925, 1742, 1633,
1538, 1490, 1443, 1218, 1184, 1081, 1034, 972, 855, 745, 699 cm�1;
HRMS (FAB): m/z calcd for C65H69N4O7S2 (MþþH) 1081.4608, found1081.4601.

4.1.40. (7S,11R,14S,17R,E)-17-Benzyl-7-hydroxy-11-isopropyl9,12,15,18-tetraoxo-1,1,1-triphenyl-14-tritylthiomethyl-2-thia10,13,16,19-tetraazahenicos-5-en-21-oic acid (14e)Compound 14e was synthesized from 27e (331 mg, 0.31 mmol)
in a manner similar to that described for the synthesis of 14a. Puriﬁcation by column chromatography (CHCl3/MeOH 20:1 to 4:1)
gave 14e (327 mg, 100%) as a colorless amorphous solid. [a]25
D �17.1(c 1.0, CHCl3); 1H NMR (400 MHz, CD3OD/CDCl3 10:1): d 0.83e0.85(6H, m), 1.97e2.03 (3H, m), 2.15e2.19 (2H, m), 2.32 (1H, dd, J ¼ 14.0,
4.8 Hz), 2.40 (1H, dd, J ¼ 13.8, 8.7 Hz), 2.48 (2H, d, J ¼ 6.8 Hz), 2.91
(1H, dd, J ¼ 14.0, 8.7 Hz), 3.15 (1H, dd, J ¼ 13.8, 4.8 Hz), 3.66 (1H, d,
J ¼ 17.4 Hz), 3.77 (1H, d, J ¼ 17.9 Hz), 3.98e4.01 (1H, m), 4.11 (1H, d,
J ¼ 5.8 Hz), 4.28 (1H, dd, J ¼ 12.3, 5.8 Hz), 4.59e4.62 (1H, m), 5.36
(1H, dd, J ¼ 15.9, 6.3 Hz), 5.46 (1H, dt, J ¼ 15.5, 6.3 Hz), 7.07e7.32(35H, m); 13C NMR (100 MHz, CD3OD/CDCl3 10:1): d 18.4, 19.8, 31.1,
32.3, 32.4, 34.2, 38.1, 42.0, 44.3, 54.0, 55.7, 60.2, 67.6, 67.9, 70.3,
127.6 (4C), 127.8 (3C), 128.7 (6C), 128.9 (6C), 129.3 (2C), 130.0, (2C),
130.45 (6C), 130.54 (6C), 130.7, 133.9, 138.0, 145.5 (3C), 146.0 (3C),
171.7, 172.8, 172.9, 173.3, 174.0; IR (neat): 3299, 3059, 3029, 2964,
2927, 1723, 1636, 1532, 1490, 1444, 1219, 1033, 972, 751, 700 cm�1;
HRMS (FAB): m/z calcd for C64H67N4O7S2 (MþþH) 1067.4451, found1067.4442.

4.1.41. (6R,9S,12R,16S)-6-benzyl-12-isopropyl-16-[(E)-4-(tritylthio)but-1-en-1-yl]-9-tritylthiomethyl-1-oxa-4,7,10,13tetraazacyclohexadecane-2,5,8,11,14-pentaone (28e)Compound 28e was synthesized from 14e (206 mg, 0.19 mmol)
in a manner similar to that described for the synthesis of 28a. Puriﬁcation by column chromatography (hexane/EtOAc 2:1) gave 28e(184 mg, 91%) as a colorless amorphous solid. [a]25
D �2.8 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 0.83 (3H, d, J ¼ 6.8 Hz), 0.89
(3H, d, J ¼ 6.8 Hz), 1.89e2.02 (3H, m), 2.13e2.16 (2H, m), 2.26e2.32(2H, m), 2.39e2.43 (1H, m), 2.84e2.90 (1H, m), 3.00 (1H, dd,
J ¼ 13.5, 9.7 Hz), 3.22e3.29 (2H, m), 3.58 (1H, d, J ¼ 14.0 Hz),
4.00e4.10 (2H, m), 4.38e4.44 (1H, m), 5.26 (1H, dd, J ¼ 15.5, 6.3 Hz),5.40e5.52 (2H, m), 6.10 (1H, br d, J ¼ 6.8 Hz), 6.97 (5H, m), 7.10e7.26(26H, m), 7.32e7.34 (6H, m), 7.57 (1H, br s); 13C NMR (100 MHz,CDCl3): d 18.0, 19.4, 29.7, 31.04, 31.06, 31.2, 31.3, 41.7, 42.1, 55.1, 56.4,
58.2, 66.6, 67.0, 72.2, 126.5, 126.6 (3C), 126.8 (3C), 127.8 (6C), 127.9(6C), 128.4 (2C), 129.3 (2C), 129.4 (6C), 129.5 (7C), 132.3, 137.3, 144.3(3C), 144.8 (3C), 168.9, 169.4, 170.6, 170.8, 173.1; IR (neat): 3309,
3058, 3029, 2963, 2929, 1745, 1651, 1540, 1490, 1444, 1274, 1216,
1033, 973, 849, 745, 700 cm�1; HRMS (FAB): m/z calcd for
C64H65N4O6S2 (MþþH) 1049.4346, found 1049.4342.

4.1.42. FK-A5 (13e)Compound 13e was synthesized from 28e (184 mg, 0.18 mmol)
in a manner similar to that described for the synthesis of 13a. Puriﬁcation by column chromatography (CHCl3/MeOH 40:1) gave 13e(92.0 mg, 93%) as a colorless amorphous solid. [a]25
D �16.3 (c 1.1,
CHCl3); 1H NMR (400 MHz, CDCl3): d 1.05 (3H, d, J ¼ 6.4 Hz), 1.06
(3H, d, J ¼ 6.3 Hz), 2.12e2.21 (1H, m), 2.52e2.61 (2H, m), 2.68e2.73(1H, m), 2.94e3.11 (4H, m), 3.30e3.36 (2H, m), 3.46 (1H, dd, J ¼ 4.4,
14.1 Hz), 3.81e3.89 (2H, m), 4.00 (1H, dd, J ¼ 4.9, 6.1 Hz), 4.51 (1H,
dd, J ¼ 6.8, 17.6 Hz), 4.59e4.65 (1H, m), 5.71e5.75 (2H, m),
5.80e5.86 (1H, m), 6.41 (1H, d, J ¼ 4.4 Hz), 7.10e7.11 (3H, m), 7.21
(1H, d, J ¼ 9.8 Hz), 7.26e7.30 (2H, m), 7.37 (1H, d, J ¼ 6.8 Hz), 7.58
(1H, d, J ¼ 7.3 Hz); 13C NMR (100 MHz, CDCl3): d 19.36, 19.42, 29.2,
31.0, 33.9, 34.0, 37.5, 37.9, 43.1, 54.9, 57.5, 61.8, 69.5, 126.6, 128.6(2C), 129.0 (2C), 129.6, 130.5, 138.6, 168.1, 169.78, 169.81, 170.2,
172.3; IR (neat): 3332, 3024, 2966, 2932, 1743, 1659, 1522, 1260,
1184, 979, 753, 700, 666 cm�1; HRMS (FAB): m/z calcd for
C26H35N4O6S2 (MþþH) 563.1998, found 563.2000.
4.1.43. Fmoc-L-PheeGly-OMe (30f)
Compound 30f was synthesized from L-phenylalanine (L-Phe)(18f) (500 mg, 3.0 mmol) in a manner similar to that described forthe synthesis of 21. Puriﬁcation by column chromatography (CHCl3/
MeOH 30:1) gave 30f (1.03 g, 73%, 2 steps) as a colorless amorphous
1
solid. [a]25
D �10.0 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3):
d 3.10e3.11 (2H, m), 3.72 (3H, s), 390e4.05 (2H, m), 4.18 (1H, t,
J ¼ 6.8 Hz), 4.34e4.47 (3H, m), 5.35 (1H, br d, J ¼ 5.3 Hz), 6.33 (1H,
br s), 7.21e7.32 (7H, m), 7.40 (2H, t, J ¼ 7.8 Hz), 7.52 (2H, t,
J ¼ 7.8 Hz), 7.76 (2H, d, J ¼ 7.3 Hz); 13C NMR (100 MHz, CDCl3):
d 38.4, 41.1, 47.0, 52.3, 55.9, 67.0, 119.9 (2C), 125.0, 126.95 (2C),
126.98 (2C), 127.6 (2C), 128.6 (2C), 129.2 (2C), 136.3, 141.2 (2C),
143.62, 143.64, 156.0, 169.8, 171.3; IR (neat): 3300, 3063, 3029,
2950, 1748, 1661, 1540, 1449, 1259, 1214, 1036, 758, 740, 701 cm�1;
HRMS (FAB): m/z calcd for C27H27N2O5 (MþþH) 459.1920, found459.1924.
4.1.44. Boc-D-Cys(Tr)eL-PheeGly-OMe (32f)Compound 32f was synthesized from 30f (1.01 g, 2.2 mmol) in amanner similar to that described for the synthesis of 23. Puriﬁcation by column chromatography (hexane/EtOAc 1:1) gave 32f(1.13 g, 75%, 2 steps) as a colorless amorphous solid. [a]25
D �29.8 (c1.0, CHCl3). 1H NMR (400 MHz, CDCl3): d 1.37 (9H, s), 249 (1H, dd,
J ¼ 13.0, 5.3 Hz), 2.63 (1H, dd, J ¼ 13.0, 7.7 Hz), 3.04e3,14 (2H, m),
3.43 (1H, dd, J ¼ 12.8, 7.3 Hz), 3.66 (3H, s), 3.76 (1H, dd, J ¼ 17.9,
5.3 Hz), 397 (1H, dd, J ¼ 17.6, 5.3 Hz), 4.74 (1H, dd, J ¼ 14.5, 6.8 Hz),4.93 (1H, d, J ¼ 6.8 Hz), 6.41 (1H, d, J ¼ 8.7 Hz), 7.10e7.18 (5H, m),
7.21e7.27 (10H, m), 7.36e7.37 (6H, m); 13C NMR (100 MHz, CDCl3):
d 28.2 (3C), 33.4, 37.2, 41.1, 52.2, 53.8, 54.0, 67.3, 80.5, 126.9, 127.0(3C), 128.1 (6C), 128.6 (2C), 129.2 (2C), 129.5 (6C), 136.2, 144.2 (3C),
155.5, 169.7, 170.6, 170.8; IR (neat): 3297, 3059, 3028, 2977, 1749,
1651, 1495, 1443, 1391, 1366, 1213, 1168, 1033, 867, 744, 700 cm�1;
HRMS (FAB): m/z calcd for C39H44N3O6S (MþþH) 682.2951, found682.2957.
4.1.45. H2N-D-Cys(Tr)eL-PheeGly-OMe (15f)Compound 15f was synthesized from 32f (1.13 g, 1.7 mmol) in amanner similar to that described for the synthesis of 15a. Puriﬁcation by column chromatography (CHCl3/MeOH 40:1) gave 15f(846 mg, 88%) as a colorless amorphous solid. [a]25
D �51.6 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 1.26 (2H, br s), 2.55 (1H, dd,
J ¼ 12.6, 4.3 Hz), 2.61 (1H, dd, J ¼ 12.6, 7.3 Hz), 2.85 (1H, dd, J ¼ 7.2,
4.4 Hz), 3.00 (1H, dd, J ¼ 14.0, 7.7 Hz), 3.14 (1H, dd, J ¼ 14.0, 5.8 Hz),3.64 (3H, s), 3.71 (1H, dd, J ¼ 18.3, 5.3 Hz), 3.89 (1H, dd, J ¼ 17.9,
5.8 Hz), 4.67 (1H, dd, J ¼ 14.0, 7.7 Hz), 6.98 (1H, t, J ¼ 5.6 Hz),7.13e7.27 (14H, m), 7.37e7.40 (7H, m); 13C NMR (100 MHz, CDCl3):— 220 —

K. Narita et al. / European Journal of Medicinal Chemistry 121 (2016) 592e609

d 37.1, 37.3, 40.9, 52.1, 53.8, 53.9, 67.0, 126.8, 126.9 (3C), 128.0 (6C),
128.5 (2C), 129.2 (2C), 129.5 (6C), 136.5, 144.4 (3C), 169.8, 171.0,
173.0; IR (neat): 3298, 3059, 2950, 1750, 1654, 1509, 1492, 1442,
1370, 1209, 1032, 743, 700 cm�1; HRMS (FAB): m/z calcd for
C34H36N3O4S (MþþH) 582.2427, found 582.2421.
4.1.46. (7S,11R,14S,17S,E)-Methyl 17-benzyl-11-isopropyl-7-(4methoxybenzyloxy)-9,12,15,18-tetraoxo-1,1,1-triphenyl-14tritylthiomethyl-2-thia-10,13,16,19-tetraazahenicos-5-en-21-oate(26f)
Compound 26f was synthesized from 15f (200 mg, 0.34 mmol)
in a manner similar to that described for the synthesis of 26a. Puriﬁcation by column chromatography (CHCl3/MeOH 40:1) gave 26f(400 mg, 97%) as a colorless amorphous solid. [a]25
D �5.0 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3/CD3OD 10:1): d 0.68 (3H, d,
J ¼ 6.8 Hz), 0.77 (3H, d, J ¼ 6.8 Hz), 1.87e1.95 (1H, m), 2.07e2.16(2H, m), 2.21e2.25 (2H, m), 2.41e2.48 (3H, m), 2.65 (1H, dd, J ¼ 8.3,
13.2 Hz), 2.94 (1H, dd, J ¼ 8.8, 14.1 Hz), 3.20 (1H, dd, J ¼ 5.4, 14.4 Hz),3.51 (1H, dt, J ¼ 5.4, 7.8 Hz), 3.65 (3H, s), 3.76e3.83 (4H, m),
3.94e4.05 (3H, m), 4.20 (1H, d, J ¼ 10.7 Hz), 4.37 (1H, d, J ¼ 10.7 Hz),4.71e4.77 (1H, m), 5.28 (1H, dd, J ¼ 7.8, 15.1 Hz), 5.54 (1H, dt, J ¼ 6.8,
15.5 Hz), 6.44 (1H, brd, J ¼ 8.3 Hz), 6.59 (1H, d, J ¼ 5.4 Hz),
6.79e6.83 (2H, m), 7.07 (1H, d, J ¼ 6.8 Hz), 7.06e7.12 (26H, m),
7.34e7.37 (6H, m), 7.39e7.42 (6H, m); 13C NMR (100 MHz, CDCl3/
CD3OD 10:1): d 17.5, 18.9, 30.3, 31.05, 31.14, 32.5, 36.9, 40.8, 42.2,
51.9, 52.8, 53.9, 55.0, 58.4, 66.4, 66.9, 69.7, 76.2, 113.6 (2C), 126.4(3C), 126.5, 126.6 (3C), 127.6 (6C), 127.8 (6C), 128.2 (2C), 128.5, 128.9(2C), 129.27 (6C), 129.31 (6C), 129.5 (2C), 129.8, 133.1, 136.4, 144.1(3C), 144.6 (3C), 159.0, 170.0 (2C), 171.4, 171.6, 171.8; IR (neat): 3290,
3060, 3029, 2957, 1753, 1676, 1632, 1539, 1515, 1250, 1215, 1180,
1034, 825, 744, 700 cm�1; HRMS (FAB): m/z calcd for C73H77N4O8S2(MþþH) 1201.5183, found 1201.5183.
4.1.47. (7S,11R,14S,17S,E)-Methyl 17-benzyl-7-hydroxy-11isopropyl-9,12,15,18-tetraoxo-1,1,1-triphenyl-14-tritylthiomethyl-2thia-10,13,16,19-tetraazahenicos-5-en-21-oate (27f)Compound 27f was synthesized from 26f (400 mg, 0.33 mmol)
in a manner similar to that described for the synthesis of 27a. Puriﬁcation by column chromatography (CHCl3/MeOH 50:1) gave 27f(343 mg, 95%) as a colorless amorphous solid. [a]25
D �20.8 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3/CD3OD 10:1): d 0.92 (3H, d,
J ¼ 7.3 Hz), 0.97 (3H, d, J ¼ 6.8 Hz), 2.03e2.10 (2H, m), 2.20e2.30(4H, m), 2.43 (1H, dd, J ¼ 3.4, 13.2 Hz), 2.52e2.63 (2H, m), 3.01 (1H,
dd, J ¼ 8.3, 14.1 Hz), 3.26 (1H, dd, J ¼ 5.9, 13.9 Hz), 3.54 (3H, s), 3.62
(1H, dd, J ¼ 4.9, 17.6 Hz), 3.95 (1H, br s), 4.05 (1H, dd, J ¼ 6.8,
18.1 Hz), 4.11e4.17 (2H, m), 4.47e4.56 (2H, m), 5.40 (1H, dd, J ¼ 5.9,
15.4 Hz), 5.58e5.65 (1H, m), 6.12 (1H, br d, J ¼ 6.8 Hz), 6.94e7.01(2H, m), 7.11e7.34 (30H, m), 7.39e7.42 (6H, m); 13C NMR (100 MHz,CDCl3/CD3OD 10:1): d 17.4, 19.1, 29.3, 31.1, 31.3, 32.6, 36.7, 40.9, 43.3,
52.0, 52.3, 54.4, 59.5, 66.4, 66.8, 69.1, 126.45 (3C), 126.54, 126.6 (3C),
127.6, 127.7 (6C), 127.8 (6C), 128.3 (2C), 128.5, 128.9 (2C), 129.3 (6C),
129.4 (6C), 136.6, 144.2 (3C), 144.7 (3C), 170.2 (2C), 170.4, 171.7,
171.7; IR (neat): 3289, 3059, 3029, 2962, 2961, 2926, 1744, 1634,
1532, 1491, 1443, 1392, 1217, 1183, 1034, 972, 744, 700 cm�1; HRMS
(FAB): m/z calcd for C65H69N4O7S2 (MþþH) 1081.4608, found1081.4601.
4.1.48. (7S,11R,14S,17S,E)-17-Benzyl-7-hydroxy-11-isopropyl9,12,15,18-tetraoxo-1,1,1-triphenyl-14-tritylthiomethyl-2-thia10,13,16,19-tetraazahenicos-5-en-21-oic acid (14f)Compound 14f was synthesized from 27f (343 mg, 0.32 mmol)
in a manner similar to that described for the synthesis of 14a. Puriﬁcation by column chromatography (CHCl3/MeOH 20:1 to 4:1)
gave 14f (338 mg, 100%) as a colorless amorphous solid. [a]25
D �4.0(c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3/CD3OD 10:1): d 0.84e0.88605(6H, m), 2.02e2.08 (3H, m), 2.19e2.22 (2H, m), 2.35e2.42 (3H, m),
2.47e2.53 (1H, m), 2.88e2.94 (1H, m), 3.20e3.24 (1H, m),
3.69e4.09 (4H, m), 4.39e4.43 (1H, m), 4.53e4.57 (1H, m), 5.41 (1H,
dd, J ¼ 15.1, 5.9 Hz), 5.54 (1H, dt, J ¼ 15.1, 5.9 Hz), 7.09e7.42 (35H,m); 13C NMR (100 MHz, CDCl3/CD3OD 10:1): d 17.5, 18.9, 29.4, 31.1,
31.2, 32.3, 36.8, 42.3, 43.1, 52.6, 54.6, 59.2, 66.4, 66.8, 68.8, 126.4(3C), 126.5, 126.6, (3C), 127.6 (6C), 127.8 (6C), 128.2 (2C), 128.5, 128.8(2C), 129.2 (6C), 129.3 (6C), 132.5, 136.5, 144.1 (3C), 144.5 (3C), 170.6,
171.5, 172.3, 172.9, 173.9; IR (neat): 3318, 3059, 3029, 2962, 2928,
1643, 1535, 1490, 1444, 1316, 1218, 1083, 1034, 972, 746, 700 cm�1;
HRMS (FAB): m/z calcd for C64H67N4O7S2 (MþþH) 1067.4451, found1067.4442.
4.1.49. (6S,9S,12R,16S)-6-Benzyl-12-isopropyl-16-[(E)-4tritylthiobut-1-en-1-yl]-9-tritylthiomethyl-1-oxa-4,7,10,13tetraazacyclohexadecane-2,5,8,11,14-pentaone (28f)Compound 28f was synthesized from 14f (95.7 mg, 90 mmol) in amanner similar to that described for the synthesis of 28a. Puriﬁcation by column chromatography (Hexane/EtOAc 2:1) gave 28f(82.0 mg, 87%) as a colorless amorphous solid. [a]25
D �22.2 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 0.84 (3H, d, J ¼ 6.8 Hz), 0.91
(3H, d, J ¼ 6.8 Hz), 1.96e2.08 (3H, m), 2.14e2.22 (2H, m), 2.47e2.54(2H, m), 2.72e2.86 (2H, m), 2.92e3.04 (2H, m), 3.38 (1H, dd,
J ¼ 14.6, 4.4 Hz), 3.52 (1H, dd, J ¼ 16.8, 3.4 Hz), 4.10e4.13 (1H, m),
4.33 (1H, dd, J ¼ 17.1, 8.3 Hz), 4.74 (1H, dt, J ¼ 9.5, 4.4 Hz), 5.40e5.44(2H, m), 5.54e5.62 (1H, m), 6.16 (1H, br d, J ¼ 6.8 Hz), 6.98e7.00(2H, m), 7.06e7.39 (36H, m); 13C NMR (100 MHz, CDCl3/CD3OD
10:1): d 17.3, 19.0, 30.5, 30.8, 31.0, 31.9, 36.0, 40.7, 41.3, 53.1, 53.6,
58.7, 66.4, 66.7, 72.1, 126.3 (3C), 126.5 (3C), 127.4, 127.5 (6C), 127.6(6C), 128.1 (2C), 128.36, 128.43 (2C), 129.1 (6C), 129.2 (6C), 132.5,
137.0, 144.1 (3C), 144.5 (3C), 168.7, 170.0, 170.5, 171.3, 172.4; IR(neat): 3304, 3058, 3029, 2963, 2928, 1735, 1647, 1540, 1490, 1444,
1217, 1033, 978, 852, 744, 700 cm�1; HRMS (FAB): m/z calcd for
C64H65N4O6S2 (MþþH) 1049.4346, found 1049.4342.
4.1.50. FK-A6 (13f)Compound 13f was synthesized from 28f (82.0 mg, 78 mmol) in amanner similar to that described for the synthesis of 13a. Puriﬁcation by column chromatography (CHCl3/MeOH 40:1) gave 13f(41.5 mg, 94%) as a colorless amorphous solid. [a]25
D �93.0 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 1.08 (3H, d, J ¼ 6.8 Hz), 1.10
(3H, d, J ¼ 6.8 Hz), 2.29e2.37 (1H, m), 2.48e2.52 (1H, m), 2.60e2.69(2H, m), 2.86e3.03 (4H, m), 3.07 (1H, dd, J ¼ 4.4, 15.4 Hz), 3.17e3.26(2H, m), 4.04e4.09 (2H, m), 4.20 (1H, dd, J ¼ 17.8, 5.4 Hz), 4.62e4.73(2H, m), 5.67e5.75 (2H, m), 5.81e5.88 (1H, m), 6.60 (1H, d,
J ¼ 3.9 Hz), 7.01 (1H, t, J ¼ 4.6 Hz), 7.20e7.32 (5H, m), 7.37 (1H, d,
J ¼ 7.3 Hz), 7.53 (1H, d, J ¼ 8.3 Hz); 13C NMR (100 MHz, CDCl3):
d 18.6, 19.6, 29.2, 31.4, 37.4, 37.5, 37.6, 39.5, 42.4, 55.4, 55.6, 62.1,
69.4, 126.8, 128.5 (2C), 129.0. 129.4 (2C), 129.8, 136.5, 167.4, 169.8,
170.6, 170.7, 170.8; IR (neat): 3296, 2965, 2930, 1741, 1653, 1525,
1252, 1191, 1024, 978, 752, 700, 667 cm�1; HRMS (FAB): m/z calcd
for C26H35N4O6S2 (MþþH) 563.1998, found 563.2000.
4.1.51. Fmoc-D-LeueGly-OMe (30g)Compound 30g was synthesized from D-leucine (D-Leu) (18g)(500 mg, 3.8 mmol) in a manner similar to that described for thesynthesis of 21. Puriﬁcation by column chromatography (Hexane/
EtOAc 1:1) gave 30g (1.16 g, 72%, 2 steps) as a colorless amorphous
1
solid. [a]25
D þ19.4 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3):
d 090e0.99 (6H, m), 1.54e1.67 (3H, m), 3.74 (3H, s), 4.03e4.07 (2H,m), 4.20e4.26 (2H, m), 4.44 (2H, d, J ¼ 5.4 Hz), 5.19 (1H, d,
J ¼ 7.3 Hz), 6.52 (1H, br s), 7.29e7.35 (2H, m), 7.40 (2H, t, J ¼ 7.1 Hz),7.57e7.63 (2H, m), 7.76e7.79 (2H, m); 13C NMR (100 MHz, CDCl3):
d 21.9, 22.9, 24.7, 41.2, 41.3, 47.2, 52.4, 65.2, 67.0, 120.0 (2C), 124.7(2C), 127.1 (2C), 127.7 (2C), 141.3 (2C), 143.8 (2C), 156.0, 170.1, 172.3;— 221 —

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IR (neat): 3298, 2953, 2358, 1700, 1663, 1540, 1449, 1211, 1043, 758,
740 cm�1; HRMS (FAB): m/z calcd for C24H29N2O5 (MþþH)425.2076, found 425.2067.
4.1.52. Boc-D-Cys(S-Tr)-D-Leu-Gly-OMe (32g)Compound 32g was synthesized from 30g (1.62 g, 3.8 mmol) in amanner similar to that described for the synthesis of 23. Puriﬁcation by column chromatography (hexane/EtOAc 1:1) gave 32g(2.01 g, 81%, 2 steps) as a colorless amorphous solid. [a]25
D þ8.0 (c1.0, CHCl3); 1H NMR (400 MHz, CDCl3): d 0.84e0.94 (6H, m), 1.40
(9H, s), 1.48e1.55 (1H, m), 1.60e1.67 (1H, m), 1.75e1.81 (2H, m),
2.59 (1H, dd, J ¼ 12.2, 5.9 Hz), 2.74 (1H, dd, J ¼ 12.7, 6.8 Hz), 3.68
(3H, s), 3.71e3.79 (1H, m), 3.98 (1H, dd, J ¼ 18.1, 5.9 Hz), 4.44e4.50(1H, m), 4.81 (1H, d, J ¼ 6.9 Hz), 6.28 (1H, d, J ¼ 7.8 Hz), 7.00 (1H, brs), 7.21e7.41 (15H, m); 13C NMR (100 MHz, CDCl3): d ¼ 21.4, 23.1,
24.6, 28.2 (3C), 33.2, 40.2, 40.9, 51.5, 52.1, 53.9, 67.2, 80.7, 127.0 (3C),
128.1 (6C), 129.4 (6C), 144.2 (3C), 155.7, 169.9, 170.6, 171.9; IR (neat):3331, 3271, 1675, 1536, 1280, 1175, 1023, 745, 701 cm�1; HRMS
(FAB): m/z calcd for C36H46N3O6S (MþþH) 648.3107, found;648.3104.
4.1.53. H2N-D-Cys(Tr)eD-LeueGly-OMe (15g)Compound 15g was synthesized from 32g (2.01 g, 3.1 mmol) in amanner similar to that described for the synthesis of 15a. Puriﬁcation by column chromatography (CHCl3/MeOH 40:1) gave 15g(1.34 g, 78%) as a colorless amorphous solid. [a]25
D þ30.1 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 0.87 (3H, dd, J ¼ 6.3 Hz), 0.91
(3H, dd, J ¼ 5.9 Hz), 1.44e1.73 (5H, m), 2.55 (1H, dd, J ¼ 12.7, 7.8 Hz),2.65 (1H, dd, J ¼ 12.5, 4.4 Hz), 3.11 (1H, dd, J ¼ 7.6, 3.9 Hz), 3.69 (3H,s), 3.86e3.98 (2H, m), 4.38e4.44 (1H, m), 6.98 (1H, t, J ¼ 5.4 Hz),
7.19e7.44 (16H, m); 13C NMR (100 MHz, CDCl3): d 21.8, 22.9, 24.6,
37.0, 40.2, 40.9, 51.1, 52.2, 53.8, 66.8, 126.8 (3C), 127.9 (6C), 129.5(6C), 144.5 (3C), 170.0, 172.1, 173.5; IR (neat): 3310, 2955, 1749, 1652,
1520, 1444, 1208, 1181, 744, 701 cm�1; HRMS (FAB): m/z calcd for
C31H38N3O4S (MþþH) 548.2583, found 548.2577.
4.1.54. (7S,11R,14S,17R,E)-Isobutyl 11-isopropyl-7-(4methoxybenzyloxy)-17-isobutyl-9,12,15,18-tetraoxo-1,1,1-triphenyl14-tritylthiomethyl-2-thia-10,13,16,19-tetraazahenicos-5-en-21oate (26g)Compound 26g was synthesized from 15g (172 mg, 0.31 mmol)
in a manner similar to that described for the synthesis of 26a. Puriﬁcation by column chromatography (hexane/EtOAc 1:2) gave 26g(316 mg, 86%) as a colorless amorphous solid. [a]25
D �4.1 (c 1.1
CHCl3); 1H NMR (400 MHz, CDCl3): d 0.60 (3H, d, J ¼ 6.8 Hz), 0.75
(3H, d, J ¼ 6.8 Hz), 0.80e0.84 (6H, m), 1.59e1.70 (3H, m), 1.87e1.97(1H, m), 1.99e2.06 (2H, m), 2.12e2.18 (2H, m), 2.22e2.32 (3H, m),
2.88 (1H, dd, J ¼ 12.2, 5.4 Hz), 3.58 (3H, s), 3.72e3.76 (4H, m),
3.87e3.96 (3H, m), 4.07 (1H, dd, J ¼ 12.2, 5.9 Hz), 4.10 (1H, d,
J ¼ 10.7 Hz), 4.37 (1H, d, J ¼ 10.2 Hz), 4.40e4.46 (1H, m), 5.17e5.23(1H, m), 5.35e5.42 (1H, m), 6.57 (1H, d, J ¼ 6.4 Hz), 6.75 (2H, d,
J ¼ 8.3 Hz), 6.87 (1H, d, J ¼ 7.8 Hz), 7.05e7.34 (34H, m); 13C NMR(100 MHz, CDCl3): d 17.6, 19.4, 21.2, 23.2, 24.8, 29.5, 31.2, 31.3, 33.3,
38.6, 39.8, 41.0, 42.3, 52.0, 52.4, 55.3, 59.9, 66.6, 66.9, 70.1, 76.5,
113.9 (2C), 126.6 (3C), 127.0 (3C), 127.82 (6C), 128.1 (6C), 129.3 (6C),
129.40, 129.45, 129.48 (6C), 129.9 (2C), 133.7, 144.1 (3C), 144.8 (3C),
159.4, 169.6, 169.9, 171.5, 172.2, 172.3; IR (neat): 3274, 2955, 1749,
1637, 1540, 1514, 1490, 1443, 1247, 1213, 1181, 1078, 1034, 744,
700 cm�1; HRMS (FAB): m/z calcd for C70H79N4O8S2 (MþþH)1167.5339, found 1167.5356.
4.1.55. (7S,11R,14S,17R,E)-Isobutyl 7-hydroxy-11-isopropyl-17methyl-9,12,15,18-tetraoxo-1,1,1-triphenyl-14-tritylthiomethyl-2thia-10,13,16,19-tetraazahenicos-5-en-21-oate (27g)Compound 27g was synthesized from 26g (258 mg, 0.22 mmol)

in a manner similar to that described for the synthesis of 27a. Puriﬁcation by column chromatography (Hexane/EtOAc 1:3) gave 27g(209 mg, 90%) as a colorless amorphous solid. [a]25
D �9.8 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 0.84e0.93 (12H, m), 1.62e1.67(2H, m), 1.73e1.75 (1H, m), 2.03e2.22 (5H, m), 2.25e2.39 (2H, m),
2.46 (1H, dd, J ¼ 12.2, 5.4 Hz), 2.63e2.68 (1H, m), 3.19 (1H, br s),
3.63 (3H, s), 3.75e3.90 (2H, m), 4.21e4.24 (1H, m), 4.26e4.34 (2H,m), 4.49e4.55 (1H, m), 5.35 (1H, dd, J ¼ 15.1, 5.9 Hz), 5.44 (1H, dt,
J ¼ 15.1, 6.3 Hz), 6.68 (1H, brs), 7.14e7.42 (33H, m); 13C NMR(100 MHz, CDCl3): d 17.8, 19.3, 21.4, 23.1, 24.8, 29.8, 31.3, 33.4, 38.6,
40.0, 40.9, 43.6, 52.0, 52.1, 52.5, 59.5, 66.6, 66.8, 69.6, 126.6 (3C),
126.9 (3C), 127.8 (6C), 128.1 (6C), 129.3 (6C), 129.5 (6C), 130.1, 132.4,
144.1 (3C), 144.8 (3C), 170.0, 170.1, 171.6, 172.3, 172.5; IR (neat):3279, 3059, 2957, 2926, 1753, 1637, 1543, 1491, 1443, 1216, 1034,
745, 700, 676 cm�1; HRMS (FAB): m/z calcd for C62H71N4O7S2(MþþH) 1047.4764, found 1047.4758.
4.1.56. (7S,11R,14S,17R,E)-7-Hydroxy-11-isopropyl-17-isobutyl9,12,15,18-tetraoxo-1,1,1-triphenyl-14-tritylthiomethyl-2-thia10,13,16,19-tetraazahenicos-5-en-21-oic acid (14g)Compound 14g was synthesized from 27g (250 mg, 0.24 mmol)
in a manner similar to that described for the synthesis of 14a. Puriﬁcation by column chromatography (CHCl3/MeOH 10:1 to 8:1)
gave 14g (230 mg, 93%) as a colorless amorphous solid. [a]25
D �13.4(c 1.1, CHCl3); 1H NMR (400 MHz, CD3OD/CDCl3 10:1): d 0.85e0.94(12H, m), 1.50e1.66 (3H, m), 2.05e2.62 (7H, m), 2.54e2.76 (2H, m),
3.65e3.78 (2H, m), 4.10e4.19 (2H, m), 4.32e4.38 (2H, m),
5.39e5.54 (2H, m), 7.19e7.40 (30H, m); 13C NMR (100 MHz, CD3OD/
CDCl3 10:1): d 18.1, 19.7, 21.7, 23.6, 25.4, 30.4, 30.7, 32.1, 32.3, 33.8,
41.1, 44.0, 53.0, 53.8, 60.3, 67.4, 67.8, 70.1, 127.4 (3C), 127.7 (3C),
128.5 (6C), 128.8 (6C), 130.2 (6C), 130.3 (6C), 130.6, 133.7, 145.2 (3C),
145.8 (3C), 170.0, 171.6, 171.7, 173.4, 174.1; IR (neat): 3296, 3057,
2958, 2928, 1637, 1538, 1490, 1444, 1219, 1034, 744, 700, 675,
617 cm�1; HRMS (FAB): m/z calcd for C61H68N4O7S2Na (MþþNa)1055.4427, found 1055.4436.
4.1.57. (6R,9S,12R,16S)-12-Isopropyl-6-isobutyl-16-[(E)-4tritylthiobut-1-enyl]]-9-tritylthiomethyl-1-oxa-4,7,10,13tetraazacyclohexadecane-2,5,8,11,14-pentaone (28g)Compound 28g was synthesized from 14g (230 mg, 0.22 mmol)
in a manner similar to that described for the synthesis of 28a. Puriﬁcation by column chromatography (hexane/EtOAc 1:3) gave 28g(157 mg, 69%) as a colorless amorphous solid. [a]25
D �3.6 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 0.84e0.91 (12H, m), 1.62e1.68(1H, m), 1.79e1.85 (2H, m), 1.95e2.03 (3H, m), 2.14e2.17 (2H, m),
2.33e2.56 (3H, m), 2.87e2.92 (1H, m), 3.67e369 (2H, m), 4.12e4.17(2H, m), 4.25e4.31 (1H, m), 5.31 (1H, dd, J ¼ 14.6, 6.8 Hz), 5.53e5.57(2H, m), 6.29e6.31 (1H, m), 7.14e7.40 (32H, m), 7.66e7.76 (1H, m);
13
C NMR (100 MHz, CDCl3): d 18.2, 19.5, 21.3, 23.2, 24.9, 29.7, 30.8,
31.1, 31.3, 31.8, 39.3, 41.8, 42.4, 52.5, 58.4, 66.6, 67.0, 72.3, 126.6 (3C),
126.8 (3C), 127.8 (6C), 128.0 (6C), 129.4 (6C), 129.5 (7C), 132.4, 144.3(3C), 144.8 (3C), 169.1, 169.6, 170.9, 172.0, 173.2; IR (neat): 3305,
3056, 2958, 2927, 1743, 1655, 1542, 1490, 1444, 1278, 1215, 1035,
744, 700, 667 cm�1; HRMS (FAB): m/z calcd for C61H67N4O6S2(MþþH) 1015.4502, found; 1015.4503.
4.1.58. FK-A7 (13g)Compound 13g was synthesized from 28g (157 mg, 0.16 mmol)
in a manner similar to that described for the synthesis of 13a. Puriﬁcation by column chromatography (CHCl3/MeOH 20:1) gave 13g(67.7 mg, 83%) as a colorless amorphous solid. [a]25
D þ26.7 (c 0.56,
CHCl3); 1H NMR (400 MHz, CDCl3): d 0.89 (3H, d, J ¼ 6.8 Hz), 0.93
(3H, d, J ¼ 5.9 Hz), 1.09e1.11 (6H, m), 1.55e1.66 (1H, m), 1.82e1.89(1H, m), 2.03e2.10 (1H, m), 2.18e2.26 (1H, m), 2.52e2.65 (1H, m),
2.64 (1H, d, J ¼ 12.7 Hz), 2.70e2.74 (1H, m), 2.92e3.02 (3H, m),— 222 —

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3.06e3.12 (1H, m), 3.38 (1H, d, J ¼ 15.9, 10.7 Hz), 3.64e3.69 (1H, m),
3.92 (1H, dd, J ¼ 17.6, 2.4 Hz), 4.06 (1H, t, J ¼ 5.1 Hz), 4.38 (1H, dd,
J ¼ 17.8, 6.3 Hz), 4.77e4.82 (1H, m), 5.73e7.76 (2H, m), 5.85e5.90(1H, m), 6.60 (1H, d, J ¼ 3.4 Hz), 7.13 (1H, d, J ¼ 1.0 Hz), 7.54 (1H, d,
J ¼ 7.3 Hz), 7.72 (1H, d, J ¼ 6.3 Hz); 13C NMR (100 MHz, CDCl3):
d 19.4, 19.5, 21.5, 23.1, 25.0, 29.3, 31.3, 34.6, 36.7, 37.5, 38.3, 43.0,
54.5, 54.9, 62.0, 69.4, 129.8, 130.3, 168.0, 170.0, 170.2, 171.0, 172.5; IR(neat): 3340, 2960, 2927, 2871, 1756, 1653, 1523, 1260, 1187, 981,
756, 668 cm�1; HRMS (FAB): m/z calcd for C23H37N4O6S2 (MþþH)529.2155, found 529.2155.
4.1.59. Fmoc-L-LeueGly-OMe (30h)Compound 30h was synthesized from L-luecine (L-Leu) (18h)(500 mg, 3.8 mmol) in a manner similar to that described for thesynthesis of 21. Puriﬁcation by column chromatography (Hexane/
EtOAc 1:1) gave 30h (1.58 g, 97%, 2 steps) as a colorless amorphous
1
solid. [a]25
D �21.0 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3):
d 0.92e0.95 (6H, m), 1.54e1.67 (3H, m), 3.72 (3H, s), 4.01e4.03 (2H,m), 4.18e4.26 (2H, m), 4.37e4.43 (2H, m), 5.33 (1H, br s), 6.67 (1H, brs), 7.25e7.31 (2H, m), 7.37e7.40 (2H, m), 7.57 (2H, d, J ¼ 7.3 Hz), 7.75
(2H, d, J ¼ 7.3 Hz); 13C NMR (100 MHz, CDCl3): d 21.9, 22.9, 24.6, 41.1,
41.3, 47.2, 52.3, 53.3, 67.0, 120.0 (2C), 125.0 (2C), 127.1 (2C), 127.7 (2C),
141.3 (2C), 143.8 (2C), 156.2, 170.1, 172.3; IR (neat): 3302, 2956, 2361,
1749, 1704, 1665, 1539, 1449, 1213, 1045, 758, 740 cm�1; HRMS
(FAB): m/z calcd for C24H29N2O5 (MþþH) 425.2076, found 425.2071.
4.1.60. Boc-D-Cys(Tr)eL-LeueGly-OMe (32h)Compound 32h was synthesized from 30h (1.62 g, 3.8 mmol) ina manner similar to that described for the synthesis of 23. Puriﬁcation by column chromatography (hexane/EtOAc 1:1) gave 32h(1.85 g, 83%, 2 steps) as a colorless amorphous solid. [a]25
D �25.5 (c1.0, CHCl3); 1H NMR (400 MHz, CDCl3): d 0.87 (3H, d, J ¼ 6.8 Hz),0.89 (3H, d, J ¼ 6.3 Hz), 1.48 (9H, s), 1.46e1.55 (1H, m), 1.55e1.64(1H, m), 1.72e1.79 (1H, m), 2.56 (1H, dd, J ¼ 12.7, 5.4 Hz), 2.76 (1H,
dd, J ¼ 12.7, 6.8 Hz), 3.69 (3H, s), 3.71e3.80 (2H, m), 4.01 (1H, dd,
J ¼ 14.9, 4.4 Hz), 4.43e4.48 (1H, m), 4.89 (1H, d, J ¼ 6.4 Hz), 6.32
(1H, d, J ¼ 6.3 Hz), 7.04 (1H, brs), 7.16e7.40 (15H, m); 13C NMR(100 MHz, CDCl3): d 21.5, 23.0, 24.6, 28.1 (3C), 33.5, 40.2, 40.9, 51.5,
52.1, 54.1, 67.2, 80.5, 127.0 (3C), 128.0 (6C), 129.4 (6C), 144.2 (3C),
155.3, 167.0, 170.7, 171.9; IR (neat): 3290, 2953, 1753, 1653, 1540,
1490, 1445, 1366, 1212, 1168, 1036, 743 cm�1; HRMS (FAB): m/z
calcd for C36H46N3O6S (MþþH) 648.3107, found 648.3109.
4.1.61. H2N-D-Cys(Tr)eL-LeueGly-OMe (15h)Compound 15h was synthesized from 32h (1.85 g, 2.9 mmol) ina manner similar to that described for the synthesis of 15a. Puriﬁcation by column chromatography (CHCl3/MeOH 40:1) gave 15h(1.31 g, 77%) as a colorless amorphous solid. [a]25
D �55.0 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 0.88 (3H, d, J ¼ 6.3 Hz), 0.91
(3H, d, J ¼ 6.8 Hz), 1.48e1.64 (4H, m), 1.72e1.79 (1H, m), 2.65 (1H,
dd, J ¼ 12.2, 3.9 Hz), 2.73 (1H, dd, J ¼ 12.7, 6.8 Hz), 3.10 (1H, dd,
J ¼ 7.8, 6.8 Hz), 3.67 (3H, s), 3.78 (1H, dd, J ¼ 18.1, 5.4 Hz), 3.96 (1H,
dd, J ¼ 18.1, 5.9 Hz), 4.37e4.43 (1H, m), 6.86 (1H, t, J ¼ 5.4 Hz),7.20e7.45 (16H, m); 13C NMR (100 MHz, CDCl3): d 21.8, 22.9, 24.8,
37.5, 40.1, 40.9, 51.3, 52.2, 53.9, 67.0, 126.9 (3C), 128.0 (6C), 129.5(6C), 144.5 (3C), 169.9, 172.1, 173.4; IR (neat): 3286, 3060, 2954,
1752, 1650, 1555, 1490, 1444, 1208, 1035, 743,700 cm�1; HRMS
(FAB): m/z calcd for C31H38N3O4S (MþþH) 548.2583, found548.2575.
4.1.62. (7S,11R,14S,17S,E)-Isobutyl 11-isopropyl-7-(4methoxybenzyloxy)-17-isobutyl-9,12,15,18-tetraoxo-1,1,1-triphenyl14-tritylthiomethyl-2-thia-10,13,16,19-tetraazahenicos-5-en-21oate (26h)Compound 26h was synthesized from 15h (167 mg, 0.30 mmol)

607

in a manner similar to that described for the synthesis of 26a. Puriﬁcation by column chromatography (hexane/EtOAc 1:2) gave 26h(195 mg, 50%) as a colorless amorphous solid. [a]25
D þ7.6 (c 1.1,
CHCl3); 1H NMR (400 MHz, CDCl3): d 0.69 (3H, d, J ¼ 6.8 Hz), 0.79
(3H, d, J ¼ 6.8 Hz), 0.84 (3H, d, J ¼ 5.8 Hz), 0.87 (3H, d, J ¼ 6.3 Hz),1.53e1.72 (3H, m), 1.93e1.96 (1H, m), 2.04e2.18 (2H, m), 2.21e2.25(2H, m), 2.46 (2H, d, J ¼ 5.4 Hz), 2.56 (1H, dd, J ¼ 12.7, 5.4 Hz), 2.79
(1H, dd, J ¼ 12.7, 7.3 Hz), 3.63 (3H, s), 3.78 (3H, s), 3.78e3.84 (1H,m), 3.88e3.95 (2H, m), 4.06 (1H, dd, J ¼ 13.4, 5.9 Hz), 4.14 (1H, t,
J ¼ 6.3 Hz), 4.21 (1H, d, J ¼ 10.7 Hz), 4.40 (1H, d, J ¼ 10.7 Hz),4.49e4.54 (1H, m), 5.29 (1H, dd, J ¼ 14.6, 7.3 Hz), 5.54 (1H, dt,
J ¼ 15.1, 6.8 Hz), 6.60 (1H, d, J ¼ 7.8 Hz), 6.82 (2H, d, J ¼ 8.7 Hz), 6.96
(1H, br s), 7.16e7.31 (34H, m); 13C NMR (100 MHz, CDCl3): d 17.8,
19.4, 21.5, 23.1, 24.6, 30.3, 31.28, 31.34, 33.7, 40.7, 41.0, 42.4, 51.7,
52.0, 52.6, 55.3, 59.1, 66.6, 67.1, 70.0, 76.5, 113.8 (2C), 126.6 (3C),
126.9 (3C), 127.8 (6C), 128.0 (6C), 129.47 (6C), 129.52 (6C), 129.7,
129.8 (2C), 129.9, 133.2, 144.3 (3C), 144.8 (3C), 159.3, 169.7, 170.0,
171.2, 171.7, 172.3; IR (neat): 3237, 3058, 2956, 2929, 1755, 1633,
1539, 1514, 1489, 1443, 1248, 1206, 1180, 1034, 744, 700, cm�1;
HRMS (FAB): m/z calcd for C70H79N4O8S2 (MþþH) 1167.5339, found1167.5322.

4.1.63. (7S,11R,14S,17S,E)-Methyl 7-hydroxy-11-isopropyl-17isobutyl-9,12,15,18-tetraoxo-1,1,1-triphenyl-14-tritylthiomethyl-2thia-10,13,16,19-tetraazahenicos-5-en-21-oate (27h)Compound 27h was synthesized from 26h (160 mg, 0.14 mmol)
in a manner similar to that described for the synthesis of 27a. Puriﬁcation by column chromatography (hexane/EtOAc 1:3) gave 27h(97 mg, 68%) as a colorless amorphous solid. [a]25
D �36.7 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 0.85 (3H, d, J ¼ 5.9 Hz), 0.92
(3H, d, J ¼ 5.9 Hz), 0.95 (3H, d, J ¼ 7.3 Hz), 1.00 (3H, d, J ¼ 7.3 Hz),1.64e1.78 (3H, m), 2.00e2.09 (2H, m), 2.16e2.23 (2H, m), 2.25e2.36(2H, m), 2.44 (1H, dd, J ¼ 13.2, 3.4 Hz), 2.58e2.68 (2H, m), 3.54 (3H,s), 3.64 (1H, dd, J ¼ 18.0, 4.9 Hz), 4.03e4.14 (2H, m), 4.29e4.34 (1H,m), 4.39e4.45 (2H, m), 5.39 (1H, dd, J ¼ 15.4, 6.3 Hz), 5.59 (1H, dt,
J ¼ 15.1, 6.8 Hz), 6.05 (1H, d, J ¼ 6.3 Hz), 6.91e6.99 (2H, m),
7.15e7.42 (31H, m); 13C NMR (100 MHz, CDCl3): d 17.5, 19.6, 21.6,
23.0, 24.6, 29.7, 31.2, 31.4, 33.7, 38.6, 40.8, 44.0, 51.7, 52.0, 52.3, 60.2,
66.6, 66.8, 69.5, 126.6 (3C), 126.7 (3C), 127.85 (6C), 127.90 (6C), 129.5(6C), 129.5 (6C), 129.8, 132.8, 144.5 (3C), 144.8 (3C), 170.7 (2C), 171.0,
172.8, 173.1; IR (neat): 3288, 3058, 2957, 2926, 1747, 1633, 1539,
1489, 1444, 1215, 1183, 1034, 744, 699 cm�1; HRMS (FAB): m/z calcd
for C62H71N4O7S2 (MþþH) 1047.4764, found 1047.4791.
4.1.64. (7S,11R,14S,17S,E)-7-Hydroxy-11-isopropyl-17-isobutyl9,12,15,18-tetraoxo-1,1,1-triphenyl-14-tritylthiomethyl-2-thia10,13,16,19-tetraazahenicos-5-en-21-oic acid (14h)Compound 14h was synthesized from 27h (147 mg, 0.13 mmol)
in a manner similar to that described for the synthesis of 14a. Puriﬁcation by column chromatography (CHCl3/MeOH 10:1 to 8:1)
gave 14h (115 mg, 89%) as a colorless amorphous solid. [a]25
D �19.6(c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3/CD3OD 10:1): d 0.83e0.94(12H, m), 1.59e1.69 (3H, m), 2.01e2.23 (5H, m), 2.37e2.39 (2H, m),
2.50e2.55 (1H, m), 2.59e2.64 (1H, m), 3.64e3.92 (2H, m),
4.08e4.12 (2H, m), 4.30e4.35 (1H, m), 4.40e4.45 (1H, m), 5.42 (1H,
dd, J ¼ 15.4, 5.4 Hz), 5.55 (1H, dt, J ¼ 15.1, 6.3 Hz), 7.17e7.45 (30H,m); 13C NMR (100 MHz, CDCl3/CD3OD 10:1): d 17.5, 19.1, 21.1, 22.9,
24.5, 29.5, 31.2, 31.3, 32.7, 39.7, 41.4, 43.1, 51.9, 52.6, 59.4, 66.5, 66.8,
68.9, 126.5 (3C), 126.7 (3C), 127.7 (6C), 127.8 (6C), 128.5, 129.3 (6C),
129.4 (6C), 132.6, 144.2 (3C), 144.7 (3C), 170.6, 170.7, 171.98, 172.03,
172.9; IR (neat): 3287, 3057, 2959, 2928, 1726, 1636, 1538, 1490,
1468, 1444, 1218, 1034, 744, 700 cm�1; HRMS (FAB): m/z calcd for
C61H69N4O7S2 (MþþH) 1033.4608, found 1033.4608.

— 223 —

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4.1.65. (6S,9S,12R,16S)-12-Isopropyl-6-isobutyl-16-[(E)-4tritylthiobut-1-enyl]-9-tritylthiomethyl-1-oxa-4,7,10,13tetraazacyclohexadecane-2,5,8,11,14-pentaone (28h)Compound 28h was synthesized from 14h (49.6 mg, 0.14 mmol)
in a manner similar to that described for the synthesis of 28a. Puriﬁcation by column chromatography (CHCl3/MeOH 40:1) gave 28h(56.0 mg, 50%) as a colorless amorphous solid. [a]25
D �24.1 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): d 0.80e0.85 (9H, m), 0.91 (3H, d,
J ¼ 6.8 Hz), 1.36e1.44 (1H, m), 1.51e1.57 (1H, m), 1.71e1.74 (1H, m),
1.93e2.07 (3H, m), 2.09e2.15 (2H, m), 2.49e2.55 (2H, m),
2.71e2.75 (1H, m), 2.82e2.87 (1H, m), 3.53e3.59 (2H, m),
4.22e4.29 (2H, m), 4.41e4.47 (1H, m), 5.38 (1H, dd, J ¼ 15.6, 6.3 Hz),
5.48e5.41 (1H, m), 5.59 (1H, dt, J ¼ 14.6, 6.8 Hz), 6.42 (1H, brs),
7.12e7.38 (33H, m); 13C NMR (100 MHz, CDCl3): d 17.8, 19.7, 21.0,
21.3, 23.2, 24.7, 28.2, 31.0, 31.2, 31.4, 32.3, 41.9, 51.5, 54.0, 58.9, 66.7,
66.8, 71.8, 126.6 (3C), 126.7 (3C), 127.8 (6C), 128.0 (6C), 129.4 (6C),
129.5 (7C), 133.0, 144.4 (3C), 144.7 (3C), 168.8, 169.5, 170.0, 172.2,
172.3; IR (neat): 3292, 3059, 2959, 2928, 1739, 1653, 1537, 1492,
1444, 1214, 1034, 750, 700 cm�1; HRMS (FAB): m/z calcd for
C61H67N4O6S2 (MþþH) 1015.4502, found 1015.4501.
4.1.66. FK-A8 (13h)Compound 13h was synthesized from 28h (56.0 mg, 56 mmol) ina manner similar to that described for the synthesis of 13a. Puriﬁcation by column chromatography (CHCl3/MeOH 20:1) gave 13h(19.0 mg, 64%) as a colorless amorphous solid. [a]25
D �41.1 (c 1.4,
CHCl3); 1H NMR (400 MHz, CDCl3): d 0.92 (3H, d, J ¼ 5.9 Hz), 0.96
(3H, d, J ¼ 5.9 Hz), 1.09 (3H, d, J ¼ 2.4 Hz), 1.11 (3H, d, J ¼ 2.4 Hz),1.59e1.74 (3H, m), 2.28e2.34 (1H, m), 2.48e2.51 (1H, m), 2.65e2.68(2H, m), 2.89e2.95 (2H, m), 3.08e3.13 (1H, m), 3.20 (2H, d,
J ¼ 7.3 Hz), 4.02e4.07 (2H, m), 4.24 (1H, dd, J ¼ 17.1, 4.9 Hz), 4.52
(1H, td, J ¼ 9.3, 4.4 Hz), 4.79 (1H, dd, J ¼ 14.4, 6.8 Hz), 5.72e5.88(3H, m), 6.51 (1H, br s), 7.61 (1H, br s), 8.29 (1H, d, J ¼ 8.3 Hz), 7.45
(1H, d, J ¼ 6.8 Hz); 13C NMR (100 MHz, CDCl3): d 18.8, 19.5, 21.6,
23.0, 24.8, 29.3, 31.0, 37.1, 37.4, 39.6, 40.8, 42.5, 52.7, 56.1, 62.3, 69.4,
129.0, 129.7, 167.3, 169.6, 171.0, 171.1, 171.8; IR (neat): 3299, 2960,
2930, 1743, 1653, 1530, 1468, 1437, 1254, 1220, 1189, 770, 665 cm�1;
HRMS (FAB): m/z calcd for C23H37N4O6S2 (MþþH) 529.2155, found529.2158.
4.2. Biological evaluation
4.2.1. HDACs preparation and enzyme inhibition assay [26]In a 100-mm dish, 293T cells (1e2 x 107) were grown for 24 hand transiently transfected with 10 mg each of the vector pcDNA3HDAC1 for human HDAC1 or pcDNA3-mHDA2/HDAC6 for mouse
HDAC6, using the LipofectAMINE2000 reagent (Invitrogen). After
successive cultivation in DMEM for 24 h, the cells were washedwith PBS and lysed by sonication in lysis buffer containing 50 mM
TriseHCl (pH 7.5), 120 mM NaCl, 5 mM EDTA, and 0.5% NP40. The
soluble fraction collected by microcentrifugation was precleared byincubation with protein A/G agarose beads (Roche). After thecleared supernatant had been incubated for 1 h at 4 � C with 4 mg ofan anti-FLAG M2 antibody (SigmaeAldrich Inc.) for HDAC1 andHDAC6, the agarose beads were washed three times with lysis
buffer and once with histone deacetylase buffer consisting of
20 mM TriseHCl (pH 8.0), 150 mM NaCl, and 10% glycerol. The
bound proteins were released from the immune complex by incubation for 1 h at 4 � C with 40 mg of the FLAG peptide (SigmaeAldrich Inc.) in histone deacetylase buffer (200 mL). The
supernatant was collected by centrifugation. For the enzyme assay,
10 mL of the enzyme fraction was added to 1 mL of ﬂuorescent
substrate (2 mM Ac-KGLGK(Ac)-MCA) and 9 mL of histone deacetylase buffer, and the mixture was incubated at 37 � C for 30 min.
The reaction was stopped by the addition of 30 mL of trypsin(20 mg/mL) and incubated at 37 � C for 15 min. The released aminomethylcoumarin (AMC) was measured using a ﬂuorescence plate
reader. The 50% inhibitory concentrations (IC50) were determinedas the means with SD calculated from at least three independent
doseeresponse curves. The HDAC inhibitory activity of all test
compounds was measured in the presence of 0.1 mM DTT.
4.2.2. Cell-growth inhibition assay [24]This experiment was carried out at the Cancer Chemotherapy
Center, Japanese Foundation for Cancer Research. The screening
panel consisted of the following 39 human cancer cell lines (HCC
panel): breast cancer HBC-4, BSY-1, HBC-5, MCF-7, and MDA-MB231; brain cancer U-251, SF-268, SF-295, SF-539, SNB-75, andSNB-78; colon cancer HCC2998, KM-12, HT-29, HCT-15, and HCT116; lung cancer NCI-H23, NCI-H226, NCI-H522, NCI-H460, A549,
DMS273, and DMS114; melanoma LOX-IMVI; ovarian cancer
OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, and SK-OV-3; renal cancer
RXF-631L and ACHN; stomach cancer St-4, MKN1, MKN7, MKN28,
MKN45, and MKN74; prostate cancer DU-145 and PC-3. The GI50
(50% cell growth inhibition) value for these cell lines was determined by using the sulforhodamine B colorimetric method.
Acknowledgements
We are grateful to the Screening Committee of New Anticancer
Agents, which is supported by a Grant-in-Aid for Scientiﬁc Research
onInnovative Area ‘Scientiﬁc Support Programs for Cancer Research(No. 221S0001, 2010e2014)’ from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT), for assistancewith the biological evaluation of compounds 1 and 13aeh. This
study was also supported by a Grant-in-Aid for the Strategic
Research Foundation Program at Private Universities (No.
S1511001L, 2015e2019), a Grant-in-Aid for Scientiﬁc Research (C)(No. 15K07865, 2015e2017), and a Grant-in-Aid for ScientiﬁcResearch (S) (No. 26221204, 2014e2018) from MEXT.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2016.05.031.
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DOI: 10.1002/ejoc.201600949

Full Paper

Natural Product Synthesis

Enantioselective Total Synthesis of (–)-Siphonodictyal B and(+)-8-epi-Siphonodictyal B with Phosphatidylinositol 3-Kinase α(PI3Kα) Inhibitory Activity
Takuya Kikuchi,[a] Koichi Narita,[a] Ken Saijo,[b] Chikashi Ishioka,[b] and Tadashi Katoh*[a]Abstract: The biologically interesting marine meroterpenoids(–)-siphonodictyal B and (+)-8-epi-siphonodictyal B were efficiently synthesized in 29–40 % overall yield in a longest linear
sequence of 11 steps, starting from commercially available (+)sclareolide. The synthesis involved the following crucial steps:(i) stereodivergent hydrogenation of a homoallylic decalin alcohol to install the requisite C8 stereogenic centre present in the
decalin fragments; (ii) coupling of the decalin fragments withan aromatic moiety to assemble the desired carbon skeletons;and (iii) deprotection from multiple O-protective groups on thearomatic ring to complete the project synthesis. Both (–)-siphonodictyal B and (+)-8-epi-siphonodictyal B showed PI3Kα inhibitory activity, with potencies comparable to that of liphagal, anaturally occurring PI3Kα inhibitor. New structure–activity relationships for this class of marine meroterpenoids were also revealed.

Introduction

total synthesis of this marine natural product.[4] Siphonodictyal B has a trans-fused decalin ring connected to a highly
substituted aromatic ring through a methylidene linkage; thedecalin unit contains three asymmetric carbon centres.[4,5] Aclosely related meroterpenoid, liphagal (2), which comprises a
fused 6,7,5,6-tetracyclic skeleton (ABCD ring system) with thesame carbon framework and substitution pattern on the aromatic ring, was also isolated from the same Aka coralliphagum
marine sponge by Andersen et al. in 2006.[6] George et al. proposed a hypothesis that siphonodictyal B (1a) is a biogenetic

In recent years, numerous biologically active meroterpenoids(terpenoid–polyketide hybrid structures) having unique structural features have been isolated from marine organisms, particularly from algae and marine sponges.[1] Several of these meroterpenoids have received considerable attention owing to theirpotential for use as new therapeutic agents. In most cases, however, biological studies – including those focussing on structure–activity relationships (SARs) – have been severely restricted, probably because of the scarcity of samples and/or the
structural diversity of the natural products derived from marineorganisms. Consequently, the development of efficient and flexible methods for the synthesis of biologically active meroterpenoids and their analogues is desirable and worthwhilefrom the viewpoint of medicinal/pharmaceutical chemistry.
Siphonodictyal B (1a; Figure 1) was originally isolated in 1981
by Faulkner et al. from the marine sponge Aka coralliphagum(also known as Siphonodictyon coralliphagum), and its structure
was proposed as shown in formula 1c by the same researchers.[2] Subsequently, the first proposed structure (i.e., 1c) was
revised to formula 1b (8-epi-siphonodictyal B) by Faulkner and
Clardy et al. in 1986.[3] Quite recently, the second proposed
structure (i.e., 1b) was revised again to formula 1a, the true
structure (C8 epimer of 1b), by George et al. (2015) through[a] Laboratory of Synthetic and Medicinal Chemistry, Faculty of
Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University,
4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan
E-mail: katoh@tohoku-mpu.ac.jp
http: www.tohoku-mpu.ac.jp/laboratory/iyakugo/index.html[b] Department of Clinical Oncology, Institute of Development,
Aging and Cancer, Tohoku University,
4-1 Seiryomachi, Aoba-ku, Sendai 980-8575, Japan
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/ejoc.201600949.
Eur. J. Org. Chem. 2016, 5659–5666

Figure 1. Structure of siphonodictyal B (1a), earlier proposed structures for1a (1b and 1c), and structure of liphagal (2).

5659

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Full Paper
precursor of liphagal (2), and this hypothesis was strongly supported by their successful biomimetic conversion of 1a into 2.[4]Liphagal has been reported to show potent and selective inhibitory activity against phosphatidylinositol 3-kinase α (PI3Kα),
which has become a new potential target for cancer chemotherapy.[6,7] Siphonodictyal B has been reported to show biological properties such as antimicrobial and antiproliferative activities;[2,3,5] however, its PI3Kα inhibitory activity has not yet beenassessed.
The interesting structural features, attractive biological properties, and limited availability from natural resources of(–)-siphonotictyal B (1a) and (+)-8-epi-siphonodictyal B (1b)prompted us to undertake a project directed towards their total
synthesis in enantiomerically pure form. Recently, while ourstudies into the synthesis of 1a and 1b were in progress,
George et al.[4] reported the first and elegant total synthesis of
1a and 1b, which was achieved in a longest linear sequence of
10 steps, in 11 and 8 % overall yields, respectively. An approachto the total synthesis of 1b has also been reported by Seifert
et al.[8]In this paper, we describe our total synthesis of 1a and 1bby applying a synthetic strategy developed previously in ourlaboratory.[9] The synthesized compounds 1a and 1b were subjected to a PI3Kα inhibition assay to evaluate their biologicalpotency as compared to liphagal (2). This evaluation represents
the first report on the PI3Kα inhibition efficacy of siphonodictyal B (1a) and its C8 epimer (1b).

ported by us,[9] we envisaged that target molecules 1a and 1b
could be synthesized by site-specific formylation of the aromatic ring at the C3′ position in the corresponding intermediates (i.e., 3a and 3b), followed by deprotection from multiple
O-protective groups on the aromatic ring. Intermediates 3a and3b would be formed by condensation of the corresponding
optically active decalin aldehydes (i.e., 4a and 4b) with trioxysubstituted arylcerium reagent 5, which is accessible from trioxyaryl bromide 6. Two sets of C8 diastereomeric isomers 4aand 4b would be separately prepared from common homoallylic decalin alcohol 7 by stereodivergent hydrogenation of theC7–C8 double bond.

Synthesis of Aromatic Fragment 6
We initially pursued the synthesis of aromatic fragment 6,
which is a condensation partner for decalin fragments 4a and4b, starting from commercially available 2-hydroxy-4-methoxybenzaldehyde (8), as shown in Scheme 2. Trioxybenzene 9 was
prepared from 8 in two steps (82 % overall yield) according toa known procedure.[8] Site-specific monobromination at the C5
position in 9 was carried out by exposure to N-bromosuccinimide (NBS; 1.1 equiv.) in N,N-dimethylformamide (DMF) atroom temperature, giving the requisite aromatic fragment (i.e.,6) in 86 % yield.

Results and Discussion
Synthetic Plan for Siphonodictyal B (1a) and 8-epiSiphonodictyal B (1b)Our retrosynthetic analysis is outlined in Scheme 1. With reference to the synthetic approach to liphagal (2) previously re-

Scheme 2. Synthesis of aromatic fragment 6. (a) NBS, DMF, room temp., 13 h,
86 %. NBS = N-bromosuccinimide, DMF = N,N-dimethylformamide.

Synthesis of Decalin Fragments 4a and 4b

Scheme 1. Retrosynthetic analysis of siphonodictyal B (1a) and 8-epi-siphonodictyal B (1b).
Eur. J. Org. Chem. 2016, 5659–5666

www.eurjoc.org

The synthesis of 4a and 4b was efficiently achieved in a stereodivergent manner from a common intermediate, homoallylic
decalin alcohol 7. Intermediate 7 was prepared from commercially available (+)-sclareolide (10) in four steps and 64 % overall
yield according to our reported method,[9] as shown in
Scheme 3. Thus, hydroxy-group-directed hydrogenation of 7 using Crabtree's catalyst {[Ir(COD)(PCy3)(py)]+[PF6]–}[10] (1.0 mol-%)gave C8 α-methyl compound 11a as the major product (89 %yield), along with a small amount of C8 �-methyl compound
11b (9 % yield; 11a/11b, 10:1). Conversely, when the hydrogenation of 7 was carried out using PtO2 as the catalyst, C8 �methyl compound 11b was produced as the major product(90 % yield) along with a small amount of C8 α-methyl compound 11a (8 % yield; 11a/11b, 1:11). These stereoisomers wereseparated by silica-gel column chromatography. The hydrogenation of 7 using PtO2 as a catalyst must have mainly takenplace at the sterically less hindered α-face of the molecule topreferentially give 11b. To take the synthesis forward, Swern

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Full Paper
oxidation of 11a and 11b gave decalin fragments 4a and 4b,
respectively, in almost quantitative yield.

tained, and unidentified decomposition products were generated. Site-specific formylation of the aromatic ring at the C3′
position in 3a (nBuLi, DMF, THF, –20 °C) gave the corresponding
aldehyde (i.e., 13a) in 95 % yield.

Scheme 3. Synthesis of decalin fragments 4a and 4b. (a) H2 (1 atm), Crabtree's
catalyst (1.0 mol-%), CH2Cl2, 0 °C, 2 h, 89 % for 11a, 9 % for 11b; (b) H2(1 atm), PtO2 (5.0 mol-%), EtOAc, 0 °C, 30 min, 90 % for 11b, 8 % for 11a;(c) (COCl)2, DMSO, iPr2NEt, CH2Cl2, –78 °C, 30 min; –78 to 0 °C, 1 h, 97 % for
4a, 99 % for 4b. Crabtree's catalyst = [Ir(COD)(PCy3)(py)]+[PF6]– (COD = 1,5cyclooctadiene, Cy = cyclohexyl, py = pyridine), DMSO = dimethyl sulfoxide.

Synthesis of (–)-Siphonodictyal B (1a)Having obtained the aromatic fragment 6 and the two decalinfragments 4a and 4b, we next carried out the synthesis of thefirst target molecule, (–)-siphonodictyal B (1a), as shown in
Scheme 4. The crucial coupling reaction of sterically hinderedand sensitive decalin aldehyde 4a, which has the C8 α-methylgroup in an equatorial orientation, with highly functionalizedaromatic fragment 6 was efficiently achieved by using anorganocerium reagent.[11] Thus, treatment of aryl bromide 6
with nBuLi in THF at –78 °C, followed by addition of the organolithium reagent generated in situ to anhydrous cerium chloridein THF at –78 °C, resulted in the production of the corresponding organocerium reagent (i.e., 5). This was then allowed toreact with 4a at –78 °C to give the desired coupling product(i.e., 12a) in excellent yield (98 %) as a single stereoisomer at
the C10 position. The stereochemistry at C10 in 12a was notdetermined, because this stereogenic centre would disappear
in the following dehydration step. The dehydration of 12a wasbest achieved by MgBr2-catalysed acetylation[12] of the C10hydroxy group followed by elimination of the resulting acetate
in a one-pot operation. This gave olefin 3a in 82 % yield. The(E) configuration of the olefinic double bond in 3a was confirmed by a NOESY experiment (see Supporting Information).
When the dehydration of alcohol 12a was attempted underconventional conditions (e.g., MsCl, Et3N, CH2Cl2, 0 °C to reflux
temp.), the requisite dehydration product (i.e., 3a) was not obEur. J. Org. Chem. 2016, 5659–5666

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Scheme 4. Synthesis of (–)-siphonodictyal B (1a) and (+)-8-epi-siphonodictyal B (1b). (a) 6, nBuLi, THF, –78 °C, 30 min; CeCl3, –78 °C, 1 h; additionof 4a or 4b, –78 °C, 1 h, 98 % for both 12a (single diastereomer) and 12b(ca. 5:3 mixture of C10 diastereomers); (b) conversion of 12a into 3a: MgBr2,
Ac2O, CH2Cl2, room temp., 30 min, 82 %; conversion of 12b into 3b: MsCl,
Et3N, CH2Cl2, 0 °C, 30 min, 94 %; (c) nBuLi, DMF, THF, –20 °C, 30 min, 95 % for
13a, 97 % for 13b; (d) nBuSLi, HMPA, 0 °C to room temp., 1 h, 91 % for 14a,
88 % for 14b; (e) BCl3, CH2Cl2, –40 °C, 1 h, 75 % for 1a, 91 % for 1b. MsCl =methanesulfonyl chloride, HMPA = hexamethylphosphoramide.

In the final stage of the synthesis, the deprotection of thetwo types of protective groups (i.e., O-methyl ether and acetonide moieties) in 13a was thoroughly investigated. Thus, treatment of 13a with nBuSLi[13] in hexamethylphosphoramide(HMPA) at 0 °C rising to room temperature efficiently deprotected the aromatic O-methyl group, leading to the free phenol
derivative 14a in 91 % yield. It is noteworthy that the sensitive
formyl group on the aromatic ring remained intact during Odemethylation. Finally, exposure of 14a to BCl3 in CH2Cl2 at–40 °C resulted in deprotection of the acetonide moiety to givethe first target, (–)-siphonodictyal B (1a), in 75 % yield after purification by gel-filtration chromatography on Sephadex LH-20(using MeOH as an eluent). When the purification of 1a was
attempted using conventional silica-gel column chromatogra-

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Full Paperphy (with hexane/EtOAc as an eluent), a lower yield of 1a (ca.
50 %) was obtained, probably because partial oxidative decomposition occurred during the operation, due to the presence ofthe highly oxygenated aromatic ring. The spectroscopic data(IR, 1H and 13C NMR, and HRMS) of our synthetic 1a are identicalwith those reported for natural 1a[2,3] and synthetic 1a.[4] The
optical rotation of our synthetic 1a, [α]25
D = –78.8 (c = 0.20 in
CHCl3), is in good agreement with that reported for George's[4]synthetic 1a, [α]25
no optical rotaD = –61.2 (c = 0.67 in CHCl3);[2,3]
tion data was reported for natural 1a.

Table 1. PI3Kα inhibitory activity of siphonodictyal B (1a) and 8-epi-siphonodictyal B (1b).

Synthesis of (+)-8-epi-Siphonodictyal B (1b)Using decalin fragment 4b (C8 �-Me in axial orientation) insteadof 4a (C8 α-Me in equatorial orientation) as the starting material, the second target, (+)-8-epi-siphonodictyal B (1b), was synthesized in a manner similar to that described for the synthesisof (–)-siphonodictyal B (1a) (Scheme 4). This sequence of reactions deserves comment. In the coupling reaction (4b + 5 →12b), the product (i.e., 12b; 98 % yield) was formed as a mixtureof diastereomers (ca. 5:3 ratio) at the C10 hydroxy group, andthe mixture could not be separated by silica-gel column chromatography. Furthermore, in the subsequent dehydration step
(12b → 3b), the reaction proceeded well under conventional
conditions (MsCl, Et3N, CH2Cl2, 0 °C, 30 min), and the requisite
dehydration product (i.e., 3b) was obtained in excellent yield(94 %). These differences in reactivity between the α- and �methyl series (i.e., 4a + 5 → 12a vs. 4b + 5 → 12b; and 12a→ 3a vs. 12b → 3b) may be caused by steric effects aroundthe C8 position in the trans-decalin scaffold.
The spectroscopic data (IR, 1H and 13C NMR, and HRMS) of
our synthetic 1b were identical to those reported for George's
synthetic 1b.[4] The optical rotation of our synthetic 1b, [α]25D =+42.1 (c = 0.33 in CHCl3), is essentially consistent with that reported for George's synthetic 1b, [α]25
D = +5.3 (c = 0.75 in
CHCl3).[4]Biological Evaluation and PI3Kα Inhibition Assay
Phosphatidylinositol 3-kinases (PI3Ks) are key enzymes that
control the signalling pathways used by a wide variety of cellsurface receptors on neutrophils.[14] There are several isoformsof PI3Ks, including PI3Kα, PI3K�, PI3Kγ, and PI3Kδ, that show
different expression patterns and different pathophysiologicalroles.[15] PI3Kα is considered to be a potential anticancer target,[16] and PI3K�, PI3Kγ, and PI3Kδ are expected to be promising targets for other pathogenic states, such as cardiovasculardisorders (PI3K�)[17] and inflammation and autoimmune diseases (PI3Kγ and PI3Kδ).[18] Therefore, the potent and selective
inhibition of PI3Kα is highly desirable in cancer chemotherapy.
The synthesized siphonodictyal B (1a) and its C8 epimer (1b)were tested for their PI3Kα inhibitory activity to elucidate theirpotency. In this assay, liphagal (2) was used as a reference compound, and the results are summarized in Table 1. In multiple
side-by-side assays, 1a and 1b showed remarkable PI3Kα inhibitory activity, with IC50 values of 2.6 and 3.3 μM, respectively.
Their potency is equal to or better than that of liphagal (IC50 =Eur. J. Org. Chem. 2016, 5659–5666

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4.1 μM). These results indicate that the characteristic ring-fused
bicyclic cyclohepta[b]furan ring (the BC system) present inliphagal is not responsible for the pronounced PI3Kα inhibitory
activity. In addition, the C8 stereochemistry in siphonodictyal B
dose not significantly affect its PI3Kα inhibition efficacy. To thebest of our knowledge, the PI3Kα inhibitory activity of siphonodictyal B has not been previously reported in the literature;
thus, these findings provide new knowledge that the structurally less complex siphonodictyal B is a useful seed compoundfor designing new synthetic PI3Kα inhibitors.

CompoundIC50[a] [μM]

Siphonodictyal B (1a)8-epi-Siphonodictyal B (1b)Liphagal (2)[b]

2.6
3.3
4.1

[a] Concentration that induces 50 % inhibition against PI3Kα. [b] Positive control used in this study, synthesized in our laboratory.[9]Conclusions
We have achieved the enantioselective total synthesis of (–)siphonodictyal B (1a) and (+)-8-epi-siphonodictyal B (1b) in aconvergent and unified manner in 29–40 % overall yield over alinear sequence of 11 steps, starting from commercially available (+)-sclareolide (10). Compared to the previously reported
method, the main advantage of the synthesis reported here is
the higher overall yields [George's synthesis:[4] 8–11 % overall
yield over 10 steps]. A preliminary biological evaluation of 1aand 1b against PI3Kα revealed their inhibitory potency (1a:IC50 = 2.6 μM; 1b: IC50 = 3.3 μM). New aspects of the SAR of thisclass of marine meroterpenoids were also revealed. These results may be useful for the design and development of anticancer agents that target the inhibition of PI3Kα. Further studies
concerning the synthesis of unnatural siphonodictyal B analogues and the study of their SAR are currently underway in
our group, and the results will be reported in due course.

Experimental Section
General Methods: All reactions involving air- and moisture-sensitive reagents were carried out using oven-dried glassware and standard syringe/septum-cap techniques. Routine monitoring of reactions was carried out using glass-supported Merck silica gel 60 F254
TLC plates. Flash column chromatography was carried out on Kanto
Chemical Silica Gel 60N (spherical, neutral, 40–50 nm) with the solvents indicated. All solvents and reagents were used as supplied,with the following exceptions: tetrahydrofuran (THF) was freshly
distilled from Na metal/benzophenone under argon; dimethyl sulfoxide (DMSO), N,N-diisopropylethylamine, N,N-dimethylformamide(DMF), and CH2Cl2 were distilled from calcium hydride under argon.
Optical rotations were measured with a JASCO DIP-370 and an Anton Paar MCP 100 automatic digital polarimeter. Melting points
were measured with a Yanaco MP-3 micro melting-point apparatus.
1
H and 13C NMR spectra were measured with a JEOL AL-400(400 MHz) spectrometer. Chemical shifts are expressed in ppm, andMe4Si (δ = 0 ppm) was used as an internal standard. The following

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abbreviations are used: singlet (s), doublet (d), triplet (t), quartet(q), multiplet (m), and broad (br.). Infrared (IR) spectroscopic measurements were carried out with a JASCO FTIR-4100 spectrometer.
Low- and high-resolution mass (HRMS) spectra were measured witha JEOL JMS-700 MStation high-resolution mass spectrometer.
5-Bromo-6-methoxy-2,2-dimethylbenzo[d][1,3]dioxole (6): NBromosuccinimide (NBS; 1.65 g, 9.3 mmol) was added to a stirred
solution of 5-methoxy-2,2-dimethylbenzo[d][1,3]dioxole (9)[8](1.52 g, 8.4 mmol) in DMF (8 mL) at room temperature. After 13 h,
the reaction was quenched with water (10 mL), and the mixture
was extracted with Et2O (2 × 40 mL). The combined extracts were
washed with brine (2 × 20 mL), then dried with MgSO4. Concentration in vacuo gave a residue, which was purified by column chromatography (hexane/EtOAc, 100:1) to give 6 (1.88 g, 86 %) as a colourless viscous liquid. 1H NMR (400 MHz, CDCl3): δ = 1.66 (s, 6 H), 3.81
(s, 3 H), 6.49 (s, 1 H), 6.90 (s, 1 H) ppm. 13C NMR (100 MHz, CDCl3):
δ = 25.6 (2 C), 57.2, 96.0, 100.7, 112.3, 119.1, 141.9, 147.5,
150.7 ppm. IR (neat): ν̃ = 2991, 2938, 2864, 2833, 1625, 1604, 1495,
1396, 1384, 1351, 1272, 1191, 1164, 1084, 1040, 980, 965, 859, 819,
786, 669, 641 cm–1. HRMS (EI): calcd. for C10H11BrO3 [M]+ 257.9892;
found 257.9883.
[(1S,2R,4aS,8aS)-2,5,5,8a-Tetramethyldecahydronaphthalen-1yl]methanol (11a) and [(1S,2S,4aS,8aS)-2,5,5,8a-Tetramethyldecahydronaphthalen-1-yl]methanol (11b)
Method A: Stereocontrolled Hydrogenation of 7 Using
Crabtree's Catalyst, Leading to 11a as the Major Product:[Ir(COD)(PCy3)(py)]+[PF6]– (Crabtree's catalyst; 46.9 mg, 58 μmol) was
added to a solution of [(1S,4aS,8aS)-2,5,5,8a-tetramethyl1,4,4a,5,6,7,8,8a-octahydronaphthalen-1-yl]methanol (7)[9] (1.30 g,
5.8 mmol) in CH2Cl2 (58 mL) at room temperature. The mixture
was degassed using ultrasound, then it was stirred under hydrogen(balloon) at 0 °C for 2 h. The reaction mixture was concentrated invacuo to give a residue, which was purified by column chromatography (hexane/EtOAc, 10:1) to give 11a (1.17 g, 89 %, less polar)
and 11b (118 mg, 9 %, more polar).
Data for 11a: White solid; m.p. 63–65 °C. [α]D27 = +1.5 (c = 1.0,
CHCl3). 1H NMR (400 Hz, CDCl3): δ = 0.67 (dt, J = 3.4, 11.2 Hz, 1 H),
0.82 (s, 3 H), 0.85 (s, 3 H), 0.87 (s, 3 H), 0.97 (d, J = 6.8 Hz, 3 H),
1.00–1.19 (m, 4 H), 1.24–1.49 (m, 4 H), 1.54–1.65 (m, 3 H), 1.79 (ddd,
J = 3.9, 7.3, 12.9 Hz, 1 H), 1.88 (br. d, J = 12.7 Hz, 1 H), 3.63 (dd, J =3.4, 11.2 Hz, 1 H), 3.79 (dd, J = 2.9, 11.7 Hz, 1 H) ppm. 13C NMR(100 Hz, CDCl3): δ = 15.6, 18.8, 21.0, 21.8, 21.9, 30.8, 33.3, 33.6, 36.8,
37.6, 39.5, 42.1, 55.1, 60.7, 61.9 ppm. IR (KBr): ν̃ = 3356, 2923, 2869,
2844, 1457, 1387, 1366, 1231, 1205, 1118, 1088, 1066, 980, 940, 839,
815, 666 cm–1. HRMS (EI): calcd. for C15H28O [M]+ 224.2140; found224.2147.
Data for 11b: White solid; m.p. 104–105 °C. [α]D27 = +17.5 (c = 1.1,
CHCl3). 1H NMR (400 Hz, CDCl3): δ = 0.82 (s, 3 H), 0.85 (d, J = 2.4 Hz,
1 H), 0.86 (s, 6 H), 0.88 (d, J = 2.4 Hz, 1 H), 0.96 (d, J = 7.3 Hz, 3 H),
1.00–1.05 (m, 2 H), 1.16 (dt, J = 4.4, 13.7 Hz, 1 H), 1.34–1.70 (m, 8H), 2.12–2.17 (m, 1 H), 3.59 (t, J = 9.8 Hz, 1 H), 3.86 (dd, J = 4.4,
10.5 Hz, 1 H) ppm. 13C NMR (100 Hz, CDCl3): δ = 15.6, 17.1, 17.5,
18.4, 21.6, 28.6, 33.2, 33.6, 34.5, 37.6, 39.9, 42.0, 55.8, 56.5, 61.1 ppm.
IR (KBr): ν̃ = 3298, 2991, 2920, 2864, 1683, 1521, 1455, 1368, 1215,
1084, 1040, 984, 839, 756, 668 cm–1. HRMS (EI): calcd. for C15H28O[M]+ 224.2140; found 224.2139.
Method B: Stereocontrolled Hydrogenation of 7 Using Pt2O asa Catalyst, Leading to 11b as the Major Product: PtO2 (39.5 mg,
0.17 mmol) was added to a solution of 7 (773 mg, 3.5 mmol) in
EtOAc (35 mL) at 0 °C. The mixture was stirred under hydrogen(balloon) at 0 °C for 30 min. The reaction mixture was diluted with
Eur. J. Org. Chem. 2016, 5659–5666

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EtOAc (50 mL), and the catalyst was removed by filtration througha small pad of Celite®. The filtrate was concentrated in vacuo to givea residue, which was purified by column chromatography (hexane/
EtOAc, 100:1 → 20:1) to give 11a (65.8 mg, 8 %, less polar) and 11b(706 mg, 90 %, more polar). The spectroscopic data (IR, 1H and 13C
NMR, and HRMS) for these samples are identical with those recorded for 11a and 11b as described before.
(1S,2R,4aS,8aS)-2,5,5,8a-Tetramethyldecahydronaphthalen-1carbaldehyde (4a): DMSO (0.41 mL, 6.2 mmol) was added dropwise to a stirred solution of oxalyl chloride (0.30 mL, 3.6 mmol) in
CH2Cl2 (12 mL) at –78 °C under argon. After 20 min, a solution of
11a (525 mg, 2.3 mmol) in CH2Cl2 (12 mL) was slowly added to the
mixture at –78 °C. After 20 min, N,N-diisopropylethylamine (2.3 mL,
13 mmol) was added to the mixture at –78 °C. The resulting mixture
was gradually warmed to 0 °C and stirred for a further 1 h. The
reaction was quenched with water (20 mL) at 0 °C, and the mixture
was extracted with CH2Cl2 (3 × 50 mL). The combined extracts were
washed with brine (2 × 50 mL), then dried with MgSO4. Concentration in vacuo gave a residue, which was purified by column chromatography (hexane/EtOAc, 30:1) to give 4a (502 mg, 97 %) as a colourless viscous liquid. [α]D30 = +14.4 (c = 1.2, CHCl3). 1H NMR (400 Hz,
CDCl3): δ = 0.79 (s, 3 H), 0.84 (s, 3 H), 0.86 (s, 3 H), 0.90–1.05 (m, 3H), 1.09 (s, 3 H), 1.18–1.64 (m, 7 H), 1.76–1.93 (m, 2 H), 2.03–2.15(m, 1 H), 9.69 (d, J = 4.8 Hz, 1 H) ppm. 13C NMR (100 Hz, CDCl3):
δ = 15.9, 18.3, 20.6, 21.6, 21.8, 27.6, 33.3, 33.5, 35.5, 38.2, 40.2, 41.9,
54.2, 70.3, 207.7 ppm. IR (neat): ν̃ = 3366, 2946, 2922, 2864, 2864,
1456, 1441, 1387, 1364, 1270, 1213, 1136, 1079, 1033, 983, 963, 812,
771, 678, 643 cm –1 . HRMS (FAB): calcd. for C 15 H 25 O [M – H]+
221.1900; found 221.1902.
(1S,2S,4aS,8aS)-2,5,5,8a-Tetramethyldecahydronaphthalen-1carbaldehyde (4b): DMSO (0.24 mL, 3.4 mmol) was added dropwise to a stirred solution of oxalyl chloride (0.15 mL, 1.7 mmol) in
CH2Cl2 (6 mL) at –78 °C. After 20 min, a solution of 11b (254 mg,
1.1 mmol) in CH2Cl2 (6 mL) was added to the mixture at –78 °C.
After 20 min, N,N-diisopropylethylamine (2.0 mL, 11 mmol) was
added to the mixture at the same temperature. The resulting mixture was gradually warmed to 0 °C and stirred for a further 1 h. The
reaction was quenched with water (10 mL) at 0 °C, and the mixture
was extracted with CH2Cl2 (3 × 30 mL). The combined extracts were
washed with brine (2 × 20 mL), then dried with MgSO4. Concentration in vacuo gave a residue, which was purified by column chromatography (hexane/EtOAc, 30:1) to give 4b (248 mg, 99 %) as a colourless viscous liquid. [α]D30 = +22.9 (c = 1.2, CHCl3). 1H NMR (400 Hz,
CDCl3): δ = 0.85 (s, 3 H), 0.87 (s, 3 H), 0.81–0.89 (m, 1 H), 1.04 (d,J = 7.3 Hz, 3 H), 0.96–1.08 (m, 1 H), 1.18 (s, 3 H), 1.15–1.26 (m, 1 H),
1.37–1.73 (m, 7 H), 1.96–1.99 (m, 2 H), 2.36–2.41 (m, 1 H), 9.88 (s, 1H) ppm. 13C NMR (100 Hz, CDCl3): δ = 17.2, 17.3, 17.9, 18.0, 21.5,
29.8, 33.2, 33.4, 34.2, 37.1, 39.7, 41.9, 55.7, 65.6, 206.5 ppm. IR (neat):ν̃ = 2924, 2870, 2849, 2681, 1868, 1771, 1715, 1684, 1653, 1636,
1557, 1541, 1522, 1507, 1457, 1387, 1365, 1339, 1113, 1068, 1038,
638 cm –1 . HRMS (EI): calcd. for C 15 H 26 O [M] + 222.1984; found222.1982.
( 6 - M e t h ox y - 2 , 2 - d i m e t h y l b e n z o [ d ] [ 1 , 3 ] d i oxo l - 5 - y l ) [(1S,2R,4aS,8aS)-2,5,5,8a-tetramethyldecahydronaphthalen-1yl]methanol (12a): Anhydrous CeCl3 (1.24 g, 5.0 mmol) was prepared by heating CeCl 3 ·H 2 O (finely ground powder; 1.87 g,
5.0 mmol) at 140 °C for 12 h under reduced pressure. The material
was then cooled to room temperature, and THF (12 mL) was added.
The resulting suspension was stirred at room temperature underargon for 4 h. Separately, a solution of nBuLi (1.6 M in n-hexane;
3.3 mL, 5.0 mmol) was added dropwise to a stirred solution of 6
(1.30 g, 5.0 mmol) in THF (12 mL) at –78 °C under argon. After

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30 min, the resulting solution was added to the stirred solution ofanhydrous CeCl3 in THF at –78 °C under argon. After 1 h, a solutionof 4a (312 mg, 1.4 mmol) in THF (1 mL) was added to the organocerium reagent, prepared in situ before, at –78 °C, and stirring wascontinued at the same temperature for a further 1 h. The reaction
was quenched with saturated aqueous NH4Cl (15 mL) at –78 °C,
and the resulting mixture was extracted with EtOAc (3 × 50 mL).
The combined extracts were washed with brine (2 × 30 mL), thendried with MgSO4. Concentration in vacuo gave a residue, which
was purified by column chromatography (hexane/EtOAc, 50:1 →30:1) to give 12a (548 mg, 98 %) as a colourless amorphous solid.
[α]D25 = –6.8 (c = 1.1, CHCl3). 1H NMR (400 Hz, CDCl3): δ = 0.84 (d,J = 7.8 Hz, 3 H), 0.85 (s, 3 H), 0.86 (s, 3 H), 0.90 (d, J = 2.4 Hz, 1 H),
0.98–1.08 (m, 2 H), 1.09 (s, 3 H), 1.11–1.19 (m, 1 H), 1.25–1.46 (m, 4H), 1.55–1.61 (m, 2 H), 1.64 (s, 3 H), 1.66 (s, 3 H), 1.74–1.79 (m, 1 H),
1.91–1.99 (m, 2 H), 2.48 (d, J = 4.4 Hz, 1 H), 3.77 (s, 3 H), 5.26 (d, J =4.4 Hz, 1 H), 6.40 (s, 1 H), 6.91 (s, 1 H) ppm. 13C NMR (100 Hz, CDCl3):
δ = 15.0, 19.0, 22.0 (2 C), 23.4, 25.6 (2 C), 28.7, 33.5, 33.8, 37.8, 39.1,
39.4, 42.3, 55.1, 56.0, 59.3, 67.2, 94.3, 107.7, 117.8, 126.9, 140.7,
146.0, 150.9 ppm. IR (neat): ν̃ = 3461, 2989, 2933, 2868, 2843, 1627,
1493, 1464, 1418, 1385, 1375, 1345, 1298, 1268, 1244, 1216, 1191,
1156, 1124, 1081, 1065, 1009, 980, 958, 934, 886, 844, 820, 802, 788,
758, 667 cm–1. HRMS (EI): calcd. for C25H38O4 [M]+ 402.2770; found402.2773.
6-Methoxy-5-{(E)-[(2R,4aS,8aS)-2,5,5,8a-tetramethyloctahydronaphthalen-1(2H)-ylidene]methyl}-2,2-dimethylbenzo[d][1,3]dioxole (3a): MgBr2 (432 mg, 2.4 mmol) and acetic anhydride (1.0 mL,
10.6 mmol) were slowly added to a stirred solution of 12a (189 mg,
0.47 mmol) in CH2Cl2 (4 mL) at room temperature. After 30 min, thereaction mixture was diluted with CH2Cl2 (30 mL). The organic layer
was washed successively with water (2 × 10 mL), saturated aqueous
NaHCO3 (2 × 10 mL), and brine (2 × 10 mL), and then dried with
MgSO4. Concentration in vacuo gave a residue, which was purified
by column chromatography (hexane/EtOAc, 100:1) to give 3a(148 mg, 82 %) as a colourless viscous liquid. [α]D25 = –75.3 (c = 0.88,
CHCl3). 1H NMR (400 Hz, CDCl3): δ = 0.87 (s, 3 H), 0.90 (s, 3 H), 0.91
(d, J = 6.8 Hz, 3 H), 1.16 (s, 3 H), 1.18–1.26 (m, 2 H), 1.32–1.57 (m, 6H), 1.66 (s, 3 H), 1.65 (s, 3 H), 1.68–1.79 (m, 2 H), 1.85 (d, J = 11.2 Hz,
1 H), 2.66–2.70 (m, 1 H), 3.71 (s, 3 H), 6.14 (s, 1 H), 6.39 (s, 1 H), 6.52
(s, 1 H) ppm. 13C NMR (100 Hz, CDCl3): δ = 19.6, 19.9, 21.7, 22.1,
23.1, 25.7 (2 C), 31.7, 32.2, 33.2, 34.0, 39.7, 40.8, 42.4, 49.5, 56.9, 95.2,
110.0, 115.4, 117.6, 121.3, 140.6, 146.0, 151.6, 156.9 ppm. IR (neat):ν̃ = 2989, 2932, 2869, 2844, 1615, 1492, 1463, 1416, 1385, 1374,
1349, 1268, 1247, 1216, 1190, 1158, 1067, 1013, 980, 888, 842, 828,
818, 789, 759 cm–1. HRMS (EI): calcd. for C25H36O3 [M]+ 384.2665;
found 384.2663.
5-Methoxy-6-{(E)-[(2R,4aS,8aS)-2,5,5,8a-tetramethyloctahydronaphthalen-1(2H)-ylidene]methyl}-2,2-dimethylbenzo[d][1,3]dioxole-4-carbaldehyde (13a): nBuLi (2.6 M in n-hexane; 0.43 mL,
1.2 mmol) was added dropwise to a stirred solution of 3a (148 mg,
0.49 mmol) in THF (4 mL) at –20 °C under argon. After 30 min, DMF(0.29 mL, 3.9 mmol) was added dropwise to the reaction mixture at–20 °C, and the mixture was stirred at the same temperature for afurther 30 min. The reaction was quenched with saturated aqueous
NH4Cl (5 mL) at –20 °C, and the resulting mixture was extractedwith Et2O (3 × 10 mL). The combined extracts were washed withbrine (2 × 5 mL), then dried with MgSO4. Concentration in vacuo
gave a residue, which was purified by column chromatography(hexane/EtOAc, 20:1) to give 13a (151 mg, 95 %) as a yellow amorphous solid. [α]D25 = –71.4 (c = 1.0, CHCl3). 1H NMR (400 Hz, CDCl3):
δ = 0.88 (s, 3 H), 0.92 (s, 3 H), 0.93 (d, J = 6.8 Hz, 3 H), 1.18 (s, 3 H),
1.15–1.23 (m, 1 H), 1.31 (dd, J = 4.9, 12.2 Hz, 1 H), 1.36–1.59 (m, 6H), 1.73 (s, 3 H), 1.74 (s, 3 H), 1.65–1.81 (m, 2 H), 1.85 (d, J = 12.2 Hz,
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1 H), 2.66–2.72 (m, 1 H), 3.75 (s, 3 H), 6.17 (s, 1 H), 6.72 (s, 1 H),
10.27 (s, 1 H) ppm. 13C NMR (100 Hz, CDCl3): δ = 19.5, 20.1, 21.7,
22.1, 22.8, 25.9 (2 C), 32.1, 32.5, 33.2, 34.0, 39.7, 41.0, 42.4, 49.9, 62.2,
113.9, 114.0, 115.4, 120.6, 125.8, 143.8, 146.3, 152.9, 158.5,
188.8 ppm. IR (neat): ν̃ = 2931, 2868, 1689, 1616, 1599, 1469, 1386,
1282, 1225, 1202, 1088, 1048, 1004, 978, 883, 836, 790, 756, 712 cm–1.
HRMS (EI): calcd. for C26H36O4 [M]+ 412.2613; found 412.2604.
5-Hydroxy-6-{(E)-[(2R,4aS,8aS)-2,5,5,8a-tetramethyloctahydronaphthalen-1(2H)-ylidene]methyl}-2,2-dimethylbenzo[d][1,3]dioxole-4-carbaldehyde (14a): nBuLi (2.6 M in n-hexane; 0.69 mL,
1.8 mmol) was added to a stirred solution of nBuSH (0.20 mL,
1.8 mmol) in hexamethylphosphoramide (HMPA; 5 mL) at room
temperature under argon. After 15 min, a solution of 13a (151 mg,
0.37 mmol) in HMPA (2 mL) was added to the mixture at 0 °C. The
resulting mixture was warmed to room temperature, and stirring
was continued for 1 h. The reaction was quenched with saturated
aqueous NH4Cl (5 mL) at 0 °C, and the resulting mixture was extracted with Et2O (3 × 20 mL). The combined extracts were washedwith brine (2 × 20 mL), then dried with MgSO4. Concentration in
vacuo gave a residue, which was purified by column chromatography (hexane/EtOAc, 200:1 → 100:1) to give 14a (133 mg, 91 %) as
an orange-yellow solid; m.p. 99–101 °C. [α]D25 = –112.5 (c = 1.2,
CHCl3). 1H NMR (400 Hz, CDCl3): δ = 0.87 (s, 3 H), 0.90 (s, 3 H), 0.91
(d, J = 7.3 Hz, 3 H), 1.17 (s, 3 H), 1.19–1.23 (m, 1 H), 1.30–1.57 (m, 7H), 1.71 (s, 3 H), 1.71 (s, 3 H), 1.63–1.79 (m, 2 H), 1.85 (d, J = 12.7 Hz,
1 H), 2.64–2.70 (m, 1 H), 6.09 (s, 1 H), 6.76 (s, 1 H), 10.08 (s, 1 H),
10.59 (s, 1 H) ppm. 13C NMR (100 Hz, CDCl3): δ = 19.5, 20.0, 21.7,
22.1, 23.1, 25.7 (2 C), 32.0, 32.5, 33.2, 34.0, 39.6, 41.0, 42.4, 49.8,
106.5, 113.4, 118.1, 119.1, 120.4, 139.4, 148.4, 151.5, 158.9,
191.5 ppm. IR (KBr): ν̃ = 3435, 2930, 2869, 1655, 1639, 1610, 1475,
1467, 1459, 1397, 1377, 1300, 1253, 1221, 1200, 1163, 1162, 1020,
1002, 701 cm–1. HRMS (EI): calcd. for C25H34O4 [M]+ 398.2457; found398.2464.
(–)-Siphonodictyal B (1a): BCl 3 (1.0 M in CH 2 Cl 2 ; 0.62 mL,
0.62 mmol) was added to a stirred solution of 14a (49.7 mg,
0.13 mmol) in CH2Cl2 (2 mL) at –40 °C under argon. After 30 min,the reaction was quenched with saturated aqueous NaHCO3 (2 mL)at –40 °C, and the resulting mixture was extracted with CH2Cl2 (3 ×15 mL). The combined extracts were washed with brine (2 × 10 mL),
then dried with MgSO 4 . Concentration in vacuo gave a residue,
which was purified by gel-filtration chromatography on Sephadex
LH-20 using MeOH as an eluent to give 1a (33.4 mg, 75 %) as ayellow solid; m.p. 118–120 °C. [α]D25 = –78.8 (c = 0.20, CHCl3) {ref.[4][α]D25 = –61.2 (c = 0.67, CHCl3)}. 1H and 13C NMR spectra of 1a wererecorded in three different solvents (i.e., CDCl 3 , [D 6 ]DMSO, andCD3OD) to compare with the data reported in the literature.[4] The
1
H and 13C NMR, IR, and MS data (see below) are identical to thosereported[4] (see also the Supporting Information). 1H NMR (400 Hz,
CDCl3): δ = 0.75 (d, J = 6.8 Hz, 3 H), 0.86 (s, 3 H), 0.89 (s, 3 H), 1.16
(s, 3 H), 1.11–1.30 (m, 3 H), 1.41–1.82 (m, 8 H), 2.52–2.61 (m, 1 H),
5.12 (br. s, 1 H), 5.41 (br. s, 1 H), 5.90 (s, 1 H), 6.83 (s, 1 H), 10.29 (s,1 H), 11.45 (br. s, 1 H) ppm. 13C NMR (100 Hz, CDCl3): δ = 19.4, 21.1,
21.2, 21.7, 22.6, 33.2, 33.9, 34.1, 34.7, 39.3, 41.9, 42.3, 52.3, 109.1,
109.5, 116.9, 123.7, 136.9, 147.6, 148.5, 165.8, 194.5 ppm. 1H NMR(400 Hz, [D6]DMSO): δ = 0.84 (s, 3 H), 0.86 (d, J = 4.8 Hz, 3 H), 0.87
(s, 3 H), 1.11 (s, 3 H), 1.12–1.18 (m, 1 H), 1.25–1.41 (m, 4 H), 1.44–
1.52 (m, 2 H), 1.58–1.78 (m, 4 H), 2.56–2.65 (m, 1 H), 6.02 (s, 1 H),
6.83 (s, 1 H), 9.00 (s, 1 H), 10.02 (s, 1 H), 10.24 (s, 1 H), 10.89 (s, 1 H)
ppm. 13C NMR (100 Hz, [D6]DMSO): δ = 19.0, 19.3, 21.4, 21.8, 22.9,
31.3, 31.7, 32.9, 33.6, 39.1, 40.3, 41.9, 49.0, 110.2, 113.4, 116.6, 126.2,
136.1, 147.8, 151.2, 157.0, 194.7 ppm. 1H NMR (400 Hz, CD3OD): δ =0.86 (d, J = 6.8 Hz, 3 H), 0.88 (s, 3 H), 0.93 (s, 3 H), 1.19 (s, 3 H),
1.20–1.26 (m, 1 H), 1.29–1.48 (m, 3 H), 1.50–1.62 (m, 3 H), 1.68–1.82

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Full Paper(m, 4 H), 2.61–2.69 (m, 1 H), 6.05 (s, 1 H), 6.80 (s, 1 H), 10.34 (s, 1 H)
ppm. 13C NMR (100 Hz, CD3OD): δ = 20.6, 21.7, 22.1, 22.4, 23.2, 33.7,
34.1, 34.2, 34.9, 40.5, 42.2, 43.6, 51.9, 111.5, 114.4, 119.1, 127.6,
137.3, 149.5, 152.9, 159.8, 196.0 ppm. IR (KBr): ν̃ = 3383, 2929, 2870,
1644, 1458, 1389, 1375, 1306, 1270, 938, 757 cm–1. HRMS (EI): calcd.
for C22H30O4 [M]+ 358.2144; found 358.2159.
( 6 - M e t h ox y - 2 , 2 - d i m e t h y l b e n z o [ d ] [ 1 , 3 ] d i oxo l - 5 - y l ) [(1S,2S,4aS,8aS)-2,5,5,8a-tetramethyldecahydronaphthalen-1yl]methanol (12b): Anhydrous CeCl3 (1.20 g, 4.9 mmol) was prepared by heating CeCl 3 ·H 2 O (finely ground powder; 1.82 g,
4.9 mmol) at 140 °C under reduced pressure for 4 h. The material
was then cooled to room temperature, and THF (12 mL) was added.
The resulting suspension was stirred at room temperature underargon for 4 h. Separately, a solution of nBuLi (1.6 M in n-hexane;
3.2 mL, 4.9 mmol) was added dropwise to a stirred solution of 6
(1.26 g, 4.9 mmol) in THF (12 mL) at –78 °C under argon. After
30 min, the resulting solution was added to the above stirred solution of anhydrous CeCl3 in THF at –78 °C under argon. After 1 h, asolution of 4b (272 mg, 1.2 mmol) in THF (1 mL) was added to theorganocerium reagent, prepared in situ above, at –78 °C, and the
mixture was stirred at the same temperature for a further 1 h. The
reaction was quenched with saturated aqueous NH4Cl (20 mL) at–78 °C, and the resulting mixture was extracted with EtOAc (2 ×100 mL). The combined extracts were washed with brine (2 ×50 mL), then dried with MgSO4. Concentration in vacuo gave aresidue, which was purified by column chromatography (hexane/
EtOAc, 50:1 → 10:1) to give 12b (483 mg, 98 %) as a yellow amorphous solid. This product comprised a mixture of diastereomers (ca.
5:3 ratio) at the hydroxy group, and the mixture could not be separated by silica gel column chromatography. 1H NMR (400 Hz, CDCl3):
δ = 0.67–2.23 (m, 32 H), 3.76 (s, 15/8 H), 3.78 (s, 9/8 H), 4.91–4.95(m, 3/8 H), 5.36–5.38 (m, 5/8 H), 6.39 (s, 5/8 H), 6.41 (s, 3/8 H), 6.66
(s, 3/8 H), 6.85 ppm (s, 5/8 H). 13C NMR (100 Hz, CDCl3): δ = 16.4
(5/8 C), 17.2 (3/8 C), 17.5 (3/8 C), 17.8 (5/8 C), 17.9 (5/8 C), 18.3 (5/8 C), 18.5 (3/8 C), 18.6 (3/8 C), 21.6 (5/8 C), 21.7 (5/8 C), 22.6 (3/8
C), 22.8 (3/8 C), 25.66 (3/8 C), 25.69 (10/8 C), 28.7, 30.7 (3/8 C), 31.0
(5/8 C), 33.7 (5/8 C), 33.8 (3/8 C), 35.3 (3/8 C), 36.0 (5/8 C), 39.3 (3/8 C), 39.6 (5/8 C), 42.0 (5/8 C), 42.2 (3/8 C), 56.1 (3/8 C), 56.3 (5/8
C), 56.9 (3/8 C), 57.0 (5/8 C), 57.2 (5/8 C), 58.8 (3/8 C), 69.2 (5/8 C),
72.4 (3/8 C), 94.4 (5/8 C), 94.5 (3/8 C), 107.5 (5/8 C), 108.6 (3/8 C),
117.8 (5/8 C), 118.0 (3/8 C), 124.5 (3/8 C), 126.6 (5/8 C), 140.9 (3/8
C), 141.0 (5/8 C), 146.4 (3/8 C), 146.7 (5/8 C), 150.2 (5/8 C),
151.3 ppm (3/8 C). IR (neat): ν̃ = 3550, 2991, 2993, 2869, 2848, 1626,
1494, 1464, 1421, 1385, 1376, 1342, 1224, 1216, 1191, 1156, 1112,
1065, 1038, 1010, 980, 909, 886, 843, 822, 804, 787, 759, 737,
667 cm –1 . HRMS (EI): calcd. for C 25 H 38 O 4 [M] + 402.2770; found402.2773.
6-Methoxy-5-{(E)-[(2S,4aS,8aS)-2,5,5,8a-tetramethyloctahydronaphthalen-1(2H)-ylidene]methyl}-2,2-dimethylbenzo[d][1,3]dioxole (3b): Methanesulfonyl chloride (0.18 mL, 2.3 mmol) was
added dropwise to a stirred solution of 12b (308 mg, 0.77 mmol)in CH2Cl2 (7 mL) containing triethylamine (0.53 mL, 3.8 mmol) at
0 °C. After 30 min, the reaction was quenched with saturated aqueous NH4Cl (3 mL) at 0 °C, and the resulting mixture was extractedwith Et2O (2 × 50 mL). The combined extracts were washed withbrine (2 × 30 mL), then dried with MgSO4. Concentration in vacuo
gave a residue, which was purified by column chromatography(hexane/EtOAc, 200:1) to give 12b (277 mg, 94 %) as a white solid;
m.p. 96–98 °C. [α]D25 = +106.2 (c = 0.98, CHCl3). 1H NMR (400 Hz,
CDCl3): δ = 0.87 (s, 3 H), 0.89 (s, 3 H), 1.03 (dd, J = 2.4, 11.7 Hz, 1
H), 1.18 (s, 3 H), 1.20–1.25 (m, 5 H), 1.40 (d, J = 12.7 Hz, 1 H), 1.48–
1.61 (m, 6 H), 1.65 (s, 3 H), 1.68 (s, 3 H), 1.78 (d, J = 12.2 Hz, 1 H),
3.01–3.09 (m, 1 H), 3.73 (s, 3 H), 6.13 (s, 1 H), 6.43 (s, 1 H), 6.59 (s, 1
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H) ppm. 13C NMR (100 Hz, CDCl3): δ = 17.8, 18.9, 21.8, 22.6, 22.8,
25.7, 25.8, 30.7, 33.3, 34.0, 34.1, 38.6, 40.9, 42.1, 54.6, 57.0, 95.3,
109.4, 115.1, 117.7, 120.4, 140.7, 146.0, 152.2, 155.5 ppm. IR (KBr):ν̃ = 2934, 2869, 2854, 1615, 1493, 1463, 1420, 1385, 1375, 1348,
1273, 1248, 1217, 1190, 1157, 1068, 1011, 980, 890, 841, 818, 759,
736 cm –1 . HRMS (EI): calcd. for C 25 H 36 O 3 [M] + 384.2665; found384.2663.
5-Methoxy-6-{(E)-[(2S,4aS,8aS)-2,5,5,8a-tetramethyloctahydronaphthalen-1(2H)-ylidene]methyl}-2,2-dimethylbenzo[d][1,3]dioxole-4-carbaldehyde (13b): nBuLi (2.6 M in n-hexane; 0.49 mL,
1.3 mmol) was added dropwise to a stirred solution of 3b (165 mg,
0.43 mmol) in THF (5 mL) at –20 °C under argon. After 30 min, DMF(0.32 mL, 4.3 mmol) was added dropwise to the reaction mixture at–20 °C, and stirring was continued at the same temperature for
30 min. The reaction was quenched with saturated aqueous NH4Cl(2 mL) at –20 °C, and the resulting mixture was extracted with Et2O
(2 × 25 mL). The combined extracts were washed with brine (2 ×10 mL), then dried with MgSO4. Concentration in vacuo gave aresidue, which was purified by column chromatography (hexane/
EtOAc, 20:1) to give 13b (172 mg, 97 %) as a yellow amorphoussolid. [α]D25 = +59.4 (c = 1.12, CHCl3). 1H NMR (400 Hz, CDCl3): δ =0.89 (s, 3 H), 0.91 (s, 3 H), 0.98 (dd, J = 2.9, 11.7 Hz, 1 H), 1.21 (s, 3H), 1.24 (d, J = 7.3 Hz, 3 H), 1.16–1.28 (m, 2 H), 1.42–1.69 (m, 7 H),
1.73 (s, 3 H), 1.75 (s, 3 H), 1.82 (d, J = 12.2 Hz, 1 H), 2.95–3.02 (m, 1
H), 3.75 (s, 3 H), 6.15 (s, 1 H), 6.74 (s, 1 H), 10.28 (s, 1 H) ppm. 13C
NMR (100 Hz, CDCl3): δ = 17.8, 18.9, 21.8, 22.4, 22.5, 26.0 (2 C), 31.0,
33.4, 34.0, 34.1, 38.8, 41.0, 42.1, 55.0, 62.6, 114.2, 114.3, 114.7, 120.7,
124.8, 144.0, 146.2, 153.6, 157.6, 188.7 ppm. IR (neat): ν̃ = 2981,
2933, 2869, 2854, 1691, 1614, 1600, 1470, 1386, 1284, 1253, 1221,
1203, 1089, 1049, 999, 979, 757 cm–1. HRMS (EI): calcd. for C26H36O4[M]+ 412.2613; found 412.2604.
5-Hydroxy-6-{(E)-[(2S,4aS,8aS)-2,5,5,8a-tetramethyloctahydronaphthalen-1(2H)-ylidene]methyl}-2,2-dimethylbenzo[d][1,3]dioxole-4-carbaldehyde (14b): nBuLi (2.6 M in n-hexane; 0.82 mL,
2.2 mmol) was added to a stirred solution of nBuSH (0.23 mL,
2.2 mmol) in hexamethylphosphoramide (HMPA; 5 mL) at room
temperature under argon. After 15 min, a solution of 13b (172 mg,
0.43 mmol) in HMPA (2 mL) was added to the above mixture at
0 °C. The resulting mixture was warmed to room temperature, andstirring was continued for 1 h. The reaction was quenched withsaturated aqueous NH4Cl (3 mL) at 0 °C, and the resulting mixture
was extracted with Et2O (2 × 25 mL). The combined extracts were
washed with brine (2 × 10 mL), then dried with MgSO4. Concentration in vacuo gave a residue, which was purified by column chromatography (hexane/EtOAc, 200:1 → 100:1) to give 14b (151 mg, 88 %)as an orange-yellow solid; m.p. 167–169 °C. [α]D25 = +175.7 (c = 0.96,
CHCl3). 1H NMR (400 Hz, CDCl3): δ = 0.87 (s, 3 H), 0.89 (s, 3 H), 1.05
(dd, J = 2.9, 11.7 Hz, 1 H), 1.19 (s, 3 H), 1.25 (d, J = 7.3 Hz, 3 H),
1.21–1.26 (m, 2 H), 1.40 (d, J = 13.2 Hz, 1 H), 1.49–1.69 (m, 6 H),
1.71 (s, 3 H), 1.74 (s, 3 H), 1.80 (d, J = 11.7 Hz, 1 H), 2.95–2.98 (m, 1
H), 6.11 (s, 1 H), 6.83 (s, 1 H), 10.09 (s, 1 H), 10.71 (s, 1 H) ppm. 13C
NMR (100 Hz, CDCl3): δ = 17.8, 18.9, 21.8, 22.5, 22.9, 25.8 (2 C), 31.0,
33.3, 34.0, 34.1, 38.5, 41.0, 42.0, 54.5, 106.5, 113.3, 117.4, 118.0,
120.5, 139.5, 148.4, 152.2, 157.4, 191.5 ppm. IR (KBr): ν̃ = 3435, 2984,
2962, 2929, 2869, 1658, 1641, 1478, 1459, 1389, 1376, 1317, 1301,
1255, 1237, 1159, 1065, 1029, 1003, 691, 676 cm–1. HRMS (EI): calcd.
for C25H34O4 [M]+ 398.2457; found 398.2464.
(+)-8-epi-Siphonodictyal B (1b): BCl3 (1.0 M in CH2Cl2; 0.76 mL,
0.76 mmol) was added to a stirred solution of 14b (60.7 mg,
0.15 mmol) in CH2Cl2 (3 mL) at –40 °C under argon. After 30 min,the reaction was quenched with saturated aqueous NaHCO3 (3 mL)at –40 °C, and the resulting mixture was extracted with CH2Cl2 (2 ×5665

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Full Paper
25 mL). The combined extracts were washed with brine (2 × 10 mL),
then dried with MgSO4. Concentration in vacuo gave a residue,
which was purified by gel-filtration chromatography on Sephadex
LH-20 using MeOH as an eluent to give 1b (49.4 mg, 91 %) as ayellow solid; m.p. 86–88 °C. [α]D25 = +42.1 (c = 0.33, CHCl3) {ref.[4][α]D25 = +5.3 (c = 0.75, CHCl3)}. The 1H and 13C NMR, IR, and MS data
(see below) are identical to those reported[4] (see also the Supporting Information). 1H NMR (400 Hz, CDCl3): δ = 0.88 (s, 6 H), 1.93–
1.96 (m, 1 H), 1.09 (d, J = 7.3 Hz, 3 H), 1.20 (s, 3 H), 1.14–1.22 (m, 1
H), 1.40–1.76 (m, 8 H), 1.83 (d, J = 12.6 Hz, 1 H), 2.66–2.73 (m, 1 H),
5.08 (br. s, 1 H), 5.51 (br. s, 1 H), 5.83 (s, 1 H), 6.81 (s, 1 H), 10.31 (s,1 H), 11.35 (br. s, 1 H) ppm. 13C NMR (100 Hz, CDCl3): δ = 17.7, 18.8,
21.8, 22.5, 22.7, 31.6, 33.4, 34.0, 34.1, 38.0, 41.3, 42.0, 55.0, 109.4,
110.7, 115.7, 123.7, 137.0, 147.6, 148.9, 165.6, 194.6 ppm. IR (KBr):ν̃ = 3398, 2921, 2869, 1655, 1638, 1609, 1459, 1439, 1399, 1388,
1254, 1204, 1018, 947 cm–1. HRMS (EI): calcd. for C22H30O4 [M]+358.2144; found 358.2146.
PI3Kα Inhibition Assay: The PI3Kα (p110α/p85α) activity was evaluated using the mobility shift assay (Carna Biosciences, Kobe, Japan). Compound solutions containing the kinase and substrate
were prepared with assay buffer (Carna Biosciences). The mixed solutions were incubated in a 384-well microplate at room temperature for 5 h. The reaction mixtures were then applied to the LabChip(Caliper Life Sciences, MA, USA), and the product and substrate
peaks were separated and quantified. The PI3K reaction was evaluated from the product ratio, calculated from the peak heights ofthe product and substrate. Data are the mean of two independent
experiments carried out in duplicate. IC50 values were determinedfrom concentration vs. %-inhibition curves by fitting to a fourparameter logistic curve.

Acknowledgments
This study was supported by the Ministry of Education, Culture,
Sports, Science and Technology, Japan (MEXT) through a Grantin-Aid for the Strategic Research Foundation Program at Private
Universities (no. S1511001L, 2015–2019) and a Grant-in-Aid for
Scientific Research (C) (no. 15K07865, 2015–2017).
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Suzuki, M. Inoue, T. Katoh, Org. Lett. 2002, 4, 4483–4486; c) T. Taishi, S.
Takechi, S. Mori, Tetrahedron Lett. 1998, 39, 4347–4350; d) P. A. Aristoff,
A. W. Harrison, A. M. Huber, Tetrahedron Lett. 1984, 25, 3955–3958; e)
S. C. Welch, A. S. C. P. Pao, Tetrahedron Lett. 1977, 18, 505–508; f) G. I.
Feutrill, R. N. Mirrington, Aust. J. Chem. 1972, 25, 1719–1729; g) P. A.
Bartlett, W. S. Johnson, Tetrahedron Lett. 1970, 11, 4459–4412.
[14] For reviews, see: a) B. Vanhaesebroeck, L. Stephens, P. Hawkins, Nat. Rev.
Mol. Cell Biol. 2012, 13, 195–203; b) M. P. Wymann, C. Schultz, ChemBioChem 2012, 13, 2022–2035.
[15] For reviews, see: a) K. Blajecka, A. Borgström, A. Arcaro, Curr. Drug Targets
2011, 12, 1056–1081; b) S. G. Ward, P. Finan, Curr. Opin. Pharmacol. 2003,
3, 426–434; c) S. Ward, Y. Sotsios, J. Dowden, I. Bruce, P. Finan, Chem. Biol.
2003, 10, 207–213.
[16] For reviews, see: a) I. Brana, L. L. Siu, BMC Med. 2012, 10, 161; b) Q.-W.
Fan, W. A. Weiss, Cell Cycle 2006, 5, 2301–2305.
[17] For reviews, see: a) B. A. McLean, P. Zhabyeyev, E. Pituskin, I. Paterson,
M. J. Haykowsky, G. Y. Oudit, J. Card. Failure 2013, 19, 268–282; b) A.
Eisenreich, U. Rauch, Cardiovasc. Ther. 2011, 29, 29–36.
[18] For reviews, see: a) L. So, D. A. Fruman, Biochem. J. 2012, 442, 465–481;b) M. D. Blunt, S. G. Ward, Curr. Opin. Pharmacol. 2012, 12, 444–451; c)
P. Norman, Expert Opin. Ther. Pat. 2011, 21, 1773–1790; d) C. Rommel,
M. Camps, H. Ji, Nat. Rev. Immunol. 2007, 7, 191–201.
Received: August 1, 2016
Published Online: November 2, 2016

5666

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Communication
pubs.acs.org/JACS

Enantioselective Total Synthesis of Mandelalide A andIsomandelalide A: Discovery of a Cytotoxic Ring-Expanded Isomer
Nagarathanam Veerasamy,† Ankan Ghosh,† Jinming Li,† Kazuhiro Watanabe,†,‡ Jeﬀrey D. Serrill,§
Jane E. Ishmael,§ Kerry L. McPhail,§ and Rich G. Carter*,††

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Department of Chemistry and §Department of Pharmaceutical Sciences, Oregon State University, Corvallis, Oregon 97331, UnitedStates‡Laboratory of Synthetic and Medicinal Chemistry, Tohoku Pharmaceutical University, Sendai 981-8558, JapanS Supporting Information*

intriguing strategic premise was the possibility that themacrocyclization event could be conducted with both the C23
and C24 alcohols exposed and governed by strong, preferential
translactonization to the desired C23 macrolactone. Support forthis premise can be seen in the observations that translactonization was operable with the originally proposed
structure 14 as well as with other macrolides such asamphidinolides G/H;8 however, the revised structure 2 exists
exclusively as the C23 macrolactone. If this hypothesis proved
problematic, the C24 position could be masked prior tomacrolactonization.
Synthesis of the northern C15−C24 fragment 4 commenced
from the alkyne 96b,d and iodide 109 (Scheme 2). Thesebuilding blocks were readily available from (S)-malic acid (8).
Sonogashira coupling provided the cis-enyne in excellent yieldusing diisopropylamine as the solvent. Sharpless asymmetricdihydroxylation of cis-alkenes10 has seen limited use in total
synthesis11 despite the widespread utility and prevalence ofanti-diols. We were pleased to observe that Sharplessdihydroxylation using commercially available (DHQD)2PHAL
provided the desired diastereomer 12 in good yield andreasonable diastereoselectivity (76%, 3.5:1 dr). Use of thepseudo-enantiomeric ligand (DHQ)2PHAL gave the opposite
diastereomer in similar selectivity (1:3.6 dr). Interestingly, theSharpless ligand speciﬁcally designed for cis-alkenes (DHQDIND)10 proved less eﬀective (2.2:1 dr). Bisesteriﬁcation using
benzoyl chloride followed by desilylation generated the AgCC
precursor. While our laboratory has explored the utility of diols
as viable nucleophiles in the AgCC process,6b,d the alternate
combination of using dibenzoates (e.g., 6) has not beenreported. We were pleased to discover that this substrate
rapidly and smoothly undergoes cyclization to provide theunstable enol benzoate 13. Subsequent in situ treatment with
MeLi·LiBr revealed ketone 14 which also proved unstable until
protection as its silyl ether 15 in good overall yield withexcellent stereoselectivity (68%, > 10:1 dr). Ketone 15 was next
oleﬁnated with the Petasis reagent followed by diastereoselective reduction using Rh/Al2O3 to provide the 18S stereochemistry. Finally, removal of the Piv ester followed byconversion to Wittig salt 4 proceeded smoothly.

ABSTRACT: The total synthesis of mandelalide A and its
ring-expanded macrolide isomer isomandelalide A has
been achieved. Unexpected high levels of cytotoxicity wereobserved with the ring-expanded isomandelalide A with arank order of potency: mandelalide A > isomandelalide A> mandelalide B. Key aspects of the synthesis include Agcatalyzed cyclizations (AgCC’s) to construct both the
THF and THP rings present in the macrocycle,
diastereoselective Sharpless dihydroylation of a cis-enyne,
and lithium acetylide coupling with a chiral epoxide.

In 2012, McPhail et al. reported the isolation of a series ofnovel macrolides in small quantities from a new species ofLissoclinum ascidian found in Algoa Bay, South Africa.1 These
compounds were named mandelalides A−D, and theirstructures were proposed by extensive spectroscopic analysis.
Given the small quantities of material obtained from isolation,
only preliminary evaluation of their biological properties could
be performed. Mandelalides A and B showed low nanomolar
cytotoxicity to lung (NCI-H460) and neuroblastoma (Neuro2A) cancer cell lines. Not surprisingly, these structurally
complex, cytotoxic compounds have attracted considerable
synthetic attention from multiple laboratories. In 2014,
Willwacher and Fürstner reported the total synthesis of the
proposed structure 1, which did not match the spectroscopic
data of the natural product.2 Shortly thereafter, Ghosh et al.
reported the synthesis of the agylcone of the proposed structureof mandelalide A.3 Ye et al. synthesized a series of structuralvariants of mandelalide A, which allowed them to unequivocallyestablish the absolute structure of mandelalide A as compound
2.4,5 Given the scarcity of 2 and our laboratory’s long-standing
interest in the synthesis of cytotoxic macrolides,6 we sought todevelop a robust synthesis of compound 2 to enable additional
biological evaluation. Herein, we report the enantioselective
total synthesis of mandelalide A (2).
As shown in Scheme 1, our synthesis strategy builds on theobservation that both the THF and the THP rings present inmacrolide 2 could be accessed by a silver-catalyzed cylization(AgCC) from the requisite propargylic benzoates. Ourlaboratory had previously demonstrated the utility of thisgeneral concept in our synthesis of amphidinolides C and F;6b,dhowever, we are unaware of any examples for accessing sixmembered oxygenated heterocycles using AgCC.7 Another© 2016 American Chemical Society

Received: November 24, 2015
Published: January 13, 2016770

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DOI: 10.1021/jacs.5b12318
J. Am. Chem. Soc. 2016, 138, 770−773

Journal of the American Chemical Society

Communication

Scheme 1. Retrosynthetic Analysis of Mandelalide A (2)Scheme 2. Synthesis of Northern Fragment 4

Scheme 3. Synthesis of Southern Fragment 5

With the northern phosphonium salt in hand, we shifted our
focus to the southern C1−C14 subunit (Scheme 3). Startingfrom the readily available epoxide 20 and alkyne 21 (eachaccessible in 5 steps from commercially available sources), BF3·
Et2O-mediated coupling under optimized conditions provided
the homopropargylic alcohol 7. While examples exist of alkyne/
epoxide couplings in the presence of an ester,12 propargylic
benzoates have not been used before in the process. With thecyclization precursor 7 in hand, we explored the key AgCC toaccess the pyran 22. While we are unaware of prior examples ofrelated silver-catalyzed cyclizations for extending the AgCC
process to larger ring systems,7 we were gratiﬁed to discoverthat the transformation proceeded smoothly to yield 22 in a 6:1
dr at C 5 based on crude 1 H NMR. This level ofdiasteroselectivity may be explained by the modest level ofenantiopurity (7:1 er) in the alkyne precursor 21. The crude
benzoate was immediately submitted to hydrolysis conditions(NaOMe, MeOH, rt) to generate the desired C7 ketone 23 ingood yield with no loss in stereochemical integrity. It is
important to note that alternate protocols to convert the enol
benzoate 22 into the ketone 23 (including MeLi·LiBr)6b,d led
to inferior yields and/or reduced dr. Removal of the C12 silyl
ether followed by oxidation and Wittig oleﬁnation generated
the ester 24. Borohydride reduction followed by Schmidt
glycosidation5 produced the target glycoside 26 in good yieldand reasonable dr at C1′. Finally, conversion of the C14 ester
into its corresponding aldehyde and cross metathesis13
generated the southern fragment 5.
Our ﬁrst generation approach to coupling and macrocyclization is shown in Scheme 4. Treatment of phosphonium
salt 4 with NaHMDS followed by addition of the aldehyde 5771— 235 —

DOI: 10.1021/jacs.5b12318
J. Am. Chem. Soc. 2016, 138, 770−773

Journal of the American Chemical Society

Communication

Scheme 4. Total Synthesis of Isomandelalide A (31)

Scheme 5. Total Synthesis of Mandelalide A (2)Table 1. Cytotoxic Eﬀects of Compounds 2, 31, and 33
Against Human Cancer Cell Lines
HeLa cervix

cleanly provided the C12−C15 E,Z-diene. Next, selective
deprotection of the C23−C24 acetonide in the presence of the
C7 glycoside and the acid-sensitive E,Z-diene motif wasaccomplished using TFA to generate the C23,24 diol in excellent
yield. Yamaguchi conditions smoothly induced macrolactonization; however, the ring-expanded, 25-membered isomer 29 was
the primary isolable product from this reaction. Removal of thethree silyl moieties in 29 using TAS-F provided the previously
unknown, 25-membered macrolide called isomandelalide A(31). A second product 30 was also observed in themacrolactonization. Subsequent TAS-F treatment of thecrude mixture from the macrolactonization revealed the desired
mandelalide A (2); however, only minor amounts of 2 could beaccessed through this route. The preference for 29 over 30 issurprising based on the observation by Ye et al. that 2 appeared
to prefer the smaller, 24-membered macrolide.4,14
In order to selectively access the desired natural product, theC24 position was masked as its TBS ether on diol 27 (Scheme
5). Subsequent conversion of the methyl ester into the acid 32
was accomplished by a redox process. Attempted direct
saponiﬁcation of the C1 methyl ester on the C24 silylated
product led to extensive silyl migration. We were pleased to seethat Yamaguchi macrolactonization of the seco acid 32 proved
eﬀective on the C24 silyl ether series to give the known Yeintermediate.4 Finally, global desilylation generated the natural
product, the data for which matched with the literature values(1H, 13C, [α]D).1 A modest amount of a second macrolide wasobserved under the cyclization process, which we hypothesize
to be the C21 macrolactone (likely due to C21/C23 silyl
migration); however, this minor product decomposed under
the TAS-F conditions.
Isomandelalide A (31) showed potent cytotoxicity incomparative testing with synthetic mandelalide A (2) andrelative to the natural mandelalide B (33) (Table 1). Using
assay conditions comparable to those used in the original

H460 lungcompd. no.

EC50 (nM)

95% C.I. (nM)

EC50 (nM)

95% C.I. (nM)

2
31
33

10.5
17.1
35.5

8.6−12.2
13.1−21.7
16.1−52.4

13.2
30.3
35.5

8.6−18.0
15.0−42.5
26.3−61.2

bioassay-guided drug discovery screen,1 synthetic 2 showedbiological activity that was entirely consistent with thecytotoxicity observed in earlier testing of the natural product.
Synthetic 2 was approximately 3-fold more potent than 33
against human HeLa cervical and H460 lung cancer cells.
Although slightly less potent than 2, isomandelalide A (31) was
also a fully eﬃcacious cytotoxin against both human cancer cell
types with EC50 values of 17.1 and 30.3 nM for HeLa and NCIH460 cells, respectively.
In conclusion, eﬃcient total syntheses of mandelalide A (2)and its ring-expanded isomer isomandelalide A (31) have been
completed. The unexpected potency of isomandelalide A raises
intriguing questions about the biology of these natural
products, given that variation in ring size of a macrocycle
often leads to conformational changes that can have a notable
impact on biological activity (e.g., amphidinolide G/H15 andisoapoptolidin/apoptolidin16). The disclosed synthetic campaign demonstrated the utility of the AgCC for theconstruction of oxygenated heterocycles, including the ﬁrst
reported example for accessing a pyran ring system. The E,Zdiene motif was incorporated smoothly through a Wittig
reaction. The macrocyclization preferences for the C23,24 diol
were explored. Further application of this methodology toaccess other members of the mandelalide family will be
disclosed in due course.

■

ASSOCIATED CONTENT

S Supporting Information*

The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.5b12318.
772— 236 —

DOI: 10.1021/jacs.5b12318
J. Am. Chem. Soc. 2016, 138, 770−773

Journal of the American Chemical Society

Communication(14) Blakemore, P. R.; Browder, C. C.; Hong, J.; Lincoln, C. M.;
Nagorney, P. A.; Robarge, L. A.; Wardrop, D. J.; White, J. D. J. Org.
Chem. 2005, 70, 5449−5460.
(15) Kobayashi, J.; Shigemori, H.; Ishibashi, M.; Yamasu, T.; Hirota,
H.; Sasaki, T. J. Org. Chem. 1991, 56, 5221−5224.
(16) Wender, P. A.; Gulledge, A. V.; Jankowski, O. D.; Seto, H. Org.
Lett. 2002, 4, 3819−3822.

Complete experimental details and biological protocols(PDF)
1
H and 13C NMR spectra (PDF)■

AUTHOR INFORMATION

Corresponding Author*rich.carter@oregonstate.edu
Funding

No competing ﬁnancial interests have been declared.
Notes

The authors declare no competing ﬁnancial interest.

■

ACKNOWLEDGMENTS
Financial support provided by Oregon State University.
Tohuku Pharmaceutical University is acknowledged for theirfellowship for K.W. Prof. Claudia Maier and Jeﬀ Morré (OSU)
are acknowledged for mass spectra. Dr. Mohamed F. El-Mansy(OSU) is acknowledged for his assistance with organizing thespectral data. Finally, the authors are grateful to Prof. Paul HaYeon Cheong (OSU) and Dr. Roger Hanselmann (Akebia
Therapeutics) for their helpful discussions.

■

REFERENCES(1) (a) Sikorska, J.; Hau, A. M.; Anklin, C.; Parker-Nance, S.; DaviesColeman, M. T.; Ishmael, J. E.; McPhail, K. L. J. Org. Chem. 2012, 77,
6066−6075. (b) Sikorska, J.; Hau, A. M.; Anklin, C.; Parker-Nance, S.;
Davies-Coleman, M. T.; Ishmael, J. E.; McPhail, K. L. J. Org. Chem.
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8587−8598.
(3) Reddy, K. M.; Yamini, V.; Singarapu, K. K.; Ghosh, S. Org. Lett.
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(5) (a) Wilwacher, J.; Heggen, D.-I. B.; Wirtz, C.; Thiel, W.; Fürtsner,
A. Chem. - Eur. J. 2015, 21, 10416−10430. (c) Brütsch, T. M.; Bucher,P.; Altmann, K.-H. Eur. J. Chem. DOI: 10.1002/chem.201504918.
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H.; Endo, H.; Kobayashi, Y. Tetrahedron Lett. 1986, 27, 6103−6106.
(10) Wang, L.; Sharpless, K. B. J. Am. Chem. Soc. 1992, 114, 7568−
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(11) (a) Dong, S.; Paquette, L. A. J. Org. Chem. 2005, 70, 1580−
1596. (b) Halim, R.; Brimble, M. A.; Merten, J. Org. Lett. 2005, 7,2659−2662. (c) Hicks, J. D.; Flamme, E. M.; Roush, W. R. Org. Lett.
2005, 7, 5509−5512. (d) Ermolenko, L.; Sasaki, N. A. J. Org. Chem.
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(12) (a) Ramana, C. V.; Raghupathi, N.; Gujar, M. K.; Chorghade,
M. S. Tetrahedron Lett. 2005, 23, 4073−4075. (b) Tinsley, J. M.;
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773— 237 —

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Letterpubs.acs.org/OrgLett

Stereoselective, Ag-Catalyzed Cyclizations To Access Polysubstituted
Pyran Ring Systems: Synthesis of C1−C12 Subunit of Madeirolide A
Kazuhiro Watanabe,†,‡ Jinming Li,‡ Nagarathanam Veerasamy, Ankan Ghosh, and Rich G. Carter*
Department of Chemistry, Oregon State University, 153 Gilbert Hall, Corvallis, Oregon 97331, United States

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S Supporting Information*

ABSTRACT: The exploration into the scope of a silvercatalyzed cyclization (AgCC) of propargyl benzoates foraccessing pyran ring systems has been reported. The impact ofthe degree of substitution, nature of the substitution on the
carbon backbone/benzoate moiety, and stereochemistry has
been evaluated. The application of this methodology to thesynthesis of the C1−C12 southern fragment of madeirolide A isdisclosed.

N

Scheme 1. Background of Metal-Catalyzed THF Synthesis
and Select Natural Product Examples

ature produces a diverse array of polyketide natural
products that have shown interesting biological activity.
Many of these polyketides contain stereodeﬁned, polysubstituted tetrahydrofurans and tetrahydropyrans (Scheme 1).
Consequently, a broad array of synthetic tools have beendeveloped to access these structural classes,1 including varioustransition-metal-mediated processes. The use of allenyl alcohols
as suitable precursors has proven eﬀective in metal-catalyzed(primarily Au and Ag) methods for accessing dihydrofurans (eq1)2 and dihydropyrans.3 Our laboratory has had a long-standing
interest in macrolides including those containing furan andpyran ring systems.4 More recently, we have been focused on
the potential of silver-catalyzed cyclizations (AgCCs) withpropargyl benzoates for accessing furanyl ring systems (eq 2).5
This work was inspired by Shigemasa and other pioneering
authors in the area.6,7 In these transformations, the propargyl
benzoate 4 must ﬁrst undergo metal-catalyzed rearrangement toan allenyl enol ether 6 which then undergoes a second metalcatalyzed process, this time a 5-endo cyclization to access apresumed vinyl metallo intermediate 7 which subsequently
protodemetallates to give the ﬁnal, dihydrofuranyl enol ether
product 8. We became intrigued by the potential of extending
this concept to access tetrahydropyrans using the corresponding homopropargylic alcohols. Toste and co-workers demonstrated the potential of phenolic-containing propargylic
benzoates for accessing enantioenriched benzopyrans.8 Kotikalapudi and Swamy utilized highly unsaturated propargyl
benzoate systems for access to dienyl pyran systems.9 In ourrecent total synthesis of mandelalide A (9), we demonstrated
the ﬁrst example of using a AgCC to access a cis-disubstituted
pyran moiety.5c Concurrent with our mandelalide A synthesis,
Fürstner and co-workers reported a exo-cyclic version of thistransformation in their synthesis of enigmazole A.10 Herein, we
disclose the exploration of a general method for accessing enol
benzoate-containing dihydropyrans and its application to the© 2016 American Chemical Society

Received: February 11, 2016
Published: March 31, 2016
1744

— 238 —

DOI: 10.1021/acs.orglett.6b00414
Org. Lett. 2016, 18, 1744−1747

Organic Letters

Lettersynthesis of the C1−C12 fragment of the antifungal macrolide
madeirolide A (10).11
We ﬁrst sought to explore the impact of substitution on the
propargylic benzoate carbon12 in the AgCC process (Table 1).

Table 2. Exploration of Diastereoselective AgCC

Table 1. Exploration of Scope of AgCC

entry

R

yield (%)

a
b
c
d
e
f
ga

Me
Et
Ph
−(CH2)4−−(CH2)5−−(CH2)2−O−(CH2)2−
H

73
76
decomp89
83
73
decompaThis result was observed in reﬂuxing toluene (3 h) and xylenes (1 h).
No reaction was observed under reﬂuxing benzene (4 h).

The optimum conditions for these cyclizations were in reﬂuxing
benzene. The reaction proceeded faster at higher temperatures(e.g., reﬂuxing toluene or xylenes); however, slightly lower
yields were typically observed (ca. 5−10% lower). We werepleased to see clean conversion with both the dimethyl
benzoate 11a and the diethyl variant 11b to their desired
products in 73% and 76% yield, respectively. Interestingly, thediphenyl benzoate 11c proved problematic, as decomposition
was observed under the reaction conditions. We attribute thisoutcome to the strong propensity of the benzoate moiety toionize to the corresponding 3° carbocation which would behighly stabilized by the two phenyl substituents as well as thealkyne. Cyclic systems (e.g., 11d−11f) worked smoothly toprovide the spirocyclic products in good to excellent yields(73−89%). The absence of any substitution at the propargylic
benzoate carbon was problematic as the primary benzoate 11g
led to no reaction under reﬂuxing benzene conditions anddecomposition at higher temperatures.
We next set out to study the impact of substituents on the
benzoate moiety in the cyclization (Table 2). We focused on
the disubstituted AgCC, as we recently demonstrated the utilityof this transformation in our mandelalide A synthesis.5c These
cyclization precursors were accessed from known chiralbuilding blocks.13 We observed a modest temperature dependent stereoerosion in the cyclization process. Interestingly,
higher reaction temperatures appeared to have a slight, positive
impact on the stereochemical outcome of the process. The useof more electron-rich benzoates (e.g., 13d) led to an increase in
the reaction rate, likely due to the increased nucleophilicity ofthe benzoate to attack the silver-activated alkyne (entries 10−12); however, a decrease in the overall selectivity was observed.
In contrast, the use of a more electron-deﬁcient benzoate (13e)
led to dramatically lower yields and slightly reduced
diastereoselectivity (entries 13−15).
Varying substitution of the carbon backbone led tointeresting results (Scheme 2).12 The use of a primary alcohol
as the terminal nucleophile (compound 15) in the AgCC
worked smoothly and in high yield. This result is in contrast to
the primary propargylic benzoate 11g which led to decom-

entrysubstrate

conditions% yield (dr)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

13a
13a
13a
13b
13b
13b
13c
13c
13c
13d
13d
13d
13e
13e
13e

PhH, reﬂux, 3.5 hPhMe, reﬂux, 1.5 hxylenes, reﬂux, 50 min
PhH, reﬂux, 4 hPhMe, reﬂux, 5 hxylenes, reﬂux, 50 min
PhH, reﬂux, 3.5 hPhMe, reﬂux, 1.5 hxylenes, reﬂux, 50 min
PhH, reﬂux, 2.5 h
PhMe, reﬂux, 1.5 hxylenes, reﬂux, 30 min
PhH, reﬂux, 7 hPhMe, reﬂux, 5 hxylenes, reﬂux, 1 h

92 (4.6:1)
93 (5.4:1)
93 (5.5:1)
79 (4.2:1)
87 (4.8:1)
86 (4.6:1)
93 (3.8:1)
94 (4.0:1)
90 (4.3:1)
77 (2.6:1)
93 (3.8:1)
91 (3.8:1)
13 (3.0:1)
45 (3.2:1)
62 (3.5:1)

position. A series of cyclization precursors containing multiple
stereochemistries were studied. The stereoselectivities on
alcohols 17b−c were modest (5−6:1 dr), but the reactions
proceeded in generally good yields. Interestingly, the epimericalcohol stereochemistry 19 did produce the expected transpyran ring 20, but in low diastereoselectivity (2:1 dr). This
example clearly demonstrates that signiﬁcant stereochemical
scrambling is occurring on a similar time frame to cyclization.
The presence of a second propargylic stereocenter proved
beneﬁcial, leading to excellent stereoconversion (20:1 dr) foralcohol 21 to enol benzoate 22; however, the formation of adihydrofuran (DHF) byproduct 23 was also observed.14 The
use of tertiary alcohols produced nearly equal amounts of ﬁveand six-membered products. Finally, the use of the stereochemically dense alcohol 27 did provide the target dihydropyran 28.14 Crude NMR indicated a similar DHF byproduct wasalso formed in nearly equal amounts (approximately 1:1);
however, this presumed DHF compound proved too unstableto verify. Subsequent hydrolysis of the enol benzoate 28
provided the pyranone 29 in 20:1 dr.
With a ﬁrm knowledge of the reaction scope, we applied the
AgCC protocol to the synthesis of the C1−C12 fragment ofmadeirolide A (Scheme 3). Starting from readily available vinyl
iodide 3015 and alkyne 31,5b Sonogashira cross-coupling
generated the enyne 32 in good yield. Shi epoxidation ofenyne 32 proceeded in excellent diastereoselectivity and goodoverall yield. Subsequent ring opening at the propargylic
position was best accomplished using LiAlMe4/BF3·Et2O toprovide clean stereoconversion to alcohol 35. We were pleased
to see that the key AgCC proceeded in excellent
diastereoselectivity to provide the enol p-methoxybenzoate 36
in >20:1 dr along with a minor amount of the undesired DHF
37 in an approximately 1.4:1 ratio (36:37). The p-methoxy
1745— 239 —

DOI: 10.1021/acs.orglett.6b00414
Org. Lett. 2016, 18, 1744−1747

Organic Letters

LetterScheme 3. AgCC for Accessing the C3−C7 THP Ring in
Madeirolide A

Scheme 2. Exploration of Stereochemistry in AgCC

The completion of the southern, C1−C12 fragment ofmadeirolide A is shown in Scheme 4. NaOMe cleavage5c ofthe enol benzoate revealed the desired ketone with no
Scheme 4. Synthesis of the C1−C12 Subunit of Madeirolide Agroup on the aromatic ring of the benzoate was essential for
obtaining reasonable yields. Interestingly, the DHF product14
was formed in greater quantities at lower temperatures,indicating a higher activation barrier for isomerization of the
propargylic benzoate to the allene as compared to direct
cyclization by the alcohol to the activated alkyne. The DHF 37
proved unstable and decomposed partially under SiO 2
puriﬁcation conditions and upon storage in CDCl3.
1746— 240 —

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Org. Lett. 2016, 18, 1744−1747

Organic Letters

LetterOrg. Chem. 2015, 2015, 220−225. (n) Roy, A.; Bhat, B. A.; Lepore, S.
D. Org. Lett. 2015, 17, 900−903.
(3) (a) Gockel, B.; Krause, N. Org. Lett. 2006, 8, 4485−4488.
(b) Zriba, R.; Gandon, V.; Aubert, C.; Fensterbank, L.; Malacria, M.
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M.; Augé, M.; Desmarets, C.; Moussa, J.; Ollivier, C.; Aubert, C.;
Fensterbank, L.; Gandon, V.; Malacria; Chamoreau, L. M.; Amouri, H.
Organometallics 2010, 29, 6636−6638. (d) Kilpin, K. J.; Paul, U. S. D.;
Lee, A.-L.; Crowley, J. D. Chem. Commun. 2011, 47, 328−330.
(e) Dupré, N.; Brazel, C.; Fensterbank, L.; Malacria, M.; Thorimbert,
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(f) Gockel, B.; Krause, N. Eur. J. Org. Chem. 2010, 2010, 311−316.
(g) Mundal, D. A.; Lutz, K. E.; Thomson, R. J. J. Am. Chem. Soc. 2012,
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- Eur. J. 2011, 17, 5796−5799.
(4) (a) Carter, R. G.; Bourland, T. C.; Zhou, X.-T.; Gronemeyer, M.
A. Tetrahedron 2003, 59, 8963−74. (b) Zhou, X.-T.; Carter, R. G.
Angew. Chem., Int. Ed. 2006, 45, 1787−1790. (c) Zhou, X.-T.; Lu, L.;
Furkert, D. P.; Wells, C. E.; Carter, R. G. Angew. Chem., Int. Ed. 2006,
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7948−7951. (b) Mahapatra, S.; Carter, R. G. J. Am. Chem. Soc. 2013,
135, 10792−10803. (c) Veerasamy, N.; Ghosh, A.; Li, J.; Watanabe,K.; Serrill, J. D.; Ishmael, J. E.; McPhail, K. L.; Carter, R. G. J. Am.
Chem. Soc. 2016, 138, 770−773.
(6) Shigemasa, Y.; Yasui, M.; Ohrai, S.-I.; Sasaki, M.; Sashiwa, H.;
Saimoto, H. J. Org. Chem. 1991, 56, 910−912.
(7) (a) Buzas, A.; Istrate, F.; Gagosz, F. Org. Lett. 2006, 8, 1957−
1959. (b) Yeom, H.-S.; Yoon, S.-J.; Shin, S. Tetrahedron Lett. 2007, 48,4817−4820.
(8) Wang, Y.-M.; Kuzniewski, C. N.; Rauniyar, V.; Hoong, C.; Toste,
F. D. J. Am. Chem. Soc. 2011, 133, 12972−12975.
(9) Kotikalapudi, R.; Swamy, K. C. K. Tetrahedron 2013, 69, 8002−
8012.
(10) Ahlers, A.; de Haro, T.; Gabor, B.; Fürstner, A. Angew. Chem.,
Int. Ed. 2016, 55, 1406−1411.
(11) (a) Winder, P. L. Ph.D. Thesis, Florida Atlantic University,
2009. (b) Paterson, I.; Haslett, G. W. Org. Lett. 2013, 15, 1338−1341.
(12) The synthetic routes for each one of the cyclization precursors
provided in the Supporting Information.
(13) (a) Yue, Y.; Turlington, M.; Yu, X.-Q.; Pu, L. J. Org. Chem.
2009, 74, 8681−8689. (b) Bae, H. J.; Jeong, W.; Lee, J. H.; Rhee, Y. H.
Chem. - Eur. J. 2011, 17, 1433−1436.
(14) A mechanistic explanation for the formation of unwanted DHP
byproduct is provided in the Supporting Information.
(15) (a) Ni, Y.; Kassab, R.; Chevliakov, M.; Montgomery, J. J. Am.
Chem. Soc. 2009, 131, 17714−17718. (b) Huang, Z.; Negishi, E. Org.
Lett. 2006, 8, 3675−3678.

stereoerosion at C3 or C6. Stereoselective reduction of the C5
ketone followed by attachment of the glycoside 4011b provided
product 41. Finally, buﬀered TBAF deprotection of the C10
OTIPS ether followed by DMP oxidation and Wittig
oleﬁnation introduced the key trans C10,11 alkene 42.
In summary, the scope of AgCC to access pyran ring systems
has been explored. The impact of the degree of substitution,
nature of substituents, and stereochemistry has been studied.
Extension of this work to the synthesis of the southern, C1−C12
subunit of madeirolide A has been demonstrated. Further work
exploring the AgCC reaction and its application in synthesis
will be reported in due course.

■

ASSOCIATED CONTENT* Supporting InformationS

The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.6b00414.
Complete experimental procedures (PDF)
Copies of 1H and 13C spectra for all new compounds
(PDF)

■

AUTHOR INFORMATION

Corresponding Author*E-mail: rich.carter@oregonstate.edu.
Present Address†

Tohoku Pharmaceutical University, 4-1-1 Komatsushima,
Aoba-ku, Sendai, 981-8558, Japan.
Author Contributions‡

K.W. and J.L. contributed equally.

Notes

The authors declare no competing ﬁnancial interest.

■

ACKNOWLEDGMENTS
Financial support was provided by Oregon State University.
K.W. acknowledges Tohoku Pharmaceutical University for
fellowship support. We thank Professor Claudia Maier and Dr.
Jeﬀ Morré (OSU) for mass spectrometric data.

■

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1747— 241 —

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Org. Lett. 2016, 18, 1744−1747

1521-0103/359/1/102–109$25.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

http://dx.doi.org/10.1124/jpet.115.231589
J Pharmacol Exp Ther 359:102–109, October 2016

Activation of p38 Mitogen-Activated Protein Kinase by
Clotrimazole Induces Multidrug Resistance–Associated Protein
3 Activation through a Novel Transcriptional Element
Takamitsu Sasaki, Keita Inami, Yoshihiro Numata, Kodai Funakoshi, Midori Yoshida,
Takeshi Kumagai, Shuichi Kanno, Satomi Matsui, Takayoshi Toriyabe, Yasushi Yamazoe,
Kouichi Yoshinari, and Kiyoshi Nagata

Received February 23, 2016; accepted August 2, 2016

ABSTRACTMultidrug resistance–associated protein 3 (MRP3) is a basolaterally localized transporter in the liver and contributes to thetransport of various metabolites such as conjugates of endogenous compounds and drugs from hepatocytes. MRP3 expression in the human liver is low under normal physiologic
conditions but is induced by drug treatment. Although several
studies have identified a region necessary for the basal transcription of MRP3, no region that responds to drugs has beenreported. To identify the xenobiotic-responsive elements of
MRP3, we constructed a luciferase reporter plasmid containing
the MRP3 59-flanking region up to 210 kb upstream from thetranscription start site. Among typical nuclear receptor ligands,
clotrimazole dramatically enhanced MRP3 reporter activity in
HepG2 cells, whereas rifampicin had no effect. We thenconducted MRP3 reporter assays with deletion or mutation
constructs to identify a clotrimazole-responsive element. The

Introduction
Multidrug resistance–associated proteins (MRPs) belong to
the ATP-binding cassette family of membrane transporters
and transport endogenous and exogenous compounds acrossbiologic membranes (Borst and Elferink, 2002). Eight MRP
isoforms have been identified as organic anion transporters.
Among them, MRP2 and MRP3 are primary transporters in
the liver, which transport various metabolites (e.g., conjugatesof endogenous compounds and drugs) from hepatocytes. Both
This research was supported by the Japanese Ministry of Education,
Culture, Sports, Science and Technology–supported Program for the Strategic
Research Foundation at Private Universities (No. S1511001L) and Grant-inAid for Scientific Research (C) (No. 25460195).
dx.doi.org/10.1124/jpet.115.231589.

element was located approximately 26.8 kb upstream from the
MRP3 transcription start site. Overexpression of the pregnane X
receptor did not enhance clotrimazole-mediated transcription.
We found that clotrimazole was toxic to HepG2 cells and wetherefore investigated whether mitogen-activated protein kinase(MAPK) activation is involved in the transactivation of MRP3 byclotrimazole. p38 MAPK inhibitor SB203580 [4-(4-fluorophenyl)2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] suppressed MRP3 mRNA expression induced by clotrimazole,
whereas c-Jun N-terminal kinase inhibitor SP600125 (1,9pyrazoloanthrone) and extracellular signal-regulated kinase
inhibitor PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran4-one] did not. Phosphorylated p38 MAPK was detected in
HepG2 cells treated with clotrimazole. These results suggestthat activation of the p38 MAPK pathway induces the transcriptional activation of MRP3.

isoforms show similar substrate specificity, including several
sulfate, glutathione, and glucuronate conjugates, but havedifferent physiologic roles (Bodo et al., 2003; Borst et al., 2007;
Nies and Keppler, 2007; Bachour-El Azzi et al., 2015). MRP2 islocated at the apical membrane of hepatocytes and transports
conjugated metabolites into the bile canaliculus, whereas
MRP3 is expressed on the basolateral membrane of hepatocytes and eliminates its substrates toward blood circulation(König et al., 1999; Borst et al., 2006).
MRP3 expression is induced by treatment with pregnane X
receptor (PXR) activators, including rifampicin and clotrimazole, in human hepatoma cell lines (Teng et al., 2003). Arylhydrocarbon receptor activators omeprazole and b-naphtoflavone
also appear to induce MRP3 expression (Hitzl et al., 2003).
Recent studies in PXR or nuclear factor E2–related factor-2

ABBREVIATIONS: CAR, constitutive androstane receptor; CDCA, chenodeoxycholic acid; DMSO, dimethylsulfoxide; DR, direct repeat; GAPDH,
glyceraldehyde-3-phosohate dehydrogenase; hPXR, human pregnane X receptor; hRXRa, human retinoid X receptor a; JNK, c-Jun N-terminal
kinase; LDH, lactase dehydrogenase; MAPK, mitogen-activated protein kinase; MRP, multidrug resistance–associated protein; PCR, polymerase
chain reaction; PD98059, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; PXR, pregnane X receptor; SB203580, 4-(4-fluorophenyl)-2-(4methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; SP600125, 1,9-pyrazoloanthrone; TNFa, tumor necrosis factor a; VD3, 1a, 25-dihydroxyvitamin D3.
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Department of Environmental and Health Science (T.S., K.I., Y.N., K.F., M.Y., T.K., K.N.) and Clinical Pharmacotherapeutics(S.K.), Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Aoba-ku, Sendai, Miyagi, Japan;
Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan(T.S., K.Y.); and Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku
University, Aoba-ku, Sendai, Miyagi, Japan (S.M., T.T., Y.Y.)

Novel Transactivation of the MRP3 Gene

Materials and Methods
Materials. Restriction and DNA modification enzymes wereobtained from Takara Bio Inc. (Shiga, Japan). Rifampicin, clotrimazole,chenodeoxycholic acid (CDCA), and 1a,25-dihydroxyvitamin D3 (VD3)
were purchased from Sigma-Aldrich (St. Louis, MO). Anisomycin,
SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1Himidazole], PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4one], and SP600125 (1,9-pyrazoloanthrone) were purchased from
Wako Pure Chemicals (Osaka, Japan). Dimethylsulfoxide (DMSO)
was obtained from Nacalai Tesque (Kyoto, Japan).
Reporter Gene Constructs and Expression Vector. The
luciferase reporter plasmid pGL3-Basic was purchased from Promega(Madison, WI). An approximately 10-kb fragment (nucleotides from 116
to 210,136) of the MRP3 59-flanking region was isolated by polymerase

chain reaction (PCR) amplification from human genomic DNA using theforward primer 59-GCGACGCGTGCCCAGGGTCATGCCTATCTGG-39
and the reverse primer 59-GCGGTCGACGCGGCTGCAAGGAAGGCGAGC-39. The underlined sequences represent the MluI and SalI
restriction sites, respectively. The fragment was subcloned into a pCRXL-TOPO vector (Invitrogen, Carlsbad, CA). The plasmid was thendigested with the MluI and SalI restriction enzymes and then ligatedinto the MluI and XhoI sites of the pGL3-Basic vector (pGL3-MRP310k). The resultant construct was used as a template to generatevarious reporter plasmids lacking portions of the 59-flanking region of
MRP3. To generate an internal deletion construct (pGL3-MRP3Acc65), a region (nucleotides from 25913 to 2837) was removed fromthe pGL3-MRP3-10k vector by EcoRI digestion. In addition, theconstruct was digested with Acc65 and the nucleotides from 210,136
to 28021 were deleted. Successive deletions of the reporter plasmid
to 27.9, 27.6, 27.4, 27.2, 27.1, 26.8, and 26.4 kb were created frompGL3-MRP3-Acc65 as a template. The 26.8- and 26.4-kb deleted fragments were isolated by PCR amplification using the forward primers forthe 26.8-kb fragment (59-GCGACGCGTGCTGACTGGCATTACAGGCACTGTCC-39) and the 26.4-kb fragment (59-GCGACGCGTCACCTGTCTGGTGCTACTGCAGAC-39) and the reverse primer(59-GCGGTCGACGCGGCTGCAAGGAAGGCGAGC-39). The underlined sequences represent the MluI restriction sites in the forward primer
and the SalI restriction site in the reverse primer. The fragments were
subcloned into the pGEM-T vector (Promega). The plasmids were digested
with MluI and SalI and then ligated into the MluI and XhoI sites ofthe pGL3-Basic vector (pGL3-MRP3-6.8k and pGL3-MRP3-6.4k). The
27.9-kb, 27.6-kb, 27.4-kb, 27.2-kb, and 27.1-kb deleted fragments wereisolated by PCR amplification using the forward primers for the 27.9-kb
fragment (59-GCGACGCGTGGTTCCCGACACCAGAAGCTGTTGG-39),
the 27.6-kb fragment (59-GCGACGCGTATTTTGGAAACTGTAATGTAAC-39), the 27.4-kb fragment (59-GCGACGCGTTGATGAGAGAGAGATCCTGGAGATGG-39), the 2 7.2-kb fragment(59-GCGACGCGTTCTGAGAGTGAGAGAACCTGAAC-39), and the27.1-kb fragment (59-GCGACGCGTCTCTAGCTAGGCAGCTCAGTGAATGG-39) and the reverse primer (59-AACATGTGTGAATTCTACAAAAATATAAAAG-39). The underlined sequences represent the
MluI restriction sites in the forward primes and EcoRI restriction site
in the reverse primer. These fragments were subcloned into thepGEM-T vector. The plasmids were then digested with MluI andEcoRI and then ligated into the MluI and EcoRI sites of the pGL3MRP3-10k vector (resulting in pGL3-MRP3-7.9k, pGL3-MRP37.6k, pGL3-MRP3-7.4k, pGL3-MRP3-7.2k, and pGL3-MRP3-7.1k).
Mutated reporter plasmids were constructed by the QuikChange II
site-directed mutagenesis kit (Stratagene, La Jolla, CA) using pGL3MRP3-Acc65 as a template. Preparation of the human pregnane X
receptor (hPXR) expression plasmid was previously described(Takada et al., 2004). All plasmids were sequenced to confirm theirintegrity.
Cell Culture and Reporter Assays. The human hepatoma cellline HepG2 was obtained from American Type Culture Collection(Manassas, VA) and cultured in Dulbecco’s modified Eagle’s medium(Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal
calf serum (Boehringer, Mannheim, Germany) and minimum Eagle’s
medium nonessential amino acids (Invitrogen). Cells were seeded in
12-well tissue culture plates (BD Biosciences, Franklin Lakes, NJ) at
1.2  105 cells per well 1 day before transfection. Each reporter
plasmid and a pSV-b-galactosidase control vector (Promega) were
cotransfected using the CellPhect Transfection Kit (GE Healthcare,
Little Chalfont, UK) according to the manufacturer’s instructions. In
overexpression analyses of hPXR, either the hPXR expression vector
or the pCMV4 control plasmid was simultaneously transfected into
HepG2 cells with a reporter plasmid. After transfection, the cells wereincubated in culture medium containing various compounds (rifampicin, clotrimazole, CDCA, or VD3) dissolved in DMSO (final concentration 0.1%) for 48 hours. Control cells were treated with 0.1% DMSO.
Subsequently, cells were washed with phosphate-buffered saline andsuspended in 0.1 ml passive lysis buffer (Promega). Luciferase— 243 —

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(Nrf2) knockout mouse models have examined the roles of thetranscription factors in the regulation of Mrp3 and suggestthat these factors are involved in the induction of Mrp3
expression (Teng et al., 2003; Maher et al., 2007). Constitutive androstane receptor (CAR) activators 1,4-bis[2-(3,5dichloropyridyloxy)]benzene and phenobarbital have also
been reported to induce Mrp3 expression in mice (Cherrington
et al., 2003; Maher et al., 2005). Taken together, MRP3/Mrp3
expression is thought to be induced via nuclear receptor–
mediated pathways in human and mouse livers.
MRP3 expression in the human liver is low under normal
physiologic conditions, but the expression is markedly upregulated in patients with cholestasis and Dubin–Johnson
syndrome as a result of deficient MRP2 expression (König
et al., 1999; Chai et al., 2012). In cholestasis, the enhancementof MRP3 expression is mediated via the activation of c-Jun
N-terminal kinase (JNK), a member of mitogen-activatedprotein kinase (MAPK) family. Mrp3 expression was also
found to be upregulated in a rat model of cholestasis andDubin–Johnson syndrome (Soroka et al., 2001; Kuroda et al.,2004). These results suggest that MRP3/Mrp3 expression is
induced by signal pathways, including MAPK, independentlyof nuclear receptor–mediated pathways and that MRP3/Mrp3
may work as a compensatory transporter for MRP2.
Few studies have examined the molecular mechanism oftranscriptional regulation of human MRP3. In reporter assays
with a MRP3 59-flanking region up to 24235 bp from thetranscriptional start site, a GC-rich region located between
2127 and 223 bp was reported to be important for its basal
expression (Takada et al., 2000). MRP3 gene expression isunder the control of a TATA-less promoter, and some putative
binding sites for transcription factors such as Sp1 are found in
this GC-rich region. Another study demonstrated that a MRP3
59-flanking region up to 21287 bp from the transcriptional
start site includes regions involved in basal expression(Stöckel et al., 2000). However, xenobiotic-responsive elements of the MRP3 59-flanking region have not been identified, indicating that the elements may be further upstream.
In this study, we constructed a luciferase reporter plasmid
containing the MRP3 59-flanking region up to 210 kb toinvestigate nuclear receptor ligand-induced MRP3 expression
using reporter assays and we identified a novel xenobioticresponsive element involved in clotrimazole-induced MRP3
expression. We demonstrate that MRP3 induction by clotrimazole occurs through the p38 MAPK pathway. Our results
suggest an association between drug-induced hepatotoxicity
and transcriptional activation of human MRP3.

103

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Sasaki et al.
(Millipore, Billerica, MA), which were then incubated for 16 hours
with a phospho-p38 MAPK (Thr180/Tyr182) antibody (Cell Signaling Technology, Beverly, MA) diluted in Tris-buffered saline (1:1000).
The membrane was also incubated with p38 MAPK antibody (dilution,
1:500; Cell Signaling Technology) or b-actin antibody (dilution,
1:1000; Cell Signaling Technology) for 1 hour. The membranes were
subsequently incubated for 1 hour with horseradish peroxidase–
conjugated secondary antibody diluted in Tris-buffered saline(1:5000; Santa Cruz Biotechnology, Santa Cruz, CA). After development with the SuperSignal West Pico chemiluminescent substrate(Thermo Fisher Scientific, Waltham, MA), the membrane wasscanned using a Lumino Imaging Analyzer FAS-1000 (Toyobo,
Osaka, Japan).
Statistical Analysis. Data are presented as means 6 S.E.M. Data
were analyzed with the Welch t test or one-way analysis of variancefollowed by the Dunnett or Bonferroni post hoc test. All statisticalanalyses were performed using Prism software (version 6.0; GraphPad
Software Inc., San Diego, CA). A P value less than 0.05 was considered
statistically significant.

Results
Effect of Typical Nuclear Receptor Ligands on MRP3
Reporter Activity in HepG2 Cells. Previous studies
demonstrated that the MRP3 59-flanking region up to 21287 bp
or 24235 bp including the GC-rich region regulates MRP3
basal expression (Stöckel et al., 2000; Takada et al., 2000).
However, to our knowledge, a role of the far upstream region
in the drug-induced expression of MRP3 has not been investigated. To identify xenobiotic-responsive elements of
MRP3, we constructed a luciferase reporter plasmid containing the MRP3 59-flanking region up to 210 kb (pGL-MRP310k) and transfected the plasmid into HepG2 cells. We
investigated whether treatment with typical ligands of PXR(rifampicin and clotrimazole), the vitamin D receptor (VD3),
and the farnesoid X receptor (CDCA) increased MRP3 reporter
activity. Clotrimazole treatment resulted in a 132-fold increase in MRP3 reporter activity (Fig. 1). In contrast, treatment with rifampicin and other compounds did not increase
the luciferase activities in HepG2 cells.
We also used the CYP3A4 reporter plasmid (pCYP3A4-3627.7k), including everted repeat separated by six nucleotides

Fig. 1. Effects of typical nuclear receptor ligands on MRP3 reporter
activity in HepG2 cells. HepG2 cells seeded in 12-well tissue culture plates
at 1.2  105 cells/well 1 day before transfection were transfected with eachreporter plasmid and pSV-b-galactosidase as an internal control. The cells
were treated with vehicle (0.1% DMSO), rifampicin (10 mM), clotrimazole(5 mM), VD3 (10 nM), or CDCA (100 mM) for 48 hours. Relative luciferase
activities are shown as means 6 S.E.M. (n = 4). *P , 0.05; **P , 0.01,
significantly different from the activities in corresponding vehicle-treatedcells based on the Welch t test.

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activities were determined using a luciferase assay system (Promega).
To normalize transfection efficiency, b-galactosidase assays were
performed as previously described (Herbomel et al., 1984).
Electrophoretic Mobility Shift Assays. hPXR and humanretinoid X receptor-a (hRXRa) were synthesized in vitro with thepTNT-hPXR and pTNT-hRXRa plasmids, respectively, using the TNT
Quick Coupled Transcription/Translation System (Promega) following
the manufacturer’s protocol. Preparation of the pTNT-hPXR and pTNThRXRa plasmids was previously described (Toriyabe et al., 2009).
Double-strand oligonucleotides (Fig. 4) were labeled with [g-32P]ATPusing T4 polynucleotide kinase (New England BioLabs, Ipswich, MA)
and purified using NAP5 columns (GE Healthcare). The binding
reactions were carried out as previously described (Toriyabe et al., 2009).
Cytotoxicity Assays. HepG2 cells were seeded in 96-well tissue
culture plates (BD Biosciences) at 1.5  104 cells per well. The culture
medium was replaced with fresh medium containing clotrimazole orrifampicin 24 hours after cell seeding. After 72 hours of exposure, therelease of lactase dehydrogenase (LDH) was determined using the
Cytotoxicity Detection Kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s instructions.
HepaRG Cells and Cell Culture. Differentiated HepaRG cells
were obtained from Life Technologies (Carlsbad, CA). The cells were
thawed and cultured in William’s medium E containing GlutaMAXIand HepaRG Thaw, Plate, and General Purpose Medium Supplement (all from Life Technologies) according to the manufacturer’s
protocol. The cells were seeded in type I collagen–coated 48-welltissue culture plates (Corning, Corning, NY) at 2.0  105 cells perwell and cultured for 72 hours before drug treatment. The cells werethen cultured in William’s medium E containing GlutaMAXI,
HepaRG Induction Medium Supplement, and clotrimazole or rifampicin for 48 hours.
Small Interfering RNA Transfection. Trypsinized HepG2 cells
were reverse transfected with 40 nM ON-TARGETplus SMART Pool
MAPK14 or 40 nM ON-TARGETplus Non-Targeting Pool (GEHealthcare) using Lipofectamine RNAiMAX (Invitrogen). After
24 hours, the medium was changed to fresh medium containing
clotrimazole and the cells were cultured for an additional 24 hours.
Next, the cells were forward transfected with the small interfering
RNAs for 6 hours and subsequently treated with clotrimazole for an
additional 48 hours.
Quantitative Analysis of mRNA Levels. HepG2 cells wereseeded in 24-well tissue culture plates (BD Biosciences) at 1.0  105cells per well. The culture medium was replaced with fresh medium
containing clotrimazole or rifampicin 24 hours after cell seeding. In
MAPK inhibitor experiments, the cells were pretreated with
SB203580, PD98059, or SP600125 1 hour before exposure to thevarious compounds. After 72 hours of exposure, total RNA wasextracted using TRI Reagent (Molecular Research Center, Cincinnati,
OH). First-strand cDNA was synthesized from 2 mg total RNA in a
25-ml reaction mixture using Moloney murine virus reverse transcriptase (Promega), oligo (dT) 20 primers, and ribonuclease inhibitor(Takara Bio Inc.). cDNA was used to carry out real-time PCR using
SYBR premix ExTaq (Takara Bio Inc.) to measure the mRNA levels of
MRP3, activating transcription factor 3 (ATF3), and glyceraldehyde-3phosphate dehydrogenase (GAPDH). The amplification reaction was
performed using specific primers for MRP3 (forward: 59-GTCCGCAGAATGGACTTGAT-39; reverse: 59-TCACCACTTGGGGATCATTT39), ATF3 (forward: 59-AGCCTGGAGCAAAATGATGCTT-39; reverse:
59-AGGTTAGCAAAATCCTCAAACAC-39), and GAPDH (forward: 59GAAGGTGAAGGTCGGAGTCAAC-39; reverse: 59-CAGAGTTAAAAGCAGCCCTGGT-39). The relative mRNA levels in each sample werenormalized according to those of GAPDH.
Immunoblot Analysis. Immunoblot analysis was performed toevaluate the expression of phospho-p38 MAPK proteins. Whole celllysates were size fractionated by gel electrophoresis on a 10%
polyacrylamide/0.1% sodium dodecyl sulfate gel after denaturationby heating in loading buffer containing 2-mercaptoethanol. The
proteins were electrotransferred onto Immobilon-P membranes

Novel Transactivation of the MRP3 Gene

105

Fig. 2. Effects of deletions of the MRP3 59-flanking
region on reporter activity. HepG2 cells seeded in
12-well tissue culture plates at 1.2  105 cells/well
1 day before transfection were transfected with various
reporter plasmids lacking portions of the MRP3 59flanking region of pGL3-MRP3-10k and with pSVb-galactosidase as an internal control. The cells weretreated with vehicle (0.1% DMSO) or clotrimazole(5 mM) for 48 hours. The reporter activity of cells treatedwith vehicle was set to 1.0. The data represent means 6
S.E.M. (n = 4). *P , 0.05; **P , 0.01, significantly
different from the reporter activity of the cells transfected with pGL3-MRP3-10k based on one-way analysis
of variance with the Dunnett post hoc test.

Mutation Analysis of the Clotrimazole-Responsive
Element in the MRP3 59-Flanking Region. We next
performed mutation analysis to identify a clotrimazoleresponsive element located between 27.1 and 26.8 kb. Several
nuclear receptor binding half-sites, AG(G/T)TCA-like sequence,are present between 26831 and 26810 bp. To assess the roles of
the half-sites in the transcriptional activation of MRP3 in
response to clotrimazole, a series of reporter plasmids constructed by site-directed mutagenesis were used. The introductionof mutations into and surrounding the half-sites significantly
reduced the reporter activities (Fig. 3). These results suggest thatthe clotrimazole-responsive element including half-sites is present in this region between 26831 and 26810 bp.
Role of PXR in the Transcriptional Activation of
MRP3. We investigated whether PXR was involved in the
clotrimazole-mediated activation of MRP3. CYP3A4 reporter
activity was significantly increased in hPXR-overexpressing
HepG2 cells (173-fold) and clotrimazole treatment further
increased the reporter activity (328-fold). However, overexpression of PXR did not increase MRP3 reporter activity (Fig. 4A).
On the contrary, PXR suppressed the reporter activity induced
by clotrimazole. The hPXR/hRXRa heterodimer binding site
located within prER6 of CYP3A4 was recognized by the
hPXR/hRXRa heterodimer (Fig. 4B). The DNA-protein complex
was competed out by an 100-fold excess of unlabeled prER6.

Fig. 3. Mutation analysis of the clotrimazole-responsive
element in the MRP3 59-flanking region. HepG2 cells
seeded in 12-well tissue culture plates at 1.2  105cells/well 1 day before transfection were transfectedwith various mutation constructs of pGL3-MRP3-Acc65
and with pSV-b-galactosidase as an internal control.
The cells were treated with vehicle (0.1% DMSO) or
clotrimazole (3 mM) for 48 hours. The reporter activity ofcells treated with vehicle was set to 1.0. The data
represent means 6 S.E.M. (n = 4). The mutations areindicated as M and all mutations changed the original
nucleotide (G or C) to A. **P , 0.01; ***P , 0.001,
significantly different from the reporter activity of thecells transfected with pGL3-MRP3-Acc65 based on oneway analysis of variance with the Dunnett post hoc test.

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located in the CYP3A4 proximal promoter (prER6; 111 to2362 bp) and essential distal nuclear receptor-binding element for CYP3A4 induction and distal nuclear receptorbinding element 1 (27.2 to 27.8 kb) as the positive control.
CYP3A4 reporter activities were significantly increased byclotrimazole and rifampicin treatment. The reporter activity
was also enhanced by VD3.
Effect of Deletions of the MRP3 59-Flanking Region
on Reporter Activity. To identify a clotrimazole-responsive
region in MRP3, reporter assays were carried out with various
plasmids lacking portions of the MRP3 59-flanking region. The
reporter plasmid, in which the regions from 210 to 28.0 kb
and 26.0 to 20.8 kb were removed (pGL3-MRP3-Acc65), wasmore responsive to clotrimazole treatment compared withpGL-MRP3-10k, although the difference was not significant(Fig. 2).
Deletion constructs of pGL3-MRP3-Acc65 were transiently
transfected into HepG2 cells. Reporter activities of pGL3MRP3-Acc65 with successive deletions from the 59 end to
27.9, 27.6, 27.4, 27.2, and 27.1 kb were not affected byclotrimazole. However, further deletion from 27.1 to 26.8 kb
dramatically reduced the clotrimazole-induced MRP3 reporter activity. These results suggest that an unidentified
region responding to clotrimazole is present between 27.1
and 26.8 kb on MRP3.

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Sasaki et al.

However, binding of the hPXR/hRXRa heterodimer to theclotrimazole-responsive element of MRP3 was not observed.
These results suggest that PXR is not likely to be involved in
MRP3 transactivation via the clotrimazole-responsive element.
Effect of Clotrimazole on Cytotoxicity and MRP3mRNA Levels in HepG2 Cells. Our results suggest thatthe activation of PXR by clotrimazole is less effective on thetranscriptional activation of MRP3 in HepG2 cells. We
therefore focused on the hepatotoxicity of azole antifungal
agents (García Rodríguez et al., 1999; Kuroda et al., 2004;
Girois et al., 2005), because it has been reported that Mrp3

Discussion
In this study, we investigated whether ligands of PXR andother nuclear receptors induced MRP3 expression (Fig. 1).
Among the PXR ligands tested, clotrimazole dramaticallyenhanced MRP3 reporter activity in HepG2 cells, whereas
rifampicin had no effect. However, these compounds significantly increased CYP3A4 reporter activity. Our results— 246 —

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Fig. 4. Role of PXR in the transcriptional activation of MRP3. (A) HepG2
cells seeded in 12-well tissue culture plates at 1.2  105 cells/well 1 day
before transfection were transfected with each reporter plasmid and with
pSV-b-galactosidase as an internal control. The hPXR expression plasmidor pCMV4 was simultaneously transfected into the HepG2 cells. The cells
were treated with vehicle (0.1% DMSO) or clotrimazole (3 mM) for
48 hours. The reporter activity of HepG2 cells treated with vehicle was set
to 1.0. The data represent means 6 S.E.M. (n = 4). *P , 0.05; **P , 0.01;***P , 0.001, significantly different from the activities of the vehicletreated/pCMV4-transfected cells based on the Welch t test. (B) Sequencesof the oligonucleotides used for the gel electrophoresis mobility shift
assays are shown. The assays were carried out with radiolabeled prER6
and the clotrimazole-responsive element. Incubations were carried out
with hPXR and/or hRXRa synthesized in vitro as indicated. Competition
assays were performed with radiolabeled prER6 as probes. Ten- or 100fold molar excesses of unlabeled competitors were added to the reaction
solution. CLO, clotrimazole.

expression is significantly increased in cholestasis-induced liver
injury (Wagner et al., 2003). To confirm the cytotoxicity of
clotrimazole, LDH activity was measured in HepG2 cells treatedwith clotrimazole or rifampicin. Clotrimazole resulted in asignificantly high LDH activity level compared with that incontrol cells (Fig. 5A). In contrast, the LDH release from HepG2
cells treated with rifampicin was similar to that from control
cells. To investigate the effect of clotrimazole and rifampicin on
intrinsic MRP3 mRNA expression, HepG2 cells were treatedwith these drugs. MRP3 mRNA levels were markedly induced byclotrimazole (8.7-fold) (Fig. 5B). In contrast, MRP3 mRNA levels
were not affected by rifampicin treatment. We also tested
whether MRP3 mRNA expression was induced by treatment
with troglitazone, which is known to be a hepatotoxicity-inducing
drug. MRP3 mRNA levels were significantly increased by thetreatment (5.9-fold) (Fig. 5C). Furthermore, we confirmed that
the MRP3 mRNA levels were increased in HepaRG cells, which
retain many characteristics of primary human hepatocytes,
treated with clotrimazole but not with rifampicin (Fig. 5D).
Effect of MAPK Inhibitors on Clotrimazole-Induced
MRP3 mRNA Expression in HepG2 Cells. We next investigated whether MRP3 transcriptional activation by clotrimazole was associated with the activation of MAPKs, which
are essential for the cellular response to injuries caused bydrug treatment and inflammatory diseases, including cholestasis (Hanawa et al., 2008; Boaglio et al., 2012). To assess theeffects of MAPK inhibitors on MRP3 mRNA expression
induced by clotrimazole, HepG2 cells were preincubated with
a p38 MAPK inhibitor (SB203580), JNK inhibitor (SP600125),or extracellular signal-regulated kinase inhibitor (PD98059)
for 1 hour, followed by with clotrimazole. SB203580 pretreatment suppressed MRP3 mRNA expression induced byclotrimazole, whereas SP600125 and PD98059 did not (Fig.
6A). We also investigated whether ATF3, which is known to be
induced by the stress-activated p38 signaling pathway (Luet al., 2007), was enhanced by clotrimazole treatment. Clotrimazole induced ATF3 mRNA expression as well as MRP3mRNA expression, and the increased expression was inhibited
only by pretreatment with SB203580. Furthermore, treatment with anisomycin, a p38 MAPK activator (Saito et al.,2013), resulted in a 12.8-fold increase in MRP3 mRNA levels,and the induced expression was also suppressed by pretreatment with SB203580 (Fig. 6B). To confirm the activation of p38 MAPK by clotrimazole, Western blotting was
carried out to detect phosphorylated p38 MAPK. Increased
phosphorylated p38 MAPK protein levels were detected in
HepG2 cells treated with clotrimazole (Fig. 6C). In contrast,
phosphorylated p38 MAPK was not detected in HepG2 cells
treated with rifampicin, which did not induce MRP3 mRNA
expression. We also demonstrated that clotrimazole-induced
MRP3 mRNA expression was significantly suppressed in
HepG2 cells transfected with p38 MAPK small interfering
RNA (Fig. 6D).

Novel Transactivation of the MRP3 Gene

107

suggest that transcriptional activation of MRP3 and CYP3A4
by clotrimazole might be regulated via different pathways.
To identify the clotrimazole-responsive element, we conductedreporter assays with various plasmids lacking portions of the
MRP3 59-flanking region. The responsive element was locatedbetween the 27.1 and 26.8 kb region, far upstream from thetranscription start site of MRP3 (Fig. 2). Furthermore, we confirmed that the clotrimazole-responsive element including halfsite–like sequences were present between 26831 and 26810 bp(Fig. 3). However, our results showed that the activation of PXR
by clotrimazole was less effective on the transcriptional activationof MRP3 (Fig. 4). Several studies have demonstrated that Mrp3mRNA expression was increased in wild-type and PXR knockout
mice upon cholestasis induced by a-naphthyl isothiocyanate orcholic acid (Teng and Piquette-Miller, 2007; Cui et al., 2009),
suggesting that PXR-independent pathways are involved in the
induction of Mrp3 expression.
Azole antifungal agents have been reported to induce
hepatotoxicity in patients (Somchit et al., 2009; Kao et al.,2014). We confirmed that clotrimazole caused significant
cytotoxicity in HepG2 cells (Fig. 5A). Furthermore, we demonstrated that intrinsic MRP3 mRNA levels increased in
HepG2 cells treated with clotrimazole or troglitazone, two
drugs that are known to induce hepatotoxicity (8.7- and 5.9fold, respectively) (Fig. 5, B and C). These results suggest thatthe enhancement of MRP3 mRNA expression by clotrimazole
in HepG2 cells is associated with drug-induced hepatotoxicity.
MAPKs are activated under hepatotoxicity caused by drug
treatment and inflammatory diseases, including cholestasis(Hanawa et al., 2008; Boaglio et al., 2012). We therefore
investigated whether MAPK activation was involved in thetransactivation of MRP3 by clotrimazole. In our study, the p38
MAPK inhibitor and p38 MAPK knockdown suppressed the
MRP3 mRNA expression induced by clotrimazole, whereas

the JNK or extracellular signal-regulated kinase inhibitor
showed no effect on MRP3 mRNA expression levels (Fig. 6, A
and D). We also found that anisomicin, a p38 MAPK activator,
significantly increased MRP3 mRNA levels (Fig. 6B). Furthermore, phosphorylated p38 MAPK was detected in HepG2
cells treated with clotrimazole but not with rifampicin (Fig.
6C). These results suggest that the p38 MAPK pathway is
involved in clotrimazole-induced MRP3 expression.
MRP3 expression is reported to be markedly upregulated ina rat model of bile duct ligation and in human cholestatic liverdisease (Soroka et al., 2001; Schaap et al., 2009). LDH releasefrom the liver is observed in cholestatic conditions. Furthermore, the expression levels of tumor necrosis factor a (TNFa)
and fibroblast growth factor 19 are significantly elevated(Bohan et al., 2003; Schaap et al., 2009). TNFa induces theactivation of stress-activated protein kinase, JNK, and p38
MAPK (Wajant et al., 2003). Chai et al. (2012) reported that
MRP3 mRNA levels in liver are significantly correlated withplasma TNFa levels in patients with obstructive cholestasis
and that the induction involves activated JNK. These results
suggest that MAPKs are involved in inducing MRP3 expression in liver injury.
A novel link between nuclear receptors, such as PXR and
CAR, and the p38 MAPK pathway was recently demonstrated(Kodama and Negishi, 2011; Saito et al., 2013). In addition,
another study reported that PXRs have anti-inflammation
functions via suppression of signal transduction pathways(Sun et al., 2015). Although we did not observe the binding of
PXR to the clotrimazole-responsive element, the increase inclotrimazole-induced MRP3 reporter activity was suppressed
by PXR overexpression (Fig. 4). These findings suggest thatPXR might suppress clotrimazole-induced cytotoxicity, resulting in the suppression of MRP3 reporter activity induced byclotrimazole. In addition, it has been reported that activated— 247 —

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Fig. 5. Effects of clotrimazole on cytotoxicity and MRP3 mRNA expression in HepG2 cells and HepaRG cells. HepG2 cells were seeded in 24-well tissue
culture plates at 1.0  105 cells/well 1 day before drug treatment. The cells were treated with vehicle (0.1% DMSO), rifampicin (20 mM), clotrimazole(20 mM), or troglitazone (50 mM) for 72 hours. (A) The medium in each well was collected and LDH activities were measured. The media from Triton
X-100–treated cells were used as a positive control for cytotoxicity. (B and C) Total RNA was extracted and the MRP3 mRNA levels were determined. (D)HepaRG cells were seeded in type I collagen–coated 48-well tissue culture plates at 2.0  105 cells/well 3 days before drug treatment. The cells were treatedwith vehicle (0.1% DMSO), rifampicin (20 mM), or clotrimazole (20 mM). Total RNA was extracted and the MRP3 mRNA levels were determined. The mRNAlevels were normalized to those of GAPDH. The relative LDH activity and mRNA levels in HepG2 cells treated with vehicle were set to 1.0. The data
represent means 6 S.E.M. (n = 3). *P , 0.05; **P , 0.01; ***P , 0.001, significantly different from the activity and mRNA levels of the vehicle-treated cells
based on the Welch t test or one-way analysis of variance with the Dunnett post hoc test. CLO, clotrimazole; RIF, rifampicin; TRO, troglitazone.

108

Sasaki et al.

Nrf2, known as a cellular sensor for oxidative stress, binds to
the regulatory region of Mrp3 and activates gene expression(Maher et al., 2007). Further studies are required to reveal
whether Nrf2 is activated by clotrimazole treatment.
In conclusion, we identified a novel clotrimazole-response
element, located at 26.8 kb upstream from the transcription
start site of MRP3, and we found that there are differencesin the transactivation mechanism by clotrimazole between
MRP3 and CYP3A4. Furthermore, our results suggest thatthe transcriptional activation of MRP3 via the clotrimazoleresponsive element involves the p38 MAPK pathway.
Authorship Contributions

Participated in research design: Sasaki, Toriyabe, Yamazoe,
Yoshinari, Nagata.
Conducted experiments: Sasaki, Inami, Numata, Funakoshi,
Yoshida, Kanno, Matsui, Toriyabe.
Contributed new reagents or analytic tools: Sasaki, Toriyabe,
Nagata.
Performed data analysis: Sasaki, Kumagai, Toriyabe.
Wrote or contributed to the writing of the manuscript: Sasaki,
Inami, Yoshinari, Nagata.
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Fig. 6. Effects of MAPK inhibitors on clotrimazoleinduced MRP3 mRNA expression in HepG2 cells.
HepG2 cells were seeded in 24-well tissue culture
plates at 1.0  105 cells/well 1 day before drug
treatment. The cells were pretreated with orwithout MAPK inhibitors (5 mM) for 1 hour. (A)The cells were treated with vehicle (0.1% DMSO) or
clotrimazole (20 mM) for 72 hours and the MRP3
and ATF3 mRNA levels were determined. (B) The
cells were treated with anisomycin (1 mM) for
12 hours and the MRP3 mRNA levels were determined. (C) Whole cell lysates were subjected toimmunoblot analyses with an antibody for phospho-p38 MAPK, p38 MAPK, or b-actin. (D) HepG2
cells were transfected with control or p38 MAPK
small interfering RNA. The cells were treated withvehicle (0.1% DMSO) or clotrimazole (20 mM) for
72 hours and the MRP3 mRNA levels were determined. The mRNA levels were normalized tothose of GAPDH. The relative mRNA levels in
HepG2 cells treated with vehicle were set to 1.0.
The data represent means 6 S.E.M. (n = 3). *P ,0.05; **P , 0.01; ***P , 0.001, significantly
different from the expression levels of the corresponding clotrimazole-treated cells based on oneway analysis of variance with the Dunnett or
Bonferroni post hoc test. ANI, anisomycin; CLO,
clotrimazole; PD, PD98059; RIF, rifampicin; SB,
SB203580; siRNA, small interfering RNA; SP,
SP600125.

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Address correspondence to: Kiyoshi Nagata, Department of Environmentaland Health Science, Faculty of Pharmaceutical Sciences, Tohoku Medical andPharmaceutical University, 4-4-1, Komatsushima, Aoba-ku, Sendai 981-8558,
Japan. E-mail: nagataki@tohoku-mpu.ac.jp— 249 —

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Drug Metabolism and Pharmacokinetics 31 (2016) 139e145

Contents lists available at ScienceDirect

Drug Metabolism and Pharmacokinetics
journal homepage: http://www.journals.elsevier.com/drug-metabolism-andpharmacokinetics

Regular article

Indirubin, a component of Ban-Lan-Gen, activates CYP3A4 genetranscription through the human pregnane X receptor
Takeshi Kumagai a, *, Yusuke Aratsu a, b, Ryosuke Sugawara a, Takamitsu Sasaki a, c,
Shinichi Miyairi d, Kiyoshi Nagata aaDepartment of Environmental Health Science, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, Japan
Drug Metabolism and Pharmacokinetics Research Laboratories, Central Pharmaceutical Research Institute, Japan Tobacco Inc., 1-1 Murasaki-cho,
Takatsuki, Osaka 569-1125, Japanc
Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, JapandLaboratory of Organic Chemistry, School of Pharmacy, Nihon University, 7-7-1 Narashino, Funabashi, Chiba 274-8555, Japanba r t i c l e i n f o

a b s t r a c t

Article history:
Received 22 October 2015
Received in revised form
14 January 2016
Accepted 14 January 2016
Available online 22 January 2016

Ban-Lan-Gen is the common name for the dried roots of indigo plants, including Polygonum tinctorium,
Isatis indigotica, Isatis tinctoria, and Strobilanthes cusia. Ban-Lan-Gen is frequently used as an antiinﬂammatory and an anti-viral for the treatment of hepatitis, inﬂuenza, and various types of inﬂammation. One of the cytochrome P450 (CYP) enzymes, CYP3A4, is responsible for the metabolism of a wide
variety of xenobiotics, including an estimated 60% of all clinically used drugs. In this study, we investigated the effect of Ban-Lan-Gen on the transcriptional activation of the CYP3A4 gene. Ban-Lan-Gen
extract increased CYP3A4 gene reporter activity in a dose-dependent manner. Indirubin, one of thebiologically active ingredients in the Ban-Lan-Gen, also dose-dependently increased CYP3A4 gene reporter activity. Expression of short hairpin RNA for the human pregnane X receptor (hPXR-shRNA)
inhibited CYP3A4 gene reporter activity, and overexpression of human PXR increased indirubin- andrifampicin-induced CYP3A4 gene reporter activity. Furthermore, indirubin induced CYP3A4 mRNA
expression in HepG2 cells. Taken together, these results indicate that indirubin, a component of Ban-LanGen, activated CYP3A4 gene transcription through the activation of the human PXR.

Keywords:
AhR activator
Indirubin
CYP3A4 induction
PXR
Herbedrug interactions

Copyright © 2016, Published by Elsevier Ltd on behalf of The Japanese Society for the Study of Xenobiotics.

1. Introduction
Chinese herbal medicines are an integral part of health care in
Asia, and many herbal medicines are extensively used as alternative
and/or complementary therapies. However, the clinical efﬁcacy,mechanisms of action, and safety proﬁles of most herbal medicines
are not well characterized. Moreover, herbal medicines are often
Abbreviations: AdCont, b-galactosidase-expressing adenovirus; AdPXR, human
PXR-expressing adenovirus; AdhPXR-shRNA, AdhPXR-short hairpin RNA; AhR, arylhydrocarbon receptor; Ct, threshold PCR cycle; CAR, constitutive androstane receptor; CYP, cytochrome P450; DMEM, Dulbecco's modiﬁed Eagle's medium; dNR1,
distal nuclear receptor-binding protein 1; DMSO, dimethyl sulfoxide; D-PBS, Dulbecco's phosphate buffered saline; DR3, direct repeats spaced by 3 bases; eNR3A4,
essential distal nuclear receptor-binding element; ER6, everted repeats separated
by 6 bases; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GBE, Ginkgo
biloba extract; GRa, glucocorticoid receptor-a; MOI, multiplicity of infection; PAHs,
polycyclic aromatic hydrocarbons; prER6, ER6 in the CYP3A4 proximal promoter;
PXR, pregnane X receptor; RXRa, retinoid X receptor a; SJW, St. John's wort; TCID50,
50% titer culture infectious dose; VDR, vitamin D receptor.
* Corresponding author.
E-mail address: ta-kuma@tohoku-pharm.ac.jp (T. Kumagai).

co-administered with therapeutic drugs, creating the potential forherbedrug interactions [1,2]. While the underlying mechanism of
most herbedrug interactions is not known, induction and inhibition of drug-metabolizing enzymes or drug transporters have been
implicated as potential mechanisms.
Ban-Lan-Gen is the common name for the dried roots of indigo
plants, including Polygonum tinctorium, Isatis indigotica, Isatis tinctoria, and Strobilanthes cusia. Indigo plants have been an importantsource of dyes in Asia since ancient times, and today, they arecommonly used as therapeutics in traditional Chinese medicine.
Ban-Lan-Gen is frequently used as an anti-inﬂammatory and antiviral for the treatment of hepatitis, inﬂuenza, and various kinds ofinﬂammation [3,4]. Several biologically active ingredients havebeen identiﬁed in Ban-Lan-Gen, including indigoid alkaloids (e.g.,
indigo and indirubin) and quinazolinone alkaloids (e.g., tryptanthrin) [5]. In fact, indigo, indirubin, and tryptanthrin are threemarker compounds found in Ban-Lan-Gen [6]. Among these marker
compounds, it has been reported that indirubin causes transactivation of cytochrome P450 1A1 (CYP1A1) and CYP1A2 genes via

http://dx.doi.org/10.1016/j.dmpk.2016.01.002
1347-4367/Copyright © 2016, Published by Elsevier Ltd on behalf of The Japanese Society for the Study of Xenobiotics.

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T. Kumagai et al. / Drug Metabolism and Pharmacokinetics 31 (2016) 139e145

the aryl hydrocarbon receptor (AhR) [7]. In addition, a content of
indirubin have been reported in the roots of I. indigotica as main
raw materials of Ban-Lan-Gen (0.224e34.4 mg/g) [5,6].
Members of the CYP supergene family of monooxygenases play
an important role in detoxiﬁcation by converting pollutants, plant
toxins, carcinogens, and drugs to products that are then excreted into urine or bile [8,9]. Human CYP3A4 is of particular signiﬁcance inthis respect, because it is involved in the metabolism of approximately two-thirds of clinically relevant drugs [10]. A number ofcompounds, including pesticides, herbal supplements, vitamins,
and drugs, activate CYP3A4 gene transcription both in the liver andin the small intestine [11,12]. This induction process is the molecular basis for a number of important drug interactions that occur inpatients taking multiple medications.
The pregnane X receptor (PXR; NR1I2) is the principal regulator
of CYP3A4 gene expression and binds as a heterodimer with retinoid X receptor a (RXRa) to regulatory DNA sequences. These sequences include: (1) AG(G/T)TCA-like direct repeats spaced by 3
bases (DR3) located 8 kb upstream from the transcription startpoint and identiﬁed as distal nuclear receptor-binding element 1(dNR1), and (2) everted repeats separated by 6 bases (ER6) locatedin the CYP3A4 proximal promoter (prER6) [13]. Recently, Toriyabe
et al. identiﬁed a distinct PXR response element as an essential
distal nuclear receptor-binding element (eNR3A4) for CYP3A4 geneinduction [14]. PXR is activated by a number of structurally andchemically diverse ligands, such as drugs [rifampicin (RIF), clotrimazole] [15], pesticides (pyributicarb, endosulfan) [16,17], naturaland synthetic steroids (dexamethasone) [15], bile acids (lithocholicacid) [18], and herbal medicines (St. John's wort [SJW], Ginkgo
biloba, and Sophora ﬂavescens) [19e21]. CYP3A4 induction by these
PXR activators leads to accelerated metabolism of the activatorsthemselves and concomitantly-administered drugs that are
metabolized by CYP3A4.
Induction and inhibition of CYP3A4 by Ban-Lan-Gen are not fully
understood. In this study, we investigated the effect of Ban-LanGen on transcriptional activation of the CYP3A4 gene.
2. Materials and methods
2.1. Materials
Ban-Lan-Gen (lot. 9115476) was purchased from Beijing Tong
Ren Group Co., Ltd. (Beijing, China). SJW was purchased from
ChromaDex (Irvine, CA). Indirubin and tryptanthrin were purchased from Enzo Life Sciences, Inc. (Farmingdale, NY). Indican,
isatin, and indigo were purchased from Tokyo Chemical Industry
Co., Ltd. (Tokyo, Japan). Ginkgo biloba extract (GBE) was purchasedfrom Tama Biochemicals Co., Ltd (Tokyo, Japan). RIF was purchasedfrom SigmaeAldrich (St. Louis, MO). The chemicals used for thisstudy were dissolved in dimethyl sulfoxide (DMSO). Ban-Lan-Gen,
SJW, and GBE were extracted with culture medium used for thecell culture in this study, for 2 h at 37  C. These extract solutions
were centrifuged at 2,000  g for 15 min at 4  C and the supernatants were used for this study. All other reagents used were of thehighest quality available. Dulbecco's modiﬁed Eagle's medium(DMEM) and fetal bovine serum were obtained from Wako Pure
Chemical Industries, Ltd. (Osaka, Japan) and Hyclone Laboratories(Logan, UT), respectively. MEM non-essential amino acids andantibiotic-antimycotic were purchased from Invitrogen (Paisley,UK).
2.2. Cell culture
HepG2 cells were obtained from RIKEN cell bank (Tsukuba,
Japan). The HepG2-derived cell line stably expressing the CYP3A4-

luciferase reporter gene, clone 3-1-20, was maintained as reported
previously [22]. Cells were cultured in DMEM supplemented with
10% fetal bovine serum, MEM non-essential amino acids, andantibiotic-antimycotic. The cells were seeded at 3  104 cells perwell onto 48-well tissue culture plate (BD Biosciences, Heidelberg,
Germany) for luciferase gene reporter assay, and 5  104 cells perwell onto 24-well tissue culture plate (BD Biosciences) for real-timepolymerase chain reaction (PCR). After 24 h, the cell medium waschanged with the extract solutions or various subjects dissolved in
DMSO (ﬁnal concentration, 0.1%) and cultured for 48 h.
2.3. Luciferase gene reporter assay
3-1-20 cells were washed with Dulbecco's phosphate bufferedsaline (D-PBS) and suspended in passive lysis buffer (Promega,
Madison, WI) in a microcentrifuge tube. The cell suspension wascentrifuged at 12,000  g for 5 min at 4  C, and the cell extract wasused for the luciferase assay. The luciferase assay was performed
using the Luciferase Assay System and a GloMax™ 96 Microplate
Luminometer (Promega) according to the manufacturer's instructions. The resulting data are presented as the ratio of luminescence of treated cell samples to that of control. The
luminescence of each sample was normalized by its protein concentration as determined with the Protein Assay Kit (Bio-Rad
Laboratories, Hercules, CA).
2.4. Reporter gene constructs and transient transfection
The luciferase reporter plasmids, pGL3-Basic and pGL4.70, werepurchased from Promega. Preparation of the CYP3A4 luciferase reporter gene constructs, including pCYP3A4-362-7.7k, pCYP3A4362-7.7km, pCYP3A4-362m-7.7k, pCYP3A4-362m-7.7km, andpCYP3A4-362-7.7kmDa, was described previously [14]. One day
before transfection, cells were seeded in 48-well plates. Each reporter plasmid and pGL4.70 was transfected using Targefect F-1(Targeting System, El Cajon, CA) according to the manufacturer's
protocol. pGL4.70 was used for the normalization of transfection
efﬁciency. After transfection, cells were cultured in medium in thepresence of various chemicals for 48 h. Control cells were cultured
with vehicle (0.1% DMSO) alone. Subsequently, the cells were harvested and suspended in passive lysis buffer (Promega). Luciferase
activities were determined with Dual-Luciferase® Reporter Assay
System (Promega).
2.5. Construction of recombinant adenovirus and infection
Construction of the human PXR-expressing adenovirus(AdhPXR) and AdhPXR-short hairpin RNA (AdhPXR-shRNA) was
described previously [17]. Control adenovirus, a b-galactosidaseexpressing adenovirus (AdCont; AxCALacZ), was provided by Dr.
Izumi Saito (Tokyo University, Japan) [23]. The titer of the adenoviruses, 50% titer culture infectious dose (TCID50), was determined
as reported previously [17]. Multiplicity of infection (MOI) was
calculated by dividing the TCID50 by the number of cells. One day
before transfection, cells were seeded in 24-well plates. Adenovirus
infection was carried out as described previously [24].
2.6. Isolation of RNA and analysis of quantitative real-timepolymerase chain reaction
Total RNA was isolated from HepG2 cells using TRI Reagent(Molecular Research Center, Inc., Montgomery, AL) according to the
manufacturer's protocol. cDNA was prepared from 2.0 mg of total
RNA with Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega) using oligo(dT)20 primer (Greiner Japan, Japan)— 251 —

T. Kumagai et al. / Drug Metabolism and Pharmacokinetics 31 (2016) 139e145141

and porcine RNase inhibitor (TaKaRa Bio, Japan). Quantitative realtime PCR was performed using Permix Ex Taq (Perfect Real Time,
TaKaRa Bio) in a Thermal Cycler Dice Real Time System (TaKaRa
Bio). All samples were quantiﬁed using a comparative threshold
PCR cycle (Ct) method for relative quantiﬁcation of gene expression, normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sequences of the primers used are shown in
Table 1.
2.7. Statistical analysis
Data are presented as mean ± standard deviation (S.D.) andwere evaluated by paired Student's t-test. A P value < 0.05 wasconsidered to be signiﬁcant.
3. Results
3.1. Ban-Lan-Gen increases CYP3A4 gene reporter activity
The effects of Ban-Lan-Gen, SJW, and GBE on CYP3A4 gene reporter activity were conﬁrmed in HepG2-derived cells stably
expressing the CYP3A4 gene reporter (3-1-20 cells) as reported
previously [22]. SJW and GBE served as positive controls for CYP3A4
induction. Similar to SJW and GBE, Ban-Lan-Gen extract increased
CYP3A4 gene reporter activity, increasing luminescence 11-foldover control levels (Fig. 1). Furthermore, Ban-Lan-Gen extract
increased CYP3A4 gene reporter activity in a dose-dependent
manner (Fig. 2).
3.2. Indirubin, a component of Ban-Lan-Gen, activates CYP3A4 genetranscription
We next examined the effect of components of Ban-Lan-Gen on
CYP3A4 gene reporter activity. RIF was used as a positive control. 31-20 cells were treated with RIF (5 mM) or various Ban-Lan-Gen
components (5 mM), such as indirubin, indigo, isatin, indicant,and tryptanthrin, for 48 h. The CYP3A4 gene reporter activity wassigniﬁcantly elevated in response to indirubin, and the inductionlevel reached the same level as that of RIF (Fig. 3). Indirubinincreased reporter activity in a dose-dependent manner (Fig. 4). We
further investigated the effect of indirubin on intrinsic CYP3A4mRNA expression in HepG2 cells. In these cells, indirubin increased
CYP3A4 mRNA expression to a similar magnitude as that observedwith RIF (Fig. 5). Previous studies reported that indirubin is a prototypical AhR ligand [25]. In this study, indirubin also resulted in an
increase in CYP1A1 mRNA expression. Furthermore, we investigated the effect of indirubin on CYP3A4 mRNA expression in humanhepatocyte. When human cryopreserved hepatocyte was incubatedwith 5 mM of RIF and indirubin, CYP3A4 mRNA expression wasincreased 6.3-fold by RIF compared with untreated cells, but not byindirubin (data not shown).

Fig. 1. Effects of SJW, GBE, and Ban-Lan-Gen on CYP3A4 gene reporter activity in 3-120 cells. Clone 3-1-20 was seeded at 3 � 104 cells in 24-well tissue culture plates with
0.3 mL of DMEM, 24 h before SJW, GBE, and Ban-Lan-Gen extract treatment. The cells
were treated with SJW (0.1 mg/mL), GBE (0.5 mg/mL), and Ban-Lan-Gen extract(10 mg/mL) for 48 h, and the reporter activity was measured by luciferase assay. Reporter activities are expressed as fold increase compared with that in the vehicletreated cells. Results represent the mean ± S.D. of three separate experiments.
*P < 0.005, difference from the vehicle-treated cells.

3.3. Transcriptional activation of CYP3A4 reporter genes byindirubin
To elucidate the molecular mechanism of CYP3A4 gene transcriptional activation by indirubin, we performed transient transfection assays with reporter gene constructs containing mutationsin the hPXR binding elements. As shown in Fig. 6, treatment of
HepG2 cells with 5 mM indirubin or RIF resulted in an increase inluciferase activity (18.1-fold and 32.9-fold, respectively) after
transfection of pCYP3A4-362-7.7k, which included the CYP3A4 50 ﬂanking regions dNR1, eNR3A4, and prER6, all known to be highly
involved in the transcriptional regulation of the CYP3A4 gene [14].
Mutation of either dNR1 (pCYP3A4-362-7.7km) or prER6(pCYP3A4-362m-7.7k) of pCYP3A4-362-7.7k did not abolish theindirubin response completely (6.8- and 13.9-fold, respectively).

Table 1
Sequences of the oligonucleotide primers used for PCR ampliﬁcation.
Primer sequence
Human CYP3A4
Sense
Antisense
Human CYP1A1
Sense
Antisense
Human GAPDH
Sense
Antisense

50 -CTGTGTGTTTCCAAGAGAAGTTAC-30
50 -TGCATCAATTTCCTCCTGCAG-30
50 -CATCCCCCACAGCACAACA-30
50 -CAGGGGTGAGAAACCGTTCA-30
50 -CATGGGTGTGAACCATGAGAA-30
50 -GGTCATGAGTCCTTCCACGAT-30

Fig. 2. Effect of various concentrations of Ban-Lan-Gen on the CYP3A4 gene reporter
activity in 3-1-20 cells. Clone 3-1-20 was seeded at 3 � 104 cells in 24-well tissue
culture plates with 0.3 mL of DMEM 24 h before Ban-Lan-Gen treatment. The cells
were treated with 1e30 mg/mL Ban-Lan-Gen for 48 h, and the reporter activity wasmeasured by luciferase assay. Reporter activities are expressed as fold increase
compared with that in the vehicle-treated cells. Results represent the mean ± S.D. of
three separate experiments.

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T. Kumagai et al. / Drug Metabolism and Pharmacokinetics 31 (2016) 139e145

Fig. 3. Effect of various components of Ban-Lan-Gen on CYP3A4 gene reporter activity in 3-1-20 cells. Clone 3-1-20 was seeded at 3  104 cells in 48-well tissue culture plates with
0.3 mL DMEM, 24 h before Ban-Lan-Gen treatment. The cells were treated with each 5 mM of indirubin, indigo, isatin, indicant, tryptanthrin, and RIF for 48 h, and the reporter
activity was measured by luciferase assay. Reporter activities are expressed as fold increase compared with that in the vehicle-treated cells. Results represent the mean ± S.D. of
three separate experiments. *P < 0.005, **P < 0.05, difference from the vehicle-treated cells.

However, the response was signiﬁcantly decreased by the introduction of a mutation into the a half-site of eNR3A4 (pCYP3A4-3627.7kmDa) of pCYP3A4-362-7.7k (2.5-fold). RIF, in contrast, signiﬁcantly decreased the response in cells transfected with the mutated
pCYP3A4-362-7.7ks in dNR1, prER6, or eNR3A4 motifs (3.4-, 9.4and 0.8-fold, respectively).
3.4. Indirubin increases CYP3A4 gene reporter activity through the
PXR pathway
PXR is widely known as a major transcription factor mediating
CYP3A4 induction. To clarify whether indirubin activates the
CYP3A4 reporter gene via this pathway, we utilized knockdown of
PXR using AdhPXR-shRNA and overexpression of PXR using
AdhPXR (hPXR-expressing adenovirus). Indirubin-induced CYP3A4
gene reporter activities were signiﬁcantly increased by the introduction of AdhPXR (Fig. 7). In addition, CYP3A4 gene reporter
activation after treatment with indirubin, as well as RIF, wassigniﬁcantly decreased by the introduction of AdhPXR-shRNA in 31-20 cells (Fig. 8).

the CYP3A4 reporter gene (Figs. 1 and 2). These results indicate that
Ban-Lan-Gen extract induces CYP3A4 gene expression. In addition,
indirubin, a biological ingredient found in Ban-Lan-Gen, increased
CYP3A4 gene reporter activity in a dose-dependent manner (Fig. 4)
and increased CYP3A4 mRNA expression (Fig. 5). Although indirubin activated CYP3A4 gene transcription in HepG2 cells, it did notincrease CYP3A4 mRNA expression in human hepatocytes. A majordifference between these two cells types is cell proliferation.
HepG2 cells grow without limitation, but human hepatocytes dosenot. It is known that indirubin is a potent inhibitor of cyclindependent kinase 2 (CDK2), a modulator of cell cycle [28]. In
addition, Sivertsson et al. reported that the CYP3A4 induction in
conﬂuent Huh 7 cells was regulated by the endogenous activationof PXR as a result of decreased CDK2 activity, which was linked toreduced cell proliferation in the conﬂuent cell culture [29]. Therefore, the difference in CYP3A4 induction by indirubin between
HepG2 cells and the human hepatocytes may be due to CDK2 activity. Further studies are needed to resolve this issue.
CYP3A4 induction by xenobiotics and hormones is mediated by
the PXR [30e32], the constitutive androstane receptor (CAR, NR1I3)[33], the vitamin D receptor (VDR, NR1I1) [34], and the

4. Discussion
Herbal medicines and supplements are used worldwide [26].
Because the safety and efﬁcacy of herbedrug combinations arelargely unknown, research on herbedrug interactions is urgently
needed to guide the therapeutic use of herbs [27]. Metabolismmediated herbedrug interactions are very common and candecrease the efﬁcacy or increase the toxicity of the drug. Althoughvarious studies have described the actions of Ban-Lan-Gen [3,4],
there are few reports on Ban-Lan-Genedrug interactions.
In this study, we investigated the effect of Ban-Lan-Gen on
transactivation of the CYP3A4 gene using 3-1-20 cells, which have a
CYP3A4 reporter gene incorporated into their chromosomes. In
general, there is a plurality of the extraction method of herbal
medicine such as Ban-Lan-Gen. Thus, it is considered that the difference of the extraction method of Ban-Lan-Gen might be reﬂected on the CYP3A4 induction activity. However, to mimic
extraction of components from herbal medicine and their absorption in human intestine, we used the extraction from the Ban-LanGen product extracted with culture medium for CYP3A4 induction
study. As a result, Ban-Lan-Gen extract dose-dependently activatedFig. 4. Dose effect of indirubin on the CYP3A4 gene reporter activity in 3-1-20 cells.
Clone 3-1-20 was seeded at 3  104 cells in 24-well tissue culture plates with 0.3 mL of
DMEM 1 day before indirubin treatment. The cells were treated with 1e20 mM
indirubin for 48 h, and the reporter activity was measured by luciferase assay. Reporter
activities are expressed as fold increase compared with that in the vehicle-treatedcells. Results represent the mean ± S.D. of three separate experiments.

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143

Fig. 5. Effect of indirubin on CYP mRNA expression in HepG2 cells. HepG2 cells were seeded at 5 � 104 cells in 24-well tissue culture plates with 0.5 mL DMEM, 24 h before RIF andindirubin treatment. The cells were treated with 5 mM RIF and 5 mM indirubin for 48 h and then assayed using quantitative polymerase chain reaction (qPCR). CYP3A4 mRNA or
CYP1A1 mRNA expression was normalized by expression of GAPDH housekeeping gene and presented as fold increase compared with that of vehicle-treated cells. Results represent
the mean ± S.D. of three separate experiments. (A) CYP3A4 mRNA level, (B) CYP1A1 mRNA level. *P < 0.005, **P < 0.01, difference from the vehicle-treated cells.

glucocorticoid receptor-a (GRa, NR3C1) [35] in the liver.
Chemically-induced activation of the CYP3A4 gene is mainly
mediated by PXR binding to the CYP3A4 50 -ﬂanking region[30,32,36]. Therefore, we knocked down hPXR expression using
AdhPXR-shRNA. When AdhPXR-shRNA was introduced into the 31-20 cells, the activation of the CYP3A4 reporter gene by indirubin,
as well as by RIF, was signiﬁcantly decreased (Fig. 8). Furthermore,
the overexpression of PXR using AdhPXR increased CYP3A4 reporter activity by indirubin in an infection dose-dependent manner(Fig. 7). These results strongly suggest that indirubin enhanced thetransactivation of the CYP3A4 gene through PXR activation.

Since indirubin is a natural AhR ligand isolated from humanurine [7], it is possible that indirubin activation of the CYP3A4 geneis through the AhR pathway. We previously demonstrated thatpolycyclic aromatic hydrocarbons (PAHs) enhance the transactivation of the CYP3A4 gene through PXR activation but not AhR
activation [24]. Recently, Luckert et al. also reported that PAHs andtheir metabolites induce CYP3A4 gene expression via PXR [37].
Thus, indirubin and other PAHs activate CYP3A4 gene transcription
through the PXR pathway but not the AhR pathway.
A number of independent studies have examined the relative
contribution of the individual functional PXR-binding motifs (dNR1,

Fig. 6. Mutational analysis of putative human PXR responsive elements in the CYP3A4 gene. Schematic structures of reporter plasmids used are shown in the middle. Closed boxes
represent mutated PXR binding elements. HepG2 cells seeded at 3 � 104 cells in 48-well tissue culture plates pre-incubated for 24 h before transfection. Transfection into HepG2
cells was performed as described in the Materials and methods section. The cells were treated with 5 mM RIF and 5 mM indirubin for 48 h, and the reporter activity was measured byluciferase assay. Reporter activities are expressed as fold increase compared with that in the vehicle-treated cells. Results represent the mean ± S.D. of three separate experiments.
*P < 0.005, **P < 0.05, difference from the indirubin- or RIF-treated cells transfected with the pCYP3A4-362-7.7k.

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T. Kumagai et al. / Drug Metabolism and Pharmacokinetics 31 (2016) 139e145(dNR1, prER6, and eNR3A4). Notably, the introduction of a mutation
into eNR3A4 signiﬁcantly decreased the indirubin-induced transcriptional activation, whereas mutations of dNR1 and prER6 did
not (Fig. 6). These results indicate that eNR3A4 might be involved in
the activation of CYP3A4 gene through PXR by indirubin.
In conclusion, we have demonstrated that Ban-Lan-Gen activated CYP3A4 gene transcription and that indirubin, a componentof Ban-Lan-Gen, played an important role in CYP3A4 gene transcription. In addition, we have shown that indirubin activated
CYP3A4 gene through the activation of PXR in HepG2 cells. Moredetailed studies on the molecular mechanism of CYP3A4 induction
by indirubin may provide important information on the herbedrugand drugedrug interactions.

Fig. 7. Effect of PXR on CYP3A4 gene reporter activity in 3-1-20 cells. Clone 3-1-20 was
seeded at 2  104 cells in 24-well tissue culture plates pre-incubated for 24 h and thentreated with AdhPXR (MOI of 0, 1, 3, and 10). Forty-eight hours after infection, thesecells were treated with 5 mM RIF and 5 mM indirubin for 48 h, and the reporter activity
was measured by luciferase assay. Reporter activities are expressed as fold increase
compared with that in the vehicle-treated cells uninfected with AdhPXR. Results
represent the mean ± S.D. of three separate experiments. *P < 0.005, difference fromthe corresponding AdLacZ-infected cells.

prER6, and eNR3A4) on PXR-mediated transactivation of the
CYP3A4 gene. In particular, a novel functional cis-acting PXRbinding element designated eNR3A4 has been discovered to be
essential for RIF-inducible CYP3A4 transactivation in human liver[14]. This element is located approximately 7.6 kb upstream fromthe transcription initiation site of the CYP3A4 gene to which PXRbinds as a heterodimer with RXRa. Furthermore, Pavek et al. reported that eNR3A4 has negligible or no effect on CYP3A4 transactivation through VDR [38]. These ﬁndings strongly suggest thateNR3A4 is a key regulatory element for the xenobiotic induction of
CYP3A4 through PXR in the liver. Thus, we investigated the
involvement of eNR3A4 in the transactivation of the CYP3A4
through PXR activation by indirubin with the CYP3A4 reporter geneconstructs that have a mutation in three PXR response elements

Fig. 8. Effects of PXR-shRNA on CYP3A4 gene reporter activity in 3-1-20 cells. Clone 31-20 was seeded at 2  104 cells in 24-well tissue culture plates pre-incubated for 24 hand then treated with AdhPXR-shRNA (MOI of 0, 0.3, 1, and 3). Forty-eight hours after
infection, these cells were treated with 5 mM RIF and 5 mM indirubin for 48 h, and thereporter activity was measured by luciferase assay. Reporter activities are expressed as
fold increase compared with that in the vehicle-treated cells uninfected with AdhPXRshRNA. Results represent the mean ± S.D. of three separate experiments. *P < 0.005,**P < 0.01, difference from the corresponding AdLacZ-infected cells.

Conﬂict of interest
None.
Acknowledgments
This work was supported by a Health and Labour Sciences
Research Grant for Research on Food Safety (No. 22230301) from
the Ministry of Health, Labour and Welfare, Japan. We would like tothank Ms. Machi Miura for the excellent technical assistanceprovided.
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Tetrahedron Letters 56 (2015) 921–924

Contents lists available at ScienceDirect

Tetrahedron Lettersjournal homepage: www.elsevier.com/locate/tetlet

Directed synthesis of 2-spiroindolines from indole derivatives byformic acid-induced N-acyliminium ion-conjugated dienespirocyclization
Hideki Abe a,⇑, Naohiro Miyagawa a, Sho Hasegawa a, Toyoharu Kobayashi a, Sakae Aoyagi b,
Chihiro Kibayashi b, Tadashi Katoh c, Hisanaka Ito a,⇑aSchool of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japanc
Department of Chemical Pharmaceutical Science, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japanba r t i c l e

i n f oArticle history:
Received 15 November 2014
Revised 17 December 2014
Accepted 26 December 2014
Available online 7 January 2015a b s t r a c t
Synthesis of 2-spiroindolines from 2-substituted indole derivatives via acid-induced N-acyliminium ionconjugated diene spirocyclization was demonstrated. This methodology can be used to direct transformations of 2-substituted indole moieties into 3-nonsubstituted-2-spiroindoline skeletons.
 2015 Elsevier Ltd. All rights reserved.

Keywords:
2-Spiroindoline
Spirocyclization
2-Substituted indole
Formic acid
N-Acyliminium ion

Introduction
Indole and indoline frameworks are common substructures in a
number of biologically active compounds and natural products. 2Spiroindoline alkaloids, such as lapidilectines, lapidilectam, tenuisines, and grandilodines (Fig. 1 and 1–8), have been isolated fromplants, including Kopsia lapidilecta, Kopsia tenuis, and Kopsia grandifolia.1–4 Lapidilectine B (4), grandilodine C (5), and tenuisine A (6)have been found to reverse multidrug resistance in vincristineresistant KB cells without showing any cytotoxicity against either
drug-sensitive or drug-resistant cells in the absence of added vincristine.4 The only synthetic studies on these biologically active
and complex alkaloids reported to date are the total synthesis oflapidilectine B (4)5 by Pearson and the total synthesis of lundurine
B,6 from Kopsia tenuis, by Nishida. There have been several synthetic studies on the construction of the alkaloids’ indole core
fused to an azacyclooctane ring,7–10 but to our knowledge there
have as yet been no attempts to synthesize complex 2-spiroindoline alkaloids. Although some synthetic methods for 2-spiroindolines, for example, intramolecular CH insertion11,12 and spiro ring
⇑ Corresponding authors. Tel.: +81 42 676 7293; fax: +81 42 676 7282.

E-mail addresses: abehidek@toyaku.ac.jp (H. Abe), itohisa@toyaku.ac.jp (H. Ito).

formation from 2,2-disubstituted indoline,13 have been reported,
there are few known methodologies for direct transformation of
2-substituted indoles to 2-spiroindolines. Additionally, there havebeen few reports of directed alkylations at the C2 position to give
2-substituted indoles due to the high reactivity of the C3 position
toward electrophilic reagents. In fact, many reactions of indole at
the C3 position, such as Friedel–Crafts-type reactions, have beendeveloped.14 Harrowven and co-workers15 reported the only
example of direct synthesis of 2-spiroindoline from indole by 5exo or 6-exo radical cyclization of indoles tethered to aromaticrings. We previously reported formic-acid-induced spirocyclization of N-acyliminium ions with conjugated dienes and the total
synthesis of tricyclic marine alkaloids16 using a prepared spirocompound as the key intermediate. This key reaction was used
for stereoselective construction of a 1-azaspiro[4.5]decane skeleton tethered to an allyl alcohol moiety from an amide ketone ina single operation. Therefore, the possibility of synthesizing 2-spiroindoline by acid-induced spirocyclization attracted our interest.
We recently reported the spirocyclization of a 3,3-ethylenedioxy2-hydroxy indoline derivative to afford an octahydroacridine
derivative17 via spirocyclization followed by rearrangement.
Herein, we report the directed transformation of a 2-substituted
indole to 2-spiroindoline by spirocyclization.

http://dx.doi.org/10.1016/j.tetlet.2014.12.1300040-4039/ 2015 Elsevier Ltd. All rights reserved.

— 257 —

922

H. Abe et al. / Tetrahedron Letters 56 (2015) 921–924

R3
MeO2C

N

O

N

N
O

Me

N

R1 R
2
CO2Me

CH2Cl2

lapidilectine B (4):R1 = H, R2 = H2
grandilodine C (5):R1 = H, R2 = O
tenuisine A (6):R1 = OMe, R2 = H2
tenuisine C (7):R1 = OMe, R2 = O

N
R

R H
CO2Me

N
R

grandilodine A (8):R = CO2MeMe

14: R = H
15: R = Boc
16: R = CO2Me

Substrate

Temp

Time (h)Products (yield (%))

12
12
13
13

Rt
0 C
Rt
0 C

14
48
4
48

14 (36)15 (14) and 14 (18)
16 (53)16 (22)2-spiroindoline framework

Me

Me

H+

Our investigation of the construction of 2-spiroindoline beganwith the synthesis of 2-substituted indole derivatives 12 and 13.
Initially, 2-substituted indole 11 was derived from E,E-dienol 918
in 4 steps, including Smith’s synthesis method for indole,19 asshown in Scheme 1. Thus, two-step oxidation (Parikh–Doering oxidation20/Pinnick oxidation21) of alcohol 9, followed by esteriﬁcation of the resulting carboxylic acid, gave the ethyl ester 10.
Condensation of 10 with N-TMS-o-toluidine,22 obtained easily
from o-toluidine, was achieved by Smith’s method to provide 11
in moderate yield. Finally, protection of the nitrogen atom of 11
with a tert-butoxycarbonyl group or a methoxycarbonyl groupgave the requisite N-Boc indole 12 and N-methoxycarbonyl indole
13, respectively.
Having obtained the N-Boc derivative 12 and the N-methoxycarbonyl derivative 13 in an efﬁcient manner, we then investigated
the transformation to 2-spiroindolines via formic acid-induced spirocyclization of N-acyliminium ions with conjugated dienes
Me

a–c

OH

10
Me

HH

H+

N
R
12: R = Boc
13: R = CO2Me

N
R

19

+
N

+
N
R
17

Me

Me

O

H

18

N
R

+

R

O
H

N
H

Me

15: R = Boc
16: R = CO2Me

O

O
H

Me

14

Scheme 2. Proposed mechanism for spirocyclization.

d

CO2Et

9

Me

e or f

11

H

3'

1
2
3
4

Results and discussion

N
H

O

H

Entry

Figure 1. Structures of Kopsia alkaloids and 2-spiroindoline framework.

MeO

12: R = Boc
13: R = CO2Me

N

N

N
R

HCOOH

CO2Me

lapidilectine A (1):R1 = CO2Me, R2 = H, R3 = H2
lapidilectam (2):R1 = CO2Me, R2 = H, R3 = O
grandilodine B (3):R1 = H, R2 = CO2Me, R3 = O
MeO2CTable 1
Spirocyclization of 2-substituted indole derivatives 12 and 13

R2
R1

N
R
12: R = Boc
13: R = CO2Me

Scheme 1. Reagents and conditions: (a) SO3�pyridine, Et3N, DMSO, rt, 10 min; (b)NaClO2, 2-methyl-2-butene, NaH2PO4, tert-BuOH–H2O, rt, 10 min; (c) p-TsOH, EtOH,
reﬂux, 2 h, 63%, 3 steps; (d) N-TMS-o-toluidine, n-BuLi, hexane, reﬂux, 10 h, then 10,
THF, �78 C ? rt, 1 h, 58%; (e) di-tert-butyl-dicarbonate, Et3N, DMAP, CH2Cl2, rt, 1 h,
98% for 12; (f) methyl chloroformate, n-BuLi, �78 C, 1 h, then 0 C, 1 h, 96% for 13.

(Table 1).23 The ﬁrst attempt at formic-acid induced spirocyclization of 12, under the same conditions as in our previous report(HCO2H–CH2Cl2, rt),17 gave the deBoc 2-spiroindoline 14 in 36%
yield as a 1:1 mixture of diastereoisomers at the C30 position (entry1). When the reaction was performed at 0 C for 48 h (entry 2), thedesired 2-spiroindoline derivative 15 and the spiro compound 14
were detected, but in poor yields (14% and 18%, respectively), alongwith recovered 12. However, the spirocyclization of N-methoxycarbonyl derivative 13 with formic acid proceeded smoothly atroom temperature to afford the desired 2-spiroindoline 16 in 53%
yield as a 1:1 mixture of diastereoisomers at C30 (entry 3). Whenthe reaction time was lengthened and the temperature decreased
to 0 C, the spiro compound 16 was obtained in only 22% yield,
along with a complex mixture of unidentiﬁed products. Whenusing indole 11 as a starting material in this reaction, complex
mixtures were obtained.
The proposed mechanism for the transformation of N-acyl-2substituted indoles to 2-spiroindolines is shown in Scheme 2.
The 2-substituted indoles 12 and 13 are in equilibrium with— 258 —

H. Abe et al. / Tetrahedron Letters 56 (2015) 921–924

N
MeO2C

a

O

O

N
MeO2C
HO

Me

H

20

16

N
MeO2C

b

O

Me

Me

21

Scheme 3. Reagents and conditions: (a) satd K2CO3 aqueous solution, MeOH, rt,
30 min; (b) MnO2, CH2Cl2, rt, 3 h, 95% for 2 steps.

iminium ions 17, generated by protonation at the C3 position of theindole ring in the presence of formic acid.24 Spirocyclization of theiminium ion 17 with the conjugated diene occurs via the chair-like
transition state 1816 to afford spiro compound 19, which contains
an allyl cation moiety. Addition of formic acid to the C30 position of
the side chain gives the desired 2-spiroindolines 15 and 16, respectively. In the case of the reaction of N-Boc derivative 12, cleavage of
the Boc group of 12 and the desired product 15 competed with spirocyclization under acidic conditions, decreasing the yields of thedesired spiro compounds. Fortunately, the N-methoxycarbonylgroup was more suitable for use under acidic conditions than the
Boc group. Thus, spirocyclization of N-methoxycarbonyl indole
13 proceeded without cleavage of the methoxycarbonyl group in
the presence of formic acid to give the desired N-methoxycarbonyl-2-spiroindoline 16 in moderate yield.
To conﬁrm that the resulting 2-spiroindoline mixture was a
mixture of diastereoisomers at the C30 position, transformation offormate to a conjugated enone was carried out as shown in
Scheme 3. Hydrolysis of spiro formate 16 under basic conditions
gave the allyl alcohol 20 as a 1:1 mixture of diastereoisomers. Oxidation of allyl alcohol 20 with activated MnO2 afforded the conjugated enone 21 as a single diastereomer in 95% yield over 2 steps.
This result proved that the spiro compounds obtained consisted ofa mixture of diastereoisomers at the C30 position.
The substrate scope of the spirocyclization of 2-substituted
indoles25 to 2,2-spiro indoline derivatives was investigated(Table 2). Spirocyclization of 5-methyl substituted indole derivative 22 using formic acid gave the desired spiroindoline 26 in

Table 2
Spirocyclization of 2-substituted indole derivatives 22–25

R1

Me

R2
HCOOH

R1 5
R2 6

CH2Cl2

N
CO2Me

N
MeO2C

rt
O

R1 = Me, R2 = H

O
H

H
3'

Me

R1 = Me, R2 = H

22:
23: R1 = OMe, R2 = H
24: R1 = F, R2 = H
25: R1 = H, R2 = Me

26:
27: R1 = OMe, R2 = H
28: R1 = F, R2 = H
29: R1 = H, R2 = Me

Entry

Substrate

Time (h)Products (yield (%))

1
2
3
4
5

22
23
23
24
25

6
2
6
8
8

26 (67)27 (42, 66% brsm)
27 (30, 46% brsm)
28 (30, 39% brsm)
29 (48)923

67% yield as a 1:1 mixture of diastereoisomers (entry 1). In the caseof the reaction of 5-methoxy indole derivative 23 (entries 2 and 3),
the spiroindoline 27, which is a component of tenuisine-type alkaloids, was obtained in 42% (66% brsm) yield. When this reaction
time was prolonged, the yield of 27 decreased to 30% due todecomposition of the product. Spirocyclization of the 5-ﬂuorosubstituted compound 24, containing an electron-withdrawing
group (entry 4), gave 28 in low yield (30%). The reaction of a 6methyl indole derivative afforded the corresponding spiroindoline
29 in moderate yield (entry 5). Although the construction ofindole-2-spiro-20 -cyclohexane derivatives from 2-nona-5,7-dienyl
indole derivatives 12, 13, and 22–25 was achieved as stated above,
attempts to transform the 2-octa-4,6-dienyl indole to the corresponding indole-2-spiro-20 -cyclopentane derivative under thesame conditions failed.26
In conclusion, directed synthesis of 2-spiroindoline derivatives
by spirocyclization of indoles tethered to conjugated dienes at
the C2 position was demonstrated. The transformation of indoles
to spiroindolines proceeded via protonation at the C3 position in
the presence of formic acid, followed by spirocyclization of theresulting iminium ion with the conjugated diene. This synthetic
methodology is expected to be useful for total synthesis of Kopsia
alkaloids with N-methoxycarbonyl-2-spiroindoline components, aproject that is now in progress in our laboratory.
Acknowledgments
This work was supported by the Platform for Drug Discovery,
Informatics, and Structural Life Science project of the Ministry of
Education, Culture, Sports, Science, and Technology, Japan. H.A.
thanks the Takeda Science Foundation and the Uehara Memorial
Foundation for ﬁnancial support.
Supplementary data
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.tetlet.2014.12.
130.
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924

H. Abe et al. / Tetrahedron Letters 56 (2015) 921–924

23. General procedure: To a solution of 2-substituted indole (0.1 mmol) in
CH2Cl2 (1 mL) at 0 C was added formic acid dropwise (1 mL). After stirring
for the indicated times and temperatures, the reaction mixture was madebasic by addition of 28% NH3 aqueous solution, and then extracted with
CHCl3 three times. The combined organic layers were washed with waterand dried over MgSO4, and the solvent was removed in vacuo. Puriﬁcationof the resulting residue by ﬂash column chromatography gave the2-spiroindolines.
24. The spirocyclization reaction of 13 with acetic acid (room temperature for5 days) did not proceed, and 94% of 13 was recovered.
25. The 5- or 6-substituted indole derivatives used as starting materials weresynthesized by the same procedure used for the synthesis of 13, as shown in
Scheme 1, condensation of ethyl ester 10 and the corresponding N-TMS-otoluidine derivatives.

26. The reaction of methyl 2-((4E,6E)-octa-4,6-dien-1-yl)-1H-indole-1-carboxylate
i with formic acid gave only a complex mixture and did not afford the spiro
compound ii.

— 260 —

Me

HCOOH

N
CO2Me
i

N
MeO2C

CH2Cl2

O

O
H
ii

H
Me

Molecules 2015, 20, 4623-4634; doi:10.3390/molecules20034623

OPEN ACCESS

molecules

ISSN 1420-3049
www.mdpi.com/journal/molecules
ArticleConstruction of an Isonucleoside on a2,6-Dioxobicyclo[3.2.0]-heptane Skeleton
Yuichi Yoshimura 1,*, Satoshi Kobayashi 1, Hitomi Kaneko 1, Takeshi Suzuki 1 andTomozumi Imamichi 212Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University, 4-4-1 Komatsushima,
Aoba-ku, Sendai 981-8558, Japan; E-Mails: s-kobayashi@fujichemical.co.jp (S.K.);
hitomi.f528@gmail.com (H.K.); suzutake19851109@coral.plala.or.jp (T.S.)
Laboratory of Human Retrovirology, Leidos Biochemical Research Inc., Frederick National
Laboratory for Cancer Research, Frederick, MD 21702, USA; E-Mail: timamichi@mail.nih.gov* Author to whom correspondence should be addressed; E-Mail: yoshimura@tohoku-pharm.ac.jp;
Tel./Fax: +81-22-727-0144.
Academic Editors: Mahesh K. Lakshman and Fumi Nagatsugi
Received: 2 February 2015 / Accepted: 4 March 2015 / Published: 12 March 2015

Abstract:
We
have
built
a
new
isonucleosidederivativeona2,6-dioxobicyclo[3.2.0]heptane skeleton as a potential anti-HIV agent. To synthesize thetarget compound, an acetal-protected dihydroxyacetone was first converted to a 2,3-epoxytetrahydrofuran derivative. Introduction of an azide group, followed by the formation of an
oxetane ring, gave a pseudosugar derivative with a 2,6-dioxobicyclo[3.2.0]heptane
skeleton. The desired isonucleoside was obtained by constructing a purine base moiety on
the scaffold, followed by amination.
Keywords: nucleoside; bicyclo; oxetane ring; conformation

1. Introduction
Since the discovery of 3'-azidothymidine (AZT), much attention has been paid to the developmentof effective chemotherapeutic agents against the human immunodeficiency virus (HIV), a causative
agent for AIDS [1,2]. More than 20 anti-HIV drugs have now been approved and are clinically used
for the treatment of AIDS. Among them, nucleoside reverse transcriptase inhibitors (NRTIs) play a— 261 —

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critical role in the treatment of AIDS patients. In the most successful regimen for AIDS referred to as
ART (Anti-Retroviral Therapy), a cocktail of anti-HIV drugs, including NRTIs, non-nucleosidereverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs) [3], is used. Although ART
greatly contributes to increasing the lifespan of patients, drug-resistant strains of the virus are still aserious problem [4,5]. Therefore, new drugs that are effective against the resistant virus strains areconstantly needed.
Most NRTIs belong to a category of dideoxynucleosides, e.g., zalcitabine (ddC) [6] and didanosine(ddI) [6]. AZT [7] and lamivudine [8] are 3'-substituted dideoxynucleoside derivatives, and abacavir is
a carbocyclic analogue of dideoxynucleoside. Only tenofovir [9], which is a nucleoside phosphonate(Figures 1 and 2), is different. From the viewpoint of designing new anti-HIV agents, nucleosides
constructed on a novel scaffold are expected to have antiviral activity against the resistant virus strains
and may avoid cross-resistance to the known NRTIs.

Figure 1. Approved NRTIs.

Figure 2. Our previous works searching for anti-HIV nucleosides built on a new scaffold.
Thus, we have been focusing on the design and synthesis of nucleoside derivatives attached
to a pseudosugar scaffold [10–17]. Among them, nucleosides with cyclohexenyl [13],dihydrothiophenyl [15], and dihydropyranyl [17] groups in place of a furanose ring have beensynthesized as “ring-expanded” analogues of stavudine and abacavir. Dihydropyranyl derivative 1 did— 262 —

Molecules 2015, 20

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not show any activity, whereas cyclohexenyl derivative 2 showed weak anti-HIV activity [13,17]. On
the other hand, dihydrothiophenyl derivative 3 showed significant anti-HIV activity [15]. In addition,
we have applied the “ring-expanding” concept to lamivudine and synthesized isonucleosides 4
constructed on 2-oxa-6-thiabicyclo[3.2.0]heptane [14]. The isonucleoside 4 was also considered asconformationally-restricted analogue of lamivudine by introducing a fused thietane ring (vide infra).
However, 4 showed no anti-HIV activity (Figure 2). In this study, we planned to build isonucleoside 6
on a 2,6-dioxobicyclo[3.2.0]heptane skeleton, an analogue of dioxolane nucleoside 5 which exhibited
potent anti-HIV activity [18–20]. The similar conformationally-restricted analogue of d4T was known:
cyclopropane-fused carbocyclic d4T (N-MCd4T), fixed in north conformation, was originally reportedby Marquez and his colleagues and had significant anti-HIV activity with lesser cytotoxicity [21]. In
addition, D-enantiomer of 5 was known to have potent cytotoxicity [18–20]. Thus, isonucleoside 6
should be promising although the thietane-fused derivative 4 was inactive (Figure 3).

Figure 3. Design of nucleoside derivative built on a 2,6-bicyclo[3.2.0]heptane skeleton.
2. Results and Discussion
Following our previous reports [11,14], epoxide7 was synthesized. We first attempted to introduce
an adenine onto 7 by treating it with DBU [22]. However, the reaction did not give the desired product9 (Scheme 1).

Scheme 1. Attempt to introduce adenine moiety.
In addition, Lewis acid-catalyzed reactions did not afford 9 either (data not shown). Since the low
reactivity of 7 might be due to its rigid structure, we next tried nucleophilic substitution using a morereactive cyclic sulfate derivative [23]. Cis-allyl alcohol 10, a precursor of epoxide 7 [11,14], wascyclized under Mitsunobu conditions, as in the case of epoxide 7 [11,14], to give dihydrofuran 11 in
71% yield. Treatment of dihydrofuran 11 with potassium osmate in the presence ofN-methylmorpholine N-oxide afforded cis-diol 12. The desired cyclic sulfate 13 was obtained bytreatment of 12 with thionyl chloride, followed by oxidation. However, the nucleophilic substitution of
13 with adenine did not afford the desired isonucleoside 14 (Scheme 2).
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Molecules 2015, 20

4626

Scheme 2. Second attempt to introduce adenine using a cyclic sulfate.
Therefore, we revised our plan to synthesize an isoadenosine constructed on a2,6-dioxobicyclo[3.2.0]heptane scaffold, and the revised scheme is shown in Scheme 3 in a
retrosynthetic manner. Instead of the direct introduction of adenine, we decided to build the adenine
ring on the 2,6-dioxobicyclo[3.2.0]heptane pseudosugar skeleton in a stepwise manner. According tothis plan, compound 16 was thought to be a suitable intermediate for preparing 15 since it can betransformed to 6 by the formation of an imidazole ring, followed by amination. Fused oxetane
derivative 16 can be obtained from dimesylate 17. Finally, epoxide 7, described above, was selected as
the starting compound because it can be converted to 17 by the selective cleavage of the oxirane ringwith an azide anion (Scheme 2).

Scheme 3. Revised retrosynthesis of isoadenosine 6 built on a 2,6-dioxobicyclo[3.2.0]heptane skeleton.
First, regioselective cleavage of the oxirane ring of 7 with sodium azide in 2-methoxyethanol under
reflux conditions gave the desired azide-alcohol 18 as a single regioisomer in 66% yield. It is obviousthat the nucleophilic azide anion attacked from the less hindered side since similar regioselective
epoxide opening was observed in our previous report [11,14]. After benzoylation of the hydroxyl
group, the acetal group of 19 was removed by using acidic hydrolysis, and the resulting diol was— 264 —

Molecules 2015, 20

4627

mesylated to give dimesylate 20 in good yield. Deprotection of the benzoyl group and the subsequent
formation of an oxetane ring were achieved by treating 20 with sodium methoxide under reflux
conditions to give mesylate 21 in 72% yield. The structure of 21 was unambiguously determined bycomparison of 1D NMR spectrum with that of 2-oxa-6-thiabicyclo[3.2.0]heptane skeleton [14] afterconverting it to benzoate 22 by treatment with benzoic acid in the presence of cesium fluoride. In
1
H-NMR spectra of 22, the peaks corresponding to the methyl groups of the dimesylate were absent,
and only the peaks corresponding to the benzoyl group in the range of 8.1–7.4 ppm were present. In
addition, one of the methylene protons at the 2-position was observed as a doublet at 4.42 ppm,
meaning that the coupling with H-3 disappeared. This indicates that the conformation around thetetrahydrofuran ring changes and becomes fixed, which causes a loss of coupling between one pair of
H-2 and H-3 protons. A similar correlation between conformation and couplings in 1H-NMR spectra
has been reported for the 2-oxa-6-thiabicyclo[3.2.0]heptane skeleton [14]. Moreover, in the mass
spectrum of the compound, a molecular ion peak was observed at m/z = 276, further supporting theassignment of the structure. Finally, 22 was deprotected to afford azido-alcohol 23 in 88% yield(Scheme 4).

Scheme 4. Synthesis of isoadenosine 6.
Azido-alcohol 23 was reduced by catalytic hydrogenation to give key intermediate 16, which wastreated with 5-amino-4,6-dichloropyrimidine and diisopropylethylamine in refluxing n-butanol [23] togive diaminopyrimidine derivative 15 in 58% yield from 23. Formation of the imidazole ring of 15 was— 265 —

Molecules 2015, 20

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accomplished by treatment with orthoethyl formate under acidic conditions [24] to give 6-chloropurine
nucleoside 24. Finally, the isoadenosine was built on the 2,6-dioxobicyclo[3.2.0]heptane scaffold 6 byheating 24 with methanolic ammonia in a sealed tube in 69% yield (Scheme 4). Isoadenosine 6 did notshow any significant activity against HIV even at a concentration of 100 µM.
3. Experimental Section
General Information
Melting points are uncorrected. NMR spectra were recorded at 400 MHz (1H), 100 MHz (13C) using
CDCl3 as a solvent. As an internal standard, tetramethylsilane was used for CDCl3. Mass spectra wereobtained by EI or FAB mode. Silica gel for chromatography was Silica Gel 60N (spherical, neutral,
100–210 µm, Kanto Chemical Co. Inc., Tokyo, Japan). When the reagents sensitive to moisture wereused, the reaction was performed under argon atmosphere.
8,8-Dimethyl-1,7,9-trioxaspiro[4,5]dec-3-ene (11). To a solution of PPh3 (2.49 g, 9.48 mmol) in THF(10 mL) was added DEAD (4.31 mL, 9.48 mmol) and the mixture was stirred at room temperature for5 min. To this mixture, a solution of 10 [11,14] (1.04 g, 5.58 mmol) in THF (10 mL) was added. The
mixture was stirred at room temperature for 1 h. After the solvent was removed under reducedpressure, the residue was purified by silica gel column chromatography (hexane–ethyl acetate = 19:1)
to give 11 (677 mg, 71%) as a white crystal. mp 47–49 °C; 1H-NMR (CDCl3) δ 1.45 (3H, s), 1.48 (3H,s), 3.76 (2H, d, J = 11.6 Hz), 3.81 (2H, d, J = 11.6 Hz), 4.71 (2H, t, J = 1.9 Hz), 5.84-5.87 (1H, m),
6.02 (1H, d, J = 6.3 Hz); 13C-NMR (CDCl3) δ 22.6, 24.6, 66.7, 74.9, 98.0, 128.1, 129.1; IR (neat)2924.2, 2853.1, 1724.1, 1215.9, 758.3 cm−1; FAB-MS (m/z) 155 [M−15]+; Anal. Calcd for C9H14O3; C,
62.84; H, 8.32. Found; C, 62.78; H, 8.44.
(3S*,4S*)-8,8-Dimethyl-1,7,9,-trioxaspiro[4.5]decane-3,4-diol (12). To a solution of 11 (73 mg,
0.43 mmol) and NMO (0.22 mL) in acetone (4 mL), was added a solution of K2OsO4·2H2O (1 mg,
0.004 mmol) in H2O (0.4mL) at 0 °C. After stirred at room temperature for 60 h, Na2S2O3·5H2O(125 mg) was added and the mixture was stirred at room temperature for 30 min. After the whole
mixture was dried over Na2SO4, the solid materials were removed by suction and washed with ethyl
acetate. The combined filtrate was concentrated under reduced pressure. The residue was purified bysilica gel column chromatography (CHCl3–MeOH = 19:1) to give 12 (84 mg, 97%). 1H-NMR (CDCl3)
δ 1.43 (3H, s), 1.49 (3H, s), 3.59 (1H, dd, J = 11.6, 1.9 Hz), 3.80 (1H, d, J = 9.7 Hz), 3.82–3.87 (2H,m), 3.94 (1H, dd, J = 9.7, 4.9 Hz), 4.14 (1H, dd, J = 11.6, 1.9 Hz), 4.22 (1H, d, J = 5.3 Hz) 4.34 (1H, q,
J = 5.0 Hz); 13C-NMR (CDCl3) δ 21.0, 25.8, 63.3, 66.5, 71.0, 71.4, 74.9, 76.7, 98.4; IR (KBr) 3306.4,
2953.6, 2741.6, 1452.2, 524.19 cm−1; EI-MS (m/z): 204 [M+1]+; HRMS Calcd for C9H15N3O4:
204.0998, Found: 204.0992.
(3S*,4S*)-8,8-Dimethyl-1,7,9,-trioxaspiro[4.5]decane-3,4-cyclicsulfate (13). To a solution of 12 (410 mg,
2.01 mmol) and Et3N (672 µL, 4.82 mmol) in CH2Cl2 (10 mL), was added dropwise a solution of
SOCl2 (113 μL, 1.55 mmoL) in CH2Cl2 (10 mL) at 0 °C. After stirred at room temperature for 15 min,the mixture was washed with water. The water layer was extracted with CHCl3 twice and the combined— 266 —

Molecules 2015, 204629

organic layer was washed with brine, then dried over Na2SO4. After filtration, the residue was passed
through a short silica gel column (eluate: hexane–ethyl acetate = 1:1). After the solvents were removed
under reduced pressure, the residue was dissolved in CCl4–CH3CN–H2O (2:2:3, 3 mL). To thissolution, were added RuCl3·3H2O (2.7 mg) and NaIO4 (73 mg, 0.34 mmol) at 0 °C. The mixture wasstirred at the same temperature for 1.5 h. After diluted with ether, the mixture was washed with water,
sat.NaHCO3 and brine, then dried over Na2SO4. After filtration, the solvents were removed under reducepressure, the residue was purified by silica gel column chromatography (hexane-ethyl acetate = 6:1) togive 13 (82 mg, 77%). 1H-NMR (CDCl3) δ1.43 (3H, s), 1.50 (3H, s), 3.56 (1H, dd, J = 12.1, 2.4 Hz),3.82 (1H, d, J = 12.1 Hz), 3.91 (1H, d, J = 12.1 Hz), 3.97 (1H, dd, J = 12.6, 4.4 Hz), 4.06 (1H, dd,
J = 12.1, 2.4 Hz), 4.28 (1H, d, J = 12.6 Hz), 5.42 (1H, d, J = 6.3 Hz), 5.48 (1H, t, J = 5.1 Hz); 13C-NMR(CDCl3) δ 19.6, 27.1, 61.0, 62.4, 69.7, 79.0, 83.3, 84.5, 99.2; IR (KBr) 3000.8, 2892.3, 1699.7, 1380.9,
1089.9 cm−1; EI-MS (m/z): 267 [M+1]+; HRMS Calcd for C9H15N3O4: 266.0460, Found: 266.0467.
(3R*,4S*)-3-Azido-8,8-dimethyl-1,7,9,-trioxaspiro[4.5]decan-4-ol (18). A mixture of 7 [11,14](433 mg, 2.33 mmol) and NaN3 (752 mg 11.6 mmol) in 2-methoxyethanol (26 mL) was kept at 100 °C
for 5 h. After the solvent was removed under reduced pressure, the residue was dissolved in ethyl
acetate. After washed with water and brine, the organic layer was dried over Na2SO4. After filtration,
the solvents were removed under reduce pressure, the residue was purified by silica gel column
chromatography (hexane–ethyl acetate = 5:1) to give 18 (351 mg, 66%). 1H-NMR (CDCl3) δ 1.40 (3H,s), 1.49 (3H, s), 3.69-3.80 (3H, m), 3.84 (1H, d, J = 11.6 Hz), 4.01–4.08 (3H, m), 4.38 (1H, t, J = 1.9,
2.4 Hz); 13C-NMR (CDCl3) δ 19.5, 27.4, 62.3, 66.0, 67.6, 69.4, 78.2, 79.1, 98.5; IR (neat) 3419.0,
2104.8, 1086.6, 831.7 cm−1; EI-MS (m/z): 229 [M+1]+; HRMS Calcd for C9H15N3O4: 229.1063,
Found: 229.1062.
(3R*,4S*)-3-Azido-8,8-dimethyl-1,7,9,-trioxaspiro[4.5]decan-4-yl benzoate (19). To a solution of 18(432 mg, 1.88 mmol), Et3N (0.59 mL, 4.24 mmol), and DMAP (23 mg, 0.19 mmol) in CH2Cl2 (15 mL)
was added benzoyl chloride (0.40 mL, 3.39 mmol) and the mixture was stirred at room temperature for
6.5 h. The reaction was quenched by addition of MeOH, and the whole was stirred at room
temperature for 10 min. The mixture was diluted with CH2Cl2 and washed with water and brine, thendried over Na2SO4. After filtration, the solvents were removed under reduce pressure, the residue waspurified by silica gel column chromatography (hexane–ethyl acetate = 4:1) to give 19 (598 mg, 95%).
1
H-NMR (CDCl3) δ 1.37 (3H, s), 1.47 (3H, s), 3.84-3.91 (3H, m), 3.99 (1H, dd, J = 1.4, 10.6 Hz), 4.07
(1H, dd, J = 1.4, 10.6 Hz), 4.18-4.25 (2H, m) 5.47 (1H, d, J = 1.0 Hz), 7.48 (2H, t, J = 7.2 Hz), 7.62
(1H, J = 7.2 Hz), 8.03 (2H, J = 7.2 Hz); 13C-NMR (CDCl3) δ 22.4, 24.2, 62.1, 65.0, 66.2, 69.9, 78.9,
79.2, 98.4, 128.6, 129.0, 129.6, 133.6, 165.3; IR (neat) 2993.1, 2107.2, 1725.4, 1267.4, 1091.3, 711.5
cm−1; EI-MS (m/z): 333 [M]+; HRMS Calcd for C16H19N3O5: 333.1325, Found: 333.1336.
(3S*,4R*)-4-Azido-2,2-bis((methylsulfonyloxy)methyl)tetrahydrohuran-3-yl benzoate (20). A mixtureof 19 (1.01 g, 3.04 mmol) in 80% AcOH (80 mL) was stirred at room temperature for 5 h. After thesolvent was removed under reduced pressure, the residue was co-evaporated with EtOH five times toremove residual AcOH. The resulting crude product was dissolved in CH2Cl2 (40 mL). To thismixture, were added, MsCl (1.19 mL, 15.18 mmol), Et3N (2.14 mL, 15.18 mmol), and DMAP (38 mg,— 267 —

Molecules 2015, 20

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0.30 mmol). After stirred at room temperature for 1 h, the mixture was diluted with CH2Cl2 washedwith 5%HCl, sat.NaHCO3 and brine. The separated organic layer was dried over Na2SO4. After
filtration, the solvents were removed under reduce pressure, the residue was purified by silica gelcolumn chromatography (hexane–ethyl acetate = 2:1) to give 20 (1.05 g, 77%). 1H-NMR (CDCl3) δ
3.00 (3H, s), 3.12 (3H, s), 3.94 (1H, dd, J = 5.80, 4.37 Hz), 4.33~4.48 (6H, m), 5.48 (1H, d,
J = 3.4 Hz), 7.50 (2H, t, J = 7.7 Hz), 7.64 (1H, t, J = 8.0 Hz), 8.04 (2H, d, J = 7.3 Hz); 13C-NMR(CDCl3) δ 37.7, 65.5, 65.8, 67.4, 70.3, 77.2, 78.9, 82.8, 128.1, 128.8, 129.9, 134.2, 165.2; IR (near)
2110.7, 1728.8, 1360.0, 1267.0 cm−1; FAB-MS (m/z): 450 [M+1]+; HRMS Calcd for C15H20N3O9S2:
450.0641, Found: 450.0631.
((1R*,4R*,5S*)-4-Azido-2,6-dioxabicyclo[3.2.0]heptan-1-yl)methyl methanesulfonate (21). A mixtureof 20 (36 mg, 0.08 mmol) and NaOCH3 (4.6 mg, 0.08 mmol) in MeOH (2 mL) was kept at 75 °C
overnight. After the solvent was removed under reduced pressure, the residue was dissolved in CHCl3
and washed with water and brine, then dried over Na2SO4. After filtration, the solvents were removed
under reduce pressure, the residue was purified by silica gel column chromatography (hexane–ethyl
acetate = 2:1) to give 21 (14 mg, 72%). 1H-NMR (CDCl3) δ 3.09 (3H, s), 4.05 (1H, d, J = 3.4 Hz), 3.40
(1H, d, J = 11.1 Hz), 4.29~4.77 (4H, m), 4.76 (1H, d, J = 8.2 Hz), 5.06 (1H, s); 13C-NMR (CDCl3) δ37.8, 63.6, 67.4, 71.9, 77.1, 84.4, 89.9; IR (neat) 2098.2, 1360.4, 1175.0, 960.2, 815.5 cm−1; FAB-MS(m/z): 250 [M+1]+; HRMS Calcd for C7H12N3O5S: 250.0498, Found: 250.0490.
((1R*,4R*,5S*)-4-Azido-2,6-dioxabicyclo[3.2.0]heptan-1-yl)methyl benzoate (22). A mixture of CsF(332 mg, 2.19 mmol) and PhCOOH (267 mg, 2.19 mmol) in DMF (40 mL) was stirred at room
temperature for 20 min. To this mixture was added a solution of 21 (182 mg, 0.73 mmol) in DMF(20 mL). After stirred at 60 °C overnight, the mixture was partitioned between EtOAc and H2O. The
separated water layer was extracted with EtOAc, and the organic layer was washed with sat.NaHCO3,
brine, then dried over Na2SO4. After filtration, the filtrated was concentrated under reduced pressure.
The residue was purified by silica gel column chromatography (hexane-ethyl acetate = 5:1) to give 22(170 mg, 86%). 1H-NMR (CDCl3) δ 4.08 (1H, d, J = 3.4 Hz), 4.42 (1H, d, J = 10.6 Hz), 4.52 (2H, dd,
J = 10.6, 3.4 Hz), 4.60 (2H, s), 4.83 (1H, d, J = 7.7 Hz), 5.15 (1H, s), 7.61 (2H, t, J = 15.5 Hz) 7.59
(1H, t, J = 17.4 Hz), 8.10 (2H, d, J = 9.7 Hz); 13C-NMR (CDCl3) δ 63.1, 63.8, 71.5, 77.8, 85.1, 90.4,
128.4, 129.4, 129.7, 133.3, 166.1; IR (neat) 2099.7, 1723.1, 1284.3, 713.4 cm−1; FAB-MS (m/z);
276 [M+1]+; HRMS Calcd for C13H14N3O4: 276.0984, Found: 276.0977.
((1S*,4R*,5S*)-4-Azido-2,6-dioxa-bicyclo[3.2.0]heptan-1-yl)methanol (23). A mixture of 22 (184 mg,
0.67 mmol) and NaOCH3 (19 mg, 0.33 mmol) in MeOH (15 mL) was stirred at room temperature.
After the mixture was neutralized with AcOH (19 µL), the solvents were removed under reducedpressure and the residue was purified by silica gel column chromatography (hexane–ethyl acetate = 4:1)
to give 23 (90 mg, 79%). 1H-NMR (CDCl3) δ 3.91 (2H, d, J = 6.3 Hz), 4.04 (1H, d, J = 3.9 Hz), 4.36
(1H, d, J = 10.6 Hz), 4.48 (2H, dd, J = 10.6, 3.4 Hz), 4.48 (1H, d, J = 8.2 Hz), 4.68 (1H, d, J = 8.2 Hz),5.05 (1H, s); 13C-NMR (CDCl3) δ 62.3, 64.0, 71.5, 77.6, 86.9, 90.2; IR (neat) 3431.5, 2101.6, 1248.2,
870.88 cm−1; EI-MS (m/z): 171 [M]+; HRMS Calcd for C6H9N3O3: 171.0644, Found: 171.0643.

— 268 —

Molecules 2015, 20

4631((1S*,4R*,5S*)-4-(5-Amino-6-chloropyrimidin-4-ylamino)-2,6-dioxa-bicyclo[3.2.0]heptan-1-yl)methanol(15). A mixture of 23 (69 mg, 0.40 mmol) and Pd(OH)2 (6.2 mg, 0.04 mmol) in MeOH (5 mL) was
stirred at room temperature overnight under H2 atmosphere. After insoluble materials were removed byfiltration, the solvents were removed under reduced pressure. The resulting crude product wasdissolved in n-BuOH (3 mL). To this mixture, were added 5-amino-4,6-dichloropyrimidine (140.1 mg,
0.86 mmol) and i-Pr2NEt (298 µL, 1.71 mmol). The mixture was kept under reflux overnight. After thesolvents were removed under reduced pressure, the residue was purified by silica gel column
chromatography (chloroform–methanol = 19:1) to give 15 (64 mg, 58%). 1H-NMR (CD3OD) δ3.67(2H, q, J = 12.6 Hz), 4.24 (1H, d, J = 10.1 Hz), 4.38 (1H, d, J = 7.7 Hz), 4.45 (1H, d, J = 4.4 Hz), 4.50
(1H, d, J = 10.1, 4.4 Hz), 4.63 (1H, d, J = 7.2 Hz) 4.90 (1H, s), 7.72 (1H, s); 13C NMR (CD3OD) δ
58.1, 58.3, 62.7, 73.1, 88.3, 92.1, 125.5, 138.9, 147.3, 153.3; IR (KBr) 3381.3, 2926.7, 1578.6,
1056.3 cm−1; EI-MS (m/z): 272 [M]+; HRMS Calcd for C10H13ClN4O3: 272.0676, Found: 272.0673.
((1S*,4R*,5S*)-4-(6-Chloro-9H-purin-9-yl)-2,6-dioxa-bicyclo[3.2.0]heptan-1-yl)methanol (24). To asolution of 15 (18 mg, 0.07 mmol) in DMF (0.5mL), were added orthoethyl formate (0.7 mL,
4.21 mmol) and conc HCl (2 µL, 0.024 mmol) at 0 °C. After the mixture was stirred at room
temperature, the solvents were removed under reduced pressure. The residue was dissolved in 0.5 M
aqHCl (1 mL) and the mixture was stirred at room temperature for 1 h. The mixture was neutralizedwith 1M aqNaOH (0.5 mL) and concentrated under reduced pressure. The residue was extracted with asolution of chloroform–methanol = 1:1. After the insoluble materials were removed by filtration, thesolvents were removed under reduced pressure. The residue was purified by pTLC (developed bychloroform-methanol = 5:1) to give 24 (14.9 mg, 81%). 1H-NMR (CDCl3) δ 4.12 (2H, q, J = 12.6 Hz),4.59 (1H, d, J = 11.6 Hz), 4.66 (1H, d, J = 9.7 Hz), 4.68 (1H, d, J = 9.7 Hz), 4.66 (1H, d, J = 9.7 Hz),4.90 (1H, dd, J = 11.1, 4.8 Hz), 5.24 (1H, s), 5.38 (1H, d, J = 4.4 Hz), 8.68 (1H, s), 8.76 (1H, s);
13
C-NMR (CDCl3-CD3OD = 19 : 1) δ 58.6, 61.2, 71.9, 77.2, 87.8, 90.9, 130.7, 144.6, 150.7, 151.2,
151.8; IR (KBr) 3401.4, 2931.1, 1597.4, 1567.4, 1056.6 cm−1; EI-MS (m/z): 282 [M]+; HRMS Calcd
for C11H11ClN4O3: 282.0520, Found: 282.0506.
((1S*,4R*,5S*)-4-(6-Amino-9H-purin-9-yl)-2,6-dioxa-bicyclo[3.2.0]heptan-1-yl)methanol(6).
Compound 24 (29.4 mg, 0.10 mmol) was dissolved in sat. methanolic ammonia (7 mL) and the
mixture was kept at 100 °C for 21 h in a glass sealed tube. After the solvents were removed under
reduced pressure, the residue was purified by pTLC (developed by chloroform–methanol = 5:1) to give
6 (18.8 mg, 69%). 1H-NMR (CDCl3-CD3OD = 17:3) δ 3.91 (1H, d, J = 12.6 Hz), 4.00 (1H, d,
J = 12.1 Hz), 4.57 (1H, d, J = 10.6 Hz), 4.64 (1H, d, J = 7.7 Hz), 4.70 (1H, d, J = 7.7 Hz), 4.87 (1H,
dd, J = 11.1, 4.8 Hz), 5.19 (1H, s), 5.25 (1H, d, J = 4.8 Hz), 8.25 (1H, s), 8.28 (1H, s); 13C-NMR(CDCl3–CD3OD = 17:3) δ 29.5, 58.3, 61.3, 72.1, 87.6, 91.0, 118.2, 139.1, 148.9, 152.6, 155.3; IR(KBr) 3192.2, 2409.9, 1660.5, 1615.0, 1054.9 cm−1; EI-MS (m/z): 263 [M]+; HRMS Calcd for
C11H13N5O3: 263.1018, Found: 263.1021.
4. Conclusions
We constructed an isoadenosine derivative on a 2,6-dioxobicyclo[3.2.0]heptane scaffold. Since ourinitial attempt to synthesize 6 by directly introducing the adenine moiety was not successful, we— 269 —

Molecules 2015, 20

4632

synthesized it by the de novo synthesis of an adenine ring on a pseudosugar moiety. However, thisunique adenosine analogue showed no activity against HIV. Previously, we have reported that neither
thymine nor adenine analogues 4 built on a 2-oxa-6-thiabicyclo[3.2.0]heptane skeleton inhibit HIV [14].
The structural rigidities of these analogues and isoadenosine 6 due to the introduction of fused thietane
and oxetane rings, respectively, appear to inhibit anti-HIV activity. In particular, phosphorylation at
the 5'-hydroxyl group would be inhibited since deoxynucleoside kinase recognizes the puckering ofsugars [25]. Thus, we are currently preparing new substituted nucleoside derivatives based on 4 and 6,
and the results will be reported elsewhere.
Acknowledgments
This work was supported in part by a Grant-in-Aid for Scientific Research (No. 24590143, Y.Y.)
from JSPS and by a grant of Strategic Research Foundation Grant-aided Project for Private Universities
from Ministry of Education, Culture, Sport, Science, and Technology, Japan (MEXT), 2010–2014.
Author Contributions
YY and TI designed research; SK, HK and TS performed the synthesis of compounds and TI
assayed anti-HIV activity. YY wrote the paper. All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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BIOPHARMACEUTICS & DRUG DISPOSITION
Biopharm. Drug Dispos. 36: 139–147 (2015)
Published online 25 January 2015 in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/bdd.1927

Simultaneous evaluation of human CYP3A4 and ABCB1
induction by reporter assay in LS174T cells, stably expressing
their reporter genes
Keita Inami, Takamitsu Sasaki, Takeshi Kumagai, and Kiyoshi Nagata*
Department of Environmental and Health Science, Tohoku Pharmaceutical University, Sendai, Japan

ABSTRACT: The bioavailability of orally administered therapies are often signiﬁcantly limitedin the human intestine by the metabolic activities of cytochrome P450 3A4 (CYP3A4) andP-glycoprotein (P-gp). Predicting whether candidate compounds induce CYP3A4 and P-gp is a
crucial stage in the drug development process, as drug–drug interactions may result in the inductionof intestinal CYP3A4 and P-gp. However, the assay systems needed to evaluate both CYP3A4 andP-gp induction in the intestine are yet to be established. To address this urgent requirement,
LS174T cells were used to create two stable cell lines expressing the CYP3A4 or ATP-binding cassette
subfamily B member 1 (ABCB1, encoding P-gp) reporter genes. First, these stable cells were tested bytreatment with 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), all-trans-retinoic acid (ATRA) and 9-cisretinoic acid (9-cis RA) that induce CYP3A4 and P-gp in the intestines. All these compounds signiﬁcantly increased both CYP3A4 and ABCB1 reporter activities in the stable cell lines. To simultaneously
assess the induction of CYP3A4 and ABCB1, both stable cells were co-cultivated to measure theirreporter activities. The mixed cells showed a signiﬁcant increase in the CYP3A4 and ABCB1 reporter
activities following treatment with 1,25(OH)2D3, ATRA, and 9-cis RA. These activity levels were maintained after passaging more than 20 times and following multiple freeze–thaw cycles. These results
demonstrate that our established cell lines can be used to evaluate simultaneously CYP3A4 and
ABCB1 induction in the intestines, providing a valuable in vitro model for the evaluation of future
drug candidates. Copyright © 2014 John Wiley & Sons, Ltd.
Key words: CYP3A4; P-gp; stable cell line; LS174T; reporter assay

Introduction
Cytochrome P450 (CYP) plays important roles in
the metabolism of various xenobiotics and endogenous compounds [1]. Of the CYPs isoforms,
CYP3A4 is the most abundant, found in the adulthuman liver and small intestines. It is estimatedthat hepatic and intestinal CYP3A4 activity metabolizes approximately half of the prescribed drugs*Correspondence to: Department of Environmental and
Health Science, Tohoku Pharmaceutical University, 4-4-1,
Komatsushima, Aoba-ku, Sendai 981-8558, Japan.
E-mail: nagataki@tohoku-pharm.ac.jp

used today [2–4]. In addition to CYP, the ATP dependent efﬂux transporter, P-glycoprotein (P-gp)
has a crucial role in limiting intracellular drug concentrations, therefore reducing the bioavailabilityof many prescribed drugs [5]. P-gp, also known
as the multi-drug resistance protein 1, is encodedby the ATP-binding cassette subfamily B member 1
gene (ABCB1) and is expressed in various tissues
including the liver, small intestine, the blood–brain
barrier, kidney and the adrenal glands [6].
Both CYP3A4 and P-gp contribute signiﬁcantly
in limiting the bioavailability of orally administered drugs. This is due to similar substrate speciﬁcities, as well as to high levels of expression in
Received 23 June 2014
Revised 27 October 2014
Accepted 13 November 2014

Copyright © 2014 John Wiley & Sons, Ltd.

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K. INAMI ET AL.

the liver and small intestine [7]. Furthermore, theexpression of CYP3A4 and P-gp is often induced
simultaneously by many drug therapies [8]. To
date, many researchers have reported clinically relevant drug–drug interactions, mediated throughCYP3A4 and/or P-gp [9,10]. It is therefore important in the drug development process, to consider
whether candidate compounds induce CYP3A4
and ABCB1 expression.
Recently, studies have shown that expression of
the CYP3A4 and ABCB1 genes are regulated by
nuclear receptors, such as the pregnane X receptor(PXR), constitutive androstane receptor, and thevitamin D receptor (VDR) [11–13]. Nuclear receptors are ligand-activated transcription factors,
which mediate the body’s defense mechanism
against toxic compounds and xenobiotics. The
PXR is activated by a broad spectrum of xenobiotics and plays a key role in mediating the body’s
defense mechanism against toxic substances, including prescription drugs. Upon ligand-binding,the PXR receptor is translocated to the nucleus
where it forms a heterodimer with the retinoid X
receptor α [14]. The heterodimer then binds to
the regulatory cis-elements, which speciﬁcally activates a number of genes involved in xenobiotic
elimination that include CYP3A4. Hepatic andintestinal induction of CYP3A4 and P-gp occurs
via different ligand-speciﬁc, receptor-deﬁned pathways. For example, rifampicin activation of
PXR, mediates transactivation of the CYP3A4
predominantly in the liver. However, 1α,25dihydroxyvitamin D3 (1,25(OH)2D3), mediates
activation of the VDR, resulting in intestinal
transactivation of CYP3A4 [15]. As with CYP3A4,
the induction of P-gp in the liver and intestine also
occurs via different pathways. This is because theexpression of PXR is higher than VDR in the liver;
however, VDR expression is higher than PXR in
the intestines [16]. Consequently, the expression
proﬁles for nuclear receptors would need to beconsidered, when selecting a suitable cell line for
evaluating intestinal CYP3A4 and P-gp induction.
Several in vitro systems exist for the assessment of
CYP3A4 and P-gp induction in the liver [17–19].
However, an in vitro system for evaluating the effect of drug absorption on intestinal CYP3A4 andP-gp induction has yet to be established.
A reporter gene under the control of the
CYP3A4 and ABCB1 regulatory regions provides

a rapid and sensitive expression system. However,a plasmid based reporter system is both timeconsuming and cost-intensive, because transienttransfection of the reporter plasmid is required before each experiment. In addition, the data fromthese experiments often vary, due to variations in
the transfection efﬁciency. To solve this problem
several research groups, including our own, havedeveloped a stable reporter CYP gene expression
system, constructed by inserting the reporter gene
into the chromosomes of HepG2 cells [20–22].
The reporter cell lines have the advantage of being
easily maintained and readily available, as well as
increasing experimental reproducibility in comparison with the transient expression systems.
In this study, two stable cell lines were
established, expressing chromosomally integrated
reporter genes driven by CYP3A4 and ABCB1
regulatory regions. The stable cell lines are termed
1-2-10 and 3-13, respectively, and were generatedusing LS174T. The utility of these stable genereporter systems was demonstrated, using the
CYP3A4 and ABCB1 inducers, 1,25(OH)2D3,
9-cis-retinoic acid (9-cis RA), and all-trans-retinoic
acid (ATRA) [18,23]. Our results clearly demonstrate a highly sensitive and reproducible expression system for measuring the induction response
for both CYP3A4 and ABCB1. It is concluded thatthese two cell lines provide a rapid and reproducible assay for identifying compounds that simultaneously induce CYP3A4 and ABCB1.

Materials and Methods
Materials
The 1,25(OH)2D3 was purchased from SigmaAldrich (St Louis, MO). The 9-cis RA and ATRA
were purchased from Wako Pure Chemicals(Osaka, Japan). Dimethyl sulfoxide (DMSO) was
obtained from Nacalai Tesque (Kyoto, Japan). Oligonucleotides were commercially synthesized by
Fasmac (Atsugi, Japan).

Construction of the reporter plasmids
The reporter plasmid pGL3-CYP3A4-362-7.7 k forthe human CYP3A4 gene was described previously [21]. The pGL3-CYP3A4-362-7.7 k includes
the promoter region (362 bp to +11 bp) and distal

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EVALUATING INTESTINAL CYP3A4 AND P-GP INDUCTION

nuclear receptor binding elements (7836 bp to7208 bp) of the human CYP3A4 gene derived fromhuman liver. The reporter plasmid of the human
ABCB1 gene was constructed using the p-8935
construct that was reported by Tachibana et al. [24].
Brieﬂy, the p-8935 construct, which included the5′-untranslated region of the ABCB1 gene from
8935 bp to +126 bp, was digested with SpeI and
AatII (Takara Bio, Otsu, Japan), and the region
between the enhancer region (from 8935 bp to7472 bp) and the promoter region (from 601 bp
to +126 bp) was removed and then both regions
were directly ligated. The obtained construct
pSEAP2-ABCB1-601-(9-7.5 k) and pGL4.70[hRluc]
vector (Promega, Madison, MI) were digested with
Acc65I (New England Biolabs, Ipswich, MA) and
HindIII (Nippon Gene, Tokyo, Japan). These
digested constructs were ligated together to obtainthe construct pGL4.70-ABCB1-601-(9-7.5 k).

Generation of stable cell lines
In order to generate a ligation for transfection,
the reporter plasmids pGL3-CYP3A4-362-7.7 k,
pGL4.70-ABCB1-601-(9-7.5 k) were linearized with
BamHI and BglII (Nippon Gene, Tokyo, Japan).
The pQBI plasmid (Wako Pure Chemicals) was
also linearized with BamHI and BglII resulting in
a 4500 bp fragment, which included the neomycin
resistance gene. The reporter plasmid fragments
were ligated into the linearized pQBI plasmid,
using the DNA ligation kit ver. 2.1 (Takara Bio).
The human colon adenocarcinoma cell line, LS174T
was obtained from the Institute of Development,
Aging and Cancer, Tohoku University (Sendai,
Japan). The cells were cultured under 5% CO2 at
37°C in Eagle’s minimum essential medium (EMEM,
Wako Pure Chemicals), supplemented with 10%
fetal bovine serum (FBS, Biowest, Miami, FL) andAntibiotic-Antimycotic (Invitrogen, Carlsbad, CA).
The cells were seeded in 6-well tissue culture plates(BD Biosciences, Heidelberg, Germany) at 3 × 105
cells per well, one day before transfection. The
ligation fragments were transfected using TargefectF-1 (Targeting Systems, EI Cajon, CA), according to
the manufacturer’s instructions. After a 2-week incubation, the cells were selected with 500 μg/mL
geneticin (G418, Invitrogen). The positive colonies
were further subcloned into 24-well tissue culture
plates (BD Biosciences) to obtain monoclonal cells.

G418-resistant clones were chosen for induction
testing by treating the cells with 10 nM 1,25(OH)2D3.
The induction response was measured by theluciferase assay. Clones exhibiting the strongest
induction response of CYP3A4 and ABCB1 weretermed 1-2-10 cells and 3-13 cells, respectively.

Cell culture and cell extract
The stable cells were cultured in EMEM (Wako
Pure Chemicals) containing 10% FBS (Biowest)
and the Antibiotic-Antimycotic (Invitrogen). The
cells were seeded in 48-well tissue culture plates(BD Biosciences) at 8 × 104 cells per well in 0.2 mL
EMEM. After 24 h, the cell medium was changed
with the medium containing various drugs dissolved in DMSO (ﬁnal concentration, 0.1%). The
control cells were treated with 0.1% DMSO. After
6–48 h exposure to various drugs, the cells were
washed twice with Dulbecco’s phosphate bufferedsaline and suspended in 50 μL of passive lysis
buffer (Promega). The cell suspension was centrifuged at 2000 rpm for 10 min at 4°C. The supernatant was processed for luciferase assay anddetermination of protein concentration. The protein
concentration was measured using the Bio-Rad
protein assay (Bio-Rad Laboratories, Hercules, CA).

Luciferase assay
The luciferase assay system (Promega) and theRenilla luciferase assay system (Promega) wereused for 1-2-10 and 3-13 cells according to the manufacturer’s instructions. The luciferase activity was
normalized to the total protein content andexpressed as a ratio of the luminescence of 0.1%
DMSO-treated cells. The luminescence obtained
from reaction of the luciferase assay was then monitored by a GlomaxTM 96 microplate luminometer(Promega). Simultaneous measurement of ﬁreﬂy
and Renilla luciferase activity was performed using
the dual-luciferase reporter assay system (Promega)according to the manufacturer ’s instructions.

RNA puriﬁcation and real-time PCR
Total RNA was extracted using TRI reagent(Molecular Research Center, Cincinnati, OH). Firststrand cDNA was synthesized from 1 μg total
RNA in a 25 μL reaction mixture using Moloneymurine leukemia virus reverse transcriptase

Copyright © 2014 John Wiley & Sons, Ltd.

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K. INAMI ET AL.

(Promega), oligo(dT)20 primer and recombinant
RNase inhibitor (Takara Bio). The cDNA wasused as a template for real-time PCR using SYBR
Premix Ex Taq (Takara Bio) to measure the expression levels of CYP3A4 and ABCB1. The control used for all real-time PCR analysis was the
GAPDH gene, which encodes glyceraldehyde3-phosphate dehydrogenase. The ampliﬁcation
reactions were performed with the following
speciﬁc primers: CYP3A4 (forward: 5′-CTGT
GTGTTTCCAAGAGAAGTTAC-3′ and reverse: 5′TGCATCAATTTCCTCCTGCAG-3′), ABCB1 (forward: 5′-GCCAAAGCCAAAATATCAGC-3′ andreverse: 5′-TTCCAATGTGTTCGGCATTA-3′), and
GAPDH (forward: 5′-CATGGGTGTGAACCATG
AGAA-3′ and reverse: 5′-GGTCATGAGTCCTTC
CACGAT-3′). Quantitative values were obtained
above the threshold PCR cycle number (Ct) at
which an increase in signal associated with an exponential growth in PCR products was detectedusing Thermal Cycler DiceTM TP800 (Takara Bio).
Relative mRNA expression levels for each sample
were obtained using the 2-ΔΔCt method, normalized to GAPDH expression levels.

Statistical analysis
Data were evaluated using the paired Student’s
t-test. Values of p < 0.05 were considered statistically signiﬁcant.

Results
Dose-dependent induction of the CYP3A4 and
ABCB1 reporter genes by 1,25(OH)2D3
The two stable cell lines, 1-2-10 and 3-13, wereconstructed by inserting the CYP3A4 and ABCB1
reporter plasmids, into the chromosomes of LS174T
cells. To characterize the isolated stable cell lines,
a dose-dependent induction of reporter activity
was measured. The reporter activity for CYP3A4
and ABCB1 was determined at four time intervals,
6, 12, 24, and 48 h, following the addition of 1,25
(OH)2D3 to the culture medium at concentrationsin the range 1–100 nM and 3–500 nM. The results
show a dose-dependent increase in the luciferase
activity for both the CYP3A4 and ABCB1 reporter
plasmids, in response to increasing concentrationsof 1,25(OH)2D3 (Figure 1). The response peaked at
24 h, and thereafter the level decreased. The
maximum induction of CYP3A4 reporter activity
occurred when the 1-2-10 cells were exposed to
100 nM of 1,25(OH)2D3 for 24 h. This resulted in
an 80-fold increase in the reporter gene activity
when compared with the control (Figure 1A). The
CYP3A4 reporter activity was readily detectablein the 1-2-10 cells, even when a low concentrationof 1,25(OH) 2D3 (1 nM) for 24 h was used (Figure 1A).
A maximum increase in ABCB1 reporter activity
was seen when the 3-13 cells were treated with

Figure 1. Dose-dependent induction of the CYP3A4 and ABCB1 reporter genes by 1,25(OH)2D3. The 1-2-10 or 3-13 cells were seeded4
at 8 × 10 cells per well in 48-well tissue culture plates one day before drug treatment. The following day, the cell medium was
exchanged to a medium containing the various concentrations of 1,25(OH)2D3. The cell lysate was harvested at 6 h (closed diamonds), 12 h (closed squares), 24 h (closed triangles), and 48 h (closed circles) after drug treatment. The lysate was used to assay
for luciferase activity and to determine protein concentration. The luciferase activity was normalized with protein concentration,and the reporter activity in 0.1% DMSO-treated cells was set equal to 1.0. Each point represents mean ± SD (n = 3). *, **, †, and††, p < 0.05 in the cells treated with 1,25(OH)2D3 for 6 h, 12 h, 24 h, and 48 h versus 0.1% DMSO-treated cells. (A) CYP3A4 reporter
activity, (B) ABCB1 reporter activity
Copyright © 2014 John Wiley & Sons, Ltd.

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EVALUATING INTESTINAL CYP3A4 AND P-GP INDUCTION

500 nM for 24 h. This gave a 14-fold increase inreporter activity in comparison with the control(Figure 1B). Generally, Renilla luciferase shows ahigher background than ﬁreﬂy luciferase becauseof the autoluminescence. Consistent with this, the3-13 cells showed higher background luminescence than that of the 1-2-10 cells.

The effect of cell density on 1,25(OH)2D3 induced
reporter activity in the stable cell lines
The effect of various cell densities on luciferase
activity was also examined. The stable cells wereseeded at densities of 3 × 104, 4 × 104, 6 × 104,
8 × 104, 10 × 104, and 12 × 104 cells per well in
48-well tissue culture plates. CYP3A4 and ABCB1
reporter activities were induced with 10 nM or
300 nM 1,25(OH)2D3 and measured after 24 h.
Increases in the CYP3A4 reporter activity occurredin a cell density-dependent manner with thehighest cell density (12 × 104 cells per well)
exhibiting the greatest induction (Figure 2A).
However, the ABCB1 reporter activity decreased
in a cell density-dependent manner (Figure 2B).

Transcriptional activation of the CYP3A4 and
ABCB1 genes by retinoids
Wang et al. (2008) have reported that retinoids suchas 9-cis RA and ATRA, induce CYP3A4 activity
through activation of the VDR in the humancolorectal adenocarcinoma cell line Caco-2 [25].
The study tested whether these compounds could
induce the reporter activity in the stable cells. The
results show that 9-cis RA at a concentration of
100 μM caused a 2.3-fold increase in luciferase
activity in 1-2-10 cells (Figure 3A). Similarly, ATRA
at a concentration of 20 μM increased the luciferase
activity by 1.8-fold in 1-2-10 cells. With respect tothe 3-13 cells, treatment with 100 μM 9-cis RA and20 μM ATRA resulted in an increase of luciferase
activity by 3.1-fold and 2.2-fold, respectively(Figure 3B).

The effect of 1,25(OH)2D3 and retinoids on
CYP3A4 and ABCB1 expression levels
Real-time PCR was performed to examine the effect of 1,25(OH)2D3 and retinoids on the expression of intrinsic CYP3A4 and ABCB1 in the stable
cell lines. When the 1-2-10 cells were treated with
10 nM 1,25(OH)2D3, CYP3A4 expression increasedby 76-fold in comparison with the control(Figure 4A). Treatment of the 1-2-10 cells with
100 μM 9-cis RA and 20 μM ATRA resulted in agreater than 3-fold increase in CYP3A4 expression
compared with the control (Figure 4A). The effectof these compounds on ABCB1 expression in 3-13
cells was also examined. Induction with 300 nM
1,25(OH)2D3, 100 μM 9-cis RA and 20 μM ATRA
resulted in an increase of ABCB1 expression by
5.9-, 2.3-, and 3.6-fold, respectively (Figure 4B).

Figure 2. The effect of cell density on 1,25(OH)2D3 induced reporter activity in the stable cell lines. The 1-2-10 or 3-13 cells were
4
4
4
4
4
4
seeded in 48-well tissue culture plates at various cell densities; 3 × 10 , 4 × 10 , 6 × 10 , 8 × 10 , 10 × 10 and 12 × 10 cells per well
24 h before drug treatment. The cells were treated with 1,25(OH)2D3 (10 or 300 nM) for 24 h, and then the lysate was harvested.
The lysate was used to assay for luciferase activity and to determine protein concentration. The luciferase activity was normalizedusing protein concentration, and the reporter activity in 0.1% DMSO-treated cells was set equal to 1.0. The values represent mean
± SD (n = 4). *, p < 0.05 versus 0.1% DMSO-treated cells. (A) CYP3A4 reporter activity, (B) ABCB1 reporter activity
Copyright © 2014 John Wiley & Sons, Ltd.

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K. INAMI ET AL.

4

Figure 3. Transcriptional activation of the CYP3A4 and ABCB1 genes by retinoids. The 1-2-10 or 3-13 cells were seeded at 8 × 10
cells per well in 48-well tissue culture plates and treated with 1,25(OH)2D3 (10 or 300 nM), 9-cis RA (100 μM) or ATRA (20 μM)
for 48 h. The cells were harvested and the lysate was used to assay for luciferase activity and to determine protein concentration.
The luciferase activity was normalized with protein concentration, and the reporter activity in 0.1% DMSO-treated cells was set
equal to 1.0. The values represent mean ± SD (n = 3). *, p < 0.05 versus 0.1% DMSO-treated cells. (A) CYP3A4 reporter activity,(B) ABCB1 reporter activity

Figure 4. Effect of 1,25(OH)2D3 and retinoids on the expression levels of CYP3A4 and ABCB1 mRNA. The 1-2-10 or 3-13 cells were5
seeded at 1.5 × 10 cells per well in 24-well tissue culture plates 24 h before drug treatment. The cells were treated with 1,25(OH)2D3(10 or 300 nM), 9-cis RA (100 μM) or ATRA (20 μM). After 48 h, the total RNA was extracted and real-time PCR was carried out asdescribed in Materials and Methods. CYP3A4 and ABCB1 mRNA levels were normalized to those of GAPDH. The mRNA expression level in 0.1% DMSO-treated cells was set equal to 1.0. The values represent mean ± SD (n = 4). *, p < 0.05 versus 0.1% DMSOtreated cells. (A) CYP3A4 mRNA level, (B) ABCB1 mRNA level

Simultaneous measurement of CYP3A4 and
ABCB1 reporter activity
The 1-2-10 and 3-13 stable cells were seeded atvarious ratios 3:5, 1:1, 5:3, and 3:1 respectively, toa total cell number of 8 × 104 cells per well. The cells
were exposed to 300 nM 1,25(OH)2D3 for 24 h, after
which a dual-luciferase reporter assay was performed. As expected the 1-2-10 cells demonstrated
an increase in CYP3A4 reporter activity that
increased in a density-dependent manner. Whereas
the 3-13 cells showed a reduction in the ABCB1

reporter activity which decreased in a cell densitydependent manner (data not shown). These results
are consistent with the results described above,shown in Figure 2. The reporter activities for bothCYP3A4 and ABCB1 showed the greatest induction
when the 1-2-10 and 3-13 cells were mixed at a ratioof 3:1. Consequently, 1-2-10 and 3-13 cells weremixed at a ratio of 3:1, and subcultured continuallyfor more than 20 passages. The mixed cells showeda signiﬁcant increase in CYP3A4 and ABCB1 reporter
activity following treatment with 1,25(OH)2D3 andCopyright © 2014 John Wiley & Sons, Ltd.

Biopharm. Drug Dispos. 36: 139–147 (2015)
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EVALUATING INTESTINAL CYP3A4 AND P-GP INDUCTION

Figure 5. Simultaneous measurement of CYP3A4 and ABCB1 reporter activity. 1-2-10 and 3-13 cells were mixed at a ratio of 3:1, and4
the mixed cells were seeded at 8 × 10 cells per well in 48-well tissue culture plates 24 h before drug treatment. The cells were treatedwith 1,25(OH)2D3 (300 nM), 9-cis-RA (100 μM) and ATRA (20 μM) for 48 h. The cells were harvested and the lysate was used fordual-luciferase reporter assay. The reporter activity in 0.1% DMSO-treated cells was set equal to 1.0. The values represent mean
± SD (n = 3). *, p < 0.05 versus 0.1% DMSO-treated cells. (A) CYP3A4 reporter activity, (B) ABCB1 reporter activity

retinoids. Induction with 300 nM 1,25(OH)2D3,
100 μM 9-cis RA and 20 μM ATRA resulted in a4.6-, 1.3-, and 1.3-fold increase of CYP3A4 reporter
activity, respectively, in comparison with the DMSO
control (Figure 5A). These activators also increased
the ABCB1 reporter activity by 3.3-, 2.0-, and 2.7-foldabove the DMSO control (Figure 5B).

Discussion
In the drug development process, the efﬁcient
screening systems are required for shortening thedevelopment period and to reduce cost. Cell-based
reporter assay systems have been used extensively
to screen CYP inducers as reliable and highthroughput methods [26]. Predicting whether
drug candidates induce intestinal CYP3A4 andP-gp is crucial as well as in the liver. In fact,
Greiner et al. (1999) have reported clinically
relevant drug interaction between digoxin andrifampicin caused by intestinal P-gp [27]. However, the in vitro intestinal models for predicting
CYP3A4 and P-gp are yet to be established. In
this study, LS174T cells were chosen for constructing the stable cell lines expressing CYP3A4 or
ABCB1 reporter genes, because LS174T cells
were used widely as an in vitro human intestinalmodel [24].
The stable cells were ﬁrst characterized by measuring the transactivation of CYP3A4 and ABCB1
by 1,25(OH)2D3, which is a strong inducer of thesegenes in the intestine [28]. As shown in Figure 1,
1,25(OH)2D3 induced the reporter activity in adose-dependent manner in both the 1-2-10 and
3-13 cells, with the response reaching a peak at
24 h. The effect of cell density on the reporter
activity was also examined. The results showedthat the reporter activity increased in a celldensity-dependent manner in the 1-2-10 cells,
whereas reporter activity gradually diminishedin the 3-13 cells (Figure 2). One reason for thismay be due to a cell density-dependent increase
in reporter activity in non-treated 3-13 cells
resulting in a high background compared withthe 1-2-10 cells. However, the 3-13 cells did
display a signiﬁcant reporter activity above the background level needed to assess ABCB1 induction.
To gauge the usefulness of these stable cells,
the response of the reporter activity for known
inducers of CYP3A4 and ABCB1 in the intestine
was investigated. In both the 1-2-10 and 3-13
cells, the compounds tested, which included
9-cis RA (100 μM) and ATRA (20 μM), caused
signiﬁcant increases in reporter activity (Figure 3),
as well as increasing the expression of CYP3A4/
ABCB1 (Figure 4). Other researchers have demonstrated the transactivation of CYP3A4 and ABCB1
genes caused by 1,25(OH)2D3 using transient
expression systems. Pavek et al. (2010) and Saeki
et al. (2008) have reported that approximately
30- and 9-fold induction of CYP3A4 and ABCB1
reporter activity, respectively, has been achievedby transducing human VDR into LS174T or

Copyright © 2014 John Wiley & Sons, Ltd.

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Caco-2 cells [18,29]. Our data from this study iscomparable to these results, without the overexpression of VDR. In addition, the stable cells created in this study, have the advantage of being
more convenient than transient transfection andtransduction methods.
CYP3A4 and P-gp are often co-induced and act
synergistically to limit oral drug bioavailability.
As one example, co-induction of CYP3A4 andP-gp by corticosteroids reduced the tacrolimus
blood concentration [30]. Therefore, simultaneousassessment of CYP3A4 and P-gp induction has
implications for preventing drug–drug interactions. For this reason, our group attempted toinsert both the CYP3A4 and ABCB1 reporter plasmids into LS174T cells, to create a double-stable
cell line. Unfortunately, the resulting cells showed
little reporter activity in response to inducers.
Therefore, the 1-2-10 and the 3-13 cells were cocultivated at a ratio of 3:1. The mixed cells wereused for a dual-luciferase reporter assay where
they showed a signiﬁcant increase in bothCYP3A4 and ABCB1 reporter activity, following
treatment with 1,25(OH)2D3 and retinoids(Figure 5). In addition, the responsiveness of themixed cells was sustained for more than 20
subculturing passages, and after freeze-thawing.
These results indicate that the mixed cells may
be applicable for the simultaneous assessment of
CYP3A4 and P-gp, and may be suitable for highthroughput screening of candidate compounds.

Conclusion
In this study, two stable cell lines were establishedto assess intestinal CYP3A4 and ABCB1 induction. These cells showed highly sensitive andreproducible induction in response to 1,25(OH)
2D3, 9-cis RA, and ATRA, and the responsivenessof the stable cell lines was comparable to thoseof the assay systems previously reported. Furthermore, the stable cells have the advantage of being
more readily available, as well as being applicablefor simultaneous measurement of CYP3A4 and
ABCB1 reporter activity by co-cultivation. In
conclusion, these cells have the potential topredict which candidate drugs cause intestinal
CYP3A4 and P-gp induction, providing a useful
tool in the drug development process.

Acknowledgements
This work was supported by a Health and Labor
Sciences Research Grant for Research on Food
Safety (No. 22230301) from the Ministry of Health,
Labor and Welfare of Japan; and by the Japanese
Ministry of Education, Culture, Sports, Scienceand Technology (MEXT)-Supported Program for
the Strategic Research Foundation at Private
Universities.

Conﬂict of Interest
The authors have no conﬂicts of interest to
declare.

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Biopharm. Drug Dispos. 36: 139–147 (2015)
DOI: 10.1002/bdd— 281 —J Pharm Pharm Sci (www.cspsCanada.org) 18(4) 316 - 327, 2015

Development of a Highly Reproducible System to Evaluate Inhibition of
Cytochrome P450 3A4 Activity by Natural Medicines
Yu Sato, Takamitsu Sasaki, Shogo Takahashi, Takeshi Kumagai, Kiyoshi Nagata
Department of Environmental and Health Science, Tohoku Pharmaceutical University, Miyagi, Japan.
Received, June 2, 2015; Revised, July 17, 2015; Accepted, July 28, 2015; Published, August 4, 2015.

ABSTRACT – PURPOSE: In recent years, a number of natural medicines have been reported to haveinductive or inhibitive effects on the activity of drug metabolizing enzymes, upon co-administration withprescribed medicines. However, information regarding natural medicine-drug interactions that influencedrug metabolism is limited owing to the lack of efficient screening method for such interactions. Therefore,
to understand whether P450 activity is affected by natural medicine, we have established frozen recombinant
P450-expressing cells infected with human CYP3A4 expressing adenovirus (Ad-CYP3A4) to evaluate theeffect of natural medicines on CYP3A4 activity. METHODS: Ad-CYP3A4 cells were created by infecting
HepG2 cells with Ad-CYP3A4 at 10 multiplicity of infection (MOI) and these cells were stored using
cryopreservation medium (fAd-CYP3A4 cells) to obtain long-term consistent data and stable supplies ofcells expressing a constant level of CYP3A4 activity. RESULTS: The CYP3A4 activity in fAd-CYP3A4
cells remained unaffected at the end of each frozen period (0, 1, 2, and 6 months). Inhibitory effect on
CYP3A4 activity by typical inhibitors (ketoconazole, hyperforin) and natural medicines (Cat’s Claw, Devil’s
Claw, Feverfew, Peppermint Oil, Red Clover, and Siberian Eleuthero) were evaluated. The inhibitors had
nearly equal IC50 values in fAd-CYP3A4 cells, Ad-CYP3A4 cells and recombinant CYP3A4 microsomes.
Cat’s Claw, Peppermint Oil and Siberian Eleuthero inhibited CYP3A4 activity more potently than 0.1 μM
ketoconazole in fAd-CYP3A4 cells. CONCLUSIONS: In the present study, we have successfully
developed a highly reproducible system to evaluate CYP3A4 inhibition by natural medicines.
This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For
Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.
_______________________________________________________________________________________INTRODUCTION

of felodipine (5-7). GFJ reduces the first-pass effectof felodipine by inhibition of its metabolism by
CYP3A4. Therefore, it is important to evaluate not
only drug-drug interaction but also drug-dietary
substance or drug-natural product interaction.
Natural medicines have been used for various
health conditions worldwide. These have been used
for self-medication without medical supervision
and are believed to be safe. In recent years, anumber of natural medicines have been reported tohave inductive or inhibitive effects on the activityofdrug
metabolizing
enzymes,upon
co-administration with prescribed medicines. A
typical example is St. John’s wort (SJW), which
increases the activity of CYP3A4 and reduces
plasma concentrations of a number of drugs (7-9).
However, the information regarding natural
medicine-drug interactions that influence drug_________________________________________

Cytochromes P450 (P450) belong to a supergene
family of monooxygenases and play an importantrole in the oxidative metabolism of endogenous
compounds such as steroids, fatty acids, andprostaglandins and exogenous compounds such as
drugs, environmental pollutants, and foodcomponents (1, 2). Of all the CYPs, CYP3A4,CYP2D6, CYP2C19, CYP2C9, and CYP1A2 aremainly expressed in human livers and are involvedin ~90% of the oxidative metabolism of drugs (1).
In particular, CYP3A4 is the most abundant CYP
form found in livers and small intestines,
comprising approximately 30% and 70% of thetotal P450 content, respectively (3). CYP3A
metabolizes more than 50% of the currently usedtherapeutic drugs, for example, alprazolam, andnifedipine (3, 4). Thus, compounds that inhibit the
CYP3A4 activity could potentially affect thepharmacokinetics/pharmacodynamics of these
therapeutic drugs. For example, grapefruit juice(GFJ) has been found to increase the bioavailability

Corresponding Author: Kiyoshi Nagata; Department ofEnvironmental and Health Science, Tohoku Pharmaceutical
University; 4-4-1 Komatsushima, Aoba-ku, Sendai, Miyagi,
Japan; E-mail: nagataki@tohoku-pharm.ac.jp316— 282 —J Pharm Pharm Sci (www.cspsCanada.org) 18(4) 316 - 327, 2015

METHODS

metabolism is limited. Therefore, one can assume
that natural medicine-drug interactions pose animportant problem for clinical therapeutics and it isessential to understand whether P450 activity isaffected by natural medicine.
At present, human liver microsomes andprimary human hepatocytes are the most desirable
enzyme sources for drug metabolism experiments
since they retain majority of the biological
functions of human livers as well abundantly
express drug-metabolizing enzymes (10-12).
However, these show large donor-to-donor
variability in drug metabolizing activity, whichmake date interpretation from different sourcesdifficult (10-12). Human hepatoma-derived celllines such as HepG2 cells have been used for invitro drug metabolism studies owing to have an
unlimited lifespan, but have drawback the limit ofliver-specific functions including expression of
drug-metabolizing enzymes (13-15). In order tocompensate for the shortcomings of HepG2 cells,various transient P450 expression systems are
generally used for drug metabolism studies.
On the other hand, viral and non-viral methods
have been used as tools for intracellular genetransfer (16). In particular, adenoviral vectors arecapable of introducing target genes into a variety ofcells efficiently (17). The expression level of arecombinant protein can be easily controlled bytransfection, depending on the amount of
adenovirus. However, this process requires
infection of adenovirus with biosafety regulation
before each experiment and, a well-to-welldifference in enzyme activity is observed within thesame experiment, thus, making this system
unsuitable for high-throughput screening. In orderto overcome these problems, we have establishedfrozen recombinant P450-expressing cells infectedwith human CYP3A4 expressing adenovirus(Ad-CYP3A4) to evaluate the effect of natural
medicines on CYP3A4 activity. By create large
amounts of the cells simultaneously, it is possible toobtain long-term consistent data and stable supplies
of cells expressing a constant level of P450 activity.
In the present study, we examined thedifference in enzymatic activity between the frozen
Ad-CYP3A4-infected cells (fAd-CYP3A4 cells)
and non-frozen Ad-CYP3A4-infected cells(Ad-CYP3A4 cells), and by using our new method
based on fAd-CYP3A4 cells, we evaluated theextent of CYP3A4 inhibition by natural medicines.

Materials
Ketoconazole was obtained from LKT Laboratories(St. Paul, MN). Hyperforin was obtained from
Sigma-Aldrich (St. Louis, MO). Dimethyl sulfoxide(DMSO) was obtained from Nacalai Tesque (Kyoto,
Japan). Oligonucleotides were commerciallysynthesizedbyFasmac(Atsugi,
Japan).
Microsomes expressing human CYP3A4 preparedfrom baculovirus-infected insect cells (recombinant
CYP3A4 microsomes) were purchased from BD
Gentest (Woburn, MA). Cat’s Claw, Devil’s Claw,
Feverfew, Peppermint Oil, Red Clover, andSiberian Eleuthero were obtained from Nature’sway (Springville, UT).
Cell Culture
HepG2 and HEK293 cells were obtained from
Riken cell bank (Tsukuba, Japan). Cells were
cultured in Dulbecco’s modified Eagle’s medium(DMEM; WAKO Pure Chemicals, Osaka, Japan)
supplemented with 10% fetal bovine serum (FBS;
Biowest, Miami, FL), minimum essential medium
nonessential amino acids (MEM NEAA; Invitrogen,
Carlsbad, CA), and Antibiotic-Antimycotic(Invitrogen), under 5% CO2 at 37°C. Before 24 hadenovirus infection, HepG2 cells were seeded at
1.5 × 105 cells per well onto 24-well tissue culture
plates (BD Biosciences, Heidelberg, Germany) for
real-time PCR, 1.0 × 105 cells per well onto 48-welltissue culture plates (BD Biosciences) for
immunoblotting, and 0.5 × 105 cells per well onto
96-well tissue culture plates (BD Biosciences) for
P450-GloTM assay. The cells were subjected tocorresponding experiments after 48 h.
Sample Preparation
Ketoconazole and hyperforin were dissolved in
DMSO and the final assay concentration ranges
were 0.01-10 μM (ketoconazole) and 0.1-100 μM(hyperforin). For the assessment of inhibitory
activities, the daily dose of the natural medicines
was extracted in 10 mL ethanol 70% (v/v) for 2 h inwater-bath at 37°C. The resulting solution wascentrifuged at 3,500 rpm for 15 min at 4°C and thesupernatants were used as natural medicine extracts.
These were added to the culture medium (0.1%,
0.25%, and 1% extracts) for CYP3A4 activity
assay.
Construction of Recombinant Adenovirus
The open reading frame of CYP3A4 DNA was
isolated by amplification of cDNA obtained from317

— 283 —J Pharm Pharm Sci (www.cspsCanada.org) 18(4) 316 - 327, 2015

human hepatocyte mRNA by PCR using theforwardprimer,5’-CACCATGGCTCTCATCCCAGACTTGGC-3’,
andthereverseprimer,5’-TCAGGCTCCACTTACGGTGCCATC-3’. The
synthesis was carried out according to the
manufacturer’s protocol for the ViraPowerTM
Adenoviral
Expression
System,pENTRTM
Directional
TOPO
Cloning
andpAd/CMV/V5-DESTTM Gateway® Vector kits
(Invitrogen). The plasmid was purified by CsCl
density gradient centrifugation, linearized with
PacI (New England Biolabs Inc, Ipswich, MA) andthen transfected into HEK293 cells using TargefectF-1 (Nacalai Tesque). The control adenovirus, aβ-galactosidase-expressing adenovirus (Ad-LacZ)
was produced using the pAd/CMV/V5-GW/lacZ
control plasmid (Invitrogen). The titer of
adenovirus, 50% titer culture infectious dose
(TCID50), was determined in HEK293 cells. TCID50
has been reported almost equivalent to that of theplaque-forming unit (18). Multiplicity of infection(MOI) was calculated by dividing TCID50 by the
number of cells at the time of seeding.

(TaKaRa Bio, Shiga, Japan). The cDNA was used
to carry out real-time polymerase chain reaction(PCR) using SYBR® Premix ExTaq (TaKaRa Bio)in order to measure mRNA levels of CYP3A4 andglyceraldehyde-3-phosphate
dehydrogenase(GAPDH). The amplification reactions were
performed with specific primers for CYP3A4(forward:
5’-CTGTGTTTCCAAGAGAAGTTAC-3’
andreverse: TGCATCAATTTCCTCCTGCAG-3’), andGAPDH(forward:
5’-CATGGGTGTGAACCATGAGAA-3’
andreverse: 5’-GGTCATGAGTCCTTCCACGAT-3’)(19). Quantitative values were obtained above thethreshold PCR cycle number (Ct) at which theincrease in signal associated with an exponential
growth in PCR products was detected using
Thermal Cycler DiceTM TP800 (TaKaRa Bio). The
relative mRNA expression levels in each sample
were normalized to those of GAPDH.
Determination of CYP3A4 Enzymatic Activities
using P450-GloTM Assay
CYP3A4 activity was measured using a
P450-GloTM CYP3A4 assay kit (Promega)according to the manufacturer’s protocol.
Ad-CYP3A4 and fAd-CYP3A4 cells were rinsed
twice with phosphate buffer saline. Fresh medium
containing 3 μM luciferin-IPA and/or test
compound was added to the cells and incubated at
37°C for 1 h. Subsequently, 40 μL of the medium
from each well was transferred to a 96-well white
luminometer plate (BD Biosciences), and theluminescence was detected by using a GlomaxTM
96 microplate luminometer (Promega). The
luminescence signals were calculated bysubtracting background luminescence values(no-cell control) from the values of the test
compound and the blank (without the test
compound).
The reaction mixture consisted of recombinant
CYP3A4 microsomes, potassium phosphate buffer,
luciferin-IPA (final concentration is 2 nM, 100 mM
and 3 μM, respectively) and/or test compounds in a
final volume of 25 μL. The mixture was incubatedat 37°C for 20 min. At the end of the reaction
period, reaction mixture was transferred to a
96-well white luminometer plate, and the CYP3A4
activity was measured as described above.

Preparation of Frozen Ad-CYP3A4 Infected
HepG2 Cells
HepG2 cells were seeded at 3 × 106 cells in
100-mm2 culture dish (BD Biosciences) 24 h before
Ad-CYP3A4 infection at 10 MOI. After 24 h, thecells were harvested, media was replaced withcryopreservation medium (Bambanker; Nippon
Genetics, Tokyo), and cells were stored in liquid
nitrogen. fAd-CYP3A4 cells were thawed in a 37°C
water-bath and then washed with cultured medium.
The cells were seeded into 60-mm2 culture dish(BD Biosciences) and incubated in fresh medium
for 24 h. The cells were seeded at 1.5 × 105 cells
per well onto 24-well tissue culture plates forreal-time PCR, 1.0 × 105 cells per well onto 48-welltissue culture plates for immunoblotting, and 0.5 ×
105 cells per well onto 96-well tissue culture plates
for P450-GloTM assay, respectively, for 24 h prior toexperiments.
Quantitative Analysis of CYP3A4 mRNAContents
Total RNA was extracted by using TRI(Molecular
ResearchCenter,REAGENT®
Cincinnati, OH). First-strand cDNA wassynthesized from 2 μg total RNA in a 20 μL
reaction mixture using Moloney Murein Virus
Reverse Transcriptase (Promega, Madison, MI),oligo(dT)20 primer, and Ribonuclease Inhibitor

Immunoblot Analysis
Immunoblot analysis was performed to detect theexpression of CYP3A4 proteins in HepG2,
Ad-CYP3A4, and fAd-CYP3A4 cells. Whole cell318

— 284 —J Pharm Pharm Sci (www.cspsCanada.org) 18(4) 316 - 327, 2015

lysates from these cells were size-fractionated bygel electrophoresis on a 10% polyacrylamide/0.1%
sodium dodecyl sulfate gel after being denatured byheating in a 2-mercaptoethanol-containing loading
buffer. The proteins were electrotransferred to
Immobilon®-P membranes (Millipore, Billerica,MA), which were then incubated for 1 h withpolyclonal goat anti-human CYP3A4 antibody(Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
that had been diluted in Tris-buffered saline(1:1,000). The membranes were subsequently
incubated for 1 h with a horseradish
peroxidase-conjugated secondary antibody diluted
in Tris-buffered saline (1:10,000; Santa Cruz
Biotechnology, Inc.). After development with
SuperSignal West Pico chemiluminescent substrate(Thermo Fisher Scientific Inc., Waltham, MA), themembrane was scanned using LAS-4000 (GEHealthcare, Milwaukee, WI).

CYP3A4 expression in fAd-CYP3A4 cells, we
compared CYP3A4 mRNA, activity and protein
expression between fAd-CYP3A4 cells andnon-frozen Ad-CYP3A4 cells at 10 MOI. The
mRNA, activity, and protein expression levels of
CYP3A4 were upregulated in fAd-CYP3A4 cells,
as compared to the HepG2 cells (Figure 2). In
fAd-CYP3A4 cells, these levels showed higher
values as compared to the Ad-CYP3A4 cells.
Moreover,toconfirmtheeffectsofcryopreservation period on CYP3A4 activity in
fAd-CYP3A4 cells, we measured the activity after
cryopreservation at the end of 0, 1, 2, and 6 months.
As the results, CYP3A4 activity remained
unaffected at the end of each frozen period (Figure3). In those periods, the activities were notsignificantly different.
Effects of specific inhibitors on CYP3A4 activity
In order to demonstrate the utility of fAd-CYP3A4
cells for the evaluation of inhibition of CYP3A4
enzyme activity, we used ketoconazole andhyperforin as typical inhibitors (20). When
fAd-CYP3A4 cells, Ad-CYP3A4 cells, andrecombinant CYP3A4 microsomes were treatedwith ketoconazole or hyperforin, enzyme activity of
CYP3A4wasdecreasedinaconcentration-dependent manner (Figure 4). IC50
values of 0.077, 0.105, and 0.096 μM forketoconazole were determined in fAd-CYP3A4
cells, Ad-CYP3A4 cells, and recombinant CYP3A4
microsomes, respectively. Hyperforin, a compoundof SJW, were inhibited CYP3A4 activity at IC50
values of 9.212, 6.738, and 4.411 μM, respectively(Table 1). The IC50 values of ketoconazole andhyperforin showed nearly equal values in each
evaluation systems.

STATISTICAL ANALYSIS
Data are expressed as the mean ± standard (S.D.)
error from three determinations. Statisticalcomparison of two groups was performed using a
two-tailed Student’s t-test. P-values less than 0.05
were considered to be statistically significant.
RESULTS
Determination of optimal conditions for
CYP3A4 expression in Ad-CYP3A4-infected
HepG2 cells
To optimize the conditions of CYP3A4 expressionin Ad-CYP3A4 cells, HepG2 cells were infectedwith various amounts (0, 3, 5, 10, and 20 MOI) of
Ad-CYP3A4 and measured mRNA expression,
enzyme activity, and protein expression. The levelsof CYP3A4 mRNA expression, activity, and proteinincreased corresponding to the infection amount of
Ad-CYP3A4, but not in Ad-LacZ infected cells at
20 MOI (Figure 1). The highest activity wasmeasured in cells infected with Ad-CYP3A4 at 20
MOI, however this infection level was found to becytotoxic. Therefore, we used Ad-CYP3A4 at 10
MOI in order to prepare the Ad-CYP3A4 cells.

Effects of natural medicines on CYP3A4 activity
In order to substantiate the ability of fAd-CYP3A4
cells to assess CYP3A4 activity inhibition by
natural medicines, the cells were treated withnatural medicine extracts. As shown in Figure 5,each natural medicine extract inhibited the activity
in a concentration-dependent manner. 1% extractsof Cat’s Claw, Peppermint Oil, and Siberian
Eleuthero strongly inhibited CYP3A4 activity in
fAd-CYP3A4 cells (Figure 5A). The inhibitorylevels of Devil’s Claw, Feverfew, and Red Clover
were found to be comparable to 0.1 μM
ketoconazole (positive control) in fAd-CYP3A4
cells.

Effects of cryopreservation on CYP3A4
expression
fAd-CYP3A4 cells were prepared by infecting
HepG2 cells with Ad-CYP3A4 at 10 MOI and thesecells were stored using cryopreservation medium.
To confirm the effects of cryopreservation on
319— 285 —J Pharm Pharm Sci (www.cspsCanada.org) 18(4) 316 - 327, 2015

Figure 1. MOI-dependent increase of CYP3A4 levels in HepG2 cells infected with Ad-CYP3A4.
HepG2 cells were seeded in 24-well tissue culture plates at 1.5 × 105 cells per well for real-time PCR, 96-well tissue
culture plates at 0.5 × 105 cells per well for P450-GloTM assay, and 48-well tissue culture plates at 1.0 × 105 cells perwell for immunoblotting. The next day, the cells were infected with Ad-CYP3A4 (MOI of 3, 5, 10, and 20) or Ad-LacZ(MOI of 20). After 48 h, mRNA expression (A), enzyme activity (B), and protein levels (C) were measured using
real-time PCR, P450-GloTM assay, and immunoblotting. Results represent the mean ± S.D. (n = 3). Student’s t-test:**p< 0.01, compared with non-infection cells (0 MOI).
320— 286 —J Pharm Pharm Sci (www.cspsCanada.org) 18(4) 316 - 327, 2015

Figure 2. Comparison of CYP3A4 levels in fAd-CYP3A4 cells and Ad-CYP3A4 cells.
HepG2 cells were seeded in 24-well tissue culture plates at 1.5 × 105 cells per well for real-time PCR, 96-well tissue
culture plates at 0.5 × 105 cells per well for P450-GloTM assay, and 48-well tissue culture plates at 1.0 × 105 cells perwell for immunoblotting. The next day, the cells were infected with Ad-CYP3A4 (MOI of 10) or Ad-LacZ (MOI of 10).
After 48 h, mRNA expression (A), enzyme activity (B), and protein levels (C) were measured using real-time PCR,
P450-GloTM assay, and immunoblotting. fAd-CYP3A4 cells were seeded in the same way as HepG2 cells before 24 hof real-time PCR, P450-GloTM assay, and immunoblotting. Results represent the mean ± S.D. (n = 3). Student’s t-test:**p< 0.01, *p< 0.05.
321— 287 —J Pharm Pharm Sci (www.cspsCanada.org) 18(4) 316 - 327, 2015

Figure 3. Effect of the frozen period on CYP3A4 activity.
After cryopreservation for 0, 1, 2 and 6 months, fAd-CYP3A4 cells were seeded in 96-well tissue culture plates at 0.5 ×
105 cells per well. After 24 h, enzyme activity was measured using P450-GloTM assay. Results represent the mean ± S.D.
(n = 3).

Figure 4.
322— 288 —J Pharm Pharm Sci (www.cspsCanada.org) 18(4) 316 - 327, 2015

Figure 4. Inhibition of CYP3A4 activity by ketoconazole (A) and hyperforin (B).
HepG2 cells were seeded in 96-well tissue culture plates at 0.5 × 105 cells per well prior to 24 h Ad-CYP3A4 (MOI of20) infection. After 48 h, enzyme activity was measured using P450-GloTM assay. fAd-CYP3A4 cells were seeded in
96-well tissue culture plates at 0.5 × 105 cells per well density before 24 h measurement of enzyme activity.
Recombinant CYP3A4 microsomes were incubated with ketoconazole or hyperforin before measurement of enzyme
activity. Results represent the mean ± S.D. (n = 3). Student’s t-test: **p< 0.01, *p< 0.05, compared between
Ad-CYP3A4 cells and fAd-CYP3A4 cells. ##p< 0.01, #p< 0.05, compared between Ad-CYP3A4 cells andrecombinant CYP3A4 microsomes.

Table 1. IC50 values of CYP3A4 activity using each evaluation systems

IC50 values were determined with data shown in Figure 4.

323— 289 —J Pharm Pharm Sci (www.cspsCanada.org) 18(4) 316 - 327, 2015

Figure 5. Inhibitory effect of natural medicines on CYP3A4 activity in fAd-CYP3A4 cells (A) and CYP3A4
microsomes (B).
fAd-CYP3A4 cells were seeded in 96-well tissue culture plates at 0.5 × 105 cells per well. After 24 h, enzyme activity
of CYP3A4 was measured. Reaction mixture of recombinant CYP3A4 microsomes was incubated with ketoconazole(0.1 μM) and test natural medicine compounds. The enzyme activity was measured using P450-GloTM assay. The
values of enzyme activity in untreated groups were set equal to 100%. Results represent the mean ± S.D. (n = 3) ND,
not detected. Student’s t-test: **p< 0.01, *P<0.05, compared with control.

On the other hand, in recombinant CYP3A4
microsomes, CYP3A4 activity was strongly
inhibited by 1% extracts of Cat’s Claw, Feverfew,
Red Clover, and Siberian Eleuthero, which wasstronger than that of the finding in fAd-CYP3A4
cells (Figure 5B). However, this was not observedin case of Peppermint Oil.

expression levels of the target protein can becontrolled by the infection amount of the
adenovirus. As shown in Figure 1, Ad-CYP3A4
infection leads to increased CYP3A4 mRNA,
protein levels, and enzyme activity in HepG2 cells
and this increase corresponds to the infection
amount of Ad-CYP3A4. However, it may cause awell-to-well difference in the enzyme activity by
the virus infection in every experiment. Ugai et al.
(21) previously reported that the function of
adenoviral vectors usually does not change unlessthey are exposed to excessive freeze/thaw cycles.

DISCUSSION
Adenoviral vectors are highly efficient tools forgene transfer into various cell types. Furthermore,324— 290 —J Pharm Pharm Sci (www.cspsCanada.org) 18(4) 316 - 327, 2015

We therefore assume that recombinant P450
activity in adenovirus-infected cells is not alteredby limited cycles of freeze / thawing.
The fAd-CYP3A4 cells had higher CYP3A4
activity than Ad-CYP3A4 cells (Figure 2). The
difference in activity levels among these cells
corresponded to the mRNA and protein levels. Thismay be owing to the difference in total culture
period after seeding the cells (fAd-CYP3A4 cells:
96 h, non-frozen Ad-CYP3A4 cells: 72 h). For the
inhibition assays with ketoconazole, a potent
inhibitor of CYP3A4-mediated metabolism, theIC50 values in fAd-CYP3A4 cells, Ad-CYP3A4
cells, and recombinant CYP3A4 microsomes werein good agreement with what has been reported (20,22) (Figure 4 and Table 1). Moreover, hyperforin(one of SJW components) inhibited CYP3A4
activity and IC50 values were comparable on eachexperiment. Obach et al (23) previously reported
that hyperforin potently inhibited of CYP3A4
activity in studies using recombinant CYP3A4
microsomes and hepatocytes (24). Moreover, Qi etal. (25) reported that oral clearance of midazolam
in rats decreased with SJW treatment of shortperiod. Therefore, our results suggest that
fAd-CYP3A4 cells are a useful system to evaluate
P450 inhibition and are comparable to humanhepatocytes and microsomes.
Further, we have compared that inhibition
effect to CYP3A4 enzymes activity by natural
medicines on fAd-CYP3A4 cells and recombinant
CYP3A4 microsomes. A previously publishedclinical study has shown that Cat’s Claw interacts
with HIV protease inhibitors, which are known
CYP3A4 inhibitors (26, 27). Unger et al (28)reported that Devil’s Claw, Feverfew, Peppermint
Oil, and Red clover were inhibitors of CYP
enzymes including CYP3A4. Although extent ofinhibition of CYP3A4 activity by these natural
medicines differed, all the natural medicine extracts
exhibited this inhibitory effect (Figure 5). The
inhibitory levels in recombinant CYP3A4
microsomes were stronger than those in case offAd-CYP3A4 cells. In contrast, the CYP3A4
activity was strongly inhibited in fAd-CYP3A4
cells treated with Peppermint Oil extracts comparedto that in the recombinant CYP3A4 microsomes.
This difference might be involved in thepermeability of the CYP3A4 inhibitors includingthese natural medicines into the cells. It was notpossible to evaluate the cell membrane permeabilityof the compounds in microsomes unlike cell-basedin vitro test systems for P450 inhibitors. In fact,
Englund et al (29) have reported that IC50 values of

elacrider for midazolam (CYP3A substrate)
metabolism in hepatocytes were found to be higher
than those found in liver microsomes. They havedescribed that this difference may be due to thedifference in membrane permeability of theinhibitor into the cells. Therefore, our results
suggest that our evaluation system has reflected
membrane permeability of compounds into the cellsof livers. Our cell based-inhibitory assay systemhave mimicking human drug metabolism
intracellular considered for membrane permeability.
Primary human hepatocytes are the mostdesirable enzyme source, but there are lot to lot（donor to donor）variation and limitation to use
the large amount of the each lot. Our results
indicated that Ad-CYP3A4-infected cells could becryopreserved and their activity did not decrease
upon long-term storage. Thus, easy andreproducibly assessment is possible by storing alarge volumes of fAd-CYP3A4 cells. Aoyama et al.
(30) also have reported that HepG2 cells infectedwith two different varieties of P450-expressing
adenovirus had no effect on each expression levels.
Therefore, our model can be developed to evaluatea system that considers metabolic pathways inaddition to other P450- and transporter-expressing
adenoviruses.
In the present study, we have successfully
developed a highly reproducible system, which
provideslarge
amountsoffrozenCYP3A4-expressing cells after Ad-CYP3A4
infection. Our results suggested that this evaluation
system become more similar to hepatocytes byexpressing of multiple P450s and is more useful for
screening inhibitory effect of natural medicine on
P450 activity.
ACKNOWLEDGMENTS
This work was supported by a Health and Labour
Sciences Research Grant for Research on Food
Safety (No. 22230301) from the Ministry of Health,Labour and Welfare of Japan; and by the Japanese
Ministry of Education, Culture, Sports, Science andTechnology (MEXT)-Supported Program for theStrategic Research Foundation at Private
Universities.
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— 294 —研究テーマ２
老年性神経系疾患（老人性認知症、高齢者うつ病）改善薬の開発— 295 —

— 296 —Behavioural Brain Research 377 (2020) 112226

Contents lists available at ScienceDirect

Behavioural Brain Research
journal homepage: www.elsevier.com/locate/bbr

Research report

Role of prefrontal cortical 5-HT2A receptors and serotonin transporter in the
behavioral deficits in post-pubertal rats following neonatal lesion of theventral hippocampus
Satoru Mitazakia,b,1, Osamu Nakagawasaia,⁎,1, Hiroshi Onogia,c, Kenya Watanabea,g,Kohei Takahashia,d, Koichi Tan-Noa, Rémi Quirione, Lalit K Srivastavae, Takeshi Tadanoa,fa
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai, 981-8558,
Japanb
Laboratory of Molecular Neuroscience, Faculty of Pharmacy, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki, 370-0033, Japanc
Faculty of Health Science, Tohoku Fukushi University, 1-8-1 Kunimi, Aoba-ku, Sendai, 981-8522, Japand
Department of Pharmacology, School of Pharmacy, International University of Health and Welfare, 2600-1 Kitakanemaru, Ohtawara, 324-8501, Japane
Douglas Mental Health University Institute, Department of Psychiatry, McGill University, Montreal, Québec, H4H 1R3, CanadafComplementary and Alternative Medicine Clinical Research and Development, Graduate School of Medicine Sciences, Kakumamachi, Kanazawa University, Kanazawa,
920-8640, Japang
Department of Pharmacy, Fukushima Medical University Hospital, Hikarigaoka 1, Fukushima, 960-1295, JapanART I CL E I NF O

ABS TRACT

Keywords:
5-HT2A receptor
Serotonin transporter
Prepulse inhibition
Schizophrenia
Receptor autoradiography
Prefrontal cortex

Neonatal ventral hippocampal-lesioned (NVHL) rats have been shown to display neurochemical and behavioral
abnormalities at adulthood, analogous to some of those seen in schizophrenia. Serotonergic neurotransmission is
implicated the pathophysiology and treatment of schizophrenia. In this study, we evaluated possible role ofserotonergic transmission is the behaviors of NVHL-lesioned rats. Bilateral lesions to the ventral hippocampus(VH) in rat pups were made using the excitotoxin ibotenic acid. We investigated 5-HT2A-receptor and SERT
binding sites in cortical and subcortical areas in post-pubertal NVHL and sham-lesioned rats, using quantitative
receptor autoradiography. We compared a 5-HT-dependent behavior in NVHL and sham animals, the wet-dog
shake response (WDSr) to a 5-HT2A receptor agonist DOI. In addition, we studied prepulse inhibition (PPI) of
startle responses in NVHL and Sham-lesioned animals treated with antipsychotic drugs haloperidol, risperidone
and clozapine and 5-HT2A antagonists ketanserin or MDL100907. The WDSr elicited by DOI was enhanced in
post-pubertal NVHL rats compared to sham-lesioned controls. Moreover, post-pubertal NVHL rats exhibited PPI
deficits which was reversed by atypical antipsychotics, ketanserin and MDL100907. A significant increase in 5HT2A-like receptor binding was observed in the medial prefrontal cortex (mPFC) in post-pubertal NVHL rats
without any significant change in the striatum and ventral pallidum. A significant increase in SERT-like binding
was also observed in the mPFC and striatum of NVHL rats at pre-pubertal period; however, at post-pubertal age,the binding remained elevated in mPFC only. These data suggest that increased prefrontal cortical 5-HTtransmission may play a role in the behavioral deficits observed in this neurodevelopmental model of schizophrenia.

Abbreviations: 5-HT, serotonin; ANOVA, analysis of variance; ASR, acoustic startle response; Cg1, cingulate cortex area 1; CPu, caudate putamen; DA, dopamine;
DOI, ( ± )-2,5-dimethoxy-4-iodoamphetamine hydrochloride; HTR, head twitch response; i.p., intraperitoneally; IL, infralimbic cortex; MIA, maternal immune
activation; NAcC, nucleus accumbens core; NAcSh, nucleus accumbens shell; NVHL, Neonatal ventral hippocampal-lesioned; mPFC, medial prefrontal cortex; PD,
postnatal day; PLSD, Protected Least Significant Difference; PrL, prelimbic cortex; PPI, prepulse inhibition; SEM, standard error of the mean; SERT, serotonintransporter; VP, ventral pallidum; WDSr, Wet-dog shakes response⁎
Corresponding author at: Department of Pharmacology, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1
Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, Japan.
E-mail address: osamun@tohoku-mpu.ac.jp (O. Nakagawasai).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bbr.2019.112226
Received 3 June 2019; Received in revised form 23 August 2019; Accepted 10 September 2019
Available online 12 September 20190166-4328/ © 2019 Elsevier B.V. All rights reserved.

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1. Introduction

2.1. Neonatal ventral hippocampal lesion

Neonatal ventral hippocampal-lesion (NVHL) in rats have beenshown to induce post-pubertal emergence of behavioral abnormalities
thought to simulate some aspects of positive, negative and cognitive
symptoms classically observed in schizophrenic patients. These include
hyper-reactivity to stress and amphetamine, deficits in prepulse inhibition (PPI) of startle and latent inhibition, and impaired social behavior and working memory [1].
It is suggested that the serotonergic system may be related to thepathophysiology of schizophrenia due to its role in many physiologic
processes related to mood, cognition, and perception [2]. Alternationsin the expression of serotonin (5-HT) receptors and transporter havebeen reported in the brains of patients with schizophrenia [3,4] andmay play a role in the differential action of atypical as compared totypical antipsychotic drugs [5]. In addition, polymorphisms in serotonergic receptor genes are related to the prediction of response toatypical antipsychotics [6]. Furthermore, the effectiveness of clozapine,
an atypical antipsychotic drug with significant 5-HT receptor binding
affinity, and the observation that 5-HT modulates the activity of dopaminergic neurons constitute important evidence implying a role of 5HT in schizophrenia [7]. While genetic association studies of 5-HT2A
receptor and the serotonin transporter (SERT) in schizophrenia havenot been conclusive, given the above-evidence, it is imperative to better
explore the roles of serotonergic systems in schizophrenia [8–11].
Schizophrenia has been associated with abnormalities in information processing, attentional mechanisms and sensorimotor gating [12].
One well-established method for evaluating sensory gating is theparadigm of PPI of startle which refers to the inhibition of a startle
reflex by the presentation of a weak prepulse immediately before astartling stimulus. Interestingly, PPI is a translatable phenomenon
which can be measured using virtually identical techniques and parameters across species and it shows similar sensitivity to stimulus
parameters in rats and human beings [13]. Disruption of PPI in schizophrenia patients has been well described in several studies [14,15].
PPI is regulated at by various brain regions and by many neurotransmitters. Previous studies have suggested that atypical antipsychotics, in contrast to typical antipsychotics, were able to attenuate
NVH lesion-induced PPI deficits [16]. Given the significant affinity ofatypical antipsychotics to serotonin receptors, it is possible that the PPI
deficits in NVHL rats may also involve 5-HT system, in addition to otherneurotransmitters as suggested previously [16–18].
In order to assess possible alterations in serotonergic system, thepresent investigation was designed to evaluate the levels of 5-HT2A
receptor and SERT binding sites at pre- and post-pubertal ages in cortical and subcortical areas of NVHL rats, using quantitative receptor
autoradiography. In addition, we assessed the role of serotonergictransmission in the behaviors of NVHL rats using the Wet-dog shakes
response (WDSr), a 5-HT2A receptor-associated behavior, and the effects of 5-HT2A antagonists in PPI deficits observed in NVHL rats. Ourdata show that increased prefrontal cortical serotonergic system maycontribute to some of the behavioral deficits observed in this neurodevelopmental model of schizophrenia.

Lesions of the ventral hippocampus were performed as previously
described [19]. Pregnant Sprague-Dawley rats at 14–16 days of gestation were obtained from Japan SLC (Hamamatsu, Shizuoka, Japan),housed under conditions of constant temperature (23 ± 1 °C) and humidity (55 ± 5%), on a 12:12 h light-dark cycle (light: 7–19 h). The
rats
were
housedindividuallyinplastic
cages(20 cm × 25 cm × 40 cm). On postnatal day (PD) 7, male pups
(14–18 g) within each litter (4–10 males/litter) were randomly divided
to sham or lesion status. Pups were anesthetized by hypothermia byplacing them on ice for 20 min and were immobilized on a platform
fixed on a stereotaxic frame. An incision in the skin overlaying the skull
was made and two 1 mm holes were drilled. A needle connected to an
infusion pump through a Hamilton syringe was lowered into the eachventral hippocampus at the coordinates: AP -3.0 mm ML ± 3.5 mm
relative to bregma and -5.0 mm relative to the surface of the skull.
Ibotenic acid (0.3 μl, 10 μg/μl; Sigma, St. Louis, MO, USA) in 0.15 M
phosphate buffer (PB) pH 7.4 was infused bilaterally at a flow rate of
0.15 μl/min. Sham operated animals received the same volume of PB.
The needle was withdrawn 2 min after completion of the infusion. Pups
were placed on a warming pad (MUROMACHI KIKAI CO., LTD., Tokyo,
Japan) and then returned to their mothers. On PD 21 to 25, rats were
weaned and grouped 2 to 3 per cage (20 cm × 25 cm × 40 cm). Experiments were performed in pre- (PD 35) and post-pubertal (PD 60)
animals.
2.2. Drugs
Ketanserin (Sigma Chemical Co., St. Louis, MO, USA) and ( ± ) - 2, 5
- dimethoxy - 4 - iodoamphetamine hydrochloride (DOI) (Sigma) weredissolved in saline, and MDL 100907 (ABX, Radeberg, Germany) was
dissolved in tween 80 (0.5%) in saline. Drugs were administered in a
volume of 1 ml/kg intraperitoneally (i.p.; ketanserin) or subcutaneously(s.c.; MDL100907, DOI).
2.3. Histology
Histology was conducted in a cohort of animals used for the behavioral study. To assess the size and location of the lesion, adult rats
were sacrificed by decapitation and brains were removed and frozen in
isopentane at −40 °C and stored at −80 °C until sectioning using acryostat. Coronal sections (20 μm) were mounted onto gelatin-coatedslides and stained with cresyl violet. Lesion size was confirmed by digital camera. As shown in Fig. 1, bilateral damage including neuronalloss, atrophy and cavitation of the ventral hippocampus was observedin ibotenic acid-treated rats.
2.4. Wet-dog shakes response (WDSr)
Each rat was allowed to adapt for 1 h in an observation cage
(20 cm × 25 cm × 40 cm) before injection of the DOI (1.0 mg/kg, s.c.)or vehicle. The number of wet-dog shakes (rapid right and left movements of the head with little or no involvement of the trunk) was
counted for 30 min after injection of DOI or vehicle. All groups had
9–13 animals per condition.

2. Materials and methods
All experiments were performed following the approval of the
Ethics Committee of Animal Experiment in Tohoku Medical andPharmaceutical University and according to the National Institutes of
Health Guide for the Care and Use of Laboratory Animals. Efforts weremade to minimize suffering and to reduce the number of animals used.
Measurements of the behaviors and post-mortem analyses were conducted by an observer blind to treatment conditions. Behavioral testing
occurred between 10:00 and 18:00.

2.5. PPI of the acoustic startle response2

Tests were conducted using SR-LAB system (SR-LAB, San Diego
Instruments, San Diego, CA, USA) that comprised five sound-attenuating chambers each equipped with a cylindrical plexiglas animal enclosure (length, 16 cm; inner diameter, 8.2 cm). Ventilation was provided by a small electric fan that also generated a 70 dB backgroundnoise. Tone pulses were presented by a speaker positioned 24 cm directly above the animal enclosure. A piezoelectric accelerometer affixed— 298 —

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Fig. 1. Experimental time course for protocol (A). Diagram showing the dorsal to ventral hippocampus (B). (C): Sham rat. (D): NVHL rat. A typical cresyl violet
stained coronal sections at the level of ventral hippocampus of a PD 60 rat indicating ibotenic acid induced neuron loss (arrow) in the ventral hippocampus (D).

3

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ketanserin binding and 10 days for [3H] citalopram binding. Specificlabelling was quantified (nCi/mg wet weight) using a computer-assisted
Image Reader BAS 5000 ver. 1.12 (Fuji Film).

to the animal enclosure frame was used to detect and transduce motion
resulting from the animal’s response. Tone pulse parameters were
controlled by a computer using a commercial software package (SRLAB) and interface assembly that also digitized (0–4095), rectified, andrecorded stabilimeter readings.
Measures of both acoustic startle response (ASR) and PPI were obtained in a single session as described by Brake et al [20] with minor
modifications. We investigated the effects of 5-HT2A/2C antagonist ketanserin (0.2, 1.0 and 2.0 mg/kg, i.p.) and selective 5-HT2A antagonist
MDL100907 (0.1 and 0.5 mg/kg, s.c.). Thirty minutes after drug injections, rats were placed in the Plexiglas enclosure and allowed toacclimatize to the environment for 5 min before being tested during 42
discrete trials. On the first two trials, the magnitude of the ASR to a
50 ms duration of 120 dB tone was measured. These first two startle
tones were presented in order to habituate the animals to the testingprocedure. Therefore, the ASR magnitude of these two trials wasomitted from the statistical analysis of the mean ASR amplitude. On thesubsequent 40 trials, the startle tone was either presented alone or
100 ms after presentation of a 30 ms duration prepulse. Prepulse intensity ranged from 3 to 15 dB above background noise and was varied
randomly between trials in 3 dB steps. Measures were taken at each of
the five prepulse intensities on 5 trials; animals were randomly presented with the startle tone alone during the other 10 trials; null trials(background tone alone) during the other 5 trials. The same stimulus
condition was never presented on more than two consecutive trials. Theinterval between each trial was programmed to a variable time schedule with an average duration of 15 s (range 5–30 s). A measure ofstartle response amplitude was derived from the mean of 100 digitizeddata points collected from stimulus onset at a rate of 1 kHz. Prepulse
effectiveness in suppressing the startle response was expressed as apercentage based on the mean amplitude of responses to the startle tone
alone (10 startle tones) relative to those recorded under the five prepulse conditions (5 startle tones/condition): PPI = 100 － [(meanstartle amplitude for prepulse + pulse trials/mean startle amplitude forpulse-alone trials) × 100]%. The responses to five different prepulse
intensities were averaged and used for statistical analysis.

2.7. Statistical analysis
Results are expressed as mean ± standard error of the mean (SEM).
Normality and homoscedasticity assumptions were verified prior to theuse of any parametric tests (Shapiro-Wilk normality test and equality ofvariances F-test). An unpaired t-test was used to analyze the effect of
the lesion on the average PPI, startle amplitude, and on the WDSr. A
two-way ANOVA (group × treatment or brain region) was used to assess the effect of drug on the average PPI and startle amplitude at PD 60
and the quantitative receptor autoradiography. Bonferroni test wasused to analyze autoradiography data at both PD 35 and 60, while
Tukey-kramer test was used for evaluating the effect of drugs on the
average PPI and startle amplitude. In some cases, when a main effectwas significant without interaction effect, we did an exploratory andlimited pairwise post-hoc comparison consistent with our a priori hypothesis. All significance levels were set at a P value of < 0.05.
3. Results
3.1. Histological examination of the ventral hippocampal lesion
In PD 60 (post-pubertal period) NVHL, bilateral cell loss and cavitation of the ventral hippocampus was observed while the dorsal hippocampus was spared. Fig. 1 shows a typical bilateral lesion at PD 60.
Animals exhibiting damage in the dorsal hippocampus, thalamus orcortex were excluded from the study. Sham-lesioned control animals
did not show any obvious damage in ventral hippocampal areas.
3.2. Changes of DOI-induced WDSr in post-pubertal NVHL rats
The administration of DOI induced WDSr in all animals; however, it
was significantly greater in NVHL rats compared to sham animals at
post-puberty [t (22) = 3.092, p = 0.0053, Fig. 2(B)], but not at prepubertal age [t (16) = 0.6765, p = 0.5084, Fig. 2(A)].

2.6. Quantitative receptor autoradiography
Two cohorts of sham and NVHL rats (drug-free) were sacrificed bydecapitation: one at PD 35 (pre-pubertal) and the other at PD 60 (postpubertal). Brains were removed and frozen in isopentane at −40 °C andstored at −80 °C. Using a cryostat (Leica 1720, Germany), 20 μm thickcoronal sections from the PFC (corresponding to plates 8–9 of the atlasof Paxinos and Watson [21]), caudate putamen (CPu) and nucleus accumbens (NAc) and ventral pullidum (VP) (plates 10–12 of the atlas of
Paxinos and Watson [21]) were obtained on gelatin-coated slides andstored at −80 °C until use.
5-HT2A receptors and SERT binding sites were visualized using [3H]
ketanserin (72.2 Ci/mmol) and [3H] citalopram (79.0 Ci/mmol) respectively (PerkinElmer Life and Analytical Sciences, Boston, MA,
USA). The protocol was carried out as previously described with minor
modifications [22,23]. Briefly, sections were preincubated for 15 min in
50 mM Tris-HCl buffer (NaCl 120 mM, CaCl2 4 mM, pH 7.4) at room
temperature before a 60 min incubation into the same buffer containing
either 2 nM [3H] ketanserin with 1 μM prazosin hydrochloride, and1 μM tetrabenazine or 2 nM [3H] citalopram. Non-specific binding wasdetermined by inclusion of 10 μM mianserin (Sigma) or 10 μM fluoxetine (TOCRIS, Ellisville, MO, USA) in the assay buffer already containing 2 nM [3H] ketanserin with 1 μM prazosin hydrochloride and1 μM tetrabenazine or 2 nM [3H] citalopram, respectively. At the end of
the incubation, sections were rinsed four times, 2 min each in ice-coldsame buffer, then dipped in deionized water to remove salts and rapidly
dried. All slides were exposed to tritium sensitive imaging plate (FujiFilm, Tokyo, Japan) together with [3H] microscale standards (GEHealthcare UK Ltd., Buckinghamshire, UK) for 8 days for [3H]

3.3. Effects of antipsychotics drugs on PPI in post-pubertal NVHL rats

4

Post-pubertal, but not pre-pubertal, NVHL rats exhibited significant
reduction in the PPI [Pre-pubertal period: average PPI: t (18) = 1.742,
p = 0.0986, startle amplitude: t (18) = 0.4093, p = 0.6871, Fig. 3(A).
Post-pubertal period: average PPI: t (18) = 3.664, p = 0.0018, startle
amplitude: t (18) = 1.088, p = 0.2909, Fig. 3 (B)] consistent withprevious studies [20,24]. We evaluated whether administration of haloperidol (a typical antipsychotic drug) or risperidone and clozapine(atypical antipsychotics) reversed the PPI deficits in NVHL rats. The
results show that NVHL-induced PPI deficits were reversed by all tested
compounds except for haloperidol. The drugs produced no effect insham animals, and had no significant effect on the startle amplitude[Fig. 3(C–E) Two-way ANOVA: haloperidol (C): average PPI: group: F(1, 40) = 46.62, p < 0.0001, treatment: F (1, 40) = 0.02024,
p = 0.8876, group × treatment: F (1, 40) = 0.4077, p = 0.5268; startle
amplitude: group: F (1, 40) = 0.4876, p = 0.4890, treatment: F (1,40) = 2.913, p = 0.0956, group × treatment: F (1, 40) = 0.08907,
p = 0.7669. Two-way ANOVA: risperidone (D): average PPI: group: F(1, 23) = 7.668, p = 0.0109, treatment: F (1, 23) = 17.19, p = 0.0004,
group × treatment: F (1, 23) = 4.631, p = 0.0421; startle amplitude:group: F (1, 23) = 0.2102, p = 0.6509, treatment: F (1, 23) = 0.7925,
p = 0.3826, group × treatment: F (1, 23) = 1.094, p = 0.3064. Posthoc test (average PPI): p < 0.0001. Two-way ANOVA: clozapine (E):average PPI: group: F (1, 26) = 8.07, p = 0.0086, treatment: F (1,26) = 9.01, p = 0.0059, group × treatment: F (1, 26) = 4.075,
p = 0.0439; startle amplitude: group: F (1, 26) = 0.06324, p = 0.8034,— 300 —

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Fig. 2. Changes of DOI-induced WDSr in NVHL rats at both pre- and post-pubertal periods. Each column shows the total number of WDSr for 30 min. Vertical bars
represent standard errors of means (SEM). **p < 0.01 compared with sham group.

treatment: F (1, 26) = 0.3448, p = 0.5622, group × treatment: F (1,26) = 0.6531, p = 0.4263. Post-hoc test (average PPI): p = 0.0010].

p = 0.0355, treatment: F (1, 45) = 1.711, p = 0.1975, group × treatment: F (1, 45) = 0.2233, p = 0.6388. Post-hoc test (average PPI):p = 0.0050. Two-way ANOVA: MDL100907 (B): average PPI: group: F(1, 37) = 68.83, p < 0.0001, treatment: F (1, 37) = 8.438,
p = 0.0062, group × treatment: F (1, 37) = 13.81, p = 0.0007; startle
amplitude: group: F (1, 37) = 0.5083, p = 0.4804, treatment: F (1,37) = 1.67, p = 0.2043, group × treatment: F (1, 37) = 0.3584,
p = 0.5530. Post-hoc test (average PPI): p = 0.0002].

3.4. Effects of 5-HT2A antagonists on PPI in post-pubertal NVHL rats
In order to know whether 5-HT2A receptors are involved in the
impaired PPI observed in post-pubertal NVHL rats, the effects of ketanserin and MDL100907, two 5-HT2A receptor antagonists, were
evaluated in this behavioral test. The data show that ketanserin and
MDL100907 attenuated PPI deficits in post-pubertal NVHL rats but
produced no effect in sham animals. The drugs had no significant effecton the startle amplitude [Fig. 4(A) and (B) Two-way ANOVA: ketanserin (A): average PPI: group: F (1, 45) = 29.51, p < 0.0001,
treatment: F (1, 45) = 7.438, p = 0.0091, group × treatment: F (1,45) = 6.306, p = 0.0157; startle amplitude: F (1, 45) = 4.699,

3.5. Quantitative receptor autoradiography of 5-HT2A receptor andserotonin transporter binding sites
We investigated the levels of 5-HT2A receptor binding sites in NVHL
rats at both pre-and post-pubertal periods. Our observed pattern of
5HT2A binding in different brain regions is consistent with previous

Fig. 3. Influence of NVHL rats on PPI deficits in startle reflex tested at both pre- and post-pubertal periods. Vertical bars represent SEM. **p < 0.01 compared withsham/vehicle group. ##p < 0.01 compared with NVHL/vehicle group.
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Fig. 4. Effect of ketanserin or MDL100907 in NVHL rats induced deficits in PPI of startle reflex. Vertical bars represent SEM. **p < 0.01 compared with sham/vehicle group. ##p < 0.01 compared with NVHL/vehicle group.

literatures [3,22]. The 5-HT2A receptor binding level was not significantly altered in NVHL rats at pre-pubertal period. However, significant increases in 5-HT2A receptor binding sites were found in the
medial prefrontal cortex (mPFC), regions viz., infralimbic cortex (IL),prelimbic cortex (PrL) and cingulate cortex area 1 (Cg1) of the postpubertal NVHL rats compared to sham animals [Fig. 5 Two-way
ANOVA: group: F (1, 63) = 0.190, p = 0.6640, brain region: F (6,63) = 53.422, p < 0.0001 and group × brain region: F (6,63) = 0.817, p = 0.5608, (A); group: F (1, 133) = 15.384,
p < 0.0001, brain region: F (6, 133) = 277.873, p < 0.0001 andgroup × brain region: F (6, 133) = 12.376, p < 0.0001, post-hoc test:
IL: p = 0.0065, PrL: p = 0.0012, Cg1: p = 0.0002, CPu: p > 0.9999,
NAcC: p > 0.9999, NAcSh: p > 0.9999, VP: p = 0.4161, (B)].
The changes of SERT binding sites in different brain regions of preand post-pubertal NVHL rats are shown in Fig. 5(C) and (D). We observed a significant main effect of groups (sham/lesion) by ANOVA,
while interaction (group × brain region) was not significantly different
at post-pubertal period [Two-way ANOVA: group: F (1, 98) = 123.380,
p < 0.0001, brain region: F (6, 98) = 25.537, p < 0.0001 andgroup × brain region: F (6, 98) = 2.903, p = 0.0479, post-hoc test: IL:
p = 0.0002, PrL: p = 0.0013, Cg1: p = 0.0263, CPu: p = 0.0082,
NAcC: p = 0.1842, NAcSh: p < 0.0001, VP: p < 0.0001, (C); group: F(1,98) = 27.608, p < 0.0001, brain region: F (6, 98) = 29.601,
p < 0.0001, and group × brain region: F (6, 98) = 0.909, p = 0.4919,
post-hoc test: IL: p = 0.1121, PrL: p = 0.0050, Cg1: p = 0.0595, CPu:p = 0.7401, NAcC: p = 0.0599, NAcSh: p = 0.0619, VP: p > 0.9999,(D)]. Thus, we focused on the effect of lesion, but not brain region at
post-pubertal period. We found a significant increase in SERT binding
all areas except NAcC at pre-pubertal period compared to sham control
animals. In contract to pre-pubertal period, increases in SERT bindingof the post-pubertal NVHL rats were found PrL and Cg1 only compared
to sham control animals.

antidepressants has been shown to lead to a reduction in DOI-induced
WDSr, which correlates with a downregulation of central 5-HT2A receptors [27]. Furthermore, it is well known that mPFC is an importantregion in the generation of WDSr. For example, Willins and Meltzer
have found that a WDSr occurs via stimulation of 5-HT2A receptor in
mPFC [28]. Accordingly, the present data indicate functional hypersensitivity of 5-HT2A receptors in mPFC in post-pubertal NVHL rats.
The actual hyperactivation of the receptor and associated signaling
pathways will be presented in a subsequent paper.
In agreement with previous reports [24,29], we have shown that
NVHL rats showed significant PPI deficits at PD 60 but not PD 35. The
PPI deficits in the NVHL rats are attenuated by atypical antipsychotics
such as clozapine, risperidone but not by a typical antipsychotic haloperidol consistent with previous study [16]. The atypical antipsychotics, such as clozapine and risperidone, have been shown to havea high affinity for 5-HT2A receptors [30]. Similar results are found inschizophrenia patients, where atypical antipsychotic drugs, but not
typical antipsychotic drugs, attenuate PPI deficits in the patients [31].
Since the levels of 5-HT2A binding sites were altered in several prefrontal cortical areas of post-pubertal NVHL rats, the capacity of ketanserin to attenuate the PPI deficits of NVHL rats was investigated. We
observed that NVHL-induced PPI deficits were attenuated by the administration of ketanserin. However, ketanserin has affinity not only for
5-HT2A/2C receptors but also α1 and dopamine (DA) 1/2 receptors[32,33]. It has been reported that alterations in prefrontal α1 adrenergic receptors may contribute to altered PPI responses observed in
NVHL rats [17]. However, we observed that the NVHL-induced PPI
deficits were also attenuated by the administration of a more selective
5-HT2A receptor antagonist MDL100907. Previous study reported thatboth increases and decreases in cortical 5-HT activity disrupt PPI [34].
These findings suggest that cortical serotonergic system play an important role in PPI deficit of NVHL rats.
It is possible that 5-HT system in the PFC interacts with the dopaminergic system to modulate the PPI deficit in the NVHL rats. It has
been suggested that abnormalities in dopaminergic transmission in the
PFC might be a critical component of abnormal behaviors in postpubertal NVHL rats [35]. A role of prefrontal cortical DA in PPI has
been reported previously. For example, reduction of dopaminergic activity in the PFC by local injection of the neurotoxin 6-hydroxydopamine was shown to induce PPI deficits [36]. Further, intra-PFC
injection of a dopamine D1 receptor antagonist enhanced DA agonist
apomorphine-induced PPI deficits [37]. Systemic administration of
MDL100907 has been shown to increase medial prefrontal DA efflux
and 5-HT2A receptor suppresses dopaminergic transmission in the PFC[38,39]. Taken together, we suggest that PPI deficits in NVHL animals
may be related to increased sensitivity or activity of 5-HT2A receptors
which may disrupt the balance between multiple neurotransmitter

4. Discussion
In the present study, we have demonstrated that post-pubertal
NVHL rats exhibit increases in the levels of both 5-HT2A receptors and
SERT binding sites in mPFC. These increases are likely to be functionally relevant as the administration of 5-HT2A agonist DOI significantly
enhanced WDSr in post-pubertal NVHL rats. Moreover, we have observed that 5-HT2A antagonists were able to attenuate the PPI deficits inpost-pubertal NVHL rats.
The administration of DOI elicits a characteristic behavioral response that is manifested as an induction of WDSr in rats and mice [25].
The WDSr is mediated by selective activation of 5-HT2A receptors, andis a useful and non-invasive marker of alterations in endogenous 5-HT2A
receptor activity [26]. For example, chronic administration of
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Fig. 5. Quantitative autoradiography of [3H] ketanserin or [3H] citalopram binding sites in various regions of sham and NVHL rats at PD 35 (A and C) and 60 (B and
D). Non-specific binding was defined in the presence of 10 μM mianserin for quantitative autoradiography of [3H] ketanserin and 10 μM fluoxetine for quantitative
autoradiography of [3H] citalopram. Specific binding is expressed as nCi/mg wet weight. Vertical bars represent SEM. *p < 0.05, **p < 0.01 compared with shamgroup.

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systems that are involved in modulating sensorimotor gating.
We found that NVHL rats have increased levels of prefrontal cortical
SERT binding sites at both pre- and post-pubertal period. SERT exists atpresynaptic serotonergic nerve terminal [40]. Increased SERT bindingsites may indicate increased 5-HT reuptake and/or the number of 5-HT
nerve terminals [41]. The present study found that SERT level in theraphe nuclei at post-pubertal period shows a tendency toward of increase (p = 0.07). The raphe nuclei which has 5-HT-producing neurons
projects to the PFC and ventral hippocampus, respectively [42]. Thus,
NVHL may induce an increase of 5-HT neurons in the PFC through theraphe nuclei. We hypothesized that increased 5-HT reuptake in the PFC
results in the reduction of 5-HT levels in the subsynaptic interval, while
this phenomenon induces an increase of 5-HT2A receptors in the post
synapse as compensatory mechanism. Interestingly, increase of SERT
has been reported in PFC in schizophrenia patients [4] and genetic
animal model of schizophrenia [43]. Further, an enhanced DOI-inducedhead twitch response (HTR) has also been shown in SERT overexpressing transgenic mice [44]. In contrast, the SERT knockout mice
showed reducing HTR to DOI [45]. Thus, we consider that altered SERT
binding sites in NVHL may be associated with alternation of cortical 5HT2A receptors bindings, and may indirectly induce PPI deficits.
The exact mechanism by which NVHL leads to serotonergic changes
is unknown at present. However, observations in NVHL animals of altered morphology and electrophysiological properties of prefrontal
pyramidal neurons [46] suggest that the lesion may initiate a reorganization within PFC with 5-HT2A and SERT changes being part ofthis remodeling. Regardless of the mechanisms of serotonergic changes,
our data in NVHL animals suggest that the alterations in serotonergictransmission may contribute to some of the behavioral changes observed in this neurodevelopmental animal model of schizophrenia.
Taken together, our data also suggest that at least some of the sensorimotor gating deficits in schizophrenia patients may be due to overstimulation of 5-HT2A receptors.

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Declaration of Competing Interest
Authors report no conflicts of interest.
Acknowledgments
This study was supported in part by Grants-in-Aid for Scientific
Research (Grant number 18K06687) and Matching Fund Subsidy for
Private University (Grant number S1511001L) from the Ministry of
Education, Culture, Sports, Science and Technology of Japan.
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Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Lettersjournal homepage: www.elsevier.com/locate/bmcl

ER subtype selectivity of m-carborane-containing phenols: C-alkyl groups on
the m-carborane cage enhance ERα selectivity
Kiminori Ohtaa, , Takumi Ogawab, Koichi Katoc,d, Akifumi Odac, Yasuyuki Endob⁎a

School of Pharmacy, Showa University, 1-5-8, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan
Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tenpaku-ku, Nagoya 468-8503, Japand
Department of Pharmacy, Kinjo Gakuin University, 2-1723 Omori, Moriyama-ku, Nagoya, Aichi 463-8521, Japanbc

ART I CL E I NF O

ABS TRACT

Keywords:
Carborane
Estrogen receptor
Erα
Subtype selectivity

Estrogen receptor (ER) exhibits two subtypes, ERα and ERβ, whose biological functions are quite different
despite expression in the same tissues. We developed diiodo-m-carborane derivative 3a, which showed 14-foldselectivity for ERβ with high binding affinity toward ERβ. Interestingly, introduction of an alkyl group into thecarbon atom of the m-carborane cage of 3a markedly enhanced the binding affinity toward ERα and decreased
affinity toward ERβ. C-n-propyl derivative 3d showed 28-fold selectivity for ERα in an ER binding assay andpromoted proliferation of MCF-7 breast cancer cells. Docking simulation studies suggest that the directions of then-propyl group and the diiodo substituent introduced on the m-carborane cage play important roles for thecontrol of ER subtype selectivity. As 3a and 3d showed ERβ and ERα selectivity with high binding affinity,
respectively, these ligands may be useful as biological tools to aid in understanding the different roles of ER
subtypes.

Since the discovery of estrogen receptor α (ERα) in 1986,1 ardent
studies on ER have continued in a variety of research fields, such as
biology,2 pharmacology,2 physiology,2 physical chemistry,3 as well as
medicinal chemistry.4 ERα is a well known member of the nuclear receptor superfamily and mainly present in female reproductive organs
and bone.1,2 Additionally, transmembrane ER has also been discovered
and belongs to the G-protein coupled receptor (GPCR) family.5 ERα is
activated by endogenous estrogen, 17β-estradiol (E2, 1, Fig. 1), andregulates various important physiological processes, including development and function of the female reproductive system, maintenance ofbone mineral density, tumor repressive effects, cardiovascular health,and protection of the central nervous system.2 Decrease of E2 production after menopause induces ERα deactivation and may lead to osteoporosis and menopause. ERα agonists are often used for estrogen
replacement therapy but increased risk of breast cancer. Recently,
several tissue selective estrogen receptor modulators (SERMs), whichshow tissue selective agonist or antagonist activities by selective recruitment of co-repressors or co-activators toward ERα, have been
discovered as potential therapeutic agents for osteoporosis, and menopause, in addition to breast cancer.6
ER-subtype β (ERβ) was cloned from a rat ventral prostate cDNA
library as a second form of ERα.7 ERβ is also activated by E2, but⁎

exhibits quite different biological functions from ERα despite expression in the same tissues.8 Recent studies suggest that ER subtype selective modulators offer potential as therapeutic agents for ER-relateddiseases, such as depression,9 Alzheimer’s disease,10 multiple
sclerosis,11 metabolic disorders,12 cardiovascular diseases,13 and various type of cancers.14 The subtype selective ligands can be used toelicit beneficial estrogen activities and reduce side effects.15 Thus, thediscovery of subtype selective ER modulators is of particular interest to
medicinal chemists, for the ultimate benefit of patients with ER-dependent diseases. However, development of ER subtype selective ligands is a major challenge, because the structural differences within thehydrophobic ligand binding domains (LBDs) of ERs occur at only two
amino acid residues: Leu384 and Met421 in ERα, which are replaced by
Met336 and Ile373, respectively, in ERβ.16
We have developed several ERβ-selective ligands using a carborane
cage as a hydrophobic pharmacophore.17 Recently, we reported that Biodo-m-carboranyl phenols 2a and 3a showed high ERβ-selectivity withpotent binding affinity toward ERβ (Fig. 1).18 Table 1 summarizes therelative binding affinity (RBA) and the selectivity index (SI) values of
m-carborane-containing ERβ-selective ligands toward ERα and ERβ.
The SAR studies revealed that the iodine atom on the boron atom of the
m-carborane cage enhanced the binding affinity toward ERβ, while theCorresponding author.
E-mail address: ohta@pharm.showa-u.ac.jp (K. Ohta).

https://doi.org/10.1016/j.bmcl.2019.06.025
Received 25 April 2019; Received in revised form 17 June 2019; Accepted 18 June 2019
Available online 19 June 20190960-894X/ © 2019 Elsevier Ltd. All rights reserved.

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K. Ohta, et al.

Fig. 1. Structures of 17β-estradiol (E2, 1) and mcarborane-containing ERβ-selective ligands 2a and3a.

Cu to obtain the key intermediate 7 at a 65% yield.20 The C-Li salt
obtained from the reaction of 7 with n-BuLi was reacted with an alkyl
halide and I2, followed by demethylation with BBr3, to afford 3b-3f at
12–56% yields over two steps. The iodine atoms of 7 were transformed
into methyl and ethyl groups using the corresponding alkyl Grignard
reagents under conditions of Pd-catalyzed coupling.21 When methylmagnesium bromide was used in this coupling reaction, the B,B-dimethyl derivative was obtained, which was dimethylated to produce 4a
at a 61% yield over two steps. Unexpectedly, use of ethylmagnesium
bromide instead of methylmagnesium bromide afforded the B-monoethylated derivative owing to steric hinderance of the ethyl group,
which was transformed into the corresponding phenol 4b at a 14%
yield over two steps. The C-Li salt obtained from the reaction of 6 with
n-BuLi was reacted with 4-methoxybenzyl chloride, followed by demethylation with BBr3, to afford 5 at a 2% yield over two steps. The
benzylation of m-carborame cage gave mono- and di-substituted intermediates which are hard to separate, and thus the yield of 5 is quitelow.
The binding affinity of the synthesized compounds toward ERα andERβ was evaluated by means of a competitive binding assay using[2,4,6,7-3H]17β-estradiol.17 Table 2 summarizes the RBA and SI values
toward both ERs. Surprisingly, introduction of a methyl group onto thecarbon atom of the m-carborane cage of 3a markedly enhanced thebinding affinity toward ERα, but reduced affinity toward ERβ. Thus, theC-methylated derivative 3b exhibited 4.4-fold higher selectivity towardERα vs Erβ; nevertheless, the lead compound 3a acted as an ERβ-selective ligand. The C-ethyl derivative 3c exhibited similar binding affinity toward both ERα and E2. The binding affinity of the C-propyl
derivative 3d toward ERβ was markedly decreased, yet 3d exhibitedhigh ERα binding affinity, and thus showed the highest SI values among
the tested compounds, and a 28-fold selectivity for ERα vs ERβ. Although an alkyl group on the carbon atom of the m-carborane cage of
3a is important for enhancing the ERα binding affinity, likely via hydrophobic interaction with the amino acid residues of the ERα LBD, ann-butyl group is likely too large for similar interactions with ERα LBD.
Thus, 3e exhibited lower selectivity toward ERα than 3d. 3f showedsimilar RBA and SI values to those of 3b, owing to the similar van der
Waals radii of the iodine atom and methyl group. The RBA values of
7,9-dimethyl-m-carboranylphenol (4a) were similar to those of theparent compound (3a) but the ER subtype selectivity was reduced. The
RBA values of 4b toward both ERs were not high, and the B-ethyl groupof 4b may be too large for interaction with both ER LBDs. Thus, 4b
showed little ER subtype selectivity. A methylene group between the mcarborane cage and the phenol group of 5 enhanced flexibility of thehydrophobic pharmacophore, and the binding affinities toward bothERα and ERβ were markedly decreased. Additionally, the SI values of 5
were similar to those of 1 and the ER subtype selectivity was diminished.
The biological profiles, namely agonistic and antagonistic activities,of compound 3d were evaluated by means of a cell proliferation assay
using the MCF-7 cell line, which shows estrogen-dependent cell growth(Fig. 3).17 Compound 3d promoted MCF-7 cell proliferation in a concentration-dependent manner, and acted as an ER agonist, similar toendogenous estrogen (E2).
To understand the effect of alkyl substituents on the carbon atom of
the m-carborane ring in 3 on ER subtype selectivity, we performed

Table 1
Relative binding affinity (RBA) and selectivity index (SI) values of m-carborane
derivatives 2a-2e and 3a.
RBA
Compound

R

ERα

ERβ

SI for ERβ

2a
2b
2c
2d
2e
3a

H
I
Allyl
n-Butyl
Benzyl
–

13
8.4
17
29
28
8.0

129
81
62
157
19
111

9.9
9.6
3.6
5.4
0.68
14

C-substituents of the m-carborane cage reduced and enhanced thebinding affinities toward ERβ and ERα, respectively. In particular,
compound 2e, with a benzyl group, showed little selectivity. As substituents on the m-carborane cage greatly affected the ER subtype selectivity, the m-carborane cage seems both suitable and attractive as ahydrophobic pharmacophore for the discovery of ER subtype-selective
ligands.
Thus, novel m-carborane-containing ER subtype-selective ligands,
which exhibit high ERα or ERβ selectivity with high binding affinity
toward either ER subtype, were designed using 3a as a lead compound.
Development of novel ERα selective agonist is quite important for the
discovery of new functions of ERα. We were interested in the substitution on the carbon atom of the m-carborane cage, the transformation of the B-I bonds into B-alkyl groups, and the introduction of amethylene linker between the phenol ring and the m-carborane cage
(Fig. 2). Herein, we describe the syntheses of several m-carboranecontaining phenols, 3–5, evaluation of their ER binding affinities andselectivity, biological functions of 3d against the estrogen-dependent
breast cancer cell line MCF-7, and results of a docking simulation studyof both the parent 3a and 3d toward ERα and ERβ.
The synthesis of the m-carborane-containing phenol derivatives 3–5
is summarized in Scheme 1. 7,9-Diiodo-m-carborane, 6, which was
prepared by iodination of m-carborane with I2 in the presence of
AlCl3,19 was treated with n-BuLi to obtain the C-Li salt, which wastransformed into the C-Cu salt in the presence of CuCl (I) and reactedwith 4-iodoanisole in the presence of pyridine as a base and a ligand forFig. 2. Design of the ER subtype-selective ligand candidates 3–5.
2291

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K. Ohta, et al.

Scheme 1. Synthesis of novel m-carborane derivatives 3–5. Reagent and conditions: (a) (i) n-BuLi, DME, 0 °C; (ii) CuCl, rt; (iii) 4-iodoanisole, pyridine, 100 °C; (b) (i) nBuLi, THF, 0 °C; (ii) alkyl halides or I2, rt; (c) BBr3, CH2Cl2, 0 °C; (d) CH3MgBr, PdCl2(PPh3)2, CuI, THF, reflux; (e) C2H5MgBr, PdCl2(PPh3)2, CuI, THF, reflux; (f) (i) nBuLi, THF, 0 °C; (ii) 4-methoxybenzyl chloride, rt.
Table 2
Binding affinity and subtype selectivity of the synthesized compounds 3–5 toward ERα and ERβ.a

RBAb

b

SIc

Compound

R

ERα

ERβ

ERα/ERβ

3a
3b
3c
3d
3e
3f
4a
4b
5

H
CH3
C2H5
n-C3H7n-C4H9
I
–
–
–

8
61
99
65
36
59
12
18
0.005

111
14
14
2.3
2.9
26
75
30
0.004

0.07
4.4
7.1
28
12
2.3
0.16
0.6
1.3

a
All binding assays were treated with the test compounds (0.4 nM–4 μM) in the presence of [2,4,6,7-3H]17β-estradiol (4 nM) and performed in triplicate (n = 3).
RBA values were calculated from the IC50 values of E2 and test compounds, taking that of E2 as 100%.
c
Subtype selectivity was estimated from the RBA values toward ERα and ERβ.

guanidino group of Arg394 and the carbonyl group of Leu384 in the
backbone of the α-helix in ERα (Fig. 4A and B). Upon binding to ERβ,each phenol group of 3a and 3d formed hydrogen bonds with the aminoacid residues of Arg346 and Glu304 (Fig. 4C and D). The diiodo substituent of 3a was located at the hydrophobic pocket of ERα, whichcontains sufficient space and is comprised of Leu387, Met421, Ile424,
and Leu525 (Fig. 4A). Upon binding to ERβ, the diiodo substituent of
3a was located near the hydrophobic amino acid residues Ile373 andIle376 (Fig. 4C). It is suggested that the diiodo-m-carborane cage is of asuitable size for interaction with the hydrophobic pocket of ERβ. The npropyl group of 3d is much larger than the diiodo substituent and isclose to both the Met421 and Ile424 residues of ERα. Thus, the diiodo
substituent of 3d was turned within the hydrophobic pocket to allow
the substituted-m-carborane structure of 3d to fit well within the hydrophobic pocket of ERα (Fig. 4B). As the alkyl substituents of 3 wasmade longer, the RBA values toward ERα increased, and the ethyl derivative 3c showed the highest RBA value. Methyl group of 3b would beslightly small for the hydrophobic space formed by both the Met421
and Ile424 residues of ERα. On the other hand, the n-propyl group of 3d
exhibited steric repulsion with Ile373 of ERβ and was swung backwards
against the binding mode of 3d with ERα (Fig. 4D). Compounds 3b and3c with a methyl and ethyl groups, respectively, would be slightly large
for ERβ LBD. The substituted-m-carborane structure of 3d is too large
for interaction with the hydrophobic pocket of Erβ, and thus thebinding affinity of 3d for ERβ was low. Results of the calculation explain the effects of the alkyl group of 3 on the binding affinity for ERα
and ERβ.

Fig. 3. Concentration-dependent MCF-7 cell proliferation induced by 3d. MCF7 cells were incubated with 3d (100 nM–1 nM) for 5 days, and the results areshown as fold cell number, with the value for 0.1 nM E2 taken as 1. Assays were
performed in triplicate (n = 3).

docking simulation studies of 3a and 3d for ERα22 and ERβ22 using adocking program (GOLD 5.2).23 Results are summarized in Fig. 4. Eachphenol group of 3a and 3d formed hydrogen bonds with both the2292— 308 —

Bioorganic & Medicinal Chemistry Letters 29 (2019) 2290–2293

K. Ohta, et al.

Research Program for Private Universities (2015-2019) and a Grant-inAid for Scientific Research (C) (15K08029) from the Japanese Ministryof Education, Culture, Sports, Science, and Technology (MEXT, Japan).
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Fig. 4. Comparisons of docking models of 3a and 3d with ERα and ERβ (atomcolors: H (white), B (pink), C (gray), N (purple), O (red), S (yellow) and I(purple)). (A) Binding mode of 3a with the ERα LBD (baggy). (B) Binding modeof 3d with the ERα LBD (well fitted). (C) Binding mode of 3a with the ERβ LBD(well fitted). (D) Binding mode of 3d with the ERβ LBD (too tight).

(b) Mauvais-Jarvis F, Clegg DJ, Hevener AL. Endocr Rev. 2013;34:309.
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2013;111:E283.
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In conclusion, we designed and synthesized various m-carboranecontaining phenol derivatives as ER subtype-selective ligand candidates
using 3a as the lead compound. Although introduction of a hydrophobic substituent on the carbon atom of the m-carborane cage greatly
affected the binding affinity toward ERs, the other chemical transformations of 3a showed no improvements in binding affinity. Thus, wefound that the 7, 9-diiodo-m-carboranylphenol structure acts as a suitable pharmacophore for ER subtype-selective ligands. Compound 3d
showed the highest ERα selectivity and ER agonistic activity in a MCF-7
cell proliferation assay. Docking simulation studies of 3a and 3d forERα and ERβ suggest that the directions of the n-propyl group and the
diiodo substituent introduced into the m-carborane cage play importantroles for the control of ER subtype selectivity. These findings offer
useful information for the potential design of ER modulators, as well as
SERMs.

(b) Pravettoni A, Mornati O, Martini PGV, et al. Mol Cell Endocrinol. 2007;263:46;(c) Kim IY, Kim B-C, Seong DH, et al. Cancer Res. 2002;62:5365.
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Chem. 1993;462:19.
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1989;59:607.
22. The docking simulation study were performed using 3D structures of human ERα andERβ which were retrieved from the Protein Data Bank (PDB ID: 1ERE and 2I0G,
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Acknowledgments
This research was supported by a Grant-in-Aid for the Strategic

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https://doi.org/10.1186/s12974-019-1580-7(2019) 16:201

RESEARCH

Open Access

Effect of Enterococcus faecalis 2001 oncolitis and depressive-like behavior in
dextran sulfate sodium-treated mice:
involvement of the brain–gut axis
Kohei Takahashi1,2, Osamu Nakagawasai1* , Wataru Nemoto1, Takayo Odaira1, Wakana Sakuma1, Hiroshi Onogi3,
Hiroaki Nishijima4, Ryuji Furihata5, Yukio Nemoto6, Hiroyuki Iwasa7, Koichi Tan-No1 and Takeshi Tadano1,8
Abstract
Background: Patients with inflammatory bowel disease (IBD), including those with ulcerative colitis and Crohn’sdisease, have higher rates of psychiatric disorders, such as depression and anxiety; however, the mechanism of
psychiatric disorder development remains unclear. Mice with IBD induced by dextran sulfate sodium (DSS) in
drinking water exhibit depressive-like behavior. The presence of Lactobacillus in the gut microbiota is associatedwith major depressive disorder. Therefore, we examined whether Enterococcus faecalis 2001 (EF-2001), a biogenic
lactic acid bacterium, prevents DSS-induced depressive-like behavior and changes in peripheral symptoms.
Methods: We evaluated colon inflammation and used the tail suspension test to examine whether EF-2001
prevents IBD-like symptoms and depressive-like behavior in DSS-treated mice. The protein expression of tumor
necrosis factor-α (TNF-α), interleukin-6 (IL-6), X-linked inhibitor of apoptosis protein (XIAP), and cleaved caspase-3 inthe rectum and hippocampus was assessed by western blotting. Hippocampal neurogenesis, altered nuclear
factor-kappa B (NFκB) p65 morphometry, and the localization of activated NFκB p65 and XIAP were examinedby immunohistochemistry.
Results: Treatment with 1.5% DSS for 7 days induced IBD-like pathology and depressive-like behavior, increased TNF-α
and IL-6 expression in the rectum and hippocampus, activated caspase-3 in the hippocampus, and decreased
hippocampal neurogenesis. Interestingly, these changes were reversed by 20-day administration of EF-2001. Further,
EF-2001 administration enhanced NFκB p65 expression in the microglial cells and XIAP expression in the hippocampusof DSS-treated mice.
Conclusion: EF-2001 prevented IBD-like pathology and depressive-like behavior via decreased rectal and hippocampal
inflammatory cytokines and facilitated the NFκB p65/XIAP pathway in the hippocampus. Our findings suggest a close
relationship between IBD and depression.
Keywords: Antidepressant, Apoptosis, EF-2001, Inflammatory bowel disease, Neurogenesis, Neuroinflammation* Correspondence: osamun@tohoku-mpu.ac.jp
1
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Tohoku
Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai
981-8558, Japan
Full list of author information is available at the end of the article© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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Background
Inflammatory bowel disease (IBD), which comprises
ulcerative colitis and Crohn’s disease, affects approximately 2.2 million people in Europe and 1.4 million
people in the USA. Recent studies have demonstrated aconnection between intestinal inflammation and changes
in brain function [1]. Inflammation in the bowel is associated with alterations in the central nervous system, as
revealed by the activation of tumor necrosis factor-α(TNF-α) signaling and microglia in the brain [2]. Other
researchers have demonstrated that chronic experimental colitis increases anxiety behavior in mice [3]. Further,
peripheral inflammation may account for at least someof the neurological and behavioral changes associatedwith chronic inflammatory diseases. Indeed, patientswith IBD have higher rates of obsessive–compulsive disorder, panic disorder, depression, and anxiety [4–7].
A well-characterized mouse model of IBD is produced
by repeated administration of dextran sulfate sodium(DSS) in drinking water [8]. DSS does not cross the
blood–brain barrier because of its higher molecularweight. Epithelial cell toxicity, increased intestinal permeability, and macrophage activation are implicated in
the deleterious effects of DSS. The DSS model is characterized by colonic epithelial cell lesions and acute (7–14
days after the beginning of the treatment) intestinal inflammation [9]. Repeated DSS cycling in combination withtreatment with azoxymethane, a genotoxic agent, induced
colitis-dependent neoplasia, generating a commonly usedmodel for colorectal neoplasia and cancer in humans [10].
Recent studies have reported that DSS-treated rodents
exhibit anxiety- and depressive-like behavior [11] andreduction of hippocampal neurogenesis [12].
Decreased adult hippocampal neurogenesis is associatedwith depression in rodents and humans [13–15]. Moreover, depression is associated with altered inflammation[16], which manifests due to increased inflammatory cytokine expression [17]. Pro-inflammatory cytokines also inhibit adult neurogenesis in the hippocampus [18–20].
Therefore, cytokine-induced disruption of neurogenesis
might be a key link between inflammation and depression.
Antidepressants enhance hippocampal neurogenesis [21]and regulate several apoptotic factors, which are involvedin cell survival pathways [22]. Treatment of mood disorders, including depression and anxiety, is criticallydependent on intact adult neurogenesis in the hippocampal dentate gyrus (DG) [23, 24]. Thus, the stimulation ofneurogenesis and reduction of apoptosis may constituteimportant drug targets in the modulation of depressive
symptoms [25].
Enterococcus faecalis 2001 (EF-2001) is a biogenic lactic acid bacterium that is used as a biological response
modifier (BRM). Live E. faecalis 2001 can be heattreated to produce a BRM containing high levels of β-glucan, named EF-2001. β-Glucan, one constituent of
EF-2001, is a ligand for toll-like receptor 2 (TLR2) andactivates nuclear factor-kappa B (NFκB) p65, which controls spontaneous apoptosis and anti-apoptotic effects.
NFκB p65 activation inhibits apoptosis by increasing Xlinked inhibitor of apoptosis protein (XIAP), an antiapoptotic factor that exerts its action by regulating
caspase-3 activity [26, 27]. EF-2001 can decrease serum
inflammatory cytokines in a contact dermatitis model
mouse [28], has anti-tumor effects [29], and protects
chemical-induced colitis [30]. Therefore, we hypothesized that EF-2001 may attenuate inflammation andapoptosis in DSS-treated mice. Additionally, reports indicate that E. faecalis modulates inflammation in colitis
models [31, 32]. However, the effect of EF-2001 oncolitis-induced depression is unclear.
Taken together, we examined whether EF-2001 prevents
DSS-induced depressive-like behaviors and changes inperipheral symptoms and investigated the neuroprotective
molecular mechanisms underlying these effects.

Materials and methods
All experiments were performed following approval of
the Ethics Committee of Animal Experiments in Tohoku
Medical and Pharmaceutical University (approval numbers: 16023 cn, 17015 cn, and 18031 cn) and according
to the National Institutes of Health Guide for the Careand Use of Laboratory Animals. All efforts were made tominimize suffering and reduce the number of animals
used.
Animals

We used male ddY mice (weight, 28–32 g; Japan SLC,
Shizuoka, Japan) for all experiments (total: n = 239; behavioral tests: n = 127; immunohistochemistry: n = 55;
western blot analysis: n = 24; mRNA quantification: n =33). Mice were housed in cages with free access to foodand water under conditions of constant temperature(22 ± 2 °C) and humidity (55 ± 5%), on a 12-h light todark cycle (lights on: 07:00–19:00).
Compounds

Commercially available heat-treated EF-2001 was originally isolated from healthy human feces. It was supplied asa heat-killed, dried powder by Nihon BRM Co. (Tokyo,
Japan). DSS (0.75%, 1.5%, or 3%; Wako Pure Chemical Industries Ltd., Osaka, Japan) and EF-2001 (250 mg/kg) weredissolved in drinking water. Mice were given drinking
water containing DSS ad libitum for 7 days to induce colitis. Dexamethasone (Dex; 0.1 mg/kg; Aspen Japan, Tokyo,
Japan) and imipramine (Imi; 20 mg/kg; Sigma–Aldrich,
St-Louis, USA) were dissolved in saline. EF-2001 wasadministered orally (per os [p.o.]) from 14 days before thebeginning of DSS administration until the day prior to the— 311 —

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last DSS treatment. Dex and Imi were administered intraperitoneally (i.p.) starting on the same day as the first DSS
administration until the last day of DSS treatment. The
dose for each drug used was calculated from previous reports [29, 33, 34].

is the combined score of stool consistency and bleeding,as detailed in Table 1. When mice were sacrificed, thecolon length, starting above the anus to the top of thececum, was measured. All parameters were scored ondays 3, 5, and 7 during DSS treatment.

Evaluation of colon inflammation

Tail suspension test

This evaluation was conducted according to the experimental protocol shown in Fig. 1a, b. Disease activity
index (DAI) scores are well correlated with pathologicalfindings in a DSS-induced model of IBD [35]. DAIscores were calculated as described previously [36]. DAIThis test was conducted according to the experimental
protocol shown in Fig. 1a, b. The tail suspension test
was conducted to assess depressive-like behaviors andantidepressant effects. The procedure was performed as
described previously [37]. Mice were considered

Fig. 1 Experimental time course for the behavioral tests, assessment of inflammation, histology, western blotting, and immunohistochemical of
the experimental protocol A, experimental protocol B, and experimental protocol C— 312 —

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Table 1 Score of stool consistency and rectal bleeding
Score

Stool consistency

Rectal bleeding

1

NormalAbsent2

Loose stool (tangible stool withhigh moisture content)

Visible blood stool3

Diarrhea stool (collapsed stool
with high moisture content)

Blood is attached tothe anus

4

Watery diarrhea (almost intangible
liquid stool)

Always bleeding fromthe anus

immobile only when they hung completely and passively
motionless. Mice were suspended 30 cm above the floor
by means of adhesive tape placed approximately 1 cm
from the tip of the tail. The duration of time spent immobile was quantified during a test period of 10 min.
Locomotor activity

This test was conducted according to the experimental
protocol shown in Fig. 1a. The locomotor activity ofmice was evaluated using the multichannel activitycounting system SUPERMEX (Muromachi Kikai Co.,Tokyo, Japan). The procedure and instrument have beenreported previously [38]. This instrument can monitor
even minute movements in all three planes of motion(sagittal, vertical, and horizontal) as one movement,
owing to its infrared sensor with multiple Fresnel lenses
that can be moved close enough to the cage to capture
multidirectional locomotor alterations in a single mouse.
This system interprets each movement as a count. Thus,
the vertical movements, such as jumping, and horizontal
movements, such as walking and running, can becounted. Activity measurements were conducted between 11:00 and 15:00 during the light phase. Mice weredivided into three groups (water, DSS 1.5%, and DSS
3%) and were placed in activity boxes during 15 min foradaptation. Locomotor activity was recorded for 60 min.
Histology

The histological analysis was conducted according to theexperimental protocol shown in Fig. 1b. Histology wasassessed in mice following DAI and behavioral evaluations. The entire colon was collected and fixed in 4%
buffered formalin for 24 h at room temperature, imbedded in paraffin, and sliced. Samples were sectioned into
5-μm slices and subjected to staining with hematoxylin
and eosin (HE). Finally, sections were examined under alight microscope to evaluate the histopathologic changes
in colon tissue.
Western blotting

Western blotting was performed according to the experimental protocol shown in Fig. 1b. Mice were divided into
four groups (water/water, water/EF-2001, DSS/water, andDSS/EF-2001). Mice were sacrificed by decapitation after

20 days of water or EF-2001 administration. Protein isolation and western blots were performed as described previously [37, 39]. Sectioning was performed as described
previously [40, 41]. After sacrifice, the rectum of eachmouse was washed in ice-cold phosphate-buffered saline(PBS). The rectal tissue, 8 mm from the edge of the cecum(side of the anus), was carefully cut into 5-mm slices on
ice. The brain was removed and sectioned on ice using amouse brain slicer (Muromachi Kikai) to produce 1-mmthick coronal sections. To ensure precise regions, the cerebral peduncle was regarded as a landmark, and the fiveedge blades were anteriorly placed from this landmark.
We visually confirmed the dorsal hippocampal location
using Paxinos and Franklin mouse brain atlas [42]. Afterelectrophoresis, proteins were transferred to a PVDF
membrane, which was then incubated with blocking solution [10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.01%
Tween 20, and 5% skim milk] for 1 h. Next, membranes
were probed with antibodies against TLR2 (1:100; Cell
Signaling Technology, Danvers, USA), TNF-α (1:1000;
Cell Signaling Technology), interleukin-6 (IL-6; 1:1000;
Cell Signaling Technology), XIAP (1:200; Abcam Ltd.,
Cambridge, UK), brain-derived neurotrophic factor(BDNF; 1:100; Abcam Ltd.), and β-actin (1:1000; Cell Signaling Technology) overnight at 4 °C. Membranes were
washed with blocking solution without milk and incubatedwith horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology) for 2 h, followed byvisualization of the immunoreactive species with ECLWestern Blotting Detection Reagent (Amersham Life Science, Piscataway, USA). Band densities were analyzed with
ImageJ 1.43 (National Institutes of Health).
Immunohistochemical analysis

Immunohistochemical analysis was conducted according
to the experimental protocol shown in Fig. 1c. To assess
neurogenesis, on day 20 after EF-2001 administration, 5bromo-2′-deoxyuridine (BrdU; Sigma–Aldrich; 75 mg/
kg, i.p.) was injected three times every 2 h after the last
administration of water or EF-2001. Animals were subsequently sacrificed 24 h after the last injection. Brainsamples were collected as described previously [37, 38].
The brains were cut into 40-μm sections from bregma −2.20 to − 2.80 mm using a cryostat (MICROM HM560,
Mikron Instrument, Inc., California, USA).
Frozen sections were mounted on glass slides (Matsunami Glass, Osaka, Japan). Sections were treated withHCl (2 N) at 37 °C for 30 min, followed by neutralization
with sodium borate buffer (0.15 M) at room temperature
twice every 10 min. After three washes every 5 min, thesections were incubated with PBS containing 1% normal
goat serum (Life Technologies Corporation, Carlsbad,
USA) and 0.3% Triton X-100 (PBSGT) at room
temperature for 2 h. The sections were incubated— 313 —

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overnight at 4 °C with rat anti-BrdU monoclonal antibody (1:100; Harlan SeraLab, Loughborough, UK) andmouse anti-doublecortin (DCX) monoclonal antibody (1:50; Santa Cruz Biotech, Santa Cruz, CA). Sections were
washed and incubated for 2 h at room temperature withgoat anti-rat IgG Alexa Fluor 568 (1:200; Molecular
Probes, Eugene, USA) and goat anti-mouse IgG Alexa
Fluor 488 (1:200; Molecular Probes) with PBSGT. DAPI
was used to identify the nuclei. Finally, sections were
washed and coverslipped with Dako fluorescence mounting medium (Dako, Carpinteria, USA). Labeled sections
were analyzed using a confocal laser-scanning microscope(A1Rsi; Nikon, Tokyo, Japan). Eight sections per mousewere used, and two images (left and right hemisphere,
640 × 640 μm) of the DG region of the hippocampus wereobtained from each section. To assess neurogenesis, wecounted the number of BrdU+/DCX+ cells in the DG. A
mean number of eight images were analyzed for eachmouse, and each group contained 6–9 mice.
Double immunofluorescence staining

Immunofluorescence analysis was conducted according
to the experimental protocol shown in Fig. 1b. The brain
samples were collected as described previously [43, 44].
The sections were incubated overnight at 4 °C withrabbit anti-TLR2 (1:100; Cell Signaling Technology),
rabbit anti-NFκB p65 (1:500; Cell Signaling Technology),
rabbit anti-XIAP (1:200; Abcam Ltd.), mouse anti-DCX
monoclonal antibody (1:50; Santa Cruz Biotech), mouseanti-neuronal nuclear antigen (NeuN; 1:500; Millipore
Corporation), rabbit anti-ionized calcium-binding adaptor
molecule 1 (Iba1; 1:200; Wako Pure Chemical Industries
Ltd., Osaka, Japan), and mouse anti-glial fibrillary acidic
protein (GFAP; 1:200; Millipore Corporation) antibodies.
When double labeling was performed using two primary
antibodies from different host species (rabbit and mouse),
sections were washed and incubated for 2 h at room
temperature with goat anti-rabbit IgG Alexa Fluor 568 (1:200; Molecular Probes) and goat anti-mouse IgG Alexa
Fluor 488 (1:200; Molecular Probes) in PBSGT.
When double labeling was performed using two primary antibodies from the same host species (rabbit
anti-TLR2, rabbit anti-NFκB p65, rabbit anti-XIAP,and rabbit anti-Iba1 antibodies), the detection ofeach antigen was performed sequentially and labeledgoat anti-rabbit IgG Alexa Fluor 488 AffiniPure Fabfragments (Jackson ImmunoResearch Laboratories,USA), instead of whole antibodies, were used in thefirst detection (Iba1). The immunohistochemical
staining with two primary antibodies from the same
host species was carried out as described previously
in detail [45]. Immunofluorescent images were analyzed with a confocal laser-scanning microscope(A1Rsi; Nikon).

Neuromorphometrical study

Morphometric assessment of the brain was conductedaccording to the experimental protocol shown in Fig. 1b.
The brain samples were collected as described previously[37, 38]. The sections were incubated overnight at 4 °C
with rabbit anti-NFκB p65 antibody (1:500; Cell Signaling Technology). Sections were washed and incubatedfor 2 h at room temperature with goat anti-rabbit IgG
Alexa Fluor 568 (1:200; Molecular Probes) in PBSGT.
We observed alterations in activation of NFκB p65positive cells in the hippocampal DG area with a confocal laser-scanning microscope. We then evaluated theactivation of NFκB p65-positive cells by observing translocation to cell nuclei.
Reverse transcription polymerase chain reaction (RT-PCR)

RT-PCR was performed according to the experimental
protocol shown in Fig. 1b. Total RNA was isolated fromthe rectum and hippocampus of mice using TRI Reagent
according to the manufacturer’s protocol. Total RNA wasreverse transcribed using ReverTra Ace and oligo (dT)primers. PCR was conducted using the following primer
sequences: IL-6 sense primer 5′-AGGAGTGGCTAAGGACCAAGA-3′ and antisense primer 5′-CATAACGCACTAGGTTTGCCG-3′, TNF-α sense primer 5′-GGCAGG
TCTACTTTGGAGTCATTGC-3′ and antisense primer
5′-ACATTCGAGGCTCCAGTGAATTCGG-3′,
and
TATA- binding protein (TBP) sense primer 5′-ACCGTG
AATCTTGGCTGTAAAC-3′ and antisense primer 5′GCAGCAAATCGCTTGGGATTA-3′. For quantificationof mRNA expression, real-time PCR was carried out in a
20-μl solution containing Go Taq quantitative PCR
Master mix (10 μl), RT template (2 μl), water (7 μl), andprimers (1 μl) using the StepOnePlus Real-Time PCR
System (Applied Biosystems, California, USA). The
amount of each PCR product was normalized to TBP.
Statistical analysis

Normality and homoscedasticity assumptions were verified before the use of any parametric tests (Shapiro–
Wilk normality test and equality of variances F test).
Results are expressed as mean ± standard error of themean (SEM). The significance of differences was determined by the Student’s t test for two-group comparisonsor by one or two-way analysis of variance (ANOVA),
followed by Tukey–Kramer tests for multigroup comparisons using GraphPad Prism 7 (GraphPad Software,
California, USA) and StatView 5.0 (HULINKS, Tokyo,
Japan). For the DAI scores, statistical significance ofdifferences was assessed with a non-parametric Mann–
Whitney test for two-group comparisons or Kruskal–
Wallis test followed by Steel’s test for multigroupcomparisons. In some cases, when a main effect was significant without interaction effect, we did an exploratory— 314 —

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and limited pairwise post hoc comparison consistent
with our a priori hypothesis. Results were considered
statistically significant if p < 0.05.

Results
Concentration-dependent effect of DSS on DAI scores,
colon length, immobility time, and locomotor activity inmice

DAI scores of both stool consistency and rectal bleedingin DSS-treated mice (1.5% and 3%) were significantly increased compared with those in the control group[Fig. 2a, b, Kruskal–Wallis test, stool consistency: p <0.0001, rectal bleeding: p < 0.0001]. The colon length in
DSS-treated mice (0.75%: p = 0.0007, 1.5%: p < 0.0001,
and 3%: p < 0.0001) was significantly shorter than in control mice [Fig. 2c, one-way ANOVA, F (3, 41) = 16.2, p <0.0001]. There was a significantly prolonged duration of
immobility in DSS-treated mice (0.75%: p = 0.0337, 1.5%:
p = 0.0165, and 3%: p < 0.0001) compared with controls
in the tail suspension test [Fig. 2d, one-way ANOVA, F(3, 44) = 9.626, p < 0.0001]. Furthermore, DSS (1.5% and3%) did not affect locomotor activity in mice [Fig. 2e,
one-way ANOVA, F (2, 27) = 0.142, p = 0.8683].
We observed that DSS 3% caused the death of a few
mice (data not shown). Based on these results, DSS 1.5%
was the appropriate dose to investigate in the IBD modelwith depression.

Time-dependent effects of DSS on DAI scores, colon
length, and immobility time in mice

As shown in Fig. 3, diarrhea and shortened colon length
were observed on day 7 of DSS treatment, but not ondays 3 and 5 [Fig. 3a, Mann–Whitney test, day 3: p >0.9999, day 5: p = 0.3173, day 7: p = 0.0009; Fig. 3c, twoway ANOVA, group: F (1, 65) = 14.51, p = 0.0003, time:F (2, 65) = 0.8248, p = 0.4429, group × time: F (2, 65) =16.31, p < 0.0001]. In contrast, bloody stool and prolonged duration of immobility were observed on days 5
and 7 of DSS treatment, but not on day 3 [Fig. 3b,
Mann–Whitney test, day 3: p > 0.9999, day 5: p = 0.0139,
day 7: p = 0.0008; Fig. 3d, two-way ANOVA, group: F (1,66) = 23.4, p < 0.0001, time: F (2, 66) = 3.874, p = 0.0257,
group × time: F (2, 66) = 6.591, p = 0.0025]. Based onthese results, day 7 after the beginning of DSS treatment
was the best time point to investigate changes in the
IBD model with depression.
Effects of Imi, Dex, or EF-2001 on DAI scores, colon
length, and immobility time in DSS-treated mice

We investigated the effects of Imi, Dex, or EF-2001 on
DSS-induced changes in mice. Imi reversed the DSSinduced prolonged duration of immobility time, whileother changes were not affected. In contrast, Dex and
EF-2001 prevented DSS-induced diarrhea, bloody stool(Dex showed a tendency toward prevention of bloody

Fig. 2 Changes in stool consistency (a), rectal bleeding (b), colon length (c), immobility time (d), and locomotor activity (e) in dextran sulfate
sodium (DSS)-treated mice at day 7. Bars represent means ± standard error of mean (SEM). *p < 0.05 and **p < 0.01 vs. water group (n = 6–12
per group)— 315 —

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Fig. 3 Time-course of stool consistency (a), rectal bleeding (b), colon length (c), and immobility time (d) in dextran sulfate sodium (DSS; 1.5%)treated mice at days 3, 5, and 7. Bars represent means ± standard error of mean (SEM). *p < 0.05 and **p < 0.01 vs. water group (n = 11–12
per group)

stool), and colon atrophy. Further, it reversed the prolonged duration of immobility time [Kruskal–Wallis test,
Fig. 4a, stool consistency: p = 0.0012; Fig. 4b, rectal
bleeding: p = 0.0181; Fig. 4c, one-way ANOVA: F (2,31) = 5.089, p = 0.0123; Fig. 4d, F (2, 31) = 12.17, p =0.0001; Mann–Whitney test, Fig. 4e, stool consistency:
p = 0.0231; Fig. 4f, rectal bleeding: p = 0.0535; Student’s ttest, Fig. 4g, colon length: t (22) = 3.632, p = 0.0015;
Fig. 4h, immobility time: t (22) = 2.939, p = 0.0076]. In
the histological study, EF-2001 prevented DSS-inducedcolon erosion similar to treatment with Dex (Fig. 4i–l).

Effect of EF-2001 on TNF-α and IL-6 levels in the rectumand hippocampus of DSS-treated mice

As shown in Fig. 5, the immunocontent of TNF-α
and IL-6 in the rectum and hippocampus of DSStreated mice was significantly increased comparedwith controls. Interestingly, these changes were reversed by treatment with EF-2001 [two-way ANOVA,
Fig. 5b, group: F (1, 17) = 10.01, p = 0.0057, treatment:
F (1, 17) = 10.26, p = 0.0052, group × treatment: F (1,17) = 10.27, p = 0.0052; Fig. 5c, group: F (1, 14) = 6.676,
p = 0.0216, treatment: F (1, 14) = 5.352, p = 0.0364,
group × treatment: F (1, 14) = 5.813, p = 0.0302; Fig. 5e,group: F (1, 22) = 3.273, p = 0.0841, treatment: F (1,22) = 19.13, p = 0.0002, group × treatment: F (1, 22) =8.049, p = 0.0096; Fig. 5f, group: F (1, 22) = 8.157, p =0.0092, treatment: F (1, 22) = 16.3, p = 0.0006, group ×
treatment: F (1, 22) = 4.728, p = 0.0407].
Effect of EF-2001 on TNF-α and IL-6 mRNA levels in the
hippocampus of DSS-treated mice

We investigated the changes in the expression of TNF-α
and IL-6 mRNA levels in the hippocampus. The hippocampal TNF-α and IL-6 mRNA levels in DSS-treatedmice did not change as compared to those in control
mice [Fig. 6a, two-way ANOVA, group: F (1, 17) =0.1801, p = 0.6766, treatment: F (1, 17) = 0.2614, p =0.6157, group × treatment: F (1, 17) = 0.3334, p = 0.5713;
Fig. 6b, group: F (1, 29) = 3.335, p = 0.0781, treatment: F(1, 29) = 0.2765, p = 0.6030, group × treatment: F (1,29) = 0.1689, p = 0.6841].
Effect of EF-2001 on reduced neurogenesis in the
hippocampal DG of DSS-treated mice

To determine any change in hippocampal neurogenesis
in DSS-treated mice, animals were injected with BrdU.
The incorporation of BrdU indicates that cells were replicating at the time of the BrdU injection. Further, antiDCX staining was used to identify immature neurons in
the DG. DSS-treated mice had a significantly lower
number of BrdU+/DCX+ cells compared with the control
group, which was reversed by administration of EF-2001[Fig. 7b, two-way ANOVA, group: F (1, 27) = 5.927, p =— 316 —

Takahashi et al. Journal of Neuroinflammation(2019) 16:201

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Fig. 4 Effect of subchronic dexamethasone (Dex) or imipramine (Imi) treatment or chronic Enterococcus faecalis 2001 (EF-2001) treatment on stool
consistency (a, e), rectal bleeding (b, f), colon length (c, g), and immobility time (d, h) in dextran sulfate sodium (DSS)-treated mice. Effect of EF2001 and Dex on histopathologic changes in colon tissue in DSS-induced colitis. Colon tissue of the control (i), DSS 1.5% (j), DSS 1.5% + EF-2001(k), and DSS 1.5% + Dex (l) groups. The red arrow indicates thinning of the mucous membrane. Bars represent means ± standard error of mean
(SEM). #p < 0.05 and ##p < 0.01 vs. vehicle-treated DSS group (n = 10–12 per group)

0.0218, treatment: F (1, 27) = 1.663, p = 0.2082, group ×
treatment: F (1, 27) = 7.613, p = 0.0103].
NFκB p65-positive astrocytes and microglia in the
hippocampal DG after treatment with EF-2001

NFκB p65 may control spontaneous apoptosis, antiapoptotic gene expression, and translocation to cell nuclei
during activation. Two-way ANOVA showed significant
main effects for the treatment factor but not an interaction [Fig. 8f, two-way ANOVA, group: F (1, 15) = 3.237,
p = 0.0922, treatment: F (1, 15) = 34.35, p < 0.0001, group× treatment: F (1, 15) = 0.4169, p = 0.5282]. Therefore, we
focused our analysis on the effects of treatment. EF-2001
treatment significantly increased activation of NFκB p65positive cells in the DG compared with the water-treatedgroup. To determine which cell types were involved, dual
immunofluorescence staining for NFκB p65 was performed in conjunction with cell-specific markers, such as

DCX, NeuN (a marker for mature neurons), GFAP (anastrocyte marker), and Iba1 (a microglia marker). Activated NFκB p65-positive cells were identified as astrocytes
and microglia (Fig. 8g).

Effect of EF-2001 on TLR2 levels in the hippocampus of
DSS-treated miceTwo-way ANOVA showed statistical significance for themain effects of treatment [F (1, 14) = 16.49, p = 0.0012]
but no significance for the TLR2 interaction [group ×
treatment: F (1, 14) = 0.13, p = 0.7239] (Fig. 9a). Thus,
we focused our analysis on the major effects of EF-2001
administration. EF-2001 significantly increased TLR2 expression in the hippocampus of DSS-treated mice.
Moreover, to identify the cell types involved in the production of TLR2, dual immunofluorescence staining was
performed for the localization of TLR2 and cell-specific— 317 —

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Fig. 5 Altered levels of TNF-α and IL-6 in the rectum and hippocampus after Enterococcus faecalis 2001 (EF-2001) administration. a Representative
immunoblots probed with antibodies against rectal TNF-α, IL-6, and β-actin, as indicated. b, c Quantification of normalized values of TNF-α andIL-6 levels with β-actin in the rectum. d Representative immunoblots probed with antibodies against hippocampal TNF-α, IL-6, and β-actin, as
indicated. e, f Quantification of normalized values of TNF-α and IL-6 levels with β-actin in the hippocampus. Bars represent means ± standard
error of mean (SEM). *p < 0.05 and **p < 0.01 vs. vehicle-treated water group. #p < 0.05 and ##p < 0.01 vs. vehicle-treated DSS group (n = 4–7
per group)

markers, such as DCX, NeuN, GFAP, and Iba1. TLR2
was localized in all assessed cell types (Fig. 9b).
Effect of EF-2001 on the enhancement ofneuroinflammation and apoptosis in the hippocampus of
DSS-treated mice

As shown in Fig. 10, two-way ANOVA showed significant main effects for treatment [F (1, 20) = 11.92, p =0.0025] but no interaction for XIAP [group × treatment:
F (1, 20) = 1.23, p = 0.2805] (Fig. 10b). Thus, we focused

our analysis on the effects of treatment. EF-2001 significantly increased XIAP in the hippocampus of DSStreated mice. Cleaved caspase-3 in the hippocampus of
DSS-treated mice was significantly increased comparedwith controls, while EF-2001 treatment significantly decreased cleaved caspase-3 levels in DSS-treated mice
[Fig. 10c, two-way ANOVA, group: F (1, 15) = 7.46, p =0.0155, treatment: F (1, 15) = 15.51, p = 0.0013, group ×
treatment: F (1, 15) = 13.91, p = 0.0020]. Moreover, todetermine the cell types that are involved in theFig. 6 Hippocampal expression of TNF-α (a) and IL-6 (b) mRNA in dextran sulfate sodium-treated mice. Bars represent means ± standard error ofmean (SEM) (n = 3–9 per group)— 318 —

Takahashi et al. Journal of Neuroinflammation(2019) 16:201

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Fig. 7 Influence of Enterococcus faecalis 2001 (EF-2001) on hippocampal neurogenesis in dextran sulfate sodium (DSS)-treated mice. Microscopy
images of BrdU (red), DAPI (blue), and DCX (green) immunostaining in the dentate gyrus region of the hippocampus (a). Arrows indicate BrdU/
DCX double-positive cells. Quantitative analysis of the number of BrdU/DCX double-positive cells in control and DSS-treated mice afteradministration of vehicle or EF-2001 (b). Bars represent means ± standard error of mean (SEM). **p < 0.01 vs. vehicle-treated water group. #p < 0.05
vs. vehicle-treated DSS group (n = 6–9 per group)

production of XIAP, dual immunofluorescence staining
for XIAP was performed in conjunction with cellspecific markers, such as GFAP and Iba-1. XIAP was localized in the microglia (Fig. 10d).

Discussion
Patients with IBD have higher rates of psychiatric disorders, such as depression and anxiety; however, the mechanisms underlying a link between intestinal inflammationand depressive-like symptoms are largely unknown. In thisstudy, we investigated the effect of EF-2001 in IBD-like
physiological changes and depressive-like behavior in
DSS-treated mice. Chronic administration of EF-2001 prevented such changes. In addition, EF-2001 attenuated theincrease of inflammatory cytokines in the rectum andhippocampus, attenuated the reduction of neurogenesis in
the hippocampus, and facilitated the NFκB p65/XIAP
pathway in the hippocampus of DSS-treated mice. This is
the first report that the antidepressant effect of EF-2001
may involve hippocampal neuroprotection via decreased
inflammatory cytokine expression in the rectum andhippocampus as well as apoptotic cell death regulation via
inhibition of caspase-3 activity through facilitation of the
NFκB p65/XIAP pathway in the hippocampus.
IBD, including Crohn’s disease and ulcerative colitis, isa chronic relapsing condition characterized by intestinal
damage (barrier disruption, altered microbiota) and highlevels of inflammation such as elevated inflammatory
cytokines [43, 46, 47]. Cytokines induce extensive inflammation in the colon, which has a negative impact onepithelial cells, resident and recruited immune cells, andstromal cells [44]. Specifically, inflammation can cause
damage to epithelial cells, and activate and recruit

immune and stromal cells, ultimately leading to nonresolving chronic inflammation and the development of
IBD [44]. Moreover, during acute or chronic inflammation, inflammatory cytokines can induce the development of depression [48, 49]. Clinical studies havereported that patients with IBD often exhibit obsessive–
compulsive disorder, panic disorder, depression, andanxiety [4–7]. DSS treatment induces colonic epithelial
cell lesions and intestinal inflammation, including elevated inflammatory cytokines, via epithelial cell toxicity,
increased intestinal permeability, and macrophage activation [9]. It has been suggested that E. faecalis improves colitis by increasing interleukin-10 (IL-10), a
factor that inhibits cytokine synthesis, in colonic epithelial cells [50, 51]. The present study showed that EF2001 reduced inflammatory cytokines in the rectum(Fig. 5). This effect was similar to that of steroids, which
are commonly prescribed for treating IBD [52]. Recently,
other researchers have reported that EF-2001 protects
dinitrobenzene sulfonic acid-induced colitis, a chemically induced colitis model, via a decrease in inflammatory cytokines [30]. Thus, the anti-inflammatory effect of
EF-2001 may also be related to reduced inflammatory
cytokines. Peripheral inflammation is a risk factor for developing mood or psychotic disorders, such as depression [53–56], and may affect hippocampal neurogenesis,
including the proliferation, differentiation, and survival
of newborn neurons [54, 55]. Adult neurogenesis occurs
in two main regions of the brain, one of which is the
subgranular zone of the DG [57, 58]. In the present
study, we observed that DSS treatment significantly
increased TNF-α and IL-6 levels in the rectum andhippocampus. A previous study has reported that DSS— 319 —

Takahashi et al. Journal of Neuroinflammation(2019) 16:201

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Fig. 8 (See legend on next page.)— 320 —

Takahashi et al. Journal of Neuroinflammation(2019) 16:201

Page 12 of 16(See figure on previous page.)
Fig. 8 Influence of Enterococcus faecalis 2001 (EF-2001) on hippocampal activation of NFκB p65-positive cells in dextran sulfate sodium (DSS)treated mice. Microscopy images of NFκB p65 (red) and DAPI (blue) immunostaining in the dentate gyrus region of the hippocampus (a). The
fluorescence intensity profile of DAPI (blue line) and NFκB p65 (red line) in the immunostaining indicated by the white dashed lines in theinactive NFκB p65 (b, c) and active NFκB p65 (d, e). Quantitative analysis of the number of activated NFκB p65-positive cells in control and DSStreated mice after administration of vehicle or EF-2001 (f). Activated NFκB p65 is expressed in astrocytes and microglia in the hippocampus of
DSS-treated mice treated with EF-2001. Microscopy images of NFκB p65 (red), DAPI (blue), and DCX, NeuN, GFAP, or Iba1 (green) immunostaining
in the dentate gyrus region of the hippocampus (g). The boxed area is shown in higher magnification. Bars represent means ± standard error ofmean (SEM). **p < 0.01 vs. vehicle-treated water group. ##p < 0.01 vs. vehicle-treated DSS group (n = 4–5 per group)

treatment significantly increases rectal TNF-α and IL-6
levels [59]. Interestingly, hippocampal TNF-α and IL-6
mRNA in DSS-treated mice were unchanged comparedwith controls (Fig. 6). Depression is closely associatedwith altered inflammation [16], manifested by increased
expression of inflammatory cytokines such as TNF-α
and IL-6 [17]. Neuroinflammatory factors, such as TNFα and IL-6, can negatively affect many stages of neurogenesis in the adult mammalian brain, including theproliferation, differentiation, and survival of newborn
neurons [18–20, 54, 55]. Therefore, cytokine-induced
reduction of neurogenesis might establish a key link
between inflammation and depression. In this study,
DSS-treated mice showed a significant decrease inneurogenesis in the DG, consistent with a previous study[12]. Likewise, DeCarolis and Eisch reported a reduction
in neurogenesis in the hippocampus of patients with depression [60]. These findings suggest that DSS-induced
depressive-like behavior may be associated with the reduction of neurogenesis in the DG via the release of

inflammatory cytokines derived from peripheral inflammation. Moreover, several studies have suggested that
antidepressant effects are critically dependent on intact
adult neurogenesis and may be mediated by the enhancement of neurogenesis in the hippocampal DG [21, 23, 24].
We observed that administration of EF-2001 significantly
attenuated the enhancements of rectal and hippocampal
inflammation and reduction of newborn neurons in the
hippocampus of DSS-treated mice. Therefore, we suggestthat the antidepressant effect and enhanced neurogenesis
observed upon EF-2001 administration are partly independent effects, resulting from the EF-2001-mediated reduction of peripheral inflammation. Although themechanism by which pro-inflammatory cytokines reduce
neurogenesis is not fully understood, we believe that inflammatory cytokines in peripheral tissue might be key
mediators of this process.
In human neutrophils, the activation of NFκB p65 seems
to control spontaneous apoptosis and anti-apoptotic
effects. Unexpectedly, we found that the activation ofFig. 9 Altered TLR2 protein level in the hippocampus after Enterococcus faecalis 2001 (EF-2001) administration. a TLR2 is expressed in all cell types
in the hippocampus of dextran sulfate sodium (DSS)-treated mice administered with EF-2001. Fluorescence microscopy images of TLR2 (red); DAPI
(blue); and DCX, NeuN, GFAP, or Iba1 (green) immunostaining in the dentate gyrus region of the hippocampus. b The insets (boxed area) are imagesof higher magnification. Bars represent means ± standard error of mean (SEM). #p < 0.05 vs. vehicle-treated DSS group (n = 4–6 per group)— 321 —

Takahashi et al. Journal of Neuroinflammation(2019) 16:201

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Fig. 10 Altered levels of XIAP and cleaved caspase-3 in the hippocampus after Enterococcus faecalis 2001 (EF-2001) administration. a Representative
immunoblots probed with antibodies against hippocampal XIAP, cleaved caspase-3, and β-actin, as indicated. b, c Quantification of normalized valuesof XIAP and cleaved caspase-3 levels with β-actin in the hippocampus. XIAP is expressed in microglia in the hippocampus of dextran sulfate sodium(DSS)-treated mice treated with EF-2001. Microscopy images of XIAP (red), DAPI (blue), and GFAP or Iba1 (green) immunostaining in the dentate gyrus
region of the hippocampus (d). The boxed area is shown in higher magnification. Bars represent means ± standard error of mean (SEM). **p < 0.01 vs.
vehicle-treated water group. #p < 0.05 and ##p < 0.01 vs. vehicle-treated DSS group (n = 4–6 per group)

NFκB p65-positive cells were increased by EF-2001 administration in water- and DSS-treated groups comparedwith control groups. Moreover, we found that activationof NFκB p65 was localized to hippocampal astrocytes andmicroglia; however, the mechanisms underlying this activation remain unclear. We hypothesized that β-glucan
may be associated with activation of NFκB p65 via TLR2.
β-Glucan activates TLR2 and increases TLR2 expression
via NFκB p65 activation [61–63]. Interestingly, we foundthat hippocampal TLR2 was activated by EF-2001, which
was localized in all cell types, including immature and mature neurons, astrocytes, and microglia (Fig. 9). We did
not assess the migration of EF-2001 or β-glucan into thebrain; we will examine them in a future study. Modulationof this pathway most likely regulates the balance between
pro- and anti-apoptotic factors [64], thus affecting neutrophil survival. In this study, EF-2001 attenuated DSSinduced neuroinflammation and we hypothesized that
NFκB p65 may play a role in anti-apoptosis. The activation of NFκB p65 inhibits apoptosis via a mechanism involving upregulation of various anti-apoptotic genes, suchas cellular FLICE-inhibitory protein, Bcl-xL, and XIAP
[26, 27]. XIAP, a key member of the inhibitors of apoptosis protein family, can inhibit apoptosis by directly binding to the initiator caspases: caspase-3, -7, and -9 [65].
Moreover, hippocampal XIAP regulates synaptic plasticity,which is associated with the development of depression[66, 67]. In this study, XIAP was significantly increased by
EF-2001. Further, XIAP was localized in hippocampal
microglia. Moreover, cleaved caspase-3, which is crucial in
the process of apoptosis and contributes to the irreversible
stage of apoptosis [68], was significantly increased in the
hippocampus of DSS-treated mice. In contrast, EF-2001
suppressed the increased levels of cleaved caspase-3 in the
hippocampus of DSS-treated mice. These results suggested that EF-2001 might partly modulate apoptosis via
regulation of the microglial NFκB p65/XIAP pathway andcaspase-3 in the hippocampus of DSS-treated mice.
As summarized in Fig. 11, EF-2001 had antiinflammatory and antidepressant effects in DSS-treatedmice, and hippocampal neuroprotection is a key factor
mediating the antidepressant effect of EF-2001. Other— 322 —

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Fig. 11 Schematic representation of the anti-inflammatory and antidepressant effects of Enterococcus faecalis 2001 (EF-2001). The inflammatory
cytokines derived from peripheral inflammation induced by treatment with dextran sulfate sodium may induce depressive-like symptoms via
reduction of neurogenesis in the hippocampus (red arrow). The inhibition of inflammatory cytokines in the rectum may explain the antiinflammatory and antidepressant effects of EF-2001 (blue arrow). Moreover, the antidepressant effect may be caused by neuroprotection via themodulation of XIAP in the hippocampus (blue arrow)researchers have reported that administration of Lactobacillus strains had antidepressant effects and enhanced
neurogenesis in the hippocampus via the vagus nerve[69, 70]. Hence, further extensive experiments examining the relationship between the vagus nerve and effectsof EF-2001 will be presented in a subsequent paper.

Conclusions
The present study showed that DSS-treated mice exhibited IBD-like physiological changes and depressive-likebehaviors (Figs. 2 and 3). This result was consistent withprevious studies [11, 12]. In this study, we evaluated thepredictive validity of the IBD animal model by using aclassic antidepressant drug, Imi, or a steroid, Dex. Administration of Imi significantly improved depressivelike behavior, whereas Dex significantly improved both
IBD-like peripheral symptoms and depressive-like behavior. These results demonstrated that DSS-treatedmice provided a model of IBD with depression.
Our results indicate that EF-2001 attenuated IBD-like
symptoms and depressive-like behavior in DSS-treatedmice. EF-2001 prevented DSS-induced colitis and themechanism may also involve the suppression of inflammatory cytokines in the rectum. The antidepressanteffect of EF-2001 may involve neuroprotection in the
hippocampus via decreased TNF-α and IL-6 expression
in the rectum and hippocampus and facilitation of the
NFκB p65 pathway in the hippocampus. This process islikely mediated by modulation of XIAP, which is involved in the regulation of apoptotic cell death via
caspase-3 activity. Moreover, our findings suggest a close
relationship between IBD and depression.
Abbreviations
ANOVA: Analysis of variance; BDNF: Brain-derived neurotrophic factor;
bp: Base pair; BrdU: 5-bromo-2′-deoxyuridine; BRM: Biological response
modifier; DAI: Disease activity index; DCX: Doublecortin; Dex: Dexamethasone;
DG: Dentate gyrus; DSS: Dextran sulfate sodium; EF-2001: Enterococcus faecalis
2001; GFAP: Glial fibrillary acidic protein; HE: Hematoxylin and eosin;
i.p: Intraperitoneally; Iba1: Ionized calcium-binding adaptor molecule 1;
IBD: Inflammatory bowel disease; IL-6: Interleukin-6; Imi: Imipramine;
NFκB: Nuclear factor-kappa B; p.o.: Per os; PBS: Phosphate-buffered saline;
PBSGT: PBS containing 1% normal goat serum and 0.3% Triton X-100;
SEM: Standard error of the mean; TBP: TATA-binding protein; TLR2: Tolllike receptor 2; TNF-α: Tumor necrosis factor-α; XIAP: X-linked inhibitor of
apoptosis protein

Acknowledgements
The authors would like to thank Mr. Takehiko Tanaka and Ms. Momona
Nagasaki of the Tohoku Medical and Pharmaceutical University for theirtechnical assistance.

— 323 —

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Page 15 of 16

Authors’ contributions
TK performed the experiments (evaluation of colon inflammation, behavioral
testing, western blotting, and immunohistochemical study), analyzed thedata, and wrote the manuscript; NO designed the project; NW and OH
performed the experiments (histology and RT-PCR) and analyzed the data;
OT and SW performed the experiments (evaluation of colon inflammation
and behavioral testing); and NH, FR, NY, IH, T-NK, and TT supervised theexperiments. All authors critically reviewed the manuscript and approved thefinal version for publication.

4.

Funding
This study was supported in part by the Grants-in-Aid for Scientific Research(Grant number 18 K06687) and Matching Fund Subsidy for Private University
from the Ministry of Education, Culture, Sports, Science and Technology ofJapan (grant number S1511001 L).

8.

5.

6.
7.

9.
10.

Availability of data and materials
The datasets used and/or analyzed in the current study are available fromthe corresponding author on reasonable request.

11.

Ethics approval and consent to participate
All experiments were performed following the approval of the Ethics
Committee for Animal Experiments at Tohoku Medical and Pharmaceutical
University (approval numbers: 16023 cn, 17015 cn, and 18031 cn) andaccording to the tenets of the National Institutes of Health Guide for theCare and Use of Laboratory Animals. All efforts were made to minimize thesuffering of the animals and reduce the number of animals used.

12.

13.
14.

Consent for publication
Not applicable.

15.
16.

Competing interests
Hiroyuki Iwasa is an employee of Nihon Berm Co., Ltd. All other authors
declare that they have no competing interests.

17.

Author details
1
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Tohoku
Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai
981-8558, Japan. 2Department of Pharmacology, School of Pharmacy,
International University of Health and Welfare, 2600-1 Kitakanemaru,
Ohtawara, Tochigi 324-8501, Japan. 3Faculty of Health Science, Tohoku
Fukushi University, 1-8-1 Kunimi, Aoba-ku, Sendai, Miyagi 981-8522, Japan.
4
Department of Healthcare and Regulatory Sciences, School of Pharmacy,
Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan.
5
Department of Psychiatry, Nihon University School of Medicine, 30-1
Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan. 6Kampo and Herbal
Medicine Research Center, Yokohama University of Pharmacy, 601
Matanocho, Totsuka-ku, Yokohama City, Kanagawa 245-0066, Japan. 7Nihon
Berm Co, Ltd, 2-14-3 Nagatachou, Chiyoda-ku, Tokyo 100-0014, Japan.
8Complementary and Alternative Medicine Clinical Research andDevelopment, Graduate School of Medicine Sciences, Kanazawa University,
Kanazawa 920-8640, Japan.

18.

19.
20.

21.

22.

Received: 7 June 2019 Accepted: 10 September 201923.

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Contents lists available at ScienceDirect

Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm

Mechanisms underpinning AMP-activated protein kinase-related effects on
behavior and hippocampal neurogenesis in an animal model of depression
Takayo Odaira, Osamu Nakagawasai∗, Kohei Takahashi, Wataru Nemoto, Wakana Sakuma,
Jia-Rong Lin, Koichi Tan-No
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558,
Japan

HIGHL I G HTS

activator AICAR ameliorates depressive behavior in OBX mice.
• ・AMPK
activates PKCζ/NF-κB/BDNF/TrkB/CREB pathway in the hippocampus of OBX mice.
• ・AICAR
promotes neurogenesis, and this effect is attenuated by PKCζ inhibitor.
• ・AICAR
is detected in neurons and microglia; NF-κB is detected in neurons.
• ・Phospho-AMPK• ・AMPK activators may serve as new therapeutic targets in depression.
ABST R A CT

Adenosine monophosphate-activated protein kinase (AMPK) is critical for whole-body energy metabolism regulation. Recent studies have suggested that physicalexercise ameliorates depressive-like behaviors via AMPK activation; however, the underlying mechanism is unclear. Here, we examined the effects and underlying
mechanisms of AMPK activation on depressive-like behavior in olfactory bulbectomized (OBX) mice. We treated OBX mice with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleotide (AICAR) on the 7th or 14th day after bilateral bulbectomy and evaluated depressive-like behavior using the tailsuspension test (TST) and forced swimming test (FST) on the 21st day. The expression of phosphorylated AMPK, protein kinase C ζ (PKCζ), nuclear factor-kappa B(NF-κB), brain-derived neurotrophic factor (BDNF), and cAMP response element-binding protein (CREB) in the hippocampus was assessed by western blotting.
Hippocampal neurogenesis and localization of AMPK and phosphorylated NF-κB were examined by immunohistochemistry. Chronic AICAR treatment suppressed theprolonged immobility of OBX mice in the TST and FST, and increased the levels of phosphorylated AMPK, PKCζ, NF-κB, CREB, and BDNF. Hippocampal neurogenesis
in OBX mice was promoted by chronic AICAR treatment. Co-administration of AICAR with the PKCζ inhibitor or the neurotrophic tyrosine kinase receptor type 2(TrkB) antagonist, ANA-12, inhibited these effects. Phosphorylated AMPK was detected in mature and immature hippocampal neurons and microglia, whilephosphorylated NF-κB was detected only in neurons in AICAR-treated OBX mice. These data indicate that AMPK activation produces anti-depressant effects, which
are mediated by elevated hippocampal neurogenesis potentially via PKCζ/NF-κB/BDNF/TrkB/CREB signaling in neurons.

1. Introduction
Adenosine monophosphate (AMP)-activated protein kinase (AMPK)
is a serine-threonine kinase and a heterotrimer composed of a catalytic
subunit, α, and two regulatory subunits, β and γ (Shaw et al., 2004).
AMPK is activated by physical exercise (Winder and Hardie, 1996) andan increasing AMP/ATP ratio (Hardie and Ashford, 2014), while theactivity of AMPK contributes to the expression of glucose transporters
and improvement in insulin sensitivity (Kong et al., 2013). Recently, we
reported that an AMPK activator promotes recovery from physical fatigue in mice (Nakagawasai et al., 2015). AMPK activators, such as

metformin and resveratrol, elicit anti-depressant effects in patients withdepression (Guo et al., 2014) and animal models of depression (Aliet al., 2015). However, the detailed mechanism underlying these effects
in the central nervous system, via AMPK, remains unclear.
AMPK binds to and directly phosphorylates protein kinase C ζ(PKCζ) in an isoform-specific manner in alveolar epithelial cells(Gusarova et al., 2009). Both metformin and 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleotide (AICAR), an analog of AMP that directly
activates AMPK, activate PKCζ in an AMPK-dependent manner inmuscle cells (Sajan et al., 2010). The PKC family is classified into threegroups comprising conventional (α, β, and γ), novel (δ, ε, θ, μ, and η),∗
Corresponding author. Department of Pharmacology, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima,
Aoba-ku, Sendai, Miyagi 981-8558, Japan
E-mail address: osamun@tohoku-mpu.ac.jp (O. Nakagawasai).

https://doi.org/10.1016/j.neuropharm.2019.03.026
Received 16 October 2018; Received in revised form 1 March 2019; Accepted 19 March 2019
Available online 23 March 20190028-3908/ © 2019 Elsevier Ltd. All rights reserved.

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and atypical (λ and ζ) PKCs. In particular, PKCζ plays an important rolein neuronal function; it was demonstrated to be involved in long-term
potentiation (Sacktor et al., 1993) and the release of neurotrophic
factors, such as nerve growth factor (NGF), via the transcription factor,
nuclear factor-kappa B (NF-κB) (Obara et al., 2001). Moreover,blockade of atypical PKCs resulted in decreased NF-κB activation(Dominguez et al., 1993). The latter promotes the expression of neurotrophic factors, such as NGF and brain-derived neurotrophic factor(BDNF) (Obara et al., 2001). BDNF is known to have high affinity forneurotrophic tyrosine kinase receptor type 2 (TrkB). The intracellular
TrkB signaling cascade results in the phosphorylation of cyclic AMP
response element-binding protein (CREB) and the subsequent activationof CREB-mediated gene transcription, which plays a critical role in the
improvement in depressive-like behavior and promotion of neurogenesis (Berry et al., 2012). Previous studies have reported that both BDNF
and CREB expression is lower in patients and animal models of depression than in healthy subjects (Chen et al., 2001a; Karege et al.,
2002; Patki et al., 2013; Zhang et al., 2017). Moreover, chronic administration of anti-depressants, such as tricyclic anti-depressants(TCAs) and selective serotonin reuptake inhibitors (SSRIs), increased
BDNF expression in human sera and rodent brains (Lee and Kim, 2008;
Larsen et al., 2010), as well as CREB expression and activity in rodents(Nibuya et al., 1996; Thome et al., 2000). Recently, some groups havereported that physical exercise ameliorates depressive-like behavior via
increased BDNF expression and promotion of neurogenesis in the dentate gyrus (DG) of the hippocampus (Widenfalk et al., 1999; Bjørnebekk
et al., 2005; Yau et al., 2011). Newly generated neurons are incorporated into the hippocampal circuitry and contribute to hippocampal functions, including mood and memory (Petrik et al., 2012).
Indeed, chronic anti-depressant administration promotes neurogenesis
in the rodent hippocampus (Mostany et al., 2008). However, the detailed mechanism underlying the anti-depressant effects and promotionof neurogenesis via AMPK activation remains unclear.
In the present study, we used olfactory bulbectomized (OBX) miceas an animal model of depression which is caused by non-inflammatory
mechanism and inflammatory. A previous study reported that patientswith depression exhibit reduced olfactory bulb (OB) volumes and olfaction (Negoias et al., 2010). These reductions negatively correlatewith the depression scale, and impaired olfaction recovers after medical
treatment, such as with TCAs and SSRIs (Pause et al., 2001). OBX mice
exhibit depressive-like behavior, which is evidenced by their prolonged
immobility in the tail-suspension test (TST) and the forced swimming
test (FST) (Han et al., 2009), as well as their decreased sucrose preference (Sato et al., 2010; Nakagawasai et al., 2016). Moreover, somegroups have reported that OBX rodents exhibit alternation of the serotonergic (Nakagawasai et al., 2003), noradrenergic (Cairncross et al.,1975), and dopaminergic (Takahashi et al., 2016) systems in the brain.
The behavioral alternations in OBX rodents are improved by chronic,
but not acute, administration of anti-depressants, such as TCAs, SSRIs,
and serotonin-noradrenaline reuptake inhibitors (SNRIs). Such effects
have also been observed in clinical patients. OBX has been used as an
animal model of depression because it fulfills many of the necessary
criteria for a depression model, which are comparable to the features
observed in patients with major depression (Cryan and Mombereau,2004). Therefore, OBX mice are an appropriate animal model for thescreening of novel anti-depressants and for clarification of the mechanism of depression.
The hippocampus is divided into the ventral and dorsal hippocampus. Several groups, including our group, have reported that thebilateral infusion of BDNF into the dorsal hippocampus produces antidepressant-like effects in behavioral depression models (Shirayama
et al., 2002; Scharfman et al., 2005) and that conventional anti-depressant effects occurred via neurogenesis in the dorsal hippocampus(Mostany et al., 2008; Takahashi et al., 2018). Moreover, irradiation of
the dorsal hippocampus caused a reduction in neurogenesis and a lossof fluoxetine's antidepressant effects (Santarelli et al., 2003). Therefore,

this study aimed to identify molecular and cellular changes in the dorsal
hippocampus.
In this study, we examined the effects of AICAR on depressive-likebehavior in OBX mice to clarify the role of AMPK in the anti-depressanteffects. To identify possible mechanisms involved in the promotion ofneurogenesis in the dorsal hippocampus, we measured the effect of
AICAR on the PKCζ/NF-κB/BDNF/CREB signaling pathway.
2. Materials and methods
All experiments were performed following approval of the Ethics
Committee of Animal Experiments in Tohoku Medical and Pharmaceutical
University (Approval number: 16023 cn, 17015 cn) and according to the
National Institutes of Health Guide for the Care and Use of Laboratory
Animals. Efforts were made to minimize suffering and reduce the number ofanimals used.
2.1. Animals
We used male ddY mice (age: 6–7 weeks, weight: 26–28 g; Japan
SLC, Japan) for all experiments (total: n = 251; behavioral test:n = 187; western blotting analysis: n = 29; immunohistochemical
study: n = 35). We chose this strain of mice partly based on previous
work showing that ddY mice are more vulnerable to the development ofstress-induced behavioral disturbances (Mouri et al., 2012). This strain
is also more responsive to treatment with anti-depressants than othermice (Sugimoto et al., 2011). We selected this age range because thepeak period of first onset in most mental disorders is adolescence andyoung adulthood (Ströhle et al., 2007; Patel et al., 2007). The mice
were housed in cages of 5–6 mice, under steady conditions (23 ± 1 °C,
55 ± 5%, 12/12 h light-dark cycle with lights on at 7:00 a.m.) withfree access to food or water. All behavioral tests were performed between 9:00 and 17:00. All mice were numbered by earmarks and randomly divided into groups using a table of random numbers.
2.2. Surgical procedure
OBX surgery was performed as described previously (Hozumi et al.,2003). The mice were anesthetized with sodium pentobarbital (50 mg/
kg; Kyoritsu Seiyaku, Tokyo, Japan) and subsequently placed on astereotaxic instrument. The head was incised and drilled on the sagittal
midline, and the OBs were aspirated using a micropipette tip connected
to a water vacuum pump. The mice were occluded in their anteriorholes using a hemostatic sponge. All mice were euthanized at the end of
the experiment and it was visually confirmed that two-thirds of the OB
had been lesioned. Mice were excluded from the data if the lesion did
not extend to more than two-thirds of the OB or if it extended to thecortex. Sham operations followed the same surgical procedure without
the removal of the OB. The OBs of sham and OBX showed in Fig. 1A.
2.3. Compounds

122

AICAR (50 and 100 mg/kg; Toronto Research Chemicals, Inc.; Cat#
A611700) was dissolved in saline and injected intraperitoneally at a volumeof 0.1 mL/10 g body weight. Previously Liu et al. reported that administration of 100 mg/kg of AICAR ameliorates depressive-like behavior in ahigh-fat diet and corticosterone co-treated mice (Liu et al., 2014). The
protocol for AICAR administration is provided in Figs. 1, 2, 4 and 5. Myristoylated PKC pseudosubstrate (zeta-inhibitory peptide [ZIP]; PKCζ inhibitor; AnaSpec, Inc.; Cat# AS-63361) was dissolved in Ringer's solution(Fuso Pharmaceutical Industries, Ltd, Osaka, Japan), and N-[2-[[(Hexahydro-2-oxo-1H-azepin-3-yl)amino]carbonyl]phenyl]-benzo[b]thiophene2-carboxamide (ANA-12; Trk B receptor antagonist; Sigma-Aldrich; Cat#
SML0209) was dissolved in saline containing 0.15% Tween 80. ZIP wasinjected into OBX mice using cannulas (Brain infusion kit 3; Muromachi
Kikai, Tokyo, Japan) and an osmotic pump (Alzet 2002; Muromachi Kikai)— 327 —

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Fig. 1. Effect of AICAR on the tail-suspension test (TST) in olfactory bulbectomized (OBX) mice. (A) Representative image of whole brain in sham mice and OBX
mice. (B, D, F) Time course of the experimental protocol. (C)The immobility time was evaluated by the TST 30 min after vehicle or AICAR intraperitoneal (i.p.)
administration on the 21st day after surgery. (E, G) The immobility time was measured in the TST 24 h after vehicle or AICAR administration for 7 days (E) or for 14
days (G). (H) The immobility time was measured by the FST 24h after vehicle or AICAR administration for 14 days. Vertical lines above bars represent mean ±standard error of the mean. **, p < 0.01 as compared between sham group and OBX group; #, p < 0.05 compared to sham + Vehicle group; $, p < 0.05
compared to OBX + Vehicle group. n = 9–12 animals in each group.

for 14 days (5 μg/12 μL/day) (Migues et al., 2010; Bogard and Tavalin.,2015). The protocol for ZIP microinfusion is provided in Fig. 4. ANA-12 wasinjected intraperitoneally at a dose of 0.5 mg/kg (Ma et al., 2016). The
protocol for ANA-12 administration is provided in Fig. 5.

performed as described by DeVos (DeVos and Miller, 2013). On the 7th
day after surgery, OBX mice were anesthetized with sodium pentobarbital (50 mg/kg) and placed on a stereotaxic instrument. A hole wasdrilled in the skull, and a cannula was implanted and placed at 1.0 mm
lateral, 0.22 mm posterior, and 3 mm deep in relation to thebregma. The cannula was fixed with dental cement on the skull andconnected to an osmotic pump that was inserted into the lateral back of
OBX mice.

2.4. Osmotic pump implantation for intracerebral ZIP microinfusion
Implantation of the osmotic pump and brain infusion were123

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Fig. 2. AICAR administration increases phosphorylation and expression levels of proteins in the hippocampus of olfactory bulbectomized (OBX) mice. (A)Representative immunoblots probed with antibodies against p-AMPK, AMPK, p-PKCζ, peNFeκB, BDNF, p-CREB, and β-actin, as indicated. (B–F) Quantification of thenormalized to total AMPK values of p-AMPK (B), and of the normalized to β-actin values of p-PKCζ (C), peNFeκB (D), BDNF (E) and p-CREB (F). (G) Time course of
the experimental protocol. Vertical lines above bars represent mean ± standard error of the mean. n = 6–9 per group. #, p < 0.05 compared to OBX + Vehicle
group. n = 6–9 animals in each group.

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2.5. Tail-suspension test

euthanized under deep anesthesia 24 h after the final injection of salineor AICAR and intracardially perfused with 15 mL of 4 °C phosphatebuffered saline (PBS), followed by 45 mL of 4% paraformaldehyde(PFA; Sigma–Aldrich) in 0.1 M PBS. Brains were post-fixed in 4% PFA0.1 M PBS for 1 h at 4 °C, followed by immersion in 20% sucrose-0.1 M
PBS for 48 h. The brains were sliced into 40 μm sections from−1.40 mm to −2.00 mm relative to the bregma using a cryostat (MICROM HM560, Microm International GmbH, Walldorf, Germany).
Three sections were confirmed visually dorsal hippocampus following
mouse brain stereotaxic coordinates (Paxinos and Franklin, 2001) toevaluate neurogenesis in the same location of each mouse. Frozen
sections were mounted on glass slides (Matsunami Glass, Osaka, Japan).
Sections were treated with HCl (2 N) at 37 °C for 30 min, followed byneutralization with sodium borate buffer (0.15 M) at room temperature,
twice for 10 min each. After three 5 min washes, the sections were incubated with PBS containing 1% normal goat serum and 0.3% Tri-ton
X-100 (PBSGT) at room temperature for 2 h and then incubated overnight at 4 °C with primary monoclonal antibodies against rat anti-BrdU(1:100; Bio Rad; Cat# OBT0030) and mouse anti-doublecortin (DCX)(1:50; Santa Cruz Biotechnology; Cat# sc-271390). After incubation for
2 days, the sections were washed twice with 0.1% PBS and incubatedfor 2 h with goat anti-rat IgG Alexa fluor 568 (1:200; Molecular Probes;
Cat# A11077) and goat anti-mouse IgG Alexa fluor 488 (1:200; Molecular Probes Cat# A11001) as secondary antibodies, and DAPI (1:100;
Wako Pure Chemical Industries, Ltd; Cat# 34207431) to identify nuclei.
The sections were then washed twice with 0.1% PBS and coverslippedwith Dako fluorescent mounting medium (Dako, Carpinteria, USA). The
labeled sections were analyzed using a confocal laser-scanning microscope (A1Rsi: Nikon, Tokyo, Japan). Three sections per mouse wereused, and two images (left and right hemisphere, 640 × 640 μm) of the
DG region of the hippocampus were obtained from each section. We
calculated the BrdU/DCX positive cells following methods outlined by
Liu et al. and Ouchi et al. (Liu et al., 2017; Ouchi et al., 2013). Positive
cells were counted in the region observed under a fluorescence microscope. The positive cells in 2 images × 3 sections per mouse wereadded, and this total value was considered to be the total number of
BrdU/DCX positive cells in the whole dorsal hippocampus. A total of six
images were analyzed per mouse, and each group contained 5–6 mice.

The TST was used to observe the effect of AICAR on depressive-likebehavior in OBX mice. The procedure was performed as described
previously (Takahashi et al., 2018). The mice were taped from the tip of
their tail and suspended at a height of 30 cm from the floor for 10 min.
The immobility time was measured for 10 min by an observer who was
blinded to the treatment.
2.6. Forced swimming test
The FST was also conducted to evaluate the anti-depressant effect of
AICAR. The procedure was performed as described previously (Hanet al., 2009; Takahashi et al., 2018). After the TST, OBX and sham mice
were individually placed on plastic cylinders (height: 25 cm; diameter:
20 cm) containing water maintained at 25 ± 0.5 °C up to 14 cm for
5 min. The immobility time was measured for 5 min by an observer who
was blinded to the treatment.
2.7. Western immunoblotting
Western immunoblotting analysis was performed using hippocampal samples from mice that had not undergone any behavioral
testing. Mice were euthanized by decapitation 24 h after the final
AICAR administration. Sectioning was performed as described by Liu
and Smith (2003). Briefly, the brain was removed and sectioned on ice,using a mouse brain slicer (Muromachi Kikai), to produce 1 mm thickcoronal sections. We visually confirmed the dorsal hippocampal location using mouse brain stereotaxic coordinates (Paxinos and Franklin,
2001). The two (left and right) dorsal hippocampi were isolated, frozen
in liquid nitrogen, and then stored at −80 °C until use. The brain
samples were homogenized in 150 μL of CelLytic ™ MT Cell Lysis Reagent (Sigma-Aldrich; Cat# C3228-50 ML) and centrifuged at
15,000×g for 15 min at 4 °C. The supernatants were diluted in
4 × Laemmli sample buffer (300 mM Tris-HCl [pH 6.8], 8% sodium
dodecyl sulfate [SDS], 40% glycerol, 12% 2-mercaptoethanol, and0.012% bromophenol blue) and then incubated at 95 °C for 10 min.
These extracted samples were loaded onto 10% SDS-polyacrylamidegel. After electrophoresis, proteins were transferred to polyvinylidene
difluoride membranes and incubated with blocking solution (10 mM
Tris-HCl [pH 7.4], 100 mM NaCl, 0.01% Tween 20, and 5% skim milk).
The membranes were probed with antibodies against AMPK (1:1000;
Cell Signaling Technology; Cat# 2532), phosphorylated (p)-AMPK(1:1000; Cell Signaling Technology; Cat# 2531), p-PKCζ (1:1000; Santa
Cruz Biotechnology; Cat# sc-12894-R), peNFeκB (Ser311, 1:200;
Santa Cruz Biotechnology; Cat# sc-135769), p-CREB (1:1000; Cell
Signaling Technology; Cat# 9198), BDNF (1:100; Santa Cruz Biotechnology; Cat# sc-546), and β-actin (1:1000; Cell Signaling Technology; Cat# 4967) overnight at 4 °C. The membranes were then washed with blocking solution without milk, incubated with horseradish
peroxidase-conjugated secondary antibody (1:5000; Cell Signaling
Technology; Cat# 7074) for 2 h, and visualized using an enhanced
chemiluminescence western blotting detection reagent (Amersham Life
Science; Cat# RPN2109). The band density was measured by densitometry (Image-J 1.43μ, National Institute of Health).

2.9. Immunohistochemistry for localization of p-AMPK and peNFeκB
Brain samples were collected on day 21 after surgery. The sections
were incubated overnight at 4 °C with the following primary antibodies:
rabbit anti-p-AMPK monoclonal (1:50; Cell Signaling Technology; Cat#2535), peNFeκB (Ser311, 1:200; Santa Cruz Biotechnology; Cat# sc135769), mouse anti-neuronal nuclei (NeuN; 1:500; Millipore
Corporation; Cat# MAB377), rabbit anti-NeuN (1:100; Cell Signaling
Technology; Cat# 24307), rabbit anti-DCX (1:500; Abcam Ltd.; Cat#ab18723), rabbit anti-ionized calcium binding adaptor molecule 1 (Iba1; 1:100; Wako; Cat# 019–19741), and mouse anti-glial fibrillary acidic
protein (GFAP; 1:200; Millipore Corporation; Cat# MAB360). The
sections were washed twice with 0.1% PBS and incubated for 2 h at
22 ± 2 °C with the following secondary antibodies: goat anti-rabbit
IgG Alexa fluor 568 (1:200; Molecular Probes; Cat# A11011) for anti-pAMPK, goat anti-rabbit IgG Alexa fluor 488 (1:200, Molecular Probes;
Cat# A11008) for rabbit anti-NeuN and anti-DCX, goat anti-mouse IgG
Alexa fluor 568 (1:200 Molecular Probes; Cat# A11004) for antipeNFeκB, goat anti-mouse IgG Alexa fluor 488 (1:200 Molecular
Probes; Cat# A11001) for mouse anti-NeuN and anti-GFAP, goat antirabbit IgG Alexa fluor 488 (1:200 Molecular Probes; Cat# A11008) for
rabbit anti-Iba-1, and DAPI (1:100; Wako). Lastly, the sections were
washed twice with 0.1% PBS and coverslipped with Dako medium. The
fluorescence intensity was visualized on a confocal laser-scanning microscope (Nikon).

2.8. Immunohistochemistry for neurogenesis
To investigate whether AICAR promotes cell proliferation in hippocampus, sham or OBX mice were injected with 5-bromo-2′-deoxyuridine (BrdU; 75 mg/kg intraperitoneal injection; Nacalai Tesque;
Cat# 05650-24) three times every 2 h after the final administration ofsaline or AICAR on day 20 after surgery. The procedures from perfusion
to immunohistochemical staining were performed as described previously (Nakagawasai et al., 2016). The mice were subsequently
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Fig. 3. Effect of AICAR on neurogenesis in the hippocampal dentate gyrus of olfactory bulbectomized (OBX) mice. (A, B) Confocal images of brain slices stained withDAPI (blue) and BrdU (red) (A) or DAPI, BrdU and DCX (green) (B). Arrowheads point to double or triple positive cells in the subgranular zone (SGZ). (C, D)
Quantitative analysis of the number of BrdU+ and BrdU+/DCX+ cells in SGZ sham- and OBX-operated mice treated with saline or AICAR. #, p < 0.05 compared to
OBX + Vehicle group. n = 5–6 animals in each group.

2.10. Statistical analyses

than in sham-operated mice. Two-way ANOVA of administration of
AICAR at 100 mg/kg for 14 days in TST indicated a marginally significant main effect of group [F (1, 37) = 3.595, p = 0.0658], AICAR
treatment [F (1, 37) = 5.044, p = 0.0308], and group × treatment interaction [F (1, 37) = 5.955, p = 0.0196]. Post-hoc analysis revealed
prolonged immobility time in saline-treated OBX mice compared tosaline-treated sham mice (p < 0.05) (Fig. 1G). Immobility time of OBX
mice chronically treated with AICAR at 100 mg/kg for 14 days was
significantly lower than the vehicle-treated OBX mice (p < 0.05)(Fig. 1G). Two-way ANOVA of the immobility time of FST showed amain effect of AICAR treatment [F (1, 43) = 7.034, p = 0.0112] and agroup × treatment interaction [F (1, 43) = 10.97, p = 0.0019], but nomain effect of group [F (1, 43) = 0.4614, p = 0.5006]. Post-hoc tests
confirmed the immobility time of OBX mice chronically treated with
100 mg/kg of AICAR was lower than that of vehicle-treated OBX mice(p < 0.05) (Fig. 1H). In addition, OBX mice that were chronicallytreated with AICAR at 50 mg/kg for 14 days did not significantly differ
from OBX mice in the TST and FST [TST: one-way ANOVA, F (2,30) = 7.820, p = 0.0018, post-hoc test, p > 0.05 at 50 mg/kg,
p < 0.05 at 100 mg/kg; FST: one-way ANOVA, F (2, 32) = 8.842,
p = 0.0009, post-hoc test, p > 0.05 at 50 mg/kg, p < 0.05 at 100 mg/kg] (Supplement Fig. 1). Thus, we investigated the mechanism underlying the AICAR-induced (at 100 mg/kg for 14 days) anti-depressanteffect.

Results are expressed as the mean ± standard error of the mean.
Normality and homoscedasticity assumptions were verified prior to theuse of any parametric test (Shapiro–Wilk normality test and equality ofvariances F-test). The significance of the differences was determined bya one- or two-way analysis of variance (ANOVA) to compare groups,
followed by the Tukey–Kramer test to correct for multigroup comparisons using GraphPad Prism 6 (GraphPad Software, California, USA)and StatView 5.0 (HULINKS, Tokyo, Japan), and a significant difference
was defined as p < 0.05. When a main effect of group or treatment wassignificant without a group × treatment interaction, we conducted an
exploratory and limited pairwise post-hoc comparison, consistent with
our a priori hypothesis. All statistical analyses, including the behavioral
test, western blotting analysis, and immunohistochemistry for neurogenesis was performed by investigators other than the experimenters to
avoid bias and to ensure blinding.
3. Results
3.1. AICAR exerts an anti-depressant effect in OBX mice
In TST, two-way ANOVA for the acute study of AICAR showed asignificant main effect of group [F (1, 36) = 9.014, p = 0.0048] but nottreatment [F (1, 36) = 2.047, p = 0.1611] or group × treatment interaction [F (1, 36) = 0.4639, p = 0.5002] (Fig. 1C). Similarly, twoway ANOVA analysis of sub-chronic study of AICAR revealed a significant main effect of group [F (1, 39) = 12.33, p = 0.0011] but nottreatment [F (1, 39) = 0.06799, p = 0.7957] or group × treatment
interaction [F (1, 39) = 0.1238, p = 0.7269] (Fig. 1E). The immobility
time in OBX mice 21 days after the operation was significantly longer

3.2. AICAR enhances p-AMPK, p-PKCζ, peNFeκB, p-CREB, and BDNF
levels in the hippocampi of OBX mice
To investigate whether chronic administration of AICAR changed
the phosphorylation levels of AMPK, PKCζ, NF-κB, and CREB, and theexpression level of BDNF in the hippocampus, we performed western126— 331 —

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Fig. 4. Effect of ZIP on the anti-depressant effect of AICAR in olfactory bulbectomized (OBX) mice. (A) Timeline of the experimental protocol. (B) Immobility timemeasured with the tail-suspension test on the 21st day after OBX surgery. (C, D) Confocal images of brain slices stained DAPI (blue), BrdU (red) and DCX (green).
Arrowheads point to BrdU+ or BrdU+/DCX+ cells in the SGZ. (E, F) Quantitative analysis of the number of BrdU+ and BrdU+/DCX+ cells in SGZ of OBX micetreated with AICAR [administered intraperitoneally (i.p.)] and ZIP (administered via an osmotic pump). (G) Representative immunoblots probed with antibodies
against BDNF and β-actin. Quantification of normalized values of BDNF relative to β-actin levels. Vertical lines above bars represent mean ± standard error of themean. #, p < 0.05 compared to OBX + Vehicle group; $, p < 0.05 compared to OBX + AICAR group. n = 6–9 animals in each group.

p = 0.0807; BrdU+/DCX+: F (1, 19) = 3.791, p = 0.0665] and a significant effect of treatment [BrdU+: F (1, 19) = 13.41, p = 0.0017;
BrdU+/DCX+: F (1, 19) = 14.64, p = 0.0011], but no group × treatment interaction [BrdU+: F (1, 19) = 1.393, p = 0.2525; BrdU+/
DCX+: F (1, 19) = 2.233, p = 0.1515]. Post-hoc analysis revealed that
BrdU+ and BrdU+/DCX+ cells in the chronic AICAR-treated OBX mice
were significantly greater than in the vehicle-treated OBX mice(p < 0.05), whereas no change was observed in sham mice (Fig. 3C
and D). These results indicate that AICAR promotes neurogenesis in the
DGs of OBX mice.

blot analysis. Analysis of p-AMPK levels showed a significant main effect of treatment [F (1, 20) = 10.92, p = 0.0035] but not group [F (1,20) = 0.005776, p = 0.9402] or group × treatment interaction [F (1,20) = 0.2606, p = 0.6153]. Similarly, ANOVA analysis of p-PKCζ levels
showed a significant main effect of treatment [F (1, 20) = 15.15,
p = 0.0009] but not group [F (1, 20) = 2.586, p = 0.1235] orgroup × treatment interaction [F (1, 20) = 1.966, p = 0.1762].
Analysis of peNFeκB levels also showed a significant main effect oftreatment only [F (1, 19) = 11.86, p = 0.0027], without a significant
effect of group [F (1, 19) = 1.257, p = 0.2763], or group × treatment
interaction [F (1, 19) = 1.931, p = 0.1807]. BDNF expression showed asignificant main effect of treatment [F (1, 20) = 4.615, p = 0.0441] but
not group [F (1, 20) = 1.459, p = 0.2411] or group × treatment interaction [F (1, 20) = 1.658, p = 0.2126]. ANOVA of p-CREB levels
demonstrated a significant main effect of treatment [F (1, 20) = 4.777,
p = 0.0409] but not group [F (1, 20) = 0.1917, p = 0.6662] or twoway interaction [F (1, 20) = 2.140, p = 0.1590]. Post-hoc analysis revealed that protein levels in the hippocampus of AICAR-treated OBX
mice are higher than in those vehicle-treated mice (p < 0.05), whereas
no significant changes were observed in sham mice. These data arepresented in Fig. 2 BeF.

3.4. AICAR-induced anti-depressant effect and neurogenesis are attenuatedby ZIP
To investigate whether PKCζ is involved in the anti-depressant effect of AICAR, we co-administrated AICAR and ZIP, a PKCζ inhibitor, to
OBX mice for 14 days. Post-hoc analysis revealed that the reduction
observed in the immobility time of OBX mice treated with AICAR was
significantly attenuated by co-administration of AICAR and ZIP [oneway ANOVA; F (3, 38) = 4.306, p = 0.0104; Fig. 4B]. Next, we investigated whether the PKCζ inhibitor altered neurogenesis in OBX
mice. In this experiment, as a control, we used OBX mice implantedwith a cannula, and treated with ringer's solution. Post-hoc analysis
revealed that the increase in the number of BrdU+ and BrdU+/DCX+
cells in OBX mice treated with AICAR was significantly attenuated byco-administration of AICAR and ZIP [BrdU+ cells: one-way ANOVA, F(2, 13) = 31.89, p < 0.0001; BrdU+/DCX+ cells: one-way ANOVA, F

3.3. AICAR promotes neurogenesis in the hippocampal DG
We examined whether AICAR altered neurogenesis in the DG of the
hippocampus. ANOVA of BrdU+ and BrdU+/DCX+ cells showed amarginally significant main effect of group [BrdU+: F (1, 19) = 3.404,
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Fig. 5. Effect of ANA-12 on the anti-depressant effect of AICAR in olfactory bulbectomized (OBX) mice. (A) Timeline of theexperimental protocol. (B) OBX mice immobility time was measured by the tailsuspension test on the 21st day after OBX
surgery. (C, D) Confocal images of brainslices stained DAPI (blue), BrdU (red) andDCX (green). Arrowheads point to BrdU+ or
BrdU+/DCX+ cells in the SGZ. (E, F)
Quantitative analysis of the number of
BrdU+ and BrdU+/DCX+ cells in SGZ of
OBX mice treated with AICAR (administered intraperitoneally (i.p.)) and ANA-12(i.p.). Vertical lines above bars represent
mean ± standard error of the mean. #,p < 0.05 compared to OBX + Vehicle
group; $, p < 0.05 compared to
OBX + AICAR group. n = 6–9 animals ineach group. i.p., intraperitoneally.

(2, 13) = 17.88, p = 0.0002; Fig. 4C–F]. These results indicate that
PKCζ is indeed involved in the anti-depressant effect and promotion ofneurogenesis induced by AICAR.

3.7. AMPK is activated in mature hippocampal nerve cells and microglia
We performed dual immunofluorescence staining for p-AMPK anddifferent markers, such as NeuN and DCX for neurons, GFAP for astrocytes, and Iba-1 for microglia, to determine which types of cells areactivated by AICAR. We found that p-AMPK was localized in NeuN+,
DCX+, and Iba-1+ cells, but was absent from GFAP+ cells (Fig. 6),
indicating that AICAR activates mature neurons, immature neurons andmicroglia but not astrocytes in the hippocampi of OBX mice.

3.5. ZIP attenuates the AICAR-induced increase in BDNF levels in the
hippocampi of OBX mice
We next examined whether administration of ZIP changed the expression levels of BDNF in the hippocampi of OBX mice. Post-hoc
analysis revealed that co-administration of AICAR and ZIP to OBX micesignificantly attenuated the increase in BDNF levels in the hippocampiof OBX mice treated only with AICAR (p < 0.05) [one-way ANOVA; F(2, 13) = 9.897, p = 0.0024; Fig. 4G]. This data indicates that PKCζ
mediates the production of BDNF.

3.8. NF-κB is activated in the mature nerve cells of the hippocampus
Next, we performed immunofluorescence staining to determine thecell type in which NF-κB is activated. As illustrated in Fig. 7, peNFeκB
was localized in NeuN+ and DCX+ cells but not Iba-1+ cells, indicatingthat AICAR activates AMPK/PKCζ/NF-κB signaling in mature and immature hippocampal neurons in OBX mice.

3.6. ANA-12 attenuates the anti-depressant effect of AICAR

4. Discussion

To investigate whether the TrkB receptor is involved in the antidepressant effect of AICAR, we co-administrated AICAR and the TrkB
antagonist, ANA-12, to OBX mice for 14 days. Post-hoc analysis revealed that co-administration of the two drugs significantly attenuatedthe observed reduction in immobility time of OBX mice treated onlywith AICAR [one-way ANOVA; F (3, 41) = 5.432, p = 0.0031; Fig. 5B].
To investigate whether the TrkB receptor mediates neurogenesis, we
examined neurogenesis in OBX mice treated with AICAR and ANA-12.
Post-hoc analysis revealed that the increase in the number of BrdU+
and BrdU+/DCX+ cells in OBX mice treated with AICAR was blocked
by co-administration of AICAR and ANA-12 [BrdU+ cells (Fig. 5E): oneway ANOVA, F(2, 15) = 28.02, p < 0.0001; BrdU+/DCX+ cells(Fig. 5F): one-way ANOVA, F(2, 15) = 20.71, p < 0.0001]. These data
indicate the involvement of TrkB in the anti-depressant effect of AICAR.

In this study, we investigated the role of AMPK in depressive-likebehavior in OBX mice. Chronic administration of AICAR caused a reduction in the immobility time in the TST and FST. In addition, p-AMPK
induced PKCζ/NF-κB/BDNF/CREB signaling and increased neurogenesis in the hippocampi of OBX mice. Inhibition of PKCζ or the TrkB
receptor attenuated these effects of AICAR in OBX mice. Therefore, wereport for the first time that activation of AMPK contributes to theimprovement in depressive-like behavior via the PKCζ/NF-κB/BDNF/
CREB signaling pathway and enhancement of neurogenesis in OBX
mice.
In our study, OBX mice exhibited depressive-like behavior, as assessed by the TST and FST, 21 days after surgery. Chronic AICAR
treatment for 14 days, but not acute or subchronic treatment for 7 days,
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Fig. 6. Phosphorylated AMPK is expressed in mature and immature neurons and microglia in the hippocampus of olfactory bulbectomized (OBX) mice treated withAICAR. Double immunofluorescence staining for p-AMPK and either NeuN (A–D), DCX (E–I), Iba-1 (J–N), or GFAP (O–S). (A) Low magnification image of the
hippocampus stained with p-AMPK and NeuN antibodies. The boxed area is shown in higher magnification in B. (C, D) Independent photomicrographs of NeuN
(green; C) and p-AMPK positive cells (red; D). (E) Low magnification image of the hippocampus stained with p-AMPK and DCX antibodies. The boxed area is shown inhigher magnification in F. (G–I) Independent photomicrographs of DAPI (blue; G), DCX (green; H) and p-AMPK positive cells (red; I). (J) Low magnification image ofthe hippocampus stained with p-AMPK and Iba-1 antibodies and counterstained with DAPI. The boxed area is shown in higher magnification in K. (L–N) Independent
photomicrographs of fluorescent DAPI (blue; L), Iba-1 (green; M), and p-AMPK positive cells (red; N). (O) Low magnification image of the hippocampus stained with
p-AMPK and GFAP antibodies and counterstained with DAPI. The boxed area is shown in higher magnification in P. (Q–S) Independent photomicrographs offluorescent DAPI (blue; Q), GFAP (green; R) and p-AMPK positive cells (red; S).

resulted in an anti-depressant effect in OBX mice, which manifested as areversal of the reduced immobility in the TST. A recent study reported
that the depressive-like behaviors induced by a combination of high-fat
diet and corticosterone treatment were reversed with AICAR 100 mg/kg
administration for 4 weeks (Liu et al., 2014). These results indicate that
AICAR requires several days to exert its anti-depressant effect. Chronic
administration of conventional anti-depressants, such as SSRIs andnoradrenergic and specific serotonergic antidepressants (NaSSA), takes
several weeks to exhibit any clinical effect (Stahl et al., 2001). Moreover, chronic anti-depressant treatment promotes neurogenesis in the
hippocampus (Zhao et al., 2008). Recently, we also reported thatchronic treatment with memantine, an NMDA receptor antagonist,
enhances neurogenesis in the hippocampus and ameliorates depressivelike behavior in OBX mice (Takahashi et al., 2018). It is believed thatthe effects of anti-depressants may be mediated by the enhancement ofhippocampal neurogenesis (Santarelli et al., 2003; Kino, 2015). Moreover, AICAR at 100 mg/kg is more effective than 50 mg/kg in both TST
and FST. Liu et al. also reported that the same dosage of AICAR induced
anti-depressant effects (Liu et al., 2014). Therefore, we focused on themolecular mechanism that might lead to the increased neurogenesisthat mediates the anti-depressant effects of AICAR (100 mg/kg).
It was previously demonstrated that the activation of AMPK by
exercise or AICAR promotes glucose transport via PKCζ activity (Chenet al., 2002). Previous studies have reported a negative correlation
between depressive-like behavior and AMPK activation (Liu et al.,2014; Yau et al., 2014). In addition, other groups have reported that the
AMPK activator, metformin, promotes neurogenesis in the hippocampus via PKCζ signaling (Wang et al., 2012), while Obara et al. foundthat PKCζ activation enhances the secretion of neurotrophic factors,
such as NGF and BDNF, via NF-κB (Obara et al., 2001). The transcription factor, NF-κB, is a heterodimer composed of p50 and p65 and plays
important roles in neuronal growth and survival. Indeed, it was reported that the activated PKCζ/NF-κB pathway contributes to neuronalsurvival in PC12 cells (Macdonald et al., 1999). BDNF is known to playa critical role in neuronal survival and is believed to modulate neuroplasticity, including neurogenesis (Noble et al., 2011). Previous studies
have led to the formulation of the neurotrophic hypothesis of depression (Duman and Monteggia, 2006; Groves, 2007), based on evidence
that low levels of BDNF lead to a depressive condition (Chen et al.,2001b; Karege et al., 2002). Medical treatments, such as SSRIs and
SNRIs, increase BDNF levels in the brains of depressive patients (Leeand Kim, 2008; Larsen et al., 2010). In the present study, BDNF levels of
OBX mice increased after chronic treatment with AICAR. The activationof BDNF/TrkB signaling leads to the phosphorylation of CREB (Yanet al., 2016; Ye et al., 2017). In addition, overexpression of CREB in the
hippocampus results in anti-depressant effects (Chen et al., 2001a). The
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Fig. 7. Phosphorylated NF-κB is expressed in mature and immature neurons in the hippocampus of olfactory bulbectomized (OBX) mice treated with AICAR. Double
immunofluorescence staining for peNFeκB and either NeuN (A–D) DCX (E–H) or Iba-1 (I–L). (A) Low magnification image of the hippocampus stained with NeuNand p- NF-κB antibodies. The boxed area is shown in higher magnification in D. (B, C) Independent photomicrographs of fluorescent NeuN (green; B) and p- NF-κB
positive cells (red; C). (E) Low magnification image of the hippocampus stained with DCX and p- NF-κB antibodies. The boxed area is shown in higher magnificationin H. (F, G) Independent photomicrographs of fluorescent DCX (green; F) and p- NF-κB positive cells (red; G). (I) Low magnification image of the hippocampus stainedwith p- NF-κB and Iba-1 antibodies. The boxed area is shown in higher magnification in L. (J, K) Independent photomicrographs of Iba-1 (green; J) and p- NF-κB
positive cells (red; K).

present study revealed that AICAR significantly increased p-CREB levelsin the hippocampi of OBX mice. Thus, we consider that activation of the
AMPK/PKCζ/NF-κB pathway by AICAR may promote the secretion of
BDNF, which in turn activates the TrkB/CREB signaling pathway.
The stimulation of BDNF/TrkB/CREB signaling is critical for the
regulation of hippocampal neurogenesis (Ji et al., 2010), which improves depressive-like behavior. Indeed, the volume of the hippocampus is decreased in patients with depression (MacQueen et al.,2003) and rodent models of depression (Luo et al., 2014). Chronic SSRI
and SNRI treatment induces neurogenesis and subsequently ameliorates
depressive-like behavior in rodents (Luo et al., 2014). In addition,neurogenesis ablation by x-ray irradiation attenuates the effect of antidepressants in animal models of depression (Santarelli et al., 2003).
Recent studies have demonstrated that activation of AMPK enhances
neurogenesis (Wang et al., 2012; Arya et al., 2016) and ameliorates
depressive-like behavior (Liu et al., 2014; Yau et al., 2014). We also
observed enhancement of neurogenesis and improvements in depressive-like behavior in AICAR-treated OBX mice. Considering the previous findings and our current results, we suggest that AMPK activation
promotes neurogenesis in a similar manner to anti-depressants, such as
SSRIs and SNRIs.
Our hypothesis that activated AMPK promotes neurogenesis via
PKCζ/NF-κB/BDNF/TrkB/CREB signaling was further confirmed using
ZIP and ANA-12. In our study, inhibition of PKCζ blocked the AICAR-

induced anti-depressant effect and promotion of neurogenesis in OBX
mice, suggesting that PKCζ activation mediates such effects of AICAR.
In agreement with this notion, Wang et al. reported that fluoxetineinduced activation of atypical PKC and promotion of neurogenesis areblocked by ZIP (Wang et al., 2014). Furthermore, we found that cotreatment with AICAR and ANA-12 blocks the AICAR-induced anti-depressant effect and neurogenesis in OBX mice. Consistently, ANA-12
also blocks the anti-depressant effect of fluoxetine (Ma et al., 2016).
Jiang et al. reported that the anti-depressant effect and enhancement ofcell proliferation induced by ginsenoside Rg1 in the hippocampus areblocked by co-treatment with the TrkB antagonist, K252a (Jiang et al.,2012). Altogether, these results indicate that TrkB may be involved in
the improvement in depressive-like behavior and enhancement ofneurogenesis by inducing PKCζ phosphorylation via the activation of
AMPK.
We previously reported that OBX resulted in memory dysfunction
induced by alternation of the dopaminergic system. This dysfunction
was ameliorated by aripiprazole via stimulation of D1 receptors in theprefrontal cortex and increases in NGF and neurogenesis in the hippocampus (Takahashi et al., 2017). It has been suggested that the prefrontal cortex and hippocampus are associated with not only memory,
but also depression (Pan et al., 2014; Petrik et al., 2012); however, we
focused on only the hippocampus due to the observed neurogenesis
dysfunction in the DGs of OBX mice in this study. Therefore, further
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study will be required to examine the influence of AMPK activation inother brain regions involved in the pathogenesis of depression.
In the present study, we found that p-AMPK exhibits a nuclear
punctate distribution in NeuN+ and DCX+ cells, thus reflecting thetranslocation of AMPK to the nucleus (McGee et al., 2003) or the localization of the α-subunit of AMPK in the nucleus (Salt et al., 1998). A
previous study demonstrated that the nuclear localization of the AMPK
α2 subunit regulates gene expression (Salt et al., 1998). Therefore, it ispossible that AMPK, like NF-κB, regulates gene expression in the hippocampi of OBX mice. Moreover, we found that p-AMPK was expressedin microglia but not in astrocytes. Previously, Xu et al. reported that
AMPK reduces cytokines and neuroinflammation, thus promoting microglia conversion towards the M2 phenotype (Xu et al., 2015).
Therefore, p-AMPK expressed in microglia may contribute to anti-neuroinflammation. Further work is required to examine the anti-neuroinflammation effects of AMPK in OBX mice and the detailed underlying mechanisms. Moreover, we found that AICAR-induced peNFeκB
was localized in NeuN+ cells. Consistently, another group also reported
phosphorylation of NF-κB at Ser311 in the DG of the hippocampus (Kimet al., 2013). PKCζ directly phosphorylates NF-κB, and this phosphorylation contributes to neuronal survival and promotes transcription(Obara et al., 2001; Duran et al., 2003). PKCζ, which is upstream of NFκB, is widely distributed in the brain and can be detected in nerve cells,
microglia, and astrocytes (Slepko et al., 1999; Wang et al., 2012).
However, PKCζ expression levels and basal activity of NF-κB in microglia are very low (Slepko et al., 1999; Kaltschmidt and Kaltschmidt,2009). These results suggest that activation of AMPK/PKCζ/NF-κB occurs in nerve cells and then secrets BDNF. Samuels et al. found that theeffects of the anti-depressant fluoxetine are, at least in part, mediatedby 5-HT1A receptor activation in mature granule cells of the ventral
hippocampus but not in adult-born neurons (Samuels et al., 2015). In
contrast, we observed peNFeκB expression, not only in mature neurons, but also in immature neurons. Consistently, other groups reported
that inhibition of NF-κB in neonatal nodose neurons and cortical neurons caused an impairment in the growth of these cells (Gutierrez et al.,2005). The PKC/CREB binding protein (CBP; co-activator of NF-κB)
pathway was also detected in hippocampal precursor cells, and thispathway also regulates the maturation of neurons (Gouveia et al.,2016). Moreover, autocrine function of BDNF was required for the
regulation of synaptogenesis, survival, and maturation in dorsal root
ganglion neurons and adult-born neurons (Acheson et al., 1995; Davies
and Wright, 1995; Wang et al., 2015). From these reports, the AMPK/
PKCζ/NF-κB/BDNF/TrkB pathway in hippocampal immature neuronsof OBX mice may play an important role in neurogenesis. Therefore, it
is possible that the effects of AMPK on neurogenesis are direct effects
that act on adult-born neurons, or indirect effects that possibly act onmature neurons and microglia, which may lead to BDNF release from
cells that could stimulate neurogenesis. Future investigation is necessary to further delineate these possibilities.
In conclusion, our data indicate that persistent AMPK activation
may induce anti-depressant effects and enhancement of neurogenesis
via the PKCζ/NF-κB/BDNF/TrkB/CREB signaling pathway. We thus
provide evidence on the molecular mechanism of AMPK activation inan animal model of depression. Our findings suggest that AMPK activators may be used as novel therapeutic agents in the treatment ofneuropsychiatric disorders, such as depression.

Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.org/10.1016/j.neuropharm.2019.03.026.
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Acknowledgements and conflict of interest disclosureThis study was supported in part by the Nagai Memorial Research
Scholarship from the Pharmaceutical Society of Japan, Grants-in-Aid
for Scientific Research (Grant number 18K06687), and a Matching Fund
Subsidy for Private University from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan (Grant number S1511001L).
The authors have no conflicts of interest to declare.
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Contents lists available at ScienceDirect

Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharmPrenatal treatment with methylazoxymethanol acetate as aneurodevelopmental disruption model of schizophrenia in mice
Kohei Takahashia,1, Osamu Nakagawasaia,∗, Wakana Sakumaa,1, Wataru Nemotoa,
Takayo Odairaa, Jia-Rong Lina, Hiroshi Onogib, Lalit K. Srivastavac, Koichi Tan-NoaaDepartment of Pharmacology, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai, Miyagi 9818558, Japanb
Faculty of Health Science, Tohoku Fukushi University, 1-8-1 Kunimi, Aoba-ku, Sendai, Miyagi 981-8522, Japanc
Department of Psychiatry, Douglas Mental Health University Institute, McGill University, Montreal, QC, Canada

HIGHL I G HTS

mice showed schizophrenia-like behaviors.
• MAM
mice exhibited neuroanatomical changes in the hippocampus.
• MAM
concentrations and DOPAC/DA increased in the mPFC of MAM treated mice.
• DA
PPI deficits and social withdrawal improved with atypical antipsychotics.
• MAM-induced• Atypical and typical antipsychotics improved hyperactivity by MK-801 in MAM mice.
ART I CL E I NF O

ABS TRACT

Keywords:
Antipsychotic drugs
Methylazoxymethanol acetate
Monoamine transmission
Neurogenesis
Prepulse inhibition
Schizophrenia

Methylazoxymethanol (MAM)-treated pregnant rat at gestation day (GD) 17 has been shown to be a valuable
developmental animal model for schizophrenia. Yet, this model remains to be established in mice. In the present
study, we examined behavioral, cytoarchitectural, and neurochemical changes in the offspring of MAM-treatedmice and validated the model's face, construct and predictive validities. We found that in contrast to a single
injection of MAM to dams at GD 15, 16 or 17, its daily administration from GD 15 to 17 led to deficits in prepulse
inhibition (PPI) of startle in the post-pubertal offspring. In addition, we observed behavioral deficits in working
memory and social interactions, as well as an increase in locomotor activity induced by the NMDA antagonist
MK-801 in GD15-17 MAM offspring. These animals also showed a reduction in the volume of the prefrontal
cortex (PFC) and hippocampus, neuroanatomical changes such as discontinuities and heterotopias in the hippocampus, and an increase of DA level and DOPAC/DA ratio in the medial PFC. Atypical antipsychotic drugs
clozapine, risperidone, and aripiprazole, but not the typical drug haloperidol, reversed the deficit in PPI andsocial withdrawal in the offspring of MAM-treated dams. In contrast, MK-801-induced hyperactivity in MAM
mice was reversed by both and typical or atypical antipsychotic drugs. Taken together, the treatment of pregnant
mice with MAM during GD 15–17 offers a new approach to study neurobiological mechanisms involved in thepathogenesis of schizophrenia.

1. Introduction
Schizophrenia is a frequent psychiatric disorder that appears during
the late adolescent stage or early adulthood and is regarded as a neurodevelopmental disorder in which alterations start early indevelopment. Epidemiological research (Waddington et al., 1999)
suggest that during the second trimester of pregnancy, the fetus could
be exposed to environmental factors that increase the risk of developing
schizophrenia (Waddington et al., 1999). During this period, extensive
neuronal cell migration to the cortex occurs in the human brain

Abbreviations: ANOVA, analysis of variance; BrdU, 5-bromo-2′-deoxyuridine; CA, Cornu Ammonis; DA, dopamine; DG, dentate gyrus; GD, gestation day; MAM,
methylazoxymethanol; mPFC, medial prefrontal cortex; NVH, neonatal ventral hippocampal lesions; SEM, standard error of the mean∗
Corresponding author.
E-mail address: osamun@tohoku-mpu.ac.jp (O. Nakagawasai).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.neuropharm.2019.02.034
Received 16 April 2018; Received in revised form 12 February 2019; Accepted 25 February 2019
Available online 01 March 20190028-3908/ © 2019 Elsevier Ltd. All rights reserved.

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(Sidman and Rakic, 1973). The current view is that during adolescence
brain maturation could be inhibited by the primal developmental alterations and thus contribute to the pathophysiology of schizophrenia(Rapoport et al., 2005). A number of animal models have been described to explore this hypothesis.
Current schizophrenia models are not intended to serve as thecomplete animal equivalent of the human disorder. Rather, they areoften designed to test specific causative or mechanistic hypotheses.
In accordance with the neurodevelopmental hypothesis of schizophrenia, animal models have been proposed using pre- or early postnatal brain damages to induce schizophrenia-like behavioral changes(Lipska and Weinberger, 2000; Boksa, 2004; Laplante et al., 2005).
Prenatal toxin treatment can induce alterations in the structures of thebrain, with a relative specificity given by the neurogenic timetable(Cattabeni and Di Luca, 1997). To induce neurodevelopmental damages
in the hippocampus and cortex, methylazoxymethanol (MAM) can beused to briefly interfere with brain cellular proliferation during lategestation, a time when the neurons that will migrate to these areas areundergoing major cell division (Bayer and Altman, 2004). MAM administration to pregnant rats at gestation day (GD) 17 was recently used
as an experimental model for schizophrenia (Moore et al., 2006). This
treatment leads to aberrant methylation of DNA and interference withneurogenesis (Hoareau et al., 2006) and induces many characteristics
consistent with schizophrenia, including neurochemical changes, behavioral changes [in prepulse inhibition (PPI) of startle reflex, working
memory and social interaction] and increased locomotor activity to
psychotropic drugs (Gomes et al., 2016). Furthermore, MAM-treatedrats display an increase in the firing of dopamine (DA) neurons that isdriven by hyperactivity in the ventral hippocampus (Chen et al., 2014;
Gomes et al., 2016). Thus, this model is appropriate for studying thelink between altered embryonic neurogenesis and transition to schizophrenia-like deficits in the adult.
Given the general advantage of mice models (for example, theavailability of transgenic strains) the MAM-treated mice could enable
researchers to analyze genetic influences in the pathophysiology ofschizophrenia. Guo et al. (2013) have reported the effect of MAM given
to adolescent mice. More recent studies have reported establishment ofmodels based on pregnant mice treated with MAM at GD16 or 17(Chalkiadaki et al., 2018; Huo et al., 2018). However, these models failto exhibit many schizophrenia-like phenotypes, such as behavioral andbrain changes, in the offspring.
Our validation experiments using GD 16 or 17 MAM-treated mice
did not reveal significant schizophrenia-related behaviors in the offspring, e.g., deficits in prepulse inhibition of startle. Therefore, wedeveloped a protocol in which mice are treated with MAM on GD15-17.
According to species comparison of Carnegie stages of embryonic development (https://embryology.med.unsw.edu.au/embryology/index.
php/Mouse_Development), GD15.5 of mice may be in accordancewith GD17 of rats, regarding the morphological development of theembryo. In addition, the latest predictive model (Workman et al., 2013)
proposes that equivalent maturation stages of brain development between mouse and rat differ by 1–2days, with the latter maturing later.
We anticipated that a prenatal treatment with MAM during this period
would induce neuronal loss (Gaspard et al., 2008; Alsiö et al., 2013) andbehavioral, pathological and neurochemical changes relevant to schizophrenia. Our data shows that a number of these changes induced byprenatal MAM treatment are detectable in the offspring at post-pubertal
period and can be reversed by typical and atypical antipsychotic drugs.
We believe that the mice MAM model may serve as a new heuristic
paradigm to further investigate the neurodevelopmental bases of schizophrenia.

Pharmaceutical University, and according to the National Institutes of
Health Guide for the Care and Use of Laboratory Animals. Efforts weremade to minimize suffering and to reduce the number of animals used.
Measurement of the behaviors and post-mortem analyses were done inblinded manner.
2.1. Animals
Pregnant female ddY mice were obtained from Japan SLC(Shizuoka, Japan) on GD 11–13 and were housed individually in plastic
cages (height: 12 cm, width: 17 cm, length: 27 cm). Our choice of thisstrain of mice was partly based on previous work showing that ddYmice are more vulnerable to the development of behavioral disturbanceby stress or phencyclidine treatments (Mouri et al., 2012). This strain isalso more responsive to treatment with antipsychotics than otherstrains (Sugimoto et al., 2008). The dams were injected once daily withsaline or MAM (10 mg/kg, i.p.) for 3 days, between GD 15 and 17 (damstreated with higher dose of MAM, 20 mg/kg, died almost immediately
after delivery and the pups didn't survive). Within 10 days after birth,
all female offspring were culled. The remaining pups were weaned 21days after birth and were housed in groups of 4–6 mice. Only the malepups were included in the studies, and each animal was tested only for
one behavior at pre-pubertal age (5 weeks) and then one behavior at
post-pubertal age (8 weeks). The onset of pubertal age has been described previously (Palanza et al., 2001; Naert et al., 2013). The experiments were performed on several groups of saline or MAM-treatedanimals. They consisted of: (1) behavioral testing (n = 492), (2) measurement of body and brain weights (n = 10), (3) determination ofneurotransmitterconcentrations(n = 14),and(4)
immunohistochemistry (n = 28). The animals were housed in plastic
cages with free access to food and water under conditions of constanttemperature (22 ± 2 °C) and humidity (55 ± 5%), on a 12 h lightdark cycle (lights on at 07:00).
2.2. Drugs
MAM (Wako Pure Chemical Industries Ltd, Osaka, Japan) and MK801 (Sigma-Aldrich, St-Louis, USA) were dissolved in saline.
Haloperidol (Sigma-Aldrich), Clozapine (Sigma-Aldrich), Risperidone(Sigma-Aldrich) and Aripiprazole (Wako Pure Chemical Industries Ltd)
were dissolved in 0.5% Tween80 (Wako Pure Chemical Industries Ltd).
These drugs were administered intraperitoneally (i.p.) at a volume of
0.1 ml/10 g of mouse body weight. The antipsychotic drugs were administered 30 min before the behavioral tests.
2.3. PPI of the acoustic startle response

2. Materials and methods
All experiments were performed following the approval of the
Ethics Committee of Animal Experiment in Tohoku Medical and2

Sensorimotor gating processes which are frequently impaired inschizophrenia patients were evaluated using the PPI of the startle response which is one of the paradigms to detect this deficit in patients(Braff et al., 2001). Tests were conducted using the commercial software package SR-LAB system (SR-LAB, San Diego Instruments, San
Diego, USA) that comprised two sound-attenuating chambers eachequipped with a cylindrical Plexiglas animal enclosure (length: 6.7 cm;
inner diameter: 3.8 cm). Ventilation was provided by a small electric
fan that also generated a 70 dB background noise. Tone pulses were
presented by a speaker positioned 24 cm directly above the animal
enclosure. A piezoelectric accelerometer affixed to the animal enclosure
frame was used to detect and transduce motion resulting from the animal's response. Tone pulse parameters were controlled by a computer
using a SR-LAB and interface assembly that also digitized, rectified andrecorded stabilimeter readings.
Measures of both acoustic startle response (ASR) and PPI were obtained in a single session. Mice were placed in the Plexiglas enclosureand allowed to acclimatize to the environment for 5 min before being
tested during 42 discrete trials. When antipsychotic drugs or vehicle— 340 —

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were administered, mice were placed in the Plexiglas enclosure 30 min
after the administration. On the first two trials, the magnitude of the
ASR to a 120 dB tone lasting 50 ms was measured. These first two
startle tones were presented in order to habituate the animals to thetesting procedure. Therefore, the ASR magnitude of these two trials wasomitted from the statistical analysis of the mean ASR amplitude. On thesubsequent 40 trials, the startle tone was either presented alone or
100 ms after the presentation of a 30 ms duration prepulse. Prepulse
intensity ranged from 3 to 15 dB above background noise and wasvaried randomly between trials in 3 dB steps. Measures were taken ateach of the five prepulse intensities during five trials; animals wererandomly presented with the startle tone alone during another ten
trials; null trials (background tone alone) were conducted during theother five trials. The same stimulus condition was never presented on
more than two consecutive trials. The interval between each trial was
programmed to a variable time schedule with an average duration of
15 s (range 5–30 s). A measure of startle response amplitude was derived from the mean of 100 digitized data points collected from stimulus onset at a rate of 1 kHz. Prepulse effectiveness in suppressing thestartle response was expressed as a percentage based on the mean
amplitude of response to the startle tone alone (10 startle tones) relative
to those recorded under the five prepulse conditions (5 startle tones/
condition): PPI = 100 - [(mean startle amplitude for prepulse + pulse
trials/mean startle amplitude for pulse-alone trials) × 100]%. The fivedifferent prepulse intensities were averaged and used for statisticalanalysis.

behaviors were analyzed.
2.6. Y-maze test
In addition to positive and negative symptoms, schizophrenia patients consistently present cognitive deficits in working memory(Heinrichs and Zakzanis, 1998). We assessed cognitive deficits in MAMtreated using spontaneous alternation in a Y-maze. The Y-maze apparatus consisted of three compartments (height: 25 cm, width: 3 cm,
length: 40 cm) radiating out from the center. The mice were placed in
one of the compartments and allowed to move freely for 8 min. Experiments were performed at a light intensity of 35 lux. An arm entry
was defined as three legs entering one of the arms, and the sequence ofentries was manually recorded. An alternation was defined as entry into
all three arms on consecutive trials. Thus, the maximum number ofalternations was the total number of entries minus 2, and the percent
alternation was calculated as (actual alternations/maximum alternations) × 100. The percent spontaneous alternation behavior of themouse was taken as a measure of spatial short-term memory.
2.7. Measurement of body and brain weights
We examined regional brain weights in MAM mice as schizophrenia
patients consistently show a decrease in cortical and hippocampal volumes (Brugger and Howes, 2017). On postnatal day 56, mice were
weighed and then killed by decapitation. The brain (including the
cerebellum) was removed and weighed. On a separate group of saline
and MAM treated mice, the prefrontal cortex (PFC), hippocampus andstriatum were dissected out and weighed. The PFC corresponded to anarea that included the rostral pole of the brain, and was delimited
medially by the interhemispheric fissure, laterally by the corpus callosum and caudally extended to Bregma +2.68. The striatum andhippocampus were dissected from the remaining bloc of tissue.

2.4. Locomotor activity
Positive symptoms of schizophrenia develop around adulthood(Tandon et al., 2009) and are exacerbated by NMDA glutamatergictransmission in brain (Lahti et al., 1995). An increase in locomotor
activity has been proposed as an animal behavioral equivalent to thepositive symptoms seen in schizophrenia patients (Tandon et al., 2009).
The locomotor activity of mice was evaluated using the multichannel
activity-counting system SUPERMEX (Muromachi Kikai Co., Tokyo,
Japan). This instrument can monitor even minute movements in all
three planes of motion (vertical and horizontal) as one movement,
owing to its infrared sensor with multiple Fresnel lenses that can be
moved close enough to the cage to capture multidirectional locomotor
alterations in a single mouse. Thus, vertical movement such as jumping
as well as horizontal movements such as walking and running could becounted. The Supermex instrument was connected to a behavioral
analyzing system (CompACT AMS) (Muromachi Kikai Co.) which interpreted the movements as one count. Measurements of activity wereconducted between 11:00 and 15:00 during the light phase. Mice weredivided into two groups (saline and MAM), placed in activity boxes for
60 min. Mice were then injected with saline or MK-801 (0.1 mg/kg, i.p.)
and locomotor activity was recorded for another 60 min.

2.8. Immunohistochemical study (cell proliferation and neurogenesis)

2.5. Social interaction test
Social withdrawal is frequently observed as a negative symptom ofschizophrenia (Mueser and McGurk, 2004) that can be modeled in rodents. The general design of the model was adapted from Tanda et al.
(2009). The test was performed in a Plexiglas cage (height: 18 cm,
width: 26 cm, length: 30 cm). The measurement of social interaction
was done by video recording without any observer in the room. Experiments were performed at a light intensity of 35 lux. The arena wasplaced in a dimly lit room and was cleaned after each test. Two unfamiliar mice that had approximately the same weight and had received
an identical prenatal treatment were placed simultaneously in the opposite corners of the arena and the behavior of each mouse was recorded for a 10 min period. Behaviors such as sniffing, grooming andchasing at a distance closer than 2 cm of one another were counted associal interaction. Frequency as well as the time spent in these3

Some studies performed on postmortem brains revealed that the
proliferation of hippocampal neural stem cell was significantly reducedin schizophrenic patients (Allen et al., 2016; Iannitelli et al., 2017). On
postnatal day 42, 5-bromo-2′-deoxyuridine (BrdU) (Nacalai Tesque, Inc,
Kyoto, Japan; 75 mg/kg i.p.) was injected three times every 24 h foranalysis of neurogenesis. These mice were sacrificed 2 weeks after the
first BrdU injection. On postnatal day 56, BrdU was injected three times
every 2 h for analysis of cell proliferation. These mice were sacrificed
24 h after the first BrdU injection. Brains were fixed with 4% paraformaldehyde. The brains were cut into 40 μm sections from bregma−1.60 mm to −2.0 mm using a cryostat (MICROM HM560, Mikron
Instrument, Inc., Simi Valley, USA). For BrdU immunodetection, tissue
sections were first incubated at 37 °C for 30 min in HCl (2 N) followedby primary antibody addition. The primary antibodies used were rat
anti-BrdU monoclonal antibody (1:100; Harlan SeraLab, Loughborough, UK), mouse anti-doublecortin (DCX) monoclonal antibody (1:50;
Santa Cruz Biotech, Santa Cruz, CA) and mouse anti-neuronal nuclear
antigen (NeuN) monoclonal antibody (1:500; Millipore, Temecula,
USA). The following fluorescence conjugated secondary antibodies
were used: goat anti-rat IgG Alexa Fluor 568 (1:200; Molecular Probes,
Eugene, USA) and goat anti-mouse IgG Alexa Fluor 488 (1:200; Molecular Probes). DAPI was used to identify nuclei. Finally, sections were
washed and coverslipped with Dako fluorescence mounting medium(Dako, Carpinteria, USA). Labeled sections were analyzed using a
confocal laser-scanning microscope (A1Rsi: Nikon, Tokyo, Japan).
Eight sections per mouse were used, and two images (640 × 640 μm) of
the DG region of the hippocampus were obtained from each section. We
counted the number of BrdU+/DCX + cells and BrdU+/NeuN + cells
in the DG for analysis of cell proliferation and neurogenesis using NISElements AR analysis system, respectively. A mean number of eight— 341 —

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Fig. 1. Behavioral changes in mice exposed to MAM at GD 15–17. Average PPI and acoustic startle response in pre-pubertal (A and B) and post-pubertal (C and D)
mice treated prenatally with saline or MAM. PPI deficits in MAM mice developed at post-pubertal, but not pre-pubertal age. Locomotor activity in pre-pubertal (E–G)
and post-pubertal (H–J) mice treated prenatally with saline or MAM. (E) and (H) show the temporal profiles of MK-801-induced locomotion. (F, G, I and J) Analysisof total activity scores following MK-801 treatment reveals that MAM mice become more active than control animals only at post-pubertal period. Social interactionin pre-pubertal and post-pubertal mice treated prenatally with saline or MAM (K–N). Social withdrawal in MAM mice began development in pre-pubertal age.
Working memory in the Y-maze in pre-pubertal and post-pubertal mice treated prenatally with saline or MAM (O–R). Memory deficits in MAM mice developed in
post-pubertal, but not pre-pubertal age. Bars represent means ± S.E.M. *: p < 0.05 and **: p < 0.01 vs. prenatal treatment with saline group.

images were analyzed for each mouse, and each group contained 4–5
mice.

ventral hippocampus were obtained from each section. We counted thenumber of DAPI positive cells in each sector of the hippocampus i.e.
CA1, CA2, CA3 and DG. An average of eight images from each of thethree mice per group was used for the analysis. Heterotopias, defined asa group of misplaced neurons, were counted in the hippocampus from
MAM and control animals. Discontinuities were defined as a disruption
in the CA layers characterized by the presence of interruption in theneuronal layer continuity and dispersed neurons on any side of thecellular layer. Heterotopias, defined as a group of misplaced neurons,
were counted in the dorsal hippocampus from MAM and control animals.

2.9. Immunohistochemical study (cytoarchitectural andneuromorphometric alteration)
One of the structural abnormality observed in the brains of schizophrenic patients is the hippocampal cellular disarray such as selective
decrease in the number of pyramidal cells in the cornu ammonis (CA)2
area and neuron of discontinuities and heterotopias (Benes et al., 1998;
Heckers, 2004; Nelson et al., 1998). Brain sections were prepared as
described in the preceding section (cell proliferation and neurogenesis).
Antigen retrieval was performed for RGS-14 staining. Briefly, freefloating sections were incubated at 80 °C for 30 min in 50 mM sodium
citrate (pH = 9.0). We used the following primary antibodies: rabbit
anti-Prox1 monoclonal antibody (1:500; Abcam Ltd., Cambridge, UK),
mouse anti-RGS14 monoclonal antibody (1:100; NeuroMab, Davis,
USA), and rat anti-Ctip2 monoclonal antibody (1:500; Abcam Ltd). The
fluorescence conjugated secondary antibodies were: goat anti-mouse
IgG Alexa Fluor 488 (1:200; Molecular Probes), goat anti-rabbit IgG
Alexa Fluor 568 (1:200; Molecular Probes) and goat anti-rat IgG Alexa
Fluor 647 (1:200; Molecular Probes). For analyses, eight sections per
mouse were used and two images (640 × 640 μm) of the dorsal and2.10. DA and metabolite measurements with high-performance liquid
chromatography (HPLC)
It has been suggested that a disturbance in the dopaminergic
system, such as an increased presynaptic DA function in the various
brain regions, underlies schizophrenia (Kesby et al., 2018). Mice weredivided into two groups (saline and MAM) and sacrificed by decapitation on postnatal day 56. Each brain was rapidly removed and the
medial PFC (mPFC), striatum and hippocampus were dissected on an
ice-cold plate. These brain tissues were homogenized by sonication in
ice-cold 0.1 M perchloric acid with an internal standard (Isoproterenol)
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added at a concentration of 100 ng/ml. The homogenates were centrifuged at 10000×g then each supernatant was filtered through a0.45 μm pore size membrane filter. The filtrate was used for thequantification of DA (Sigma-Aldrich) by HPLC coupled with electrochemical detection. The HPLC system was comprised of a CCPM pump,
an auto-sampler equipped with a cooling plate maintained at 4 °C, andCO-8010 column oven (Tosoh, Tokyo, Japan). Separation was achieved
on a TSK gel ODS-100s (Tosoh, 250 × 4.6 mm i.d.). The mobile phase
was 95% 50 mM sodium acetate, 10 mM citric acid, 0.15 mM EDTA,
0.45 mM SOS and 5% acetonitrile adjusted to pH 3.5 with glacial acetic
acid then filtered through a 0.45 μm membrane. The flow rate was
1.0 ml/min. The electrochemical detection was accomplished using anelectrochemical detector (model EC8020, Tosoh) with a glass working
electrode at a potential of 1700 mV. The ratio of DOPAC/DA and HVA/
DA were used as indices of DA turnover.

pubertal age in MAM treated mice compared to saline treated mice.
[Fig. 1 (E-J) Three-way ANOVA: prenatal treatment: F (1, 43) = 0.93,
p = 0.76, treatment: F (1, 43) = 8.80, p = 0.005, time: F (3,143) = 35.32, p < 0.0001, prenatal treatment × treatment: F (1,43) = 0.004, p = 0.95, time × prenatal treatment: F (3, 143) = 1.67,
p = 0.17, time × treatment: F (3, 143) = 0.21, p = 0.91, time × prenatal treatment × treatment: F (3, 143) = 0.68, p = 0.58, (E); Student's
t-test: t (14) = 0.50, p = 0.62, (F); t (29) = 0.26, p = 0.80, (G); Threeway ANOVA: prenatal treatment: F (1, 41) = 4.88, p = 0.033, treatment: F (1, 41) = 9.28, p = 0.004, time: F (2, 86) = 35.99,
p < 0.0001, prenatal treatment × treatment: F (1, 41) = 1.61,
p = 0.21, time × prenatal treatment: F (2, 86) = 3.55, p = 0.031,
time × treatment: F (2, 86) = 1.18, p = 0.31, time × prenatal treatment × treatment: F (2, 86) = 0.88, p = 0.43, (H); Student's t-test: t(19) = 1.75, p = 0.096, (I); t (22) = 2.56, p = 0.018, (J)]. These results indicated that prenatal MAM treatments induced MK-801-induced
hyperactivity in mice during the post-pubertal period.

2.11. Statistical analysis
Normality and homoscedasticity assumptions were verified prior to
the use of any parametric tests (Shapiro-Wilk normality test andequality of variances F-test). Results are expressed as mean ± standard
error of the mean (SEM). The statistical significance of differences wasdetermined by the Student's t-test for two-group comparisons. One-way,
two-way or three-way analysis of variance (ANOVA) followed by
Tukey-kramer test were employed for multigroup comparisons. In somecases, when a main effect was significant without interaction effect, we
did an exploratory and limited pairwise post-hoc comparison consistent
with our a priori hypothesis. Brain weights were analyzed by ANCOVA
with body weight as a covariate. p < 0.05 represented a significant
difference.

3.3. Social interaction test
The effect of prenatal MAM on social behavior was evaluated at preand post-pubertal periods between pairs of unfamiliar mice that received the same prenatal treatments [Fig. 1 (KeN)]. Compared tosaline-treated mice, MAM-treated mice showed significantly reduced
time spent in social interactions at both developmental periods [Student's t-test: t (14) = 2.38, p = 0.032, (L); t (18) = 5.91, p < 0.0001,(N)]. Moreover, at the post-pubertal period, the number of interactions
significantly increased in MAM treated mice compared to saline treatedmice [Student's t-test: t (14) = 1.38, p = 0.19, (K); t (18) = 3.12,
p = 0.0059, (M)]. These results indicate that prenatal MAM treatments
induced social deficits in mice at both pre- and post-pubertal periods.

3. Results

3.4. Y-maze test

3.1. Acoustic startle and PPIMemory functions were evaluated in saline and MAM-treated miceat pre- and post-pubertal periods in the Y-maze test [Fig. 1 (O-R)]. Totalarm entries were not significantly different between saline and MAM
treated mice at both periods [Student's t-test: t (35) = 0.57, p = 0.57,(P); t (24) = 0.58, p = 0.57, (R)]. However, spontaneous alternation
was significantly decreased in MAM-treated mice compared to salinetreated mice at the post-pubertal, but not the pre-pubertal period[Student's t-test: t (35) = 1.82, p = 0.077, (O); t (24) = 2.48,
p = 0.021, (Q)]. The data indicates that prenatal MAM treatments induced post-pubertal cognitive deficits in mice.

There was no significant differences in the startle amplitude measured at either pre- or post-pubertal periods between mice treatedprenatally (GD 15–17) with saline or MAM. MAM-treated mice displayed significant average PPI deficits at the post-pubertal period, but
not pre-pubertal period [Fig. 1 (A-D) Pre-pubertal: Student's t-test of
average PPI: t (32) = 1.19, p = 0.24, (A); startle amplitude: t(32) = 0.61, p = 0.55, (B). Post-pubertal: Student's t-test of average
PPI: t (30) = 3.01, p = 0.0053, (C); startle amplitude: t (30) = 0.94,
p = 0.35, (D)]. These results indicated that the prenatal administrationof MAM at GD 15–17 induced schizophrenia-like behavioral deficits in
PPI at adulthood, similar to that reported for MAM-treated rats at GD
17 (Gomes et al., 2016). In preliminary experiments, we found thatmice prenatally treated with single injection of MAM (10 mg/kg) oneither GD 15, GD 16 or GD 17 did not display PPI deficits during thepost-pubertal period [Fig. S1 GD 15 (A): Student's t-test: average: t(27) = 0.88, p = 0.39. GD 16 (B): Student's t-test of average PPI: t(49) = 0.029, p = 0.98. GD 17 (C): Student's t-test of average PPI: t(35) = 1.28, p = 0.21]. Based on these results, we chose GD 15–17 as
the time period to treat pregnant mice with MAM for further behavioral, pathological and neurochemical studies.

3.5. Measurement of body and brain weights
Our data show that the weight of the PFC and hippocampus, but not
the striatum, were significantly reduced in MAM-treated mice compared to saline controls on postnatal day 56 [Fig. 2 (A-E) Student's ttest: PFC: t (8) = 3.26, p = 0.012, Fig. 2 (A); striatum: t (8) = 1.53,
p = 0.17, Fig. 2 (B); hippocampus: t (8) = 4.51, p = 0.002, Fig. 2 (C);whole brain: t (8) = 4.92, p = 0.0012, Fig. 2 (D); whole body: t(8) = 3.05, p = 0.016, Fig. 2 (E)]. Total brain weight was also significantly reduced by approximately 9.5% in MAM treated mice while
the average body weight was smaller by 16.2%. However, an analysis
by ANCOVA revealed that the body weight contribution to brain weight
was not significant [ANCOVA: whole brain: F (1, 6) = 11.23,
p = 0.015, whole body: F (1, 6) = 0.013, p = 0.91].

3.2. Locomotor activity
Changes in locomotor activity induced by the NMDA antagonist MK801 was analyzed at both pre- and post-pubertal periods. For either
periods, the activities of MAM and saline treated mice were not significant different following the saline treatment of the test. ANOVA
showed significant main effects of treatment and prenatal treatment
factors but not an interaction. Thus, we focused our analysis on theeffects of MAM and drug treatments. A significant increase in total locomotor activity by treatment with MK-801 was only observed at post-

3.6. Cytoarchitectural alterations in the hippocampus of MAM treated mice5

We have performed multiple immuno-labeling in order to evaluate
the pyramidal/granule cell density and morphology in the dorsal andventral hippocampus [Fig. 3 (A and B)]. Anti-Ctip2, anti-RGS14 andanti-Prox1 antibodies were used to identify the CA1, CA2 and DG areas,— 343 —

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Fig. 2. Prefrontal cortex (PFC) (A), striatum (B), hippocampus (C), whole brain (D) and whole body (E) weight comparisons at postnatal day 56 between mice treatedprenatally with saline or MAM. The weight of the PFC, hippocampus and whole brain, but not the striatum, were significantly reduced in MAM-treated micecompared to saline controls on postnatal day 56, while body weight contribution to brain weight was not significant. Bars represent means ± S.E.M. *: p < 0.05
and **: p < 0.01 vs. prenatal treatment with saline group.

respectively. The pyramidal cell density in CA2 was significantly decreased in MAM-treated mice compared to saline treated mice, whereas
the subfields CA1, CA3 or DG in both the dorsal and ventral hippocampus showed no significant differences [Fig. 3 (C and D) Two-way
ANOVA: prenatal treatment: F (1, 32) = 20.43, p < 0.0001, brain region: F (3, 32) = 282.1, p < 0.0001, prenatal treatment × brain region: F (3, 32) = 11.66, p < 0.0001, Post-hoc test: CA1: p > 0.9999,
CA2: p < 0.0001, CA3: p > 0.9999, DG: p = 0.75, (C); prenatal
treatment: F (1, 32) = 9.27, p = 0.0046, brain region: F (3,32) = 67.87, p < 0.0001, prenatal treatment × brain region: F (3,32) = 3.32, p = 0.032, Post-hoc test: CA1: p > 0.9999, CA2:
p = 0.0007, CA3: p > 0.9999, DG: p = 0.77, (D)]. Quantitative analyses of discontinuities and heterotopias in the dorsal hippocampus[Fig. 3 (E, F and H)] showed that, compared to control saline-treatedmice. Main effect of prenatal treatment was significant by ANOVA,
while interaction was not significantly different. Thus, we focused on
the effect of MAM, but not brain regions. The mice treated with MAM
exhibited more discontinuities in CA1, CA2 and CA3 compared tocontrol mice [Fig. 3 (G) Two-way ANOVA: prenatal treatment: F (1,24) = 26.59, p < 0.0001, brain region: F (2, 24) = 0.39, p = 0.68,
prenatal treatment × brain region: F (2, 24) = 0.86, p = 0.44, Post-hoc
test: CA1: p = 0.015, CA2: p = 0.013, CA3: p = 0.025, (G)]. In addition, the number of heterotopias was also significantly increased in the
MAM mice [Fig. 3 (I) Student's t-test: t (8) = 2.43, p = 0.042, (I)].
These results indicate that the prenatal treatment with MAM induced adecrease in pyramidal cell density in the CA2 area which was accompanied with an increase in discontinuities and heterotopias.

compared to the saline-treated group [Fig. 4 (B) and (D) Student's t-test:t (8) = 8.52, p < 0.0001, (B); t (6) = 5.70, p = 0.0013, (D)]. These
results indicate that the prenatal treatment with MAM in mice induced
a reduction in cell proliferation and neurogenesis in the DG of the
hippocampus.
3.8. Alterations of dopaminergic system in the brain of MAM-treated mice
Tissue levels of DA, its metabolites and their turnover in the mPFC,
striatum and hippocampus of mice treated prenatally with saline or
MAM are shown in Fig. 5. ANOVA showed a significant main effect ofprenatal treatment but not of interaction in the content and turnover inthe mPFC area. Thus, we focused on the effect of MAM only. In themPFC, the DA concentration and DOPAC/DA ratio in MAM treated mice
was significantly increased compared to the saline treated group [Twoway ANOVA: prenatal treatment: F (1, 36) = 12.56, p = 0.0011, concentration: F (2, 36) = 19.52, p < 0.0001, prenatal treatment × concentration: F (2, 36) = 0.60, p = 0.55, Post-hoc test: DA: p = 0.038,
DOPAC: p = 0.059, HVA: p = 0.12, (A); Two-way ANOVA: prenatal
treatment: F (1, 24) = 6.53, p = 0.017, turnover: F (1, 24) = 7.08,
p = 0.014, prenatal treatment × turnover: F (1, 24) = 2.33, p = 0.14,
Post-hoc test: DOPAC/DA: p = 0.032, HVA/DA: p = 0.36, (B); Twoway ANOVA: prenatal treatment: F (1, 36) = 1.54, p = 0.22, concentration: F (2, 36) = 120.7, p < 0.0001, prenatal treatment × concentration: F (2, 36) = 1.28, p = 0.29, (C); prenatal treatment: F (1,24) = 0.11, p = 0.75, turnover: F (1, 24) = 77.22, p < 0.0001, prenatal treatment × turnover: F (1, 24) = 0.19, p = 0.66, (D); prenatal
treatment: F (1, 36) = 3.19, p = 0.083, concentration: F (2,36) = 10.29, p = 0.0003, prenatal treatment × concentration: F (2,36) = 0.46, p = 0.64, (E); prenatal treatment: F (1, 24) = 1.63,
p = 0.21, turnover: F (1, 24) = 3.64, p = 0.068, prenatal treatment × turnover: F (1, 24) = 0.25, p = 0.62, (F)]. These results indicate that the treatment with MAM induced an increase in the tissuelevels of DA and a disturbance in the homeostasis of the dopaminergic
system.

3.7. Cell proliferation and neurogenesis in the DG of the hippocampal
formation
To determine the rate of hippocampal cell proliferation and neurogenesis in our model, animals were injected with BrdU. Anti-DCX
antibody was used to identify immature neurons while anti-NeuN antibody was used to identify mature neurons in the DG area. The incorporation of BrdU into a cell indicates that it was dividing at the timeof the BrdU injection. MAM-treated mice had a significantly lower
number of both BrdU+/DCX + cells and BrdU+/NeuN + cells
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3.9. Effect of antipsychotic drugs on MAM-induced PPI disruption

tested compounds except for haloperidol [Fig. 6 (A) Two-way ANOVA:
average PPI: prenatal treatment: F (1, 78) = 18.17, p < 0.0001, antipsychotic: F (3, 78) = 1.36, p = 0.26, prenatal treatment × antipsychotic: F (3, 78) = 0.66, p = 0.58, startle amplitude: prenatal
treatment: F (1, 78) = 3.30, p = 0.073, antipsychotic: F (3, 78) = 0.82,
p = 0.49, prenatal treatment × antipsychotic: F (3, 78) = 0.91,

We evaluated whether haloperidol (a typical antipsychotic), risperidone and clozapine (atypical antipsychotics) or aripiprazole (dopamine-serotonin stabilizer) reversed the PPI deficits (average PPI) in
MAM-treated mice. MAM-induced PPI deficits were reversed by all(caption on next page)7— 345 —

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Fig. 3. Alteration of pyramidal/granule cell density and cytoarchitecture in different areas of the hippocampus in mice exposed to MAM at GD 15–17. Representative
tissue sections used for cell counting in the dorsal (A) and ventral (B) hippocampus. Color of the neuronal layer in the different areas after merging of labeled images:
CA1 and DG (magenta), CA2 (white), CA3 (blue). An example of labeling with individual antibodies is shown on Fig. S2. Cells were counted in the pyramidal/granulecell layer (C and D). Bar designated total was the average of all hippocampal subregions and DG. Coronal slices at Bregma −2.2 mm to −3.0 mm of the dorsal andventral hippocampus: CA1, CA2 and CA3 from MAM treated mice stained with anti-Ctip2 antibody, anti-RGS14 antibody, anti-Prox1 antibody and DAPI. The
pyramidal cell density in CA2 was significantly decreased in MAM-treated mice compared to saline treated mice, whereas the subfields CA1, CA3 or DG in both thedorsal and ventral hippocampus showed no significant differences. Arrows point to discontinuities in the dorsal hippocampus (E and F). Discontinuity differences
between MAM and saline treated mice in hippocampal subfields CA1, CA2 and CA3 (G). The prenatal treatment with MAM exhibited more discontinuities in CA1 andCA2. The arrow points to heterotopia in the dorsal hippocampal CA1 area (H). Comparison between the number of heterotopias in the hippocampus of MAM treatedmice and controls (I). The prenatal treatment with MAM induced an increase in heterotopias in the dorsal hippocampus. Bars represent means ± S.E.M. *: p < 0.05
and **: p < 0.01 vs. prenatal treatment with saline group.

Fig. 4. Microscopy images of BrdU (red), DAPI (blue), and DCX or NeuN (green) immunostaining in the DG region of the hippocampus. The prenatal treatment with
MAM in mice induced a reduction in cell proliferation and neurogenesis in the DG of the hippocampus. Bars represent means ± S.E.M. **: p < 0.01 vs. prenatal
treatment with saline group.

p = 0.44, (A)]. Risperidone (0.03 mg/kg), clozapine (0.5, 1 and 3 mg/kg) and aripiprazole (1 and 5 mg/kg) attenuated the MAM-induced PPI
deficit for the average PPI, respectively [Fig. 6 (B-D) Two-way ANOVA:
average PPI: prenatal treatment: F (1, 75) = 10.28, p = 0.0020, antipsychotic: F (2, 75) = 4.21, p = 0.019, prenatal treatment × antipsychotic: F (2, 75) = 4.076, p = 0.021, startle amplitude: prenatal
treatment: F (1, 75) = 0.00047, p = 0.98, antipsychotic: F (2,75) = 1.73, p = 0.19, prenatal treatment × antipsychotic: F (2,75) = 0.18, p = 0.84. Post-hoc test (average PPI): 0.01 mg/kg:
p = 0.99; 0.03 mg/kg: p = 0.0027, (B); Two-way ANOVA: average PPI:
prenatal treatment: F (1, 102) = 6.76, p = 0.011, antipsychotic: F (3,102) = 7.97, p < 0.0001, prenatal treatment × antipsychotic: F (3,102) = 3.79, p = 0.013, startle amplitude: prenatal treatment: F (1,102) = 0.30, p = 0.59, antipsychotic: F (3, 102) = 0.59, p = 0.62,
prenatal treatment × antipsychotic: F (3, 102) = 0.45, p = 0.72. Posthoc test (average PPI): 0.5 mg/kg: p = 0.034; 1 mg/kg: p = 0.0041;
3 mg/kg: p < 0.0001, (C); Two-way ANOVA: average PPI: prenatal
treatment: F (1, 70) = 1.91, p = 0.17, antipsychotic: F (3, 70) = 2.12,
p = 0.11, prenatal treatment × antipsychotic: F (3, 70) = 3.077,
p = 0.033, startle amplitude: prenatal treatment: F (1, 70) = 2.86,
p = 0.095, antipsychotic: F (3, 70) = 0.26, p = 0.85, prenatal treatment × antipsychotic: F (3, 70) = 0.21, p = 0.89. Post-hoc test

(average PPI): 0.2 mg/kg: p = 0.68; 1 mg/kg: p = 0.027; 5 mg/kg:
p = 0.013, (D)]. These results indicate that prenatal MAM-induced PPI
deficits in mice were improved with atypical antipsychotics but notwith the typical antipsychotic drug haloperidol.
3.10. Effect of antipsychotic drugs on MK-801-induced hyperactivity in
MAM-treated mice
We evaluated whether haloperidol, risperidone, clozapine or aripiprazole reversed the hyperactivity by MK-801 in MAM-treated mice.
MAM-induced hyperactivity by MK-801 were reversed by all tested
compounds without influence on control group [Fig. 7 (A-D) Haloperidol: Two-way ANOVA: prenatal treatment: F (1, 47) = 6.076,
p = 0.017, antipsychotic: F (3, 47) = 19.69, p < 0.0001, prenatal
treatment × antipsychotic: F (3, 47) = 3.86, p = 0.015, Post-hoc test:
0.05 mg/kg: p = 0.0002, 0.1 mg/kg: p < 0.0001, 0.5 mg/kg:
p < 0.0001, (A); Risperidone: Two-way ANOVA: prenatal treatment: F(1, 32) = 1.89, p = 0.18, antipsychotic: F (2, 32) = 4.12, p = 0.026,
prenatal treatment × antipsychotic: F (2, 32) = 3.99, p = 0.028, Posthoc test: 0.01 mg/kg: p = 0.0097, 0.03 mg/kg: p = 0.034, (B); Clozapine: Two-way ANOVA: prenatal treatment: F (1, 40) = 4.75,
p = 0.035, antipsychotic: F (3, 40) = 3.30, p = 0.030, prenatal
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Fig. 5. Alternation of DA, DOPAC and HVA concentrations and DOPAC/DA and HVA/DA in the mPFC, striatum and hippocampus in MAM mice. Concentrations areindicated as ng per g fresh weight of brain tissue. The treatment with MAM induced an increase in the tissue levels of DA and a disturbance in the homeostasis of the
dopaminergic system in the mPFC. Bars represent means ± S.E.M. *: p < 0.05 vs. prenatal treatment with saline group.

treatment × antipsychotic: F (3, 40) = 2.47, p = 0.049, Post-hoc test:
0.5 mg/kg: p = 0.14, 1 mg/kg: p = 0.0091, 3 mg/kg: p = 0.18, (C);
Aripiprazole: Two-way ANOVA: prenatal treatment: F (1, 45) = 2.71,
p = 0.11, antipsychotic: F (3, 45) = 11.47, p < 0.0001, prenatal
treatment × antipsychotic: F (3, 45) = 2.95, p = 0.043, Post-hoc test:
0.2 mg/kg: p = 0.0006, 1 mg/kg: p = 0.0001, 5 mg/kg: p < 0.0001,(D)]. These results indicate that prenatal MAM-induced hyperactivity
by MK-801 were improved with typical and atypical antipsychotics.

3.11. Effect of antipsychotic drugs on MAM-induced deficits in social
behavior
We evaluated whether haloperidol, risperidone, clozapine or aripiprazole reversed the social deficits in MAM treated mice. Haloperidol(0.05–0.5 mg/kg) did not significantly improve MAM-induced social
deficits [One-way ANOVA: F (3, 25) = 5.88, p = 0.0035, Post-hoc test:
0.05 mg/kg: p = 0.47, 0.1 mg/kg: p = 0.66, 0.5 mg/kg: p = 0.0020,
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Fig. 6. Effect of haloperidol (A), risperidone (B), clozapine (C) and aripiprazole (D) on average PPI and startle amplitude in saline and MAM-treated mice. Prenatal
MAM-induced PPI deficits in mice were improved with atypical antipsychotics but not with the typical antipsychotic drug haloperidol. Bars representmeans ± S.E.M. *: p < 0.05 and **: p < 0.01 vs. vehicle-treated saline group. #: p < 0.05 and ##: p < 0.01 vs. vehicle-treated MAM group.

Fig. 8 (A)]. Risperidone at 0.01 mg/kg improved the social deficits in
MAM mice [One-way ANOVA: F (2, 19) = 19.27, p < 0.0001, Post-hoc
test: 0.01 mg/kg: p < 0.0001, 0.03 mg/kg: p = 0.075, Fig. 8 (B)].
Clozapine at 0.5 mg/kg also improved the MAM-induced social deficit[One-way ANOVA: F (3, 23) = 7.52, p = 0.0011, Post-hoc test: 0.5 mg/
kg: p = 0.0046, 1 mg/kg: p = 0.090, 3 mg/kg: p = 0.99, Fig. 8 (C)].
Similarly, aripiprazole at 0.2 mg/kg improved the MAM-induced social
deficits [One-way ANOVA: F (3, 24) = 4.72, p = 0.010, Post-hoc test:
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Fig. 7. Effect of haloperidol (A), risperidone (B), clozapine (C) and aripiprazole (D) on hyperactivity in saline and MAM-treated mice. Prenatal MAM-induced
hyperactivity by MK-801 in mice were improved with typical and atypical antipsychotics. Bars represent means ± S.E.M. *: p < 0.05 and **: p < 0.01 vs. vehicletreated saline group. #: p < 0.05 and ##: p < 0.01 vs. vehicle-treated MAM group.

0.2 mg/kg: p = 0.045, 1 mg/kg: p = 0.87, 5 mg/kg: p = 0.99, Fig. 8(D)]. These results indicate that prenatal MAM-induced social withdrawal were improved with atypical antipsychotics but not with thetypical antipsychotic drug haloperidol.

withdrawal is considered as one of the negative symptoms of schizophrenia (Mueser and McGurk, 2004) which is frequently observed before the onset of positive symptoms and is persistent (Olin and Mednick,1996). Fourthly, we found that mice prenatally treated with MAM
presented working memory deficits in the Y-maze test at post-pubertal
period [Fig. 1 (M-P)]. Working memory deficits in the Y-maze test has
also been reported at post-pubertal period for rats treated with MAM(Moore et al., 2006) and for NVH lesioned mice (Naert et al., 2013). In
agreement, schizophrenia patients consistently present cognitive deficits involving working memory (Heinrichs and Zakzanis, 1998). From
our results we can infer that treating mice with MAM on GD 15–17 canlead to behavioral deficits of relevance to positive and negative symptoms as well as cognitive dysfunctions.
Meta-analysis studies reveal that schizophrenia patients present a
decrease in the volume of prefrontal cortex and hippocampus (Brugger
and Howes, 2017). In the present study, the treatment with MAM resulted in a marked decrease in brain tissue weight for both the PFC andhippocampus which appears to be consistent with a previous study in
rat MAM model (Flagstad et al., 2004). Studies on postmortem hippocampi of schizophrenic patients suggest that there may be a selective
decrease in the number of pyramidal cells in the CA2 area which areessential for social memory (Benes et al., 1998; Hitti and Siegelbaum,2014). In accordance, social memory deficits have been observed inpatients with schizophrenia (Okruszek et al., 2017). Our MAM-treatedmice also displayed a decrease in pyramidal cell density in the CA2 area
compared to the control group (Fig. 3). Changes in hippocampal
structure in schizophrenia patients are consistently reported in postmortem and neuro-imaging studies (Heckers, 2004; Nelson et al.,1998). Neuronal discontinuities and heterotopias in the dorsal hippocampus observed in schizophrenia patients have also been observed in

4. Discussion
In the present study, we have examined the face, constructive andpredictive validities of GD 15-17 MAM-treated mice to determine
whether this model exhibits schizophrenia-like features. We initiallyevaluated the face validity with respect to behavioral changes such as
PPI, locomotor activity, social interaction and working memory.
First, we found that mice exposed to MAM at GD 15–17 present PPI
deficits during post-pubertal period, which is one of the endophenotypes of schizophrenia patients (Braff et al., 2001) and also avalid marker for sensorimotor gating in rodent models (Geyer et al.,2001). Interestingly, PPI deficits in prenatal MAM-treated rats were also
observed only at post-pubertal period (Gomes et al., 2016), a findingthat parallels the onset of the disorder in schizophrenia patients(Neumann et al., 1995). Secondly, we found that MK-801-induced locomotor activity was enhanced in MAM-treated mice at post-pubertal
period in a similar fashion to what was observed with the neonatal
ventral hippocampal (NVH) lesion rat model (Al-Amin et al., 2000).
Clinical studies suggest that the antagonism of NMDA glutamatergictransmission in brain exacerbates positive symptoms of schizophrenia(Lahti et al., 1995). Thirdly, we found that GD 15–17 MAM treated micespent less time in social interactions at both pre- and post-pubertal ages
[Fig. 1 (I-L)]. Such changes have previously been observed at both preand post-pubertal periods in MAM-treated rats (Hazane et al., 2009) as
well as in NVH lesioned rats (Sams-Dodd et al., 1997). Social
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Fig. 8. Effect of haloperidol (A), risperidone (B), clozapine (C) and aripiprazole (D) on social withdraw in saline and MAM-treated mice. Prenatal MAM-inducedsocial withdrawal in mice were improved with atypical antipsychotics but not with the typical antipsychotic drug haloperidol. Bars represent means ± S.E.M. #:p < 0.05 and ##: p < 0.01 vs. vehicle-treated MAM group.

the offspring of MAM-treated rat dams (Singh, 1977; Chevassus-AuLouis et al., 1998). Consistent with these observations, we found neuronal discontinuities and heterotopias in the dorsal hippocampus in theoffspring of MAM-treated pregnant mice.
Our studies show that prenatal MAM-treated mice display a decrease in cell proliferation and neurogenesis in the DG compared to thecontrol group (Fig. 5). Some studies have suggested that a reduction in
hippocampal neural stem cell proliferation may be involved in the pathogenesis of schizophrenia and an association between decreased
neurogenesis and schizophrenia has been demonstrated (Allen et al.,2016; Eriksson, 2006; Iannitelli et al., 2017). Interestingly, it has been
demonstrated in rodents that a reduction in hippocampal neurogenesis
may lead to PPI deficits (Osumi et al., 2015), memory deficits and social
withdrawal (Iwata et al., 2008). Together, these results may indicatethat the schizophrenia-like behaviors induced by prenatal MAM may berelated to the reductions in cell proliferation and neurogenesis we observed.
It has been demonstrated that a dysfunction in central DA efflux
may be related to the positive and negative symptoms as well as the
cognitive deficits of schizophrenia (Grace, 2016). DA levels and
DOPAC/DA ratio in the PFC were found to be increased in the mice
offspring of MAM-treated dams, which is in agreement with the enhanced cortical DA levels seen in the offspring of pregnant rats treatedwith MAM (Hallman and Jonsson, 1984) or after prenatal treatment
with mitotic inhibitor Ara-C in rats which is proposed as a neurodevelopmental model of schizophrenia (Brown et al., 2012). Belujon et al.
(2014) reported that MAM rats leaded to a hyperdopaminergic state
and abnormally increased activity in the mPFC induced suppression of
the nucleus accumbens and long-term potentiation via D2 receptor in
the mPFC. Long-term potentiation in the ventral subiculum–mPFC-

nucleus accumbens pathway plays a significant role in the performanceof goal-directed behavior (Kelley, 2004; Goto and Grace. 2005a,
2005b). It is thus possible that the abnormal increased activity by hyperdopaminergic state in the mPFC may be associated with some abnormal behaviors related to schizophrenia. Interestingly, similar to ourfinding in the MAM mice, a clinical study has reported that schizophrenia patients show increased DA synthesis rate in the mPFC
(Lindström et al., 1999). However, Abi-Dargham and colleagues suggestthat schizophrenia patients exhibit reduced dopaminergic transmission
in the dorsolateral PFC as revealed by neuroimaging studies (AbiDargham et al., 2012; Slifstein et al., 2015; Roffman et al., 2016). These
paradoxical results may possibly be associated with the different of PFC
regions studied in the rodents versus humans. Taken together, wesuggest that hyperdopaminergic state in the mPFC of MAM mice may be
associated with altered behaviors relevant to schizophrenia.
Other researchers have reported the effects of antipsychotic drugs
on prenatal MAM rat model in multiple paradigms (Brown et al., 2013;
Du and Grace. 2013, 2016; Goda et al., 2015; Grace and Gomes. 2018;
Le Pen et al., 2011; Sonnenschein et al., 2018). This study was undertaken to evaluate the predictive validity of our mice model whether
typical or atypical antipsychotic drugs could reverse the deficits in the
PPI of startle reflex, hyperactivity by MK-801 and social withdrawal
observed in MAM-treated mice. Using the PPI of startle paradigm orsocial interaction test, we showed that atypical antipsychotics such as
clozapine, risperidone and the dopamine-serotonin stabilizer aripiprazole all reversed the MAM-induced sensorimotor gating deficits
and social withdrawal. In contrast the typical antipsychotic haloperidol
could not reverse the PPI deficits and social withdrawal in these mice.
On the other hand, we found that hyperactivity by MK-801 in MAM
mice was reversed by both typical and atypical antipsychotics, results
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that are consistent with previous studies in MAM rat model (Le Pen
et al., 2011). These results suggest that PPI deficits and social withdrawal may be particularly sensitive to some critical characteristics
related to the “atypicality” of those antipsychotic drugs while alterations of DA neurotransmission may be responsible for hyperactivity by
MK-801. Clinical studies reported that atypical antipsychotics such as
clozapine or ziprasidone, but not typical antipsychotic haloperidoltreated schizophrenic patients occasionally showed normalization of
PPI (Kumari et al., 1999; Braff et al., 1992) and negative symptoms(Stahl et al., 2010). Moreover, clinical efficacy of antipsychotic drugs
for positive symptoms is correlated with their ability to block subcortical D2 receptors (Creese et al., 1976; Seeman and Lee, 1975),
suggesting DA signal is important. Thus, atypical antipsychotics such as
clozapine, risperidone and aripiprazole probably interact with brain
systems affected by the MAM treatment through their action on receptors other than DA receptors to normalize PPI deficits and social
withdrawal, while all typical and atypical antipsychotics may improve
hyperactivity by MK-801 via inhibition of D2 receptors. In contrast to
haloperidol, atypical antipsychotics also interact with 5HT2A, α2 adrenergic, muscarinic and histaminergic receptors, among others(Bymaster et al., 2003; Citrome, 2014; Kusumi et al., 2015). Any or allof these receptors could play an important role for the unique action of
atypical antipsychotics in our mice model. Taken together, the datasuggest that the MAM mice model may be useful for the screening ofnovel antipsychotic drugs for negative symptoms of schizophrenia.
In conclusion, we have established a MAM model in mice that displays behavioral changes similar to what has been found in MAM
treated rats including neurodevelopment-dependent onset of behavioral
alterations such as PPI deficits, hypersensitivity to MK-801, social interaction deficits and cognitive deficits. Moreover, the prenatal treatment with MAM led to a decrease in brain weight (notably in the PFC
and hippocampus), cytoarchitectural alterations such as discontinues
and heterotopias, reductions in cell proliferation and neurogenesis, aswell as an increase of DA content and turnover in the PFC. The behavioral, anatomical and neurophysiological changes are more consistent
with those observed in schizophrenia patients than prenatal MAM rat
models. Moreover, we found for the first time that atypical antipsychotic drugs reversed MAM-induced PPI deficits and social withdrawal. We believe that prenatal GD 15-17 administration of MAM inmice may offer a new means to study neurobiological mechanisms involved in the pathophysiology of schizophrenia.

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Acknowledgments
This study was supported in part by Grants-in-Aid for Scientific
Research (Grant number 18K06687) and Matching Fund Subsidy for
Private University from the Ministry of Education, Culture, Sports,
Science and Technology of Japan (Grant number S1511001L). The
authors would like to thank Ms. Anna Mori, Ms. Kaoru Takano, Mr.
Daiki Hasegawa and Mr. Daisuke Tezuka of Tohoku Medical andPharmaceutical University for their technical assistance and Prof. Remi
Quirion of McGill University for experimental advice.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.org/10.1016/j.neuropharm.2019.02.034.
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Contents lists available at ScienceDirect

Neurochemistry International
journal homepage: www.elsevier.com/locate/neuint

Involvement of catecholaminergic and GABAAergic mediations in the
anxiety-related behavior in long-term powdered diet-fed mice
Fukie Yaoitaa,∗, Masahiro Tsuchiyab, Yuichiro Araic, Takeshi Tadanod, Koichi Tan-NoaaDepartment of Pharmacology, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai, 981-8558,
Japanb
Department of Nursing, Tohoku Fukushi University, 1-8-1 Kunimi, Aoba-ku, Sendai, 981-8522, Japanc
Tokyo Ariake University of Medical and Health Science, 2-9-1 Ariake, Koto-Ku, Tokyo, 135-0063, JapandComplementary and Alternative Medicine Clinical Research and Development, Graduate School of Medicine Sciences, Kanazawa University, Kakumamachi, Kanazawa,
920-1192, JapanA R T I C LE I N FO

A B S T R A C T

Keywords:
Atomoxetine
Methylphenidate
PD168077
Anxiety-related behavior
Low anxiety
Bicuculline
Attention deﬁcit/hyperactivity disorder

Dietary habits are important factors which aﬀect metabolic homeostasis and the development of emotion. We
have previously shown that long-term powdered diet feeding in mice increases spontaneous locomotor activity
and social interaction (SI) time. Moreover, that diet causes changes in the dopaminergic system, especially
increased dopamine turnover and decreased dopamine D4 receptor signals in the frontal cortex. Although theincreased SI time indicates low anxiety, the elevated plus maze (EPM) test shows anxiety-related behavior andimpulsive behavior. In this study, we investigated whether the powdered diet feeding causes changes in anxietyrelated behavior. Mice fed a powdered diet for 17 weeks from weaning were compared with mice fed a standard
diet (control). The percentage (%) of open arm time and total number of arm entries were increased in powdereddiet-fed mice in the EPM test. We also examined the eﬀects of diazepam, benzodiazepine anti-anxiety drug,
bicuculline, GABA-A receptor antagonist, methylphenidate, dopamine transporter (DAT) and noradrenalinetransporter (NAT) inhibitor, atomoxetine, selective NAT inhibitor, GBR12909, selective DAT inhibitor, andPD168077, selective dopamine D4 receptor agonist, on the changes of the EPM in powdered diet-fed mice.
Methylphenidate and atomoxetine are clinically used to treat attention deﬁcit/hyperactivity disorder (ADHD)
symptoms. The % of open arm time in powdered diet-fed mice was decreased by treatments of atomoxetine,
methylphenidate and PD168077. Diazepam increased the % of open arm time in control diet-fed mice, but not in
powdered diet-fed mice. The powdered diet feeding induced a decrease in GABA transaminase, GABA metabolic
enzymes, in the frontal cortex. Moreover, the powdered diet feeding induced an increase in NAT expression, but
not DAT expression, in the frontal cortex. These results suggest that the long-term powdered diet feeding may
cause low anxiety or impulsivity, possibly via noradrenergic and/or dopaminergic, and GABAAergic mediations
and increase the risk for onset of ADHD-like behaviors.

1. Introduction
Various changes brought on by modern lifestyle have caused anincreased risk of disease. Dietary habits are particularly importantfactors which aﬀect development of the central nervous system as wellas emotion (Aoki et al., 2005; Kushida et al., 2008; Mitome et al., 2005;
Niijima-Yaoita et al., 2013; Yamamoto and Hirayama, 2001). It has
been suggested that evolutionary changes in dietary habit to softer

foods in part explain lesser developed masticatory function (Yamanaka
et al., 2009). We recently reported that long-term powdered dietfeeding in mice increases spontaneous locomotor activity and social
interaction (SI) time of SI test. Moreover, this diet causes changes in the
dopaminergic system, in particular, increased dopamine turnover anddecreased dopamine D4 receptor signals in the frontal cortex (NiijimaYaoita et al., 2013). However, some reports suggest that the impairmentof dopaminergic activity in the hippocampus may be related to theAbbreviations: ADHD, attention deﬁcit/hyperactivity disorder; ANOVA, analysis of variance; BZD, benzodiazepines; DAT, dopamine transporter; EPM, elevated plus
maze; GABA-A, γ−amino butyric acid receptor type A; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; i.p., intraperitoneally; NA, noradrenaline; NAT, noradrenaline transporter; % of open arm time, percentage of time spent in the open arm; SI, social interaction; T-PBS, Tween-20 in phosphate-buﬀered saline∗
Corresponding author.
E-mail address: niijima@tohoku-mpu.ac.jp (F. Yaoita).
https://doi.org/10.1016/j.neuint.2018.12.002
Received 25 September 2018; Received in revised form 30 November 2018; Accepted 4 December 2018
Available online 07 December 20180197-0186/ © 2018 Elsevier Ltd. All rights reserved.

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masticatory dysfunction induced by powdered diet feeding (Kushida
et al., 2008; Yoshino et al., 2012).
In addition to the SI test, the elevated plus maze (EPM) test is usefulfor evaluating anxiety-related behavior (File, 1992). It is known thatγ−amino butyric acid type A (GABA-A) receptor plays a critical role inanxiolytic activity. GABA-A receptor is a chloride channel composed ofa pentameric hetero-oligometric protein with binding sites for GABA,
benzodiazepines (BZD), barbiturates, and steroids, etc. The activationof the GABA-A receptor induced by BZD including diazepam increases
the percentage (%) of open arm time in the EPM test, indicating low
anxiety (i.e. anxiolytic eﬀect) (for review see Jacob et al., 2008; Möhler
et al., 2002). On the other hand, Hwang et al. (2004) reported that theincrease in expression of GABA transaminase and GABA metabolic
enzymes, provide important information about the brain with GABA
dysfunction. Moreover, the GABA transaminase has been primarily investigated in relation to epilepsy, but it has also been found that adecrease in GABA neurotransmission appears to be involved in the
aetiology of several neurological disorders such as anxiety (for review
see Kowalczyk and Kulig, 2014).
Attention deﬁcit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by inattention, hyperactivity, andimpulsivity. Using the ADHD animal model, impulsive behavior can beevaluated by the EPM test (Kishikawa et al., 2014; Niimi et al., 2011;
Ueno et al., 2002). This test is designed to detect anxiety-related behavior but it also reﬂects impulsivity for novelty seeking.
The frontal cortex is an important brain region which is related to
ADHD symptoms, including impulsive behavior (for reviews see
Arnsten and Pliszka, 2011; Brevet-Aeby et al., 2016; Herman et al.,2018). The etiology and pathophysiology of ADHD have not been
completely clariﬁed, however the prefrontal cortex is known to beparticularly sensitive to the neurochemical environment; relatively
slight changes in the levels of noradrenaline (NA) and dopamine canproduce signiﬁcant changes in its function (for review see Arnsten andPliszka, 2011). Although newer drugs have been developed, methylphenidate and atomoxetine remain the predominantly prescribed drugs
for the treatment of ADHD. Methylphenidate increases extracellular
dopamine and NA indirectly by blocking the transporters, dopaminetransporter (DAT) and NA transporter (NAT), while atomoxetine preferentially inhibits NAT (for review see Arnsten and Pliszka, 2011).
The aims of the present mice study on were to investigate the following: (a) whether long-term powdered diet feeding induces changes
in anxiety-related behavior in the EPM test, (b) the eﬀect of diazepam,
benzodiazepine anti-anxiety drug, bicuculline, a GABA-A receptor antagonist, GBR12909, a selective DAT inhibitor, methylphenidate, DAT
and NAT inhibitor, atomoxetine, a selective NAT inhibitor and
PD168077, a selective dopamine D4 receptor agonist, on the changes inthe EPM test, and (c) GABA transaminase expressions in the frontal
cortex, DAT and NAT expressions in the frontal cortex and hippocampus.

2.2. Drugs and treatment
The following drugs were used: diazepam (Sigma–Aldrich, St. Louis,
MO, USA), bicuculline (GABA-A receptor antagonist, Tocris Bioscience,
Bristol, UK), methylphenidate (Novartis Pharma, Tokyo, Japan), atomoxetine (selective NET inhibitor, Sigma–Aldrich) and GBR12909(selective DAT inhibitor, Tocris Bioscience). Diazepam, bicuculline andPD168077 (selective dopamine D4 receptor agonist, Sigma–Aldrich)were suspended in saline containing 0.5% Tween 80. Methylphenidate,
atomoxetine and GBR12909 were dissolved in saline. All drugs wereadministered intraperitoneally (i.p.) (volume: 0.1 ml/10 g body weight)
at 30 min before the behavioral test. The drug doses used in the present
study were within the ranges widely used in experiments on mice andrats (Gould et al., 2005; Hiraide et al., 2013; Kanegawa et al., 2010;
Leggio et al., 2011; Niijima et al., 2006; Niijima-Yaoita et al., 2013;
Robinson et al., 2012; Ueno et al., 2002).
2.3. EPM test
At 20 weeks of age, the anxiety-related behavior was evaluatedusing the EPM test. The apparatus consisted of two open arms
(6 cm × 30 cm) opposite each other, crossed by two closed arms
(6 cm × 30 cm) (walls 10 cm) with an open roof and central platform(9 cm × 9 cm). The maze ﬂoor and walls were constructed from acrylic
plate and elevated 40 cm from the ground ﬂoor. Initially, the animals
were placed on the central platform of the maze in front of a closed arm.
The animal had 5 min to explore the apparatus. Activity of mice wasmonitored via video camera positioned above the apparatus. The timespent in each arm and the number of entries into each arm were automatically analyzed oﬄine using the ANY-maze video tracking software (Stoelting Company, Wood Dale, IL, USA). The test area wascleaned with 20% ethanol between each session.
2.4. Western blot analysis of GABA transaminase in the frontal cortex andboth NAT and DAT expressions in the frontal cortex and hippocampus
After 17 weeks of treatment, animals were sacriﬁced by decaptation
without anesthesia. The brains were rapidly resected and two brain
regions (frontal cortex and hippocampus) were dissected, and placed on
an ice-cold plate. Each brain sample was quickly frozen and stored at−80 °C, until use. The dissected tissues were homogenized in
CelLyticTM MT Cell Lysis Reagent (Sigma–Aldrich) containing 1%
protease inhibitor cocktail (Sigma-Aldrich) and centrifuged at
15,000×g for 15 min at 4 °C. Supernatants were isolated and protein
concentrations were measured using the Pierce BCA protein assay kit(Pierce, Rockford, lL). Equivalent amounts of protein lysates (15 μg)
were separated by electrophoresis on a sodium dodecylsulphate polyacrylamide gel (10% e-PAGEL, ATTO Corp., Tokyo, Japan) and transferred onto polyvinylidene ﬂuoride membranes (Immobilon-P,
Millipore, Bedford, MA). Blots were blocked for 1 h with Blocking One(Nacalai Tesque, Kyoto, Japan) at room temperature. The membranes
were sequentially incubated overnight with antibodies to NAT(Norepinephrine Transporter Monoclonal Antibody, dilution 1: 2000,
P21934, Thermo Scientiﬁc, Rockford, lL), DAT (Anti-Dopamine
Transporter N-terminal Antibody, dilution 1:1000, D6944,
Sigma–Aldrich), GABA transaminase (ABAT Antibody, dilution
1:10000, NBP1-95517, Novus Biologicals, CO) or glyceraldehyde-3phosphate dehydrogenase (GAPDH, dilution 1:1000, 5714, Cell
Signaling Technology, Danvers, MA). After repeated washing with
0.05% Tween-20 in phosphate-buﬀered saline (T-PBS), a peroxideconjugated goat anti-mouse antibody (dilution 1:5000, NA931,
Amersham, GE Healthcare, Buckinghamshire, UK) for the membrane of
NAT, a peroxide-conjugated goat anti-rabbit antibody (dilution 1:5000,
7074, Cell Signaling Technology) for DAT, GABA transaminase or
GAPDH, was added for 1 h at room temperature. After repeatedwashing with T-PBS, the immunoreactive bands were visualized using

2. Materials and methods
2.1. Animal treatment
Male Balb/c mice (3 weeks old) were obtained from Japan CREA
(Tokyo, Japan). The animals were housed under conditions of constanttemperature (23 ± 1 °C) and humidity (55 ± 5%), on a 12/12 hlight–dark cycle (light from 7 to 19 h; dark from 19 to 7 h). The mice
were housed in groups of 5–6 in standard plastic cages (30 cm × 20 cm
x 14 cm high) with wire mesh lids and a bedding of wood shavings.
Mice were fed either a control diet (pellet type Labo MR stock, Nihon
Nosan, Kanagawa, Japan) or powdered diet (powder type Labo MR
stock, Nihon Nosan) containing the same ingredients with free access towater for 17 weeks. All experiments complied with the Guidelines forCare and Use of Laboratory Animals issued by Tohoku Medical andPharmaceutical University.
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ECL Western Blotting Detection Reagents (GE Healthcare) and detectedby Image Quant LAS4010 (GE Healthcare). Immunoblots were quantiﬁed by Image Quant TL software (GE Healthcare).

(p < 0.01, Fig. 2A). In contrast to the % of open arm time, two-way
ANOVA revealed the main eﬀect for diet on the total number of arm
entries [F (1, 42) = 8.119, p = 0.0068], the main eﬀect for drug [F (1,42) = 7.985, p = 0.0072] and diet × drug interaction [F (1,42) = 0.1310, p = 0.7192] (Fig. 2B). The post hoc Tukey's test revealed
that the total number of arm entries in the vehicle-treated powdereddiet-fed mice signiﬁcantly increased, compared with vehicle-treatedcontrol diet-fed mice (p < 0.05, Fig. 2B).

2.5. Statistical analyses
Statistical analyses were performed with the computer program,
GraphPad Prism (GraphPad Software, Inc., San Diego, CA, USA).
Results are expressed as means ± standard errors of the mean (SEM).
To analyze the drug's eﬀect on powdered diet group, the results wereanalyzed by a one-way analysis of variance (ANOVA) using GraphPad
Prism, followed by Dunnett's test. To evaluate the eﬀects of diazepamon the EPM test in both control diet and powdered diet groups, two-way
ANOVA was used. Signiﬁcant main eﬀects or interactions were followed by Tukey's test. Statistical evaluations between two groups were
performed using Student's t-test. Statistical signiﬁcance was set at
p < 0.05.

3.3. Eﬀect of bicuculline, a GABA-A receptor antagonist, on the anxietyrelated behavior of powdered diet-fed mice in the EPM test
In the experiments described above, treatment with diazepam increased the % of open arm time in control diet-fed group, but not in the
powdered diet-fed group, suggesting that the alteration of GABA-A receptor function. Fig. 3 shows the % of open arm time and the total
number of arm entries after treatment with bicuculline, a GABA-A receptor antagonist, on powdered diet-fed mice. The increase in the % ofopen arm time elicited by powdered diet feeding was attenuated bytreatment with bicuculline at a dose of 5 mg/kg, compared with that ofvehicle-treated powdered diet-fed group (unpaired t-test: p = 0.0181,
Fig. 3A). However, the total number of arm entries in powdered diet-fed
group [unpaired t-test: p = 0.312, Fig. 3B], after treatment with bicuculline (5 mg/kg) did not show any behavioral changes as comparedwith the vehicle-treated powdered diet-fed group. There were no signiﬁcant diﬀerences between vehicle-treated control diet-fed mice andbicuculline (5 mg/kg)-treated control diet-fed mice, in the EPM test
(data not shown).

3. Results
3.1. Anxiety-related behavior of powdered diet-fed mice assessed by theEPM test
At 20 weeks of age, the anxiety-related behavior of powdered dietfed group was measured for 5 min in the EPM apparatus. Fig. 1 shows
the inﬂuence of powdered diet feeding on the % of open arm time andtotal number of arm entries. The powdered diet group had a signiﬁcantly increased % of open arm time (unpaired t-test: p = 0.0017)(Fig. 1A) and the total number of arm entries (unpaired t-test:
p = 0.0111) (Fig. 1B).

3.4. Eﬀect of GBR12909, a selective DAT transporter inhibitor, on the
anxiety-related behavior of powdered diet-fed mice in the EPM test

3.2. Eﬀect of diazepam on the anxiety-related behavior of powdered dietfed mice in the EPM test

We recently reported that mice subjected to long-term feeding on
powdered diet induces the alteration of dopaminergic regulation, and inparticular, it decreases the expression of dopamine D4 receptor mRNAand increases dopamine turnover in the frontal cortex (Niijima-Yaoita
et al., 2013). To study the inﬂuence of selective DAT inhibitor on the
anxiety-related behaviors elicited by powdered diet, the % of open arm
time and the total number of arm entries after treatment with selective
DAT transporter inhibitor were measured in powdered diet group.
Fig. 4 shows the % of open arm time and the total number of arm entries after treatment with GBR12909, a selective DAT inhibitor, in
powdered diet-fed mice. One-way ANOVA of the inﬂuence of
GBR12909 on the % of open arm time [F (2, 35) = 1.551; p = 0.2263](Fig. 4A) and total number of arm entries [F (2, 35) = 1.395;
p = 0.2612] (Fig. 4B) in powdered diet-fed group revealed no signiﬁcant diﬀerences. There were no signiﬁcant diﬀerences between vehicle-treated control diet-fed mice and GBR 12909 (3 mg/kg)-treatedcontrol diet-fed mice, in the EPM test (data not shown).

To study the implication of inﬂuence of anxiolytic agent on the
anxiety-related behaviors elicited by powdered diet, mice at 20 weeks
of age were used; the % of open arm time and the total number of arm
entries were measured in powdered diet- and control diet groups. Fig. 2
shows the % of open arm time and the total number of arm entries after
treatment with diazepam, an anxiolytic agent, on powdered diet- andcontrol diet-fed mice. Two-way ANOVA revealed the main eﬀect fordiet on the % of open arm time [F (1, 42) = 16.21, p = 0.0002], themain eﬀect for drug [F (1, 42) = 18.33, p = 0.0001] and diet × druginteraction [F (1, 42) = 4.004, p = 0.0519] (Fig. 2A). The post hoc
Tukey's test revealed that diazepam (1 mg/kg) signiﬁcantly increased
the % of open arm time in control diet-fed group, compared with that ofvehicle-treated control diet-fed mice (p < 0.01, Fig. 2A). The % ofopen time in vehicle-treated powdered diet-fed mice signiﬁcantly increased, compared with vehicle-treated control diet-fed mice

Fig. 1. Inﬂuence of powdered diet feeding for 17 weeks on the elevated plus maze test in mice. The left and right panels show time spent in the open arm (A) and totalarm entries (B), respectively. The data are expressed as the mean ± S.E.M. for 14–18 mice/group. *p < 0.05, **p < 0.01; vs. control diet-fed mice (unpaired ttest).
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Fig. 2. Eﬀect of diazepam on the elevated plus
maze test in control diet- and powdered diet-fed
mice. The left and right panels show time spentin the open arm (A) and total arm entries (B),respectively. The data are expressed as themean ± S.E.M. for 8–15 mice/group.
*p < 0.05, **p < 0.01; vs. vehicle-treatedcontrol diet-fed mice (Tukey's test).

Fig. 3. Eﬀect of bicuculline on the elevated plus maze test in powdered diet-fed mice. The left and right panels show time spent in the open arm (A) and total arm
entries (B), respectively. The data are expressed as the mean ± S.E.M. for 6–15 mice/group. *p < 0.05; vs. vehicle-treated mice (unpaired t-test).

diﬀerences [F (4, 58) = 3.851; p = 0.0077] (Fig. 5A). The post hoc
Dunnett's test revealed that atomoxetine at a dose of 0.18 mg/kg, signiﬁcantly attenuated the % of open arm time in powdered diet-fed
group, compared with that of saline-treated powdered diet-fed group(p < 0.01, Fig. 5A). One the other hand, one-way ANOVA revealed
that the total number of arm entries in powdered diet-fed group [F (4,58) = 0.2623; p = 0.9009, Fig. 5B] was not inﬂuenced by treatment
with atomoxetine. There were no signiﬁcant diﬀerences between salinetreated control diet-fed mice and atomoxetine (0.18 and 0.5 mg/kg)treated control diet-fed mice, in the EPM test (data not shown).

3.5. Eﬀect of atomoxetine, a selective NAT inhibitor, on the anxiety-related
behavior of powdered diet-fed mice in the EPM test
To study the inﬂuence of selective NAT inhibitor on the anxietyrelated behaviors elicited by powdered diet, the % of open arm time
and the total number of arm entries after treatment with selective NATtransporter inhibitor were measured in powdered diet group. Fig. 5
shows the % of open arm time and the total number of arm entries after
treatment with atomoxetine, a selective NAT inhibitor, in powdereddiet-fed mice. One-way ANOVA of the inﬂuence of atomoxetine on the% of open arm time in powdered diet-fed group revealed signiﬁcantFig. 4. Eﬀect of GBR12909 on the elevated plus maze test in powdered diet-fed mice. The left and right panels show time spent in the open arm (A) and total arm
entries (B), respectively. The data are expressed as the mean ± S.E.M. for 10–18 mice/group.
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Fig. 5. Eﬀect of atomoxetine on the elevated plus maze test in powdered diet-fed mice. The left and right panels show time spent in the open arm (A) and total arm
entries (B), respectively. The data are expressed as the mean ± S.E.M. for 10–18 mice/group. **p < 0.01; vs. saline-treated mice (Dunnett's test).

(Niijima-Yaoita et al., 2013). To study the inﬂuence of PD168077 on
the anxiety-related behaviors elicited by powdered diet, the % of open
arm time and the total number of arm entries after treatment with
PD168077 (0.1 and 0.3 mg/kg) were measured in powdered diet group.
Fig. 7 shows the % of open arm time and the total number of arm entries after treatment with PD168077 on powdered diet-fed mice. Oneway ANOVA of the inﬂuence of PD168077 on the % of open arm time
revealed signiﬁcant diﬀerences in powdered diet-fed mice [F (2,23) = 5.651; p = 0.0101, Fig. 7A]. The post hoc Dunnett's test revealed
that PD168077 (0.3 mg/kg) signiﬁcantly attenuated the % of open arm
time in powdered diet-fed group, compared with that of vehicle-treatedpowdered diet-fed mice (vehicle vs. 0.3 mg/kg; p < 0.05, Fig. 7A). In
contrast to the % of open arm time in powdered diet-fed group, the total
number of arm entries in powdered diet-fed group [F (2, 23) = 0.8001;
p = 0.4614, Fig. 7B] was not inﬂuenced by treatment with PD168077.
There were no signiﬁcant diﬀerences between vehicle-treated control
diet-fed mice and PD168077 (0.3 mg/kg)-treated control diet-fed mice,in the EPM test (data not shown).

3.6. Eﬀect of methylphenidate, a DAT and NAT inhibitor, on the anxietyrelated behavior of powdered diet-fed mice in the EPM test
To study the inﬂuence of methylphenidate, a DAT and NAT inhibitor, on the anxiety-related behaviors elicited by powdered diet, the% of open arm time and the total number of arm entries after treatment
with methylphenidate (0.133, 0.2 and 0.3 mg/kg) were measured in
powdered diet group. Fig. 6 shows the % of open arm time and the total
number of arm entries after treatment with methylphenidate in powdered diet-fed mice. One-way ANOVA of the inﬂuence of methylphenidate on the % of open arm time in powdered diet-fed group revealed
signiﬁcant diﬀerences [F (3, 46) = 2.839; p = 0.0482] (Fig. 6A). The
post hoc Dunnett's test revealed that methylphenidate (0.2 mg/kg),
signiﬁcantly attenuated the % of open arm time in powdered diet-fed
group, compared with that of saline-treated powdered diet-fed group(saline vs. 0.2 mg/kg; p < 0.05, Fig. 6A). In contrast, one-way ANOVA
revealed that the total number of arm entries in the powdered diet-fed
group [F (3, 46) = 1.408; p = 0.2525, Fig. 6B] was not inﬂuenced bytreatment with methylphenidate. There were no signiﬁcant diﬀerences
between saline-treated control diet-fed mice and methylphenidate (0.2
and 0.3 mg/kg)-treated control diet-fed mice, in the EPM test (data notshown).

3.8. Inﬂuence of long-term powdered diet feeding on the expression of
GABA transaminase in the frontal cortex
In the experiments described above, the increase in the % of open
arm time elicited by long-term powdered diet feeding was attenuatedby treatment with bicuculline, GABA-A receptor antagonist. Hwanget al. (2004) suggested that the increase in expression of GABA transaminase, GABA metabolic enzymes (for review see Kowalczyk andKulig, 2014), provides important information about the brain with

3.7. Eﬀect of PD168077, a selective dopamine D4 receptor agonist, on the
anxiety-related behavior of powdered diet-fed mice in the EPM test
We recently reported that PD168077, a selective D4 receptor agonist, attenuates the increased SI time in powdered diet-fed mice

Fig. 6. Eﬀect of methylphenidate on the elevated plus maze test in powdered diet-fed mice. The left and right panels show time spent in the open arm (A) and totalarm entries (B), respectively. The data are expressed as the mean ± S.E.M. for 10–18 mice/group. *p < 0.05; vs. saline-treated mice (Dunnett's test).
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Fig. 7. Eﬀect of PD168077 on the elevated plus maze test in powdered diet-fed mice. The left and right panels show time spent in the open arm (A) and total arm
entries (B), respectively. The data are expressed as the mean ± S.E.M. for 5–15 mice/group. *p < 0.05; vs. vehicle-treated mice (Dunnett's test).

expression in the frontal cortex and hippocampus of powdered diet-fed
mice were higher and lower in control diet-fed mice, respectively(unpaired t-test: p = 0.0452, Fig. 9B and p = 0.003, Fig. 10B). There
were no signiﬁcant diﬀerences in the expressions of DAT in the frontal
cortex and hippocampus (unpaired t-test: p = 0.4672, Fig. 9A andp = 0.4293, Fig. 10A).
4. Discussion
The ﬁndings of the present study may be summarized as follows: (i)Long-term powdered diet increased the % of open arm time and thetotal number of arm entries, indicating that the mice have low anxiety,hyperactivity and impulsivity behaviors (EPM test) (Fig. 1), (ii) The %of open arm time in control diet-fed mice was increased by treatment
with anxiolytic agent, while the anxiolytic agent did not inﬂuence the% of open arm time in powdered diet-fed mice (Fig. 2), (iii) GABA-A
receptor antagonist attenuated the increase in the % of open arm timeby powdered diet feeding (Fig. 3), (iv) selective DAT inhibitor did not
inﬂuence the % of open arm time and the total number of arm entries inpowdered diet-fed mice. In contrast, the increased the % of open arm
time by powdered diet feeding was attenuated by treatment with selective NAT inhibitor, DAT and NAT inhibitor and dopamine D4 receptor agonist (Figs. 4–7), (v) GABA transaminase expressions in thefrontal cortex of powdered diet-fed mice were decreased (Fig. 8), (vi)powdered diet feeding induced an increase and a decrease in NAT expression, but not DAT expression, in the frontal cortex and hippocampus, respectively (Figs. 9 and 10). These ﬁndings are discussed
below.
We have previously reported that mice subjected to long-term
feeding on powdered diet exhibits increased SI behavior (NiijimaYaoita et al., 2013), elevation of blood catecholamine and corticosterone, hyperglycemia, hypertension and no changes in growth curves
between diet types; further, we found that among daily dietary habits,
mastication of food of adequate hardness is very important for the
maintenance of systemic (physical and mental) health (Tsuchiya et al.,2014). Although the SI test has been widely documented as an anxiety
test, mainly because of a novel partner/place, the EPM test was also
developed to detect anxiety-related behavior. In this study, we chose
the EPM test as an approach to study anxiety-related behavior; the longterm powdered diet-fed mice showed an increase in the % of open arm
time and total number of arm entries, indicating that the mice subjected
to long-term feeding on powdered diet have low anxiety behavior.
It is known that GABA-A receptor plays a critical role in anxiolytic
activity and the activation of the GABA-A receptor induced by BZD
including diazepam reduces anxiety (for review see Jacob et al., 2008;
Möhler et al., 2002). Thus, in this study, to investigate the implicationof GABA-A receptor on these behaviors, the eﬀects of diazepam andFig. 8. Inﬂuence of powdered diet feeding for 17 weeks on the expression of
GABA transaminase in the frontal cortex (FC) of mice. The data are given as themean ± SEM for a group of 5 mice/group. *p < 0.05; vs. control diet-fed mice
(unpaired t-test).

GABA dysfunction. Therefore, we compared the diﬀerences in the expression levels of GABA transaminase in the frontal cortex between the
powdered diet group and control diet group (Fig. 8). The levels of GABA
transaminase expression in the frontal cortex of powdered diet-fed mice
were lower than that of control diet-fed mice (unpaired t-test:
p = 0.026, Fig. 8).
3.9. Inﬂuence of long-term powdered diet feeding on the expression of DAT
and NAT in the frontal cortex and hippocampus
In the experiments described above, increase in the % of open arm
time elicited by long-term powdered diet feeding was attenuated bytreatment with atomoxetine, selective NAT inhibitor, and methylphenidate, DAT and NAT inhibitor, but not treatment with GBR12909, a
selective DAT inhibitor (Figs. 4–6). Moreover, we recently reported thealteration of dopaminergic regulation, especially the increase in dopamine turnover in the frontal cortex, but not but in the hippocampus of
powdered diet-fed mice (Niijima-Yaoita et al., 2013). Therefore, we
compared the diﬀerences in the expression levels of both DAT and NAT
in the frontal cortex and hippocampus between the powdered dietgroup and control diet group (Figs. 9 and 10). The levels of NAT
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Fig. 9. Inﬂuence of powdered diet feeding for 17 weeks on the expression of dopamine transporter (DAT) (A) and noradrenaline transporter (NAT) (B) in the frontal
cortex of mice. The data are given as the mean ± SEM for a group of 5 mice/group. *p < 0.05; vs. control diet-fed mice (unpaired t-test).

bicuculline on the behaviors in powdered diet- and control diet-fed
mice, were evaluated. We showed that the increase in the % of open
arm time in powdered diet-fed mice was attenuated by treatment with
GABA-A receptor antagonist bicuculline, not by treatment with anxiolytic diazepam. However, the % of open arm time in control diet-fed
mice was increased by treatment with diazepam. Moreover, both diazepam and bicuculline did not aﬀect the total number of arm entries inpowdered diet- and control diet-fed groups. These data indicated thatthe low anxiety elicited by long-term powderd-diet feeding was mediated via the GABAAergic transmission.
Recent evidence suggests that GABAergic neurotransmission within
the medial prefrontal cortex may be central to the regulation of
amygdala activity related to emotion and anxiety-related behavior
processing (Delli Pizzi et al., 2017). Solati et al. (2013) reported thatusing rats, bilateral medial prefrontal cortex injection of the GABA-A
receptor agonist muscimol produces a low anxiety in the EPM test.
Majewska et al. (1986) reported that one of the corticosterone metabolites is allotetrahydrodeoxycorticosterone, a neuroactive 3α 5α-reduced steroid, which are potent positive allosteric modulator of GABAA receptors. In our previous study, we demonstrated that the plasma
corticosterone level of powdered diet-fed mice is higher than that ofcontrol diet-fed mice (Tsuchiya et al., 2014), suggesting that the increased corticosterone metabolites may stimulate GABA-A receptor in

the powdered diet-fed mice and the low anxiety behavior may be elicited.
Kazi and Oommen (2014) reported that using rats, chronic noise
stress induced an increase in GABA levels and a decrease in activity of
GABA transaminase in cortex. Moreover, to investigate the relationshipbetween low anxiety behavior elicited by long-term powdered dietfeeding and the leading cause of activated GABA-A receptors, wemeasured the expression of GABA transaminase, GABA metabolic enzymes, in frontal cortex of powdered diet- and control diet-fed mice. In
this study, the expression levels of GABA transaminase of the frontal
cortex in powdered diet-fed mice were lower than that in control dietfed mice, indicating that the GABA-A receptor in the frontal cortex of
powdered diet-fed mice may be stimulated by an increase in the concentration of GABA via attenuated GABA transaminase.
In our previous study, we proposed that the increase in SI behaviors(i.e. it prolonged SI time) in powdered diet-fed mice might be mediatedvia alteration of dopaminergic system, especially a decrease in dopamine D4 receptor signals in the frontal cortex (Niijima-Yaoita et al.,2013). Interestingly, dysregulation of dopamine D4 receptor signaling
is linked to several pathological conditions, such as ADHD (for review
see Rondou et al., 2010). Moreover, the exploratory behavior in open
arms may reﬂect impulsivity for novelty-seeking (for review see Steimer
and Driscoll (2003)]. Indeed, the increase in the % of open arm time

Fig. 10. Inﬂuence of powdered diet feeding for 17 weeks on the expression of dopamine transporter (DAT) (A) and noradrenaline transporter (NAT) (B) in the
hippocampus of mice. The data are given as the mean ± SEM for a group of 6 mice/group. *p < 0.05; vs. control diet-fed mice (unpaired t-test).
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(low anxiety behavior) and total number of arm entries elicited bypowdered diet feeding may relate to the impulsivity and hyperactivitylike symptoms in ADHD, indicating that the long-term powdered dietfeeding may increase the risk for the onset of ADHD-like behavior orsymptoms.
To investigate the implication of these behaviors in the EPM test on
these symptoms in ADHD, we measured whether the ADHD therapeutic
agent, methylphenidate and atomoxetine, attenuate the low anxiety or
impulsivity-like behavior and hyperactivity-like behavior elicited bypowdered diet feeding. In this study, treatments of methylphenidate
and atomoxetine improved the impulsivity-like behavior, but not thehyperactivity-like behavior in powdered diet-fed group. Moreover, the% of open arm time and the total number of arm entries in control dietfed group were not aﬀected by a similar dose of methylphenidate andatomoxetine. Methylphenidate and atomoxetine appear to enhance
prefrontal cortex function through inhibition of both DAT and NAT and
inhibition of NAT, respectively (for review see Arnsten and Pliszka,2011). Andrews and Lavin (2006) reported that methylphenidate acts
to increase catecholaminergic tone in the prefrontal cortex, and activation of alpha-2 adrenoceptors located in GABA interneurons. Indeed,
the inhibition of GABA function may inﬂuence the amygdala activity
related to emotion and anxiety-related behavior processing and attenuate the low anxiety or impulsivity-like behavior. In this study, we
showed an increase in the expression of NAT in the frontal cortex of
powdered diet-fed mice. In contrast, DAT expression was not changedin the frontal cortex of powdered diet-fed mice. These ﬁndings may
suggest the relationship between improvement of methylphenidate andatomoxetine on the low anxiety behavior or impulsive-like behavior
elicited by long-term powdered diet feeding and the activation of alpha2 adrenoceptors by an increase in the concentration of NA mainly via
inhibition of NAT in the synaptic cleft. In addition, the involvement oflong-term powdered diet feeding on an increased risk ADHD-like behavior onset or symptoms was suggested.
However, in this study, improvement by methylphenidate and atomoxetine on the low anxiety behavior or impulsive-like behavior elicited by long-term powdered diet feeding was not dose dependent.
Berridge and Spencer (2016) reported that NA is targeted by pharmacological treatments used to treat prefrontal cortex dysfunction associated with a range of psychopathologies, including ADHD, and modulating the distinct processes via alpha-1 and alpha-2 adrenoceptors,
respectively. Moreover, they described that the diﬀerential activation ofadrenoceptor subtypes within the prefrontal cortex is reﬂected in the
inverted-U shaped function. In addition to the receptor speciﬁc actions,the diﬀerences of concentration, such as low doses that are within aclinically-relevant range or doses without clinically-relevant range, arereﬂected in the inverted-U shaped function. These ﬁndings may support
our data showing that improvements of methylphenidate and atomoxetine were not dose dependent.
Although the low anxiety behavior or impulsive-like behavior in
powdered diet-fed mice may be related to changes in noradrenergic
functions, i.e., decrease in the concentration of NA in the synaptic cleft
via elevated NAT expressions in the frontal cortex, in the previous study
we suggested that the powdered diet feeding may cause the increased SI
time and the changes in the dopaminergic system, especially the decrease in dopamine D4 receptor signals in the frontal cortex (NiijimaYaoita et al., 2013). Moreover, in this study, we showed that
PD168077, a dopamine D4 receptor agonist, attenuated the low anxiety
behavior or impulsive-like behavior in powdered diet-fed mice. In
contrast, GBR12909, a DAT inhibitor, which increases the concentration of dopamine in the synaptic cleft, did not inﬂuence the low anxiety
behavior or impulsive-like behavior in powdered diet-fed mice. Interestingly, agonism of endogenous neurotransmitters at the dopamine D4
receptor has been described not only for dopamine (Asghari et al.,1995), but also for NA and adrenaline (Lanau et al., 1997; NewmanTancredi et al., 1997). Moreover, it is reported that in the prefrontal
cortex, where the DAT density of dopaminergic neurons is low (Sesack

et al., 1998), NAT discharges dopamine reuptake (Carboni et al., 1990;
Morón et al., 2002). In addition, it is well known that emotional stressinduces an elevation of serum glucocorticoids. Ayada et al. (2002)
observed that improved mastication behavior might reduce the stressinduced enhancement of serum cortisol. In our previous study, we
showed the elevation of serum corticosterone in powdered diet-fed
mice (Tsuchiya et al., 2014) and the decrease in the expression of dopamine D4 receptor mRNA, but not in D1, D2, D3 and D5 receptor
mRNA in powdered diet-fed mice (Niijima-Yaoita et al., 2013). Barros
et al. (2003) reported that corticosterone down-regulates dopamine D4
receptor in a mouse cerebral cortex neuronal cell line. These ﬁndings
suggest that the masticatory dysfunction induced by long-term powdered diet feeding may cause the low anxiety behavior or impulsive likebehavior, the decrease in not only NA but also dopamine in the synaptic
cleft of the frontal cortex, and the decreased dopamine D4 receptor
signaling via the elevated NAT expression and corticosterone. Indeed,
the decreased expression of dopamine D4 receptor may be stimulatedby the selective dopamine D4 receptor agonist PD168007, but not by anincrease in the concentration of dopamine via inhibition of DAT by
GBR12909.
In addition, Shah et al. (2004) reported that using rats, intra-medial
prefrontal cortex infusions of the highly selective dopamine D4 receptor
antagonist, increased the % of open arm time in the EPM test, indicating
low anxiety. Moreover, rodent dopamine D4 receptor activation in
prefrontal cortex pyramidal neurons inhibits GABAAnergic functions(Wang et al., 2002). Indeed, the decreased dopamine D4 signals in thefrontal cortex of powdered diet-fed mice may attenuate the inhibitory
eﬀects of dopamine D4 receptors on GABAAnergic function in regulating amygdala activity related to emotion and anxiety-related behavior processing.
Zhang et al. (2017) reported that chronic social stress up-regulated
expression of NAT in the brain is mediated by glucocorticoid receptor(GR) and the GR expressional alterations in response to chronic stressare complex and brain region-speciﬁc. However, the measurement of
GR protein levels after chronic stress was inconsistent (Kohda et al.,2007; Liberzon et al., 1999; Makino et al., 2002; Trujillo et al., 2016). In
this study, we showed the increase and decrease in NAT expressions in
the frontal cortex and hippocampus of powdered diet-fed mice, respectively. We previously observed that long-term powdered dietfeeding markedly increased the serum level of corticosterone (Tsuchiya
et al., 2014). These ﬁndings may support the diﬀerences in NAT expressions between the frontal cortex and hippocampus in our study.
In conclusion, the present study demonstrated that the dietary habits, e.g. a long-term powdered diet feeding, increased the % of open
arm, the total number of arm entries, the expression of NAT, and decrease in expression of GABA transaminase in the frontal cortex. In
addition, the increased the % of open arm time in powdered diet-fed
mice was attenuated by GABA-A receptor antagonist, ADHD therapeutic agents and selective dopamine D4 receptor agonist, but not by
anxiolytic agent or DAT inhibitor. These results suggest that the longterm powdered diet feeding may cause low anxiety or impulsivity,
possibly via an increase in NAT expression, decrease in dopamine D4
receptor signals and increase in GABA-A receptor functions in thefrontal cortex of mice. Moreover, the long-term powdered diet feeding
may increase the risk for onset of ADHD-like behavior or symptoms andthe eﬀect of ADHD therapeutic agents on the impulsive behavior might
be inhibited mainly by NAT and by the increased NA and/or dopaminelevels in the synaptic cleft.
Acknowledgements
We would like to express our deepest gratitude to Mr. Shunsuke
Nishidate, Mr. Kota Namura, Mr. Hiroki Imaizumi and Mr. Keigo
Kawanami of the Department of Pharmacology, Faculty of
Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical
University for their excellent technical assistance.
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This study was supported by JSPS KAKENHI Grant Numbers
24700433, 26500014 and 17K00768 and the MEXT (Ministry of
Education, Culture, Sports, Science and Technology)-Supported
Program for the Strategic Research Foundation at Private Universities(No. S1511001L, 2015–2019).

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disorder. Behav. Pharmacol. 13, 1–13.
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hippocampus and parietal cortex of senescence-accelerated mice. Brain Res. 902,
255–263.
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force and occlusal contact area in Japanese children. J. Oral Rehabil. 36, 584–591.
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Neurochem. Int. 108, 397–409. https://doi.org/10.1016/j.neuint.2017.05.021.

Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.org/10.1016/j.neuint.2018.12.002.
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Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistryjournal homepage: www.elsevier.com/locate/bmc

Novel androgen receptor full antagonists: Design, synthesis, and a docking
study of glycerol and aminoglycerol derivatives that contain p-carborane
cages⁎

Asako Kaisea, Kiminori Ohtab, , Shinya Fujiic, Akifumi Odad, Tokuhito Gotoa, Yasuyuki Endoaa

Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan
School of Pharmacy, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japanc
Institute of Molecular and Cellular Bioscience, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, JapandFaculty of Pharmacy, Meijo University, 150 Yagotoyama, Tenpaku-ku, Nagoya 468-8503, Japanb

A R T I C LE I N FO

A B S T R A C T

Keywords:
Androgen receptor
Hydrophobic pharmacophore
Carborane
Anti-androgen withdrawal syndrome

Based on the co-crystal structure of bicalutamide with a T877A-mutated androgen receptor (AR), glycerol andaminoglycerol derivatives were designed and synthesized as a novel type of carborane-containing AR modulators. The (R)-isomer of 6c, whose chirality is derived from the glycerol group, showed 20 times more potent
cell inhibitory activity against LNCaP cell lines expressing T877A-mutated AR than the corresponding (S)isomer. Docking studies of both isomers with AR suggested that (R)-6c is in closer spatial proximity to helix-12 of
the AR than (S)-6c, which is the most important common motif in the secondary structure of AR for the expression of antagonistic activity.

1. Introduction
Worldwide, prostate cancer (PC) is one of the major causes ofcancer-related death in men.1 The molecular basis of this disease involves an irregular behavior of the functions mediated by the androgen
receptor (AR), which belongs to the nuclear receptor (NR) superfamilyof transcription factors that regulate gene transcription upon ligand
binding.2 Synthetic AR modulators can be structurally classiﬁed as either steroidal or non-steroidal, and may exhibit various types of functional (androgenic, anti-androgenic, or anabolic) activity.3 The nonsteroidal ligand ﬂutamide (1),4 which is metabolized into the morepotent AR antagonist hydroxyﬂutamide (OH-Flu, 2),4 is a well-known
AR antagonist used for the treatment of PC (Fig. 1), while bicalutamide(Bic, 3) is used for the treatment of D2-stage metastatic PC (Fig. 1).5 In
contrast to steroidal ligands, these two typical non-steroidal AR antagonists exhibit a high selectivity for AR.
Anti-androgens are particularly useful for the early-stage treatmentof PC. However, PC often advances to a hormone-refractory state, inwhich the disease progresses despite continued androgen ablation or
anti-androgen therapy due to the appearance of androgen-independent
PC cells or the ability of adrenal androgens to support tumor growth.6
Instances of anti-androgen-withdrawal syndrome (AWS), i.e., a clinicalimprovement upon withdrawal of the anti-androgen therapy, have also
been reported after prolonged treatment with anti-androgens.7 Thus,⁎

the development of pure AR full antagonists which are eﬀective for
AWS represents an attractive drug discovery goal in the context of AR.8
Although the molecular mechanism of operation for AR is not fully
understood yet, one proposed mechanism is based on the hypothesisthat AR may be subject to a mutation, which has been discovered inpatients treated with ﬂutamide.9 AR mutations that result in receptor
promiscuity and the ability of anti-androgens to exhibit agonist activity
might at least partially account for this phenomenon. In particular, apoint mutation at T877 has been discovered in the AR-ligand-binding
domain (AR LBD) in AWS patients, and this mutation has since been
considered as a hotspot for the critical pathology.9 Bic, which is themost widely used pure anti-androgen agent, acts as an anti-androgenfor the T877A-mutated AR. However, Bic also exhibits signiﬁcant androgenic activity towards W741L- or W741C-mutated AR, which is
known as Bic-withdrawal syndrome (BWS).9
Carborane-containing ligands are of interest in medicinal chemistry,
as they exhibit a diﬀerent spectrum of ligand responsiveness relative toother therapeutic agents due to their unique chemical and structuralfeatures.10 We have reported several in vivo drug candidates, such as
estrogen-receptor modulators11 and retinoid-X-receptor antagonists12
that contain a carborane cage as a hydrophobic pharmacophore.
Moreover, we have obtained several useful carborane-containing AR
ligands as PC-treatment-drug candidates and AR-related biochemical
tools, e.g. BA341 (4) and BA632 (5).13,14 Compound 4 showed more

Corresponding author.
E-mail address: k-ohta@pharm.showa-u.ac.jp (K. Ohta).

https://doi.org/10.1016/j.bmc.2018.06.007
Received 16 April 2018; Received in revised form 6 June 2018; Accepted 6 June 2018

Available online 07 June 20180968-0896/ © 2018 Elsevier Ltd. All rights reserved.

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A. Kaise et al.
R

H
N
O2N

H
N

O

O

NC

CF3

derivatives with glycerol and aminoglycerol groups bind to the vitamin
D receptor (VDR) and AR LBD, the speciﬁc roles of glycerol and aminoglycerol remain unclear.16 Thus, we designed novel p-carborane derivatives with glycerol (6) and aminoglycerol groups (7) at the para
position to mimic the binding mode of Bic with AR LBD containing awater molecule, and to bring the p-carborane cage closer to helix-12 in
the AR (Fig. 2). In this paper, we describe the synthesis of novel pcarborane derivatives with glycerol (6) and aminoglycerol groups (7) atthe para position and their biological activity, i.e., AR binding, theireﬀect on AR-dependent proliferation of SC-3 and LNCaP cells, and an
AR-ligand docking simulation study.

OH O
S
O
F

CF3
Bicalutamide (Bic, 3)

R = H; Flutamide (1)R = OH; Hydroxyflutamide (OH-Flu, 2)

OH
O

OH

F
CN

BA341 (4)CN

BA632 (5)Fig. 1. Structures of non-steroidal and carborane-containing AR ligands.

2. Results and discussion

potent AR-binding and AR-antagonistic activity levels than 2, but acted
as an agonist toward LNCaP cells, which express the T877A-mutated AR(EC50 = 6.3 × 10−7 M). Compound 5, which contains a Bic-like side
chain, acted as an AR full antagonist for LNCaP cells, i.e., the carborane
cage acted as an eﬀective hydrophobic pharmacophore for the AR LBD
for both agonist and antagonist.
It seems that the steric repulsion between the carborane cage andseveral amino acid residues around helix-12 is able to directly control
helix-12, which is crucial for expressing AR-antagonistic activity.2 Our
strategy for the development of AR full antagonists is based on a permanent AR antagonistic activity that is unaﬀected by the point mutation of AR LBD, using the direct repulsion between helix-12 and thecarborane cage. Bohl et al. have reported that the cyano group of Bic
interacts with the amino-acid residues Arg752, Glu711, and Met745 of
AR LBD via a water molecule in co-crystal structure (Fig. 2a).15
Glycerol and aminoglycerol groups contain three hydrogen-bondacceptors and two or three hydrogen-bond donors, respectively. We
anticipated that compounds with these groups could eﬀectively bind to
the target protein through the formation of multivalent hydrogenbonds. Although Hosoda et al. have reported that diphenylmethane

2.1. Chemistry
Scheme 1 summarizes the synthesis of the target molecules 6 and7. p-Carborane derivative 8,11 which is protected with a tert-butyldimethylsilyl (TBS) group was consecutively treated with n-BuLi and
CuCl to aﬀord the corresponding Cu reagent, which was reacted with
4-iodoanisole or 4-iodonitrobenzene under Ullmann coupling conditions to furnish the corresponding aryl-p-carborane derivatives 9 and
11 in 54% and 80% yield, respectively.17 The methoxy and TBSgroups of 9 were deprotected with BBr3 to aﬀord phenol derivative
10, in which a phenol group between two hydroxy groups is selectively transformed with 3-chloro-1,2-propanediol to generate thecorresponding glycerol derivative 6a in 91% yield over two steps. The
reduction of the nitro group of 11 was accomplished by a catalytichydrogenation using Pd/C under an atmosphere of hydrogen. The thus
quantitatively obtained aniline (12) was reacted with glycidol, andthe TBS group was subsequently deprotected with tetra-n-butylammonium ﬂuoride (TBAF) to furnish the corresponding aminoglycerol derivative (7) in 29% yield over two steps.
Scheme 2 summarizes the synthesis of 6b, 6c, and 14, whereby theFig. 2. (A) X-ray crystal structure of bicalutamide with AR-LBD (PDBID: 1Z95); (B) Design of novel carborane-containing AR full antagonist drug candidates 6 and 7.
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A. Kaise et al.

OTBS

a
H3CO

OTBS

OH

b
HO

9

OH

c
HO

10

OH

6a

O

H
8

OTBS

a
O2N

OTBS

d
H2N

11

OH

e, f
HO

12

OH

N
H

7

Scheme 1. Synthesis of hydroxymethyl derivatives 6a and 7. Reagents and conditions: (a) n-BuLi, DME, CuCl, pyridine; then 4-iodoanisole or 4-iodonitrobenzene,
100 °C; (b) BBr3, CH2Cl2; (c) 3-chloro-1,2-propanediol, K2CO3, DMF; (d) Pd/C, H2, MeOH; (e) glycidol, EtOH; (f) TBAF, THF.

latter is the acetonide-protected derivative of 6c. p-Carboranyl phenol
1311 was treated with 3-chloro-1,2-propanediol in the presence of potassium carbonate to aﬀord the corresponding glycerol derivative (6c)in 82% yield. In this reaction, using of (R)- or (S)-3-chloro-1,2-propanediol instead of the corresponding racemic reagent aﬀorded chiral
glycerol derivatives (R)-6c and (S)-6c in 76% and 97% yield, respectively. Both compounds showed an optical purity of > 99% (HPLC).
The diol moiety of 6c was initially protected with acetone to aﬀord thecorresponding acetonide (14) in 88% yield, which was transformed into
the boronic ester that was subjected to an oxidative rearrangement with
H2O2 in the presence of acetic acid, followed by deprotection of theacetonide to aﬀord p-carboranol 6b in 69% yield.18

were removed by the charcoal stripping treatment and these cells were
only proliferated in the presence of 1 nM of testosterone. That is, we candirectly understand AR-dependent cell proliferation inhibitory activityof the tested compounds in these assay systems. To further determine
whether the cell proliferation activity of 6, 7, and 14 depends on AR,
we also evaluated cell proliferation inhibitory activity against AR-independent prostate cancer cell line PC-3. OH-Flu, as an active metabolite of ﬂutamide, showed the most potent antagonistic activity in the
SC-3-proliferation assay, but acted as an agonist for LNCaP cells. On theother hand, (R)-Bic, an active enantiomer, showed antagonistic activity
towards LNCaP cell lines (IC50 = 0.43 μM). Glycerol derivatives 6a–6c
showed weaker AR antagonistic activity than OH-Flu in the SC-3-proliferation assay, but acted as AR antagonists toward LNCaP cells.
Compound 6a, which contains a hydroxymethyl group on anothercarbon atom of the p-carborane cage, showed the most potent AR-antagonistic activity toward LNCaP cell lines. Its IC50 value (0.39 μM) was
slightly lower than that of (R)-Bic. The IC50 values of 6a–6c toward
LNCaP cells were aﬀected by the size of the substituent at the carbon
atom of the p-carborane (H > OH > CH2OH). It seems that OH and
CH2OH groups form no strong hydrogen bond interactions with theamino acid residues of AR LBD. This result is probably due to the repulsion between the substituents and helix-12.We also examined theeﬀects of the chirality of the secondary hydroxyl group in the glycerol
group on the biological activity related to AR by using the chiral derivatives (R)-6c and (S)-6c. Interestingly, the (R)-isomer showed an ARantagonistic activity in the LNCaP-proliferation assay that was 20 times
more potent than that of the corresponding (S)-isomer, even thoughboth enantiomers showed a similar binding aﬃnity to hAR. As expected
for 6a–6c, the p-carborane cage of the (R)-isomer should be locatedcloser to helix-12 in the AR LBD than that of the (S)-isomer.
Aminoglycerol derivative 7 exhibited a similar bioactivity proﬁle tothat of the corresponding glycerol derivative 6a in the AR-binding assay
and the SC-3- and LNCaP-proliferation assays. None of the synthesizedcompounds showed cell proliferation inhibitory activity against AR

2.2. Biological activities of the synthesized compounds
Binding aﬃnities of 6, 7, and 14 for human AR (hAR) were evaluated by means of a competitive binding assay using [1,2-3H]-dihydrotestosterone ([3H]-DHT) and hAR.13,14 The results showed that
6a–6c and 7 bind in a dose-dependent fashion to the AR LBD and that
their aﬃnity values are comparable to or slightly weaker than that of
OH-Flu. The binding aﬃnity of (R)-6c for hAR is slightly higher thanthat of (S)-6c, which suggests that the orientation of the secondaryhydroxyl group in both enantiomers has little eﬀect on the formation ofhydrogen bonds with the amino acid residues Arg752 and Glu711 in the
AR LBD. Our hypothesis was supported by the fact that 14, in which thecentral functional group is masked by an acetonide, showed no binding
aﬃnity to the AR LBD. The glycerol or aminoglycerol groups of 6 and 7
play an important role for the binding to the AR LBD. Thus, these results
strongly support the validity of our drug-design concept based on glycerol and aminoglycerol groups.
The anti-androgenic activity of 6, 7, and 14 was evaluated by cellproliferation assays using SC-3 and LNCaP cell lines, which show ARdependent proliferation. In these assay systems, endogenous steroid
hormones, growth factors, and cytokines in fetal bovine serum (FBS)
H

H

H

H

a
HO

13

HO

O

HO

6c

OH

OH

O

HO

(R)-6cbOHO(S)-6c

H

OH

c
O

O

O
14

HO

O
OH

6b

Scheme 2. Synthesis of glycerol derivatives 6b and 6c and acetonide 14. Reagents and conditions: (a) rac-3-chloro-1,2-propanediol, K2CO3, DMF for 6c; (R)- or (S)-3chloro-1,2-propanediol, K2CO3, DMF for (R)- or (S)-6c, respectively; (b) acetone, p-TsOH; (c) n-BuLi, Et2O, B(OMe)3, H2O2, AcOH.
3807— 364 —

Bioorganic & Medicinal Chemistry 26 (2018) 3805–3811

A. Kaise et al.

Surprisingly, we observed a diﬀerence regarding the location of bothcarborane cages. The p-carborane cage of (R)-6c is closer to the aminoacid residue Met895 on helix-12 than that of the other enantiomer(Fig. 3B). We propose that the potent anti-androgenic activity of (R)-6c
toward LNCaP cells is caused by the steric repulsion between the pcarborane cage as a hydrophobic pharmacophore and helix-12 of the
AR LBD. As expected, the glycerol groups of (R)-6c and (S)-6c bind to
the amino acid residues Arg752 and Gln711 of AR (Fig. 3C and D).
Interestingly, the supposed interaction modes of the glycerol group of(R)-6c with the amino acid residues of Arg752 and Gln711 in the AR
LBD are diﬀerent from those obtained for (S)-6c. In the interaction
mode of (R)-6c, an oxygen atom of the ether moiety and the secondaryhydroxy group interacts with Gln711, whereas the secondary and primary hydroxy groups of (S)-6c interacts with Gln711. For Arg752 inAR, (R)-6c and (S)-6c interact with the secondary and primary hydroxygroups, respectively. Based on the results from the docking study andthe biological activity, we suggest that the diﬀerence of the arrangement of the p-carborane cage between (R)-6c and (S)-6c should becaused by the diﬀerent interaction modes of each glycerol group withthe amino acid residues of Arg752 and Gln711 of AR, which was induced by the chirality of the secondary hydroxy group. It seems that theglycerol group can partially mimic the binding mode observed in theco-crystal structure of Bic with the AR LBD. These groups should thus
be useful as novel functional groups for the development of AR ligands.

Table 1
Biological proﬁles of 6, 7, and 14.
Compound

OH-Flu(R)-Bic
6a6b6c(R)-6c(S)-6c714IC50(μM)for hARbindinga

10.6
nt
15.9
16.1
12.5
10.3
12.2
16.1
Inactive

IC50(μM) for cell proliferationb
SC-3 cell(AR+)

LNCaP cell(AR+)

PC-3 cell
(AR−)

0.02
nt
0.27
0.70
0.58
1.0
0.50
0.21
Inactive

Agonist
0.43
0.39
1.6
5.7
0.66
11
0.42
Inactive

Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactivea
All competitive binding assays were carried out in duplicate (n = 2) andIC50 values were calculated based on the dose-response curves obtained fromthe binding assays.
b
SC-3 and LNCaP cells were incubated for 3 and 5 days, respectively, withthe test compounds (0.1 nM–10 μM) in the presence of testosterone (1 nM). All
assays were performed in triplicate (n = 3).

non-expression PC-3 cells. Therefore,cell proliferation inhibitory activity of 6 and 7 against SC-3 and LNCaP cells should be caused by the
inhibition of AR activation by 1 nM of testosterone. As expected fromthe results of the AR-binding assay, acetonide 14 did not show any cellproliferation activity toward the SC-3 or LNCaP cell lines. As theacetonide protecting group is stable under these assay conditions, suchderivatives may be considered prodrugs of the glycerol and aminoglycerol derivatives (Table 1).19

3. Conclusion
In summary, we have designed and synthesized p-carborane-containing glycerol and aminoglycerol derivatives based on the co-crystal
structure of Bic with AR LBD. Glycerol and aminoglycerol groups canpartial mimic the AR-Bic binding mode via a water molecule. Thus, thesynthesized compounds bind to the AR LBD and exhibit anti-androgenic
activity towards LNCaP cells by expressing the T877A-mutated receptor
AR. The chiral secondary hydroxy group also plays an important role
for the anti-androgenic activity toward LNCaP cells. Recently, it has
been reported that Bic exhibits signiﬁcant androgenic activity toward
W741L- and W741C-mutated AR, which is called the Bic-withdrawal
syndrome (BWS).9 We hope that 6 and 7 act as anti-androgens towards
AWS and BWS owing to their signiﬁcant structural diﬀerences relative
to Bic. Biological assays of 6 and 7 would be performed using BWS

2.3. Docking simulation study of (R)-6c and (S)-6c with AR
Docking simulation studies of (R)-6c and (S)-6c with T877A-mutated AR, using the co-crystal structure of T877A-mutated AR withcyproteron acetate (PDBID: 2OZ7), were performed with a docking
program (GOLD 5.2).20 The overall structure of the docking results isshown in Fig. 3A. The p-carborane cages of (R)-6c and (S)-6c are accommodated in a deep position of the hydrophobic pocket of the AR
LBD close to helix-12, which is the most important common motif in thesecondary structures of AR for the expression of antagonistic activity.

Fig. 3. Comparisons of the docking models for (R)6c and (S)-6c with AR. (A) Overall view of thebinding mode of (R)-6c (aqua) and (S)-6c (brown)with AR. (B) The arrangement of (R)-6c (aqua) and(S)-6c (brown) relative to the amino acid residue of
Met895 on helix-12 in the AR LBD. (C) Proposedinteraction of the glycerol group of (R)-6c with theamino acid residues of Arg752 and Gln711 in the AR
LBD. (D) Proposed interaction of the glycerol groupof (S)-6c with the amino acid residues of Arg752 and
Gln711 in the AR LBD.

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was heated at 80 °C for 8 h, before 3-chloro-1,2-propanediol (456 μL,
5.47 mmol) and K2CO3 (455 mg, 3.19 mmol) were further added. The
reaction was continued for 40 h until 10 was disappeared on TLC. Aftercooling, the reaction was quenched with water and the reaction mixture
was extracted with AcOEt. The organic layer was washed with brine,
dried over Na2SO4, and concentrated. Puriﬁcation by column chromatography on silica gel (eluent: n-hexane/AcOEt = 10/1 to 5/1, v/v)
gave 6a (236 mg, 0.69 mmol) as a colorless solid in 91% yield; Colorlessprisms (MeOH); mp 144.0–146.0 °C; 1H NMR (400 MHz, CD3OD)
1.00–3.50 (brm, 10H), 3.46 (s, 2H), 3.58–3.66 (m, 2H), 3.88–3.94 (m,2H), 3.97–4.02 (m, 1H), 6.77 (d, J = 9.3 Hz, 2H), 7.12 (d, J = 9.3 Hz,2H); 13C{1H} NMR (100 MHz, CD3OD) 64.1, 66.2, 70.4, 71.7, 83.3,
115.0, 115.9, 129.4, 130.0, 160.6. MS (EI) m/z 340 (M+), 266 (100%);
HRMS Calcd for C12H24B10O4: 340.2678, Found: 340.2677; Anal. Calcd
for C12H24B10O4: C, 42.35; H, 7.06; Found: C, 42.34; H, 7.11.

model cell lines. Further investigations, especially into the synthesis ofanalogues, the biological evaluation for other mutated ARs, and thetissue selectivity, are currently in progress.
4. Experimental section
4.1. General considerations
Melting points were determined on a Yanaco micro melting point
apparatus and are uncorrected. 1H NMR and 13C{1H} NMR spectra wererecorded on JEOL JNM-EX-270 and JNM-LA-400 spectrometers.
Chemical shifts (δ) are expressed in ppm relative to tetramethylsilane(0.0 ppm; 1H NMR) as an internal standard, or against the signals of thedeuterated solvents (13C NMR). The splitting patterns are designated asfollows: s (singlet), d (doublet), t (triplet), m (multiplet), and br(broad). Mass spectra were recorded on a JEOL JMS-DX-303 spectrometer. Column chromatography was carried out using Fuji Silysia
BW80s, while TLC was performed on Merck silica gel F254. p-Carborane
was purchased from Katchem s.r.o. Other chemicals were purchasedfrom Aldrich or Tokyo Kasei Ltd. and used without further puriﬁcation.
All solvents were commercial products of reagent grade, which wereused without further puriﬁcation.

4.2.4. 1-tert-Butyldimethylsiloxymethyl-12-(4-nitrophenyl)-1,12-dicarbacloso-dodecaborane (11)Compound 11 was prepared by the same method as described forthe synthesis of 9 using 4-iodonitrobenzene (0.33 g, 1.31 mmol) insteadof 4-iodoanisole; 330 mg (0.80 mmol, 80% yield); 1H NMR (400 MHz,CDCl3) −0.01 (s, 6H), 0.86 (s, 9H), 1.00–3.50 (brm, 10H), 3.50 (s, 2H),
7.38 (d, J = 8.8 Hz, 2H), 8.03 (d, J = 8.8 Hz, 2H); 13C{1H}NMR(100 MHz, CDCl3) −5.7, 18.2, 25.6, 66.0, 79.9, 83.0, 123.2, 128.4,
142.9, 147.6; MS (EI) m/z 411 (M+), 73 (100%).

4.2. Synthesis
4.2.1. 1-tert-Butyldimethylsiloxymethyl-12-(4-methoxyphenyl)-1,12dicarba-closo-dodecaborane (9)A 2.64 M solution of n-BuLi in hexane (3.96 mL, 10.5 mmol) was
added dropwise to a solution of 8 (2.0 g, 6.94 mmol) in DME (5 mL) at
0 °C under argon (Ar) atmosphere.11 The mixture was stirred at room
temperature for 30 min, before CuCl (920 mg, 9.02 mmol) was added in
one portion. Stirring was continued at room temperature for 1 h, before
pyridine (4.13 mL, 51.3 mmol) and 4-iodoanisole (1.85 g, 7.92 mmol)
were added in one portion. Subsequently, the mixture was heated to
100 °C for 45 h. After cooling, the reaction was quenched with 10% aq.
HCl, before the insoluble materials were removed by ﬁltration throughCelite. The ﬁltrate was extracted with AcOEt, and the organic fractions
were washed with 10% aq. Na2S2O3 and brine, dried over Na2SO4, andconcentrated. Puriﬁcation by column chromatography on silica gel(eluent: n-hexane to n-hexane/AcOEt = 10/1, v/v) gave 10 (1.47 g,
3.72 mmol) as a colorless solid in 54% yield; 1H NMR (400 MHz, CDCl3)−0.02 (s, 6H), 0.86 (s, 9H), 1.00–3.50 (brm, 10H), 3.49 (s, 2H), 3.74 (s,3H), 6.67 (d, J = 8.8 Hz, 2H), 7.14 (d, J = 8.8 Hz, 2H); 13C{1H} NMR(100 MHz, CD3OD) −5.7, 18.2, 25.7, 55.2, 66.5, 81.1, 81.9, 113.2,
128.3, 128.5, 159.5; MS (EI) m/z 394 (M+), 337 (100%).

4.2.5. 4-(12-tert-Butyldimethylsiloxymethyl-1,12-dicarba-closododecaborane-1-yl)aniline (12)A catalytic amount of Pd/C (80 mg) was added to a solution of 11(400 mg, 0.97 mmol) in methanol (10 mL), before the mixture wasstirred at room temperature for 3 h under H2 atmosphere. Pd/C wasremoved by ﬁltration through Celite, before the ﬁltrate was evaporated.
Puriﬁcation by column chromatography on silica gel (eluent: n-hexane/
AcOEt = 100/1 to 20/1, v/v) gave 12 (401 mg, 1.06 mmol) as a colorless solid in quantitative yield; 1H NMR (400 MHz, CDCl3) 1.00–3.50(brm, 10H), 3.49 (s, 2H), 3.63 (brs, 2H), 6.44 (d, J = 8.8 Hz, 2H), 6.97
(d, J = 8.8 Hz, 2H); 13C{1H} NMR (100 MHz, CDCl3) -5.7, 18.1, 25.7,
66.0, 80.6, 82.4, 114.1, 126.5, 128.1, 146.5; MS (EI) m/z 379 (M+),
322 (100%).
4.2.6. N-2,3-Propanediol-4-(12-hydoxymethyl-1,12-dicarba-closododecaborane-1-yl)aniline (7)To a solution of 12 (50 mg, 0.13 mmol) in ethanol (3 mL) was added
glycidol (11.7 mg, 0.16 mmol) at room temperature. The mixture wasstirred at 80 °C for 48 h, before the solvent was removed under reducedpressure. Puriﬁcation by column chromatography on silica gel (eluent:n-hexane/AcOEt = 5/1 to 2/1, v/v) gave N-2,3-propanediol-4-(12-tertbutyldimethylsiloxy-methyl-1,12-dicarba-closo-dodecaborane-1-yl)aniline (23 mg, 0.05 mmol) as a colorless solid in 39% yield; 1H NMR(400 MHz, CDCl3) −0.02 (s, 6H), 0.85 (s, 9H), 1.00–3.50 (brm, 10H),
3.12 (dd, J = 7.3 Hz, 13.2 Hz, 1H), 3.22 (dd, J = 4.4 Hz, 13.2 Hz, 1H),
3.49 (s, 2H), 3.60 (dd, J = 5.6 Hz, 11.2 Hz, 1H), 3.74 (dd, J = 3.5 Hz,
11.2 Hz, 1H), 3.88–3.95 (m, 1H), 6.41 (d, J = 8.8 Hz, 2H), 7.00 (d,J = 8.8 Hz, 2H); 13C{1H} NMR (100 MHz, CDCl3) −5.6, 18.2, 25.7,
46.4, 64.7, 66.1, 70.2, 80.7, 82.4, 112.4, 126.3, 128.1, 147.7; MS (EI)m/z 453 (M+), 393 (100%). A 1 M solution of TBAF in THF (0.07 mL,
0.07 mmol) was added to a solution of N-2,3-propanediol-4-(12-tertbutyldimethylsiloxy-methyl-1,12-dicarba-closo-dodecaborane-1-yl)aniline (20 mg, 0.04 mmol) in 1 mL of dry THF, before the reaction mixture
was stirred at room temperature for 24 h. The reaction was quenchedwith water and the reaction mixture was extracted with AcOEt. The
organic layer was washed with brine, dried over MgSO4, and concentrated. Puriﬁcation by column chromatography on silica gel (eluent:n-hexane/AcOEt = 1/1 to 1/2, v/v) gave 7 (10 mg, 0.03 mmol) as acolorless solid in 75% yield; Colorless cubes (CHCl3); mp
137.5–139.0 °C; 1H NMR (400 MHz, CDCl3) 1.00–3.50 (brm, 10H), 3.13

4.2.2. 1-Hydroxymethyl-12-(4-hydroxyphenyl)-1,12-dicarba-closododecaborane (10)A 1 M solution of BBr3 in CH2Cl2 (0.36 mL, 0.36 mmol) was added
dropwise to a solution of 9 (20 mg, 0.05 mmol) in CH2Cl2 (1 mL) at 0 °C
under Ar atmosphere, before the reaction mixture was stirred at room
temperature for 16 h. The reaction was quenched with water and thereaction mixture was extracted with CH2Cl2. The organic layer waswashed with brine, dried over Na2SO4, and concentrated. Puriﬁcationby column chromatography on silica gel (eluent: n-hexane to n-hexane/
AcOEt = 10/1, v/v) gave 10 (16.6 mg, 0.07 mmol) as a colorless solidin quantitative yield; 1H NMR (400 MHz, CD3OD) 1.00–3.50 (brm,
10H), 3.55 (s, 2H), 4.96 (brs, 1H), 6.61 (d, J = 8.8 Hz, 2H), 7.06 (d,J = 8.8 Hz, 2H); 13C{1H} NMR (100 MHz, CD3OD) 66.1, 82.7, 83.6,
115.6, 128.6, 129.6, 129.3, 158.6; MS (EI) m/z 266 (M+), 250 (100%).
4.2.3. 1-Hydroxymethyl-12-{4-(2,3-dihydroxypropyloxy)phenyl}-1,12dicarba-closo-dodecaborane (6a)To a solution of 10 (200 mg, 0.76 mmol) in DMF (5 mL) was added
3-chloro-1,2-propanediol (76 μL, 0.91 mmol) and K2CO3 (130 mg,
0.91 mmol) at room temperature under Ar atmosphere. The mixture3809— 366 —

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A. Kaise et al.

(dd, J = 7.8 Hz, 13.2 Hz, 1H), 3.24 (dd, J = 4.4 Hz, 13.2 Hz, 1H), 3.54
(brs, 2H), 3.61 (dd, J = 5.9 Hz, 11.2 Hz, 1H), 3.75 (dd, J = 3.3 Hz,
11.2 Hz, 1H), 3.89–3.95 (m, 1H), 6.42 (d, J = 8.8 Hz, 2H), 7.00 (d,J = 8.8 Hz, 2H); 13C{1H} NMR (100 MHz, CD3OD) 47.3, 65.4, 66.2,
71.6, 82.5, 84.5, 112.9, 125.7, 128.9, 150.3; MS (EI) m/z 339 (M+),
278 (100%); HRMS Calcd for C12H25B10NO3: 339.2839; Found:339.2836; Anal. Calcd for C12H25B10NO3: C, 42.46; H, 7.42; N, 4.13;
Found: C, 42.17; H, 7.27; N, 4.32.

4.3. A competitive binding assay against AR
Binding aﬃnities of test compounds for hAR (human androgen receptor) were measured in competition experiments using [3H]-DHT and
cytosolic fraction of hAR-LBD (hAR ligand-binding domain)-transformed Escherichia coli as described previously. A hAR-LBD expression
plasmid vector that encodes GST-hARLBD (627–919 aa, EF domain)
fusion protein under the lac promoter (provided by Prof. S. Kato,
University of Tokyo) was transfected into E. coli strain HB-101. An
overnight culture (10 mL) of the bacteria was added to 1 L of LB
medium and incubated at 27 °C until its optical density reached 0.6–0.7
at 600 nm. Following the addition of IPTG to a concentration of 1 mM,
incubation was continued for an additional 4.5 h. Cells were harvested
by centrifugation at 4000 g at 4 °C for 15 min and stored at −80 °C until
use. All subsequent operations were performed at 4 °C. The bacterial
pellet obtained from 40 mL of culture was resuspended in 1 mL of icecold TEGDM buﬀer (10 mM Tris-HCl pH 7.4, 1 mM EDTA, 10% glycerol, 10 mM DTT, 10 mM sodium molybdate). This suspension was
subjected to sonication (10 × 10 s bursts) on ice, and crude GSThARLBD fraction was prepared by centrifugation of the suspension at
12,000 g for 30 min at 4 °C. This crude receptor fraction was diluted
with buﬀer (20 mM Tris–HCl pH 8.0, 0.3 M KCl, 1 mM EDTA) to aprotein concentration of 0.3–0.5 mg/mL and used in binding assays as
hAR-LBD fraction. Aliquots of the hAR-LBD fraction were incubated in
the dark at 4 °C with [3H]-DHT (NEN, 4 nM ﬁnal concentration),triamcinolone acetonide (1 µM ﬁnal concentration), and reference ortest compounds (dissolved in DMSO). Nonspeciﬁc binding was assessedby addition of a 200-fold excess of nonradioactive DHT. After 15 h, a
Dextran T-70/c-globulin-coated-charcoal suspension was added to theligand/protein mixture (1% Norit A, 0.05% c-globulin, 0.05% Dextran
T-70 ﬁnal concentration each) and the whole was incubated at 4 °C for
10 min. The charcoal was removed by centrifugation for 5 min at
1300 g, and the radioactivity of the supernatant was measured in
Atomlight (NEN) by using a liquid scintillation counter.

4.2.7. 1-{4-(2,3-Dihydroxypropyloxy)phenyl}-1,12-dicarba-closododecaborane (6c)Compound 6c was prepared by the same method as described forthe synthesis of 6a using 1311 (0.33 g, 1.31 mmol) instead of 10;
330 mg (0.80 mmol, 80% yield); Colorless needles (n-hexane/CH2Cl2);
mp 81.0–83.0 °C; 1H NMR (400 MHz,CDCl3) 1.00–3.50 (brm, 10H),
1.89 (t, J = 5.9 Hz, 1H), 2.45 (d, J = 4.9 Hz, 1H), 2.76 (brs, 1H), 3.71
(dd, J = 5.9 Hz, 11.2 Hz, 1H), 3.78–3.84 (m, 1H), 3.97–3.98 (m, 2H),
4.05–4.08 (m, 1H), 6.69 (d, J = 8.8 Hz, 2H), 7.12 (d, J = 8.8 Hz, 2H);
13
C{1H} NMR (100 MHz, CDCl3) 59.3, 63.5, 69.1, 70.2, 86.1, 113.8,
128.3, 130.0, 158.3; MS (EI) m/z 310 (M+), 236 (100%); HRMS Calcd
for C11H22B10O3: 310.2573; Found: 340.2566; Anal. Calcd for
C11H22B10NO3: C, 42.56; H, 7.14; Found: C, 42.66; H, 7.17.
4.2.8. 1-{4-(1,3-Dioxolane-2,2-dimethyl-4-methyloxy)phenyl}-1,12dicarba-closo-dodecaborane (14)A solution of 6c (1.0 g, 3.23 mmol) and TsOH (80 mg, 0.46 mmol) in
acetone (10 mL) was stirred at room temperature for 4 h, before thereaction was quenched with saturated aq. NaHCO3 and the mixture was
extracted with AcOEt. The organic layer was washed with brine, driedover Na2SO4, and concentrated. Puriﬁcation by column chromatography on silica gel (eluent: n-hexane/AcOEt = 10/1 to 1/1, v/v) gave
14 (994 mg, 2.84 mmol) as a colorless solid in 88% yield. Colorless thinneedles (n-hexane); mp 85.0–86.0 °C; 1H NMR (400 MHz, CDCl3) 1.38
(s, 3H), 1.43 (s, 3H), 1.00–3.50 (brm, 10H), 2.75 (brs, 1H), 3.83–3.88(m, 2H), 3.96–4.00 (m, 2H), 4.39–4.45 (m, 1H), 6.69 (d, J = 8.8 Hz,
2H), 7.11 (d, J = 9.2 Hz, 2H); 13C{1H} NMR (100 MHz CDCl3) 25.2,
26.6, 59.1, 66.6, 68.6, 73.6, 86.1, 109.5, 113.7, 128.0, 129.6, 158.4;
MS (EI) m/z 350 (M+), 101 (100%); HRMS Calcd for C14H26B10O3:
350.2886; Found: 350.2894; Anal. Calcd for C14H26B10O3: C, 47.98, H,
7.48; Found: C, 48.05; H, 7.53.

4.4. Cell growth inhibitory assay
4.4.1. SC-3 cell proliferation Assay14
SC-3 cells were cultivated in MEMα (Wako Co.) supplemented with
2% FBS, 100 IU/mL penicillin, 100 mg/mL streptomycin, and 1 nM
testosterone at 37 °C in a 5% CO2 humidiﬁed incubator. Cells weretrypsinized from the maintenance dish with trypsin-EDTA and seeded ina 96-well plate at a density of 2000 cells per well (ﬁnal volume: 100 μL)
with MEMα supplemented with 2% charcoal-stripped FBS. After 1 day,
10 μL of the medium was removed from each well and 10 μL of solution
containing serial dilutions of the test compounds or DMSO (dilutecontrol) in the presence or absence of 1 nM testosterone were added tomicrocultures in triplicate. Cells were incubated at 37 °C under 5% CO2
for 3 days and the proliferation was evaluated by using 10 μL WST-8(DOJINDO), which was added to each well of microcultures before thecells were incubated for 4 h. Subsequently, the absorbance at 450 nm
was measured with a model 680 microplate reader (BIO-RAD). Thisparameter is related to the number of living cells in the culture. The
concentration required for 50% cell growth inhibition (GI50) was calculated using the Prism 4 software.

4.2.9. 1-Hydroxy-12-{4-(2,3-Dihydroxypropyloxy)phenyl}-1,12-dicarbacloso-dodecaborane (6b)A 2.64 M solution of n-BuLi in hexane (1.2 mL, 3.33 mmol) was
added dropwise to a solution of 14 (1.0 g, 2.86 mmol) in dry ether(20 mL) at 0 °C under Ar atmosphere. The mixture was cooled to−30 °C, before trimethyl borate (3.6 mL, 3.33 mmol) was added in one
portion. The reaction mixture was warmed to 0 °C over 1 h. A mixture of
10 mL of 30% H2O2 and 7 mL AcOH was added to the reaction mixture,
before the reaction was continued at room temperature for 11 h.
Subsequently, saturated aq. NaHSO3 (28 mL) and 10% aq. NaOH
(48 mL) were added to the reaction mixture, before the solution was
stirred at room temperature for 1 h. The reaction mixture was extractedwith ether and the organic layer was washed with water and brine,
dried over MgSO4, and concentrated. Puriﬁcation by column chromatography on silica gel (eluent: n-hexane/AcOEt = 10/1 to 1/1, v/v)
gave 6b (0.65 mg, 1.98 mmol) as a colorless solid in 69% yield;
Colorless sheets (ether/n-hexane); mp 200.0–201.5 °C; 1H NMR(400 MHz, CDCl3) 1.0–3.5 (brm, 10H), 3.58–3.63 (m, 2H), 3.87–3.92(m, 2H), 3.96–4.00 (m, 1H), 6.75 (d, J = 8.8 Hz, 2H), 7.11 (d,J = 8.8 Hz, 2H); 13C{1H} NMR (100 MHz, CDCl3) 59.3, 63.5, 69.1,
70.2, 86.1, 113.8, 128.3, 130.0, 158.4; MS (EI) m/z 326 (M+), 252(100%); HRMS Calcd for C11H22B10O4: 326.2522; Found; 326.2525;
Anal. Calcd for C11H22B10O4: C, 40.48; H, 6.79; Found: C, 40.53; H,
6.75

4.4.2. LNCaP cell proliferation Assay14
LNCaP cells were cultivated in RPMI-1640 (Wako Co.) supplemented with 10% FBS at 37 °C under 5% CO2 humidiﬁed incubator.
Cells were trypsinized from the maintenance dish with trypsin-EDTA
and seeded in a 96-well plate at a density of 3000 cells per well (ﬁnalvolume: 100 μL) with RPMI-1640 supplemented with 10% charcoalstripped FBS. After 1 day, 10 μL of the medium was removed from eachwell and 10 μL of solution containing serial dilutions of the test compounds or DMSO (dilute control) in the presence or absence of 1 nM
testosterone were added to microcultures in triplicate. Cells were3810

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A. Kaise et al.

incubated at 37 °C under 5% CO2 for 6 days. During this period, 50 μL of
the medium was replaced from each well to the corresponding medium
once after 3 days. The proliferation was evaluated by using 10 μL WST-8(DOJINDO), which was added to each well of microcultures before thecells were incubated for 4 h. Subsequently, the absorbance at 450 nm
was measured with a model 680 microplate reader (BIO-RAD). Thisparameter is related to the number of living cells in the culture. The
concentration required for 50% cell growth inhibition (GI50) was calculated using the Prism 4 software.

A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmc.2018.06.007.
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22. Ohta K, Goto T, Fujii S, et al. Bioorg Med Chem. 2011;19:3540.

4.4.3. PC-3 cell proliferation Assay21
PC-3 cells were cultivated in RPMI-1640 (Wako Co.) supplemented
with 10% FBS at 37 °C under 5% CO2 humidiﬁed incubator. Cells weretrypsinized from the maintenance dish with trypsin-EDTA and seeded ina 96-well plate at a density of 3000 cells per well (ﬁnal volume:
100 μL); for the cell culture, the same media were used. After 1 day, themedium was removed from each well and 100 μL of solution containing
serial dilutions of the test compounds or DMSO (dilute control) wereadded to microcultures in triplicate. Cells were incubated at 37 °C under
5% CO2 for 3 days and the proliferation was evaluated by using 10 μL
WST-8 (DOJINDO), which was added to each well of microcultures
before the cells were incubated for 4 h. Subsequently, the absorbance at
450 nm was measured with a model 680 microplate reader (BIO-RAD).
This parameter is related to the number of living cells in the culture.
The concentration required for 50% cell growth inhibition (GI50) was
calculated using the Prism 4 software.
4.5. Computational study
Three-dimensional (3D) structures of protein-ligand complexes werepredicted by GOLD 5.2 software with default settings.20 The 3D structure of human AR used in this study was retrieved from the Protein
DataBank (PDB) (PDB ID: 2OZ7). The active site of hAR LBD was deﬁned as the collection of amino acids for which at least one atom wascloser than 6.5 Å to any atom of the bound cyproteron acetate. The
structural optimizations of ligands were carried out by B3LYP/631G(d,p) using Gaussian 09 (Gaussian, Inc., Wallingford, CT). Default
values were used for other parameters, and the treatment of the ligand
molecule was the same as in a previous study.22
Acknowledgments
This research was supported by a grant-in-aid from the Strategic
Research Program for Private Universities (2015–2019) and a grant-inaid for Scientiﬁc Research (C) (26460151 and 15K08029) from theJapanese Ministry of Education, Culture, Sports, Science, andTechnology (MEXT).

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Contents lists available at ScienceDirect

Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharmMemantine ameliorates depressive-like behaviors by regulating
hippocampal cell proliferation and neuroprotection in olfactorybulbectomized mice
Kohei Takahashi a, Osamu Nakagawasai a, *, Wataru Nemoto a, Shogo Kadota a,
Jinichi Isono a, Takayo Odaira a, Wakana Sakuma a, Yuichiro Arai b, Takeshi Tadano c,
Koichi Tan-No aa

Department of Pharmacology, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai
981-8558, Japan
Course of Judo-therapy, Faculty of Health Science, Tokyo Ariake University of Medical and Health Science, 2-9-1 Ariake, Koto-Ku, Tokyo 135-0063, JapancComplementary and Alternative Medicine Clinical Research and Development, Graduate School of Medical Sciences, Kanazawa University, Kanazawa 9208640, Japanba r t i c l e i n f o

a b s t r a c t

Article history:
Received 31 October 2017
Received in revised form
5 April 2018
Accepted 10 April 2018
Available online 4 May 2018

Our previous study suggested that the non-competitive N-methyl-D-aspartate receptor antagonist
memantine (MEM) inhibits dopamine (DA) reuptake and turnover by inhibiting brain monoamine oxidase. Clinical studies have reported that MEM may improve depressive symptoms; however, speciﬁcmechanisms underlying this effect are unclear. We performed emotional behavior, tail suspension, andforced swimming tests to examine whether MEM has antidepressant effects in olfactory bulbectomized(OBX) mice, an animal model of depression. Subsequently, we investigated the effects of MEM on the
distribution of tyrosine hydroxylase (TH), altered microglia morphometry, and astrocyte and cell proliferation in the hippocampus with immunohistochemistry. We also investigated MEM effects on thelevels of norepinephrine (NE), DA, and their metabolites with high performance liquid chromatography,
and of neurotrophic, proinﬂammatory, and apoptotic molecules in the hippocampus with western
blotting. Forty-two days after surgery, OBX mice showed depressive-like behaviors, as well as decreased
levels of monoamines, reduced cell proliferation, and lower levels of TH, phospho(p)-TH (ser31 andser40), p-protein kinase A (PKA), p-DARPP-32, p-ERK1/2, p-CREB, brain-derived neurotrophic factor(BDNF), doublecortin, NeuN, and Bcl-2 levels. In contrast, the number of activated microglia and astrocytes and the levels of Iba1, GFAP, p-IkB-a, p-NF-kB p65, TNF-a, IL-6, Bax, and cleaved caspase-3 wereincreased in the hippocampus. These changes (except for those in NE and Bax) were reversed withchronic administration of MEM. These results suggest that MEM-induced antidepressant effects areassociated with enhanced hippocampal cell proliferation and neuroprotection via the PKA-ERK-CREBBDNF/Bcl-2-caspase-3 pathway and increased DA levels.
© 2018 Elsevier Ltd. All rights reserved.

Keywords:
Apoptosis
Cell proliferation
Depression
Emotional behavior
Olfactory bulbectomy
Neuroinﬂammation

Abbreviations: ANOVA, analysis of variance; Bax, Bcl-2-associated protein X; Bcl-2, B cell lymphoma/leukemia gene 2; BDNF, brain-derived neurotrophic factor; BrdU, 5bromo-20 -deoxyuridine; CREB, cAMP-responsive element binding protein; DA, dopamine; DARPP-32, dopamine- and cAMP-regulated phosphoprotein-32; DOPAC, dihydroxyphenylacetic acid; DG, dentate gyrus; ERK, extracellular signal-regulated protein kinase; GFAP, glial ﬁbrillary acidic protein; HVA, homovanillic acid; Iba1, ionized
calcium binding adaptor molecule 1; IL-6, interleukin-6; i.p., intraperitoneally; IkB-a, NF-kB inhibitor-a; MAO, monoamine oxidase; MEM, memantine; MHA, 3-methoxy-4hydroxymandelic acid; NMDA, N-methyl-D-aspartate; NE, norepinephrine; NeuN, neuronal nuclear antigen; NF-kB, nuclear factor-kappa B; OBX, olfactory bulbectomized;
p.o., per os; PBS, phosphate-buffered saline; PFC, prefrontal cortex; PKA, protein kinase A; PLSD, Protected Least Signiﬁcant Difference; ROI, region of interest; SEM, standard
error of the mean; TH, tyrosine hydroxylase; TNF-a, tumor necrosis factor-a.
* Corresponding author. Department of Pharmacology, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku,
Sendai, Miyagi 981-8558, Japan.
E-mail address: osamun@tohoku-mpu.ac.jp (O. Nakagawasai).
https://doi.org/10.1016/j.neuropharm.2018.04.013
0028-3908/© 2018 Elsevier Ltd. All rights reserved.

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1. Introduction

mechanisms underlying this from the perspective of cell proliferation and neuroprotection.

Olfactory bulbectomized (OBX) mice are a useful experimental
animal model for depression, as reported by us and several otherresearchers. OBX mice express abnormal behaviors, including
cognitive deﬁcits and depressive-like and hyperemotional behaviors (Kelly et al., 1997; Hozumi et al., 2003; Takahashi et al., 2011).
These abnormal behaviors improve with chronic, but not acute,administration of antidepressant drugs (Breuer et al., 2009a;
2009b). Furthermore, physiological and neurochemical changes in
OBX models, such as the reduction of hippocampal monoamines,
cell proliferation, and brain-derived neurotrophic factor (BDNF)
levels, were similar to those of clinical depression (Nakagawasai
et al., 2003a, 2003b; 2016; Takahashi et al., 2016, 2017; Thakare
et al., 2017). BDNF is known to play an important role in neuronalsurvival and is believed to regulate neuroplasticity, including
neurogenesis (Waterhouse et al., 2012). Decreased adult hippocampal neurogenesis is associated with depression in both rodents
and humans (Scorza et al., 2005; Duman and Monteggia, 2006;
�n et al., 2007). Moreover, it has been reported that the effects
Castreof chronic antidepressant drug administration may be exerted
through the enhancement of hippocampal neurogenesis (Santarelli
et al., 2003). Thus, in some cases, enhanced neurogenesis in the
hippocampus via BDNF signaling pathway activation may mediate
the effects of antidepressants.
In contrast, OBX induces neuroinﬂammation in the hippocampus via activation of microglia, and this inﬂammation may be
associated with depressive-like behavior (Rinwa and Kumar,2013). The available evidence indicates that depression is closely
associated with altered inﬂammation and microglia activation(Yirmiya et al., 2015), which manifest due to increased inﬂammatory cytokine levels (Milior et al., 2016). Microglia function isaffected by signaling systems associated with depression, namely
BDNF (Gomes et al., 2013), glucocorticoids (Ros-Bernal et al., 2011),€rber et al., 2005). These
norepinephrine (NE), or dopamine (DA) (Fareports suggest that microglia-induced inﬂammation is an
important factor in depression. Furthermore, it has been reported
that OBX-induced depressive-like behavior is associated withincreased inﬂammatory cytokine and apoptotic factor levels(Rinwa et al., 2013). Antidepressants regulate several apoptotic
factors, such as B cell lymphoma/leukemia gene 2 (Bcl-2)
expression, which are involved in cell survival pathways (Engelet al., 2013). Thus, neurogenesis and apoptosis may constituteimportant drug targets for the modulation of depressive symptoms (Lucassen et al., 2006).
Recent clinical and preclinical studies have demonstrated that asubanesthetic single-dose of the N-methyl-D-aspartate (NMDA)
receptor antagonist ketamine produces a rapid and sustained antidepressant effect in 70% of treatment-resistant patients with bipolar and major depressive disorder (Berman et al., 2012; Zarate
et al., 2006; Diazgranados et al., 2010). Furthermore, our previous
study suggested that a non-competitive NMDA receptor antagonist
memantine (MEM) inhibits the reuptake and turnover of DA byinhibiting brain monoamine oxidase (MAO) (Onogi et al., 2009). In
addition to the already described afﬁnities for the NMDA and D2
receptors, MEM binds with relatively high afﬁnity to the a7 nicotinic and 5-HT3 receptors (Berman et al., 2012; Kishi and Iwata,
2013; Parsons et al., 2007; Rammes et al., 2001) and possibly interacts with D1 and 5-HT2A receptors (Ishida et al., 2010; Nakaya
et al., 2011). Clinical studies have reported that MEM may
improve depressive symptoms, including emotional behavior(Matsunaga et al., 2015; Omranifard et al., 2017); however, thespeciﬁc detailed mechanisms underlying this effect are unclear.
Therefore, we examined whether MEM improves OBX-induced
depressive-like behaviors and investigated the molecular

2. Materials and methods
All experiments were performed following the approval of the
Ethics Committee of Animal Experiments at Tohoku Medical andPharmaceutical University, and according to the National Institutesof Health Guide for the Care and Use of Laboratory Animals. Efforts
were made to minimize suffering and to reduce the number ofanimals used. The scores on the behavioral tests were determinedmanually and measurements were conducted by a blinded
observer.
2.1. Animals
We used male ddY strain mice (weighing 28e32 g; Japan SLC,
Shizuoka, Japan) for all experiments (total: n ¼ 330; behavioral
tests: n ¼ 250; HPLC: n ¼ 16; immunohistochemistry: n ¼ 34;
western blot analysis: n ¼ 30). Mice were housed in groups in eachcage with free access to food and water under conditions of constant temperature (22 ± 2 � C) and humidity (55 ± 5%), on a 12-h
light-dark cycle (lights on: 07:00e19:00).
2.2. Olfactory bulbectomized mice
OBX surgery was performed as previously described(Nakagawasai et al., 2003a). All mice were sacriﬁced at the end of
the experiment and visually examined to conﬁrm that two thirds ofthe olfactory bulb (OB) had been lesioned. We excluded data frommice in which the lesion was either not extensive enough or
extended to the cortex. Sham operations were performed with thesame procedure but without the removal of the OB.
2.3. Drugs
MEM (10 and 20 mg/kg; Sigma-Aldrich, St-Louis, USA) was
dissolved in physiological saline and chronically intraperitoneally(i.p.) administered once daily between days 14e42 after surgery.
Acute treatment of MEM was performed on the 42nd day after
surgery.
2.4. Emotional assessments
The evaluation of hyperemotional behaviors in OBX rodents
provide a suitable model for evaluating antidepressant efﬁciency(Saitoh et al., 2003, 2007). At 14e42 days after surgery, emotional
behavior was measured with a modiﬁed procedure, which has beenpreviously described (Takahashi et al., 2011). All animals underwent emotional behavior observation 30 min after a single drug
injection on day 42, or 24 h after the last drug injection after 1, 3,
and 4 weeks of administration. Each animal was tested within
10 min.
The study was conducted according to the experimental protocols shown in Fig. 1 (A and B). The emotional behavior of mice
was measured by scoring their responses to the following stimuli:
attack response, scored by presenting a rod of 4e5 cm in front of
the snout; startle response, scored by blowing air on the dorsum
using a 5-mL syringe; struggle response, scored by handling with agloved hand; and ﬁght response, scored by pinching the tail withforceps. The attack response was graded as follows: 0, no reaction;
1, slight; 2, moderate; 3, marked; or 4, extreme reaction. Other
responses and vocalization during the test were scored and gradedas follows: 0, no reaction or vocalization; 2, moderate; 4, extreme.
Fewer gradations were used because it was difﬁcult to discriminate— 370 —

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143

Fig. 1. Effects of memantine (MEM) on depressive-like behaviors of olfactory bulbectomized (OBX) mice. A, B: Experimental time course for behavioral tests of experimental
protocol 1 (A) and of experimental protocol 2 (B). MEM effects were evaluated with an emotional behavior (C and F), tail-suspension (D and G), and forced swim (E and H) tests. The
emotional behavior test was conducted after an acute, 1-week, 3-week, and 4-week administration of MEM, while the tail suspension and forced swim tests were conducted only
after the acute or 4-week treatment. CeH: Quantiﬁcation of the total emotional score (C and F) in the emotional test and immobility time in the tail suspension (D and G) and forced
swim (E and H) tests. Bars represent means ± SEM. KruskaleWallis test: (C), acute administration, p < 0.01; (F), chronic administration, p < 0.01. Two-way ANOVA [(D), group: F(1,38) ¼ 11.88, p < 0.01; treatment: F(1, 38) ¼ 2.70, p > 0.05; group � treatment: F(1, 38) ¼ 0.071, p > 0.05; (E), group: F(1, 37) ¼ 13.29, p < 0.01; treatment: F(1, 37) ¼ 1.33, p > 0.05;
group � treatment: F(1, 37) ¼ 0.0048, p > 0.05; (G), group: F(1, 50) ¼ 0.90, p > 0.05; treatment: F(2, 50) ¼ 4.93, p < 0.05; group � treatment: F(2, 50) ¼ 3.06, p > 0.05; and (H),group: F(1, 52) ¼ 2.08, p > 0.05; treatment: F(2, 52) ¼ 17.79, p < 0.01; group � treatment: F(2, 52) ¼ 6.66, p < 0.01]. *p < 0.05 vs. saline-treated sham group, #p < 0.05 and##p < 0.01 vs. saline-treated OBX group (n ¼ 7e21 per group).

differences in vocalization. Finally, we evaluated the totalemotional score.
2.5. Tail suspension test
The tail suspension test was conducted to assess depressive-likebehavior and the effects of antidepressants. Mice were suspended
30 cm above the ﬂoor by means of adhesive tape placed approximately 1 cm from the tip of the tail, 30 min after the acute or 24 hafter the chronic administration. Mice were considered immobile
only when they were hanging completely and were passively
motionless. The duration of time spent immobile was quantiﬁed
during a test period of 10 min.
2.6. Forced swimming test
The forced swimming test was conducted according to a

modiﬁed method of Porsolt et al. (1978). As a pre-test, mice wereindividually placed in vertical glass cylinders (height, 16 cm;
diameter, 10 cm) that contained 8 cm of water, maintained at 25 � C,
for 15 min. After 24 h, the time during which mice remained
immobile was measured during a test period of 5 min. Mice wereconsidered immobile when they passively ﬂoated in the water andonly made movements necessary to keep their heads above thewater line. The forced swimming test was performed 30 min after
the acute or 24 h after the chronic drug administration on the 42nd
day after surgery.
2.7. Western blotting
Mice were divided into four groups (sham/vehicle, sham/MEM
20 mg/kg, OBX/vehicle, OBX/MEM 20 mg/kg). The mice weresacriﬁced by decapitation after 4 weeks of saline or MEM administration. Protein isolation and western blots were performed as— 371 —

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K. Takahashi et al. / Neuropharmacology 137 (2018) 141e155

described previously (Nakagawasai et al., 2016). After electrophoresis, proteins were transferred to a PVDF membrane, which wasthen incubated with blocking solution [10 mM TriseHCl (pH 7.4),
100 mM NaCl, 0.01% Tween 20, and 5% skim milk] for 1 h andprobed with antibodies against tyrosine hydroxylase (TH; 1:100;
Millipore Corporation, Billerica, USA), phosphorylated-TH (p-TH,
Ser31; 1:1000; Cell Signaling Technology, Danvers, USA), p-TH
(Ser40; 1:500; Millipore Corporation), p-protein kinase A (PKA;
1:1000; Cell Signaling Technology), p-DA- and cAMP-regulated
phosphoprotein-32 (DARPP-32; 1:1000; Cell Signaling Technology), extracellular signal-regulated protein kinase (ERK; 1:1000;
Cell Signaling Technology), p-ERK (1:1000; Cell Signaling Technology), cAMP-responsive element binding protein (CREB; 1:1000;
Cell Signaling Technology), p-CREB (1:1000; Cell Signaling Technology), BDNF (1:100; Abcam Ltd., Cambridge, UK), doublecortin(DCX; 1:100; Santa Cruz Biotech, Santa Cruz, CA), neuronal nuclear
antigen (NeuN; 1:1000; Millipore Corporation), ionized calcium
binding adaptor molecule 1 (Iba1; 1:500; Abcam Ltd.), glial ﬁbrillary acidic protein (GFAP; 1:200; Millipore Corporation), tumor
necrosis factor-a (TNF-a; 1:1000; Cell Signaling Technology),interleukin-6 (IL-6; 1:1000; Cell Signaling Technology), p-nuclear
factor-kappa B (NF-kB) p65 (1:500; Cell Signaling Technology), pNF-kB inhibitor-a (IkB-a; 1:1000; Cell Signaling Technology), Bcl-2(1:200; Santa Cruz Biotechnology), Bcl-2-associated protein X (Bax;
1:1000; Abcam Ltd.), cleaved caspase-3 (1:1000; Cell Signaling
Technology), and b-actin (1:1000; Cell Signaling Technology)
overnight at 4 � C. The membrane was then washed with blocking
solution without milk, incubated with horseradish peroxidaseconjugated secondary antibody (Cell Signaling Technology/Abcam
Ltd.) for 2 h, and the immunoreactive species were visualized with
ECL Western blotting detection reagent (Amersham Life Science,
Piscataway, USA). The densities of the bands were analyzed withdensitometry (Image-J 1.43 m, National Institute of Health).
2.8. High-performance liquid chromatography (HPLC)
Mice were divided into four groups (sham/saline, sham/MEM
20 mg/kg, OBX/saline, OBX/MEM 20 mg/kg). Sample isolation andmeasurement of monoamines were performed as described previously (Fukuda et al., 2011). The sample was homogenized in 0.1 M
perchloric acid containing 100 ng/mL of isoproterenol as an internal
standard. The homogenates were centrifuged at 10,000 � g for
10 min. Supernatants were ﬁltered through a 0.45-mm pore size
membrane ﬁlter. The ﬁltrate was used for quantiﬁcation of NE(Sigma-Aldrich), 3-methoxy-4-hydroxymandelic acid (MHA;
Sigma-Aldrich), DA (Sigma-Aldrich), dihydroxyphenylacetic acid
(DOPAC; Sigma-Aldrich), and homovanillic acid (HVA; SigmaAldrich) with HPLC coupled with electrochemical detection. In
the mobile phase, we used 95% of 50 mM sodium acetate, 10 mM
citric acid, 0.15 mM EDTA, 0.45 mM SOS, and 5% acetonitrile,adjusted to pH 3.6 with glacial acetic acid and ﬁltered to 0.45 mm.
The ﬂow rate was 0.4 mL/min. Electrochemical detection was
accomplished with an electrochemical detector (model EC8020,
Tosoh, Tokyo, Japan) with a glassy working electrode at a potentialof 1700 mV. The ratios of MHA/NE, DOPAC/DA, and HVA/DA wereused as an index of NE and DA turnover.

to �2.80 mm using a cryostat (MICROM HM560, Mikron Instrument, Inc., California, USA).
Frozen sections were mounted on glass slides (Matsunami Glass,
Osaka, Japan). Sections were treated with HCl (2 N) at 37 � C for
30 min, followed by neutralization with sodium borate buffer(0.15 M) at room temperature twice every 10 min. After threewashes every 5 min, the sections were incubated with phosphatebuffered saline (PBS) containing 1% normal goat serum (Life Technologies Corporation, Carlsbad, USA) and 0.3% Triton X-100 (PBSGT)at room temperature for 2 h. The sections were incubated overnightat 4 � C with rat anti-BrdU (1:100; Harlan SeraLab, Loughborough,UK) and mouse anti-NeuN (1:500; Millipore Corporation) monoclonal antibodies. Sections were washed and incubated for 2 h atroom temperature with goat anti-rat IgG Alexa Fluor 568 (1:200;
Molecular Probes, Eugene, USA) and goat anti-mouse IgG Alexa
Fluor 488 (1:200; Molecular Probes) in PBSGT. Finally, sections
were washed and coverslipped with Dako ﬂuorescent mounting
medium (Dako, Carpinteria, USA).
Immunoﬂuorescent images were analyzed with a confocal laserscanning microscope (A1Rsi: Nikon, Tokyo, Japan). Eight sections
per mouse were analyzed. We counted the number of BrdU positive
cells in both the right and left sides of the dentate gyrus (DG) images (640 � 640 mm) from each section. The total number of BrdU
positive cells was extrapolated for the entire volume of the DG.
2.10. Neuromorphometrical study
The brain samples were collected as described previously(Nakagawasai et al., 2016) and in paragraph 2.9. The sections wereincubated overnight at 4 � C with rabbit anti-TH (1:100; Millipore
Corporation), rabbit anti-Iba1 (1:200; Wako Pure Chemical Industries Ltd, Osaka, Japan), and mouse GFAP (1:200; Millipore
Corporation) antibodies. Sections were washed and incubated for
2 h at room temperature with goat anti-rabbit IgG Alexa Fluor 568(1:200; Molecular Probes) and goat anti-mouse IgG Alexa Fluor 488(1:200; Molecular Probes) in PBSGT. We observed alterations in THpositive cells in OBX mice with a confocal laser-scanning microscope. We then evaluated the activation of microglia and astrocytes
by observing and counting cells that typically exhibited hypertrophy with thicker processes, and larger and densely stained cellbodies.
2.11. Statistical analysis
Results are expressed as mean ± standard error of the mean
(SEM). The signiﬁcance of differences was determined by a twoway analysis of variance (ANOVA), followed by Fisher's Protected
Least Signiﬁcant Difference (PLSD) test for multigroup comparisons. Regarding the emotional behavioral test, the statistical signiﬁcance of differences was assessed with a non-parametric
KruskaleWallis test followed by Steel's test (non-parametric statistical analysis). We considered p < 0.05 for representing signiﬁcant differences.
3. Results
3.1. Effects of MEM on OBX-induced depressive-like behaviors

2.9. Immunohistochemistry
On day 42 after surgery, 5-bromo-20 -deoxyuridine (BrdU; SigmaeAldrich; 75 mg/kg) was injected i.p. three times every 2 h after
the last administration of saline or MEM. Animals were subsequently sacriﬁced 24 h after the last injection. Brain samples werecollected as described previously (Nakagawasai et al., 2016). The
brains were cut into 40 mm sections from bregma �2.20

We measured emotional behavior to clarify whether emotional
responses in OBX mice were affected by an acute, 1-week, 3-week,or 4-week treatment with MEM (20 mg/kg). There was a signiﬁcant
increase in the total emotional score in the OBX þ saline group on
day 42 after surgery compared with the sham þ saline group [Fig. 1(C-F)]. This was reversed after the 4-week chronic administration of
MEM (20 mg/kg) [Fig. 1 (F)], but not after the acute administration— 372 —

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145

Fig. 2. Effects of memantine (MEM) on the levels of total and activated tyrosine hydroxylase (TH). A: Microscopy images of TH (red) and DAPI (blue) immunostaining in the
hippocampus. Images show alterations in TH, p-THSer31, and p-THSer40 levels in the hippocampus after MEM administration. B: Representative immunoblots probed with antibodies
against TH, p-THSer31, p-THSer40, and b-actin. CeE: Quantiﬁcation of the normalized to b-actin levels of TH, p-THSer31, and p-THSer40 after saline or MEM treatment in sham-operated
or olfactory bulbectomized (OBX) mice. Bars represent means ± SEM. Two-way ANOVA [(C), group: F(1, 12) ¼ 11.72, p < 0.01; treatment: F(1, 12) ¼ 6.05, p < 0.05; group � treatment:
F(1, 12) ¼ 0.9, p > 0.05; (D), group: F(1, 12) ¼ 34.14, p < 0.01; treatment: F(1, 12) ¼ 19.28, p < 0.01; group � treatment: F(1, 12) ¼ 0.011, p > 0.05; and (E), group: F(1, 12) ¼ 6.83,
p < 0.05; treatment: F(1, 12) ¼ 25.23, p < 0.01; group � treatment: F(1, 12) ¼ 0.11, p > 0.01]. *p < 0.05 and **p < 0.01 vs. saline-treated sham group, #p < 0.05 and ##p < 0.01 vs.
saline-treated OBX group (n ¼ 3e5 per group).

[Fig. 1 (C)]. On days 21 (1 week of drug treatment) and 35 (3 weeks
of drug treatment) after surgery, we observed no differences between the OBX þ saline and OBX þ MEM groups (data not shown).
Furthermore, on day 42 after surgery, OBX control mice remained

immobile for a signiﬁcantly longer period than sham controls inboth the tail suspension and forced swimming tests. In contrast,
OBX mice, chronically treated with MEM (10 and 20 mg/kg),
remained immobile for signiﬁcantly shorter periods than the OBX— 373 —

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K. Takahashi et al. / Neuropharmacology 137 (2018) 141e155

controls in both tests [Fig. 1 (G and H)], whereas acute administration of MEM (20 mg/kg) did not have this effect [Fig. 1 (D and E)].
In contrast, there was no change in the duration of immobility ineither test between sham mice treated with MEM (10 and 20 mg/kg) and sham controls. These results suggest that chronic MEMtreatment improves OBX-induced depressive-like behaviors.
3.2. Changes in TH and monoamine levels in the hippocampus of
OBX mice
Our previous study demonstrated that OBX induces a decreasein striatal TH levels, a biosynthetic rate-controlling enzyme ofcatecholamines, such as NE and DA (Takahashi et al., 2016). Thakare
et al. (2017) demonstrated a decrease in monoamines in the hippocampus of OBX mice. As the pathophysiology of depression isassociated with low levels of monoamines in the central nervous
system, like NE and DA, (Carlsson et al., 1969; Schildkraut and Kety,1967), we examined whether OBX can decrease TH levels and activity in the hippocampus.
As shown in Fig. 2, the TH, p-THser31, and p-THser40 immunocontent was signiﬁcantly lower in OBX mice than in sham controls.
Compared with saline, MEM treatment signiﬁcantly increased TH
levels in the hippocampus of OBX mice, and signiﬁcantly increased
p-THser31 and p-THser40 levels in the hippocampus of both groups.

Fig. 3. Altered norepinephrine (NE), MHA (3-methoxy-4-hydroxymandelic acid), dopamine (DA), DOPAC (dihydroxyphenylacetic acid), and HVA (homovanillic acid) concentrations
and turnover in the hippocampus of olfactory bulbectomized (OBX) mice. AeE: Graphs showing the quantiﬁcation of the concentrations of NE (A), MHA (B), DA (C), DOPAC (D), andHVA (E) in the hippocampus of sham or OBX mice treated with saline or MEM. Concentrations are expressed as ng per g weight of fresh brain tissue. Bars represent means ± SEM.
Two-way ANOVA [(A), group: F(1, 12) ¼ 23.37, p < 0.01; treatment: F(1, 12) ¼ 1.56, p > 0.05; group � treatment: F(1, 12) ¼ 0.62, p > 0.05; (B), group: F(1, 12) ¼ 19.9, p < 0.01;
treatment: F(1, 12) ¼ 0.076, p > 0.05; group � treatment: F(1, 12) ¼ 1.16, p > 0.05; (C), group: F(1, 12) ¼ 28.47, p < 0.01; treatment: F(1, 12) ¼ 23.1, p < 0.01; group � treatment: F(1,12) ¼ 2.41, p > 0.05; (D), group: F(1, 12) ¼ 11.04, p < 0.01; treatment: F(1, 12) ¼ 3.8, p > 0.05; group � treatment: F(1, 12) ¼ 6.29, p < 0.05; and (E), group: F(1, 12) ¼ 14.04, p < 0.01;
treatment: F(1, 12) ¼ 0.12, p > 0.05; group � treatment: F(1, 12) ¼ 2.87, p > 0.05]. FeH: Graphs showing the MHA/NE (F), DOPAC/DA (G), and HVA/DA (H) ratios. Bars representmeans ± SEM. Two-way ANOVA [(F), group: F(1, 12) ¼ 6.28, p < 0.05; treatment: F(1, 12) ¼ 1.50, p > 0.05; group � treatment: F(1, 12) ¼ 0.34, p > 0.05; (G), group: F(1, 12) ¼ 0.08,
p > 0.05; treatment: F(1, 12) ¼ 7.92, p < 0.05; group � treatment: F(1, 12) ¼ 14.42, p < 0.01; and (H), group: F(1, 12) ¼ 0.43, p > 0.05; treatment: F(1, 12) ¼ 7.19, p < 0.05;
group � treatment: F(1, 12) ¼ 3.70, p > 0.05]. *p < 0.05 and **p < 0.01 vs. saline-treated sham group, #p < 0.05 and ##p < 0.01 vs. saline-treated OBX group (n ¼ 4 per group).

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From these results, we hypothesized that monoamines weredecreased in the hippocampus of OBX mice, and that this could bereversed by MEM.
Next, we measured the levels of NE, DA, and their metabolites in
the hippocampus of OBX mice [Fig. 3 (AeE)]. Sham mice treatedwith MEM had signiﬁcantly higher levels of DA and DOPAC than thesham control group. We observed signiﬁcantly lower NE, MHA, andDA concentrations in the OBX than the sham control group, while
OBX mice treated with MEM had signiﬁcantly higher DA levels.
Moreover, we observed that the DOPAC/DA ratio was signiﬁcantly
higher in OBX mice than in sham controls, while the ratios of
DOPAC/DA and HVA/DA were reduced after the administration of
MEM in OBX mice [Fig. 3 (G and H)]. These results suggest that theantidepressant effects of MEM may underlie, at least in part, theincrease in DA concentration in the hippocampus, via the inhibition
of DA turnover.
3.3. Changes in p-PKA, p-DARPP-32, p-ERK, p-CREB, and BDNF
levels in the hippocampus of OBX mice
In the present study, MEM led to increased DA levels in the
hippocampus of OBX mice. We examined whether the magnitudeof this increase is functionally meaningful. The stimulation of DA
receptors activates PKA, DARPP-32, and the ERK-CREB-BDNF
pathway (Bozzi et al., 2011). As shown in Fig. 4, the levels of pPKA, p-DARPP-32, p-ERK, p-CREB, and BDNF were signiﬁcantly
lower in OBX mice than in sham controls. Compared with saline,
MEM treatment signiﬁcantly increased p-PKA and p-DARPP-32

147

levels in the hippocampal DG of both the sham and OBX groups.
Similarly, MEM treatment increased p-ERK, p-CREB, and BDNF
levels in the hippocampus of OBX mice. These results suggest that
increased DA, induced by MEM, is functionally meaningful and that
MEM-induced antidepressant effects might underlie the increased
BDNF levels in the hippocampus, via the PKA-ERK-CREB pathway,
by increasing DA.
3.4. Effect of MEM on reduced cell proliferation in the hippocampal
DG of OBX mice
In the present study, MEM enhanced BDNF levels, which regulate forms of plasticity including neurogenesis in the hippocampus(Waterhouse et al., 2012). Therefore, animals were injected with
BrdU on the 42nd day after surgery to determine the rate of hippocampal cell proliferation. Anti-NeuN antibody was used to
identify mature neurons in the DG area. The incorporation of BrdU
into a cell indicates that it was cycling at the time of the BrdU injection. We found a signiﬁcantly lower number of BrdU positive
cells in OBX than in sham mice [Fig. 5 (A and B)]. However, thechronic administration of MEM (20 mg/kg) signiﬁcantly increased
the number of BrdU positive cells in OBX mice.
In order to examine whether the excess of newborn cells differentiates into immature and mature neurons in the hippocampus,
we investigated changes in DCX and NeuN, markers of immatureand mature neurons, respectively, after MEM treatment. As shownin Fig. 5 (CeE), the immunocontents of DCX and NeuN in OBX mice
were signiﬁcantly lower than in sham controls. Compared with

Fig. 4. Altered levels of the phosphorylated (p) forms of PKA, DARPP-32, ERK, and CREB, and of BDNF in the hippocampus after memantine (MEM) administration. A: Representative
immunoblots probed with antibodies against p-PKA, p-DARPP-32, p-ERK, t-ERK, p-CREB, t-CREB, BDNF, and b-actin, as indicated. BeF: Quantiﬁcation of the normalized values of pPKA (B), p-DARPP-32 (C), and BDNF (F) levels with b-actin and of p-ERK (D) and p-CREB (E) with total (t)-ERK and t-CREB, respectively. Bars represent means ± SEM. Two-way
ANOVA [(B), group: F(1, 9) ¼ 227.5, p < 0.01; treatment: F(1, 9) ¼ 137.3, p < 0.01; group � treatment: F(1, 9) ¼ 54.15, p < 0.01; (C), group: F(1, 12) ¼ 125.8, p < 0.01; treatment: F(1, 12) ¼ 96.87, p < 0.01; group � treatment: F(1, 12) ¼ 23.98, p < 0.01; (D), group: F(1, 14) ¼ 11.54, p < 0.01; treatment: F(1, 14) ¼ 10.51, p < 0.01; group � treatment: F(1,14) ¼ 0.20, p > 0.05; (E), group: F(1, 12) ¼ 11.72, p < 0.01; treatment: F(1, 12) ¼ 6.05, p < 0.05; group � treatment: F(1, 12) ¼ 0.90, p > 0.05; and (F), group: F(1, 15) ¼ 9.19, p < 0.01;
treatment: F(1, 15) ¼ 7.67, p < 0.05; group � treatment: F(1, 15) ¼ 0.73, p > 0.05]. *p < 0.05 and **p < 0.01 vs. saline-treated sham group, #p < 0.05 and ##p < 0.01 vs. saline-treated
OBX group (n ¼ 3e5 per group).

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Fig. 5. Inﬂuence of memantine (MEM) on hippocampal neurogenesis in olfactory bulbectomized (OBX) mice. A: Microscopy images showing BrdU (red) and NeuN (green) immunostaining in the dentate gyrus region of the hippocampus. Arrows indicate BrdU-labeled cells. B: Quantitative analysis of the number of BrdU positive cells in sham and OBX
mice treated with saline or MEM. Bars represent means ± SEM. Two-way ANOVA [(B), group: F(1, 16) ¼ 47.59, p < 0.01; treatment: F(1, 16) ¼ 13.15, p < 0.01; group � treatment: F(1,16) ¼ 0.16, p > 0.05]. C: Representative immunoblots probed with antibodies against DCX, NeuN, and b-actin, as indicated. Altered DCX and NeuN levels in the hippocampus wereobserved after MEM administration. D, E: Quantiﬁcation of normalized values of DCX and NeuN levels with b-actin. Bars represent means ± SEM. Two-way ANOVA [(D), group: F(1,18) ¼ 16.85, p < 0.01; treatment: F(1, 18) ¼ 17.5, p < 0.01; group � treatment: F(1, 18) ¼ 0.27, p > 0.05; and (E), group: F(1, 17) ¼ 3.84, p > 0.05; treatment: F(1, 17) ¼ 1.53, p > 0.05;
group � treatment: F(1, 17) ¼ 4.83, p < 0.05]. *p < 0.05 and **p < 0.01 vs. saline-treated sham group, #p < 0.05 vs. saline-treated OBX group (n ¼ 5e6 per group).

saline, MEM treatment signiﬁcantly decreased DCX and NeuN
levels in the hippocampus of OBX mice. In contrast, DCX but not
NeuN levels in the hippocampus were signiﬁcantly higher in shammice treated with MEM. We suggest that MEM-induced antidepressant effects in OBX mice may be associated with enhanced cellproliferation in the DG and the enhancement of BDNF levels.
3.5. Effect of MEM on the enhancement of inﬂammation in the
hippocampus of OBX mice
Acute and chronic neuroinﬂammation can negatively affectmany stages of neurogenesis in the adult mammalian brain,including proliferation, differentiation, and survival of newborn
neurons (Hashimoto, 2015; Miller et al., 2009). To examine whether
OBX induces the activation of microglia and astrocytes in the hippocampus, we performed immunostaining and western blotting byusing antibodies for Iba1 and GFAP [Fig. 6 (AeD)]. Compared withsham controls, the microglia marker Iba1 and the astrocyte marker
GFAP were signiﬁcantly increased in the hippocampus of OBX mice.
We observed that activated microglia and astrocytes typicallyexhibited hypertrophy, with thicker processes, larger and more
densely stained cell bodies, while the number of activated glial cells
was increased in the hippocampus of OBX mice. These phenomena
were also observed in the DG area of OBX mice. In contrast,

activation of microglia and astrocytes in the hippocampus of MEMtreated OBX mice was attenuated. As shown in Fig. 6 (C and D), Iba1
and GFAP immunocontent was signiﬁcantly higher in OBX micethan in sham controls. Compared with saline, MEM treatment
signiﬁcantly decreased Iba1 and GFAP levels in the hippocampus of
OBX mice. Moreover, MEM reversed the effects of OBX on glial cell
activation, including on cell body size, processes, cell count, and celldensity [Fig. 6 (I-P)]. These results suggest that OBX induces neuroinﬂammation in the hippocampus via activation of microglia andastrocytes, while MEM appears to have an anti-neuroinﬂammatory
effect.
3.6. Changes in p-IkB-a, p-NF-kB p65, TNF-a, and IL-6 levels in the
hippocampus of OBX miceMicroglia are important resident immunoreactive cells in thecentral nervous system. The available evidence indicates that
depression is intimately associated with altered inﬂammation andmicroglia (Yirmiya et al., 2015), manifesting with an increased
proinﬂammatory proﬁle, involving increased TNF-a and IL-6 levels(Milior et al., 2016). Thus, we examined whether OBX-induced
neuromorphometrical alterations of microglia and astrocytes arefunctionally meaningful.
As shown in Fig. 7, the immunocontent of p-IkB-a, p-NF-kB p65,

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TNF-a, and IL-6 was signiﬁcantly higher in OBX mice than in shamcontrols. Compared with saline, MEM treatment signiﬁcantly
decreased p-IkB-a, p-NF-kB p65, TNF-a, and IL-6 levels in the hippocampus of OBX mice. Moreover, OBX-induced neuroinﬂammation, leading to increased TNF-a and IL-6 levels via theactivation of p-IkB-a and p-NF-kB p65, was reversed by MEM. These
results suggest that OBX-induced neuromorphometrical alterationsof microglia and astrocytes are functionally meaningful.
3.7. Changes in Bcl-2, Bax, and cleaved caspase-3 levels in the
hippocampus of OBX mice
Modulation of apoptosis may play an important role in themodulation of depressive symptoms (Lucassen et al., 2006). Since
the balance between Bcl-2 and Bax is involved in the regulation of
apoptotic cell death (Cory and Adams, 2002) and cleaved caspase-3
is a crucial mediator of apoptosis (Fiandalo and Kyprianou, 2012),
we next investigated whether OBX inﬂuences Bcl-2, Bax, andcleaved caspase-3 expression.
As shown in Fig. 8 (A), the Bcl-2 immunocontent was signiﬁcantly lower in OBX than in sham mice. Compared with saline,
MEM treatment signiﬁcantly increased Bcl-2 levels in the hippocampus of OBX mice. In contrast, the Bax immunocontent wassigniﬁcantly higher in OBX mice treated with either saline or MEM
than in sham controls [Fig. 8 (B)]. Similarly, cleaved caspase-3 levels
were signiﬁcantly higher in OBX than in sham mice, and thischange was reversed by MEM. In contrast, cleaved caspase-3
immunocontent signiﬁcantly increased in sham mice treated with
MEM compared to sham controls [Fig. 8 (C)]. These results
demonstrate that neuronal apoptosis is induced by OBX and thattreatment with MEM signiﬁcantly reverses this effect.
4. Discussion
The present study showed that OBX mice exhibit depressive-likebehaviors in the tail suspension, forced swimming, and emotional
behavior tests, 42 days after surgery. Furthermore, chronic, but notacute, administration of MEM improved OBX-induced depressivelike behavior. We observed that MEM (10 or 20 mg/kg, i.p.) has
antidepressant effects when administered chronically for 4 weeks
after OBX surgery in mice. In contrast, Borre et al. reported that
MEM [20 mg/kg, per os (p.o.)] has antidepressant effects whenadministered before the OBX surgery in rats (Borre et al., 2012). We
propose that the administration method, species differences, orevaluation of speciﬁc behavioral aspects may be responsible forthese discrepancies. For example, drugs reach the brain more efﬁciently with i.p. than p.o. administration, suggesting that the concentration of MEM in the brain examined in our study may behigher than that examined in the study by Borre et al. Moreover, it
has been reported that OBX in different species occasionally exhibits different outcomes (Hendriksen et al., 2015). In the striatum,
an area of the dopaminergic system associated with motivation andanhedonia in depression patients, OBX mice exhibit decreased TH
levels (Takahashi et al., 2016). On the other hand, OBX rats exhibit
increased DA levels in this area (Masini et al., 2004). Moreover, OBX
mice but not OBX rats exhibit depressive-like behavior, including
longer immobility time during the forced swimming test (Hanet al., 2009; Kelly and Leonard, 1999). Regarding the behavioral
aspects, Borre et al. evaluated the effects of MEM on OBX-induced
hyperactivity, which may reﬂect psychomotor retardation oragitation. In contrast, our study evaluated MEM effects on OBXinduced hyperemotional response and immobility duration,
which may reﬂect mood, motivation, or remissness (Abelaira et al.,2013). These factors may be responsible for the different results.
Clinical and preclinical studies have reported that MEM

149

ameliorates depressive-like symptoms, including emotional
behavior (Matsunaga et al., 2015; Omranifard et al., 2017; Kos andPopik, 2005). Other studies have reported that the antidepressanteffects of MEM are associated with the 5-HT3 receptor and antagonism of NMDA receptors in the central nervous system (Rammes
us et al., 2010). However, we previously reported
et al., 2001; Rethat MEM inhibits DA reuptake and turnover by inhibiting brain
MAO activity (Onogi et al., 2009). Thus, in this study, we focused on
the role of the dopaminergic system in the antidepressant effects of
MEM.
Consistent with our results, previous studies have suggested
that NE and DA levels are decreased in the hippocampus of OBX
mice (Thakare et al., 2017). These changes might be, at least in part,
attributed to the decreased TH levels and activity in the hippocampus of these mice. The pathophysiology of depression involves
decreased 5-HT, NE, and DA levels in the central nervous system(Carlsson et al., 1969; Schildkraut and Kety, 1967). Clinical studies
have reported that monoamine replacement is an important factor
towards the effective treatment of patients with depression(Carroll, 1971). In the present study, MEM treatment signiﬁcantly
enhanced TH activity and increased DA concentration in the hippocampus of mice in both the sham and OBX groups. These effects
have also been demonstrated in studies using primary midbrainneuron-glial cultures, in which MEM was shown to increase THpositive cells and DA levels (Wu et al., 2009). Preclinical and clinical studies have reported that increased DA levels in the brainshow antidepressant effects (Hosenbocus and Chahal, 2013; Chenet al., 2016). We also observed a signiﬁcant increase in the
DOPAC/DA ratio of OBX mice compared with the one in shamcontrols. Previously, we demonstrated that OBX increases MAOB
activity, which leads to DA metabolism in the forebrain(Nakagawasai et al., 2003b). Thus, we suggest that the increased
DOPAC/DA ratio in OBX mice might be induced by activation of
MAOB. Moreover, DA turnover (DOPAC/DA and HVA/DA) was
inhibited in OBX mice treated with MEM. This might be explainedby our previous study in which we showed that MEM inhibits thereuptake of DA and MAOB in the forebrain (Onogi et al., 2009).
Taken together, these results suggest that DA is involved in themodulation of MEM-induced antidepressant effects.
Takamura et al. (2014) suggested that DA might play a role inadult hippocampal neurogenesis via DA receptors, and that activation of these receptors holds a therapeutic potential for depression. We observed that the low levels of p-PKA, p-DARPP-32, pERK1/2, p-CREB, and BDNF in the hippocampus of OBX mice werereversed after chronic treatment with MEM. Phosphorylation of
DARPP-32 plays an important role in mediating dopaminergictransmission (Walaas et al., 1983). Furthermore, the state of phosphorylation of this protein in dopaminergic neurons is regulated byboth DA and cAMP. Thus, we suggest that increased DA levels,
induced by MEM, stimulate DA receptors. Moreover, enhanced DA
activates the PKA and ERK1/2 pathways (Bozzi et al., 2011). In turn,these pathways lead to the activation of CREB, important for neuralplasticity, as well as to the transcription of BDNF (Finkbeiner et al.,1997) and TH genes (Gueorguiev et al., 2006). Results from several
studies have led to the formulation of the neurotrophic hypothesisof depression, based on which low levels of BDNF lead to a
n et al., 2007; Duman and Monteggia,
depressive condition (Castre2006). Neurotrophins are trophic proteins essential for neuronalsurvival and differentiation, while high hippocampal BDNF levels
attenuate depression symptoms (Chen et al., 2001). Moreover,
ERK1/2 pathway promotes the activation of TH by increasing its
phosphorylation on ser31 (Haycock et al., 1992), while PKA induces
TH phosphorylation on ser40 (Funakoshi et al., 1991). The present
study revealed that MEM signiﬁcantly increases p-PKA, p-ERK1/2,
p-THser31, and p-THser40 levels in both the sham and OBX groups (p-— 377 —

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151

Fig. 7. Altered levels of phosphorylated (p)-IkBa, p-NF-kB p65, TNF-a, and IL-6 in the hippocampus after memantine (MEM) administration. A: Representative immunoblots probedwith antibodies against p-IkBa, p-NF-kB p65, TNF-a, IL-6, and b-actin, as indicated. BeE: Quantiﬁcation of normalized values of p-IkBa, p-NF-kB p65, TNF-a, and IL-6 levels with bactin. Bars represent means ± SEM. Two-way ANOVA [(B), group: F(1, 15) ¼ 0.86, p > 0.05; treatment: F(1, 15) ¼ 4.36, p > 0.05; group Х treatment: F(1, 15) ¼ 4.73, p < 0.05; (C),group: F(1, 14) ¼ 6.11, p < 0.05; treatment: F(1, 14) ¼ 12.96, p < 0.01; group Х treatment: F(1, 14) ¼ 30.75, p < 0.01; (D), and group: F(1, 14) ¼ 2.01, p > 0.05; treatment: F(1, 14) ¼ 8.55,
p < 0.05; group Х treatment: F(1, 14) ¼ 5.85, p < 0.05; and (E), group: F(1, 15) ¼ 4.83, p < 0.05; treatment: F(1, 15) ¼ 5.35, p < 0.05; group Х treatment: F(1, 15) ¼ 2.73, p > 0.05].
*p < 0.05 and **p < 0.01 vs. saline-treated sham group, #p < 0.05 and ##p < 0.01 vs. saline-treated OBX group (n ¼ 3e5 per group).

ERK1/2 levels in the sham group were slightly increased (p ¼ 0.08)
by MEM compared to saline). Thus, our results suggest that MEMinduced antidepressant effects might underlie the increased BDNF
levels in the hippocampus and that MEM-induced enhancement of
TH levels and activity in the hippocampus might be associated with
PKA-ERK-CREB pathway via increasing DA.
It is well known that adult neurogenesis occurs mainly in two
regions of the brain, one of which is the subgranular zone of the DG
in the hippocampal formation (Alvarez-Buylla and Garcia-Verdugo,
2002; Cayre et al., 2002). In the present study, OBX mice showed asigniﬁcant decrease in cell proliferation in the DG, consistently with
our previous study (Nakagawasai et al., 2016; Takahashi et al.,2017). Moreover, DCX and NeuN levels in the hippocampus weresigniﬁcantly lower in OBX than in sham mice. Likewise, DeCarolisand Eisch (2010) reported a reduction in neurogenesis in the hippocampus of patients with depression. Moreover, Santarelli et al.
(2003) suggested that antidepressant effects may be mediated by
the enhancement of neurogenesis in the hippocampus. Theseﬁndings suggest that enhancement of neurogenesis in the hippocampus may ameliorate depressive-like behaviors in the OBX

model. We observed that chronic administration of MEM to OBX
mice signiﬁcantly increases the number of newborn cells in the
hippocampus. Thus, we suggest that MEM-induced antidepressanteffects might be mediated by the induction of neurogenesis in the
hippocampal DG of OBX mice.
Neuroinﬂammation can negatively affect many stages of neurogenesis in the adult mammalian brain, including proliferation,differentiation, and survival of newborn neurons (Hashimoto, 2015;
Miller et al., 2009). Current evidence points to a role of inﬂammatory processes in the pathophysiology of depression (Hashimoto,2009, 2015; Miller et al., 2009). Patients with depression show
increased blood concentrations of pro-inﬂammatory cytokines,
such as IL-1b, IL-6, and TNF-a (Howren et al., 2009; Kim et al.,2007). Moreover, antidepressant treatment attenuates the expression of inﬂammatory biomarkers in depression (Maes et al., 2009),
while depression is intimately associated with altered inﬂammation and microglia (Yirmiya et al., 2015). Indeed, results from manystudies converge to suggest that microglia are morphologicallyaltered in frontal limbic regions of patients with depression andsuicide completers (Steiner et al., 2008; Schnieder et al., 2014;

Fig. 6. Effects of memantine (MEM) on microglia and astrocyte activation in the hippocampus of olfactory bulbectomized (OBX) mice. A, B: Microscopy images of Iba1 (green), GFAP
(red), and DAPI (blue) immunostaining in the hippocampus (A) and dentate gyrus (B). Altered Iba1 and GFAP levels in the hippocampus were observed after MEM administration. C,
D: Representative immunoblots probed with antibodies against Iba1 (C), GFAP (D), and b-actin are shown. Graphs in panels C and D indicate the quantiﬁcation of the normalized
values of Iba1 and GFAP levels with b-actin. Bars represent means ± SEM. Two-way ANOVA [(C), group: F(1, 14) ¼ 0.016, p > 0.05; treatment: F(1, 14) ¼ 5.04, p < 0.05;
group � treatment: F(1, 14) ¼ 15.3, p < 0.01; (D), group: F(1, 13) ¼ 2.80, p > 0.05; treatment: F(1, 13) ¼ 4.24, p > 0.05; group � treatment: F(1, 13) ¼ 4.98, p < 0.05]. EeH: Microscopy
images showing normal and activated microglia (E and F) and astrocytes (G and H), as indicated. IeP: Quantiﬁcation of soma size (I and M), process length (J and N), Iba1 positive
cells (K), GFAP positive cells (O), and cell density in the region of interest (ROI), using representative tissue sections stained with Iba1 and GFAP antibodies. Bars representmeans ± SEM. Two-way ANOVA [(I), group: F(1, 9) ¼ 38.93, p < 0.01; F(1, 9) ¼ 17.47, p < 0.01; group � treatment: F(1, 9) ¼ 28.98, p < 0.01; (J), group: F(1, 9) ¼ 92.22, p < 0.01;
treatment: F(1, 9) ¼ 21.12, p < 0.01; group � treatment: F(1, 9) ¼ 16.97, p < 0.01; (K), group: F(1, 9) ¼ 55.9, p < 0.01; treatment: F(1, 9) ¼ 44.7, p < 0.01; group � treatment: F(1,9) ¼ 34.75, p < 0.01; (L), group: F(1, 9) ¼ 45.58, p < 0.01; treatment: F(1, 9) ¼ 19.57, p < 0.01; group � treatment: F(1, 9) ¼ 14.4, p < 0.01; (M), group: F(1, 9) ¼ 10.01, p < 0.01;
treatment: F(1, 9) ¼ 13.67, p < 0.01; group � treatment: F(1, 9) ¼ 14.66, p < 0.01; (N), group: F(1, 9) ¼ 11.93, p < 0.01; treatment: F(1, 9) ¼ 15.95, p < 0.01; group � treatment: F(1,9) ¼ 12.66, p < 0.01; (O), group: F(1, 9) ¼ 17.19, p < 0.01; treatment: F(1, 9) ¼ 23.79, p < 0.01; group � treatment: F(1, 9) ¼ 21.93, p < 0.01; and (P), group: F(1, 9) ¼ 20.4, p < 0.01;
treatment: F(1, 9) ¼ 18.34, p < 0.01; group � treatment: F(1, 9) ¼ 16.77, p < 0.01]. *p < 0.05 and **p < 0.01 vs. saline-treated sham group, #p < 0.05 and ##p < 0.01 vs. saline-treated
OBX group (n ¼ 3e5 per group).

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Fig. 8. Altered Bcl-2, Bax, and cleaved caspase-3 levels in the hippocampus after memantine (MEM) administration. A-C: Representative immunoblots probed with antibodies
against Bcl-2, Bax, cleaved caspase-3, and b-actin, as indicated. Graphs indicate the quantiﬁcation of the normalized values of Bcl-2, Bax, and cleaved caspase-3 levels with b-actin.
Bars represent means ± SEM. Two-way ANOVA [(A), group: F(1, 15) ¼ 29.13, p < 0.01; treatment: F(1, 15) ¼ 1.89, p > 0.05; group � treatment: F(1, 15) ¼ 13.83, p < 0.01; (B), group:
F(1, 15) ¼ 9.61, p < 0.01; treatment: F(1, 15) ¼ 0.048, p > 0.05; group � treatment: F(1, 15) ¼ 0.21, p > 0.05; and (C), group: F(1, 18) ¼ 3.08, p > 0.05; treatment: F(1, 18) ¼ 7.06,
p < 0.05; group � treatment: F(1, 18) ¼ 84.44, p < 0.01]. *p < 0.05 and **p < 0.01 vs. saline-treated sham group, ##p < 0.01 vs. saline-treated OBX group (n ¼ 4e6 per group).

Torres-Platas et al., 2014; Setiawan et al., 2015). Thus, the available
evidence indicates that dysfunction of microglia is a core event indepression. In the present study, we observed for the ﬁrst time that
OBX mice show signiﬁcantly increased TNF-a and IL-6 levels,
possibly via the activation of p-IkB-a and p-NF-kB p65 and reﬂectedby the increased immunoreactivity for Iba1 (a marker of microglia)
and GFAP (a marker of astrocytes) in the hippocampus. DominguezMeijide et al. (2017) demonstrated that DA inhibits activatedmicroglia in vitro. Moreover, the neuroprotective effects of MEM
observed in primary midbrain neuron-glia culture studies are
partly mediated via alternative mechanisms, which reduce
microglia-associated inﬂammation (Wu et al., 2009). We found thatmicroglia-induced inﬂammation, observed in the hippocampus of
OBX mice, was attenuated by MEM, suggesting that MEM exhibits
anti-inﬂammatory effects by inhibiting microglial activity in thesemice.
In human neutrophils, activation of NF-kB seems to control
spontaneous apoptosis and antiapoptotic effects. The modulation ofthis pathway most likely regulates the balance between pro- andanti-apoptotic factors, especially members of the Bcl-2 family,(Akgul et al., 2001), thus affecting neutrophil survival. The balance
between Bcl-2 (an anti-apoptotic protein) and Bax (a pro-apoptotic
protein) regulates apoptotic cell death. Moreover, cleaved caspase3 is crucial in the process of apoptosis, and its activation contributes
to the irreversible stage of apoptosis (Fiandalo and Kyprianou,2012). Our results showed that MEM reverses the decreased
levels of Bcl-2 in the hippocampus of OBX mice, but not theincreased levels of Bax. In contrast, MEM reverses the increased
levels of cleaved caspase-3 in the hippocampus of OBX mice.
Interestingly, sham mice treated with MEM also showed enhanced
cleaved caspase-3 levels in the hippocampus. These paradoxical
results may be associated with the increased DCX and unchanged
NeuN expression levels in the hippocampus of sham mice. Indeed, aprevious study reported that the disequilibrium between neuronal
proliferation and apoptosis may be involved in the pathogenesis ofneuropsychiatric disorders (Genius et al., 2012). We hypothesizethat the MEM-induced excess of immature neurons in the hippocampus of sham mice did not differentiate to mature neurons, butinstead underwent apoptosis. These results suggest that MEM
might partly modulate apoptosis by regulating caspase-3 and thebalance between Bcl-2 and Bax in the hippocampus of OBX mice.

Besides the hippocampus, the pathogenesis of depression affects various brain regions, including the prefrontal cortex, striatum, and amygdala (Treadway and Zald, 2011). In this study, weonly examined the antidepressant effects of MEM on the hippocampus. Moreover, the regulation of emotional behavior is thought
to rely more on ventral hippocampal neurogenesis (Tanti et al.,2012). Hence, further experiments examining the relationship between other brain areas involved in depressive-like behavior,including the ventral hippocampus, and the effects of MEM will bepresented in a subsequent paper.
When we investigated the time-dependency of MEM effects in
OBX mice by emotional behavioral testing, we observed that the
OBX-induced emotional behavior increase was reduced after 4
weeks of MEM administration. A previous study suggested that
antidepressants may exert their action by stimulating neurogenesis
in the hippocampus (Santarelli et al., 2003). Moreover, enhancement of neurogenesis was observed after chronic but not acuteadministration, consistently with the time course for the therapeutic action of antidepressants (Malberg et al., 2000). Chiu et al.
(2015) reported that the enhancement of neurogenesis and antidepressant effects of L-DOPA (L-3,4-dihydroxyphenylalanine), theprecursor of the neurotransmitter DA, or of pramipexole, a D2/D3
receptor agonist, were also observed after chronic, but not acuteadministration. These ﬁndings suggest that the dopaminergic
activity-related enhancement of neurogenesis, as well as the antidepressant effects, develop later (Chiu et al., 2015). Thus, we suggest that the delayed antidepressant effect of MEM might
contribute to the enhancement of neurogenesis via the enhancement of dopaminergic activity. Moreover, recent short-term clinicaland meta-analysis studies have reported that MEM as adjunct tosertraline, but not MEM alone, efﬁciently improves depressive
symptoms in patients with major depressive disorder (Amidfar
et al., 2017; Kishi et al., 2017). In contrast, a 12-week course of an
open-label, ﬂexible-dose study of MEM revealed its efﬁcacy in suchpatients (Ferguson and Shingleton, 2007). In the present study,
MEM exhibited an antidepressant effect 4 weeks after the ﬁrst
treatment in OBX mice. Previous studies have reported that OBXinduced hyperemotional behaviors improve by administration ofclassic antidepressants, such as desipramine, milnacipran, andﬂuvoxamine, for 7 days (Saitoh et al., 2003, 2007). Antidepressants
are the mainstay of pharmacological treatment for patients with— 380 —

K. Takahashi et al. / Neuropharmacology 137 (2018) 141e155

depressive disorders; however, evidence supporting their use indementia is unclear (Ford and Almeida, 2017). Thus, we consider
that MEM may be effective for depression and emotional disturbances in dementia but its effects might develop later than in thecase of classic antidepressants. Therefore, long-term studies on
MEM effects on depression are required. However, we suggest that
MEM can reduce polypharmacy, a frequent problem in the elderly,
which can lead to worsened cognition, increased risk of adverse
drug reactions, and falls (Beer et al., 2011).
In conclusion, our results indicate that MEM has antidepressanteffects in OBX mice. The mechanism of action may involve theenhancement of cell proliferation in the hippocampus, stimulated
by increased BDNF levels and mediated by the activation of the DA
receptor signaling pathway (PKA-ERK-CREB). This leads toincreased DA via the enhancement of TH activity and inhibition of
DA turnover following MEM treatment. In addition, we showedthat MEM exerts neuroprotective effects in the hippocampus, bydecreasing TNF-a and IL-6 levels and activating the IkB-a and NF-kB
p65 signaling pathways. This process is likely mediated by the
suppression of microglial activity, and the modulation of the balance between Bcl-2 and Bax, involved in the regulation of apoptotic
cell death via caspase-3 activity. Our results provide the basis forthe development of MEM strategies not only towards treating dementia, but also depression.
Conﬂicts of interest
We do not have any conﬂicts of interest in connection with thismanuscript.
Acknowledgments
This study was supported in part by JSPS KAKENHI (Grant
Number: JP26460102) and Matching Fund Subsidy for Private
Universities from the Ministry of Education, Culture, Sports, Scienceand Technology of Japan (Grant Number: S1511001L). The authors
would like to thank Ms. Yurika Yamamoto, Mr. Kazuya Sano, andMr. Kotaro Yabuki of Tohoku Medical and Pharmaceutical University for their technical assistance.
Appendix A. Supplementary data
Supplementary data related to this article can be found athttps://doi.org/10.1016/j.neuropharm.2018.04.013.
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Bioorganic & Medicinal Chemistry Letters 27 (2017) 4030–4033

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Lettersjournal homepage: www.elsevier.com/locate/bmcl

Design and synthesis of iodocarborane-containing ligands with highaffinity and selectivity toward ERb
Kiminori Ohta ⇑, Takumi Ogawa, Yasuyuki Endo
Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japana r t i c l e

i n f oArticle history:
Received 21 June 2017
Revised 19 July 2017
Accepted 20 July 2017
Available online 25 July 2017
Keywords:
Carborane
Estrogen receptor
ERb
Subtype selectivity

a b s t r a c t
The selectivity and the binding affinity of previously reported carborane-containing ligands 2 and 3
toward ERb remains to be optimized. To improve their biological profiles, a series of iodinated carboranyl
phenol derivatives (4–6) were designed and synthesized as prospective ERb-selective ligands with highaffinity. Several iodinated carboranyl phenols showed high relative binding affinity (RBA) values for bothERs, and especially for ERb, due to suitable hydrophobic interactions of the iodine atoms with thehydrophobic amino acid residues of the ERb ligand-binding domains. Among these derivatives, 9,10diiodo-m-carborane 5f exhibited a more than 100% increase of the RBA values toward ERb, a 14-foldincreased selectivity for ERb over ERa, and ER-agonistic activity in MCF-7 cell proliferation assays.
 2017 Elsevier Ltd. All rights reserved.

Estrogen receptor b (ERb)1 was initially cloned from a rat ventral prostate cDNA library as a second form of ERa,2 and has sinceshown quite different biological functions compared to ERa.3
Endogenous estrogen 17b-estradiol (E2, 1) modulates various
physiological processes, including the development and functionof the female reproductive system, as well as the maintenance ofbone mineral density.4 However, the therapeutic use of 1 is limited
by an increased risk of breast cancer, which has been linked to theactivation of ERa (Fig. 1).5 Nevertheless, the activation of ERb
induces an anti-proliferative effect on breast cancer.6 ERb-selective
agonists down-regulate the expression of the androgen receptor(AR),7 inhibit the proliferation and migration of prostate cancer celllines,8 and delay the progression of Alzheimer’s disease by including the degradation of Ab aggregates.9 Thus, ERb-selective ligands
are of interest as potential therapeutic agents for Alzheimer’s
disease and several other types of cancer, and as probes for ERbrelated molecular biology.10
The development of ER-subtype-selective ligands remains amajor challenge, as there are only two different amino acid residues in the hydrophobic pocket of the ER-ligand-binding domains(LBDs),11 i.e., Leu384 and Met421 in ERa are substituted in ERb by
Met336 and Ile373, respectively.11 Most ERb-selective ligands
obtain their subtype selectivity through electronic, hydrophobic,
or steric interactions with these key amino acid residues.12
Based on the above theory, we have recently developed carborane-containing ERb-selective ligands 213 and 3,14 which showed⇑ Corresponding author.

E-mail address: k-ohta@tohoku-mpu.ac.jp (K. Ohta).

high RBA values and high selectivity toward ERb, respectively(Fig. 1). Although 3 showed much better ERb selectivity than 2, itis still not sufficient for the development of drug candidates or biological tools on account of the low binding affinity toward ERb. An
SAR study on the ERb selectivity of 2 revealed that the iodine atomon the carbon atom of the m-carborane cage in 2 increases thebinding affinity toward ERb. Thus, we designed novel iodinatedcarborane-containing ERb-selective ligands with high affinity
toward ERb, using 2 as a lead compound. In this paper, we describe
the syntheses of several iodinated carboranyl phenols (4–6) andthe evaluation of their ER-binding affinity and selectivity.
The synthesis of iodinated-o-carborane 4 is summarized in
Scheme 1. 3-Iodo-o-carborane 7, which was prepared from the o15
carborane-reconstructing reaction of nido-7,8-C2B9H
12 with BI3,
was treated with 4-methoxyphenylmagnesium bromide in thepresence of CuI under Pd-catalyzed coupling conditions to afford
3-(4-methoxyphenyl)-o-carborane 8 in 98% yield.16 Two iodine
atoms were introduced on two carbon atoms of the o-carborane
cage of 8 by consecutive exposure to n-BuLi and I2, which afforded
9 in 91% yield. Subsequently, 9 was treated with BBr3 to furnish
1,2-diiodo-o-carboranylphenol 4 in 96% yield.
Iodinated m-carboranes 5a–5e were synthesized from 11(Scheme 2), which was obtained in 28% yield from the iodinationof m-carborane 10 with ICl in the presence of AlCl3,17 followed
by a transformation into a copper salt and an Ullman coupling.18
In this coupling reaction, 7,9-diiodo derivative 12 was also
obtained unexpectedly in 23% yield, which suggests that the copper salt of 11 reacts with the iodine liberated during the reaction.
The iodinated m-carboranes 11 and 12 were treated with BBr3 to

http://dx.doi.org/10.1016/j.bmcl.2017.07.053
0960-894X/ 2017 Elsevier Ltd. All rights reserved.

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K. Ohta et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 4030–4033

Fig. 1. Structures of 17b-estradiol and carborane-containing ERb-selective ligands.

Scheme 1. Synthesis of iodinated o-carborane 4. Reagents and conditions: (a)4-methoxyphenylmagnesium bromide, PdCl2(PPh3)2, CuI, THF, reflux; (b) (i) n-BuLi,
Et2O, 0 C; (ii) I2, rt; (c) BBr3, CH2Cl2, 0 C.

Scheme 2. Synthesis of iodinated m-carborane derivatives 5a–f. Reagents andconditions: (a) (i) n-BuLi, DME, 0 C; (ii) CuCl, rt; (iii) 4-iodoanisole, pyridine, 100 C;(b) (i) n-BuLi, Et2O, 0 C; (ii) R-X, rt; (c) BBr3, CH2Cl2, 0 C.

4031

afford 9-iodo-m-carboranylphenol 5a and 7,9-diiodo-m-carboranylphenol 5b in 90% and quantitative yield, respectively. Allyl,
n-butyl, and benzyl groups were introduced into the C–H moietyof the m-carborane cage of 11 using the corresponding halide
reagents in 68–83% yield, which were treated with BBr3 to afford
the corresponding 9-iodophenol derivatives 5c, 5d, and 5e in
68%, 75%, and 93% yield, respectively. Scheme 2 also includes thesynthesis of 9,10-diiodo-m-carboranylphenol 5f. 9,10-Diiodo-mcarborane 13,19 prepared from the iodination of the corresponding
m-carborane, was transformed into 14 (65%) under Cu-mediatedcoupling conditions, followed by a deprotection of the methylgroup with BBr3 to afford 5f in 61% yield.
Scheme 3 summarizes the synthesis of iodinated p-carboranyl
phenols 6a–6c. Compound 16, which was prepared by the Cumediated coupling reaction of p-carborane 15 with 4-iodoanisole,
was consecutively treated with n-BuLi and I2, before a demethylation with BBr3 afforded 12-iodo-p-carboranylphenol 6a in 33%
overall yield. A Pd-catalyzed B-arylation of 2-iodo-p-carborane
18, which was obtained from the iodination of p-carborane with
4-methoxymagnesium bromide, afforded 19 in 90% yield.16 Compound 19 was subsequently treated with 2 equivalents of n-BuLi,
quenched with I2, and demethylated to afford 6b in 73% overall
yield. One of the less hindered carbon atoms of the p-carborane
cage of 19 was protected with a triphenylsilyl group,20 while
another carbon atom was iodinated to afford 22 in 20% overall
yield. The two protecting groups, i.e., the triphenylsilyl and methylgroups, were separately removed with TBAF and BBr3, respectively,
to afford 6c in 90% overall yield.
The relative binding affinity (RBA) values toward ERa, ERb, andthe ER-subtype selectivity of the compounds synthesized in thisstudy are summarized in Table 1.21 Interestingly, several derivatives showed RBA values of more than 100% toward ERb. Althougho-carborane 4 showed a high ERb-binding affinity, the ERb selectivity was moderate due to the potent binding affinity toward ERa.
We found that iodinated m-carborane 5a is an ERb-selective ligand
with high binding affinity toward ERb. The introduction of an
iodine atom onto the carbon atom of the m-carborane cage
reduced the RBA values toward ERa and ERb, but the ERb selectivity of 5b was almost similar to that of 5a. On the other hand, theintroduction of an alkyl substituent, such as an allyl (5c), n-butyl(5d), or benzyl (5e) group, enhanced the ERa-binding affinity
under concomitant decrease of the ERb selectivity. Benzyl derivative 5e exhibited a remarkable decrease of the binding affinity

Scheme 3. Synthesis of iodinated p-carboranes 6a–c. Reagents and conditions: (a) (i) n-BuLi, DME, 0 C; (ii) CuCl, rt; (iii) 4-iodoanisole, pyridine, 100 C; (b) (i) n-BuLi, Et2O,
0 C; (ii) I2, rt; (c) BBr3, CH2Cl2, 0 C; (d) 4-methoxyphenylmagnesium bromide, PdCl2(PPh3)2, CuI, THF, reflux; (e) (i) n-BuLi, Et2O, 0 C; (ii) Ph3SiCl, rt; (f) TBAF, THF, rt.

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K. Ohta et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 4030–4033

Table 1
RBA values toward ERa and ERb, and ER-subtype selectivity of the iodinated carboranyl phenols 7–9.aCompoundCarborane

R

RBAb
ERa

ERb

ERa/ERb

ERb/ERa

4
5a
5b
5c
5d
5e
5f
6a
6b
6c

o-Carborane
m-Carborane

–
H
I
Allyl
n-Butyl
Benzyl
–
–
I
H

42
13
8.4
17
29
28
8.0
574
18
10

242
129
81
62
157
19
111
888
107
38

0.17
0.10
0.10
0.27
0.18
1.50
0.07
0.65
0.17
0.26

5.8
9.9
9.6
3.6
5.4
0.68
14
1.5
5.9
3.8

p-Carborane

Selectivityca

In all binding assays, which were performed in triplicate (n = 3), the test compounds (0.4 nM–4 lM) were examined in the presence of [2,4,6,7-3H]17b-estradiol (4 nM).
RBA values were calculated from the IC50 values of E2 and the test compounds, whereby that of E2 was set to 100%.
c
The ER-subtype selectivity was estimated from the RBA values toward ERa and ERb.

busing the MCF-7 cell line, which shows estrogen-dependent
growth (Fig. 2).21 5f promoted the MCF-7 cell proliferation in aconcentration-dependent manner and acted as an ER agonist.
In conclusion, we designed and synthesized various iodinatedcarboranyl phenols containing o-, m-, and p-carborane cages asprospective ligands with high selectivity and affinity toward ERb.
A competitive binding assay revealed that the introduction ofiodine atoms enhanced their ERb selectivity, which might becaused by a suitable hydrophobic interaction of the iodine atomwith the amino acid residues of ERb LBD. 5f showed the highest
ERb selectivity, potent ERb binding affinity, and ER agonistic activity in an MCF-7 cell proliferation assay.
Acknowledgments

Fig. 2. Concentration-dependent MCF-7 cell proliferation induced by 5f. MCF-7
cells were incubated with 5f (100 nM–1 nM) for 5 days; the results are shown as
the fold cell number, whereby the value for 0.1 nM E2 was set to 100%; assays were
performed in triplicate (n = 3).

toward ERb, and the RBA value of 5e toward ERa surpassed that ofERb. 9,10-Diiodo-m-carborane 5f showed a 14-fold higher selectivity toward ERb than toward ERa with an RBA value of more than
100% for ERb. Surprisingly, the RBA value of C-iodo-p-carboranyl
phenol 6a toward ERb was more than 800%. Unfortunately, theRBA value toward ERa was also more than 500%, resulting in an
overall poor ERb selectivity. These results suggest that the rectilinear C-iodo-p-carboranyl phenol structure is particularly suitable
for the hydrophobic pocket of both ERs. p-Carboranes 6b and 6c
exhibit a bent core structure comparable to that of 4 and 5, andshowed 5.9- and 3.8-fold selectivity toward ERb, respectively.
The biological profiles, as well as the agonistic and antagonistic
activity of 5f were evaluated by means of a cell proliferation assay

This research was supported by a grant-in-aid from theStrategic Research Program for Private Universities (2015–2019)
and Grants-in-Aid for Scientific Research (C) (26460151 and
15K08029) from the Japanese Ministry of Education, Culture,
Sports, Science, and Technology (MEXT).
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Biochemical and Biophysical Research Communications 478 (2016) 279e285

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

BA321, a novel carborane analog that binds to androgen and estrogen
receptors, acts as a new selective androgen receptor modulator ofbone in male mice
Kenta Watanabe a, b, Michiko Hirata a, Tsukasa Tominari a, c, Chiho Matsumoto a,
Yasuyuki Endo d, Gillian Murphy e, Hideaki Nagase c, f, Masaki Inada a, b, c,
Chisato Miyaura a, b, c, *a

Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei, Tokyo 184-8588, Japan
Cooperative Major in Advanced Health Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei, Tokyo 184-8588, Japan
Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei, Tokyo 184-8588, JapandFaculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1, Komatsushima, Aoba-ku, Sendai, 981-8558, Japane
Department of Oncology, University of Cambridge, Cancer Research UK, Cambridge Institute, Li Ka Shing Centre, Cambridge, CB2 0RE, United KingdomfKennedy Institute of Rheumatology, Nufﬁeld Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, OX3
7FY, United Kingdombca r t i c l e i n f o

a b s t r a c t

Article history:
Received 26 June 2016
Accepted 4 July 2016
Available online 9 July 2016

Carboranes are a class of carbon-containing polyhedral boron cluster compounds with globular geometry
and hydrophobic surface that interact with hormone receptors such as estrogen receptor (ER) andandrogen receptor (AR). We have synthesized BA321, a novel carborane compound, which binds to AR.
We found here that it also binds to ERs, ERa and ERb. In orchidectomized (ORX) mice, femoral bone mass
was markedly reduced due to androgen deﬁciency and BA321 restored bone loss in the male, whilst thedecreased weight of seminal vesicle in ORX mice was not recovered by administration of BA321. In female mice, BA321 acts as a pure estrogen agonist, and restored both the loss of bone mass and uterine
atrophy due to estrogen deﬁciency in ovariectomized (OVX) mice. In bone tissues, the trabecular boneloss occurred in both ORX and OVX mice, and BA321 completely restored the trabecular bone loss in both
sexes. Cortical bone loss occurred in ORX mice but not in OVX mice, and BA321 clearly restored corticalbone loss due to androgen deﬁciency in ORX mice. Therefore, BA321 is a novel selective androgen receptor modulator (SARM) that may offer a new therapy option for osteoporosis in the male.
© 2016 Elsevier Inc. All rights reserved.

Keywords:
Osteoporosis
Bone mass
Carborane compound
EstrogenAndrogenOrchidectomized mice
Selective androgen receptor modulator

1. Introduction
Osteoporosis is accompanied by severe bone loss due to adeﬁciency of sex hormones associated with advanced ageing.
Postmenopausal osteoporosis in women is mainly due to increased
trabecular bone resorption caused by estrogen deﬁciency. Ovariectomy (OVX) in rats and mice is commonly used to study estrogendeﬁciency in females, and it has been shown that estrogen treatment can prevent bone loss in OVX animals, as in patients with
postmenopausal osteoporosis by suppressing bone resorption* Corresponding author. Department of Biotechnology and Life Science, Tokyo
University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei, Tokyo 1848588, Japan. Tel.: þ81 42 388 7390.
E-mail address: miyaura@cc.tuat.ac.jp (C. Miyaura).

[1e4]. Osteoporosis in elderly men is thought to be due to androgendeﬁciency and aging. Like estrogen, androgen suppresses bone
resorption to maintain bone mass, but androgen also stimulates
new bone formation. Similarly, orchidectomy (ORX) in mice causes
marked bone loss by increased bone resorption and decreased boneformation. Administration of testosterone recovers bone loss in
ORX mice, furthermore estrogen can also restore the bone loss due
to androgen deﬁciency. Since testosterone may be partly converted
to estrogen by aromatase, both androgen and estrogen may beeffective for the maintenance of bone tissues in the male. Indeed,
we have reported that aromatase-deﬁcient (ArKO) mice showedmarked bone loss due to increased osteoclastic bone resorption
because of estrogen deﬁciency in both sexes [5].
Androgen and estrogen exert potent inﬂuences on skeletal
shape, mass, and homeostasis in both men and women [6].

http://dx.doi.org/10.1016/j.bbrc.2016.07.027
0006-291X/© 2016 Elsevier Inc. All rights reserved.

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K. Watanabe et al. / Biochemical and Biophysical Research Communications 478 (2016) 279e285

Estrogen and androgen suppress bone resorption by ER- and ARdependent mechanisms, respectively, and androgen also stimulates bone formation in an AR-dependent manner [6]. However,replacement therapy with sex steroids is not generally used forpostmenopausal osteoporosis patients due to the risk of uterine
cancer. Some synthetic compounds such as raloxifene preferentially act on bone and cardiovascular systems as agonists, whereas
they antagonize the effects of estrogen in reproductive tissues[4,7,8]. These compounds are known as selective estrogen receptor
modulators (SERM) and raloxifene is now available for the treatment of osteoporosis in women. For osteoporosis in men, selective
androgen receptor modulators (SARM), which act as androgens inbone and muscle without androgenic action in male sex organ areuseful for male patients. Various SARMs such as GTx-024, GTx-027,
MK-4541, TSAA-291, NEP28, and S-101479 have been developed,
but no SARM is available for clinical use [9e13].
Carboranes (dicarba-closo-dodecaboranes) are a class of carboncontaining polyhedral boron-cluster compounds with remarkable
thermal stability and exceptional hydrophobicity, and have been
applied in the ﬁeld of boron neutron capture therapy in cancer[14e16]. On the other hand, we have focused on the binding afﬁnityof carboranes to steroid hormone receptors and possible biologicalactivity in various cells [17]. Previously, we reported that BE120, a
carborane compound, bound to ER and acted as a potent estrogenagonist in both bone and reproductive tissues [18], and that BE360,
another carborane, exhibited anti-estrogenic properties as a hydrophobic pharmacophore in ER-dependent luciferase reporter
gene assays [19] and recovered bone loss in OVX mice without any
action in reproductive tissues [20], indicating that BE360 is apossible SERM in female osteoporosis. We also developed several
carborane compounds, which bind the AR and some of the compounds, BA321 and BA341, act as anti-androgens [21,22].
In this study, we examined the effect of the carborane compound, BA321 on bone mass and sex organs in ORX and OVX mice.
BA321 showed a high binding afﬁnity to both AR and ERs, andrecovered bone loss in both ORX and OVX mice. BA321 did not
inﬂuence the sex organs in male mice. On the other hand, it acted asa pure estrogen in female mice. Therefore BA321 is a novel SARM
which acts as a selective agonist in bone tissue, and may be a new
candidate in the treatment of osteoporosis in men.
2. Materials and methods

assay with [3H]-DHT, and the IC50 value determined was comparedwith that of DHT. For in vitro binding assay for ERa and ERb, therecombinant LBDs of human ERa (hrERa-LBD) and human ERb(hrERb-LBD) prepared by the CERI, Japan were used. The binding
afﬁnity of BA321 to ERs was measured by the competitive binding
assay with [3H]-17b-estradiol, and the IC50 values of BA321 for
hrERa and for hrERb were determined and compared with those for17b-estradiol.
2.3. Radiographic analysis of the femur
The bone mineral density (BMD) of the femurs was measured bydual X-ray absorptiometry (model DCS-600R; Aloka, Tokyo, Japan),as reported previously [23]. The bone mineral content (BMC) of themouse femur was closely correlated with the ash weight [23]. The
BMD was calculated by dividing the BMC of the measured area by
the area. The scanned area was dividing equally into three regions
comprising the proximal, middle and distal femur to assess regional
differences. The BMD in the total area of the femur was alsomeasured.
2.4. Micro CT analysis
The CT scanning of the femurs was performed using a microfocus X-ray CT system (inspeXio SMX-90T; Shimadzu). Threedimensional microstructural image data were reconstructed usinga cross-section of the distal femur for trabecular bone. The structural indices were calculated for trabecular bone, bone volume/
tissue volume [BV/TV], bone surface of bone volume [BS/BV],
trabecular number [Tb. N] and trabecular separation [Tb. S] using
the TRI/3D-BON software program (Ratoc System Engineering Co.,
Ltd.), as reported [23].
2.5. Statistical analysis
Data were analyzed using one-way ANOVA, followed by Tukey’s
test for post hoc analysis. All data are presented as the means ± SEM,
and all statistical analyses were performed using IBM SPSS Statistics Ver.23 software.

2.1. Animals and drugs

3. Results

Eight-week-old female and male mice ddy strains were obtained
from Japan SLC Inc. (Shizuoka, Japan). Male mice were either shamoperated or ORX. Some of the ORX mice were administered subcutaneously with BA321 dissolved in polyethyleneglycol-300(Wako, Osaka, Japan) using a mini-osmotic pump (Alza Co., Palo
Alto, CA) immediately after surgery. Female mice were shamoperated or OVX. Some of the OVX mice were administered subcutaneously with BA321 as described above. The mice were fed alaboratory chow containing 1.12% calcium and 1.07% phosphorus(Nippon Clea, Tokyo, Japan) for 4 weeks after the surgery. BA321
was synthesized in our laboratory as reported in our previous
studies [22].

3.1. Design and synthesis of carborane BA321, and its afﬁnity to AR
and ERs

2.2. Binding analysis of BA321 to androgen receptor and estrogen
receptors
The in vitro AR binding assay used the recombinant ligand
binding domain (LBD) of human AR (hrAR-LBD) prepared by the
Chemicals Evaluation Research Institute (CERI), Japan. The binding
afﬁnity of BA321 to AR was measured by the competitive binding

Carboranes are a class of carbon-containing polyhedral boroncluster compounds, having globular geometry and a hydrophobic
surface (Fig. 1A). We focused on the hydrophobic feature of carboranes which interact with steroid hormone receptors because
their shape may be suitable as an AR or ER ligand. The size of the
carborane cage seems to be appropriate for a hydrophobic structure
in place of the C, D rings of sex steroid, DHT and 17b-estradiol(Fig. 1B). We synthesized a novel carborane compound, BA321(Fig. 1A), which has p-carborane skeletons and exhibits antiandrogen properties in AR-dependent luciferase reporter gene assays [22]. We ﬁrst investigated the binding afﬁnity of BA321 to AR
and both ERa and ERb, in comparison with DHT and 17b-estradiol
using a binding competition assay. BA321 clearly bound to AR, andits binding afﬁnity was 1/220 of that of DHT (Fig. 1C). Surprisingly,
BA321 also bound to ERs, ERa and ERb, and its binding afﬁnity to
ERa and ERb was about 1/340 and 1/48 of that of 17b-estradiol,
respectively (Fig. 1C).

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K. Watanabe et al. / Biochemical and Biophysical Research Communications 478 (2016) 279e285

281

Fig. 1. Structures of the carborane cage, carborane compounds, BA321 and binding afﬁnity to AR or ER. (A) In icosahedral cage structures, closed circles (C) represent carbon atoms
and other vertices represent BH units. (B) Structures of dihydrotestosterone (DHT) and 17b-estradiol (E2). (C) Binding of BA321 (B) and DHT (C) to human AR. The binding afﬁnities
of BA321 and DHT to AR were determined by measuring their abilities to compete the binding of radiolabeled [3H]DHT to hrAR-LBD in the presence of increasing concentrations of acompetitor, taking the binding of [3H]DHT to hrAR-LBD without a competitor as 100%. Log IC50 values (M) of DHT and BA321 for AR were 7.82 and 5.48, respectively. (D) Bindingof BA321 ( ) and E2 (-) to human ERa and ERb. The binding afﬁnities of BA321 and E2 were determined by measuring their abilities to compete the binding of radiolabeled [3H]
17b-estradiol to hrER-LBD in the presence of increasing concentrations of a competitor, taking the binding of [3H]17b-estradiol to hrER-LBD without a competitor as 100%. Log IC50
values (M) of E2 and BA321 for ERa were 9.20 and 6.67, respectively. Log IC50 values (M) of the two ligands for ERb were 8.74 (E2) and 7.06 (BA321), respectively.

▫

3.2. Effects of BA321 in sex organs in ORX and OVX mice

3.3. Effects of BA321 on bone mass in ORX mice

We ﬁrst examined the effects of BA321 on the weight of sexorgans in mice of both sexes. The weight of the seminal vesicles
decreased markedly in ORX mice due to androgen deﬁciency(Fig. 2A). When ORX mice were given androgens, the weight ofseminal vesicles returned to the sham level (data not shown).
Administration of BA321 (10 and 30 mg/day) showed no effects on
the weight of seminal vesicles in ORX mice (Fig. 2A). In OVX mice,the uterine weight decreased markedly due to estrogen deﬁciency,
and the administration of BA321 (10 and 30 mg/day) dosedependently restored the uterine weight to the sham level(Fig. 2B). These results indicate that BA321 exhibits estrogenic action in female sex organs, but does not act as androgen in male sexorgans.

Androgen deﬁciency caused by ORX resulted in a marked
decrease in bone mass due to the stimulation of bone resorption
and a concomitant decrease in bone formation. To determine theeffects of BA321 on bone mass in ORX mice, male mice were shamoperated or ORX, and some ORX mice were treated with BA321.
Four weeks after the surgery, we measured the femoral BMD of thetotal area and of three divided areas; proximal, central and distal.
Total BMD was signiﬁcantly reduced by ORX, and BA321 administration (10 and 30 mg/day) caused dose-dependent recovery of bonemass to the sham level (Fig. 3A). All areas showed a decreased BMD
in ORX mice, and BA321 restored the BMD to the sham level in the
all area (Fig. 3A). BA321 may stimulate cortical bone formation in
the central area of the femurs. Using mCT, we examined the effects— 390 —

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K. Watanabe et al. / Biochemical and Biophysical Research Communications 478 (2016) 279e285

administration of BA321 dose-dependently recovered the all CT
markers to the sham levels (Fig. 4B).
4. Discussion

Fig. 2. Effects of BA321 on the weight of the seminal vesicles in ORX mice and theuterine weight in OVX mice. (A) Male mice were sham-operated or ORX, and some of
the ORX mice were treated with 10 and 30 mg/day/mouse of BA321. At 4 weeks after
the surgery, the weight of the seminal vesicles was measured. Signiﬁcantly different
from sham mice, ***P < 0.001. Data are expressed as the means ± SEM of 6e8 mice. (B)Female mice were sham-operated or OVX, and some of the OVX mice were treatedwith 10 and 30 mg/day/mouse of BA321 after the surgery. The uterine weight wasmeasured at 4 weeks after the operation. Signiﬁcantly different from sham mice,***P < 0.001, and from OVX mice, ###P < 0.001. Data are expressed as the means ± SEM
of 8e11 mice.

of BA321 on the trabecular bone architecture in ORX mice. In ORX
mice, we detected a marked loss of trabecular bone in the distal
femurs, which was accompanied by a decrease in BV/TV and Tb.N.
and an increase in BS/BV and Tb.Sp. (Fig. 3B). Administration ofBA321 (10 and 30 mg/day) dose-dependently recovered all CT
markers to the sham levels, and trabecular bone architecture also
recovered (Fig. 3B).
3.4. Effects of BA321 on bone mass in OVX mice
To determine the effects of BA321 on bone mass in OVX mice,
OVX mice were treated for 4 weeks with BA321 and femurs were
subjected to radiographic analysis. We measured the femoral BMD
of total area and three divide proximal, central and distal areas.
Total BMD was signiﬁcantly reduced by OVX, and BA321 (10 and
30 mg/day) dose-dependently caused bone mass recovery (Fig. 4A).
The distal BMD was clearly decreased by OVX, but the central BMD
showed no change on OVX, and was not inﬂuenced by theadministration of BA321 (Fig. 4A). In mCT analysis, the loss of distaltrabecular bone was detected in OVX mice, and BA321 dosedependently recovered the trabecular bone architecture to thesham levels (Fig. 4B). In OVX mice, we detected a decrease in BV/TV
and Tb.N. and an increase in BS/BV and Tb.Sp. (Fig. 3B). The

In the present study, we showed that the carborane BA321 binds
to the AR, and acts as an androgen in bone tissues without androgenic action in sex organs. Thus BA321 acts as a novel SARM which
may be useful for the maintenance of bone mass in elderly men
with accompanying androgen deﬁciency. In addition to AR, BA321
also binds to ERa and ERb, and exhibits a pure estrogenic action inboth bone and uterus in female mice. This is unusual because
SARMs generally bind to AR but not ERs and exhibit the tissues
selective androgenic or anti-androgenic function. The mechanismof the tissue-speciﬁc action of SARMs is thought to depend on the
AR-related recruitment of numerous cofactors, and SARMs induce
recruitment of fewer cofactors than DHT [11,13]. BA321 was originally designed based on potent AR antagonists that exhibited antiandrogenic activity in reporter gene assay using NIH3T3 cells
transfected with a human AR expression plasmid [22]. However, weshow that BA321 also binds to ERs and exhibits estrogenic action in
OVX mice. The mechanisms of action of BA321 mediated by AR andER are unknown, and have to be examined in a future study.
Androgens and estrogens exert potent inﬂuences on the bonehomeostasis in both men and women [6]. In the present study, thetrabecular bone in the femur was clearly decreased with increasedbone resorption in both ORX and OVX mice, and the administrationof BA321 restored the trabecular bone loss to sham levels (Figs. 3Band 4B). The distal area of the femur is rich in trabecular bone, andBA321 restored the loss of distal BMD in the femur (Figs. 3A and
4A). The BMD in central area of the femur consists only of corticalbone. In ORX mice, the central BMD clearly suppressed, and theadministration of BA321 restored the cortical bone loss. However,the signiﬁcant loss of central BMD was not detected in OVX mice,
and BA321 did not inﬂuenced the cortical bone mass in the female(Fig. 4A). These results suggest that BA321 binds to AR and acts on
trabecular bone to suppress bone resorption, and on cortical bone
to stimulate bone formation in the male, and that it binds to ERs
and suppresses bone resorption in trabecular bone in the female.
BA321 may not be useful for osteoporosis in the female, since it
exhibits an estrogenic action in the uterus, which is thus a cancer
risk. On the other hand, BA321 may be a potential candidate SARM
for male osteoporosis without a risk of cancer in the sex organs.
Kawano et al. [24] have reported that global deletion of AR inmale mice results in high bone turnover with increased trabecularbone resorption, as well as decreased cortical bone volume due tosuppressed bone formation. On the other hand, data in the previous
reports using global deletion of ERs are complex [6]. Cell-speciﬁc
ERa knockout mice have been generated, and suggested that the
ERa-dependent apoptosis occurs in osteoclasts [6,25], but themechanisms of bone loss by the cell-speciﬁc ERs depletion are still
not clear. Further studies are needed to deﬁne the mechanisms of
AR-dependent and ER-dependent bone turnover in both sexes.
Loss of musculoskeletal mass and function is a natural ageing
trait, and osteoporosis and sarcopenia are increasing in older
people [26], but androgen therapy is not generally used due to theperceived side effects, such as prostate cancer. Therefore SARMs arepotential candidate anabolic compounds that maintain bone massand muscle function. In addition to osteoporosis and sarcopenia,
SARMs are thought to be useful for the therapy of AR-dependent
prostate cancer, the muscle wasting associated with cancer, andchronic illness, as well as AR-positive breast cancer [9,10,12,27]. We
examined the effects of BA321 on the cell growth of AR-positive
prostate cancer LNCap, and found that BA321 suppressed the
DHT-dependent growth of LNCap cells (Inada, M. et al., unpublished— 391 —

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283

Fig. 3. Effects of BA321 on bone mass of femurs in ORX mice. (A) Mice were sham-operated or ORX, and some of the ORX mice were treated with 10 and 30 mg/day/mouse of BA321.
After 4 weeks, femurs were collected and the femoral bone mineral density (BMD) in each area (total, distal, central and proximal) was measured by dual X-ray absorptiometry(DEXA). Signiﬁcantly different from sham mice, *P < 0.05, **P < 0.01, ***P < 0.001 and from ORX mice, #P < 0.05, ##P < 0.01, ###P < 0.001. Data are expressed as the means ± SEM of
6e8 mice. (B) A three-dimensional (3D) mCT analysis of femoral trabecular bone collected from sham, ORX, and ORX mice treated with BA321 (10 and 30 mg/day) at 4 weeks after
surgery. The upper panel shows a 3D mCT image of the femoral distal metaphysis in each group. The Bone Volume/Total tissue Volume (BV/TV), Trabecular Number (Tb.N.), Bone
Surface/Bone Volume (BS/BV), and Trabecular Separation (Tb.Sp.) were calculated by a 3D mCT analysis. The data are expressed as the mean ± SEM of 6e8 mice. Signiﬁcantly
different from sham mice, *P < 0.05, **P < 0.01, ***P < 0.001 and from ORX mice, ##P < 0.01, ###P < 0.001.

— 392 —

Fig. 4. Effects of BA321 on bone mass of femurs in OVX mice. (A) Mice were sham-operated or OVX, and some of the OVX mice were treated with 10 and 30 mg/day/mouse of BA321
immediately after surgery. After 4 weeks, their femurs were collected and the femoral BMD in each area (total, distal, central and proximal) was measured by DEXA. Signiﬁcantly
different from sham mice, **P < 0.01, ***P < 0.001 and from OVX mice, #P < 0.05, ##P < 0.01, ###P < 0.001. Data are expressed as the means ± SEM of 8e11 mice. (B) A 3D mCT analysisof femoral trabecular bone collected from sham, OVX, and OVX mice treated with BA321 (10 and 30 mg/day) at 4 weeks after surgery. The upper panel shows a 3D mCT image of thefemoral distal metaphysis in each group. The BV/TV, Tb.N., BS/BV, and Tb.Sp. were calculated by a 3D mCT analysis. The data are expressed as the mean ± SEM of 8e11 mice.
Signiﬁcantly different from sham mice, *P < 0.05, ***P < 0.001 and from OVX mice, #P < 0.05, ##P < 0.01, ###P < 0.001.

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K. Watanabe et al. / Biochemical and Biophysical Research Communications 478 (2016) 279e285

data). Therefore, it is possible that BA321 exhibits anti-androgenic
action for prostate cancer as a SARM in vivo without the loss ofmusculoskeletal mass, a typical risk of anti-androgen therapy.
In conclusion, we have demonstrated that BA321, a novel carborane compound, bound to not only AR, but also ERs with a highbinding afﬁnity, and completely restored the bone loss in both ORX
and OVX mice. Since BA321 did not inﬂuence the sex organ in themales, it may act as a potential SARM to prevent the loss ofmusculoskeletal mass due to androgen deﬁciency in men.
Acknowledgments
This work is partly supported by Grants-in-Aid for ScientiﬁcResearch 25460062 (C.M.) and 23590069 (M.I.), National Instituteof Health grant AR40994 (H.N.), Arthritis Research UK (H.N.), Cancer Research UK (G.M.) and Institute of Global Innovation Research
in TUAT (M.I., H.N.). This project was supported by Open Partnership Joint Projects of JSPS Bilateral Joint Research Projects.
Transparency document
Transparency document related to this article can be foundonline at http://dx.doi.org/10.1016/j.bbrc.2016.07.027.
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Bioorganic & Medicinal Chemistry 24 (2016) 1089–1094

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistryjournal homepage: www.elsevier.com/locate/bmc

Symmetric 4,40 -(piperidin-4-ylidenemethylene)bisphenol derivatives
as novel tunable estrogen receptor (ER) modulators
Manabu Sato, Kiminori Ohta ⇑, Asako Kaise, Sayaka Aoto, Yasuyuki Endo
Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japana r t i c l e

i n f oArticle history:
Received 16 December 2015
Revised 14 January 2016
Accepted 18 January 2016
Available online 18 January 2016
Keywords:
Estrogen receptor modulator
Piperidine
2,2,6,6-Tetramethylpiperidine
McMurry coupling
Hydrophobicity

a b s t r a c t
We designed and synthesized 4,40 -(piperidin-4-ylidenemethylene)bisphenol derivatives as novel tunableestrogen receptor (ER) modulators. The introduction of hydrophobic substituents on the nitrogen atom of
the piperidine ring enhanced ERa binding affinity. In addition, the introduction of four methyl groups
adjacent to the piperidine ring nitrogen atom remarkably enhanced ERa binding affinity. N-Acetyl2,2,6,6-tetramethylpiperidine derivative 3b showed high ERa binding affinity, high MCF-7 cell proliferation inducing activity, and high metabolic stability in rat liver S9 fractions.
 2016 Elsevier Ltd. All rights reserved.

1. Introduction

N

Tamoxifen (Fig. 1) has been used worldwide for more than
40 years for the treatment of breast cancer.1 It exhibits competitive
antagonism against endogenous estrogen 17b-estradiol (E2, Fig. 1)
on the estrogen receptor (ER). Tamoxifen produces a powerful
effect against ER-positive but not ER-negative breast cancer.1 In
addition, since E2 is an endogenous estrogen that plays importantroles in the female and male reproductive systems as well as inbone maintenance, the central nervous system, and the cardiovascular system, tamoxifen also exerts some biological actions in
those tissues.2 Interestingly, tamoxifen acts as either an agonist
or an antagonist depending on tissue type; it exhibits antiestrogenic action in breast cancer and hot flashes, and estrogenic
action in bone and cholesterol metabolism, and is a selective estrogen receptor modulator (SERM).3
Following the success of tamoxifen, several triphenylethylene
derivatives, such as toremifene4 and clomifene5, were developed
as novel SERMs (Fig. 1). An alkylamino chain is attached to the terminal of benzene ring where it plays a strategic role in the expression of antagonistic activity.6 The three SERMs show quite varied
biological activities because of the different hydrophobic side
chains attached to the ethylene moiety.7 The hydrophobic side
chain plays an important role in controlling SERM activity. The
triphenylethylene structure has geometric isomers E and Z, which⇑ Corresponding author.

E-mail address: k-ohta@tohoku-pharm.ac.jp (K. Ohta).

O
OH

HO

R
17β-Estradiol (E2)

R = H Tamoxifen
R = OH 4-Hydroxytamoxifen

N
O

N
OAc

O

Cl
Toremifene

Cl
Clomifene

AcO
Cyclofenil

Figure 1. Chemical structures of 17b-estradiol (E2) and clinically used SERMs.

are due to the key alkylamino chain and the asymmetrichydrophobic part. As the isomers easily isomerize between the E
and Z forms, problems arise in the synthesis, purification, andpreservation of the SERMs.8 Indeed, 4-hydroxytamoxifen, an active

http://dx.doi.org/10.1016/j.bmc.2016.01.035
0968-0896/ 2016 Elsevier Ltd. All rights reserved.

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M. Sato et al. / Bioorg. Med. Chem. 24 (2016) 1089–1094

tion of the cyclohexane ring of 1a into the 3,3,5,5-tetramethylcyclohexane ring enhanced ERa binding affinity because of thefavorable hydrophobic interaction with the ERa ligand binding
domain (LBD). 3,3,5,5-Tetramethylcyclohexane derivative 1b acts
as an ER partial agonist, whereas compound 1a is an ER agonist.
The cyclohexyl moiety in 1a and 1b plays an important role in thehydrophobic interactions with the hydrophobic amino acid residues
of ERa LBD, but is not easy tunable. A multi-tunable symmetricstructure would be a very attractive tool for ER ligand studies
because it would enable detailed SAR studies and not require isomer
separation, and its physicochemical properties would be controllable for optimum ADME. Therefore, we focused on the discoveryof readily available ER modulators with multi-tunable symmetricstructures and designed novel ER ligand candidates 2a–2d, 3a,and 3b containing a piperidine ring (Fig. 3). In this paper, we
describe the synthesis of those compounds, their biological activities, such as ERa binding affinity and ER-dependent proliferationof MCF-7 cell line, and a metabolic study in rat liver S9 fractions.

OH

OH

HO

HO

1b

1a
RBA for ERα = 6.71

RBA for ERα = 28.4

ER agonist

ER partial agonist

Figure 2. Chemical structures of cyclofenil derivatives 1a and 1b.

OH

HO

OH

N
2

R

HO

N
3

R

R = H, CH3, COCH3, CO2Et

2. Results and discussion

Figure 3. Novel ER modulator candidates 2 and 3 having a tunable piperidine ring.

2.1. Chemistry
A structure common to compounds 2a–2d was synthesized frompiperidinone derivatives and 4,40 -dimethoxybenzophenone bymeans of the McMurry coupling.16 Scheme 1 summarizes the synthesis of N-substituted piperidine derivatives 2a–2d. Commerciallyavailable N-ethoxycarbonyl piperidin-4-one 4 was reacted with
4,40 -dimethoxybenzophenone to afford 5 in 90% yield, which wasthen demethylated with BBr3 to afford 2d in 78% yield. McMurry
coupling product 5 was hydrolyzed with KOH and then subjectedto spontaneous decarboxylation to afford key intermediate 6 in
90% yield. Compound 6 was transformed into 2a with BBr3 in 54%
yield. N-Methylated derivative 2b was obtained by reductive
amination using paraformaldehyde and NaBH4 in trifluoroethanol,

metabolite of tamoxifen, easily isomerizes from the active Z form
to the inactive E form in solution and in vitro studies (Fig. 1).9 In
this regard, novel SERMs having other skeletons, such as benzothiophene,10 dihydronaphthalene,11 benzopyrane,12 and steroid,13
have been developed and used as antagonistic SERMs.
On the other hand, cyclofenil shows weak estrogenic activity
because of the absence of the alkylamino chain, and thus it has
been developed as an agonistic SERM (Fig. 1).14 Cyclofenil has a
C-2 symmetric structure and no E and Z isomer. Recently, we havereported an SAR study of the cyclohexane ring of cyclofenil
reductant 1a, in which the diphenylmethane skeleton and thecyclohexane ring are linked by a single bond (Fig. 2).15 Transforma-

OH

OMe

N

CO2Et

a

b

O
4

MeO

5

N

HO

CO2Et

N
2d

CO2Et

c
OMe

MeO
d, b

OH

6

N

H
b

e, b

OH
OH

HO

2c

N

COCH3

HO
HO

2b

N

2a

N

H

Me

Scheme 1. Synthesis of the designated derivatives 2a–2d. Reagents and conditions: (a) TiCl4, Zn, 4,40 -dimethoxybenzophenone, THF, 90%; (b) BBr3, CH2Cl2, 23–90%; (c) KOH,
EtOH, 90%; (d) Ac2O, 93%; (e) (CHO)n, NaBH4, CF3CH2OH, 87%.

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M. Sato et al. / Bioorg. Med. Chem. 24 (2016) 1089–1094

1.1

N

H

a

N

O

COCH3

b, c

O

N

HO

8

7

Relative cell number

OH

3b

COCH3

b
OH

OMe

H

N

HO

9

H

3a

Scheme 2. Synthesis of 2,2,6,6-tetramethylpiperidine derivatives 3a and 3b.
Reagents and conditions: (a) Ac2O, 88%; (b) TiCl4, Zn, 4,40 -dimethoxybenzophenone,
THF, 48–75%; (c) BBr3, CH2Cl2, 75–82%.

1.2

Fraction of specific [3H]E2
binding remaining

0.8
0.7
0.6
0.5
0.4
-12

-11

-10

-9

-8

-7

-6

-5

Log concentration (M)Figure 5. Dose–response curve of 3b in the MCF-7 cell proliferation assay. MCF-7
cells were incubated with 3b (1 � 10�6 to 1 � 10�12 M) for 5 days, and the results
are shown by relative cell number with the value for E2 taken as 1. Cell proliferation
assay was performed in triplicate (n = 3). Values are means ± SD of separate
experiments.

Percentage of unmetabolized
test compounds (%)

N

MeO

0.9

0.3
-13

c

3b

1.0

E2
2a
2b
2c
2d
3a
3b

1.0
0.8
0.6
0.4
0.2

Testosterone
2c
3b

120
100
80
60
40
20
0

10

20

30

40

50

Time (min)
0

0.1

1

10

100

1000

Figure 6. Percentages of 2c and 3b remaining in rat liver S9 fractions. The
percentages were estimated from peak area ratios of corticosterone as an internal
standard to the tested compounds.

Relative concentration
Figure 4. Binding affinities of test compounds 2a–2d, 3a, and 3b. Competitive
binding assay of compounds 2a–2d, 3a, and 3b with [3H]E2 (4 nM) for ERa. Binding
assays of the test compounds (0.4–4 lM) were conducted in the presence of[6,7-3H]17b-estradiol (4 nM). The assays were performed in duplicate (n = 2).

followed by deprotection with BBr3 in 20% yield over two steps.
N-Acetylated derivative 2c was synthesized by acetylation andsubsequent demethylation in 84% yield over two steps.
The McMurry coupling of 4,40 -dimethoxybenzophenone with
N-acetyl tetramethylpiperidinone 8, which was obtained throughthe acetylation of commercially available 2,2,6,6-tetramethylpiperidin-4-one 7 followed by deprotection with BBr3, afforded
3b in 38% yield over three steps (Scheme 2). To prepare 3a, we tried
to remove the N-acetyl group from 3b. However, deacetylation did
not proceed under both acidic and basic conditions due to thesteric hindrance of the four methyl groups adjacent to the nitrogen
atom of the piperidine ring. Therefore, direct McMurry coupling of
2,2,6,6-tetramethylpiperidine with 4,40 -dimethoxyphenylbenzophenone was carried out to afford corresponding couplingproduct 9 in 75% yield. Compound 9 was transformed into 3a bydemethylation with BBr3 in 82% yield.
2.2. Biological evaluations
The ERa-binding affinity of the synthesized compounds wasevaluated in a competitive binding assay using tritium-labeled E2
and recombinant hERa (Fig. 4).17 Unsubstituted piperidine derivative 2a showed no binding affinity for ERa LBD, whereas N-methyl
and N-acetyl derivatives 2b and 2c bound very weakly to ERa LBD.
Aliphatic amines 2a and 2b would be present in the protonatedform in this assay buffer. Therefore, the extremely low binding
affinity of 2a and 2b might be caused by the polarity around theprotonated nitrogen atom on the piperidine ring rather than thebasicity of the nitrogen atom. N-Acetylated derivative 2c also
showed low binding affinity because of the high polarity (C Log P:
1.72).18 Compound 2d having an ethyl carbamate group dosedependently bound to ERa and showed higher ERa binding affinity
than 2a–2c owing to the increase in hydrophobicity caused by the
ethyl group. Indeed, compound 2d had the highest C Log P value at
3.90 among 2a–2d.18 In addition, ethyl group of 2d would beaccommodated in the hydrophobic pocket of ERa. The sterichinderance of the four methyl groups in 3a and 3b obscured thephysicochemical properties of the nitrogen atom, such as basicity
and hydrophilicity. Thus, 3a and 3b would show higher binding
affinity than corresponding normal piperidine derivatives 2a and2c, if the sterically bulky tetramethyl moiety were tolerated in
the hydrophobic space of ERa LBD. Although the binding affinityof 3a was low, it seemed that the four methyl groups improved
incompatibility between the piperidine ring and ERa LBD. Interestingly, compound 3b showed more than 100 times higher binding
affinity than corresponding piperidine derivative 2c. The four
methyl groups exerted a strong positive effect on the binding of
the piperidine derivatives to ERa LBD.
Next, cell functional assays of 3b were performed using theMCF-7 cell line that showed ERa-dependent growth.17 Compound3b demonstrated high cell proliferation inducing activity in a dose
dependent manner (Fig. 5), but no inhibition of cell proliferation
activity inducing by 0.1 nM of E2 (data not shown). The EC50 value— 397 —

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M. Sato et al. / Bioorg. Med. Chem. 24 (2016) 1089–1094

of 3b estimated from the sigmoidal dose response curve was
0.22 nM. The maximal efficacy for MCF-7 cell proliferation inducing activity of 3b was similar to that of E2. Interestingly, 3b acted
as ER full agonist, unlike the lead compound 1b. We suggested that
ER full agonist activity of 3b would be caused by the nitrogen atomor the spatial configuration of 2,2,6,6-tetramethylpiperidine ring.
To examine the effect of the four methyl groups on metabolicstability, we measured the elimination rates of piperidine derivatives 2c and 3b in rat liver S9 fractions (Fig. 6).19 The percentage(%) of unmetabolized test compounds was estimated from the peakareas of 2c and 3b in HPLC analyses. Approximately 80% of testosterone used as a positive control was metabolized at 50 min under
the assay conditions. Compounds 2c and 3b showed low metabolic
rates; approximately 10% and 20% were metabolized at 50 min,respectively. Compound 3b was more rapidly metabolized than2c and the tetramethyl moiety had no effect on the metabolic stability of these compounds. It seems that the piperidine rings of 2cand 3b are quite stable under the assay conditions.
3. Conclusion
In conclusion, we designed and synthesized novel tunable ER
modulators having the 4,40 -(piperidin-4-ylidenemethylene)bisphenol structure. In contrast to the unsubstituted piperidine
ring that was unfavorable for ERa LBD, the introduction ofhydrophobic substituents on the nitrogen atom or the four methylgroups adjacent to the nitrogen atom improved the binding affinityof the piperidine ring for ERa LBD. Compound 3b showed highbinding affinity for ERa, high MCF-7 cell proliferation inducing
activity, and good metabolic stability in rat liver S9 fractions. We
are of the opinion that 2,2,6,6-tetramethylpiperidine ring is apromising core skeleton for ER modulator studies. Further investigations focusing on the chemical modification of the substituentson the nitrogen atom of the 2,2,6,6-tetramethylpiperidine ring,the introduction of alkylmino chains on the phenol group for thedevelopment of ER antagonists and SERMs, and the biological evaluation of ER subtype and tissue selectivity, are in progress.
4. Experimental section
4.1. General
Melting points were determined with a Yanaco micro melting
point apparatus and were uncorrected. 1H NMR and 13C NMR spectra were recorded with JEOL JNM-LA-400 spectrometers. Chemical
shifts for 1H NMR spectra were referenced to tetramethylsilane(0.0 ppm) as an internal standard. Chemical shifts for 13C NMR
spectra were referenced to residual 13C present in deuterated solvents. The splitting patterns are designed as follows: s (singlet), d(doublet), t (triplet), and q (quartet). Mass spectra were recorded
on a JEOL JMS-DX-303 spectrometer. Elemental analyses were performed with a Perkin Elmer 2400 CHN spectrometer. Column chromatography was carried out using Fuji Silysia silica gel BW-80S
and TLC was performed on Merck silica gel F254. Reagents werepurchased from Wako Pure Chemical Industries, Ltd, Sigma-Aldrich
Co., and Tokyo Chemical Industry, Ltd (TCI). All solvents were ofreagent quality, purchased commercially, and were used without
further purification.
4.2. Synthesis
4.2.1. Methyl 4-[(4-methoxyphenyl)(N-ethyloxy-carbonylpiperidin-4-ylidene)methyl]phenoxide (5)To a stirred suspension of Zn powder (2.83 g, 43.2 mmol) in
30 mL of dry THF was added slowly titanium(IV) tetrachloride(2.37 mL, 21.6 mmol) under an argon (Ar) atmosphere. A mixture
was refluxed for 2.5 h and cooled to room temperature. To the
mixture was added a solution of 4,40 -dimethoxybenzophenone(1.42 g, 5.86 mmol) and ethyl 4-oxopiperidine-1-carboxylate(1.01 g, 5.90 mmol) in 20 mL of dry THF, and it was refluxed for
12 h. The reaction mixture was poured into saturated NaHCO3
aqueous solution. Ether was added to the aqueous solution withvigorous stirring, and insoluble materials were filtered off throughCelite. The filtrate was extracted with ether, dried over MgSO4, andconcentrated. The residue was purified by column chromatography
on silica gel with 40:1 n-hexane/AcOEt to afford the title compound 5 (2.23 g, 99%) as a yellow liquid; 1H NMR (395 MHz, CDCl3)d (ppm) 7.01 (d, 2H, J = 7.9 Hz), 6.82 (d, 2H, J = 7.9 Hz), 4.14 (q, 2H,
J = 7.9 Hz), 3.79 (s, 6H), 3.49 (t, 4H, J = 4.0 Hz), 2.35 (t, 4H,
J = 4.0 Hz), 1.26 (t, 3H, J = 7.9 Hz); 13C NMR (100 MHz, CDCl3) d(ppm) 158.1, 155.5, 136.5, 134.8, 132.9, 130.7, 113.3, 61.2, 55.0,
44.8, 31.5, 14.6; MS (EI) m/z 381 (M+, 100%).
4.2.2. Methyl 4-[(4-methoxyphenyl)(piperidin-4-ylidene)
methyl]phenoxide (6)A mixture of 5 (7.54 g, 19.8 mmol) and potassium hydroxide(92.9 g, 1.66 mol) in 300 mL of ethanol was refluxed for 6 h underan Ar atmosphere. The solvent was evaporated, water (50 mL) was
added to the residue, and then the aqueous mixture was extractedwith AcOEt. The organic phase was dried over Na2SO4 andconcentrated to afford the title compound 6 (5.52 g, 90%) as ayellow solid. 1H NMR (395 MHz, CDCl3) d (ppm) 7.02 (d, 4H,
J = 7.9 Hz), 6.82 (d, 4H, J = 7.9 Hz), 3.79 (s, 6H), 2.90 (t, 4H,
J = 4.0 Hz), 2.33 (t, 4H, J = 4.0 Hz); 13C NMR (100 MHz, CDCl3) d(ppm) 157.9, 136.3, 135.2, 130.9, 129.2, 113.3, 55.1, 48.5, 33.5;
MS (EI) m/z 309 (M+), 267 (100%).
4.2.3. 4-[(4-Hydroxyphenyl)(piperidin-4-ylidene)methyl]phenol(2a)
To a solution of 6 (2.95 g, 9.54 mmol) in 20 mL of CH2Cl2 wasadded 1 M of BBr3 solution (17.5 mL, 17.5 mmol) at 0 C under Ar
atmosphere. The mixture was stirred for 12 h at 0 C, the solvent
was removed, water was added to the residue, and the mixture
was extracted with AcOEt. The organic phase was dried over
Na2SO4 and concentrated to afford the title compound 2a (1.44 g,
54%) as a yellow solid; colorless cubes (MeOH); mp 205–207 C;
1
H NMR (395 MHz, CD3OD) d (ppm) 6.88 (d, 4H, J = 7.9 Hz), 6.69
(d, 4H, J = 7.9 Hz), 2.83 (t, 4H, J = 4.0 Hz), 2.32 (t, 4H, J = 4.0 Hz);13
C NMR (100 MHz, CD3OD) d (ppm) 157.2, 137.6, 135.3, 134.3,
131.9, 115.8, 33.6, 32.2; MS (EI) m/z 281 (M+, 100%); Anal. Calcd
for C18H19NO2: C, 76.84; H, 6.81; N, 4.98. Found: C, 76.84; H,
6.81; N, 4.91.
4.2.4. 4-[(4-Hydroxyphenyl)(N-methylpiperidin-4-ylidene)
methyl]phenol (2b)
A solution of paraformaldehyde (191 mg, 6.4 mmol) in 30 mL oftrifluoroethanol was stirred at 35–40 C. To the stirred solution
was added 6 (1.18 g, 3.88 mmol), and NaBH4 (185 mg, 4.89 mmol)
was added 5 min later. After 30 min, the mixture was filteredthrough Celite and the filtrate was concentrated. The residue waspurified by column chromatography on silica gel with AcOEt toafford N-methylated compound (1.09 g, 87%) as a yellow solid.
1
H NMR (395 MHz, DMSO) d (ppm) 6.96 (d, J = 7.9 Hz, 4H), 6.85
(d, 4H, J = 7.9 Hz), 3.72 (s, 6H), 2.33 (t, 4H, J = 4.0 Hz), 2.25 (t, 4H,
J = 4.0 Hz), 2.15 (s, 3H); 13C NMR (100 MHz, DMSO) d (ppm)
159.8, 137.7, 136.2, 133.3, 131.8, 114.4, 58.0, 55.7, 45.9, 32.1; MS(EI) m/z 323 (M+, 100%). The N-methylated compound wastransformed into 2b by the same method that described thesynthesis of 2a; 23% yield; colorless needles (MeOH–CHCl3)
mp 255–257 C; 1H NMR (395 MHz, CD3OD) d (ppm) 6.99 (d, 4H,
J = 8.0 Hz), 6.70 (d, 4H, J = 8.0 Hz), 2.52 (t, 4H, J = 8.0 Hz), 2.41

— 398 —

M. Sato et al. / Bioorg. Med. Chem. 24 (2016) 1089–1094(t, 4H, J = 4.0 Hz), 2.32 (s, 3H); 13C NMR (100 MHz, CD3OD) d (ppm)
157.1, 138.0, 135.3, 132.9, 131.9, 115.7, 58.1, 46.1, 32.2; MS (EI)m/z 295 (M+, 100%).; Anal. Calcd for C19H21NO2: C, 76.45; H,
7.17; N, 4.74; Found: C, 76.45; H, 7.15; N, 4.69.
4.2.5. 4-[(4-Hydroxyphenyl)(N-acetylpiperidin-4-ylidene)
methyl]phenol (2c)
A mixture of 6 (218 mg, 0.71 mmol) and 3 mL of acetic anhydride was heated at 100 C for 6 h. After cooling to room temperature, 10% NaOH aqueous solution was added and stirred. The
mixture was extracted with CH2Cl2, washed with brine, dried over
Na2SO4, and then concentrated. The residue was purified bycolumn chromatography on silica gel with 2:1 n-hexane/AcOEt toafford N-acetylated compound (230 mg, 93%) as a yellow solid.
1
H NMR (395 MHz, CD3OD) d (ppm) 7.02 (d, J = 7.9 Hz, 4H),
6.85 (d, 4H, J = 7.9 Hz), 3.77 (s, 6H), 3.60 (t, 2H, J = 4.0 Hz), 3.55
(t, 2H, J = 4.0 Hz), 2.43 (t, 2H, J = 4.0 Hz), 2.35 (t, 2H, J = 4.0 Hz),2.11 (s, 3H); 13C NMR (100 MHz, CDCl3) d (ppm) 168.7, 158.0,
136.7, 134.5, 132.0, 130.5, 113.2, 54.9, 47.3, 42.7, 31.8; MS (EI)m/z 351 (M+, 100%). The N-acetylated compound was transformed
into 2c by the same method that described the synthesis of 2a; 22%
yield; colorless leaflets (MeOH–CHCl3) mp 255–257 C; 1H NMR(395 MHz, CD3OD) d (ppm) 6.99 (d, 4H, J = 8.0 Hz), 6.70 (d, 4H,
J = 8.0 Hz), 2.52 (t, 4H, J = 8.0 Hz), 2.41 (t, 4H, J = 4.0 Hz), 2.32 (s,3H); 13C NMR (100 MHz, CD3OD) d (ppm) 157.1, 138.0, 135.3,
132.9, 131.9, 115.7, 58.1, 46.1, 32.2; MS (EI) m/z 295 (M+, 100%).
Anal. Calcd for C20H21NO3: C, 74.28; H, 6.55; N, 4.33; Found: C,
74.28; H, 6.58; N, 4.25.
4.2.6. 4-[(4-Hydroxyphenyl)(N-ethyloxycarbonyl-piperidin-4ylidene)methyl]phenol (2d)Compound 2d was prepared by the same method that described
the synthesis of 2a; 90% yield; a yellow prisms (CH2Cl2) mp 105 C;
1
H NMR (395 MHz, CDCl3) d (ppm) 6.96 (d, 4H, J = 7.9 Hz), 6.75 (d,4H, J = 7.9 Hz), 4.14 (q, 2H, J = 4.0 Hz), 3.48 (t, 4H, J = 4.0 Hz), 2.34
(t, 4H, J = 4.0 Hz), 1.26 (t, 3H, J = 7.9 Hz); 13C NMR (100 MHz,
CD3OD) d (ppm) 157.3, 157.2, 139.0, 135.2, 133.0, 131.9, 115.8,
62.7, 46.4, 32.6, 15.0; MS (EI) m/z 353 (M+, 100%). Anal. Calcd for
C21H23NO4: C, 71.37; H, 6.56; N, 3.96; Found: C, 71.11; H, 6.59;
N, 3.93.
4.2.7. N-Acetyl-2,2,6,6-tetramethyl-4-piperidone (8)A mixture of 2,2,6,6-tetramethyl-4-piperidone (4.91 g,
31.6 mmol) and 20 mL of acetic anhydride was heated at 100 C
for 7 h. Solvent was removed, 10% NaOH aqueous solution wasadded, and the mixture was stirred. The mixture was extractedwith CH2Cl2, washed with brine, dried over Na2SO4, and then concentrated. The residue was purified by column chromatography on
silica gel with 3:1 n-hexane/AcOEt to afford the title compound 8(5.49 g, 88%) as a colorless liquid. 1H NMR (395 MHz, CDCl3) d(ppm) 2.59 (s, 4H), 2.23 (s, 3H), 1.54 (s, 12H); MS (EI) m/z 197(M+), 140 (100%).
4.2.8. Methyl 4-[(4-methoxyphenyl)(2,2,6,6-tetramethylpiperidin-4-ylidene)methyl]phenoxide (9)Compound 9 was prepared by the same method that described
the synthesis of 5; 75% yield; 1H NMR (395 MHz, CD3OD) d (ppm)
7.07 (d, 4H, J = 8.0 Hz), 6.82 (d, 4H, J = 8.0 Hz), 3.78 (s, 6H), 2.14
(s, 4H), 1.18 (s, 12H); 13C NMR (CD3OD) d (ppm) 157.8, 137.3,
135.4, 132.4, 130.2, 113.4, 55.1, 52.8, 43.7, 31.2; MS (EI) m/z 365(M+), 98 (100%).
4.2.9. 4-[(4-Hydroxyphenyl)(2,2,6,6-tetramethylpiperidin-4ylidene)methyl]phenol (3a)Compound 3a was prepared by the same method that described
the synthesis of 2a; 82% yield; colorless cubes (MeOH–CHCl3) mp

1093

110–112 C; 1H NMR (395 MHz, CD3OD) d (ppm) 6.96 (d, 4H,
J = 8.0 Hz), 6.70 (d, 4H, J = 8.0 Hz), 2.18 (s, 4H), 1.18 (s, 12H); 13C
NMR (100 MHz, CD3OD) d (ppm) 182.2, 176.4, 157.2, 140.4,
134.7, 131.9, 115.7, 58.4, 46.1, 30.3, 28.4; MS (EI) m/z 379 (M+),
224 (100%). Anal. Calcd for C22H27NO2 0.1 H2O: C, 77.88; H, 8.08;
N, 4.13. Found: C, 77.92; H, 8.19; N, 4.10.
4.2.10. 4-[(4-Hydroxyphenyl)(2,2,6,6-tetramethyl-N-acetylpiperidin-4-ylidene)methyl]phenol (3b)The product obtained from McMurry coupling reaction of compound 9 with 4,40 -dimethoxybenzophenone was transformed into
3b by the same method that described the synthesis of 2a; 43%
yield over two steps; colorless cubes (Et2O) mp 138–140 C; 1H
NMR (395 MHz, CD3OD) d (ppm) 6.93 (d, 4H, J = 7.9 Hz), 6.72
(d, 4H, J = 7.9 Hz), 2.64 (s, 4H), 2.17 (s, 3H), 1.45 (s, 12H); 13C
NMR (100 MHz, CD3OD) d (ppm) 182.2, 176.4, 157.2, 140.4,
134.7, 131.9, 115.7, 58.4, 46.1, 30.3, 28.4; MS (EI) m/z 379 (M+),
224 (100%). Anal. Calcd for C24H29NO3: C, 75.96; H, 7.70; N, 3.69.
Found: C, 75.91; H, 7.83; N, 3.73.
4.3. ERa binding assay
The ligand binding affinity of ERa was determined by the previously reported method.17 Briefly, ERa was diluted with an assay
buffer, which contains 20 mM Tris–HCl, 0.3 M NaCl, 10 mM 2-mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM EDTA,
and incubated with 4 nM [6,7-3H]17b-estradiol in the presence orabsence of an unlabeled competitor at 4 C for 16 h. The incubation
mixture was absorbed by suction onto a nitrocellulose membranethat had been soaked in assay buffer. The membrane was washedtwice with buffer, and then with 25% EtOH in distilled water.
Radioactivity that remained on the membrane was measured in
Atomlight using a liquid scintillation counter.
4.4. MCF-7 cell proliferation assay
The human breast adenocarcinoma cell line, MCF-7 wasroutinely cultivated in DMEM supplemented with 10% FBS,
100 IU/mL penicillin and 100 mg/mL streptomycin at 37 C in a
5% CO2 humidified incubator. Before an assay, MCF-7 cells were
switched to DMEM (low glucose phenol red-free supplemented
with 5% FBS, 100 UI/mL penicillin and 100 mg/mL streptomycin).
Cells were trypsinized from the maintenance dish with phenol
red-free trypsin–EDTA and seeded in a 96-well plate at a densityof 2000 cells per final volume of 100 lL DMEM supplemented with
5% FBS, 100 UI/mL penicillin and 100 mg/mL streptomycin. After
24 h, the medium was exchanged into 90 lL of fresh DMEM and
10 lL of the drug solution, supplemented with serial dilutions of
3b or DMSO as dilute control in the presence or absence of 1 nM
E2, was added to triplicate microcultures. Cells were incubatedfor 5 days, and medium with 3b or DMSO as dilute control in thepresence or absence of 1 nM E2 was exchanged once after 3 days.
At the end of the incubation time, proliferation was evaluated byusing the WST-8. 10 nM of WST-8 was added to microculturesand cells were incubated for 4 h. The absorbance at 450 nm wasmeasured. This parameter relates to the number of living cells in
the culture.
4.5. Metabolism study of compounds 2c and 3b in rat liver S9
fractions
For the evaluation of metabolic stability, 40 lL of compound
was incubated with 1 mg/mL pooled rat liver S9 fractions in
0.1 M potassium phosphate buffer (pH 7.4) containing 3.3 mM
MgCl2, 1.3 mM NADP+, 3.3 mM glucose-6-phosphate, and
0.4 U/mL glucose-6-phosphatedehydrogenase at 37 C for 0, 5, 10,— 399 —

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M. Sato et al. / Bioorg. Med. Chem. 24 (2016) 1089–1094

20, 30, 40, and 50 min. The incubation was stopped by the precipitation of S9 enzymes with the same volume of cold acetonitrile
containing corticosterone as the internal standard. The percentage
of unmetabolized test compound at the different time points wasestimated from the peak area in HPLC spectrum.
Acknowledgments
This research was supported by a Grant-in-Aid of Strategic
Research Program for Private Universities (2010–2014), a Grantin-Aid for Young Scientists (B) (No. 21790116), and a Grant-inAid for Scientific Research (C) (No. 26460151) from the Ministryof Education, Culture, Sports, Science, and Technology, Japan.
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Behavioural Brain Research 297 (2016) 315–322

Contents lists available at ScienceDirect

Behavioural Brain Research
journal homepage: www.elsevier.com/locate/bbr

Research report

BE360, a new selective estrogen receptor modulator, produces
antidepressant and antidementia effects through the enhancement ofhippocampal cell proliferation in olfactory bulbectomized mice
Osamu Nakagawasai a,∗ , Wataru Nemoto a , Hiroshi Onogi b , Takahiro Moriya c ,Jia-Rong Lin a , Takayo Odaira a , Fukie Yaoita a , Takumi Ogawa d , Kiminori Ohta d ,Yasuyuki Endo d , Koichi Tan-No aa

Department of Pharmacology, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan
Faculty of Health Science, Tohoku Fukushi University, 1-8-1 Kunimi, Aoba-ku, Sendai 981-8522, Japanc
Department of Cellular Signaling, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba 6-3, Aramaki, Aoba-ku, Sendai 980-8578, JapandFaculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University, 4-4-1 Komatushima, Aoba-ku, Sendai 981-8558, Japanbh i g h l i g h t s

g r a p h i c a l

a b s t r a c t• BE360 showed beneﬁcial effects overdepressive-like behavior and cognitive deﬁcit in OBX mice.
• BE360 enhanced hippocampal cellproliferation in OBX mice.
• BE360 increased expression of pCREB
and BDNF in hippocampus.
• BE360 has antidepressant andantidementia effect by hippocampal neurogenesis via CREB/BDNF
signaling pathways.

a r t i c l e

i n f oArticle history:
Received 2 May 2015
Received in revised form 2 September 2015
Accepted 15 October 2015
Available online 20 October 2015
Keywords:
Depression
MemoryCarborane compound
Olfactory bulbectomy
Cell proliferation
Selective estrogen receptor modulatora b s t r a c t
We have reported that the carborane compound BE360 is a novel selective estrogen receptor modulator and new therapy option for osteoporosis. The aim of this study was to explore the effects andunderlying mechanisms of BE360 on depressive-like behavior and memory impairment in the olfactory bulbectomized (OBX) mice, an experimental animal model of depression and dementia. BE360 was
administered subcutaneously to mice using a mini-osmotic pump for 2 weeks. Depressive-like behavior
was measured as the reduced intake of a sweet solution in the sucrose preference test. Short-term memory was assessed using the Y-maze test. Cell proliferation was assessed by the analysis of cells expressing
5-bromo-2� -deoxyuridine (BrdU) uptake. The expression of phosphorylated cyclic-AMP response elementbinding protein (pCREB) and brain-derived neurotrophic factor (BDNF) were measured by immunoblot.
The depressive-like behavior and memory impairment in OBX mice were improved by the chronic treatment with BE360. Immunohistochemical analysis showed that the number of BrdU-positive cells in thedentate gyrus of the hippocampus signiﬁcantly decreased in OBX mice whereas they increased after
the chronic treatment with BE360. Immunoblotting studies revealed that pCREB and BDNF were significantly increased in the hippocampus of OBX mice treated with BE360. The present study has shownthat BE360 has antidepressant and antidementia effects characterized by hippocampal cell proliferation
potentially activated via CREB/BDNF signaling pathways. These results indicate that BE360 may havevaluable therapeutic potential against depression and neurodegenerative diseases.
© 2015 Elsevier B.V. All rights reserved.

∗ Corresponding author. Fax: +81227270123.

E-mail address: osamun@tohoku-pharm.ac.jp (O. Nakagawasai).

http://dx.doi.org/10.1016/j.bbr.2015.10.033
0166-4328/© 2015 Elsevier B.V. All rights reserved.

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1. Introduction

2.2. Estrous cycle monitoring

Estrogens play important roles in numerous health issues
including cardiovascular disease, osteoporosis, depression, cognition, and sexual function [1,2]. For example, several of thesesystem dysfunctions including osteoporosis, depression and cognitive deﬁcits frequently arise during the postmenopausal period.
Recently, Hirata et al. have reported that the carborane compound
1,2-bis(4-hydroxypheny)-o-carborane, BE360, showed high binding afﬁnity to the estrogen receptors (ER), ER and ER, andrecovered bone loss in ovariectomized (OVX) and orchidectomized(ORX) mice, while not affecting the sex organs [3]. We have thus
suggested that this novel selective estrogen receptor modulator(SERM) BE360 could represent a new therapy option for osteoporosis.
Accumulating data conﬁrms the existence of olfactory dysfunctions in brain diseases, including Alzheimer’s Disease (AD) anddepression, much of which appear at early stages [4]. In rodents,
olfactory bulbectomy (OBX) leads to numerous behavioral deﬁcits,
including cognitive deﬁcits and anhedonia that are used to model
major depression, but may also represent a valuable tool in the
study of neurodegenerative disorders like AD [5–7]. Indeed, OBX
has been reported to decrease hippocampal neurogenesis, a putative pathogenic mechanism in AD and depression [8,9].
Some SERMs such as tamoxifen and raloxifene are neuroprotective in a variety of experimental models of neurodegeneration,
reduce anxiety and depression, promote cognition and modulate
synaptic plasticity in the hippocampus of rodents [10–12]. Estradiol modulate hippocampus-related behaviors and cognition byenhancing neurogenesis in the dentate gyrus (DG) of adult animals [13]. In this region, as in the subventricular zone, neurogenesis
continues into adulthood. Estrogens increase the release of brainderived neurotrophic factor (BDNF) from the DG. BDNF is a potent
modulator of neuronal functions in the hippocampus, including
neurotransmission, memory formation and neurogenesis [14]. It
was shown that estrogen-mediated neurogenesis following global
ischemia requires the activation of the cyclic-AMP response element binding protein (CREB) pathway [15–17]. The CREB is an
upstream transcription factor for BDNF expression. Thus, the effects
of estrogens in hippocampal neurogenesis involve the CREB-BDNF
signaling cascade.
In the present study, we examined the effects of a novel carborane compound, BE360, on depressive-like behavior and cognitive
impairment in experimental animal model of dementia and depression, the OBX female mice [5]. In a ﬁnal set of experiments to
identify possible mechanisms involved in the increase in cell proliferation, we assessed the effect of BE360 on the expression of theneurotrophic factor BDNF and the transcription factor CREB.

To determine the phase of the estrous cycle for each female,vaginal smears were taken daily for two consecutive weeks before
operation and on the behavioral testing day. Samples were collected by inserting the tip of a clean glass pipette ﬁlled with 10 l ofsaline into the mouse’s vagina. One drop of ﬂuid was collected fromeach mouse, placed onto a glass slide, air dried, and then stainedwith Giemsa‘s solution. The cell morphology was assessed under alight microscope. The metestrus phase was deﬁned as the presence of approximately equal proportions of nucleated epithelial
cells, non-nucleated corniﬁed epithelial cells and leukocytes. The
diestrus phase was deﬁned as a minimum amount of cells, including leukocytes and occasional epithelia. The proestrus phase wasdeﬁned by the presence of more than 75% nucleated epithelial cells.
The estrus phase was deﬁned as the presence of more than 75%
non-nucleated corniﬁed epithelial cells. Only females showing four
consecutive regular estrous cycles before operation were used inthis study.
2.3. Olfactory bulbectomized miceMice, 10 weeks of age, were anesthetized with sodium pentobarbital were placed in a stereotaxic frame. The scalp was incised
and two holes were drilled to expose the olfactory bulb (OB), which
was then bilaterally aspirated using a suction pump. All animals
were sacriﬁced at the end of the experiment and the lesions wereveriﬁed visually. It was conﬁrmed that at least two thirds of the
OB had been removed and that some parts of the olfactory nuclei
also had also been lesioned. If the lesion was either not extensive
enough or extended to the cortex, data from these animals were
excluded. Sham operations were performed in the same manner
but without the removal of the OB. Behavioral tests (sucrose preference test and Y-maze test) were performed on the 28th day after
OBX surgery.
2.4. Drugs
17 -estradiol (E2; Sigma–Aldrich, St.-Louis, MO, USA), Tamoxifen (TAM; Sigma–Aldrich), Raloxifen hydrochloride (RAL; LKTLaboratories, Inc., St. Paul, MN, USA) and BE360 were dissolvedin polyethyleneglycol-300 (Wako Pure Chemical Industries Ltd.,
Osaka, Japan). BE360 was synthesized in our laboratory as reported
in our previous studies [18]. These compounds were administeredusing a mini-osmotic pump (model 2002; Alza Co., Paolo Alto, CA,
USA) that was implanted subcutaneously on the back of the mouse
2 weeks after OBX surgery.
2.5. Sucrose preference test

2. Materials and methods
All experiments were performed according to the Guide for Careand Use of Laboratory Animals at Tohoku Pharmaceutical University.

2.1. Animals
Female ddY mice, 8 weeks of age, were obtained from Japan SLC(Hamamatsu, Japan) and acclimated for 2 weeks. The mice weremaintained under conditions of constant temperature (23 ± 1 ◦ C)
and humidity (55 ± 5%) on a 12 h/12 h light-dark cycle (light from 9
to 21 h; dark from 21 to 9 h). The mice had free access to food andwater throughout the experimental period.

This experiment was conducted for four days, starting 28 days
post OBX surgery. The training phase lasted at least 24 h and took
place before the testing phase to allow the animals to adapt to
the novel solution. When starting the training phase, animals weretransferred into single housing with free access to food and twobottles, one of which contained 1% sucrose solution and the second
bottle contained tap water. To prevent possible effects of side preference in drinking, the position of the bottles was switched twice aday. Bottles were weighed to record the consumption of tap waterand sucrose ﬂuid every morning (9:00–11:00). A sucrose preference
score was calculated using the following formula and the averageof the four days was calculated. % preference = (sucrose intake/total
intake) × 100. Reduction of sucrose preference, i.e., anhedonia, is avalidated index of a depression-like state in animals [19].

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317

Fig. 1. Effects of estrogen receptors ligands on OBX mice in the sucrose preference test. * p < 0.05 vs. sham + vehicle group. # p < 0.05 vs. OBX + vehicle group.

2.6. Y-maze test
The Y-maze apparatus consisted of three compartments (3 cm(W) × 40 cm (D) × 25 cm (H)) radiating out from the center. The
mice were placed in one of the compartments and allowed to movefreely for 8 min. Experiments were performed at a light intensityof 35 lux. An arm entry was deﬁned as three legs entering one of
the arms, and the sequence of entries was manually recorded. An
alternation was deﬁned as entry into all three arms on consecutive
choices. Thus, the maximum number of alternations was the total
number of entries minus 2, and the percent alternation was calculated as (actual alternations/maximum alternations) × 100. The
percent alternation was designated as the spontaneous alternation
behavior of the mouse, and was taken as a measure of short-term
memory.
2.7. Immunohistochemistry
On days 27 after OBX surgery, 5-bromo-2� -deoxyuridine (BrdU)(Sigma–Aldrich; 75 mg/kg i.p.) was injected three times once every
two hours for a total of three injections. Eighteen hours after
the last BrdU injection, animals were deeply anesthetized withsodium pentobarbital and intracardially perfused with 15 ml of
4 ◦ C phosphate-buffered saline (PBS), followed by 45 ml of 4%paraformaldehyde (PFA; Sigma–Aldrich) in 0.1 M PBS. Brains were
post-ﬁxed in 4% PFA-0.1 M PBS for 1 h at 4 ◦ C, followed by immersion in 20% sucrose-0.1 M PBS for 48 h. The brains were cut into
40 m sections that included the dorsal DG from bregma −1.40 mm
to −2.00 mm using a cryostat (MICROM HM560, Microm International GmbH, Walldorf, Germany). Frozen sections were mounted
on glass slides (Matsunami Glass, Osaka, Japan). Sections weretreated with HCl (2 N) at 37 ◦ C for 30 min, followed by neutralization with sodium borate buffer (0.15 M) at room temperature,
twice for 10 min each. After three 5 min washes, the sections were

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incubated with PBS containing 1% normal goat serum and 0.3% Triton X-100 (PBSGT) at room temperature for 2 h. Then, the sections
were incubated overnight at 4 ◦ C with rat anti-BrdU monoclonal
antibody (1:100; Harlan SeraLab, Loughborough, UK) and mouseanti-NeuN monoclonal antibody (1:500; Millipore, Temecula, CA,
USA). Sections were washed then incubated for 2 h at room temperature with goat anti-rat IgG Alexa Fluor 568 (1:200; Molecular
Probes, Eugene, OR, USA) and goat anti-mouse IgM Alexa Fluor 488(1:500; Molecular Probes) in PBSGT. Finally, sections were washedand coverslipped with Dako ﬂuorescent mounting medium (Dako,
Carpinteria, CA, USA). Immunoﬂuorescent images were analyzed
using a confocal laser-scanning microscope (A1Rsi: Nikon, Tokyo,
Japan). Four sections per mouse were used, and two ﬂuorescentimages (640 × 640 m) of DG region of the hippocampus wereobtained from each section. The number of BrdU -positive cells wascounted in each image. Non-speciﬁc binding of the secondary antibodies was barely detectable when tested in the absence of primary
antibodies. A mean value of eight images was used for each mouse.
Each group contained 8–10 mice.
2.8. Western blotting
BE360-treated mice were used for Western blotting experiments. Twelve mice were divided into four groups (Sham + vehicle,
Sham + BE360, OBX + vehicle, and OBX + BE360). The mice weresacriﬁced by decapitation after 2 weeks of vehicle or drug administration. After sacriﬁce, the brains were immediately removed
and washed with ice-cold PBS (pH 7.4). Then, 1 mm-thick coronal sections were cut using a mouse brain slicer (Muromachi
Kikai, Tokyo, Japan) and immersed into PBS. The hippocampi were
removed, frozen in liquid nitrogen, and stored at −80 ◦ C until use.
The samples were homogenized in 150 l of CelLyticTM MT Cell
Lysis Reagent (Sigma–Aldrich) and centrifuged at 15,000 × g for
15 min at 4 ◦ C. Supernatants were collected and their protein con-

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O. Nakagawasai et al. / Behavioural Brain Research 297 (2016) 315–322

Fig. 2. Effects of estrogen receptors ligands on the impairment of spontaneous alternation behavior induced by OBX in the Y-maze test. * p < 0.05 vs. sham + vehicle group.
#p < 0.05 vs. OBX + vehicle group.

centration determined using the Advanced Protein Assay Reagent(Cytoskeleton Inc., USA). Supernatants were then diluted in 4×
Laemmli sample buffer (300 mM Tris–HCl (pH 6.8), 8% SDS, 40%
glycerol, 12% 2-mercaptoethanol, 0.012% bromophenol blue), andincubated at 95 ◦ C for 10 min. Aliquots of the obtained extracts(30 g of proteins/well for each extract) were loaded onto a
10% SDS–polyacrylamide gel. After electrophoresis, proteins weretransferred to a PVDF membrane, which was then incubated withblocking solution [10 mM Tris–HCl (pH 7.4), 100 mM NaCl, 0.01%
Tween20 and 5% skim milk] for 1 h and probed overnight at 4 ◦ Cwith antibodies against rabbit pCREB (1:1000, Cell Signaling Technology Inc., USA) or rabbit BDNF (1:1000, Abcam Ltd., UK) and rabbit
-actin (1:1000, Cell Signaling Technology Inc.). The membrane
was then washed with blocking solution without milk, incubatedwith horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Cell Signaling Technology) for 2 h followed by visualizationof the immunoreactive species with the ECL select Western blotting
detection reagent (Amersham Life Sciences, USA). The densities ofthe bands were analyzed by densitometry (Image-J 1.43 , National
Institute of Health).

2.9. Statistical analysis
Results are expressed as mean ± standard error of the mean
(SEM). The signiﬁcance of differences was determined by a oneway analysis of variance (ANOVA), followed by Fisher’s PLSD test

for multigroup comparisons, respectively. p < 0.05 represented asigniﬁcant difference.

3. Results
3.1. Effect of estrous cycle on behavior
Due to the small size of mouse samples for each of proestrus,
estrus, metestrus, or diestrus phases at the time of testing (n < 5 perphase), we could not verify the inﬂuence that individual phasesmight have on behavior. To enable a preliminary evaluation of
the inﬂuence of ovarian hormones, female sham and OBX mice
were split into two groups: proestrus-estrus (high ovarian hormones) and metestrus-diestrus (low ovarian hormones) to increase
stastical power [20–22]. In this study, we have used mice in themetestrus-diestrus phase, except for the estradiol treated group.
Effects of BE360 on sucrose preference in OBX mice Administration of RAL and BE360, but not E2 nor TAM, signiﬁcantly increased
the preference for sucrose in OBX mice as compared to the vehicle [E2: F(3, 31) = 4.36, p = 0.01 by one-way ANOVA, p < 0.05 by
Fischer’s PLSD, Fig. 1a; TAM: F(3, 29) = 3.62, p = 0.02 by one-way
ANOVA, p < 0.05 by Fischer’s PLSD, Fig. 1b; RAL: F(3, 32) = 3.82,
p = 0.02 by one-way ANOVA, p < 0.05 by Fischer’s PLSD, Fig. 1c;
BE360: F(3, 35) = 3.85, p = 0.02 by one-way ANOVA, p < 0.05 by Fischer’s PLSD, Fig. 1d]. Moreover, in sham mice, sucrose preference
was not increased by E2, TAM, RAL or BE360 compared to vehicle.

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319

Fig. 3. Effects of estrogen receptors ligands on hippocampal cell proliferation in OBX mice. (a) Indicate NeuN+ (Green) and BrdU+ (Red) -postive cells in DG regions of the
hippocampus. (b) Indicate quantitative analysis of the number of BrdU-positive cells in DG regions of the hippocampus. Numbers in parentheses indicate the number ofanimals in each group. * p < 0.05 vs. sham + vehicle group. # p < 0.05 vs. OBX + vehicle group. (For interpretation of the references to color in this ﬁgure legend, the reader isreferred to the web version of this article.)— 405 —

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Fig. 4. Effects of chronic BE360 treatment on the hippocampal immunocontent of phosphorylated CREB (panels a and b) and BDNF (panels c and d) in OBX mice. Representative
images of western blots probed with antibodies against phosphorylated CREB (P-CREB), BDNF and -actin in the hippocampus (upper panels). Densitometric quantitations of
P-CREB and BDNF are shown (lower panels). Data are expressed as fold expression relative to the sham control. N = 4–5 per group. ** p < 0.01 vs. sham + vehicle group. # p < 0.05
and ## p < 0.01 vs. OBX + vehicle group.

3.2. Effects of BE360 on spontaneous alternation behavior in OBX
mice in the Y-maze test

BE360 showed signiﬁcantly higher BDNF levels than the OBX mice
given vehicle [F(3, 8) = 6.28, p < 0.05 by one-way ANOVA, Fig. 4d].

Both the spontaneous alternation behavior and total number ofarms entered were measured in this short-term memory test. The
percentage of alternation behavior signiﬁcantly decreased, without
any change in the total number of arms entered (data not shown),
in OBX compared with sham mice (Fig. 2). Administration of BE360
signiﬁcantly increased the percentage of alternation behavior in
OBX mice [E2: F(5, 45) = 3.44, P = 0.01 by one-way ANOVA, p < 0.05
by Fischer’s PLSD, Fig. 2a; TAM: F(5, 45) = 2.50, p = 0.04 by oneway ANOVA, p < 0.05 by Fischer’s PLSD, Fig. 2b; RAL: F(5, 37) = 3.54,
p = 0.01 by one-way ANOVA, p < 0.05 by Fischer’s PLSD, Fig. 2c;
BE360: F(3, 30) = 3.73, p = 0.02 by one-way ANOVA, p < 0.05 by Fischer’s PLSD, Fig. 2d].

4. Discussion

3.3. Effects of BE360 on the decrease in cell proliferation in thedentate gyrus of OBX mice
Confocal microscopy analysis demonstrated the presence of
BrdU-positive cells (red) in the granular cell layer of the DG in mice.
The number of BrdU-positive cells was signiﬁcantly decreased in
OBX mice. However, chronic treatments of these mice with estrogen receptors ligands increased the number of BrdU-positive cells
compared with vehicle [E2: F(3, 31) = 4.53, p = 0.01 by one-way
ANOVA, p < 0.05 by Fischer’s PLSD; RAL: F(3, 30) = 6.77, p < 0.01 by
one-way ANOVA, p < 0.05 by Fischer’s PLSD; BE360: F(3, 31) = 80.32,
p < 0.01 by one-way ANOVA, Fig. 3b]. BrdU-positive cells wereobserved predominately in the deepest layers of the subgranularzone (SGZ) and hilus of hippocampal DG (Fig. 3a).
3.4. Effects of BE360 on phospho-CREB and BDNF levels in OBX
mice
Chronic administration of BE360 in OBX mice signiﬁcantly
increased CREB phosphorylation in the hippocampus when compared with vehicle-treated OBX mice [F(3, 8) = 19.89, p < 0.01 by
one-way ANOVA, p < 0.01 by Fischer’s PLSD, Fig. 4b]. The BDNF
immunocontent in vehicle-treated OBX mice was signiﬁcantly
decreased compared with the sham group. The OBX mice given

We have previously demonstrated that OBX female mice showa dysfunction in the brain’s reward system when submitted tothe sucrose preference test [7]. This validated behavioral test for
depression was used in the present study to evaluate anhedonic
response [23]. Here, chronic treatment with raloxifene improved
the anhedonic response in OBX mice. Interestingly, we observedthat chronic treatment with BE360 also improved the reduction
in sucrose preference in OBX female mice. Thus, the fact that
BE360 treatment was efﬁcient in behavioral test above supports
the hypothesis that this compound may be useful in modulating
the depressive symptoms associated with major depression.
We have previously shown that OBX induces memory impairments in both the passive avoidance and Y-maze tasks [5,6]. In thepresent study, we demonstrate that a chronic treatment with BE360
improved the OBX-induced memory impairment in the Y-maze
test. The Y-maze task is largely dependent on working memory,a form of short-term memory [24]. Thus, it is likely that BE360 has
the potential to improve working memory deﬁcits.
Failure in adult hippocampal neurogenesis is accompanied bycognitive deﬁcits [25,26] and depression [27,28]. It is known that
estradiol and SERMs can increase cell proliferation in the DG ofthe hippocampus [29–31], and that hippocampal neurogenesis canlead to changes in cognitive function and mood [32]. In this study,
we also demonstrated that estradiol and RAL increases cell proliferation in the DG. Chronic treatment with antidepressants enhances
hippocampal neurogenesis in OBX rodents [33,34]. Conversely,
X-irradiation of the hippocampus, which reduces hippocampal
neurogenesis, abolishes the behavioral and proliferative effects ofmany different antidepressants [35]. The OBX mice model exhibit
not only characteristics of depression but also characteristics ofAD, such as amyloid- (A) accumulation in the neocortex andhippocampus, cholinergic neuron loss in the basal forebrain, andspatial memory impairment [5,36]. Some studies have revealed adysregulation of adult neurogenesis in AD patients and in various
AD mouse models [37–39]. In the present study, BE360 enhanced
hippocampal cell proliferation in OBX mice. The enhancement— 406 —

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of hippocampal cell proliferation by the chronic administrationof BE360 could underlie the beneﬁcial effects of BE360 on OBXinduced depressive-like behavior and cognitive deﬁcits.
Previous reports have pointed to a role for BDNF in the control of hippocampal neurogenesis [40–42]. For example, infusionof exogenous BDNF into the DG leads to increased genesis of granule cells [43]. BDNF has been shown to promote the survival anddifferentiation of neurons during development, in adult brain andin cultured cells [44]. Furthermore, BDNF increases the synthesis ofmany proteins used in translation [45] which is required for memory formation [46] and reduce depression [47]. Moreover, pCREB isessential for memory consolidation and storage in the hippocampus [48] and increases in the rat hippocampus following a chronic
administration of antidepressants [49]. Using transgenic mice overexpressing a dominant negative mutant of CREB, Nakagawa et al.
reported a signiﬁcant decrease in BrdU-labeled cells suggestingthat CREB induces a positive effect on cell proliferation in the adulthippocampus [50]. In addition, it has been determined that increasing CREB activity may enhance cell proliferation, memory [51] andreduce depression [52]. It has been suggested that the activationof CREB-BDNF signaling may produce antidepressant and antidementia effects. We showed that BE360 increased the expression of
BDNF and pCREB in the hippocampus. Therefore, it might be suggested that the activation of CREB and BDNF signaling pathways
could mediate the effects of BE360 on cell proliferation. However,
we have not examined whether BE360 shows high binding afﬁnity
to the membrane ER. Further studies are needed to determine themechanism of action of BE360.
The present study has demonstrated that a new carborane compound BE360 has antidepressant and antidementia
effects characterized by hippocampal cell proliferation potentiallymediated through CREB/BDNF signaling pathways. These results
indicate that BE360 may have a valuable therapeutic potential
against depression and neurodegenerative diseases such as AD.
Conﬂicts of interest
None
Acknowledgments
This study was supported in part by JSPS KAKENHI (Grant Number 26460102) and Matching Fund Subsidy for Private Universities
from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The authors would like to thank Ms. Chie Saito, Ms.
Rei Fukazawa, Ms. Anna Hoshi and Mr. Toshihiro Sasaki of Tohoku
Pharmaceutical University for their technical assistance.
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Bioorganic & Medicinal Chemistry Letters 25 (2015) 4174–4178

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Lettersjournal homepage: www.elsevier.com/locate/bmcl

Design and synthesis of carborane-containing estrogen receptor-beta(ERb)-selective ligands
Kiminori Ohta a,⇑, Takumi Ogawa a, Akifumi Oda b, Asako Kaise a, Yasuyuki Endo a,⇑abFaculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan
Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japana r t i c l e

i n f oArticle history:
Received 9 July 2015
Revised 1 August 2015
Accepted 4 August 2015
Available online 7 August 2015
Keywords:
Carborane
Estrogen receptor
Subtype selectivity
Docking studya b s t r a c t
Candidates for highly selective estrogen receptor-beta (ERb) ligands (6a–c, 7a–c, 8a and 8b) were
designed and synthesized based on carborane-containing ER ligands 1 and 2 as lead compounds.
Among them, p-carboranylcyclohexanol derivatives 8a and 8b exhibited high ERb selectivity in competitive binding assay: for example, 8a showed 56-fold selectivity for ERb over ERa. Docking studies of 8aand 8b with the ERa and ERb ligand-binding domains (LBDs) suggested that the p-carborane cage ofthe ligands is located close to key amino acid residues that influence ER-subtype selectivity, that is,
Leu384 in the ERa LBD and Met336 in the ERb LBD. The p-carborane cage in 8a and 8b appears to playa crucial role in the increased ERb selectivity.
 2015 Elsevier Ltd. All rights reserved.

Since estrogen receptor (ER) was cloned in 1986,1 its biological
functions have been extensively studied.2 ER is activated by thebinding of endogenous estrogen, 17b-estradiol (E2, Fig. 1), andmediates the activity of estrogens in the regulation of various
physiological processes, including development and function of
the female reproductive system and maintenance of bone mineraldensity.1,2 Ten years later, a second form of ER, ERb, was discoveredin rat prostate.3 ERb has quite different biological functions fromthe first form of ER, ERa. In general, the two ER subtypes show
divergent roles and sometimes antagonize each other under physiological conditions.4 The therapeutic use of E2 is limited by estrogenic effects and an increased risk of cancer, which seem to becaused by the ERa activation.5 On the other hand, over-expression
or activation of ERb has an anti-proliferative effect on breast cancer
cells.6 Moreover, activation of ERb inhibits proliferation and migration of prostate cancer cell lines and stimulates differentiation of
prostate.7 Recently, George et al. reported that an ERb-selective
agonist delayed the progression of Alzheimer’s disease by including degradation of Ab aggregates.8 Thus, ERb-selective ligands areof interest as potential clinical candidates for several cancers andAlzheimer’s disease, and also as probes for ERb-related molecularbiology.

⇑ Corresponding authors. Tel./fax: +81 22 727 0143 (K.O.); tel.: +81 22 727 0142;
fax: +81 22 275 2013 (Y.E.).
E-mail addresses: k-ohta@tohoku-pharm.ac.jp (K. Ohta), yendo@tohoku-pharm.
ac.jp (Y. Endo).

Carboranes, a group of carbon-containing boron clusters, havebeen studied as boron carriers for boron neutron capture therapy(BNCT), based on their high boron content.9 On the other hand,
we have focused on the carborane cage as a novel hydrophobic
pharmacophore for drug development, and have reported carborane derivatives with various biological activities.10 During ourstudies on carborane-containing ER ligands, we found that many
carboranyl phenol derivatives showed more potent ER-binding
affinity than E2 because the high hydrophobicity and sphericalstructure of carborane result in strong binding to the hydrophobic
pocket of the ER ligand-binding domain (LBD).10,11
1-(4-Hydroxyphenyl)-12-hydroxymethyl-p-carborane (BE120,
1, Fig. 1) shows greater binding affinity for ERa and more potent
estrogenic activity than E2.10 Recently, we also reported that introduction of a fluorine atom or an alkyl group on a boron atom of the
carborane cage enhanced the ERb selectivity of the parent estrogenic carboranyl phenols.12 For example, BE701 (2, Fig. 1), whichcontains a B-fluorinated m-carborane cage, showed 8.5 times
greater binding affinity for ERb than for ERa, although its selectivity is still not sufficient for development of drug candidates or biological tools.
The two ERs have quite similar LBDs, but in the hydrophobic
pocket of the LBDs, Leu384 and Met421 of ERa are substituted
by Met336 and Ile373, respectively, in ERb.13 E2 is accommodated
equally well by the two LBDs because it has little interaction withthese amino acid residues.13 Several ERb-selective ligands, including 2, have been designed based on the idea of specifically decreasing the affinity for ERa by introducing ligand substituents that

http://dx.doi.org/10.1016/j.bmcl.2015.08.007
0960-894X/ 2015 Elsevier Ltd. All rights reserved.

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K. Ohta et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4174–4178
H

OH
OH

unmarkedvertices = BH
=C

a

F
HO

HO

E2

H

HO

BE120 (1)

OH

9

3

9

HO

4

F

b
HO

5

6a

Figure 1. Structures of 17b-estradiol (E2), carborane-containing ER ligands 1 and 2,
and ERb-selective ligands 3–5.

OH

9

14

interfere sterically with Met421 of ERa (Fig. 1). For example,
compounds 315 and 416 have a methyl group and a fluorine atom
at the ortho position of the phenolic hydroxyl group, respectively;
these substituents moved the phenol ring closer to Met336 of ERb
and facilitate C-H. . .p interaction, resulting in preferential binding
to ERb. An endogenous dihydrotestosterone metabolite, 3b-androstanediol,17 and 5,18 which contains an androstane skeleton,show high ERb-selectivity. The androstane skeleton appears to
intrinsically favor binding to ERb rather than to ERa, and an axial
substituent such as the vinyl group of 5 at the ring junction interacts sterically with Leu384 of ERa.18 In general, chemical modifications of the phenol ring of ER ligands appear to greatly influence
the ER subtype selectivity.
Therefore, based on the structures of the selective ERb ligands
3–5, we designed carborane-containing ligands 6a–c, 7a–c, 8a
and 8b. Here, we describe synthesis of 6a–c, 7a–c, 8a and 8b, evaluation of their binding affinity and selectivity for ERa and ERb, anddocking simulation studies of 8a and 8b with both ERs (Fig. 2).
Synthesis of p-carborane-containing o-methylated phenol
derivatives 6a–c is summarized in Scheme 1. p-Carborane wastreated with n-BuLi, and then transformed into C-copper-p-carborane, which was reacted with 2-methyl-4-iodoanisole in the presence of pyridine as a ligand of copper to afford monoarylated
p-carborane (9) and diarylated p-carborane (10) in 41% and 34%
yields, respectively.19 The methoxy group of compound 9 was
demethylated with BBr3 to afford the corresponding phenol derivative 6a in quantitative yield. Compound 9 was treated with n-BuLi,
and then reacted with paraformaldehyde, followed by demethylation with BBr3 to afford the o-methylated BE120 derivative 6b in
97% yield over two steps. Compound 6c was obtained by demethylation reaction of 10 with BBr3 in 93% yield.
Scheme 2 summarizes the synthesis of o-halogenated-m- carboranyl phenol derivative 7. B-Fluoro-m-carboranyl anisole 11, which
was obtained as a synthetic intermediate in our previous study,12
was nitrated with potassium nitrate and concentrated sulfuric acid,followed by catalytic hydrogenation of the nitro group to afford
key intermediate 12 in 77% yield over two steps. The amino groupof 12 was directly transformed into a fluorine atom by treatment
with nitrosonium tetrafluoroborate under Balz–Schiemann
reaction conditions20 and the product was demethylated with
BBr3 to afford o-fluorinated phenol 7a in 23% yield over two steps.
H
R

R
F

HO
CH3

6

HO
X

10

H

N

HO

H3 CO

OH

O
HO

+
H3 CO

BE701 (2)

OH

OCH 3

H
H

7

HO

Figure 2. Structures of the target compounds 6–8.

8

c, b
HO

6b
OH

10

d, b

HO

6c

Scheme 1. Synthesis of o-methylated phenol derivatives 6a–c. Reagents andconditions: (a) n-BuLi, DME, then CuCl, pyridine, 4-iodo-2-methylanisole, 41% for
9, 34% for 10; (b) BBr3, CH2Cl2, 93%–quant; (c) n-BuLi, ether, then paraformaldehyde, 97%.

o-Chlorinated derivative 7b and o-brominated derivative 7c weresynthesized via Sandmeyer reaction21 using tert-butyl nitrite andthe corresponding copper halide, followed by demethylation with
BBr3, in 49% and 29% yield, respectively, over two steps.
Scheme 3 summarizes the synthesis of p-carborane-containing
cyclohexanol derivative 8a and 8b. After treatment of p-carboranewith n-BuLi, the resultant C-lithio-p-carborane was reacted with
1,4-cyclohexanedione monoethyleneketal. The tertiary alcohol
was eliminated and the ketal group was deprotected under acidic
conditions, and then catalytic hydrogenation of the double bondafforded 4-(p-carboranyl)-cyclohexanone 13 in 59% yield overthree steps. Reduction of the carbonyl group of 13 with sodium
borohydride afforded cyclohexanol derivative 8a as a single
trans-isomer.22 A hydroxyl group of 8a was protected with atetrahydropyran (THP) group, and then treatment with n-BuLi, followed by deprotection of THP, afforded completely reduced BE120
derivative 8b in 51% yield over three steps.
Binding affinity for both ERs was evaluated by means of competitive binding assay using [2,4,6,7-3H]17b-estradiol and humanrecombinant ERa and ERb, and the selectivity was estimated fromthe relative binding affinity (RBA) values.23 The binding profiles of
6a–c, 7a–c, 8a and 8b are summarized in Table 1. The parent compounds 1 and 2 showed ERb-selectivity ratios of 1.4 and 8.5,
respectively. o-Methylphenol derivative 6a showed a remarkabledecrease of binding affinity to both ERs, but the hydroxymethyl
derivatives 6b retained high binding affinity for both ERs. Thismay be explained in terms of hydrogen bond formation of thehydroxymethyl group with His524. However, 6a and 6b showedno selectivity. Interestingly, however, symmetric bisphenol-pcarborane 6c showed 10 times higher binding affinity for ERa thanERb. Introduction of a halogen atom into the phenol ring of
m-carboranyl phenol 2 led to decreased binding affinity for ERb.
The binding affinity of o-chlorinated derivative 7b for ERb wasdecreased more than 100 times compared to that of the parent
compound 2, and thus 7b showed ERa selectivity. A bromine atom
is too large to be tolerated at the ortho-position of phenol ring.

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K. Ohta et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4174–4178
H

F
HO

7a

F
H

H

F
H3 CO

c, d

a, b

e, d

F
H3 CO

11

NH2

H

F
HO

12

7b

Cl

f, d

H

F
HO

7c

Br

Scheme 2. Synthesis of o-halogenated phenol derivatives 7a–c. Reagents and conditions: (a) KNO3, concd H2SO4, CH2Cl2, 79%; (b) H2, Pd/C, AcOEt/MeOH, 98%;(c) NOBF4, CH2Cl2, 33%; (d) BBr3, CH2Cl2, 70–89%; (e) tBuONO, CuCl2, CH3CN, 56%; (f) tBuONO, CuBr2, CH3CN, 33%.

H
H

a, b, c

H

H

d
13

O

8a

HO

H

8a

OH

e

f, g
14

THPO

HO

8b

Scheme 3. Synthesis of cyclohexanol derivatives 8a and 8b. Reagents andconditions: (a) n-BuLi, ether, then 1,4-cyclohexanedione monoethylene-ketal,
71%; (b) concd H2SO4, 85%; (c) H2, Pd/C, MeOH, 98%; (d) NaBH4, MeOH, 74%; (e)3,4-dihydro-2H-pyran, TsOH, CH2Cl2, 68%; (f) n-BuLi, ether, then paraformaldehyde,
78%; (g) TsOH, MeOH, 96%.

Various modifications of the phenol ring of m- or p-carboranyl phenol structures failed to enhance the binding affinity for ERb
through C-H  p interaction, as observed in compounds 3 and 4.
Therefore, we concluded that ortho-substitution of carboranyl phenols is unsuitable for increasing ERb selectivity. On the other hand,
we found that p-carboranyl cyclohexanol derivative 8a showednearly 60 times higher binding affinity for ERb than ERa; this is
the highest ERb selectivity among known carborane-containing
ER ligands. As expected, the binding affinity of compound 8b for
both ERs was greatly enhanced by hydrogen bond formation of
the hydroxymethyl group on a carbon atom of the p-carborane
cage, as seen with BE120. However, the ERb selectivity ratio of
8b was only 7. By analogy with 5, we anticipate that introductionof a medium-size substituent onto carbon of the p-carborane cageof 8b would enhance the ERb selectivity. Further structural modifications and biological studies of 8b are in progress.

Table 1
Binding affinity for ERa and ERb and subtype selectivity of the phenol ring modified compounds 6–8
H

H

R

R

OH
F

F

1

HO

HO

2

CH3

HO

6

X

7

HO

8

Relative binding affinitya

Compound

R

X
ERa

ERb

ERb/ERa

6a
6b

H
CH2OH

—
—

6.3
1.2  102

6.3
1.4  102

1.0
1.2

—

83

5.9

7.1  102

F
Cl
Br
—
—
—
—

3.4
2.2
4.5  103
5.2  103
1.4
1.6  102
4.8

13
0.30
2.7  103
0.29
9.8
2.2  102
41

3.8
0.14
0.60
56
7.0
1.4
8.5

CH3

6c
7a
7b
7c
8a
8b
1
2
a

HO

—
—
—
H
CH2OH
—
—

OH

Selectivity

All binding assays were performed in triplicate (n = 3). RBA values are calculated from the IC50 values of E2 and test compounds, taking the binding affinity of E2 as 100.

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K. Ohta et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4174–4178

4177

imidazole ring of histidine are 2.623 Å or 3.536 Å, respectively(Fig. 3D). Although the hydrogen bond between the hydroxymethyl group of 8b and His524 of ERa should be stronger thanthe corresponding hydrogen bond with His475 of ERb, the binding
affinity of 8b with ERb was higher than that with ERa owing to theinteraction with the hydrophobic amino acid residues at the opposite side to Met336.
In conclusion, various carborane derivatives were designed andsynthesized as candidate ERb-selective ligands, and their ER binding affinities were evaluated by means of a competitive binding
assay. Introductions of a methyl group or halogen at the ortho position to the hydroxyl group on the phenol ring had little effect on ER
subtype selectivity. Interestingly, cyclohexanol derivative 8abound to ERb with nearly 60 times greater affinity than to ERa
owing to a decrease in the binding affinity for ERa. Docking studiesof 8a with both ER LBDs suggested effective hydrophobic interactions of 8a with ERb LBD. Further structure–activity relationshipstudies of 8a and ERb-related biological evaluations are in progress. These findings should be helpful for molecular design of further highly ERb-selective carborane-containing ligands.
Acknowledgments
This research was supported by a Grant-in-Aid for theStrategic Research Program for Private University (2015-2019)
and a Grant-in-Aid for Scientific Research (B) (No. 26460151)
from the Ministry of Education, Culture, Sports, Science andTechnology, Japan.
References and notes

Figure 3. Comparisons of docking models of 8a and 8b with ERa and ERb. (A)Binding mode of 8a with the ERa LBD. (B) Binding mode of 8b with the ERa LBD. (C)The arrangements of 8a and Leu384 in ERa LBD (aqua) and 8a and Met336 in ERb
LBD (brown). (D) The arrangements of 8b and His524 in ERa LBD (aqua) and 8b andHis475 in ERb LBD (brown).

Binding modes of 8a and 8b with ERa24 and ERb24 were calculated using a docking program (GOLD 5.2).25 The results for ERa
LBD are shown in Figure 3A and B. The hydrophobic cyclohexylp-carborane skeleton of 8a and 8b was accommodated in thehydrophobic pocket of ERa. The hydroxyl group of 8a forms hydrogen bonds with Arg394 and the backbone carbonyl group of
Leu387 (Fig. 3A), whereas the hydroxyl group of 8b forms a hydrogen bond with Glu353 instead of Leu387. The hydroxymethylgroup of 8b also forms a hydrogen bond with His524, which may
account for the enhanced ERa-binding affinity and different hydrogen-bonding mode of the hydroxyl group. The p-carborane cage of
8a is located close to Leu384 in the ERa LBD and Met336 in the ERb
LBD (Fig. 3C). Since Met336 of ERb squeezes the p-carborane cageof 8a into the hydrophobic pocket, the p-carborane cage
approaches hydrophobic amino acid residues, such as Phe356,
Leu298, Ile373, Ile376, and Phe377, located on the opposite sideto Met336, and this may enhance the ERb-binding affinity. The
hydroxymethyl group of 8b forms hydrogen bonds with His524
in the ERa LBD and His475 in the ERb LBD, and the distances
between oxygen of the hydroxyl group and nitrogen of the

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15106; (b) Malamas, M. S.; Manas, E. S.; McDevitt, R. E.; Gunawan, I.; Xu, Z. B.;
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22. Compound 8a: Colorless leaflets (AcOEt/n-hexane); mp 183.5–185.0 oC; 1H
NMR (400 MHz, CDCl3) d (ppm): 0.90 (m, 2H), 1.09 (m, 2H), 1.40 (tt, J = 2.9 Hz,
12.1 Hz, 1H), 1.50–1.75 (m, 2H), 1.60–3.60 (brm, 10H), 1.80–1.95 (m, 2H), 2.71
(brs, 1H), 3.39 (tt, J = 4.3 Hz, 11. 1 Hz, 1H); MS (EI) m/z 242 (M+), 57 (100%).
23. Ohta, K.; Chiba, Y.; Ogawa, T.; Endo, Y. Bioorg. Med. Chem. Lett. 2008, 18, 5050.
The ligand-binding activity of ERa and ERb was determined by thenitrocellulose filter binding assay method. ERa and ERb (0.5 lg/tube) werediluted with a binding assay buffer (20 mM Tris–HCl, pH 8.0, 0.3 M NaCl, 1 mM
EDTA, pH 8.0, 10 mM 2-mercaptoethanol, 0.2 mM phenylmethylsulfonyl
fluoride) and incubated with 4 nM [6,7-3H]17b-estradiol in the presence orabsence of an unlabeled competitor at 4 C for 16 h. The incubation mixture
was absorbed by suction onto a nitrocellulose membrane that had been soakedin binding assay buffer. The membrane was washed twice with buffer (20 mM
Tris–HCl, pH 8.0, 0.3 M NaCl), and then with 25% ethanol in distilled water.
Radioactivity that remained on the membrane was measured in Atomlightusing a liquid scintillation counter.
24. The 3D structures of human ERa and ERb used in this study were retrieved
from the Protein Data Bank (PDB ID: 1ERE and 2I0G, respectively).
25. Jones, G.; Willett, P.; Glen, R. C. J. Mol. Biol. 1995, 245, 43.

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Bioorganic & Medicinal Chemistry Letters 25 (2015) 3213–3216

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Lettersjournal homepage: www.elsevier.com/locate/bmcl

Synthesis and biological evaluation of novel m-carborane-containing
estrogen receptor partial agonists as SERM candidates
Kiminori Ohta, Takumi Ogawa, Asako Kaise, Yasuyuki Endo ⇑Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japana r t i c l e

i n f oArticle history:
Received 12 May 2015
Revised 25 May 2015
Accepted 27 May 2015
Available online 3 June 2015
Keywords:
Carborane
Estrogen receptor
Partial agonist
SERM

a b s t r a c t
We designed and synthesized novel m-carborane-containing selective estrogen receptor modulator(SERM) candidates using previously reported m-carborane-containing ER partial agonist 1 as the leadcompound. Biological activities were evaluated by means of ERa competitive binding assay and MCF-7
cell proliferation assay. Re-positioning the N,N-dimethylaminoethyloxy group at the para position of 1
to the meta position enhanced the ERa-binding afﬁnity, and 4c showed the highest relative binding
afﬁnity (RBA: 83 vs 17b-estradiol = 100) among the tested compounds. Compound 4b showed the mostpotent ER-agonist activity (EC50: 1.4 nM) and the lowest maximal efﬁcacy (Emax: 50%) in MCF-7 cell proliferation assay. Inhibition of 0.1 nM 17b-estradiol-induced MCF-7 cell proliferation by 4b (IC50: 0.4 lM)
was at least 10 times more potent than that of the lead compound 1.
 2015 Elsevier Ltd. All rights reserved.

Estrogens are involved in regulation of the female and male
reproductive systems, bone metabolism, and the cardiovascularsystem, as well as the central nervous system, and these activities
are expressed through binding to and activation of nuclear estrogen receptor (ER).1 ER has two subtypes (a and b), which show different patterns of tissue expression and mediate two different
signaling pathways: transcriptional regulation and non-genomic
membrane-associated transduction.1 Many of the physiologicaleffects of ER are subtype-speciﬁc. Non-steroidal and non-hormonal
ER antagonists, such as tamoxifen2 and raloxifene,3 are widely used
for treatment of breast cancer (Fig. 1). Tamoxifen and its active
metabolite, 4-hydroxytamoxifen, show ER agonism in endometrium and bone, whereas raloxifene acts as an antagonist in
endothelium and as an agonist in bone.4 Therefore, raloxifene hasno risk for cancer of the female reproductive system, and is used
as a protective agent against osteoporosis in post-menopausal
women.5 Compounds having tissue-speciﬁc ER agonist or antagonist activity are called selective estrogen receptor modulators(SERMs).6 They exhibit considerable functional diversity: for example, bazedoxifene contains an alkylamino side chain, which is critical for agonist and antagonist activities of SERMs, but its biologicalactivities are different from those of tamoxifen and raloxifene.7 In
addition, the binding mode of bazedoxifene to ERa is different
from that of 4-hydroxytamoxifen, as evaluated by docking simulation study.8 The relative agonist/antagonist activities of SERMs,⇑ Corresponding author. Tel.: +81 22 727 0142; fax: +81 22 275 2013.
E-mail address: yendo@tohoku-pharm.ac.jp (Y. Endo).

including ER partial agonists, seem to be controlled by the overall
shape of the ER homodimer formed after ER-ligand binding.
Therefore, binding of different ligands to ER can facilitate or
impede the interaction of ER homodimer with various co-regulators.9 The hydrophobic core structure of SERMs plays an importantrole in determining their elaborate biological and pharmacokinetic
proﬁles. Therefore, development of ER modulators with novelhydrophobic core structures is expected to afford unique SERMs
with distinctive biological properties.
We have developed several ER modulators having a carborane
cage as a novel hydrophobic pharmacophore.10 They exhibit
unique estrogen-related biological activities, different from thoseof the endogenous estrogen 17b-estradiol (E2), or the abovementioned SERMs. BE360, an o-carborane-containing ER
modulator without an alkylamino side chain, showed partial
agonist activity in MCF-7 cell proliferation assay, and it increasedbone density with no effect on the uterus in ovariectomized mice
(Fig. 2).11 That is, BE360 is a carborane-containing SERM. We havealso reported that m-carborane derivative 1 bearing an alkylaminogroup acted as a potent ER partial agonist in ER transactivation
assays (Fig. 2).12 However, the maximal efﬁcacy of 1 is not low,and so there is a risk that 1 might induce breast cancer. SAR studiesof the alkylamino chain of m-carborane derivative 2 revealed thatcompounds with an alkyl, carbamate, or thiocarbamate groupinstead of the alkylamino group acted as ER full agonists.13
Thus, the alkylamino group of 1 is essential for expression of ER
partial agonist activity, as has been observed with other SERM
candidates (Fig. 2).14 Recently, we have reported that the

http://dx.doi.org/10.1016/j.bmcl.2015.05.083
0960-894X/ 2015 Elsevier Ltd. All rights reserved.

— 414 —

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K. Ohta et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3213–3216
H3C
N

N

N
O

N
CH3

HO
O

HO

R = H Tamoxifen
R = OH 4-Hydroxytamoxifen

N
OH

S

HO

Raloxifene

Figure 3. Structures of novel ER partial agonist candidates 4.

Bazedoxifene

9,10-dimethyl-m-carborane cage is effective for obtaining ER partial agonist activity with very low maximal efﬁcacy, as well as
for increasing ER-binding afﬁnity (compound 3, Fig. 2).15 Thismay be due to a geometry change of the alkylamino group remotely induced by steric repulsion between the bulky hydrophobic
structure and amino acid residues surrounding the ER ligand-binding domain (LBD). Although the 9,10-dimethyl-m-carborane cageof 3 seems to be more promising as a hydrophobic structure for
ER partial agonist discovery than m-carborane, preparation of
1,7-diaryl-9,10-dimethyl-m-carborane derivatives is synthetically
difﬁcult in that coupling reaction of 9,10-dimethyl-m-carboranewith aryl iodides affords the products in very low yield.
Therefore, we selected the m-carborane cage as a hydrophobic
structure for the preparation of ER partial agonists and designed
derivatives 4 in which the alkylamino side chain of 1 is transferred
to a neighboring carbon (Fig. 3).
Scheme 1 summarizes the synthesis of m-carborane-containing
ER partial agonist candidates 4. m-Carborane 5 was treated with nBuLi, and then transformed into C-copper-p-carborane, which wasreacted with 4-iodoanisole in the presence of pyridine as a ligandof copper to afford 4-methoxyphenyl-m-carborane 6 in 71%
yield.16 Next, coupling reaction of 6 with TBS-protected iodophenol
under the same conditions, followed by deprotection of the TBS
group, afforded key intermediate diaryl-m-carborane 7 in 71%
yield. Dimethylaminoethyl and dimethylaminopropyl groups wereintroduced into 7 by using the corresponding alkyl halides in 33%
and 24% yields, respectively. Demethylation of 8 with BBr3 afforded
the desired compounds 4a and 4b in 45% and 74% yields, respectively. Compound 7 was reacted with BBr3 to afford bisphenol 9
HO

OH

=C
unmarked = BH
vertices
HO

HO

BE360

17β-estradiol (E2)

ER partial agonist (in vitro)
SERM (in vivo)

HO

CH3
N

R

O

1

ER partial agonist

HO

O

2
ER agonist

H3C

HO

4

ER partial agonist candidatesO
H

Figure 1. Structures of clinically used SERMs.

H3C

n=0~2

O

O

R

O
n

CH3
N

O

CH3
CH3

3

Potent ER partial agonist

Figure 2. Structures of 17b-estradiol (E2) and m-carborane-containing ER
modulators.

in 94% yield; its biological activity was evaluated and comparedwith that of bisphenol 12, an intermediate of 1 (Scheme 1).
Compound 4c was obtained by stepwise synthesis involving two
SN2 reactions of dibromobutane. One bromine atom was changed
to a phenolic hydroxyl group (7) in 62% yield, and the other was
reacted with dimethylamine to afford 11 in 78% yield.
Demethylation of 11 with BBr3 afforded 4c in 34% yield.
ER-binding afﬁnity was evaluated by means of competitive
binding assay using human recombinant ERa and [6,7-3H]
17b-estradiol. Relative binding afﬁnity (RBA) values of the test
compounds are summarized in Table 1.17 Compounds 1 and 12
showed low and high RBA values of 1.5 and 110, respectively,
which are close to the previously reported values of 1.1 and 106,
respectively.12 Although the binding afﬁnity of the parent bisphenol 9 is similar to that of 12, compound 4a with an N,N-dimethylaminoethoxy side chain showed 5 times more potent ERabinding than the corresponding p-substituted derivative 1. Our
previous results showed that extension of the alkylamino chainof 1 has little inﬂuence on RBA values, but the same modiﬁcationof the alkylamino chain of 4 led to a remarkable enhancement of
RBA value, and the RBA of 4c was 83.18 These results suggest thatan alkylamino side chain at the meta position ﬁts well into the cavity of the ERa LBD, and the terminal tertiary amino group of 4cforms hydrogen bonds with amino acid residues of the ERa LBD.
Next, the functional activities of the test compounds were evaluated by means of cell proliferation assays using MCF-7 cell linesthat show ER-dependent growth.17 Table 2 summarizes EC50 and
IC50 values as parameters of the agonist and antagonist activitiesof the test compounds, respectively. Agonist activity is also shown
as relative maximal efﬁcacy (Emax), based on estradiol as 100%.
Bisphenol 9 showed similar EC50 and Emax values to 12. Bothbisphenols showed no ER-antagonist activity and acted as ER fullagonists, not as side-chainless partial agonists like BE360. The leadcompound 1 showed moderate agonist activity and its Emax value
was 78%, which means it has lower maximal efﬁcacy than E2.
Compound 1 antagonized MCF-7 cell proliferation induced by
0.1 nM of E2 with an IC50 value of 4.4 lM. Compound 4a, which
has the same side chain as the lead compound 1, but at the meta
position, showed potent ER agonist activity (EC50 = 4.7 nM) and a
low Emax value of 63%. The IC50 value of 4a was 6.5 lM, which issimilar to that of 1. Compound 4b with a dimethylaminopropylgroup showed the lowest EC50 value and the lowest Emax value
among the tested compounds. In addition, compound 4b showed
10 times more potent ER antagonist activity than the lead compound 1. Although dimethylaminobutyl derivative 4c showed thegreatest ERa-binding afﬁnity, its biological activities parameters,
EC50, Emax, and IC50, are similar to those of compound 4a. These
results conﬁrm that the dimethylaminopropyl group is the mostsuitable ER partial agonist activity-inducing substituent in theseries of m-carborane-containing m-substituted derivatives 4.
The ER-antagonist activity of 4b was more potent than that of 3,
which contains the 9,12-dimethyl-m-carborane cage (IC50 of
3 = 0.88 lM). The low IC50 value of 4b suggested that the side chain
serves to inhibit binding of co-activators by moving helix-12 of ER
to an unfavorable position. However, compound 4b has a higher— 415 —

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K. Ohta et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3213–3216
HO
H
a

H

N

n

O

N

n

O

H
b, c
6

H3CO

5

d

H3CO

8

H3CO

e

OH

e

7

HO

f

HO

O

Br

N

O

12

HO

9

10

H3CO

O

N

g

HO

n = 1 4an = 2 4b

e

H3CO

11

HO

4c

Scheme 1. Synthesis of novel ER modulators 4. Reagents and conditions: (a) n-BuLi, DME, then CuCl, pyridine, 4-iodoanisole, 71%; (b) n-BuLi, DME, then CuCl, pyridine, 3-tertbutyldimethylsiloxyiodobenzen; (c) TBAF, THF, 71% over 2 steps; (d) N,N-dimethylaminoalkyl chloride, K2CO3, acetone, 24–38%; (e) BBr3, CH2Cl2, 34–94%; (f) 1,4dibromobutane, K2CO3, acetone, 62%; (g) dimethylamine, THF, 78%.

Table 1
Relative binding afﬁnity (RBA) of test compounds versus speciﬁc [3H]estradiol (4 nM)
binding with human recombinant ERa
CompoundPosition

Substituent

RBAa

1
4a
4b
4c
9
12

pmmmmp-–O–(CH2)2–N(CH3)2–O–(CH2)2–N(CH3)2–O–(CH2)3–N(CH3)2–O–(CH2)4–N(CH3)2
–OH
–OH

1.5
7.4
8.4
83
131
110

a
All binding assay were treated with the test compounds (0.4 nM to 4 lM) in thepresence of [6,7-3H]17b-estradiol (4 nM). The relative binding afﬁnity is calculated
from IC50 values of E2 and test compounds taking that of E2 as 100%. Values represent the average of duplicate experiments.

Emax value than compound 3 (Emax of 3 = 23%), and might show
moderate estrogenic activity in breast tissue. A terminal cyclic
alkylamino group, such as piperidine or azepane (used in raloxifene and bazedoxifene, respectively), can often enhance ER-antagonist activity, and thus introduction of these substituents mightafford better partial agonists or SERM candidates. Carborane cages
are promising hydrophobic core structure for the development of
ER modulators,10–13,15,17 and carborane-containing ER modulators
may show unique biological properties.

Table 2Biological activities of the test compounds on MCF-7 cell proliferation

Novel ER modulator 3 showed better partial agonist activity
than 1, even though it contains the same alkylamino chain andhas the same substitution position.15 Further syntheses of a seriesof diaryl-m-carborane derivatives, docking simulation studies, andbiological investigations with other ER-expressing tissues, determination of ER expression levels, and examination of the inﬂuenceof 9,12-dimethyl-m-carborane structure on ER partial agonist
activity, as well as in vivo experiments, are in progress.
In conclusion, novel m-carborane-containing ER partial agonists
were synthesized and their biological activities were evaluated bymeans of competitive ER binding assay and MCF-7 cell proliferation assay. All tested compounds 4 showed higher RBA values thanthe corresponding para-substituted derivatives, suggesting that analkylamino side chain at the meta position ﬁts well into the cavityof the ERa LBD. Compound 4b showed the most potent ER-agonist
activity and the lowest Emax value among the tested compounds.
Moreover, the ER-antagonist activity of 4b was 10 times morepotent than that of the lead compound 1. As observed in the caseof bazedoxifene, the unique hydrophobic carborane cage structure
provide an entry into carborane-containing ER modulators withvarious characteristics of ER up- or down-regulation, as well as distinctive pharmacokinetic properties. These ﬁndings should be
helpful for molecular design of further carborane-containing ER
modulators, including agonists, partial agonists, antagonists, andSERMs as biological tools or candidate therapeutic agents for ERrelated diseases.
Acknowledgments

CompoundPosition

Substituent

EC50a
(nM)

Emaxb
(%)

IC50c
(lM)

1

p-

8.2

78

4.4

4a

m-

4.7

63

6.5

4b

m-

1.4

50

0.4

4c

m-

2.6

62

4.3

9
12

mp-

–O–(CH2)2–
N(CH3)2–O–(CH2)2–
N(CH3)2–O–(CH2)3–
N(CH3)2–O–(CH2)4–
N(CH3)2
–OH
–OH

1.8
1.4

107
90

Inactive
Inactive

This research was supported by a Grant-in-Aid for the Strategic
Research Program for Private University (2010-2014) and a Grantin-Aid for Scientiﬁc Research (C) (No. 26460151) from the Ministryof Education, Culture, Sports, Science, and Technology, Japan.
References and notesaMCF-7 cells were treated with the test compounds (1  1013 to 1  105 M)
alone. EC50 values were estimated from the sigmoidal dose-response curves using
GraphPad Prism software.
b
Emax values indicate efﬁcacy for cell proliferation, based on the value for E2
taken as 100%.
c
MCF-7 cells were treated with the test compounds (1  1011 to 1  105 M) in
the presence of 0.1 nM E2. IC50 values were estimated from the sigmoidal doseresponse curves for competitive antagonism against cell proliferation activity
induced by 0.1nM E2, using GraphPad Prism software.

— 416 —

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Kagechika, H. Bioorg. Med. Chem. Lett. 2003, 13, 4089; (e) Ogawa, T.; Ohta, K.;
Iijima, T.; Suzuki, T.; Ohta, S.; Endo, Y. Bioorg. Med. Chem. 2009, 17, 1109; (f)Ohta, K.; Ogawa, T.; Kaise, A.; Endo, Y. Bioorg. Med. Chem. Lett. 2013, 23, 6555.

11. (a) Endo, Y.; Yoshimi, T.; Miyaura, C. Pure Appl. Chem. 2003, 75, 1197; (b) Hirata,
M.; Inada, M.; Matsumoto, C.; Takita, M.; Ogawa, T.; Endo, Y.; Miyaura, C.
Biochem. Biophys. Res. Commun. 2009, 380, 218.
12. Ogawa, T.; Ohta, K.; Yoshimi, T.; Yamazaki, H.; Suzuki, T.; Ohta, S.; Endo, Y.
Bioorg. Med. Chem. Lett. 2006, 16, 3943.
13. Ohta, K.; Ogawa, T.; Suzuki, T.; Ohta, S.; Endo, Y. Bioorg. Med. Chem. 2009, 17,7958.
14. Shiau, A. K.; Barstad, D.; Loria, P. M.; Cheng, L.; Kushner, P. J.; Agard, D. A.;
Greene, G. L. Cell 1998, 95, 927.
15. Ohta, K.; Ogawa, T.; Kaise, A.; Endo, Y. Bioorg. Med. Chem. 2014, 22, 3508.
16. (a) Coult, R.; Fox, M. A.; Gill, W. R.; Herbertson, P. L.; MacBride, J. A. H.; Wade, K.
J. Organomet. Chem. 1993, 462, 19; (b) Ohta, K.; Goto, T.; Endo, Y. Inorg. Chem.
2005, 44, 8569.
17. Ohta, K.; Chiba, Y.; Ogawa, T.; Endo, Y. Bioorg. Med. Chem. Lett. 2008, 18, 5050.
18. RBA values of the corresponding m-carborane-containing p-substituted
derivatives
areasfollows:
N,N-dimethylaminoethyl = 1.1,
N,Ndimethylaminopropyl = 1.5, and N,N-dimethylaminobutyl = 1.7.

— 417 —

— 418 —研究テーマ３
難治性疼痛に対する緩和医療法の確立— 419 —

— 420 —1

東北医科薬科大学研究誌，65，1−6（2018）
Journal of Tohoku Medical and Pharmaceutical university，65，1−6（2018）

総説
μオピオイド受容体の多様性
溝口広一

Variability of μ-Opioid Receptors
Hirokazu MizogucHi
Department of Physiology and Anatomy, Faculty of Pharmaceutical Sciences,
Tohoku Medical and Pharmaceutical university.
（Received November 20, 2018）
The μ-opioid receptors are target receptors for narcotic analgesics. Although there is critical variability on theirpharmacological eﬀects between narcotic analgesics, only one μ-opioid receptor（MoR-1）DNA has been cloned.
After the cloning of MoR-1 cDNA, many exons have been identiﬁed in the MoR-1 gene. With many exons in theirgene, the MoR-1 has synthesized with a large number of splice variants. in the present review, the splice variantsof MoR-1 and the possible physiological functions for speciﬁc splice variants have been discussed.
Key words ── μ-opioid receptor, splice variants, analgesia, narcotic analgesics, pain

１．はじめにド鎮痛薬は共にμオピオイド受容体の作動薬であ疼痛は，非常に不快な感覚刺激であると共に，生り，その鎮痛作用はμオピオイド受容体を介して体に侵害が加えられたことを知らせる重要な警告信発現する．これらμオピオイド受容体作動薬であ号でもある．しかし，持続する強い痛みは苦痛以外

るオピオイド鎮痛薬は，その鎮痛作用や鎮咳作用の何物でもなく生活の質を著しく低下させることかといった主作用および，嘔気・嘔吐，便秘，呼吸ら，鎮痛薬による疼痛緩和が行われる．臨床におけ抑制，依存性などの副作用において，薬物間でる疼痛緩和においては，痛みの質や程度により様々

様々な特徴的違いを示す．現在臨床では，オピオな鎮痛薬が使用されており，比較的軽度な疼痛には

イド鎮痛薬間の薬理作用におけるこの相違を逆に

非ステロイド性消炎鎮痛薬（NSAiD）が，中等度の利用したオピオイドローテーションが行われてい疼痛には非麻薬性オピオイド鎮痛薬や弱オピオイド

る．しかし，同じμオピオイド受容体を介して発の麻薬性鎮痛薬が，ガン性疼痛や術後痛などの重篤現する薬理作用に，オピオイド鎮痛薬間で多様なな疼痛には強オピオイドの麻薬性鎮痛薬が用いられ差が生じる原因については，μオピオイド受容体ている．現在日本で用いられている麻薬性鎮痛薬にの多様性による可能性が一部示唆されているもの

は，morphine，oxycodone，fentanyl，remifentanyl，の，いまだ明確にはなっていない．

hydromorphone，methadone，tapentadol など鎮痛
活 性 の 非 常 に 強 力 な 強 オ ピ オ イ ド と codeine，

２．μオピオイド受容体

pethidine など鎮痛活性が比較的低い弱オピオイド

オピオイド受容体は，morphine をはじめとするが存在し，また，非麻薬性オピオイド鎮痛薬としオピオイドが作用する受容体の総称である．薬物ては，tramadol，buprenorphine，pentazocine などの作用点として受容体の概念が導入されて以来，が用いられている．オピオイドの作用するオピオ

morphine の作用点としてその存在は想定されていイド受容体は，主にμオピオイド受容体，δオピ

たが，その存在が初めて実験的に実際に確認され

オ イ ド 受 容 体 ， κオ ピ オ イ ド 受 容 体 ， oRL-1

たのは 1970 年代初頭である．1971 年から 1973 年（opioid receptor like-1）受容体の 4 種のサブタイプ

にかけて複数の研究グループが，放射性標識をしに分類されるが，pentazocine 以外の上記オピオイ

た各種オピオイドの結合実験を行うことにより，オピオイド受容体の存在を確認した．1971 年にgoldstein ら 1）は，マウス脳に［3H］levorphanol の東北医科薬科大学薬学部機能形態学教室
e-mail: mizo@tohoku-mpu.ac.jp

特異的結合部位が存在することを明らかにし，そ— 421 —

2

溝口

広一

の後 1973 年に，Snyder ら 2）は［3H］
naloxone を，いる．9 ）しかし，μ 1 オピオイド受容体および μ 2

Simon ら 3）は［3H］
etorphin を，そして Terenius 4）

オピオイド受容体に相当する受容体の遺伝子は，

は［ H］
dihydromorphine をそれぞれ用い，ラット

いまだ発見されていない．

3

脳中にその特異的結合部位の存在を明らかにした．
３．μオピオイド受容体の遺伝子クローニングと彼らが発見した特異的結合部位が，今でいうμオ

その多様性

ピオイド受容体であり，morphine の頭文字の Mを用いてμオピオイド受容体と名付けられている．

1992 年に Evans ら 10）および Kieﬀer ら 11）がδオ

上記放射性標識オピオイドは，μオピオイド受容

ピオイド受容体（DoR-1）遺伝子をクローニング

体の他にδオピオイド受容体やκオピオイド受容して以来，オピオイド受容体に関する研究におい体にも親和性を示すことから，1981 年にμオピオても遺伝子工学の実験手法が多く取り入れられる

イド受容体に最も選択的な薬物として DAMgo

ようになった．DoR-1 がクローニングされた翌年

5（［D-Ala ,NMePhe ,gly-ol ］
enkephalin）が合成され1993 年には，Yasuda ら 12）によりκオピオイド受た 5）後は，［3H］
DAMgo の特異的結合部位をμオ

容 体 （KoR-1）遺 伝 子 が ，さ ら に chen ら ，1 3 ）ピオイド受容体とするようになった．この［3 H］

Wang ら 14）や Fukuda ら 15）によりμオピオイド受DAMgo を用いた結合実験により，μオピオイド

容体（MoR-1）遺伝子がクローニングされ，また，受容体は脳や脊髄といった中枢神経系のみならず，

1994 年には oRL-1 受容体（oRL1）遺伝子がク

心臓，肺，腸管などの末梢神経系にも広範に分布

ローニングされた 16）ことにより，現在同定されてしていることが明らかになっている．Morphine ないる 4 種のオピオイド受容体の構造（アミノ酸配どのμオピオイド受容体作動薬は，特異的部位に列）が全て明らかとなった．MoR-1 は 398 個のア存在するμオピオイド受容体を刺激することによ

ミノ酸残基からなる受容体であり，DoR-1，KoR-

り，鎮痛作用（脊髄，大脳皮質，視床，中脳水道

1，oRL1 と共にその構造中に 7 つの細胞膜貫通領

周囲灰白質），呼吸抑制作用（孤束核），腸管運動域を持つ．細胞膜貫通領域のアミノ酸残基は他の抑制作用（腸管），自発運動亢進作用（黒質），鎮オピオイド受容体と相同性が極めて高く，受容体

静作用（大脳皮質），陶酔作用（腹側被蓋野），心全体での相同性も約 60％と高い．MoR-1 は他のオ抑制作用（孤束核，心臓）など様々な薬理効果を

ピオイド受容体と同様に，gi/go 蛋白と共役した発現すると考えられている．

受容体であり，その受容体刺激により抑制性のシ

24μオピオイド受容体の発見から約 10 年の時を経グナルを発生させることが確認されている．また，て，μオピオイド受容体にはサブクラスが存在す

クローニングされた MoR-1 遺伝子は，4 つのエキ

は，

ソン（エキソン 1〜エキソン 4）を含んでおり，エオピオイドペプチドとオピオイドアルカロイドのキソン 1 は細胞外 N 末端領域と 1 回目細胞膜貫通

双方に等しく高い親和性を示すμオピオイド受容

領域を，エキソン 2 は 2 回目細胞膜貫通領域〜4 回体をμ1 オピオイド受容体，オピオイドペプチドよ目細胞膜貫通領域を，エキソン 3 は 5 回目細胞膜

りもオピオイドアルカロイドにより高い親和性を貫通領域〜7 回目細胞膜貫通領域を，そしてエキソ示すμオピオイド受容体を μ2 オピオイド受容体とン 4 は細胞内 c 末端領域をそれぞれコードしてい

して，μオピオイド受容体を 2 つのサブクラスにることが明らかとなっている．

ることが報告された．1980 年に Pasternak ら

6）分類した．また彼らは，μ1 オピオイド受容体選択

前述したように，従来 μオピオイド受容体は，的拮抗薬である naloxonazine を開発し， μ1 オピ

作動薬・拮抗薬に対する感受性の違いからμ1 オピ

オイド受容体は上位中枢（脳）鎮痛作用，陶酔作

オイド受容体とμ2 オピオイド受容体に細分化され用，acetylcholine や prolactin の放出制御作用，摂ていた．しかし，実際に遺伝子が発見され，ク

食促進作用に，一方，μ2 オピオイド受容体は脊髄

ローニング・単離・同定が行われたμオピオイド

鎮痛作用，呼吸抑制作用，腸管運動抑制作用に関受容体は，いまだ MoR-1 のみである．それでは，与することを明らかにしている． μ1 オピオイド

各種オピオイド鎮痛薬で認められている薬理作用

受容体・μ2 オピオイド受容体の理論は，自然発症の多様性は，どのようにして発現するのか．その的にμ 1 オピオイド受容体が著しく減少（ほぼ欠疑問は，MoR-1 遺伝子がクローニングされた翌年損）している cXBK マウスにおいても確認されて1994 年に，Bare ら 17）により MoR-1 のスプライス

7）

8）— 422 —

μオピオイド受容体の多様性

3Fig. 1. Schematic of MoR-1 splicing in the mouse.

バリアントとして MoR-1A が発見されたことに端からイントロンが切り落とされて mRNA が産生さを発し，早急に解決へと向かった．スプライスバれる際に，エキソンも切り落とされることによりリアントとは，遺伝子に複数のエキソンが含まれ発現する多様性のことである．前述したように，ている場合，遺伝子から転写された mRNA 前駆体

MoR-1 は発見された当初，その遺伝子にエキソン— 423 —

4

溝口

広一

1〜エキソン 4 の 4 つのエキソンを含む受容体とさて，MoR-1 スプライスバリアント同士あるいはれていたが，Bare らはエキソン 4 相応部分が欠落

種々の受容体との様々な二量体によっても生じてしたスプライスバリアントとして MoR-1A を発見いるのかもしれない．

した．一方 Höllt ら

18）

は，MoR-1 の遺伝子中に新しいエキソンとしてエキソン 5 を発見し，エキソ

４．各種 MOR-1 スプライスバリアントの生理機能

ン 5 を含むスプライスバリアントとして MoR-1B

MoR-1 スプライスバリアントの存在が明らかとにより，MoR-1 のなってから，既に 20 年以上経過しているが，その遺伝子中に大量のエキソンが発見され，それらエ生理機能に関してはいまだほとんど明らかとなっキソンに関連した大量の MoR-1 スプライスバリアていない．個別の MoR-1 スプライスバリアントにントが発見されている．MoR-1 のスプライスバリ

選択的な薬物が存在しないことと，各 MoR-1 スプ

アントは，特にマウスにおいて研究が進んでおり，

ライスバリアントを個別に機能低下させることが現在 19 個のエキソンに基づく 34 種類の MoR-1 ス

技術的に困難であることが原因である．MoR-1 ス

プライスバリアントの存在が報告されている（Fig.

プライスバリアントは，数種以外のほぼ全てがエ1）． その他にも，配列の一部が発見されただけ

キソン 2 とエキソン 3 を含んでおり，エキソン 1

でその全配列がいまだ確定していない新規 MoR-1

による 1 回目細胞膜貫通領域ならびに細胞外 N 末

スプライスバリアントの候補も多数存在しており，

端領域とエキソン 4 以降の c 末端細胞内領域に多

現在も新たなエキソンが発見される毎に MoR-1 ス

様性が存在する．また，大部分の MoR-1 スプライ

プライスバリアントの種類も増加している．一方，スバリアントはエキソン 1 を含んでおり，ラットやヒトにおいてもマウスの MoR-1 スプライ

morphine を筆頭とした全ての麻薬性鎮痛薬やμオ

スバリアントに相当するスプライスバリアントはピオイド受容体作動薬は，このエキソン 1 を含ん存在し，現時点でラットでは 17 種類，ヒトでは 20

だ MoR-1 スプライスバリアントに選択的である．

種類の MoR-1 スプライスバリアントが報告されてそのため，エキソン 1 を含んだ MoR-1 スプライス

いる．23）

バリアントの発現細胞を用いても，既存の麻薬性を発見した．その後 Pan ら

19-22）

23）

発見された MoR-1 スプライスバリアントの大部

鎮痛薬やμオピオイド受容体作動薬の間に際立っ分は，MoR-1 と同様に 7 回細胞膜貫通構造を持つ

た選択性の違いは認められない．27）また，エキソ受容体であるが，一部の MoR-1 スプライスバリア

ン 1 を含んだ MoR-1 スプライスバリアントに含まントはエキソン 1 が欠損した 6 回細胞膜貫通構造

れている各種エキソンは，必ず複数の MoR-1 スプを持つ受容体である．一方，エキソン 2 およびエライスバリアントに含まれているため，エキソン

キソン 3 が欠損したり，エキソンの途中にストッ

選択的に個別の MoR-1 スプライスバリアントを機

プコドンが存在することにより，結果的に 1 回細能低下させることは不可能であり，また個々の胞膜貫通構造となってしまう MoR-1 スプライスバ

MoR-1 スプライスバリアントに対する選択的抗体

リアントも少数ながら存在する．これら 7 回細胞も存在しない．これらの要因から，MoR-1 スプラ

膜貫通構造を持たない MoR-1 スプライスバリアン

イスバリアントの機能解析は不可能と判断され，

トが，MoR-1 と同様に単量体で機能するか否かにその研究は一時下火となっていた．しかし著者ら関しては，いまだ明らかになっていない．MoR-1，

は，エキソン 1 を含まない MoR-1 スプライスバリ

DoR-1 や KoR-1 といったオピオイド受容体は，単アント内には，単独の MoR-1 スプライスバリアン量体（モノマー）で機能する一方，g 蛋白共役型

トにしか含まれていない 3 種類のエキソン（エキの同種受容体や異種受容体と二量体（ホモダイ

ソン 12，エキソン 13，エキソン 14）が存在するこ

マーやヘテロダイマー）を形成し，それら二量体とに着目して研究を行うことにより，3 種の MoR-は単量体と比較して，薬物の結合特性や受容体と1 ス プ ラ イ ス バ リ ア ン ト （MoR-1J，MoR-1K，しての機能特性が著しく異なることが知られていMoR-1L）の生理機能の一端を見いだすことに成功

このような二量体は，オピオイド受容体同した．これら 3 種の MoR-1 スプライスバリアント

士だけでなく，β受容体など全く異種の受容体とは，既存のμオピオイド受容体作動薬に非感受性の間でも存在する．

MoR-1 の複雑な多様性は，の受容体であり，これら MoR-1 スプライスバリア

MoR-1 スプライスバリアント自身の多様性に加えントをエキソン選択的に発現抑制しても，既存のる．

24,25）

26）— 424 —

μオピオイド受容体の多様性

5μオピオイド受容体作動薬の脊髄鎮痛作用は影響

体作動薬の併用による「依存性のない夢の鎮痛薬」

を受けない． 一方，新規ペプチド性μオピオイの開発が試みられたが，当時上梓されていた κオ

ド受容体作動薬 amidino-TAPA（Nα-amidino-Tyr-

ピオイド受容体作動薬は全て μオピオイド受容体

D-Arg-Phe-β-Ala）は，既存の μオピオイド受容体拮抗作用を持っていたことから，「夢の鎮痛薬」と作動薬とは異なり，脊髄で内因性オピオイドペプチ

はならなかった．一方，新規μオピオイド受容体

ド（dynorphin A，dynorphin B，α-neo-endorphin，作動薬 amidino-TAPA は，前出したように既存の［Leu ］
enkephalin）を遊離する作用を持つが，麻薬性鎮痛薬とは異なり，内因性κオピオイドペ

28）

5

29）

その脊髄鎮痛作用はこれら 3 種の MoR-1 スプライ

プ チ ド （dynorphin A，dynorphin B，α-neo-

スバリアントをエキソン選択的に発現抑制するこ

endorphin）を遊離する作用を持つ．μオピオイドとにより著しく抑制される．28）詳細な検討により，受容体刺激により側坐核での dopamine 遊離を増加

amidino-TAPA は脊髄のこれら 3 種の MoR-1 スプさせる一方，遊離した内因性κオピオイドペプチ

ライスバリアントを介して鎮痛作用を発現するが，ドが側坐核のκオピオイド受容体を刺激すること

その鎮痛作用には MoR-1J を介した dynorphin A

によってその遊離増加を打ち消すため，amidino-

遊離，MoR-1K を介した dynorphin B 遊離と α-

TAPA はμオピオイド受容体作動薬であるにもか

neo-endorphin 遊離，MoR-1L を介した dynorphin

かわらず，精神依存性を示さないことが明らかとA 遊離と［Leu ］
enkephalin 遊離が関与しているこなっている．32）この内因性 κオピオイドペプチドとが明らかになっている．30）このように，既存のの遊離に関わる amidino-TAPA 感受性の MoR-1 ス

μオピオイド受容体作動薬に非感受性の特異的プライスバリアントは，MoR-1J と MoR-1L であ

MoR-1 スプライスバリアントは，エキソン 1 を含り，これら MoR-1 スプライスバリアントは，依存有する麻薬性鎮痛薬感受性の MoR-1 スプライスバ

性のない夢の鎮痛薬を開発する上でのターゲット

リアントとは異なる，特異的生理機能を持っていとなり得る受容体と考えられる．

5

るかもしれない．
６．おわりに
５．夢の鎮痛薬を創製するためのターゲット受容体

MoR-1 スプライスバリアントの機能解析は，実

麻薬性鎮痛薬は，μオピオイド受容体を介して質的にはまだ始まったばかりである．しかし数少

強力な鎮痛作用を発現する一方，強い精神依存性ない先行研究において，morphine 等の麻薬性鎮痛を持っており，この精神依存性による乱用が世界

薬に非感受性の MoR-1 スプライスバリアントは，的に問題となっている．この麻薬性鎮痛薬の精神従来μオピオイド受容体で認められていた生理機依存性は，μオピオイド受容体刺激を介して発現能とは異なる生理機能に関与していることが示唆する陶酔感により形成される．中脳腹側被蓋野かされている．MoR-1 スプライスバリアントの機能

ら大脳辺縁系側坐核へ投射している dopamine 神経

解析は，臨床応用可能な新たな生理機能の発見には，快・不快に関与しており，麻薬性鎮痛薬は，つながるかもしれない．

腹側被蓋野の dopamine 神経細胞体を抑制的に制御謝辞しているγアミノ酪酸（gABA）神経上に存在す

本研究を遂行するにあたりご指導を賜るμオピオイド受容体を刺激し，gABA 神経抑制

りました，機能形態学教室の前教授である櫻田忍による脱抑制により側坐核での dopamine 遊離を増客員教授に衷心より御礼申し上げます．また，同加させる． この側坐核での dopamine 遊離増加が教室の渡辺千寿子准教授をはじめご協力いただき

陶酔感を惹起し，精神依存性が形成される．一方，ました同教室の歴代諸氏に厚く御礼申し上げます．

側坐核の dopamine 神経終末には dynorphin 神経が

本論文の文献 28〜30 および 32 に関わる研究は，投射しており，dopamine 遊離を抑制的に制御して科学研究費補助金基盤研究 c（21600013，24590731，

い る ． そ れ 故 κオ ピ オ イ ド 受 容 体 作 動 薬 は ，

15K08678）の助成により行われました．

31）

dopamine 遊離抑制による嫌悪作用を引き起こし，
利益相反

また併用することによりμオピオイド受容体作動薬の陶酔感を打ち消すことが知られている． こ

開示すべき利益相反はない．

31）の現象を基に，麻薬性鎮痛薬とκオピオイド受容— 425 —

6

溝口

広一

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Fitoterapia 129 (2018) 20–24

Contents lists available at ScienceDirect

Fitoterapia
journal homepage: www.elsevier.com/locate/ﬁtote

Antinociceptive eﬀect of inhalation of the essential oil of bergamot in mice
Damiana Scuteria, Michele Crudoa, Laura Rombolàa, Chizuko Watanabeb, Hirokazu Mizoguchib,
Shinobu Sakuradab, Tsukasa Sakuradac, Rosaria Grecod, Maria Tiziana Corasanitie,⁎
Luigi Antonio Morronea, Giacinto Bagettaa,aPreclinical and Translational Pharmacology, Department of Pharmacy, Health Science and Nutrition, University of Calabria, 87036 Rende, CS, Italy
Department of Physiology and Anatomy, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai
981-8558, Japanc
Daiichi College of Pharmaceutical Sciences - First Department of Pharmacology Fukuoka, JapandLaboratory of Neurophysiology of Integrative Autonomic Systems, Headache Science Centre, IRCCS Mondino Foundation, Pavia, Italye
Department of Health Sciences, University "Magna Graecia" of Catanzaro, Catanzaro, Italyb

A R T I C LE I N FO

A B S T R A C T

Keywords:
Aromatherapy
Bergamot essential oil
Pain
Inhalatory route
BPSDs

Bergamot essential oil (BEO) has proven wide evidence of pharmacological antinociceptive eﬀectiveness both innociceptive and in neuropathic pain models. The antinociceptive properties of BEO for inhalation have not been
investigated. The purpose of this study is to evaluate the eﬀects of the inhalation of BEO on formalin-induced
nociceptive response in mice.
Male ddY-strain mice (Japan SLC, Hamamatsu, Japan) of 23–25 g of weight at the time the experiments
underwent the formalin test. Twenty μl of formalin (2% in saline) were administered into the plantar surface ofthe mice hindpaw and the time of licking/biting was observed and recorded at intervals of 5 min. The device for
BEO inhalatory delivery consisted in a ﬁlter paper disc soaked with known volume of BEO placed on the edge ofthe cage.
Inhalation of BEO exerted antinociceptive activity. In particular, it reduced the formalin-induced licking/biting behaviour in a manner that was dependent on the volume of BEO used in the device for its release and on
the time of exposure to the phytocomplex.
The results support the use of BEO in aromatherapy for complementary management of chronic pain relief ina stepwise therapeutic programme.

1. Introduction

nonvolatile residue is a natural odor ﬁxative which inﬂuences the olfactory properties of the oil; however, it contains about 0.2% bergapten
which is responsible for the phototoxicity of BEO [6, 7]. Therefore, abergapten-free extract of the essence (BEO-BF) together with a naturalessence deprived of the hydrocarbon fraction and of bergapten (BEOHF/BF) are prepared by extractive industries for perfumery and cosmetic uses. Recently, this essential oil has been rigorously studied andsome pharmacological activities of the utmost importance have been
deciphered. In particular, strong evidence has been gathered for BEO to
be endowed with analgesic activity, both in nociceptive and in neuropathic pain models (see [8]). In fact, intraplantar (i.pl.) BEO, or its
components linalool and linalyl acetate, reduced the nociceptive response as assayed by the capsaicin test [9]. The latter antinociceptive
eﬀects were antagonized by the ipsilateral i.pl. injection of naloxonehydrochloride and by intraperitoneal (i.p.) naloxone methiodide, an

According to the Farmacopea Uﬃciale Italiana (1991) bergamot
essential oil (BEO) is obtained by cold pressing of the epicarp and,partly, of the mesocarp of the fresh fruit of bergamot (Citrus bergamia
Risso et Poiteau). BEO comprises a volatile fraction (93–96% of total)
containing monoterpene and sesquiterpene hydrocarbons (such as limonene) and oxygenated derivatives (such as linalool) and a nonvolatile fraction (4–7% of total) containing waxes, polymethoxylated
ﬂavones, coumarins and psoralens such as bergapten (5-methoxypsoralen) and bergamottine (5-geranyloxypsoralen) [1, 2]. The mostabundant compounds found in the volatile fraction are the monoterpene hydrocarbons limonene, γ-terpinene, and β-pinene, the monoterpene alcohol, linalool, and the monoterpene ester, linalyl acetate,
which altogether constitute > 90% of the whole oil [3–5]. The

Abbreviations: BPSDs, Behavioural and psychological symptoms of dementia; BEO, Bergamot Essential Oil; BEO-BF, Bergapten-free extract of the essence; BEO-HF/BF, Essence deprivedof the hydrocarbon fraction and of bergapten; i.p., Intraperitoneal; i.pl., Intraplantar; i.t., Intrathecal; PSNL, Partial Sciatic Nerve Ligation⁎
Corresponding author.
E-mail address: g.bagetta@unical.it (G. Bagetta).
https://doi.org/10.1016/j.ﬁtote.2018.06.007
Received 14 May 2018; Received in revised form 9 June 2018; Accepted 11 June 2018
Available online 12 June 20180367-326X/ © 2018 Published by Elsevier B.V.

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antagonist acting at the peripheral opioid receptors; morphine-induced
antinociception after i.p. and intrathecal (i.t.) injections was markedly
enhanced by the combined injection of i.pl. BEO or linalool, its main
oxygenated monoterpene [9]. Interestingly, for i.pl. injection BEO orlinalool reduced partial sciatic nerve ligation (PSNL)-induced neuropathic pain symptoms in mice and inhibition of spinal ERK phosphorylation seems to be involved in this anti-allodynic eﬀect [10]. Thesesame Authors [9] have shown that BEO or linalool modulate morphineinduced anti-allodynic eﬀect under neuropathic pain, a condition
known to be resistant to opioid treatment (see [11]). More recently, fori.pl. injection BEO and linalool have been reported to reduce behavioural signs of formalin-induced nociception in a dose-dependent
manner. The formalin test is characterized by formalin-induced biphasic nocifensive behaviour of licking/biting, with the early nociceptive phase being followed by a late, second, phase that involves peripheral inﬂammation and central sensitization (see [12]). Due to thelack of relevant information about the eﬀects of the inhalation of BEO
on nocifensive behaviour, the purpose of this study was to investigate
the antinociceptive action of BEO via the inhalatory route of administration. Indeed, according to the literature, BEO inhalation was found to
produce anxiolytic-like behaviour [13], but there are no data available
about its eﬀect on nociception and this may be relevant to the use of
BEO in aromatherapy.

intervals of 5 min: during the early phase, beginning immediately after
formalin administration and lasting for 10 min (0–10 min), and during
the late phase, starting 10 min after formalin injection and lasting for
20 min (10–30 min).
2.4. BEO composition
BEO was obtained from “Capua Company1880 S.r.l.,” CampoCalabro, Reggio Calabria (Italy). According to chromatographic analysis provided in the certiﬁcate of analysis, this batch of BEO contains:
D-limonene (39.60%), linalyl acetate (31.09%), linalool (9.55%).
2.5. Statistical analysis
The results are presented as mean ± s.e.m. duration (seconds) of
nociceptive response and evaluated statistically for diﬀerences by
ANOVA followed by Bonferroni's test and considered signiﬁcant whenp < 0.05.
3. Results
3.1. Eﬀect of BEO inhalation as post-treatment on formalin test evokedlicking/biting

2. Materials and methods

In the post-inhalation group (see treatment scheme in Fig. 1 a) theﬁlter paper disc, applied on the edge of the cage, was soaked with 200,
400 or 800 μl of BEO and the mice were subjected to BEO inhalationfrom the time of formalin injection for the following 30 min, during
which the formalin test was carried out. Under these experimental
conditions, BEO did not show signiﬁcant eﬀects on the early phase(0–10 min) (Fig. 2). However, administration of 400 and 800 μl of BEO
signiﬁcantly reduced the time of licking/biting in the late phase(10–30 min) in a dose-dependent manner (Fig. 2).

2.1. Animals
For the study, male ddY-strain mice (Japan SLC, Hamamatsu,
Japan) of 23–25 g of weight at the time of these experiments were used.
Mice were individually housed in a colony maintained in a controlledenvironment (12 h light/dark cycle, room temperature 23 °C, 50–60%
relative humidity), with food and water ad libitum. All of the experiments were performed in agreement with the Guidelines on EthicalStandards for Investigation of Experimental Pain in Animals and approved by the Committee of Animal Care and Use of Tohoku Medical
and Pharmaceutical University, for minimizing animal suﬀering and touse only the number of animals necessary to produce reliable results.

3.2. Eﬀect of BEO inhalation as pre-treatment on licking/biting
Mice were subjected to the inhalation of BEO (ﬁlter paper discsoaked with 200, 400 or 800 μl) for the whole habituation period of 1 h(see treatment scheme in Fig. 1 b) but not during the formalin test.

2.2. Experimental protocol for BEO administration via inhalatory route
The habituation of mice was carried out in a transparent cage(22.0 cm × 15.0 cm × 12.5 cm), that also served as observationchamber. Another plexiglass cage was turned upside down and placed
over the ﬁrst cage, in order to avoid any leaks of BEO. A ﬁlter paper dry
disc (control) or soaked with diﬀerent volumes of BEO according to theexperiment (100, 200, 400, 800 μl) was applied on the edge of the cage
5 min before placing the mice in the observation chamber, so that it was
saturated with BEO. Mice were divided into three experimental groups(post-inhalation, pre-inhalation and double-inhalation). In the post-inhalation group the inhalation of BEO was carried out immediately after
the i.pl. injection of formalin and for the whole duration of the formalintest. In the pre-inhalation group the inhalation of BEO was carried out
as pre-treatment for 1 h, during the mice habituation, at the end ofwhich BEO-releasing ﬁlter paper was removed and the formalin test
was performed. In the double-inhalation group the inhalation of BEO
was carried out both as pre-treatment for 1 h during the habituation and
immediately after formalin administration for the whole duration of theformalin test, in order to assess the total eﬀects of these two diﬀerent
options of delivery of BEO.
2.3. Formalin test

Fig. 1. BEO inhalation scheme. Schematic representation of the administration
scheme of BEO as: a) post-inhalation, b) pre-inhalation and c) double-inhalationin relation to formalin intraplantar (i.pl.) administration.

After 1 h of habituation 20 μl of formalin (2% in saline) were i.pl.
administered to the mice, using a microsyringe with 26-gauge needle.
The time of licking/biting was recorded with a handheld stop-watch at
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D. Scuteri et al.

Fig. 2. Eﬀect of BEO inhalation on the time of licking/biting in the post-inhalation group. Time of licking/biting is represented as mean ± s.e.m. duration expressed in seconds (sec). Statistical signiﬁcance was by ANOVA followed
by Bonferroni's test (n = 8 mice per group). *p < 0.05, **p < 0.01,***p < 0.001 vs control (Cont).

Fig. 3. Eﬀect of BEO inhalation as pre-treatment (pre-inhalation group) on thetime of licking/biting. Time of licking/biting is represented as mean ± s.e.m.
duration expressed in seconds (sec). Statistical signiﬁcance was by ANOVA
followed by Bonferroni's test (n = 8 mice per group). *p < 0.05, **p < 0.01,***p < 0.001 vs control (Cont).

Under these experimental conditions, the eﬀects of BEO were dosedependent and even the smaller volume, which had previously resulted
ineﬀective in the post-inhalation group, resulted eﬀective (Fig. 3).

4. Discussion
BEO is often used in aromatherapy (see [13]), a specialized form ofphytotherapy recorded for thousand years, used nowadays in industrialized countries as complementary medicine to control symptomsof anxiety, depression, pain and sleep disorders, among others. At
variance with the psychological responses, most reliant on the individual experience, pharmacological mechanisms for inhalatory aromatherapy stem from components of the phytocomplex entering the
body via the bloodstream by absorption through the lungs or olfactory
mucosa [14]. In line with the latter concept, our data demonstrate that
BEO is endowed with antinociceptive eﬀects when administered via theinhalatory route in mice. In fact, under our present experimental conditions, a paper ﬁlter disc soaked with diﬀerent volumes of BEO andapplied at the edge of a plexiglass cage allowed the eﬀects of inhalationof BEO to be studied in the formalin-induced nocifensive response in
ddY-strain mice. Exposure to BEO via inhalation occurred according tothree diﬀerent treatment schedules (Fig. 1): the post-inhalation groupof mice was exposed to BEO immediately after formalin injection, thepre-inhalation group was exposed for the whole habituation, but notduring the formalin test, and the double-inhalation group was exposed

3.3. Eﬀect of the inhalation of BEO on licking/biting after formalin
administration (post-inhalation group) as compared with the eﬀect reported
in the pre-inhalation and the double-inhalation groups
Comparison of the eﬀects of the inhalation of BEO (800 μl) on thetime of licking/biting, recorded at 5 min intervals, in the 3 experimental groups (post-, pre- and double-inhalation groups) unraveled
that, while in the early phase BEO was eﬃcacious in a statisticallysigniﬁcant manner only at 5 min and if administered as pre-treatment(pre-inhalation group) or both as pre-treatment and after formalin injection (double-inhalation group) (p < 0,001; Fig. 4 a), in the latephase it resulted eﬀective also when administered just immediately
after formalin injection (post-inhalation group) (Fig. 4 a and b). However, BEO administered in pre-inhalation and in double-inhalation
scheme resulted more eﬀective than when administered after formalin
injection (post-inhalation group).
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D. Scuteri et al.

recapitulates the antinociception of BEO (given i.pl.) and that opioid
receptor antagonists (e.g. naloxone hydrochloride and methiodide, thelatter being unable to cross the blood brain barrier) could attenuate
BEO- or linalool-induced antinociception, suggesting that the peripheral opioid system takes part in the analgesic activity of the essential oil[12]. Indeed, for systemic administration BEO interferes with exocytotic and membrane transporter-mediated release of glutamate in the
rat hippocampus and this eﬀect is lost when the monoterpene hydrocarbon-free fraction of the essential oil is used [15]. Most recently, BEO
has been shown to enhance basal and induced autophagy [16], a mechanism whose derangement has been implicated in pain sensitization[17]. On the other hand, linalool inhibited the licking/biting response
induced by i.t. injection of proinﬂammatory cytokines, e.g. IL-1β or
TNF-α [18]. Furthermore, it has been reported that linalool could
produce antinociception through interactions with opioid, muscarinic
M2 or adenosine A1 receptors, or by modulating nitric oxide (NO)synthesis [19–22]. There is also evidence to suggest that linalool may
modulate glutamatergic neurotransmission via NMDA receptors [9, 23,24]. Accordingly, it is conceivable that antinociceptive properties ofsystemic administration of BEO and linalool may involve additional,
central, mechanisms to the demonstrated activation of the opioid
system in the periphery [8, 9], [11]. Altogether, our ﬁndings could
support the development of aromatherapy clinical trials for the inhalatory administration of BEO for the treatment of chronic pain and of
age-related pathological conditions often associated with chronic pain(see osteoarthritis, post-diabetic and post-herpetic neuropathic pain
and behavioural and psychological symptoms of dementia, BPSDs).
Indeed, the data gathered so far demonstrating a strong analgesic effectiveness of BEO do support aromatherapy with BEO for the control ofchronic pain and a reduced use of drugs like opioids, bearing serious
side eﬀects. The latter are very often prescribed despite the lack ofstrong evidence for their eﬃcacy in chronic non-cancer pain [25]. Some
40–60% among care homes demented patients suﬀer from BPSDs [26]and these are still not adequately controlled through atypical antipsychotics; in fact, BPSDs are strongly associated with pain states [27]and atypical antipsychotics are devoid of analgesic eﬀects. At variancewith the latter concept, aromatherapy with BEO, known to be endowedwith analgesic activity, may be a promising management option for thecontrol of BPSDs. Quite importantly, aromatherapy for inhalation might
have its eﬀects on pain in the absence of any psychological perceptionof the fragrance and this is of primary importance, since most demented
people may be anosmic because of the early loss of olfactory neurons[28]. Recently, the inhalatory administration of BEO in clinical setting
was found to exert an eﬀect on mental health, assessed through the
Positive and Negative Aﬀect Scale, improving mood states [29]. Furthermore, the recent ﬁnding that the anxiolytic-like/relaxant eﬀects of
BEO are devoid of sedation, in contrast with benzodiazepines [30],strengthens the usefulness of aromatherapeutic treatment with BEO in
the elderly. Therefore, additional studies are needed for a deeper
knowledge of the eﬀects of BEO given via inhalation, both from a basic
and clinical perspective.

Fig. 4. Eﬀect of the inhalation of BEO (800 μl) after formalin administration(post-inhalation group) compared with its inhalation as pre-treatment (pre-inhalation group) and with its inhalation during habituation and for the whole
duration of the formalin test (double inhalation group) on the duration oflicking/biting. Time of licking/biting is represented as mean ± s.e.m. duration
expressed in seconds (sec). Statistical signiﬁcance was by ANOVA followed by
Bonferroni's test (n = 8 mice per group). *p < 0.05, **p < 0.01,***p < 0.001 vs control (Cont).

to BEO both during habituation and immediately after formalin injection, in order to evaluate the total eﬀect of either inhalation times. The
results demonstrate that, when administered immediately after formalin, BEO reduced in a dose-dependent and statistically signiﬁcantmanner the nocifensive behaviour in the late phase. When administeredas pre-treatment, during the habituation of the mice, BEO was dosedependently eﬀective; interestingly, the lower volume used, inactive inthe post-treatment (post-inhalation group), here resulted active. Finally, BEO resulted eﬀective in the late phase when administered immediately after formalin and this is at variance with the eﬀect on theearly phase that has been observed only at 5 min, after pre-treatment
administration or if administered both as pre-treatment and after formalin. Nevertheless, the administration of BEO carried out as pretreatment and, both as pre-treatment and immediately after formalin
for the whole duration of the formalin test, remained more eﬀective
than BEO given immediately after formalin injection. These results
demonstrate that BEO is endowed with consistent and reproducible
antinociceptive properties when administered via the inhalatory route.
It has been previously reported that in the formalin test i.pl. linalool

Conﬂict of interest
The authors declare that there is no conﬂict of interests regarding
the publication of this paper.
Acknowledgements
Dr. Michele Crudo was a post-doc recipient of a research bursary in
the frame of a research project (Principal Investigator: Prof. Giacinto
Bagetta) on “Analgesic properties of the essential oil of bergamot”(PADOEB) funded by Calabria Region and carried out at the Tohoku
Medical and Pharmaceutical University (Sendai, Japan). Financial
support from the Tohoku Medical and Pharmaceutical University andfrom Fondazione Istituto Neurologico Nazionale (IRCCS) “Casimiro
23— 430 —

Fitoterapia 129 (2018) 20–24

D. Scuteri et al.

Mondino” (Ricerca Corrente 2017, Ministry of Health, Rome), Pavia(Italy) is gratefully acknowledged.

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Experimental Allergy – Research Article
Int Arch Allergy Immunol
DOI: 10.1159/000488289

Received: December 29, 2017
Accepted after revision: March 9, 2018
Published online: June 6, 2018

Increased Susceptibility to Allergic Asthma with
the Impairment of Respiratory Tolerance Causedby Psychological Stress
Tasuku Kawano a Ryusuke Ouchi a Takahiro Ishigaki a Chiaki Masuda a
Tomomitsu Miyasaka a Yuichi Ohkawara b Nobuo Ohta c Motoaki Takayanagi a
Tomoko Takahashi a Isao Ohno daDivision of Pathophysiology, Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences,
Tohoku Medical and Pharmaceutical University, Sendai, Japan; b Division of Experimental Allergy and Immunology,
Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Tohoku Medical Pharmaceutical
University, Sendai, Japan; c Division of Otorhinolaryngology, Faculty of Medicine, Tohoku Medical andPharmaceutical University, Sendai, Japan; d Center for Medical Education, Faculty of Medicine, Tohoku Medical andPharmaceutical University, Sendai, Japan

Abstract
Background: Bronchial asthma is characterized by type 2
T helper (Th2) cell inflammation, essentially due to a breakdown of immune tolerance to harmless environmental allergens. Etiologically, experiences of psychological stress canbe associated with a heightened prevalence of asthma.
However, the mechanisms underlying stress-related asthma
development are unclear. In this study, we examined whether psychological stress increases susceptibility to allergic
asthma by downregulating immune tolerance. Methods:
Female BALB/c mice were sensitized with ovalbumin/alum,
followed by ovalbumin inhalation. Ovalbumin inhalation induced immune tolerance before sensitization occurred.
Some mice were exposed to restraint stress during tolerance
induction or sensitization. Asthma development was evaluated by airway responsiveness, inflammation, cytokine expression, and IgE synthesis. Sensitization was evaluated by© 2018 S. Karger AG, Basel
E-Mail karger@karger.com
www.karger.com/iaa

measuring proliferation and cytokine production by splenocytes. The effects of stress exposure on the numbers andfunctions of dendritic cells and regulatory T (Treg) cells in
bronchial lymph nodes and spleens were evaluated. To investigate the role of endogenous glucocorticoid in inhibiting immune tolerance after stress exposure, we examinedthe effects of (i) a glucocorticoid-receptor antagonist administered prior to stress exposure, and (ii) exogenous glucocorticoid (instead of stress exposure). Results: Asthmatic responses and Th2-biased sensitization, which were suppressed in tolerized mice, re-emerged in tolerized micestressed during tolerance induction in association with decreased tolerogenic dendritic and Treg cell numbers. The ef
effects of stress exposure on tolerized mice were abolished byadministering a glucocorticoid-receptor antagonist and reproduced by administering exogenous glucocorticoid without stress. Conclusions: Our findings suggested that psychological stress can potentially increase allergic asthma
susceptibility by inhibiting immune tolerance.
© 2018 S. Karger AG, Basel

Edited by: H.-U. Simon, Bern.

Correspondence to: Dr. Tasuku Kawano
Division of Pathophysiology, Department of Pharmaceutical Sciences
Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University
4-4-1 Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558 (Japan)
E-Mail t-kawano@tohoku-mpu.ac.jp— 432 —

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Keywords
Asthma · Psychological stress · Immune tolerance ·Regulatory T cell · Glucocorticoid

Bronchial asthma is a common cause of chronic illness
that is estimated to affect more than 300 million people
worldwide. Asthma is caused by various environmental
factors, such as allergens, infection, and stress. Asthma
manifests with airway inflammation, airway hyperresponsiveness (AHR), and tissue remodeling. Asthmatic
inflammatory responses are caused by eosinophil infiltration, increased mucous production, and elevated serum
IgE levels, which are orchestrated by type 2 T helper (Th2)cells and Th2 cytokines, such as interleukin (IL)-4, IL-5,
and IL-13. Airway inflammation leads to AHR and mucosal remodeling [1–6].
Regulatory T (Treg) cells play a critical role in balancing the immune network by suppressing or limiting ef
effector immune responses against internal and external
insults, for example, by preventing organ-specific autoimmunity and allograft rejection, and maintaining selftolerance [7–9]. Treg cells are generated by plasmacytoid
dendritic cells (pDCs) characterized by CD11cmid and
PDCA-1 expression, or IL-10-producing myeloid DCs(mDCs) with CD11chigh and IL-10 expression [9–11].
Their generation appears to be dependent on microenvironmental factors such as IL-10 and TGF-β [12, 13]. Patients with severe asthma show reduced numbers of Treg
cells in the blood and sputum, with impaired suppressive
activities as compared with healthy subjects [14]. Moreover, the percentage of Treg cells in bronchoalveolar lavage fluid (BALF) of children [15] and adults [16] withasthma is lower than that in healthy subjects. The failureof Treg cells to suppress Th2-mediated immune responses to innocuous inhaled antigens may lead to an imbalance between allergen activation of Treg cells and effector
Th2 responses, resulting in the development and maintenance of asthmatic airway inflammation characterized byuncontrolled Th2-biased immune responses [13, 17, 18].
Recent studies have investigated the mechanisms underlying the impairment of Treg cells in maintaining immune tolerance and the association with increased susceptibility to allergic asthma. Respiratory infections withviruses such as rhinovirus and respiratory syncytial virus(RSV) in early life are risk factors for asthma developmentin adult life. RSV infection impairs immune tolerance by
inducing phenotypic changes in Foxp3+ Treg cells, including GATA-3 expression, Th2 cytokine production,and loss of suppressive functions, in an IL-4-dependent
pathway [19]. However, how exactly ineffective tolerogenic immune responses develop in asthmatic patientsremains unclear.
2

Several epidemiological studies have demonstrated an
association between psychological stress and asthma development [20–23]. For example, perceived stress wasstrongly associated with the development of adult-onset
asthma and hospitalization [24]. Experiences of abuse
during childhood and adolescence were positively associated with adult-onset asthma in African American women [25]. Stressful life events, such as a broken life partnership, also contributed to asthma development [26]. However, the pathogenic mechanism of asthma developmentby psychological stress is unknown.
Various kinds of stressors have been reported to decrease Treg cell production. The number of Treg cells in
the peripheral blood of patients with PTSD (posttraumatic stress disorder) was significantly lower than that in
non-PTSD subjects [27, 28]. An acute laboratory stressor
decreased the number of CD4+FOXP3+ Treg cells in the
peripheral blood of human subjects [29].
Psychological stress upregulates the release of glucocorticoid (GC) by activating the hypothalamic-pituitaryadrenal cortex axis. GC evokes defensive responses tostress that help to maintain homeostatic balance, including regulatory effects on immune-system activities. By af
affecting the immune system, GC can potentially enhanceTh2 immune responses through inhibiting the development of Treg cells essential for respiratory tolerance [30,31].
Therefore, we hypothesized that exposure to psychological stress prevents the development of respiratory tolerance by inhibiting Treg cell generation via the enhanced
release of GC, resulting in Th2-biased immune responses
followed by increased susceptibility to allergic asthma. To
test this hypothesis, we examined the inhibitory effects ofpsychological stress on respiratory tolerance induction
with or without a GC-receptor antagonist in a murine
model of allergic asthma.
Materials and Methods
Mice
Specific pathogen-free female BALB/c mice were purchasedfrom CLEA Japan (Tokyo, Japan). OVA-specific TCR transgenic
DO11.10 (Rag2–/–) mice were kindly provided by Dr. Ryo Abe(Tokyo University of Science, Noda, Japan). Animals were housed
under a 12-h light/dark cycle at a constant temperature (22 ±2 °C). Sterilized food and water were available ad libitum. All experiments described below were approved by the Committee ofAnimal Experiments at Tohoku Medical and Pharmaceutical
University (approval numbers: 14002-cn, 15001-cn, and 16002cn). We took the utmost care to alleviate any pain and sufferingin the mice.

Kawano et al.

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DOI: 10.1159/000488289— 433 —

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IntroductionBALB/c ♀ 7 weeks
Tolerization
OVA or saline
inhalation 30 min

Challenge
OVA or saline inhalation
60 min × 2

Sensitization
OVA/alum i.p.

–7 –6 –5 –4 –3 –2
+
+

0

*

5

17 18

Restraint stress 6 h+
0 OVA 0.5

a

*0

6 (h)

Restraint stress

Tolerization
OVA or saline
inhalation 30 min

SpleenOVA

1

Sensitization
OVA/alum i.p.

–7 –6 –5 –4 –3 –2
+
+

0

5

Restraint stress 6 h

5

OVA

BAL

6 (h)

22 (day)
AHR
BAL
Lung tissue
Serum

Challenge
OVA inhalation
60 min × 2*

17
Spleen22 (day)BAL

RU-486 (6.25 mg/kg)
or solvent s.c.
1 h before stress exposureb
Tolerization
OVA or saline
inhalation 30 min

Sensitization
OVA/alum i.p.

–7 –6 –5 –4 –3 –2++

tocols used for tolerization, stress exposure, sensitization, and antigen challenge.

5

DEX (15.6–1,000 µg/kg)
or PBS i.p.

17 *

22 (day)
BAL

cProtocols for Tolerization, Sensitization, Antigen Challenge,
and Stress Exposure
Seven-week-old mice were sensitized and made to inhale antigen, as described previously [32], with a minor modification(Fig. 1a). Briefly, mice were sensitized by intraperitoneal injections of chicken ovalbumin (OVA; grade V; Sigma-Aldrich, St.
Louis, MO, USA; 8 µg/mouse) adsorbed onto aluminum hydroxide (Wako Pure Chemical Industries, Osaka, Japan; 4 mg/
mouse) on days 0 and 5. Respiratory tolerance was induced by
OVA inhalation without aluminum hydroxide on days –6 and–3, as described previously [33]. For the first 30 min of the period of stress exposure described below, aerosolized OVA (5 mg/
mL in saline) flowed through the chambers for OVA exposure(tolerized mice). As a control for tolerance, saline alone wasflowed through the chambers (asthmatic mice). For restraint
stress, each mouse was placed in a 50-mL conical centrifuge tube

with multiple ventilation holes [32]. This restraint allowed miceto rotate from a supine to a prone position, but not to turn theirheads toward their tails or consume food and water. Restraint
stress is generally considered to induce psychological stress inanimals [34]. During the tolerance phase, stressed/tolerized andstressed/asthmatic mice in 50-mL conical centrifuge tubes wereleft in plastic chambers for 6 h for 6 consecutive days (–7 to –2),
at the same time each day. In another set of experiments, during
the sensitization phase, restraint-stress exposure was performedon days 0–5 in each case. Nonstressed mice were only deprivedof food and water during the period when stressed mice were
exposed to stress. On day 17, all mice were challenged with aerosolized OVA (5 mg/mL) or saline. Before and after the challenge,
bronchoalveolar lavage (BAL) was performed [32]. Briefly, thelungs were lavaged twice by injecting 0.25 mL of cold phosphatebuffered saline (PBS), and the recovered PBS was pooled. Ap-

Allergic Asthma Susceptibility andPsychological Stress

Int Arch Allergy Immunol
DOI: 10.1159/000488289— 434 —

3

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Fig. 1. Schematic representation of the pro-0

Challenge
OVA inhalation
60 min × 2

Administration of a GC-Receptor Antagonist andDexamethasone
In experiments using a GC-receptor antagonist, mifepristone(RU-486; Sigma-Aldrich) was dissolved in polyethylene glycol 400(Nacalai Tesque, Kyoto, Japan) and subcutaneously administered(6.25 mg/kg body weight) 1 h before each stress exposure (Fig. 1b).
In experiments involving GCs, dexamethasone (DEX; Sigma-Aldrich) in PBS was intraperitoneally injected at the indicated doseto substitute for restraint stress (Fig. 1c). The DEX dose administered was determined based on previous studies [35–38] to makethe serum GC contents reach levels comparable to those after restraint-stress exposure. The solvents RU-486 and DEX were usedas vehicle controls for these agents.
Plasma Corticosterone Measurements
Before and immediately after restraint-stress exposure on day–7 and immediately after restraint-stress exposure on days –5 and–2, blood was collected from stressed and nonstressed mice as described [32]. Plasma samples separated from blood were stored at–80 °C for corticosterone concentration measurements using an
enzyme immunoassay kit (Enzo Life Sciences, Farmingdale, NY,
USA) according to the manufacturer’s protocol. The sensitivity ofdetection was 32 pg/mL.
AHR Measurements
Measurements of airway resistance (RL) were conducted using
the Buxco FinePointe Resistance and Compliance system (DSI,
Minneapolis, MN, USA), as described [32] (Fig. 1a). Mice weresequentially challenged with aerosolized PBS (baseline) followed
by increasing doses of methacholine (Sigma-Aldrich), ranging
from 0.625 to 5 mg/mL. Maximum-resistance values were recorded during a 3-min period following each challenge. The concentration of methacholine that provoked a 200% increase in RL (PC200)
was calculated by linear interpolation of the dose-response curves.
Histological Analysis of the Lungs
Sections of lung tissues collected on day 22 were stained withperiodic acid-Schiff (PAS) to evaluate mucus-containing goblet
cells in the bronchial epithelium, or with hematoxylin and eosin to
study eosinophil infiltration in peribronchial areas. The number of
PAS-positive cells and eosinophils were determined as described
previously [32].
OVA-Specific IgE Measurement
The concentrations of OVA-specific IgE in serum collected on
day 22 were measured by ELISA, as described previously [32]. The
sensitivity of detection was 1.9 × 10–2 EU/mL for IgE.
Preparation of Bronchial Lymph Node Cells
Bronchial lymph nodes (BLNs) obtained on day –2 from 10
nontolerized mice and from 3 tolerized mice with or without thestress exposure were pooled and weighed. BLN cells were prepared4

as described previously [39], processed for flow cytometric analysis, and used for the transfer and coculture experiments described
below.
Adoptive Transfer of BLN DCs
DCs as CD11c-positive cells were isolated from BLN cells fromtolerized and stressed/tolerized mice on day –2 using an autoMACS Pro Separator (Miltenyi Biotec, Bergisch Gladbach, Germany) with anti-mouse CD11c (N418) MicroBeads (Miltenyi Biotec) and adoptively transferred (day –2) into naïve mice as described previously [39]. The mice were subsequently immunized(days 0–5) with OVA/alum and challenged with OVA (day 17),and BAL was performed (day 22) as described above.
Coculture of BLN DCs with Naïve DO11.10 T Cells
CD11c-positive DCs were isolated from BLN cells of tolerized
and stressed/tolerized mice as mentioned above. CD4+ T cells wereisolated from splenocytes of naïve DO11.10 (Rag2–/–) female miceusing an autoMACS Pro Separator (Miltenyi Biotec) with antimouse CD4 (L3T4) MicroBeads (Miltenyi Biotec). DCs (1 × 104
cells/well) were cocultured with CD4+ T cells (2 × 105 cells/well) in
the presence of OVA (100 μg/mL). After culture for 3 days, cells
were processed for flow-cytometric analysis.
Adoptive Transfer of CD4+ T Splenocytes
Splenocytes were prepared from the 4 mouse groups 1 day after
the last stress exposure. CD4+ T cells were purified from the splenocytes, as described above. CD4+ T cells (2 × 107 cells/0.2 mL ofsaline for each mouse) were transferred into naïve BALB/c mice
via the tail vein (day –1). The mice were subsequently immunized(days 0–5) with OVA/alum and challenged (day 17) with OVA,
and BAL was performed (day 22) as mentioned above.
Culture of Splenocytes
Splenocytes were prepared from mice of all 4 experimentalgroups on day 17. The cells were cultured at 1 × 106 cells in 200 µL/
well in 96-well culture plates (MICROPlate; Asahi Techno Glass,
Chiba, Japan) in the presence or absence of OVA (200 μg/mL) for
72 h. The samples were then centrifuged, and the supernatants
were stored at –80 °C for subsequent cytokine measurements. Cell
proliferation was measured by performing WST-8 assays with cellcount reagent SF (Nacalai Tesque) according to the manufacturer’s protocol.
Culture of Bone Marrow-Derived DCs
Bone marrow-derived DCs (BM-DCs) were prepared from BM
cell cultures from female BALB/c mice (6–10 weeks old) incubatedwith recombinant mouse GM-CSF for 8 days in RPMI 1640 [40].
The cultured cells were more than 95% CD11c+. BM-DCs (2 × 104
cells in 200 µL/well in 96-well culture plates; MICROPlate, Asahi
Techno Glass) were stimulated with OVA (100 μg/mL) in the absence or presence of DEX at the indicated concentration for 24 h.
Cell proliferation was measured by WST-8 assay as mentioned
above.
Flow Cytometric Analysis
Cells were preincubated with anti-CD16/CD32 (FC gamma III/II receptor; BD Biosciences, Franklin Lanes, NJ, USA) to reduce
nonspecific binding of the subsequent antibodies. Dead cells were
excluded using a LIVE/DEAD Fixable Blue Dead Cell Stain Kit

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proximately 0.4 mL of instilled fluid was consistently recoveredfrom each mouse. BALFs were processed to measure cytokine
contents and to count the total cell numbers and cell differentials. After centrifugation, BALF supernatants were stored at–80 ° C before performing cytokine measurements by an enzyme-linked immunosorbent assay (ELISA). Lung tissues were
processed as described below.

Measurements of Cytokine Contents
The concentrations of IL-4, IL-5, and IL-13 in BALFs, and theconcentrations of IL-4 and IFN-γ in cell culture supernatants were
measured using ELISA kits (R&D Systems, Minneapolis, MN,
USA) according to the manufacturer’s protocol. The sensitivity ofdetection was 2 pg/mL for IL-4, 7 pg/mL for IL-5, 1.5 pg/mL for
IL-13, and 8 pg/mL for IFN-γ.
Statistical Analysis
Data are expressed as the mean ± standard deviation (SD) frommultiple independent experiments (as indicated by n values). Significant differences between 2 groups were determined by the nonparametric Mann-Whitney U test. For comparisons between multiple groups, analysis of variance and Bonferroni post hoc tests
were performed. These analyses were performed using Prism5(GraphPad Software, San Diego, CA, USA). p < 0.05 was considered statistically significant.

costerone concentrations after stress exposure were significantly higher at all time points than before stress exposure, while no such increases were observed in nonstressed mice (online suppl. Fig. 2).
Effects of Stress on Asthmatic Responses in TolerizedMice
To investigate the effects of restraint stress on asthmatic responses in tolerized mice, we compared airway
responsiveness, inflammation, cytokine expression, andIgE synthesis after antigen challenge in the 4 mousegroups (Fig. 2). As expected, tolerized mice showed significantly lower AHR, mucus production, numbers of total cells, eosinophils, and lymphocytes in BALFs, peribronchial eosinophil accumulation, Th2 cytokine content in BALF, and OVA-specific IgE in serum thanasthmatic mice. In contrast, those airway responses andIgE levels were significantly higher in stressed/tolerized
mice than in tolerized mice. However, no significant dif
differences occurred in airway responses or IgE levels between asthmatic and stressed/asthmatic mice. The IFN-γ
content in BALFs did not significantly differ between the
4 groups. Significant differences were not observed in total cell and cell differential counts in BALFs before OVA
challenge (day 17) or after saline challenge (day 22) between the 4 groups (data not shown). These data indicated that stress indirectly enhanced allergic responses by
inhibiting the development of respiratory tolerance.
However, tolerized mice with stress exposure during sensitization (after immune tolerance establishment) showed
low numbers of inflammatory cells in the BALF, similar
to tolerized mice without stress exposure. No significant
differences in the numbers of inflammatory cells were observed between asthmatic mice (with or without stress
exposure) during sensitization (online suppl. Fig. 3).

Effects of Stress on Plasma Corticosterone
Concentrations
Corticosterone concentrations (in ng/mL) in stressed
mice were significantly higher than those in nonstressed
mice after the 1st, 3rd, and 6th stress exposures on days–7, –5, and –2, respectively (1st: 327.2 + 250.3 [n = 9] vs.
88.9 ± 29.8 [n = 8], p < 0.05; 3rd: 547.4 ± 256.0 [n = 7] vs.
90.0 ± 95.3 [n = 5], p < 0.05; 6th; 365.5 ± 205.7 [n = 11] vs.
85.1 ± 63.7 [n = 8], p < 0.01). Before stress exposure, nosignificant differences in corticosterone concentrations
occurred between stressed and nonstressed mice. Corti-

Effects of Stress on Sensitization in Tolerized Mice
Because restraint stress caused a re-emergence of antigen-induced asthmatic responses in tolerized mice, we
next investigated the effects of psychological stress on immune tolerance. As expected, pretreating mice with aerosolized OVA inhibited splenocyte proliferation upon
OVA stimulation in vitro (vs. pretreatment with aerosolized saline). However, restraint stress exposure during
the induction phase of tolerance significantly increased
splenocyte proliferation in vitro compared with findings
in tolerized mice (Fig. 3a). Moreover, we compared the
OVA-induced cytokine expression (IFN-γ and IL-4) by
the splenocyte in the 4 groups (Fig. 3b, c). With regard to
IFN-γ (Fig. 3b), tolerance had no effects on the expression

Allergic Asthma Susceptibility andPsychological Stress

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Results— 436 —

5

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172.16.6.87 - 6/12/2018 3:54:14 PM(Life Technologies/Thermo Fisher Scientific Corporation, Grand
Island, NY, USA). The cells were stained for surface antigens with
anti-CD11c-PE (clone N418; BioLegend, San Diego, CA, USA),anti-PDCA1-APC (clone 927; BioLegend), anti-CD3ε-FITC(clone 145–2C11; Miltenyi Biotec), anti-CD4-AF700 (clone RM45; BD Biosciences), anti-DO11.10-TCR-APC (clone KJ1-26; eBioscience), or isotype-control antibodies. For intracellular staining,cells were stimulated with phorbol 12-myristate 13-acetate (5 ng/mL), ionomycin (500 ng/mL), and monensin (2 mM) for 4 h before
surface-antigen staining. After fixation and permeabilization, thecells were incubated with an anti-IFN-γ-PE (clone XMG1.2; eBioscience), anti-IL-4+-PE-CF594 (clone 11B11; BD Biosciences), anti-IL-10-FITC (clone JES5–16E3; BioLegend), anti-Foxp3-PE or
PE-Cy7 (clone FJK-16s; eBioscience), or isotype-control antibody.
IL-10-producing mDCs were identified as CD11chigh MHC IIhigh
IL-10+ cells, pDCs as CD11cmid PDCA-1+ cells, DO11.10 Th1 cells
as KJ1-26+IFN-γ+ cells, Th2 cells as KJ1-26+IL-4+ cells, and Treg
cells as CD3+CD4+Foxp3+ cells. The cells were counted on a FACSAria II flow cytometer (BD Biosciences), and the analyses were
performed using FACSDiva software (BD Biosciences). The flow
cytometric gating strategies used to evaluate cell subsets are shownin online supplementary Figure 1 (for all online suppl. material, see
www.karger.com/doi/10.1159/000488289).

of IFN-γ either with or without stress exposure, whereas
the stress exposure decreased the cytokine expression intolerized mice but not in nontolerized mice. On the other
hand, tolerance significantly decreased IL-4 expression
either with or without stress exposure, while the stress
exposure did not significantly alter the cytokine expres-15

Tolerized

Stressed/tolerized

10

#

5
0

0

0.625

**

**
2.5

5

80
70
60
50
40
30
20
10
0
Tolerization
Stress**Methacholine, mg/mL(Tolerization)

+

–
–

1,000

(Tolerization)***

OVA IgE, EU/mL

1,400
1,200
1,000
800
600
400
200
0
Tolerization –Stress
–

g

+

2

0
Tolerization ––b Stress

–
+

120

*
***

+
+

d

100

80

+
–

+
+

AsthmaStressed

***

60
40

*

600

Total

10,000

400
200
–
–

–
+

+
–

***
**

***
***
**

Mφ

Eo

Neu

Lym

Asthma

*

+
+

1,000
100

Stressed/tolerized

***

20

0

Tolerized

***
***

***

pg/mL

Cells/mm2–(Stress)
+

–

e

+
–

800

0
Tolerization
Stress

4

**

–
+

###

***

6

×104, cells/mL–(Stress)
+

–

c

#
*
1.25

PAS-positive cells, %a8Stressed

Color version available online

Asthma

PC200, mg/mL

RL, cmH2O/mL/s

20sion in either nontolerized or tolerized mice (Fig. 3c).
Therefore, the deviation of the Th1/Th2 balance, as seenby the IFN-γ/IL-4 ratios, toward Th2-dominant responses caused by the stress exposure in tolerized mice (Fig. 3d)
could be mainly due to the decreased expression of IFN-γ.

Stressed

***
*

***

*

Tolerized

Stressed/tolerized
***
***

10
0

IL-4

IL-5

IL-13

f

***

***
–
+

+
–

+
+

2

6

Kawano et al.

Int Arch Allergy Immunol
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172.16.6.87 - 6/12/2018 3:54:14 PM(For legend see next page.)Effects of Stress on the Development of Tolerogenic
DCs and Tregs
Because restraint stress inhibited the induction of respiratory tolerance, we analyzed Treg cells, pDCs, and IL10-producing mDCs as Treg-generating DCs in BLNs.
The numbers and percentages of pDCs (Fig. 4a, b) andTreg cells (Fig. 4c, d) were significantly increased in toler-ized mice compared with nontolerized mice under the
nonstress condition. The stress exposure significantly decreased the numbers (Fig. 4a), but not the percentages(Fig. 4b), of pDCs and both the numbers (Fig. 4c) andpercentages (Fig. 4d) of Treg cells in stressed/tolerized
mice compared with tolerized mice. Few IL-10-producing mDCs were detected in BLN cells.

***

0.9
0.8

6,000

0.6

IFN-γ, pg/mL

WST-8
optical density

*

***

0.7
0.5
0.4
0.3

4,000

2,000

0.2
0.1
–
–

200

+
–

0

+
+

b

IL-4/IFN-γ

**

100
50

0
Tolerizationc Stress

–
–

–
+

–
–

0.06

***

150
IL-4, pg/mL

Fig. 3. Effects of stress on sensitization. On
day 17, splenocytes were prepared frommice of each group of sensitized mice andstimulated in vitro with OVA. a After a
72-h culture, cell proliferation was measured in WST-8 assays. The content of
IFN-γ (b) and IL-4 (c), and the content ratios of IL-4/IFN-γ (d) in culture supernatants were determined by ELISA. Data areexpressed as the mean ± SD of triplicate
cultures from 3 independent experiments.
* p < 0.05, ** p < 0.01, and *** p < 0.001.

–
+

+
–

+
+

+
–

+
+

***
***

0.04

*

0.02

0

d

–
+

–
–

–
+

+
–

+
+

Fig. 2. Effects of stress during the tolerance induction phase on allergic airway responses. Airway reactivity (RL; a) and airway sensitivity (PC200; b) were measured 5 days after the last OVA challenge (day 22). Data are expressed as the mean ± SD (n = 9–13 from
4 independent experiments). * p < 0.05, ** p < 0.01, and *** p <0.001, compared with asthmatic mice; # p < 0.01 and ### p < 0.001,
compared with tolerized mice. c Lungs were isolated from mice ofeach group 5 days after the last OVA challenge (day 22). Lung sections were prepared and stained with PAS. Representative imagesof lung sections (left; scale bar, 100 μm) and the percentage of PASpositive cells (right) from the 4 mouse groups are shown. Data areexpressed as the mean ± SD (n = 5–7 mice from 3 independent
experiments). d BALFs were obtained from mice of each group 5days after the last OVA challenge (day 22). Total cells (Total), macrophages (MΦ), eosinophils (Eo), neutrophils (Neu), and lymphocytes (Lym) were counted. Data are expressed as the mean ± SD

(n = 7–9 from 3 independent experiments). e Lung sections were
stained with hematoxylin and eosin. Representative images of lungsections (left; scale bar, 100 μm) and the eosinophil counts in the
peribronchial area (right) are shown. Data are expressed as theEfmean ± SD (n = 5–8 mice from 3 independent experiments). f Effects of stress on BALF cytokine contents. BALFs were obtained
from mice of each group 1 day after the last OVA challenge (day18). BALF IL-4, IL-5, and IL-13 levels were measured by ELISA.
Data are expressed as the mean ± SD (n = 3–9 from 3 independent
experiments). g Effects of stress on OVA-specific IgE in serum.
Sera were obtained from mice of each treatment group 5 days after
the last OVA challenge (day 22). OVA-specific IgE in serum wasmeasured by ELISA. Data are expressed as the mean ± SD (n = 5–7
from 3 independent experiments). * p < 0.05, ** p < 0.01, and*** p < 0.001.

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0Tolerizationa Stress

c

8,000
6,000
4,000
2,000
0
Tolerization
Stress––90,000

–
+

**

+
–

+
+

***

60,00030,0000Tolerization
StressTransfer of the Inhibitory Effects of Stress on the
Acquisition of Respiratory Tolerance by CD4+ T Cells
Analyses of BAL cells after sensitization followed by
OVA inhalation in mice adoptively transferred with
CD4+ T cells showed similar numbers of total cells andeosinophils between the 4 groups (Fig. 5a) compared to
the variable levels observed between groups using theconventional mouse model (Fig. 2d). Adoptively trans-

–
–

–
+

+
–

+
+

PDCA-1+ cells/BLN cells, %10,000An adoptive transfer experiment was performed to examine the effects of stress exposure on DC functions that
promote the development of respiratory tolerance in ourstress/tolerance model. As expected, airway inflammation was suppressed in mice transferred with CD11c+
DCs from tolerized mice as compared with mice withouttransfer, although mice transferred with CD11c+ DCs
from stressed/tolerized mice showed no significant dif
difference compared to mice transferred with CD11c+ DCs
from tolerized mice (online suppl. Fig. 4A). Furthermore,
we investigated the effects of stress on the function of DCs
in Treg development. Treg, Th2, and Th1 cell development promoted by DCs from the tolerized and stressed/
tolerized mouse groups were not different (online suppl.
Fig. 4B).

8

**

1.5
1.0

*

0.5
0

b

–
–

12

–
+

+
–

*

+
+

**

8
4
0

d

–
–

–
+

+
–

+
+

ferring CD4+ T cells from tolerized mice resulted in significant decreases in the numbers of total cells and eosinophils in BALFs, relative to those numbers observed
after transferring CD4+ T cells from asthmatic mice or
mice injected with saline (vehicle control). The cellnumbers in BALFs measured after adoptively transferring CD4+ T cells from stressed/tolerized mice were significantly higher than those after transferring CD4+ T
cells from tolerized mice. However, no significant differences were found in the total numbers of cells or eosinophils between mice transferred with CD4+ T cells fromasthmatic and stressed/asthmatic mice. The number oflymphocytes in BALFs showed a tendency similar tothat of the numbers of total cells and eosinophils, although the differences between groups were not significant. In addition, the percentage of Treg cells in splenocytes from tolerized mice was significantly higher thanthat in asthmatic mice. In contrast, the percentage ofTreg cells in splenocytes was significantly lower instressed/tolerized mice than in tolerized mice (Fig. 5b).
The percentage of CD3+CD4+ cells in splenocytes was
not significantly different between the 4 groups (datanot shown).

Kawano et al.

Int Arch Allergy Immunol
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Fig. 4. Effects of stress on tolerogenic DCs
and Treg cells in BLN. BLNs were collected
after the final stress exposure (day –2). The
numbers and percentages of PDCA-1+ cells(a, b), and CD3+CD4+Foxp3+ cells (c, d)were determined. Data from 3 independent
experiments for nontolerized groups and 4
independent experiments for tolerizedgroups are expressed as the mean ± SD.
* p < 0.05, ** p < 0.01, and *** p < 0.001.

CD3+CD4+Foxp3+ cells/mouse, na*

12,000Foxp3+/CD3+CD4+ cells, %PDCA-1+ cells/mouse, n

*

150
100
50
0

Sal.

a

–
–

–
+

+
–

+
+

250
200
150
100
50
0

Sal.

Tolerization
Stress––CD4+ cells
–
+

+
–

150
100
50
0

Sal.

Tolerization
StressLymphocytes, ×104 cells/mL

Neutrophils, ×104 cells/mL

Tolerization
StressCD4+ cells

200

+
+

–
–

CD4+ cells
–
+

+
–

+
+

150

200
150
100
50
Sal.

Tolerization
Stress––CD4+ cells
–
+

+
–

+
+

**

100

b

*

50
0

Sal.

Tolerization
Stress

250

0

200

–
–

CD4+ cells
–
+

+
–

*

15

+
+

*

10

5

0
Tolerization
Stress

–
–

–
+

+
–

+
+

Fig. 5. Adoptive transfer of spleen CD4+ T cells. CD4+ T cells were

OVA/alum and challenged with OVA. BALFs were obtained frommice of each group 5 days after the last OVA challenge. a Total
cells, macrophages, eosinophils, neutrophils, and lymphocytes
were counted. Data are expressed as the mean ± SD (n = 3–9 from
2 independent experiments). * p < 0.05 and ** p < 0.01.

Effects of a GC-Receptor Antagonist on Stress-Induced
Inhibition of Respiratory Tolerance
The sizes and weights of BLNs and number of BLN
cells in stressed/tolerized mice were lower than those in
nonstressed/tolerized mice (Fig. 6a–c in solvent-treatedgroups). The atrophy of BLNs caused by exposure to restraint stress was abolished by treatment with RU-486(Fig. 6a, b). Furthermore, the numbers of BLN cells, Treg
cells, and tolerogenic DCs in stressed/tolerized mice recovered to the levels in nonstressed/tolerized mice after
RU-486 treatment (Fig. 6c, d, f). Stress exposure appeared
to affect the numbers of total cells, eosinophils, neutrophils, and lymphocytes in BALFs observed in tolerized
and stressed/tolerized mice treated with solvent, but notin those treated with RU-486 (Fig. 7).

Effects of GC as a Substitute for the Stress on
Respiratory Tolerance
Because the effects of stress exposure were diminishedby administering the GC-receptor antagonist just before
stress exposure, we examined whether exogenous GC
could reproduce the effects of stress exposure. DEX administration significantly increased the numbers of total
cells (250, 1,000 µg/kg), eosinophils (250, 1,000 µg/kg),
and lymphocytes (250 µg/kg) in BALFs as compared to
PBS administration in tolerized mice, but not in nontolerized mice (Fig. 8).

Allergic Asthma Susceptibility andPsychological Stress

Int Arch Allergy Immunol
DOI: 10.1159/000488289

isolated from spleens of mice of each mouse 1 day after the final
stress exposure (day –1). b Spleen CD3+CD4+Foxp3+ cells wereanalyzed by flow cytometry. CD4+ T cells were intravenously injected into naïve BALB/c mice. Saline (Sal.) was injected as a control. One day postinjection, mice of all groups were sensitized with

Effects of GC on BM-DC Proliferation
The proliferation of BM-DCs stimulated with OVA
was reduced by DEX in a dose-dependent manner (Fig. 9).

— 440 —

9

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172.16.6.87 - 6/12/2018 3:54:14 PM*

250

Foxp3+/CD3+CD4+ cells, %

200

250

Eosinophils, ×104 cells/mL*

Macrophages, ×104 cells/mL

Total cells, ×104 cells/mL

250

Color version available online

1,000,000–––+
Solventc
60,000

0.5

–
–

–
+

Solvent+–++

RU-486**++–+
RU-486*40,00020,0000–––+
SolventfFig. 6. Effects of a GC-receptor antagonist on BLN enlargement

and cellular changes induced by stress. Tolerized mice with orwithout stress exposure were administered a GC-receptor antagonist, RU-486, or the solvent as a control before each stress exposure. BLNs were collected after the final stress exposure (day –2)
and weighed. Representative images (a; scale bar, 5 mm) andweights (b) of BLNs in each mouse group are shown. Data are expressed as the mean ± SD (n = 7–8 from 3 independent experi-

Discussion

In this study, we demonstrated for the first time that
exposure to psychological stress during the induction
phase of respiratory tolerance, but not during the sensitization phase, promoted the development of Th2-biased
sensitization and, thereby, asthmatic airway responses to10*

500,000
0++–+
RU-486

1.0

e

**

+
+
–
+
RU-486

RU-486++40,00030,000**20,00010,000
0

d

–
–
–
+
Solvent

*

15
10

+
+
–
+
RU-486

*

5
0

g

–
–
–
+
Solvent++–+
RU-486

ments). Cell populations of BLN cells were analyzed by flow cytometry. The numbers of total BLN cells (c), PDCA-1+ cells (d),and CD3+CD4+Foxp3+ cells (f) were determined and are expressedas percentages of PDCA-1+ cells (e) and CD3+CD4+Foxp3+ cells(g) in BLN cells. Data are expressed as the mean ± SD (n = 6 from
2 independent experiments). * p < 0.05, ** p < 0.01, and *** p <0.001.

inhaled allergen, while restraint-stress exposure had noeffect on sensitization and allergic airway inflammation(Fig. 2; online suppl. Fig. 3). Preventing the developmentof immune tolerance by stress exposure was confirmed by
the re-emergence of Ag-specific splenocyte proliferation
and Th2-predominant cytokine secretion in stressed/tolerized mice, both of which were suppressed in tolerized
Kawano et al.

Int Arch Allergy Immunol
DOI: 10.1159/000488289— 441 —

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172.16.6.87 - 6/12/2018 3:54:14 PM–––+
Solvent

1.5

0Tolerization
Stress+–Foxp3+/CD3+CD4+ cells, %5bPDCA-1+ cells/BLN
cells, %*100Tolerization
Stress++1,500,000**

BLN cells/mouse, n

15

Solvent

CD3+CD4+Foxp3+
cells/mouse, n

Weight of BLN, mg

a+–

PDCA-1+ cells/mouse, nTolerization
StressSolvent, ×104 cells/mL

**

150

***

100
50
0

150

Total

50

250

***
***

MĴ

Eo

104 cells/mL

Stressed/tolerized

**

**
Neu

***

Lym

***
***

***
**

###

###
Total

MĴ

***
***

##

Eo

Neu

##

Lym

**
**

200

Fig. 8. Effects of DEX on the numbers ofinflammatory cells. Solvent or 15.6, 62.5,
250, or 1,000 µg DEX/kg body weight wasadministered instead of stress exposure to
nontolerized (open columns) and tolerized(closed columns) mice. BALFs were obtained from mice of each treatment group5 days after the last OVA challenge (day22). Total cells (Total), macrophages (MΦ),
eosinophils (Eo), neutrophils (Neu), andlymphocytes (Lym) were counted. Data areexpressed as the mean ± SD (n = 5–7 from
2 independent experiments). * p < 0.05 and** p < 0.01.

Tolerized

***
**

100

0

*

AsthmaStressed

150

**
*

100

*
*

50

**
**

0
Tolerization – – – + + + + +
DEX –

–
Total

– – – +++++– – – +++++– – – +++++– – – +++++

–

–

–

–

–
MĴ

–
Eo

–
Neu

–
Lym

mice as compared with asthmatic mice (Fig. 3). Stress exposure might inhibit the development of respiratory tolerance by dampening the tolerogenic DC-Treg cell axis
(Fig. 4, 5), which could result from GC release upon stress
exposure (Fig. 5–9).

In this study, the induction of immune tolerance by
OVA inhalation correlated with BLN swelling, which was
inhibited by stress exposure (Fig. 6). BLN atrophy following stress exposure has been shown in recent reports, revealing that stress leads to adverse physiological effects,
such as atrophy of the thymus and lymph nodes [41, 42].

Allergic Asthma Susceptibility andPsychological Stress

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172.16.6.87 - 6/12/2018 3:54:14 PMon the stress-induced inhibition of respiratory tolerance. A GC-receptor antagonist,
RU-486, or solvent was administered before each stress exposure. BALFs were obtained from mice of each treatment group5 days after the last OVA challenge (day22). Total cells (Total), macrophages (MΦ),
eosinophils (Eo), neutrophils (Neu), andlymphocytes (Lym) were counted. Data areexpressed as the mean ± SD (n = 5–8 from
4 independent experiments). * p < 0.05,** p < 0.01, and *** p < 0.001; # p < 0.05,## p < 0.01, and ### p < 0.001, compared withsolvent-treated control mice.

RU-486, ×104 cells/mL

Fig. 7. Effects of a GC-receptor antagonist

200

*

WST-8
optical density

0.20
0.15
0.10
0.05
0

0

10–9 M

10–8 M

10–7 M

10–6 M
DEX

Fig. 9. Effects of DEX on BM-DC proliferation. BM-DCs were generated from female BALB/c mice. BM-DCs (2/104 cells in 200 µL/well) in 96-well culture plates were stimulated with OVA (100 μg/mL) in the absence or presence of DEX at the indicated concentrations for 24 h. Cell proliferation was measured in WST-8 assays.

Stress exposure caused the reduction of the absolute
number of pDCs in BLNs (Fig. 4a), whereas the percentage of pDCs (Fig. 4b) and the ability of DCs to generate
Treg cells (online suppl. Fig. 4B) were not altered and,furthermore, the effect of stress was not transferred byDCs (online suppl. Fig. 4A). Therefore, it is assumed thatthe potential roles for tolerogenic DCs in this model
might not be strong. However, the transfer of CD4+ T
cells from the spleen of stressed/tolerized mice caused there-emergence of allergic airway inflammation, which was
suppressed by the transfer of CD4+ T cells from tolerized
mice (Fig. 5a), and the percentage of Treg cells in transferred splenocytes was significantly lower in stressed/tolerized mice than in tolerized mice (Fig. 5b). Thus, stress
exposure during the tolerance-induction phase may havedisturbed the induction, but not the function, of tolerogenic DCs, leading to insufficient differentiation of Treg
cells, resulting in the failure to prevent Th2-biased immune sensitization. The inhibitory effects of stress exposure on Treg cell development are supported by studies
showing that traumatic stress and a mental stressor reduced Treg cell numbers in peripheral blood in humans[27–29].
In airway immune responses, the stress exposure significantly increased IL-4 expression (Fig. 2f), although
the stress had no effects on IFN-γ expression (data notshown), in BALF in tolerized mice. Furthermore, thenumber (Fig. 4c) and percentage (Fig. 4d) of Treg cells in
BLNs were decreased by the stress exposure. On the oth12

er hand, in the spleen, the deviation of the Th1/Th2 balance toward Th2-dominant responses caused by thestress exposure in tolerized mice (Fig. 3d) could be mainly due to the decreased expression of IFN-γ, while thepercentage of Treg cells was decreased by the stress exposure (data not shown). Together with these findings, although the stress exposure has a potential to inhibit thedevelopment of tolerance leading to Th2-dominant immune responses, the main mechanisms underlying thedeviation towards Th2-dominant immune responses
might be different depending on the site of immune responses, for example systemic immunity in spleen andlocal immunity in airways.
To investigate the mechanisms underlying the inhibitory activity of stress on immune tolerance, we assessed
the contribution of GC released upon stress exposure(online suppl. Fig. 2). The anti-tolerogenic effect of stress
exposure was abolished by administering RU-486 (a GCreceptor antagonist) before each stress exposure during
the tolerance-induction phase. RU-486 administration
caused a re-emergence of BLN swelling (Fig. 6a) and retrieved the numbers of BLN cells, pDCs, and Treg cells in
BLNs (Fig. 6) and inflammatory cells in BALFs after antigen challenge (Fig. 7) to the levels in tolerized but notstressed mice. Furthermore, the administration of DEX
as a substitute for restraint stress during the toleranceinduction phase showed a similar effect as stress exposure, as demonstrated by the significant increase in thenumbers of total cells, eosinophils, and lymphocytes in
BALFs, compared with those in tolerized mice administered PBS (Fig. 8). These results indicated that GC signaling played a critical role in the stress exposure-induced
perturbation of immune tolerance. GC can induce apoptosis of several types of immune cells [43], including
mDCs [44] and pDCs [45]. Because RU-486 caused a reemergence of BLN swelling and restored the number of
pDCs, we investigated the effect of GC on DC proliferation in vitro. As expected, DEX reduced the proliferationof BM-DCs in a dose-dependent fashion (Fig. 9). Therefore, it is likely that inhibiting the acquisition of immune
tolerance by stress exposure was mediated by GC, which
decreased the number of pDCs and impaired Treg differentiation.
GC is recognized worldwide as a gold standard fortreating and preventing asthma by its anti-allergic activities, including the reduction of airway inflammationand improvement of AHR through limiting cytokine
production by T cells and epithelial cells, and impairing
the recruitment, survival, and activation of eosinophils
and other inflammatory cells [2, 46]. However, GC has
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0.25

been shown to amplify Th2 immune responses via several mechanisms. GC treatment can potentiate Th2 difdifferentiation, by either reducing IL-12 secretion from antigen-presenting cells [47] or directly suppressing Th1
cell polarization [48]. GC administration during sensitization enhanced Th2 responses in an allergic mousemodel [31]. In addition, GC treatment during antigen
inhalation (to promote the development of respiratory
tolerance) eliminated IL-10-producing DCs required forthe development of IL-10-producing Treg cells, leading
to Th2-biased sensitization and allergic airway responses
upon antigen exposure [30]. Interestingly, the development of respiratory tolerance was prevented by systemic,
but not local, GC administration [49], in accordance with
our finding that the GC level in the circulation had increased. Systemic administration of GC to immunocompetent humans and naïve mice significantly reduced thenumber, but not the suppressive function, of Foxp3+
Treg cells in blood [50]. More importantly, either systemic or local GC administration during the sensitization and allergen-induced inflammation phases decreased the number of Foxp3+ Treg cells in mouse lungs[51]. In contrast, both GC inhalation and systemic GC
treatment increased the number and function of
CD4+CD25high Treg cells in blood and BALF in pediatricasthma [15], and Foxp3+ Treg cells in blood in adult asthma [52]. Whether the effect of GC on human Treg cells
is inhibitory or stimulatory might depend on the immunocompetence status, such as healthy (immunocompetent) or asthmatic. The studies mentioned above and thisstudy involved the use of wild-type, naïve mice so thatthe effect of GC treatment on Treg cells would be inhibitory, as observed with healthy subjects. The reason forthe dependency was not determined and is beyond theaims of this study.
To our knowledge, it has not yet been reported thatpsychological stress prevents the development of respiratory tolerance. However, some studies have investigatedthe mechanisms underlying the impairment of immune
tolerance associated with increased susceptibility to allergic asthma. Respiratory infections with viruses, suchas rhinovirus and RSV, in early life are risk factors forasthma development in adult life. Simultaneous rhinovirus infection and tolerance induction in mice inhibitedthe acquisition of immune tolerance induced by intranasal OVA, attenuating the development of Foxp3+ Treg
cells through OX40 ligand expression on lung DCs andthymic stromal lymphopoietin and IL-33 expression by
airway epithelial cells, which increased the susceptibility
to allergic airway responses [53]. Krishnamoorthy et al.

[19] demonstrated that after OVA tolerization by breast
milk from maternal mice exposed to OVA, recurrent
RSV infection in pups induced allergic responses, while
the allergic responses were not observed in uninfectedpups, in adult life (6 weeks old). RSV infection impaired
immune tolerance by inducing phenotypic changes inFoxp3+ Treg cells, including GATA-3 expression, Th2
cytokine production, and loss of suppressive functions,in an IL-4-dependent pathway [19]. The potential of IL33 in overcoming acquired immune tolerance was confirmed recently [54]. It was revealed that Foxp3+ Treg
cells express the IL-33 receptor ST2, the expression ofwhich was enhanced by the ligand, that IL-33 evoked
Th2-like properties in those cells by inducing GATA-3
expression and the synthesis of Th2 cytokines associatedwith the loss of suppressive function, and that intranasal
IL-33 administration to tolerized mice (during or after
sensitization) caused allergen-induced airway inflammation.
In conclusion, psychological stress exposure inhibited the development of respiratory tolerance by GC-dependent suppression of tolerogenic DC and Treg cell induction, resulting in increased susceptibility to allergic
asthma through Th2-biased immune sensitization. It isnecessary to further explore the neuroendocrine pathway linking stress exposure to GC release, including
neuropeptides such as opioids involved in stress-related
asthma exacerbations [55, 56], as well as the endocrineimmune pathway, especially the GC-airway epithelium interaction leading to Th2-dominant immune responses.

Allergic Asthma Susceptibility andPsychological Stress

Int Arch Allergy Immunol
DOI: 10.1159/000488289

Acknowledgements
We thank Dr. Ryo Abe (Tokyo University of Science, Noda,
Japan) for providing the DO11.10 (Rag2–/–) mice.

Statement of Ethics
Studies on animal subjects were approved by the Committee ofAnimal Experiments at Tohoku Medical and Pharmaceutical University (approval numbers: 14002-cn, 15001-cn, and 16002-cn).
We took the utmost care to alleviate any pain and suffering in themice.

Disclosure Statement— 444 —

13

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The authors have no conflicts of interest to declare.

Funding Sources

Author Contributions

This work was supported in part by a Grant-in-Aid for Scientific Research (C; No. 25461164, 17K09624), a Grant-in-Aid forYoung Scientists (B; No. 16K19608, 17K16212), and the Matching
Fund Subsidy for Private Universities (No. S1001002, S1511001L)from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. The funders played no role in the study
design, the collection, analysis, and interpretation of data, or the
preparation of the report.

T.K. and I.O. designed the study. T.K., R.O., and T.I. performed
the majority of experiments. C.M. contributed to the flow cytometric analysis of lung cells and lymph node cells. T.M. contributed tothe analysis of histology. Y.O. contributed to the analysis of AHR.
N.O., T.T., and M.T. suggested key experiments and edited themanuscript. T.K. and I.O. wrote the manuscript.

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original article

The expression of 11 β−hydroxysteroid
dehydrogenase in severe allergic rhinitis
Nobuo OhtaMD, PhD1, Yusuke SuzukiMD, PhD2, Naoya NoguchiMD, PhD1, RisakoKakutaMD, PhD1, Takahiro SuzukiMD, PhD1, Toshiichi
AwataguchiMD, PhD1, Yukiko TakahashiMD, PhD1, Sachiko Tomioka-MatsutaniMD, PhD1, Yusuke IshidaMD, PhD3, Ryokichi IkedaMD,
PhD1, Muneharu YamazakiMD, PhD1, Yusuke KusanoMD1, Yutarou SaitoMD1, Fumi ShojiMD, PhD1,
Yuji YaginumaMD, PhD1, Tatsutoshi SuzukiMD, PhD4, Hiroshi OsafuneMD5, Tasuku KawanoPhD6,
Tomomitsu MiyasakaPhD6, Tomoko TakahashiMD, PhD6, Isao OhnoMD, PhD7, KotaWadaMD, PhD5Division of Otolaryngology, Tohoku Medical and Pharmaceutical University Hospital 1-12-1, Fukumuro, Miyaginoku, Sendai, Japan
Department of Otolaryngology, Head and Neck Surgery, Yamagata University Faculty of Medicine, Yamagata, Japan
3
Division of Anatomy and Cell Biology, Tohoku Medical and Pharmaceutical University, Sendai, Japan
4
Department of Otolaryngology, Head and Neck Surgery, Kitasato University Faculty of Medicine, Kanagawa, Japan
5
Department of Otolaryngology, Head and Neck Surgery, Toho University Faculty of Medicine, Tokyo, Japan
6
Division of Pathophysiology, Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical
University, Sendai, Japan
7
Center for Medical Education, Faculty of Medicine, Tohoku Medical and Pharmaceutical University, Sendai, Japan
1

2

Article history: Received: 15.09.2018

AbstrAct:

Accepted: 05.10.2018 Published: 05.10.2018

objective: To clarify the roles of 11 β-HSD in resistance to glucocorticoid therapy for allergic rhinitis, a case series study was
conducted.
Methods: The patient group consisted of 20 subjects with allergic rhinitis, aged from 21 to 46 years (mean age 26.5), whoshowed persistent GC resistance necessitating surgical removal of the inferior turbinate after 6 months of GC treatment. The
patients with poor response to GC treatment for 6 months were defined as GC-resistant. The control group consisted of 10
subjects aged from 16 to 39 years (mean age 24.5) who underwent maxillofacial surgery, from whom nasal tissues were taken
and who did not receive GC treatment. Nasal mucosal tissues from patients and control subjects were examined immunohistochemically. The sections were washed with 0.01 M phosphate-buffered saline (PBS; pH 7.2) containing 0.15 M NaCl and
0.01% Triton X-100, and incubated for 2 h with rabbit polyclonal anti-11 β−HSD1 and 11 β−HSD2 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), each diluted 1:200 in PBS containing 0.1% bovine serum albumin. Immunostained sections
were assessed under an Olympus microscope with an eyepiece reticule at 200× magnification. Cell counts are expressed asmeans per high-power field (0.202 mm2). Control group means (arithmetic mean ± SD) were compared with patient groupmeans by Mann–Whitney U-test at P = 0.05.
results: Although 11 β−HSD1 was expressed to a similar extent in patients and controls, 11 β−HSD2 was expressed more significantly in patients with severe allergic rhinitis, resulting in an increased HSD-1/HSD-2 ratio. The significantly increased
expression of 11 β−HSD2 in the nasal epithelium and submucosal inflammatory cells of patients with severe nasal allergy wasobserved in the present study.
conclusion: Our findings suggest that 11 β-HSD2 plays an important role in resistance to glucocorticoid therapy for allergic
rhinitis, and its expression might be used as an additional parameter indicating steroid resistance in allergic rhinitis.

Keywords:

allergic rhinitis, 11 β-hydroxysetroid dehydrogenase, glucocorticoid resistance, glucocorticoid therapy, glucocorticoid response

IntroductIon
Glucocorticoids (GCs) are commonly used as anti-inflammatory agents in the treatment of chronic allergic diseases, including
allergic rhinitis. However, these clinical benefits are sometimes
limited because some patients demonstrate persistent tissue inflammation despite repeated and high doses of GC treatment. Studies of GC receptors (GR), NF-κB and other mediators in underlying mechanism of GC resistance have been reported (1,2). The
11β-hydroxysteroid dehydrogenase (11β-HSD) is a tissue-specificregulator of glucocorticoid responses, inducing the interconversion of inactive and active glucocorticoids. The 11β-HSD has two
isoforms, 11β-HSD1, which converts inactive 11-keto corticosteroids into active 11-hydroxy corticosteroids, thereby potentiating theeffects of endogenous glucocorticoids, and 11β-HSD2,which acts
as a classic dehydrogenase by converting active 11-hydroxylated
cortisol and corticosterone into inactive 11-keto forms of cortisone and 11-dehydrocorticosterone, respectively (3). Here, to clarify
the roles of 11 β-HSD in resistance to glucocorticoid therapy forallergic rhinitis, a case series study was conducted.

OtOlaryngOl POl 2019; 73 (2): 9-13

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9

original article

Fig. 1. Representative pathological findings of 11β-HSD1 immunoreactivities in the nasal mucosa of patients with severe nasal allergy and controls. (original magnification ×100).

subjects And Methods
All patients were treated by experienced ENT doctors at Yamagata
City Hospital Saiseikan, Yamagata University Faculty of Medicine, and Tohoku Medical and Pharmaceutical University between
February 2010 and August 2017. Informed consent was obtained
under protocols approved by the Institutional Review Board. The
patient group consisted of 20 subjects with allergic rhinitis, aged
from 21 to 46 years (mean age 26.5), who showed persistent GC
resistance necessitating surgical removal of the inferior turbinate
after 6 months of GC treatment. The patients with poor response to GC treatment for 6 months were defined as GC-resistant.
The control group consisted of 10 subjects aged from 16 to 39 years(mean age 24.5) who underwent maxillofacial surgery, from whom
nasal tissues were taken and who did not receive GC treatment.
Nasal mucosal tissues were fixed in 10% formalin for 1 to 2 days,
dehydrated through an ethanol series, and embedded in paraffin
wax. Sections 3 μm thick were dewaxed in xylene and dehydrated.
The sections were washed with 0.01 M phosphate-buffered saline(PBS; pH 7.2) containing 0.15 M NaCl and 0.01% Triton X-100,
and incubated for 2 h with rabbit polyclonal anti-11 β−HSD1 and11 β−HSD2 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz,
CA, USA), each diluted 1:200 in PBS containing 0.1% bovine serum
albumin. Controls for nonspecific staining were incubated with 10
μg/mL mouse IgG1 (DAKO, Glostrup, Denmark). Sections were

washed and incubated with biotinylated rabbit antibody to mouse
IgG, IgA, and IgM (Immunotech, Tokyo, Japan) for 1 h.
The sections were incubated with Vectastain reagent (ABC Elite;
Vector Laboratories, Burlingame, CA, USA), followed by 3.3-diaminobenzidine (Dojin Chemicals, Kumamoto, Japan) as the chromogen. Finally, the slides were counterstained with hematoxylin.
Immunostained sections were assessed under an Olympus microscope with an eyepiece reticule at 200× magnification. Cellcounts are expressed as means per high-power field (0.202 mm2).
At least 2 sections were immunostained, and more than 5 areas
were evaluated via the graticule. Results are expressed as positive cell ratio per field. Control group means (arithmetic mean ±SD) were compared with patient group means by Mann–Whitney U-test at P = 0.05.

results
Although 11 β−HSD1 was expressed to a similar extent in patientsand controls, 11 β−HSD2 was expressed more significantly in patients with severe allergic rhinitis, resulting in an increased HSD-1/
HSD-2 ratio. The significantly increased expression of 11β−HSD2
in the nasal epithelium and submucosal inflammatory cells of patients with severe nasal allergy was observed in the present study(Figure 1 and Figure 2).

10

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original article

Fig. 2. Representative pathological findings of 11β-HSD2 immunoreactivities in the nasal mucosa of patients with severe nasal allergy and controls. (original magnification ×100).

dIscussIon
The tissue levels of bioactive GCs are modulated by 11β-HSD
which interconverts corticosteroids between inactive and active
states; only the active forms of corticosteroids are able to interact
with GR (3). Previous studies demonstrated that the expressionof 11β-HSD1 was up-regulated and 11β-HSD2 was decreased inallergic rhinitis and chronic rhinosinusitis (3, 4). On the other
hand, a recent report suggests that 11β-HSD1 and 11β-HSD2 areassociated with steroid sensitivity in childhood nephrotic syndrome, acute lymphoblastic leukemia and 11β-HSD2 may contribute to steroid resistance (5). The 11β-HSD2 is also expressedin glucocorticoid-resistant leukemic cell lines where it contributes to prednisolone resistance (6).
The expression of 11β-HSD was regulated by Th2 type cytokines,
chemical mediators and reagents (7,8). Taken together, our results
strongly suggest that 11β-HSD2 expression in nasal mucosal epithelium and inflammatory cells could influence a patient’s sensitivity toGC, and explain why some patients exhibit persistent GC resistancedespite high doses and repeated treatment. The mechanisms underlying the development of steroid resistance remain unclear, andthere are currently no reliable biomarkers to identify patients whowill go on to develop steroid dependence and resistance.

conclusIon
Our findings suggest that 11β-HSD2 plays an important role inresistance to glucocorticoid therapy for allergic rhinitis, and its
expression might be used as an additional parameter indicating
steroid resistance in allergic rhinitis.

AcKnowledgMents
This work was supported by JSPS KAKENHI Grant Number
JP17K11363 and the Ministry of Health, Labor, and Welfare ofJapan. We express our sincere thanks to Mrs. Yuko Ohta, Uyo
Gakuen college, for her editorial assistance.

11

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original article

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by Th2 cytokines: asthma and rhinitis. Clin Exp Allergy. 2014;44(2):197-211.

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Jun YJ, Park SJ, Kim TH, Lee SH, Lee KJ, Hwang SM, et al. Expression of
11β-hydroxysteroid dehydrogenase 1 and 2 in patients with chronic rhinosinusitis and their possible contribution to local glucocorticoid activation in sinus mucosa. J Allergy Clin Immunol. 2014;134(4):926-934.

5.

Sai S, Yamamoto M, Yamaguchi R, Chapman KE, Hongo T. Reciprocal Regulation of 11β-HSDs May Predict Steroid Sensitivity in Childhood Nephrotic
Syndrome. Pediatrics. 2016 Sep;138(3).

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Tables: – Figures: 2

6.

Sai S, Nakagawa Y, Yamaguchi R, et al Expression of 11beta-hydroxysteroid
dehydrogenase 2 contributes to glucocorticoid resistance in lymphoblastic
leukemia cells. Leuk Res. 2011;35(12):1644–1648

7.

Park SJ, Kook JH, Kim HK, Kang SH, Lim SH, Kim HJ, et al Macrolides increase the expression of 11β-hydroxysteroid dehydrogenase 1 in human sinonasal
epithelium, contributing to glucocorticoid activation in sinonasal mucosa. Br
J Pharmacol. 2015;172(21):5083-95.

8.

Randall MJ, Kostin SF, Burgess EJ, Hoyt LR, Ather JL, Lundblad LK, et al.
Anti-i nflammatory effects of levalbuterol-induced 11β-hydroxysteroid dehydrogenase type 1 activity in airway epithelial cells. Front Endocrinol (Lausanne). 2015; 12;5:236.

9.

Hu A, Fatma S, Cao J, Grunstein JS, Nino G, Grumbach Y, et al Th2 cytokine-induced upregulation of 11beta-hydroxysteroid dehydrogenase-1 facilitates
glucocorticoid suppression of proasthmatic airway smooth muscle function.
Am J Physiol Lung Cell Mol Physiol. 2009;296(5):L790-803.

References: 9

Access the article online: DOI: 10.5604/01.3001.0012.6143

Table of content: https://otolaryngologypl.com/issue/11772

Corresponding author: Dr. Nobuo Ohta, Division of Otolaryngology, Tohoku Medical and Pharmaceutical University Hospital, 1-12-1, Fukumuro Miyaginoku,
Sendai 983-8512, Japan; Email: noohta@hosp.tohoku-mpu.ac.jp
Copyright © 2019 Polish Society of Otorhinolaryngologists Head and Neck Surgeons. Published by Index Copernicus Sp. z o.o. All rights reserved.
Competing interests: The authors declare that they have no competing interests.
Cite this article as: Ohta N., Suzuki Y., Noguchi N., Kakuta R., Suzuki T., Awataguchi T., Takahashi Y., Tomioka-Matsutani S., Ishida Y., Ikeda R., Yamazaki M., Kusano Y., Saito Y., Shoji F.,
Yaginuma Y., Suzuki T., Osafune H., Kawano T., Miyasaka T., Takahashi T., Ohno I., Wada K.: The expression of 11 β−hydroxysteroid dehydrogenase in severe allergic rhinitis;
Otolaryngol Pol 2019; DOI: 10.5604/01.3001.0012.6143

12

www.otolaryngologypl.com— 450 —

original article

13

OtOlaryngOl POl 2019; 73 (2): 9-13

— 451 —

— 452 —— 453 —— 454 —— 455 —— 456 —— 457 —— 458 —— 459 —— 460 —— 461 —— 462 —Received: 14 June 2017

| Revised: 13 December 2017 | Accepted: 17 December 2017

DOI: 10.1111/cea.13081

ORIGINAL ARTICLE
Basic Mechanisms in Allergic DiseaseSex-based differences in CD103+ dendritic cells promote
female-predominant Th2 cytokine production during allergic
asthmaC. Masuda1 | T. Miyasaka1 | K. Kawakami1 | J. Inokuchi2 | T. Kawano1 |K. Dobashi-Okuyama1 | T. Takahashi1 | M. Takayanagi1 | I. Ohno3
1
Division of Pathophysiology, Departmentof Pharmaceutical Sciences, Faculty of
Pharmaceutical Sciences, Tohoku Medical
and Pharmaceutical University, Sendai,Japan

Summary
Background: Gender disparities in adult patients with asthma regarding its prevalence and severity are mainly due to enhanced type 2 T-helper (Th2) cytokine pro-

Division of Glycopathology, Institute of
Molecular Biomembranes and Glycobiology,
Tohoku Medical and Pharmaceutical
University, Sendai, Japan

duction in female patients compared to that in male patients. However, the3(DCs) in females, specifically those displaying CD11b or CD103, during enhanced

2

Center for Medical Education, Faculty of
Medicine, Tohoku Medical andPharmaceutical University, Sendai, Japan
Correspondence
Tomomitsu Miyasaka, Division of
Pathophysiology, Department ofPharmaceutical Sciences, Faculty of
Pharmaceutical Sciences, Tohoku Medical
and Pharmaceutical University, Sendai,
Japan.
Email: t-miya13@tohoku-mpu.ac.jp

pathways mediating this effect remain unclear.
Objective: We aimed to determine the roles of two major subsets of dendritic cells
Th2 priming after allergen exposure, using an ovalbumin-induced asthma mousemodel.
Methods: Sex-based differences in the number of DCs at inflamed sites, costimulatory molecule expression on DCs, and the ability of DCs to differentiate na€ıve CD4+
T cells into Th2 population were evaluated after allergen exposure in asthmaticmice. In addition, we assessed the role of 17b-oestradiol in CD103+ DC function
during Th2 priming in vitro.
Results: The number of CD11bhigh DCs and CD103+ DCs in the lung and bronchial

Present address
K. Dobashi-Okuyama, Department ofMicrobiology, Faculty of Medicine,Interdisciplinary Graduate School of
Medicine and Engineering, University of
Yamanashi, Yamanashi, Japan.
Funding information
Ministry of Education, Culture, Sports,
Science and Technology of Japan, Grant/Award Number: Scientific Research (C) (No.
25461164), Research Activity Start-up (No.
25893222), Young Scientists (B) (No.
16K19608), Matching Fund Subsidy for
Private Universities

lymph node (BLN) was increased to a greater extent in female mice than in malemice at 16 to 20 hours after ovalbumin (OVA) inhalation. In BLNs, CD86 and I-A/
I-E expression levels and antigen uptake ability in CD103+ DCs, but not inCD11bhigh DCs, were greater in female mice than in male mice. Furthermore, CD4+
T cells cultured with CD103+ DCs from female mice produced higher levels of interleukin (IL)-4, IL-5, and IL-13, compared with CD4+ T cells cultured with CD103+
DCs from male mice. The 17b-oestradiol-oriented enhancement of CD86 expression
on CD103+ DCs after allergen exposure induced the enhanced IL-5 production from
CD4+ T cells.
Conclusions and Clinical Relevance: These findings suggest that with regard toasthma, enhanced Th2 cytokine production in females might be attributed to 17boestradiol-mediated Th2-oriented CD103+ DCs in the BLN.
KEYWORDS

bronchial asthma, CD103+ dendritic cells, oestradiol, sex-based differences, Th2

Clin Exp Allergy. 2018;48:379–393.

wileyonlinelibrary.com/journal/cea— 463 —© 2017 John Wiley & Sons Ltd

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1 | INTRODUCTION

allergen challenge increases the BDCA3 on mDC levels in the sputum of subjects with asthma, and the frequency of BDCA3+ mDCs

In medicine, sex has been recognized as a biological characteristic

and extent of allergy to house dust mites are reportedly positively

that defines cellular, molecular, and immunological differences, and iscorrelated,23 suggesting both BDCA1+ and BDCA3+ DC subsets are

an important modulator of disease severity and response to thera-

involved in asthmatic immune responses. However, sex-related dif-peutic intervention.1,2 Additionally, sex is deeply associated with theferences in DC subsets responsible for enhanced-Th2 cytokine pro-prevalence and severity of asthma, effectiveness of asthma treat-

duction in female patients with asthma remain unclear.

ment and adverse reactions to asthma treatment. The relapse rate

Recent studies in mice have shown that the CD11b+ CD103

after discharge from the emergency department is evidently higher(CD11b+) and CD11b CD103+ (CD103+) DCs, homologs of human3

for female patients than for male patients. Moreover, sex was foundCD1c (BDCA1)+ and CD141 (BDCA3)+ DCs, respectively, play crucial

to be significantly associated with bronchial hyperresponsiveness inroles in priming the Th2 response after allergen encounter.24-26

5systemic inflammatory conditions. Meanwhile, the prevalence andUpon allergen inhalation, conditional depletion of total DCs abol-

severity of asthma are greater in boys than in girls; however, this dif-ished Th2 cytokine production by lymph node cells and accumulation

ference is reversed after puberty due to age-related changes, partic-of airway eosinophils in a murine asthma model.27 Moreover, the4addition,

selective depletion of either CD11b+ DCs or CD103+ DCs signifi-

postmenopausal use of oestrogen alone was associated with ancantly reduced these asthmatic responses.28,29 Therefore, both DC

9,10

increased rate of newly diagnosed asthma in menopausal women.

subsets are required to induce a complete immune response to

Gender-based epidemiological differences4-8 are frequently associ-inhaled allergen.

ularly,alterations

in

sex

hormonelevels.6-8

In

ated with the specific overproduction of type 2 T-helper (Th2)

In this study, we aimed to clarify the role of DCs in sex-related

cytokines in females. Actually, more interleukin (IL)-13–producing T

enhanced Th2 cytokine production upon allergen inhalation using acells are observed in female patients with atopic asthma than in male

mouse model.

patients.11 Thus, increasing evidence has indicated that symptombased features of asthma in female patients are based on the differences in pathogenic features, including enhanced Th2-type immune
responses.12
Animal studies have verified the influence of sex on the patho-

2 | MATERIALS AND METHODS
2.1 | Micegenesis of asthma. Using an ovalbumin (OVA)-induced asthma mouse

Male and female C57BL/6 (wild-type; WT) mice, and OT-I and OT-II

model, an increased number of CD4+ T cells and eosinophils, higher

transgenic (Tg) mice were obtained from CLEA Japan (Osaka, Japan)

Th2 cytokine content in broncho-alveolar lavage (BAL) fluid andand the Jackson Laboratory (Bar Harbor, ME, USA), respectively. All

higher Th2 cytokine production by the spleen and bronchial lymph

mice were maintained under specific pathogen-free conditions at thenode (BLN) cells were noted in female asthmatic mice compared toInstitute for Animal Experimentation, Tohoku Medical and Pharma-

Furthermore, ovariectomy before

ceutical University (Sendai, Japan). All experimental procedures

OVA inhalation significantly inhibited lung eosinophil infiltration.

involving animals were approved by the Committee of Animal Exper-

Moreover, oestradiol treatment in ovariectomized mice partiallyiments at Tohoku Medical and Pharmaceutical University (approval

restored the number of eosinophils in the BAL fluids of asthmatic

numbers: 14002-cn, 15001-cn, and 16002-cn). We took utmost caremice.16

to alleviate any pain and suffering of the mice.

13-15

those in male asthmatic mice.

Dendritic cells (DCs), the most proficient antigen-presenting cells,
bridge the gap between innate and adaptive immune responses bypriming Th cells, including Th1, Th2, and Th17 cells, to respiratory

2.2 | Sensitization and antigen challengeallergens, which is a critical step for the development and exacerba-

Mice, 6-8 weeks old, were sensitized through intraperitoneal injec-tion of allergic asthma.17 In patients with asthma, circulating myeloid

tion of 8 lg OVA (Grade V; Sigma-Aldrich, St Louis, MO, USA)

DCs extravasate from the circulation within 4-5 hours after allergenadsorbed with 4 mg aluminium hydroxide (Wako Pure Chemical

facili-Industries, Osaka, Japan) in 500 lL saline, on days 0 and 5. On day+

tating acquired immunity to the inhaled allergen. In humans, BDCA1

26, mice were challenged with aerosolized OVA (0.5% in saline) for

myeloid (m) DCs accumulate to a greater extent in the airway

1 hour on two occasions 4 hour apart.15,30

18,19

inhalation to the bronchial wall during the first 24 hours,

epithelia of patients with Th2-high asthma, compared to that inpatients with Th2-low asthma or healthy controls.20 Mature BDCA1+
DCs from patients with atopic dermatitis induce fewer interferon

2.3 | Preparation of the lung white blood cells(IFN)-c–producing and more IL-4–producing helper T cells, compared

Mice were sacrificed before or at various time intervals after OVA

21

In patients with allergic rhinitis,

challenge. Pulmonary leucocytes were prepared as previously

local allergen challenge resulted in an increase in mDC and plasma-described.31 Briefly, the lung vascular bed was flushed by injectingwith DCs from healthy controls.

22

cytoid DC numbers in the nasal mucosa and skin.

Meanwhile,

5 mL chilled saline into the right ventricle. The entire lungs were— 464 —

| 381

MASUDA ET AL.

teased with a 40-lm cell strainer (BD Falcon, Bedford, MA, USA) andincubated in RPMI 1640 medium (NakaraiTesque, Kyoto, Japan) with

2.6 | Preparation of the lung and BLN homogenate

10% foetal calf serum (FCS; Thermo Fisher Scientific, Waltham, MA,

For RT-PCR, the entire lungs were excised from WT mice beforeUSA), 100 U/mL penicillin G, 100 lg/mL streptomycin, 10 mmol/Land 3 and 16 hours after OVA challenge. BLNs were excised from

HEPES and 2 mmol/L L-glutamine, containing 20 U/mL collagenase D

WT mice before and 16 and 20 hours after OVA inhalation. Theseand 1 lg/mL DNase I (Roche Diagnostics GmbH, Mannheim, Ger-tissues were homogenized in buffer RLT (Qiagen, Valencia, CA, USA)many). After incubation for 60 minutes at 37°C with vigorous shaking,supplemented with 1% 2-mercaptoethanol. For cytokine assay, thecells were resuspended in 4 mL 40% (v/v) Percoll (Pharmacia, Upp-

entire lungs were excised 1 day after OVA inhalation and homoge-sala, Sweden) and layered onto 4 mL 80% (v/v) Percoll. After centrifu-

nized in chilled 0.1% Triton-X PBS with 1% protease inhibitor

gation at 6009 g for 20 minutes at 15°C, cells at the interface were

(Sigma-Aldrich). After centrifugation at 15 0009 g for 15 minutes atcollected. For some experiments, the cells were centrifuged and

4°C, the supernatants were stored at 80°C.15

mounted onto a glass slide, stained with Diff-Quick (Sysmex Corp.,
Kobe, Japan), and then observed under a microscope. The number ofleucocyte fractions was estimated by multiplying the total leucocyte

2.7 | Culture of DCs with T cells
CD4+ T cells or CD8+ T cells were isolated from the spleen cell pop-

number by the proportion of each fraction, for 200 cells.

ulation of na€ıve male and female OT-II or OT-I Tg mice using an
auto MACS Separator (Miltenyi Biotec) and anti-mouse CD4 or CD8

2.4 | Preparation of the BAL fluids

Micro Beads (Miltenyi Biotec), respectively. Furthermore, CD3+
Briefly, BAL

CD4+ CD11c and CD3+ CD8+ CD11c cells were purified fromsamples collected on day 3 or 5 after OVA inhalation with

CD4+ and CD8+ T cell population by cell sorting using a BDAriaII

2 9 0.25 mL chilled PBS through a cannula inserted in the trachea

flow cytometer. The proportion of CD4+ T cells or CD8+ T cells inwere centrifuged at 4509 g for 10 minutes at 4°C, and the super-

the sorted cell population was >99%. For the culture experiment of

natants were stored at 80°C for the cytokine assay. Cells (2 9 105)

DCs with T cells, T cell population was prepared as a mixture of T

were stained with Diff-Quick solution, and cell differential percent-cells from both male and female mice at the ratio of 1:1 to exclude

age was determined by counting a minimum of 200 cells under lightthe influence of sex-related differences in the ability of T cells, based

microscopy. The number of effector Th2 cells in BAL fluids wason our previous observation that the potential of CD4+ T cells todetermined by flow cytometry.

produce Th2 cytokine is different between male and female mice.32

BAL fluids were prepared as previously described.

15

CD4+ T cells from OT-II mice or CD8+ T cells from OT-I mice were2.5 | Preparation of DCs

cultured with each DC subset at a ratio of 3:1 in the presence ofEntire lungs and BLNs were extracted from sensitized and chal-

1640 medium supplemented with 10% FCS, 100 U/mL penicillin G,

lenged WT mice 16 or 20 hours after OVA inhalation, respectively.

100 lg/mL streptomycin, 2 mmol/L L-glutamine, and 50 lmol/L 2-

Pulmonary leucocytes and BLN cells were prepared as previously

mercaptoethanol. Following primary culture, T cells were further cul-

described.14 The number of each DC subset per lung and BLN was

tured in microwells coated with 1 lg/mL purified anti-CD3e (Clone

calculated based on the total number of leucocytes, and percentage

145-2C11; BioLegend, San Diego, CA, USA) and 5 lg/mL anti-CD28

of each DC subset in those populations was estimated using a(Clone 37.51; BioLegend). For some experiments, CD4+ T cells were

BDAriaII flow cytometer (BD Biosciences, San Jose, CA, USA). In

labelled with 1 lmol/L 5,6-carboxyfluorescein diacetate succinimidyl

another experiment, CD11c+ cells were enriched using an autoMACS

ester (CFSE; Dojindo Laboratories, Kumamoto, Japan) for 10 minutes

Separator (Miltenyi Biotec, Bergish Gladbach, Germany) and anti-

before coculture with CD103+ DCs for 2 or 4 days.

OVA323-339 or OVA257-264 peptide, respectively, for 4 days in RPMI

lowmouse Pan DC MicroBeads (Miltenyi Biotec). SSChihi

CD11c I-A/I-E

CD11bhi CD103 DCs and SSClow CD11chi I-A/I-Ehi CD103+ DCs
were purified by cell sorting using a BDAriaII flow cytometer. The

2.8 | CD103+ bone marrow-derived DCs

proportion of CD11chi I-A/I-Ehi cells in the sorted cells was consis-

CD103+ bone marrow-derived DCs (BM-DCs) were prepared as

tently 95%-98%. Furthermore, the proportion of CD11bhi anddescribed previously by Mayer et al33 In brief, BM-derived cells+

CD103 cells in DCs was consistently 96%-98%. Following centrifu-

from WT male mice were cultured at a density of 1.5 9 106 cells/gation, morphologies were assessed using a microscope by mounting

mL in 10 mL RPMI 1640 medium without phenol red supplemented

cells onto a glass slide and subsequent staining with Diff-Quick. To

with 10% FCS, 100 U/mL penicillin G, 100 lg/mL streptomycin,assess antigen uptake ability of lung DCs and migration into the

2 mmol/L L-glutamine, and 50 lmol/L 2-mercaptoethanol, and con-

BLN, Alexa Fluor 647-conjugated OVA (100 lg; Thermo Fisher Sci-

taining 3 ng/mL murine granulocyte-macrophage colony-stimulating

entific) and LPS (0.1 lg; Sigma-Aldrich) or non-labelled endotoxin-

factor (GM-CSF; Wako pure Chemical Industries, Ltd., Osaka,

free OVA (Hyglos GmbH, Bernried am Starnberger See, Germany)

Japan), and 200 ng/mL murine FMS-like tyrosine kinase 3 ligand

and LPS in 50 lL saline were injected intratracheally into sensitized(Flt3L; PeproTech Inc., Rocky Hill, NJ, USA) in the presence of vehi-

WT mice.

cle, 17b-oestradiol (1 9 107 M; Sigma-Aldrich), or 17b-oestradiol— 465 —

382|

MASUDA ET AL.

plus ICI182,780 (Tocris Bioscience, Ellisville, MO, USA). ICI182,780

BioLegend), PE-conjugated anti-CD11c, and PerCP-conjugated anti-

is an oestrogen receptor antagonist. On day 9, the non-adherent

CD3e (Clone 145-2C11; BioLegend). In the isolation of CD4+ T cells

cells were harvested and replated at a density of 3 9 106 cells/mL

for division-tracking experiments with CFSE, the spleen cells fromin 10 mL complete medium supplemented with GM-CSF and Flt3L

OT-II Tg mice were stained with PE-conjugated anti-CD11c, APC/

in the presence of the vehicle, 17b-oestradiol, or 17b-oestradiol

Cy7-conjugated anti-CD4 (Clone GK1.5; BioLegend), and PerCP-con-plus ICI182,780, as on day 0. On day 15, non-adherent cells werejugated anti-CD3e or APC-conjugated anti-CD3e (Clone 145-2C11;

collected and used as CD103+ BM-DCs. CD103+ BM-DCs, at a

BioLegend). In addition, to isolate CD8+ T cells for coculture experi-

5cells/mL, were stimulated for 24 hours withments, the spleen cells from OT-I Tg mice were stained with FITC-

OVA and/or murine recombinant IL-33 (R&D Systems, Inc., Min-

conjugated anti-CD8a (Clone 53-6.7; BioLegend), PE-conjugatedneapolis, MN, USA) in the presence of vehicle, 17b-oestradiol, or

anti-CD11c, and PerCP-conjugated anti-CD3e. Furthermore, to eval-

17b-oestradiol plus ICI182,780. For some experiments, 0.25 lg/mLuate ST2 expression on BLN CD4+ T cells after the culture withrecombinant anti-B7-2/CD86 antibody (R&D Systems, Inc.) or iso-CD103+ BM-DCs, cells were stained with FITC-conjugated anti-ST2

density of 5 9 10

type-matched control antibody was used to block CD86/CD28 cos-and APC-conjugated anti-CD3e. Cells in the BAL fluid were stained

timulatory signals.

with FITC-conjugated anti-CD3e (Clone 145-2C11; BioLegend) or
APC/Cy7-conjugated anti-CD3 (Clone 17A2; BioLegend), PE-conju-

2.9 | Measurement of cytokine and chemokine
concentration

gated anti-CD44 (Clone IM7; BioLegend), PerCP-conjugated anti-

Levels of IL-4, IL-5, IL-13, IL-17A, IL-33, and IFN-c were assayed

RM4-5; BD Biosciences) to identify effector Th2 cells. The positive

using enzyme-linked immunosorbent assay (ELISA) kits (eBioscience,

populations or mean values of the fluorescence intensity are

San Diego, CA, USA). The detection limits were 4 pg/mL for IL-4, IL-5,

defined based on isotype-matched control IgG for each Ab or

IL-13, and IL-17A; 25 pg/mL for IL-33; and 15 pg/mL for IFN-c. The

fluorescence minus one (FMO) controls. To distinguish false-

CCL20 content in the lung homogenate was measured using ELISA kit

positive signals from real positive ones, delta mean fluorescence(R&D Systems, Inc.). The limit of detection was 0.30 pg/mL. Total pro-intensity (MFI), defined as the ratio of MFI of sample histogram

tein levels of the lung homogenates were assayed using a detergent-

to MFI of control histogram, was used in evaluation of thecompatible protein assay kit (Bio-Rad Laboratory, Hercules, CA,

results in some experiments. Dead cells, stained with the LIVE/

USA). The cytokine and chemokine concentrations in the lung were

DEAD fixable blue dead cell stain kit (Thermo Fisher Scientific),

adjusted for the protein levels of each lung.15

were excluded from analysis. Data showed that 95%-99% of the

CD62L (Clone MEL-14; BioLegend), APC-conjugated anti-ST2 (Clone
DIH9; BioLegend), and Alexa Fluor 700-conjugated anti-CD4 (Clone

lung leucocytes were viable. Data from these studies were anal-

2.10 | Flow cytometric analysis

ysed using Diva (BD Biosciences) and FlowJo (Tree Star Inc.,

Cells were diluted to attain a density of 1 9 106/100 lL, and pre-by multiplying the leucocyte number, calculated as mentioned

incubated with anti-FccRII and III mAb (Clone 93; BioLegend) on ice

previously, by the proportion of each subset. However, thefor 15 minutes in PBS containing 1% FCS and 0.1% sodium azide.

interpretation of flow cytometry data presents limitations owing

To identify DC subsets, the lung cells and BLN cells were stained

to various factors, such as the types of antibodies included andwith FITC-conjugated anti-I-A/I-E (Clone M5/114.15.2; BioLegend),

the gating strategy.

Ashland, OR, USA) software. The number of DCs was estimatedPE-conjugated anti-CD11c (Clone N418; BioLegend), PerCP-conjugated anti-CD11b (Clone M1/70; BioLegend), and PE/Cy7-conjugated anti-CD103 (Clone 2E7; BioLegend). In addition, to evaluate

2.11 | Real-time RT-PCR analysis

surface molecule expression levels on DCs, APC-conjugated anti-

Total RNA was extracted from DCs, entire lung homogenates, andCD40 (Clone 3/23; BioLegend), CD80 (Clone 16-10A1; BioLegend),

BLN homogenates using a ReliaPrep RNA cell miniprep system (Pro-CD86 (Clone GL-1; BioLegend), or Alexa Fluor 700-conjugated anti-mega Corporation, WI, USA) or an RNeasy micro kit (Qiagen, Valen-

CCR7 (Clone 4B12; eBioscience, San Diego, CA, USA) were used. In

cia, CA, USA). First-strand cDNA was synthesized using theanother experiment, to evaluate ST2 expression levels on DCs, they

PrimeScript RT reagent kit with gDNA Eraser (TaKaRa Bio Inc., Otsu,

were stained with FITC-conjugated anti-ST2 (Clone DJ8; MD Bio-

Japan). Real-time reverse transcription (RT)-PCR was performed

sciences, Oakdale, MN, USA), PE-conjugated anti-CD11c, PerCP-con-using gene-specific primers and Power SYBR Green PCR Master Mix

jugated anti-CD11b, PE/Cy7-conjugated anti-CD103, and APC/Cy7-(Applied Biosystems, Foster City, CA, USA) and a Step One Plus

conjugated anti-I-A/I-E (Clone M5/114.15.2; BioLegend). In the

Real-Time PCR system (Applied Biosystems).15 The primer sequencesin vitro experiments, to evaluate CD86 and I-A/I-E expression levels

used for amplification are shown in Table S1. The expression levels+

on CD103 BM-DCs, they were stained with FITC-conjugated anti-I-of target genes and hypoxanthine-guanine phosphoribosyltransferase

A/I-E and APC-conjugated anti-CD86. Furthermore, to isolate CD4+

(Hprt) as a reference gene were calculated for each sample using the

T cells used in coculture experiments, the spleen cells from OT-II Tg

reaction efficiency, as determined by performing amplifications using

mice were stained with FITC-conjugated anti-CD4 (Clone GK1.5;

standards.

— 466 —

| 383

MASUDA ET AL.

2.12 | Confocal microscopic analysisof total cells 24 hours after OVA inhalation in female mice was

DCs were prepared from the BLN of male and female mice sensitizedicance (Figure 1A). In this model, increased accumulation of eosino-with non-labelled OVA and then intratracheally administered withphils (Figure 1C) and effector Th2 cells (Figure 1D) in the BAL fluids

Alexa Fluor 647-conjugated OVA. Cells were stained with Cell Mask

on day 5, and higher IL-4, IL-5, and IL-13 production (Figure 1E) ingreen plasma membrane stain (Thermo Fisher Scientific) and SYTO 82

the BAL fluids on day 3 post-OVA inhalation were observed inorange fluorescent nucleic acid stain (Thermo Fisher Scientific) accord-female mice compared with those in male mice. In addition, the lunging to manufacturer’s instructions. After incubation, the cells wereresistance in response to inhaled methacholine increased in a dose-

diluted to a density of 1 9 105/200 lL in Opti-MEM (Thermo Fisher

dependent manner in both male and female mice. Moreover, the

Scientific) containing 10% FCS, 0.1% sodium azide, and 10 mmol/Llung resistance was significantly enhanced in female mice compared

HEPES and then seeded in 35-mm glass bottom dishes (Matsunami

to that in male mice, 1 day after OVA inhalation (Figure 1F).

lower than that in male mice, although there was no statistical signif-

Glass Ind., Ltd, Osaka, Japan). Confocal studies were performed using

Next, to address sex-specific differences in the number of DCs inan oil immersion objective (60 9 UPLSAPO, numerical aperture 1.35)

response to inhaled allergens, two major DC subsets from alveolar

and Fluoview FV1000 confocal laser scanning microscopy (Olympus,

macrophages (CD11chi I-A/I-Elow SSChi cells) and monocytes (CD11c+

Tokyo, Japan). FV1000 software version 4.2 (Olympus) was used to

I-A/I-E cells) were distinguished using internal complexity [side scat-acquire and process the confocal images. Tri-colour images wereter (SSC) parameters], and the expression levels of I-A/I-E on CD11c+

acquired using a sequential acquisition mode to avoid cross-excitation.

cells, followed by the expression levels of CD11b and CD103, were
determined (Figure 2A). The number of total DCs and CD11bhi DCs
in male mice significantly increased 20 hours after allergen inhalation

2.13 | Measurement of airway responsivenessand was maintained up to 24 hours after inhalation, whereas the34,35

Airway responsiveness was measured as previously described.

In

number of these cells in female mice was significantly elevated,brief, to measure airway responsiveness, lung resistance at the base-peaked 20 hours after allergen inhalation, and decreased thereafter.

line and in response to aerosolized saline and acetyl-b-methylcholine

The number of total DCs and CD11bhi DCs was significantly higher(methacholine; Sigma-Aldrich) was measured for 3 minutes underin female mice than in male mice at 16 hours, but was not signifi-

each condition using Buxco whole body plethysmography (Buxco

cantly different at 20 and 24 hours post-OVA inhalation. In contrast,

Electronics, Sharon, CT, USA).

the number of CD103+ DCs in the lung was not significantly changed
within 24 hours of OVA inhalation in both male and female mice,

2.14 | Statistical analysis

although the number at 16 hours post-OVA inhalation was signifi-

Statistical analysis was performed using GraphPad Prism 5 software

results suggest that initial pulmonary cell responses after allergen(GraphPad Software, La Jolla, CA, USA). Differences between the

exposure, particularly, the increase in DCs occurring within 20 hours

two groups were tested using a two-tailed analysis and an unpaired

after allergen inhalation, are enhanced in the lungs of female miceStudent’s t test. Differences among the three groups or more werecompared to that in male mice.

cantly higher in female mice than in male mice (Figure 2B,C). These

tested using ANOVA with a post hoc analysis (Turkey’ multiple comparison test). A P-value of <.05 was considered significant.

To further address this possibility, we next examined the mRNA
expression of CCL2, CCL7, CCL19, CCL20, and CCL21 in the lung,which attract monocytes, immature DCs, and precursors of DCs to
the inflammatory sites.36 As shown in Figure 3A, CCL20 mRNA

3 | RESULTS

levels in the lung at 3 and 16 hours post-OVA inhalation were signif-

3.1 | Sex-based differences in the accumulation of
the lung DCs

icantly higher in female mice than in male mice, although other che-

To investigate sex-associated differences during the initial pulmonary

are associated with DC differentiation and activation in the lung dur-cell responses within 24 hours of allergen inhalation in asthmatic

ing an asthma attack,24,37 was significantly increased in the lung of

mice, the number of lung inflammatory cells between male andfemale mice compared to that in male mice at 3 or 16 hours post-

female mice was compared. The number of total cells and mononu-

OVA inhalation (Figure 3B). In addition, CCL20 and IL-33 protein

clear cells, such as monocytes, macrophages, and DCs, in the lungs

levels in the lung at 16 hours post-OVA inhalation were also signifi-

16 hours after OVA inhalation was higher in female mice than incantly higher in female mice than in male mice (Figure 3C).

mokine levels in the lung were not different between the sexes.
Furthermore, mRNA expression of Flt3L and IL-33, cytokines thatmale mice (Figure 1A,B). A similar tendency was also observed withrespect to the number of eosinophils and lymphocytes, although thedifferences between male and female mice did not reach significanceat the time points examined (Figure 1B). Meanwhile, the number of

3.2 | Sex-based differences in DC migration into
BLN

lung leucocytes 20 hours after OVA inhalation was not significantly

To define sex-associated differences in the number of DCs in the

different between male and female mice. Furthermore, the number

BLN after allergen inhalation, the number of CD11bhi and CD103+— 467 —

384|

MASUDA ET AL.

× 106 cells/lung(A)

Total cell

2.0
1.5
1.0
0.5
0
Pre 4

8 12 16 20 2 4Time after inhalation (h)

16 h

Pre
(D)

15
10

NS

5

N.D.

0

NeutrophilNS× 106 cells/lung

1.0
0.8
0.6
0.4
0.2
0

Pre

16 h

20
10
0

EffectorTh2 cell

12

100
0

IL-4

IL-5

IL-13

16 h

Male OVA/OVA
Female OVA/OVA
Male OVA/Saline
Female OVA/Saline

16

200

Lymphocyte
NS

Pre

(F)
300

30

1.0
0.8
0.6
0.4
0.2
0

16 h

(E)

(pg/mL)(C)

× 105 cells/mL

× 106 cells/lung× 106 cells/lung
Pre

1.0
0.8
0.6
0.4
0.2
0

× 103 cells/mL

× 106 cells/lung

1.0
0.8
0.6
0.4
0.2
0

EosinophilNS

RL (cmH2O/mL/s)

Mononuclear cell

(B)

8

NS

4
0

0

1.25 2.5

5

10

20

Methacholine (mg/mL)

F I G U R E 1 Sex-based differences in inflammatory cell infiltration in the lung. (A) The number of white blood cells in the mice lungs wascounted before (Pre) and 12, 16, 20, and 24 h after (post) ovalbumin (OVA) challenge. (B) The lung white blood cells pre- and 16 h post-OVA
challenge were stained with Diff-Quick and then observed using a microscope. The number of leucocyte fractions was estimated bymultiplying the total leucocyte number by the proportion of each fraction. (C) The amount of cells in each leucocyte fraction and (D) the
number of effector Th2 cells in the BAL fluids 5 days post-OVA challenge were measured. (E) IL-4, IL-5, and IL-13 levels in the BAL fluids
3 days post-OVA challenge were measured by ELISA. (F) Airway responsiveness in male and female sensitized mice were measured 1 day after
OVA (OVA/OVA) or saline (OVA/Saline) challenge using whole body plethysmography. Data are shown as the mean  SEM based on at least
two independent experiments (n = 7-13). Open bar and circles, male mice; closed bar and circles, female mice. *P < .05 compared to male
mice; **P < .01 compared to male mice; ##P < .01 compared to the level before OVA inhalation; NS, not significant

DCs in the BLN was initially examined, as shown in Figure 4A.

sex-associated differences in the migratory properties of each DC

Female mice exhibited an increasingly robust accumulation of DCs

subset. The percentage of CCR7-expressing cells in CD103+ DCs

from 16 to 20 hours after OVA inhalation compared to that in malewas significantly increased in the lungs of female mice, but not inmice (Figure 4B,C). The number of total DCs, CD11bhi DCs, andmale mice, after OVA inhalation (Figure 5A). However, such sex-

CD103+ DCs in BLN had reached to peak levels 20 to 24 hours

associated differences were not observed in the case of CD11bhi

after OVA inhalation and then further decreased to basal levels at

DCs (Figure 5A). Meanwhile, total number of CCR7+ CD11bhi DCs

72 hours after OVA inhalation in both male and female mice. The

and CCR7+ CD103+ DCs in the lung 16 hours after OVA inhalationhi

DCs at 16 hours post-OVA inhalation was sig-

was significantly higher in female mice than in male mice (Figure 5B).

nificantly higher in female mice than in male mice (Figure 4C), similar

In the BLN, the mRNA expression levels of ligands for CCR7, includ-

to the time-dependent change that was observed in the lung (Fig-ing CCL19 and CCL21, were not statistically different between maleure 2C). In contrast, the number of CD103+ DCs in the BLN 16 toand female mice either before or 16 and 20 hours after OVA inhala-

20 hours after OVA inhalation was significantly higher in femaletion (Figure 5C). These results suggest that increase in the number

mice than in male mice (Figure 4C); nevertheless, the number of theand proportion of CCR7+ DCs together results in the sex difference

cells in the lung was not altered in both male and female mice afterin the kinetics of each DC subsets in the BLN; DCs from the female

allergen inhalation (Figure 2C). To address the discrepancy in the dis-mice responded earlier and resolved sooner than those from thetribution of CD103+ DCs after allergen exposure, we next examinedmale mice did in the lungs of asthmatic mice.

number of CD11b— 468 —

| 385MASUDA ET AL.

Lung

104
CD103

103

SSC

SSC

SSC

(A)

102
101

FMO

SSC
CD11c
FemaleI-A/I-E

FMO

CD103

CD103

Male

FMO

FMO

SSC
(B)

CD11b
CD11b

(C) CD11chi I-A/I-Ehi cell

4
2
0
Pre

8

16

24

Time after inhalation (h)CD103+ DC
3

4

× 103 cells/lung6

CD11bhi DC
× 104 cells/lung× 104 cells/lung

F I G U R E 2 Sex-based differences in thenumber of dendritic cells in the lung. (A)Flow cytometric gating for SSClow CD11chi
I-A/I-Ehi dendritic cells (DCs) from themouse lungs. Positive populations aredefined based on fluorescence minus one(FMO) controls. (B) Representative profileof the expression of CD11b and CD103
on DCs in the lung of male and female
mice 16 h after OVA challenge. (C) Total
DCs, CD11bhi DCs, and CD103+ DCs in
the lung were counted pre- and 12, 16, 20
and 24 h post-OVA challenge. Data areshown as the mean  SEM based on atleast three independent experiments(n = 8-12). Open circle, male mice; closed
circle, female mice. *P < .05 compared tomale mice; #P < .05 compared to the level
before OVA inhalation

100
100 101 102 103 104
CD11b

100 101 102 103 104 100 101 102 103 104
CD11c
I-A/I-E

FSC

3
2
1
0
Pre

8

16

24

Time after inhalation (h)

2
1
0
Pre

8

16

24

Time after inhalation (h)female mice, compared to those in CD103+ DCs from male mice,

3.3 | Sex-based differences in the antigenpresenting potential of BLN DCs

although sex-associated differences were not observed for costimulatory molecule expression on CD11bhi DCs (Figure 6D). Further-

The pathogenesis of asthma is directed by allergen-specific Th2 cells

more, IL-33 receptor expression on BLN DCs was significantly

that are generated in the secondary lymphoid organs, such as theenhanced in CD103+ DCs compared to that in CD11bhi DCs in

BLN, where DC antigen presentation and signalling through costimu-

female mice, whereas such differences were not observed between

latory molecules are critical for the generation of Th2 cells. There-

CD11bhi and CD103+ DCs in male mice (Figure 6E). These resultsfore, to test sex-associated differences in the ability of BLN DCs tosuggest that enhanced activation and antigen uptake in BLN

act as antigen-presenting cells, we initially examined the antigen

CD103+ DCs from female mice, as compared to those in correspond-

uptake ability. As shown in Figure S1, OVA was actually taken up

ing DCs from male mice, are responsible for sex-based differences inintracellularly by DCs of both male and female mice. Furthermore, aTh2 cytokine production during asthma.

high frequency of OVA-positive DCs was observed in migrated DCs(male mice vs. female mice: 74.6  2.2% vs. 84.2  3.2%; P = .054),

but low levels were found in resident DCs in the BLNs from bothmale and female mice (Figure 6A). In the BLN, 81.3  2.8% of DCs

3.4 | Sex-based differences in BLN DC-induced T
cell differentiationin male mice and 79.2  5.6% of DCs in female mice were migratory

To directly address the contribution of DCs to sex-related Th2 cyto-(Figure 6B). Moreover, fluorescent OVA signals were more intensekine production, we initially purified CD11bhi and CD103+ DCs fromin migrated CD103+ DCs from female mice than those from male

BLNs. These sorted populations were consisted of DCs, although

mice, although such sex-associated differences were not observed inthere is the possibility of contamination with CD3+ cells in thesehi

DCs (Figure 6C). Next, the analysis of costimula-populations (Figure 7A). In addition, sex-associated differences in the

tory molecule expression on BLN DCs revealed enhanced expression

morphology of each DC subset were not observed between maleof CD86 and I-A/I-E, but not CD40 and CD80, in CD103+ DCs fromand female mice (Figure 7A). Next, we cultured each BLN DC subset

migrated CD11b— 469 —

|

Relative expression

(B)

3h

16 h

GM-CSF

2.0
1.5
1.0
0.5
0

Pre

3h

16 h

0

Pre

3h

16 h

M-CSF
3.0
2.0
1.0
0

Pre

3h

16 h

2.5
0

Pre

3h

16 h

Flt3L

3.0
2.0
1.0
0

Pre

3h

16 h

8.0

CCL20

75
50
25
0

Pre

3h

16 h

IL-33

6.0
4.0
2.0
0

Pre

3h

16 h

CC21

Relative expression

Pre

200

7.5
5.0

100

2.0

Relative expression

0400

Relative expression

50

600

CCL19
10

Relative expression

100

CCL7

800

Relative expression

CCL2

150

Relative expression

Relative expression(A)

Relative expression

MASUDA ET AL.

Relative expression

386

2.0

1.5
1.0
0.5
0

Pre

3h

16 h

TSLP

1.5
1.0
0.5
0

Pre

3h

16 h

IL-33 (pg/mg protein)

CCL20 (pg/mg protein)

(C)
3
2
1
0

100
75
50
25
0

F I G U R E 3 Sex-based differences in cytokine and chemokine expression in the lung. The mouse lungs were excised pre-, and 3 and 16 h
post-OVA challenge. (A) Chemokine mRNA expression and (B) cytokine mRNA expression in the lungs were determined by real-time RT-PCR.
(C) CCL20 and IL-33 protein levels in the lung 16 h post-OVA challenge were measured by ELISA. Data are shown as the mean  SEM basedon two to three independent experiments (n = 6-14). Open bar, male mice; closed bar, female mice. *P < .05 compared to male mice;**P < .01 compared to male micewith OVA peptide-specific T cell receptor-expressing CD4+ T cells.

(Figure 7D,E). These results suggest that CD103+ DCs, but not

As shown in Figure 7B, IL-4, IL-5, and IL-13 levels in the super-

CD11bhi DCs, promote female-predominant Th2 cell differentiation,+natants were higher after culturing with CD103

DCs than withfollowed by female-predominant Th2 cytokine production.

CD11bhi DCs in case of both male and female mice. In addition,
CD103+ DCs from female mice induced higher IL-4, IL-5, and IL-13
production from CD4+ T cells than that by CD103+ DCs from male
mice; however, sex-associated differences in Th2 cytokine produc-

3.5 | Oestradiol induces enhanced Th2-type
immunity in CD103+ DCs

tion were not observed after culturing these cells with CD11bhi DCs

Female hormones can reportedly enhance Th2-type immune

from male and female mice. IFN-c was also produced from CD4+ T

responses via their effects on DCs.38,39 We tested the role of 17b-

cells, the levels of which were 10- to 100-fold higher than those ofoestradiol, the most common circulating form of oestrogen, in theTh2 cytokine in both male and female mice; however, no sex-asso-

associated Th2-type immune response in CD103+ DCs. Oestradiolhi+or CD103

enhanced the expression of CD86 and I-A/I-E on CD103+ BM-DCs

DCs. Furthermore, CD11bhi DCs and CD103+ DCs from male andstimulated with OVA, which was further enhanced by IL-33 (Fig-female mice induced similar levels of IL-17 production from CD4+

ure 8A). However, in the case of CD103+ DCs developed from theT cells, which were 40- to 4000-fold lower than those of other cyto-

BM cells in the absence of 17b-oestradiol, enhanced expression ofkines. For cytokine production in CD8+ T cells, CD103+ DCs from

CD86 and I-A/I-E was not observed upon stimulation with OVA plus

female mice induced higher levels of IL-5, but not IFN-c, production

17b-oestradiol (Figure S2).

ciated differences were observed with either CD11b

than that by CD103+ DCs from male mice (Figure 7C). Finally, sex+

associated differences in CD4+

T cell proliferation induced by+

CD103 DCs were assessed. As expected, CD103 DCs from female+

CD86 and I-A/I-E expression on CD103+ BM-DCs was not different between oestradiol-primed CD103+ BM-DCs and vehicleprimed CD103+ BM-DCs on IL-33 stimulation (Figure 8A). Oestra-

mice induced greater T cell proliferation than CD103 DCs fromdiol-primed CD103+ BM-DCs also induced ST2 expression on CD4+

male mice did, as determined by CFSE dilution in dividing cells

T cells in the presence of OVA (Figure 8B). An oestrogen receptor— 470 —

| 387MASUDA ET AL.

Male

Bronchial LN
SSC

CD11b

× 104 cells/BLN

F I G U R E 4 Sex-based differences in thenumber of DCs in the bronchial lymph
node. (A) Flow cytometric gating for
SSClow CD11chi I-A/I-Ehi DCs frombronchial lymph nodes (BLNs) of mice.
Positive populations are defined based on
FMO controls. (B) Representative profile ofthe expression of CD11b and CD103 on
DCs in the BLN of male and female mice
20 h after OVA challenge. (C) Numbers of
total DCs, CD11bhi DCs, and CD103+ DCs
in the BLN were counted pre- and 12, 16,
20, 24, 48, and 72 h post-OVA challenge.
Data are shown as the mean  SEM basedon two to three independent experiments(n = 8-9). Open circle, male mice; closed
circle, female mice. *P < .05 compared tomale mice; **P < .01 compared to male
mice; #P < .05 compared to the level
before OVA inhalation

FSC

104
103

103

CD103

CD11c

104

CD11chi I-A/I-Ehi cell

(C)

102

102

101

101

100 0 1 2 3 4 100 0 1 2 3 410 10 10 10 10
10 10 10 10 10
I-A/I-E
CD11b
FMO

FMO

8
6
4
2
0
Pre 8

16 24 32 40 48 56 64 72

Time after inhalation (h)× 104 cells/BLN(A)

Female

CD103(B)

CD11bhi DC

4
3
2
1
0
Pre 8

16 24 32 40 48 56 64 72

Time after inhalation (h)FMO

I-A/I-E

CD11b

× 104 cells/BLN

CD11c

CD103

FMO

CD103+ DC

4
3
2
1
0
Pre 8

16 24 32 40 48 56 64 72

Time after inhalation (h)antagonist abolished the expression of these molecules on oestra-draining lymph node reached a peak 24 hours after OVA inhalation

diol-primed CD103+ BM-DCs and CD4+ T cells (Figure 8A,B). In

and then decreased to almost half of their peak levels at the end of+addition, enhanced IL-5 production from CD4 T cells was observed+

48 hours.40 Furthermore, Plantinga et al41 reported, using a house

after culture with oestradiol-primed CD103 BM-DCs, compared todust mite–induced asthma mouse model, that 60% of BLN DCs on

that after culture with vehicle-treated CD103+ BM-DCs, and an

day 3 post–house dust mite challenge were migratory (CD11chianti-CD86 antibody completely abolished this effect (Figure 8C).

MHCIIhi) DCs, and 40% of BLN DCs were resident (CD11chi MHCIIdim)
DCs, although the sex of mice used was not described in these studies. Our data regarding the proportions of migratory DCs in the BLN

4 | DISCUSSION

20 hours after allergen exposure (Figure 6A,B) indicate a similar tendency, in either sex, to those of migratory DCs in the BLN reported

This study reports the first evidence of CD103+ DCs being involved

previously as mentioned above.40,41 Regarding the sex difference inin the development of sex-based differences in Th2 cytokine produc-

the number of DCs, Melgert et al42 showed that T cell expansion intion in asthma. We previously demonstrated that BLN cells fromfemale mice appeared to be driven by a greater number of inflamma-

female mice with OVA-stimulated asthma, but not those treated withtory mDCs, defined as CD11chi MHC-IIhi cells, migrating from theanti-CD3/CD28 antibodies, produced higher levels of IL-5 and IL-13

lungs to the lymph nodes, during asthma. Consistent with this obser-

than those from male mice, suggesting the involvement of antigen-vation, we showed that the total number of DCs, defined as CD11chipresenting cells in the sex-associated differences in Th2 cytokine pro-

I-A/I-Ehi cells in the lung and BLN, was increased in female mice32

duction during asthma.

With regard to migratory DCs in the BLN,

compared to those in male mice.

Nembrini et al40 showed, using an OVA-induced asthma mouse5

Chemokines, such as CCL2 and CCL20, from the airway epithelial

model, that 1-2 9 10 DCs were observed in the lung-draining lymph

cells activated in response to allergens, attract monocytes and DC

node 24 hours after OVA inhalation, of which 1.5-4 9 104 DCs wereprecursors to the lung.43 In contrast, TSLP, IL-25, and IL-33 in theidentified as migratory DCs based on their uptake of FITC-conjugatedlung confer a Th2 polarizing profile to DCs through the upregulation

OVA. In their study, the numbers of migratory DCs in the lung-of B7-1, B7-2, and OX40L, which instructs naive T cells to— 471 —

|

MASUDA ET AL.

CCR7+ CD11bhi DCs (%)

(A)

NS

CCR7+ CD103+ DCs (%)

388

50
40
30
20
10
0

Pre

NS
50
40
30
20
10
0

16 h

Pre

16 h

CCR7+ CD103+ DCs(cells/lung)

3000
2000
1000
0

(C)

Relative expression

Pre

80

NS

NS

NS

40
0

Pre

750
500
250
0

Pre

CCL19

120

1000

16 h

16 h

20 h

Relative expression

CCR7+ CD11bhi DCs(cells/lung)

(B)

16 h
CCL21

2000

NS

1500
1000

NS

NS

Pre

16 h

500
0

20 h

F I G U R E 5 Sex-based differences in the
migration capacity of pulmonary DCs tothe BLN. (A) The percentages of CCR7+
population in the pulmonary CD11bhi DCs
and CD103+ DCs, and (B) the total
numbers of CCR7+ CD11bhi DCs andCCR7+ CD103+ DCs in the lungs pre- and16 h post-OVA challenge. (C) CCL19 and
CCL21 mRNA expression in the BLN preand 16 and 20 h post-OVA challenge wasquantitated by RT-PCR. Data are shown asmean  SEM based on three to six mice.
Experiments were repeated twice withsimilar results. Open bar and circles, male
mice; closed bar and circles, female mice.
**P < .01; NS, not significant

differentiate into Th2 cells.44-47 Development of CD11b+ DCs from

DCs express higher levels of Ccr7 in the lung 24 hours after LPS

precursors in the lung depends on cytokines M-CSF and GM-CSF

instillation, indicating that migratory capacities of CD103+ DCs arelevels, but does not require an interaction between Flt3 and Flt3L,

superior to those of CD11b+ DCs.25 Furthermore, Besnard et al37

whereas CD103+ DC development from common DC precursors

demonstrated that IL-33 induces CCR7 expression on DCs and DC

relies on the stepwise activity of Flt3 signalling.48 In contrast, Plan-

activation in the lung. Recently, Chen et al49 suggested the possibil-

41

/

mice lacked both CD11b andity that airway exposure to IL-33 might increase the number of cells

CD103+ DCs, and exhibited significantly reduced numbers of eosino-

expressing ST2, a receptor for IL-33, in the lung. Here, the enhanced

phil, neutrophil, and lymphocytic infiltration in the BAL fluids com-

population of ST2-expressing CD103+ DCs in female mice waspared to that in the asthmatic WT mice, after exposure to house dust

accompanied by increased expression of IL-33 mRNA and protein in

mites. In the present study, we showed that Flt3L and IL-33 expres-the lungs of these animals (Figures 3B,C and 6E). Consistent withsion in the lung tissues was enhanced in female mice compared tothis observation, the increased CCR7+ cell population in CD103+

male mice (Figure 3), whereas TSLP expression levels were similar

DCs and the increased number of CCR7+ CD103+ DCs in the lung

between the sexes, and IL-25 was not detected in either sex (datawere observed in female mice (Figure 5A,B). These results suggest

not shown). These observations suggest that close proximity of lungthat the development of the CD103+ DC lineage, in association with

DCs to IL-33- or Flt3L-secreting cells might be responsible, at least in

CCR7 expression and further activation by enhanced IL-33 inpart, for the sex-associated differences in DC development and infemales, might be involved in the sex-related differences in CCR7

conferring a Th2 polarizing profile to DCs in the asthmatic lung.

expression on CD103+ DCs, which might lead to the corresponding

tinga et alreported that Flt3L+

Developmental lineage affects the differential expression ofdifferences in the number of CD103+ DCs in the BLN (Figure 4B,C).

CCR7, a chemokine receptor required for migration to lung-draining

CD103+ DCs comprise a primary DC subset in the lung and medi-hi+mediastinal lymph nodes; Ly-6C CD11b DCs, and CD14 CD11b

astinal lymph nodes that prime Th2 responses upon allergen encoun-

DCs, which are Flt3-independently derived from monocytes in theter, whereas CD11bhi DCs prime Th1 responses.28 Furthermore,

24

lungs,+

hi+

express lower levels of Ccr7, whereas pulmonary CD103

CD103+ DCs in the BLN are specialized for the cross-presentation of— 472 —

| 389MASUDA ET AL.

Male Res-DC

NS

Female Mig-DC

25
0

20

16

0

Male

Female

NS

15
10
5
0

Male

Female

20

20

12

NS

20
10
0

Male

Female

NS

15
10
5
0

Male

FemaleMale

FemaleMale

FemaleMale Mig-DC

75
50

10

delta MFI CD40

NS

20

30

delta MFI CD80

100

20

NS

delta MFI CD86

102

100
80
60
40
20
0
101 102 103 104 105
OVA-AF647

30

CD103+ DC

delta MFI I-A/I-E

103

100
80
60
40
20
0

delta MFI CD40

Max (%)

104

101
101 102 103 104 105
I-A/I-E

FSC% of DCs

102
105

CD11c

SSC

103
101

OVA–AF647

(B)

104

Max (%)

SSC

CD11c

105

delta MFI CD80

Non-labelled OVA

CD11bhi DC(D)

Mig-DCsdelta MFI CD86

Res-DCsdelta MFI I-A/I-E(A)Female Res-DC
DC es-DC
R

Mig-

NS

15
10
5
0

Male

Female151050CD103+ DC
100
(E)
NS
80
50
60
40
30
40
20
20
10
0
0
101 102 103 104 105
Male
OVA-AF647
ST2+ cells (%)

CD11bhi DC
100
80
60
40
20
0
101 102 103 104 105
OVA-AF647

Max (%)Max (%)(C) Gated on alive, CD11chi, I-A/I-Ehi, Mig-DCs

Female

NS

12
8
4
0

Male

Female8
40

F I G U R E 6 Sex-based differences in Th2 priming ability of DCs in the BLN. (A) Sex-based differences in OVA uptake by CD11chi I-A/
I-Edim BLN-resident DCs and CD11chi I-A/I-Ehi migratory DCs (from the lung to the BLN). Solid blue line, migratory DCs from male
mice; solid red line, migratory DCs from female mice; dotted blue line, resident DCs from male mice; dotted red line, resident DCs fromfemale mice. (B) The percentages of resident DCs and migratory DCs in BLN DCs of male and female mice. (C) Sex-based differencesin OVA uptake by CD11bhi and CD103+ DCs in the BLN. Representative results of three independent experiments are shown. Greyline, DCs that took up non-conjugated OVA; blue line, migratory DCs of each subset from male mice; red line, migratory DCs of eachsubset from female mice. (D) Sex-based differences in mean fluorescent intensity of CD40, CD80, CD86, and I-A/I-E on migratory
CD11bhi DCs and CD103+ DCs in the BLN. (E) Differences in ST2 expression on migratory BLN CD11bhi DCs and CD103+ DCs frommale and female mice. Horizontal line bars, CD11bhi DCs; vertical line bars, CD103+ DCs. Open bar, male mice; closed bar, female
mice. Data are shown as the mean  SEM based on four to six mice. Experiments were repeated twice with similar results. *P < .05;
NS, not significant
inhaled antigens to CD8+ T cells, in contrast to CD11b+ DCs, which

differentiation and activation of Th1, Th2, and Th17 cells.52 More-are characterized by the differential expression of MHC-I and MHC-II

over, OX40L, Jagged2, ICOSL, and TSLPR expression on DCs has

processing pathways.50,51In accordance with the cross-presentation

been associated with Th2 induction by DCs during Th2 cell differenti-

activity of CD103+ DCs, these cells mediated sex-associated differ-ation.53-55 In the current study, mRNA expression of OX40L,

ences in the Th2 cytokine production, but not IFN-c production, from

Jagged2, ICOSL, and TSLPR in CD103+ DCs is not different between++

not only CD4 T cells (Figure 7B), but also CD8 T cells (Figure 7C).

male and female mice (Figure S3). Therefore, the enhanced expres-In the enhanced Th2 cytokine production, the expression of CD40,

sion of CD86 on CD103+ DCs may be directly responsible for the

CD80, CD86, or OX40L is required for directional T cellcorresponding enhanced Th2 cell differentiation (Figures 6D and 8C).

— 473 —

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MASUDA ET AL.

SSC

CD11c

(A) Bronchial LN

104

104

103

103

103

102

102

102

101

101

104

Post-sort DCs

Male

FemaleCD11bhi

96%

100
101
100 0 1 2 3 410 10 10 10 10101 102 103 104 105 100 101 102 103 104
104
I-A/I-E
CD103+
3
10
CD103

FSC

Pre-sort DCs

105

98%

102
101

CD11b

100 0 1 2 3 410 10 10 10 10

400
300
200
100

0 N.D.

NS

0 N.D.

50
40
30
20
10
0 N.D.

NS
NS

NS

100
0 N.D.

(D) 100

Day 2

2000

NS

1000
0

80
60
40
20
0
101 102 103 104 105
CFSE

N.D.

IL-17A (ng/mL)

10

200

1.00

NS

0.75

NS

0.50
0.25

NS NS

0

(E)
100 Day 4
80
60
40
20
0
101 102 103 104 105
CFSE

CFSElow cells (%)

IL-5 (pg/mL)(C)

IFN-γ (pg/mL)

0

NS

NS

3000

Max (%)NS

20

IFN-γ (ng/mL)

50

30

300

Max (%)

100

40

IL-13 (ng/mL)

150

IL-5 (ng/mL)

IL-4 (ng/mL)

(B)

100
75
50
25
0

Day 2 Day 4

F I G U R E 7 Sex-based differences in T cell differentiation promoted by BLN DCs. (A) Purity of sorted BLN CD11bhi DCs and CD103+ DCs.
Representative images of each population are shown. Scale bar = 10 lm. (B) Cytokine levels in cultures of CD4+ T cells from the spleen of
OT-II transgenic (Tg) mice with CD11bhi DCs or CD103+ DCs from the BLN of male or female mice. (C) Cytokine production in CD8+ T cells
from OT-I transgenic (Tg) mice cultured with each DC subset from the BLN of male or female mice. Cytokine levels in the culture
supernatants were determined by enzyme-linked immunosorbent assay (ELISA). (D, E) 5,6-carboxyfluorescein diacetate succinimidyl ester(CFSE)-labelled CD4+ T cells were cultured with CD103+ DCs from male or female mice. (D) The dilution of the CFSE signal on day 2 or 4
post-culture was assayed by flow cytometry. Representative results of two independent experiments are shown. Grey line, CFSE-labelled
CD4+ T cells cultured without DCs; blue line, CD4+ T cells cultured with CD103+ DCs from male mice; red line, CD4+ T cells cultured with
CD103+ DCs from female mice. (E) Percentage of CFSElow cells in CD4+ T cells cultured with CD103+ DCs from the BLN of male and female
mice after 2 or 4 days of culture. Grey bar, CFSE-labelled CD4+ T cells cultured without DCs; open bar, CFSE-labelled CD4+ T cells cultured
with CD103+ DCs from male mice; closed bar, CFSE-labelled CD4+ T cells cultured with CD103+ DCs from female mice. Data are shown as
the mean  SD based on triplicate cultures. Experiments were repeated twice with similar results. *P < .05; **P < .01; NS, not significant;N.D., not detected

Oestrogen likely induces enhanced immune responses in

exposure to 17b-oestradiol, including that during the period offemales through interaction with its receptors.38,56 Yang et al39

CD103+ DC development, might enhance CD103+ DC activation

showed that 17b-oestradiol upregulates costimulatory moleculesand maturation in females upon allergen exposure, which could

and MHC-II on CD11c-positive murine splenic DCs, which pro-lead to female-enhanced Th2 cell differentiation and activation (Fig-motes T cell priming. In the present study, CD86 and I-A/I-E

ure 8 and Figure S2). Meanwhile, 17b-oestradiol enhanced theexpression on CD103+ DCs was not enhanced through culture withexpression of CD86 and I-A/I-E on DCs after stimulation with

17b-oestradiol alone, but rather through incubation with IL-33, as

OVA, but not with IL-33 (Figure 8A). Our results suggest that 17b-

previously suggested by Rank et al57 Furthermore, sustained

oestradiol has a limited role in the IL-33-ST2 axis-mediated— 474 —

| 391

MASUDA ET AL.

VehicleOestradiol

Oestradiol + ICI182,780

(A)

1500

4000

NS

NS

MFI I-A/I-E

MFI CD86

2000

1000
500
0

Medium

OVA

IL-33

2000

0

Medium

OVA

IL-33

OVA +
IL-33

(C)
150

IL-5 (pg/mL)

1600

MFI ST2

NS

1000

OVA +
IL-33

(B)

NS

3000

1200
800
400

0
OVA
DCs
Oestradiol
ICI182,780

–
–
–
–

+
–
–
–

+
+
–
–

+
+
+
–

100

0
OVA
DCs
Oestradiol
α-CD86 Ab

+
+
+
+

NS

50

–
–
–
–

+
–
–
–

+
+
–
–

+
+
+
–

+
+
–
+

+
+
+
+

F I G U R E 8 17b-oestradiol enhances Th2 cell priming by bone marrow-derived CD103+ DCs. CD103+ bone marrow (BM)-DCs were induced
from BM cells of male mice in the presence of 17b-oestradiol, 17b-oestradiol plus ICI182,780, or vehicle. (A) Each set of CD103+ BM-DCs
was cultured in the presence of OVA, IL-33, or OVA plus IL-33 for 24 h. CD86 and I-A/I-E expression levels on CD103+ BM-DCs wereassayed by flow cytometry. (B) BLN CD4+ T cells were cultured with 17b-oestradiol-primed, 17b-oestradiol plus ICI182, 780-primed, orvehicle-primed CD103+ BM-DCs in the presence or absence of OVA. ST2 expression on CD4+ T cell from BLNs was determined by flow
cytometry. (C) BLN CD4+ T cells were cultured with 17b-oestradiol-primed or vehicle-primed CD103+ BM-DCs in the presence or absence of
OVA and recombinant anti-mouse CD86 antibody. IL-5 levels in the culture supernatants were determined by enzyme-linked immunosorbent
assay (ELISA). Data are shown as the mean  SD based on triplicate cultures. Experiments were repeated twice with similar results. *P < .05;**P < .01; NS, not significant

activation of CD103+ DCs in female mice; however, this requires

16K19608), and the Matching Fund Subsidy for Private Universities

further investigation.

from the Ministry of Education, Culture, Sports, Science and Tech-

The present study has important implications for understanding

nology of Japan. The funders played no role in the study design, thethe pathophysiological role of DCs in sex-associated asthma pheno-collection, analysis, and interpretation of data, or the preparation oftypes. Recently, the possibility that CD103+ DCs might even havethe manuscript.

tolerogenic ability towards inhaled allergens through the induction ofregulatory T cells in the absence of DC maturation stimulus has beenreported.58,59 Further research is needed to completely investigate
the precise role of CD103+ DCs in the pathogenesis of asthma. Fur-

CONFLICT OF INTEREST
The authors declare no conflict of interests.

thermore, it still remains unclear whether BDCA3+ DCs, the humanhomolog of mice CD103+ DCs, play an important role in enhanced
Th2 cytokine production in female patients with asthma. Therefore,

AUTHOR CONTRIBUTIONS

the results of this study have limitations regarding their application

IO and TM conceived and designed the experiments; CM, KK, TM,to humans. Additional clinical studies are already ongoing in our lab-

and KO-D performed the experiments; TM, IO, TT, CM, KK, TK, andoratory to confirm the results, which could lead to the development

JI analysed the data; IO, JI, and MT contributed reagents/materials/of more effective strategies to treat female patients with asthma.

analysis tools; TM, CM, IO, TT, and MT contributed to the writing of
the manuscript.

ACKNOWLEDGEMENTS

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et al. Sex-based differences inCD103+ dendritic cells promote
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asthma. Clin Exp Allergy. 2018;48:379–393. https://doi.org/
10.1111/cea.13081

European Journal of Pharmacology 800 (2017) 9–15

Contents lists available at ScienceDirect

European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar

The involvement of spinal release of histamine on nociceptive behaviors
induced by intrathecally administered spermine
Hirokazu Mizoguchia, Chizuko Watanabea, Takafumi Hayashib, Yoko Iwataa,
Hiroyuki Watanabec, Soh Katsuyamad, Kengo Hamamurae, Tsukasa Sakuradae, Hiroshi Ohtsuf,⁎
Kazuhiko Yanaig, Shinobu Sakuradaa,aDepartment of Physiology and Anatomy, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aobaku, Sendai 981-8558, Japanb
Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aobaku, Sendai 981-8558, Japanc
Department of Pharmaceutical Biosciences, Division of Biological Research on Drug Dependence, Uppsala University, Husargatan 3, Uppsala 751 24,
SwedendCenter for Experiential Pharmacy Practice, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 1920392, JapaneFirst Department of Pharmacology, Daiichi College of Pharmaceutical Sciences, 22-1 Tamagawa-cho, Minami-ku, Fukuoka 815-8511, Japanf
Department of Quantum Science and Energy Engineering, Graduate School of Engineering, Tohoku University, 6-6-01-2 Aobayama, Aoba-ku, Sendai, 9808579, Japang
Department of Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan

ARTICLE INF O

A BS T RAC T

Keywords:
Spermine
Histamine
ReleaseNMDA receptor
Nociception
Spinal cord

The involvement of spinal release of histamine on nociceptive behaviors induced by spermine was examined inmice. Intrathecal spermine produced dose-dependent nociceptive behaviors, consisting of scratching, biting andlicking. The nociceptive behaviors induced by spermine at 0.02 amol and 10 pmol were markedly suppressed byi.t. pretreatment with antiserum against histamine and were abolished in histidine decarboxylase-deﬁcientmice. In histamine H1 receptor-deﬁcient mice, the nociceptive behaviors induced by spermine were completely
abolished after treatment with 0.02 amol of spermine and signiﬁcantly suppressed after treatment with 10 pmolof spermine. The i.t. pretreatment with takykinin NK1 receptor antagonists eliminated the nociceptive behaviors
induced by 0.02 amol of spermine, but did not aﬀect the nociceptive behaviors induced by 10 pmol of spermine.
On the other hand, the nociceptive behaviors induced by spermine at both 0.02 amol and 10 pmol were
suppressed by i.t. pretreatment with antagonists for the NMDA receptor polyamine-binding site. The present
results suggest that the nociceptive behaviors induced by i.t. administration of spermine are mediated throughthe spinal release of histamine and are elicited via activation of NMDA receptors.

1. Introduction
The polyamines spermine, spermidine and putrescine are endogenous aliphatic amines that are widely distributed (Gilad and Gilad,
1992). Since they have a polycationic structure, they electrically bind tonegatively charged cellular molecules, including nucleic acids, acidic
lipids and proteins (Ahern et al., 2006; Scott et al., 1993). Therefore,polyamines are considered to be closely involved with cellular functions(Ekegren et al., 2004; Gilad and Gilad, 1992; Scott et al., 1993; Tanabe
et al., 2004). Polyamines are mainly synthesized from ornithine byornithine decarboxylase, spermidine synthase and spermine synthase(Gilad and Gilad, 1992; Laube et al., 2002; Silva et al., 2011; Wolﬀ⁎

et al., 2003). Putrescine is formed from ornithine by ornithine
decarboxylase, and then, putrescine is converted to spermidine andspermidine is converted to spermine by spermidine synthase andspermine synthase, respectively. Polyamine synthesis has been observed in both neurons and glial cells in the central nervous system(Bernstein and Müller, 1999; Ekegren et al., 2004; Laube 2002).
Immunoreactivity for spermine/spermidine was detected in bothneurons and glial cells in most regions in the central nervous system(Laube, 2002). Immunoreactivity for ornithine decarboxylase was
detected in neurons and glial cells in the brains under several
pathophysiological conditions (Bernstein and Müller, 1999). The
physiological functions of polyamines in the central nervous system

Corresponding author.
E-mail address: s-sakura@tohoku-mpu.ac.jp (S. Sakurada).

http://dx.doi.org/10.1016/j.ejphar.2017.01.031
Received 27 August 2016; Received in revised form 6 January 2017; Accepted 24 January 2017
Available online 26 January 20170014-2999/ © 2017 Elsevier B.V. All rights reserved.

— 478 —

European Journal of Pharmacology 800 (2017) 9–15

H. Mizoguchi et al.

are considered to include the control of neuronal excitability involvedin the variable physiological responses related to pathophysiologicalconditions (Gilad and Gilad, 1992).
In the neurons and glial cells of the central nervous system, themolecular target for polyamines is considered to be the N-methyl-Daspartate (NMDA) receptor. The NMDA receptor is a hetero-tetramer
consisting of 7 NMDA receptor subunits (NR1, NR2A-D and NR3A-B)
that form a Ca2+/Na+ channel (Dingledine et al., 1999). The NR1
subunit is an essential subunit for NMDA receptors and is ubiquitously
distributed in the central nervous system (Nagy et al., 2004). It
contains a polyamine-binding site, through which endogenous polyamines positively modulate the functions of the NMDA receptors(Dingledine et al., 1999). On the other hand, the NR2A-D subunits
contain binding sites for the endogenous ligand glutamate, and theactivation of the glutamate-binding site opens the Ca2+/Na+ channel
and results in neural excitation. This response plays an important rolein pain transmission in the spinal cord (Dickenson et al., 1997).
Glutamate is contained in and released from primary aﬀerent nerves(Ueda et al., 1994) and binds to NMDA receptors on the cell body of thesecond order neuron for pain transmission.
It has been reported that intrathecal (i.t.) administration of
spermine induced nociceptive behaviors, consisting of scratching,biting and licking (Tan-No et al., 2000). However, the mechanism ofspermine-induced nociceptive behaviors is not fully understood. We
reported the possible interactions among spinal neurotransmitters/
neuromodulators, histamine, substance P and polyamines in spinal
pain transmission (Sakurada et al., 2003, 2004; Watanabe et al., 2008).
Therefore, in the present study, the mechanism of nociceptive behaviors induced by i.t. administration of spermine was investigated, witha focus on the involvement of histamine and NMDA receptors in themouse spinal cord.

attached to a 50-μl Hamilton microsyringe. The volume of the i.t.
injections was 5 μl.
2.3. Behavioral procedures
The nociceptive behaviors of mice were evaluated according to themethods described in our previous report (Mizoguchi et al., 2011).
Approximately 1 h before i.t. injection, mice were adapted to individual
plastic cages (22.0×15.0×12.5 cm), which also served as observationchambers. Immediately after i.t. injection of spermine, each mouse wasplaced in the transparent cage, and the nociceptive behaviors inducedby spermine were observed for 30 min at 5 min intervals. The
nociceptive behaviors observed included caudally directed biting andlicking along with reciprocal hindlimb scratching. The total response
times of these nociceptive behaviors were pooled and recorded as asingle value for each animal.
2.4. Drugs
The drugs used were spermine tetrahydrochloride (Nacalai Tesque,
Kyoto, Japan), arcaine sulfate (Tocris Cookson Ltd., Bristol, UK),
agmatine sulfate (Tocris Cookson Ltd.), (5R,10S)-(+)−5-methyl10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-imine (MK-801) hydrogen maleate (Sigma-Aldrich Chemical Co., St. Louis, MO), D-(-)−2amino-5-phosphonovaleric acid (D-APV) (Sigma-Aldrich ChemicalCo.),
3-((+)−2-carboxypiperazin-4-yl)-propyl-1-phosphonicacid(CPP) (Sigma-Aldrich Chemical Co.), rabbit antiserum against histamine (Progen Biotechnik GMBH, Heidelberg, German), normal rabbit
serum (Vector Laboratories, Burlingame, CA). [Tyr6,D-Phe7,D-His9]
substance P-(6−11) (sendide) and [D-Phe7,D-His9]substance P-(6−11)
were synthesized by solid-phase peptide methodology. (+)-[(2S,3S)−3(2-methoxy-benzyl-amino)−2-phenylpiperidine] (CP-99,994) was a giftfrom Pﬁzer Pharmaceuticals (New York, NY). These drugs weredissolved or diluted in sterile artiﬁcial cerebrospinal ﬂuid (aCSF)
containing 126.6 mM NaCl, 2.5 mM KCl, 2.0 mM MgCl2, and 1.3 mM
CaCl2.

2. Materials and methods
All experiments were performed following the approval of the
Ethics Committee for Animal Experiments at Tohoku Medical andPharmaceutical University and according to the National Institutes of
Health Guide for the Care and Use of Laboratory Animals. Every eﬀort
was made to minimize the number of animals and any suﬀering by the
animals used in the following experiments.

2.5. Statistical analysis of data
The time spent performing nociceptive behaviors (s) was presented
as the mean ± S.E.M for 10 mice. The statistical signiﬁcance of thediﬀerences between groups was assessed with Student's t-test and oneway analysis of variance (ANOVA) followed by Bonferroni's test or twoway ANOVA followed by Bonferroni's test.

2.1. Animals
Male ddY mice (Japan SLC, Hamamatsu, Japan), histidine decarboxylase-deﬁcient mice (supplied by Dr. Ohtsu: Ohtsu et al., 2001),
histamine H1 receptor-deﬁcient mice (supplied by Dr. Yanai: Inoueet al., 1996), histamine H2 receptor-deﬁcient mice (supplied by Dr.
Kobayashi: Kobayashi et al., 2000) and respective wild-type miceweighing 22–25 g were used. The histidine decarboxylase-deﬁcient
mice have 129/Sv and CD-1 mixed genetic backgrounds. The histamine
H1 receptor-deﬁcient mice have C57BL/6 and 129/ola mixed genetic
backgrounds. The histamine H2 receptor-deﬁcient mice have C57BL/6
and 129/ola mixed genetic backgrounds and were backcrossed eighttimes to the C57BL/6 mice. The genotypes of histidine decarboxylasedeﬁcient mice, histamine H1 receptor-deﬁcient mice, histamine H2
receptor-deﬁcient mice and wild-type mice, which have the same
genetic backgrounds as the respective knockout mice, were identiﬁedby the polymerase chain reaction. The animals were housed in a room
maintained at 22–23 °C and 50–60% relative humidity with analternating 12-h light/dark cycle. Food and water were available adlibitum. Mice only were used once.

3. Results
3.1. Involvement of histamine release in spermine-induced
nociceptive behaviors
Spermine-induced nociceptive behaviors were observed in mice.
Groups of mice were treated with various i.t. doses of spermine (from0.001 amol to 10 pmol) or aCSF, and nociceptive behaviors wereobserved for 30 min at 5 min intervals. The i.t. administration of
spermine at a dose of 0.001 amol to 1 amol dose-dependently evokedcharacteristic nociceptive behaviors mainly consisting of vigorous
biting and/or licking with a little scratching (Fig. 1). However, at doses
over 1 amol, the nociceptive behavior response elicited by spermine
was saturated, and no additional increase in nociceptive behaviors wasobserved with 10 pmol of spermine. The nociceptive behaviors peaked
at 5–10 min and disappeared at 25–30 min after the injection (datanot shown).
To elucidate the involvement of the spinal release of histamine on
spermine-induced nociceptive behaviors, the eﬀect of an antiserum
against histamine on spermine-induced nociceptive behaviors wasdetermined. Groups of mice were pretreated with i.t. antiserum against

2.2. Intrathecal injections
I.t. injection was performed following the method described by
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Fig. 1. Spermine-induced nociceptive behaviors in mice. Groups of mice were treatedwith i.t. spermine (0.001 amol-10 pmol) or aCSF, and nociceptive behaviors induced byspermine were observed for 30 min. Each column represents the mean ± S.E.M. for 10
mice. The statistical signiﬁcance of the diﬀerences between groups was assessed with
one-way ANOVA followed by Bonferroni's test. The F-value of one-way ANOVA wasF[6,63] =33.18 (P < 0.001). ***, P < 0.001 vs. aCSF.

Fig. 3. Spermine-induced nociceptive behaviors in histidine decarboxylase-deﬁcient
mice (HDC-KO mice). Groups of HDC-KO mice and corresponding wild-type mice weretreated with i.t. spermine (0.02 amol or 10 pmol), and nociceptive behaviors induced byspermine were observed for 30 min. Each column represents the mean ± S.E.M. for 10
mice. The statistical signiﬁcance of the diﬀerences between groups was assessed with
two-way ANOVA followed by Bonferroni's test. The F-value of two-way ANOVA wasF[1,36] =171.2 (P < 0.001). ***, P < 0.001 vs. wild-type mice.

Fig. 4. Spermine-induced nociceptive behaviors in histamine H1 receptor-deﬁcient mice(H1-KO mice), histamine H2 receptor-deﬁcient mice (H2-KO mice) and respective wildtype mice. Groups of H1-KO mice (A), H2-KO mice (B) and respective wild-type mice
were treated with i.t. spermine (0.02 amol or 10 pmol), and nociceptive behaviors
induced by spermine were observed for 30 min. Each column represents the mean ±S.E.M. for 10 mice. The statistical signiﬁcance of the diﬀerences between groups wasassessed with two-way ANOVA followed by Bonferroni's test. (A) The F-value of two-way
ANOVA was F[1,36] =29.70 (P < 0.001). **, P < 0.01 and ***, P < 0.001 vs. wild-type
mice.

Fig. 2. Eﬀect of histamine antiserum on spermine-induced nociceptive behaviors inmice. Groups of mice were treated with i.t. histamine antiserum (1:200-1:50 dilution) or
normal rabbit serum (NRS: 1:50 dilution) 5 min prior spermine treatment, andnociceptive behaviors induced by i.t. spermine injection (A: 0.02 amol or B: 10 pmol)
were observed for 30 min. Each column represents the mean ± S.E.M. for 10 mice. The
statistical signiﬁcance of the diﬀerences between groups was assessed with one-way
ANOVA followed by Bonferroni's test. (A) The F-value of one-way ANOVA was F[3,36]=13.80 (P < 0.001). ***, P < 0.001 vs. NRS. (B) The F-value of one-way ANOVA wasF[3,36] =17.83 (P < 0.001). *, P < 0.05 and ***, P < 0.001 vs. NRS.

30 min. The nociceptive behaviors induced by spermine at both 0.02
amol and 10 pmol were suppressed by i.t. pretreatment with antiserum
against histamine in a concentration-dependent manner (Fig. 2). In
particular, the nociceptive behaviors induced by 0.02 amol of spermine
were eliminated by the 1:50 histamine antiserum dilution (Fig. 2A).

histamine (1:200-1:50 dilution) or normal rabbit serum (1:50 dilution)
5 min prior i.t. treatment with spermine (0.02 amol or 10 pmol), andthe nociceptive behaviors induced by spermine were measured for11— 480 —

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Fig. 5. Eﬀects of the takykinin NK1 receptor antagonists on spermine-induced nociceptive behaviors in the mouse spinal cord. Groups of mice were co-administered i.t. sendide (A and
D: 0.5–2 pmol), CP99,994 (B and E: 0.5–2 nmol) or [D-Phe7,D-His9]SP(6−11) (C and F: 0.5–2 nmol) with spermine (A, B and C: 0.02 amol or D, E and F: 10 pmol), and nociceptive
behaviors induced by spermine were observed for 30 min. Each column represents the mean ± S.E.M. for 10 mice. The statistical signiﬁcance of the diﬀerences between groups wasassessed with one-way ANOVA followed by Bonferroni's test (A, B and C) or with Student's t-test (D, E and F). (A) The F-value of one-way ANOVA was F[4,45] =9.937 (P < 0.001). **, P< 0.01 and ***, P < 0.001 vs. spermine alone. (B) The F-value of one-way ANOVA was F[3,36] =7.192 (P < 0.001). **, P < 0.01 vs. spermine alone. (C) The F-value of one-way ANOVA
was F[3,36] =12.28 (P < 0.001). ***, P < 0.001 vs. spermine alone.

The contribution of endogenous spinal histamine to the nociceptive
behaviors induced by spermine was conﬁrmed using histidine decarboxylase-deﬁcient mice. Groups of histidine decarboxylase-deﬁcient
mice and corresponding wild-type mice were treated with spermine(0.02 amol or 10 pmol) i.t., and nociceptive behaviors were observedfor 30 min. The nociceptive behaviors induced by spermine at both0.02 amol and 10 pmol were abolished in histidine decarboxylasedeﬁcient mice (Fig. 3).

3.2. Involvement of histamine receptors in spermine-induced
nociceptive behaviors
The involvement of histamine receptor subtypes in spermineinduced nociceptive behaviors was determined using histamine H1
receptor-deﬁcient mice and histamine H2 receptor-deﬁcient mice.
Groups of histamine H1 receptor-deﬁcient mice, histamine H2 receptor-deﬁcient mice and corresponding wild-type mice were treated withi.t. spermine (0.02 amol or 10 pmol), and nociceptive behaviors wereobserved for 30 min. The nociceptive behaviors induce by 0.02 amol of
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Fig. 6. Eﬀects of the antagonists for the polyamine-binding site of the NMDA receptor on the spermine-induced nociceptive behaviors in mice. Groups of mice were co-administered i.t.
agmatine (A and C: 2.5–40 pmol) or arcaine (B and D: 15–240 pmol) with spermine (A and B: 0.02 amol or C and D: 10 pmol), and nociceptive behaviors induced by spermine wereobserved for 30 min. Each column represents the mean ± S.E.M. for 10 mice. The statistical signiﬁcance of the diﬀerences between groups was assessed with one-way ANOVA followed
by Bonferroni's test. (A) The F-value of one-way ANOVA was F[3,36] =15.14 (P < 0.001). ***, P < 0.001 vs. spermine alone. (B) The F-value of one-way ANOVA was F[3,36] =12.09 (P <0.001). *, P < 0.05 and ***, P < 0.001 vs. spermine alone. (C) The F-value of one-way ANOVA was F[3,36] =11.10 (P < 0.001). *, P < 0.05 and ***, P < 0.001 vs. spermine alone. (D) The
F-value of one-way ANOVA was F[3,36] =10.71 (P < 0.001). ***, P < 0.001 vs. spermine alone.

3.4. Involvement of NMDA receptors in spermine-induced nociceptive
behaviors

spermine were abolished in histamine H1 receptor-deﬁcient mice,
whereas the nociceptive behaviors induced by 10 pmol of spermine
were suppressed in histamine H1 receptor-deﬁcient mice (Fig. 4A). On
the other hand, the nociceptive behaviors induced by spermine at both0.02 amol and 10 pmol were not aﬀected in histamine H2 receptordeﬁcient mice (Fig. 4B).

The NMDA receptor has multiple ligand-binding sites, including a
glutamate-binding site, a glycine-binding site, an ion channel modulator-binding site and a polyamine-binding site. Therefore, theinvolvement of each ligand-binding site of the NMDA receptor in
spermine-induced nociceptive behaviors was determined. Groups ofmice were co-administered i.t. agmatine (2.5–40 pmol) or arcaine (15–240 pmol), antagonists for the polyamine-binding site of the NMDA
receptor, with spermine (0.02 amol or 10 pmol), and the nociceptive
behaviors induced by spermine were observed for 30 min. The
nociceptive behaviors induced by both 0.02 amol and 10 pmol ofspermine were dose-dependently attenuated by the co-administrationof antagonists for the polyamine-binding site of the NMDA receptor(Fig. 6A–D).
Other groups of mice were co-administered i.t. D-APV (0.25–1nmol) or CPP (1.95–31.25 pmol), antagonists for the glutamatebinding site of the NMDA receptor, or MK-801 (5–10 nmol), an
antagonist for the ion channel modulator-binding site of the NMDA
receptor, with spermine (0.02 amol), and the nociceptive behaviors
induced by spermine were observed for 30 min. The nociceptive
behaviors induced by 0.02 amol of spermine were dose-dependently
reduced by the co-administration of these antagonists for the NMDA
receptor (Fig. 7A-C).

3.3. Involvement of takykinin NK1 receptors in spermine-induced
nociceptive behaviors
The involvement of the takykinin NK1 receptor in spermineinduced nociceptive behaviors was determined. Groups of mice wereco-administered the i.t. takykinin NK1 receptor antagonists sendide(0.5–2 pmol), CP99,994 (0.5–2 nmol) or [D-Phe7,D-His9]SP(6−11)(0.5–2 nmol) with spermine (0.02 amol or 10 pmol), and nociceptive
behaviors induced by spermine were observed for 30 min. The
nociceptive behaviors induced by 0.02 amol of spermine were dosedependently attenuated by the co-administration of takykinin NK1
receptor antagonists (Fig. 5A–C). The highest dose of takykinin NK1
receptor antagonists eliminated the nociceptive behaviors induced by
0.02 amol of spermine. In contrast, the nociceptive behaviors inducedby 10 pmol of spermine were not aﬀected by the co-administration of
takykinin NK1 receptor antagonists (Fig. 5D–F).

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spinal transmission of pain, endogenous polyamines are thought tobind to the polyamine-binding site of the NMDA receptor and tomodulate the Ca2+ channel function of this receptor to potentiate
neural excitation (Williams et al., 1991). In fact, i.t. -administration of
spermine leads to nociceptive behaviors in mice (Tan-No et al., 2000).
In the present study, the mechanism of spermine-induced nociceptive
behaviors was investigated in the mouse spinal cord. Intrathecal
administration of spermine was previously reported to induce dosedependent nociceptive behaviors at doses of 100 amol-10 fmol andshowed saturated responses at doses of 10 fmol–10 pmol (Tan-No
et al., 2000). In the present study, we found that i.t. -administered
spermine showed dose-dependent nociceptive behaviors at much lower
doses (0.001–1 amol) (Fig. 1). Surprisingly, 0.02 amol of spermine wasadequate to stimulate signiﬁcant nociceptive behaviors. It may be alowest dose to elicit pharmacological eﬀect in any drugs or compounds.
At present, there is unfortunately no evidence to explain the mechanism of this extraordinarily high potency of spermine. The extensive
research should be required to explain this phenomenon. To describe
the mechanism of spermine-induced nociceptive behaviors, the present
study was conducted using 0.02 amol (a very low dose) and 10 pmol (ahigh dose) of spermine. As a result, we found evidence that the
nociceptive behaviors induced by i.t. administration of spermine atboth 0.02 amol and 10 pmol were eliminated by i.t. pretreatment withhistamine antiserum (Fig. 2), suggesting that the nociceptive behaviors
induced by i.t. administration of spermine are mediated by the spinal
release of histamine. This hypothesis was supported by the ﬁnding thatthe nociceptive behaviors induced by i.t. administration of spermine atboth 0.02 amol and 10 pmol were abolished in histidine decarboxylasedeﬁcient mice lacking endogenous histamine (Fig. 3). Herein, we report
the remarkable physiological function of spermine to regulate thespinal release of histamine. In contrast to the results of the present
study, we previously reported that i.t. administration of histamine
stimulates the polyamine-binding site of the NMDA receptor to induce
nociceptive behaviors (Watanabe et al., 2008). Taken together with thepreviously reported results, the current ﬁndings indicate that paintransmission in the spinal cord involves a complicated mutual regulation between spermine and histamine.
In the present study, the nociceptive behaviors induced by i.t.
administration of spermine at both 0.02 amol and 10 pmol were
suppressed in histamine H1 receptor-deﬁcient mice (Fig. 4A), but notin histamine H2 receptor-deﬁcient mice (Fig. 4B), suggesting that thehistamine released by i.t. administration of spermine stimulates thehistamine H1 receptor, but not the histamine H2 receptor, in the spinal
cord. It is noteworthy that the nociceptive behaviors induced by 0.02
amol of spermine were abolished in histamine H1 receptor-deﬁcient
mice, whereas the nociceptive behaviors induced by 10 pmol ofspermine were only partially suppressed in histamine H1 receptordeﬁcient mice. We previously reported that the nociceptive behaviors
induced by low doses of i.t. histamine are mediated by the activation of
the histamine H1 receptor in the spinal cord (Sakurada et al., 2003),
whereas the nociceptive behaviors induced by high doses of i.t.
histamine are mediated by the activation of the NMDA receptor via
the binding of histamine to the polyamine-binding site of the NMDA
receptor in the spinal cord (Watanabe et al., 2008). Although there isno direct evidence to demonstrate that the i.t. administration of
spermine dose-dependently increases the release of histamine in the
spinal cord, the nociceptive behaviors induced by 0.02 amol and 10
pmol of spermine may be primarily mediated by the activation of thehistamine H1 receptor and NMDA receptor in the spinal cord,
respectively, via the release of histamine.
It is well established that histamine-containing neurons projectfrom the tuberomammillary nucleus of the posterior hypothalamus to avariety of brain regions and to the dorsal spinal cord (Haas and Panula,2003; Panula et al., 1984, 1989a, 1989b; Watanabe et al., 1984).
Histamine released at the dorsal spinal cord stimulates histamine H1
receptors on the terminals of primary aﬀerent nerves and subsequently

Fig. 7. Eﬀects of the antagonists for the glutamate-binding site and ion channel
modulator-binding site of the NMDA receptor on spermine-induced nociceptive behaviors in the mouse spinal cord. Groups of mice were co-administered i.t. MK-801 (5–10nmol), D-APV (0.25–1 nmol) or CPP (1.95–31.25 pmol) with spermine (0.02 amol), andnociceptive behaviors induced by spermine were observed for 30 min. Each column
represents the mean ± S.E.M. for 10 mice. The statistical signiﬁcance of the diﬀerences
between groups was assessed with one-way ANOVA followed by Bonferroni's test. (A)The F-value of one-way ANOVA was F[4,45] =7.577 (P < 0.001). *, P < 0.05 and **, P <0.01 vs. spermine alone. (B) The F-value of one-way ANOVA was F[3,36] =11.67 (P <0.001). ***, P < 0.001 vs. spermine alone. (C) The F-value of one-way ANOVA wasF[3,36] =14.83 (P < 0.001). *, P < 0.05 and ***, P < 0.001 vs. spermine alone.

4. Discussion
Polyamines, including spermine, are aliphatic molecules that are
widely distributed in the body and are involved in a variety ofphysiological functions and responses (Gilad and Gilad, 1992). In the14— 483 —

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causes the release of substance P and glutamate from primary aﬀerent
nerves, which bind to takykinin NK1 receptors and NMDA receptors on
the dorsal horn of the spinal cord, respectively (Sakurada et al., 2003,
2004). In the present study, the nociceptive behaviors induced by i.t.
administration of 0.02 amol of spermine were dose-dependently
reduced by the co-administration of antagonists for the polyaminebinding site of the NMDA receptor (Fig. 6A,B), antagonists for theglutamine-binding site of the NMDA receptor (Fig. 7A,B) or anantagonist for the ion channel modulator-binding site of the NMDA
receptor (Fig. 7C). Moreover, the nociceptive behaviors induced by i.t.
administration of 0.02 amol of spermine were suppressed by the coadministration of takykinin NK1 receptor antagonists (Fig. 5A–C).
These results clearly suggest that the nociceptive behaviors associatedwith i.t. administration of 0.02 amol of spermine are due to the spinal
release of histamine, which stimulates histamine H1 receptors on the
primary aﬀerent nerves and leads to the release of substance P andglutamate from the primary aﬀerent nerves, which bind to thetakykinin NK1 receptor and the NMDA receptor on the dorsal spinal
cord, respectively.
In the present study, the nociceptive behaviors induced by i.t.
administration of 10 pmol of spermine were suppressed by coadministration of antagonists for the polyamine-binding site of the
NMDA receptor (Fig. 6C-D). Moreover, it has been reported that the
nociceptive behaviors induced by i.t. administration of 10 pmol ofspermine were dose-dependently suppressed by the co-administrationof the antagonists for the glutamine-binding site of the NMDA receptor
or the antagonist for the ion channel modulator-binding site of the
NMDA receptor (Tan-No et al., 2000). These results clearly suggestthat the nociceptive behaviors induced by 10 pmol of spermine are
mediated by the activation of the spinal NMDA receptor. However, incontrast to the 0.02 amol spermine-induced nociceptive behaviors, themechanism for the nociceptive behaviors induced by 10 pmol ofspermine may be more complicated. In the present study, administration of takykinin NK1 receptor antagonists failed to suppress thenociceptive behaviors induced by 10 pmol of spermine (Fig. 5D–F).
Taken together with the ﬁnding that the nociceptive behaviors inducedby 10 pmol of spermine were completely abolished in histidine
decarboxylase-deﬁcient mice (Fig. 3), but only partially suppressed inhistamine H1 receptor-deﬁcient mice (Fig. 4A), the nociceptive behaviors associated with i.t. administration of 10 pmol of spermine may be
due to the spinal release of histamine, which preferentially and directly
stimulates the polyamine-binding site of the NMDA receptor, but not
the histamine H1 receptor, on the dorsal spinal cord.
In conclusion, the nociceptive behaviors induced by i.t. administration of spermine are mediated by the spinal release of histamine.
Histamine indirectly activates the takykinin NK1 receptor and NMDA
receptor via the spinal release of substance P and glutamate anddirectly stimulates the polyamine-binding site of the NMDA receptor.
The receptor that is indirectly or directly stimulated by the released
histamine may depend on the dose of spermine injected.
Acknowledgements
This work was supported by JSPS KAKENHI Grant numbers
15K08678 and 25460725, and a MEXT (Ministry of Education,
Culture, Sports, Science and Technology)-Supported Program for theStrategic Research Foundation at Private Universities (2015–2019).
References
Ahern, G.P., Wang, X., Miyares, R.L., 2006. Polyamines are potent ligands for thecapsaicin receptor TRPV1. J. Biol. Chem. 281, 8991–8995.
Bernstein, H.G., Müller, M., 1999. The cellular localization of the L-ornithine
decarboxylase/polyamine system in normal and diseased central nervous systems.

15— 484 —

Send Orders for Reprints to reprints@benthamscience.ae
444

Current Neuropharmacology, 2017, 15, 444-456

REVIEW ARTICLE

Opioids Resistance in Chronic Pain Management
Luigi A. Morronea,b,*, Damiana Scuteria, Laura Rombolàa, Hirokazu Mizoguchic andGiacinto Bagettaa,baDepartment of Pharmacy, Health and Nutritional Sciences, University of Calabria, Rende, Italy; bUniversity
Consortium for Adaptive Disorders and Head Pain (UCADH), Section of Neuropharmacology of Normal andPathological Neuronal Plasticity, University of Calabria, Rende, Italy; cDepartment of Physiology and Anatomy,
Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, JapanA R T I C L E H I S T O R Y	
  Received: June 27, 2015
Revised: August 11, 2016
Accepted: October 24, 2016
DOI:
10.2174/1570159X14666161101092
822	
  Abstract: Chronic pain management represents a serious healthcare problem worldwide. Chronic pain
affects approximately 20% of the adult European population and is more frequent in women and older
people. Unfortunately, its management in the community remains generally unsatisfactory and rarely
under the control of currently available analgesics. Opioids have been used as analgesics for a long
history and are among the most used drugs; however, while there is no debate over their short term use
for pain management, limited evidence supports their efficacy of long-term treatment for chronic noncancer pain. Therapy with opioids is hampered by inter-individual variability and serious side effects andsome opioids often result ineffective in the treatment of chronic pain and their use is controversial.
Accordingly, for a better control of chronic pain a deeper knowledge of the molecular mechanisms
underlying resistance to opiates is mandatory.

Keywords: Chronic pain, opiate resistance, polymorphisms.
INTRODUCTION
Chronic pain management is one of the most debatedissues in pharmacology and public healthcare. Epidemiologicalstudies show that in Europe one in five adults suffer fromchronic pain [1], often of unknown etiology and rarely under
the control of currently available analgesics [2]. Opioids
have been used as analgesics for a long history and areamong the most used drugs [3]. Interestingly, they have beenused for centuries for the treatment of pain but their moleculartargets were discovered only about forty years ago, in theearly 70s, when the full blossom of receptor binding studies
made it possible [4]. Opioid receptors belong to the family of
G-protein coupled receptors and, in particular, they arecoupled to pertussis toxin (PTX)-sensitive or PTX-insensitive
Gi/o proteins. The discovery of the second messenger system
coupled to the receptor binding of opioid drugs led to theunderstanding of their mechanism of action. These receptors
mediate an inhibitory signal of neural transmission involvedin the analgesic action of opioids. The discovery of opioid
receptors prompted the isolation of the first two endogenous
opioid neurotransmitters called Met-enkephalin and Leuenkephalin [5]. Since then, several opioid peptides wereidentified and the following studies led to a better
understanding of their properties [6]. However, while there is*Address correspondence to this author at the Department of Pharmacy,
Health and Nutritional Sciences, University of Calabria, Rende, Italy;
Tel: 0039 0984493054; Fax: 0039 0984493107; E-mail: luigimorron@libero.it
1570-159X/17 $58.00+.00— 485 —

no debate over the short term use of opioids for pain
management, there is limited evidence to support the efficacy
of long-term treatment for chronic non-cancer pain [7, 8] andsome of them are often ineffective in the treatment ofchronic pain and their use is controversial. This is likely due
to the inter-individual variability originating from the presenceof several polymorphisms which attract genes involved in
the actions of the opioid system. In addition, the use ofchronic opioid therapy is limited by a set of problems. These
include tolerance, addiction, pseudo-addiction, opioid induced
hyperalgesia, bowel dysfunction, suppression of testosterone,cognitive impairment, substance abuse and diversion [9]. In
this article we review the literature to draw a comprehensive
picture of the mechanisms underlying resistance to opioids
for a better control in chronic pain management.
CAUSES OF CHRONIC PAIN
Chronic pain is a multifactorial condition, caused by thecomplex interplay of several pathogenic mechanisms [10,11]. This condition can be triggered by lesions or diseases
affecting the somatosensory nervous system [12]. Costigan
and colleagues described it as an expression of maladaptive
plasticity within the nociceptive system [13]. Particularly,
several changes, e.g., facilitation and disinhibition of synaptictransmission and neuroimmune interactions, spreaded acrossthe nervous system contribute to dysregulation of painneurocircuitry and neurochemistry resulting in complex pain
phenotypes. Chronic pain is associated with specific andnonspecific medical conditions and generally, it is broadly©2017 Bentham Science Publishers

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Current Neuropharmacology, 2017, Vol. 15, No. 3

categorised as cancer pain and non-cancer pain [11]. Noncancer pain can be caused by specific chronic medical
conditions such as osteoarthritis, back pain, fibromyalgia,
diabetic neuropathy and migraine headaches [11, 14].
However, several pathophysiological situations might also
induce it, such as alcoholism [15], HIV/AIDS [16, 17] andneurodegenerative disorders, such as multiple sclerosis [18,19]. All these diseases have a different impact on the opioid
system. For example, several animal and human studies haveshown a decreased analgesic potency of µ opioid agonists indiabetic neuropathic pain [20-24]. Chen and Pan reported
that the inhibitory effect of systemic morphine on spinothalamic
tract neurons is substantially reduced in diabetic ratssuggesting a reduction in or dysfunction of opioid receptors
in the spinal cord dorsal horn in diabetes [25]. Interestingly,
some studies demonstrated a reduction in spinal µ opioid
receptors [26, 27] whereas others suggested that the reduced
analgesic action of opioid agonists in diabetic neuropathic
pain is due, at least in part, to impaired receptor-G protein
coupling [28]. Following nerve injury, primary afferents
reduce their expression of µ opioid receptors (MOR), anddorsal horn neurons are less sensitive to inhibition by µ
opioid agonists [29]. Some authors reported a deficient
functioning of MOR at the supraspinal level and k-opioid
receptors (KOR) at the spinal level in diabetic mice related
to the activation of δ opioid receptors (DOR) at both thesupraspinal level and spinal levels [30]. Moreover, thedysfunction of adenosine triphosphate-sensitive potassiumchannels may contribute to the reduction in MOR-mediatedanalgesia in diabetic mice [31]. Alcoholism also impacts theopioid system. In fact, prolonged exposure to ethanol can
promote an upregulation of functional DOR in the spinal
cord and modulate MOR-mediated analgesia [32]. In
addition, Hull and colleagues suggested that ethanol may
reduce opioid tolerance in mice at the cellular level acting on
the γ-aminobutyric acid (GABA)ergic system either at thelevel of GABA release or GABA receptors [33]. Furthermore,
the HIV-1 envelope glycoprotein 120 (gp120) has been shownto increase MOR mRNA expression in human vascularendothelium [34] and also in HL-60 human promyelocytic
leukemia cells differentiated into macrophage-like cells by
12-O-tetradecanoylphorbol-13-acetate (TPA) [35]. In addition,
more recently, Dever and colleagues reported that theexpression of MOR-1 and other MOR variants may bedifferentially regulated by HIV-1 [36]. Interestingly, thecombination of opioids and HIV-1 infection may promote
the damage of neurons and glia in the pain-processing neuralpathway [17]. Preclinical investigations utilizing animalmodels, as well as clinical observations with multiple sclerosis
patients, also suggested alteration of endogenous opioid
systems in the disease. Particularly, Gironi and colleagues
found reduced β-endorphin concentrations in peripheral
blood mononuclear cells of patients with multiple sclerosis[37]. More recently, in a Theiler's murine encephalomyelitis
virus model of multiple sclerosis, Lynch and colleagues
reported that mRNA levels of MOR, KOR and DOR are
significantly decreased in the spinal cord [38].
LIFE STRESSFUL EVENTS AND CHRONIC PAIN
Several evidences in rodents and humans havehighlighted a crucial role of early life stressful events (such— 486 —

445

as early maternal separation, physical violence, sexual or
psychological abuses) in the development and worsening ofchronic pain [39-44]. The neurobiological mechanisms
underlying the relationship between early-life stress anddevelopment of chronic pain are unclear, however, clinicaland preclinical data suggested a key role for some neurobiological substrates, e.g. the hypothalamic-pituitary-adrenalaxis, neurotransmissions (monoaminergic, opioidergic, endocannabinoid) and immune systems [45-47]. In this regard,
Interestingly, in maternal separation, one of the most commonlyused models of early-life stress, Ploj and colleagues reported
altered expression of the endogenous opioids dynorphin andenkephalin in the hypothalamus, substantia nigra, amygdala,and periaqueductal gray key brain areas in the modulation ofemotional and nociceptive processes [48]. Moreover, Alexander
and colleagues reported that stress potentiates nerve injuryinduced tactile allodynia through a mechanism involving
glucocorticoids acting at glucocorticoid receptors andglutamate receptor-mediated extracellular signal-regulated
kinase (ERK) activation in dorsal horn neurons suggestingthat these pathways converge to cause central sensitization[42]. Epigenetic alterations may also represent one of the key
mechanisms underlying the development of chronic pain inlater life [43, 47, 49-51].
Recently, several data supported a relationship between
early life stressful events and increased oxidative stress inthe central nervous system (CNS) suggesting a crucial role in
the etiopathogenesis of psychiatric disorders such as anxiety,depression, drug abuse or psychosis [52, 53] and alsochronic pain [54-57]. Interestingly, some reports have shownthat impaired mitochondrial energy production reduced MOR
but not DOR or KOR function in neuronal SK-N-SH cells[58, 59]. Moreover, DOR agonists may exert neuroprotective
effects on cells [60] and rat brain [61] attenuating intracellularoxidative stress. In particular, Wallace and colleagues
demonstrated that δ agonists can act in human SK-N-SH
cells in part through a receptor-mediated mechanism [60],whereas Yang and colleagues reported that DOR activation
attenuates oxidative injury in the ischemic brain by enhancing
the activity of antioxidant enzymes such as superoxide
dismutase and glutathione peroxidase and reduces free
radicals, malondialdehyde and nitric oxide (NO) [61]. Morerecently, Chao and colleagues, reported that DOR activation
may exert neuroprotective effects against hypoxic/ischemic
Na+ influx through Na+ channels via a protein kinase C(PKC)-dependent pathway in the cortex [62]. Xe and colleagues
also suggested that DOR signaling could act at multiple
levels to confer neuronal tolerance to harmful insult [63].
PAIN: PHARMACOLOGY AND CLINIC USE OFOPIOID DRUGSThe control of pain is the most used therapeutic action of
opioids. Opioids are commonly prescribed because they are
effective in relieving many types of pain. At the moment,
opioids represent the most effective treatment for chronic
cancer pain conditions [64]. Moreover, in these years,
despite limited strong scientific evidences, use of opioids forchronic non-cancer pain has increased remarkably [8, 65].
However, the use of opioids in pain management requires
careful dose escalation and empirical adjustments based on

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Morrone et al.

clinical response and the presence of side effects or adverse
drug reactions. The forefather of opioid analgesics, that still
remains the most used drug in the management of chronic
pain conditions, is morphine. Morphine is extracted fromopium obtained through Papaver somniferum because it
accounts for 10% of the alkaloids contained in this latex. The
first firm reference to opium employment is traceable inTeofrasto’s writings dating back to the third century b.C.
[66]. The pioneer study of Snyder and his colleagues led in
the 1970s to identify high affinity binding sites for opioids inintestine and brain [4]. Opioid receptors belong to the large
superfamily of seven transmembrane spanning G proteincoupled receptors and are classified as µ (µ1, µ2, µ3), δ (δ1,δ2), k (k1, k2, k3) and ORL1 [67]. These receptors have beencloned and their cDNAs described in the years from 1992 to1994 demonstrating that their corresponding mRNAs present
more than 60%-homology [68]. Opioid receptors activation
inhibits adenylate cyclase (AC)-cyclic adenosine 3',5'monophosphate (cAMP) – protein kinase-A (PKA) signaltransduction pathway thus modulating a wide series ofeffectors up to mitogen-activated protein kinase (MAPK)
family [69, 70]. For a long time it has been thought that
opioid receptors could be coupled only to PTX-sensitive
Gi/o proteins but, after several studies, it was demonstrated
that all of these receptors can transduce their signal eventhrough the PTX-insensitive subunits Gz, G14 and G16
which also stimulate G protein-coupled inwardly rectifying
K+ channel (GIRK) and inhibit AC [71, 72]. Inhibition of
the signal transduction of the pathway AC-cAMP-PKA
by opioid receptors activation leads to reduced neuronal
excitability and consequently nociceptive stimuli transmission.
It was demonstrated that opioid receptors were particularly
expressed in pain-modulating descending pathways, which
include the medulla, locus coeruleus, medial thalamus andperiaqueductal gray area. They were also expressed inlimbic, midbrain, cortical structures and in the spinal cord
substantia gelatinosa [73]. Pain stimuli are perceived by
nociceptors and are inserted at level of the dorsal horn of
spinal cord [74]. At this point opioid drugs come into action
because the cells of the substantia gelatinosa are inhibitory
interneurons rich of opioid receptors that are activated by theantinociceptive descending system and regulate painfulstimuli transmission from primary afferents to spino-thalamic
neurons. Characterization of the properties of opioid receptors
sharpened the interest for identifying endogenous opioid-likeneurotransmitters. In 1975 Hughes and Kosterlitz isolated
two pentapeptides endowed with high affinity for opioid
receptors, i.e. Met-enkephalin and Leu-enkephalin [5].
Additional opioid peptides were successively isolated andclassified as enkephalins, endorphins, dynorphins andendomorphins according to their structure [73]. Several othernon-mammalian opioid peptides, which show affinity to
opioid receptors, have been discovered to date. These
include opioid peptides derived from amphibian skin [75],opioid peptides derived from plant proteins [76] and opioid
receptor ligands derived from food proteins [77]. Today,
opioids can be classified in different groups comprehending
morphine analogues, thebaine analogues, phenylpiperidines,
methadone analogues and benzomorphanes [78]. In the classof morphine analogues we can recognize heroin, codeine,
nalorphine, naloxone, naltrexone etc. The baine analogues,— 487 —

like buprenorphine, are synthetic derivatives of which thechemical structure is unrelated to morphine. Amongphenylpiperidines we can find fentanyl and the methadone
analogues (e.g. dextropropoxyphene). The main representativesof the class of benzomorphanes are pentazocine andcyclazocine. For what concerns the pharmacodynamic of
opioids, they are distinguished in agonists (morphine, codeine,meperidine, methadone, tramadol, tapentadol, fentanyl,
oxycodone), partial agonists (buprenorphine), mixed agonistantagonsts (pentazocine) and antagonists (naloxone,
naltrexone). For the clinical use the most employed drugs aretramadol, codeine and oxycodone (often in combination
therapy with paracetamol), that are classified as mild opioids,
and morphine and fentanyl categorized as strong opioids.
Opioids are strong or mild according to the pain conditions
in which they are used [79, 80]. Tramadol is a synthetic
analogue of codeine very useful both in the treatment of
nociceptive chronic pain and in neuropathic pain syndromes[81], in which it represents a second line therapy whengabapentin, pregabalin or tryciclic antidepressants seem not
to work anymore. It is very interesting to know that this drug
is sold in the shape of racemic mixture since this is more
effective than the single enantiomers. It is well known that inthis mixture (+) enantiomer binds µ receptor and inhibits
serotonin reuptake, while (-) enantiomer inhibits noradrenaline
uptake and stimulates α2-adrenergic receptors [82]. Tramadol
has a better potency ratio relative to morphine in neuropathic
pain than in nociceptive pain models suggesting that
this increase in potency of tramadol is likely due to its
monoaminergic mechanism [83]. Tapentadol is a novel
MOR agonist whose activity is several-fold greater thantramadol, with prominent norepinephrine reuptake inhibition(NRI) and minimal serotonin effect [84]. Tapentadol showedantinociceptive and antihyperalgesic activity in various
models of acute and chronic inflammatory pain, and both
MOR agonism and NRI were found to contribute to theseeffects [85]. In addition, tapentadol has the benefit of greater
gastrointestinal tolerability compared to classical strong
opioids. Codeine, a mild µ opioid agonist, has gained much
popularity as a single agent or in combination with a nonopioid agent, such as paracetamol, for the treatment of mild
to moderate pain. Codeine has a very low affinity for opioid
receptors and its analgesic effect is dependent on its
conversion to morphine through the cytochrome P-450
enzyme 2D6, enzyme highly polymorphic (see below) and
responsible of variability in inter-individual response to thisopioid [86]. Oxycodone is a semisynthetic opioid analgesic
derived from thebaine that acts at MOR and KOR. Oxycodone
has a high oral bioavailability and produces more predictable
plasma concentrations than morphine [87]. The clinicalefficacy of oxycodone is similar to that of morphine and it,alone and in combination with paracetamol, is a useful
opioid analgesic in acute postoperative pain, cancer pain,
visceral pain and chronic nonmalignant pain [88]. Oxycodone
combined with a µ receptor antagonist may improve pain
control, reduce physical tolerance and withdrawal, minimizing
opioid-related bowel dysfunction and act as an abuse
deterrent [89]. Morphine and fentanyl have strong agonist
activity for µ receptors and they bind more selectively these
opioid receptors compared to the others. Morphine iscommonly used as a reference for all other opioids. Its main

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Current Neuropharmacology, 2017, Vol. 15, No. 3

indications of use are for postoperative and chronicmalignant pain, however, it is also used for other severe pain
conditions (e.g. colic pain, angina pectoris) [90]. Fentanyl isa potent synthetic µ-opioid receptor agonist with a rapid
onset and short duration of action [91]. This compound is75-125 times more active than morphine [92] and isextensively used for anesthesia and analgesia in intensive
care units in combination with propofol and midazolam [93].
At present, fentanyl is the only rapid-onset analgesic that issuitable for the treatment of breakthrough pain [91]. The
pure agonists have no apparent ceiling effect for analgesia,
however, meperidine is associated with excitatory side
effects with a risk of seizures and it is not recommended forthe treatment of chronic pain [81]. Partial agonists with
mixed agonist-antagonist action are generally not indicatedfor the treatment of chronic pain [94], however, in 2011 theFood and Drug Administration has approved a transdermal
formulation of buprenorphine for treatment of moderate tosevere chronic pain [86]. Interestingly, buprenorphine may
also act as a potent local anesthetic and blocks voltage-gatedsodium channels via the local anesthetic binding site [95]and this property is likely to be relevant when buprenorphineis used for pain treatment and for local anesthesia. Ideally,
the greatest analgesic activity would be obtained by a drug
able to activate µ receptors and to inhibit k receptors.
Activation of µ receptors present on GABA-ergic interneurons
in the nucleus of the raphe magnus reduces GABA release,
removing the inhibition of the primary neurons that give
origin to the discendent pathway inhibiting painful stimulitransmission at spinal level. On the contrary, k receptors
localized on primary neurons of the nucleus of the raphemagnus cause hyperpolarization and consequent blockade of
the inhibitory descending pathway. Opioid receptors may
interact with each other to form heteromeric complexes andthese interactions affect morphine signaling [96, 97]. Sincechronic morphine administration leads to an enhanced
level of these heteromers, these opioid receptor heteromericcomplexes represent novel therapeutic targets for thetreatment of pain and opioid addiction [98]. At the cellularlevel, opioid receptors are inhibitory and prevent the presynaptic
release of a number of neurotransmitters. Of particularinterest were the observations that opioids inhibited therelease of glutamate, calcitonin gene related protein (CGRP),
and substance P in view of their established roles in pain
circuitry and nociceptive transmission [99]. Glutamate has aunique place in nociception since activation of N-methylaspartate (NMDA) receptors has been associated withcentrally mediated chronic neuropathic pain and hyperalgesia
and ‘wind up’, which is induced by sustained depolarizationof wide dynamic range (WDR) neurons found in deeper
layers of the dorsal horn [100]. Substance P is known tocontribute to chronic inflammatory pain and participate incentral sensitization and associated hyperalgesia [101-103].
CGRP is released from primary afferents and facilitates theactivity of substance P within the dorsal horn [100]. Recently,
in in vivo experiments, Endres-Becker and colleagues [104]found that locally applied morphine reduced capsaicininduced thermal allodynia, suggesting that MOR activation
can also inhibit the activity of the transient receptor potential
vanilloid type 1 (TRPV1) via G(i/o) proteins and the cAMP
pathway. Opioids show a wealth of side effects, among— 488 —

447

which one of the most dangerous is respiratory depression
due to a reduction of sensitivity of the brainstem respiratory
centers to CO2 tension. Moreover, these drugs depress
pontine and bulbar centers involved in the modulation of therespiratory rhythm [105]. Other side effects are constipation,
vomiting, myosis, cough reflex suppression and modulationof the immune system. In particular, opioids have thecapability to modulate immune system both through direct
effects on immune cells and via indirect effects mediated bycentral neuronal mechanisms [106]. However, it has beensuggested that not all the opioids affect immune function in
the same way [107] in particular, morphine and tramadol atanalgesic doses induce different effects on immune system[108]. A repeated or prolonged use of opioids causes adaptive
modifications that lead to tolerance, craving and addiction[109]. These adaptive modifications range from the receptors
modulation and uncoupling with G protein to the hyperactivation of the cAMP-pathway, and so of the AC, withconsequent increase of the proteins CREB (cAMP response
element-binding protein) and fos. Hyperactivation of AC isencountered in tolerance and addiction [81]. One of the
future challenges in this field is to obtain non-addictive
opioids. To pursue this purpose, Mizoguchi and colleagues[110] synthesized compounds like amidino-TAPA. This drugprobably exerts its pharmacologic action via the release ofendogenous k opioid peptides and appears to be nonaddictive and more effective on neuropathic pain [111].
OPIOIDS RESPONSIVENESS IN NEUROPATHIC
PAIN
Neuropathic pain is a clinical manifestation characterized
by the presence of allodynia and hyperalgesia, and it isdifficult to treat with the most potent analgesic compounds[112]. Since the fundamental pathophysiologic mechanismsof neuropathic pain remain unclear, the exact mechanisms
which may account for the weak efficacy of opioids incertain neuropathic pain states remain elusive [80, 113]. It
has been suggested that this reduced opioid responsivenessmay be related to inter-individual variability (see below) but
also to multiple factors including desensitization of opioid
receptors [114], functional changes in glutamate receptors[115] and transporters [116] and uncoupling of G-proteinfrom opioid receptors [117]. β-arrestin has been also
demonstrated as playing an important role in regulating
opioid receptors [118, 119]. It has been suggested that
reduced opioid responsiveness may be related to the lack ofsupraspinal/spinal synergy that is normally associated withmorphine efficacy in conditions of acute pain determining a
disturbance of normal opioid mechanisms/signaling in the
spinal cord [120, 121]. In fact, the analgesic efficacy of
opioids is significantly reduced from an intrathecal opioid
injection compared with an intraperitoneal opioid injection
for neuropathic pain states [122, 123]. Particularly, thefailure of intrathecal opioids to produce antiallodynic effects
may be due, in part, to the lack of available functional spinal
opioid µ-receptors which may occur following nerve injury.
A reorganization of MORs in the dorsal horn spinal cordfollow peripheral axotomy and the reduced effectiveness of
opioids may be related to a crucial functional change involving
downregulation or desensitization of µ-opioid receptors.
Functional downregulation and/or desensitization of these448 Current Neuropharmacology, 2017, Vol. 15, No. 3

Morrone et al.

receptors in the dorsal horn of the spinal cord andparticularly in laminae I and II has been observed in nerveinjury neuropathy [121, 124] and diabetic neuropathy [125,126]. The reduced analgesic effect of intrathecal morphine indiabetes is probably due to impairment of µ-opioid receptorG protein coupling rather than reduction in µ-opioid receptor
number in the spinal cord dorsal horn and may be related toincreased production of PKC [127, 128]. Neuroadaptation of
MOR in the brain may also contribute to the reduced
efficacy of opioids in neuropathic pain [129]. A reducing µopioid receptor-mediated G-protein activity has been
demonstrated in a model of neuropathic pain in the thalamic
region of mice [130] and impairment of G transducer proteins
Gi2α, Gi3α, and Gzα function led to weaker analgesic responses
to various opioids (e.g., methadone, buprenorphine) [131].
Opioid receptor heterodimerization may be an additional
contributing factor to clinical variability of opioids. In fact,opioid receptor dimerization can alter opioid receptor
selectivity and trafficking [132]. Heterodimers may havedifferent opioid binding profiles compared with monomers,
as shown by the association of DORs-1 and KORs-1 [132,133] to form a receptor consistent with the KORs-2 first
proposed from binding assays [134]. Perhaps the mostprominent change in ligand selectivity within the opioid fieldis the dimerization of MORs and the orphanin FQ receptor,
ORL-1 [135]. OFQ/N binds to its own receptor with very highaffinity and is insensitive to traditional opioids. Coexpressionof opioid receptors has been shown to alter opioid ligand
properties and affect receptor signaling in cell culture
model systems [97, 132, 136-138] and these differences arehypothesized to occur as a consequence of receptor
heteromerization.
Sustained agonist activation of MOR initiates rapid
regulatory events, including receptor desensitization andtrafficking, that are thought to contribute to the behavioral
opioid tolerance that develops during prolonged opioid
administration [139]. β-arrestins, including β-arrestin 1 andβ-arrestin 2, are predominantly expressed in neuronal tissues
and regulate G-protein-coupled receptor coupling andsignaling [140]. In β-arrestin 2 knockout mice, the tolerance
to the antinociceptive effects was significantly attenuated in
the tail-flick test [119]. The capacity of opioids to alleviate
inflammatory pain is also negatively regulated by theglutamate-binding NMDA receptor (R) [141]. µ-opioid
receptors and NMDAR NR1 subunits are associated in thepostsynaptic structures of PAG neurons and MOR
desensitization secondary to neuropathic pain appears to
involve PKA. Therefore, PKA may be responsible for the
dissociation of NR1 subunits from MORs, which occurs
as a result of NMDAR activation leading to MOR Ser
phosphorylation and uncoupling from G-proteins [141].
NMDA receptor and PKC translocation are importantly
involved in neuropathic pain and morphine tolerance [142].
In fact, the development of the hyperalgesia and allodynia inneuropathic pain states is suppressed by administration of
NMDAR antagonists or PKC inhibitors [110, 143, 144]. The
development of morphine analgesic tolerance by increased
NMDA receptor activity seems to occur via neural nitricoxide synthase (nNOS) [145]. On the other hand, several
studies have shown that NO mediates numerous neuropathic
pain symptoms [146] and modulates the peripheral— 489 —

antinociceptive effects induced by certain drugs during
inflammatory pain, including opioids [147-149]. NO also
regulates the transcription of µ- and k-opioid receptor genes
under basal and inflammatory conditions [150, 151]. In thebrain, morphine increases the production of NO via thePI3K/Akt/nNOS pathway [152]. Subsequently, NO enhances
NMDAR/calmodulin-dependent protein kinase II (CaMKII)
cascade, promotes MOR phosphorylation and its uncouplingfrom regulated G-proteins [141, 145] diminishing the strengthof morphine-activated µ-opioid receptor signaling. An
increase in dynorphin A has also been suggested to be
involved in the diminished opioid responsiveness that may
be seen in neuropathic pain states [153]. Despite dynorphin
A is an endogenous opioid with activity at k-opioid receptors[154], many of its effects are blocked by MK-801 but notnaloxone, implicating direct or indirect interaction with
NMDAR [155]. Neuropathic pain may also inhibit endogenous
analgesia in PAG through an increase in presynaptic GABA
release [156].
The evidence of increased rates of opioid tolerance
development in neuropathic pain states has been examined
and found to involve an opioid-induced increase in central
immune signaling [157-160]. Opioid exposure induces
profound short- and long-term modulations of central
immune signaling and recently, it has been suggested that theactivation of glial cells, including astrocytes and microglia,
at the level of the spinal cord plays an important role in thedevelopment of opioid tolerance [160-163]. The oppositionof opioid analgesia by acute central immune signaling seems
started, at least in part, by an opioid-induced toll-likereceptor 4 (TLR4) response. TLR4 knockout mice exhibit athree-fold leftward shift in the systemic dose of morphine
necessary for analgesia when compared with wild-type mice[164]. The hypothesis of opioid-induced TLR4 signaling isfurther supported by the potentiation of acute morphineanalgesia after blockade of TLR4 activity [165, 166].
However, recent findings suggest that microglial activationin the development of morphine tolerance is not mediated by
TLR4 [167]. The activation of microglia and astrocytes by
repeated morphine administration also increased TNFα, IL1β and IL-6 expression in the spinal cord [168, 169].
Particularly, IL-1β (by intrathecal administration) produces
mechanical and thermal hyperalgesia [170, 171] and has
been shown to oppose opioid-induced analgesia [172],decreasing morphine efficacy and contributing to thedevelopment of morphine tolerance.
Pharmacogenetic of Opioids
The individual variability of opioid pharmacology
suggests that the patients' genetic disposition influences the
response to opioids. Several studies suggest a genetic variability
between individuals and in their ability to metabolize andrespond to drugs [113]. Some drug-metabolizing enzymes
and transporters (including cytochrome P450 [CYP], uridine
5′-diphosphate [UDP]-glucuronosyltransferases [UGT],
and adenosine triphosphate (ATP)-binding cassette [ABC]transporters) may play a significant role in opioid metabolism
and affect inter-individual differences in opioid concentrationsin the human body and brain. For example, codeine isconverted in morphine through a particular isoform of theOpioids Resistance in Chronic Pain Management

Current Neuropharmacology, 2017, Vol. 15, No. 3

cytochrome P450, the CYP2D6 that is involved in themetabolism of several drugs. A well characterized genetic
polymorphism of CYP2D6, that affects at least 10% of
caucasic population, leads to the incapability to carry out thisconversion and so makes codeine ineffective [173]. Chinese
population is also less sensitive to codeine. Moreover, in thispopulation even morphine is less effective, likely because ofa decreased production of morphine-6-glucuronide [174]. On
the other end of the spectrum, those with gene variants
resulting in extra copies of CYP2D6 may metabolize codeine
to morphine more rapidly and completely than others [175].
Tramadol undergoes metabolism by CYP2D6 to an active
metabolite (O-desmethyl tramadol), which has greateraffinity for the µ-opioid receptor than the parent compound[176]. The modulation of CYP2D6 also affects oxycodone
pharmacodynamics [177]. In addition to CYP2D6, CYP2B6,
2C19, 3A4, and 3A5 isoforms are involved in opioids
metabolism.
The CYP2B6 gene is highly polymorphic, with at least
50 allelic variants identified [178] and its polymorphism
influence the plasma concentration and clearance of themethadone S-enantiomer [179]. CYP3A4 polymorphism isrelated to the pharmacokinetics of fentanyl and patients withCYP3A4*1G variant A allele have a lower metabolic rate of
drug [180]. In Japanese patients with CYP3A5*3 polimorphism
the plasma disposition of noroxycodone is altered [181]. In
addition, multiple single nucleotide polymorphisms in
the promoter region of uridine diphosphateglucuronosyltransferase 2B7 (UGT2B7), the predominant enzyme thatcatalyzes morphine glucuronidation, have been reported[182]. Presence of the UGT2B7-840G allele is associatedwith significantly reduced glucuronidation of morphine andthus contributes to the variability in hepatic clearance ofmorphine in sickle cell disease [183]. The efficacy of opioids
in humans may be affected by allelic variants in the genes oftransporters, structural proteins that can influence theabsorption, distribution, and elimination of opioids [184].
The most characterized of the ATP binding cassette (ABC)superfamily of efflux transporters is ABCB1 encoding Pglycoprotein. Opioid induced analgesia is increased andprolonged in mice lacking P-glycoprotein [185]. Pglycoprotein can limit the concentration of pain management
drugs, such as morphine, in the brain because it actively
pumps drugs out of the CNS. Also methadone, loperamide,
and fentanyl have all been confirmed as P-glycoprotein
substrates [186-188]. ABCB1 gene is highly polymorphic.
The most investigated ABCB1 genetic polymorphism is thenon-synonymous exon 26 SNP, C3435T, which is observedwith a frequency of 50-60% in Caucasians, 40-50% in
Asians, and 10-30% in Africans [189, 190]. Genetic
variation in the multidrug-resistance gene MDR-1 (which
encodes for P-glycoprotein), may account for the genetic
variability in P-glycoprotein activity [191]. However, about
opioids there are not the only genetic variations of themetabolic system to take into account. For example, A118G
single nucleotide polymorphism (SNP) in exon 1 of the µopioid receptor gene (OPRM1) has been linked to thevariability of the analgesic effect of morphine. Individuals
with G118 polymorphism have a reduced or variable response
to morphine and increased opioid dose requirements during— 490 —

449

opioid therapy [192]. The SNPs interesting OPRM1 underlie
the expression of µ-opioid receptors variants in humans. In
particular, A118G SNP causes an asparagine to an aspartate
substitution in the extracellular domain of the µ-opioid
receptor. This allele has a frequency from 4% to 48%
according to the population [193]. Recent studies suggestthat this amino acid substitution causes a different capabilityof the receptor to inhibit native Cav2.2 calcium currents witha consequent difference in pain perception. It seems thatpatients endowed with the mutated µ-opioid receptor
containing the aspartate show higher sensitivity to theanalgesic action due to this receptor activation [194]. A118G
SNP has been associated with elevated pain responses anddecreased pain threshold in a variety of populations and it
seems that A118G genotypes may influence migraineassociated head pain in females [195]. It has also beenshown that this polymorphism A118G could be a clinicalmarker of the outcome of the progression of pain intensityand disability in patients affected by sciatic pain after lumbar
disc herniation, since it seems to increase pain stimuli
sensitivity in female patients and to protect male patients inthe first year following to herniation [196].
In human studies, low catechol-O-methyltransferase(COMT) activity has been associated with increased
sensitivity to acute clinical preoperative or postoperative
pain [197]. As a result, genetic variability in the COMT genecan contribute to differences in pain sensitivity and responseto analgesics. Low COMT activity also increases opioid
receptors and enhances opioid analgesia and adverse effects
in some cancer [198, 199]. Environmental and/or genetic
influences that alter central immune signaling may also
contribute to altered acute opioid analgesia. For example,
single-nucleotide polymorphisms in the genes encoding IL-6[200] or IL-1ra [201], which are associated with increased
proinflammation, lead to increased opioid requirements after
surgery, suggesting a possibly reduced opioid analgesic
efficacy combined with or separate from increased pain.
CONCLUSION
Actually, some of the most powerful analgesics belong tothe opioid drugs family which is, thus, object of intense
study. Opioid receptors field is remarkable among the otherfields in pharmacology because of the fact that the earliest
findings, made without the aid of the modern molecularbiology tools, still remain irrecusable, both about receptors
and about endogenous opioid peptides, and the opioid
binding sites identified in the 1973 are responsible for themain pharmacological actions of the most clinically used
opioids [68]. It remains to clarify the mechanisms of
addiction in order to develop non-addictive opioids and todissect more and more the pharmacogenetic of the opioid
system to obtain always the best clinical outcome with opioids
therapy. In this way, through translational pharmacology, we
would be able to use better the opioid drugs already available
and to produce new drugs more effective in pain control andless addictive. The achievement of opioid drugs really active
even in neuropathic pain syndromes is a very interesting
topic. Indeed, neuropathic pain control is a so debatedquestion that in recent years great effort has been put inunderstanding the mechanisms of spinal cord synaptic

450 Current Neuropharmacology, 2017, Vol. 15, No. 3

Morrone et al.

plasticity which may contribute to neuropathic pain [202]. In
fact, it is now clear that pain shares a lot of similarities withother neurobiological processes such as learning andmemory [203] and the participation of cellular and molecularmechanisms typical of conditions in which these processes
are altered, i.e. in neurodegenerative diseases, seems to play
an important role in the development and maintenance of
pain states. In this regard, autophagy, a major intracellulardegradation pathway lately implicated in several pathologicalconditions and brain diseases [204], represents a novel
pathway regulating neuroinflammation and neuropathic pain
[205, 206] thus offering a new target for the development of
more effective treatments and that could help us understandthe different sensitivity to opioids in some pain states.

[13]

[14]

[15]

[16]

CONFLICT OF INTEREST
The authors confirm that this article content has noconflict of interest.

[17]

ACKNOWLEDGEMENTS[18]

Declared none.
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Pharmacology, Biochemistry and Behavior 140 (2016) 68–74

Contents lists available at ScienceDirect

Pharmacology, Biochemistry and Behavior
journal homepage: www.elsevier.com/locate/pharmbiochembeh

Intrathecal morphine-3-glucuronide-induced nociceptive behavior via
Delta-2 opioid receptors in the spinal cord
Takaaki Komatsu a,b, Soh Katsuyama c, Hiroshi Nagase d, Hirokazu Mizoguchi e, Chikai Sakurada f,
Minoru Tsuzuki f, Shinobu Sakurada e, Tsukasa Sakurada a,⁎a

Department of Pharmacology, Daiichi University of Pharmacy, 22-1 Tamagawa-cho, Minami-ku, Fukuoka, Fukuoka 815-8511, Japan
School of Pharmacy, Duquesne University, 600 Forbes Avenue, Pittsburgh, PA 15282, USA
Center for Experiential Pharmacy Practice, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japand
Department of Medicinal Chemistry, Life Science Center of Tsukuba Advanced Research Alliance, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japane
Department of Physiology and Anatomy, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japanf
Department of Biochemistry, Nihon Pharmaceutical University, 10281 Komuro Ina-Machi Kitaadachi-gun, Saitama 362-0806, Japanbca r t i c l e

i n f oArticle history:
Received 24 January 2015
Received in revised form 11 October 2015
Accepted 13 October 2015
Available online 22 October 2015
Keywords:
Morphine-3-glucuronide (M3G)
Dynorphin
Leucine-enkephalinδ2-opioid receptor
Extracellular signal-regulated kinase (ERK)
Dorsal spinal cordMicea b s t r a c t
Intrathecal (i.t.) injection of morphine-3-glucuronide (M3G), a major metabolite of morphine without analgesic
actions, produces severe hindlimb scratching followed by biting and licking in mice. The M3G-induced behavioral
response was inhibited dose-dependently by pretreatment with an antisera against dynorphin. However, the selective κ-opioid receptor antagonist, nor-BNI did not prevent the M3G-induced behavioral response. Dynorphin
is rapidly degraded by a dynorphin-converting enzyme (cystein protease), to leucine-enkephalin (Leu-ENK). The
M3G-induced behavioral response was inhibited dose-dependently by pretreatment with the antisera against
Leu-ENK. We also showed that M3G co-administered with Leu-ENK-converting enzyme inhibitors,
phosphoramidon and bestatin produced much stronger behavioral responses than M3G alone. Furthermore,
the M3G-induced behavioral responses were inhibited dose-dependently by i.t. co-administration of the nonselective δ-opioid receptor antagonist, naltrindole or the selective δ2-opioid receptor antagonist, naltriben,
whereas the selective δ1-opioid receptor antagonist, BNTX had no effect. An i.t. injection of M3G also produced
a deﬁnite activation of ERK in the lumbar dorsal spinal cord. Western blotting analysis revealed that antisera
against dynorphin, antisera against Leu-ENK, naltrindole or naltriben resulted in a signiﬁcant blockade of ERK activation induced by M3G in the spinal cord. Taken together, these results suggest that M3G-induced nociceptive
responses and ERK activation may be triggered via δ2-opioid receptors activated by Leu-ENK, which is formed
from dynorphin in the spinal cord.
© 2015 Elsevier Inc. All rights reserved.

1. Introduction
Morphine, with its potent analgesic property, has been widely used
for the treatment of various types of acute pain and for the long-term
treatment of severe chronic pain. However, the clinical use of morphine
is complicated by unwanted side-effects, including a paradoxical increase in pain sensitivity (i.e., hyperalgesia and allodynia) (Arner et al.,
1988; DeConno et al., 1991; Sakurada et al., 2005). These clinical observations have been conﬁrmed in laboratory studies. At doses far higher
than those required for antinociception, morphine injected intrathecally(i.t.) into the spinal subarachnoid space of mice produces a spontaneous
vocalization/squeaking and agitation as well as hyperalgesia, allodynia
and scratching, biting and licking compared with antinociception at
low doses (Yaksh et al., 1986; Sakurada et al., 1996, 2005; Komatsu
et al., 2007). Previous studies have also demonstrated that these pain⁎ Corresponding author.
E-mail address: tsukasa@daiichi-cps.ac.jp (T. Sakurada).

related behaviors evoked by i.t. high-dose morphine are not an μ opioid
receptor-mediated event because behaviors evoked by i.t. high-dose
morphine are not reversed by pretreatment with naloxone, an opioid
receptor antagonist. Morphine is known to be metabolized by the conjugation of glucuronide to two major metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in humans (Boerner
et al., 1975; Christrup, 1997). Most rodents do not form M6G but only
form M3G (Handal et al., 2002; Lötsch, 2009). M6G has a high afﬁnity
for the μ-opioid receptor and appears to be a more potent opioid agonist
than morphine. In contrast, M3G does not bind to μ-, δ-, or κ-opioid receptors, NMDA, GABAA or glycine receptors and appears to be devoidof analgesic activity. However, despite these apparent lacks of activity,
i.t. and intracerebroventricular (i.c.v.) administrations of M3G havebeen reported to evoke a range of excitatory behaviors in rodents(Smith, 2000; Komatsu et al., 2009a,b, Hemstapat et al., 2009). Despite
the increasing amount of evidence for the involvement of M3G inmorphine-induced nociceptive responses, very few studies haveaddressed the underlying signaling mechanism. In the present set of

http://dx.doi.org/10.1016/j.pbb.2015.10.010
0091-3057/© 2015 Elsevier Inc. All rights reserved.

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studies, we employed behavioral and biochemical approaches to examine the mechanism of i.t. M3G in morphine-induced nociceptive responses using speciﬁc components affecting the signaling pathway.
Elevation in spinal dynorphin content has also been observed in the
opioid-induced pain state (Vanderah et al., 2000). Although dynorphin
was originally identiﬁed as an endogenous κ-opioid receptor agonist
and may act as an endogenous antinociceptive peptide under speciﬁc
conditions, considerable evidence indicates that enhanced expressionof spinal dynorphin is pronociceptive. Furthermore, i.t. administrationof 15 nmol of dynorphin A(1–17), dynorphin A(2–17), or dynorphin
A(2–13) in rats produced evoked allodynia. Similarly, dynorphin
A(2–17) (3 nmol, i.t.) in mice also induced allodynia (Laughlin et al.,
1997; Vanderah et al., 1996a). Pain-related behavior associated withnerve injury is also blocked by an antiserum to dynorphin (Bian et al.,
1999; Malan et al., 2000; Wagner and Deleo, 1996; Wang et al., 2001).
Dynorphin-converting enzymes, belonging to the cysteine proteasefamily, cleave dynorphin A and dynorphin B between Leu5–Arg6 andArg6–Arg7 bonds, thereby generating leucine-enkephalin (Leu-ENK)
and Leu-ENK-Arg, which are primarily active in δ-opioid receptors(Silberring et al., 1992). The i.t. administration of dynorphin A(1–17)
also produces an anti-analgesic activity against morphine via activationof the δ2-opioid receptor by the increased release of Leu-ENK in the
spinal cord (Rady et al., 1999, 2001; Tseng et al., 1994). Furthermore,
i.t. Leu-ENK in combination with peptidase inhibitors produces nociceptive behavior via activation of the glutamate receptor, which results in
the release of nitric oxide via the δ2-opioid receptor in the spinal cord
(Komatsu et al., 2014). Thus, nociceptive behavior induced by M3G inmorphine may potentially occur via δ2-opioid receptors activated by
Leu-ENK, which is formed from dynorphin in the spinal cord.
Extracellular signaling-regulated kinase (ERK) is activated in thedorsal spinal cord by nociceptive stimuli, including formalin, capsaicinor carrageenan injection (Galan et al., 2002; Ji et al., 1999; Karim et al.,2001). Inhibition of ERK signaling reduces nociceptive behavior after
nociceptive stimuli, suggesting that ERK activation contributes toacute nociceptive processing in the spinal cord (Ji et al., 1999; Karimet al., 2001).
The purpose of the present research study was to determine whether δ2-opioid receptor activation by Leu-ENK, which is formed fromdynorphin, is involved in M3G-induced nociceptive behavior and ERK
activation in the spinal cord.
2. Materials and methods
2.1. Animals
Pathogen-free adult male ddY-strain mice weighing an average of
24 g (Shizuoka Laboratory Center, Japan) were used in all experiments.
The mice were maintained in a controlled 12 h light–dark cycle withfood and water ad libitum. Room temperature and humidity were controlled at 22–24 °C and 50–60%, respectively.
2.2. Intrathecal injections
The i.t. injections were administered by percutaneous lumbar
puncture through an intervertebral space at the level of the 5th or 6th
vertebrae using the Hylden and Wilcox technique (Hylden andWilcox, 1980). The drugs were administered i.t. in a volume of 5 μlwith a 50-μl Hamilton microsyringe. A tail ﬂick was used as an indication that the needle had penetrated the dura.
2.3. Behavioral experiments
Mice were acclimatized initially for 1 h in an individual plastic cage(22.0 × 15.0 × 12.5 cm) which also served as the observation chamber.
The animals were challenged i.t. with M3G observed for 5 min. The
observation of items of the induced behaviors was the total response69

time (s) of the following behaviors: hindlimb scratching, biting or licking of the hindpaw.
2.4. Drugs
The following drugs were used: M3G, bestatin, naltrindole,
naltriben, nor-binaltorphimine dihydrochloride (nor-BNI), 7benzylidenenaltrexone (BNTX), 4-(hydroxymercuri) benzoic acidsodium salt (PHMB) (Sigma Chemical Co., St. Louis, MO, USA),phosphoramidon (Nakalai tesq, Kyoto, Japan), dynorphin A antibody(Phenix Pharmaceutical, Inc., USA), Leucine-enkephalin polyclonal antibody (Millipore Corporation, USA), 1,4-diamino-2,3-dicyano-1,4-bis(2aminophenylthio) butadiene (U0126) (Calbiochem, Darmstadt,
Germany). Monoclonal anti-phospho-p44/42 MAP kinase antibody
and anti-p44/42 MAP kinase antibody were obtained from Cell Signaling Technology, Inc. U0126 was initially dissolved in 100% DMSO as
stock solution, further diluted by artiﬁcial CSF and adjusted to 6.71%
DMSO as the ﬁnal concentration. The other drugs were dissolved in
50% dimethylsulfoxide (DMSO) to prepare the concentrated stock solution and working solutions were then diluted in artiﬁcial cerebrospinal
ﬂuid (CSF), containing NaCl 7.4 g, KCl 0.19 g, MgCl2 0.19 g and CaCl2 0.14g/1000 ml of distilled and sterilized water, in a stepwise fashion. The
highest concentrations of drugs used contained 0.9% and 1.4% DMSO, respectively. Low concentrations of DMSO resulted in no substantial effecton M3G-induced behavioral changes.
All antagonists were co-administered i.t. with M3G in a volume of
5 μl. Antiserum against dynorphin or leucine-enkephalin were injected
i.t. 5 min prior to i.t. M3G.
2.5. Sample preparation
At 3 min after i.t. injection, the mice were decapitated and the entire
spinal cord was obtained by pressure expulsion with physiological saline. The dorsal part of lumbar spinal cord was dissected quickly on an
ice-cooled glass dish for Western blotting analysis.
2.6. Western blotting analysis
Tissue samples were homogenized in 0.1 ml of lysis buffer reagent(150 mM NaCl, 1.0% NP-40, 50 mM Tris–HCl pH 8.0, 1 mM
phenylmethylsulfonyl ﬂuoride, 1 mg/ml aprotinin, 1 mM sodium vanadate and 1 mM EDTA pH 8.0) and centrifuged at 15,000 × g for 30 min at
4 °C. Supernatants were collected and total protein amounts were measured using the Protein Assay (BIO-RAD, Hercules, CA). An equal volumeof 2× sample buffer (100 mM Tris–HCl pH 6.8, 2.5% SDS, 20% glycerol,
0.006% bromophenol blue and 10% β-mercaptoethanol) was added to
30 μg of total protein. The samples were boiled, electrophoresed in a
10% SDS-polyacrylamide gel (BIO-RAD, Hercules, CA) and then transferred to a Hybond-P membrane (Amersham Biosciences).
The blotted membrane was then incubated overnight with 5% skim
milk (Wako Pure Chemical Industries, LTD, Osaka, Japan) in T-PBS(PBS containing 0.1% v/v Tween 20). All antibody applications were performed in T-PBS. After the membranes were washed, primary antibody
incubations were performed for 2 h at room temperature using theappropriate dilutions (anti-phospho-p44/42 MAP kinase 1:1000 andanti-p44/42 MAPK antibody 1:1000). The membranes were extensively
washed with T-PBS and incubated for 2 h with the secondary antibody (anti-rabbit or anti-mouse IgG peroxidase-conjugated antibody
1:5000) (Amersham Biosciences). After washing, the proteins weredetected using the ECL-Plus Western blotting detection system(Amersham Biosciences) and visualized using the Dolphine-Chemi
Image System (Wealtec). MagicMark western protein standard(Invitrogen) was simultaneously resolved on the gel, and the molecularweight of the proteins was estimated.

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2.7. Data analyses
Statistical analyses of the results were performed using Dunnett's
test for multiple comparison, following analysis of variance (ANOVA).
Differences were considered to be signiﬁcant if P b 0.05. All values are
expressed as the mean ± S.E.M.
3. Results
3.1. Effects of antiserum against dynorphin and a selective κ-opioid receptor
antagonist on i.t. M3G-induced behavioral response
An i.t. injection of M3G (2.5 nmol) into the spinal lumbar space
evoked reciprocal hindlimb scratching toward the ﬂanks in mice. Biting
and licking of the hindpaw were also observed after the incidence ofscratching. These behavioral characterizations of i.t. M3G conﬁrms previously reported data (Komatsu et al., 2009a). Antiserum against
dynorphin, injected i.t. 5 min prior to M3G, inhibited i.t. M3G-induced
behavioral responses in a dilution-dependent manner (Fig. 1A). Treatment with nor-BNI, a selective κ-opioid receptor antagonist did not prevent the behavioral responses induced by i.t. M3G (Fig. 1B).
3.2. Effects of antiserum against Leu-ENK and δ-opioid receptor antagonists
on i.t. M3G-induced behavioral response
Antiserum against Leu-ENK, injected i.t. 5 min prior to M3G, inhibited
i.t. M3G-induced behavioral responses in a dilution-dependent manner(Fig. 2A). Treatment with peptidase inhibitors (Leu-ENK-converting

enzyme inhibitors), phosphoramidon, (an endopeptidase 24.11 inhibitor) (2.0 nmol) and bestatin, (a general amino-peptidase inhibitor)(0.25 nmol), signiﬁcantly enhanced the efﬁcacy of the i.t. M3G-induced
responses (Fig. 2B). The non-selective δ-opioid receptor antagonist,
NTI (50–200 pmol) and the selective δ2-opioid receptor antagonist,
NTB (50–200 pmol) showed a dose-dependent inhibition of i.t.
M3G-evoked nociceptive responses (Fig. 2C and D). NTI (200 pmol)and NTB (200 pmol) alone did not induce a signiﬁcant behavioral
response compared to CSF-injected controls. Treatment with BNTX
(200 pmol), a selective δ1-opioid receptor antagonist, did not prevent
an i.t. M3G-evoked nociceptive response (Fig. 2E). The doses of the antagonists used have been previously reported to completely block theantinociception induced by the respective selective opioid agonists(Tseng et al., 1997).
3.3. Effects of the MEK inhibitor on ERK activation in the dorsal spinal cordand on nociceptive responses after i.t. injection of M3G
We further examined whether spinal ERK is activated by i.t. M3G inthe lumbar dorsal cord. Activation of ERK in the lumbar dorsal cord wasquantiﬁed using the Western blotting assay (Fig. 3A). We compared theeffect of i.t. M3G (2.5 nmol) with that of the artiﬁcial CSF treatment in
the lumbar dorsal cord extracted 3 min after i.t. injection. Western blotting analyses revealed that M3G signiﬁcantly increased phospho-ERK
expression (Fig. 3A).
Next, we examined whether upstream effectors of ERK were necessary for the induction of the behavioral responses induced by i.t. M3G.
The MEK inhibitor U0126 (5.0 nmol) reduced a signiﬁcant ERK
activation 3 min after i.t. M3G (Fig. 3A). Total spinal ERK expression
was unchanged when M3G alone or in combination with U0126 wasadministered (Fig. 3A). In the behavioral test, i.t. injection of U0126(2.5–5.0 nmol) caused a dose-dependent inhibition of the nociceptive
response induced by i.t. M3G when compared with the artiﬁcial CSFtreated controls (Fig. 3B). ERK activation and behavioral characterizations of i.t. M3G conﬁrms previously reported data (Komatsu et al.,2009a).
3.4. Effects of antiserum against dynorphin, antiserum against Leu-ENK andδ-opioid receptor antagonists on M3G-induced ERK activation
To investigate whether the release of dynorphin, Leu-ENK and activation of the δ-opioid receptor is necessary for upstream activators ofphospho-ERK, effects of antiserum against dynorphin, antiserum
against Leu-ENK, δ-opioid receptor antagonist NTI, selective δ2-opioid
receptor antagonist NTB and the selective δ1-opioid receptor antagonist
BNTX were injected i.t. with M3G. Consistent with the behavioral results(Fig. 1A–E), all treatments except BNTX were effective in blocking
phospho-ERK activation induced by M3G (Fig. 4A–E).
4. Discussion

Fig. 1. Effects of antiserum against dynorphin (A) and nor-binaltorphimine dihydrochloride (nor-BNI) (B) on the nociceptive behavior induced by i.t. injection of M3G. Antiserum against dynorphin was pre-injected i.t. 5 min prior to i.t. injection with M3G. The
nor-BNI was co-administered i.t. with M3G in a total volume of 5 μl. The duration ofscratching, biting and licking responses induced by M3G was determined over a 5 min period starting immediately after the i.t. injection. Each value represents the mean ± S.E.M.
of ten mice in each group. Statistically signiﬁcant difference compared with M3G alone isindicated by **P b 0.01, *P b 0.05.

The present results demonstrate for the ﬁrst time that i.t. injection of
M3G evoked nociceptive responses and that ERK activation may be triggered via δ2-opioid receptors activated by Leu-ENK formed fromdynorphin in the spinal cord. Several studies have suggested an important role for spinal dynorphin in abnormal pain. First, blocking the effects of dynorphin with antiserum clearly blocks opioid-induced
allodynia and hyperalgesia, and second, continuous intrathecal infusion
or subcutaneous administration of the μ-opioid agonist morphin andspinal nerve ligation result in an increase in spinal dynorphin content(Kajander et al., 1990; Draisci et al., 1991; Dubner and Ruda, 1992;
Rattan and Tejwani, 1997; Vanderah et al., 2000). In addition, the antianalgesic activities of i.t. administration of dynorphin were inhibitedby naltriben, a δ2-opioid receptor antagonist, or by treatment with aδ-opioid receptor antisense oligodeoxynucleotide, which downregulates spinal δ2-opioid receptors (Rady et al., 1999, Tseng et al.,— 500 —

T. Komatsu et al. / Pharmacology, Biochemistry and Behavior 140 (2016) 68–74

71

Fig. 2. Effects of antiserum against Leu-ENK(A), Leu-ENK-converting enzyme inhibitors (phosphoramidon and bestatin) (B), naltrindole (NTI) (C), naltriben (NTB) (D) and 7benzylidenenaltrexone (BNTX) (E) on the nociceptive behavior induced by i.t. injection of M3G. Antiserum against Leu-ENK was pre-injected i.t. 5 min prior to i.t. injection of M3G.
Each antagonist was co-administered i.t. with M3G in a total volume of 5 μl. The duration of scratching, biting and licking responses induced by M3G was determined over a 5 min period
starting immediately after i.t. injection. Each value represents the mean ± S.E.M. of ten mice in each group. Statistically signiﬁcant difference compared with M3G alone is indicated by**P b 0.01, *P b 0.05.

Fig. 3. Effect of U0126, a MEK inhibitor, on spinal ERK activation and nociceptive response
induced by i.t. injection of M3G. (A) Western blotting analyses of the dorsal spinal homogenates for pERK and tERK. Dorsal spinal cord samples were obtained 3 min after i.t. injection of M3G. tERK was used as a loading control. U0126 (5.0 nmol) was administered i.t.
5 min prior to i.t. M3G. This Western blot is representative of four independent experiments. (B) Inhibitory effects of i.t. U0126 (2.5–5.0 nmol) on nociceptive behavior inducedby i.t. injection of M3G. The duration of scratching, biting and licking responses induced by
M3G was determined over a 5 min period starting immediately after the i.t. injection. Eachvalue represents the mean ± S.E.M. of ten mice in each group. Statistically signiﬁcant difference compared with M3G is indicated by **P b 0.01, *P b 0.05.

1994). Leu-ENK antiserum also inhibited i.t. administration of a low doseof Leu-ENK, which produced anti-analgesia activity against morphineinduced antinociception (Rady et al., 2001). Taken together, nociceptive
responses evoked by i.t. dynorphin may result from an increasing
accumulation of Leu-ENK in the spinal cord. Leu-ENK has a relatively
high afﬁnity for the δ-opioid receptor and may act as an endogenous
antinociceptive peptide. A genuine effect of enkephalin has not been observed because it is degraded easily by enzymes in the body (Hambrocket al., 1976). Thus, a synthetic inhibitor for enkephalin-degrading enzymes is used. It has been shown that peptidase inhibitors, such as
amastatin, captopril, phosphoramidon and spinorphin prevent the degradation of enkephalin, and these peptidase inhibitors enhanced the effects of enkephalin administered at doses required for antinociception(Taguchi et al., 1998; Honda et al., 2001). However, a previous study
showed that i.t. injection of a low dose of Leu-ENK, co-administeredwith peptidase inhibitors, produced behaviors consisting of biting andlicking of the hindpaw and tail along with hindlimb scratching directed
toward the ﬂank (Komatsu et al., 2014). For this reason, Leu-ENK could
act as an inverse agonist to the δ2-opioid receptor. This inverse agonist
activity was obtained at a low concentration, which is approximately
1/1000 of the amount necessary to produce analgesic synergism withmorphine (Vanderah et al., 1996b). Although inverse agonists havebeen applied to opioid receptor antagonists (Szekeres and Traynor,
1997), they have dual actions, as opioid agonists and antagonists(Crain and Shen, 1990, 1998, 2000a). Low concentrations of opioids (μ,δ, κ opioid peptides) elicit excitatory prolongation of the action potential
duration in mouse sensory dorsal root ganglion neurons, whereas— 501 —

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T. Komatsu et al. / Pharmacology, Biochemistry and Behavior 140 (2016) 68–74

Fig. 4. Effect of antiserum against dynorphin (A), antiserum against Leu-ENK(B), NTI (C), NTB (D) and BNTX (E) on spinal ERK activation induced by i.t. injection of M3G. Western blotting
analyses of the dorsal spinal homogenates for pERK and tERK. Representative Western blots are shown on top, and quantiﬁcation of pERK1 and pERK2 levels are shown in the bar graphs.
Dorsal spinal cord samples were obtained 3 min after the i.t. injection of M3G. tERK was used as a loading control. NTI, NTB and BNTX were co-administered i.t. with M3G. Antiserum
against dynorphin and antiserum against Leu-ENK were pre-injected i.t. 5 min prior to i.t. injection on M3G. Each value represents the mean ± S.E.M. of four mice in each group. Statistically signiﬁcant difference compared with A-CSF controls is indicated by **P b 0.01, *P b 0.05. ##P b 0.01, #P b 0.05 compared to M3G alone.

higher concentrations result in inhibitory shortening of the action potential duration (Shen and Crain, 1994). Shortening of the action potential of primary sensory neurons by opioids has generally been
considered to be a useful model of their inhibition of calcium inﬂux
and transmitter release at presynaptic terminals in the dorsal spinal
cord, thereby accounting for opioid-induced analgesia in vivo (Shenand Crain, 1994). However, the opioid-induced prolongation of action
potentials as evidence of the excitatory effects of opioids on sensory
neurons, which may result in enhanced calcium inﬂux and transmitter
release at presynaptic terminals, may account for some types ofhyperalgesia and allodynia elicited by opioids in vivo (Shen and Crain,1994). These results are consistent with the results of the analgesic activity and the mechanism obtained in the previous study at a low dose
(0.5–1.0 fmol) of Leu-ENK in combination with peptidase inhibitors(Komatsu et al., 2014). The nociceptive behavior evoked by i.t. LeuENK in combination with peptidase inhibitors was inhibited dosedependently by co-administration of the non-selective δ-opioid receptor
antagonist naltrindole or the selective δ2-opioid receptor antagonist
naltriben, but not by the selective δ1-opioid receptor antagonist BNTX
(Komatsu et al., 2014).
Our studies have shown previously that i.t. M3G-evoked nociceptive
responses contributes to the activation of spinal ERK signaling in the

NO-cGMP-PKG pathway via the release of a primary afferent neurotransmitter such as glutamate (Komatsu et al., 2009a). Dynorphininduced allodynia was blocked by MK-801, an NMDA antagonist(Laughlin et al., 1997, Vanderah et al., 1996a,b). The release of excitatory
amino acids, such as glutamate and aspartate, increased with i.t.
dynorphin and was blocked by MK-801 (Faden, 1992; Skilling et al.,1992). The elimination of morphine-induced antinociception producedby i.t. Leu-ENK may potentially occur by the increased release of glutamate from primary afferent terminals via activation of spinal δ2-opioid
receptors because i.t. administration of naltriben, a δ2-opioid receptor
antagonist or MK801, a non-competitive NMDA antagonist inhibitedthe anti-analgesic action of Leu-ENK (Rady et al., 2001). Furthermore,in an analysis of 129S6/SvEv mice lacking responsiveness to NMDA, i.t.
injected Leu-ENK did not inhibit i.t. morphine-induced analgesia(Rady et al., 2001). The anti-analgesic action of dynorphin and LeuENK through NMDA receptors suggest that the signaling pathway involved in producing anti-analgesia is different from that for analgesia(Crain and Shen, 1998, 2000b; Fundytus and Coderre, 1999). Previous
studies have also indicated that nociceptive behaviors induced by LeuENK co- injected with peptidase inhibitors were inhibited by MK-801(Komatsu et al., 2014). In view of these ﬁndings, we speculate that i.t. injection of M3G could release primary afferent neurotransmitters,— 502 —

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potentially glutamate or activate glutamate receptors, via δ2-opioid receptors activated by Leu-ENK formed from dynorphin in the spinal
cord. Indeed, i.t. injection of high-doses of morphine in rats evoked amarked increase in glutamate and nitric oxide (NO) release in dorsal
spinal cord extracellular ﬂuid (Watanabe et al., 2003). On the basis of
our previous results, glutamate released from presynaptic sites in response to i.t. M3G activated NMDA receptors, which triggered a feedforward mechanism of stimulation of nNOS activity via a mechanismthat is largely dependent on calcium. In addition, activation of NMDA
receptors can stimulate nNOS activity via a calcium calmodulindependent mechanism (Li et al., 1994, Wu et al., 1994). Extracellular calcium also appears essential for noxious stimulation-induced ERK activation (Lever et al., 2003). NMDA receptor functions as a calcium channel
and has been widely implicated in ERK activation (Ji et al., 1999; Leveret al., 2003; Kawasaki et al., 2004). Recently, accumulating evidence
has suggested that ERK has important roles in the modulation of nociceptive signaling. ERK is activated in the dorsal spinal cord in modelsof pain, such as formalin, capsaicin or carrageenan injection (Thomasand Hunt, 1993; Ji et al., 1999; Karim et al., 2001; Cruz et al., 2005). Furthermore, inhibition of ERK phosphorylation in the dorsal spinal cord by
the MEK inhibitor reduces the pain behavioral response after formalin,capsaicin or carrageenan injection. Western blotting studies had also
shown that i.t. M3G evoked a strong activation of spinal phospho-ERK,
which is consistent with the nociceptive behaviors induced by i.t.
M3G. The MEK inhibitor U0126 in the lumbar spinal cord reveals thatthe signal transduction pathway through MEK and phospho-ERK canbe activated in the lumbar spinal cord after M3G injection (Komatsu
et al., 2009a). Importantly, treatment with the non-selective δ opioid receptor antagonist, NTI or the selective δ2-opioid receptor antagonist,
NTB, could clearly block phospho-ERK, while the selective δ1-opioid receptor antagonist BNTX (7-benzylidenenaltrexone), was not effectively
blocked. These results support a critical role of ERK activation via theδ2-opioid receptor in the establishment of nociception induced by i.t.
M3G. Activation of this nociception signaling pathway was observed
after M3G injection and supports the heuristic possibility of several independent concepts. Several lines of evidence indicate that the δopioid receptor is essential for analgesic tolerance, which could be consistent with the sensitization, resulting in an excitatory action (Kestet al., 1996; Chakrabarbarti et al., 1998; Zhu et al., 1999; Crain andShen, 2000a). Indeed, morphine analgesic tolerance does not develop
in either δ-opioid receptor or preproenkephalin knock-out mice (Zhu
et al., 1999; Joshua et al., 2002). δ-opioid receptor antagonists with morphine reduced analgesic tolerance to morphine and restored morphineanalgesic potency. (Abdelhamid et al., 1991, Fundytus et al., 1995, AbulHusn et al., 2007). More recently, a compound of a μ opioid receptor agonist with a δ opioid antagonist was reported to produce reduced tolerance compared to fentanyl (Mosberg et al., 2014). Moreover, in several
studies, blockade of the NMDA receptor-NOS cascade was shown to attenuate the development of tolerance to morphine (Trujillo and Aki,
1991; Kolensnikov et al., 1993; Elliott et al., 1994; Mao et al., 1994;
Pasternak et al., 1995; Gonzalez et al., 1997). Subsequent analyticalstudies of the δ-opioid receptor and preproenkephalin knock-outs
mice, and the NMDA receptor-deﬁcient 129S6 inbred mouse strain
have supported these hypotheses, implicating the signaling pathway involved in that release of preproenkephalin-derived peptides during
chronic morphine exposure, the δ-opioid receptor and the NMDA receptor are necessary for the expression of morphine tolerance (Joshua et al.,2002). Overall, these data suggest that the development of morphine
tolerance may result from activation of the NMDA receptor-NOS cascade, via the activation of δ2-opioid receptors followed by a continuous
release of Leu-ENK during chronic morphine exposure. In conclusion,
the current study suggests that nociception and spinal ERK activation
evoked by i.t. M3G, major metabolite of morphine may be mediatedvia δ2-opioid receptors activated by Leu-ENK, which is formed fromdynorphin in the spinal cord. This ﬁnding suggests that inhibitorsof δ2-opioid receptors can enhance the analgesic response to spinal

73

morphine, diminishing the development of tolerance to morphine andthus represents a promising lead for the development of opioid analgesics with reduced side effects.
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Contents lists available at ScienceDirect

Neuroscience Lettersjournal homepage: www.elsevier.com/locate/neulet

Research paper

Involvement of spinal glutamate in nociceptive behavior induced byintrathecal administration of hemokinin-1 in mice
Chizuko Watanabe a , Hirokazu Mizoguchi a , Giacinto Bagetta b , Shinobu Sakurada a,∗ab

Department of Physiology and Anatomy, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan
Department of Pharmacobiology, University of Calabria, Arcavacata di Rende, Cosenza 87036, Italyh i g h l i g h t s• Hemokinin-1-induced nociceptive behavior was inhibited by NMDA receptor antagonists.
• Hemokinin-1 produced release of glutamate in the spinal cord.
• Spinal release of glutamate was inhibited by NMDA receptor antagonists.
• The release of glutamate may be involved in the nociceptive behavior of hemokinin-1.

a r t i c l e

i n f oArticle history:
Received 25 December 2015
Received in revised form 10 February 2016
Accepted 14 February 2016
Available online 17 February 2016
Keywords:
Hemokinin-1
Pain
Glutamate
NMDA receptor
Microdialysis
Spinal corda b s t r a c t
The most recently identiﬁed tachykinin, hemokinin-1, was cloned from mouse bone marrow. While several studies indicated that hemokinin-1 is involved in pain and inﬂammation, the physiological functionsof hemokinin-1 are not fully understood. Our previous research demonstrated that the intrathecal (i.t.)
administration of hemokinin-1 (0.00625–1.6 nmol) dose-dependently induced nociceptive behaviors,consisting of scratching, biting and licking in mice, which are very similar with the nociceptive behaviors
induced by the i.t. administration of substance P. Low-dose (0.0125 nmol) hemokinin-1-induced nociceptive behavior was inhibited by a speciﬁc NK1 receptor antagonist; however, high-dose (0.1 nmol)
hemokinin-1-induced nociceptive behavior was not affected.
In the present study, we found that the nociceptive behaviors induced by hemokinin-1 (0.1 nmol)
were inhibited by the i.t. co-administration of MK-801 or D-APV, which are NMDA receptor antagonists.
Moreover, we measured glutamate in the extracellular ﬂuid of the mouse spinal cord using microdialysis.
The i.t. administration of hemokinin-1 produced a signiﬁcant increase in glutamate in the spinal cord,
which was signiﬁcantly reduced by co-administration with NMDA receptor antagonists. These results
suggest that hemokinin-1-induced nociceptive behaviors may be mediated by the NMDA receptor in the
spinal cord.
© 2016 Published by Elsevier Ireland Ltd.

1. Introduction
Substance P, neurokinin A, neurokinin B and hemokinin-1 aremembers of the mammalian tachykinin family. They are derived
from distinct preprotachykinin genes: TAC1 encodes substance P
and neurokinin A, TAC3 encodes neurokinin B and TAC4 encodes
hemokinin-1 [1,2]. Speciﬁcally, substance P is mainly distributedin both the peripheral and central nervous systems as a neurotransmitter or neuromodulator in pain processing. Moreover,∗ Corresponding author. Department of Physiology and Anatomy, Tohoku Pharmaceutical University, 4−4−1 Komatsushima, Aoba−ku, Sendai 981-8558, Japan.
E-mail address: s-sakura@tohoku-pharm.ac.jp (S. Sakurada).

substance P is released from small primary afferents and binds tothe neurokinin-1 (NK1 ) receptor to induce various pharmacological responses. The NK1 receptor is expressed in the superﬁcial layerof the spinal dorsal horn, which is an important site for pain transmission [3]. Hemokinin-1 is the most recently identiﬁed tachykinin
peptide, and it resembles substance P in amino acid sequence andhas a very high afﬁnity to the NK1 receptor compared with the NK2and NK3 receptors [4–6]. Hemokinin-1 mRNA is localized in the
spinal cord and dorsal root ganglion, as is substance P mRNA [7].
In a previous report, the intrathecal administration of hemokinin-1
induced pain-related behaviors, such as scratching, in rats, and theNK1 receptor antagonist inhibited pain-related behavior [8]. Interestingly, we demonstrated, in a previous report, that the intrathecal
administration of hemokinin-1 induced pain-related responses,

http://dx.doi.org/10.1016/j.neulet.2016.02.027
0304-3940/© 2016 Published by Elsevier Ireland Ltd.

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consisting of scratching, biting and licking in mice, are very similar with substance P-induced pain behavior; however, the NK1
receptor antagonist failed to inhibit some of the dose-induced painrelated behaviors [9]. In recent reports, hemokinin-1 is described
as a tachykinin with a distinct function compared to substance P inpain and inﬂammatory processes [8,10–13]. However, these details
have not yet been elucidated. On the other hand, glutamate is an
important transmitter, as is substance P. Glutamate is the excitatorytransmitter that is released from primary afferent synapses in the
spinal cord dorsal horn and activates the postsynaptic glutamate
receptor. Accordingly, the glutamate receptor in the spinal cord,
which is classiﬁed into two types, an ionotropic and metabotropic
glutamate receptor, plays a crucial role in nociceptive signaling,
and both glutamate receptors are also expressed on presynaptic
terminals. There are three main classes of ionotropic glutamate receptors, the N-methyl-D-aspartate receptor (NMDAR), the-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor(AMPAR) and the kainate receptor (KAR) [14,15]. Speciﬁcally, the
NMDA receptor is suggested to be involved in nociceptive transmission and processing within the spinal cord. Behavior studies
indicate that pain-related responses, consisting of scratching, biting and licking, induced by the intrathecal administration of NMDA
are inhibited by selective NMDA receptor antagonists [16,17].
In this study, we examined the involvement of the NMDA
receptor on the spinally mediated behavioral response induced byhemokinin-1 in mice.
2. Materials and methods
The experiments were performed according to a protocol
approved by the Ethics Committee for Animal Experiments in the
Tohoku Pharmaceutical University and the National Institutes of
Health Guide for the Care and Use of Laboratory Animals. Everyeffort was made to minimize suffering and the number of animals
used.
2.1. Animals
All of the experiments were carried out with male ddY miceweighing 23–25 g (Japan SLC, Hamamatsu, Japan). The mice weremaintained in a controlled 12-h light-dark cycle, at a constant temperature of 22–24 ◦ C, at a relative humidity of 55 ± 5%, and had freeaccess to food and water. They were used only once.
2.2. Behavioral observation
The experiments were performed essentially according to
the method described by Watanabe et al. [9]. Before drug
administration, the mice were adapted to the plastic cage(22.0 cm × 15.0 cm × 12.5 cm) for 60 min, which was used as theobservation chamber. The intrathecal administrations (i.t.) weremade in conscious mice at the L5 and L6 space in a volume at 5 Lusing the method described by Hylden and Wilcox [18]. The drugs
were administered i.t. with a 28-gauge needle attached to a 50-L
Hamilton syringe. The puncture of the dura was signaled by a slightﬂick of the tail. Immediately following the i.t. administration, eachmouse was returned to its cage and the accumulated time for the
nociceptive responses, including scratching, biting and licking, wasmeasured at 5 min intervals.

237

of 0.8 cm, with a 0.2-mm inner diameter, a 0.24-mm outer diameter and a 5-kDa molecular weight cut-off). The i.t. catheter was
made of a silicon-coated polyethylene tube, which was previously
tapered to the appropriate size by heating. Under anesthesia, themicrodialysis probe and the i.t. catheter were inserted simultaneously into the mouse spinal cord from L1 to L2, and the tip wasplaced at L5–L6. After implantation, the dialysis experiment was
performed under 1.0% isoﬂurane anesthesia. The inlet tube of theprobe was connected to a syringe pump (EP-800, EICOM, Kyoto,
Japan) and was perfused with artiﬁcial cerebrospinal ﬂuid (ACSF),
containing 126.6 mM NaCl, 2.5 mM KCl, 2.0 mM MgCl2 and 1.3 mM
CaCl2, at a ﬂow rate of 10 L/min, and the dialysate was sampledfrom the dialysis tube. After the initial 45 min washout period, twosamples were collected at 5-min intervals as a control, and the samples were collected at 5-min intervals after treatment with ACSF,
hemokinin-1 and the NMDA receptor antagonists with hemokinin1. Immediately after sample collection, glutamate was analyzed in
the dialysate by high-performance liquid chromatography (HPLC:
ECD-300, EICOM). The chromatogram was recorded for glutamate
on a personal computer using a Data Processor (EPC-500, EICOM).
2.4. Drugs
The following drugs were used: hemokinin-1 (Tocris Cookson, Inc., Bristol, UK); (5S,10R)-(+)-5-Methyl-10,11-dihydro-5Hdibenzo[a,d] cyclohepten-5,10-imine hydrogen maleate (MK-801)
and D(−)-2-Amino-5-phosphonopentanoic acid (D-APV) (SigmaAldrich, St. Louis, MO, USA). All of the drugs were dissolved in
ACSF. MK-801 and D-APV, the NMDA receptor antagonists, wereco-administered with hemokinin-1.
2.5. Analyses of data
The total time spent scratching, biting and licking for 5 min isexpressed as the mean ± S.E.M. The statistical signiﬁcance of thedifferences between the groups was assessed with one-way or twoway analysis of variance (ANOVA) followed by the Bonferroni’s test.
3. Results
3.1. Behavioral responses induced by the i.t. administration ofhemokinin-1
The i.t. administration of hemokinin-1 produced nociceptive
behaviors, consisting of scratching, biting and licking. The timecourse of the nociceptive responses after the administration ofhemokinin-1 peaked at 0–5 min and disappeared within 15 min
post-injection. The accumulated nociceptive response time for the
5 min observation following the administration of hemokinin-1
was 58.6 ± 3.7 s. In contrast, the i.t. administration of ACSF did nothave an effect (Fig. 1A).
3.2. Antinociceptive effect of the NMDA receptor antagonists
The nociceptive responses elicited by hemokinin-1 were dosedependently attenuated by the co-administration of MK-801(2.5–10.0 nmol) or D-APV (0.25–1.0 nmol). The ID50 values of MK801 and D-APV for the nociceptive responses were 6.10 and 0.72
nmol, respectively (Fig. 2A and B).
3.3. Spinal release of glutamate evoked by the i.t. administrationof hemokinin-1

2.3. Spinal cord microdialysis
Spinal cord microdialysis was conducted according to the modiﬁed method of Watanabe et al. [19]. The microdialysis loop-probe
consisted of 1.0-cm long microdialysis ﬁbers (active dialysis ﬁbers— 506 —The mean of the baseline samples collected before hemokinin-1
was used for the determination of glutamate in the dorsal spinal

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C. Watanabe et al. / Neuroscience Letters 617 (2016) 236–239

Fig. 1. The time-course of the nociceptive responses (A) and the spinal releaseof glutamate (B) following the i.t. administration of hemokinin-1. The nociceptive
behaviors induced by hemokinin-1 or ACSF are presented as a time-course of thenociceptive response time at 5 min intervals. The concentration of glutamate in the
spinal microdialysate is expressed as the percentage change from baseline. Eachvalue represents the mean ± S.E.M. for 10 or 6 mice. ***P < 0.001 compared with
ACSF-treated control group.

cord extracellular ﬂuid. The i.t. administration of hemokinin-1 produced over a 1.5-fold increase of glutamate during the ﬁrst 5 mincompared to ACSF injection. However, the glutamate level during the time interval of 5–15 min was not signiﬁcantly increased(Fig. 1B).

Fig. 2. Effect of MK-801 (A) and D-APV (B) on the behavioral responses induced by
the i.t. administration of hemokinin-1. The duration of the nociceptive behaviors
induced by hemokinin-1 was measured over a 5-min period starting immediately
after administration. MK-801 or D-APV were co-administered i.t. with hemokinin-1.
The data represents the mean ± S.E.M. for 10 mice. ***P < 0.001, **P < 0.01, comparedwith the hemokinin-1 alone-treated group.

3.4. Effect of the NMDA receptor antagonists on the spinal releaseof glutamate
The NMDA receptor antagonists MK-801 (10.0 nmol) and DAPV (1.0 nmol), which completely blocked hemokinin-1-induced
the nociceptive responses, when co-administered with the i.t.
hemokinin-1, revived the hemokinin-1-evoked increase of glutamate to the control level during the ﬁrst 5 min (Fig. 3).
4. Discussion
In a previous report, we demonstrated that the intrathecal administration of hemokinin-1 (0.0125–1.6 nmol) dosedependently produced pain-related responses, which were very
similar to substance P-induced pain behaviors. The nociceptive
behaviors, after administration of hemokinin-1, peaked at 0–5 min
and disappeared within 15 min post-injection. Interestingly,
the nociceptive behaviors evoked by a low-dose (0.0125 nmol)of hemokinin-1 were dose-dependently inhibited by the i.t.
co-administration of CP-99,994, a non-peptidic NK1 receptor
antagonist, whereas at a high-dose (0.1 nmol), hemokinin-1induced behaviors were not affected [9]. In the present study,
we examined the effects of the NMDA receptor antagonists on
the spinally mediated nociceptive behaviors induced by the i.t.
administration of high-dose hemokinin-1. Hemokinin-1-induced
scratching, biting and licking were dose-dependently inhibited

Fig. 3. Effect of MK-801 and D-APV on the increased spinal release of glutamate evoked by the i.t. administration of hemokinin-1. MK-801 or D-APV wereco-administered i.t. with hemokinin-1. The concentration of glutamate in the
spinal microdialysate is expressed as the percentage change from baseline after
hemokinin-1. Each value represents the mean ± S.E.M. for 6 mice. ***P < 0.001 compared with the hemokinin-1 alone-treated group.

by the co-administration of MK-801 or D-APV, which are the
NMDA receptor antagonists. These results suggest that hemokinin1-induced nociceptive behaviors are mediated through the NMDA
receptor. In addition, we measured the release of glutamate using a
spinal microdialysis evoked by the i.t. administration of hemokinin1. As the results indicate, glutamate in the spinal cord wasmarkedly increased in the ﬁrst 5 min after the i.t. administration ofhemokinin-1. The increased release of glutamate in spinal dialysate
completely corresponded with the appearance of the nociceptive— 507 —

C. Watanabe et al. / Neuroscience Letters 617 (2016) 236–239

responses. Additionally, the co-administration of MK-801 or D-APV
completely blocked the increase of glutamate by the i.t. administration of hemokinin-1.
Hemokinin-1 is the newest member of the tachykinin family andwas identiﬁed in mouse bone marrow in 2000 [20]. The sequenceof hemokinin-1 closely resembles substance P, and pharmacological studies demonstrate that substance P and hemokinin-1 are thepreferred ligands of the NK1 receptor. Moreover, hemokinin-1 may
also have functions that are similar to those of substance P at thelevel of the spinal cord [4–6]. On the other hand, several reports, aswell as our previous report, suggested that the effects of substance
P and hemokinin-1 are slightly different, and the existence of an
undeﬁned hemokinin-1 receptor that is not NK1 receptor has been
proposed [8,10]. In the present study, we ﬁrst demonstrated that
hemokinin-1-induced nociceptive behaviors are mediated by therelease of glutamate. In other words, hemokinin-1-induced nociceptive behaviors are unrelated to the NK1 receptor.
Glutamate is the predominant excitatory transmitter in the
spinal dorsal horn released from nociceptive primary afferents.
The glutamate receptors, both ionotropic and metabotropic, areexpressed on the presynaptic and postsynaptic terminals. Althoughreleasing glutamate from nociceptive primary afferents activatesthe postsynaptic glutamate receptors on the spinal dorsal horn,
the presynaptic glutamate receptors in the primary afferent ﬁbers
modulate neurotransmitter release. The expression of presynaptic glutamate receptors has been demonstrated in interneurons,particularly on the GABAergic terminus or glycinergic terminus[14,15,21]. In this study, we directly measured the release of glutamate in the spinal dorsal horn evoked by the i.t. administrationof hemokinin-1. Interestingly, a signiﬁcant increase of glutamatein the ﬁrst 5 min after administration of hemokinin-1 was completely suppressed by the NMDA receptor antagonists as well as in
the behavioral study. The present behavioral and microdialysis data
indicate that glutamate in the primary afferent ﬁbers is released byhemokinin-1 and activates the postsynaptic glutamate receptor.
However, it has not yet been elucidated how hemokinin-1 releases
glutamate via the activation of the NMDA receptor in this result.
In conclusion, the present study demonstrated that the nociceptive responses induced by the i.t. administration of hemokinin-1,
especially a high-dose of hemokinin-1, may be mediated by therelease of glutamate in the spinal dorsal horn. These ﬁndings suggest that hemokinin-1 may act an important mediator in spinal pain
pathways.
Acknowledgements
This study was supported by JSPS KAKENHI Grant Numbers
23590718 and the MEXT (Ministry of Education, Culture, Sports,
Science and Technology)-Supported Program for the Strategic
Research Foundation at Private Universities (2015–2019).

239

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Contents lists available at ScienceDirect

Allergology International
journal homepage: http://www.elsevier.com/locate/alit

Original article

The involvement of central nervous system histamine receptors in
psychological stress-induced exacerbation of allergic airway
inﬂammation in mice
Tomomitsu Miyasaka a, *, Kaori Okuyama-Dobashi a, c, Chiaki Masuda a, Shunya Iwami a,
Miki Sato a, Hirokazu Mizoguchi b, Tasuku Kawano a, Yuichi Ohkawara a,
Shinobu Sakurada b, Motoaki Takayanagi a, Isao Ohno aabDepartment of Pathophysiology, Tohoku Pharmaceutical University, Sendai, Japan
Department of Physiology and Anatomy, Tohoku Pharmaceutical University, Sendai, Japana r t i c l e i n f o

a b s t r a c t

Article history:
Received 29 January 2016
Received in revised form
17 May 2016
Accepted 25 May 2016
Available online 11 August 2016

Background: Psychological stress is one of the major risk factors for asthma exacerbation. Although
histamine in the brain acts as an excitatory and inhibitory neurotransmitter associated with psychological stress, the contribution of brain histamine to psychological stress-induced exacerbation of asthma
remains unclear. The objective of this study was to investigate the role of histamine receptors in the CNS
on stress induced asthma aggravation.
Methods: We monitored the numbers of inﬂammatory cells and interleukin (IL)-13 levels in bronchoalveolar lavage ﬂuid, airway responsiveness to inhaled methacholine, mucus secretion in airway
epithelial cells, and antigen-speciﬁc IgE contents in sera in a murine model of stress-induced asthma
treated with epinastine (an H1R antagonist), thioperamide (an H3/4R antagonist), or solvent.
Results: All indicators of stress-induced asthma exacerbation were signiﬁcantly reduced in stressed micetreated with epinastine compared with those treated with solvent, whereas treatment with thioperamide did not reduce the numbers of inﬂammatory cells in the stressed mice.
Conclusions: These results suggest that H1R, but not H3/4R, may be involved in stress-induced asthma
exacerbations in the central nervous system.
Copyright © 2016, Japanese Society of Allergology. Production and hosting by Elsevier B.V. This is an open access

Keywords:
Bronchial asthma
Epinastine
Histamine receptors
Psychological stressThioperamide
Abbreviations:IL, interleukin; HR, histamine receptor;
Th2, type 2 T-helper; Th1, type 1 T-helper;
FEV1, forced expiratory volume in 1 s; TNFa, tumor necrosis factor-alpha; CNS, central
nervous system; OVA, ovalbumin;
RS, restraint stress; FSS, forced swimming
stress; PBS, phosphate-buffered saline;
ELISA, enzyme-linked immunosorbent
assay; BSA, bovine serum albumin;PAS, periodic acideSchiff; PC200, lung
resistance to 200% above baseline;
SEM, standard error of the mean;
HPA, hypothalamic-pituitary-adrenal;
CRH, corticotropin-releasing hormone

article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction* Corresponding author. Department of Pathophysiology, Tohoku Pharmaceutical
University, 4-4-1 Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, Japan.
E-mail address: t-miya13@tohoku-mpu.ac.jp (T. Miyasaka).
Peer review under responsibility of Japanese Society of Allergology.
cPresent address: Department of Microbiology, Division of Medicine,Interdisciplinary Graduate School of Medicine and Engineering (Faculty of
Medicine), University of Yamanashi, Yamanashi, Japan.

Asthma is characterized by chronic airway inﬂammation in
response to various types of inherited and environmental factors,
which leads to wheezing, coughing, tightness in the chest, andshortness of breath. Increasing evidence has indicated that asthma
is not a disease but a syndrome with heterogeneous pathobiology,
clinical course, and therapeutic response to medicines. Therefore,
asthma phenotypes based on pathological and clinical features

http://dx.doi.org/10.1016/j.alit.2016.05.015
1323-8930/Copyright © 2016, Japanese Society of Allergology. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).

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T. Miyasaka et al. / Allergology International 65 (2016) S38eS44

such as types of airway inﬂammation and risk factors for exacerbations have been proposed to identify more effective asthma
therapies speciﬁc for each phenotype.1
In asthma pathophysiology, type 2 T-helper (Th2) cytokines,
such as interleukin (IL)-4, IL-5, and IL-13, play pivotal roles inregulating the behavior of inﬂammatory cells; inducing B cell isotype switching to produce IgE; and promoting the accumulation,
activation, and prolonged survival of eosinophils. These events
initiate and maintain the cardinal features of asthma, such asasthmatic airway inﬂammation, thereby accelerating airway
responsiveness and epithelial mucus secretion.2
Psychological and psychosocial stressors have been longrecognized as important universal risk factors for asthma exacerbations,3e9 which are accompanied by aggravation of airway
inﬂammation attributable to further skewing towards a Th2dominant cytokine proﬁle.10e14 The cognitive processes accompanying psychological stress are deeply connected to asthma exacerbations. Activation of the anterior cingulate cortex and insula bypsychological stimuli exhibited a greater decrease in forced expiratory volume in 1 s (FEV1), increased recruitment of eosinophils,
and diminished glucocorticoid inhibition of TNF-a production.15
The intensity of activation in the anterior insula in response toasthma-relevant psychological stimuli is correlated with theseverity of airway inﬂammation evoked by allergen inhalation.16
Stress-induced brain activation results in the production andrelease of stress neuropeptides, such as opioid peptides, substanceP, and histamine, which bind to membrane receptors in the central
nervous system (CNS).17,18 However, the molecular mechanism
linking psychological stress perceived in the CNS to exacerbationsof asthma pathophysiology in airways is not yet fully understood.
Histamine receptors (HRs) are a group of seven-transmembrane
G protein-coupled receptors classiﬁed to four major subtypes (H1R,
H2R, H3R, and H4R), which are expressed throughout the body
including in immune cells, gastric mucosal cells, and neurons.19
Histamine in the brain is produced by histaminergic neurons
comprising the tuberomammillary nucleus in the posterior hypothalamus. Projections from the tuberomammillary nucleus extend
to almost all areas in the brain and are implicated in basic homeostatic and higher brain functions such as arousal, cognition,
nociception, and responses to perceived stress.19e21
In the present study, we aimed to determine the role of HRs in
the CNS in psychological stress-induced asthma exacerbations using a murine model of stress asthma. We found that H1Rs in the CNS
were required for exacerbation of asthmatic airway responses
induced by the stress exposure. In contrast, H3/4Rs might be irrelevant in this process or function in the negative feedback control ofstressor signals in asthma exacerbation.
Methods

S39

Sensitization, antigen challenge, and stress exposure
A schematic of the experimental protocol is shown in Figure 1.
Six- to eight-week-old mice were sensitized and made to inhale
an aerosolized antigen as previously described.22 Brieﬂy, mice
were sensitized by intraperitoneal injections of 8 mg ovalbumin(OVA; Grade V, SigmaeAldrich, St. Louis, MO, USA) adsorbedwith aluminum hydroxide (Wako Pure Chemical Industries,
Osaka, Japan) on days 0 and 5. On days 17 and 24, the mice werechallenged with aerosolized OVA (0.5% in saline) for 1 h. Psychological stressors were applied on the following schedule to
avoid habituation to psychological stress: 6 h restraint per day
from day 17 to day 19, and 3 min forced swim per day from day
20 to day 23. For restraint stress (RS), each mouse was placed ina 50-mL conical centrifuge tube with multiple ventilation holes.
For forced swimming stress (FSS), the mice were placed in aplastic tank (19 cm in diameter, 27 cm in height) containing
15 cm water at 32  C. Non-stressed mice were deprived of foodand water, which has been used as a non-stress condition inother rodent experiments that investigated the effect ofstress.23,24 RS and FSS are category D procedures, as are othertypes of psychological stress, such as electric foot shock stress,
sound stress, and communication box-induced stress, that havebeen used to investigate the exacerbation of stress-induced
allergic airway inﬂammation by many groups including us.
Category D procedures are used in these studies because exposure to stress is required to induce asthma exacerbation, andalternative methods for reducing stress fail to exacerbate thecondition.
To test the effects of HR antagonists, 2 mL epinastine as a selective H1R antagonist (Tokyo Chemical Industry, Tokyo, Japan),25
ranitidine as a selective H2R antagonist (SigmaeAldrich, St. Louis,
MO, USA),25 or thioperamide as a selective H3/4R antagonist(Sigma-Aldrich)26,27 in calcium- and magnesium-free phosphatebuffered saline (PBS) was intracerebroventricularly administered to
selectively block HRs in the CNS 1 h before each stress exposure at
the dose indicated. Solvent was used as a negative control. Mice
were placed in a stereotactic device under anesthesia induced byinhalation of 2% isoﬂurane in oxygen (DS Pharma Animal Health,Tokyo, Japan). A 25G microinjection needle tip was aligned with thebregma and then inserted into the ventricle using the following
coordinates from bregma: 1.0 mm anterior and 1.0 mm lateral.
Intracerebroventricular injection has been used generally to
selectively administer agents to the CNS.22 In our preliminary experiments, the intracerebroventricularly injected solution was notdetected outside the CNS within 1 h for volumes below 5 mL. The
doses of HR antagonists used herein were based on those used inprevious studies.28,29 At the end of the experiments, the mice wereeuthanized by an overdose of pentobarbital (Somnopentyl; Kyoritsu Seiyaku, Tokyo, Japan).

Mice and ethical statement
Preparation of bronchoalveolar lavage (BAL) ﬂuids
Speciﬁc pathogen-free female C57BL/6 mice were purchasedfrom CLEA Japan (Osaka, Japan). All mice were kept under speciﬁc
pathogen-free conditions at the Institute for Animal Experimentation, Tohoku Pharmaceutical University, Sendai, Japan. Mice werehoused under a 12 h/12 h light/dark cycle at a constant temperature(22 ± 2  C). Sterilized food and water were available ad libitum. All
experimental procedures involving animals were approved by theCommittee of Animal Experiments at Tohoku Pharmaceutical
University (approval numbers: 13001-cn-a, 14002-cn, and 15001cn). We took the utmost care to alleviate any pain and sufferingon the part of the mice.

BAL samples were collected with 2  0.25 mL chilled PBS
through a cannula inserted in the trachea. Total cell numbers
recovered from BAL ﬂuid were counted with a hemocytometer.
After centrifugation of the BAL ﬂuids, supernatants were storedat 80  C for cytokine assay. For each sample, 2  105 cells were
centrifuged onto a glass slide using a Cytospin 4 (Thermo Scientiﬁc,Waltham, MA, USA) and stained with Diff-Quick solution (International Reagents, Kobe, Japan). The fractions of leukocytes wereanalyzed by counting a minimum of 200 cells under a lightmicroscope.

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T. Miyasaka et al. / Allergology International 65 (2016) S38eS44

Treatment with HR antagonist
RS05

Sensitization
OVA/Alum

FSS

17 18 19 20 21 22 23 24 25
1st challengewith OVA

2nd challengewith OVA

27
Sample collection

Measurement of
AHR
Fig. 1. Schematic of the experimental protocol.

Measurement of airway responsivenessResults

Lung resistance at the baseline and in response to aerosolizedsaline and acetyl-b-methylcholine (methacholine; SigmaeAldrich)were measured for 3 min under each condition.22 To measure
airway sensitivity, the concentration of acetyl-b-methylcholine
needed to increase lung resistance to 200% above baseline (PC200)
was calculated by interpolating the log concentration-lung resistance curve from individual mice.

Effects of HR antagonists on stress-induced exacerbations of airway
inﬂammation

Lung histology
Lungs were isolated from mice 3 days after the last OVA challenge, ﬁxed in 10% buffer formalin, dehydrated, and embedded inparafﬁn. Sections were cut at 4-mm-thick and stained with periodic
acideSchiff (PAS). Mucin production was estimated as the proportion of PAS-positive cells in the total airway epithelium of bronchioles in the PAS-stained section. The percentage of PAS-positive
cells was calculated in each of ﬁve random bronchioles in threelung sections from each mouse.
Measurement of OVA-speciﬁc IgE and cytokine
Serum samples were collected 3 days after the last OVA challenge. The quantities of OVA-speciﬁc IgE in serum were measured
by enzyme-linked immunosorbent assay (ELISA). Brieﬂy, microtiterplates (Nunc A/S, Roskilde, Denmark) were coated with 10 mg/mL
OVA in bicarbonate buffer for 1 h at 37  C and then blocked with 1%
bovine serum albumin (BSA) in PBS at 4  C overnight. Prior to
testing, serum samples were diluted with 1% BSA/PBS. Horseradish
peroxidase-conjugated goat anti-mouse IgE antibodies (Bethyl
Laboratories, Montgomery, TX, USA) diluted 1:2500 were used for
detection. IgE concentrations were determined from the absorbance at 450 nm using that at 650 nm for reference and calculated
based on a reference standard of pooled sera from sensitized female
mice assigned as 1000 experimental units (EU)/mL. The sensitivity
was 1.9  102 EU/mL for IgE. The concentrations of IL-13 in BAL
ﬂuids were measured using an ELISA kit (eBioscience, San Diego,
CA, USA) according to the manufacturer's instructions. The limit ofdetection was 1.5 pg/mL.
Statistical analysis
Statistical analysis was conducted using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA). Differences between
the two groups were tested using two-tailed analysis in an unpaired
Student's t-test. Differences among four or more groups were tested
using ANOVA with post hoc analysis (Tukey's multiple comparisontest). A p-value less than 0.05 was considered signiﬁcant. Data areexpressed as the mean ± standard error of the mean (SEM).

The numbers of total cells, eosinophils, and lymphocytes in BAL
ﬂuid were signiﬁcantly increased by the stress exposure but
showed a dose-dependent reduction with epinastine treatment(Fig. 2). Treatment with 3.0  109 mol epinastine signiﬁcantly
reduced the numbers of inﬂammatory cells in stressed mice to thelevels observed in non-stressed mice treated with the same dose of
epinastine and tended to reduce the numbers of total cells, eosinophils, and lymphocytes in BAL ﬂuids of non-stressed micecompared with solvent treatment, although the difference was notstatistically signiﬁcant (Fig. 2). Intracerebroventricular administration of 3.0  109 mol ranitidine signiﬁcantly reduced theincreased numbers of total cells, eosinophils, and lymphocytes in
BAL ﬂuids of stressed mice, whereas treatment with a higher dose
(3.0  108 mol) of ranitidine did not reduce the stress-induced
increases (Supplementary Fig. 1). The effects of ranitidine instressed mice were not dose-dependent but reverse bell-shaped.
The maximal suppressive effect was observed at 3.0  109 mol.
In contrast, the effects of ranitidine in non-stressed mice seemed to
be dose-dependent but were not statistically signiﬁcant(Supplementary Fig. 1). Treatment with thioperamide, an H3/4R
antagonist, did not signiﬁcantly reduce the stress-induced
increased numbers of inﬂammatory cells in BAL ﬂuid (Fig. 3),even at a higher dosage (4.9  108 mol; data not shown).
Effects of an H1R antagonist, epinastine, on stress-induced
exacerbations of asthmatic airway responses
Lung resistance increased dose-dependently in response toinhaled methacholine in all groups of mice. The lung resistanceto 200% above baseline (PC200) with solvent treatment tended
to be lower in stressed mice than in non-stressed mice,although the difference was not signiﬁcant (Fig. 4). In the
stressed mice, epinastine treatment signiﬁcantly increased thePC200 to a level similar to that in the non-stressed mice treatedwith epinastine. However, in the non-stressed mice, no significant difference in PC200 was observed between solvent andepinastine treatments.
Effects of epinastine on stress-induced exacerbations of airway
mucus secretion
The number of PAS-positive cells under solvent treatment wassigniﬁcantly higher in the stressed mice than in the non-stressed
mice (Fig. 5). In the stressed mice, treatment with epinastinesigniﬁcantly reduced the number of PAS-positive cells to the level— 516 —

T. Miyasaka et al. / Allergology International 65 (2016) S38eS44

S41

Fig. 2. Effects of epinastine on airway inﬂammation. Mice were sensitized and challenged with ovalbumin (OVA) in the absence (open bars) or presence (closed bars) of psychological stress. Solvent or 0.3  109, 1.0  109, or 3.0  109 mol epinastine was administered intracerebroventricularly before each stress exposure. The numbers of inﬂammatory cells in bronchoalveolar lavage ﬂuid were counted on day 3 after the last OVA challenge. Each group consisted of 7e17 mice from at least two independent experiments.
Data are shown as the mean ± SEM.*p < 0.05 compared with non-stressed mice treated with solvent using an unpaired Student's t-test. Differences among the data on the stressed
mice or non-stressed mice were tested using ANOVA with Tukey's multiple comparison test. #p < 0.05 compared with the stressed mice treated with solvent. NS, not signiﬁcantly
different.

Fig. 3. Effects of thioperamide on airway inﬂammation. Mice were sensitized and challenged with ovalbumin (OVA) in the absence (open bars) or presence (closed bars) of
psychological stress. Solvent or 0.01  109, 0.1  109, 0.3  109, 1.0  109, or 3.0  109 mol thioperamide was administered intracerebroventricularly before each stress
exposure. The numbers of inﬂammatory cells in bronchoalveolar lavage ﬂuid were counted on day 3 after the last OVA challenge. Each group consisted of three to four mice from
two independent experiments. Data are shown as the mean ± SEM. *p < 0.05 compared with the non-stressed mice treated with solvent using an unpaired Student's t-test.
Differences among the data on the stressed mice were tested using ANOVA with Tukey's multiple comparison test. NS, not signiﬁcantly different.

observed for non-stressed mice treated with epinastine. However,in the non-stressed mice, no signiﬁcant difference in cell number
observed between solvent and epinastine treatments.
Effects of epinastine on the stress-induced increase of IL-13 in BAL
ﬂuid
The IL-13 contents in BAL ﬂuids under solvent treatment weresigniﬁcantly higher in the stressed mice than in the non-stressed
mice (Fig. 6). In the stressed mice, treatment with epinastinesigniﬁcantly reduced the IL-13 contents to the levels measured fornon-stressed mice treated with epinastine. In the non-stressed

mice, there was no signiﬁcant difference in IL-13 contents between solvent and epinastine treatments.
Effects of epinastine on the stress-induced increase of OVA-speciﬁc
IgE in sera
OVA-speciﬁc IgE concentrations in sera under solvent treatment
tended to be higher in the stressed mice than in the non-stressed
mice, although the difference was not statistically signiﬁcant(Fig. 7). In the stressed mice, treatment with epinastine signiﬁcantly decreased IgE concentrations to the levels of those in the
non-stressed mice treated with epinastine. In the non-stressed— 517 —

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T. Miyasaka et al. / Allergology International 65 (2016) S38eS44

Fig. 4. Effects of epinastine on methacholine-induced airway hyperresponsiveness.
Mice were sensitized and challenged with ovalbumin (OVA) in the absence (open bars)
or presence (closed bars) of psychological stress. Solvent or 3.0  109 mol epinastine
was administered intracerebroventricularly before each stress exposure. Airway
sensitivity was measured 1 day after the last OVA challenge. Each group consisted ofsix to eight mice from two independent experiments. Data are shown as themean ± SEM. #p < 0.05 compared with the stressed mice treated with solvent using an
unpaired Student's t-test.

Fig. 6. Effects of epinastine on interleukin (IL)-13 contents in bronchoalveolar lavage(BAL) ﬂuid. Mice were sensitized and challenged with ovalbumin (OVA) in the absence(open bars) or presence (closed bars) of psychological stress. Solvent or 3.0  109 mol
epinastine was administered intracerebroventricularly before each stress exposure.
BAL samples were collected on day 3 after the last OVA challenge. The concentration of
IL-13 in BAL ﬂuids was measured by enzyme-linked immunosorbent assay. Each groupconsisted of 4e10 mice from two independent experiments. Data are shown as themean ± SEM. *p < 0.05 compared with the non-stressed mice treated with solvent;#p < 0.05 compared with the stressed mice treated with solvent using an unpaired
Student's t-test.

mice, epinastine treatment yielded no signiﬁcant difference in IgEconcentrations compared with solvent treatment.
Discussion
To our knowledge, this is the ﬁrst report demonstrating that HRs
in the CNS are involved in both the development and psychologicalstress-induced exacerbations of allergic asthma. Histamine is aneurotransmitter whose release is upregulated in response toperceived stress.19 Ito et al. showed that chronic RS increased histamine turnover in the nucleus accumbens and striatum in Fisher
rats.30 In addition, Gadek-Michalska et al. showed that intracerebroventricular administration of an H1R agonist in rats
increased the levels of corticosterone, one of the stress hormones
released in the neuroendocrine response to psychological stress.31
The increase of serum corticosterone after stress exposure waspreviously observed in this model of stress asthma.22 Therefore, it islikely that histamine was released upon stress exposure in thepresent study.
Epinastine seemed to increase the PC200 (Fig. 4), and reduce theeosinophil counts (Fig. 2), the IL-13 contents in BAL ﬂuid (Fig. 6) andthe OVA-speciﬁc IgE levels in sera (Fig. 7) of non-stressed micecompared with solvent treatment, although the differences were

Fig. 7. Effect of epinastine on ovalbumin (OVA)-speciﬁc IgE production in serum. Mice
were sensitized and challenged with OVA in the absence (open bars) or presence(closed bars) of psychological stress. Solvent or 3.0  109 mol epinastine wasadministered intracerebroventricularly before each stress exposure. Serum samples
were collected on day 3 after the last OVA challenge. The concentrations of OVAspeciﬁc IgE in sera were measured by enzyme-linked immunosorbent assay. Eachgroup consisted of 8e12 mice from two independent experiments. Data are shown as
the mean ± SEM. #p < 0.05 compared with the stressed mice treated with solvent
using an unpaired Student's t-test.

Fig. 5. Effects of epinastine on airway mucus secretion. Mice were sensitized and challenged with ovalbumin (OVA) in the absence (open bars) or presence (closed bars) of
psychological stress. Solvent or 3.0  109 mol epinastine was administered intracerebroventricularly before each stress exposure. Lungs were isolated from mice 3 days after thelast OVA challenge. Lung sections were prepared and stained with periodic acideSchiff (PAS). (A) Representative images of lung sections from the non-stressed and stressed micetreated with solvent or epinastine (scale bar, 20 mm). (B) The percentage of PAS-positive cells was calculated in each of ﬁve random bronchioles in three lung sections from eachmouse. Each group consisted of 8e13 mice from two independent experiments. Data are shown as the mean ± SEM. *p < 0.05 compared with the non-stressed mice treated withsolvent; #p < 0.05 compared with the stressed mice treated with solvent using an unpaired Student's t-test.

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T. Miyasaka et al. / Allergology International 65 (2016) S38eS44

not statistically signiﬁcant. In addition, ranitidine tended to dosedependently reduce the eosinophil counts in non-stressed micecompared with solvent treatment. Previous studies demonstrated
that H1Rs mediated the excitatory CNS functions of brain histaminesuch as maintenance of wakefulness, acceleration of locomotor
activity, enhancement of learning and memory processes, andincreased pain perception, and H2Rs are thought to play similarroles.19,32 Therefore, histamine physiologically released in the CNS
might be partially involved in the development of allergic airway
responses through the activation of H1Rs and H2Rs, which could
explain the (non-signiﬁcant) epinastine- and ranitidine-mediateddecreases in the development of allergic asthma under stress-free
conditions. However, we cannot rule out the possibility that theprocedure for intracerebroventricular administration of solvent
could induce stress via HR activation in the non-stressed mice. In
our previous study investigating the effect of a glucocorticoid receptor antagonist using the same stress model,22 subcutaneousadministration of the solvent tended to aggravate allergic airway
responses in non-stressed mice compared to untreated nonstressed mice (unpublished data). Thus, epinastine and ranitidine
might attenuate the aggravation of asthma-related features causedby stress from handling in non-stressed mice.
Epinastine (3.0 � 10�9 mol) and ranitidine (3.0 � 10�9 mol)signiﬁcantly attenuated the stress-induced enhancement of asthmatic airway responses and Th2 immune responses to the levels of
non-stressed mice treated with the antagonists (Supplementary
Fig. 1, Fig. 2,4,5,6,7). These results suggest that the stress exposure exaggerated asthmatic airway responses by increasing histamine release, leading to the further activation of H1Rs and H2Rs in
the CNS.
On the other hand, the intracerebroventricular administration ofan H3/4R antagonist, thioperamide, exhibited no signiﬁcant effecton the airway inﬂammation at the same dose (Fig. 3) or at a higher
dose (4.9 � 10�8 mol; data not shown). However, within the group
receiving thioperamide administration, a bell-shaped enhancement effect was observed, and the maximal effect was observed at
0.3 � 10�9 mol. H3Rs act as inhibitory auto- and hetero-receptors in
the CNS, which inhibit the synthesis and release of histamine19,33
and a number of neurotransmitters, including acetylcholine,
dopamine, noradrenaline, and serotonin, in a pathway-dependent
manner, leading to suppression of convulsion, stress-induced
excitation, and feeding behavior.20,21 The structureeactivity relationships of H3R and H4R overlap as Gi/o protein-coupled receptors.34 Therefore, the activation H3Rs and H4Rs by the stress
exposure might play a suppressive role in stress-induced asthma
exacerbations.
Taken together, our ﬁndings and the physiological properties ofeach HR subtype suggest that the interaction of histamine with HR
subtypes playing distinctive roles in the CNS is associated with theseverity of allergic airway responses, particularly in the case ofpsychological stress-induced exacerbations. Further studies will be
required to identify the exact roles of each HR subtype in the CNS
and to elucidate how the stressor signal via HRs in the CNS modulates Th2 immune responses in asthmatic pathophysiology. The
present study presents a new insight into the global role of thehistamine-regulated CNS in allergic asthma, which might lead tothe development of more effective strategies for the managementof asthmatic patients with the stress-related asthma phenotype.
Acknowledgments
This work was supported in part by a Grant-in-Aid for ScientiﬁcResearch (No. 25461164) for IO and by the Matching Fund Subsidy
for Private Universities from the Ministry of Education, Culture,
Sports, Science, and Technology (No. S1511001L) for IO, TK, and TM.

S43

Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.alit.2016.05.015.
Conﬂict of interest
The authors have no conﬂict of interest to declare.
Authors' contributions
Conceived and designed the experiments: IO, TM; performed the experiments:
TM, KOD, CM, SI, MS, TK; analyzed the data: TM, IO, KOD; contributed reagents/
materials/analysis tools: IO, YO, HM, SS, MT; contributed to the writing of themanuscript: TM, IO, HM.

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World Journal of Vaccines, 2016, 6, 34-41
Published Online May 2016 in SciRes. http://www.scirp.org/journal/wjv
http://dx.doi.org/10.4236/wjv.2016.62005

Human CD4− CD8− Invariant Natural Killer T
Cells Promote IgG Secretion from B Cells
Stimulated by Cross-Linking of Their
Antigen Receptors
Tomomitsu Miyasaka1*, Yurie Watanabe1, Yukiko Akahori1*, Namiko Miyamura1,
Keiko Ishii1, Yuki Kinjo2, Yoshitsugu Miyazaki2, Tian-Yi Liu3,4, Yasushi Uemura3,5,Kazuyoshi Kawakami1#
1Department of Medical Microbiology, Mycology and Immunology, Tohoku University Graduate School of
Medicine, Sendai, Japan2
Department of Chemotherapy and Mycoses, National Institute of Infectious Diseases, Tokyo, Japan
3
Division of Immunology, Aichi Cancer Center Research Institute (ACCRI), Nagoya, Japan
4
Key Laboratory of Cancer Center, Chinese PLA General Hospital, Beijing, China
5
Division of Cancer Immunotherapy, Exploratory Oncology Research & Clinical Trial Center, National Cancer
Center (NCC), Kashiwa, Japan
Received 30 January 2016; accepted 9 May 2016; published 12 May 2016
Copyright © 2016 by authors and Scientific Research Publishing Inc.
This work is licensed under the Creative Commons Attribution International License (CC BY).
http://creativecommons.org/licenses/by/4.0/

Abstract
Immunoglobulin (Ig) M production can be induced by the interaction of thymus-independent
type-2 (TI-2) antigen (Ag) with B cell Ag receptors (BCRs) without the involvement of conventional
T cells; for IgG production through the same process, however, a second signal is required. Previous studies have reported that invariant natural killer T (iNKT) cells may be responsible for thesecond signal involved in IgG production. In the present study, we addressed whether human iNKT
cells could participate in the production of Ig against TI-2 Ag in vitro. Two major distinct subsets ofhuman iNKT cells, CD4+ CD8β− (CD4) and CD4− CD8β− [double negative (DN)] cells, were generated
from peripheral blood monocytes from a healthy volunteer. BCR engagement, triggered by anti-IgM antibody stimulation, examined here as a model of BCR engagement triggered by TI-2 Ag,
induced abundant IgM production by B cells. Both CD4 and DN iNKT cells reduced IgM production
and conversely enhanced IgG production in a dose-dependent manner. In addition, IgG production
by CD19+CD27− (naïve) and CD19+CD27+ (memory) B cells was predominantly promoted by DN*

Present address: Tomomitsu Miyasaka, Department of Pathophysiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan;
Yukiko Akahori, Department of Medical Science Technology, International University of Health and Welfare, Narita, Japan.
#
Corresponding author.

How to cite this paper: Miyasaka, T., Watanabe, Y., Akahori, Y., Miyamura, N., Ishii, K., Kinjo, Y., Miyazaki, Y., Liu, T.-Y.,−−
Uemura, Y. and Kawakami, K. (2016) Human CD4 CD8 Invariant Natural Killer T Cells Promote IgG Secretion from B Cells
Stimulated by Cross-Linking of Their Antigen Receptors. World Journal of Vaccines, 6, 34-41.
http://dx.doi.org/10.4236/wjv.2016.62005— 521 —

T. Miyasaka et al.

iNKT cells rather than CD4 iNKT cells; nevertheless, IgM production by both B cell subsets was similarly reduced by either subset of iNKT cells. These results suggest that the DN iNKT subsets may
preferentially promote Ig class switching by B cells upon stimulation with TI-2 Ag.

Keywords
Invariant Natural Killer T Cells, TI-2 Antigen, B Cells, IgM, IgG

1. Introduction
The main causative bacteria of invasive infection, including Streptococcus pneumoniae and Haemophilus influenzae, possess thick polysaccharide capsules which confer the ability to resist phagocytosis by polymorphonuclear leukocytes [1]. Immunoglobulins specific for these polysaccharide capsules enhances opsonophagocytic
killing (OPK) activity, which plays an important role in host protection against infections caused by these encapsulated bacteria [2] [3]. Recently, we reported that immunization with pneumococcal polysaccharide vaccine
led to an increase in the serum level of serotype 3-specific IgG3, which facilitates survival after pneumococcal
infection in mice [4]. Immunization with pneumococcal polysaccharide vaccine also generates polysaccharide-specific IgG response in humans [5] [6].
Polysaccharide capsule, a thymus-independent type 2 (TI-2) antigen (Ag), has highly repetitive structureswith simultaneous cross-linking of B cell receptors (BCRs) and induces B cell proliferation and IgM production[7]. The antibody response induced by TI-2 Ag is smaller than that induced by thymus-dependent (TD) Ag, andconsists largely of the production of low-affinity IgM by B cells without the conventionally necessary T cell involvement. In addition, however, IFN-γ induces T cell-independent IgG production in response to TI-2 Ag [8],as this cytokine triggers the secondary stimulatory signals for T cell-independent B cell activation and isotype
switching to produce IgG [9] [10].
Human invariant natural killer T (iNKT) cells express only two αβ T cell Ag receptors, namely, Vα24-Jα18
and Vβ11, and have been identified as a unique lymphocyte population playing a critical role in both innate andadaptive immune responses [11] [12]. Although Vα24+Vβ11+ iNKT cells are present only in very small proportions (<0.01% - 1%) in human blood [13], these cells recognize glycolipids from bacteria and/or self in context
with CD1d, a nonpolymorphic MHC class I-like molecule, which leads to the production of large quantities ofcytokines such as IFN-γ, IL-4, IL-10 and IL-17A [14]. Human Vα24+ iNKT cells comprise two distinct major
subpopulations, one expressing CD4+CD8β− (CD4) and the other CD4−CD8− [double negative (DN)] [13].
These two subsets of iNKT cells differ in terms of the cytokines they produce to regulate various immune responses [15]. Mice lacking iNKT cells exhibit defective IgG response to pneumococcal polysaccharide Ags, intact
response to TD Ags [16] and impaired host defense against pneumococcal infection [17]. These previous observations suggest that iNKT cells may secrete the IFN-γ that triggers isotype switching in TI-2-induced IgG production.
In the present study, we examined the in-vitro effect of human iNKT cells on Ig production by human B cells
stimulated via cross-linking of their Ag receptors, which mimics BCR engagement by TI-2 Ags. We found thatco-culture with iNKT cells reduced IgM production but increased IgG production by B cells stimulated via
cross-linking of BCRs, and that this activity was higher in DN iNKT cells than in CD4 iNKT cells. These findings suggest that iNKT cells may contribute to the class-switching from IgM to IgG that occurs upon stimulation
with TI-2 Ags.

2. Materials and Methods
2.1. Ethical Statement
All experimental protocols described in this study were reviewed and approved by the Ethics Committee for
Human Experimentation at Tohoku University, Sendai, Japan (approval numbers: 2012-1-20, 2013-1-496).

2.2. Ab and Reagents
Alpha-galactosylceramide (α-GalCer; KRN7000) was purchased from Funakoshi (Tokyo, Japan). Recombinant

35— 522 —

T. Miyasaka et al.

human (rh) IL-2 was from PeproTech (Rocky Hill, NJ, USA). Polyclonal hIgM and hIgG were from Rockland
Immunochemicals (Limerick, PA, USA). Anti-human IgM, anti-human IgG, HRP-conjugated anti-human IgMand HRP-conjugated anti-human IgG for ELISA were from Acris Antibodies (San Diego, CA, USA). GoatF(ab’)2 anti-hIgM and rabbit F(ab’)2 anti-goat IgG for B cell stimulation were from Beckman Coulter (Fullerton,
CA, USA). Anti-Vα24Jα18 (Clone; 6B11), anti-Vβ11 (C21), isotype-matched control and 7-amino-actinomycin
D (7-AAD) were from BD Pharmingen (San Diego, CA, USA). Anti-CD4 (RPA-T4) and anti-CD8 (RPA-T8)
were from eBioscience (San Diego, CA, USA). Memory B cell isolation kit was from Miltenyi Biotec (Bergisch
Gladbach, Germany).

2.3. Generation of Vα24Jα18+ Invariant NKT Cells
Human iNKT cells were separated from peripheral blood mononuclear cells (PBMCs) obtained from peripheral
blood of healthy volunteers as described previously [18]. After 15 days of expansion, CD4+CD8− (CD4) andCD4−CD8− [double negative (DN)] iNKT subsets were sorted using a FACSAria cell sorter (Becton Dickinson,
San Diego, CA, USA). The CD4 and DN iNKT cells (2 × 106 cells/well) were stimulated with irradiated allogenic PBMC (1 × 107 cells/well) prepulsed for 5 h with α-GalCer (100 ng/ml) in RPMI 1640 medium supplemented with 10% human serum, 100 U/ml penicillin G, 100 μg/ml streptomycin, 2 mM L-glutamine, and 25
mM HEPES containing 20 U/ml rhIL-2. From day 3 to day 9, cells were split into two fractions once or twice
daily. The cultures were expanded by adding medium containing rhIL-2. On day 11 or 12, expanded cells werecollected and used as iNKT cells. The surface phenotypes of expanded iNKT cells were identified by flow cytometry (FACSCant II; BD Biosciences). The presence of dead cells was excluded by running parallel
7-AAD-stained samples. After one or two passages of each primary cell culture, the remaining cells were usedin the experiments.

2.4. Human Peripheral Blood B Cells
PBMCs were isolated from heparinized blood of one healthy adult volunteer by standard density gradient concentration over Ficoll-Paque PLUS (GE Healthcare Life Sciences, Piscataway, NJ, USA). Interface PBMCs
were pelleted, washed twice, and resuspended in MACS buffer (Miltenyi Biotec). The naïve (CD19+CD27−) andmemory phenotype (CD19+CD27+) B cells were isolated from PBMCs by a memory B cell isolation kit according to the manufacturer’s protocol.

2.5. NKT and B Cell Cultures
The B cells used in the current assays were derived from a single donor. Initially, CD27− and CD27+ B cells (2.5
× 104 cells/well) were stimulated with goat F(ab’)2 anti-human IgM (1 μg/ml) for 15 min on ice. The B cells
were washed three times with culture medium, and then co-cultured with rabbit F(ab’)2 anti-goat IgG (3 μg/ml)
in the presence or absence of CD4 or DN iNKT cells (2.5× 103 - 2.5× 104 cells/well) for five days. The culture
supernatants were stored at −80˚C until assayed for immunoglobulins by ELISA.

2.6. Measurement of Total IgG and IgM
The quantities of IgM and IgG in the culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Microtiter plates (Nunc A/S, Roskilde, Denmark) were coated with 250 ng/ml of anti-human IgM
or 192 ng/ml of anti-human IgG Ab in PBS for 1 h at 37˚C, and blocked with 1% FCS PBS at 4˚C overnight.
Prior to testing, samples were diluted with culture medium supplemented with 0.05% Tween 20. Next, serial
two-fold dilution of hIgM or hIgG to 1:1024 was performed arbitrarily; resulting solutions were added to thewells and incubated at room temperature for 2 h. HRP-conjugated anti-human IgM or IgG antibodies diluted
with 1:4000 were used as detection Ab. The concentrations of IgM and IgG were determined based on the absorbance at 450 nm. The detection limit was 0.2 ng/ml.

2.7. Statistical Analysis
Data are presented as mean values ± standard deviation (SD). Differences between the two groups were tested
using two-tail analysis in an unpaired Student’s t-test. Differences among three or more groups were tested using
ANOVA with post-hoc analysis (Student-Newman-Keuls test).

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3. Results
3.1. Human CD4+CD8− (CD4) and CD4−CD8− (DN) Vα24 iNKT Cells

To investigate the functional differences between the human iNKT subsets in terms of immunoglobulin production against TI-2 Ags, we initially expanded α-GalCer-specific CD4 and DN iNKT cells separated from healthy
individuals. More than 94% - 97% of the cultured iNKT cells, including both CD4 and DN subsets, expressed aninvariant TCR, consisting of Vα24Jα18 CDR3 loop and Vβ11. The expanded-CD4 iNKT cells were CD4+ CD8−
at 98% - 99% and Vα24Jα18+ CD4+ at 92%. By contrast, 98% - 99% of the DN iNKT cells were negative forexpression of CD4 and CD8, whereas 97% of these cells expressed Vα24Jα18 (data not shown).

3.2. iNKT Cell-Induced Enhancement of Immunoglobulin Production by B Cells upon
Stimulation with Antigen Receptor-Engagement
To investigate the effect of co-culture with iNKT cells on Ig production by B cells activated via cross-linking of
BCRs, B cells were stimulated with anti-IgM Ab in the presence or absence of CD4 or DN iNKT cells, and the
production of IgM and IgG in the culture supernatants was analyzed. As shown in Figure 1(a), B cells produced
large quantities of IgM under BCR cross-linking alone, whereas the addition of CD4 iNKT cells resulted in the
reduction of IgM production in a dose-dependent manner. A similar pattern was observed upon co-culture with
DN iNKT cells (Figure 1(c)). By contrast, IgG production by B cells was not clearly increased when activated
via BCR cross-linking alone (Figure 1(b) and Figure 1(d)). The synthesis of IgG by B cells stimulated via
cross-linking of BCRs was significantly enhanced by co-culture with CD4 and DN iNKT cells in a dose- dependent manner. In addition, this activity was much higher in DN iNKT cells than in CD4 iNKT cells (Figure1(b) and Figure 1(d)).

3.3. Role of iNKT Cells in Immunoglobulin Production by Naïve and Memory B Cells upon
Stimulation with Antigen Receptor-Engagement
IgM+CD27+ memory B cells in PBMCs play an important role in anti-pneumococcal polysaccharide IgG pro-

Figure 1. Effect of co-culture with iNKT cells on IgM and IgG production by B cells stimulated with anti-IgM Ab. B cells
were stimulated with anti-IgM Ab in the presence or absence of CD4 iNKT cells or DN iNKT cells for five days, and theconcentrations of Ig in the culture supernatants were measured. IgM (a) and IgG (b) production by B cells co-cultured with
CD4 iNKT cells; IgM (c) and IgG (d) production by B cells co-cultured with DN iNKT cells. Similar results were obtainedin two independent experiments. α-μ, anti-IgM Ab. **, p < 0.01.

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duction in humans [19]. Therefore, to address the effect of co-culture with iNKT cells on Ig production by naïve
(CD27−) and memory (CD27+) B cells, we separated B cells into two subsets according to the expression of
CD27 and stimulated each subset with anti-IgM Ab in the presence or absence of CD4 or DN iNKT cells. As
shown in Figure 2(a) and Figure 2(c), both CD27+ and CD27− B cells produced similar levels of IgM upon
BCR cross-linking, and IgM production by both subsets was significantly reduced when co-cultured with either
CD4 or DN iNKT cells. The cross-linking of BCRs induced low levels of IgG production by naïve and memory
B cells in the absence of iNKT cells, and IgG production by CD27+ B cells was significantly higher than that by
CD27− B cells. In addition, IgG production by naïve and memory B cells was significantly enhanced when they
were co-cultured with either CD4 iNKT cells or DN iNKT cells; this enhancement effect was much stronger
with DN iNKT cells than with CD4 iNKT cells (Figure 2(b) and Figure 2(d)).

4. Discussion
In the present study, we evaluated the effect of co-culture with iNKT cells on Ig production by B cells upon stimulation via crosslinking of BCRs. Our data demonstrated that CD4 iNKT cells and DN iNKT cells accelerated
the isotype switching from IgM to IgG, as shown by decreased IgM and increased IgG, in both naïve and memory B cells. DN iNKT cells accelerated this response even further than CD4 iNKT cells did. These results suggest that activation of iNKT cells may serve as a potent adjuvant, eliciting TI-2 Ag-induced IgG production in
the development of more effective vaccination strategies for prevention of the infectious diseases caused by encapsulated bacteria.
Kobrynski and coworkers [16] demonstrated that TI-2 Ag-specific IgG production was completely abrogatedin CD1d- or β2-microglobulin-deficient mice, suggesting that NKT cells may potentially promote IgG production in response to TI-2 Ags. In addition, we previously reported that activation of iNKT cells by α-GalCer increased IFN-γ-producing NKT cells, and that this increase was correlated with enhanced production of the poly-

Figure 2. Effect of co-culture of iNKT cells on IgM and IgG production by naïve and memory B cells stimulated with anti-IgM Ab. CD27+ or CD27− B cells were stimulated with anti-IgM Ab in the presence or absence of CD4 iNKT cells or DN
iNKT cells for five days, and the concentrations of Ig in the culture supernatants were measured. IgM (a) and IgG (b) production by B cells co-cultured with CD4 iNKT cells; IgM (c) and IgG (d) production by B cells co-cultured with DN iNKT
cells. Similar results were obtained in two independent experiments. α-μ, anti-IgM Ab. *, p < 0.05; **, p < 0.01; NS, not significant.

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saccharide-specific IgG3 after immunization with pneumococcal polysaccharide vaccine in mice [4]. In ourclinical study, immunization with anti-pneumococcal polysaccharide vaccine led to the production of serotype-specific IgG, which was correlated with an increase in DN iNKT cells in peripheral blood [5]. Thus iNKT
cells are suggested to play an important role in the production of serotype-specific IgG after immunization with
TI-2 Ags.
Co-culture with iNKT cells accelerated the production of IgG by B cells upon stimulation with BCR
cross-linking without any exogenously added agonists of iNKT cells. In addition, α-GalCer did not induce any
further increase in IgG production promoted by iNKT cells alone (data not shown). In earlier studies by Galli etal. [20], iNKT cell-induced promotion of B cell activation was demonstrated to depend on CD1d expressed on avariety of B cell subsets and to be delivered in the absence of α-GalCer [21]. These findings suggest that iNKT
cells may recognize some endogenous ligand presented in context with CD1d on B cells, although the responsible molecule remains to be identified.
IgG production by B cells was more dramatically accelerated by DN iNKT cells than by CD4 iNKT cells.
While CD4 iNKT cells have the potential to produce large amounts of Th2 cytokines such as IL-4 and IL-13,
DN iNKT cells have a Th1-biased profile, enabling increased IFN-γ production and prominent expression of NK
lineage receptors [15] [22]-[24]. In addition, chemokine receptors such as CCR6 and CXCR6 are preferentiallyexpressed on DN iNKT cells rather than CD4 iNKT cells [22] [25], though the latter are the more common type
among Th1 cells [26]. CD4 co-receptor potentiates the activation of human CD4 iNKT cells by engaging CD1d
molecules [24]. Previously, Galli and co-workers [20] demonstrated that, compared to DN iNKT cells, humanCD4 iNKT cells induced higher levels of IgM and IgG production in α-GalCer-pulsed B cells. In that study, B
cells were considered to receive activation signals during cognate interaction with activated iNKT cells without
BCR cross-linking. The current study differed from theirs in terms of the primary B cell activation method. Thus
the phenotypic and functional properties of iNKT cells may be associated with IgG production by B cells stimulated with TI-2 Ags, though further investigation is required to define the molecular mechanism mediating thefunctional difference between CD4 iNKT cells and DN iNKT cells in T cell-independent Ig production.
TI-2 Ags are reported to generate memory B cells, although they do not elicit the Ab booster response or thegerminal center formation following secondary immunization [27]. Moens and coworkers [19] reported that
CD19+CD27+ IgM+ B cells were predominantly associated with an anti-polysaccharide IgG response afterpneumococcal polysaccharide vaccination. In keeping with these previous observations, in the current study,
cross-linking of BCRs induced the production of IgG by CD27+ (memory type) B cells at a higher level than thatby CD27− (naïve) B cells in the absence of iNKT cells. Yet iNKT cells promoted IgG production not only bymemory B cells but also by naïve B cells. In a previous study by Bai and co-workers [28], iNKT cell activation
through cognate interaction with dendritic cells induced isotype switching by B cells and promoted long-term
memory response to pneumococcal capsular polysaccharides. In addition, CD4 iNKT cells and DN iNKT cells
are reported to promote the proliferation of naïve and memory B cells derived from peripheral blood in vitro[20]. Thus, iNKT cells might be involved in the potentiation of IgG production by naïve and memory B cells
upon stimulation with TI-2 Ags.
Maddur and coworkers demonstrated that B cells activated via BCR cross-linking enhanced the expression of
OX-40L and co-stimulatory molecules such as CD80, CD86 and CD40 on DCs [29] [30]. In our previous study,
DCs with increased expression of OX-40L caused NKT cells to produce substantial levels of IFN-γ [31]. Considered collectively, B cells activated by TI-2 Ags may amplify IFN-γ production by iNKT cells through the enhanced Th1 response induced by DCs in vivo.

5. Conclusion
In conclusion, we demonstrated that iNKT cells promoted the production of IgG by human CD27+ and CD27− Bcells upon stimulation via cross-linking of BCRs and that IgG production was more strongly promoted by DN
iNKT cells than by CD4 iNKT cells. The present study provides important implications for understanding thecontribution of iNKT cells to IgG production by TI-2 Ag-stimulated B cells, which is expected to be helpful inthe development of more effective vaccination strategies for prevention of pneumococcal infection.

Acknowledgements
This work was supported in part by Grants from the Ministry of Health, Labour and Welfare of Japan (22-39
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SHINKOU-IPPAN-014 to KK, H25-SHINKOU-WAKATE-005 to YK), a Grant from the Japan Agency for
Medical Research and Development, AMED (the Research Program on Emerging and Re-emerging Infectious
Diseases), and Aid Funding from the Takeda Science Foundation to YK.

Conflicts of Interest
The authors have no financial conflicts of interest.

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