Patent Application: US-63442906-A

Abstract:
the subject invention provides compounds having the structure : wherein r 1 is h , oh , a photoactivatable moiety , a fluorescent moiety , or a radioactive moiety ; r 2 is h , oh , a photoactivatable moiety , a fluorescent moiety , or a radioactive moiety ; r 3 is h or oh ; r 4 is h , oh , a photoactivatable moiety , a fluorescent moiety , or a radioactive moiety ; and wherein at least one of r 1 , r 2 , r 3 , or r 4 is a photoactivatable moiety , a fluorescent moiety , or a radioactive moiety , or an optically pure enantiomer of the compound or wherein r1 is h or oh ; r2 is h , oh , halogen , unsubstituted or substituted , straight or branched alkyl group , alkenyl , or a alkynyl , alkoxy , alkenyloxy , or alkynyloxy , — n3 , — cor5 , — conr5r6 , — co2r5 , — ocor5 , — nh , — nr5r6 , — nhcor5 , — ncor5 , — ch2or5 , — och2co2r5 , — ch2sr5 , — ch2nr5r6 , — sr5 , — osr5 , or — nr5so2r6 , where r5 and r6 are each independently hydrogen , substituted or unsubstituted alkyl , alkenyl , or alkynyl , or a cycloalkyl or aryl group having 3 to 10 carbon atoms ; r3 is h or oh ; r4 is h , alkyl , alkenyl , alkynyl , - a - ar , - a - z - ar , — so 2 — ar , or - a - nr 5 , or — r 7 , where a , z and ar are as defined herein , and the use of the compounds for detecting or identifying a receptor which binds the compounds of the invention or for treating a paf associated condition in a subject .

Description:
wherein r 1 is h , oh , a photoactivatable moiety , a fluorescent moiety , or a radioactive moiety ; wherein r 2 is h , oh , a photoactivatable moiety , a fluorescent moiety , or a radioactive moiety ; wherein r 3 is h or oh ; wherein r 4 is h , oh , a photoactivatable moiety , a fluorescent moiety , or a radioactive moiety ; and wherein at least one of r 1 , r 2 , r 3 , or r 4 is a photoactivatable moiety , a fluorescent moiety , or a radioactive moiety , or an optically pure enantiomer of the compound . in the compound , r 1 may be a fluorescent moiety and each of r 2 and r 4 may be h or oh ; r 2 may be a fluorescent moiety or a radioactive moiety and each of r 1 and r 4 may be h or oh ; or r 4 may be a photoactivatable moiety or a radioactive moiety and each of r 1 and r 2 may be h or oh . the photoactivatable moiety may be a phenylazide , a purine or pyrimidine azides , a diazoacetate , a diazoketone , a nitrobenzene , or an aryldiazonium salt . in some embodiments , the photoactivatable moiety may be benzophenone , trifluoromethyldiazirine tetrafluorophenyl , 8 - azidoadenosine , 2 - azidoadenosine , or 3h , 3 - aryldiazirine . the fluorescent moiety may be a fluorescent amine . in some embodiments , the fluorescent moiety may be 5 -( dimemethylamino ) naphthalene - sulfonyl chloride , 1 -( bromoacetyl ) pyrene , 3 - bromoacetyl - 7 - diethylaminocoumarin , 3 - bromomethyl - 6 , 7 - dimethoxycoumarin , 8 - bromomethyl - 4 , 4 - difluoro - 1 , 3 , 5 , 7 - tetramethyl - 4 - bora - 3a , 4a - diaza - s - indacene , 3 - bromomethyl - 6 , 7 - dimethoxy - 1 - methyl - 2 ( 1h )- quinoxazolinone , 6 - bromoacetyl - 2 - dimethylaminonaphthalene , or 4 -( 9 - anthroyloxy ) phenacyl bromide . the radioactive moiety may be 11 c , 13 n , 15 o , 3 h or 18 f . in specific embodiments of the compounds of this invention , the photoactivatable moiety may be benzophenone , trifluoromethyldiazirine or tetrafluorophenyl ; the fluorescent moiety may be 5 -( dimemethylamino ) naphthalene - sulfonyl (“ dansyl ”); and the radioactive moiety may be 18 f , 11 c or 3 h . in a specific embodiment the invention also provides compounds having the structure : where r 1 , r 2 , r 3 and r 4 are defined as above . in specific embodiments , r 1 may be h , oh , a fluorescent moiety ; r 2 may be h , oh , a fluorescent moiety , or a radioactive moiety ; r 3 may be h or oh ; and r 4 may be h , oh , a photoactivatable moiety , or a radioactive moiety . in yet further embodiments , r 1 may be — o - dansyl ; or r 2 may be — o - dansyl ; or r 2 may be — 11 ch 3 ; or r 2 may be — ch 2 ch 2 18 f ; or r 2 may be 18 f ; or r 2 may be 3 h ; or r 4 may be a benzophenone moiety ; or r 4 may be a trifluoromethyldiazirine moiety ; or r 4 may be a tetrafluorophenyl azide moiety ; or r 4 may be — 11 ch 3 ; or r 4 may be — ch 2 ch 2 18 f . wherein r 2 is h , oh , halogen , unsubstituted or substituted , straight or branched ( c 1 - c 5 ) alkyl group , ( c 2 - c 5 ) alkenyl , or a ( c 2 - c 5 ) alkynyl , ( c 1 - c 5 ) alkoxy , ( c 2 - c 5 ) alkenyloxy , or ( c 2 - c 5 ) alkynyloxy , — n 3 , — cor 5 , — conr 5 r 6 , — co 2 r 5 , — ocor 5 , — nh ( oh ), — nr 5 r 6 , — nhcor 5 , — n ( oh ) cor 5 , — ch 2 or 5 , — och 2 co 2 r 5 , — ch 2 sr 5 , — ch 2 nr 5 r 6 , — sr 5 , — osr 5 , or — nr 5 so 2 r 6 , where r 5 and r 6 are each , independently , hydrogen , substituted or unsubstituted ( c 1 - c 5 ) alkyl , ( c 2 - c 5 ) alkenyl , or ( c 2 - c 5 ) alkynyl , or a cycloalkyl or aryl group having 3 to 10 carbon atoms ; wherein r 4 is h , oh , ( c 1 - c 10 ) alkyl , ( c 2 - c 10 ) alkenyl , ( c 2 - c 10 ) alkynyl , - a - ar , - a - z - ar , — so 2 — ar , or - a - nr 6 , or — r 6 , where a is ( c 1 - c 8 ) alkyl , ( c 2 - c 8 ) alkenyl , ( c 2 - c 8 ) alkynyl , which is unsubstituted or substituted by a straight or branched alkyl chain group having 1 to 5 carbon atoms ; z is carbon , oxygen , sulfur or nitrogen ; ar is a phenyl group , a pyridyl group , a naphthyl group , a pyrimidyl group , or a quinolyl group , each of which may contain heteroatoms and may be unsubstituted or substituted by one to five substituents selected from the group consisting of hydrogen , halogen , a hydroxy group , a carboxylic acid group , substituted or unsubstituted ( c 1 - c 10 ) alkyl , ( c 2 - c 10 ) alkenyl , ( c 2 - c 10 ) alkynyl , ( c 1 - c 10 ) haloalkyl , ( c 1 - c 10 ) alkoxy , ( c 2 - c 10 ) alkenyloxy , ( c 2 - c 10 ) alkynyloxy , ( c 1 - c 10 ) haloalkoxy , a phenyl group , a phenoxy group , an aralkyl group , an aralkyloxy group , a substituted phenyl group , a substituted phenoxy group , a substituted aralkyl group , a substituted aralkyloxy group , — cor 6 , — conr 6 r 6 , — co 2 r 6 , — nhcor 6 , — nh ( oh ), — n ( oh ) cor 6 , — ch 2 or 6 , — och 2 co 2 r 6 , — ch 2 sr 6 , — ch 2 nr 6 r 6 , — sr 6 , — osr 6 , — nr 6 r 6 , — nr 6 so 2 r 6 , where r 6 is hydrogen , ( c 1 - c 10 ) alkyl , ( c 3 - c 10 ) cycloalkyl , — scx 3 in which x is a halogen , — cn , — no2 or - z - a - z ′- in which z and a are as defined above and z ′ represents carbon , oxygen , sulfur , or nitrogen ; or an optically pure enantiomer , or a tautomer , or a salt of the compound . wherein r 2 is cl , f , oh , a substituted or unsubstituted , straight or branched ( c 1 - c 5 ) alkyl , ( c 2 - c 5 ) alkenyl , ( c 2 - c 5 ) alkynyl , ( c 1 - c 5 ) alkoxy , ( c 2 - c 5 ) alkenyloxy , or ( c 2 - c 5 ) alkynyloxy , — n 3 , — cor 5 , — conr 5 r 6 , — co 2 r 5 , — ocor 5 , — nh ( oh ), — nr 5 r 6 , — nhcor 5 , — n ( oh ) cor 5 , — ch 2 or 5 , — och 2 co 2 r 5 , — ch 2 sr 5 , — ch 2 nr 5 r 6 , — sr 5 , — osr 5 , or — nr 5 so 2 r 6 , where r 5 and r 6 are each , independently , hydrogen , substituted or unsubstituted ( c 1 - c 5 ) alkyl , ( c 2 - c 5 ) alkenyl , or ( c 2 - c 5 ) alkynyl , or a cycloalkyl or aryl group having 3 to 10 carbon atoms ; wherein r 8 is ( c 1 - c 10 ) alkyl , ( c 2 - c 10 ) alkenyl , ( c 2 - c 10 ) alkynyl , - a - ar , - a - z - ar , — so 2 — ar , or - a - nr 6 , where a is an unsubstituted , straight chain ( c 1 - c 5 ) alkyl , ( c 2 - c 5 ) alkenyl , ( c 2 - c 5 ) alkynyl ; z is carbon , oxygen , sulfur or nitrogen ; ar is a phenyl group , a pyridyl group , a naphthyl group , a pyrimidyl group , or a quinolyl group , each of which may contain heteroatoms and may be unsubstituted or substituted by one to five substituents selected from the group consisting of hydrogen , halogen , hydroxy , substituted or unsubstituted ( c 1 - c 10 ) alkyl , ( c 2 - c 10 ) alkenyl , ( c 2 - c 10 ) alkynyl , ( c 1 - c 10 ) alkoxy , ( c 2 - c 10 ) alkenyloxy , ( c 2 - c 10 ) alkynyloxy , phenyl , phenoxy , aralkyl , or aralkyloxy , — cor 6 , — conr 6 r 6 , — co 2 r 6 , — nhcor 6 , — nh ( oh ), — n ( oh ) cor 6 , — ch 2 or 6 , — och 2 co 2 r 6 , — ch 2 sr 6 , — ch 2 nr 6 r 6 , — sr 6 , — osr 6 , — nr 6 r 6 , or — nr 6 so 2 r 6 , where r 6 is hydrogen , ( c 1 - c 10 ) alkyl , ( c 3 - c 10 ) cycloalkyl , — scx 3 in which x is a halogen , — cn , — no 2 or - z - a - z ′- in which z and a are as defined above and z ′ represents carbon , oxygen , sulfur , or nitrogen , or an optically pure enantiomer , or a tautomer , or a salt of the compound . in another embodiment , r 4 is h or — ch 2 c 6 h 5 . in another embodiment , r 2 is oh , cl , — n 3 , — ocor 3 , or — nr 3 r 4 . in another embodiment , r 2 is oh , cl , — ococh 3 , — ococh 2 c 6 h 5 , — n 3 , — nh 2 , — nhch 3 , — nhch 2 ch 3 . in another embodiment , r 2 is oh , cl , — ococh 3 , — ococh 2 c 6 h 5 , — n 3 , — nh 2 , — nhch 3 , — nhch 2 ch 3 ; and wherein r 4 is h or — ch 2 c 6 h 5 . in another embodiment , r 1 is oh , r 2 is f , r 3 is oh , and r 4 is h . in another embodiment , r 2 is cl , and r 4 is h . in another embodiment , r 2 is — n 3 , and r 4 is h . in another embodiment , r 2 is — nhch 3 , and r 4 is h . in another embodiment , r 2 is — nhch 2 ch 3 , and r 4 is h . in another embodiment , r 2 is — ococh 2 c 6 h 5 , and r 4 is h . in another embodiment , r 2 is f , and r 4 is — ch 2 c 6 h 5 . in another embodiment , r 2 is cl , and r 4 is — ch 2 c 6 h 5 . in another embodiment , r 2 is h , and r 4 is — ch 2 c 6 h 5 . in another embodiment , r 2 is oh , and wherein r 4 is — ch 2 c 6 h 5 . wherein r 2 is h , f , br , unsubstituted or substituted , straight or branched ( c 1 - c 5 ) alkyl group , ( c 2 - c 5 ) alkenyl , or a ( c 2 - c 5 ) alkynyl ; wherein r 4 is h , - a - ar , - a - z - ar , — so 2 — ar , or - a - nr 6 , or — r 6 , where a is an alkylene group having 1 to 8 carbon atoms , which is unsubstituted or substituted by a straight or branched alkyl chain group having 1 to 5 carbon atoms ; z is carbon , oxygen , sulfur or nitrogen ; ar is a phenyl group , a pyridyl group , a naphthyl group , a pyrimidyl group , or a quinolyl group , each of which may contain heteroatoms and may be unsubstituted or substituted by one to five substituents selected from the group consisting of hydrogen , halogen , a hydroxy group , a carboxylic acid group , ( c 1 - c 10 ) alkyl , ( c 2 - c 10 ) alkenyl , ( c 2 - c 10 ) alkynyl , a ( c 1 - c 10 ) haloalkyl , an ( c1 - c 10 ) alkoxy , an ( c 2 - c 10 ) alkenyloxy , an ( c 2 - c 10 ) alkynyloxy , a ( c 1 - c 10 ) haloalkoxy , a phenyl group , a phenoxy group , an aralkyl group , an aralkyloxy group , a substituted phenyl group , a substituted phenoxy group , a substituted aralkyl group , a substituted aralkyloxy group , — cor 6 , — conr 6 r 6 , — co 2 r 6 , — nhcor 6 , — nh ( oh ), — n ( oh ) cor 6 , — ch 2 or 6 , — och 2 co 2 r 6 , — ch 2 sr 6 , — ch 2 nr 6 r 6 , — sr 6 , — osr 6 , — nr 6 r 6 , — nr 6 so 2 r 6 , where r 6 is hydrogen , ( c 1 - c 10 ) alkyl , ( c 3 - c 10 ) cycloalkyl , — scx 3 in which x is a halogen , — cn , — no 2 or - z - a - z ′- in which z and a are as defined above and z ′ represents carbon , oxygen , sulfur , or nitrogen , wherein r 2 is h , oh , f , br , unsubstituted or substituted , straight or branched ( c 1 - c 5 ) alkyl , ( c 2 - c 5 ) alkenyl , or ( c 2 - c 5 ) alkynyl ; wherein r 4 is h , oh , - a - ar , - a - z - ar , — so 2 — ar , or - a - nr 5 , or — r 6 , where a is an ( c 1 - c 8 ) alkyl group , which is unsubstituted or substituted by a straight or branched ( c 1 - c 5 ) alkyl chain ; z is carbon , oxygen , sulfur or nitrogen ; ar is a phenyl group , a pyridyl group , a naphthyl group , a pyrimidyl group , or a quinolyl group , each of which may contain heteroatoms and may be unsubstituted or substituted by one to five substituents selected from the group consisting of hydrogen , halogen , a hydroxy group , a carboxylic acid group , ( c 1 - c 10 ) alkyl , ( c 2 - c 10 ) alkenyl , ( c 2 - c 10 ) alkynyl , ( c 1 - c 10 ) haloalkyl , ( c 1 - c 10 ) alkoxy , ( c 2 - c 10 ) alkenyloxy , ( c 2 - c 10 ) alkynyloxy , ( c 1 - c 10 ) haloalkoxy , a phenyl group , a phenoxy group , an aralkyl group , an aralkyloxy group , a substituted phenyl group , a substituted phenoxy group , a substituted aralkyl group , a substituted aralkyloxy group , — cor 5 , — cor 6 , — conr 5 r 6 , — co 2 r 5 , — nhcor 5 , — nh ( oh ), — n ( oh ) cor 5 , — chor 5 , — och 2 co 2 r 5 , — ch 2 sr 5 , — ch 2 nr 5 r 6 , — sr 5 , — osr 5 , — o 2 nr 5 r 6 , — nr 5 r 6 , — nr 5 so 2 r 6 , in which r 5 and r 6 are the same or different and each is hydrogen , ( c 1 - c 10 ) alkyl or a ( c 3 - c 10 ) cycloalkyl , — scx 3 in which x is a halogen , — cn , — no 2 or - z - a - z ′- in which z and a are as defined above and z ′ represents carbon , oxygen , sulfur , or nitrogen , wherein r 4 is a photoactivatable moiety , a fluorescent moiety , or a radioactive moiety . in one embodiment , the photoactivatable moiety is a phenylazide , a purine or pyrimidine azides , a diazoacetate , a diazoketone , a nitrobenzene , or an aryldiazonium salt . in another embodiment , the photoactivatable moiety is benzophenone , trifluoromethyldiazirine tetrafluorophenyl , 8 - azidoadenosine , 2 - azidoadenosine , or 3h , 3 - aryldiazirine . in another embodiment , the fluorescent moiety is 5 -( dimemethylamino ) naphthalene - sulfonyl ( dansyl ), 1 -( bromoacetyl ) pyrene , 3 - bromoacetyl - 7 - diethylaminocoumarin , 3 - bromomethyl - 6 , 7 - dimethoxycoumarin , 8 - bromomethyl - 4 , 4 - difluoro - 1 , 3 , 5 , 7 - tetramethyl - 4 - bora - 3a , 4a - diaza - s - indacene , 3 - bromomethyl - 6 , 7 - dimethoxy - 1 - methyl - 2 ( 1h )- quinoxazolinone , 6 - bromoacetyl - 2 - dimethylaminonaphthalene , or 4 -( 9 - anthroyloxy ) phenacyl bromide . in another embodiment , the radioactive moiety is 18 f , 11 c , 3 h . wherein r 4 is h , oh , - a - ar , - a - z - ar , — so 2 — ar , or - a - nr 5 , or — r 6 , where a is a ( c 1 - c 8 ) alkyl group , which is unsubstituted or substituted by a straight or branched ( c 1 - c 5 ) alkyl chain ; z is carbon , oxygen , sulfur or nitrogen ; ar is a phenyl group , a pyridyl group , a naphthyl group , a pyrimidyl group , or a quinolyl group , each of which may contain heteroatoms and may be unsubstituted or substituted by one to five substituents selected from the group consisting of hydrogen , halogen , a hydroxy group , a carboxylic acid group , ( c 1 - c 10 ) alkyl , ( c 2 - c 10 ) alkenyl , ( c 2 - c 10 ) alkynyl , ( c 1 - c 10 ) haloalkyl , ( c 1 - c 10 ) alkoxy , ( c 2 - c 10 ) alkenyloxy , ( c 2 - c 10 ) alkynyloxy , ( c 1 - c 10 ) haloalkoxy , a phenyl group , a phenoxy group , an aralkyl group , an aralkyloxy group , a substituted phenyl group , a substituted phenoxy group , a substituted aralkyl group , a substituted aralkyloxy group , — cor 5 , — cor 6 , — conr 5 r 6 , — co 2 r 5 , — nhcor 5 , — nh ( oh ), — n ( oh ) cor 5 , — chor 5 , — och 2 co 2 r 5 , — ch 2 sr 5 , — ch 2 nr 5 r 6 , — sr 5 , — osr 5 , — o 2 nr 5 r 6 , — nr 5 r 6 , — nr 5 so 2 r 6 , in which r 5 and r 6 are the same or different and each is hydrogen , ( c 1 - c 10 ) alkyl or a ( c 3 - c 10 ) cycloalkyl group , — scx 3 in which x is a halogen , — cn , — no 2 or - z - a - z ′- in which z and a are as defined above and z ′ represents carbon , oxygen , sulfur , or nitrogen , in one embodiment of the above compounds , if any group is substituted , the substituent is halogen , hydroxyl , straight chain ( c 1 - c 5 ) alkyl , branched chain ( c 3 - c 5 ) alkyl , ( c 3 - c 10 ) cycloalkyl , straight chain ( c 1 - c 5 ) alkylcarbonyloxy , branched chain ( c 3 - c 5 ) alkylcarbonyloxy , arylcarbonyloxy , straight chain ( c 1 - c 5 ) alkoxycarbonyloxy , branched chain ( c 3 - c 5 ) alkoxycarbonyloxy , aryloxycarbonyloxy , carboxylate , straight chain ( c 1 - c 5 ) alkylcarbonyl , branched chain ( c 3 - c 5 ) alkylcarbonyl , straight chain ( c 1 - c 5 ) alkoxycarbonyl , branched chain ( c 3 - c 5 ) alkoxycarbonyl , aminocarbonyl , straight chain ( c 1 - c 5 ) alkylthiocarbonyl , branched chain ( c 3 - c 5 ) alkylthiocarbonyl , straight chain ( c 1 - c 5 ) alkoxyl , branched chain ( c 1 - c 5 ) alkoxyl , phosphate , phosphonate , cyano , amino , straight chain ( c 1 - c 5 ) alkylamino , branched chain ( c 3 - c 5 ) alkylamino , straight chain ( c 1 - c 5 ) dialkylamino , branched chain ( c 3 - c 5 ) dialkylamino , arylamino , diarylamino , straight chain ( c 1 - c 5 ) alkylarylamino , branched chain ( c 3 - c 5 ) alkylarylamino , acylamino , straight chain ( c 1 - c 5 ) alkylcarbonylamino , branched chain ( c 3 - c 5 ) alkylcarbonylamino , arylcarbonylamino , carbamoyl , ureido , amidino , imino , sulfhydryl , straight chain ( c 1 - c 5 ) alkylthio , branched chain ( c 3 - c 5 ) alkylthio , arylthio , thiocarboxylate , sulfates , sulfonato , sulfamoyl , sulfonamido , nitro , trifluoromethyl , azido , 4 - 10 membered heterocyclyl , straight chain ( c 1 - c 30 ) alkylaryl , branched chain ( c 3 - c 30 ) alkylaryl , or an aromatic or 5 - 6 membered heteroaromatic moiety , which substituent may be further substituted by any of the above . the subject invention also provides a pharmaceutically acceptable salt of above compounds , wherein the salt is the chloride , mesylate , maleate , fumarate , tartarate , hydrochloride , hydrobromide , esylate , p - toluenesulfonate , benzoate , acetate , phosphate and sulfate salts . the subject invention also provides a pharmaceutical composition comprising a therapeutically effective amount of one of the above compounds and a pharmaceutically acceptable carrier . the subject invention also provides a process for the manufacture of a pharmaceutical composition comprising admixing any of the above compounds with a pharmaceutically acceptable carrier . the subject invention also provides a method of inhibiting activation of the platelet - activating factor receptor ( pafr ) which comprises contacting the pafr with any of the above compounds so as to thereby inhibit activation of the pafr . the subject invention also provides a method of treating a platelet - activating factor ( paf ) associated condition in a subject comprising administering to the subject an amount of any of the above compounds effective to treat the paf - associated disease . in one embodiment , the paf - associated condition is a neurodegenerative disease , alzheimer &# 39 ; s disease , senile dementia , stroke , nerve cell damage due to ischemia , asthma , abnormal blood clot formation , end toxic shock , myocardial ischemia or hyperacute rejection arising from post - renal transplant or xenoperfusion . the subject invention also provides a process of preparing the above compound comprising the steps of : wherein r 1 , r 3 and r 4 are as defined above , with trifluoromethanesulfonic anhydride under inert conditions to form a triflate ; and ii ) reacting the triflate of step i ) with a nucleophilic reagent in a polar - aprotic solvent to substitute the triflate with the nucleophile and to form the compound . in one embodiment of the above process , the nucleophilic reagent is sodium acetate , sodium phenylacetate , sodium azide , tetrabutylammonium fluoride hydrate , or tetrabutylammonium chloride . in another embodiment , the polar - aprotic solvent is dichloromethane , pyridine , dimethylformamide , methylsulfoxide , or acetonitrile . in another embodiment , the nucleophilic reagent is sodium acetate or sodium phenylacetate , further comprising hydrolyzing the product of step ii ) in the presence of 1n hydrochloric acid . in another embodiment , the nucleophilic reagent is sodium azide , further comprising reacting the product of step ii ) with hydrogen in the presence of palladium and carbon in methanol or ethanol . in another embodiment , the nucleophile is tetrabutylammonium fluoride hydrate , further comprising reacting the product of step ii ) with benzyl chloride . the subject invention also provides a process of forming a secondary amine compound from an azide of the compound by contacting the azide of the compound with hydrogen , palladium and carbon in a polar - protic solvent . wherein r 1 , r 3 and r 4 are as defined above and r 9 is an alkyl group , in another embodiment , the alcohol is ch 3 oh and r 9 is ch 3 . in another embodiment , the alcohol is ch 3 ch 2 oh and r 9 is ch 3 ch 2 . the subject invention also provides a process for detecting the binding of compound ( i ) to a target , comprising contacting the compound with the target and detecting the binding of the compound to the target . the subject invention also provides a process for detecting the localization of a receptor in a subject comprising administering compound ( i ) to the subject and detecting at any location in the subject &# 39 ; s body to identify a point of accumulation of the compound so as to thereby localize the receptor in the subject , wherein localization of a receptor means a higher concentration of that receptor then at other points in the subject &# 39 ; s body . the subject invention also provides a process of identifying a target that binds compound ( i ), comprising contacting the compound with the target , isolating the target so as to thereby identify the target . the subject invention also provides a process of identifying a receptor that binds compound ( i ) in a subject , comprising administering the compound to the subject , imaging the subject &# 39 ; s body to identify the point of accumulation of the compound in the subject , and identifying the receptor present at the point of accumulation of the compound , so as to thereby identify the receptor in the subject . the invention also provides a process for detecting the binding of any of the described the compounds to a target , comprising contacting the compound with the target and detecting the binding of the compound to the target the target may be a dna , enzyme or a receptor . the invention also provides a process for detecting the localization of a receptor in a subject comprising administering any of the described compounds to the subject and detecting at any location in the subject &# 39 ; s body to identify a point of accumulation of the compound so as to thereby localize the receptor in the subject , wherein localization of a receptor means a higher concentration of that receptor then at other points in the subject &# 39 ; s body . the invention also provides a process of identifying a target that binds any of the described compounds , comprising contacting the compound with the target and identifying target . the target may be a dna , enzyme or a receptor . the invention also provides a process of identifying a receptor that binds to any of the described compounds in a subject , comprising administering the compound to the subject , imaging the subject &# 39 ; s body to identify the point of accumulation of the compound in the subject , and identifying the receptor present at the point of accumulation of the compound , thereby identifying the receptor in the subject . the photoactivatable moieties react with a receptor , enzyme or other target upon irradiation and enable researchers to identify the targets of compounds , to determine the affinity and selectivity of the drug - target interaction , and to identify the binding site on the target . examples are presented from three fundamentally different approaches : ( 1 ) photoaffinity labeling of target macromolecules ; ( 2 ) photoactivation and release of “ caged ligands ”; and ( 3 ) photoimmobilization of ligands onto surfaces . a number of photoactivatable moieties are described in the literature , for example , aryl azides , which , when photoactivated to yield aryl nitrenes , can label any binding site containing carbon - hydrogen bonds by insertion into the c — h bond ( galardy , et al ., j . biol . chem ., 249 : 350 ( 1974 ); u . s . pat . no . 4 , 689 , 310 ; and u . s . pat . no . 4 , 716 , 122 ); and a number of others are described in u . s . pat . no . 6 , 077 , 698 , the contents of which are hereby incorporated by reference . the photoactivatable groups can be used for treatment as well as screening studies and diagnostics . photoactivatable groups can be used to irreversibly bind compounds to their targets . thus , the subject invention also provides compounds useful in methods of treatment where a desired compound is irreversibly bound to its target . the photoactivatable groups may be phenylazides , purine and pyrimidine azides , 8 - azidoadenosine , 2 - azidoadenosine , diazoacetates , diazoketones , nitrobenzenes , aryldiazonium salts , or 3h , 3 - aryldiazirines . the preferred photoactivatable moieties for use with ginkgolides are benzophenone , trifluoromethyldiazirine and tetrafluorophenyl . the fluorescent moiety , for example , may be 5 -( dimemethylamino ) naphthalene - sulfonyl chloride ( dansyl chloride ), a fluorescent amine such as 1 - pyrenemethylamine , or any number of other groups readily available from molecular probes — http :// www . probes . com /, the contents of which are hereby incorporated by reference . other specific groups which are useful in this invention are 1 -( bromoacetyl ) pyrene , 3 - bromoacetyl - 7 - diethylaminocoumarin , 3 - bromomethyl - 6 , 7 - dimethoxycoumarin , 8 - bromomethyl - 4 , 4 - difluoro - 1 , 3 , 5 , 7 - tetramethyl - 4 - bora - 3a , 4a - diaza - s - indacene , 3 - bromomethyl - 6 , 7 - dimethoxy - 1 - methyl - 2 ( 1h )- quinoxazolinone , 6 - bromoacetyl - 2 - dimethylaminonaphthalene , and 4 -( 9 - anthroyloxy ) phenacyl bromide . radioactive moieties are widely known in the art and include radionuclides , radionuclides covalently attached to other groups , and metal chelates . the appropriate ginkgolide - based radioligands can be prepared using known radioactive moieties to suit the environment of use and detection method . gamma - emitter and positron - emitter radionuclides are well - known in the art and include 111 in , 198 au , 113 ag , 111 ag , 123 i , 125 i , 130 i , 131 i , 133 i , 135 i , 47 sc , 72 as , 72 se , 90 y , 88 y , 97 ru , 100 pd , 109 pd , 105 rh , 128 ba , 197 hg , 203 pb , 212 pb , 67 ga , 68 ga , 64 cu , 67 cr , 97 ru , 75 br , 76 br , 77 br , 99m tc , 11 c , 13 n , 15 o , 3 h and 18 f . for positron emission tomography ( pet ) studies contemplated by this disclosure , ginkgolide derivatives labeled with the radionuclides [ 18 f ]- and [ 11 c ] possessing half lives of 110 min and 20 min , respectively , are preferred . while [ 3 h ] is preferred for other radioactivity based studies . this invention will be better understood from the experimental details which follow . however , one skilled in the art will readily appreciate that the specific , methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter . ginkgolides and bilobalide ( 1 - 6 ) were obtained as previously described ( nakanishi , 1967 ). the syntheses of ginkgolide derivatives are outlined in fig3 - 7 , and details appear below : anhydrous solvents were dried by eluting through alumina columns . triethylamine was freshly distilled from naoh pellets . unless otherwise noted , materials were obtained from a commercial supplier and were used without further purification . all reactions were performed in predried glassware under argon or nitrogen , and all reactions of involving azides or diazirines were performed in dim red light . flash column chromatography was performed using icn silica gel ( 32 - 63 mesh ) or icn silica gel ( 32 - 63 mesh ) impregnated with sodium acetate . [ van beek , t . a . & amp ; lelyved , g . p . ( 1993 ) phytochem . anal . 4 , 109 - 114 ]. thin - layer chromatography was carried out using pre - coated silica gel 60 f 254 plates with thickness of 0 . 25 μm . spots were observed at 254 nm , and by staining with acetic anhydride , or cerium / molybdenum in h 2 so 4 . 1 h and 13 c nmr spectra were obtained on bruker dmx 300 or 400 mhz spectrometers and are reported in parts per million ( ppm ) relative to internal solvent signal , with coupling constants ( j ) in hertz ( hz ). for 19 f nmr spectra hexafluorobenzene (− 162 . 9 ppm ) was used as internal standard . high resolution mass spectra ( hrms ) were measured on a jeol jms - hx110 / 100a hf mass spectrometers using a 3 - nitrobenzyl alcohol ( nba ) matrix and xe ionizing gas . synthesis of 8a - c and 9a - c — general synthetic procedure . gb ( 2 ) or gc ( 3 ) ( 0 . 07 mmol ) was dissolved in thf ( 4 ml ) and kh ( 0 . 008 g , 0 . 24 mmol ) was added at room temperature . the reaction mixture was stirred for 10 min ., when a solution of 7a , 7b , or 7c ( 0 . 212 mmol ) in thf ( 1 ml ) was added dropwise . the reaction was stirred at room temperature for 4 hours . the solution was then cooled to 0 ° c . and concentrated hcl ( 0 . 3 ml ) was added . the mixture was diluted with h 2 o ( 10 ml ), extracted with etoac ( 3 × 10 ml ) and washed with sat . aq . nh 4 cl - solution ( 30 ml ), brine ( 30 ml ) and water ( 30 ml ). the organic phase was dried ( mgso 4 ) and removed in vacuo . purification . the crude material is purified by flash column chromatography using either a : chcl 3 / meoh ( 100 : 1 and 50 : 1 ), b : chcl 3 / meoh ( 30 : 1 and 20 : 1 ), or c : cyclohexane / acetone ( 3 : 1 and 2 : 1 ) giving a white solid . 10 - o - benzophenone ginkgolide b ( 8a ). purified by method b . yield : 0 . 035 g ( 78 %). 1 h nmr ( 400 mhz , cd 3 od ): δ 1 . 13 ( s , tert - butyl ), 1 . 24 ( d , j = 7 . 1 , ch 3 ), 1 . 92 ( dd , j = 14 . 3 , 4 . 5 , 8 - h ), 2 . 07 ( td , j = 13 . 9 , 4 . 4 , 7α - h ), 2 . 27 ( dd , j = 13 . 5 , 4 . 6 , 7β - h ), 3 . 06 ( q , j = 7 . 1 , 14 - h ), 4 . 31 ( d , j = 7 . 2 , 1 - h ), 4 . 55 ( d , j = 7 . 2 , 2 - h ), 4 . 85 ( d , j = 11 . 5 , benzylic - h , 1h ), 5 . 28 ( s , 10 - h ), 5 . 42 ( d , j = 4 . 0 , 6 - h ), 5 . 59 ( d , j = 11 . 5 , benzylic - h , 1h ), 6 . 15 ( s , 12 - h ), 7 . 53 - 7 . 60 ( m , ar — h , 4h ), 7 . 65 - 7 . 67 ( m , ar — h , 1h ), 7 . 77 - 7 . 82 ( m , ar — h , 4h ). 13 c nmr ( 100 mhz , cd 3 od ): δ 7 . 25 , 28 . 46 ( 3c ), 32 . 18 , 37 . 26 , 42 . 29 , 49 . 61 , 68 . 21 , 72 . 59 , 72 . 80 , 74 . 45 , 76 . 76 , 79 . 48 , 83 . 53 , 93 . 15 , 99 . 78 , 110 . 83 , 127 . 96 ( 2c ), 128 . 58 ( 2c ), 130 . 03 ( 2c ), 130 . 52 ( 2c ), 132 . 94 , 137 . 76 ( 2c ), 141 . 67 , 171 . 52 , 172 . 70 , 177 . 33 , 196 . 45 . hrms : c 34 h 34 o 11 requires m + na at m / z 641 . 1999 , found 641 . 2018 . 10 - o -( trifluoromethyl - 3h - diazirine ) benzyl ginkgolide b ( 8b ). purified by method b . yield : 0 . 024 g ( 59 %). 1 h nmr ( 400 mhz , cd 3 od ): δ 1 . 11 ( s , tert - butyl ), 1 . 23 ( d , j = 7 . 1 , ch 3 ), 1 . 89 ( dd , j = 14 . 3 , 4 . 3 , 8 - h ), 2 . 01 ( td , j = 13 . 9 , 4 . 3 , 7α - h ), 2 . 25 ( dd , j = 13 . 4 , 4 . 4 , 7β - h ), 3 . 05 ( q , j = 7 . 1 , 14 - h ), 4 . 27 ( d , j = 7 . 3 , 1 - h ), 4 . 53 ( d , j = 7 . 3 , 2 - h ), 4 . 77 ( d , j = 11 . 2 , benzylic - h , 1h ), 5 . 24 ( s , 10 - h ), 5 . 39 ( d , j = 3 . 9 , 6 - h ), 5 . 51 ( d , j = 11 . 2 , benzylic - h , 1h ), 6 . 14 ( s , 12 - h ), 7 . 29 and 7 . 53 ( aa ′ bb ′ system , ar — h , 4h ). 13 c nmr ( 75 mhz , cdcl 3 ): δ 7 . 67 , 21 . 57 ( q , 2 j cf = 40 . 9 , ccf 3 ), 29 . 56 ( 3c ), 32 . 65 , 37 . 49 , 49 . 31 , 68 . 07 , 72 . 88 , 73 . 57 , 74 . 57 , 76 . 56 , 77 . 65 , 80 . 08 , 83 . 90 , 90 . 90 , 99 . 05 , 110 . 68 , 122 . 33 ( q , 1 j cf = 274 . 3 , cf 3 ), 127 . 83 ( 2c ), 129 . 53 ( 2c ), 131 . 06 , 136 . 44 , 171 . 25 , 171 . 50 , 175 . 87 . 19 f nmr ( 282 mhz , cdcl 3 ): δ - 66 . 23 ( s , 3f ). hrms : c 29 h 29 f 3 n 2 o 10 requires m + 1 at m / z 623 . 1853 , found 623 . 1834 . 10 - o - tetrafluorobenzylazide ginkgolide b ( 8c ). purified by method b . yield : 0 . 023 g ( 50 %). 1 h nmr ( 400 mhz , cdcl 3 ): δ 1 . 13 ( s , tert - butyl ), 1 . 32 ( d , j = 7 . 0 , ch 3 ), 1 . 84 - 1 . 97 ( m , 8 - h and 7α - h ), 2 . 27 - 2 . 33 ( m , 7β - h ), 2 . 84 ( d , j = 3 . 5 , 1 - oh ), 2 . 99 ( s , 3 - oh ), 3 . 06 ( q , j = 7 . 0 , 14 - h ), 4 . 29 ( dd , j = 7 . 9 , 3 . 5 , 1 - h ), 4 . 61 ( d , j = 7 . 9 , 2 - h ), 4 . 81 ( d , j = 10 . 7 , benzylic - h , 1h ), 4 . 94 ( s , 10 - h ), 5 . 39 ( d , j = 3 . 4 , 6 - h ), 5 . 64 ( d , j = 10 . 7 , benzylic - h , 1h ), 6 . 03 ( s , 12 - h ); 13 c nmr ( 100 mhz , cdcl 3 ): δ 7 . 70 , 29 . 52 ( 3c ), 32 . 62 , 37 . 37 , 42 . 03 , 49 . 30 , 61 . 21 , 68 . 11 , 72 . 79 , 74 . 65 , 80 . 07 , 83 . 89 , 91 . 00 , 99 . 12 , 108 . 95 , 110 . 73 , 139 . 71 , 142 . 24 , 144 . 45 , 147 . 10 , 170 . 69 , 171 . 45 , 175 . 83 . 19 f nmr ( 282 mhz , cdcl 3 ) δ − 143 . 31 ( m , 2f ), − 150 . 85 ( m , 2f ). hrms : c 27 h 25 f 4 n 3 o 10 requires m + 1 at m / z 628 . 1554 , found 628 . 1565 . 10 - o - benzophenone ginkgolide c ( 9a ). purified by method a . yield : 0 . 023 g ( 64 %). 1 h nmr ( 400 , mhz , cd 3 od ): δ 1 . 20 ( s , tert - butyl ), 1 . 24 ( d , j = 7 . 1 , ch 3 ,), 1 . 78 ( d , j = 12 . 5 , 8 - h ), 3 . 04 ( q , j = 7 . 1 , 14 - h ), 4 . 21 ( dd , j = 12 . 5 , 4 . 3 , 7 - h ), 4 . 28 ( d , j = 7 . 0 , 1 - h ), 4 . 54 ( d , j = 7 . 0 , 2 - h ), 4 . 87 ( d , j = 11 . 6 , benzylic - h , 1h ), 5 . 13 ( d , j = 4 . 3 , 6 - h ), 5 . 28 ( s , 10 - h ), 5 . 60 ( d , j = 11 . 6 , benzylic - h , 1h ), 6 . 17 ( s , 12 - h ) 7 . 53 - 7 . 61 ( m , ar — h , 4h ), 7 . 65 - 7 . 67 ( m , ar — h , 1h ), 7 . 77 - 7 . 83 ( m , ar — h , 4h ). 13 c nmr ( 100 mhz , cd 3 od ): δ 7 . 34 , 28 . 50 ( 3c ), 32 . 12 , 42 . 26 , 50 . 00 , 64 . 48 , 67 . 40 , 72 . 77 , 74 . 28 , 75 . 14 , 76 . 74 , 79 . 49 , 83 . 55 , 93 . 28 , 99 . 54 , 110 . 63 , 127 . 95 ( 2c ), 128 . 59 ( 2c ), 130 . 03 ( 2c ), 130 . 53 ( 2c ), 132 . 96 , 137 . 68 ( 2c ), 141 . 65 , 171 . 41 , 172 . 55 , 177 . 27 , 197 . 03 . hrms : c 34 h 34 o 12 requires m + 1 at m / z 635 . 2129 , found 635 . 2098 . 10 - o -( trifluoromethyl - 3h - diazirine ) benzyl ginkgolide c ( 9b ). purified by method a . yield : 0 . 023 g ( 51 %). 1 h nmr ( 400 mhz , cd 3 od ): δ 1 . 17 ( s , tert - butyl ), 1 . 24 ( d , j = 7 . 1 , ch 3 ,), 1 . 76 ( d , j = 12 . 5 , 8 - h ), 3 . 02 ( q , j = 7 . 1 , 14 - h ), 4 . 15 ( dd , j = 12 . 5 , 4 . 3 , 7 - h ) 4 . 24 ( d , j = 7 . 0 , 1 - h ), 4 . 52 ( d , j = 7 . 0 , 2 - h ), 4 . 79 ( d , j = 11 . 3 , benzylic - h , 1h ), 5 . 10 ( d , j = 4 . 3 , 6 - h ), 5 . 23 ( s , 10 - h ), 5 . 52 ( d , j = 11 . 3 , benzylic - h , 1h ), 6 . 15 ( s , 12 - h ), 7 . 29 and 7 . 54 ( aa ′ bb ′ system , aromatic - h , 4h ). 13 c nmr ( 75 mhz , cdcl 3 ): δ 7 . 65 , 23 . 77 ( q , 2 j cf = 38 . 9 , ccf 3 ), 29 . 52 ( 3c ), 32 . 65 , 41 . 94 , 50 . 92 , 64 . 43 , 67 . 48 , 73 . 89 , 74 . 30 , 76 . 03 , 76 . 34 , 79 . 63 , 83 . 90 , 90 . 91 , 98 . 94 , 110 . 53 , 122 . 32 ( q , 1 j cf = 275 . 0 , cf 3 ), 127 . 94 ( 2c ), 129 . 68 ( 2c ), 131 . 26 , 136 . 07 , 170 . 97 , 171 . 07 , 175 . 69 . 19 f nmr ( 282 mhz , cdcl 3 ): δ - 66 . 23 ( s , 3f ). hrms : c 29 h 29 f 3 n 2 o 11 , requires m + 1 at m / z 639 . 1802 , found 639 . 1790 . 10 - o - tetrafluorobenzylazide ginkgolide c ( 9c ). purified by method c . yield : 0 . 080 g ( 54 %). 1 h nmr ( 400 mhz , cdcl 3 ): δ 1 . 22 ( s , tert - butyl ), 1 . 33 ( d , j = 7 . 0 , ch 3 ,), 1 . 71 ( d , j = 12 . 4 , 8 - h ), 2 . 33 ( d , j = 10 . 6 , 7 - oh ), 2 . 88 ( d , j = 3 . 4 , 1 - oh ), 3 . 01 ( s , 3 - oh ), 3 . 08 ( q , j = 7 . 0 , 14 - h ), 4 . 08 ( m , 7 - h ) 4 . 27 ( dd , j = 7 . 8 , 3 . 4 , 1 - h ), 4 . 62 ( d , j = 7 . 8 , 2 - h ), 4 . 83 ( d , j = 10 . 7 , benzylic - h , 1h ), 4 . 96 ( s , 10 - h ), 5 . 09 ( d , j = 4 . 4 , 6 - h ), 5 . 58 ( d , j = 10 . 7 , benzylic - h , 1h ), 6 . 04 ( s , 12 - h ). 13 c nmr ( 75 mhz , cdcl 3 ): δ 7 . 64 , 29 . 42 ( 3c ), 32 . 59 , 42 . 08 , 50 . 64 , 51 . 16 , 61 . 47 , 64 35 , 67 . 32 , 74 . 27 , 75 . 88 , 79 . 64 , 83 . 88 , 91 . 26 , 99 . 14 , 110 . 71 , 120 - 150 ( m , 6c ), 170 . 72 , 171 . 17 , 176 . 29 . 19 f nmr ( 282 mhz , cdcl 3 ): δ - 143 . 56 ( m , 2f ), − 151 . 08 ( m , 2f ); hrms : c 27 h 25 f 4 n 3 o 11 , requires m + 1 at m / z 644 . 1503 , found 644 . 1527 . 10 - o - benzophenone - 7 - o - dansyl ginkgolide c ( 10a ). a solution of dansyl chloride ( 0 . 010 g , 0 . 035 mmol ) in acetonitrile ( 0 . 3 ml ) was added to a solution of 9a ( 0 . 020 g , 0 . 032 mmol ) and dmap ( 0 . 008 g , 0 . 063 mmol ) in acetonitrile ( 1 . 5 ml ). the reaction mixture was stirred for 16 h at room temperature , then a sat . aq . nh 4 cl0 - solution ( 2 ml ) was added , and the mixture was extracted with etoac ( 3 × 5 ml ). the combined organic phases were washed with sat . aq . nacl - solution ( 3 × 15 ml ), dried ( mgso 4 ) and removed in vacuo . the crude product was purified by flash column chromatography eluting with cyclohexane / acetone ( 2 : 1 ) to give the product as a slightly yellow solid ( 0 . 015 g , 56 %). 1 h nmr ( 400 mhz , dmso - d 6 ): δ 0 . 83 ( s , tert - butyl ), 1 . 09 ( d , j = 7 . 2 , ch 3 ,), 1 . 94 ( d , j = 12 . 5 , 8 - h ), 2 . 81 [ m , 14 - h and n ( ch 3 ) 2 ], 4 . 26 ( t , j = 5 . 3 , 1 - h ), 4 . 53 ( d , j = 5 . 4 , 2 - h ), 4 . 79 ( d , j = 13 . 2 , benzylic - h , 1h ), 4 . 89 ( dd , j = 12 . 5 , 4 . 0 , 7 - h ), 5 . 19 ( d , j = 4 . 0 , 6 - h ), 5 . 23 ( s , 10 - h ), 5 . 46 ( d , j = 13 . 2 , benzylic - h , 1h ), 6 . 07 ( d , j = 5 . 3 , 1 - oh ), 6 . 21 ( s , 12 - h ), 6 . 51 ( s , 3 - oh ), 7 . 28 - 7 . 30 ( m , ar — h , 1h ), 7 . 50 - 7 . 70 ( m , ar — h , 7h ), 7 . 79 - 7 . 82 ( m , ar — h , 4h ), 8 . 18 - 8 . 20 ( m , ar — h , 2h ), 8 . 54 - 8 . 56 ( m , ar — h , 1h ); ). hrms : c 46 h 45 no 14 s requires m + 1 at m / z 868 . 2639 , found 868 . 2642 . 10 - o - benzophenone - 1 - o - dansyl ginkgolide c ( 10b ). synthesized as 10a , but using 2 equivalents of dansyl chloride ( instead of 1 . 1 equivalent ) give rise to a 1 : 1 mixture of 10a and 10b . the two products were separated on analytical tlc giving 10b ( 0 . 008 g , 30 %) as a slightly yellow solid . 1 h nmr ( 400 mhz , dmso - d 6 ): δ 0 . 91 ( s , tert - butyl ), 1 . 16 ( d , j = 7 . 6 , ch 3 ,), 1 . 78 ( d , j = 12 . 5 , 8 - h ), 2 . 80 [ s , n ( ch 3 ) 2 ], 2 . 97 ( q , j = 7 . 6 , 14 - h ), 4 . 22 ( d , j = 3 . 8 , 1 - h ), 4 . 26 ( m , 7 - h ), 4 . 57 ( d , j = 3 . 9 , 6 - h ), 4 . 80 ( d , j = 13 . 2 , benzylic - h , 1h ), 5 . 20 ( d , j = 3 . 8 , 2 - h ), 5 . 30 ( s , 10 - h ), 5 . 31 ( d , j = 4 . 9 , 7 - oh ), 5 . 49 ( d , j = 13 . 2 , benzylic - h , 1h ), 5 . 95 ( s , 12 - h ), 5 . 98 ( s , 3 - oh ), 7 . 25 - 7 . 27 ( m , ar — h , 1h ), 7 . 54 - 7 . 79 ( m , ar — h , 11h ), 8 . 18 - 8 . 24 ( m , ar — h , 2h ), 8 . 47 - 8 . 49 ( m , ar — h , 1h );). hrms : c 46 h 45 no 14 s requires m + 1 at m / z 868 . 2639 , found 868 . 2668 . 10 - o - benzoylbenzoic ginkgolide c ( 11 ). 4 - benzoylbenzoic acid ( 0 . 018 g , 0 . 08 mmol ) and 2 ( 0 . 028 g , 0 . 07 mmol ) was dissolved in thf ( 5 ml ), and the mixture cooled to 0 ° c . 1 -[ 3 -( dimethylamino ) propyl ]- 3 - ethylcarbodiimide hcl ( edc ) ( 0 . 018 g , 0 . 092 mmol ) and dmap ( 0 . 002 g , 0 . 01 mmol ) was added , and the reaction mixture stirred at 0 ° c . for 1 h , and continued overnight at room temperature . the solvent was removed in vacuo , the crude product dissolved in etoac ( 20 ml ), and washed with a sat . 5 % nahco 3 - solution ( 20 ml ) and brine ( 20 ml ). the organic fraction was dried ( mgso 4 ) and the solvent evaporated in vacuo . the crude product was purified by flash column chromatography eluting with hexane / etoac ( 2 : 1 ) to give the product as white crystals ( 0 . 026 g , 62 %). 1 h nmr ( 400 mhz , cd 3 od ): δ 1 . 07 ( s , tert - butyl ), 1 . 26 ( d , j = 7 . 1 , ch 3 ), 1 . 98 - 2 . 10 ( m , 8 - h and 7α - h ), 2 . 30 - 2 . 36 ( m , 7β - h ), 3 . 12 ( q , j = 7 . 1 , 14 - h ), 4 . 37 ( d , j = 6 . 5 , 1 - h ), 4 . 55 ( d , j = 6 . 5 , 2 - h ), 5 . 66 ( d , j = 3 . 2 , 6 - h ), 6 . 32 ( s , 10 - h ), 6 . 45 ( s , 12 - h ), 7 . 54 - 7 . 58 ( m , ar — h , 2h ), 7 . 67 - 7 . 69 ( m , ar — h , 1h ), 7 . 80 - 7 . 83 ( m , ar — h , 2h ), 7 . 86 - 7 . 88 ( m , ar — h , 2h ), 8 . 42 - 8 . 44 ( m , ar — h , 2h ). 13 c nmr ( 100 mhz , cd 3 od ): δ 7 . 42 , 28 . 22 ( 3c ), 32 . 16 , 37 . 27 , 42 . 29 , 49 . 42 , 67 . 81 , 70 . 64 , 72 . 74 , 74 . 42 , 79 . 29 , 83 . 64 , 95 . 13 , 100 . 51 , 111 . 12 , 128 . 73 ( 2c ), 129 . 92 ( 2c ), 130 . 17 ( 2c ), 130 . 58 ( 2c ), 131 . 61 , 133 . 41 , 137 . 06 , 142 . 66 , 164 . 56 , 168 . 93 , 171 . 41 , 177 . 33 , 196 . 48 . hrms : c 34 h 31 , o 12 requires m + na at m / z 655 . 1791 , found 655 . 1790 . 7 - trifluoromethanesulfonyloxy ginkgolide b ( 12 ). trifluoromethanesulfonic anhydride ( 0 . 2168 ml , 1 . 281 mmol ) was added dropwise to a cooled solution of ch 2 cl 2 ( 2 . 84 ml ) and pyridine ( 0 . 115 ml , 1 . 405 mmol ) at − 20 ° c . the solution was added dropwise to a solution of 3 ( 0 . 500 g , 1 . 134 mmol ) in pyridine ( 4 . 83 ml ) and the reaction was stirred for 2 hours at − 20 °. the reaction mixture was heated to room temperature and the solvent was in vacuo . the residue was dissolved in etoac ( 30 ml ) and washed with 1m hcl ( 30 ml ), and an aqueous , saturated nacl solution ( 30 , ml ). the organic phase was dried ( mgso 4 ), then treated with activated carbon and filtered through celite . the solvent was removed in vacuo and remaining solid was precipitated from heptane / methyl tert - butyl ether ( 2 : 1 ) to give a colorless solid . the solid was purified by flash column chromatography eluting with chcl 3 / meoh / etoac ( 30 : 1 : 1 , 20 : 1 : 1 , 10 : 1 : 1 ) give the product as white crystals ( 0 . 61 g , 93 %). 1 h nmr ( 400 mhz , dmso - d 6 ): δ 1 . 11 ( s , tert - butyl ), 1 . 13 ( d , j = 9 . 6 hz , ch 3 ,), 2 . 22 ( d , j = 16 . 5 , 8 - h ), 2 . 82 ( q , j = 9 . 6 hz , 14 - h ), 4 . 15 ( dd , j = 8 . 0 , 5 . 9 hz , 1 - h ), 4 . 73 ( d , j = 8 . 0 hz , 2 - h ), 5 . 08 ( d , j = 7 . 4 hz , 10 - h ), 5 . 24 ( dd , j = 16 . 5 , 5 . 6 hz , 7 - h ) 5 . 41 ( d , j = 5 . 6 hz , 6 - h ), 5 . 54 ( d , j = 5 . 9 hz , 1 - oh ), 6 . 20 ( s , 12 - h ), 6 . 62 ( s , 3 - oh ), 7 . 63 ( d , j = 7 . 4 hz , 10 - oh ). 13 c nmr ( 100 mhz , dmso - d 6 ): δ 9 . 1 , 29 . 2 ( 3c ), 32 . 6 , 42 . 1 , 49 . 0 , 64 . 2 , 68 . 1 , 69 . 4 , 74 . 4 , 75 . 2 , 84 . 0 , 86 . 3 , 93 . 2 , 99 . 9 , 109 . 4 , 118 . 6 ( q , 1 j cf = 316 . 6 , cf 3 ), 173 . 9 , 176 . 9 , 179 . 2 . hrms : c 21 h 23 f 3 o 13 s requires m + 1 at m / z 573 . 0890 , found 573 . 0872 . 7 - fluoro ginkgolide b ( 13 ). 7 - trifluoromethanesulfonyloxy - ginkgolide b ( 12 ) ( 0 . 035 g , 0 . 061 mmol ) and tetrabutylammonium fluoride hydrate ( 0 . 038 g , 0 . 145 mmol ) were dissolved in acetonitrile ( 0 . 2 ml ) and heated at 80 ° c . for 30 min . the crude product was applied on an hplc semi - preparative c18 column ( 7 . 8 mm × 30 cml ) with a mobile phase of meoh / h 2 o ( 40 : 60 ) at a flow of 2 ml / min . the hplc purified product was purified by flash column chromatography eluting with chcl 3 / meoh ( 25 : 1 ) to give the final product as a white solid ( 0 . 016 g , 60 %). 1 h nmr ( 400 mhz , cd 3 od ): δ 1 . 25 ( s , tert - butyl ), 1 . 26 ( d , j = 7 . 1 , ch 3 ), 1 . 94 ( dd , j hf = 45 . 5 , j = 2 . 3 , 8 - h ), 3 . 05 ( q , j = 7 . 1 , 14 - h ), 4 . 24 ( d , j = 8 . 0 , 1 - h ), 4 . 59 ( d , j = 8 . 0 , 2 - h ), 5 . 18 ( s , 10 - h ), 5 . 35 ( d , j hf = 10 . 9 , 6 - h ), 5 . 38 ( dd , j hf = 48 . 8 , j = 2 . 3 , 7 - h ), 6 . 14 ( s , 12 - h ). 13 c nmr ( 75 mhz , cd 3 od ): δ 7 . 0 , 29 . 5 ( 3 c ), 32 . 9 , 42 . 4 , 53 . 7 ( 2 j cf = 20 . 4 ), 68 . 8 , 69 . 1 , 71 . 5 , 74 . 5 , 79 . 5 ( 2 j cf = 36 . 0 ), 83 . 7 , 91 . 7 , 96 . 9 ( 1 j cf = 184 . 2 ), 98 . 8 , 111 . 6 , 171 . 2 , 173 . 9 , 177 . 1 . hrms : c 20 h 23 fo 10 requires m + 1 at m / z 443 . 1354 , found 443 . 1370 . 10 - o - methyl ginkgolide b ( 14 ). to a suspension of ginkgolide b ( 0 . 030 g , 0 . 071 mmol ) and k 2 co 3 ( 0 . 030 g , 0 . 212 mmol ) in acetonitrile ( 0 . 5 ml ) was added iodomethane ( 0 . 020 g , 0 . 141 mmol ): the reaction mixture was heated under reflux for 30 min . this reaction resulted in a mixture of 10 - methoxy - ginkgolide b and 1 - methoxy ginkgolide b in a ratio of 4 to 1 . 10 - methoxy - ginkgolide b was separated using an hplc semi - preparative c18 column ( 7 . 8 mm × 30 cml ) with a mobile phase of meoh / h 2 0 ( 40 : 60 ) at a flow of 2 ml / min to give the final product as a white solid ( 0 . 017 g , 56 %). 1 h nmr ( 400 mhz , cd 3 od ): δ 1 . 14 ( s , tert - butyl ), 1 . 24 ( d , j = 7 . 1 , ch 3 ), 1 . 90 ( dd , j = 14 . 2 , 4 . 6 , 8 - h ), 2 . 06 ( td , j = 13 . 9 , 4 . 3 , 7α - h ), 2 . 25 ( dd , j = 13 . 6 , 4 . 6 , 7β - h ), 3 . 05 ( q , j = 7 . 1 , 14 - h ), 3 . 80 ( s , och 3 ), 4 . 24 ( d , j = 7 . 4 , 1 - h ), 4 . 57 ( d , j = 7 . 4 , 2 - h ), 4 . 93 ( s , 10 - h ), 5 . 43 ( d , j = 4 . 1 , 6 - h ), 6 . 10 ( s , 12 - h ). 13 c nmr ( 75 mhz , cd 3 od ): δ 7 . 6 , 7 . 0 , 29 . 6 ( 3 c ), 32 . 9 , 37 . 6 , 41 . 8 , 48 . 8 , 68 . 6 , 70 . 5 , 71 . 5 , 74 . 4 , 80 . 4 , 82 . 2 , 84 . 8 , 90 . 9 , 98 . 2 , 110 . 3 , 171 . 1 , 173 . 0 , 175 . 4 . [ hu , l ., chen , z ., xie , y ., jiang , y ., & amp ; zhen , h . ( 2000 ) bioorg . med . chem . 8 , 1515 - 1521 ] 10 - o -( 2 - fluoroethyl ) ginkgolide b ( 15 ). ginkgolide b ( 2 ) ( 0 . 031 g , 0 . 073 mmol ) and 1 - bromo - 2 - fluoroethane ( 0 . 102 g , 0 . 803 mmol ) were dissolved in acetonitrile / dmf ( 4 : 1 0 . 25 ml ) and heated at 80 ° c . for 30 min in the presence of tetrabutylammonium hydroxide ( 1m in meoh , 0 . 125 ml ). the crude product was a mixture of 10 - fluoroethoxy ginkgolide b and 1 - fluoroethoxy ginkgolide b in a 5 to 1 ratio ( as determined by 1 h nmr ). the crude product was applied on an hplc semi - preparative c18 column ( 7 . 8 mm i . d .× 30 cml ) with a mobile phase of meoh / h 2 0 ( 50 : 50 ) at a flow of 2 ml / min the hplc purified product was purified by flash column chromatography eluting with chcl 3 / meoh ( 50 : 1 and 25 : 1 ) to give the final product as a white solid ( 0 . 022 g , 63 %). 1 h nmr ( 400 mhz , cd 3 od ): δ 1 . 14 ( s , tert - butyl ), 1 . 25 ( d , j = 7 . 1 , ch 3 ), 1 . 93 ( dd , j = 14 . 3 , 4 . 7 , 8 - h ), 2 . 10 ( td , j = 14 . 0 , 4 . 3 , 7α - h ), 2 . 28 ( dd , j = 13 . 6 , 4 . 7 , 7β - h ), 3 . 05 ( q , j = 7 . 1 , 14 - h ), 3 . 90 - 4 . 01 ( m , 1h , fch 2 ch 2 o ), 4 . 27 ( d , j = 7 . 4 , 1 - h ), 4 . 53 - 4 . 78 ( m , 3h , fch 2 ch 2 o ), 4 . 60 ( d , j = 7 . 4 , 2 - h ), 5 . 15 ( s , 10 - h ), 5 . 45 ( d , j = 4 . 1 , 6 - h ), 6 . 13 ( s , 12 - h ). 13 c nmr ( 75 mhz , cd 3 od ): δ 7 . 1 , 28 . 4 ( 3 c ), 32 . 1 , 37 . 1 , 42 . 3 , 49 . 6 , 68 . 1 , 70 . 4 ( 2 j cf = 21 . 0 ), 72 . 7 , 74 . 5 , 77 . 8 ( 1 j cf = 182 . 6 ), 81 . 7 , 83 . 4 , 83 . 9 , 92 . 9 , 99 . 6 , 110 . 8 , 171 . 5 , 172 . 6 , 177 . 3 . hrms : c 22 h 27 fo 10 requires m + 1 at m / z 471 . 1667 , found 471 . 1687 . initially , nbu 4 nb 3 h 4 was synthesized as follows : nab 3 h 4 ( 4 . 69 mg , 100 μmol specific activity 100 ci / mmol , total activity 100 mci ) and naoh ( 0 . 20 mg , 5 μmol ) is dissolved in 3 h 2 o ( 100 μl , specific activity 5 ci / g , total activity 500 mci ) in a vial . nbu 4 ncl ( 18 . 53 mg , 66 . 7 μmol ) in 3 h 2 o ( 100 μl , specific activity 5 ci / g , total activity 500 mci ) is added and the reaction mixture is stirred for 1 min . ch 2 cl 2 ( 500 μl ) is added to the vial , the mixture is shaken , the water layer is removed , mgso 4 is added and the suspension stirred . the ch 2 cl 2 solution is filtered through mgso 4 ( in a syringe ) into vial , and the solvent is removed by flowing nitrogen over the solution , and heating . then , the synthesis of [ 3 h ]- ginkgolide b followed ( fig7 ). nbu 4 nb 3 h 4 ( 1 . 1 mg ) in dry thf ( 20 μl ) is added to a solution of 7 - triflate - ginkgolide c ( 14 . 5 mg , in dry thf ( 100 μl ) precooled to 0 ° c . the mixture is stirred for 1 . 5 h at room temperature . meoh ( 25 μl ) is added , the mixture is shaken , and the solvent removed by flowing nitrogen over the solution , and heating . the residue is dissolved in acetonitrile ( 50 μl ) and a mixture of h 2 0 / ch 3 cn ( 1 : 1 , 50 μl ), and the solution is injected into the ( preparative ) hplc ( the product has a retention time of ca . 9 . 6 min .). fractions is collected every 30 s ( until 8 min .) then every 20 s , and an aliquot ( 5 μl ) is taken into a scintillation vial , scintillation liquid is added and the vials is placed in a scintillation counter . fractions corresponding to the peak of [ 3 h ]- gb are collected and diluted with water , and passed through a c18 sep - pak column , that is washed with water and [ 3 h ]- gb is eluted with absolute ethanol into a vial , and the solvent is removed to give the product as a white solid ( total activity 2 . 4 mci , specific activity 3 . 8 mci / μmol ). bilobalide ( 6 ) derivatives . bilobalide derivatives having modifications at the 10 - oh corresponding position are prepared following the procedures used herein to prepare ginkgolide derivatives having modifications at the 10 - oh position . derivatization of bilobalide ( bb , 6 ) is performed as follows : for the synthesis of derivatives with variation at c - 7 , a useful intermediate was 7β - otf - gb ( 16 ). gc ( 2 ) reacted with high selectivity at 7 - oh with trifluoromethanesulfonic ( tf ) anhydride giving 16 in very high yield , with no reactions occurring at other hydroxyl groups ( teng , b .- p ., u . s . pat . no . 5 , 599 , 950 ). this selectivity is noteworthy , as 10 - oh , and in some cases 1 - oh , of gc ( 2 ) is generally the more reactive hydroxyl group ( u . s . pat . no . 5 , 541 , 183 ; hu , 2000 ), although we recently observed higher reactivity of 7 - oh when acetylation was performed under strong acidic conditions ( jaracz , 2002 ). 7β - otf - gb ( 16 ) was reacted with various nucleophiles as depicted in scheme 1 to give derivatives 17 - 22 . the inverted configuration at c - 7 was reflected by considerable changes in coupling constants in 1 h nmr spectra , i . e ., 3 j 7 , 8 and 3 j 6 , 7 are 12 and 4 hz in gc ( 2 ), whereas they are 3 - 5 hz and ca . 0 hz , respectively , when the configuration at c - 7 is inverted . the reactions shown in scheme 1 generally proceeded in good yield , but in several other cases the nucleophilic substitution did not proceed as expected . when reacting with a soft nucleophile such as nascn only starting material was recovered . increase in the basicity of the nucleophiles , as in nacn and aliphatic amines , resulted in a complex mixture of products , probably due to reaction at c - 11 , as previously described ( hu , 2001 ). in addition to the presence of multiple electrophilic sites in 16 it is believed that the steric hindrance of the bulky tert - butyl group , which is in close proximity to the reaction site , is responsible for lack of reaction . this assumption is corroborated by reaction of 16 with halogens ; incorporation of fluorine ( 7α - f - gb , 21 ) proceeded in high yield , chlorine ( 7α - cl - gb , 22 ) in slightly lower yield , whereas the larger bromine was introduced in trace amounts only , while no iodine product could be detected . these results were not affected by changing the solvent or the halide counterion . steric hindrance may also be the prerequisite for two remarkable products arising from reaction of triflate 16 with meoh and nascoch 3 , respectively ( scheme 2 ). in the former case 16 was dissolved in meoh and 2 , 6 - lutidine , and reacted for three days at 70 ° c . expecting to provide 7α - ome - gb ; instead a new product with a molecular weight similar to gc ( 2 ), but with a different 1 h nmr spectrum was obtained . extensive nmr studies revealed a new relactonized structure , neoginkgolide c ( 23 ) ( scheme 2 ), arising from opening of lactone c , followed by displacement of the triflate group . the structure of this compound 23 with a new rearranged ginkgolide skeleton not encountered earlier was determined by high resolution mass spectrometry , rotating frame nuclear overhauser and exchange spectroscopy ( roesy ), correlation spectroscopy ( cosy ) and heteronuclear single - quantum coherence ( hsqc ) nmr experiments ( see supporting information ). moreover , treatment of compound neoginkgolide c ( 23 ) with 1 m naoh followed by acidic work - up resulted in a clean conversion to the thermodynamically more favorable 7 - epi - gc ( 18 ). the reaction between triflate 16 and nascoch 3 did not give the expected 7α - scoch 3 - gb , but instead the 10 - acetate , while the 7 - triflate group remained intact to give 24 ( scheme 2 ). this product might arise from a reaction by thioacetate at c - 11 , followed by a transfer of the acetate to 10 - oh , tautomerization to thionic acid and relactonization to give the final product ( scheme 2 ). another interesting feature was the reduction of azide 20 using pd / c in meoh under hydrogen . the reaction did not provide the expected amine , but instead gave n - methylamine 25 in quantitative yield ( table 3 ). to investigate this further , the reaction was carried out in etoh , which gave n - ethyl amine 26 as the major product . the desired primary amine 27 was obtained when thf was used as solvent ( table 3 ). this intriguing reaction might provide a convenient way to convert azides directly into various alkylamines , an aspect which is under further investigation . for the synthesis of 7 - epi - gc ( 18 ) ( scheme 1 ) various approaches were attempted ; gc ( 2 ) and 4 - nitrobenzoic acid was treated with diethyl azodicarboxylate ( dead ) and ph 3 p in a mitsunobu reaction , but no reaction was observed . instead , 7β - otf - gb ( 16 ) was reacted with kno 2 and 18 - crown - 6 ether in a reaction that could potentially lead to 7 - epi - gc ( 18 ) directly from 16 ( moriarty , 1993 ), but only starting material was recovered . instead , the inversion of 7 - oh of gc ( 2 ) was accomplished using acetate as the nucleophile , followed by basic hydrolysis of the acetate . acetylation of 7β - otf - gb ( 16 ) was achieved by reaction with naoac ; attempts to use the more reactive csoac led to decomposition of 16 , while using csoccf 3 did not lead to any reaction . the hydrolysis was accomplished by treating 17 with 2n naoh to give 7 - epi - gc ( 18 ) in 95 % yield ( scheme 1 ). to further investigate the importance of stereochemistry at c - 7 , we planned a series of corresponding 7β - substituted derivatives . thus 7 - epi - gc ( 18 ) was reacted with tf anhydride , but only starting material was recovered . this lack of reaction is most likely due to a change in steric environment of 7α - oh , relative to 7β - oh , due to the tert - butyl group . finally , the observation that an aromatic substituent at 10 - oh of gb ( 1 ) and gc ( 2 ) increases the antagonistic effect at pafr ( u . s . pat . no . 5 , 541 , 183 ; hu , 2000 ; strømgaard , 2002 ) led us to investigate whether a similar increase would be observed for 7α - gb derivatives . benzylated derivatives 28 - 31 ( table 5 ) were therefore prepared , following previously described procedures ( u . s . pat . no . 5 , 541 , 183 ; hu , 2000 ). gb ( 1 ) and gc ( 2 ) was obtained by extraction of leaves from g . biloba , purification by column chromatography and recrystallization as previously described ( lichtblau , 2002 ; van beek , 1997 ). the purity was & gt ; 98 % as estimated by 1 h nmr . unless otherwise noted , materials were obtained from a commercial supplier and were used without further purification . solvents were dried by eluting through alumina columns . flash column chromatography was performed using icn silica gel ( 32 - 63 mesh ). thin - layer chromatography was carried out using pre - coated silica gel 60 f 254 plates with thickness of 0 . 25 mm . plates were heated and spots were detected by monitoring at 254 nm . 1 h and 13 c nmr spectra were obtained on bruker dmx 300 mhz or bruker dmx 400 mhz spectrometers and are reported in parts per million ( ppm ) relative to internal solvent signal , with coupling constants ( j ) in hertz ( hz ). hsqc , cosy and roesy spectra were obtained on a bruker dmx 400 mhz or bruker dmx 500 mhz spectrometer . analytical and preparative high performance liquid chromatography ( hplc ) were performed on a hp 1100 lc instrument with detection by uv at 219 and 254 nm . preparative hplc was performed using a 10 μm c18 reversed - phase vydac column ( 250 × 22 mm ) with a flow of 4 ml / min and eluting with either eluent a or b . a : water / ch 3 cn / tfa ( 60 : 40 : 0 . 1 ), raising to ( 40 : 60 : 1 ) after 20 min . b : water / ch 3 cn / tfa ( 65 : 35 : 0 . 1 ), raising to ( 40 : 60 : 1 ) after 20 min . analytical hplc were performed using a 5 μm c18 reversed - phase phenomenex luna column ( 150 × 4 . 60 mm ), with a flow of 1 ml / min eluting with water / ch 3 cn / tfa 70 : 30 : 0 . 1 . compounds 18 and 23 were eluted with water / ch 3 cn / tfa 80 : 20 : 0 . 10 and compounds 25 - 27 with water / ch 3 cn 90 : 10 . accurate mass determinations were performed on a jeol jms - hx110 / 100a hf mass spectrometer using a 3 - nitrobenzyl alcohol ( nba ) matrix and xe ionizing gas , and are within ± 10 ppm of theoretical values . all were crystalline compounds that decompose above 200 ° c . 7 - trifluoromethanesulfonyloxy ginkgolide b ( 16 ). in a mixture of dry ch 2 cl 2 ( 1 . 0 ml ) and dry pyridine ( 1 . 5 ml ) gc ( 2 ) ( 184 mg , 0 . 42 mmol ) was dissolved . the solution was cooled to − 20 ° c . under argon and trifluoromethanesulfonic anhydride ( 78 μl ) was added dropwise . the reaction was stirred at − 20 ° c . for 2 h and allowed to warm to room temperature over 1 h . the solvent was removed in vacuo , and the residue was dissolved in etoac ( 30 ml ) and washed with 1 n hcl ( 3 × 20 ml ), brine ( 10 ml ), dried ( mgso 4 ) and the solvent removed in vacuo . the crude product was purified by flash chromatography eluting with chcl 3 / ch 3 oh / etoac ( 30 : 1 : 1 and 20 : 1 : 1 ) to obtain 3 as white crystals ( 232 mg , 97 %). 1 h nmr ( 400 mhz , dmso - d 6 ): δ 1 . 11 ( s , tert - butyl ), 1 . 13 ( d , j = 9 . 6 , ch 3 ), 2 . 22 ( d , j = 16 . 5 , 8 - h ), 2 . 82 ( q , j = 9 . 6 , 14 - h ), 4 . 15 ( dd , j = 8 . 0 , 5 . 9 , 1 - h ), 4 . 73 ( d , j = 8 . 0 , 2 - h ), 5 . 08 ( d , j = 7 . 4 , 10 - h ), 5 . 24 ( dd , j = 16 . 5 , 5 . 6 , 7 - h ) 5 . 41 ( d , j = 5 . 6 , 6 - h ), 5 . 54 ( d , j = 5 . 9 , 1 - oh ), 6 . 20 ( s , 12 - h ), 6 . 62 ( s , 3 - oh ), 7 . 63 ( d , j = 7 . 4 , 10 - oh ). 13 c nmr ( 100 mhz , dmso - d 6 ): δ 9 . 1 , 29 . 2 ( 3c ), 32 . 6 , 42 . 1 , 49 . 0 , 64 . 2 , 68 . 1 , 69 . 4 , 74 . 4 , 75 . 2 , 84 . 0 , 86 . 3 , 93 . 2 , 99 . 9 , 109 . 4 , 118 . 6 ( q , 1 j cf = 316 . 6 , cf 3 ), 173 . 9 , 176 . 9 , 179 . 2 . hrms : c 21 h 23 f 3 o 13 s requires m + 1 at m / z 573 . 0890 ; found , 573 . 0872 . 7α - o - acetate ginkgolide b ( 17 ). sodium acetate ( 163 mg , 1 . 99 mmol ) and 16 ( 228 mg , 0 . 39 mmol ) were dissolved in dmso ( 3 ml ) and the solution was stirred at 65 ° c . for 17 h . the solvent was removed in vacuo , and the residue was partitioned between 1 n hcl ( 20 ml ) and etoac ( 25 ml ). the aqueous phase was extracted with etoac ( 3 × 25 ml ) and the combined organic phases were washed with brine ( 2 × 10 ml ), dried ( mgso 4 ), and the solvent removed in vacuo . the crude product was purified by flash chromatography eluting with chcl 3 / etoac / meoh ( 10 : 1 : 1 ) to obtain 17 as white crystals ( 144 mg , 75 %). a portion ( 20 mg ) of this was recrystallized ( meoh / h 2 o ) for pharmacological evaluation ( 9 mg ). 1 h nmr ( 400 mhz , dmso - d 6 ): δ 1 . 12 ( d , j = 7 . 1 , ch 3 ), 1 . 10 ( s , tert - butyl ), 1 . 91 ( d , j = 3 . 3 , 8 - h ), 2 . 06 ( s , coch 3 ), 2 . 84 ( q , j = 7 . 1 , 14 - h ), 4 . 03 ( dd , j = 7 . 7 , 3 . 6 , 1 - h ), 4 . 66 ( d , j = 7 . 7 , 2 - h ), 4 . 86 ( d , j = 3 . 6 , 1 - oh ), 5 . 01 ( s , 6 - h ), 5 . 15 ( d , j = 6 . 8 , 10 - h ), 5 . 25 ( d , j = 3 . 3 , 7 - h ), 6 . 16 ( s , 12 - h ), 6 . 52 ( s , 3 - oh ), 7 . 42 ( d , j = 6 . 8 , 10 - oh ). 13 c nmr ( 100 mhz , cd 3 od ): δ 8 . 0 , 21 . 0 , 30 . 7 ( 3c ), 33 . 8 , 43 . 4 , 53 . 2 , 70 . 1 , 70 . 3 , 72 . 5 , 75 . 3 , 79 . 6 , 80 . 9 , 84 . 6 , 92 . 6 , 99 . 8 , 112 . 7 , 171 . 3 , 171 . 5 , 175 . 0 , 178 . 1 . hplc - uv : 96 %. hrms : c 22 h 27 o 12 requires m + 1 at m / z 483 . 1503 ; found , 483 . 1525 . 7α - o - phenylacetate ginkgolide b ( 19 ). sodium phenylacetate ( 44 mg , 0 . 28 mmol ) and 16 ( 32 mg , 0 . 07 mmol ) were dissolved in dmso ( 0 . 8 ml ) and heated at 65 ° c . for 5 h and the solvent was removed in vacuo , and the residue was partitioned between 1 n hcl ( 10 ml ) and etoac ( 15 ml ) and the aqueous phase was extracted with etoac ( 3 × 15 ml ). the combined organic phases were washed with 1 n hcl ( 2 × 10 ml ), water ( 5 × 10 ml ) and brine ( 2 × 10 ml ), dried ( mgso 4 ) and the solvent removed in vacuo . the crude product was purified by flash column chromatography eluting with chcl 3 / meoh / etoac ( 30 : 1 : 1 ), recrystallized ( meoh ) and further purified by preparative hplc ( eluent a ) to give 5 ( 10 mg , 26 %) as white crystals . 1 h nmr ( 400 mhz , dmso - d 6 ): δ 1 . 07 ( s , tert - butyl ), 1 . 12 ( d , j = 6 . 9 , ch 3 ), 1 . 93 ( d , j = 2 . 9 , 8 - h ), 2 . 84 ( q , j = 6 . 9 , 14 - h ), 3 . 70 ( s , ch 2 ), 4 . 04 ( dd , j = 3 . 6 , 7 . 7 , 1 - h ), 4 . 62 ( d , j = 7 . 7 , 2 - h ), 4 . 72 ( d , j = 3 . 6 , 1 - oh ), 4 . 99 ( s , 6 - h ), 5 . 16 ( d , j = 6 . 6 , 10 - h ), 5 . 26 ( d , j = 2 . 9 , 7 - h ), 6 . 16 ( s , 12 - h ), 6 . 48 ( s , 3 - oh ), 7 . 25 - 7 . 35 ( m , aromatic , 5h ) 7 . 46 ( d , j = 6 . 6 , 10 - oh ). 13 c nmr ( 100 mhz , cd 3 od ): δ 8 . 0 , 30 . 8 ( 3c ), 33 . 8 , 42 . 2 , 43 . 4 , 53 . 3 , 70 . 1 , 70 . 3 , 72 . 5 , 75 . 2 , 80 . 2 , 81 . 0 , 84 . 6 , 92 . 6 , 99 . 9 , 112 . 7 , 128 . 4 , 129 . 7 , 130 . 5 , 134 . 7 , 171 . 4 , 172 . 2 , 175 . 0 , 178 . 1 . hplc - uv : 98 %. hrms : c 28 h 31 o 12 requires m + h at m / z 559 . 1816 ; found , 559 . 1826 . 7α - azido ginkgolide b ( 20 ). sodium azide ( 87 mg , 1 . 34 mmol ) and 16 ( 153 mg , 0 . 27 mmol ) were dissolved in dmso ( 2 . 5 ml ) and the solution was heated at 65 ° c . for 26 h . the solvent was removed in vacuo . the solid was partitioned between saturated aqueous nh 4 cl ( 20 ml ) and etoac ( 20 ml ) and the aqueous phase was extracted with etoac ( 3 × 20 ml ). the combined organic phases were washed with brine ( 2 × 10 ml ), dried ( mgso 4 ), and the solvent removed in vacuo . the crude product was purified by flash chromatography eluting with chcl 3 / meoh / etoac ( 30 : 1 : 1 ) to give the 20 as white crystals ( 109 mg , 88 %). a portion ( 23 mg ) of this was recrystallized ( meoh / h 2 o ) for pharmacological evaluation ( 12 mg ). 1 h nmr ( 300 mhz , dmso - d 6 ): δ 1 . 12 ( d , j = 7 . 1 , ch 3 ), 1 . 13 ( s , tert - butyl ), 1 . 80 ( d , j = 4 . 0 , 8 - h ), 2 . 73 ( q , j = 7 . 1 , 14 - h ), 4 . 05 ( dd , j = 7 . 6 , 3 . 6 , 1 - h ), 4 . 70 ( d , j = 7 . 6 , 2 - h ), 4 . 74 ( d , j = 4 . 0 , 7 - h ), 4 . 97 ( d , j = 3 . 6 , 1 - oh ), 5 . 06 ( d , j = 6 . 0 , 10 - h ), 5 . 25 ( s , 6 - h ), 6 . 10 ( s , 12 - h ), 6 . 52 ( s , 3 - oh ), 7 . 05 ( d , j = 6 . 0 , 10 - oh ). 13 c nmr ( 100 mhz , dmso - d 6 ): δ 7 . 8 , 30 . 1 ( 3c ), 32 . 7 , 41 . 6 , 51 . 6 , 67 . 2 , 68 . 4 , 68 . 5 , 71 . 0 , 73 . 7 , 79 . 6 , 82 . 9 , 92 . 9 , 98 . 2 , 110 . 3 , 169 . 5 , 173 . 4 , 176 . 2 . hplc - uv : 99 %. hrms : c 20 h 24 o 10 n 3 requires m + 1 at m / z 466 . 1462 ; found , 466 . 1445 . 7α - fluoro ginkgolide b ( 21 ). tetrabutylammonium fluoride hydrate ( 37 mg , 0 . 14 mmol ) and 16 ( 62 mg , 0 . 11 mmol ) were dissolved in ch 3 cn ( 1 ml ) and heated at 80 ° c . for 1 . 5 h . the solvent was removed in vacuo , and the residue was partitioned between 1 n hcl ( 10 ml ) and etoac ( 15 ml ) and the aqueous phase was extracted with etoac ( 3 × 15 ml ). the combined organic phases were washed with water ( 2 × 15 ml ), brine ( 2 × 15 ml ), dried ( mgso 4 ), and the solvent removed in vacuo . the crude product was purified by flash column chromatography eluting with chcl 3 / meoh / etoac ( 30 : 1 : 1 ) followed by preparative hplc ( eluent b ) to give 21 as white crystals ( 34 mg , 71 %). 1 h nmr ( 400 mhz , cd 3 od ): δ 1 . 25 ( s , tert - butyl ), 1 . 26 ( d , j = 7 . 1 , ch 3 ), 1 . 94 ( dd , 2 j hf = 45 . 5 , j = 2 . 3 , 8 - h ), 3 . 05 ( q , j = 7 . 1 , 14 - h ), 4 . 24 ( d , j = 8 . 0 , 1 - h ), 4 . 59 ( d , j = 8 . 0 , 2 - h ), 5 . 18 ( s , 10 - h ), 5 . 35 ( d , 2 j hf = 10 . 9 , 6 - h ), 5 . 38 ( dd , 1 j hf = 48 . 8 , j = 2 . 3 , 7 - h ), 6 . 14 ( s , 12 - h ). 13 c nmr ( 75 mhz , cd 3 od ): δ 7 . 0 , 29 . 5 ( 3c ), 32 . 9 , 42 . 4 , 53 . 7 ( 2 j cf = 20 . 4 hz ), 68 . 8 , 69 . 1 , 71 . 5 , 74 . 5 , 79 . 5 ( 2 j cf = 36 . 0 hz ), 83 . 7 , 91 . 7 , 96 . 9 ( 1 j cf = 184 . 2 hz ), 98 . 8 , 111 . 6 , 171 . 2 , 173 . 9 , 177 . 1 . hrms : c 20 h 23 fo 10 requires m + 1 at m / z 443 . 1354 ; found , 443 . 1370 . 7α - chloro ginkgolide b ( 22 ). tetrabutylammonium chloride ( 86 mg , 0 . 31 mmol ) and 16 ( 36 mg , 0 . 06 mmol ) were dissolved in ch 3 cn ( 1 . 4 ml ) and heated at 80 ° c . for 12 h . the solvent was removed in vacuo and the residue partitioned between 1 n hcl ( 20 ml ) and etoac ( 20 ml ). the aqueous phase was extracted with etoac ( 3 × 20 ml ). the combined organic phases were washed with water ( 4 × 10 ml ) and brine ( 2 × 10 ml ), dried ( mgso 4 ), and the solvent removed in vacuo . the crude product was purified by preparative hplc ( eluent b ) and recrystallized ( ch 3 cn / chcl 3 ) to give 22 as white crystals ( 9 mg , 30 %). 1 h nmr ( 400 mhz , dmso - d 6 ): δ 1 . 12 ( d , j = 7 . 0 , ch 3 ), 1 . 17 ( s , tert - butyl ), 2 . 19 ( d , j = 4 . 2 , 8 - h ), 2 . 85 ( q , j = 7 . 0 , 14 - h ), 4 . 03 ( dd , j = 7 . 7 , 3 . 3 , 1 - h ), 4 . 63 ( d , j = 3 . 3 , 1 - oh ), 4 . 68 ( d , j = 7 . 8 , 2 - h ), 4 . 84 ( d , j = 4 . 2 , 7 - h ), 5 . 13 ( d , j = 6 . 4 , 10 - h ), 5 . 26 ( s , 6 - h ), 6 . 17 ( s , 12 - h ), 6 . 53 ( s , 3 - oh ), 7 . 57 ( d , j = 6 . 4 , 10 - oh ). 13 c nmr ( 100 mhz , cd 3 od ): δ 8 . 7 , 31 . 2 ( 3c ), 34 . 5 , 42 . 5 , 54 . 2 , 65 . 1 , 69 . 0 , 70 . 0 , 72 . 2 , 74 . 4 , 83 . 7 , 84 . 2 , 90 . 7 , 99 . 4 , 111 . 3 , 169 . 9 , 174 . 4 , 177 . 1 . hplc - uv : 98 %. hrms : c 20 h 24 o 10 cl requires m + 1 at m / z 459 . 1058 ; found , 459 . 1052 . neoginkgolide c ( 23 ). triflate 16 ( 27 mg , 0 . 05 mmol ) was dissolved in dry meoh ( 470 μl ) and 2 , 6 - lutidine ( 150 μl ) was added and the reaction mixture was heated at 65 ° c . for 3 days . the solvent was removed in vacuo and the residue purified by flash chromatography eluting with chcl 3 / meoh / etoac ( 20 : 1 : 1 ) to give the crude product , which was further purified by preparative hplc ( eluent a ) to give 23 as white crystals ( 6 mg , 29 %). 1 h nmr ( 400 mhz , cd 3 od ): δ 1 . 20 ( m , ch 3 and tert - butyl ), 1 . 64 ( dd , j = 1 . 4 , 1 . 2 , 8 - h ), 3 . 72 ( q , j = 7 . 1 , 14 - h ), 4 . 46 ( d , j = 8 . 0 , 2 - h ), 4 . 59 ( d , j = 1 . 2 , 10 - h ), 4 . 72 ( d , j = 8 . 0 , 1 - h ), 5 . 00 ( dd , j = 1 . 4 , 1 . 3 , 7 - h ), 5 . 14 ( d , j = 1 . 3 , 6 - h ), 5 . 96 ( s , 12 - h ). 13 c nmr ( 100 mhz , dmso - d 6 ): δ 7 . 6 , 30 . 2 ( 3c ), 32 . 7 , 41 . 3 , 47 . 8 , 60 . 2 , 66 . 9 , 67 . 8 , 73 . 6 , 75 . 6 , 82 . 5 , 83 . 4 , 92 . 7 , 93 . 7 , 104 . 9 , 170 . 2 , 171 . 7 , 177 . 6 . hplc - uv : 98 %. hrms : c 20 h 24 o 11 , requires m + na at m / z 463 . 1216 ; found , 463 . 1245 . 10 - o - acetate - 7 - trifluoromethanesulfonyloxy ginkgolide b ( 24 ). potassium thioacetate ( 4 mg , 0 . 035 mmol ) and 16 ( 3 mg , 0 . 006 mmol ) were dissolved in dry dmf ( 35 μl ) and heated at 40 ° c . for 3 h . the solvent was removed in vacuo , and the residue was partitioned between water ( 10 ml ) and etoac ( 15 ml ) and the aqueous phase was extracted with etoac ( 3 × 15 ml ). the combined organic phases were washed with water ( 5 × 10 ml ) and brine ( 2 × 10 ml ), dried ( mgso 4 ), and the solvent removed in vacuo . the crude product was purified by flash chromatography eluting with chcl 3 / meoh / etoac ( 20 : 1 : 1 ) to give 24 ( 1 . 3 mg , 18 %) as white crystals . 1 h nmr ( 400 mhz , dmso - d 6 ): δ 1 . 08 ( s , tert - butyl ), 1 . 13 ( d , j = 7 . 1 , ch 3 ), 2 . 21 ( s , coch 3 ), 2 . 31 ( d , j = 12 . 6 , 8 - h ), 2 . 85 ( q , j = 7 . 1 , 14 - h ), 4 . 08 ( dd , j = 5 . 9 , 5 . 8 , 1 - h ), 4 . 75 ( d , j = 5 . 9 , 2 - h ), 5 . 09 ( dd , j = 12 . 6 , 4 . 2 , 7 - h ), 5 . 48 ( d , j = 4 . 2 , 6 - h ), 6 . 13 ( s , 10 - h ), 6 . 33 ( s , 12 - h ), 6 . 53 ( s , 3 - oh ), 6 . 71 ( d , j = 5 . 8 , 1 - oh ). hrms : c 23 h 26 o 14 f 3 s requires m + 1 at m / z 615 . 0995 ; found , 615 . 1016 . 7α - n - methylamino ginkgolide b ( 25 ). azide 20 ( 38 mg , 0 . 08 mmol ) was dissolved in dry meoh ( 1 . 2 ml ) and pd / c ( 10 %, 12 mg ) was added . the suspension was stirred under an atmosphere of h 2 for 48 h . the solvent was removed in vacuo and etoac ( 10 ml ) was added and the solution filtered through celite . the solvent was removed in vacuo , and the crude product was purified by flash chromatography eluting with chcl 3 / meoh / etoac ( 30 : 1 : 1 ) to give white crystals , which were recrystallized ( meoh ) to give 25 ( 23 mg , 65 %) as white crystals . 1 h nmr ( 400 mhz , cd 3 od ): δ 1 . 22 ( m , tert - butyl and ch 3 ), 1 . 89 ( d , j = 4 . 4 , 8 - h ), 2 . 47 ( s , ch 3 ), 3 . 06 ( q , j = 7 . 0 , 14 - h ), 3 . 46 ( d , j = 4 . 4 , 7 - h ), 4 . 24 ( d , j = 7 . 2 , 1 - h ), 4 . 53 ( d , j = 7 . 2 , 2 - h ), 5 . 05 ( s , 6 - h ), 5 . 31 ( s , 10 - h ), 6 . 17 ( s , 12 - h ). 13 c nmr ( 100 mhz , cd 3 od ): δ 8 . 2 , 31 . 3 ( 3c ), 33 . 4 , 34 . 3 , 43 . 3 , 53 . 5 , 69 . 1 , 69 . 6 , 70 . 8 , 72 . 6 , 75 . 4 , 79 . 3 , 84 . 4 , 94 . 5 , 100 . 6 , 112 . 2 , 172 . 6 , 174 . 8 , 178 . 4 . hplc - uv : 97 %. hrms : c 21 h 28 o 10 n requires m + 1 at m / z 454 . 1713 ; found , 454 . 1719 . 7α - n - ethylamino ginkgolide b ( 26 ). azide 20 ( 48 mg , 0 . 10 mmol ) was dissolved in dry etoh ( 1 . 0 ml ) and pd / c ( 10 %, 15 mg ) was added . the suspension was stirred under an atmosphere of h 2 for 48 h . the solvent was removed in vacuo and etoac ( 10 ml ) was added and the solution filtered through celite . the solvent was removed in vacuo and the residue purified by flash chromatography eluting with chcl 3 / meoh / etoac ( 30 : 1 : 1 ) to give white crystals , which were recrystallized ( meoh ) to give 26 ( 22 mg , 47 %). 1 h nmr ( 300 mhz , cd 3 od ): δ 1 . 11 ( t , j = 7 . 1 , ch 3 ), 1 . 23 ( m , tert - butyl and ch 3 ), 1 . 89 ( d , j = 4 . 5 , 8 - h ), 2 . 57 ( dq , j = 7 . 1 , 12 . 0 , ch 2 , 1h ), 2 . 94 ( dq , j = 7 . 1 , 12 . 0 , ch 2 , 1h ), 3 . 06 ( q , j = 7 . 1 , 14 - h ), 3 . 56 ( d , j = 4 . 5 , 7 - h ), 4 . 22 ( d , j = 7 . 3 , 1 - h ), 4 . 53 ( d , j = 7 . 3 , 2 - h ), 5 . 08 ( s , 6 - h ), 5 . 27 ( s , 10 - h ), 6 . 16 ( s , 12 - h ). 13 c nmr ( 100 mhz , cd 3 od ): δ 8 . 2 , 15 . 5 , 31 . 3 ( 3c ), 34 . 4 , 41 . 6 , 43 . 3 , 53 . 5 , 67 . 5 , 69 . 2 , 70 . 8 , 72 . 6 , 75 . 3 , 80 . 0 , 84 . 4 , 94 . 4 , 100 . 6 , 112 . 3 , 172 . 6 , 174 . 9 , 178 . 4 . hplc - uv : 98 %. hrms : c 22 h 29 o 10 n requires m + 1 at m / z 468 . 1870 ; found , 468 . 1867 . 7α - amino ginkgolide b ( 27 ). azide 20 ( 10 mg , 0 . 02 mmol ) was dissolved in dry thf ( 0 . 4 ml ) and pd / c ( 10 %, 8 mg ) was added . the suspension was stirred under an atmosphere of h 2 for 14 h . etoac ( 10 ml ) was added and the solution filtered through celite . the solvent was removed in vacuo to give white crystals which were recrystallized ( meoh ) to give 27 as white crystals ( 5 mg , 49 %). 1 h nmr ( 400 mhz , cd 3 od ) δ 1 . 20 ( s , tert - butyl ), 1 . 23 ( d , j = 7 . 1 , ch 3 ), 1 . 90 ( d , j = 3 . 2 , 8 - h ), 3 . 08 ( q , j = 7 . 1 , 14 - h ), 3 . 83 ( d , j = 3 . 2 , 7 - h ), 4 . 26 ( d , j = 7 . 0 , 1 - h ), 4 . 51 ( d , j = 7 . 0 , 2 - h ), 5 . 01 ( s , 6 - h ), 5 . 04 ( s , 10 - h ), 6 . 18 ( s , 12 - h ). 13 c nmr ( 100 mhz , cd 3 od ): δ 8 . 3 , 31 . 0 ( 3c ), 34 . 1 , 43 . 2 , 54 . 7 , 60 . 3 , 68 . 9 , 70 . 9 , 72 . 6 , 74 . 9 , 83 . 9 , 84 . 4 , 95 . 0 , 100 . 3 , 111 . 8 , 172 . 3 , 174 . 4 , 178 . 4 . hplc - uv : 97 %. hrms : c 42 h 26 o 10 n requires m + h at m / z 440 . 1557 ; found , 440 . 1594 . 7 - epi - ginkgolide c ( 18 ). acetate 17 ( 42 mg , 0 . 087 mmol ) was dissolved in a mixture of meoh and 2 n naoh ( 2 : 1 , 1 . 8 ml ) and stirred for 5 h . 1 n hcl was added and the aqueous phase was extracted with etoac ( 3 × 20 ml ). the combined organic phases were washed with brine ( 2 × 10 ml ), dried ( mgso 4 ), and the solvent removed in vacuo to give 18 ( 36 mg , 95 %) as white crystals . a portion ( 15 mg ) of this was recrystallized ( meoh / h 2 o ) for pharmacological evaluation ( 6 mg ). 1 h nmr ( 400 mhz , dmso - d 6 ): δ 1 . 12 ( d , j = 7 . 0 , ch 3 ), 1 . 12 ( s , tert - butyl ), 1 . 64 ( d , j = 2 . 7 , 8 - h ), 2 . 89 ( q , j = 7 . 0 , 14 - h ), 4 . 11 ( dd , j = 4 . 5 , 6 . 8 , 1 - h ), 4 . 37 ( dd , j = 2 . 7 , 6 . 3 , 7 - h ), 4 . 59 ( d , j = 6 . 8 , 2 - h ), 4 . 98 ( s , 6 - h ), 5 . 04 ( d , j = 2 . 7 , 10 - h ), 5 . 52 ( d , j = 6 . 3 , 7 - oh ), 5 . 64 ( d , j = 4 . 5 , 1 - oh ), 6 . 13 ( s , 12 - h ), 6 . 45 ( s , 3 - oh ), 6 . 73 ( d , j = 2 . 7 , 10 - oh ). 13 c nmr ( 75 mhz , dmso - d 6 ): δ 8 . 0 , 30 . 2 ( 3c ), 32 . 6 , 41 . 4 , 52 . 3 , 68 . 8 , 69 . 6 , 71 . 5 , 73 . 4 , 76 . 2 , 80 . 8 , 82 . 8 , 92 . 9 , 98 . 6 , 109 . 7 , 170 . 0 , 172 . 7 , 176 . 3 . hplc - uv : 98 %. hrms : c 20 h 25 o 11 requires m + 1 at m / z 441 . 1397 ; found , 441 . 1395 . 10 - o - benzyl ginkgolide b ( 28 ). synthesis and analytical data as previously described ( park , p .- u ., et al . ; hu , l ., et al ., 2000 ). 10 - o - benzyl ginkgolide c ( 29 ). k 2 co 3 ( 31 mg , 0 . 22 mmol ) was added to a solution of 2 ( 12 mg , 0 . 02 mmol ) dissolved in dmf ( 0 . 2 ml ) followed by addition of benzyl chloride ( 30 μl , 0 . 26 mmol ). the suspension was stirred for 2 . 5 h at 60 ° c . the solvent was removed in vacuo , and the residue partitioned between 1 n hcl ( 10 ml ) and etoac ( 15 ml ) and the aqueous phase was extracted with etoac ( 3 × 15 ml ). the combined organic phases were washed with water ( 2 × 10 ml ) and brine nacl ( 2 × 10 ml ), dried ( mgso 4 ), and the solvent removed in vacuo . the crude product was purified by flash chromatography eluting with chcl 3 / meoh / etoac ( 20 : 1 : 1 ) and further by preparative hplc ( solvent system a ) to give 16 ( 9 mg , 77 %) as white crystals . 1 h nmr ( 400 mhz , cd 3 od ): δ 1 . 21 ( s , tert - butyl ), 1 . 23 ( d , j = 7 . 1 , ch 3 ), 1 . 76 ( d , j = 12 . 5 , 8 - h ), 3 . 01 ( q , j = 7 . 1 , 14 - h ), 4 . 13 ( dd , j = 12 . 3 , 4 . 3 , 7 - h ), 4 . 19 ( d , j = 7 . 4 , 1 - h ), 4 . 49 ( d , j = 7 . 4 , 2 - h ), 4 . 76 ( d , j = 10 , 2 , ch 2 , 1h ), 5 . 04 ( s , 6 - h ), 5 . 02 ( d , j = 4 . 3 , 6 - h ), 5 . 25 ( s , 10 - h ), 5 . 46 ( d , j = 10 . 2 , ch 2 , 1h ), 6 . 14 ( s , 12 - h ), 7 . 37 - 7 . 44 ( m , aromatic , 5h ). 13 c nmr ( 75 mhz , cdcl 3 ): δ 7 . 2 , 29 . 1 ( 3c ), 32 . 2 , 41 . 6 , 50 . 5 , 64 . 1 , 67 . 1 , 73 . 8 , 74 . 3 , 75 . 6 , 77 . 2 , 79 . 3 , 83 . 5 , 90 . 6 , 98 . 5 , 110 . 1 , 128 . 9 ( 2c ), 129 . 5 ( 2c ), 129 . 8 , 134 . 2 , 170 . 8 , 170 . 8 , 175 . 5 . hplc - uv : 98 %. hrms : c 27 h 30 o 11 requires m + na at m / z 553 . 1686 ; found , 553 . 1684 . 10 - o - benzyl - 7α - fluoro ginkgolide b ( 30 ). synthesized as described for 29 using k 2 co 3 ( 107 mg , 0 . 77 mmol ), 21 ( 34 mg , 0 . 08 mmol ), and benzyl chloride ( 89 μl , 0 . 77 mmol ) in dmf ( 1 . 8 ml ). the crude product was purified by flash chromatography eluting with chcl 3 / meoh / etoac ( 30 : 1 : 1 ) to give 30 ( 16 mg , 39 %) as white crystals . 1 h nmr ( 400 mhz , cdcl 3 ): δ 1 . 25 ( s , tert - butyl ), 1 . 30 ( d , j = 7 . 0 , ch 3 ), 1 . 88 ( dd , 2 j hf = 44 . 5 , j = 2 . 3 , 8 - h ), 2 . 94 ( d , j = 3 . 1 , 1 - oh ), 3 . 06 ( q , j = 7 . 0 , 14 - h ), 4 . 28 ( dd , j = 8 . 1 , 3 . 1 , 1 - h ), 4 . 50 ( d , j = 8 . 1 , 2 - h ), 4 . 68 ( d , j = 9 . 4 , ch 2 , 1h ), 4 . 95 ( s , 10 - h ), 5 . 29 ( d , 2 j hf = 10 . 2 , 6 - h ), 5 . 30 ( dd , 1 j hf = 50 . 1 , j = 1 . 5 , 7 - h ), 5 . 41 ( d , j = 9 . 4 , ch 2 , 1h ), 6 . 04 ( s , 12 - h ), 7 . 36 ( m , aromatic , 5h ). 13 c nmr ( 100 mhz , cdcl 3 ): δ 7 . 2 , 30 . 3 ( 3c ), 32 . 9 , 41 . 7 , 53 . 3 ( 2 j cf = 20 . 4 ), 68 . 2 , 71 . 6 , 74 . 1 , 74 . 4 , 75 . 1 , 79 . 5 ( 2 j cf = 35 . 6 ), 83 . 6 , 90 . 2 , 96 . 0 ( 1 j cf = 184 . 2 ), 98 . 2 , 110 . 9 , 128 . 7 ( 2c ), 129 . 1 ( 2c ), 129 . 3 , 134 . 8 , 170 . 2 , 170 . 8 , 175 . 1 . hplc - uv : 98 %. hrms : c 27 h 30 o 10 f . requires m + 1 at m / z 533 . 1823 ; found , 533 . 1784 . 10 - o - benzyl - 7 - epi - ginkgolide c ( 31 ). synthesized as described for 29 using k 2 co 3 ( 50 mg , 0 . 36 mmol ), 18 ( 16 mg , 0 . 04 mmol ), and benzyl chloride ( 42 μl , 0 . 36 mmol ) in dmf ( 0 . 3 ml ). the crude product was purified by flash chromatography eluting with chcl 3 / meoh / etoac ( 20 : 1 : 1 ) and further by preparative hplc ( eluent a ) to give 31 ( 11 mg , 56 %) as white crystals . 1 h nmr ( 400 mhz , cdcl 3 ): δ 1 . 23 ( s , tert - butyl ), 1 . 29 ( d , j = 7 . 0 , ch 3 ), 1 . 83 ( d , j = 3 . 1 , 8 - h ), 2 . 60 ( d , j = 3 . 6 , 1 - oh ), 2 . 66 ( d , j = 11 . 0 , 7 - oh ), 3 . 06 ( q , j = 7 . 0 , 14 - h ), 3 . 40 ( bs , 3 - oh ), 4 . 28 ( dd , j = 3 . 6 , 7 . 8 , 1 - h ), 4 . 48 ( m , 2 - h , 7 - h ), 4 . 72 ( d , j = 9 . 2 , ch 2 , 1h ), 4 . 96 ( s , 6 - h ), 5 . 51 ( s , 10 - h ), 5 . 50 ( d , j = 9 . 2 , ch 2 , 1h ), 6 . 09 ( s , 12 - h ), 7 . 39 - 7 . 44 ( m , aromatic , 5h ). 13 c nmr ( 100 mhz , cdcl 3 ): δ 7 . 3 , 30 . 6 ( 3c ), 33 . 1 , 41 . 6 , 52 . 9 , 68 . 4 , 71 . 3 , 74 . 0 , 74 . 4 , 74 . 7 , 77 . 6 , 82 . 3 , 83 . 2 , 90 . 7 , 98 . 5 , 110 . 5 , 129 . 2 ( 2c ), 129 . 6 ( 2c ), 130 . 1 , 133 . 8 , 170 . 3 , 170 . 5 , 175 . 4 . hplc - uv : 99 %. hrms : c 27 h 31 o 11 , requires m + 1 at m / z 531 . 1866 ; found , 531 . 1895 . radioligand binding assay . the radioligand binding assays were performed as previously described ( shindou , 2000 ). in brief , membrane fractions from hearts and skeletal muscles of pafr - tg mice ( 50 μl suspension containing 121 fmol of pafr ) were mixed with 2 pmol of [ 3 h ]- web in 50 μl of buffer [ 25 mm hepes / naoh ( ph 7 . 4 ), 0 . 25 m sucrose , 10 mm mgcl 2 , 0 . 1 % bsa ], and the compound to be tested in 100 μl of buffer in a 96 - well microplate in triplicate for each concentration . these mixtures were incubated at 25 ° c . for 90 min , upon which the receptor - bound [ 3 h ]— web 2086 was filtered and washed with cold buffer . the plates were then dried at 50 ° c . for at least 90 min ., 25 μl of microscint - 0 scintillation cocktail was added , and filters were placed in a topcount microplate scintillation counter . binding data were analyzed with the nonlinear curve - fitting program microplate manager iii ( bio - rad , hercules , calif .). calculated ic 50 values were then converted to k i values using the cheng - prusoff correction ( cheng , 1973 ), with the following equation : k i = ic 50 /( 1 +[ l ]/ k d ), where [ l ] is the concentration of the radioligand , and k d is the previously determined dissociation constant for [ 3 h ]- web 2086 ( 4 . 3 nm ) ( shindou , 2000 ). non - specific binding was determined using methods as previously described ( shindou , 2000 ). synthesis . a series of photoactivatable gb ( 2 ) and ginkgolide c ( gc , 3 ) derivatives were synthesized . the design of gb derivatives 8a - c and gc derivatives 9a - c ( fig3 ) was based on previous sar studies of ginkgolides which demonstrated that bulky aromatic substituents in the 10 - oh position of gb ( 2 ) increases activity at the pafr ( park , 1996 ; hu , 1999 ; hu , 2000 ). three different photoactivatable moieties , benzophenone , trifluoromethyldiazirine and tetrafluorophenyl azide ( see 7a - c , fig3 ) were chosen as they have been described as being among the most successful for labeling receptors and enzymes ( dorman , 2000 ; flemming , 1995 ; kotzyba - hilbert , 1995 ). most importantly , upon irradiation these photoactivatable groups react with the receptor via different intermediates , namely , a radical , a carbene or a ( singlet ) nitrene for the benzophenone ( 7a ), trifluoromethyldiazirine ( 7b ) and tetrafluorophenyl azide ( 7c ) moieties , respectively ( dorman , 2000 ). since it is essentially impossible to predict which group will be most readily incorporated into the receptor , use of these different groups increases the likelihood of a successful incorporation . preparation of gb derivatives 8a - c and gc derivatives 9a - c was performed by reacting gb ( 2 ) and gc ( 3 ) with 4 -( bromomethyl ) benzophenone ( 7a ), 3 -( 4 - bromomethylphenyl )- 3 - trifluoromethyl - 3h - diazirine ( 7b ) and 1 - azido - 4 -( bromomethyl )- 2 , 3 , 5 , 6 - tetrafluorobenzene ( 7c ), respectively ( fig3 ). benzophenone 7a was commercially available , whereas trifluoromethyldiazirine 7b ( nassal , 1983 ; nassal , 1984 ) and tetrafluorophenyl azide 7c ( keana , 1990 ; lei , 1998 ; lei , 2000 ) ( fig5 ) were synthesized , respectively , in 3 and 7 steps essentially as previously described . ginkgolides gb ( 2 ) and gc ( 3 ) were derivatized almost exclusively at 10 - oh when potassium hydride ( kh ) was used as base , as was previously shown for gb ( 2 ) ( park , 1996 ), whereas other bases were less selective , giving rise to products derivatized at 1 - oh as well . generally , the position of the substituent was determined from the coupling systems of the appropriate protons in dmdo - d 6 , as well as by cosy nmr spectra . the relative chemical shift of 12 - h in dmso - d 6 can also be used in differentiating 1 - and 10 - oh substitutions ( hu , 2000 ). gc derivatives 9a - c can be reacted further to incorporate fluorescent groups ; for example , benzophenone derivative 9a was reacted with one equivalent of 5 -( dimemethylamino ) naphthalene - sulfonyl chloride ( dansyl chloride ), to give 10 - o - benzophenone - 7 - o - dansyl gc ( 10a ) with almost exclusive reaction at 7 - oh ( fig4 ). interestingly , increasing the amount of dansyl chloride to two equivalents gave 10 - o - benzophenone - 1 - o - dansyl gc ( 10b ) as well as ( 10a ) in a 1 : 1 ratio . the coupling of gb ( 2 ) with 4 - benzoylbenzoic acid using 1 -[ 3 -( dimethylamino ) propyl ]- 3 - ethylcarbodiimide hcl ( edc ) and 4 - dimethylaminopyridine ( dmap ) occurred exclusively at 10 - oh to give 10 - benzophenonecarbonyl gc ( 11 ) in good yield ( fig5 ). in 10 - benzophenonecarbonyl gc ( 11 ) the photoactivatable benzophenone moiety , as in the case of 8a and 9a , is linked to the ginkgolide skeleton through an ester linkage . upon incorporation into the receptor , the ester group can be aminolysed with a fluorescent amine such as 1 - pyrenemethylamine , thus avoiding the use of radioactivity for photolabeling and sequencing ( li , 1999 ). for positron emission tomography ( pet ) studies derivatives labeled with [ 18 f ]- and [ 11 c ] possessing half lives of 110 min and 20 min , respectively , will be used . in the present work , preparation of the corresponding non - radioactive analogs has been performed . compound 13 , a 7 - fluoro analog of gb ( 2 ) which can ultimately be labeled with [ 18 f ], was prepared by nucleophilic substitution of 7 - o - triflate intermediate 12 with tetrabutylammonium fluoride ( tbaf ) ( fig6 ). as expected for nucleophilic substitution , nmr showed the relative stereochemistry at c - 7 to be reversed compared to gc ( 3 ). intermediate 12 was prepared by reaction of gc ( 3 ) with trifluoromethanesulphonic anhydride [( cf 3 so 2 ) 2 o ] with remarkable selectivity for the 7 - oh , as no substitutions at other hydroxyl groups was observed ( teng , 1997 ). two other potential pet - ligands , which can be labeled with [ 11 c ], were prepared by selective reaction of the 10 - oh of gb ( 2 ) with either methyl iodide or 2 - bromoethyl fluoride to give 10 - o - methyl gb ( 14 ) and 10 - o -( 2 - fluoroethyl ) gb ( 15 ), respectively ( fig6 ). upon reacting gb ( 2 ) with methyl iodide , reaction at 1 - oh could not be avoided , the 10 - oh : 1 - oh product ratio being highly sensitive to the use of the appropriate base , e . g ., k 2 co 3 gave primarily the 10 - substituted analog , whereas tetrabutylammonium hydroxide ( tbah ) gave mainly the 1 - substituted derivative . pharmacology . the native terpene trilactones ( 1 - 6 ), as well as ginkgolide derivatives 8a - c , 9a - c , 10a , 10b , 11 and 13 - 15 were tested for their ability to bind to pafr using radioligand binding assays with membrane fractions from hearts and skeletal muscles of pafr transgenic mice ( shindou , 2000 ). initially compounds were tested in concentrations of 5 μm against [ 3 h ]- web 2086 ( fig2 ), a potent , competitive pafr antagonist and [ 3h ]- paf ; the compounds were generally less potent against [ 3 h ]- paf , but the relative potencies were comparable with the two radioligands . the degree of non - specific binding was determined to be ca . 50 % for [ 3h ]- paf and less than 5 % for [ 3 h ]- web 2086 . accordingly , the assays were performed using [ 3 h ]- web 2086 rather than [ 3 h ]- paf as the radioligand , mainly due to the high degree of non - specific binding of the latter . all compounds were dissolved in dmso to obtain 5 mm stock solutions of test compounds . examination of the effect of dmso on the binding of [ 3 h ]- web 2086 revealed that up to 1 % dmso ( final concentration ) was acceptable , but 1 - 2 . 5 % resulted in a slight inhibition of [ 3 h ]- web 2086 binding . generally this caused no problem ; however , with very weakly binding compounds the relatively high dmso concentration in solutions above 100 μm had a small inhibitory effect , thus leading to a slight overestimation of their potencies . previous studies have reported problems specifically associated with the solubilization of ginkgolides in dmso ( maclennan , 1996 ), but similar problems were not observed in the present study . native ginkgolides ( 1 - 5 ) and bilobalide ( 6 ) were tested with the cloned pafr ( fig3 a and table 1 ). gb ( 2 ) was the most potent compound with a k i value of 0 . 56 μm , while ga ( 1 ) was slightly less potent with a k i of 1 . 46 μm . gc ( 3 ) and ginkgolide j ( gj , 4 ) were significantly less potent , while ginkgolide m ( gm , 5 ) and bilobalide ( 6 ) both had k i values larger than 50 μm . the gb - derived photoactivatable compounds 8a - c and 11 with k i values in the range 0 . 09 - 0 . 15 μm ( fig3 b and table 2 ) were all more potent than gb ( 2 ), while compounds 9a - c derived from gc ( 3 ) with k i values of 0 . 47 - 0 . 79 μm were equipotent to gb ( 2 ) ( fig3 c and table 2 ), despite the fact that gc ( 3 ) itself is only weakly potent . besides proving that aromatic groups linked to 10 - oh enhance activity in both gb ( 2 ) and gc ( 3 ) derivatives , these results also indicate that the specific type of photoactivatable group was less important . derivatives 10a and 10b possessing a fluorescent dansyl group at either 1 - or 7 - oh were both less potent than 10 - o - benzophenone gc ( 9a ) without the dansyl group ( fig3 d ). however , an important difference was observed in the activities of the two ; the 1 - and 10 - disubstituted analog ( 10b ) was ca . four times more potent than the 7 - and 10 - disubstituted analog ( 10a ) ( table 2 ). finally the potential pet analogs 13 - 15 were tested . the fluorinated analog 13 had a k i value of 0 . 99 μm ( table 2 ), thus being almost equipotent with the native compound gb ( 2 ). the c - 10 derivatized compounds 10 - o - methyl gb ( 14 ) and 10 - o -( 2 - fluoroethyl ) gb ( 15 ) were both significantly less potent than gb ( 2 ) with k i values of 3 . 16 and 4 . 87 μm for 14 and 15 , respectively ( table 2 ). nine analogs ( 8a - c , 9a - c , 10a , 10b and 11 ) with photoactivatable groups , and in the case of 10a and 10b with fluorescent dansyl groups as well , have been prepared from native ginkgolides gb ( 2 ) and gc ( 3 ) by selective derivatizations of the hydroxyl groups . furthermore , we have prepared three analogs ( 13 - 15 ), the radioactive versions of which will be used for pet studies . for the synthesis of 7 - o - triflate intermediate 12 , reaction of gc ( 3 ) with sulfonic anhydride gave rise to remarkable selectivity at 7 - oh ( teng , 1997 ). 10 - o - methyl gb ( 14 ) and 10 - o -( 2 - fluoroethyl ) gb ( 15 ) were synthesized by derivatization of gb ( 2 ), indicating that when the appropriate combination of alkylating agent and base is used , even small aliphatic groups react preferentially at the 10 - oh . generally , the increased reactivity of the 1 - oh and 10 - oh compared to 7 - oh , has been rationalized by hydrogen - bonding between 1 - oh and 10 - oh ( corey , 1992 ), but this does not explain the interesting selectivity for the 10 - oh position in reactions with benzyl bromide derivatives . notably , reaction of gc ( 3 ) with a bulky silyl chloride protection group occurs exclusively at the 1 - oh of gc ( weinges , 1991 ). all native terpene trilactones as well as the derivatized compounds were investigated with respect to their binding to cloned pafr isolated from transgenic mice ( fig3 a ). previous sar studies of pafr antagonism with terpene trilactones and derivatives was performed by monitoring inhibition of paf - induced rabbit platelet aggregation . gb ( 2 ) has generally been reported to be a potent antagonist of the pafr based on the latter assay with an ic 50 , value around 0 . 2 μm ( park , 1996 ; hu , 1999 ; hu , 2000 ). gm ( 5 ) has only been found in the root bark of the g . biloba tree ( nakanishi , 1967 ) and is not readily available . thus , the interaction between pafr and gm ( 5 ) has not previously been reported . the remaining terpene trilactones are all found in the leaf of g . biloba . however 5 , lacking the hydroxyl group at c - 3 , was devoid of pafr binding at the concentrations tested . generally the activities of gc ( 3 ), gj ( 4 ) and gm ( 5 ) with hydroxyl groups at c - 7 , compared to the activity of ga ( 1 ) and gb ( 2 ) lacking the 7 - oh , showing that the 7 - oh is not necessary for binding to pafr , whereas hydroxyl groups at other positions appear to be less important . the study also confirmed that bilobalide ( 6 ), a terpene trilactone with only one five - membered carbocycle and three lactones , is not active in concentrations up to 100 μm ( table 1 ). the seven photolabile analogs , gb derivatives 8a - c and 11 and gc derivatives 9a - c , with aromatic substituents at 10 - oh all improved the affinity to the pafr relative to the activities of gb ( 2 ) and gc ( 3 ) ( table 2 ). this is in agreement with previous sar studies of gb ( 2 ) ( park , 1996 ; hu , 1999 ; hu , 2000 ), as well as a 3d - qsar study on ginkgolides ( chen , 1998 ). however , it is interesting to note that aromatic substitutions at 10 - oh of gc ( 3 ) as in compounds 9a - c , improve the affinity to pafr ca . 20 fold ( fig3 c ) thus making them equipotent to gb ( 2 ), while the same substitutions in gb ( 2 ) increases the affinity only 6 - fold ( fig3 b ). furthermore , the similar affinities of gb derivatives 8a - c ( fig3 b ) and 11 and gc derivatives 9a - c ( fig3 c ), respectively , implies that it is the steric bulk or the lipophilicity of the substituents , rather than the specific functional groups that are important for the increase in affinity ( table 2 ). gc derivatives 10a and 10b ( fig4 ) with dansyl groups at 7 - oh and 1 - oh are less potent and equipotent , respectively to their parent compound , 10 - o - benzophenone - gc ( 9a ) ( fig3 d ). in compound 10a , which is ca . 6 times less active than 9a ( table 2 ), the bulk at the 7 position seems to be responsible for the reduction in affinity . the fact that compound 10b is equipotent to 9a suggests that once a bulky aromatic group occupies this area , further aromatic groups neither increase nor decrease the affinity . the 7 - fluoro gb ( 13 , fig6 ) was essentially equipotent to gb ( 2 ), and ca . 10 times more potent than gc ( 3 ); thus , the [ 18 f ]- labeled analog of 13 could be a useful probe for visualizing the pafr binding sites in mammalian brain . furthermore , 13 could be used for probing interactions of [ 18 f ]- 13 with targets other than the pafr . the substitution at c - 7 of ginkgolides is critical for pafr binding affinity ; generally a β - oh group significantly decreases binding affinity , as in gc ( 3 ), gj ( 4 ) and gm ( 5 ), while substitution with a dansyl group at 7 - oh , as in 10a , reduces this activity further . however , an α - fluorine at c - 7 position has an affinity 10 - fold higher than that of gc ( 3 ) and comparable to that of gb ( 2 ). the inverted stereochemistry at c - 7 , rather than electronic or steric effects of the fluorine substitution , may account for the enhanced activity . others who have made , or purported to make , ginkgolide derivatives with modifications at c - 7 did not appreciate the importance of stereochemistry at the c - 7 position ( pietry , wo 99 / 52911 ; ceazaux , gb 2 , 288 , 599 ; vasella , u . s . pat . no . 6 , 143 , 725 ). the data presented shows the unexpected improvement in pafr binding affinity when the c - 7 substituent is an α - substituent . thus , ginkgolide derivatives having both an α - substituent at c - 7 and a bulky or lipophillic substitution at 10 - oh are expected to have yet further improved activity . the two derivatives bearing alkyl substituents at c - 10 of gb , 10 - o - methyl gb ( 14 ) and 10 - o -( 2 - fluoroethyl ) gb ( 15 ), are both significantly less potent than gb ( 2 ). thus , although the [ 11 c ]- labeled derivatives are not suited for examination of interactions with the pafr , both ligands should be useful for visualizing targets for ginkgolides in the brain , other than pafr . of course , all of the described compounds are expected to be useful for visualizing their targets in the brain , other than pafr . in conclusion , investigation of the effect of terpene trilactones isolated from g . biloba on the cloned pafr have demonstrated that amongst the native compounds , ga ( 1 ) and gb ( 2 ) are the most potent . a series of photoactivatable analogs have been prepared , and pafr binding assays showed that most of these analogs were more potent antagonists than their parent compounds , thus providing promising candidates for studies of the interaction of ginkgolides with the pafr . the gingkolide derivative containing both a photoactivatable and a fluorescent group , compound 10b , retained affinity to pafr , and could therefore be useful in photolabeling and subsequent sequencing studies . finally , the syntheses and assays of analogs that can be radiolabeled and used for pet studies have been described ; in particular the radiolabeled derivative of 7 - fluoro gb ( 13 ) could be a useful probe for in vivo pet studies of the pafr , as well as potential new targets for ginkgolides . radioligand binding assay . the radioligand binding assays were performed as previously described ( shindou , 2000 ). in brief , membrane fractions from hearts and skeletal muscles of pafr - transgenic mice ( 50 μl suspension containing 158 fmol of pafr ) were mixed with 2 pmol of [ 3 h ]- web 2086 in 50 μl of buffer [ 25 mm hepes / naoh ( ph 7 . 4 ), 0 . 25 m sucrose , 10 mm mgcl 2 , 0 . 1 % bsa ], and the compound to be tested in 100 μl of buffer in a 96 - well microplate in triplicate for each concentration . these mixtures were incubated at 25 ° c . for 90 min , upon which the receptor - bound [ 3 h ]- web 2086 was filtered and washed with cold buffer . the filters were then dried at 50 ° c . for at least 90 min ., 25 μl of microscint - 0 scintillation cocktail was added , and filters were placed in a topcount microplate scintillation counter . binding data were analyzed with the nonlinear curve - fitting program microplate manager iii ( bio - rad , hercules , calif .). non - specific binding was determined using methods as previously described ( shindou , 2000 ). pharmacology . derivatives 17 - 23 and 25 - 31 were tested for their ability to displace [ 3 h ]- web 2086 binding to cloned pafr ( tables 2 and 3 ) using membrane fractions from hearts and skeletal muscles of pafr transgenic mice , as previously described ( shindou , 2000 ). in these fractions , gb ( 1 ) had a k i value of 0 . 88 μm , thus being similar to the previously determined k i value of 0 . 56 μm ( strømgaard , 2002 ) derivatives with 7α - substituents were all more potent than gc ( 2 ) ( table 4 ), but within this group of compounds there were marked differences ; 7α - oac , 7α - ocobn , 7α - oh and 7α - nh 2 ginkgolide b derivatives all had k i values between 2 . 4 - 7 . 8 μm , thus being slightly more potent than gc ( 2 ), but still significantly less potent than gb ( 1 ). compounds 20 , 21 , 25 and 26 with 7α - n 3 , 7α - f , 7α - nhme and 7α - nhet substituents , respectively , were equipotent to gb ( 1 ) with k i values in the range of 0 . 55 - 1 . 62 μm . finally , 7α - chloro ginkgolide b ( 22 ) was the most potent compound in this series with a k i value of 0 . 11 μm , thus being the most potent non - aromatic ginkgolide derivative described . the relactonized compound 23 ( scheme 2 ) was also tested for binding to pafr and was found to be essentially inactive with a k i value & gt ; 40 μm . benzyl derivatives were investigated as well ( table 3 ), and as expected a 10 - o - benzyl group significantly improved the affinity for pafr . compounds 28 and 30 were the most potent with k i values of 0 . 12 and 0 . 10 μm , respectively , while 10 - o - benzyl - gc ( 29 ) and 10 - o - benzyl - 7 - epi - gc ( 31 ) were slightly less potent with k i values of 1 . 67 and 0 . 60 μm , respectively . structure - activity relationship ( sar ) studies of ginkgolides on the pafr have primarily focused on gb ( 1 ) ( fig8 ) derivatives ( corey , 1989 ; corey , 1991 ; park , u . s . pat . no . 5 , 541 , 183 ; hu , 1999 ; hu , 2000 ) as outlined in fig8 , e . g . the importance of lactones and the tert - butyl group has been investigated , whereas the effect of stereochemistry of hydroxyl groups remains to be examined . ginkgolide c ( gc , 2 , fig8 ), having a hydroxyl group at c - 7 , is significantly less potent than gb ( 1 ) ( strømgaard , 2002 ), which has been explained by the hydrophilic 7α - oh of gc ( 2 ) being next to the lipophilic tert - butyl group , which is believed to interact with a lipophilic pocket in the pafr ( braquet , 1991 ). moreover , substitution at 7 - oh further decreases antagonistic activity , as demonstrated by 7 - o -( 4 - methylphenyl )- gb that was devoid of pafr activity ( pietri , 2001 ), and a 7 - o - dansyl - gb derivative was also less potent than the parent compound ( strømgaard , 2002 ). preliminary studies showed that 7α - f - gb was equipotent to gb , and 15 - fold more potent as a pafr antagonist as compared to gc ( 2 ) ( suehiro , m ., et al . ), despite the fact that fluorine is sterically equivalent to oh , and more polar than hydrogen ( smart , 2001 ). since configuration of the fluorine atom is α , whereas that of the 7 - hydroxyl in gc ( 2 ) is β , it is not clear whether this difference in activity is due to changes in stereochemistry , steric effects or electronic effects . in the following , we describe a series of ginkgolide derivatives with variation at the critical 7 - position , and the assessment of these derivatives for their ability to displace radioligand binding to cloned pafr . herein the effect of modification of the c - 7 position of ginkgolides has been investigated by synthesis of fifteen analogs ( 17 - 31 ), which have been prepared from native ginkgolides gb ( 1 ) and gc ( 2 ), and evaluated with the cloned pafr . the derivatives with 7α - substituents were prepared by nucleophilic substitution of 7β - otf - gb ( 16 ), but in several cases these reactions did not proceed as expected . attempts to introduce larger halogens such as bromine and iodine , as well as other nucleophiles failed . in the reaction between 16 and nascoch 3 , the 7 - otf group remained intact ; instead the reaction presumably took place at c - 11 of lactone c to give 24 ( scheme 2 ). reaction of 16 with meoh and 2 , 6 - lutidine gave rise to neoginkgolide c ( 23 ) with a novel rearranged skeleton ( scheme 2 ). this compound had a k i value & gt ; 40 μm , which is in agreement with previous studies which showed that modification of lactone c significantly reduced pafr binding ( fig8 ) ( hu , 2001 ). during the reduction of azide 20 interesting observations were made ; when carried out in meoh this reaction did not give the expected primary amine 27 , but gave n - methylamine 25 instead , and when carried out in etoh the reduction gave n - ethylamine 26 . besides being a potential novel procedure for a direct conversion of azides into alkylated amines , it also raises mechanistic considerations . treatment of 27 with pd / c in meoh gave 25 , albeit in lower yield than starting from azide 20 , and with several side products . this implies that in the preparation of 25 and 26 the azide 20 is initially reduced to amine 27 , which then reacts instantly with the oxidized solvent to form an imine , that is reduced to yield the products . further studies should confirm this pathway , as well as the generality of this reaction . the prepared derivatives were tested for binding to cloned pafr ( tables 4 and 5 ). it was observed that 7α - derivatives were slightly more potent than the 7β - derivatives , as gc ( 2 ) had a k i value of 12 . 6 μm , while for 7 - epi - gc ( 18 ) k i is 4 . 26 μm . likewise 7α - oac - gb ( 17 ) had a k i of 7 . 84 μm , while 7β - oac - gb ( i . e ., 7 - oac - gc ) had been shown to have low potency comparable to that of gc ( 2 ) ( jaracz , 2002 ). furthermore , the 7β - derivative 19 is a reasonably potent pafr antagonist with a k i value of 2 . 40 μm ( table 4 ) in contrast to 7β - o -( 4 - methylphenyl )- gb , which is devoid of pafr activity ( pietri , 2001 ). the nature of the 7α - substituent , on the other hand , had a major impact on the binding to pafr . it appears that polarizable substituents at c - 7 lead to increased potency . introduction of azide and fluorine groups yielded compounds that were equipotent to gb ( 1 ) ( table 4 ), while introduction of a chlorine as in 7α - cl - gb ( 22 ) leads to a dramatic increase in binding affinity . thus 22 with k i = 0 . 11 μm , was 115 - fold more potent than gc ( 2 ), and 8 - times more potent than gb ( 1 ), thereby being the most potent non - aromatic ginkgolide derivative described to date . on the other hand , it appears that polar groups that can form hydrogen bonds decrease activity , as seen in the case of a hydroxyl group at c - 7 to give 7 - epi - gc ( 18 ) and an amino group to give 7α - nh 2 - gb ( 27 ), compounds with binding affinities lower than gb . however , alkylation of 27 to give 7α - nhme - gb ( 25 ) and 7α - nhet - gb ( 26 ) led to significant increases in binding affinities , with k i values of 0 . 61 μm and 1 . 62 μm , respectively . it may be that such alkylations of the 7α - amino group sterically disfavors hydrogen bonding . nevertheless , rationalization of these trends requires further developments in ongoing molecular mechanistic studies of the ginkgolide / pafr interaction . introduction of benzyl groups in the 10 - oh position of ginkgolides is known to improve affinity for pafr ( park , p .- u ., et al ., u . s . pat . no . 5 , 541 , 183 ; 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