Ginkgolide derivatives for photolabeling studies: Preparation and pharmacological evaluation

Article abstract of DOI:10.1021/jm020147w

The terpene trilactones (TTLs), ginkgolides and bilobalide, are structurally unique constituents of Ginkgo biloba extracts, which exhibit various neuromodulatory properties. Although the TTLs are believed to be responsible for some of these effects, the s

Full text of DOI:10.1021/jm020147w

4038
J . Med. Chem. 2002, 45, 4038-4046
Gin k golid e Der iva tives for P h otola belin g Stu d ies: P r ep a r a tion a n d
P h a r m a cologica l Eva lu a tion
Kristian Strømgaard,† D. Roland Saito,† Hideo Shindou,‡ Satoshi Ishii,‡ Takao Shimizu,‡ and Koji Nakanishi*,†
Department of Chemistry, Columbia University, 3000 Broadway, New York, New York 10027,
Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku,
Tokyo 113-0033, J apan, and CREST of J apan Science and Technology Corporation, Tokyo 113-0033, J apan
Received April 4, 2002
The terpene trilactones (TTLs), ginkgolides and bilobalide, are structurally unique constituents
of Ginkgo biloba extracts, which exhibit various neuromodulatory properties. Although the
TTLs are believed to be responsible for some of these effects, the specific interactions with
targets in the central nervous system remain to be elucidated on a molecular level. Ginkgolides
are known antagonists of the platelet-activating factor (PAF) receptor. Herein, we describe
the first examination of the binding of native TTLs and their derivatives to the cloned PAF
receptor, confirming that of the TTLs, ginkgolide B is the most potent PAF receptor antagonist.
Ginkgolide derivatives carrying photoactivatable and fluorescent groups for the purpose of
performing photolabeling have been prepared and evaluated using the cloned PAF receptor.
These studies have shown that ginkgolide derivatives with aromatic photoactivatable substit-
uents are potent PAF receptor antagonists with Ki values of 0.09-0.79 µM and hence excellent
ligands for clarifying the binding of ginkgolides to PAF receptor by photolabeling studies.
Ginkgolide derivatives incorporating both fluorescent and photoactivatable groups still retained
binding affinity to the PAF receptor and hence should be promising ligands for photolabeling
and subsequent sequencing studies.
In tr od u ction
Ginkgo biloba L., the last surviving member of a
family of trees (Ginkgoacea) that appeared more than
250 million years ago, has been mentioned in the
Chinese Materia Medica for more than 2500 years.1 A
number of G. biloba natural products have been iso-
lated,2 the most unique being the terpene trilactones
(TTLs), i.e., ginkgolides A, B, C, J , and M (1-5) and
bilobalide (6) (Figure 1).3-6 The ginkgolides are diter-
penes with a cage skeleton consisting of six five-
membered rings, i.e., a spiro[4.4]nonane carbocycle,
three lactones, and a tetrahydrofuran (THF). The dif-
ference between the five ginkgolides lies in the variation
in the number and positions of hydroxyl groups on the
spirononane framework (Figure 1).
F igu r e 1. TTLs isolated from G. biloba. GA (1), GB (2), and
GC (3) are found in the leaves and root bark of G. biloba, but
GJ (4) is found only in the leaves, and GM (5) is found only in
the root bark.
constituents of G. biloba also influence the brain under
physiological conditions.17-20
A standardized G. biloba extract (EGb 761) containing
TTLs (5-7%) and flavonoids (22-24%) has demon-
strated neuromodulatory properties,7 and several clini-
cal studies using EGb 761 have reported positive effects
on various neurodegenerative diseases,8-13 including
Alzheimer’s disease (AD).14,15 A recent study by Schultz
and co-workers found that EGb 761 upregulated several
genes in rat hippocampus and cortex, including genes
expressing proteins such as transthyretin and neuronal
tyrosine/threonine phosphatase, both of which are be-
lieved to be involved in AD.16 Several recent studies on
healthy volunteers have shown positive effects of EGb
761 on short-term working memory indicating that
Although the molecular basis for the action of G.
biloba TTL constituents on the central nervous system
(CNS) is only poorly understood, it is known that the
ginkgolides, particularly ginkgolide B (GB, 2), is a
potent in vitro antagonist of the platelet-activating
factor receptor (PAFR).21 PAF (1-O-alkyl-2-acetyl-sn-
glycero-3-phosphocholine, Figure 2) is a phospholipid
mediator involved in numerous disorders including
acute allergy, inflammation, asthma, and ischemic
injury. These effects are manifested through binding of
PAF to the PAFR, a G protein-coupled receptor that is
found in organs such as the lungs, liver, kidneys,22-24
and brain.25,26 The function of PAF in the brain is still
not clear, although PAF has been suggested to play a
role in diseases of aging27 and in initiating human
immunodeficiency virus (HIV)-related neuropathogen-
esis.28 PAF has also been suggested as a retrograde
* To whom correspondence should be addressed. Tel: +1 212 854
2169. Fax: +1 212 932 8273. E-mail: kn5@columbia.edu.
† Columbia University.
‡ The University of Tokyo and CREST of J apan Science and
Technology Corp.
10.1021/jm020147w CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/03/2002

Ginkgolide Derivatives for Photolabeling Studies
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 4039
been prepared, and these analogues have been assessed
for their ability to displace radioligand binding to cloned
PAFR.
Resu lts
Syn th esis. A series of photoactivatable GB (2) and
ginkgolide C (GC, 3) derivatives were designed based
on previous SAR studies of ginkgolides, which demon-
strated that bulky aromatic substituents in the 10-OH
position of GB (2) increase activity at the PAFR.40-42
Three different photoactivatable moieties, benzophe-
none, trifluoromethyldiazirine, and tetrafluorophenyl
azide, were chosen as they have been described as being
among the most successful for labeling receptors and
enzymes.45-47 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, trifluoro-
methyldiazirine, and tetrafluorophenyl azide moieties,
respectively.45 Because 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.
4-(Bromomethyl)benzophenone was commercially avail-
able, whereas trifluoromethyldiazirine 1248,49 and tet-
rafluorophenyl azide 1550-52 were synthesized, respec-
tively, as outlined in Scheme 1. The bromotrifluoro-
methyldiazirine 12 was synthesized starting from readily
available 4-bromotoluene, which was reacted with N-
trifluoroacetylpiperidine48 to give trifluoroacetyl toluene
7. The trifluoroacetyl compound 7 was then reacted with
hydroxylamine hydrochloride in pyridine to form oxime
8. Reacting the latter with p-toluenesulfonyl chloride
led to the tosylated oxime 9, which was placed in a thick-
walled screw-capped flask and reacted overnight with
liquid ammonia in diethyl ether to form the diaziridine
10; under dim red light, diaziridine 10 was oxidized with
iodine to give methylphenyl-trifluoromethyl-diazirine
11. Finally, 11 was brominated with N-bromosuccinim-
ide (NBS) using a catalytic amount of 2,2′-azobisisobu-
tyronitrile (AIBN) to form 3-(4-bromomethylphenyl)-3-
trifluoromethyl-3H-diazirine (12, Scheme 1). Benzoyl
peroxide was initially used as a radical initiator, but
the reaction yielded multiple products resulting from
multiple bromine substitutions. The use of AIBN led to
a 4:1 ratio of monobromiated 12 and geminal dibromo-
substituted methylphenyl-trifluoromethyldiazirine, but
F igu r e 2. Structures of PAF, the endogenous ligand for the
PAFR, and WEB 2086, a potent and selective antagonist, both
of which have been used in radioligand binding studies.
messenger in long-term potentiation (LTP).29,30 How-
ever, studies using PAFR knock-out mice gave contra-
dictory results; one study showed attenuation of LTP
in the hippocampal dentate gyrus regions of mice
lacking the PAFR,31 whereas another study showed that
the PAFR was not required for LTP in the hippocampal
CA1 region.32 These discrepancies may be due to dif-
ferences in the hippocampal areas observed, as well as
the assay conditions used. However, it is still unclear
whether the neuromodulatory effect of TTLs or G. biloba
extract is related to the PAFR.33,34
With few exceptions, previous structure-activity
relationship (SAR) studies of TTLs on the PAFR have
focused almost entirely on derivatives of GB (2). In all
cases, the derivatives were evaluated for their ability
to prevent PAF-induced aggregation of rabbit platelets.
Corey et al. investigated various intermediates encoun-
tered during the course of the total syntheses of
ginkgolide A (GA, 1),35 GB (2),36 and bilobalide (BB, 6)37
and found that although the terminal methyl-bearing
lactone was not essential for activity and could be
replaced by other lipophilic groups,38 the tert-butyl
group was important for PAFR antagonism.39 Park et
al. synthesized over 200 derivatives of GB (2), with
particular focus on aromatic substituents at 10-OH, and
found most of them to be more potent than the parent
compound.40 Similar derivatives recently synthesized by
Hu et al. also yielded compounds more potent than GB
(2),41,42 whereas other variations in GA (1) and GB (2)
led to a decrease in activity.43,44
In the following, we describe the first evaluation of
the interaction of all native ginkgolides and bilobalide
with the cloned PAFR by a radioligand binding assay,
as well as their functional properties by intracellular
calcium measurements. A series of ginkgolide deriva-
tives with photoactivatable and fluorescent groups have
Sch em e 1a
a
Reagents: (a) N-Trifluoroacetylpiperidine, n-butyllithium. (b) Hydroxylamine. (c) p-Toluenesulfonyl chloride, pyridine. (d) NH3. (e)
I2, Et3N. (f) NBS, AIBN. (g) NaN3. (h) Me2NHBH3. (i) PBr3.

4040 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Strømgaard et al.
Sch em e 2a
Sch em e 3a
a
Reagents: (a) 4-Benzoylbenzoic acid, EDC, DMAP. (b) Dansyl
chloride, DMAP.
relative chemical shift of 12-H in DMSO-d6 can also be
used in differentiating 1- and 10-OH substitutions.42
The coupling of GB (2) with 4-benzoylbenzoic acid
using 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
HCl (EDC) and 4-(dimethylamino)pyridine (DMAP)
occurred exclusively at 10-OH to give 10-benzophenon-
ecarbonyl GB (22) in good yield (Scheme 3). In 10-
benzophenonecarbonyl GB (22), the photoactivatable
benzophenone moiety is linked to the ginkgolide skel-
eton through an ester linkage. After it is incorporated
into the receptor, the ester group can be aminolyzed
with a fluorescent amine such as 1-pyrenemethylamine,
thus avoiding the use of radioactivity for photolabeling
and sequencing.54
GC derivatives 17, 19, and 21 can be reacted further
to incorporate fluorescent groups; for example, ben-
zophenone derivative 17 was reacted with 1 equiv of
5-(dimethylamino)naphthalene-sulfonyl (dansyl) chlo-
ride to give 10-O-benzophenone-7-O-dansyl GC (23) with
almost exclusive reaction at 7-OH (Scheme 3). Interest-
ingly, increasing the amount of dansyl chloride to 2
equiv gave 10-O-benzophenone-1-O-dansyl GC (24) as
well as 23 in a 1:1 ratio.
a
Reagents: (a) 4-(Bromomethyl)benzophenone, KH. (b) Com-
pound 12, KH. (c) Compound 15, KH.
separation of the two products required several repeti-
tions of flash column chromatography to yield the
monobromo-substituted diazirine 12 in sufficient purity.
For the synthesis of tetrafluorophenyl azide 15,
pentafluorobenzaldehyde was heated with sodium azide
in an acetone-water mixture. Because pentafluorophe-
nyl groups that contain electron-withdrawing groups
undergo nucleophilic aromatic substitution regioselec-
tively in the para position, azidophenylaldehyde 13 was
obtained in high yield, and no ortho-substituted product
was observed. Aldehyde 13 was selectively reduced with
a dimethylamine-borane complex to form the benzyl
alcohol 14. The benzyl alcohol 14 was then brominated
by refluxing with phosphorus tribromide in pyridine and
chloroform to give the desired product 15 (Scheme 1).
Preparation of GB and GC derivatives 16-21 was
performed by reacting GB (2) and GC (3) with 4-(bro-
momethyl)benzophenone, 3-(4-bromomethylphenyl)-3-
trifluoromethyl-3H-diazirine (12), and 1-azido-4-(bro-
momethyl)-2,3,5,6-tetrafluorobenzene (15), respectively
(Scheme 2). Ginkgolides GB (2) and GC (3) were
derivatized almost exclusively at 10-OH when potas-
sium hydride (KH) was used as base, as was previously
shown for GB (2),40 whereas other bases were less
selective, giving rise to products derivatized at 1-OH as
well. It is noteworthy that 7-OH present in GC (3) does
not react under any of the above-mentioned reaction
conditions. Generally, the position of the substituent
All compounds were characterized by NMR spectros-
copy and high-resolution mass spectrometry (HRMS).
Purity of the derivatives 16-24, with aromatic groups,
was determined by high-performance liquid chromatog-
1
raphy (HPLC)-UV and H NMR and was in the range
of 98-100%. Because ginkgolides (1-5) and bilobalide
(6) absorb only weakly in the UV spectrum, HPLC-UV
cannot be used for determination of purity. Instead,
observation of the 12-H 1H NMR signal of 1-6 is a
particularly useful way to determine purity, as this
signal has distinct and well-separated δ-values for
ginkgolides, bilobalide, and their derivatives.
P h a r m a cology. The native TTLs (1-6), as well as
ginkgolide derivatives 16-24, were tested for their
ability to bind to PAFR using a radioligand binding
assay with membrane fractions from hearts and skeletal
muscles of PAFR transgenic mice.53 Initially, compounds
were tested in concentrations of 5 µM against [3H]WEB
1
was determined from the H NMR coupling of appropri-
ate protons in dimethyl sulfoxide (DMSO)-d6, as well
as by correlation spectroscopy (COSY) spectra. The

Ginkgolide Derivatives for Photolabeling Studies
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 4041
Ta ble 2. Ki Values of the Synthesized Derivatives
Ta ble 1. Ki Values of the Native TTLs
compd
GA (1)
GB (2)
GC (3)
GJ (4)
GM (5)
bilobalide (6)
R1
R2
R3
Ki (µM)a
H
H
H
OH
OH
OH
OH
OH
OH
OH
H
1.46
0.56
12.6
9.90
>50
>50
OH
OH
H
OH
a
Inhibition of [3H]WEB 2086 binding. Values are means of two
independent experiments performed in triplicate.
2086 (Figure 2), a potent, competitive PAFR antagonist
and [3H]PAF; the compounds were generally less potent
against [3H]PAF, but the relative potencies were com-
parable with the two radioligands. The degree of non-
specific binding was determined to be ca. 50% for
[3H]PAF and less than 5% for [3H]WEB 2086. Accord-
ingly, the assays were performed using [3H]WEB 2086
rather than [3H]PAF as the radioligand, due to the high
degree of nonspecific 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 [3H]WEB 2086
revealed that up to 1% DMSO (final concentration) was
acceptable, but 1-2.5% resulted in a slight inhibition
of [3H]WEB 2086 binding. Generally, this caused no
problem; however, with very weakly binding com-
pounds, the relatively high DMSO concentration in
solutions above 100 µM had a small inhibitory effect,
thus leading to a slight overestimation of their poten-
cies. Previous studies have reported problems associated
with the solubilization of ginkgolides specifically in
DMSO,55 but similar problems were not observed in the
present study.
Native ginkgolides (1-5) and bilobalide (6) were
tested with the cloned PAFR (Table 1). GB (2) was the
most potent compound with a Ki value of 0.56 µM, while
GA (1) was slightly less potent with a Ki 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 Ki values larger than 50 µM.
The GB-derived photoactivatable compounds 16, 18,
20, and 22 with Ki values in the range 0.09-0.15 µM
(Table 2) were all more potent than GB (2), while
compounds 17, 19, and 21 derived from GC (3) with Ki
values of 0.47-0.79 µM were equipotent to GB (2) (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 23 and 24 possessing a fluorescent dansyl
group at either 1- or 7-OH were both less potent than
10-O-benzophenone GC (17) without the dansyl group,
with Ki values of 3.94 and 0.96 µM for 23 and 24,
respectively. However, an important difference was
observed in the activities between the two; the 1- and
10-disubstituted analogue (24) was ca. four times more
potent than the 7- and 10-disubstituted analogue (23).
a
Inhibition of [3H]WEB 2086 binding. Values are means of two
independent experiments performed in triplicate.
PAFR is linked to several intracellular signal trans-
duction pathways including elevation of intracellular
calcium concentration,22 which can be used for analyzing
functional responses of PAFR ligands.56 In the present
study, the compounds with Ki values below 5 µM, i.e.,
GA (1), GB (2), and analogues 16-22, were investigated
for their ability to mobilize intracellular calcium in
Chinese hamster ovary (CHO) cells expressing the
PAFR.57 While none of them evoked calcium responses,
they all suppressed PAF-induced intracellular calcium
increase (data not shown) confirming their function as
PAFR antagonists. Because the assay is based on the
measurement of the fluorescence of a Ca2+ sensitive dye,
the fluorescence of analogues 23 and 24 interfered with
the measurement and could not be assayed.
Discu ssion
Nine analogues (16-24) with photoactivatable groups,
and in the case of 23 and 24 with fluorescent dansyl
groups as well, have been prepared from native gink-
golides GB (2) and GC (3) by selective derivatizations
of the hydroxyl groups. Generally, the increased reactiv-
ity of the 1-OH and 10-OH as compared to 7-OH has
been rationalized by stabilization of the corresponding
alkoxides by hydrogen bonding between 1-OH and 10-
OH,58 but this does not explain the interesting selectiv-
ity 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.59 Three different photoactivatable
moieties, all of them benzyl derivatives, were incorpo-

4042 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Strømgaard et al.
F igu r e 3. Concentration-displacement curves for native
TTLs, as measured by their ability to displace binding of [3H]-
WEB 2086 to the cloned PAFR. GB (2) was the most potent
analogue, while GA (1) was slightly less potent, and GC (3)
and GJ (4) were only weak antagonists. Bars represent the
standard deviation (SD).
rated into both GB and GC. The benzophenone moiety
was commercially available, whereas trifluoromethyl-
diazirine 12 and tetrafluorophenyl azide 15 moieties
were synthesized in six and three steps, respectively
(Scheme 1). The three different types of photoactivatable
groups were selected on the basis of their different
reactive intermediates when irradiated, the intermedi-
ates being a radical, a carbene, and (singlet) nitrene for
benzophenone, trifluoromethyldiazirine, and tetrafluo-
rophenyl azide, respectively.
F igu r e 4. (A) GB (2) and photoactivatable benzyl derivatives;
the three photoactivatable derivatives 16, 18, and 20 were all
ca. 6-fold more potent than GB (2), and the activities were
remarkably similar although bearing very different functional
groups. (B) GC (3) and photoactivatable benzyl derivatives (17,
All native TTLs (1-6) as well as the derivatized
compounds were investigated with respect to their
binding to cloned PAFR isolated from transgenic mice
(Figure 3 and 4). To the best of our knowledge, this is
the first report on the effect of TTLs on the cloned
PAFR, since previous SAR studies of PAFR antagonism
with TTLs 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 IC50 value around 0.2 µM,40-42 which corresponds to
the Ki value of 0.56 µM obtained in this study (Table
1). GA (1), with one hydroxyl group less than GB (2),
was only slightly less potent than GB (2, Figure 3),
indicating that the OH-1 is not essential for activity,
and neither is the hydrogen bonding between OH-1 and
OH-10. Generally, the binding of GC (3), GJ (4), and
GM (5) with hydroxyl groups at C-7, as compared to the
binding activity of GA (1) and GB (2) lacking the 7-OH,
was decreased showing that the 7-OH is not necessary
for binding to PAFR whereas hydroxyl groups at other
positions appear to be less important (Figure 3). The
study also confirmed that bilobalide (6), a TTL with only
one five-membered carbocycle and three lactones, is not
displacing [3H]WEB 2086 binding in concentrations up
to 100 µM (Table 1).
19, and 21) were all more potent than GC (3), with
a
remarkable increase in potency, thus being equipotent to GB
(2). Bars represent the SD.
them equipotent to GB (2), while the same substitutions
in GB (2) increase the affinity only 6-fold (Figure 4).
Furthermore, the similar affinities of GB derivatives 16,
18, 20, and 22 and the similar affinities of GC deriva-
tives 17, 19, and 21 (Figure 4) imply that it is the steric
bulk or the lipophilicity, including π-π interactions of
the substituents, rather than the specific functional
groups that are important for the increase in affinity.
GC derivatives 23 and 24 (Scheme 3) with dansyl
groups at 7-OH and 1-OH are less potent and equipo-
tent, respectively, to their parent compound, 10-O-
benzophenone-GC (17). In compound 23, which is ca.
six times less active than 17, the bulk at the 7-position
seems to be responsible for the reduction in affinity. The
fact that compound 24 is equipotent to 17 suggests that
once a bulky aromatic group occupies this area, further
aromatic groups neither increase nor decrease the
affinity.
In conclusion, an investigation of the effect of TTLs
isolated from G. biloba on the cloned PAFR has dem-
onstrated that among the native compounds, GA (1) and
GB (2) are the most potent. A series of photoactivatable
analogues have been prepared, and PAFR binding
assays showed that most of these analogues were more
potent antagonists than their parent compounds, thus
providing promising candidates for the planned studies
of the interaction of ginkgolides with the PAFR. The
ginkgolide derivative containing both a photoactivatable
and a fluorescent group, compound 24, retained affinity
All seven photoactivatable analogues 16-22 with
aromatic substituents at 10-OH improved the affinity
to the PAFR relative to the activities of GB (2) and GC
(3) (Figure 4). This is in agreement with previous SAR
studies of GB (2),40-42 as well as a three-dimensional
(3D)-quantitative SAR (QSAR) study on ginkgolides.60
However, it is interesting to note that aromatic substi-
tutions at 10-OH of GC (3) as in compounds 17, 19, and
21 improve the affinity to PAFR ca. 20-fold thus making

Ginkgolide Derivatives for Photolabeling Studies
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 4043
chromatography eluting with hexane/CH2Cl2 (2:1) to give a
white solid (2.90 g, 82%). H NMR (300 MHz, CDCl3): δ 2.40
to PAFR and thus could be useful in photolabeling and
subsequent sequencing studies.
1
(s, 3H), 2.48 (s, 3H), 7.26-7.34 (m, 4H), 7.37-7.40 and 7.87-
7.90 (AA′BB′ system, tosyl aromatic H, 4H). 13C NMR (75 MHz,
CDCl3): δ 21.9, 22.2, 120.0 (q, 1J CF ) 285.0, CF3), 122.0, 128.9
(2C), 129.7 (2C), 129.9 (2C), 130.2 (2C), 132.0, 142.7, 146.4,
Exp er im en ta l Section
Ch em istr y. Gen er a l P r oced u r es. Ginkgolides and bilo-
balide (1-6) were obtained by extraction of leaves from G.
biloba, purification by column chromatography, and recrys-
tallization, as previously described,3,61 and purity was >99%
as determined by 1H NMR. 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 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.61
Thin-layer chromatography (TLC) was carried out using
precoated silica gel 60 F254 plates with thickness of 0.25 µm.
Spots were observed at 254 nm and by staining with acetic
anhydride or cerium/molybdenum in H2SO4. 1H and 13C NMR
spectra were obtained on Bruker DMX 300 or Bruker DMX
400 MHz spectrometers and are reported in parts per million
(ppm) relative to internal solvent signal, with coupling con-
stants (J ) in Hertz (Hz). For 19F NMR spectra, hexafluoroben-
zene (-162.9 ppm) was used as internal standard. Analytical
HPLC was performed on a HP 1100 LC instrument using a 5
µm C18 reversed-phase Phenomenex Luna column (150 mm
× 4.60 mm), using 1 mL/min of water/acetonitrile/TFA 60:40:
0.1, raising to 50:50:0.1 after 10 min, and detecting by UV at
219 and 254 nm. Accurate mass determination was performed
on a J EOL J MS-HX110/100A HF mass spectrometer using a
3-nitrobenzyl alcohol (NBA) matrix and Xe ionizing gas, and
all are within (5 ppm of theoretical values.
2
154.0 (q, J CF ) 67.5).
3-(4-Meth ylph en yl)-3-tr iflu or om eth yldia zir id in e (10).48
In a thick-walled screw-cap tube, 9 (2.90 g, 8.12 mmol) was
dissolved in Et2O (20 mL). The solution was cooled to -78 °C,
and liquid ammonia (3.5 mL) was added. The tube was screwed
tightly, and the solution allowed rising to room temperature.
The reaction was stirred for 12 h, and the solution was cooled
to -78 °C. The cap was removed, and the mixture risen to
room temperature to remove ammonia. The solution was
partitioned between Et2O (50 mL) and H2O (50 mL), the
organic layer was dried (MgSO4), and the solvent was removed
in vacuo. The product was purified by flash column chroma-
tography eluting with chloroform to give a colorless solid (1.25
1
g, 77%). H NMR (300 MHz, CDCl3): δ 2.19 (NH, d, 1H), 2.38
(s, 3H), 2.74 (NH, d, 2H), 7.22-7.24 and 7.49-7.51 (AA′BB′
2
system, 4H). 13C NMR (75 MHz, CDCl3): δ 21.7, 57.9 (q, J CF
1
) 34.9), 125.0 (q, J CF ) 278.7, CF3), 129.3 (2C), 129.7 (2C),
130.1, 140.0.
3-(4-Meth ylp h en yl)-3-tr iflu or om eth yld ia zir in e (11).48
Diaziridine 10 (1.25 g, 6.18 mmol) was dissolved in CH2Cl2
(25 mL), and triethylamine (2.58 mL, 18.54 mmol) was added
and cooled to 0 °C. Iodine (1.73 g, 6.80 mmol) was added in
small portions until a brown color persisted. The solution was
washed with 1 M NaOH (25 mL), H2O (25 mL), and brine (25
mL). The organic phase was dried (MgSO4), and the solvent
was carefully removed in vacuo at 20 °C due to volatility of
the product. The crude product was purified by flash column
chromatography eluting with hexane/CH2Cl2 (20:1) to give a
colorless solid (1.03 g, 82%). 1H NMR (300 MHz, CDCl3): δ
2.36 (s, 3H), 7.07-7.10, 7.19 and 7.22 (AA′BB′ system, 4H).
13C NMR (100 MHz, CDCl3): δ 21.5, 28.9 (q, 2J CF ) 39.6), 123.0
2,2,2-Tr iflu or o-1-(4-m et h ylp h en yl)-1-et h a n on e (7).48
4-Bromotoluene (9.45 g, 55.25 mmol) was dissolved in Et2O
(280 mL) and cooled to -40 °C. n-Butyllithium (1.1 M in
hexane, 40.5 mL, 60.74 mmol) was added dropwise, and the
solution was warmed to 0 °C over a 2 h period. The solution
was then cooled to -60 °C and a solution of N-trifluoro-
acetylpiperidine48 (10.00 g 55.23 mmol) in dry Et2O (60 mL)
was added in portions. The reaction was allowed to stir at -60
°C for 3 h and then warmed to room temperature. The solution
was hydrolyzed with saturated NH4Cl (50 mL) and washed
with NH4Cl (5 × 50 mL) and H2O (3 × 50 mL). The organic
phase was dried (MgSO4), and the solvent was removed in
vacuo. The remaining oil was purified by flash column chro-
matography eluting with hexane/CH2Cl2 (5:1, 4:1, and 3:1) to
1
(q, J CF ) 274.7, CF3), 125.5, 127.1 (2C), 130.8 (2C), 141.1.
3-(4-Br om om e t h ylp h e n yl)-3-t r iflu or om e t h yld ia zir -
in e (12).49 A solution of diazirine 11 (0.50 g, 2.50 mmol) in
CCl4 (10 mL) was heated to 70 °C and powdered NBS (0.66 g,
3.75 mmol) was added and stirred for 10 min, then AIBN (10
mg) was added, and the reaction was refluxed for 2 h. The
precipitate was filtered, the solvent was removed in vacuo at
20 °C, and the crude product was purified by column chroma-
tography eluting with hexane/CH2Cl2 (20:1) to give an oil (0.30
g, 40%). 1H NMR (400 MHz, CDCl3): δ 4.50 (s, 2H), 7.17-
7.23, 7.43 and 7.47 (AA′BB′ system, 4H). 13C NMR (100 MHz,
2
1
CDCl3): δ 28.3 (q, J CF ) 40.5), 39.7, 122.2 (q, J CF ) 275.0,
1
give a colorless oil (5.62 g, 54%). H NMR (300 MHz, CDCl3):
CF3), 127.3 (2C), 129.6, 129.9 (2C), 139.8.
δ 2.46 (s, 3H), 7.26-7.36 and 7.96-7.98 (AA′BB′ system, 4H).
4-Azid otetr a flu or oben za ld eh yd e (13).50 To a solution of
pentafluorobenzaldehyde (2.69 g, 13.72 mmol) in acetone (24
mL) and H2O (9 mL), NaN3 (1.06 g, 14.54 mmol) was added
and the mixture was refluxed for 8 h. The solution was cooled
to room temperature and diluted with H2O (20 mL). The
solution was extracted with ether (3 × 30 mL), the organic
phase was dried (MgSO4), and the solvent was removed in
vacuo. The maroon-colored gum was sublimed at 70 °C under
reduced pressure to give a colorless solid (1.80 g, 60%). 19F
NMR (282 MHz, CDCl3): δ -152.14 (m, 2F), -146.10 (m, 2F).
4-Azid otetr a flu or oben zyl Alcoh ol (14).50 4-Azidotetraflu-
orobenzaldehyde (13, 1.80, 8.21 mmol) was dissolved in acetic
acid (27 mL), and Me2NH‚BH3 (0.58 g, 9.85 mmol) was added.
The reaction was stirred at room temperature for 1 h. The acid
versatiles were removed in vacuo at 45 °C. The residue was
dissolved in CHCl3 (10 mL) and washed with 5% Na2CO3 (3 ×
10 mL). The organic phase was dried (MgSO4), and the solvent
was removed in vacuo to give a colorless solid (1.74 g, 97%),
which was used without further purification. 1H NMR (300
MHz, CDCl3): δ 2.41 (OH, broad s, 1H), 4.79 (s, 2H). 19F NMR
(282 MHz, CDCl3): δ -150.90 (m, 2F), -143.75 (m, 2F).
13C NMR (75 MHz, CDCl3): δ 22.3, 117.0 (q, 1J CF ) 292.5, CF3),
2
127.9, 130.2 (2 C), 130.7 (2 C), 147.4, 169.1 (q, J CF ) 60.0).
2,2,2-Tr iflu or o-1-(4-m eth ylp h en yl)-1-eth a n on e Oxim e
(8).48 Ketone 7 (2.60 g, 13.80 mmol) was dissolved in pyridine
(30 mL), hydroxylamine hydrochloride (2.88 g, 41.44 mmol)
was added, and the reaction was stirred at 70 °C for 3 h.
Pyridine was removed in vacuo, and the remaining residue
was dissolved in Et2O (50 mL) and washed with 0.01 M HCl
(50 mL). The organic layer was washed with H2O (3 × 50 mL)
and dried (MgSO4), and the solvent was removed in vacuo to
give a colorless solid (2.01 g, 72%), which was used without
further purification. 1H NMR (300 MHz, CDCl3): δ 2.40 (s,
3H), 7.20-7.30 and 7.37-7.43 (m, 4H), 8.38 (bs, OH). Multiple
splitting patterns arise due to anti/syn configurations of the
1
oxime. 13C NMR (75 MHz, CDCl3): δ 21.9, 121.0 (q, J CF
)
277.5, CF3), 123.4, 129.0 (2C), 129.7 (2C), 141.4, 148.4 (q, 2J CF
) 37.5).
2,2,2-Tr iflu or o-1-(4-m et h ylp h en yl)-1-et h a n on e O-(p -
Tolu en esu lfon yl) Oxim e (9).48 To a stirred solution of oxime
8 (2.00 g, 9.85 mmol) in pyridine (36 mL), p-toluenesulfonyl
chloride (2.82 g, 14.79 mmol) was added in portions and the
reaction mixture was refluxed for 3 h. Pyridine was removed
in vacuo, and the residue was purified by flash column
1-Azid o-4-(b r om om et h yl)-2,3,5,6-t et r a flu or ob en zen e
(15).51,52 Alcohol 14 (1.74 g, 7.93 mmol) was dissolved in a

4044 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Strømgaard et al.
mixture of CHCl3 (40 mL) and pyridine (0.32 mL, 3.97 mmol).
Three portions of PBr3 (3 × 0.05 mL, 1.58 mmol) were added
dropwise every 30 min to the refluxing solution. After the last
portion of PBr3 was added, the reaction was refluxed for 2 h.
The solution was cooled to room temperature, 2-propanol (20
mL) was added, and the reaction was stirred for an additional
10 min. The solution was washed with 1 N NaHCO3 (3 × 50
mL) and dried (MgSO4), and the solvent was removed in vacuo
to give a white solid. The crude product was purified by flash
column chromatography eluting with hexane/ethyl acetate (8:
10-O-(Tr iflu or om eth yl-3H-diazir in e)ben zyl Gin kgolide
C (19). Purified by method A; yield, 0.023 g (51%). H NMR
1
(400 MHz, CD3OD): δ 1.17 (s, tert-butyl), 1.24 (d, J ) 7.1,
CH3,), 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). 13C NMR
2
(75 MHz, CDCl3): δ 7.65, 23.77 (q, J CF ) 38.9, CCF3), 29.52
(3C), 32.65, 41.94, 50.92, 64.43, 67.48, 73.89, 74.30, 76.03,
1
1
1) to give a slightly yellow solid (1.20 g, 53%). H NMR (300
76.34, 79.63, 83.90, 90.91, 98.94, 110.53, 122.32 (q, J CF
)
MHz, CDCl3): δ 4.51 (s, 2H). 19F NMR (282, MHz, CDCl3): δ
-150.50 (m, 2F), -141.80 (m, 2F).
275.0, CF3), 127.94 (2C), 129.68 (2C), 131.26, 136.07, 170.97,
171.07, 175.69. 19F NMR (282 MHz, CDCl3): δ -66.25 (s, 3F).
HPLC-UV: 97.9%. HRMS: C29H29F3N2O11 requires M + 1 at
m/z 639.1802; found, 639.1790.
Syn th esis of 16-21. Gen er a l Syn th etic P r oced u r e. 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, and then, a solution
of 4-(bromomethyl)benzophenone, 12, or 15 (0.212 mmol) in
THF (1 mL) was added dropwise. The reaction was stirred at
room temperature for 4 h. The solution was then cooled to 0
°C, and concentrated HCl (0.3 mL) was added. The mixture
was diluted with H2O (10 mL), extracted with EtOAc (3 × 10
mL), and washed with saturated aqueous NH4Cl solution (30
mL), brine (30 mL), and H2O (30 mL). The organic phase was
dried (MgSO4), and the solvent was removed in vacuo. The
crude material was purified by flash column chromatography
using either A: CHCl3/MeOH (100:1 and 50:1), B: CHCl3/
MeOH (30:1 and 20:1), or C: cyclohexane/acetone (3:1 and 2:1).
All ginkgolide derivatives were white solids that decomposed
above 250 °C.
10-O-Tetr a flu or oben zyl Azid e Gin k golid e B (20). Puri-
fied by method B; yield, 0.023 g (50%). 1H NMR (400 MHz,
CDCl3): δ 1.13 (s, tert-butyl), 1.32 (d, J ) 7.0, CH3), 1.84-
1.97 (m, 8-H and 7R-H), 2.27-2.33 (m, 7â-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). 13C NMR (100 MHz, CDCl3):
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. 19F NMR (282
MHz, CDCl3): δ -143.31 (m, 2F), -150.85 (m, 2F). HPLC-
UV: 98.8%. HRMS: C27H25F4N3O10 requires M + 1 at m/z
628.1554; found, 628.1565.
10-O-Tetr a flu or oben zyl Azid e Gin k golid e C (21). Puri-
fied by method C; yield, 0.080 g (54%). 1H NMR (400 MHz,
CDCl3): δ 1.22 (s, tert-butyl), 1.33 (d, J ) 7.0, CH3,), 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). 13C NMR (75 MHz,
CDCl3): 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. 19F NMR (282 MHz,
CDCl3): δ -143.56 (m, 2F), -151.08 (m, 2F). HPLC-UV:
99.1%. HRMS: C27H25F4N3O11 requires M + 1 at m/z 644.1503;
found, 644.1527.
10-O-Ben zop h en on e Gin k golid e
B (16). Purified by
1
method B; yield, 0.035 g (78%). H NMR (400 MHz, CD3OD):
δ 1.13 (s, tert-butyl), 1.24 (d, J ) 7.1, CH3), 1.92 (dd, J ) 14.3,
4.5, 8-H), 2.07 (td, J ) 13.9, 4.4, 7R-H), 2.27 (dd, J ) 13.5,
4.6, 7â-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). 13C NMR (100 MHz,
CD3OD): 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. HPLC-
UV: 98.5%. HRMS:
641.1999; found, 641.2018.
C34H34O11 requires M + Na at m/z
10-O-Ben zoylben zoic Gin k golid e C (22). 4-Benzoylben-
zoic acid (0.018 g, 0.08 mmol) and 2 (0.028 g, 0.07 mmol) were
dissolved in THF (5 mL), and the mixture was cooled to 0 °C.
EDC (0.018 g, 0.092 mmol) and DMAP (0.002 g, 0.01 mmol)
were added, and the reaction mixture was stirred at 0 °C for
1 h and continued overnight at room temperature. The solvent
was removed in vacuo, and the crude product was dissolved
in EtOAc (20 mL) and washed with a saturated 5% NaHCO3
solution (20 mL) and brine (20 mL). The organic fraction was
dried (MgSO4), and the solvent was evaporated in vacuo. The
crude product was purified by flash column chromatography
eluting with hexane/EtOAc (2:1) to give the product as white
10-O-Ben zop h en on e Gin k golid e
C (17). Purified by
1
method A; yield, 0.023 g (64%). H NMR (400, MHz, CD3OD):
δ 1.20 (s, tert-butyl), 1.24 (d, J ) 7.1, CH3,), 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, CD3OD): 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. HPLC-UV: 99.3%. HRMS: C34H34O12 requires
M + 1 at m/z 635.2129; found, 635.2098.
1
crystals (0.026 g, 62%). H NMR (400 MHz, CD3OD): δ 1.07
(s, tert-butyl), 1.26 (d, J ) 7.1, CH3), 1.98-2.10 (m, 8-H and
7R-H), 2.30-2.36 (m, 7â-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). 13C NMR (100 MHz, CD3-
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. HPLC-UV:
99.2%. HRMS: C34H31O12 requires M + Na at m/z 655.1791;
found, 655.1790.
10-O-(Tr iflu or om eth yl-3H-diazir in e)ben zyl Gin kgolide
1
B (18). Purified by method B; yield, 0.024 g (59%). H NMR
(400 MHz, CD3OD): δ 1.11 (s, tert-butyl), 1.23 (d, J ) 7.1, CH3),
1.89 (dd, J ) 14.3, 4.3, 8-H), 2.01 (td, J ) 13.9, 4.3, 7R-H),
2.25 (dd, J ) 13.4, 4.4, 7â-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,
2
Ar-H, 4H). 13C NMR (75 MHz, CDCl3): δ 7.67, 21.57 (q, J CF
) 40.9, CCF3), 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, 1J CF ) 274.3, CF3), 127.83 (2C), 129.53 (2C), 131.06,
136.44, 171.25, 171.50, 175.87. 19F NMR (282 MHz, CDCl3):
δ -66.23 (s, 3F). HPLC-UV: 99.1%. HRMS: C29H29F3N2O10
requires M + 1 at m/z 623.1853; found, 623.1834.
10-O-Ben zop h en on e-7-O-d a n syl Gin k golid e C (23). A
solution of dansyl chloride (0.010 g, 0.035 mmol) in acetonitrile
(0.3 mL) was added to a solution of 17 (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 saturated aqueous NH4Cl solution (2 mL) was added,

Ginkgolide Derivatives for Photolabeling Studies
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 4045
and the mixture was extracted with EtOAc (3 × 5 mL). The
combined organic phase was washed with saturated aqueous
NaCl solution (3 × 15 mL), dried (MgSO4), and the solvent
was 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%).
1H NMR (400 MHz, DMSO-d6): δ 0.83 (s, tert-butyl), 1.09 (d,
J ) 7.2, CH3,), 1.94 (d, J ) 12.5, 8-H), 2.81 [m, 14-H and
N(CH3)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). HPLC-UV: 98.9%. HRMS: C46H45NO14S requires
M + 1 at m/z 868.2639; found, 868.2642.
pound (100 µM) was applied to the cell suspension, and then,
PAF (10 nM) was added after 3 min.
Ack n ow led gm en t. We are grateful to Dr. Yasuhiro
Itagaki for performing HRMS. We thank the Alfred
Benzon Foundation (to K.S.) and the NSF REU program
(CHE 9820490, to D.R.S., Beloit College, Beloit, WI) for
financial support.
Refer en ces
(1) Drieu, K.; J aggy, H. History, development and constituents of
EGb 761. In Medicinal and Aromatic Plants-Industrial Pro-
files: Ginkgo biloba; van Beek, T. A., Ed.; Harwood Academic
Publishers: Amsterdam, 2000; Vol. 12, pp 267-277.
(2) Hasler, A. Chemical constituents of Ginkgo biloba. In Medicinal
and Aromatic Plants-Industrial Profiles: Ginkgo biloba; van
Beek, T. A., Ed.; Harwood Academic Publishers: Amsterdam,
2000; Vol. 12, pp 109-142.
(3) Nakanishi, K. The ginkgolides. Pure Appl. Chem. 1967, 14, 89-
113 and references therein.
(4) Okabe, K.; Yamada, K.; Yamamura, S.; Takada, S. Ginkgolides.
J . Chem. Soc. C 1967, 2201-2206.
(5) Nakanishi, K.; Habaguchi, K.; Nakadaira, Y.; Woods, M. C.;
Maruyama, M.; Major, R. T.; Alauddin, M.; Patel, A. R.; Weinges,
K.; Ba¨hr, W. Structure of bilobalide, a rare tert-butyl containing
seuqiterpenoid related to the C20-ginkgolides. J . Am. Chem. Soc.
1971, 93, 3544-3546.
(6) Weinges, K.; Hepp, M.; J aggy, H. Chemie der Ginkgolide, II.
Isolierung und Strukturaufkla¨rung eines neuen Ginkgolids
Liebigs Ann. Chem. 1987, 521-526.
10-O-ben zoph en on e-1-O-d a n syl Gin k golide C (24). Syn-
thesized as 23 but using 2 equiv of dansyl chloride (instead of
1.1 equiv) gave rise to a ca. 1:1 mixture of 23 and 24. The two
products were separated on analytical TLC giving 24 (0.008
1
g, 30%) as a slightly yellow solid. H NMR (400 MHz, DMSO-
d6): δ 0.91 (s, tert-butyl), 1.16 (d, J ) 7.6, CH3,), 1.78 (d, J )
12.5, 8-H), 2.80 [s, N(CH3)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). HPLC-UV: 99.0%. HRMS:
C46H45NO14S
requires M + 1 at m/z 868.2639; found, 868.2668.
(7) DeFeudis, F. V.; Drieu, K. Ginkgo biloba extract (EGb 761) and
CNS functions: basic studies and clinical applications Curr.
Drug Targets 2000, 1, 25-58.
Ra d ioliga n d Bin d in g Assa y. Heart and skeletal muscles
from 13 to 27 months old PAFR transgenic (PAFR-Tg) mice,62
which overexpress guinea pig PAFR, were homogenized in a
Polytron homogenizer in cold buffer A [25 mM Hepes/NaOH
(pH 7.4), 0.25 M sucrose, 10 mM MgCl2, 1 mM PMSF, and a
protease inhibitor cocktail Complete (Boehringer Mannheim)].
An 800g (for 10 min) supernatant of the homogenate was
centrifuged at 100 000g at 4 °C for 1 h, and the resulting pellet
was suspended in buffer A and stored at -80 °C until use.
The protein concentration of the suspended membrane fraction
was 1.37 mg/mL, as measured by the method of Bradford63
using the Bio-Rad protein assay solution and fatty acid-free
bovine serum albumin (BSA; Bayer, Kankakee, IL) as a
standard. The radioligand binding assays were performed
essentially as previously described.53 The membrane fractions
from hearts and skeletal muscles of PAFR-Tg mice (50 µL of
suspension containing 121 fmol of PAFR) were mixed with 2
pmol of [3H]WEB 2086 (NEN Life Science Products, Boston,
MA) in 50 µL of buffer B [25 mM Hepes/NaOH (pH 7.4), 0.25
M sucrose,10 mM MgCl2, 0.1% BSA], and the compound was
to be tested in 100 µL of buffer B in a 96 well microplate in
triplicate for each concentration. These mixtures were incu-
bated at 25 °C for 90 min, upon which the receptor-bound [3H]-
WEB 2086 was filtered on a UniFilter-GF/C (Packard Bio-
science, Meriden, CT) using a MicroMate 196 simultaneous
96 well harvester (Packard Bioscience). The filter was then
washed 10 times with cold buffer B and dried at 50 °C for at
least 90 min, 25 µL of MicroScint-0 scintillation cocktail
(Packard Bioscience) was added, and filters were placed in a
TopCount microplate scintillation counter (Packard Bio-
science). Binding data were analyzed with the nonlinear curve-
fitting program Microplate Manager III (Bio-Rad, Hercules,
CA). Calculated IC50 values were then converted to Ki values
using the Cheng-Prusoff correction,64 with the following
equation: Ki ) IC50/(1 + [L]/KD), where [L] is the concentration
of the radioligand, and KD is the previously determined
dissociation constant for [3H]WEB 2086 (4.3 nM).53 Nonspecific
binding was determined using methods as previously de-
scribed.53
(8) Logani, S.; Chen, M. C.; Tran, T.; Le, T.; Raffa, R. B. Actions of
Ginkgo biloba related to potential utility for the treatment of
conditions involving cerebral hypoxia. Life Sci. 2000, 67, 1389-
1396.
(9) Oken, B. S.; Storzbach, D. M.; Kaye, J . A. The efficacy of Ginkgo
biloba on cognitive function in alzheimer disease. Arch. Neurol.
1998, 55, 1409-1415.
(10) Kleijnen, J .; Knipschild, P. Ginkgo biloba. Lancet 1992, 340,
1136-1139.
(11) Søholm, B. Clinical improvement of memory and other cognitive
functions by Ginkgo biloba: review of relevant literature. Adv.
Nat. Ther. 1998, 15, 54-65.
(12) Diamond, B. J .; Shiflett, S. C.; Feiwel, N.; Mathies, R. J .; Noskin,
O.; Richards, J . A.; Schoenberger, N. E. Ginkgo biloba extract:
mechanisms and clinical indications. Arch. Phys. Med. Rehabil.
2000, 81, 668-678.
(13) van Dongen, M. C. J . M.; van Rossum, E.; Knipschild, P. Efficacy
of Ginkgo biloba special extracts - evidence from randomized
clinical trials. In Medicinal and Aromatic Plants-Industrial
Profiles: Ginkgo biloba; van Beek, T. A., Ed.; Harwood Academic
Publishers: Amsterdam, 2000; Vol. 12, pp 385-442.
(14) Le Bars, P. L.; Katz, M. M.; Berman, N.; Itil, T. M.; Freedman,
A. M.; Schatzberg, A. F. A placebo-controlled double-blind,
randomized trial of an extract of Ginkgo biloba for dementia.
North American EGb study group. J . Am. Med. Assoc. 1997, 278,
1327-1332.
(15) Kanowski, S.; Hermann, W. M.; Stephan, K.; Wierich, W.; Horr,
R. Proof of efficacy of Ginkgo biloba special extract EGb 761 in
outpatients suffering from mild to moderate primary degenera-
tive dementia of the Alzheimer type or multi-infarct dementia.
Pharmacopsychiatry 1996, 29, 47-56.
(16) Watanabe, M. H.; Wolffram, S.; Ader, P.; Rimbach, G.; Packer,
L.; Maguire, J . J .; Schultz, P. G.; Gohil, K. The in vivo
neuromodulatory effects of the herbal medicine Ginkgo biloba.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 6577-6580.
(17) Kennedy, D. O.; Scholey, A. B.; Wesnes, K. A. The dose-
dependent cognitive effects of acute administration of Ginkgo
biloba to healthy young volunteers. Psychopharmacology 2000,
151, 416-423.
(18) Polich, J .; Gloria, R. Cognitive effects of a Ginkgo biloba/
vinpocetine compound in normal adults: systematic assessment
of perception, attention and memory. Hum. Psychopharmacol.
Clin. Exp. 2001, 16, 409-416.
(19) Rigney, U.; Kimber, S.; Hindmarch, I. The effects of acute doses
of standardized Ginkgo biloba extract on memory and psycho-
motor performances in volunteers. Phytother. Res. 1999, 13,
408-415.
(20) Stough, C.; Clarke, J .; Lloyd, J .; Nathan, P. J . Neuropsychologi-
cal changes after 30-day Ginkgo biloba administration in healthy
patients. Int. J . Neuropsychopharmacol. 2001, 4, 131-134.
In tr a cellu la r Ca lciu m Mobiliza tion . CHO cells express-
ing human PAFR57 were subjected to the calcium assay as
described previously56 except that Fura-2/AM was loaded in
the presence of chromophore EL (0.01%, Sigma). Each com-

4046 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Strømgaard et al.
(21) Braquet, P.; Spinnewyn, B.; Braquet, M.; Bourgain, R. H.; Taylor,
J . E.; Etienne, A.; Drieu, K. BN 52021 and related compounds:
a new series of highly specific PAF-acether antagonists isolated
from Ginkgo biloba L. Blood Vessels 1985, 16, 559-572.
(22) Ishii, S.; Shimizu, T. Platelet-activating factor (PAF) receptor
and genetically engineered PAF receptor mutant mice. Prog.
Lipid Res. 2000, 39, 41-82.
(43) Hu, L.; Chen, Z.; Xie, Y.; J iang, Y.; Zhen, H. New products from
alkali fusion of ginkgolides A and B. J . Asian Nat. Prod. Res.
2000, 2, 103-110.
(44) Hu, L.; Chen, Z.; Xie, Y. Synthesis and biological activity of amide
derivatives of ginkgolide A. J . Asian Nat. Prod. Res. 2001, 3,
219-227.
(45) Dorman, G.; Prestwich, G. D. Using photolabile ligands in drug
discovery and development. Trends Biotechnol. 2000, 18, 64-
77.
(46) Flemming, S. A. Chemical reagents in photoaffinity labeling.
Tetrahedron 1995, 51, 12479-12520.
(47) Kotzyba-Hilbert, F.; Kapfer, I.; Goeldner, M. Recent trends in
photoaffinity labeling. Angew. Chem., Int. Ed. Engl. 1995, 34,
1296-1312.
(48) Nassal, M. 4-(1-Azi-2,2,2-trifluoroethyl)benzoic acid, a highly
photolabile carbene generating label readily fixable to biochemi-
cal agents. Liebigs Ann. Chem. 1983, 1510-1523.
(49) Nassal, M. 4′-(1-Azi-2,2,2-trifluoroethyl)phenylalanine, a pho-
tolabile carbene-generating analogue of phenylalanine. J . Am.
Chem. Soc. 1984, 106, 7540-7545.
(23) Prescott, S. M.; Zimmerman, G. A.; Stafforini, D. M.; McIntyre,
T. M. Platelet-activating factor and related lipid mediators.
Annu. Rev. Biochem. 2000, 69, 419-445.
(24) Shukla, S. D. Platelet activating factor. Biomembranes 1996, 2B,
463-479.
(25) Bito, H.; Nakamura, M.; Honda, Z.; Izumi, T.; Iwatsubo, T.;
Seyama, Y.; Ogura, A.; Kudo, Y.; Shimizu, T. Platelet-activating
factor (PAF) receptor in rat brain: PAF mobilizes intracelluar
Ca2+ in hippocampal neurones. Neuron 1992, 9, 285-294.
(26) Mori, M.; Aihara, M.; Kume, K.; Hamanoue, M.; Kohsaka, S.;
Shimizu, T. Predominant expression of platelet-activating factor
receptor in the rat brain microglia. J . Neurosci. 1996, 16, 3590-
3600.
(27) Kroegel, C.; Kortsik, C.; Kroegel, N.; Matthys, H. The patho-
physiological role and therapeutic implications of platelet acti-
vating factor in diseases of aging. Drugs Aging 1992, 2, 345-
355.
(28) Perry, S. W.; Hamilton, J . A.; Tjoelker, L. W.; Dbaibo, G.; Dzenko,
K. A.; Epstein, L. G.; Hannun, Y.; Whittaker, J . S.; Dewhurst,
S.; Gelbard, H. A. Platelet-activating factor receptor activation.
An initiator step in HIV-1 neuropathogenesis. J . Biol. Chem.
1998, 273, 17660-17664.
(50) Keana, J . F. W.; Cai, S. X. New reagents for photoaffinity
labeling: synthesis and photolysis of functionalized perfluo-
rophenyl azides. J . Org. Chem. 1990, 55, 3640-3647.
(51) Lei, H.; Marks, V.; Pasquale, T.; Atkinson, J . K. Synthesis of
photoaffinity label analogues of R-tocopherol. Bioorg. Med. Chem.
Lett. 1998, 8, 3453-3458.
(52) Lei, H.; Atkinson, J . Synthesis of phytyl- and chroman-deriva-
tized photoaffinity labels based on R-tocopherol. J . Org. Chem.
2000, 65, 2560-2567.
(29) Kato, K.; Clark, G. D.; Bazan, N. G.; Zorumski, C. F. Platelet-
activating factor as a potential retrograde messenger in CA1
hippocampal long-term potentiation. Nature 1994, 367, 175-
179.
(30) Kornecki, E.; Wieraszko, A.; Chan, J . C.; Ehrlich, Y. H. Platelet
activating factor (PAF) in memory formation: Role as a retro-
grade messenger in long-term potentiation. J . Lipid Mediators
Cell Signalling 1996, 14, 115-126.
(53) Shindou, H.; Ishii, S.; Uozumi, N.; Shimizu, T. Roles of cytosolic
phopholipase A2 and platelet-activating factor receptor in the
Ca-induced biosynthesis of PAF. Biochem. Biophys. Res. Com-
mun. 2000, 271, 812-817.
(54) Li, H.; Liu, Y.; Fang, K.; Nakanishi, K. A simple photoaffinity
labeling protocol. Chem. Commun. 1999, 365-366.
(55) Maclennan, K. M.; Smith, P. F.; Darlington, C. L. The effects of
ginkgolide
B (BN52021) on guinea pig vestibular nucleus
(31) Chen, C.; Magee, J . C.; Marcheselli, V.; Hardy, M.; Bazan, N.
G. Attenuated LTP in hippocampal dentate gyrus neurons of
mice deficient in the PAF receptor. J . Neurophysiol. 2001, 85,
384-390.
(32) Kobayashi, K.; Ishii, S.; Kume, K.; Takahashi, T.; Shimizu, T.;
Manabe, T. Platelet-activating factor receptor is not required
for long-term potentiation in the hippocampal CA1 region. Eur.
J . Neurosci. 1999, 11, 1313-1316.
(33) Smith, P. F.; Maclennan, K.; Darlington, C. L. The neuropro-
tective properties of the Ginkgo biloba leaf: a review of the
possible relationship to platelet-activating factor (PAF). J .
Ethnopharmacol. 1996, 50, 131-139.
(34) Smith, P. F.; Maclennan, K. The therapeutic potential of PAF
receptor antagonists in CNS disorders. Curr. Opin. Anti-Inflam-
matory Immunomodulatory Invest. Drugs 1999, 1, 205-218.
(35) Corey, E. J .; Ghosh, A. K. Total synthesis of ginkgolide A.
Tetrahedron Lett. 1988, 29, 3205-3206.
(36) Corey, E. J .; Kang, M. C.; Desai, M. C.; Ghosh, A. K.; Houpis, I.
N. Total synthesis of (()-ginkgolide B. J . Am. Chem. Soc. 1988,
110, 649-651.
(37) Corey, E. J .; Su, W. G. Total synthesis of a C15 ginkgolide, (()-
bilobalide. J . Am. Chem. Soc. 1987, 109, 7534-7536.
(38) Corey, E. J .; Gavai, A. V. Simple analogues of ginkgolide B which
are highly active antagonists of platelet activating factor.
Tetrahedron Lett. 1989, 30, 6959-6962.
neurons in vitro: importance of controlling for effects of dimethyl
sulfoxide (DMSO) vehicles. Neurosci. Res. 1996, 26, 395-399.
(56) Fukunaga, K.; Ishii, S.; Asano, K.; Yokomizo, T.; Shiomi, T.;
Shimizu, T.; Yamaguchi, K. Single nucleotide polymorphism of
human platelet-activating factor receptor impairs G-protein
activation. J . Biol. Chem. 2001, 276, 43025-43030.
(57) Aoki, Y.; Nakamura, M.; Kodama, H.; Matsumoto, T.; Shimizu,
T.; Noma, M. A radioreceptor binding assay for platelet-activat-
ing factor (PAF) using membranes from CHO cells expressing
human PAF receptor. J . Immunol. Methods 1995, 186, 225-
231.
(58) Corey, E. J .; Rao, K. S.; Ghosh, A. K. Intramolecular and
intermolecular hydroxyl reactivity differences in ginkgolides A,
B and C and their chemical applications. Tetrahedron Lett. 1992,
33, 6955-6958.
(59) Weinges, K.; Schick, H. Chemie der Ginkgolid IV. Herstellung
von Ginkgolid B aus Ginkgolid C. Liebigs Ann. Chem. 1991, 81-
83.
(60) Chen, J .; Hu, L.; J iang, H.; Gu, J .; Zhu, W.; Chen, Z.; Chen., K.;
J i, R. A 3D-QSAR study on ginkgolides and their analogues with
comparative molecular field analysis. Bioorg. Med. Chem. Lett.
1998, 8, 1291-1296.
(61) van Beek, T. A.; Lelyved, G. P. Preparative isolation and
separation procedure for ginkgolides A, B, C, and J and bilo-
balide. J . Nat. Prod. 1997, 60, 735-738.
(39) Corey, E. J .; Rao, K. S. Enantioselective total synthesis of
ginkgolide derivatives lacking the tert-butyl group, an essential
structural subunit for antagonism of platelet activating factor.
Tetrahedron Lett. 1991, 32, 4623-4626.
(62) Ishii, S.; Nagase, T.; Tashiro, F.; Ikuta, K.; Sato, S.; Waga, I.;
Kume, K.; Miyazaki, J .; Shimizu, T. Bronchial hyperreactivity,
increased endotoxin lethality and melanocytic tumorigenesis in
transgenic mice overexpressing platelet-activating factor recep-
tor. EMBO J . 1997, 16, 133-142.
(63) Bradford, M. M. A rapid and sensitive method for the quanti-
tation of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal. Biochem. 1976, 72, 248-254.
(64) Cheng, Y.; Prusoff, W. H. Relationship between the inhibition
constant (Ki) and the concentration of inhibitor which causes
50% inhibition (I50) of an enzymatic reaction. Biochem. Phar-
macol. 1973, 22, 3099-3103.
(40) Park, P.-U.; Pyo, S.; Lee, S.-K.; Sung, J . H.; Kwak, W. J .; Park,
H.-K.; Cho, Y.-B.; Ryu, G. H.; Kim, T. S. Ginkgolide derivatives.
U.S. Patent 5,541,183, 1996.
(41) Hu, L.; Chen, Z.; Cheng, X.; Xie, Y. Chemistry of ginkgolides:
structure-activity relationship as PAF antagonists. Pure Appl.
Chem. 1999, 71, 1153-1156.
(42) Hu, L.; Chen, Z.; Xie, Y.; J iang, H.; Zhen, H. Alkyl and
alkoxycarbonyl derivatives of ginkgolide B: synthesis and
biological evaluation of PAF inhibitory activity. Bioorg. Med.
Chem. 2000, 8, 1515-1521.
J M020147W