Preparation of a tritiated ginkgolide

Article abstract of DOI:10.1016/j.bmcl.2004.08.042

A radiolabeled constituent of Ginkgo biloba, [³H]-ginkgolide B, was prepared in high specific radioactivity, providing a tool for studying neuromodulatory properties of ginkgolides. Ginkgolide B, a constituent of the tree Ginkgo biloba, was radiolabeled with the β-emitter tritium ([ ³H]) in two steps from ginkgolide C. First, a triflate precursor was prepared utilizing the selective reactivity of 7-OH in ginkgolide C; the triflate was then reduced with sodium borotritide to yield tritiated ginkgolide B ([³H]GB) in good yield and high specific activity. The tritiated ginkgolide will be an important tool for studying neuromodulatory properties of ginkgolides.

Full text of DOI:10.1016/j.bmcl.2004.08.042

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5673–5675
Preparation of a tritiated ginkgolideI
Kristian Strømgaard,*, Makiko Suehiro and Koji Nakanishi*
Department of Chemistry, Columbia University, New York, NY 10027, USA
Received 18 June 2004;revised 17 August 2004;accepted 18 August 2004
Available online 15 September 2004
Abstract—Ginkgolide B, a constituent of the tree Ginkgo biloba, was radiolabeled with the b-emitter tritium ([3H]) in two steps from
ginkgolide C. First, a triflate precursor was prepared utilizing the selective reactivity of 7-OH in ginkgolide C;the triflate was then
reduced with sodium borotritide to yield tritiated ginkgolide B ([3H]GB) in good yield and high specific activity. The tritiated gink-
golide will be an important tool for studying neuromodulatory properties of ginkgolides.
Ó 2004 Elsevier Ltd. All rights reserved.
Ginkgo biloba L. is the last surviving member of a family
of trees, Ginkgoacea that appeared more than 250 mil-
lion years ago and has been mentioned in the Chinese
Materia Medica for more than 2500years.1 A number
of G. biloba natural products have been isolated, the
most unique being the terpene trilactones, that is, ginkgo-
lides A, B, C, J, and M (1–5, Fig. 1) and bilobalide.2 The
ginkgolides are diterpenes with a cage skeleton consist-
ing of six 5-membered rings, a spiro[4.4]nonane carbo-
cyclic ring, three lactones, and a tetrahydrofuran. The
difference of the five ginkgolides is variations in the
number and positions of hydroxyl groups at C-1, C-3,
and C-7 of the spirononane framework (Fig. 1). A
Figure 1. Structures of the five ginkgolides isolated from Ginkgo
biloba.
standardized G. biloba extract, EGb 761 containing ter-
pene trilactones (5–7%) and flavonoids (22–24%), has
demonstrated neuromodulatory properties,3 while sev-
eral clinical studies using EGb 761 have confirmed pos-
itive effects on various neurodegenerative diseases.4
of G. biloba constituents on the central nervous system
Recent studies on healthy volunteers have also shown
(CNS), in particular terpene trilactones, is poorly under-
positive effects of EGb 761 on short-term working
stood.6 Moreover, ginkgolide B (GB, 2) is a potent in vi-
memory.5
tro antagonist of the platelet-activating factor receptor
(PAFR), a G protein-coupled receptor7 that is a poten-
Although ginkgolides have been shown to be neuro-
protective in vitro, the molecular basis for the action
tial target for neurodegenerative diseases.8 PAF has
been suggested as a retrograde messenger in long-term
potentiation (LTP), although this role remains contro-
versial.9 Thus, PAFR plays an important role in the
brain, but whether the effect of ginkgolides in the CNS
Keywords: Natural products;Radiolabeling;Ginkgolides.
q Part of this work was presented at the 15th International Symposium
on Radiopharmaceutical Chemistry, August 10–14, 2003, Sydney,
is related to its effect on PAFR, or whether ginkgolides
Australia. Abstract published in J. Labelled Compd. Radiopharm.
have other targets in the CNS is currently unknown.10
2003, 46, S214.
*
Corresponding authors. Tel.: +1 212 854 2169;fax: +1 212 932
8273;e-mail: kn5@columbia.edu
Recently, a new target for ginkgolides has been
discovered;ginkgolides are highly potent and selective
antagonists of glycine receptors, which belong to the
ligand-gated ion channel (LGIC) family.11 The
Present address: Department of Medicinal Chemistry, The Danish
University of Pharmaceutical Sciences, Universitetsparken 2, DK-
2100 Copenhagen, Denmark.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.042

5674
K. Strømgaard et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5673–5675
importance of this action in relation to neuromodula-
tory properties of ginkgolides has yet to be character-
ized, and a radiolabeled ginkgolide becomes a vital
tool for such studies.
In order to explore targets for ginkgolides in CNS on a
molecular level we have prepared a tritiated ginkgolide
where the ginkgolide skeleton has been labeled with
the long-lived b-emitter tritium ([3H]). The [3H]-labeled
ligand can be used for ex vivo autoradiography, radio-
ligand binding assays, and other in vitro studies with
various cell cultures. Tritiated ginkgolides will provide
information on whether ginkgolides cross the blood
brain barrier (BBB), the distribution of specific binding
sites in the CNS, and in particular whether or not ginkgo-
lides have specific binding sites other than the PAFR.
Scheme 2. Formation of a side product during reduction, due to the
presence of base.
Previous studies have reported approaches related to
radiolabeling of G. biloba constituents, primarily with
[14C];in one study leaves from G. biloba plants fed with
[14C]-acetate was extracted, leading to a [14C]-labeled
G. biloba extract, however, the actually labeled chemical
constituents are not identified.12 Recently, an approach
to the synthesis of [14C]-labeled ginkgolide A (GA, 1)
using [14C]-labeled methyl propionate, was reported,
but synthesis of the actual radiolabeled ligand was not
described.13 Moreover, the application of a [14C]-labeled
ginkgolide may have serious limitations because of its
low specific activity. Very recently ginkgolide B has been
labeled with the positron emitter 18F to visualize its in
vivo behavior by positron emission tomography
(PET).14 Here we describe the synthesis of high specific
activity [3H]-ginkgolide B.
[3H]GB (7) could then be prepared by treating 7-OTf-
GB (6) with a tritiated reducing agent leading to intro-
duction of the radiolabel in the 7-position of GB.
Initially, the reaction was carried out with nonradioac-
tive reducing agents;triflate 6 was reduced with tetrabu-
tylammonium borohydride (nBu4NBH4) giving GB (2)
in moderate yield. Several other reducing agents such
as NaBH4, NaBH3CN, and LiBHEt3 were investigated.
However, in all cases when a 1:1 molar ratio of 6 and
reducing agent was used, several side products were
observed. Hence, we could not reproduce the results
obtained by Teng.15a [3H]GB (7) was then prepared
by reaction of triflate 6 with [3H]nBu4NBH4, the lat-
ter being prepared from [3H]-sodium borohydride
([3H]NaBH4), and tetrabutylammonium chloride
(nBu4NCl) in [3H]H2O.18 The specific activity of 7 was
3.8Ci/mmol, which is very low considering the starting
[3H]NaBH4 had a specific activity of 100Ci/mmol. The
low specific activity was presumably due to a rapid ex-
change between hydrogen in nonradioactive water con-
tained in [3H]H2O and tritium in [3H]NaBH4. The
[3H]H2O used in this study had a specific activity of
5Ci/mL corresponding to only 0.09Ci/mmol, which is
the highest specific activity commercially available.
Among the five ginkgolides, GB (2) was selected for
labeling, as GB (2) is both the most potent PAFR and
glycine receptor antagonist.2b Moreover, it was envis-
aged that tritiated GB ([3H]GB) could be prepared from
GC in only two steps using site-selective reactivity of
sulfonic anhydride, that is, trifluoromethanesulfonic
anhydride, toward 7-OH of GC in a basic medium as
originally reported by Teng.15a Thus the reaction of
ginkgolide C (GC, 3) with trifluoromethanesulfonic
anhydride in pyridine gives 7-OTf-GB (6) in high yield
(97%, Scheme 1).15 This selectivity is noteworthy, as
10-OH, and in some cases 1-OH, of GC (3) is generally
the more reactive hydroxyl group,16 although we re-
cently observed higher reactivity of 7-OH when acetyl-
ation is carried out under strong acidic conditions17
(Scheme 2).
Instead [3H]NaBH4 was used as a reducing agent to pro-
vide [3H]GB (7) with high specific activity. Although the
initial studies showed that a 1:1 molar ratio of 6 and
NaBH4 did not give GB (2), a 1:5 molar ratio gave
GB (2) in low to moderate yields, thus providing a
Scheme 1. Preparation of labeled ginkgolide B.

K. Strømgaard et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5673–5675
5675
potential means of getting [3H]GB (7) with high specific
activity. The incorporation was confirmed using NaBD4
to give [2H]GB (8), incorporation ca. 70%. However,
when the reaction was carried out with [3H]NaBH4 in
dry THF the desired [3H]GB (7) was produced only in
trace amounts. It was realized that the commercially
available [3H]NaBH4 contained substantial amounts of
NaOH, which in the presence of trace amount of water
contained in the solvents could open the lactone rings.
Moreover, a byproduct was observed;from a nonradio-
active synthesis this product was shown to be 7-epi-GC
(9), originating from a nucleophilic attack of the triflate
group by NaOH.
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9.6min), by collection of fractions every 30s. The radio-
activity of the fractions was determined by scintillation
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In conclusion, [3H]GB has been synthesized with high
specific radioactivity, thus providing an important tool
for the studies of neuromodulatory properties of
ginkgolides.
Acknowledgements
Financial supports from NIH grants MH 68817 (K.N.),
MH 6336 (M.S.) and a postdoctoral fellowship from the
Alfred Benzon Foundation (K.S.) are gratefully
acknowledged.
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