A systematic study of C-glucoside trisphosphates as myo-inositol trisphosphate receptor ligands. Synthesis of β-C-glucoside trisphosphates based on the conformational restriction strategy

Article abstract of DOI:10.1021/jm051039n

β-C-Glucoside trisphosphates having a C2 side chain (3,7-anhydro-2-deoxy-D-glycero-D-gulo-octitol 1,5,6-trisphosphate, 11) and a C3 side chain (4,8-anhydro-2,3-dideoxy-D-glycero-D-gulo-nonanitol 1,6,7-trisphosphate, 12) were designed as structurally simpl

Full text of DOI:10.1021/jm051039n

1900
J. Med. Chem. 2006, 49, 1900-1909
A Systematic Study of C-Glucoside Trisphosphates as myo-Inositol Trisphosphate Receptor
Ligands. Synthesis of â-C-Glucoside Trisphosphates Based on the Conformational Restriction
Strategy
Masaru Terauchi,† Hiroshi Abe,† Stephen C. Tovey,‡ Skarlatos G. Dedos,‡ Colin W. Taylor,‡ Michael Paul,§ Melanie Trusselle,§
Barry V. L. Potter,§ Akira Matsuda,† and Satoshi Shuto*,†
Graduate School of Pharmaceutical Sciences, Hokkaido UniVersity, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan, Department of
Pharmacology, Tennis Court Road, UniVersity of Cambridge, Cambridge, CB2 1PD, U.K., and Wolfson Laboratory for Medicinal Chemistry,
Department of Pharmacy and Pharmacology, UniVersity of Bath, Bath BA2 7AY, U.K.
ReceiVed October 17, 2005
â-C-Glucoside trisphosphates having a C2 side chain (3,7-anhydro-2-deoxy-D-glycero-D-gulo-octitol 1,5,6-
trisphosphate, 11) and a C3 side chain (4,8-anhydro-2,3-dideoxy-D-glycero-D-gulo-nonanitol 1,6,7-
trisphosphate, 12) were designed as structurally simplified analogues of a potent D-myo-inositol 1,4,5-
trisphosphate (IP3) receptor ligand, adenophostin A. Construction of the â-C-glucosidic structure, which
was the key to their synthesis, was achieved by two different methods based on the conformational restriction
strategy: (1) radical cyclization with a temporary connecting silicon tether and (2) silane reduction of
glyconolactols having an anomeric allyl substituent. Using these methods, the target â-C-glycoside
trisphosphates 11 and 12 were successfully synthesized. A structure-activity relationship was established
on a series of C-glucoside trisphosphates, including the previously synthesized related compounds, which
were a C-glycosidic analogue 3 of adenophostin A, its uracil congener 5, R-C-glucoside trisphosphates 7-9
having a C1, C2, or C3 side chain, and the â-C-glucoside trisphosphates 10-12 having a C1, C2, or C3
side chain. The O-glycosidic linkage of adenophostin A and its analogues proved to be replaced by the
chemically and biologically more stable C-glycosidic linkage. The R-C2-glucoside trisphosphate 8 stimulates
Ca2+ release with a potency similar to that of IP3 in spite of its simplified structure, indicating a better fit
to the receptor than the â-C-glucoside trisphosphates and also the R-congeners having a shorter or longer
C1 side chain, which was supported by molecular modeling using the ligand binding domain of the IP3
receptor.
Introduction
C-glucosidic trisphosphates as potential IP3 receptor ligands,
because C-glycosides can be biologically stable mimics of the
corresponding O-glycosides.8 Thus, we previously designed and
synthesized the C-glycosidic analogue 3 of adenophostin A and
its uracil congener 5.6d,e However, their biological effects have
not been reported yet.
D-myo-Inositol 1,4,5-trisphosphate (IP3, 1, Figure 1) is
produced by phospholipase C-catalyzed hydrolysis of phos-
phatidylinositol 4,5-bisphosphate, which is stimulated by activa-
tion of a diverse array of cell-surface receptors. IP3 has been
shown to mobilize Ca2+ from the intracellular stores of most
mammalian cells.1 Many IP3 analogues have been synthesized
as specific ligands of IP3 receptors. Such analogues may be
useful in investigating the mechanisms of IP3-mediated Ca2+
signaling and also as leads for developing novel clinically
effective drugs.2,3
In 1993, adenophostin A (2) was isolated from Penicillium
breVicompactum and shown to be a high-affinity agonist of IP3
receptors with 10 to ∼100 times higher affinity than IP3.4 These
results prompted the syntheses of new IP3 receptor ligands based
on the structure of adenophostin A and investigation of the
structure-activity relationships of adenophostin A and related
compounds.5-7 The results showed that the D-glucopyranose
structure is a good bioisostere of the myo-inositol backbone of
IP3, and that the adenine moiety can be replaced by other
aromatic rings: the uracil congener 4, for example, is almost
as potent as adenophostin A in stimulating release of Ca2+ from
intracellular stores.5 Consequently, we became interested in the
The three-dimensional positioning of the three phosphate
moieties and, in particular, the lone “auxiliary” phosphate group
significantly affects the activity of IP3, adenophostin A, and
their derivatives.5,6 2-Hydroxyethyl R-D-glucopyranoside 3,4,2′-
trisphosphate (6) was originally designed and synthesized as a
highly simplified analogue of adenophostin A.7a,b,e Although
the O-glycoside trisphosphate 6 is an agonist of the IP3 receptor,
its affinity is more than 10-fold lower than that of IP3.5 We
hypothesized that the lower affinity of 6 could be due to its
side-chain length, which may be too long to allow the phosphate
to achieve an effective position for binding. To test this
hypothesis, we designed a series of R-C-glucoside trisphosphates
7-9 having side chains with different lengths, as well as the
corresponding â-C-glucoside trisphosphates 10-12. The â-gly-
cosidic structure might be more effective than the R-one, because
the lone “auxiliary” phosphate group of IP3 seems to locate on
the â-face of the D-glucose backbone when the inositol and
pyranose rings are superimposed. In this strategy, it is essential
to employ a series of C-glycosides as stable mimics of the
O-glycosides since the corresponding O-glycoside trisphosphates
(Figure 2) could not be prepared because of their predictable
instability due to the “O-C-O-O-P” or “O-C-O-C-O-
* Corresponding author. Tel and fax: 81-11-706-3769. E-mail:
shu@pharm.hokudai.ac.jp.
† Hokkaido University.
‡ University of Cambridge.
§ University of Bath.
10.1021/jm051039n CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/16/2006

C-Glucoside Trisphosphates as IP3 Receptor Ligands
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 1901
Figure 1. IP3 receptor ligands.
Scheme 1
Figure 2. Putatively unstable R-O-glucoside trisphosphates.
P” structure. Syntheses of these R-C-glycoside trisphosphates
7-9 and the â-C-glucoside trisphosphate 10 with a C1 side
chain have been reported.6f,g,7c
In this paper, we describe the synthesis of the â-C-glycoside
trisphosphate 11 with a C2 side chain and 12 with a C3 side
chain, in which the key stereoselective construction of the â-C-
glucosidic structure was achieved by the conformational restric-
tion strategy using an intramolecular radical cyclization or silane
reduction at the anomeric position. The results of a systematic
biological evaluation of a series of the new and previously
synthesized R- and â-C-glucoside trisphosphates are described,
and the structure-activity-relationship is discussed together with
molecular modeling.
Results and Discussion
â-Selective Radical Cyclization Based on the Conforma-
tional Restriction Strategy. Much attention has been focused
on C-glycosides because of their importance as stable biologi-
cally active mimics for natural O-glycosides. Methods for their
synthesis have been extensively studied.8 However, synthesis
of â-C-glycosides is considerably more difficult than synthesis
of the corresponding R-C-glycosides.8 Accordingly, effective
construction of the â-C-glycosidic structure should be the key
step in the synthesis of the target compounds in this study.
The use of radical reactions is one of the most efficient
methods for constructing C-glycosidic bonds,8,9 and therefore
we have been working toward the development of stereoselec-
tive intramolecular and intermolecular radical C-glycosidation
reactions.6d-g,9 Stork and co-workers reported an efficient
synthesis of a â-C-glucoside via a stereoselective radical
cyclization using a 2,3,4-tri-O-benzyl-protected phenyl l-seleno-
â-D-glucose derivative having a phenylethynylsilyl group as a
radical acceptor tether at the 6-hydroxyl.10 As shown in Scheme
1a, heating the substrate with Bu3SnH and AIBN in benzene,
followed by treatment with TBAF, gave the 2-phenylvinyl â-C-
glucoside in 54% yield. They speculated that the radical
cyclization would proceed via a conformationally flipped
intermediate (Scheme 1a) in which the tethered hydroxymethyl
moiety assumed an axial orientation. With these results in mind,
we planned to examine the â-selective introduction of C2 and
C3 units by radical reactions with the corresponding 6-O-
vinylsilyl11 and 6-O-allylsilyl12 substrates.
We previously investigated the introduction of a C2 and a
C3 unit stereoselectively at the anomeric R-position of D-glucose
via the radical cyclization with a vinylsilyl or allylsilyl group
as a temporary connecting tether.6f,g During these studies, we
found that the conformational restriction of substrates was
effective in realizing highly R-selective reactions.6f,g,9 When the
substrate with the 2-O-allylsilyl group conformationally re-
stricted in the unusual 1C4-conformation was used, the desired
R-cyclization occurred exclusively (Scheme 1b), while the
reaction with the corresponding conformationally unrestricted
substrate gave a mixture of the R- and â-products (Scheme
1c).6g,i A similar highly R-selective radical cyclization also
1
occurred with the C4-restricted substrate with 2-O-vinylsilyl
group to produce the R-C-glycoside having an anomeric C2
chain.6f

1902 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Terauchi et al.
Scheme 2
Figure 3. Conformationally restricted and unrestricted substrates for
the radical cyclization reaction.
Scheme 3
Based on these findings, we decided to apply the conforma-
tional restriction strategy to the synthesis of â-C-glycosides via
radical cyclization reactions.13 Our synthetic plan is shown in
Scheme 2. We designed the 6-O-vinylsilyl or O-allylsilyl-D-
glucose derivative I, the conformation of which was restricted
in a 1C4-chair form, as the substrate of radical cyclization
reactions. Ab initio calculations suggest that the anomeric radical
1
intermediate preferentially assumes the substratelike C4-form
Figure 3), to clarify whether the conformational restriction of
substrates in the C4-form facilitated the â-selective radical
cyclization.
1
when the conformations of the precursors of the radical are
restricted in an unusual 1C4-chair form.9a Therefore, we expected
that the radical cyclization using the conformationally 1C4-
restricted substrate I would stereoselectively form the desired
â-cyclization product III, via the 1C4-chair-like anomeric radical
intermediate II, in which the cis-cyclization would effectively
occur without a change of conformation because of the axial
orientation of the 6-hydroxymethyl moiety bearing the tether,
as shown in Scheme 2. The conformational restriction of the
The conformations of the substrates 13-16 were investigated
1
by H NMR. The unrestricted substrates 15 and 16 had large
coupling constants (ca. 9 Hz) between the ring protons showing
4
their preference for the usual C1-chair-like conformation. On
the other hand, the considerably smaller coupling constants
between the ring protons in the 3,4-O-silyl-protected substrates
1
13 and 14 indicated that these preferred the flipped C4-like
1
conformation, as expected.13
The radical reactions of the C4-restricted substrates 13 and
substrates to the desired C4-form was thought to be possible
1
using significantly bulky silyl protecting groups, as described
below. Oxidative cleavage of the Si-C bond of III would give
the â-C-glucoside IV, which can be converted into the target
trisphosphates 11 or 12.
It is known that introducing a significantly bulky protecting
group at the 3,4-trans-hydroxyls of pyranoses causes a confor-
mational flip leading to a 1C4-form, in which the bulky
substituents are in axial positions due to mutual steric repul-
sion.9,14,19 Thus, the 3,4-bis-O-silylated substrate 13 having a
6-O-vinylsilyl group and 14 having a 6-O-allylsilyl group
(Figure 3), which should be conformationally restricted in the
1C4-form, were designed for the radical reaction.
The synthesis of the substrates 13 and 14 is summarized in
Scheme 3. Starting from D-glucose, phenyl 3,4-bis-O-TBS-6-
O-trityl-l-seleno-â-D-glucose (17) was prepared according to the
previously reported method.6f Acetylation of the 2-hydroxyl of
17 and subsequent selective removal of the 6-O-trityl group gave
19. Treatment of 19 with vinyldiphenylchlorosilane, DMAP,
and Et3N in toluene at room temperature quantitatively gave
the 6-O-vinylsilyl ether 13, the radical reaction substrate.
Similarly, the allyldimethylsilyl group was introduced at the
6-hydroxyl with allyldimethylchlorosilane to give the other
substrate 14.
14 as well as the unrestricted substrates 15 and 16 were
performed by slow addition of a mixture of Bu3SnH (1.2 equiv)
and AIBN (0.6 equiv) to a heated solution of the substrate in
benzene (80 °C). The reaction was carried out first with the
1C4-restricted vinylsilyl ether 13 and afforded the desired
1
â-endo-cyclization product 20 in 40% yield. When the C4-
restricted allylsilyl ether 14 was subjected to the reaction under
the same conditions, the endo-cyclization effectively occurred
to give the desired â-product 21 in 72% yield along with the
anomeric reduction product 22 in 20% yield. The stereochem-
istry of the cyclization products 20 and 21 was confirmed by
NOE experiments (Figure 4). On the other hand, in the treatment
of both of the conformationally unrestricted substrates 15 and
16, under similar Bu3SnH/AIBN conditions, many spots were
observed on TLC, and none of the cyclization products were
obtained.
We also prepared conformationally unrestricted substrates,
i.e., the 6-O-allyldimethylsilyl and 6-O-vinyldiphenylsilyl ethers
of phenyl 2,3,4-tri-O-benzyl-l-seleno-â-D-glucose (15 and 16,
Figure 4. NOE data of the radical cyclization products 20 and 21.

C-Glucoside Trisphosphates as IP3 Receptor Ligands
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 1903
Scheme 5
Scheme 4
Synthesis of the Target Trisphosphate 11 via Stereose-
lective Anomeric Silane Reduction with the Conformation-
ally Restricted Substrate. Synthesis of the target 11, however,
was accomplished by another route as shown in Scheme 5, via
highly stereoselective anomeric deoxygenation based on the
conformational restriction strategy.
Kishi reported an efficient synthesis of â-C-glycosides, which
involves Grignard addition to a glyconolactone to give usually
an anomeric mixture of the corresponding lactols bearing an
anomeric substituent that is reduced stereoselectively by tri-
alkylsilane under Lewis acidic conditions to the â-C-glycoside.17
However, the stereochemical outcome of the reduction depends
on the sugar structure, and the stereoselectivity is not always
high.17a,d
We have shown that, in the radical9 and also the Lewis acid
promoted18 C-glycosidation reactions, the anomeric effect19 can
be controlled by manipulating the substrate conformation, and
thus, depending on the conformation of the substrates restricted
to the 4C1- or the 1C4-form, R- or â-selective reactions can occur
highly stereoselectively.9,18 Therefore, we expected that in
Kishi’s silane reduction the stereoselectivity could also be
improved by enhancing the kinetic anomeric effect, when
conformationally restricted substrates were employed. The
conformation of the transition state and the intermediate can
be significantly influenced by conformational effects, which
stabilize the ground state conformation.9,18,20 Accordingly, as
shown in Figure 5, in the reduction of substrate A conforma-
It is worth noting that the reactions of the vinylsilyl and
allylsilyl ethers 13 and 14 produced the unusual 8-endo- and
9-endo-cyclization products 20 and 21, respectively. These
results show that the conformational restriction strategy ef-
fectively works in the radical cyclization.
Synthesis of the Target Trisphosphate 12. Conversion
of the radical reaction products 20 and 21 into the target
trisphosphates 11 and 12 was tried (Scheme 4). The 8-mem-
bered ring opening via the oxidative Si-C bond fission, with-
out removing the silyl protecting groups at the 3- and
4-hydroxyls, was achieved by treatment with AcOOH/HBr/KBr
in DMF,15 where the â-C-glucosides 23 and 24 were obtained
quantitatively and in 61% yield, respectively. Selective silyla-
tion of the primary hydroxy on the anomeric chain was next
examined. When the hydroxypropyl C-glucoside 24 was treated
with TBDPSCl/imidazole in DMF at -40 °C, selective silyla-
tion occurred to give the desired 26 in 64% yield. Treatment
of the hydroxyethyl C-glucoside 23 under the same condi-
tions gave the selectively silylated product 25 in only 11%
yield. Although we examined selective silylation of 23
under various conditions, the desired 25 was not obtained
selectively.
After benzoylation of the 6-hydroxyl of 26, the three silyl
protecting groups were simultaneously removed to give the triol
28. The phosphate units were introduced, using the phosphora-
midite method with o-xylene N,N-diethylphosphoramidite (XE-
PA).16 Thus, 28 was treated with XEPA and tetrazole in CH2Cl2,
followed by oxidation with m-CPBA to give the desired
trisphosphate derivative 29 in 58% yield. Finally, the protecting
groups of the phosphates and the hydroxyls were removed by
successive hydrogenation and basic hydrolysis to furnish the
target 12 quantitatively as a sodium salt, after treatment with
ion-exchange resin.
Synthesis of the other target 11 by this route was abandoned
because of the very low yield of the anomeric side chain
selective silylation of 23, in which the 6-hydroxyl was prefer-
entially silylated.
Figure 5. The working hypothesis for the kinetic anomeric effect
dependent R-hydride attack forming the â-C-glycosides.

1904 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Terauchi et al.
Table 1. Effects of Compounds on Ca2+ Release via Rat Type 1 IP3
Scheme 6
Receptors Expressed in DT40 Cellsa
Ca2+
release,
%
relative potency
EC50
,
Hill
slope
nM
n
1
2
1
2
3
4
5
7
8
9
24.8 ( 2.1 1.21 ( 0.06 78 ( 2 11
1
0.09 ( 0.01
1
2.1 ( 0.2 1.54 ( 0.13 76 ( 1 12 12.8( 1.3
4.0 ( 0.2 1.56 ( 0.15 81 ( 2
4.6 ( 0.4 1.31 ( 0.08 68 ( 2a
11.3 ( 2.7 1.40 ( 0.29 79 ( 3
3
4
3
3
5
5
5
5
5
6.2 ( 1.2
0.64 ( 0.05
0.43 ( 0.03
0.24 ( 0.05
0.004 ( 0.001
0.04 ( 0.01
0.009 ( 0.001
0.002 ( 0.001
6.7 ( 2.4
tionally restricted to a 4C1-chair form, the transition state would
assume the C1-chair-like form B due to the conformational
2.4 ( 0.6
4
394 ( 39
49.2 ( 4.3 1.36 ( 0.15 78 ( 1
213 ( 37 1.39 ( 0.27 69 ( 2
1.07 ( 0.19 73 ( 1
0.11 ( 0.03
0.43 ( 0.07
0.12 ( 0.01
0.04 ( 0.01
restriction of the pyranose backbone, where the anomeric center
would be pyramidal. The R-axial attack transition state B in
the 4C1-restricted form would be significantly stabilized by the
kinetic anomeric effect, i.e., the interaction between the anti-
bonding σ*q of the newly forming anomeric C-H bond and
the orbital of a nonbonded electron pair (nO) on the ring oxygen,
because of their planar arrangement, to produce the â-C-
glycoside highly selectively.21
10 1080 ( 130 0.98 ( 0.12 70 ( 1
11 2040 ( 430 1.51 ( 0.46 71 ( 3
12 4430 ( 1090 1.51 ( 0.35 64 ( 5
0.014 ( 0.003 0.001 ( 0.0002
0.007 ( 0.001 0.0006 ( 0.0002
a The experiments with 4 were performed separately from the others,
although again in parallel with 1 (IP3) and 2 (adenophostin A); the maximal
Ca2+ release evoked by 1 and 2 in these experiments was 72 ( 2% and 69
( 3%, respectively. It is therefore clear that maximal concentrations of all
the ligands listed were as effective as 1 or 2.
Based on this hypothesis, we designed the silane reduction
substrate 31 bearing a 3,4-O-cyclic-diketal group, the conforma-
and its uracil congener (4 to 5); in both cases there was a ∼2-
fold decrease in potency. These results show that, irrespective
of the attached nucleobase, changing the glycosidic linkage from
-O- to -CH2- is remarkably well tolerated. Insofar as their
interactions with IP3 receptors are concerned, the three-
dimensional structures of the C-glycosidic analogues 3 and 5
thus seem to be similar to those of the O-glycosides 2 and 4.
The simplified R- and â-C-glucoside trisphosphates 7-12
were all less potent than adenophostin A (Table 1). This accords
with previous results showing that aromatic rings such as
adenine effectively enhance the potency of adenophostin-related
compounds.5 The activities of these C-glucosides were affected
by the anomeric R/â-stereochemistry. The â-C-glucosides were
considerably less potent than the corresponding R-C-glucosides.
The EC50 values of the â-C-glucosides were > 1 µM, whereas
within the R-series, the C2-glucoside 8, with an EC50 value of
49.2 ( 4.3 nM, was almost comparable to that of IP3.
The R-C2-glucoside 8 was clearly more potent than either
its R-C1 7 (EC50 ) 394 ( 39 nM) or R-C3 9 (EC50 ) 213 (
37 nM) analogues. Thus, side-chain length markedly affects
activity with an optimum chain length of R-C2 in this series.
These results suggest that three-dimensional positioning of the
“auxiliary” phosphate group in the R-C2-glucoside 8 is suitable
for effective receptor binding. It should be noted that 8 is
significantly more potent than the simplified O-glucoside 6
reported previously, the potency of which is more than 10-fold
lower than IP3,5,9b and thus this systematic study with a series
of C-glucoside trisphosphates represents a “fine-tuning” of
activity related to the auxiliary side chain of this long established
prototype analogue.
Modeling Study. The R-C-glucoside trisphosphate 8, which
is almost as potent as IP3, can be used to address the binding
mode of adenophostin A and its analogues with a pyranose
structure. This is because the simplified structure of 8 makes
the conformational analysis easier than with adenophostin A
and its analogues reported previously.7d
We calculated the stable three-dimensional structure of the
R-C2-glucoside 8 by MacroModel and compared it with that
of IP3. The initial geometries were generated by a conforma-
tional search with the Monte Carlo method and were optimized
with MMFFs force field. The results are shown in Figure 6.
Both compounds have analogous trans-vicinal phosphates on
the six-membered chair-form ring, and these moieties are well
superimposed, as shown in Figure 6c. However, the third
phosphate of the R-C2-glucoside 8 is located at a position
significantly different from that of IP3. These results suggest
4
tion of which would be restricted in the desired C1-form due
to the trans-decalin-type ring system.6f,g,9,18,22 The substrate 31
was prepared from D-glucose via an addition of CH2dCHCH2-
MgBr on the gluconolactone 30.23
When the substrate 31 was treated with Et3SiH and TMSOTf
in CH2Cl2 at -78 °C, the hydride reduction occurred from the
R-side with complete stereoselectivity, as we expected, to give
the â-C-glycoside 32 in 92% yield. We also examined the
reduction of the corresponding conformationally unrestricted
tetra-O-benzyl substrate 34, where the stereoselectivity and also
the yield clearly decreased (R:â ) 9:91, yield 79%),21 as shown
4
in Scheme 6. These results demonstrated that the C1-confor-
mational restriction effectively improved the stereoselectivity
in the Et3SiH reduction.
Ozonolysis of 32 followed by deprotection with aqueous TFA
gave the triol product 33. Phosphorylation by the phosphora-
midite method and subsequent hydrogenation removing the
benzyl groups finally afforded the target trisphosphate 11.
Biological Activity. The ability of a series of C-glycosidic
compounds, i.e., the C-glycosidic adenophostin A analogue 3,
its uracil congener 5, and the simplified R- and â-C-glucoside
trisphosphates 7-12, to stimulate opening of the pore of IP3
receptors was examined. For these analyses, we used recom-
binant rat type 1 IP3 receptors stably expressed in chicken B
cells that otherwise lack IP3 receptors and a fluorescent Ca2+
indicator trapped within the lumen of the intracellular Ca2+
stores to measure the decrease in luminal free [Ca2+] that follows
activation of IP3 receptors.24-27 The method and its advantages
have been recently described.28 The results are summarized in
Table 1, and the potencies are presented relative to both IP3 (1)
and adenophostin A (2).
Maximal concentrations of the analogues 3-5 and 7-12, as
well as adenophostin A, examined in this study released the
same fraction of the intracellular Ca2+ stores (70-80%) as a
maximally effective concentration of IP3 (Table 1), suggesting
that each is likely to be a full agonist of the IP3 receptor. The
C-glycosidic analogue 3 of adenophostin A was a potent agonist
with an EC50 value of 4.0 ( 0.2 nM. It was thus some 6-fold
more potent than the natural ligand IP3 (EC50 ) 24.8 ( 2.1
nM) and only 2-fold less potent than adenophostin A (EC50
)
2.1 ( 0.2 nM). The C-glycosidic uracil congener 5 was also
remarkably active (EC50 ) 11.3 ( 2.7 nM), being about 2-fold
more potent than IP3 and only 5-fold less potent than adeno-
phostin A. The effect of replacing the O-glycosidic linkage with
a C-glycosidic linkage was similar for adenophostin A (2 to 3)

C-Glucoside Trisphosphates as IP3 Receptor Ligands
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 1905
IP3 also interacts with Arg504, Asp566 (not shown), and the
peptide backbone of Arg568, mediated by crystal waters. It may
be postulated that the difference in activity between the R- and
â-glucosides may be related to the interaction of the phosphate
group with different arginine residues. Furthermore, from Figure
7a, a suggestion for the higher activity of the C-glucoside 8
may be given in that this is the only glucoside trisphosphate
which shows an interaction of the phosphate group with more
than one arginine residue, i.e., Arg269 and Arg504. The other
two R-C-glucosides 7 and 9, not shown here, show an interaction
only with Arg269 and Arg504, respectively. For compound 7
the C1 chain does not seem to be long enough for the phosphate
to reach Arg269, and for compound 9 the C3 chain moves the
phosphate too far away from Arg504. However, the crystal
structure used in this study comprises only the binding domain
of the IP3 receptor. Differences in the observed biological
activity might be linked to interactions of ligands with other
parts of the receptor.
Figure 6. Stable structures of IP3 (a) and the R-C2-glucoside 8 (b)
calculated by MacroModel with MMFFs force field and their super-
imposition (c).
Conclusion. The synthesis of â-C-glucoside trisphosphates
11 and 12 having a C2 side chain or a C3 side chain, designed
as structurally simplified analogues of adenophostin A, was
achieved. In the synthesis, the key â-C-glucosidic structures
were effectively constructed based on the conformational
restriction strategy using the radical cyclization with a temporary
connecting silicon tether or the silane reduction of glyconolactols
having an anomeric allyl substituent. We showed that the
O-glycosidic linkage of adenophostin A and its analogues can
be replaced by the chemically and biologically more stable
C-glycosidic one, and that, in a series of structurally simplified
C-glucoside trisphosphates related to adenophostin A, the R-C2-
glucoside trisphosphate 8 is almost as potent as IP3 in causing
intracellular Ca2+ mobilization. This is rationalized in a
preliminary fashion using molecular modeling.
that the binding mode of the R-C2-glucoside 8 to the receptor
might also be different from that of IP3.
Potential interactions of the R- and â-C-glucoside trisphos-
phates 7-12 with the IP3 receptor binding site were subse-
quently explored. Superimposition of 8 and IP3 indicate that
the 3,4-vicinal phosphates of the C-glucosides attached directly
to the ring may bind to the receptor in the same conformation
as the 4,5-vicinal phosphates of IP3. The C-glucoside trispho-
sphates 7-12 were built directly into the IP3 receptor crystal
structure using the bound IP3 as a template. Energy minimization
of each system was performed to identify interactions between
the ligand and the receptor.
Figure 7shows the modeling of potential interactions of the
R-C2-glucoside 8 (Figure 7a) and the â-C2-glucoside 11 (Figure
7b) with arginine residues, which recognize the phosphates of
IP3 in the binding site of the IP3 receptor. IP3 bound in the crystal
structure is also shown (Figure 7c). The trans-vicinal phosphate
groups of 8 and 11 and the 4- and 5-phosphates of IP3 have
similar conformations; however, it is clear that there are
differences between the orientations and interactions of the
epimeric side-chain phosphate groups of 8 and 11 and the
1-phosphate group of IP3. Comparing the conformations of the
C-glucosides 8 and 11, it is clear that the most dominant
interaction is different between the R- and â-glucosides, in that
the phosphate group in the R-glucoside 8 points down toward
Arg504 and Arg269, whereas it points toward Arg568 in the
â-glucoside 11. IP3 in the crystal structure has a binding mode
similar to that of the compound: Arg568 forms hydrogen bonds
with the 1-phosphate of IP3 and also with the side-chain
phosphate of 11. In addition to this, the 1-phosphate group of
Experimental Section
Chemical shifts are reported in ppm downfield from tetrameth-
ylsilane (1H and 13C) or H3PO4 (31P), and coupling constants are
1
given in Hz. All of the H NMR assignments described were in
agreement with COSY spectra. Thin-layer chromatography was
done on Merck coated plate 60F254. Silica gel chromatography was
done on Merck silica gel 5715. Reactions were carried out under
an argon atmosphere.
Phenyl 2-O-Acetyl-3,4-bis-O-tert-butyldimethylsilyl-1-seleno-
â-D-glucopyranoside (19). A mixture of 17 (1.6 g, 2.0 mmol), Ac2O
(377 µL, 4.0 mmol) and DMAP (733 mg, 6.0 mmol) in MeCN (20
mL) was stirred at room temperature for 12 h. The mixture was
partitioned between AcOEt and H2O, and the organic layer was
washed with brine, dried (Na2SO4) and evaporated to give crude
18 (1.7 g, quantitative) as a colorless oil. A mixture of the oil and
aqueous TFA (80%, 500 µL) in CHCl3 (5 mL) was stirred at room
Figure 7. Calculated binding modes of R-C2-glucoside 8, â-C2-glucoside 11, and IP3: (a) modeling of potential interactions between the R-C-
glucoside 8 and arginine residues 504 and 269; (b) the â-C-glucoside 11 andArg568; and (c) IP3 with Arg568 in the ligand binding domain of the
IP3 receptor.

1906 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Terauchi et al.
temperature for 1 h and then evaporated. The residue was partitioned
between AcOEt and H2O, and the organic layer was washed with
aqueous saturated NaHCO3 and brine, dried (Na2SO4), and evapo-
rated. The residue was purified by column chromatography (SiO2,
5% AcOEt in hexane) to give 19 (1.0 g, 82%) as a white solid: 1H
NMR (400 MHz, CDCl3) δ 7.58-7.55 (m, 2 H), 7.27-7.24 (m, 3
H), 5.10 (d, 1 H, J ) 8.2), 4.95 (dd, 1 H, J ) 5.6, 8.2), 3.79-3.72
(m, 3 H), 3.63-3.58 (m, 3 H), 2.08 (s, 3 H), 0.87 (s, 9 H), 0.86 (s,
9 H), 0.10 (s, 3 H), 0.07 (s, 6 H), 0.06 (s, 3 H); 13C NMR (100
MHz, CDCl3) δ 169.6, 134.5, 133.8, 129.1, 129.0, 127.8, 82.6, 80.5,
74.8, 73.9, 71.0, 62.8, 26.1, 21.6, 18.2, -3.1, -3.3, -3.9, -4.1;
LRMS (FAB, positive) m/z 613 (MNa+). Anal. (C26H46O6SeSi2):
C, H.
Phenyl 2-O-Acetyl-3,4-bis-O-tert-butyldimethylsilyl-6-O-diphen-
ylvinylsilyl-1-seleno-â-D-glucopyranoside (13). A mixture of 19
(680 mg, 1.15 mmol), diphenylvinylchlorosilane (305 µL, 1.38
mmol), Et3N (231 µL, 1.66 mmol), and DMAP (73 mg, 0.60 mmol)
in toluene (23 mL) was stirred at room temperature for 30 min.
The mixture was partitioned between AcOEt and H2O, and the
organic layer was washed with brine, dried (Na2SO4), and evapo-
rated. The residue was purified by column chromatography (SiO2,
0-2% AcOEt in hexane) to give 13 (760 mg, 83%) as a colorless
oil: 1H NMR (400 MHz, CDCl3) δ 7.63-7.09 (m, 15 H, aromatic),
6.48 (dd, 1 H, vinyl-CH2, J ) 15.0, 20.6), 6.27 (dd, 1 H, vinyl-
CH, J ) 3.8, 15.0), 5.91 (dd, 1 H, vinyl-CH2, J ) 3.8, 20.6), 5.18
(d, 1 H, H-1, J ) 8.2), 4.95 (dd, 1 H, H-2, J ) 4.2, 8.2), 4.04 (dd,
1 H, H-6a, J ) 5.3, 10.5), 3.92 (dd, 1 H, H-5, J ) 6.5, 10.5),
3.80-3.77 (m, 3 H, H-3, -H-4, H-6b), 2.07 (s, 3 H, -COCH3),
0.84 (s, 9 H, -tBu), 0.83 (s, 9 H, -tBu), 0.07 (s, 3 H, -SiCH3),
0.05 (s, 6 H, -SiCH3), -0.02 (s, 3 H, -SiCH3); 13C NMR (100
MHz, CDCl3) δ 169.6, 134.4, 133.8, 129.1, 129.0, 127.8, 82.6, 80.5,
74.8, 73.9, 71.0, 62.8, 26.1, 21.6, 18.2, -3.1, -3.3, -3.2, -4.1;
LRMS (FAB, positive) m/z 821 (MNa+). Anal. (C40H58O6SeSi3):
C, H.
Phenyl 2-O-Acetyl-6-O-allyldimethylsilyl-3,4-bis-O-tert-bu-
tyldimethylsilyl-1-seleno-â-D-glucopyranoside (14). Compound
14 (495 mg, 90%) was obtained as a colorless oil from 19 (472
mg, 0.80 mmol) as described for the synthesis of 13 with
allyldimethylchlorosilane instead of diphenylvinylchlorosilane: 1H
NMR (400 MHz, CDCl3) δ 7.62-7.57 (m, 2 H, aromatic), 7.34-
7.22 (m, 3 H, aromatic), 5.80-5.73 (m, 1 H, allyl-CH), 5.19 (d, 1
H, H-1, J ) 7.9), 4.95 (dd, 1 H, H-2, J ) 2.0, 7.9), 4.91-4.84 (m,
2 H, allyl-CH2), 3.90 (dd, 1 H, H-6a, J ) 6.2, 9.7), 3.79-3.71 (m,
4 H, G-3, H-4, H-5, H-6b), 2.06 (s, 3 H, -COCH3), 1.62-1.54
(m, 2 H, CH2dCH-CH2), 0.91 (s, 9 H, -tBu), 0.90 (s, 9 H, -tBu),
0.12 (s, 3 H, -SiCH3), 0.11 (s, 6 H, -SiCH3), 0.09 (s, 6 H,
-SiCH3), 0.08 (s, 3 H, -SiCH3); 13C NMR (100 MHz, CDCl3) δ
169.0, 134.0, 133.8, 133.3, 129.9, 128.8, 127.3, 113.6, 83.3, 80.0,
77.2, 74.4, 74.3, 70.1, 63.2, 26.2, 26.1, 26.0 24.5, 21.4, 18.2, 18.1,
-2.3, -2.3, -3.7, -3.9, -4.2, -4.3; LRMS (FAB, positive) m/z
711 (MNa+). Anal. (C31H56O6SeSi3): C, H.
Radical Reaction Product 20. To a refluxing solution of 13
(246 mg, 0.30 mmol) in benzene (60 mL) was added a solution of
Bu3SnH (99 µL, 0.36 mmol) and AIBN (30 mg, 0.18 mmol) in
benzene (8.4 mL) dropwise with a syringe pump over 4 h. The
resulting mixture was evaporated, and the residue was partitioned
between AcOEt and H2O. The organic layer was washed with brine,
dried (Na2SO4), and evaporated. The residue was purified by column
chromatography (SiO2, 0-2% AcOEt in hexane) to give 20 (77
mg, 40%) as a colorless oil: 1H NMR (400 MHz, CDCl3) δ 7.63-
7.61 (m, 2 H, aromatic), 7.55-7.53 (m, 2 H, aromatic), 7.41-7.24
(m, 6 H, aromatic), 4.57 (br s, 1 H, 2-CH), 4.32 (dd, 1 H, H-6a, J
) 11.7, 11.7), 4.08 (m, 1 H, H-5), 3.97 (dd, 1 H, H-1, J ) 4.5,
12.1), 3.79 (br s, 1 H, H-4), 3.67 (dd, 1 H, H-6b, J ) 4.4, 11.7),
3.49 (br s, 1 H, H-3), 2.38-2.29 (m, 1 H, H-1′a), 2.07 (s, 3 H,
-COCH3), 1.99-1.89 (m, 1 H, H-1′b), 1.40-1.20 (m, 2 H, H-2′),
0.89 (s, 9 H, -tBu), 0.64 (s, 9 H, -tBu), 0.08 (s, 3 H, -CH3), 0.04
(s, 3 H, -CH3), 0.03 (s, 3 H, -CH3), -0.01 (s, 3 H, -CH3); LRMS
(FAB, positive) m/z 643 (MH+). Anal. (C34H54O6Si3): C, H.
Radical Reaction Product 21. Compound 21 (115 mg, 72%)
was obtained as a colorless oil from 14 (213 mg, 0.30 mmol) as
described for the synthesis of 20: 1H NMR (400 MHz, CDCl3) δ
6.22 (s, 1 H, H-2), 4.31 (dd, 1 H, H-6a, J ) 8.5, 8.5), 4.15 (d, 1 H,
H-5, J ) 8.5), 3.87 (d, 1 H, H-4, J ) 12.0), 3.66 (d, 1 H, H-3, J
) 12.0), 3.50 (d, 1 H, H-6b, J ) 8.5), 2.16-2.10 (m, 2 H, H-1,
H-1′a), 2.08 (s, 3 H, -COCH3), 1.70-1.65 (m, 2 H, H-2′), 1.52-
1.47 (m, 1 H, H-1′b), 0.90 (s, 9 H, -tBu), 0.88 (s, 9 H, -tBu), 0.71
(m, 1 H, H-3′a), 0.58 (m, 1 H, H-3′b), 0.14 (s, 3 H, -CH3), 0.11
(s, 3 H, -CH3), 0.09 (s, 3 H, -CH3), 0.06 (s, 3 H, -CH3), 0.05 (s,
3 H, -CH3), 0.04 (s, 3 H, -CH3); 13C NMR (100 MHz, CDCl3) δ
95.8, 77.6, 72.1, 68.3, 62.3, 51.0, 43.2, 26.6, 26.3, 21.4, 18.6, 18.3,
18.2, -0.1, -2.2, -2.8, -3.3, -3.5, -4.8; LRMS (FAB, positive)
m/z 533 (MH+). Anal. (C25H52O6Si3): C, H.
Radical Reactions of 15 and 16. Compounds 15 and 16 were
respectively treated under the radical reaction conditions described
for the synthesis of 20. In both cases, the reaction gave many spots
on TLC, and none of the desired cyclization product was obtained.
4-O-Acetyl-3,7-anhydro-5,6-bis-O-tert-butyldimethylsilyl-2-
deoxy-D-glycero-D-gulo-octitol (23). A mixture of 20 (30 mg, 47
µmol), KBr (11 mg, 94 µmol), HBr (30% in AcOH, 3 µL, 12 µmol),
and AcOOH (32% in AcOH, 127 µL, 600 µmol) in DMF (1 mL)
was stirred at room temperature for 12 h. The mixture was
partitioned between AcOEt and H2O, and the organic layer was
washed with aqueous saturated Na2S2O3, aqueous saturated NaH-
CO3, and brine, dried (Na2SO4), and evaporated. The residue was
purified by column chromatography (SiO2, 25-50% AcOEt in
hexane) to give 23 (23 mg, quantitative) as a colorless oil; 1H NMR
(400 MHz, CDCl3) δ 4.73 (dd, 1 H, J ) 6.2, 8.5), 3.80-3.54 (m,
8 H), 3.48 (ddd, 1 H, J ) 2.9, 6.2, 6.4), 2.24 (br s, 1 H), 2.06 (s,
3 H), 1.74-1.68 (m, 2 H), 0.89 (s, 9 H), 0.86 (s, 9 H), 0.10 (s, 3
H), 0.09 (s, 6 H), 0.07 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ
169.9, 81.40, 76.0, 75.5, 74.6, 72.0, 63.2, 60.1, 34.4, 26.2, 26.1,
21.6, 18.2, 18.2, -2.7, -2.8, -3.8, -3.9; LRMS (FAB, positive)
m/z 479 (MH+). Anal. (C22H46O7Si2): C, H.
5-O-Acetyl-4,8-anhydro-6,7-bis-O-tert-butyldimethylsilyl-2,3-
dideoxy-D-glycero-D-gulo-nonitol (24). Compound 24 (21 mg,
61%) was obtained as a colorless oil from 21 (35 mg, 0.07 mmol)
as described for the synthesis of 23: 1H NMR (400 MHz, CDCl3)
δ 5.90 (br s, 1 H), 3.85 (m, 1 H), 3.66-3.62 (m, 7 H), 2.09 (s, 3
H), 1.94 (br s, 1 H), 1.68-1.59 (m, 3 H), 1.23 (m, 2 H), 0.89 (s,
9 H), 0.87 (s, 9 H), 0.08 (s, 6 H), 0.07 (s, 3 H), 0.06 (s, 3 H); 13
C
NMR (100 MHz, CDCl3) δ 171.1, 93.9, 77.6, 77.2, 72.5, 62.8, 61.8,
26.2, 26.00, 25.7, 21.4, 18.3, 18.1, -3.3, -4.4, -4.5; LRMS (FAB,
positive) m/z 515 (MNa+). Anal. (C23H48O7Si2): C, H.
4-O-Acetyl-3,7-anhydro-5,6-bis-O-tert-butyldimethylsilyl-1-O-
tert-butyldiphenylsilyl-2-deoxy-D-glycero-D-gulo-octitol (25). A
mixture of 23 (135 mg, 0.28 mmol), TBDPSCl (146 µL, 0.56
mmol), and imidazole (76 mg, 1.1 mmol) in DMF (5 mL) was
stirred at -40 °C for 45 min. The mixture was partitioned between
AcOEt and H2O, and the organic layer was washed with brine,
dried (Na2SO4), and evaporated. The residue was purified by column
chromatography (SiO2, 10-20% AcOEt in hexane) to give 25 (23
mg, 11%) as a colorless oil: 1H NMR (400 MHz, CDCl3) δ 7.68
(d, 4 H, J ) 6.8), 7.41-7.35 (m, 6 H), 6.21 (s, 1 H), 3.91-3.77
(m, 4 H), 3.67-3.54 (m, 3 H), 2.13 (m, 2 H), 2.06 (s, 3 H), 2.00
(m, 1 H), 1.78 (m, 1 H), 1.26 (s, 9 H), 0.85 (s, 9 H), 0.84 (s, 9 H),
0.14 (s, 6 H), 0.02 (s, 6 H); 13C NMR (100 MHz, CDCl3) δ 169.1,
135.8, 135.6, 135.5, 135.4, 133.8, 133.3, 129.5, 129.4, 127.6, 127.4,
127.3, 91.7, 77.2, 75.5, 72.7, 69.7, 62.8, 36.6, 27.0, 26.9, 26.1, 26.0,
21.3, 19.5, 18.3, -3.7, -4.5; HRMS (FAB, positive) calcd for
C38H64O7Si3Na 739.3858 (MNa+), found 739.3828.
5-O-Acetyl-4,8-anhydro-6,7-bis-O-tert-butyldimethylsilyl-1-O-
tert-butyldiphenylsilyl-2,3-dideoxy-D-glycero-D-gulo-nonitol (26).
Compound 26 (11 mg, 64%) was obtained as a colorless oil from
24 (12 mg, 24 µmol) as described for the synthesis of 25: 1H NMR
(400 MHz, CDCl3) δ 7.66 (d, 4 H, J ) 6.5), 7.43-7.36 (m, 6 H),
5.92 (br s, 1 H), 3.88 (m, 1 H), 3.68-3.63 (m, 7 H), 2.09 (s, 3 H),
1.95 (br s, 1 H), 1.62-1.44 (m, 4 H), 1.05 (s, 9 H), 0.89 (s, 18 H),
0.10 (s, 9 H), 0.06 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ 169.9,
81.4, 76.0, 75.5, 74.6, 72.0, 63.2, 60.1, 34.4, 26.2, 26.2, 21.6, 18.2,
18.2, -2.7, -2.8, -3.8, -3.9; LRMS (FAB, positive) m/z 753
(MNa+). Anal. (C39H66O7Si3): C, H.

C-Glucoside Trisphosphates as IP3 Receptor Ligands
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 1907
5-O-Acetyl-4,8-anhydro-9-O-benzoyl-6,7-bis-O-tert-butyldi-
methylsilyl-1-O-tert-butyldiphenylsilyl-2,3-dideoxy-D-glycero-
D-gulo-nonitol (27). A mixture of 26 (17 mg, 23 µmol) and BzCl
(5.3 µL, 46 µmol) in pyridine (1 mL) was stirred at room
temperature for 30 min. The mixture was partitioned between
AcOEt and H2O, and the organic layer was washed with brine,
dried (Na2SO4), and evaporated. The residue was purified by column
chromatography (SiO2, 10% AcOEt in hexane) to give 27 (19 mg,
99%) as a colorless oil: 1H NMR (400 MHz, CDCl3) δ 8.02-8.00
(m, 2 H), 7.73-7.65 (m, 4 H), 7.56-7.50 (m, 1 H), 7.44-7.35
(m, 8 H), 6.00 (br s, 1 H), 4.58 (d, 1 H, J ) 10.3), 4.35 (m, 1 H),
3.90 (m, 2 H), 3.76 (dd, 1 H, J ) 6.5, 6.5), 3.67-3.65 (m, 3 H),
2.07 (s, 3 H), 1.99 (m, 1 H), 1.59 (m, 3 H), 1.04 (s, 9 H), 0.90 (s,
18 H), 0.11 (s, 6 H), 0.07 (s, 3 H), 0.06 (s, 3 H); 13C NMR (100
MHz, CDCl3) δ 169.1, 166.1, 135.4, 134.6, 134.4, 133.7, 133.7,
132.8, 130.4, 129.9, 129.6, 129.4, 129.4, 128.7, 128.7, 128.2, 127.6,
127.5, 94.3, 77.6, 77.2, 74.5, 72.6, 63.8, 27.0, 26.7, 26.2, 26.0, 25.9,
25.9, 25.8, 25.8, 25.8, 25.7, 21.3, 19.3, 18.4, 18.1, 18.0, -3.2, -4.3,
-4.6; LRMS (FAB, positive) m/z 857 (MNa+). Anal. (C46H70O8-
Si3): C, H.
(m, 1 H), 3.90-3.63 (m, 5 H), 3.59-3.49 (m, 3 H), 1.70-1.44
(m, 4 H); 13C NMR (100 MHz, D2O) δ 77.5, 75.2, 73.3, 73.0, 71.7,
65.5, 26.8, 21.6; 31P NMR (500 MHz, D2O, H-decoupled) δ 2.56,
2.28, 1.72 (each s); HRMS (FAB, negative) calcd for C9H17O15-
Na3P3 526.9473 (M-), found 526.9476.
â-C-Glycoside 32. A mixture of 31 (103 mg, 0.2 mmol), Et3-
SiH (35 µL, 0.22 mmol), and TMSOTf (40 µL, 0.22 mmol) in CH2-
Cl2 (10 mL) was stirred at -78 °C for 1 h, and then Et3N (100 µL)
was added. The resulting mixture was partitioned between AcOEt
and aqueous saturated NaHCO3, and the organic layer was washed
with brine, dried (Na2SO4), and evaporated. The residue was
purified by column chromatography (SiO2, 15% AcOEt in hexane)
to give 32 (92 mg, 92%) as a colorless oil: 1H NMR(400 MHz,
CDCl3) δ 7.35-7.25 (m, 10 H), 5.96-5.85 (m, 1 H), 5.11-5.05
(m, 2 H), 4.98 (d, 1 H, J ) 10.9), 4.64-4.56 (m, 3 H), 3.89 (dd,
1 H, J ) 9.1, 9.7), 3.73 (dd, 1 H, J ) 1.8, 11.1), 3.67 (dd, 1 H, J
) 5.0, 11.1), 3.66 (dd, 1 H, J ) 9.7, 10.2), 3.55 (ddd, 1 H, J )
1.8, 5.0, 10.2), 3.42 (dd, 1 H, J ) 9.1, 9.1), 3.41-3.36 (m, 1 H),
3.30 (s, 3 H), 3.21 (s, 3 H), 2.62-2.56 (m, 1 H), 2.37-2.30 (m, 1
H), 1.36 (s, 3 H), 1.29 (s, 3H); HRMS calcd for C29H38NaO7
521.2513 (MNa+), found 521.2520.
5-O-Acetyl-4,8-anhydro-9-O-benzoyl-2,3-dideoxy-D-glycero-D-
gulo-nonitol (28). A mixture of 27 (19 mg, 23 µmol), TBAF (1 M
in THF, 46 µL, 46 µmol), and AcOH (13 µL, 23 µmol) in THF (1
mL) was stirred at room temperature for 48 h. The mixture was
evaporated, and the residue was partitioned between AcOEt and
H2O. The organic layer was washed with brine, dried (Na2SO4),
and evaporated. The residue was purified by column chromatog-
raphy (SiO2, 25% AcOEt in hexane) to give 28 (8 mg, 99%) as a
colorless oil: 1H NMR (400 MHz, CDCl3) δ 8.06 (dd, 2 H, J )
7.3, 7.3), 7.58 (dd, 1 H, J ) 7.3, 7.3), 7.46 (dd, 2 H, J ) 7.3, 7.3),
4.57 (dd, 1 H, J ) 3.2, 12.3), 4.37 (dd, 1 H, J ) 1.9, 12.3), 4.12
(dd, 1 H, J ) 5.3, 9.4), 3.86 (d, 1 H, J ) 9.7), 3.68-3.58 (m, 2 H),
3.54 (dd, 1 H, J ) 9.7, 9.7), 2.11 (s, 5 H), 1.89-1.82 (m, 1 H),
1.79-1.70 (m, 1 H), 1.58-1.50 (m, 2 H), 1.37-1.25 (m, 3 H);
13C NMR (100 MHz, CDCl3) δ 169.1, 167.6, 133.4, 129.8, 129.1,
128.4, 94.4, 73.3, 70.1, 67.2, 63.6, 62.1, 43.1, 30.9, 21.3, 20.5, 0.2;
LRMS (FAB, positive) m/z 391 (MNa+). Anal. (C18H24O8): C, H.
4,8-Anhydro-2,3-dideoxy-D-glycero-D-gulo-nonitol 1,6,7-Tris-
phosphate Derivative 29. A mixture of 28 (28 mg, 76 µmol),
XEPA (73 mg, 304 µmol) and 1H-tetrazole (27 mg, 38 µmol) in
CH2Cl2 was stirred at 0 °C for 30 min. After addition of H2O (20
µL), the mixture was stirred at room temperature for 10 min. The
resulting mixture was cooled to -40 °C, and then m-CPBA (70
mg, 400 µmol) was added. The mixture was warmed to room
temperature over 30 min and then partitioned between AcOEt and
aqueous saturated Na2S2O3. The organic layer was washed with
H2O, aqueous saturated NaHCO3, and brine, dried (Na2SO4), and
evaporated. The residue was purified by column chromatography
(SiO2, 10% MeOH in CHCl3) to give 29 (40 mg, 58%) as a yellow
foam: 1H NMR (400 MHz, CDCl3) δ 8.03-8.01 (m, 2 H), 7.53-
7.11 (m, 15 H), 5.40-4.97 (m, 16 H), 4.71 (dd, 1 H, J ) 2.1,
12.3), 4.50 (dd, 1 H, J ) 4.1, 12.3), 4.23-4.14 (m, 3 H), 2.59-
2.55 (m, 1 H), 2.12 (s, 3 H), 2.12-2.08 (m, 1 H), 1.98-1.91 (m,
1 H), 1.89-1.80 (m, 1 H), 1.65-1.58 (m, 1 H); 31P NMR (500
MHz, CDCl3, H-decoupled) δ -2.18, -2.54, -5.12 (each s);
HRMS (FAB, positive) calcd for C42H46O17P3 915.1948 (MH+),
found 915.1933.
3,7-Anhydro-4,8-di-O-benzyl-2-deoxy-D-glycero-D-gulo-octi-
tol (33). Ozone-containing oxygen was bubbled into a solution of
32 (100 mg, 0.20 mmol) in MeOH (2 mL) at -20 °C until 32
disappeared on TLC (ca. 20 min). After addition of NaBH4 (23
mg, 0.60 mmol) at the same temperature, the resulting solution was
stirred at room temperature for 1 h. The mixture was evaporated,
and the residue was purified by column chromatography (SiO2, 30-
50% AcOEt in hexane) to give the crude ozonolysis product (47
mg) as an oil. A mixture of the oil and aqueous TFA (80%, 200
µL) in CHCl3 (2 mL) was stirred at 0 °C for 12 h, and then aqueous
saturated NaHCO3 (2 mL) was added. The resulting mixture was
partitioned between CHCl3 and the aqueous saturated NaHCO3, and
the organic layer was washed with brine, dried (Na2SO4), and
evaporated. The residue was purified by column chromatography
(SiO2, 10% MeOH in CHCl3) to give 33 (59 mg, 60%) as a colorless
oil: 1H NMR (400 MHz, CDCl3) δ 7.36-7.26 (m, 10 H), 4.83 (d,
1 H, J ) 10.8), 4.71 (d, 1 H, J ) 10.8), 4.57-4.55 (m, 2 H), 4.17
(br, 1 H), 3.86-3.78-3.63 (m, 9 H), 3.26-3.21 (m, 2 H), 1.98-
1.82 (m, 2 H); LRMS (FAB, positive) m/z 411 (MNa+). Anal.
(C22H28O6): C, H.
Sodium 3,7-Anhydro-2-deoxy-D-glycero-D-gulo-octitol 1,5,6-
Trisphosphate (11). A mixture of 33 (7.6 mg, 20 µmol), XEPA
(19 mg, 80 µmol), and 1H-tetrazole (7.0 mg, 10 µmol) in CH2Cl2
(1 mL) was stirred at 0 °C for 60 min. After addition of H2O (20
µL), the mixture was stirred at room temperature for 10 min. The
resulting mixture was cooled to -40 °C, and then m-CPBA (40
mg, 200 µmol) was added. The mixture was warmed to room
temperature over 30 min. The reaction mixture was partitioned
between AcOEt and aqueous saturated Na2S2O3, and the organic
layer was washed with H2O, aqueous saturated NaHCO3, and brine,
dried (Na2SO4), and evaporated. The residue was purified by
preparative TLC (SiO2, 10% MeOH in CHCl3) to give the
phosphorylation product as a foam (13 mg). A mixture of a foam
and Pd-C (10%, 17 mg) in MeOH (2 mL) was stirred at room
temperature under atmospheric pressure of H2 for 4 h. After
filtration of the catalysts with Celite, the filtrate was evaporated.
From the residue, compound 11 (sodium salt, 8 mg, 70% from 33)
was obtained as a white solid, as described for the purification of
12: 1H NMR (500 MHz, D2O) δ 3. (dd, 1 H, J ) 8.4, 17.0), 3.80
(dd, 1 H, J ) 8.4, 10.0), 3.78 (dd, 1 H, J ) 8.4, 8.8), 3.66-3.12
(m, 6 H), 2.02 (m, 1 H), 1.55 (m, 1 H); 13C NMR (100 MHz, D2O)
δ 81.4, 74.4, 73.3, 70.4, 69.7, 62.1, 33.8, 21.1; 31P NMR (500 MHz,
D2O, H-decoupled) δ 3.11, 2.67, 2.36 (each s); HRMS (FAB) calcd
C8H14O15Na6P3 580.8932 (MH+), found 580.8914.
Sodium 4,8-Anhydro-2,3-dideoxy-D-glycero-D-gulo-nonitol 1,6,7-
Trisphosphate (12). A mixture of 29 (40 mg, 44 µmol) and Pd-C
(10%, 52 mg) in MeOH (5 mL) was stirred at room temperature
under atmospheric pressure of H2 for 40 min. The catalysts were
filtered off with Celite, and the filtrate was evaporated. A mixture
of the residue and NaOMe (28%, 82 µL, 400 µmol) in MeOH (5
mL) was stirred at room temperature for 12 h. The mixture was
applied to Diaion PK-212 (H+-form), and the column was developed
with H2O. The fractions containing 12 (acidic fractions) were
evaporated. A solution of the residue was washed with CHCl3 (three
times) and was applied to Diaion WK-100 (Na+ form). The column
was developed with H2O, and the fractions containing 12 were
evaporated and dried in vacuo to give 12 (sodium salt, 35 mg,
quantitative) as a white solid: 1H NMR (500 MHz, D2O) δ 4.17
Modeling Studies. Initial investigations into binding modes of
the C-glucoside trisphosphates at the IP3 receptor (PDB code 1N4K)
were performed using a docking program (GOLD version 2.2).
Docks carried out both in the presence and in the absence of
conserved crystal waters gave poor results as the glucose ring was
in a very different orientation to the ring of the crystal structure

1908 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Terauchi et al.
(6) (a) Tatani, K.; Shuto, S.; Ueno, Y.; Matsuda, A. Synthesis of 1-O-
[(3S,4R)-3-hydroxytetrahydrofuran-4-yl]-R-D-glucopyranoside 3,4,3′-
triphosphate as a novel potent IP3 receptor ligand. Tetrahedron Lett.
1998, 39, 5065-5068. (b) Shuto, S.; Tatani, K.; Ueno, Y.; Matsuda,
A. Synthesis of adenophostin analogs lacking the adenine moiety as
novel potent IP3 receptor ligands: Some structural requirements for
the significant activity of adenophostin A. J. Org. Chem. 1998, 63,
8815-8824. (c) Kashiwayanagi, M.; Tatani, K.; Shuto, S.; Matsuda,
A. Inward current responses to IP3 and adenophostin-analogues in
turtle olfactory sensory neurons. Eur. J. Neurosci. 2000, 12, 606-
612. (d) Abe, H.; Shuto, S.; Matsuda, A. Synthesis of the C-glycosidic
analog of adenophostin A, a potent IP3 receptor agonist, using a
temporary silicon-tethered radical coupling reaction as the key step.
Tetrahedron Lett. 2000, 41, 2391-2394. (e) Abe, H.; Shuto, S.;
Matsuda, A. Synthesis of the C-glycosidic analog of adenophostin
A and its uracil congener as potential IP3 receptor ligands. Stereo-
selective construction of the C-glycosidic structure by a temporary
silicon-tethered radical coupling reaction. J. Org. Chem. 2000, 65,
4315-4325. (f) Shuto, S.; Yahiro, Y.; Ichikawa, S.; Matsuda, A.
Synthesis of 3,7-anhydro-D-glycero-D-ido-octitol 1,5,6-trisphosphate
as an IP3 receptor ligand using a radical cyclization reaction with a
vinylsilyl tether as the key step. Conformational restriction strategy
using steric repulsion between adjacent bulky protecting groups on
a pyranose ring. J. Org. Chem. 2000, 65, 5547-5557. (g) Terauchi,
M.; Yahiiro, Y.; Abe, H.; Ichikawa, S.; Stephen, C. Tovey, S. C.;
Dedos, S. G.; Taylor, C. W.; Potter, B. V. L.; Matsuda, A.; Shuto,
S. Synthesis of 4,8-Anhydro-D-glycero-D-ido-nonanitol 1,6,7-tris-
phosphate as a novel IP3 receptor ligand using a stereoselective radical
cyclization reaction based on a conformational restriction strategy.
Tetrahedron 2005, 61, 7865-7873.
(7) (a) Jenkins, A. D. J.; Potter, B. V. L. Synthesis of second messenger
mimic related to adenophostin A. J. Chem. Soc., Chem. Commun.
1995, 1169-1170. (b) Marchant, J. S.; Beecroft, M. D.; Riley, A.
M.; Jenkins, D. J.; Marwood, R. D.; Taylor, C. W.; Potter, B. V. L.
Disacharide polyphosphates based upon adenophostin A activate
hepatic D-myo-inositol 1,4,5-trisphosphate receptors. Biochemistry
1997, 36, 6, 12780-12790. (c) Rosenberg, H. J.; Riley, A. M.;
Correa, V.; Taylor, C. W.; Potter, B. V. L. C-Glycoside based mimics
of D-myo-inositol 1,4,5-trisphosphate. Carbohydr. Res. 2000, 329,
7-16. (d) Rosenberg, H. J.; Riley, A. M.; Laude, A. J.; Taylor, C.
W.; Potter, B. V. L. Synthesis and Ca2+-mobilizing activity of purine-
modified mimics of adenophostin A: a model for the adenophostin-
Ins(1,4,5)P3 receptor interaction. J. Med. Chem. 2003, 46, 4860-
4871 and references therein. (e) Wilcox, R. A.; Erneux, C.; Primrose,
W. U.; Gigg, R.; Nahorski, S. R. 2-hydroxyethyl D-glucopyranoside
2,3′,4′-trisphosphate, a novel, metabolically resistant, adenophostin
A and myo-inositol 1,4,5-trisphosphate analogue potently interacts
with the myo-inositol 1,4,5-trisphosphate receptor. Mol. Pharmacol.
1995, 47, 1201-1211. (f) de Kort, M.; Regenbogen, A. D.;
Overkleeft, H. S.; Challis, J.; Iwata, Y.; Miyamoto, S.; van der Marel,
G. A.; van Boom, J. Synthesis and biological evaluation of cyclo-
phostin: a 5′,6′′-tethered analog of adenophostin A. Tetrahedron
2000, 56, 5915-5928 and references therein. (g) Roussel, F.;
Moitessier, N.; Hilly, M.; Chre´tien, F.; Mauger, J.-P.; Chapleur, Y.
D-myo-Inositol-1,4,5-trisphosphate and adenophostin mimics: im-
portance of the spatial orientation of a phosphate group on the
biological activity. Bioorg. Med. Chem. 2002, 10, 759-768 and
references therein. (h) Hotoda, H.; Murayama, K.; Miyamoto, S.;
Iwata, Y.; Takahashi, M.; Kawase, Y.; Tanzawa, K.; Kaneko, M.
Molecular recognition of adenophostin, a very potent Ca2+ inducer
at the D-myo-inositol 1,4,5-trisphosphate receptor. Biochemistry 1999,
38, 9234-9241.
IP3 ligand. As an alternative method, simulations of the ligand
binding mode for the C-glucoside trisphosphates 7-12 were carried
out using Molecular Operating Environment (MOE 2004.03). The
basic assumption behind this study was that the two phosphate
groups attached directly to the ring bind in the same conformation
as the 4- and 5-position phosphate groups of IP3. Therefore, it was
logical to use the crystal structure conformation of IP3 bound to
the IP3 receptor as a starting point for building these molecules.
Heavy atoms of the IP3 binding site (PDB code 1N4K) and
associated water molecules were fixed, and hydrogen atoms were
added. The system was energy-minimized using the MMFF94 force
field in MOE to remove any clashes between the newly added
hydrogen atoms. A series of modifications were implemented to
transform the IP3 structure into the six C-glycoside trisphosphates.
These can be summarized as follows: (i) replacement of carbon at
position 2 with oxygen; (ii) replacement of OH attached at the
2-
3-position with CH2OH; (iii) addition of appropriate (CH2)nOPO3
,
where n ) 1, 2, or 3 in the correct position to form the three R-C-
glucoside and three â-C-glucoside polyphosphates 7-12. The
system was energy-minimized following each transformation. Once
the desired structures had been built, observation of the resulting
conformations suggested possible interactions with three arginine
residues across the R- and â-C-glucosidic compounds, namely,
Arg269 and Arg504 (for the R-structures) and Arg568 and Arg269
(for the â-structures). Therefore, a final minimization step was
simulated by allowing these three residues to move. Interactions
of 8, 11, and IP3 with these active site arginines are shown in Figure
7a-c.
Biological Assay. The ability of compounds to stimulate IP3
receptors was measured using a low-affinity Ca2+ indicator trapped
within the intracellular stores of chicken DT 40 cells expressing
only recombinant rat type 1 IP3 receptors as previously.28
Acknowledgment. We wish to acknowledge financial sup-
port from Ministry of Education, Culture, Sports, Science and
Technology, Japan, for Scientific Research 17659027 (to S.S.),
the Japan Society for Promotion of Science for Creative
Scientific Research 13NP0401 (to A.M.), and the Wellcome
Trust for Programme Grants 039662 (to C.W.T.) and 060554
(to B.V.L.P.). We thank Ms. H. Matsumoto, A. Maeda, and S.
Oka (Center for Instrumental Analysis, Hokkaido University)
for technical assistance with NMR, MS, and elemental analyses
and Dr. A. M. Riley for useful discussions.
Supporting Information Available: Analytical data of com-
pounds 13, 14, 19-21, 23, 24, 26-28, and 33. This material is
available free of charge via the Internet at http://pubs.acs.org.
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