D-myo-Inositol-1,4,5-trisphosphate and adenophostin mimics: Importance of the spatial orientation of a phosphate group on the biological activity

Article abstract of DOI:10.1016/S0968-0896(01)00329-7

Three different routes for the synthesis of heterocyclic analogues of the second messenger D-myo-inositol-1,4,5-trisphosphate (InsP₃) and the natural adenophostins, starting from allyl D-xyloside are described. The two diastereoisomers at C-2 o

Full text of DOI:10.1016/S0968-0896(01)00329-7

Bioorganic & Medicinal Chemistry 10 (2002) 759–768
D-myo-Inositol-1,4,5-trisphosphate and Adenophostin Mimics:
Importance of the Spatial Orientation of a Phosphate Group on
the Biological Activity
Fabien Roussel,a Nicolas Moitessier,a Mauricette Hilly,b Francoise Chretien,a
Jean-Pierre Maugerb and Yves Chapleura,*
a
´
´
Groupe SUCRES, UMR 7565, CNRS-Universite Henri Poincare Nancy 1, BP 239, F-54506 Vandoeuvre, France
b
´
´
Unite Inserm U 442, Universite de Paris Sud, Bat 443, F-91405 Orsay, France
ˆ
Received 17 July 2001; accepted 17 September 2001
Abstract—Three different routes for the synthesis of heterocyclic analogues of the second messenger d-myo-inositol-1,4,5-trispho-
sphate (InsP3) and the natural adenophostins, starting from allyl d-xyloside are described. The two diastereoisomers at C-2 of new
compounds, which we named xylophostins, were obtained. The preliminary biological studies shows that the presence of the adenine
residue has a beneficial effect on the affinity for the receptor. The low potency of one of the two diastereoisomeric compounds
shows that the configuration of the carbon bearing the non-vicinal phosphate group is an important requirement for a high affinity
to the receptor. These results provide evidence for the existence of a binding pocket for the adenine ring nearby the InsP3 binding
site. The consequence of these stabilizing interactions should be to place the phosphate group in a suitable position to perfectly
mimic InsP3 in the more active diastereoisomer. Obviously, in the other diastereoisomer, the phosphate cannot accommodate the
same orientation, thus explaining the low affinity. The existence of such a binding pocket for adenine is in line with the high potency
of adenophostins. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
of importance to study the receptor pharmacology, and
compounds derived from these studies would be of
interest as therapeutics to stimulate or block cells
responses. In this context, the search for new analogues
of InsP3 has become a growing field (Fig. 1).3ꢀ7 Until
recently, most of the analogues were based on mod-
ification of inositol derivatives. Some years ago, we
introduce new sugar derivatives as mimics of InsP3. Our
analogues were based on d-xylose hypothesizing that
the pyranose ring would mimic the inositol ring.8 Given
the very unimportant role, if any, of the 2- and 3-
hydroxyl groups in the biological activity of InsP3, it
was reasonable to take into account the three phos-
phates and the 6-OH. A good target was then the tris-
phosphate 8 derived from d-xylose. This compound
proved unstable and we decided to move the phosphate
group away from the anomeric position. This led to the
preparation of trisphosphate 9.8 In the mean time, an
important discovery was made during screening of micro-
organism metabolites by Japanese industry.9ꢀ11 A series of
highly potent agonists of InsP3R, the adenophostins was
then available.12,13
The understanding and the control of cell signaling
pathways is now the subject of intense interest. One of
the most important pathways is the phosphoinositide
one in which d-myo-inositol-1,4,5-trisphosphate (InsP3)
plays a pivotal role. This second messenger is released
from phosphatidyl-inositol-4,5-bisphosphate (PIP2), a
phospholipid anchored in the internal layer of cell
membrane. This release is mediated by a specific phos-
pholipase PLC activated by a G-protein coupled to an
external receptor which binds the first messenger such as
hormones, neurotransmitters etc. Hydrolysis of PIP2
produces InsP3 and another lipidic second messenger
diacylglycerol (DAG). InsP3 binds to a specific receptor
(InsP3R) located on the endoplasmic reticulum and lib-
erates calcium from internal stores.1,2 This calcium
release is responsible for the cell response by activation
of calcium sensitive proteins such as calmodulin. DAG
has its own action on cell response and both actions are
often difficult to separate and to study independently. It
is obvious that agonists and antagonists of InsP3R are
These compounds were immediately chosen as targets
for total synthesis14ꢀ16 and as models for developing
*Corresponding author. Tel.: +33-383-91-2355; fax: +33-383-91-
2479; e-mail: yves.chapleur@meseb.uhp-nancy.fr
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00329-7

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F. Roussel et al. / Bioorg. Med. Chem. 10 (2002) 759–768
other analogues. Thus, the gluco analogue 4 was pre-
pared by Potter17,18 and Gigg19 and exhibited almost
the same biological activity as 9 in terms of affinity and
of 14 and 13. Although some diastereoselectivity was
observed, epoxides 13 and 14 could not be efficiently
separated using preparative high-pressure chromato-
graphy. In order to modify the diastereoselectivity of
the epoxidation step, we changed the allylglycoside pre-
cursor and compound 17 bearing a free hydroxyl group
at position 2 was used. Treatment of 17 with mCPBA
afforded epoxide 21 as a 7:3 mixture of diastereo-
isomers. Further investigation on this mixture led to the
establishment of the (S) configuration of the major
epoxide, which corresponds to the one of adenophostin
(see structural determination). This prompted us to use
it as follows (Scheme 1).
ability to release Ca++ 19,20
Very recently more elabo-
.
rated compounds including the ribose ring of adeno-
phostin like 621,22 and 723 have been reported together
with an open chain derivatives 5 having the adenine
ring.24,25 In continuation with our own program, we
tried to enhance the affinity of our lead compound 9 by
taking into account structural informations provided by
the adenophostins.
We report here our efforts in the synthesis of analogues
10 and 11, which differ only by the stereochemistry at C-
2 of the propyl tether between the xylose ring and the
adenine but show striking different biological properties.
The mixture of epoxides 21 was benzylated and the
resulting mixture was treated with the sodio derivative
of adenine to give a mixture of N-9 and N-7 regioi-
somers (purine numbering) in a 2:1 ratio from which the
N-9 substituted derivative 23 was separated by column
chromatography. The remainder of the synthesis con-
sisted in the removal of the cyclic acetal group and
phosphitylation of the hydroxyl functions using 2-(N,N-
diisopropylamino)-5,6-benzo-1,3,2-dioxaphosphepane
(41) followed by oxidation of the corresponding phos-
phites with t-butyl hydroperoxide giving the protected
derivative 25. Deprotection of all benzyl-protecting
groups was cleanly achieved by catalytic hydrogenation
over Pd (10%) on charcoal under 20 bars to give phos-
phates 26 isolated as the sodium salt. This compound
was actually a 3:7 mixture of 10 and 11. This mixture
was tested for its affinity towards InsP3R and gave
encouraging results, which prompted us to find more ste-
reospecific routes to each compounds (R and S isomers)
(Scheme 2).
Results and Discussion
Most of the potency of InsP3 analogues is related to the
presence of the trans bisphosphate moiety and the
hydroxyl group at C-6 (inositol numbering). The third
phosphate only enhances the affinity for the receptor. In
the adenophostins, the ribose ring should play the role
of a scaffold, presenting the adenine moiety and the
phosphate group in a proper spatial arrangement. In
this context, the stereochemistry at C-2, which bears the
phosphate group should be important.
First approach
Our first synthesis began with the allylglycoside 12 pro-
tected at position 2 (carbohydrate numbering) with a ben-
zyl group. Conventional epoxidation of 12 with mCPBA
gave the expected epoxide as a 7:3 diastereomeric mixture
Scheme 1. Reagents: (i) mCPBA, CH2Cl2; (ii) NaN3, CH3OCH2-
CH2OH/H2O, NH4Cl; (iii) NaH, BnBr, DMF.
Second approach
In a second approach, we started from allyl ether 12
with the hope to introduce the diol function in a dia-
stereoselective manner by taking advantage of the
Figure 1. InsP3 and some analogues.

F. Roussel et al. / Bioorg. Med. Chem. 10 (2002) 759–768
761
asymmetric dihydroxylation (AD). If dihydroxylation
of olefin 12 with AD-mix b led to almost 1:1 mixture of
the diastereoisomeric diols, it was found that the pro-
tection of the two hydroxyl groups of 12 was beneficial
for the diastereoselectivity.26 Using modified Sharpless
conditions27 it was possible to carry out the AD of the
bisbenzoate 2726 with an 8.1:1 diastereoisomeric ratio.
The resulting diol mixture was selectively tosylated at
the primary alcohol to yield compound 29 that was
treated as before with the sodio derivative of adenine.
Again a mixture of N-9 and N-7 derivatives in a 3.5:1
ratio was obtained. Column chromatography on silica
gel allowed nice separation of the two positional iso-
mers and gave the N-9 derivative 30, which was almost
diastereomerically pure. Less than 5% of the other dia-
stereoisomer at C-2 was detectable on 1H and 13C NMR
spectra. Reaction of 30 with sodium methoxide gave the
group at C2 directs the epoxidation from one face of the
olefinic bond, by formation of an hydrogen bond with
the peracid.28,29 The presence of a benzyl group at O-2
reverts this diastereoselectivity. The benzyl group then
shielded one face of the double bond so that epoxida-
tion should take place from the other face. In each case
the formation of a 7:3 mixture should result from two
different conformations of the O-allyl group around the
C–O bond. We have undisputedly established the ste-
reochemical assignment by chemical correlation as fol-
lows. The 2(R) configuration of the major product 28 of
the AD reaction has been established by degradation of
the glycerol aglycon and comparison with literature
data.26 This configuration allowed us to establish the
stereochemistry of the above described compounds.
Tosylate 29 was treated with sodium azide to provide
the expected azido derivative, which was debenzoylated
to provide the corresponding triol of 2(R) configuration
15. This compound was identical to the major product
15 resulting from the first epoxide route. It was con-
cluded that the diastereomeric mixture of epoxide 21
obtained in our first approach contained about 70% of
the 2(S) diastereoisomer.
Scheme 2. Reagents: (i) adenine, NaH, DMF, 90 ꢁC; (ii) BnBr, NaH,
DMF; (iii) CH2Cl2/CF3COOH/H2O 20:9.5:0.5; (iv) 41,1H-tetrazole,
CH2Cl2 then tBuOOH, 0 ꢁC; (v) H2,Pd/C(10%), 20 bars, MeOH.
triol 31 and phosphitylation of 31 using (41) followed by
in situ oxidation of the corresponding phosphites gave
the protected phosphate derivative 32. Deprotection of all
the benzyl protecting groups gave compound 10 isolated
as its hexasodium salt (Scheme 3).
Scheme 3. Reagents: (i) AD-mixb, PYR(DHQD)2, tBuOH/H2O; (ii)
TsCl, pyridine; (iii) adenine, NaH, DMF, 90 ꢁC; (iv) NaN3, DMF,
90 ꢁC; (v) MeONa, MeOH; (vi) 41,1H-tetrazole, CH2Cl2 then
tBuOOH, 0 ꢁC; (vii) H2,Pd/C (10%), 20 bars, MeOH.
Structure determination
Third approach
In order to determine the diastereoselection of each
epoxidation reaction, the mixture of epoxides 14 and 13
was opened with sodium azide giving a mixture of the
expected azido triols from which pure major isomer 15
was obtained by column chromatography. Epoxides 18
and 19 cannot be completely separated by preparative
high-pressure chromatography, however small amounts
of the major compound 18 were obtained. Compound
20 was obtained by protection at position 2 of 18 with a
benzyl group. As before, treatment of 18 with sodium
azide followed by removal of the cyclic acetal group
gave the azido triol 16. 1H and 13C NMR analyses
showed that compounds 15 and 16 were diastereoi-
somers. Not unexpectedly, the presence of the hydroxyl
The second approach allowed us to obtain compound
10 contaminated with less than 5% of the other diaster-
eoisomer (NMR determination). Given the lower affinity
Table 1. Inhibition of [3H]InsP3 binding to cerebellar microsomes by
the different compoundsa
Compounds
IC50 (mM)
InsP3
9
10
0.018
0.43
3
26
11
0.087
0.066
aData are means of triplicate determinations in two different experiments.

762
F. Roussel et al. / Bioorg. Med. Chem. 10 (2002) 759–768
of the 2(R) isomer 10 for the receptor (see Table 1), it was
clear that the 2(S) isomer having the absolute config-
uration of the ribose moiety of the adenophostin models
should have a good affinity for the receptor. Thus we
searched for a new route to this 2(S) isomer starting
from the allyl glycoside 27 which was deallylated using
the catalytic procedure developed by Liu30 to provide
the free hemiacetal 33 in 68% yield. Activation of the
hemiacetalic hydroxyl via the Schmidt procedure31 gave
the trichloroacetimidate 34 as an anomeric mixture in
86% yield, the kinetically favored b anomer being
formed preferentially under these conditions b/a 3/1.
No attempts were made to separate these imidates,
which were reacted at ꢀ20 ꢁC with the enantiomerically
pure (R) (+) glycidol 35 using trimethylsilyl triflate as
the promoter. A mixture of anomeric glycosides 36 and
37 was obtained in a 1.1:1 ratio in favor of the a anomer
(Scheme 4).
We also tested the ability of the different compounds to
release 45Ca2+ from permeabilized hepatocytes (Fig. 3).
Compounds 11 released almost 100% of 45Ca2+ at a
10ꢀ5 M concentration. Compound 10 was unable to
empty calcium stores at the maximal tested concentra-
tion (10ꢀ4 M). The half maximal response for the 11
(EC50) was 465ꢂ10ꢀ9ꢂM and those for 26 was 858
10ꢀ9ꢂM. This is to be compared with the EC50 for
InsP3 that was 89ꢂ10ꢀ9 M. The rank order of potency
of the ligands (InsP3 > 11 > 26 > 10) was therefore the
same as that found with radioligand binding. The most
striking feature of these results is the high activity of the
(S) isomer 11 as compared to the (R) one 10.This abso-
lute configuration is identical to that of the natural
product adenophostin A. It is well known that the two
phosphates at C-4 and C-5 in a trans arrangement are
needed for the biological activity.32 The results descri-
bed here clearly show the importance of the spatial
orientation of the third phosphate group and show that
the presence of the adenine group strongly enhances the
potency of compound 11 as compared to our previously
described analogue 9.8 The low potency of one of the
two diastereoisomeric compounds shows that the con-
figuration of the carbon bearing the non-vicinal phos-
phate group is an important requirement for a high
affinity to the receptor and provides evidences for inter-
actions of the purine base with a binding pocket nearby
the InsP3 binding site. These interactions should place
the phosphate group in a suitable position to perfectly
mimic InsP3. In the other diastereoisomer, for several
reasons, the phosphate cannot adopt the same orienta-
tion thus explaining the low affinity. One reason should
be that stabilizing interactions of the adenine ring with a
The remainder of the synthesis from these epoxides was
identical to that described above: opening of the epox-
ide ring with adenine anion, which gave the expected
alcohols in 57% yield. Careful chromatography allowed
the separation of the N-9 and N-7 isomers formed in a
3:1 ratio. Zemplen debenzoylation of the pure a anomer
38 gave compound 39 as a pure stereoisomer. Phos-
phorylation of this compound gave the protected tris-
phosphate 40 in 74% yield and removal of the benzyl
protecting groups proceeded uneventfully to yield pure
11, which was isolated as its hexasodium salt.
The binding efficiency of the compounds was assayed in
equilibrium competition binding experiments to cere-
bellar membranes with [3H]InsP3. The analogues com-
pletely inhibited specific InsP3 binding (Fig. 2).
Concentrations that inhibited 50% of [3H]InsP3 binding
(IC50) are given in Table 1. Data indicate that the order
of potency is 11 > 26. InsP3 is about 4 times more effi-
cient that 11.The compound 10 also binds InsP3R but
with a much lower affinity.
Figure 2. Inhibition of specific [3H] InsP3 binding to rat cerebellar
microsomes by increasing concentrations of inositol phosphates ana-
logues: & for InsP3 (1); ~ for 11; * for 10 and ^ for 26.
Scheme 4. (i) CF3COOH(0.15 M), Pd/C(10%), dioxane/H2O, reflux,
20 h; (ii) CCl3CN, K2CO3, CH2Cl2; (iii) TMSOTf, CH2Cl2, MS 4 A,
ꢀ20 ꢁC; (iv) adenine, NaH, DMF, 90 ꢁC; (v) MeONa, MeOH; (vi) 41,
1H-tetrazole, CH2Cl2 then tBuOOH, 0 ꢁC; (vii) H2,Pd/C (10%),
20 bars, MeOH.
Figure 3. Analogue-induced [45Ca++] release from permeabilized
hepatocytes. Each data point represents the mean of at least three
determinations.

F. Roussel et al. / Bioorg. Med. Chem. 10 (2002) 759–768
763
possible binding pocket takes place and directs the
phosphate far from its receptor site. The existence of
such a binding pocket for adenine is in line with the high
potency of adenophostins and all analogues having the
adenine ring.33
then benzyl bromide (3 mL) was added. After 1 h, excess
NaH was destroyed by addition of MeOH, and the sol-
vents were removed by evaporation under reduced
pressure. The residue was diluted with CH2Cl2 (500 mL)
and washed with water (2 ꢂ 50 mL). The organic layer
was dried over MgSO4 and evaporated to dryness.
The residue was purified by column chromatography
(hexane/EtOAc 3:2) to give 22 (8.6 g, 86%).
Conclusion
One can conclude that the very high affinity of adeno-
phostin for InsP3R should be not only the consequence
of favorable interactions of the adenine ring at the
binding site. It is likely that the rigid adenosine ring
system largely contributes to a fine adjustment of the
phosphate position, which mimics even better that of
the 1-phosphate of InsP3. The exact nature of adenine
interactions, likely hydrophobic ones,13,34,35 at the bind-
ing site should be clarified by synthesis of adenophostin
analogues modified on the purine base.38
[2-Hydroxy-3-(6-amino-9H-purin-9-yl)]-propyl 2-O-ben-
zyl-3,4-O-[(2S,3S) 2,3-dimethoxybutane-2,3-dyl]-ꢀ-D-xy-
lopyra-noside (23). To a solution of adenine (2.025 g,
15 mmol) in DMF (70 mL) was added sodium hydride
(60% dispersion in mineral oil, 600 mg, 15 mmol). The
mixture was stirred at 80 ꢁC under inert atmosphere for
90 min then a solution of 22 (2.05 g, 5 mmol) in DMF
(15 mL) was added dropwise. After 3 h of heating, the
reaction was cooled to room temperature and filtered
through Celite. The filtrate was concentrated under
reduced pressure, and the residue was diluted with
CH2Cl2 (500 mL). The organic layer was washed twice
with water and dried over MgSO4 and concentrated.
The residue was purified by column chromatography
(CH2Cl2/MeOH 9:1) to give compound 23 (2.043 g,
75%).
Experimental
General procedures
Optical rotations were determined at 20 ꢁC with a
Perkin-Elmer model 141 automatic. NMR spectra were
recorded at 25 ꢁC with a Bruker AC250 spectrometer.
Chemical shifts (d) are given in ppm relative to the sig-
nal for internal tetramethylsilane for 1H NMR and
indirectly to the central line of CDCl3, d 77.03 for 13C
NMR spectra; those in deuterium oxide are reported
relative to external 2,3 dimethyl-2-silapentane-5 sulfate
(DSS). Infrared spectra were recorded on a Perkin-
Elmer IRFT Spectrum 1000 spectrometer. All reactions
were monitored by thin-layer chromatography on Kie-
selgel 60F254 (Merck) with detection by UV light and/or
by charring with 15% sulfuric acid in ethanol. Ele-
mental analyses and mass spectra were performed by
the Service Central d’Analyse du CNRS at Vernaison
(France). [3H]InsP3 (17-21 Ci/mmol) was obtained from
Du Pont New England Nuclear and InsP3 from Cal-
biochem.
[3-(6-Amino-9H-purin-9-yl)-2-(phosphonooxy)]propyl] 3,4
bis-O-(3,4-dihydro-3-oxido-2,4,3-benzodioxaphos-phepin-
3-yl)-ꢀ-D-xylopyranoside (25). To a solution of 23 (2 g,
3.7 mmol) in CH2Cl2 (20 mL) was added 95% aqueous
TFA (10 mL). The mixture was stirred at room tem-
perature for 10 min and then concentrated under
reduced pressure to give the desired triol 24 in 82%
yield. The crude residue (1.3 g, 3 mmol) was dissolved in
CH2Cl2 (40 mL) then 1H-tetrazole (2.52 g, 36 mmol) and
2(N,N-diisopropylamino)-5,6-benzo-1,3,2
dioxapho-
sphepane (41) (6 g, 22.5 mmol) were added to the solu-
tion. The reaction mixture was stirred under argon at
room temperature for 4 h then cooled to 0 ꢁC and
tBuOOH (70% solution in water, 10 mL) was added.
The solution was allowed to warm to room temperature
and was then stirred for 15 min. The clear solution was
diluted with CH2Cl2 (250 mL) and this solution was
washed successively with water (10 mL), saturated
aqueous NaHCO3 solution (2ꢂ10 mL) and water
(2ꢂ10 mL), dried over MgSO4 and concentrated by
evaporation under reduced pressure. The residue was
purified by column chromatography (CH2Cl2/MeOH
99:1 then 94:6) to afford 25 (2.230 g, 76%) as a white
solid.
(3-Oxypropyl) 3,4-O-[(2S,3S) (2,3-dimethoxy butane-
2,3-dyl)]-ꢀ-D-xylopyranoside (21). Compound 17 (12 g,
39.5 mmol) and 50% mCPBA (20.44 g, 59.2 mmol) in
CH2Cl2 (300 mL) was stirred at room temperature for
24 h. The reaction mixture was diluted with CH2Cl2
(400 mL) and then washed with H2O, saturated aqueous
NaHCO3 solution and H2O. The organic layer was
dried (MgSO4) and evaporated under reduced pressure.
The residue was purified by column chromatography
(hexane/EtOAc 3:4) to give compound 21 (9.85 g, 78%)
as a 3/7 diastereoisomeric mixture of 19 and 18.
[3-(6-Amino-9H-purin-9-yl)-2-(phosphonooxy) propyl]ꢀ-
D-xylopyranoside 3,4-bisphosphate hexasodium salt (26).
Compounds 25 (230 mg, 0.23 mmol) was dissolved in
MeOH (25 mL) and Pd/C (10%, 200 mg) was added.
The mixture was stirred under hydrogen atmosphere
(20 bars) during 12 h. The catalyst was removed by fil-
tration through Celite and the filtrate was concentrated
to dryness. The residue was dissolved in water (3 mL)
and applied to a column of Bio-Rad Chelex 100 resin
(Na+form). The column was eluted with water, frac-
tion containing compound 26 were combined and freeze
dried (157 mg, 94%).
(3-Oxypropyl) 2-O-benzyl-3,4-O-[(2S,3S) (2,3-di-meth-
oxybutane-2,3-dyl)]-ꢀ-D-xylopyranoside (22). A solution
of 21 (8 g, 25 mmol) in dry DMF (50 mL) was added
dropwise to a NaH (1.1 g of a 60% dispersion in oil,
27.5 mmol) suspension in dry DMF (50 mL). The mix-
ture was stirred at room temperature for 15 min, and

764
F. Roussel et al. / Bioorg. Med. Chem. 10 (2002) 759–768
2
3
[(2S) 3-Oxypropyl] 3,4-O-[(2S,3S) (2,3-dimethoxy
butane-2,3-dyl)]-ꢀ-D-xylopyranoside (18). Small
3.73 (dd, J(5a,5b)=10 Hz, 1H; H-5b), 3.63 (dd, J(1,2)
3
3.5 Hz, J(2,3)=10 Hz, 1H; H-2), 3.69 (m, 1H; H-300b),
2
3
0
0
0
0
amounts of 18 was obtained from 21 by column chro-
matography . Rf 0.44 (EtOAc/hexane, 1:9); [a]D +261.4
3.46 (dd, J(1 a-1 b)=10 Hz, J(1 a-2 )=7 Hz, 1H; H-1 b),
2.41 (s, 3H; CH3); 13C NMR (62.9 MHz, CDCl3) d
165.5, 165.4 (C¼O), 132.4, 144.8 (Ar ipso), 128.8, 129.4
(Ar ipso), 127.7, 127.8, 128.2, 128.2, 129.5, 129.7, 132.4,
132.9 (Ar), 97.5 (C-1), 69.2, 70.0 (C-10, C-30), 67.8, 69.9,
71.2, 76.7 (C-2, C-3, C-4, C-20), 58.6 (C-5), 21.3 (CH3);
C36H36O11S (676.20): calcd C 63.89, H 5.37, S 4.73;
found C 63.98, H 5.35, S 4.75.
1
(c 0.96 in chloroform); H NMR (250 MHz, CDCl3) d
4.88 (d, J(1,2)=3 Hz, 1H; H-1), 4.68 (s, 2H; CH2Ph),
3
3.52–3.91 (m, 7H; H-2, H-3, H-4, H-5b, H-5a, H-10a, H-
10b), 3.30 (s, 3H; OCH3), 3.26 (s, 3H; OCH3), 3.20 (m,
1H; H-200), 2.84 (dd, J(2 ,3 a)=4.4 Hz, J(3 a,3 b)=5.1 Hz,
3
3
0
0
0
0
1H; H-3 a), 2.66 (dd, J(2 ,3 b)=2.2 Hz, 1H; H-30b), 2.14
(d, 3J(H,OH)=9.5 Hz 1H; OH), 1.35 (s, 3H; CH3), 1.30 (s,
3H; CH3) 13C NMR (62.9 MHz, CDCl3) d 99.6, 99.2
(CH3COCH3), 98.9 (C-1), 70.4, 69.6, 65.7 (C-2, C-3, C-
4), 68.4 (C-10), 59.8 (C-5), 49.8(C-20), 47.7 (OCH3), 44.7
(C-30), 17.5 and 17.1 (CH3). C14H24O8 (320.3): calcd C
52.5, H 7.6, Found C 52.62, H, 7.42.
3
0
0
[(2R)
[2-Hydroxy-3-(6-amino-9H-purin-9-yl)]-propyl]
3,4-di-O-benzoyl-2-O-benzyl-ꢀ-D-xylo-pyranoside (30).
To a solution of adenine (398 mg, 2.94 mmol) in DMF
(6 mL) was added sodium hydride (60% dispersion in
mineral oil, 113 mg, 2.94 mmol). The mixture was stirred
at 80 ꢁC under inert atmosphere for 90 min then a solu-
tion of 29 (710 mg, 0.98 mmol) in DMF (5 mL) was
added dropwise. After 3 h of heating, the reaction was
cooled to room temperature and filtered through Celite.
The filtrate was concentrated under reduced pressure,
and the residue was purified by column chromato-
graphy (CH2Cl2/MeOH 9:1) to give compound 30
[(2S) (3-Oxypropyl)] 2-O-benzyl-3,4-O-[(2S,3S) (2,3-di-
methoxybutane-2,3-dyl)]-ꢀ-D-xylopyranoside (20). To a
solution of 18 (176 mg, 0.55 mmol) in dry THF (3 mL)
was added NaH (22 mg of a 60% dispersion in oil,
0.6 mmol). The mixture was stirred at room temperature
for 15 min, and then benzyl bromide (70 mL) was added.
After 1 h, excess NaH was destroyed by addition of
MeOH, and the solvents were removed by evaporation
under reduced pressure. The residue was diluted with
CH2Cl2 (50 mL) and washed with water (2 ꢂ 5 mL). The
organic layer was dried over MgSO4 and evaporated to
dryness. The residue was purified by column chromato-
graphy (hexane/EtOAc 3:2) to give 20 (205 mg, 91%).
Rf 0.39 (hexane/EtOAc, 3:2); [a]D=+166.4 (c 0.93 in
1
(420 mg, 63%): Rf 0.55 (CH2Cl2/MeOH 9:1); H NMR
(250 MHz, CDCl3) d 8.29 (s, 1H; H-200), 7.96 (m, 4H;
Ar), 7.89 (s, 1H; H-800), 7.51 (m, 2H; Ar), 7.38 (m, 5H;
Ar), 7.20 (m, 4H; Ar), 5.93 (dd, 3J(2,3)=10 Hz, 1H; H-3),
5.84 (m, 2H; NH2), 5.23 (ddd, J(3,4)=3J(4,5)=10 Hz,
3
3
1H; H-4), 4.90 (d, J(1,2)=3 Hz, 1H; H-1), 4.60 (s, 2H;
CH2Ph), 4.41 (m, 1H; H-30a), 4.30 (m, 2H; H-20, H-30b),
3.96 (dd, 3J(4,5a)=6 Hz, 2J(5a,5b)=11 Hz, 1H; H-5a), 3.82
(dd, J(1 a,2 )=4 Hz, J(1 a,1 b)=11 Hz, 1H; H-10a), 3.74
1
3
2
0
0
0
0
chloroform); H NMR (250 MHz, CDCl3) d 7.40–7.19
2
3
0
0
(m, 5H, Ar), 4.85 (d, J(H,H)=12.4 Hz; 1H; CH2Ph),
3
(m, 2H; H-2, H-5b), 3.41 (dd, J(1 b,2 )=6.5 Hz, 1H; H-
10b); 13C NMR (62.9 MHz, CDCl3) d 166.2, 165.9
(C¼O), 156.0 (C-600), 153.0 (C-200), 150.1 (C-400), 142.0
(C-800), 137.2 (Ar ipso), 129.2, 128.9 (Ar ipso), 133.6,
133.5, 130.0, 128.6, 128.3, 128.2 (Ar), 119.2 (C-5), 97.8
(C-1), 73.3 (CH2Ph), 70.6 (C-10), 77.3, 71.7, 70.3, 68.8
(C-2, C-3, C-4, C-20), 59.1 (C-500), 47.1 (C-30);
C34H33N5O8 (639.3): calcd C 63.83, H 5.20, N 10.95;
found C 63.95, H 5.18, N 10.92.
4.72 (d, J(1,2)=3.6 Hz, 1H; H-1), 4.65 (d, 1H; CH2Ph),
3
4.09 (dd, J(3,4)=9.4 Hz, H-3), 3.82–3.63 (m, 3H, H-4,
H-5a, H-10a), 3.62–3.44 (m, 3H, H-2, H-5b, H-10b), 3.30
(s, 3H, OCH3), 3;25 (s, 3H, OCH3), 3.16 (m, 1H, H-20),
2.75 (dd, 3J(2 ,3 a)=4.4 Hz, 3J(3 a,3 b)=4.7 Hz, 1H, H-30a),
0
0
0
0
2.65 (dd, J(2 ,3 b)=2.9 Hz, 1H, H-30b), 1.36 (s, 3H,
CH3), 1.31 (s, 3H, CH3); 13C NMR (62.9 MHz, CDCl3)
3
0
0
d
138.5 (Ar ipso), 128.1,127.4 (Ar), 99.5, 99.3
(CH3COCH3), 98.0 (C-1), 76.4 (C-2), 73.2 (CH2Ph),
69.8 (C-3), 68.0 (C-10), 66.3 (C-4), 59.3 (C-20), 47.8
(OCH3), 44.5 (C-30), 17.7 and 17.4 (CH3). C21H30O8
(410.46): calcd C 61.5, H 7.4, found C 61.38, H, 7.32
[(2R) [3-(6-Amino-9H-purin-9-yl)-2-hydroxy]propyl] 2-O-
benzyl-ꢀ-D-xylopyranoside (31). Compound 30 (370 mg,
0.58 mmol) was dissolved in MeOH (50 mL) and a cat-
alytic amount of sodium was added. After completion
of the reaction, TLC monitoring, the mixture was neu-
tralized using Amberlite IRA 120 H+. The resin was
filtered off and the filtrate was concentrated. The residue
was purified by column chromatography (EtOAc/
MeOH 9:1) to afford the triol derivative 31 (165 mg,
66%). Rf 0.12 (EtOAc/MeOH, 9:1); 1H NMR
(250 MHz, CD3OD) d 8.46 (s, 1H; H-200), 8.10 (s, 1H, H-
[(2R) (2-Hydroxy-3-tosyloxy)propyl] 2-O-benzyl-3,4-di-
O-benzoyl-ꢀ-D-xylopyranoside (29). Compound AU26
(750 mg, 1.43 mmol) was dissolved in dry pyridine
(20 mL) and tosyl chloride (300 mg, 1.57 mmol) was
added. The mixture was stirred for 2 h at room temperature
then concentrated under reduced pressure. The residue
was diluted with CH2Cl2 (200 mL) and washed with
aqueous HCl (3 N) and H2O. The organic layer was
dried over MgSO4 and concentrated. The residue was
purified by column chromatography (hexane/EtOAc 4:1
then 1:1) to give 29 (712 mg, 73%). Rf 0. 57 (H /A 1:1);
1H NMR (250 MHz, CDCl3) d 7.97 (m, 4H; Ar), 7.70
(m, 2H; Ar), 7.50 (m, 4H; Ar), 7.35 (m, 5H; Ar), 7.20
(m, 4H; Ar), 5.84 (dd, 1H; H-3), 5.19 (ddd,
800), 7.37 (m, 5H, Ar), 4.70 (d, J(H,H)=12 Hz, 1H,
2
3
CH2Ph), 4.64 (d, J(1,2)=3.5 Hz, 1H, H-1), 4.57 (d, 1H,
CH2Ph), 4.30 (dd, 3J(20,30a)=3 Hz, J(3 a,3 b)=12 Hz,
3
0
0
1H, H-30a), 4.18, (dd, J(2 ,3 b)=7 Hz, 1H, H-30b), 4.06
3
0
0
(m, 1H, H-20), 3.70 (dd, J(2,3)=3J(3,4)=9 Hz, 1H, H-3),
3
0
3
3
0
0
0
0
3.62 (dd, J(1 a,1 b)=11 Hz, J(1 a,2 )=4 Hz, 1H, H-1 a),
3.42 (m, 2H, H-4, H-5a), 3.24 (dd, 2H, H-2, H-10b), 3.18
3
3
3
3
3J(3,4)=10 Hz, J(4,5a)=5.5 Hz, J(4- 5b)=5.5 Hz, 1H; H-
4), 4.87 (d, 1H; H-1), 4.57 (s, 2H; CH2Ph), 4.10 (m, 1H;
H-10a), 3.93 (dd, 1H; H-5a), 3.85 (m, 2H; H-20, H-30a),
(dd, J(4,5b)=5.5 Hz, J(5a,5b)=10 Hz, 1H, H-5b); 13C
NMR (62.9 MHz, CD3OD) d 157.2 (C-600), 154.6 (C-200),
150.8 (C-400), 143.6 (C-800), 139.6 (Ar ipso), 128.9 128.4,

F. Roussel et al. / Bioorg. Med. Chem. 10 (2002) 759–768
765
128.2 (Ar), 119.8 (C-5), 98.9 (C-1), 74.2 (CH2Ph), 70.7
(C-10), 81.2, 74.2, 71.5, 69.5 (C-2, C-3, C-4, C-20), 62.9
(C-500), 47.6 (C-30); C20H25N5O6 (431.20): calcd C 55.7,
H 5.8, N 16.2; found C 55.95, H 5.81 N. 16.04.
The solvent was evaporated, the residue was diluted was
CH2Cl2 (100 mL). The organic layer was washed with
water, dried and concentrated under reduced pressure.
The crude residue was debenzoylated using the Zemplen
procedure to give after column chromatography (hex-
ane/EtOAc 2:3) compound 15 (170 mg, 50%). Rf 0.50
[(2R) [3-(6-Amino-9H-purin-9-yl)-2-(phosphonooxy)]pro-
pyl] 3,4 bis-O-(3,4-dihydro-3-oxido-2,4,3-benzodi-oxa-
phosphepin-3-yl)-ꢀ-D-xylopyranoside (32). The triol
derivative 31 (160 mg, 0.37 mmol) was dissolved in
CH2Cl2 (12 mL) then 1H-tetrazole (308 mg, 4.44 mmol)
and 2(N,N-diisopropylamino)-5,6-benzo-1,3,2 di-oxa-
phosphepane (41) (832 mg, 3.08 mmol) were added to
the solution. The reaction mixture was stirred under
argon at room temperature for 4 h then cooled to
0 ꢁC and tBuOOH (70% solution in water, 1.5 mL) was
added. The solution was allowed to warm to room
temperature and was then stirred for 15 min. The clear
solution was diluted with CH2Cl2 (100 mL) and this
solution was washed successively with water (10 mL),
saturated aqueous NaHCO3 solution (2 ꢂ 10 mL) and
water (2 ꢂ 10 mL), dried over MgSO4 and concentrated
by evaporation under reduced pressure. The residue was
purified by column chromatography (CH2Cl2/MeOH
99:1 then 94:6) to afford 32 (252 mg, 70%) as a white
solid. Rf 0.52 (CH2Cl2/MeOH, 9:1); 1H NMR
(250 MHz, CDCl3) d 8.42 (s, 1H; H-200), 8.12 (s, 1H; H-
800), 7.26 (m, 17H; Ar), 5.42 (m, 4H; CH2PhCH2), 5.20
(m, 8H; CH2PhCH2), 4.75 (m, 1H; H-3), 4.68 (d, 1H; H-
1), 4.63 (m, 4H; H-4, H-20, H-30a, H-30b), 4.59 (d, 1H;
1
(EtOAc); [a]D +74.0 (c 0.36 in chloroform); H NMR
(250 MHz, CDCl3) d 7.35 (m, 5H; Ar), 4.72 (d,
2
3J(1,2)=3.5 Hz, 1H; H-1), 4.71 (d, J(H,H)=11.5 Hz; 1H;
CH2Ph), 4.64 (d, 1H; CH2Ph), 3.99 (m, 1H; H-20), 3.88
3
(dd, J(2,3)=3J(3,4)=9 Hz, 1H; H-3), 3.75 (dd,
2
0
J(1 a,1 b)=3J(1 b,2 )=3.5 Hz, 1H; H-1 a), 3.56 (m, 5H; H-
0
0
0
0
4, H-5a, H-5b, 2 ꢂ OH), 3.31 (m, 5H; H-2, H-10b, H-
30a, H-30b, OH); 13C NMR (62.9 MHz, CDCl3) d 137.1
(Ar ipso), 128.6, 128.4 (Ar), 97.2 (C-1), 79.6 (C-2), 73.5
(CH2Ph), 72.7 (C-3), 69.9 (C-5), 69.7 (C-20), 69.4 (C-4),
61.4 (C-10), 52.7 (C-30), IR (neat): n=3379 (OH), 2101
(N3)cmꢀ1 ; C15H21N3O6 (339.4): calcd C 53.07, H 6.24,
N, 12.39; found C 53.17, H 6.26, N 12.35.
[(2S) 3-Azido-2-hydroxypropyl] 2-O-benzyl-ꢀ-D-xylo-
pyranoside (16). Rf 0.46 (EtOAc); [a]D +76.5. (c 0.5 in
1
chloroform); H NMR (250 MHz, CDCl3) d 7.34 (m,
5H; Ar), 4.72 (d, 2J(H,H)=11.6 Hz; 1H; CH2Ph), 4.65 (d,
3J(1,2)=3.6 Hz, 1H; H-1), 4.60 (d, 1H; CH2Ph), 4.08–
3.77 (m, 2H; H-3, H-20), 3.71–3.43 (m, 4H, H-4, H-5a,
H-5b, H-10a), 3.42–3.18 (m, 4H; H-2, H-10b, H-30a, H-
30b), 1.72 (s; 3H, OH); 13C NMR (62.9 MHz, CDCl3) d
137.0 (Ar ipso), 128.4, 128.1 (Ar), 96.8 (C-1), 79.3 (C-2),
73.3 (CH2Ph), 72.8 (C-3), 69.5 (C-4), 69.4 (C-10), 69.1
(C-20), 61.2 (C-5), 52.9 (C-30), IR (neat): n=3390 (OH),
2101 (N 3 )cmꢀ1; C15H21N3O6 (339.4): calcd C 53.07, H
6.24, N 12.39; found C 52.95, H 6.08, N 12.24.
2
CH2Ph), 4.46 (d, J(H,H)=12 Hz 1H; CH2Ph), 4.08 (dd,
2
3J(4,5a)=6, J(5a,5b)=11 Hz, 1H; H-5a), 4.01 (dd,
J(1 b,2)=1.5 Hz, 1H; H-10b), 3.76 (dd, J(4,5b)=11 Hz,
3
3
0
1H; H-5b), 3.51 (dd, 3J(1,2)=4 Hz, 3J(2,3)=10 Hz, 1H; H-
2), 3.25 (dd, J(10a,10b)=11 Hz, J(10a,20)=6.5 Hz, 1H;
H-10a); 31P NMR (100.13 MHz, CDCl3) d ꢀ1.58, ꢀ1.49,
0.94; C44H46N5O15P3 (977.80): calcd C 54.05, H 4.74, N
7.16; found C 54.22, H 4.72, N 7.18.
2
3
3,4-Di-O-benzoyl-2-O-benzyl-D-xylopyranose (33). To a
solution of 27 (1.22 g, 2.5 mmol) in dioxane/water (8:2,
23 mL) was added an aqueous TFA solution (0.15 M,
0.07 mL, 0.9 mmol) and 10% Pd/C (1.2 g). The reaction
mixture was filtered through Celite. The filtrate was
concentrated under reduced pressure. The residue was
purified by column chromatography (hexane/EtOAc
7:3) to give an anomeric mixture of 33 (760 mg, 68%) as
a white powder. Data for a anomer: Rf 0.35 (Hexane/
[(2R) 3-(6-Amino-9H-purin-9-yl)-2-(phosphonooxy)-pro-
pyl] ꢀ-D-xylopyranoside 3,4-bisphosphate hexasodium
salt (10). Compounds 32 (80 mg, 0.08 mmol) was dis-
solved in MeOH (5 mL) and Pd/C (10%, 80 mg) was
added. The mixture was stirred under hydrogen atmo-
sphere (20 bars) for 12 h. The catalyst was removed
by filtration through Celite and the filtrate was con-
centrated to dryness. The residue was dissolved in water
(3 mL) and applied to a column of Bio-Rad Chelex 100
resin (Na+ form). The column was eluted with water,
fraction containing compound 10 were combined and
1
EtOAc, 7:3); H NMR (250 MHz, CDCl3) d 7.96 (dd,
4H; Ar), 7.60–7.46 (m, 2H; Ar), 7.45–7.31 (m, 5H; Ar),
3
7.28–7.05 (m, 4H; Ar), 5.91 (dd, J(2,3)=8.0 Hz,
3J(3,4)=8.8 Hz, 1H; H-3), 5.33–5.14 (m, 2H; H-1, H-4);
4.67 (s, 2H; CH2Ph), 4.12–3.96 (m, 2H; H-5a, H-5b),
3
3
3.74 (dd, J(1,2)=3.6 Hz, J(2,3)=8.4 Hz, 1H; H-2), 3.41
(s, 1H; OH). 13C NMR (62.9 MHz, CDCl3) d 165.2,
165.1 (C¼O), 137.1 (Ar ipso), 132.9 (Ar), 129.5, 128.2,
128.1, 127.8 (Ar), 91.0 (C-1), 76.3 (C-2), 72.7 (CH2Ph),
70.4 (C-3), 69.2 (C-4), 59.4 (C-5). Data for b anomer: 1H
NMR (250 MHz, CDCl3) d 7.93 (m, 4H; Ar), 7.57–7.44
(m, 2H; Ar), 7.44–7.29 (m, 5H; Ar), 7.24–7.05 (m, 4H;
1
freeze dried (55 mg, 98%). H NMR (250 MHz, D2O) d
8.44 (s, 1H; H-200), 8.36 (s, 1H; H-800), 4.88 (d,
3J(1,2)=2.8 Hz, 1H; H-1), 4.60 (m, 3H, H-30a, H-30b, H-
20),
4.34
(ddd,
3J(2,3)=7.3 Hz,
3J(3,4)=7.7 Hz,
3J(H,P)=7.6 Hz, 1H; H-3), 4.08–3.57 (m, 5H; H-2, H-4,
H-5a, H-5b, H-10a), 3.52 (dd,
J(1 b,2 )=3.4 Hz,
3
0
0
J(1 a,1 b)=11 Hz, 1H; H-10b); 31P NMR (100.13MHz,
D2O) d 4.70, 4.17, 4.05. C13H16N5O15P3Na6 (713.15): calcd
C 21.89, H 2.26, N 9.82; found C 21.95, H 2.25, N 9.79.
Ar), 5.68 (dd, J(2,3)=8.8 Hz, J(3,4)=9.5 Hz, 1H; H-3),
3
3
3
3
0
0
5.29–5.14 (ddd, J(4,5a)=5.1 Hz, 1H; H-4), 4.93 (d,
2
3J(1,2)=6.6 Hz, 1H; H-1), 4.84 (d, J(H,H)=11.7 Hz, 1H;
CH2Ph), 4.72 (d, 1H; CH2Ph), 4.31 (dd,
2J(5a,5b)=11.70 Hz, 1H; H-5a), 3.63–3.46 (m, 2H; H-2,
H-5b), 1.67 (s, 1H; OH); 13C NMR (62.9 MHz, CDCl3)
d 165.3, 165.2 (C¼O), 137.3 (Ar ipso), 133.3, 133.1 (Ar),
129.5, 128.3, 128.1, 127.7 (Ar), 97.6 (C-1), 78.8 (C-2),
[(2R) 3-Azido-2-hydroxypropyl] 2-O-benzyl-ꢀ-D-xylo-
pyranoside (15). To a solution of 29 (475 mg, 0.7 mmol)
in DMF (10 mL) was added NaN3 (91 mg, 1.4 mmol).
The mixture was heated at 80 ꢁC and stirred overnight.

766
F. Roussel et al. / Bioorg. Med. Chem. 10 (2002) 759–768
2
73.7 (CH2Ph), 69.7 (C-3), 69.2 (C-4), 62.3 (C-5); IR
(KBr): n 3445 (OH), 1726 (C¼O) cmꢀ1. C26H24O7
(448.15): calcd C 69.62, H 5.40; found C 69.49, H 5.39.
H-4), 4.87 (d, J(H,H)=11.7 Hz, 1H; CH2Ph), 4.73–4.64
2
(m, 2H; H-1; CH2Ph), 4.30 (dd, J(5a,5b)=11.7 Hz, 1H;
3
2
0
0
0
0
H-5a), 4.14 (dd, J(1 a,2 )=2.9 Hz, J(1 a,1 b)=11.7 Hz,
1H; H-10a), 3.68–3.56 (m, 2H; H-10b, H-2), 3.51 (dd, 1H;
3
3
0
0
0
0
O-(3,4-Di-O-benzoyl-2-O-benzyl-D-xylopyranosyl)trichloro-
acetimidate (34). To a solution of compound 33
(722 mg, 1.61 mmol) and Cl3CCN (0.48 mL, 4.83 mmol)
in dry CH2Cl2 (8 mL) wad added freshly dried and
powdered K2CO3 (222 mg, 1.61 mmol). The resulting
suspension was stirred under argon at room tempera-
ture for 12 h. The mixture was concentrated under
reduced pressure. The residue was purified by column
chromatography (hexane/EtOAc 85:15) to give a b/a
mixture of 34 (820 mg, 86%) as a white powder. Data
for b isomer: Rf=0.25 (hexane/EtOAc, 85:15); [a]D
ꢀ52.6 (c 0.76 in chloroform), mp=133–134 ꢁC; 1H
NMR (250 MHz, CDCl3) d 8.78 (s, 1H; NH), 7.43–7.28
(m, 5H; Ar), 6.23 (d, 3J(1,2)=5.1 Hz, 1H; H-1), 5.70 (dd,
H-5b), 3.25 (dddd, J(2 ,3 a)=4.4 Hz, J(2 ,3 b)=2.9 Hz,
1H; H-2 ), 2.84 (dd, J(3 a,3 b)=5.1 Hz, 1H; H-30a), 2.65
(dd, 1H; H-30b); 13C NMR (62.9 MHz, CDCl3) d 165.3,
165.2 (C¼O), 137.2 (Ar ipso), 133.3, 133.2, 129.8, 129.7,
128.4, 128.3, 128.2, 128.1, 128.0 (Ar), 103.1 (C-1), 77.4
(C-2), 73.6 (CH2Ph), 72.1 (C-3), 70.1 (C-10), 69.6 (C-4),
62.0 (C-5), 50.3 (C-20), 44.0 (C-30). C29H28O8 (504.18):
calcd C 69.02, H 5.60; found C 69.19, H 5.58.
0
2
0
0
[(2S) 2-Hydroxy-3-(6-amino-9H-purin-9-yl)-propyl] 3,4-
di-O-benzoyl-2-O-benzyl-ꢀ-D-xylopyranoside (38). Com-
pound 38 was obtained from 36 as described for the
synthesis of 23 Rf 0.54 (CH2Cl2/MeOH, 9:1); [a]D ꢀ5.8
(c 0.93 in chloroform); mp 93–95 ꢁC; 1H NMR
(250 MHz, CDCl3) d 8.25 (s, 1H; H-200), 7.95 (m, 4H;
Ar), 7.73 (s, 1H; H-800), 7.58–7.45 (m, 2H; Ar), 7.44–7.28
(m, 5H; Ar), 7.07–7.24 (m, 4H; Ar), 6.10 (m, 2H; NH2),
5.95 (dd, 3J(2,3)=9.5 Hz, 3J(3,4)=10.2 Hz, 1H; H-3), 5.24
3
3J(2,3)=5.8 Hz, J(3,4)=6.6 Hz, 1H; H-3), 5.37–5.23
(ddd,
3J(4,5a)=3.6 Hz,
1H;
H-4),
4.84
(d,
2J(H,H)=11.7 Hz, 1H; CH2Ph), 4.75 (d, 1H; CH2Ph),
2
4.47 (dd, J(5a,5b)=12.4 Hz, 1H; H-5a), 4.01–3.80 (m,
2H; H-2, H-5 b); 13C NMR (62.9 MHz, CDCl3) d 165.3
(C¼O), 160.3 (C¼NH), 136.8 (Ar ipso), 133.2, 133.1
(Ar), 128.3, 128.0, 127.8 (Ar), 97.0 (C-1), 90.6 (CCl3),
74.7 (C-2), 73.4 (CH2Ph), 69.6 (C-3), 68.3 (C-4), 61.7 (C-
5); IR (KBr): n 1673 (C¼N), 1728 (C¼O) cmꢀ1. MS (IC,
CH4): m/z: 592.4 [M+]; C28H24O7NCl3 (591.06): calcd C
56.85, H 4.09, N 2.37; found C 57.01, H 4.10, N 2.39.
(ddd, J(4,5a)=5.8 Hz, J(4,5b)=10 Hz, 1H; H-4), 4.91 (d,
3J(1,2)=3.6 Hz, 1H; H-1), 4.63 (m, 2H; CH2Ph), 4.52–
4.15 (m, 3H; H-20, H-30a, H-30b), 3.98 (dd,
3
3
2
3
0
J(5a,5b)=11.0 Hz, 1H; H-5a), 3.86 (dd, J(1 a,2 )=5.1 Hz,
0
J(1 a,1 b)=10.9 Hz, 1H; H-10a), 3.81–3.68 (m, 2H; H-2,
2
0
0
3
0
0
0
H-5b), 3.40 (dd, J(1 b,2 )=5.8 Hz, 1H; H-1 b), 1.78 (s,
1H; OH); 13C NMR (62.9 MHz, CDCl3) d 165.9, 165.7
(C¼O), 155.6 (C-600), 152.7 (C-200), 150.1 (C-400), 142.0
(C-800), 137.0 (Ar ipso), 133.4, 133.3, 129.8, 129.5, 128.5,
128.4, 128.2, 128.1 (Ar), 119.4 (C-500), 97.7 (C-1), 76.7
(C-2), 73.4 (CH2Ph), 71.6 (C-3), 70.0 (C-4), 69.8 (C-10),
68.8 (C-20), 59.0 (C-5), 47.8 (C-30); IR (KBr): n
1725 cmꢀ1 (C¼O); C34H33N5O8 (639.23): calcd C 63.83,
H 5.20, N 10.95; found C 63.72, H 5.18, N 10.97.
[(2R) 3-Oxypropyl] 3,4-di-O-benzoyl-2-O-benzyl-D-xylo-
pyranoside (36 and 37). A mixture of 34 (788 mg,
1.33 mmol) and (R)-(+) glycidol 35 (0.1 mL 1.5 mmol)
in CH2Cl2 (3.6 mL) was stirred for 45 min under an argon
atmosphere in the presence of activated molecular sieves
(4 A, 200 mg). The reaction mixture was then cooled to
ꢀ20ꢁ C and a solution of TMSOTf (0.02 M in CH2Cl2,
1.3 mL) was added dropwise for 15 min. After 45 min,
the reaction mixture was quenched with saturated
aqueous NaHCO3 solution. The mixture was filtered
through Celite and the filtrate concentrated under
reduced pressure. Purification by column chromato-
graphy (hexane/EtOAc 75:25) afforded compound 36
and 37 as an anomeric mixture (362 mg, 54% a/b ratio
was 55:45 on the basis of its NMR spectrum). Data
for 36: 1H NMR (250 MHz, CDCl3) d 7.94 (dd, 4H; Ar),
7.58–7.45 (m, 2H; Ar), 7.43–7.28 (m, 5H; Ar), 7.27–7.03
[(2S) 3-(6-Amino-9H-purin-9-yl)-2-hydroxypropyl] 2-O-
benzyl-ꢀ-D-xylopyranoside (39). Compound 39 (42 mg,
95%) was obtained as white crystal from 38 (66 mg,
0.1 mmol) using the Zemplen procedure. Rf=0.13
(CH2Cl2/MeOH, 88:12); [a]D +40.1 (c 0.53 in metha-
1
nol); H NMR (250 MHz, [D4] methanol) d 8.21 (s, 1H;
H-200), 8.03 (s, 1H; H-800), 7.43–7.27 (m, 5H; Ar), 4.94–
2
4.82 (m, 3H; H-1, NH2), 4.80 (d, J(H,H)=11.7 Hz, 1H;
CH2Ph), 4.71 (d, 1H; CH2Ph), 4.39 (dd,
J(2 ,3 a)=3.0 Hz, J(3 a,3 b)=13.6 Hz, 1H; H-30a), 4.30–
3
2
0
0
0
0
4.08 (m, 2H; H-20, H-30b), 3.82 (dd, J(2,3)=8.9 Hz,
3
3
3
(m, 4H; Ar), 5.95 (dd, J(2,3)=9.5 Hz, J(3,4)=10.2 Hz,
3
3
1H; H-3), 5.22 (ddd, J(4,5a)=5.8 Hz, J(4,5b)=10 Hz,
3
3J(3,4)=9.2 Hz, 1H; H-3), 3.71 (dd, 3J(10a,20)=4.7 Hz,
J(1 a,1 b)=10.3 Hz, 1H; H-10a), 3.62–3.48 (m, 2H; H-4,
H-5a), 3.47–3.28 (m, 3H; H-2, H-5b, H-10b); 13C NMR
(62.9 MHz, CD3OD) d 157.2 (C-600), 153.7 (C-200), 151.1
(C-400), 143.7 (C-800), 139.7 (Ar ipso), 129.6, 129.5, 129.0
(Ar), 119.9 (C-500), 99.0 (C-1), 81.4 (C-2), 74.5 (CH2Ph),
74.4 (C-3), 71.6 (C-4), 70.7 (C-10), 69.8 (C-20), 63.0 (C-
2
0
0
1H; H-4), 4.93 (d, J(1,2)=3.7 Hz, 1H; H-1), 4.64 (2H;
2
CH2Ph), 3.98 (dd, J(5a,5b)=11.0 Hz, 1H; H-5a), 3.92–
3.80 (m, 2H; H-10a, H-5b), 3.77–3.62 (m, 2H; H-10b, H-
2), 3.27 (dddd, 1H; H-20), 2.88–2.77 (m, 2H; H-30a, H-
30b); 13C NMR (62.9 MHz, CDCl3) d 165.3, 165.2
(C¼O), 137.2 (Ar ipso), 133.2, 133.1, 129.8, 129.7,
128.5, 128.3, 128.2, 128.1, 127.9 (Ar), 96.8 (C-1), 76.5
(C-2), 72.4 (CH2Ph), 70.9 (C-3), 70.0 (C-4), 67.5 (C-10),
58.4 (C-5), 50.2 (C-20), 44.0 (C-30); IR (KBr): n 1727
(C¼O) cmꢀ1; MS (IC, CH4): m/z: 503.6 (Mꢀ1). Data
for 37: 1H NMR (250 MHz, CDCl3) d 7.94 (m, 4H; Ar),
7.58–7.45 (m, 2H; Ar), 7.44–7.29 (m, 5H; Ar), 7.23–7.05
5), 48.1 (C-30); IR (KBr): 3442 cmꢀ1 (OH);
n
C20H25N5O6 (431.18): calcd C 55.66, H 5.84, N 16.24;
found C 55.53, H 5.85, N 16.18.
[(2S) 3-(6-Amino-9H-purin-9-yl)-2-(phosphonooxy) pro-
pyl] 3,4 bis-O-(3,4-dihydro-3-oxido-2,4,3-benzodioxa
phosphepin-3-yl)-ꢀ-D-xylopyranoside (40). Compound
39 (40 mg, 0.09 mmol), phosphoramidite 41 (208 mg,
3
3
(m, 4H; Ar), 5.65 (dd, J(2,3)=8.7 Hz, J(3,4)=8.8 Hz, 1;
3
3
H-3), 5.20 (ddd, J(4,5a)=5.1 Hz, J(4,5b))=8.8 Hz, 1H;

F. Roussel et al. / Bioorg. Med. Chem. 10 (2002) 759–768
767
0.77 mmol) and 1H-tetrazole (77 mg, 1.11 mmol) was
dissolved in CH2Cl2 (2 mL). The reaction mixture was
stirred at room temperature for 4 h then cooled to 0 ꢁC
and tBuOOH (0.5 mL) was added. Purification by col-
umn chromatography (CH2Cl2/MeOH 99:1 then 94:6)
afforded compound 40 (67 mg, 74%) as a white powder.
Rf 0.29 (CH2Cl2/MeOH, 94:6); [a]D ꢀ1.4 (c 0.68 in
chloroform); mp 140–142 ꢁC; 1H NMR (250 MHz,
CDCl3) d 8.39 (s, 1H; H-200), 7.86 (s, 1H; H-800), 7.43–
7.03 (m, 17H; Ar), 5.69–4.88 (m, 15H; H-1, H-3, H-20,
PhCH2Ph), 4.76 (m, 2H; CH2Ph), 4.65–4.46 (m, 3H; H-
4, H-30a, H-30b), 4.08 (dd, 3J(4,5a)=5.8 Hz,
2J(5a,5b)=11.0 Hz, 1H; H-5a), 3.85–3.65 (m, 3H; H-5b,
din, 5 mg mLꢀ1 aprotinin, 50 mg mLꢀ1 trypsin inhibitor
and 1 mg mLꢀ1 orthophenantrolin, as previously descri-
bed.36 The homogenate was centrifuged for 5 min at
1500g, and the resulting supernatant was centrifuged
for 30 min at 50,000g. The pellet was washed and
resuspended at 4 mg protein/mL in washing medium
containing 250 mM sucrose, 25 mM Hepes/KOH pH
7.4, supplemented with 1 mM dithiothreitol and the
protease inhibitors cocktail.
Equilibrium [3H]InsP3-bindingstudies
Microsomes (50–100 mg) were incubated on ice in
500 mL of the binding medium mimicking the ionic
composition of the cytosol and containing 110 mM
KCl, 20 mM NaCl, 25 mM HEPES pH 7.4, 1 mM
NaH2PO4, 1 mM EDTA, supplemented with 1 mM
dithiothreitol, the protease inhibitor cocktail, 1 mg/mL
BSA and supplemented with 1 nM [3H]InsP3. Non
radio-active InsP3 and the adenophostin analogues were
added at the indicated concentrations. After a 6 min
incubation period, 400 mL of the sample was layered on
to a Whatman GF/C glass-fibre filter and washed with
1 mL of ice-cold medium consisting of 250 mM sucrose,
10 mM Na2HPO4 and adjusted to pH 8 to minimise the
dissociation of the ligand–receptor complex. Radio-
activity retained on the filter was counted by scintillation
spectrometry.
H-10a, H-10b), 3.58 (dd, J(1,2)=2.9 Hz, J(2,3)=9.5 Hz,
1H; H-2); 13C NMR (62.9 MHz, CDCl3) d 157.0 (C-600),
151.0 (C-200), 150.4 (C-400), 143.2 (C-800), 137.2, 134.9,
134.7, 134.6 (Ar ipso), 128.9, 128.8, 128.7, 128.6, 128.5,
128.3, 128.2, 128.0, 127.9 (Ar), 120.1 (C-500), 96.8 (C-1),
3
3
3
77.6 (2J(C,P)=7.6 Hz, J(C,P)=5.7 Hz, C-3), 77.2 (C-2),
69.8 (2J(C,P)=5.7 Hz, C-20), 73.6 (3J(C,P)=4.3 Hz,
2J(C,P)=3.3 Hz, C-4), 72.7 (CH2Ph), 68.1–68.6 (6C,
PhCH2Ph), 67.1 (3J(C,P)=4.3 Hz, C-10), 59.4 (C-5), 44.7
(3J(C,P)=5.2 Hz, C-30); 31P NMR (100.13 MHz, CDCl3)
d 0.56, ꢀ0.15 (P-3, P-4), ꢀ1.80 (P-20); IR (KBr): n
1284 cmꢀ1 (P¼O); C44H46N5O15P3 (977.22): calcd C
54.03, H 4.74, N 7.16; found C 54.18, H 4.75, N 7.14.
[(2S) 3-(6-Amino-9H-purin-9-yl)-2-(phosphonooxy) pro-
pyl] ꢀ-D-xylopyranoside 3,4-bisphosphate hexasodium
salt (11). Compound 40 ( 46 mg, 0.05 mmol) was dis-
solved in MeOH (5 mL) and Pd/C (10%, 60 mg) was
added. The mixture was stirred under hydrogen atmo-
sphere (20 bars) for 12 h. The reaction mixture was
filtered through a pad of Celite and the filtrate was
concentrated to dryness. The residue was dissolved in
water (3 mL) and applied to a column of Bio-Rad Che-
lex 100 resin (Na+ form). The column was eluted with
water, fraction containing compound 11 were combined
and freeze dried to give 11 (33 mg, 98%). [a]D +11.5 (c
45Ca2+ fluxes measurements
Hepatocytes were prepared as previously described.37
They were washed and resuspended at 4ꢂ106 cells mLꢀ1
in the cytosol-like medium buffered at pH 7.1 at 37 ꢁC
and containing 1 mM EGTA and 0.55 mM CaCl2. The
cells were permeabilized at 4 ꢁC by the addition of 80 mg
mLꢀ1 saponin. The medium was supplemented with
5 mM CCCP, 5 mM oligomycin, 5 mM creatine phos-
phate and 5 u mLꢀ1 creatine phosphokinase. Permeabi-
lized cells were loaded with 45Ca2+ by incubating the
cell suspension for 5 min at 37 ꢁC in the presence of
1 mM ATP, 1 mM MgCl2 and 2–3ꢂ105 cpm 45Ca2+.The
45Ca2+ release was measured at 37 ꢁC, 15 s after the
addition of the indicated concentrations of InsP3 or
adenophostin analogues. Incubations were terminated
by filtration through a Whatman GF/C glass fibber filter
and washing with ice-cold washing medium (4 ꢂ 3 mL).
1
0.5 in water); H NMR (250 MHz, D2O) d 8.42 (s, 1H;
H-200), 8.33 (s, 1H; H-800), 4.95 (d, 3J(1,2)=2.8 Hz, 1H; H-
1), 4.69–4.50 (m, 3H, H-30a, H-30b, H-20), 4.34 (ddd,
3
3
3J(2,3)=7.3 Hz, J(3,4)=7.7 Hz, J(H,P)=7.6 Hz, 1H; H-
3), 4.13–3.92 (m, 2H; H-4, H-5a), 3.90–3.53 (m, 4H; H-
2, H-5b, H-10a, H-10b); 13C NMR (62.9 MHz, D2O) d
157.0 (C-600), 155.3 (C-200), 151.8 (C-400), 146.3 (C-800),
122.2 (C-500), 101.38 (C-1), 76.8 (2J(C,P)=7.3 Hz, C-3),
74.2 (C-2), 73.1 (C-20, C-4), 70.4 (3J(C,P)=2.4 Hz, C-10),
65.1 (C-5), 47.7 (C-30); 31P NMR (100.13 MHz, D2O) d
7.76, 7.22, 7.10 (P-3, P-4, P-20); MS (ES- ): m/z: 645
[(Mꢀ4H+3Na)ꢀ], 623 [(Mꢀ3H+ 2Na)ꢀ], 601
[(Mꢀ2H+Na)ꢀ], 579 [(MꢀH)ꢀ]; C13H16N5O15P3Na6
(712.92): calcd C 21.88, H 2.26, N 9.82; found C 21.79,
H 2.27, N 9.85.
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10 mg mLꢀ1 leupeptin, 10 mM pepstatin, 2 mM benzami-

768
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34. Interestingly, good affinity is retained when replacing the
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Nagano et al. showed that malachite green- InsP3 had a good
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38. While this manuscript was completed, an interesting paper
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