Preparation of 7-substituted ginkgolide derivatives: Potent platelet activating factor (PAF) receptor antagonists

Article abstract of DOI:10.1021/jm0203985

Ginkgolides are structurally unique constituents of Ginkgo biloba extracts and are known antagonists of the platelet-activating factor (PAF) receptor. Ginkgolide C is 25-fold less potent than ginkgolide B as a PAF receptor antagonist, due to the presence of the 7fi-OH. Recently, we found that 7α-fluoro ginkgolide B was equipotent to ginkgolide B underlining the critical importance of the 7-position of ginkgolides for PAF receptor activity. Herein we describe the synthesis of a series of ginkgolide B derivatives with modifications at the 7-position and the pharmacological evaluation of these derivatives as assayed by cloned PAF receptors. In two cases nucleophilic attack on a 7β-O-triflate ginkgolide B did not lead to the expected products, but gave rise to two unprecedented ginkgolide derivatives, one with a novel rearranged skeleton. Furthermore, standard reduction of 7α-azido ginkgolide B did not give the expected primary amine, but instead yielded alkylated amines depending on the solvent employed. Pharmacological testing with cloned PAF receptors showed that ginkgolides with 7α-substitutents had increased affinity compared to 7β-substituents, in particular 7α-chloro ginkgolide B, the most potent nonaromatic ginkgolide derivative described to date with a K_i value of 110 nM.

Full text of DOI:10.1021/jm0203985

J . Med. Chem. 2003, 46, 601-608
601
P r ep a r a tion of 7-Su bstitu ted Gin k golid e Der iva tives: P oten t P la telet Activa tin g
F a ctor (P AF ) Recep tor An ta gon ists
Stine Byskov Vogensen,†,§ Kristian Strømgaard, Hideo Shindou, Stanislav J aracz, Makiko Suehiro,
†,§
‡
†
†
‡
‡
†,
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 Corp., Tokyo 113-0033, J apan
Received September 12, 2002
Ginkgolides are structurally unique constituents of Ginkgo biloba extracts and are known
antagonists of the platelet-activating factor (PAF) receptor. Ginkgolide C is 25-fold less potent
than ginkgolide B as a PAF receptor antagonist, due to the presence of the 7â-OH. Recently,
we found that 7R-fluoro ginkgolide B was equipotent to ginkgolide B underlining the critical
importance of the 7-position of ginkgolides for PAF receptor activity. Herein we describe the
synthesis of a series of ginkgolide B derivatives with modifications at the 7-position and the
pharmacological evaluation of these derivatives as assayed by cloned PAF receptors. In two
cases nucleophilic attack on a 7â-O-triflate ginkgolide B did not lead to the expected products,
but gave rise to two unprecedented ginkgolide derivatives, one with a novel rearranged skeleton.
Furthermore, standard reduction of 7R-azido ginkgolide B did not give the expected primary
amine, but instead yielded alkylated amines depending on the solvent employed. Pharmacologi-
cal testing with cloned PAF receptors showed that ginkgolides with 7R-substitutents had
increased affinity compared to 7â-substituents, in particular 7R-chloro ginkgolide B, the most
potent nonaromatic ginkgolide derivative described to date with a K
i
value of 110 nM.
In tr od u ction
Ginkgo biloba L., the last surviving member of a
family of trees (Ginkgoaceae) that appeared more than
2
50 million years ago, has been mentioned in the
1
Chinese Materia Medica for more than 2500 years. A
standardized G. biloba extract (EGb 761) containing
terpene trilactones (5-7%) and flavonoids (22-24%) has
demonstrated neuromodulatory properties.2 Several
clinical studies using EGb 761 have reported positive
effects on various neurodegenerative diseases, including
Alzheimer’s disease,4 and recent studies on healthy
,3
-6
F igu r e 1. Summary of structure-activity relationship (SAR)
studies of ginkgolides as PAFR antagonists. GB (1) is ca. 25-
fold more potent than GC (2).
volunteers have shown positive effects of EGb 761 on
short-term working memory.7,8
A number of G. biloba constituents have been isolated,
target for neurodegenerative diseases,15 while PAF (1-
O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) has been
suggested as a retrograde messenger in long-term
potentiation (LTP), thus indicating the importance of
the PAFR as a target for ginkgolides.
including the unique terpene trilactones, i.e., ginkgolides
A, B, C, J and M and bilobalide.9
-12
Ginkgolides are
diterpenes with a cage skeleton consisting of six five-
membered rings, the difference between the five ginkgo-
lides being in the variation in the number and positions
of hydroxyl groups on the spirononane framework.
Although the molecular basis for the action of G. biloba
terpene trilactone constituents in the central nervous
system (CNS) is only poorly understood, it is known that
ginkgolides, particularly ginkgolide B (GB, 1, Figure 1),
1
6
Structure-activity relationship (SAR) studies of
ginkgolides on the PAFR have primarily focused on GB
1
7-21
(1) (Figure 1) derivatives
as outlined in Figure 1,
e.g., the importance of lactones and the tert-butyl group
has been investigated, whereas the effect of stereochem-
istry of hydroxyl groups remains to be examined.
Ginkgolide C (GC, 2, Figure 1), having a hydroxyl group
at C-7, is significantly less potent than GB (1),22 which
has been explained by the hydrophilic 7â-OH of GC (2)
being next to the lipophilic tert-butyl group, which is
believed to interact with a lipophilic pocket in the
is a potent in vitro antagonist of the platelet-activating
factor receptor (PAFR).1
3,14
The PAFR is a potential
*
To whom correspondence should be addressed. Phone: +1 212 854
2
169. Fax: +1 212 932 8273. E-mail: kn5@columbia.edu.
†
Department of Chemistry, Columbia University.
Department of Biochemistry and Molecular Biology, The Univer-
‡
14
PAFR. Moreover, substitution at 7-OH further de-
sity of Tokyo and CREST of J apan Science and Technology Corp.
§
creases antagonistic activity, as demonstrated by 7-O-
Present address: Department of Medicinal Chemistry, Pharma-
ceutical University of Denmark, DK-2100 Copenhagen, Denmark.
(4-methylphenyl)-GB that was devoid of PAFR activity23
1
0.1021/jm0203985 CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/21/2003

6
02 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Vogensen et al.
Sch em e 1a
a
Reagents: (a) Tf2O, pyridine, CH2Cl2; (b) NaOCOCH3, DMSO; (c) NaOCOCH2C6H5, DMSO; (d) NaN3, DMSO; (e) TBAF, CH3CN; (f)
TBACl, CH3CN; (g) 2 N NaOH.
Sch em e 2a
a
Reagents: (a) MeOH, 2,6-lutidine; (b) KSCOCH3, DMF; (c) 1 M NaOH, acidic workup.
3
3
and a 7-O-dansyl-GB derivative was also less potent
than the parent compound.
i.e., J 7,8 and 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 nucleo-
phile 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,
2
2
Preliminary studies showed that 7R-F-GB was equi-
potent to GB and 15-fold more potent as a PAFR
antagonist as compared to GC (2),24 despite the fact that
fluorine is sterically equivalent to OH and more polar
2
5
than hydrogen. Since configuration of the fluorine
atom is R, whereas that of the 7-hydroxyl in GC (2) is
â, 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
28
as previously described. In addition to the presence
of multiple electrophilic sites in 3 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 3 with halogens; incorporation of fluorine
7
-position and the assessment of these derivatives for
their ability to displace radioligand binding to cloned
PAFR.
Resu lts
(
7R-F-GB, 7) proceeded in high yield and chlorine (7R-
Cl-GB, 8) 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.
Syn th esis. For the synthesis of derivatives with
variation at C-7, a crucial intermediate was 7â-OTf-GB
(3). GC (2) reacted with remarkable selectivity at 7-OH
with trifluoromethanesulfonic (Tf) anhydride giving 3
in very high yield, with no reactions occurring at other
2
6
Steric hindrance may also be the prerequisite for two
remarkable products arising from reaction of triflate 3
with MeOH and NaSCOCH3, respectively (Scheme 2).
In the former case 3 was dissolved in MeOH and 2,6-
lutidine and reacted for 3 days at 70 °C expecting to
provide 7R-OMe-GB; instead a new product with a
molecular weight similar to GC (2), but with a different
hydroxyl groups. This selectivity is noteworthy, as 10-
OH, and in some cases 1-OH, of GC (2) is generally the
more reactive hydroxyl group,1
9,21
although we recently
observed higher reactivity of 7-OH when acetylation was
performed under strong acidic conditions.27
7
â-OTf-GB (3) was reacted with various nucleophiles
as depicted in Scheme 1 to give derivatives 4-8. The
1
inverted configuration at C-7 was reflected by consider-
H NMR spectrum, was obtained. Extensive NMR
1
able changes in coupling constants in H NMR spectra,
studies revealed a new relactonized structure, neo-

7
-Substituted Ginkgolide Derivatives
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 603
Ta ble 1. Reduction of 7R-N3-GC (6) in Different Solvents
were interested in preparing 1- and 10-OTf derivatives
of GB. However, reaction of GB (1) with Tf anhydride
failed to give any desired product, and instead several
elimination products were obtained, originating from
elimination of 3-OH, as well as 1-OH. Similarly, reaction
of GB (2) with p-toluenesulfonyl (tosyl) chloride gave
two main products, both with a tosyl group at 10-OH,
and elimination of either 3-OH or both 3- and 1-OH,
respectively.
To further investigate the importance of stereochem-
istry at C-7, we planned a series of corresponding 7â-
substituted derivatives. Thus 7-epi-GC (14) 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 7R-OH, relative to 7â-
OH, due to the tert-butyl group.
compound
solvent
R
yield (%)a
1
1
1
1
2
3
MeOH
EtOH
THF
CH3
CH2CH3
H
95
68
98
a
Isolated yield (after flash chromatography).
ginkgolide C (9) (Scheme 2), arising from opening of
lactone C, followed by displacement of the triflate group.
The structure of this compound 9 with a new rearranged
ginkgolide skeleton not encountered earlier was deter-
mined by high resolution mass spectrometry, rotating
frame nuclear Overhauser and exchange spectroscopy
Finally, the observation that an aromatic substituent
at 10-OH of GB (1) and GC (2) increases the antago-
1
9,21,22
nistic effect at PAFR
led us to investigate whether
a similar increase would be observed for 7R-GB deriva-
tives. Benzylated derivatives 15-18 (Table 3) were
therefore prepared, following previously described pro-
(ROESY), correlation spectroscopy (COSY), and hetero-
1
9,21
nuclear single-quantum coherence (HSQC) NMR ex-
periments (see Supporting Information). Moreover,
treatment of neoginkgolide C (9) with 1 M NaOH
followed by acidic workup resulted in a clean conversion
to the thermodynamically more favorable 7-epi-GC (14).
The reaction between triflate 3 and NaSCOCH3 did not
give the expected 7R-SCOCH3-GB, but instead the 10-
acetate, while the 7-triflate group remained intact to
give 10 (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
cedures.
P h a r m a cology. Derivatives 4-9 and 11-18 were
3
tested for their ability to displace [ H]-WEB 2086
binding to cloned PAFR (Tables 2 and 3) using mem-
brane fractions from hearts and skeletal muscles of
PAFR transgenic mice, as previously described.30 In
these fractions, GB (1) had a Ki value of 0.88 µM, thus
being similar to the previously determined Ki value of
2
2
0.56 µM.
Derivatives with 7R-substituents were all more potent
than GC (2) (Table 2), but within this group of com-
pounds there were marked differences; 7R-OAc, 7R-
2
).
Another interesting feature was the reduction of azide
using Pd/C in MeOH under hydrogen. The reaction
OCOBn, 7R-OH, and 7R-NH ginkgolide B derivatives
2
all had Ki values between 2.4 and 7.8 µM, thus being
6
slightly more potent than GC (2), but still significantly
less potent than GB (1). Compounds 6, 7, 11, and 12
with 7R-N3, 7R-F, 7R-NHMe, and 7R-NHEt substituents,
respectively, were equipotent to GB (2) with Ki values
in the range of 0.55-1.62 µM. Finally, 7R-chloro
ginkgolide B (8) was the most potent compound in this
series with a Ki value of 0.11 µM, thus being the most
potent nonaromatic ginkgolide derivative described.
The relactonized compound 9 (Scheme 2) was also
tested for binding to PAFR and was found to be
essentially inactive with a Ki value > 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 15 and 17 were the most
potent with Ki values of 0.12 and 0.10 µM, respectively,
while 10-O-benzyl-GC (16) and 10-O-benzyl-7-epi-GC
did not provide the expected amine, but instead gave
N-methylamine 11 in quantitative yield (Table 1). To
investigate this further, the reaction was carried out in
EtOH, which gave N-ethylamine 12 as the major
product. The desired primary amine 13 was obtained
when THF was used as solvent (Table 1). 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 (14) (Scheme 1) various
approaches were attempted; GC (2) and 4-nitrobenzoic
acid were treated with diethyl azodicarboxylate (DEAD)
and Ph3P in a Mitsunobu reaction, but no reaction was
observed. Instead, 7â-OTf-GB (3) was reacted with
KNO2 and 18-crown-6 ether in a reaction that could
2
9
i
(18) were slightly less potent with K values of 1.67 and
potentially lead to 7-epi-GC (14) directly from 3, 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 7â-OTf-GB (3) was achieved
by reaction with NaOAc; attempts to use the more
reactive CsOAc led to decomposition of 3, while using
CsOCCF3 did not lead to any reaction. The hydrolysis
was accomplished by treating 4 with 2 N NaOH to give
0
.60 µM, respectively.
Discu ssion
Herein the effect of modification of the C-7 position
of ginkgolides has been investigated by synthesis of 15
analogues (4-18), which have been prepared from
native ginkgolides GB (1) and GC (2) and evaluated with
the cloned PAFR.
7
-epi-GC (14) in 95% yield (Scheme 1).
Since the importance of sterochemistry at 1- and 10-
OH of ginkgolides for PAFR activity is not known, we
The derivatives with 7R-substituents were prepared
by nucleophilic substitution of 7â-OTf-GB (3), but in

6
04 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Vogensen et al.
Ta ble 2. Ki Values of Ginkgolide B (1) and C (2), and 7-Epi Derivatives
compound
GB (1)
GC (2)
R
Ki (µM)a
compound
R
Ki (µM)a
H
â-OH
0.88
12.6b
7.84
4.26
2.40
0.55
7R-F-GB (7)
7R-Cl-GB (8)
7R-NHMe-GB (11)
7R-NHEt-GB (12)
7R-NH2-GB (13)
R-F
0.99
0.11
0.61
1.62
8.64
R-Cl
7
7
7
7
R-OAc-GB (4)
R-OAc
R-NHMe
R-NHEt
R-NH2
-epi-GC (14)
R-OH
R-OCOCH2Ph-GB (5)
R-N3-GB (6)
R-OCOCH2Ph
R-N3
a
3
b
Inhibition of [ H]-WEB 2086 binding. Values are means of two independent experiments performed in triplicate. Data from previous
2
2
studies.
Ta ble 3. Ki Values of Benzylated Derivatives
derivatives were slightly more potent than the 7â-
derivatives, as GC (2) had a Ki value of 12.6 µM, while
for 7-epi-GC (14), the Ki is 4.26 µM. Likewise 7R-OAc-
GB (4) had a Ki of 7.84 µM, while 7â-OAc-GB (i.e.,
7
-OAc-GC) had been shown to have low potency com-
2
7
parable to that of GC (2). Furthermore, the 7R deriva-
tive 5 is a reasonable potent PAFR antagonist with a
Ki value of 2.40 µM (Table 2) in contrast to 7â-O-(4-
methylphenyl)-GB, which is devoid of PAFR activity.23
However as these differences are relatively small, the
C-7 configuration seems to play only a minor role for
PAFR antagonistic activity.
compound
R
Ki (µM)a
1
1
1
1
0-OBn-GB (15)
0-OBn-GC (16)
0-OBn-7R-F-GB (17)
0-OBn-epi-GC (18)
H
0.12
1.67
0.10
0.60
â-OH
The nature of the 7R-substituent, on the other hand,
had a major impact on the binding to PAFR. Introduc-
tion of azide and fluorine groups yielded compounds that
were equipotent to GB (1) (Table 2), while introduction
of a chlorine as in 7R-Cl-GB (8) leads to a dramatic
increase in binding affinity. Thus 8 with Ki ) 0.11 µM,
was 115-fold more potent than GC (2) and 8-times more
potent than GB (1), thereby being the most potent
nonaromatic ginkgolide derivative described to date. It
appears that polar groups that can form hydrogen bonds
decrease activity, as seen in 7-epi-GC (14) and 7R-NH2-
GB (13), with a hydroxyl and a primary amino group,
respectively, at C-7, both having binding affinities lower
than GB. On the other hand, alkylation of 13 to give
7R-NHMe-GB (11) and 7R-NHEt-GB (12) led to signifi-
cant increases in binding affinities, with Ki values of
0.61 µM and 1.62 µM, respectively. Rationalization of
these trends requires further molecular mechanistic
studies of the ginkgolide-PAFR interaction, which are
ongoing in our laboratory.
R-F
R-OH
a
3
Inhibition of [ H]-WEB 2086 binding. Values are means of two
independent experiments performed in triplicate.
several cases these reactions did not proceed as ex-
pected. Attempts to introduce larger halogens such as
bromine and iodine, as well as other nucleophiles, failed.
In the reaction between 3 and NaSCOCH3, the 7-OTf
group remained intact; instead the reaction presumably
took place at C-11 of lactone C to give 10 (Scheme 2).
Reaction of 3 with MeOH and 2,6-lutidine gave rise to
neoginkgolide C (9) with a novel rearranged skeleton
(
Scheme 2). This compound had a Ki value > 40 µM,
which is in agreement with previous studies which
showed that modification of lactone C significantly
reduced PAFR binding (Figure 1).28
During the reduction of azide 6 interesting observa-
tions were made; when carried out in MeOH this
reaction did not give the expected primary amine 13,
but gave N-methylamine 11 instead and when carried
out in EtOH the reduction gave N-ethylamine 12.
Besides being a potential novel procedure for a direct
conversion of azides into alkylated amines, it also raises
mechanistic considerations. Treatment of 13 with Pd/C
in MeOH gave 11, albeit in lower yield than starting
from azide 6, and with several side products. This
implies that in the preparation of 11 and 12 the azide
Introduction of benzyl groups in the 10-OH posi-
tion of ginkgolides is known to improve affinity for
1
9,21,22
PAFR,
but whether this is true for 7R-substituted
derivatives was not known. The affinities of 10-O-
benzyl-7R-F-GB (17) and 10-O-benzyl-7-epi-GC (18)
(Table 3) shows 10- and 7-fold improved affinity com-
pared to their nonbenzylated derivatives. Thus 10-
benzylation of 7R-substituted derivatives improves bind-
ing affinity as previously shown for other ginkgolide
derivatives.
6
is initially reduced to amine 13, which then reacts
instantly with the oxidized solvent to form an imine that
is reduced to yield the products. Further studies are
required to confirm this pathway, as well as the
generality of this reaction. These studies are ongoing
in our laboratory.
The prepared derivatives were tested for binding to
cloned PAFR (Tables 2 and 3). It was observed that 7R-
In conclusion we have synthesized several ginkgolide
derivatives with modifications at C-7. These syntheses
have led to several unexpected products such as 9 and
10, as well as a potential novel procedure for a direct
conversion of azides into alkylamines. Moreover, con-
trary to previous convictions, we have shown that

7
-Substituted Ginkgolide Derivatives
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 605
introducing lipophilic groups in the C-7 position, in
particular chlorine, significantly improves PAFR affinity
compared to GB (1). This gives rise to new possibilities
for improving affinity of ginkgolides to PAFR, as well
as providing material for future SAR studies of ginkgo-
lides and PAFR.
with CHCl
3
/EtOAc/MeOH (10:1:1) to obtain 4 as white crystals
(
(
(
144 mg, 75%). A portion (20 mg) of this was recrystallized
1
MeOH/H
2
O) for pharmacological evaluation (9 mg). H NMR
): δ 1.12 (d, J ) 7.1, CH ), 1.10 (s, tert-
), 2.84 (q, J )
400 MHz, DMSO-d
6
3
butyl), 1.91 (d, J ) 3.3, 8-H), 2.06 (s, COCH
3
7
4
5
.1, 14-H), 4.03 (dd, J ) 7.7, 3.6, 1-H), 4.66 (d, J ) 7.7, 2-H),
.86 (d, J ) 3.6, 1-OH), 5.01 (s, 6-H), 5.15 (d, J ) 6.8, 10-H),
.25 (d, J ) 3.3, 7-H), 6.16 (s, 12-H), 6.52 (s, 3-OH), 7.42 (d, J
1
3
Exp er im en ta l Section
) 6.8, 10-OH). 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,
Ch em istr y. Gen er a l P r oced u r es. GB (1) and GC (2) was
obtained by extraction of leaves from G. biloba, purification
by column chromatography and recrystallization as previously
9
2.6, 99.8, 112.7, 171.3, 171.5, 175.0, 178.1. HPLC-UV: 96%.
HRMS: C22
83.1525.
r-O-P h en yla ceta te Gin k golid e B (5). Sodium phenyl-
27
H O
12 requires M + 1 at m/z 483.1503; found,
4
3
1,32
1
described.
The purity was >98% as estimated by H NMR.
7
Unless otherwise noted, materials were obtained from a
commercial supplier and were used without further purifica-
tion. 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 precoated silica gel 60 F254 plates with thickness of 0.25
mm. Plates were heated and spots were detected by monitoring
acetate (44 mg, 0.28 mmol) and 3 (32 mg, 0.07 mmol) were
dissolved in DMSO (0.8 mL) and heated at 65 °C for 5 h, the
solvent was removed in vacuo, 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
1
13
(5 × 10 mL), and brine (2 × 10 mL) and dried (MgSO
4
), and
at 254 nm. H and 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 chroma-
tography (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
the solvent was 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. H NMR (400 MHz, DMSO-d
1
6
): δ 1.07 (s,
), 1.93 (d, J ) 2.9, 8-H), 2.84
), 4.04 (dd, J ) 3.6, 7.7, 1-H),
tert-butyl), 1.12 (d, J ) 6.9, CH
3
(q, J ) 6.9, 14-H), 3.70 (s, CH
2
4
5
6
1
4
9
1
.62 (d, J ) 7.7, 2-H), 4.72 (d, J ) 3.6, 1-OH), 4.99 (s, 6-H),
.16 (d, J ) 6.6, 10-H), 5.26 (d, J ) 2.9, 7-H), 6.16 (s, 12-H),
.48 (s, 3-OH), 7.25-7.35 (m, aromatic, 5H) 7.46 (d, J ) 6.6,
0-OH). 13C NMR (100 MHz, CD
OD): δ 8.0, 30.8 (3C), 33.8,
2.2, 43.4, 53.3, 70.1, 70.3, 72.5, 75.2, 80.2, 81.0, 84.6, 92.6,
9.9, 112.7, 128.4, 129.7, 130.5, 134.7, 171.4, 172.2, 175.0,
3
with either eluent A or B. A: water/CH
raising to (40:60:1) after 20 min. B: water/CH
5:0.1), raising to (40:60:1) after 20 min. Analytical HPLC were
3
CN/TFA (60:40:0.1),
3
CN/TFA (65:
3
78.1. HPLC-UV: 98%. HRMS: C28
31
H O12 requires M + H
performed using a 5 µm C18 reversed-phase Phenomenex
at m/z 559.1816; found, 559.1826.
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 9 and 14 were
7r-Azid o Gin k golid e B (6). Sodium azide (87 mg, 1.34
mmol) and 3 (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
eluted with water/CH
3
CN/TFA 80:20:0.10 and compounds 11-
1
3 with water/CH CN 90:10. Accurate mass determinations
3
were performed on a J EOL J MS-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
4
between saturated aqueous NH 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) and dried (MgSO
in vacuo. The crude product was purified by flash chromatog-
raphy eluting with CHCl /MeOH/EtOAc (30:1:1) to give the 6
as white crystals (109 mg, 88%). A portion (23 mg) of this was
recrystallized (MeOH/H O) for pharmacological evaluation (12
mg). H NMR (300 MHz, DMSO-d ): δ 1.12 (d, J ) 7.1, CH ),
4
), and the solvent was removed
7
-Tr iflu or om eth a n esu lfon yloxy Gin k golid e B (3). In a
mixture of dry CH Cl (1.0 mL) and dry pyridine (1.5 mL) was
2
2
dissolved GC (2) (184 mg, 0.42 mmol). The solution was cooled
to -20 °C under argon, and trifluoromethane sulfonic anhy-
dride (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, the residue was
dissolved in EtOAc (30 mL) and washed with 1 N HCl (3 × 20
3
2
1
6
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
1
4
mL) and brine (10 mL) and dried (MgSO ), and the solvent
.25 (s, 6-H), 6.10 (s, 12-H), 6.52 (s, 3-OH), 7.05 (d, J ) 6.0,
was removed in vacuo. The crude product was purified by flash
chromatography eluting with CHCl /CH OH/EtOAc (30:1:1
and 20:1:1) to obtain 3 as white crystals (232 mg, 97%). H
NMR (400 MHz, DMSO-d ): δ 1.11 (s, tert-butyl), 1.13 (d, J )
.6, CH ), 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,
0-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 )
0-OH). 13C NMR (100 MHz, DMSO-d
): δ 7.8, 30.1 (3C), 32.7,
6
3
3
1
41.6, 51.6, 67.2, 68.4, 68.5, 71.0, 73.7, 79.6, 82.9, 92.9, 98.2,
10.3, 169.5, 173.4, 176.2. HPLC-UV: 99%. HRMS: C20
requires M + 1 at m/z 466.1462; found, 466.1445.
1
24
H -
6
10 3
O N
9
3
(
1
7r-F lu or o Gin k golid e B (7). Tetrabutylammonium fluo-
ride hydrate (37 mg, 0.14 mmol) and 3 (62 mg, 0.11 mmol)
were dissolved in CH CN (1 mL) and heated at 80 °C for 1.5
(
3
1
3
7
3
9
.4, 10-OH). C NMR (100 MHz, DMSO-d
6
): δ 9.1, 29.2 (3C),
h. The solvent was removed in vacuo, 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 ×
2.6, 42.1, 49.0, 64.2, 68.1, 69.4, 74.4, 75.2, 84.0, 86.3, 93.2,
1
9.9, 109.4, 118.6 (q, J CF ) 316.6, CF
3
), 173.9, 176.9, 179.2.
HRMS: C21
5
23 3
H F O
13S requires M + 1 at m/z 573.0890; found,
73.0872.
r-O-Aceta te Gin k golid e B (4). Sodium acetate (163 mg,
.99 mmol) and 3 (228 mg, 0.39 mmol) were dissolved in
15 mL) and brine (2 × 15 mL) and dried (MgSO ), and the
4
7
solvent was removed in vacuo. The crude product was purified
1
3
by flash column chromatography eluting with CHCl /MeOH/
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), the
combined organic phases were washed with brine (2 × 10 mL)
EtOAc (30:1:1) followed by preparative HPLC (eluent B) to give
7 as white crystals (34 mg, 71%). H NMR (400 MHz,
1
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 )
2
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).
)
1
and dried (MgSO
4
), and the solvent was removed in vacuo. The
13
crude product was purified by flash chromatography eluting
3
C NMR (75 MHz, CD OD): δ 7.0, 29.5 (3C), 32.9, 42.4, 53.7

6
06 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Vogensen et al.
(2
8
1
J
CF ) 20.4 Hz), 68.8, 69.1, 71.5, 74.5, 79.5 ( J CF ) 36.0 Hz),
2
172.6, 174.8, 178.4. HPLC-UV: 97%. HRMS: C21
requires M + 1 at m/z 454.1713; found, 454.1719.
7r-N-Eth yla m in o Gin k golid e B (12). Azide 6 (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 for 48 h. The solvent was removed in vacuo,
2
EtOAc (10 mL) was added, and the solution was filtered
through Celite. The solvent was removed in vacuo and the
28 10
H O N
1
3.7, 91.7, 96.9 ( J CF ) 184.2 Hz), 98.8, 111.6, 171.2, 173.9,
77.1. HRMS: C20 23FO10 requires M + 1 at m/z 443.1354;
found, 443.1370.
r-Ch lor o Gin k golid e B (8). Tetrabutylammonium chlo-
ride (86 mg, 0.31 mmol) and 3 (36 mg, 0.06 mmol) were
dissolved in CH CN (1.4 mL) and heated at 80 °C for 12 h.
H
7
3
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
3
residue purified by flash chromatography eluting with CHCl /
MeOH/EtOAc (30:1:1) to give white crystals, which were
1
recrystallized (MeOH) to give 12 (22 mg, 47%). H NMR (300
MHz, CD
and CH ), 1.89 (d, J ) 4.5, 8-H), 2.57 (dq, J ) 7.1, 12.0, CH
H), 2.94 (dq, J ) 7.1, 12.0, CH , 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,
-H), 5.08 (s, 6-H), 5.27 (s, 10-H), 6.16 (s, 12-H). 13C NMR (100
MHz, CD OD): δ 8.2, 15.5, 31.3 (3C), 34.4, 41.6, 43.3, 53.5,
7.5, 69.2, 70.8, 72.6, 75.3, 80.0, 84.4, 94.4, 100.6, 112.3, 172.6,
74.9, 178.4. HPLC-UV: 98%. HRMS: C22 10N requires
3
OD): δ 1.11 (t, J ) 7.1, CH
3
), 1.23 (m, tert-butyl
(2 × 10 mL) and dried (MgSO
4
), and the solvent was removed
3
2
,
in vacuo. The crude product was purified by preparative HPLC
eluent B) and recrystallized (CH CN/CHCl ) to give 8 as white
crystals (9 mg, 30%). H NMR (400 MHz, DMSO-d ): δ 1.12
), 1.17 (s, tert-butyl), 2.19 (d, J ) 4.2, 8-H),
.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),
.13 (d, J ) 6.4, 10-H), 5.26 (s, 6-H), 6.17 (s, 12-H), 6.53 (s,
1
2
(
3
3
1
6
2
(
2
d, J ) 7.0, CH
3
3
6
1
)
5
3
29
H O
1
3
M + 1 at m/z 468.1870; found, 468.1867.
-OH), 7.57 (d, J ) 6.4, 10-OH). C NMR (100 MHz,
CD3OD): δ 8.7, 31.2 (3C), 34.5, 42.5, 54.2, 65.1, 69.0, 70.0,
2.2, 74.4, 83.7, 84.2, 90.7, 99.4, 111.3, 169.9, 174.4, 177.1.
HPLC-UV: 98%. HRMS: C20 10Cl requires M + 1 at m/z
59.1058; found, 459.1052.
Neogin k golid e C (9). Triflate 3 (27 mg, 0.05 mmol) was
7r-Am in o Gin k golid e B (13). Azide 6 (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 for 14 h. EtOAc (10 mL) was added and the solution filtered
2
through Celite. The solvent was removed in vacuo to give white
7
24
H O
4
crystals which were recrystallized (MeOH) to give 13 as white
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 /MeOH/
3
EtOAc (20:1:1) to give the crude product, which was further
1
crystals (5 mg, 49%). H NMR (400 MHz, CD
3
OD): δ 1.20 (s,
), 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),
tert-butyl), 1.23 (d, J ) 7.1, CH
3
(
4
.51 (d, J ) 7.0, 2-H), 5.01 (s, 6-H), 5.04 (s, 10-H), 6.18 (s, 12-
13
H). C NMR (100 MHz, CD
3
OD): δ 8.3, 31.0 (3C), 34.1, 43.2,
4.7, 60.3, 68.9, 70.9, 72.6, 74.9, 83.9, 84.4, 95.0, 100.3, 111.8,
72.3, 174.4, 178.4. HPLC-UV: 97%. HRMS: C42
purified by preparative HPLC (eluent A) to give 9 as white
5
1
1
crystals (6 mg, 29%). H NMR (400 MHz, CD
3
OD): δ 1.20 (m,
26 10
H O N
CH
.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,
3
and tert-butyl), 1.64 (dd, J ) 1.4, 1.2, 8-H), 3.72 (q, J )
requires M + H at m/z 440.1557; found, 440.1594.
7
(
6
3
8
7
-Ep i-gin k golid e C (14). Acetate 4 (42 mg, 0.087 mmol)
-H), 5.96 (s, 12-H). 1 C NMR (100 MHz, DMSO-d
3
was dissolved in a mixture of MeOH and 2 N NaOH (2:1, 1.8
mL) and stirred for 5 h. HCl (1 N) was added, and the aqueous
phase was extracted with EtOAc (3 ×20 mL). The combined
organic phases were washed with brine (2 × 10 mL) and dried
6
): δ 7.6,
0.2 (3C), 32.7, 41.3, 47.8, 60.2, 66.9, 67.8, 73.6, 75.6, 82.5,
3.4, 92.7, 93.7, 104.9, 170.2, 171.7, 177.6. HPLC-UV: 98%.
HRMS: C20
63.1245.
0-O-Aceta te-7-tr iflu or om eth a n esu lfon yloxy Gin k go-
lid e B (10). Potassium thioacetate (4 mg, 0.035 mmol) and 3
3 mg, 0.006 mmol) were dissolved in dry DMF (35 µL) and
24
H O11 requires M + Na at m/z 463.1216; found,
4
(MgSO ), and the solvent was removed in vacuo to give 14 (36
4
mg, 95%) as white crystals. A portion (15 mg) of this was
recrystallized (MeOH/H O) for pharmacological evaluation (6
mg). H NMR (400 MHz, DMSO-d ): δ 1.12 (d, J ) 7.0, CH ),
.12 (s, tert-butyl), 1.64 (d, J ) 2.7, 8-H), 2.89 (q, J ) 7.0, 14-
1
2
1
6
3
(
1
heated at 40 °C for 3 h. The solvent was removed in vacuo,
and residue was partitioned between water (10 mL) and EtOAc
H), 4.11 (dd, J ) 4.5, 6.8, 1-H), 4.37 (dd, J ) 2.7, 6.3, 7-H),
4
5
6
.59 (d, J ) 6.8, 2-H), 4.98 (s, 6-H), 5.04 (d, J ) 2.7, 10-H),
.52 (d, J ) 6.3, 7-OH), 5.64 (d, J ) 4.5, 1-OH), 6.13 (s, 12-H),
(
×
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) and dried (MgSO ),
and the solvent was removed in vacuo. The crude product was
purified by flash chromatography eluting with CHCl /MeOH/
EtOAc (20:1:1) to give 10 (1.3 mg, 18%) as white crystals. H
NMR (400 MHz, DMSO-d ): δ 1.08 (s, tert-butyl), 1.13 (d, J )
.1, CH ), 2.21 (s, COCH ), 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),
13
.45 (s, 3-OH), 6.73 (d, J ) 2.7, 10-OH). C NMR (75 MHz,
4
DMSO-d ): δ 8.0, 30.2 (3C), 32.6, 41.4, 52.3, 68.8, 69.6, 71.5,
7
6
3.4, 76.2, 80.8, 82.8, 92.9, 98.6, 109.7, 170.0, 172.7, 176.3.
3
HPLC-UV: 98%. HRMS: C20
441.1397; found, 441.1395.
25
H O11 requires M + 1 at m/z
1
6
1
0-O-Ben zyl Gin k golid e B (15). Synthesis and analytical
7
)
5
1
3
3
19,21
data as previously described.
1
2 3
0-O-Ben zyl Gin k golid e C (16). K CO (31 mg, 0.22
.09 (dd, J ) 12.6, 4.2, 7-H), 5.48 (d, J ) 4.2, 6-H), 6.13 (s,
0-H), 6.33 (s, 12-H), 6.53 (s, 3-OH), 6.71 (d, J ) 5.8, 1-OH).
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
HRMS: C23
6
26 14 3
H O F S requires M + 1 at m/z 615.0995; found,
15.1016.
r-N-Meth yla m in o Gin k golid e B (11). Azide 6 (38 mg,
.08 mmol) was dissolved in dry MeOH (1.2 mL), and Pd/C
2
.5 h at 60 °C. The solvent was removed in vacuo, the residue
7
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 ×
0
(
10%, 12 mg) was added. The suspension was stirred under
an atmosphere of H for 48 h. The solvent was removed in
vacuo, EtOAc (10 mL) was added, and the solution was filtered
through Celite. The solvent was removed in vacuo, and the
crude product was purified by flash chromatography eluting
2
10 mL) and brine NaCl (2 × 10 mL) and dried (MgSO ), and
4
the solvent was removed in vacuo. The crude product was
purified by flash chromatography eluting with CHCl /MeOH/
3
EtOAc (20:1:1) and further by preparative HPLC (solvent
1
with CHCl
3
/MeOH/EtOAc (30:1:1) to give white crystals, which
were recrystallized (MeOH) to give 11 (23 mg, 65%) as white
system A) to give 16 (9 mg, 77%) as white crystals. H NMR
(400 MHz, CD OD): δ 1.21 (s, tert-butyl), 1.23 (d, J ) 7.1, CH ),
3
3
1
crystals. H NMR (400 MHz, CD
and CH ), 1.89 (d, J ) 4.4, 8-H), 2.47 (s, CH
.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).
3
OD): δ 1.22 (m, tert-butyl
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),
3
3
), 3.06 (q, J )
7
4.76 (d, J ) 10,2, CH
2
, 1H), 5.04 (s, 6-H), 5.02 (d, J ) 4.3,
, 1H), 6.14 (s, 12-
H), 7.37-7.44 (m, aromatic, 5H). C NMR (75 MHz, CDCl ):
δ 7.2, 29.1 (3C), 32.2, 41.6, 50.5, 64.1, 67.1, 73.8, 74.3, 75.6,
(
6-H), 5.25 (s, 10-H), 5.46 (d, J ) 10.2, CH
2
1
3
13
C NMR (100 MHz, CD
3
OD): δ 8.2, 31.3 (3C), 33.4, 34.3, 43.3,
3.5, 69.1, 69.6, 70.8, 72.6, 75.4, 79.3, 84.4, 94.5, 100.6, 112.2,
3
5

7
-Substituted Ginkgolide Derivatives
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 607
7
1
7.2, 79.3, 83.5, 90.6, 98.5, 110.1, 128.9 (2C), 129.5 (2C), 129.8,
(2) 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.
34.2, 170.8, 170.8, 175.5. HPLC-UV: 98%. HRMS: C27
30 11
H O
requires M + Na at m/z 553.1686; found, 553.1684.
0-O-Ben zyl-7r-flu or o Gin k golid e B (17). Synthesized
as described for 16 using K CO (107 mg, 0.77 mmol), 7 (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
(3) DeFeudis, F. V. Ginkgo biloba extract (EGb 761). From chemistry
to clinic; Ullstein Medical: Wiesbaden, 1998; p 401.
1
2
3
(
4) 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.
(
5) 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.
chromatography eluting with CHCl
give 17 (16 mg, 39%) as white crystals. H NMR (400 MHz,
CDCl
3
/MeOH/EtOAc (30:1:1) to
1
3
): δ 1.25 (s, tert-butyl), 1.30 (d, J ) 7.0, CH3), 1.88 (dd,
2
000, 81, 668-678.
2
J
HF ) 44.5, J ) 2.3, 8-H), 2.94 (d, J ) 3.1, 1-OH), 3.06 (q, J
(6) 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.
)
4
6
7.0, 14-H), 4.28 (dd, J ) 8.1, 3.1, 1-H), 4.50 (d, J ) 8.1, 2-H),
2
.68 (d, J ) 9.4, CH
2
, 1H), 4.95 (s, 10-H), 5.29 (d, J HF ) 10.2,
1
-H), 5.30 (dd,
J HF ) 50.1, J ) 1.5, 7-H), 5.41 (d, J ) 9.4,
, 1H), 6.04 (s, 12-H), 7.36 (m, aromatic, 5H). C NMR (100
13
CH
MHz, CDCl
2
(
(
(
7) 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.
8) 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.
9) Nakanishi, K. The ginkgolides. Pure Appl. Chem. 1967, 14, 89-
113 and references therein.
2
3
): δ 7.2, 30.3 (3C), 32.9, 41.7, 53.3 ( J CF ) 20.4),
2
6
8.2, 71.6, 74.1, 74.4, 75.1, 79.5 ( 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,
1
70.2, 170.8, 175.1. HPLC-UV: 98%. HRMS:
27 30 10
C H O F
requires M + 1 at m/z 533.1823; found, 533.1784.
0-O-Ben zyl-7-ep i-gin k golid e C (18). Synthesized as
described for 16 using K CO (50 mg, 0.36 mmol), 14 (16 mg,
.04 mmol), and benzyl chloride (42 µL, 0.36 mmol) in DMF
0.3 mL). The crude product was purified by flash chromatog-
raphy eluting with CHCl /MeOH/EtOAc (20:1:1) and further
by preparative HPLC (eluent A) to give 18 (11 mg, 56%) as
1
2
3
0
(
(10) Okabe, K.; Yamada, K.; Yamamura, S.; Takada, S. Ginkgolides.
J . Chem. Soc. C 1967, 2201-2206.
3
(
11) Nakanishi, K.; Habaguchi, K.; Nakadaira, Y.; Woods, M. C.;
Maruyama, M.; Major, R. T.; Alauddin, M.; Patel, A. R.; Weinges,
K.; B a¨ hr, W. Structure of bilobalide, a rare tert-butyl containing
sesquiterpenoid related to the C20-ginkgolides. J . Am. Chem. Soc.
1971, 93, 3544-3546.
1
white crystals. H NMR (400 MHz, CDCl
butyl), 1.29 (d, J ) 7.0, CH ), 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),
.40 (bs, 3-OH), 4.28 (dd, J ) 3.6, 7.8, 1-H), 4.48 (m, 2-H, 7-H),
.72 (d, J ) 9.2, CH , 1H), 4.96 (s, 6-H), 5.51 (s, 10-H), 5.50
, 1H), 6.09 (s, 12-H), 7.39-7.44 (m, aromatic,
H). C NMR (100 MHz, CDCl ): δ 7.3, 30.6 (3C), 33.1, 41.6,
3
): δ 1.23 (s, tert-
3
)
3
4
(12) Weinges, K.; Hepp, M.; J aggy, H. Chemie der Ginkgolide, II.
Isolierung und Strukturaufkl a¨ rung eines neuen Ginkgolids
Liebigs Ann. Chem. 1987, 521-526.
13) Braquet, P. The ginkgolides: potent platelet-activating factor
antagonists isolated from Ginkgo biloba L.: chemistry, phar-
macology and clinical applications. Drugs Future 1987, 12, 643-
699.
14) Braquet, P. Esanu, A.; Buisine, E.; Hosford, D.; Broquet, C.;
Koltai, M. Recent progress in ginkgolide research. Med. Chem.
Rev. 1991, 11, 295-355.
15) Singh, M.; Saraf, M. K. Platelet-activating factor: a new target
site for the development of nootropic agents. Drugs Future 2001,
26, 883-888.
2
(d, J ) 9.2, CH
2
(
1
3
5
5
1
3
2.9, 68.4, 71.3, 74.0, 74.4, 74.7, 77.6, 82.3, 83.2, 90.7, 98.5,
10.5, 129.2 (2C), 129.6 (2C), 130.1, 133.8, 170.3, 170.5, 175.4.
(
HPLC-UV: 99%. HRMS: C27
31.1866; found, 531.1895.
Ra d ioliga n d Bin d in g Assa y. The radioligand binding
31
H O11 requires M + 1 at m/z
5
(
3
0
assays were performed as previously described. 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 [ H]-WEB 2086 in 50 µL
of buffer [25 mM HEPES/NaOH (pH 7.4), 0.25 M sucrose, 10
mM MgCl , 0.1% BSA] and the compound to be tested in 100
2
µL of buffer in a 96-well microplate in triplicate for each
concentration. These mixtures were incubated at 25 °C for 90
(16) 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-
3
1
79.
(
17) 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.
(18) 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.
19) 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.
3
min, upon which the receptor-bound [ 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
(
(
20) Hu, L.; Chen, Z.; Cheng, X.; Xie, Y. Chemistry of ginkgolides:
structure-activity relationship as PAF antagonists. Pure Appl.
Chem. 1999, 71, 1153-1156.
Manager III (Bio-Rad, Hercules, CA). Nonspecific binding was
determined using methods as previously described.30
Ack n ow led gm en t. We are grateful to Dr. Yasuhiro
Itagaki for performing HRMS and Dr. Nasri Nesnas for
discussions. We thank the following for financial sup-
port: Memory Pharmaceuticals Corp., the Alfred Ben-
zon Foundation (to K.S.), the Welfide Medicinal Re-
search Foundation (to H.S.), the Alfred Bader Foundation
(21) 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.
(
22) Strømgaard, K.; Saito, D. R.; Shindou, H.; Ishii, S.; Shimizu, T.;
Nakanishi, K. Ginkgolide derivatives for photolabeling studies:
preparation and pharmacological evaluation. J . Med. Chem.
2002, 45, 4038-4046.
23) Pietri, S.; Liebgott, T.; Finet, J .-P.; Culcasi, M.; Billottet, L.;
Bernard-Henriet, C. Synthesis and biological studies of a new
ginkgolide C derivative: evidence that the cardioprotective effect
of ginkgolides is unrelated to PAF inhibition. Drug. Dev. Res.
(
(to S.J .), and the Ministry of Health, Labor and Welfare,
J apan (to S.I.) for a Grants-in-Aid for Comprehensive
Research on Aging and Health and Research on Allergic
Disease and Immunology.
2
001, 54, 191-201.
(
(
24) Suehiro, M.; Strømgaard, K.; Nakanishi, K. Unpublished results.
25) Smart, B. E. Fluorine substituent effects (on bioactivity) J .
Fluorine Chem. 2001, 109, 3-11.
26) Teng, B.-P. Preparation process of ginkgolide B from ginkgolide
C. U.S. Patent 5,599,950, 1997.
Su p p or tin g In for m a tion Ava ila ble: COSY, HSQC, and
ROESY spectra of compound 9. This material is available free
of charge via the Internet at http://pubs.acs.org.
(
(
27) J aracz, S.; Strømgaard, K.; Nakanishi, K. Ginkgolides: selective
acetylations, translactonization and biological evaluation. J . Org.
Chem. 2002, 67, 4623-4626.
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.
(28) 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.

6
08 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Vogensen et al.
(
29) Moriarty, R. M.; Zhuang, H.; Penmasta, R.; Liu, K.; Awasthi, A.
K.; Tuladhar, S. M.; Rao, M. S. C.; Singh, V. K. Inversion of
configuration of R-trisubstituted (neopentyl) type secondary
alcohols. Tetrahedron Lett. 1993, 34, 8029-8032.
30) 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.
(31) Lichtblau, D.; Berger, J . M.; Nakanishi, K. An efficient extraction
of ginkgolides and bilobalide from Ginkgo biloba leaves. J . Nat.
Prod. 2002, 65, 1501-1504.
(32) 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.
(
J M0203985