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Patent US7247752 - Methods for the synthesis of astaxanthin - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inPatentsA method used for synthesizing intermediates for use in the synthesis of carotenoids and carotenoid analogs, and/or carotenoid derivatives. In some embodiments, the invention includes methods for synthesizing optically active intermediates useful for the synthesis of optically active carotenoids. Synthesis...http://www.google.com/patents/US7247752?utm_source=gb-gplus-sharePatent US7247752 - Methods for the synthesis of astaxanthinAdvanced Patent SearchPublication numberUS7247752 B2Publication typeGrantApplication numberUS 11/242,643Publication dateJul 24, 2007Filing dateOct 3, 2005Priority dateOct 1, 2004Fee statusPaidAlso published asUS20060088904, US20060088905, US20060111580, US20060155150, US20060167319, US20060178538, US20060183185, US20060183947, WO2006039685A2, WO2006039685A3Publication number11242643, 242643, US 7247752 B2, US 7247752B2, US-B2-7247752, US7247752 B2, US7247752B2InventorsSamuel F. Lockwood, Peng Cho Tang, Geoff Nadolski, Henry L. Jackson, Zhiqiang Fang, Yishu Du, Min Yang, William Geiss, Richard Williams, David BurdickOriginal AssigneeCardax Pharmaceuticals, Inc.Export CitationBiBTeX, EndNote, RefManPatent Citations (84), Non-Patent Citations (71), Referenced by (3), Classifications (54), Legal Events (9) External Links: USPTO, USPTO Assignment, EspacenetMethods for the synthesis of astaxanthin
US 7247752 B2Abstract
A method used for synthesizing intermediates for use in the synthesis of carotenoids and carotenoid analogs, and/or carotenoid derivatives. In some embodiments, the invention includes methods for synthesizing optically active intermediates useful for the synthesis of optically active carotenoids. Synthesis of optically active carotenoids, in one embodiment, may be accomplished by forming an optically active dihydroxy intermediate from ketoisopherone. The optically active dihydroxy intermediate may be converted into optically active astaxanthin derivatives.
This application claims priority to U.S. Provisional Patent Application No. 60/615,032 entitled “Methods for Synthesis of Carotenoids, Including Analogs, Derivatives, and Synthetic and Biological Intermediates” filed on Oct. 1, 2004; U.S. Provisional Patent Application No. 60/675,957 entitled “Methods for Synthesis of Carotenoids, Including Analogs, Derivatives, and Synthetic and Biological Intermediates” filed on Apr. 29, 2005; U.S. Provisional Patent Application No. 60/691,518 entitled “Methods for Synthesis of Carotenoids, Including Analogs, Derivatives, and Synthetic and Biological Intermediates” filed on Jun. 17, 2005; U.S. Provisional Patent Application No. 60/692,682 entitled “Methods for Synthesis of Carotenoids, Including Analogs, Derivatives, and Synthetic and Biological Intermediates” filed on Jun. 21, 2005; U.S. Provisional Patent Application No. 60/699,653 entitled “Methods for Synthesis of Carotenoids, Including Analogs, Derivatives, and Synthetic and Biological Intermediates” filed on Jul. 15, 2005; U.S. Provisional Patent Application No. 60/702,380 entitled “Methods for Synthesis of Carotenoids, Including Analogs, Derivatives, and Synthetic and Biological Intermediates” filed on Jul. 26, 2005; and U.S. Provisional Patent Application No. 60/712,350 entitled “Methods for Synthesis of Carotenoids, Including Analogs, Derivatives, and Synthetic and Biological Intermediates” filed on Aug. 30, 2005.
Lipid soluble in natural form; may be modified to become more water soluble; Molecular weight of 597 Daltons (size <600 daltons (Da) readily crosses the blood brain barrier, or BBB); Long polyene chain characteristic of carotenoids effective in singlet oxygen quenching and lipid peroxidation chain breaking; and No pro-vitamin A activity in mammals (eliminating concerns of hypervitaminosis A and retinoid toxicity in humans). The administration of antioxidants which are potent singlet oxygen quenchers and direct radical scavengers, particularly of superoxide anion, should limit hepatic fibrosis and the progression to cirrhosis by affecting the activation of hepatic stellate cells early in the fibrogenetic pathway. Reduction in the level of “Reactive Oxygen Species” (ROS) by the administration of a potent antioxidant can therefore be crucial in the prevention of the activation of both “hepatic stellate cells” (HSC) and Kupffer cells. This protective antioxidant effect appears to be spread across the range of potential therapeutic antioxidants, including water-soluble (e.g., vitamin C, glutathione, resveratrol) and lipophilic (e.g., vitamin E, β-carotene, astaxanthin) agents. Therefore, a co-antioxidant derivative strategy in which water-soluble and lipophilic agents are combined synthetically is a particularly useful embodiment. Examples of uses of carotenoid derivatives and analogs are illustrated in U.S. patent application Ser. No. 10/793,671 filed on Mar. 4, 2004, entitled “CAROTENOID ETHER ANALOGS OR DERIVATIVES FOR THE INHIBITION AND AMELIORATION OF DISEASE” to Lockwood et al. published on Jan. 13, 2005, as Publication No. US-2005–0009758 and PCT International Application Number PCT/US2003/023706 filed on Jul. 29, 2003, entitled “STRUCTURAL CAROTENOID ANALOGS FOR THE INHIBITION AND AMELIORATION OF DISEASE” to Lockwood et al. (International Publication Number WO 2004/011423 A2, published on Feb. 5, 2004) both of which are incorporated by reference as if fully set forth herein.
M+—C≡C—R2, where M is a metal and R2 is to give an addition product having the structure:
Specific and preferred values listed below for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents. Specifically, “alkyl” includes, but is not limited to: methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl or pentadecyl; “alkenyl” includes but is not limited to vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl; 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl, 11-dodecenyl, 1-tridecenyl, 2-tridecenyl, 3-tridecenyl, 4-tridecenyl, 5-tridecenyl, 6-tridecenyl, 7-tridecenyl, 8-tridecenyl, 9-tridecenyl, 10-tridecenyl, 11-tridecenyl, 12-tridecenyl, 1-tetradecenyl, 2-tetradecenyl, 3-tetradecenyl, 4-tetradecenyl, 5-tetradecenyl, 6-tetradecenyl, 7-tetradecenyl, 8-tetradecenyl, 9-tetradecenyl, 10-tetradecenyl, 11-tetradecenyl, 12-tetradecenyl, 13-tetradecenyl, 1-pentadecenyl, 2-pentadecenyl, 3-pentadecenyl, 4-pentadecenyl, 5-pentadecenyl, 6-pentadecenyl, 7-pentadecenyl, 8-pentadecenyl, 9-pentadecenyl, 10-pentadecenyl, 1-pentadecenyl, 12-pentadecenyl, 13-pentadecenyl, or 14-pentadecenyl. “Alkoxy” includes but is not limited to methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, hexoxy, heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy, tridecyloxy, tetradecyloxy, or pentadecyloxy. “Cycloalkyl” includes, but is not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl. “Aryl” includes but is not limited to phenyl, substituted benzene, naphthyl, substituted naphthyl, anthracene, or substituted anthracene.
X, Y, and Z may be independently H, —OH or ═O.
X, Y, and Z may be independently H, —OH or ═O. R3 may be PR4 3, SO2R4, or M30 . R4 may be alkyl, phenyl, or aryl. M is Li, Na, or MgBr. Coupling of two “head units” with the C10-aldehyde yields a carotenoid. Coupling may be accomplished using a Wittig coupling (R3 is PR4 3), sulphone coupling (R3 is SO2R4), or condensation reaction (R3 is M+). The C10 aldehyde is commercially available. Described herein are various methods of synthesizing the appropriate headpiece. The following U.S. patents, all of which are incorporated herein by reference, describe the synthesis of various carotene and carotenoid synthesis intermediates: U.S. Pat. No. 4,245,109 to Mayer et al., U.S. Pat. No. 4,283,559 to Broger et al, U.S. Pat. No. 4,585,885 to Bernhard et al., U.S. Pat. No. 4,952,716 to Lukac et al., and U.S. Pat. No. 6,747,177 to Ernst et al.
In some embodiments a reduction catalyst may be a chiral catalyst. A “chiral catalyst” as defined herein is a catalyst that includes a single stereoisomer of a chiral molecule. In one embodiment, a chiral catalyst includes a transition metal and an optically active chiral ligand. Transition metals that may be used to form a chiral catalyst for reduction of ketones include Ti, Zr, Hf, V, Nb, Ta, Cr, Mo, W, Mn, Re, Fe, Ru, Os, Co, Rh, Ir, Ni, Pd, Pt, Cu, Ag, and Au. In some embodiments, a ruthenium chiral catalyst may be used to effect a stereoselective reduction of keto-α-isopherone. The ruthenium chiral catalyst may be formed from a mixture of [RuX2(η6-Ar)]2 with an optically active amine, where X represents a halogen (e.g., F, Cl, Br, I) and Ar represents benzene or a substituted benzene (e.g., alkyl substituted benzene). In some embodiments, the optically active amine includes both (S)- and (R)-amino acids, and other optically active amines such as as H2N-CHPh-CHPh-OH, H2N-CHMe-CHPh-OH, MeHN-CHMe-CHPh-OH. Reduction of keto-α-isopherone with a chiral catalyst may yield the optically active hydroxy ketone 116. While hydroxy ketone 116 is depicted in the (R)- form, it should be understood that the (S)- form may be formed by using the opposite optically active compound to form a chiral catalyst. For example, forming a ruthenium catalyst using (1R, 2S)-(−)-norephedrine leads to the (R)-form of the hydroxy ketone depicted below, while forming a ruthenium catalyst using (1S, 2R)-(+)-norephedrine leads to the (S)-form of the hydroxy ketone below. Further details regarding the use of ruthenium catalyst for the reduction of keto-α-isopherone may be found in the paper “Synthesis of (R)- and (S)-hydroxyisophorone by ruthenium-catalyzed asymmetric transfer hydrogenation of ketoisopherone” by Hennig et al., Tetrahedron:Asymmetry, 11 (2000) 1849–1858, which is incorporated herein by reference.
Addition of alkyne H—C≡C—R2 to ketone may be accomplished by forming a metal anion of the acetylene, to form the reactive nucleophilic acetylenic compound M+−C≡C—R2, where M+ may be, but is not limited to, Li, Na, MgBr, Cd, or Zn. A lithium salt of alkyne H—C≡C—R2 may be formed by reacting the alkyne with, for example, BuLi. Other metal salts of alkynes may be made using methods known to one of ordinary skill in the art. The nucleophilic acetylenic compound M+−C≡C—R2 may be reacted with ketone 108 to form a coupling product 112 as depicted below.
Examples of functionalities that may be reacted with an aldehyde include PR4 3, SO2R4, or M+ where R4 is alkyl, phenyl, or aryl and M is Li, Na, or MgBr. Coupling of two “head units” with a C10—aldehyde yields a carotenoid. Coupling may be accomplished using a Wittig coupling (R3 is PR4 3), sulphone coupling (R3 is SO2R4), or condensation reaction (R3 is M+). A phosphonium salt may be synthesized from compound 104. Phosphines and acid may be used to synthesize the phosphonium salt. Phosphines may have the general structure —PR5 3 or —CH2—P(═O)(OR5)2 where R5 is alkyl, phenyl, or aryl. Acids may include any of a number of acids known to one skilled in the art. One example of an acid which may be used is hydrogen bromide (“HBr”).
Enantiomeric excesses of over 55% may be achieved using compound 202. In some embodiments, regioselectivity and enatiomeric excess may vary with temperature, the B–H source, and/or the structure of the catalyst.
An epoxide of ketoisophorone may be prepared using reagents including, but not limited to, peroxides (e.g., hydrogen peroxide). There are many epoxidation reactions known to one skilled in the art, many of which include peroxides (e.g., m-ClC6H4CO3H). There are other epoxidation reagents described in references such as “Comprehensive Organic Transformations: A Guide to Functional Group Preparations” Larock, R. C. VCH Publishers, Inc. pages 456–461, which is incorporated herein by reference.
In some embodiments a reduction catalyst may be a chiral catalyst. In one embodiment, a chiral catalyst includes a transition metal and an optically active chiral ligand. Transition metals that may be used to form a chiral catalyst for reduction of ketones include Ti, Zr, Hf, V, Nb, Ta, Cr, Mo, W, Mn, Re, Fe, Ru, Os, Co, Rh, Ir, Ni, Pd, Pt, Cu, Ag, and Au. In some embodiments, a ruthenium chiral catalyst may be used to effect a stereoselective reduction of keto-α-isopherone. The ruthenium chiral catalyst may be formed from a mixture of [RuX2(η6-Ar)]2 with an optically active amine, where X represents a halogen (e.g., F, Cl, Br, I) and Ar represents benzene or a substituted benzene (e.g., alkyl substituted benzene). In some embodiments, the optically active amine includes both (S)- and (R)-amino acids, and other optically active amines such as as H2N—CHPh-CHPh-OH, H2N—CHMe-CHPh-OH, MeHN-CHMe-CHPh-OH, and TsNH-CHPh-CHPh-NH2.
In some embodiments, R3 may be OR5, OSiR5 3, H, alkyl, or aryl. R5 may be H, alkyl, or aryl. In some embodiments, R7 may include C—R3 or C═O.
Lycophyll was prepared by total synthesis at multiple gram scale for the current testing and derivatization to novel water-soluble, water-dispersible compounds. Isolation from natural sources demonstrates high cost, significant manpower, and generally low yields. Retrosynthetic analysis of the target xanthophyll revealed an efficient methodology utilizing at least some commercially available materials. In cases where commercial material was not available, these intermediates were synthesized in appropriate amounts. In some embodiments, commercially available materials may include geranyl acetate, a protected form of geraniol (C10), and/or crocetindialdehyde (C20). A method may include a total synthesis of acyclic carotenoids (e.g., lycophyll). In some embodiments, a synthesis of, for example, lycophyll may be realized in about 8 synthetic steps (Schemes III and IV). Synthetic steps may include an “endgame” double-Wittig olefination that successfully forms the target C40 scaffold while generating a mixture of geometric isomers (Scheme IV). The isomeric mixture may be deconvoluted to yield the target all-trans lycophyll. Deconvolution may include, but is not limited to, thermal or liquid chromatographic methods. The methodology shown in Schemes III and IV for synthesizing lycophyll may be used to synthesize other acyclic carotenoids, carotenoid derivatives, and carotenoid analogs.
In an embodiment, carotenoid derivatives may be synthesized from naturally-occurring carotenoids. The carotenoids may include structures 2A–2F depicted in FIG. 1. In some embodiments, the carotenoid derivatives may be synthesized from a naturally-occurring carotenoid including one or more alcohol substituents. In other embodiments, the carotenoid derivatives may be synthesized from a derivative of a naturally-occurring carotenoid including one or more alcohol substituents. The synthesis may result in a single stereoisomer. The synthesis may result in a single geometric isomer of the carotenoid derivative. The synthesis/synthetic sequence may include any prior purification or isolation steps carried out on the parent carotenoid. Synthesis of carotenoid derivatives can be found in U.S. Published Patent Application Nos. 2004–0162329 and 2005–0113372, both of which are incorporated herein by reference.
Preparation of (R)4-hydroxyisophorone (R)-116
All solvents were free of O2. And the reactions were done under N2. Benzeneruthenium (II) dimer (19.72 g, 39.42 mmol, 0.4 mol %) and (1R, 2S)-(−)-norephedrine (99%) (24.14 g, 159.67 mmol, 1.62 mol %) were dissolved in a 12 L three-necked flask containing 2-propanol (7.5 L). After stirring the red solution for 45 min at 80� C., the heat was removed. It was transferred to a 50 L three-necked flask containing 2-propanol (28 L). 109 (1500 g, 9.86 mol) and 0.1 M potassium hydroxide in 2-propanol (3945 ml, 0.0.395 mol, 4 mol %) were added. After 3 h (TLC showed the reaction was done), the red solution was filtered through a short silica gel pad and the filtrate was evaporated to dryness to obtain solids (about 1600 g). After five times recrystallization from iPr2O (500 ml�5), 912 g of (R)-116 was obtained. The yield: 60%. 1H NMR (CDCl3, 300 MHz): δ 1.02 (s, 3H), 1.07 (s, 3H), 1.97 (s, 1H), 2.04 (t, J=1.2 Hz, 3H), 2.21 (d, J=16.3 Hz, 1H), 2.39 (d, J=16.4 Hz, 1H), 4.03 (d, J=6.6 Hz, 1H), 5.86 (br s, 1H). 13C NMR (CDCl3, 75 MHz): δ 21.22, 21.46, 26.89, 38.48, 48.97, 76.89, 126.29, 160.81, 198.79. [α]D 23+105.34 (c=1.006, MeOH), literature [α]D 22+105.9 (c=1.00, MeOH).
Preparation of (1R,4S)-2,6,6-trimethyl-2-cyclohexen-1,4-diol (1R,4S)-118
To a solution of L-Selectride (5674 mL, 1 M in THF, 1.25 equiv), a solution of compound (R)-116 (700 g, 4.54 mol, 1 equiv) in THF (3000 mL) was added dropwise at −78� C. After stirring for 1.5 h, the mixture was sequentially treated with H2O (600 mL), 4N NaOH (1450 mL). After extractions with AcOEt (500 ml�5) and the combined organic phase was dried and concentrated. To the residue was charged 3000 mL of hexanes, then the mixture was filtered. The solid was washed with hexanes (200 mL�3). The solid crude product was purified by flash chromatography using Hexanes/AcOEt (3/1) as an eluent. 645 g of compound (1R, 4S)-118 was obtained (yield: 91%). Recrystallized from 1000 ml of EtOAc to obtain 504 g (70%) of (1R, 4S)-118. 1H NMR (CDCl3, 500 MHz): δ 0.86 (s, 3H), 1.02 (s, 3H), 1.45 (dd, J=12.8, 9.5 Hz, 1H), 1.67 (ddt, J=12.8, 6.3, 1.1 Hz, 1H), 1.84 (t, J=1.7 Hz, 3H), 3.34 (s, 1H), 4.18 (m, 1H), 5.54 (br s, 1H). [α]D 23+68.63 (c=1.6000, CHCl3), literature [α]D 24+67.4 (c=0.27, CHCl3).
Preparation of (1R,4S)4-tert-Butyidimethylsilyloxy-2,6,6-trimethyl-2-cyclohexen-1-ol (1R,4S)-120a
A mixture of enantiomerically pure (1R, 4S)-118 (1000 g, 6.40 mol), TBDMSCl (1194 g, 7.68 mol, 1.2 eq) and imidazole (566.37 g, 8.32 mol, 1.3 eq) in DMF (9 L) was stirred at room temperature for 1 hr and 20 min. Water (2 L) was added, aqueous phase was extracted with diethyl ether (2000 ml�3). The combined organic layer was dried over Na2SO4. After concentration, the crude product (1R, 4S)-120a was subjects to next step without further purification.
Preparation of (S)-4-tert-Butyidimethylsilyloxy-2-cyclohexenone (S)-108b
(1R, 4S)-120a (˜6.40 mol) was added to a mixture of PDC (3613 g, 1.5 eq) and DMF (8000 ml), which was cooled by ice-water. And then, the mixture was stirred for 1 h and 10 min at rt. Ether (8 L) was added. The mixture was passed through a pad of celite. Then solution was washed with water (3 L�2). The organic phase was dried over Na2SO4. 1718.2 g of(S)-108b (Yield: 100%, two steps from 118 to 108b) was obtained after column chromatography (hexanes/ethyl acetate, 50/1˜30/1). 1H NMR (CDCl3, 500 MHz): δ 0.12 (s, 3H), 0.13 (s, 3H), 0.92 (s, 9H), 1.11 (s, 3H), 1.14 (s, 3H), 1.78 (brs, 3H), 1.87 (dd, J=12.9, 9.8 Hz, 1H), 1.99 (ddd, J=12.9, 5.4, 1.8 Hz, 1H), 4.55 (m, 1H), 6.50 (br s, 1H).
Determine the ee Value of Compound (S)-108b
To a solution of compound 108b (40 g, 112 mmol) in THF (450 ml), nBu4NF (29.22 g, 112 mmol in 150 ml of THF was added. After 30 min, 200 ml of water and 500 ml of EtOAc was added. The organic phase was then washed with a half-saturated brine (400 ml�2) and brine (400 mL). It was dried and concentrated and subjected to column chromatography (hexane/ethyl acetate, 5/1) to give 20.25 g of (S)-108c (92%). [α]D 23−48.0 (c=1.98, EtOH), literature [α]D 20-46.7 (c=1.0, EtOH). The racemic 108c was separated using a chiral HPLC column; baseline separation was not achieved. Reverse phase HPLC column, Pirkle covalent, (S, S) Whelk-O 1, spherical silica; Eluent, 3:97 2-propanol:hexane, 1 ml/min. For racemic 108c, t1=14.07 min, t2=14.67 min. Only one peak was detected for (S)-108c using the same HPLC condition Ts(108c)=14.74 min.
Preparation of Compounds 112a
A mixture of alkyne 110a (689.3 g, 4.10 mol, 1.1 eq) and THF (13 L) was cooled to −78� C., BuLi (1640 mL, 2.5 M, 4.10 mL, 1.1 eq) was added dropwise. After 2 h, compound (S)-108b (1000 g, 3.725 mol) in 2 L of THF was added dropwise. In the 4 hrs, the temperature was allowed to raise from −78� C. to −25� C. NH4Cl saturated solution (500 mL) and brine (500 mL) was added and extracted with EtOAc (3000 mL�1). Dried. After concentration, the crude product 112a (˜1770 g) was subjected to next step without further purification.
Preparation of Compounds 114a
A solution of compounds 112a (˜3.75 mol) in DCM (2 L) was added dropwise to a mixture of PDC (2101.86 g, 5.58 mol, 1.5 eq), NaOAc (458.16 g, 5.58 mol, 1.5 eq), 4 Å MS (1000 g) and DCM (10 L). After 24 h, ethyl acetate (2000 ml) was added and it was subjected to a short silica gel pad and washed with ethyl acetate. After concentration, the crude product 114a (1710 g) was subjects to next step without further purification. 1H NMR (CDCl3, 300 MHz): δ 0.44 (s, 3H), 0.13 (s, 3H), 0.87 (s, 9H), 1.12 (td, J=6.9, 2.7 Hz, ˜3H), 1.21–1.32 (m, ˜10H), 1.59 (s, ˜1.5H), 1.62 (s, ˜1.5H), 1.83–2.2 (m, ˜4H), 3.32–3.70 (m, ˜2H), 4.29 (dd, J=11.1, 6.9 Hz, 1H), 4.87 (q, J=5.4 Hz, ˜0.5H), 4.95 (q, J=5.4 Hz, ˜0.5H), 5.18 (d, J=9.9 Hz, 1H), 5.49 (dd, J=17.4, 9.9 Hz, 1H), 5.83 (dd, J=17.1, 10.2 Hz, 0.5H), 5.95 (dd, J=17.1, 10.2 Hz, ˜0.5H). [α]D 26-106.13 (c=1.446, 1,4-dioxane)
Preparation of Compounds 114b
To a solution of compound 114a (1000 g, 2.303 mol) in 6000 mL of THF, was added 1450 ml of aq HCl (450 mL of con. HCl diluted with 1000 mL of water). The mixture was stirred for 4 hrs, sodium chloride (300 g) and 3 L of ethyl acetate were added. Organic phase was separated and washed with water (2000 mL�1), the mix solution of saturated NaHCO3 solution (2000 mL) and brine (2000 mL). Combined aqueous phase was extracted with ethyl ether (20000 mL�1). Washed with water (500 ml), brine (500 ml). Dried. 980 g of compound 114b was obtained after column chromatography (hexanes/ethyl acetate, 100/0 to 1/1). The yield was 57% (three steps, from 108b to 114b). 1H NMR (CDCl3, 300 MHz): δ 1.27 (s, 3H), 1.32 (s, 3H), 1.64 (s, 3H), 1.77 (t, J=13.8 Hz, 1H), 1.97 (s, 3H), 2.19 (dd, J=12.9, 6.0 Hz, 1H), 2.51 (br s, 3H), 3.62 (br s, 1H), 4.31 (dd, J=13.5, 5.4 Hz, 1H), 5.18 (d, J=10.2 Hz, 1H), 5.51 (br d, J=17.1 Hz, 1H), 6.02 (br dd, J=17.1, 10.2 Hz, 1H).
Preparation of Phosphonium Salt (S)-102a from Alkynediol 114b
650 g of alkynediol 114b (2.62 mol) was added to a mixture of 4300 mL of methylene chloride and 4300 mL of H2O. After cooling to 0� C., NH4Cl (280.07 g, 5.24 mol, 2 eq) and Zn (256.67 g, 3.93 mol, 1.5 eq) was added. Then the reaction mixture was stirred for 3.5 h at 0˜5� C. and the reaction was checked with HPLC. The mixture was filtered through a pad of celite, washed with DCM (500 ml�5). The organic phase was dried. To this solution, 387.6 mL of 48% aqueous HBr (3.4 mol, 1.3 eq) was added in two portions at −8� C. After 30 min, the reaction temperature was raised to −2� C. Water (1000 ml) was run in, and the organic phase was separated off. The organic phase was washed with water (1000 ml�3). To the organic solution was added 23 mL of 1,2-epoxybutane. While cooling to ˜10� C., 755.3 g (2.88 mol, 1.1 eq) of triphenylphosphine was added. After PPh3 was dissolved, another 23 mL of 1,2-epoxybutane was added and the mixture was stirred at rt for 3.5 h. Concentrated and ′BuOMe (1500 mL) was added to precipitate the phosphonium salt. The solids were filtered and washed with ′BuOMe (100 ml�2). 1000 g of 102a (66%, three steps from 114b to 102a) was obtained.
Preparation of (3S,3′S)-all-E-astaxanthin
To a mixture of phosphonium salt (S)-102a (463 g, 0.804 mol, 2.2 eq), 4 Å MS (100 g) and C10-dialdehyde (2,7-dimethyl-2,4,6-octatrienedial 122) (60 g, 0.365 mol, 1 eq) in DCM (7 L) at 0� C., MeONa in MeOH (30 wt %, 151 mL, 0.804 mol, 2.2 eq) was added dropwise. After 4 h, the additional 42 g of phosphonium salt (S)-102a (42 g, 0.2 mol) and 14 mL of MeONa in MeOH (30 wt %, 0.2 mol) was added. After 21 h, the mixture was filtered through a silica gel pad (eluents: DCM/EtOAc ˜DCM/MeOH). Concentrated and filtered to obtain 110 g of crude product. The crude product (297 g) was mixed with 1000 ml of ethyl alcohol refluxed for 3 h. After cooling, filtered and washed with ethyl alcohol (50 ml�2) to obtain 221 g of (3S, 3′S)-all-E-astaxanthin. 1H NMR (CDCl3, 300 MHz): δ 1.21 (s, 6H), 1.32 (s, 6H), 1.81 (t, J=13.2 Hz, 2H), 1.94 (s, 6H), 1.99 (s, 6H) and 2.00 (s, 6H), 2.15 (dd, J=12.6, 5.7 Hz, 2H), 4.32 (dd, J=13.8, 5.7 Hz, 2H), 6.18–6.72 (m, ˜14H).
Preparation of Compounds 110a
Ethyl vinyl ether (788 mL) was cooled to 5� C. and treated with PTSA (450 mg) followed by the slow addition of compound 110b (freshly distilled, 450 g, 4.68 mol). After the addition was done, the reaction mixture was kept at rt for 3 h, quenched with triethylamine (3 mL), and then distilled to yield acetal 110a (770 g, 97.8%). Bp: ˜80� C./20 mmHg.
Preparation of 2-(Triphenyl-phosphanylidene)-propionic acid ethyl ester
Preparation of 4-Hydroxy-2-methyl-but-2-enoic acid ethyl ester
886 g (2.44 mol) of 2-(triphenyl-phosphanylidene)-propionic acid ethyl ester in methylene chloride (4 L) was added dropwise into a refluxing solution of glycoaldehyde dimer (140 g, 1.17 mol) in methylene chloride (6 L). After refluxing for 4 h, the solvent was evaporated. Resulting crude product was fractionated (bp 108–114� C. at 2 mmHg) to give 304 g (90%) pure product as an oil. 1H-NMR (300 Hz CDCl3) δ 6.88 (t, 1H, CH), 4.35 (d, 2H, CH2OH), 4.20 (q, 2H, OCH2), 1.85 (s, 3H, CH3), 1.30 (t, 3H, CH3).
Preparation of 4-Bromo-2-methyl-but-2-enoic acid ethyl ester
Preparation of 2-Methyl-4-(triphenyl-phosphanyl)-but-2-enoic acid ethyl ester bromide salt
940 g (4.54 mol) of 4-hydroxy-2-methyl-but-2-enoic acid ethyl ester was added into the solution of triphenyl phosphine (1.34 Kg, 5.11 mol) in 10 L ethyl acetate. The reaction mixture was stirred at room temperature for 2 days. The resulting white precipitate was filtered and washed with ethyl acetate to give 2.11 kg (99%) of the 5 title compound. 1H-NMR (300 Hz DMSO-d6) δ 7.78–7.95 (m, 15H, ArH), 6.40 (q, 1H, CH), 4.76 (q, 2H, CH2P), 4.10 (q, 2H, CH2), 1.60 (d, 3H, CH3), 1.15 (t, 3H, CH3).
Preparation of 2,6,11,15-Tetramethyl-hexadeca-2,4,6,8,10,12,14-heptaenedioic acid diethyl ester
Preparation of 2,6,11,15-Tetramethyl-hexadeca-2,4,6,8,10,12,14-heptaene-1,16-diol
Preparation of 2,6,11,15-Tetramethyl-hexadeca-2,4,6,8,10,12,14-heptaenedial
Preparation of (S)-(−)-4-Hydroxy-3-methoxy-2,6,6-trimethyl-cyclohex-2-enone
To a solution of (1S,2S)-N-p-toluenesulfonyl-1,2-diphenylethylenediamine (26.8 mg, 0.073 mmol) in Argon sparged 2-propanol (10 mL) was added dichloro(p-cymene)ruthenium(II)dimer (11.2 mg, 0.018 mmol). The suspension was heated to 80� C. for 30 min during which time the solids went into solution. The reaction was cooled to room temperature, a solution of 204b (670 mg, 3.67 mmol) in degassed 2-propanol (15 mL) was added followed by 0.1 M KOH in 2-propanol (1.8 mL) and then stirred overnight. TLC analysis (1:1 ethyl acetate:hexanes) showed the reaction was complete so the reaction was neutralized with aq. citric acid, filtered through a small pad of silica gel and then concentrated under vacuum. Purification by column chromatography (silica gel, 20:80 ethyl acetate:hexanes to 40:60 ethyl acetate:hexanes over 30 min) provided compound 206b (589 mg, 87%) as a waxy solid.
Preparation of (S)-2,2,4,6,6-Pentamethyl-7,7a-dihydro-6H-benzo[1,3]dioxol-5-one
Compound 206b (587 mg, 3.18 mmol) was dissolved in acetone (5 mL), 2,2-dimethoxypropane (10 mL) and water (0.15 mL). p-Toluenesulfonic acid monohydrate (30 mg, 0.157 mmol) was added and the reaction was heated to reflux. After one hour the reaction had only gone 10% so more water (0.15 mL) was added and the reflux was continued. The reaction was monitored every hour and more water (0.15 mL) was added until a total of 0.75 mL had been added. At this point the reaction was cooled and allowed to stir over night. The next morning all the enol ether had been hydrolyzed so the reaction was heated to reflux for 1 hour to form the acetonide then cooled and quenched with saturated aq. sodium bicarbonate (0.2 mL). The volatile solvents were removed under reduced pressure then the reaction was partitioned between ethyl acetate and water then extracted with ethyl acetate, dried over sodium sulfate, filtered and concentrated under vacuum. Purification by column chromatography (silica gel, 15:85 ethyl acetate:hexanes to 25:75 ethyl acetate:hexanes over 20 column volumes) provided compound 208a (537 mg, 80%) as an oil. 1H NMR (CDCl3) δ 4.88 (m, 1H), 2.20 (dd, J=11.5 Hz, J=5.5 Hz, 1H), 1.85 (dd, J=11.5 Hz, J=11.5 Hz, 1H), 1.69 (s, 3H), 1.63 (s, 3H), 1.56 (s, 3H), 1.19 (s, 3H), 1.16 (s, 3H); ESI, m/z 211 [M+H]+, 98.2% ee by HPLC.
Preparation of Epoxyketoisophorone
A buffer may be substituted for the controlled base feed and the pH controller as described here. To a 500 ml 3-neck Morton flask fitted with a bottom outlet, an addition funnel, a magnetic stirrer and a thermometer were charged 30 g 5 wt % aqueous sodium bicarbonate, 30 g 5 wt. % aqueous sodium carbonate, and 20 g keto-isophorone (0.132 mole). To the stirred mixture was added dropwise over one hour while maintaining the temperature between 20 and 25� C. with water-ice bath cooling 15 g 35% hydrogen peroxide (0.165 mole) and the mixture stirred an additional three hours. TLC (e.g., ethyl acetate; heptane 30:70 v/v, silica, iodine visualization, ketoisophorone Rf 0.70, epoxyketoisophorone Rf 0.77) showed complete conversion. The mixture was allowed to separate, the organic phase retained and the aqueous extracted three times, each time with 100 ml dichloromethane. The combined organic and dichloromethane phases were then washed with 50 ml 5 wt % sodium bisulfite solution then with 50 ml 20 wt % sodium chloride solution and the solution dried over anhydrous sodium sulfate. The filtered solution was then concentrated in vacuo on a rotovac to furnish 19.7 g epoxyketoisophorone. Yield is estimated at 89%. NMR of this product showed it to be >95% pure. 1H NMR: 1.08 (s, 3H), 1.3 (s, 3H), 1.55 (s, 3H), 2.26 (d, j=17, 1H), 3.05 (d, j=17, 1H), 3.52 (s, 1H).
Preparation of 3-Hydroxyketoisophorone
The epoxyketoisophorone product was converted to 3-hydroxyketoisophrone. To a 500 ml round bottom flask equipped with an addition funnel, a thermometer, and a magnetic stirrer were charged 30 ml water and 19.6 g epoxyketoisophorone and the mixture stirred while adding dropwise over one hour 18 ml 28 wt % sodium hydroxide solution while keeping the temperature between 30 and 35� C. with a water ice cooling bath. The yellow mixture was stirred another two hours, cooled to room temperature then acidified by dropwise addition to pH I with 37% hydrochloric acid during which a solid precipitated. The slurry was stirred for one hour, then filtered over paper, washed to neutrality with water, then dried at 50� C. and 26 inches vacuum with a nitrogen purge to furnish 17.6 g 3-hydroxyketoisophorone as a yellowish solid. The yield is estimated at 90%. mp 137–139 (lit. 141–143).
Preparation of 3-Methoxyketoisophorone
0.17 g epoxyketoisophorone (1.01 mmole) was dissolved in 2 mL dry methanol under an argon atmosphere. Sodium methoxide was added to the reaction causing the reaction to darken, after an hour at room temperature the reaction was heated to 50� C. The solvents were removed under reduced pressure, the reaction was worked up with water and methylene chloride. The methylene chloride phase was extracted with two portions of a sodium chloride solution and dried over sodium sulphate. The product resulted as a yellow oil (128 mg) with which the NMR spectra was consistent with the desired product.
To a 3-neck round bottom flask fitted with a heating mantle, an addition funnel, a magnetic stirrer, and a reflux condenser were charged 1.68 g 3-hydroxyketoisophorone (10 mmole) and 10 mL methanol and 11 mL 1 N sodium hydroxide and the mixture stirred to furnish a yellow solution. To the solution was added dropwise 1.50 g dimethylsulfate which caused clouding. The resulting mixture was stirred vigorously for 2 hours at 20� C. then warmed to reflux. The homogeneous solution was held at reflux for 4 hours. TLC showed the reaction to be incomplete with no change after another 2 hours. On cooling the reaction mixture was combined with 25 ml water then extracted three times, each time with 50 ml dichloromethane. The combined dichloromethane phases were extracted with 25 ml 5 wt % sodium carbonate and 25 ml 20 wt % sodium chloride then dried over anhydrous sodium sulfate. The filtered solution was stripped of solvent in a rotovac to 50� C. and 26 inches vacuum to furnish a straw colored oil of 1.2 g 3-methoxyketoisophorone. Yield is estimated at 70%. NMR of the product showed it to be >90% pure. 1H NMR: 1.25 (s, 6H), 1.90 (s, 3H), 2.70 (s, 2H), 4.00 (s, 3H).
0.17 g 3-hydroxyketoisophorone (1.01 mmole) was dissolved in 3 mL methanol at 0� C. Diazomethane in ether was added dropwise to the solution to control foaming and spattering. Addition was continued until a yellow color persisted (˜8 mL). Reaction was allowed to continue for one hour, and solvents removed. A yellow oil (186 mg) resulted with an NMR consistent with the desired product.
0.2 g 3-hydroxyketoisophorone (1.19 mmole) was dissolved in methanol. 0.8 g trimethyl orthoformate and 0.04 g trifluoroacetic acid were added to the solution. The reaction was heated to 50� C.
0.107 g epoxyketoisophorone (0.64 mmole) was dissolved in 1.0 mL methanol under an argon atmosphere. Trimethyl orthoformate and trifluoroacetic acid were added to the solution. The reaction was heated to 50� C. and allowed to stir overnight.
Preparation of 4-(S)-Hydroxy-ketoisophorone
To a 50 ml round bottom flask fitted magnetic with a stirrer and a septum with Argon purge was charged 10 ml isopropanol and 11.2 mg dichloro (p-cymene) ruthenium dimer and 26.8 mg 1S, 2S (+)-N-p-luenesulfonyl, 1,2-diphenylethylenediamine and the mixture heated to 80� C. for thirty minutes then cooled to 20� C. To the mixture was charged 670 mg 3-methoxyketoisophorone in degassed 15 ml isopropanol and 1.8 ml 0.1 M potassium hydroxide in isopropanol and the reaction mixture stirred overnight. The reaction mixture was neutralized by adding a solution of 35 mg citric acid in 1 ml water, the mixture filtered through a small pad of silica gel, them stripped to dryness on a rotovac. The residue was chromatographed over 40 g silica gel using a gradient of ethyl acetate-hexanes 20:80 to 40:60 v/v. On concentration of fractions and stripping in vacuo was obtained 589 mg 4-hydroxyketoisophorone as a white crystalline solid. Yield was estimated at 87%. 1H NMR: 1.10 (s, 3H), 1.22 (s, 3H), 1.75 (s, 3H), 1.9 (m, 1H), 2.2 (d, d, 1H) 4.0 (s, 3H), 4.7 (m, 1H).
Preparation of 4-Hydroxy-ketoisophorone
Preparation of 4-Hydroyxketoisophorone acetone ketal
To a 50 ml round bottom flask fitted with a reflux condenser and a magnetic stirrer were charged 587 mg 4-hydroxyketoisophorone and 5 ml acetone and 10 ml 2,2-dimethoxypropane and 30 mg p-toluenesulfonic acid hydrate and 150 mg water and the mixture heated to reflux. At intervals of two hours an additional 150 mg water were added each time and after the fourth addition reflux continued for an additional two hours. The cooled reaction mixture was neutralized by addition of 0.2 ml saturated sodium bicarbonate solution then stripped in vacuo to near dryness. The residue was extracted with ethyl acetate and water, the organic phase dried with sodium sulfate then concentrated in vacuo. The residue was chromatographed on 40 g silica gel eluting with ethyl acetate-hexanes 15:85 to 25:75. The combined and stripped product fractions furnished 537 mg ketal as an oil which later crystallized. The yield is estimated at 80%. Chiral HPLC showed an enantiomeric excess of 98%. I H NMR: 1.18 (s, 3H), 1.20 (s, 3H), 1.55 (s, 3H), 1.63 (s, 3H), 1.70 (s, 3H), 1.85 (d, d, 1H), 2.20 (d, 1H), 4.90 (m, 1H).
Bulk Chromatographic Separation of the Diastereomeric Dicamphanic Acid Ester(s) of Astaxanthin
Bulk chromatographic separation of the diastereomeric dicamphanic acid ester(s) of synthetic astaxanthin at preparative chromatography scale was performed to subsequently make gram-scale quantities of each stereoisomer of disodium disuccinate ester astaxanthin. A total of 135 g of astaxanthin dicamphanate esters (ASTA-DCE) prepared by derivatization of racemic astaxanthin with (−)-camphanic acid chloride were fractionated by preparative HPLC (using a 77 mm i.d. 25 cm column formed by packing 550 g of 10 μm Kromasil 60 Å silica; Eka Chemicals, Marietta, GA) into a Varian RamPak column packing station. After the dry column packing material was mixed with 1200 mL of toluene/2-propanol (50/50) and the resulting slurry was transferred to the 77 mm i.d. column packing chamber, the column bed was formed using the dynamic axial compression of the RamPak unit. The packing solvent was flushed from the column bed for 50 min at a flow rate of 150 m/min using the preparative HPLC mobile phase consisting of 95% toluene and 5% methyl ethyl ketone (MEK). The preparative HPLC system consisted of a Waters Prep 4000 solvent delivery system and a Waters model 486 variable UV detector fitted with a prep cell (3 mm path length).
A total of 40 preparative injections were processed using 84 L of mobile phase. Thirty-six (36) L of effluent were collected among the five fractions. The preparative system was flushed with 100 mL of methylene chloride approximately every 6–8 injections or whenever the chromatographic separation deteriorated due to effects from mixing with mobile phase in the pump heads during the injection process. Purified materials were recovered by removing the solvents in a rotary evaporator protected from light to afford 25.4 g of 3R, 3′R ester, 47.8 g of meso-(3R, 3′S) ester, and 24.9 g of 3S, 3′S ester. The purified astaxanthin dicamphanate esters were saponified to afford 8.5 g (79.8% purity by HPLC) of 3R, 3′R-astaxanthin, 18.2 g (90.1% purity by HPLC) of meso-astaxanthin, and 9.4 g (82.0% purity by HPLC) of 3S, 3′S-astaxanthin. The major impurities of the saponification reaction were the 13- and 9-cis isomers of astaxanthin, identified by HPLC. The cis-isomers were thermally isomerized to all-trans by refluxing in heptane to afford 8.5 g (87.3% purity by HPLC) of 3R, 3′R-astaxanthin, 18.2 g (92.5% purity by HPLC) of meso-astaxanthin, and 9.4 g (86.8% purity by HPLC) of 3S, 3′S-astaxanthin.
General Preparation of Lycophyll 2H
Crocetindialdehyde (238) was obtained from SynChem, Inc. (Des Plaines, Ill.) as a brick-red solid and was used without further purification. Lycopene was obtained from ChromaDex (Santa Ana, Calif.) as a red solid and was used without further purification. Acetic acid 3,7-dimethyl-8-oxo-octa-2,6-dienyl ester (230a) (Liu and Prestwich 2002) was synthesized by literature procedures from commercially available geranyl acetate (228a). All other reagents and solvents used were purchased from Acros Organics (Morris Plains, NJ) and Sigma-Aldrich (St. Louis, Mo.) and were used without further purification. All reactions were performed under a nitrogen atmosphere. All flash chromatographic purifications were performed on Natland International Corporation 230–400 mesh silica gel using indicated solvents. LC/MS (APCI and ESI+ modes) were recorded on an Agilent 1100 LC/MSD VL system; column: Zorbax Eclipse XDB-C18 Rapid Resolution (4.6x75 mm, 3.5 μm); temperature: 25� C.; flow rate: 1.0 mL/min.; mobile phase (A=0.025% TFA in H2O, B=0.025% TFA in acetonitrile). Gradient program (for intermediates 230a–236a and 216a): 70% A/30% B (start), step gradient to 50% B over 5 minutes, step gradient to 100% B over 1.3 minutes, hold at 100% B over 4.9 minutes. Gradient program (for intermediates 218a, 2H): 70% A/30% B (start), step gradient to 50% B over 5 minutes, step gradient to 98% B over 3.3 minutes, hold at 98% B over 16.9 minutes. All-trans lycophyll was obtained from crude material using a Waters 996 Photo Diode Array detector, Millipore 600E System Controller and Waters 717 Autosampler; column: YMC C30 Carotenoid S-5, (10�250 mm, 5 μm column); temperature: 25� C.; flow rate: 4.7 mL/min; mobile phase (A=methanol (MeOH), B=methyl-t-butyl ether (MTBE)) Gradient program: 60% A/40% B (start), step gradient to 80% A over 1 minute, hold at 80% A over 119 minutes. Fractions were collected from 55-66 minutes. Fraction analysis was performed on a YMC C30 Carotenoid S-5, (4.6�250 mm, 5 μm column). Proton nuclear magnetic resonance (NMR) spectra were obtained on a Varian Unity INOVA 500 spectrometer operating at 500.111 MHz (megahertz). Electronic absorption spectra were recorded on a Cary 50 Bio UV-Visible spectrophotometer.
Preparation of 8-Acetoxy-2,6-dimethyl-octa-2,6-dienoic acid (232a)
To a solution of aldehyde 230a (19.5 g, 92.7 mmol) in 300 mL of tert-butyl alcohol was added 2-methyl-2-butene (98.0 mL, 925 mmol). To this was added a solution of sodium dihydrogen phosphate (44.5 g, 371 mmol) in 300 mL of water. Sodium chlorite (33.6 g, 371 mmol) was added in several portions. The resulting mixture was rapidly stirred overnight at room temperature. Ethyl acetate was added and the aqueous layer was acidified to pH 3 by addition of 1 M HCl. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3�200 mL). The combined organic extracts were washed with brine, dried over MgSO4, and reduced to dryness in vacuo. The crude product (27.4 g, 121 mmol, >100% yield) was used in the next step without further purification: 1H NMR (500 MHz, CDCl3) δ: 6.84(t of q, J=7.25 Hz, J=1.50 Hz, 1H, ═CH), 5.34 (t of q, J=7.00 Hz, J=1.50 Hz, 1H, ═CH), 4.56 (d, J=7.00 Hz, 2H, —CH2O—), 2.31 (q, J=7.50 Hz, 2H, —CH2—), 2.15 (t, J=7.50 Hz, 2H, —CH2—), 2.03 (s, 3H, —CH3), 1.81 (s, 3H, —CH3), 1.70 (s, 3H, —CH3). LC/MS (ESI): m/z 249 [M+Na]+.
Preparation of 8-Hydroxy-2,6-dimethyl-octa-2,6-dienoic acid (234a)
To a solution of acid 232a (20.0 g, 88.4 mmol) in 400 mL of methanol was added a solution of potassium carbonate (24.4 g, 177 mmol) in 100 mL of water. The resulting mixture was vigorously stirred overnight at room temperature. The reaction was cooled to 0� C., methylene chloride (200 mL) was added, and the aqueous layer was acidified to pH 3 with 1 M HCl. The organic layer was separated, and the aqueous layer was extracted with methylene chloride (2�200 mL). The combined organic extracts were washed with brine, dried over MgSO4, and reduced to dryness in vacuo. The crude product (9.65 g, 52.4 mmol, 59% yield) was used in the next step without further purification: 1H NMR (500 MHz, CDCl3) δ: 6.86 (t of q, J=7.25 Hz, J=1.50 Hz, 1H, ═CH), 5.43 (t of q, J=7.00 Hz, J=1.50 Hz, 1H, ═CH), 4.16 (d, J=7.00 Hz, 2H, —CH2O—), 2.33 (q, J=7.50 Hz, 2H, —CH2—), 2.16 (t, J=7.50 Hz, 2H, —CH2—), 1.83 (s, 3H, —CH3), 1.68 (s, 3H, —CH3). LC/MS (ESI): m/z 207 [M+Na]+.
Preparation of 8-Hydroxy-2,6-dimethyl-octa-2,6-dienoic acid methyl ester (234b)
To a solution of acid 234a (20.1 g, 109 mmol) in 400 mL of DMF was added a solution of potassium carbonate (16.6 g, 120 mmol) in 80 mL of water. The resulting mixture was vigorously stirred for several minutes. To the mixture was added iodomethane (7.50 mL, 120 mmol) via syringe. The resulting mixture was vigorously stirred overnight at room temperature. Ethyl acetate (400 mL) and water (400 mL) were added and the aqueous layer was acidified to pH 3 by addition of 1 M HCl. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (3�200 mL). The combined organic extracts were washed with water (3�500 mL), saturated aqueous sodium carbonate, brine, and dried over MgSO4. The solvent was removed under reduced pressure and the crude product purified by flash chromatography (MeOH/CH2Cl2, 1:49) to afford methyl ester 5 as a clear oil (19.4 g, 90% yield): 1H NMR (500 MHz, CDCl3) δ: 6.72 (t of q, J=7.50 Hz, J=1.50 Hz, 1H, ═CH), 5.43 (t of q, J=6.75 Hz, J=1.50 Hz, 1H, ═CH), 4.16 (d, J=7.00 Hz, 2H, —CH2O—), 3.73(s, 3H, —CH3), 2.31 (q, J=7.50 Hz, 2H, —CH2—), 2.15 (t, J=7.50 Hz, 2H, —CH2—),1.83 (s, 3H, —CH3),1.69 (s, 3H, —CH3). LC/MS (ESI): m/z 221 [M+Na]+.
Preparation of 8-Bromo-2,6-dimethyl-octa-2,6-dienoic acid methyl ester (236a)
To a 0� C. solution of alcohol 234b (12.9, 64.9 mmol) in 250 mL of anhydrous tetrahydofuran was added carbon tetrabromide (23.8 g, 71.4 mmol) in several portions. The mixture was stirred for a few minutes and then triphenylphosphine (18.7 g, 71.4 mmol) was added and the mixture allowed to warm to room temperature and stirred overnight. The solvent was removed under reduced pressure and the resulting residue was suspended in diethyl ether. The suspension was filtered through a pad of Celite. After solvent removal under reduced pressure the resulting crude product (contaminated with triphenylphosphine oxide) was used directly in the next step: 1H NMR (500 MHz, CDCl3) δ: 6.61 (t of q, J=7.50 Hz, J=1.50 Hz, 1H, ═CH), 547 (t of q, J=8.00 Hz, J=1.50 Hz, 1H, ═CH), 3.92 (d, J=8.50 Hz, 2H, —CH2Br), 3.63 (s, 3H, —CH3), 2.22 (q, J=8.00Hz, 2H, —CH2—),2.10 (t, J=8.00 Hz, 2H, —CH2—),1.75 (d, J=1.00 Hz, 3H, —CH3),1.66 (d, J=1.00 Hz, 3H, —CH3).
Preparation of (2,6-Dimethyl-8-octa-2,6-dienoic acid methyl ester)triphenylphosphonium bromide (216a)
To a solution of bromide 236a (9.20 g, 35.2 mmol) in ethyl acetate (200 mL) was added triphenylphosphine (10.2 g, 38.8 mmol). The resulting mixture was vigorously stirred for a few minutes, at which time an insoluble material began to oil out from the solution, adhering to the sides of the flask. The reaction solution was then decanted into a clean reaction vessel. This procedure was repeated every 5 to 10 minutes until no more oily insoluble residue was noted, at which time a white solid started to precipitate from the solution. The cloudy mixture was then stirred overnight at room temperature. The mixture was filtered and the filter cake was rinsed with ethyl acetate and dried in vacuo to afford phosphonium salt 7 as a white solid (9.60 g, 52% yield). 1H NMR (500 MHz, CDCl3) δ: 7.88–7.84 (m, 6 arom. H), 7.79–7.75 (m, 3 arom. H), 7.68–7.64 (m, 6 arom. H), 6.51 (t of q, J=5.00 Hz, J=1.00 Hz, 1H, ═CH), 5.10 (q, J=7.00 Hz, 1H, ═CH), 4.70 (d of d, J=15.0, J=8.00 Hz, 2H, —CH2PPh3Br), 3.67 (s, 3H, —CH3), 2.16 (q, J=7.00 Hz, 2H, —CH2—), 2.08 (t, J=6.00 Hz, 2H, —CH2—), 1.70 (s, 3H, —CH3),1.35 (d, J=4.00 Hz, 3H, —CH3). LC/MS (ESI): m/z 443 [M]+.
Preparation of Dimethyl ψ,ψ-Carotene-16,16′-dioate (218a)
Preparation of ψ,ψ-Carotene-16,16′-diol (I)
To a solution of dimethyl ester 218a (1.14 g, 1.83 mmol) in anhydrous tetrahydrofuran (100 mL) at 0� C. was added DIBAL (20% by wt. in toluene) (9.13 mL, 11.0 mmol) via syringe. The mixture was warmed to room temperature and stirred for one hour. The reaction was quenched by the sequential addition of H2O (440 μL), 15% aqueous NaOH (440 μL), and H2O (1.10 mL). The resulting mixture was stirred for 30 minutes and then dried over anhydrous MgSO4. After filtration and removal of solvent in vacuo, the resulting crude diol 2H (0.39 g, 38%) was used in the next step without further purification. 1H NMR (500 MHz, CDCl3) δ: 6.63 (d of d, J=15.0 Hz, J=11.5 Hz, 2H, H11, H11′), 6.63 (d, J=11.0 Hz, 2H, H15, H15′), 6.48 (d of d, J=15.0 Hz, J=11.0 Hz, 2H, H7, H7′), 6.36 (d, J=15.0 Hz, 2H, H12, H12′), 6.25 (d, J=15.0 Hz, 2H, H8, H8′), 6.19 (d, J=11.5 Hz, 2H, H10, H10′), 5.95 (d, J=11.0 Hz, 2H, H6, H6′), 5.40 (t of q, J=6.50 Hz, J=1.50 Hz, 2H, H2, H2′), 4.00 (s, 4H, —CH2O—), 2.19 (t, J=Hz, 4H, —CH2—), 2.16 (t, J=Hz, 4H, —CH2—), LC/IMS (APCI): m/z 569 [M+H]+.
General Preparation of Lycophyll Derivatives
LC/MS (APCI) and LC/MS (ESI) were recorded on an Agilent 1100 LC/MSD VL, PDA detector system; column: Zorbax Eclipse XDB-C18 Rapid Resolution (4.6�75 mm, 3.5 μm); temperature: 25� C.; flow rate: 1.0 mL/min; mobile phase (% A=0.025% trifluoroacetic acid in H2O, % B=0.025% trifluoroacetic acid in acetonitrile) Gradient program: 70% A/30% B (start), step gradient to 50% B over 5 min, step gradient to 98% B over 8.30 min, hold at 98% B over 25.20 min, step gradient to 30% B over 25.40 min. A catalytic amount of trifluoroacetic acid is used in the eluents to improve chromatographic resolution. The presence of trifluoroacetic acid facilitates the protonation of synthesized lycophyll dissucinate and diphosphate salts to give the free diacid forms (as represented by the theoretical molecular ions M+=768 for lycophyll disuccinate salt and M+=728 for lycophyll disphosphate salt). LRMS:+mode; ESI: electrospray chemical ionization, ion collection using quadrapole; APCI: atmospheric pressure chemical ionization, ion collection using quadrapole. Reverse-phase HPLC was performed on a Waters 996 HPLC with PDA detector, Millipore 600E System Controller system; column: Zorbax Eclipse XDB-C18 (9.4�250 mm, 5 μm); temperature: 25� C.; flow rate: 2.1 mU min; mobile phase (% A=0.025% trifluoroacetic acid in H2O, % B=0.025% trifluoroacetic acid in MeOH) Isocratic program: 15% A/85% B. 1H NMR analyses were performed on a Varian spectrometer (300 MHz).
Preparation of ψ,ψ-carotenyl 16,16′-disuccinate (222a)
To a solution of lycophyll (2H) (0.10 g, 0.176 mmol) in CH2Cl2 (2 mL) was added N,N-diisopropylethylamine (0.613 mL, 3.52 mmol) and succinic anhydride (0.1761 g, 1.76 mmol). The solution was stirred at room temperature overnight and then diluted with CH2Cl2 and quenched with cold water/1 M HCl (9/1). The aqueous layer was extracted two times with CH2Cl2 and the combined organic layer was washed three times with cold water/1 M HCl (9/1), dried over Na2SO4, and concentrated to yield disuccinate 222a (0.124 g, 92%) as a red hygroscopic solid; LC/MS (APCI): 11.59 min (65.17%), λmax 295 nm (28%), 362 nm (8%), 447 nm (72%), 472 nm (100%), 503 nm (93%), m/z 769 [M+H]+(100%), 668 [M—C4O3H4]+ (9%), 651 (89%), 533 (30%); 12.13 min (33.69%), λmax 295 nm (26%), 362 nm (10%), 447 nm (77%), 472 nm (100%), 503 nm (91%), m/z 769 [M+H]+ (28%), 651 (24%), 531 (8%), 261 (100%).
Preparation of ψ,ψ-carotenyl 16,16′-disuccinate sodium salt (224a)
To a solution of disuccinate 222a (0.124 g, 0.161 mmol) in methanol (3 mL) at 0� C. was added dropwise sodium methoxide (25% wt in methanol; 0.074 mL, 0.322 mmol). The solution was stirred at room temperature overnight, then cooled to 0� C., and water was added. The red mixture was stirred for 5 min at 0� C., and then methanol was removed in vacuo. The red, aqueous solution was lyophilized to afford disuccinate salt 224a (0.103 g, 88%) as a red hygroscopic solid; LC/MS (APCI): 11.58 min (71.72%), λmax 295 nm (13%), 362 nm (9%), 447 nm (68%), 472 nm (100%), 503 nm (90%), m/z 769 [M+H]+ (100%), 651 (42%), 533 (15%); 12.09 min (27.74%), λmax 295 nm (31%), 362 nm (19%), 447 nm (80%), 472 nm (100%), 503 nm (88%), m/z 769 [M+H]+ (100%), 669 [M−C4O3H4+H]+ (12%), 651 (54%), 551 (8%), 533 (11%).
Preparation of Tribenzyl phosphite (13)
To a well-stirred solution of phosphorus trichloride (1.7 mL, 19.4 mmol) in Et2O (430 mL) at 0� C. was added dropwise a solution of triethylamine (8.4 mL, 60.3 mmol) in Et2O (20 mL), followed by a solution of benzyl alcohol (8.1 mL, 77.8 mmol) in Et2O (20 mL). The mixture was stirred at 0� C. for 30 min and then at room temperature overnight. The mixture was filtered and the filtrate concentrated to give a colorless oil. Silica chromatography (hexanes/Et2O/triethylamine, 5.5/1/1%) of the crude product gave 13 (5.68 g, 83%) as a clear, colorless oil that was stored under N2 at −20� C.; 1H NMR (300 MHz, CDCl3) δ: 7.38 (15H, m), 4.90 (6H, d).
Preparation of Dibenzyl Phosphoroiodidate (14)
To a solution of tribenzyl phosphite (0.708 g, 2.01 mmol) in CH2Cl2 (5 mL) at 0� C. was added 1.93 mmol). The mixture was stirred at 0� C. for 10 min or until the solution became clear and colorless. The solution was then stirred at room temperature for 10 min and used directly in the next step.
Preparation of Mixture of 16,16′-Benzyl phosphoryloxy-ψ,ψ-carotenes (221a,221b,221c,221d)
To a solution of lycophyll (2H) (0.11 g, 0.193 mmol) in CH2Cl2 (5 mL) was added pyridine (0.624 mL, 7.72 mmol). The solution was stirred at 0� C. for 5 min and then freshly prepared 14 (1.93 mmol) in CH2Cl2 (5 mL) was added dropwise to the mixture at 0� C. The solution was stirred at 0� C. for 1 h and then diluted with CH2Cl2 and quenched with brine. The aqueous layer was extracted twice with CH2Cl2 and the combined organic layer was washed once with NaSSO4, once with brine, then dried over Na2SO4 and concentrated. Pyridine was removed from the crude red oil by azeotropic distillation using toluene to yield a mixture of benzyl-protected diphosphoric acid lycophyll derivatives 221a,221b,221c,221d used in the next step without further purification; LCtMS (ESI) for 221a: 10.15 min (7.73%), λmax 295 nm (21%), 362 nm (16%), 447 nm (72%), 472 nm (100%), 503 nm (87%), m/z 819 [M+H]+ (18%), 800 [M-H2O]+(11%), 672 (24%), 531 (10%); LC for 221b: 18.00 min (17.46%), λmax 295 nm (18%), 362 nm (13%), 447 nm (74%), 472 nm (100%), 503 nm (85%); LC for 221c: 20.08 min (20.00%), λmax 295 nm (18%), 362 nm (16%), 447 nm (74%), 472 nm (100%), 503 nm (86%); LC for 221d: 22.52 min (54.81%), λmax 295 nm (19%), 362 nm (18%), 447 nm (73%), 472 nm (100%), 503 nm (87%).
Preparation of 16,16′-Diphosphoryloxy-ψ,ψ-carotene (221e)
To a solution of a mixture of benzyl-protected diphosphoric acid lycophyll derivatives 221a, 221b, 221c, 221d (0.193 mmol) in CH2Cl2 (15 mL) at 0� C. was added dropwise N,O-bis(trimethylsilyl)acetamide (0.479 mL, 1.93 mmol) and then bromotrimethylsilane (0.203 mL, 1.54 mmol). The solution was stirred at 0� C. for 15 min, quenched with triethylamine, and stirred at 0� C. for 5 min. The red solution was then diluted with CH2Cl2, Et2O, and MeOH (2/1/1), and then concentrated. The resulting red oil was resuspended in a minimum amount of MeOH and the cloudy solution was centrifuged to remove insoluble reaction byproducts. The red supernatant was concentrated to afford a mixture of monophosphate and diphosphate lycophyll derivatives (254/221e) (1/4) contaminated with excess reagents, and reaction and decomposition byproducts; LC/MS (ESI) for 221e: 9.10 min (39.24%), λmax 295 nm (31%), 362 nm (18%), 447 nm (74%), 472 nm (100%), 503 nm (88%), m/z 849 (25%), 827 (5%), 368 (100%), 357 (11%), 317 (52%); 9.25 min (37.83%), λmax 295 nm (31%), 362 nm (18%), 447 nm (75%), 472 nm (100%), 503 nm (89%), m/z 849 (10%), 625 (8%), 581 (6%), 385 (20%), 368 (100%), 357 (28%); LC/MS (ESI) for 254: 10.21 min (18.50%), λmax 295 nm (32%), 362 nm (24%), 447 nm (78%), 472 nm (100%), 503 nm (89%), m/z 648 M+(9%), 630 [M-H2O]+ (5%), 568 (10%), 317 (100%); the crude mixture was subjected to reverse-phase HPLC purification to give diphosphate 221e (approximately 70% pure; 0.063 g, 45%) as a red oil, contaminated with excess reagents, and reaction and decomposition byproducts; LC/MS (ESI): 9.36 min (4.43%), λmax 295 nm (30%), 362 nm (25%), 447 nm (79%), 472 nm (100%), 503 nm (82%), m/z 849 (16%), 619 (7%), 399 (23%), 368 (100%), 357 (10%), 317 (8%); 9.58 min (46.42%), λmax 295 nm (30%), 362 nm (15%), 447 nm (80%), 472 nm (100%), 503 nm (92%), m/z 849 (19%), 619 (5%), (399 (21%), 368 (100%), 357 (10%), 317 (9%); 9.67 min (49.15%), λmax 295 nm (28%), 362 nm (12%), 447 nm (77%), 472 nm (100%), 503 nm (95%), nm/z 849 (15%), 619 (5%), 399 (20%), 368 (100%), 357 (8%), 317 (6%).
Preparation of 16,16′-Diphosphoryloxy-ψ,ψ-carotene sodium salt (223a)
To a solution of lycophyll diphosphate (221e) (approximately 70% pure; 0.04 g, 0.055 mmol) in methanol (2 mL) at 0� C. was added dropwise sodium methoxide (25% wt in methanol; 0.05 mL, 0.22 mmol). The solution was stirred at room temperature overnight, then cooled to 0� C., and water was added. The red mixture was stirred for 5 min at 0� C., and then methanol was removed in vacuo. The red, aqueous solution was lyophilized to yield diphosphate salt 223a (approximately 50% pure; 0.018 g, 43%) as a red hygroscopic solid; LC/MS (ESI): 9.26 min (9.34%), λmax 295 nm (28%), 362 nm (18%), 447 nm (81%), 472 nm (100%), 503 nm (87%), m/z 897 (8%), 392 (100%), 381 (10%); 9.48 min (46.98%), λmax 295 nm (29%), 362 nm (15%), 447 nm (80%), 472 nm (100%), 503 nm (91%), m/z 911 (10%), 849 (15%), 399 (87%), 368 (100%); 9.56 min (43.68%), λmax 295 nm (28%), 362 nm (12%), 447 nm (77%), 472 nm (100%), 503 nm (90%), m/z 849 (19%), 827 (5%), 368 (100%), 357 (8%).
Separation of (3S,3′S)-all-E-astaxanthin
Analysis of the stereoisomeric distribution of astaxanthin was accomplished using a chiral HPLC column. A Regis Pirkle Covalent D-phenylglycine, 5 Å, 4.6�250 nm chiral HPLC column was used. The detector was set at 474 nm. A 10 μL sample was injected into the column. The sample was passed through the column using a mobile phase of 75% Heptane, 24% dichloromethane, and 1% ethanol at a flow rate of 1.5 mL/min. Racemic astaxanthin (e.g., 3S, 3′S, meso (3R, 3′S), and 3R, 3′R in a 1:2:1 ratio) was run through the chiral HPLC column and the retention time for 3S, 3′S (“S, S”)-astaxanthin was 32.763 min, meso-astaxanthin was 31.165, and 3R,3′R (“R, R”)-astaxanthin was 29.937. The total run time was 60 minutes.
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Chirality, (2001), 13:739-744.* Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS9216164Sep 10, 2013Dec 22, 2015U.S. Nutraceuticals, LLCComposition and method to alleviate joint pain using a mixture of fish oil and fish oil derived, choline based, phospholipid bound fatty acid mixture including polyunsaturated EPA and DHAUS9238043Mar 18, 2014Jan 19, 2016U.S. Nutraceuticals, LLCComposition and method to alleviate joint pain using algae based oilsUS20140011888 *Sep 9, 2013Jan 9, 2014U.S. Nutraceuticals, Llc D/B/A Valensa InternationalComposition derived from astaxanthin and method to treat joint pain associated with osteoarthritis* Cited by examinerClassifications U.S. Classification568/343, 568/378International ClassificationC07C45/65, C07C33/14, C07C45/61, C07C45/74, C07F7/04, C07C49/753, C07C45/64, C07C29/145, C07C401/00, C07C49/713, C07C45/00Cooperative ClassificationC07C45/61, C12P23/00, C07C45/64, C07F9/113, C07C49/753, C07C2101/16, C07C29/143, C07C47/21, C07C403/08, C07F7/188, C07C45/71, C07D319/06, C07C45/70, C07B2200/07, C07C45/673, C07B2200/09, C07C45/511, C07C45/298, C07F7/1892, C07C49/713, C07C403/24, C07F9/5442European ClassificationC07F9/54A4, C07C45/29M, C07C45/67C, C07C45/70, C07C45/71, C07C45/64, C07C45/61, C07D319/06, C07F7/18C9G, C12P23/00, C07F7/18C9B, C07C45/51B, C07C47/21, C07C49/753, C07C49/713, C07C29/143, C07C403/24, C07C403/08, C07F9/113Legal EventsDateCodeEventDescriptionJan 27, 2006ASAssignmentOwner name: HAWAII BIOTECH, INC., HAWAIIFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:LOCKWOOD, SAMUEL F.;TANG, PENG CHO;NADOLSKI, GEOFF;AND OTHERS;REEL/FRAME:017511/0559Effective date: 20051219Nov 17, 2006ASAssignmentOwner name: CARDAX PHARMACEUTICALS, INC., HAWAIIFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:HAWAII BIOTECH, INC.;REEL/FRAME:018602/0423Effective date: 20060808Feb 28, 2011REMIMaintenance fee reminder mailedJul 12, 2011FPAYFee paymentYear of fee payment: 4Jul 12, 2011SULPSurcharge for late paymentJul 30, 2013ASAssignmentOwner name: CARDAX PHARMA, INC., HAWAIIFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:CARDAX PHARMACEUTICALS, INC.;REEL/FRAME:030906/0169Effective date: 20130729Mar 6, 2015REMIMaintenance fee reminder mailedJul 22, 2015FPAYFee paymentYear of fee payment: 8Jul 22, 2015SULPSurcharge for late paymentYear of fee payment: 7RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services