Synthesis, acid-base behavior, and binding properties of 6-modified myo- inositol 1,4,5-tris(phosphate)s

Article abstract of DOI:10.1021/jm991084t

myo-Inositol 1,4,5-tris(phosphate) was modified at position 6. The analogues synthesized are reported in this publication are 6-deoxy-myo- inositol 1,4,5-tris(phosphate), 6-fluoro-6-deoxy-myo-inositol 1,4,5- tris(phosphate), epi-inositol 1,4,5-tris(phosph

Full text of DOI:10.1021/jm991084t

4824
J . Med. Chem. 1999, 42, 4824-4835
Syn th esis, Acid -Ba se Beh a vior , a n d Bin d in g P r op er ties of 6-Mod ified
m yo-In ositol 1,4,5-Tr is(p h osp h a te)s
Ste´phanie Ballereau,† Philippe Gue´dat,† Ste´phane N. Poirier,‡ Gae´tan Guillemette,‡ Bernard Spiess,† and
Gilbert Schlewer*,†
Laboratoire de Pharmacochimie de la Communication Cellulaire UMR 7081 du CNRS, Faculte´ de Pharmacie, 74,
route du Rhin, 67401 Illkirch, France, and De´partement de Pharmacologie, Faculte´ de Me´decine, Universite´ de Sherbrooke,
3001, 12 avenue Nord Sherbrooke, Quebec, Canada J 1H 5N4
Received May 20, 1999
myo-Inositol 1,4,5-tris(phosphate) was modified at position 6. The analogues synthesized are
reported in this publication are 6-deoxy-myo-inositol 1,4,5-tris(phosphate), 6-fluoro-6-deoxy-
myo-inositol 1,4,5-tris(phosphate), epi-inositol 1,4,5-tris(phosphate), and 6-amino-6-deoxy-myo-
inositol 1,4,5-tris(phosphate). These derivatives showed poor affinity for the Ins(1,4,5)P3
receptors. The inframolecular acid-base behavior and the cooperative effects between the
phosphate groups could help explain the loss of affinity of these 6-modified analogues.
In tr od u ction
The fact that myo-inositol 1,4,5-tris(phosphate) (Ins-
(1,4,5,)P3, 1) (Chart 1) acts as an intracellular second
messenger is now well-established. The binding of 1 to
its endoplasmic reticulum receptors (ERR) induces the
release of calcium from intracellular stores.1-3
Several research groups have developed structure-
activity relationships (SAR) around this compound,
F igu r e 1. Pharmacophore model proposed by Kozikowski.7
especially toward its binding to the ERR.4-6 Compilation
of the SAR results allowed Kozikowski to propose a
pH variations, a series of equilibria have to be consid-
pharmacophore model showing the crucial role of the
ered. The behavior can be described by the general
vicinal bis(phosphate) in positions 4 and 5, the amplify-
equation associated with the corresponding stepwise
ing effect of the third phosphate in position 1, the slight
equilibrium constants Ky:
importance of the two hydroxyls in positions 2 and 3,
and the major role played by the hydroxyl group in
Ky
(6-y+1)-
(6-y)-
H
+ + Hy-1InsP3
{\} HyInsP3
position 6. Specifically, the hydroxyl group in position
6 seems to be involved in a hydrogen bond as the
hydrogen bond donor (Figure 1).7,8
(6-y)-
[HyInsP3
]
Ky )
In the classical approach, a pharmacophore model is
formed by considering the variation of activty as a direct
consequence of the chemical variation of a given func-
tional group. The activity is indicative of its ability to
reach its counterpart of the binding site. Since inositol
phosphates contain multiple phosphate and hydroxyl
moieties, intramolecular interactions should be consid-
ered. In fact, a chemical modification at a given position
on the inositol ring could induce changes in the other
functional groups in the ring (i.e. by changing charge
distributions, intramolecular hydrogen bond, conforma-
tions, etc.). Thus, the hydroxyl group in position 6 could
be involved in intramolecular interactions as well as in
a probable supramolecular hydrogen bond with the
receptor site. Therefore, the polyfunctionality of inositol
tris(phosphates) (InsP3) led us to investigate some
potentiometric and 31P NMR physicochemical studies
to see if intramolecular interfunctional cooperation could
help in the explanation of the SAR results.
[H+][Hy-1InsP3
]
(6-y+1)-
For polyfunctional compounds such as InsP3, the pro-
tonation-deprotonation process of each phosphate can-
not simply be described using these macroscopic con-
stants or their co-logarithm (pK). One must consider a
more detailed ionization scheme, function by function,
defining, in that manner, microspecies. This approach
will be titled as the inframolecular approach hereafter.
For InsP3, 8 microspecies have to be considered in the
studied pH range. These 8 microspecies are connected
through 12 microequilibria (Figure 2).9 In this case, 31
P
NMR is very useful to resolve such a complex system.
Because the phosphate groups can freely rotate
around the σ bond10 and no conformational changes
have been observed on the cyclohexyl ring as a function
of the pH,11 one can hypothesize that the chemical shift
observed (δiobs) for a given phosphate versus the pH is
essentially related to its ionization state. It should be
noted that for each phosphate, only one acidic proton is
concerned in the pH range of this study (2.5 < pH <
10). It is reasonable to deduce that, at a given pH, δiobs
is a weighted average of the chemical shifts of the
Potentiometric analyses give a global view of the
acid-base behavior of the molecule. According to the
† Faculte´ de Pharmacie.
‡ Universite´ de Sherbrooke.
10.1021/jm991084t CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/23/1999

6-Modified Ins(1,4,5)P3
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23 4825
Ch a r t 1
monoprotonated phosphate (δip) and of the totally
deprotonated form (δid).12 Consequently, by following the
phosphorus chemical shift variations of each phosphate
over a large pH range, it is possible to describe the
behavior, and particularly the ionization state, of the
molecule at an inframolecular level.
Finally, mathematical treatment of data obtained by
potentiometry and 31P NMR titrations by the programs
MICROPOT,13 EQUIPOT,14 SUPERQUAD,15 HYPN-
MR,16 and EASYPLOT17 yield equilibrium constants
(macroscopic and microscopic) for the protonation state
of the molecule.
behavior of the vicinal phosphate in positions 4 and 5.
This effect probably involves the neighboring hydroxyl
groups. This was verified by preparing and analyzing
the 2,3,6-trideoxy derivative 4.19,20 The compound dis-
played a 100-fold lower affinity than the parent com-
pound 1. The 31P NMR clearly showed that the three
phosphates of compound 4 were divided into two inde-
pendent groups (Figure 4). The phosphate in position 1
became an isolated phosphate and showed a classical
monophasic curve similar to that observed for Ins(1)P1,
while the phosphates in positions 4 and 5 showed curves
which mimic those of the Ins(4,5)P2 (3).19
For an inositol monophosphate, such as Ins(1)P1 (2),
the curve of the chemical shift variation versus the pH
looks like a classical titration of an acidic proton.12 For
a vicinal bis(phosphate) such as Ins(4,5)P2 (3), the
curves become more complicated. Each curve concerns
only one acidic proton but appears biphasic. These
aspects demonstrate the cooperative effect, which exists
between the two vicinal phosphates,12 a phenomenon
whose interactivity parameters have been evaluated.18
The situation becomes very complex in the case of Ins-
(1,4,5)P3 (1) (Figure 3). The curves show cooperative
effects between all three phosphates. Particularly, the
phosphate in position 1 seems to be dependent on the
According to Kozikowski’s pharmacophore model, the
hydroxyls in positions 2 and 3 do not seem important
for affinity; thus, we focused on the synthesis, acid-
base properties, and binding evaluation of 6-modified
Ins(1,4,5)P3. In this manuscript we report our studies
on (()-6-deoxy-myo-inositol 1,4,5-tris(phosphate) (5),
(()-6-deoxy-6-fluoro-myo-inositol 1,4,5-tris(phosphate)
(6), (()-epi-inositol 1,4,5-tris(phosphate) (7), and (()-6-
deoxy-6-amino-myo-inositol 1,4,5-tris(phosphate) (8).19
Syn th esis
The physicochemical analyses do not require optically
active compounds; therefore the synthesis were devel-
F igu r e 2. Microspecies and microequilibria for an IP3.

4826 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23
Ballereau et al.
Sch em e 1. Synthesis of 6-Deoxy-myo-inositol
1,4,5-Tris(phosphate) (5)a
F igu r e 3. 31P NMR titration curves for myo-inositol 1,4,5-
tris(phosphate) (1).
a
(a) NaH, CS2, MeI; (b) Bu3SnH, AIBN; (c) H2, 10% Pd/C; (d)
o-C6H4(CH2O)2PN(C2H5)2, 1H-tetrazole; (e) m-CPBA; (f) H2, 10%
Pd/C, H+.
Sch em e 2. Synthesis of 6-Deoxy-6-fluoro-myo-inositol
1,4,5-Tris(phosphate) (6)a
F igu r e 4. 31P NMR titration curves for 2,3,6-trideoxy-myo-
inositol 1,4,5-tris(phosphate) (4).
oped in the racemate series. If the binding property had
justified it, resolution of the racemates would have been
possible, as was done by the preparation of cam-
phanic,21,22 menthoxy acetic,23 or orthoesters24 diaster-
eomers.
a
(a) DAST; (b) H2, 10% Pd/C; (c) o-C6H4(CH2O)2PN(C2H5)2, 1H-
Syn th esis of (()-6-Deoxy-m yo-in ositol 1,4,5-Tr is-
(p h osp h a te) (5). The synthesis of this compound will
give us some information on the influence of the OH in
position 6 on the ionization state of the phosphate
groups and its possible influence on the cooperation
among the phosphates. Its synthesis has been previ-
ously reported starting from benzene25 or from galac-
tose,26,27 while our starting material was myo-inositol
(9) (Scheme 1). The myo-inositol was converted into
1,4,5-tri-O-benzyl-2,3-O-isopropylidene-myo-inositol (10)
using the following conditions. Massy’s procedure28 was
used to form the isopropylidene moiety. Subsequent
treatment with benzyl bromide in the presence of
dibutyltin oxide using the procedure of Gigg and Co.29,30
tetrazole; (d) m-CPBA; (e) H2, 10% Pd/C, H+.
formed the derivative 10. The hydroxyl in position 6 of
compound 10 was removed, using the method proposed
by Barton-McCombie.31 Thus, treatment of the alcohol
10 with sodium hydride followed by carbone disulfide
and finally methyl iodide formed the S-methylxanthate,
which was reduced by tributyltin hydride to yield the
deoxy derivative 11. Neutral hydrogenolysis in the
presence of palladium on charcoal (10%) gave the 1,4,5-
trihydroxy derivative 12, which was phosphorylated by
the phosphite method32 to form the protected tris-
(phosphate) 13. The final product 5 was obtained by
hydrogenolysis using palladium on charcoal (10%), but

6-Modified Ins(1,4,5)P3
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23 4827
Sch em e 3. Synthesis of epi-Inositol 1,4,5-Tris(phosphate) (7)a
a
(a) NaH, PMBCl; (b) THF, MeOH, H2O, TFA; (c) NaH, BnBr; (d) THF, MeOH, HCl; (e) DMSO, (COCl)2, Et3N; (f) EtOH, NaBH4; (g)
NaH, benzyl bromide; (h) DABCO, RhCl(PC6H5)3, then Hg(OAc)2; (i) o-C6H4(CH2O)2PN(C2H5)2, 1H-tetrazole; (j) m-CPBA; (k) H2, 10%
Pd/C.
this time in an acidic medium, leading to the simulta-
neous deprotection of the benzyl-like groups as well as
the isopropylidene moiety. The product 5 was stored as
its cyclohexylammonium salt until used for physico-
chemical or binding investigations.
and the introduction of the fluorine probably act in a
concerted mechanism to form the fluoro derivative 15
with retention of configuration.35 As for the deoxy
derivative 5 the final steps of the synthesis were the
neutral hydrogenolysis of the benzyl ethers to give the
triol 16 followed by their phosphorylation to give
compound 17. Total deprotection yielded the expected
compound 6, stored as its cyclohexylammonium salt.36
Syn t h esis of (()-ep i-In osit ol 1,4,5-Tr is(p h os-
p h a te) (7). The orientation of the hydroxyl group could
influence the ionization state or/and the binding prop-
erty and modify the ability to establish a hydrogen bond.
To probe the influence of the orientation, we prepared
an analogue where the carbon in position 6 was in-
verted. As was done for the two previous molecules, we
started the synthesis with myo-inositol (9), which was
converted into the 2,3-O-cyclohexylidene-1,4,5-tri-O-allyl
derivative 18 using a similar procedure to the one
reported above for compounds 10 and 14 (Scheme 3).
As long as a cyclohexylidene group was maintained
to protect positions 2 and 3 the reactivity of position 6
remained very low.37 For example, it was not possible
to invert the orientation of this OH by a Mitsunobu
Syn th esis of (()-6-Deoxy-6-flu or o-m yo-in ositol
1,4,5-Tr is(p h osp h a te) (6). The isosteric replacement
of the hydroxyl group by a fluorine atom could give some
information about an intramolecular receiving or donat-
ing hydrogen bond. The synthesis of this fluoro deriva-
tive was previously described by Ley and et al.25 in a
synthetic scheme requiring 12 steps. We have developed
a short, 6-step synthesis of this analogue. According to
the same procedure described above for the deoxy deriv-
ative 5, myo-inositol (9) was transformed into 2,3-O-
cyclohexylidene-1,4,5-tri-O-benzyl-myo-inositol (14)28,29,30
(Scheme 2). The use of DAST led to an unusual
retention of the myo configuration giving the fluorinated
derivative 15. Such retentions have been previously
reported33,34 and involved anchimeric assistance of
neighboring groups. In our case, this assistance could
be obtained from the benzyl ethers through the forma-
tion of benzyloxonium. The formation of the oxomium

4828 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23
Ballereau et al.
Sch em e 4. Synthesis of 6-Amino-6-deoxy-myo-inositol 1,4,5-Tris(phosphate) (8)a
a
(a) Triflic anhydride, NaN3; (b) SnCl2, C6H5SH, Et3N; (c) NaOH, C6H5CH2OCOCl; (d) ZnCl2, Pd(P(C6H5)3)4; (e) o-C6H4(CH2O)2PN(C2H5)2,
1H-tetrazole; (f) m-CPBA; (g) H2, 10% Pd/C.
reaction. It was therefore necessary to use an alterna-
tive protecting group. Thus, the last hydroxyl of com-
pound 18 was protected as a p-methoxybenzyl ether, 19.
The cyclohexylidene moiety was subsequently removed
by hydrolysis to form 20. Reprotection of the two
hydroxyls as benzyl ethers yielded the derivative 21.
The free hydroxyl in position 6 was recovered by acidic
hydrolysis to give the alcohol 22. To obtain the inverted
alcohol, it was necessary to proceed through a Swern
oxidation to give the ketone 23. Stereoselective reduc-
tion of this ketone by sodium borohydride38 yielded
mainly the alcohol 24, in the epi configuration. This
hydroxyl was protected as a benzyl ether to obtain the
fully protected derivative 25. The allyl groups were then
removed by successive treatments with tris(triphe-
nylphosphine)rhodium chloride followed by mercuric
acetate to yield the triol 26. The triol was phosphory-
lated (compound 27) and deprotected as seen above to
supply the expected epi-inositol 7.
Syn th esis of (()-6-Deoxy-6-a m in o-m yo-in ositol
1,4,5-Tr is(p h osp h a te) (8). At physiological pH the
6-amino function should be protonated. The presence
of a positive charge between the phosphates in positions
1 and 6 could induce Coulombic interactions. For this
synthesis, we used the same intermediate 24 reported
for the synthesis of the epi-inositol 1,4,5-tris(phosphate)
(7). The free alcohol of compound 24 was activated as a
trifluoromethanesulfonate and then substituted by so-
dium azide to give the azide 28 possessing, again, the
myo configuration accompanied by a significant amount
of the elimination product 29 (Scheme 4). The azido
function was reduced to the primary amine 30 according
to Barta et al. by treatment with stannyl chloride and
thiophenol in the presence of triethylamine.39 The amino
group was protected as a benzyloxycarbamate to give
the totally protected derivative 31. Treatment of the
allyl ethers with tributyltin hydride in the presence of
a catalytic amount of tetrakis(triphenylphosphine)palla-
dium and zinc chloride40,41 yielded the free 1,4,5- triol
32 which was phosphorylated (compound 33) and finally
deprotected to yield the expected compound 8 and con-
verted to the cyclohexylammonium salt as described
above.
Resu lts
Bin d in g P r op er ties. The affinity of the prepared
compounds for the Ins(1,4,5)P3 receptor was measured
by binding on bovine adrenal cortex microsomes.36,42 The
microsomes (1 mg of protein) were incubated in a buffer
solution containing 25 mM Tris/HCl, pH 8.5, 100 mM
KCl, 20 mM NaCl, 5 mM KH2PO4, 1 mM EDTA, and
0.1% bovine serum albumin. Incubations were per-
formed for 60 min at 0 °C, in a final volume of 500 µL,
with [3H]InsP3 (15 000 cpm) and concentrations of
compounds ranging from 0.1 nM to 10 µM. Nonspecific
binding was determined in the presence of 1 µM InsP3.

6-Modified Ins(1,4,5)P3
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23 4829
Ta ble 1. Binding Properties
compd
R1
R2
IC50 (µM)
5
6
7
8
1
4
H
F
H
NH2
OH
H
H
OH
H
H
1.0
2.0
3.0
5.0
14 nM
3.9
Ta ble 2. Stepwise Macroscopic Equilibrium Constants
F igu r e 5. 31P NMR titration curves for 6-deoxy-myo-inositol
1,4,5-tris(phosphate) (5).
log Ky
compd
R1
R2
y ) 1
y ) 2
y ) 3
y ) 4
5
6
7
8
1
H
F
H
NH2
OH
H
H
OH
H
H
8.30
8.76
8.01
10.69
7.85
6.65
6.64
6.39
7.35
6.40
5.53
5.56
5.40
5.58
5.31
4.62
Incubations were terminated by centrifugation at 15000g
for 15 min. The supernatant, which contained the free
ligand, was removed, and the microsomal pellet con-
taining the receptor bound radioactivity was analyzed
by liquid scintillation counting. The experimental re-
sults were analyzed with the KELL program (distrib-
uted by Biosoft, Cambridge, UK) which uses a weighted
nonlinear curve-fitting routine with the Marquardt-
Levenberg modification of the Gauss-Newton tech-
nique.43 The results are reported as IC50 values (con-
centration of compound inhibiting 50% of tracer binding)
in Table 1.
All the modifications made on the position 6 led to
compounds with dramatically lower affinities toward the
ERR. All analogues were generally 2 orders of magni-
tude less active than the parent compound 1.
Acid -Ba se Stu d ies. Acid-base analyses were run
at 37 °C, in a 0.2 M KCl medium which roughly mimics
the temperature and the ionic strength of the cell.
Ma cr oscop ic Con st a n t s. Mathemathical treat-
ments13-15 of the data obtained by potentiometric
analyses led to the stepwise equilibrium constants
reported in Table 2.
F igu r e 6. 31P NMR titration curves for 6-deoxy-6-fluoro myo-
inositol 1,4,5-tris(phosphate) (6).
Ma cr oscop ic Ap p r oa ch . In terms of the macro-
scopic protonation constants K1, the synthesized com-
pounds and the parent compound 1 can be classified
according to their decreasing basicity: 6-fluoro deriva-
tive 6 > 6-deoxy derivative 5 > epi derivative 7 > Ins-
(145)P3 (1). As the positions of the phosphate groups
always remain the same for all compounds, such a
variation involves, a fortiori, the neighboring hydroxyls.
The hydroxyls can contribute in differents ways to
diminish the basicity of the phosphate. They can modify
the solvation shell of the phosphate. They can also form
an intramolecular hydrogen bond with the phosphate
group or proceed by an attractor inducing effect through
the bond between the hydroxyls and the phosphate
groups.10 This last contribution can be discarded due
to the induction effect of the fluorine atom. Because this
effect is stronger than that of the hydroxyl, it should
lead to a decrease in basicity of the fluoro derivative 6
in comparison to the parent compound 1. In fact, the
fluoro derivative 6 is the most basic compound of this
series.
In fr a m olecu la r Resu lts. Figures 5-8 report the
inframolecular 31P NMR titration curves obtained for
the derivatives 5-8, respectively.
Discu ssion
The hydrogen bond donor character and the orienta-
tion of the hydroxyl in this position both seem important
for the affinity. To explain the decrease in affinity for
the ERR by 2 orders of magnitude, it may be necessary
to add an additional factor to the classical formation of
a hydrogen bond between position 6 and the receptor
sites.

4830 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23
Ballereau et al.
from the epimerized hydroxyl to the 6-deoxy derivative
to the fluorinated analogue is due to the reduction of
the local dielectric constant of the medium, as these
modifications diminish the hydrophilicity of the local
zone.
In fr a m olecu la r Ap p r oa ch . The shapes of the in-
framolecular titration curves obtained by 31P NMR are
relatively similar for the deoxy 5, fluoro 6, and epi 7
analogues (Figures 5-7). In all cases, the complex
pattern of the parent compound 1 is notably simplified.
The contribution of intramolecular cooperation looks
reduced, and the aspect of the curves is reduced to the
superimposition of two independent systems. One is
constituted by the phosphate in position 1 which exhib-
its a classical monophasic evolution as observed for an
independent phosphate. The other is constituted by the
two vicinal phosphates in positions 4 and 5 showing a
biphasic behavior comparable to that which we have
observed for a simple vicinal bis(phosphate). Even if
modest, the substituent variations we have made on
position 6 lead to dissociate these two groups. It is
intriguing to observe that this dissociation in the
cooperation effects seems consistent with a dramatic
loss of affinity. If we compare the different analogues,
shifts are also observed for the limit values of the
chemical shift of both the monoprotonated species (δp)
and the totally deprotonated species (δd). These mag-
netic shieldings are due to local fields linked to local
dielectric constants or to local lipophilic factors (such
as those mentioned for the macroscopic approach). A
detailed analysis of these shieldings will be published
in a future manuscript.
F igu r e 7. 31P NMR titration curves for epi-inositol 1,4,5-tris-
(phosphate) (7).
For the aminated derivative 8, the presence of the
amine, an additional protonable function, gives a more
complex problem. In addition to the titration of the
different phosphate groups, the potentiometric analysis
combined with the 31P NMR results permits the recon-
stitution of the titration of the amine in position 6
(Figure 8). It is interesting to note that, even at
relatively high pH values, a partial positive charge
remains on this ammonium. Such a charge could induce
Coulombic interactions with the neighboring phosphate
leading to an unsuitable charge distribution into the
space. In addition, the phosphate may be forced into
conformations inadequate for binding to the receptor
sites. One can also consider that such a partial charge
on the ammonium is incompatible with the correspond-
ing area of the receptor sites.
Even if tenuous, the modifications we made in posi-
tion 6 led to a dramatic loss of affinity and induced large
changes in the behavior of all three phosphate groups.
Particularly, the cooperative effects between the phos-
phate groups were greatly modified. In addition to the
interactions between Ins(1,4,5)P3 (1) and the ERR, it
seems possible that some intramolecular interactions
on Ins(1,4,5)P3 (1) could generate the right conformation
with the right charge distributions for suitable dynamic
interactions with the receptors.
F igu r e 8. 31P NMR titration curves and ammonium-amine
equilibria for 6-amino-6-deoxy-myo-inositol 1,4,5-tris(phos-
phate) (8).
If we take into account the π factor which gives a
measure of the hydrophobicity of the different substit-
uents of position 6, the classification will be as follows:
fluoro derivative 6 (+0.14) > deoxy derivative 5 (0.00)
> hydroxy derivative 1 (-0.67). Consequently, one could
presume that the observed increase in basicity going
More work is in progress to probe the preservation
or the re-establishment of the cooperative effects be-
tween the two groups of phosphates observed for Ins-
(1,4,5)P3 (1), and to examine if the cooperative effects
could be associated with the affinity, or even to the
activity, of this important second messenger.

6-Modified Ins(1,4,5)P3
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23 4831
washed with a 10% aqueous solution of Na2SO3 (2 × 50 mL)
and a saturated aqueous solution of NaHCO3 (2 × 50 mL).
The organic layer was dried over Na2SO4, evaporated to
dryness, and purified by silica gel column chromatography
(CH2Cl2-MeOH, 97:3). The tris(phosphorylated) derivative 13
was obtained as a white powder (133 mg, 70%). Rf ) 0.21 (CH2-
Cl2-MeOH, 97:3). Anal. (C33H37O14P3) C, H. 1H NMR{31P}
(CDCl3): 7.5-7.1 (m, 12H, -(C6H4)3); 5.6-5.0 (m, 12H, -(O-
CH2-C6H4-CH2-O)-3); 4.90 (dt, J ) 11.9, J ) 4.0, 1H, H-1);
4.78 (dd, J ) 9.3, J ) 7.5, 1H, H-4); 4.62 (t, J ) 4.2, 1H, H-2);
4.55 (td, J ) 10.6, J ) 4.2, 1H, H-5); 4.21 (dd, J ) 7.1, J )
4.7, 1H, H-3); 2.76 (dt, J ) 12.4, J ) 4.2, 1H, H-6eq); 2.43 (q,
J ) 11.9, 1H, H-6ax); 1.67 (s, 3H, CH3); 1.46 (s, 3H, CH3). 31P
NMR{1H} (CDCl3): -0.11 (s, 1P); -0.90 (s, 1P); -2.11 (s, 1P).
MS (FAB+) M 750.1: m/z 751.0 [M + H].
(()-6-Deoxy-myo-in ositol 1,4,5-Tr is(ph osph ate) (5). Phos-
photriester 13 (100 mg; 0.13 mmol) dissolved in 16 mL of CH2-
Cl2-MeOH-H2O (1: 2: 1) mixture was hydrogenized (5 atm)
in the presence of 10% Pd/C (0.1 g) and acetic acid (0.5 mL)
for 12 h at 20 °C. Pd/C was filtered and the filtrate was
evaporated to dryness. The crude product was dissolved in 2
mL of bidistilled water then cooled to 0 °C, and cyclohexyl-
amine (1 mL) was added. The reaction mixture was evaporated
to dryness and redissolved in 0.5 mL of bidistilled water and
the cyclohexylammonium salts were precipitated by addition
of 80 mL of acetone. The salts were filtered and precipitated
again by 80 mL of acetone. After filtration 54 mg of (()-6-
deoxy-myo-inositol 1,4,5-tris(phosphate) cyclohexylammonium
(5) were obtained41 (68%). 1H NMR{31P} (D2O): 4.5-4.1 (m,
4H, H-1, H-2, H-4, H-5); 3.78 (dd, J ) 9.2, J ) 4.2, 1H, H-3);
2.39 (dt, J ) 11.9, J ) 4.2, 1H, H-6eq); 2.14 (q, J ) 11.9, 1H,
H-6ax). 31P NMR{1H} (pH ) 11.2) (D2O): 5.45 (s, 1P, P-4); 4.11
(s, 1P, P-1); 4.01 (s, 1P, P-5). MS (FAB-) M 404.1: m/z 403.1
[M - H].
Exp er im en ta l Section
Gen er a l Meth od s. Melting points were measured on a
Mettler PF62 apparatus and are uncorrected. Microanalyses
were performed at the Service Central d’Analyses du CNRS
in Vernaison, France. Mass spectra were recorded at the
Faculte´ de Chimie de Strasbourg, France. NMR spectra were
run on a Bruker Avance DPX 300-MHz spectrometer using
the δ scale with residual CHCl3, 85% H3PO4, and CFCl3 as
references for proton, phosphorus, and fluorine, respectively.
Coupling constants are given in Hz; s, d, t, q, m, and ls means
singlet, doublet, triplet, quadruplet, multiplet, and large
singlet, respectively. THF, ether, and toluene were dried over
sodium; CH2Cl2 was distilled over CaH2; triethylamine and
pyridine were dried over KOH; acetonitrile and benzene were
kept over 3 Å molecular sieves. Column chromatographies
were performed with silca gel 60 (Merck).
(()-1,4,5-Tr i-O-b en zyl-6-d eoxy-2,3-O-isop r op ylid en e-
m yo-in ositol (11). Dry (()-1,4,5-tri-O-benzyl-2,3-O-isopro-
pylidene-myo-inositol (10)28-30,44 (800 mg; 1.63 mmol) was
dissolved in dried THF (8 mL). The reaction mixture was
cooled to 0 °C and sodium hydride (5 equiv; 326 mg; 60%
dispersion; 8.15 mmol) was added. The mixture was warmed
again to room temperature and stirred for 20 min. Carbone
disulfide (15 equiv; 1.50 mL; 24.4 mmol) was slowly added and
then the mixture was refluxed for 1 h. After cooling to room
temperature, methyl iodide (5 equiv; 220 µL; 3.60 mmol) was
added and stirring at room temperature was maintained for
16 h. The mixture was diluted with ethanol (2 mL), water (4
mL), and ethyl acetate (50 mL). The organic layer was washed
with an aqueous saturated NH4Cl solution (50 mL) then brine
(50 mL), dried over Na2SO4, filtered, and evaporated to
dryness. The crude product was dissolved in anhydrous toluene
(15 mL) and Bu3SnH (4 equiv; 1.75 mL; 6.5 mmol) and AIBN
(30 mg) were added. The reaction mixture was refluxed in an
argon atmosphere for 30 min then concentrated and the crude
product was purified by silica gel column chromatography
(ether-hexane, 1:2) giving 630 mg (82%) of compound 11 as a
colorless oil. Rf ) 0.57 (ether-hexane, 1:1). Anal. (C30H34O5)
(()-1,4,5-Tr i-O-ben zyl-2,3-O-cycloh exylid en e-6-d eoxy-
6-flu or o-m yo-in ositol (15). DMAP (2.2 equiv; 269 mg; 2.2
mmol) was added to a solution of (()-1,4,5-tri-O-benzyl-2,3-
O-cyclohexylidene-myo-inositol (14)28,30,45 (550 mg; 1.04 mmol)
in 10 mL of anhydrous toluene. The solution was cooled to -30
°C and DAST (1.9 equiv; 260 µL; 2 mmol) was added. The
reaction mixture was then heated at 60 °C for 3 h. The excess
of DAST was neutralized by adding 10 mL of a saturated
aqueous NaHCO3 solution. The reaction mixture was extracted
with AcOEt. The organic layer was dried over Na2SO4 and
evaporated to dryness. The crude product was purified by silica
gel column chromatography (ether-hexane, 1:2). The fluoro
derivative 15 was obtained (443 mg; 80.2%) as a yellowish oil.
1
C, H. H NMR (CDCl3): 7.5-7.1 (m, 15H, -(C6H5)3); 4.85 (AB,
J AB ) 11.5, ∆δ ) 0.09, 2H, -O-CH2-C6H5); 4.68 (AB, J AB
)
12.3, ∆δ ) 0.05, 2H, -O-CH2-C6H5); 4.87 (AB, J AB ) 11.7,
∆δ ) 0.02, 2H, -O-CH2-C6H5); 4.35 (t, J ) 4.0, 1H, H-2);
4.07 (dd, J ) 6.8, J ) 5.2, 1H, H-3); 3.66 (dd, J ) 9.3, J ) 6.8,
1H, H-4); 3.61 (dt, J ) 12.1, J ) 4.0, 1H, H-1); 3.33 (ddd, J )
11.5, J ) 9.3, J ) 4.0, 1H, H-5); 2.21 (dt, J ) 12.2, J ) 3.8,
1H, H-6eq); 1.96 (q, J ) 12.1, 1H, H-6ax); 1.54 (s, 3H, CH3);
1.42 (s, 3H, CH3).
1
Rf ) 0.67 (ether-hexane, 1:2). Anal. (C33H37FO5) C, H, F. H
(()-6-Deoxy-2,3-O-isopr opyliden e-myo-in ositol (12). The
tribenzylated derivative 11 (100 mg; 0.22 mmol) was dissolved
in 15 mL of ether-EtOH-H2O (2:2:1) mixture. 10% Pd/C (50
mg) was added to the reaction mixture which was hydrog-
enized (5 atm) for 16 h. The reaction mixture was filtered and
evaporated to dryness. Pure triol 12 was obtained as an oil
(44 mg, 98%). Rf ) 0.38 (CH2Cl2-MeOH, 9:1). Anal. (C9H16O5)
C, H. 1H NMR (DMSO-d6): 4.81 (d, J ) 4.2, 1H, exchangeable,
OH-1); 4.80 (d, J ) 6.0, 1H, exchangeable, OH-4); 4.60 (d, J )
4.2, 1H, exchangeable, OH-5); 4.10 (t, J ) 4.0, 1H, H-2); 3.9-
3.7 (m, 2H, containing at 4.72 (dd, J ) 6.9, J ) 5.1, 1H, H-3)
and H-1); 3.3-3.0 (m, 2H, H-4 and H-5 giving after exchange
with D2O at 3.19 (dd, J ) 9.5, J ) 6.9, 1H, H-4) and at 3.12
(td, J ) 9.9, J ) 3.8, 1H, H-5)); 1.72 (dt, J ) 11.9 and J ) 4.2,
1H, H-6eq); 1.55 (q, J ) 11.7, 1H, H-6ax); 1.42 (s, 3H, CH3);
1.26 (s, 3H, CH3).
(()-6-Deoxy-2,3-O-isop r op ylid en e-m yo-in osit ol 1,4,5-
Tr i-O-(or th oxylylen e)p h osp h a te (13). Dry triol 12 (50 mg;
0.25 mmol) and 1H-tetrazole (12 equiv; 210 mg; 3.0 mmol) were
dissolved in 5 mL of anhydrous THF. The solution was cooled
at -78 °C and N,N-diethyl O-xylylene phosphoramidite (6
equiv; 360 mg; 1.5 mmol) was added. After warming up again
to room temperature for 8 h the reaction mixture was cooled
again at -78 °C, and mCPBA (12 equiv; 740 mg; 3.0 mmol)
dissolved in 4 mL of CH2Cl2 was added. After warming up
again to room temperature and stirring at this temperature
for 30 min, CH2Cl2 (30 mL) was added and the mixture was
NMR (CDCl3-C6D6, 1:3): 7.5-7.2 (m, 15H, -(CH2C6H5)3); 5.00
(dt, J HF ) 51.2, J ) 8.1, 1H, H-6); 4.9-4.6 (m, 6H, -(CH2C6H5)3);
4.18 (dt, J ) 5.6, J ) J H2F ) 4.0, 1H, H-2); 3.97 (t, J ) 6.2,
1H, H-3); 3.77 (dd, J ) 8.6, J ) 6.4, 1H, H-4); 3.62 (ddd, J HFcis
) 11.3, J ) 8.7, J ) 3.8, 1H, H-1); 3.46 (dt, J HFcis ) 16.2, J )
8.1, 1H, H-5); 1.8-0.7 (m, 10H, C6H10). 19F NMR (CDCl3):
-195.7 (dddd, J HF ) 51.2, J HFcis ) 16.2, J HFcis ) 11.3, J H2F
4.0, 1F, F-6).
)
(()-2,3-O-Cycloh exylid en e-6-d eoxy-6-flu or o-m yo-in osi-
t ol (16). (()-1,4,5-Tri-O-benzyl-2,3-O-cyclohexylidene-6-
deoxy-6-fluoro-myo-inositol (15) (240 mg; 0.45 mmol) was
dissolved in 20 mL of ether-EtOH (1:3) mixture. 10% Pd/C
(100 mg) was added to the reaction mixture which was
hydrogenized (5 atm) for 16 h. The reaction mixture was
filtered and evaporated to dryness. Triol 16 (115 mg; 97%) was
1
obtained as an oil. Rf ) 0.25 (AcOEt). H NMR (D2O-(CD3)2-
CO, 1:1): 4.40 (dt, J ) 8.8, J HF ) 52.6, 1H, H-6); 4.33 (dd, J )
8.8, J ) 4.4, 1H); 4.0-3.8 (2H); 3.6-3.3 (m, 2H); 1.7-1.2 (m,
10H, C6H10). 19F -NMR (D2O): -194.8 (ddd, J HF ) 55.5, J HFcis
) 15.0, J HFcis ) 4.5, 1F, F-6).
(()-2,3-O-Cycloh exylid en e-6-d eoxy-6-flu or o-m yo-in osi-
tol 1,4,5-Tr i-O-(or th oxylylen e)p h osp h a te (17). (()-2,3-O-
Cyclohexylidene-6-deoxy-6-fluoro-myo-inositol (16) (80 mg;
0.31 mmol) and 1H-tetrazole (10 equiv; 215 mg; 3.05 mmol)
were dissolved in 10 mL of anhydrous THF. The solution was
cooled to -78 °C and N,N-diethyl O-xylylene phosphoramidite

4832 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23
Ballereau et al.
(5 equiv; 365 mg; 1.53 mmol) was added. After warming up to
room temperature the mixture was stirred for 8 h and then
cooled again to -78 °C and mCPBA (3.3 equiv; 500 mg; 2
mmol) dissolved in 8 mL of CH2Cl2 was added. The reaction
mixture was then kept at room temperature for 30 min. After
evaporation to dryness, the residue was redissolved in 30 mL
of CH2Cl2 and washed with a 10% Na2SO3 aqueous solution
(2 × 50 mL) then with a 5% NaHCO3 aqueous solution (2 ×
50 mL). The organic layer was dried over Na2SO4, evaporated
to dryness, and chromatographed on a silica gel column (AcOEt
+ 0.1% Et3N). The tris(phosphorylated) compound 17 (160 mg,
65%) was obtained as a white powder. Rf ) 0.25 (AcOEt +
0.1% Et3N). 1H NMR (CDCl3): 7.5-7.2 (m, 12H, (-CH2-
C6H4-CH2-)3); 5.2-4.6 (m, 13H, (-CH2-C6H4-CH2-)3 and
H-6); 4.35 (dd, J ) 6.4, J ) 3.7, 1H, H-2); 4.16 (t, J ) 6.2, 1H,
2:1) gave 2.64 g of diol 20 (84%). Rf ) 0.17 (ether-hexane,
1
2:1). Anal. (C23H32O7) C, H. H NMR (CDCl3): 7.30 and 6.88
(2d, J ) 8.6, 4H, -C6H4-OCH3); 6.1-5.8 (m, 3H, -(O-CH2-
CHdCH2)3); 5.4-4.9 (m, 6H, -(O-CH2-CHdCH2)3); 4.74 (AB,
J AB ) 10.2, ∆δ ) 0.04, 2H, -O-CH2-C6H4-OCH3); 4.5-3.9
(m, 7H, H-2 and -(O-CH2-CHdCH2)3); 3.81 (t, J ) 8.6, 1H,
H-6); 3.80 (s, 3H, -OCH3); 3.65 (t, J ) 9.5, 1H, H-4); 3.42 (ddd,
J ) 9.5, J H3OH ) 4.2, J ) 3.1, 1H, H-3); 3.30 (dd, J ) 9.5, J )
2.8, 1H, H-1); 3.24 (t, J ) 9.4, 1H, H-5); 2.78 (d, J ) 4.2, 1H,
exchangeable, OH-3); 2.77 (s, 1H, exchangeable, OH-2).
(()-1,4,5-Tr i-O-a llyl-2,3-di-O-ben zyl-6-O-p-m eth oxyben -
zyl-m yo-in ositol (21). (()-1,4,5-Tri-O-allyl-6-O-p-methoxy-
benzyl-myo-inositol (20) (2.45 g; 5.83 mmol) was dissolved in
anhydrous DMF (40 mL). The solution was placed at 0 °C and
sodium hydride (6 equiv; 1.68 g; 60% dispersion; 35.0 mmol)
and benzyl bromide (5 equiv; 3.5 mL; 30 mmol) were added.
The reaction mixture was warmed again and left at room
temperature overnight. The mixture was poured in water and
extracted with AcOEt (2 × 40 mL). The organic layer was
washed with water (3 × 25 mL), dried over Na2SO4, filtered,
and evaporated. The crude product was chromatographed on
a silica gel column (ether-hexane, 1:4). Compound 21 was
obtained in 96% yield (3.35 g) Rf ) 0.58 (ether-hexane, 1:1).
Anal. (C37H44O7) C, H. 1H NMR (C6D6): 7.6-6.8 (m, 14H,
containing at 7.32 and 6.88 (2d, J ) 8.6, 4H, -C6H4-OCH3)
and -(C6H5)2); 6.2-5.8 (m, 3H, -(O-CH2-CHdCH2)3); 5.5-5.1
(m, 6H, -(O-CH2-CHdCH2)3); 4.96 (s, 2H, -O-CH2-C6H5);
4.90 (AB, J AB ) 10.6, ∆δ ) 0.04, 2H, -O-CH2-C6H4-OCH3);
4.7-4.3 (m, 6H, containing at 4.59 (AB, J AB ) 11.9, ∆δ ) 0.10,
2H, -O-CH2-C6H5) and -(O-CH2-CHdCH2)2); 4.2-3.9 (m,
5H, containing at 4.14 (t, J ) 9.5, 1H, H-6), at 4.10 (t, J ) 9.7,
1H, H-4), at 3.98 (s, 1H, H-2) and -O-CH2-CHdCH2); 3.49
(s, 3H, -OCH3); 3.37 (t, J ) 9.3, 1H, H-5); 3.23 (dd, J ) 9.7,
J ) 1.8, 1H, H-3); 3.15 (dd, J ) 9.7, J ) 2,0. 1H, H-1).
(()-1,4,5-Tr i-O-a llyl-2,3-d i-O-ben zyl-m yo-in ositol (22).
(()-1,4,5-Tri-O-allyl-2,3-di-O-benzyl-6-O-p-methoxybenzyl-
myo-inositol (21) (3.25 g; 5.41 mmol) was dissolved in THF
(30 mL) and methanol (100 mL). 3 N HCl (50 mL) was added
and the mixture was refluxed for 2.5 h. The solvents were
evaporated and the crude product was purified by silica gel
column chromatography (ether-hexane, 1:1). (()-1,4,5-Tri-
O-allyl-2,3-di-O-benzyl-myo-inositol (22) was obtained in
96% yield (2.5 g). Rf ) 0.41 (ether-hexane, 1:1). Anal.
(C29H36O6) C, H. 1H NMR (CDCl3): 7.5-7.1 (m, 10H, -(C6H5)2);
6.2-5.8 (m, 3H, -(O-CH2-CHdCH2)3); 5.5-5.1 (m, 6H, -(O-
CH2-CHdCH2)3); 4.84 (AB, J AB ) 12.1, ∆δ ) 0.04, 2H, -O-
CH2-C6H5); 4.68 (AB, J AB ) 11.9, ∆δ ) 0.08, 2H, -O-CH2-
C6H5); 4.5-4.3 (m, 4H, -(O-CH2-CHdCH2)2); 4.2-3.9 (m, 4H,
H-6, H-2 and -O-CH2-CHdCH2); 3.88 (t, J ) 9.5, 1H, H-4);
3.33 (dd, J ) 9.9, J ) 2.2, 1H, H-3); 3.22 (t, J ) 9.2, 1H, H-5);
3.11 (dd, J ) 9.9, J ) 2.0, 1H, H-1); 2.60 (s, 1H, exchangeable,
OH-6).
H-4); 3.9-3.7 (m, 2H, H-1 and H-3) 3.57 (dt, J ) 8.8, J HFcis
)
8.0, 1H, H-5); 1.8-1.3 (m, 1H, C6H10). 19F NMR{1H} (CDCl3):
-196.0 (s, 1F, F-6). 31P NMR{1H} (CDCl3): -1.00 (s, 1P); -1.32
(s, 1P); -1.84 (s, 1P). MS (FAB+) M 808: m/z 809 [M + H].
(()-6-Deoxy-6-flu or o-myo-in ositol 1,4,5-Tr is(ph osph ate)
(6). (()-2,3-O-Cyclohexylidene-6-deoxy-6-fluoro-myo-inositol
1,4,5-tri-O-(orthoxylylene)phosphate (17) (50 mg; 0.062 mmol)
was dissolved in a EtOH-H2O-CH2Cl2 (1:1:1) mixture (25
mL). 10% Pd/C (100 mg) and AcOH (0.5 mL) were added and
the reaction mixture was hydrogenolized (5 atm H2) for 16 h
at room temperature. After filtration and evaporation to
dryness the crude product was dissolved in 5 mL of bidistilled
water and cooled to 0 °C; cyclohexylamine (1 mL) was added.
The reaction mixture was evaporated to dryness and redis-
solved in 1 mL of bidistilled water. The cyclohexylammonium
salts were precipitated by addition of 80 mL of acetone. After
filtration the salts were again dissolved in 1 mL of bidistilled
water and precipitated again by 80 mL of acetone. After
filtration 43 mg of cyclohexylammonium salts of (()-6-deoxy-
6-fluoro-myo-inositol 1,4,5-tris(phosphate) (6) were obtained.
1H NMR (D2O, pH ) 11.0, 37 °C): 4.72 (dt, J ) 8.4, J HF
)
53.5, 1H, H-6) 4.3-4.4 (m, 1H, H-2); 4.3-4.2 (m, 3H, H-1, H-4
and H-5); 3.83 (dd, J ) 9.2, J ) 2.6, 1H, H-3). 19F NMR (D2O,
pH ) 11.0): -194.4 (d, J HF ) 53.5, F-6). 31P NMR (D2O, pH )
11.0, 37 °C): 5.48 (d, J HP ) 6.1, P-4); 4.11 (d, J HP ) 8.5, P-1);
3.96 (d, J HP ) 7.9, P-5).
(()-1,4,5-Tr i-O-a llyl-2,3-O-cycloh exylid en e-6-O-p-m eth -
oxyben zyl-m yo-in ositol (19). (()-1,4,5-Tri-O-allyl-2,3-O-
cyclohexylidene-myo-inositol (18)28-30 (2.88 g; 7.57 mmol) was
dissolved in anhydrous DMF (40 mL). The solution was cooled
to 0 °C and sodium hydride (2.5 equiv; 756 mg; 60% dispersion,
19 mmol) and p-methoxybenzyl chloride (2 equiv; 2.05 mL; 15
mmol) were added. The reaction mixture was warmed again
to room temperature and stirred overnight. The mixture was
poured in cooled water and extracted with ethyl acetate (2 ×
40 mL). The organic layer was washed with water (3 × 25 mL),
dried over Na2SO4, filtered, and evaporated. The crude product
was purified by silica gel column chromatography (ether-
hexane, 1:4). p-Methoxybenzyl derivative 19 was obtained in
quantitative yield (3.8 g). Rf ) 0.59 (ether-hexane, 1:1). Anal.
(C29H40O7) C, H. 1H NMR (CDCl3): 7.32 and 6.89 (2d, J ) 8.6,
4H, -C6H4-OCH3); 6.1-5.9 (m, 3H, -(O-CH2-CHdCH2)3);
5.4-5.0 (m, 6H, -(O-CH2-CHdCH2)3); 4.74 (AB, J AB ) 10.1,
∆δ ) 0.03, 2H, -O-CH2-C6H4-OCH3); 4.4-4.1 (m, 7H,
containing at 4.38 (t, J ) 4.7, 1H, H-2) and -(O-CH2-CHd
CH2)3); 4.03 (dd, J ) 6.9, J ) 5.5, 1H, H-3); 3.82 (s, 3H,
-OCH3); 3.81 (t, J ) 8.6, 1H, H-6); 3.62 (dd, J ) 9.7, J ) 7.1,
1H, H-4); 3.57 (dd, J ) 8.8, J ) 3.8, 1H, H-1); 3.22 (dd, J )
9.5, J ) 9.0, 1H, H-5); 1.9-1.1 (m, 10H, C6H10).
(()-1,4,5-Tr i-O-a llyl-6-O-p -m et h oxyb en zyl-m yo-in osi-
tol (20). (()-1,4,5-Tri-O-allyl-2,3-O-cyclohexylidene-6-O-p-
methoxybenzyl-myo-inositol (19) (3.87 g; 7.57 mmol) was
dissolved in THF (2.5 mL) and EtOH (95%; 25 mL). Water (5
mL) and TFA (1.5 mL) were added and the mixture was
refluxed for 2 h. The solution was evaporated to dryness and
the crude product was redissolved in H2O-AcOEt. The aque-
ous phase was reextracted with AcOEt. The organic layer was
washed with water, dried over Na2SO4, filtered, and evapo-
rated. Column chromatography on silica gel (ether-hexane,
(()-1,4,5-Tr i-O-a llyl-2,3-d i-O-ben zyl-6-m yo-in osose (23).
DMSO (6.3 equiv; 1.4 mL; 20 mmol) was dissolved in CH2Cl2
(10 mL) and the solution was cooled to - 78 °C. Oxalyl chloride
(5 equiv; 1.4 mL; 16 mmol) was slowly added. The mixture
was kept at room temperature for 10 min and then cooled to
-78 °C. The alcohol 22 (1.5 g; 3.12 mmol) dissolved in CH2Cl2
(2 × 5 mL) was slowly added. After 40 min, triethylamine (20
equiv; 8.7 mL; 62.4 mmol) was added to the mixture which
was warmed again to room temperature for 20 min. The
mixture was diluted with CH2Cl2 (40 mL) and washed with
water (2 × 50 mL) and brine (2 × 50 mL). The organic layer
was dried over Na2SO4, filtered, and evaporated. The crude
product 23 (2.1 g) was used as such for the next step. An
aliquot was purified by chromatography on a silica gel column
1
(ether-hexane, 1:2). Rf ) 0.46 (ether-hexane, 1:1). H NMR
(CDCl3): 7.5-7.2 (m, 10H, -(C6H5)2); 6.1-5.8 (m, 3H, -(O-
CH2-CHdCH2)3); 5.4-5.1 (m, 6H, -(O-CH2-CHdCH2)3); 4.83
(AB, J AB ) 12.1, ∆δ ) 0.07, 2H, -O-CH2-C6H5); 4.67 (AB,
J AB ) 11.9, ∆δ ) 0.05, 2H, -O-CH2-C6H5); 4.5-4.2 (m, 4H,
-(O-CH2-CHdCH2)2); 4.2-3.8 (m, 6H, containing at 4.14 (t,
J ) 2.2, 1H. H-2), at 4.03 (t, J ) 9.3, 1H, H-4), at 3.88 (d, J )

6-Modified Ins(1,4,5)P3
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23 4833
1.6, 1H, H-1), at 3.90 (d, J ) 9.6, 1H, H-5) and -O-CH2-
CHdCH2); 3.72 (dd, J ) 9.3, J ) 2.2, 1H, H-3).
tetrazole (9 equiv; 168 mg; 2.39 mmol) were dissolved in 8 mL
of anhydrous THF. The solution was cooled to -78 °C and N,N-
diethyl O-xylylene phosphoramidite (6 equiv; 382 mg; 1.6
mmol) was added. The mixture was warmed again to room
temperature. Stirring was maintained for 8 h. The reaction
mixture was cooled again to -78 °C, and mCPBA (10 equiv;
657 mg; 2.7 mmol) dissolved in CH2Cl2 (4 mL) was added. After
warming up and stirring at room temperature for 30 min, the
mixture was evaporated to dryness, redissolved in 30 mL of
CH2Cl2, and washed with a 10% Na2SO3 aqueous solution (2
× 50 mL) and a NaHCO3 saturated aqueous solution (2 × 50
mL). The organic layer was dried over Na2SO4 and evaporated
to dryness, and the crude residue was chromatographed on a
silica gel column (CH2Cl2-MeOH, 97:3). The tris(phosphoryl-
ated) compound 27 was obtained as a white powder (238 mg;
88%). Rf ) 0.44 (CH2Cl2-MeOH, 95:5). Anal. (C51H51O15P3) C,
H, P. 1H NMR{31P} (DMSO-d6): 7.6-7.0 (m, 27H, -(C6H4)-3
and -(C6H5)3); 5.6-4.6 (m, 21H, -(O-CH2-C6H4-CH2-O)-3,
-(O-CH2-C6H5)3 and 3H cycle); 4.6-4.2 (m, 2H cycle); 4.1-
3.9 (m, 1H cycle). 31P NMR{1H} (DMSO-d6): -0.16 (s, 1P);
-2.45 (s, 1P); -3.24 (s, 1P). MS (FAB+) M 996.2: m/z 997.1
[M + H].
(()-1,4,5-Tr i-O-allyl-2,3-di-O-ben zyl-epi-in ositol (24). The
crude 23 obtained above (2.1 g) was dissolved in absolute
ethanol (50 mL) and the temperature was adjusted to 0 °C.
NaBH4 (5 equiv; 590 mg; 15.6 mmol) was added and the
mixture was stirred at room temperature for 1 h. Water (30
mL) was added and the solvents were evaporated. The residue
was redissolved in AcOEt (100 mL). The organic layer was
dried over Na2SO4, filtered, and evaporated to dryness.The
crude product was chromatographed on a silica gel column
(ether-hexane, 1:2). (()-1,4,5-Tri-O-allyl-2,3-di-O-benzyl-epi-
inositol (24) was obtained (1.09 g, 73% from 22). Rf ) 0.34
1
(ether-hexane, 1:1). Anal. (C29H36O6) C, H. H NMR (CDCl3):
7.5-7.2 (m, 10H, -(C6H5)2); 6.2-5.8 (m, 3H, -(O-CH2-CHd
CH2)3); 5.4-5.1 (m, 6H, -(O-CH2-CHdCH2)3); 4.86 (AB, J AB
) 11.2, ∆δ ) 0.05, 2H, -O-CH2-C6H5); 4.70 (AB, J AB ) 11.9,
∆δ ) 0.12, 2H, -O-CH2-C6H5); 4.5-4.3 (m, 2H, -(O-CH2-
CHdCH2)2); 4.3-3.9 (m, 8H, containing at 4.04 (t, J ) 9.7,
1H, H-4), at 4.02 (s, 1H, exchangeable, OH-6), H-6, H-2 and
-(O-CH2-CHdCH2)2); 3.30 (dd, J ) 9.7, J ) 2.8, 1H, H-3);
3.20 (t, J ) 2.8, 1H, H-1); 3.16 (dd, J ) 9.7, J ) 3.1, 1H, H-5).
Alcohol 22 (68 mg) with the myo configuration was also
obtained.
(()-epi-In ositol 1,4,5-Tr is(p h osp h a te) (7). The phospho-
triester 27 (238 mg; 0.24 mmol) dissolved in a CH2Cl2-
MeOH-H2O (1: 2: 1) mixture (6 mL) was hydrogenized in
the presence of 10% Pd/C (0.2 g) under a hydrogen atmosphere
(5 atm) for 12 h at 20 °C. Pd/C was filtered over Celite and
the filtrate was evaporated to dryness. The crude product was
dissolved in 2 mL of bidistilled water and cooled at 0 °C.
Cyclohexylamine (1 mL) was added. The reaction mixture was
evaporated to dryness and redissolved in 0.5 mL of bidistilled
water. The cyclohexylammonium salts were precipitated by
adding 80 mL of acetone. The salts were filtered and precipi-
tated again in 80 mL of acetone. After filtration 178 mg (81%)
of (()-epi-inositol 1,4,5-tris(phosphate) (7) cyclohexylammo-
(()-1,4,5-Tr i-O-a llyl-2,3,6-tr i-O-ben zyl-epi-in ositol (25).
(()-1,4,5-Tri-O-allyl-2,3-di-O-benzyl-epi-inositol (24) (250 mg;
0.52 mmol) was dissolved in anhydrous DMF (4 mL). The
solution was cooled to 0 °C and sodium hydride (1.3 mmol;
2.5 equiv; 52 mg; 60% dispersion) was added. Benzyl bromide
(1.04 mmol; 2 equiv; 124 µL) was also added. The mixture was
warmed again overnight to room temperature. The mixture
was poured in water and extracted with AcOEt (2 × 20 mL).
The organic layer was washed with water (3 × 20 mL), dried
over Na2SO4, filtered, and evaporated. The crude product was
purified on a silica gel column (ether-hexane, 1:2). The
benzylated compound 25 was obtained in 92% yield (274 mg).
Rf ) 0.54 (ether-hexane, 1:1). Anal. (C36H42O6) C, H. 1H NMR
(CDCl3): 7.5-7.2 (m, 15H, -(C6H5)3); 6.2-5.8 (m, 3H, -(O-
CH2-CHdCH2)3); 5.4-5.1 (m, 6H, -(O-CH2-CHdCH2)3); 4.93
(AB, J AB ) 12.2, ∆δ ) 0.06, 2H, -O-CH2-C6H5); 4.92 (AB,
1
nium salt were obtained H NMR{31P} (D2O, pH ) 11.3): 4.53
(t, J ) 8.7, 1H, H-4); 4.44 (s, 1H, H-6); 4.31 (s, 1H, H-2); 4.20
(s, 1H, H-1); 4.16 (t, J ) 9.3, 1H, H-5); 3.76 (dd, J ) 9.3, J )
2.4, 1H, H-3). 31P NMR{1H} (D2O, pH ) 11.3): 5.82 (s, 1P,
P-4); 4.47 (s, 1P, P-1); 4.08 (s, 1P, P-5). MS (FAB-) M 420.1:
m/z 419.1 [M - H, 100%], 509.2 [M - H + C7H7, 60%].
J AB ) 12.1, ∆δ ) 0.06, 2H, -O-CH2-C6H5); 4.69 (AB, J AB
)
12.1, ∆δ ) 0.06, 2H, -O-CH2-C6H5); 4.40 (dt, J ) 5.7, J )
2.9, 2H, -O-CH2-CHdCH2); 4.3-4.0 (m, 5H, containing at
4.22 (t, J ) 9.7, 1H, H-4), at 4.12 (t, J ) 2.6, 1H, H-2), H-6
and -O-CH2-CHdCH2); 3.93 (dt, J ) 5.1, 4J ) 2.9, 2H, -O-
CH2-CHdCH2); 3.27 (dd, J ) 9.7, J ) 2.9, 1H, H-3); 3.19 (dd,
J ) 9.9, J ) 3.1, 1H, H-5); 3.14 (t, J ) 2.7, 1H, H-1).
(()-1,4,5-Tr i-O-a llyl-6-a zid o-2,3-d i-O-b en zyl-6-d eoxy-
m yo-in ositol (28). (()-1,4,5-Tri-O-allyl-2,3-di-O-benzyl-epi-
inositol (24) (500 mg; 1.04 mmol) was dissolved in CH2Cl2 (5
mL) and the solution was cooled to 0 °C. Pyridine (0.3 mL)
and triflic anhydride (2.2 equiv; 630 µL; 2.3 mmol) were slowly
added. After 2 h at 0 °C, the mixture was diluted with CH2Cl2
(20 mL) and washed with H2O at 0 °C (20 mL). The organic
layer was separated, dried over Na2SO4, filtered, and evapo-
rated to dryness. The crude product was dissolved in DMF (6
mL) and at 0 °C, NaN3 (5 equiv; 340 mg; 5.20 mmol) was
added. After warming up again to room temperature the
mixture was stirred overnight, poured in water, and extracted
with AcOEt (2 × 20 mL). The organic layer was washed with
water, dried over Na2SO4, filtered, and evaporated. The crude
product was purified by silica gel column chromatography
(ether-hexane, 1:4). The expected compound 28 was obtained
with 59% yield (309 mg). Rf ) 0.41 (ether-hexane, 1:4). 1H
NMR (CDCl3): 7.5-7.2 (m, 10H, -(C6H5)2); 6.1-5.8 (m, 3H,
-(O-CH2-CHdCH2)3); 5.4-5.1 (m, 6H, -(O-CH2-CHdCH2)3);
4.85 (s, 2H, -O-CH2-C6H5); 4.64 (AB, J AB ) 11.9, ∆δ ) 0.08,
2H, -O-CH2-C6H5); 4.5-4.2 (m, 4H, -(O-CH2-CHdCH2)2);
4.1-4.0 (m, 2H, -O-CH2-CHdCH2); 3.98 (t, J ) 2.0, 1H,
H-2); 3.90 (t, J ) 10.0, 1H, H-6); 3.88 (t, J ) 9.3, 1H, H-4);
3.22 (dd, J ) 9.9, J ) 2.0, 1H, H-3); 3.08 (t, J ) 9.5, 1H, H-5);
3.03 (dd, J ) 10.0, J ) 2.0, 1H, H-1). IR (CHCl3): 2111.3 cm-1
(N3).
(()-2,3,6-Tr i-O-ben zyl-epi-in ositol (26). (()-1,4,5-Tri-O-
allyl-2,3,6-tri-O-benzyl-epi-inositol (25) (713 mg; 1.25 mmol)
and DABCO (0.6 equiv; 277 mg; 0.75 mmol) were dissolved in
ethanol-water (9:1, 22 mL). RhCl(PPh3)3 (0.3 equiv; 280 mg;
0.30 mmol) was added and the mixture was refluxed for 12 h.
The reaction mixture was poured in water (100 mL) and
extracted with AcOEt (3 × 60 mL). The organic layers were
dried over Na2SO4, filtered, and evaporated. The crude product
was dissolved in a mixture of THF (4.5 mL) and water (3 mL).
Under vigorous stirring a solution of mercuric acetate(II) (3.7
equiv; 1.46 g; 4.6 mmol) in water (3.5 mL) was rapidly added
to the mixture. After 20 min, the mixture was diluted with
AcOEt (40 mL). The organic layer was separated, dried over
Na2SO4, filtered, and evaporated to dryness. Pure triol 26 was
obtained by chromatography on a silica gel column (ether-
hexane, 2:1) (460 mg, 80%). Rf ) 0.48 (AcOEt). Anal. (C27H30O6)
1
C, H. H NMR (CDCl3): 7.5-7.2 (m, 15H, -(C6H5)3); 4.94 (AB,
J AB ) 11.5, ∆δ ) 0.47, 2H, -O-CH2-C6H5); 4.83 (AB, J AB
)
11.9, ∆δ ) 0.21, 2H, -O-CH2-C6H5); 4.76 (AB, J AB ) 11.9,
∆δ ) 0.10, 2H, -O-CH2-C6H5); 4.26 (t, J ) 9.3, 1H, H-4);
4.03 (t, J ) 3.1, 1H, H-2); 3.95 (t, J ) 3.5, 1H, H-6); 3.72 (dt,
Elimination product 29 (130 mg, 27%) was also obtained.
1
J H1OH ) 10.8, J ) 3.7, 1H, H-1); 3.56 (ddd, J ) 9.3, J H5OH
)
Rf ) 0.24 (ether-hexane, 1:4). H NMR (CDCl3): 7.5-7.2 (m,
6.4, J ) 3.7, 1H, H-5); 3.33 (dd, J ) 9.1, J ) 2.8, 1H, H-3);
3.02 (d, J ) 10.8, 1H, exchangeable, OH-1); 2.63 (d, J ) 6.4,
1H, exchangeable, OH-5); 2.47 (s, 1H, exchangeable, OH-4).
10H, -(C6H5)2); 6.2-5.9 (m, 3H, -(O-CH2-CHdCH2)3); 5.4-
5.2 (m, 6H, -(O-CH2-CHdCH2)3); 4.85 (AB, J AB ) 12.3, ∆δ )
0.02, 2H, -O-CH2-C6H5); 4.8-4.6 (m, 3H, containing at 4.71
(AB, J AB ) 11.9, ∆δ ) 0.05, 2H, -O-CH2-C6H5) and H-6);
4.44 (AB part of an ABMX2, J AB ) 12.5, J AM ) J BM ) 5.7, J AX
(()-2,3,6-Tr i-O-ben zyl-epi-in ositol 1,4,5-Tr i-O-(o-xylyl-
en e)p h osp h a te (27). Triol 26 (120 mg; 0.27 mmol) and 1H-

4834 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23
Ballereau et al.
) J BX ) 1.3, ∆δ ) 0.12, 2H, -O-CH2-CHdCH2); 4.3-4.0 (m,
7H, containing at 4.12 (dd, J ) 6.8, J ) 3.3, 1H, H-5), at 4.09
(dd, J ) 3.5, J ) 1.1, 1H, H-2), at 4.09 (dd, J ) 9.5, J ) 6.9,
1H, H-4) and -(O-CH2-CHdCH2)2); 3.53 (dd, J ) 9.9, J )
3.5, 1H, H-3).
(()-6-Am in o-2,3-d i-O-ben zyl-6-N-ben zyloxyca r bon yl-6-
deoxy-myo-in ositol 1,4,5-Tr i-O-(o-xylylen e)ph osph ate (33).
(()-6-Amino-2,3-di-O-benzyl-6-N-benzyloxycarbonyl-6-deoxy-
myo-inositol (32) (158 mg; 0.32 mmol) and 1H-tetrazole (9
equiv; 210 mg; 3.00 mmol) were dissolved in 15 mL of
anhydrous THF. The solution was cooled to -78 °C and N,N-
diethyl O-xylylene phosphoramidite (6 equiv; 475 mg; 2 mmol)
was added. After warming up the solution was stirred at room
temperature for 8 h. The reaction mixture was cooled again
to -78 °C, and mCPBA (10 equiv; 816 mg; 3.3 mmol) dissolved
in 5 mL of CH2Cl2 was added. The reaction mixture was then
kept at room temperature for 30 min. The solvents were
removed by evaporation and the residue was dissolved in 30
mL of CH2Cl2 and washed with 10% Na2SO3 aqueous solution
(2 × 50 mL) and saturated NaHCO3 aqueous solution (2 × 50
mL). The organic layer was dried over Na2SO4 and evaporated
to dryness, and the residue was chromatographed on a silica
gel column (CH2Cl2-MeOH, 97:3). 6-Amino-2,3-di-O-benzyl-
6-N-benzyloxycarbonyl-6-deoxy-myo-inositol 1,4,5-tri-O-(orthox-
ylylene)phosphate (33) was obtained as a white powder (295
mg; 88%). Rf ) 0.42 (CH2Cl2-MeOH, 97:3). 1H NMR{31P}
(CDCl3): 7.5-7.0 (m, 27H, -(C6H5)3 and -(C6H4)-3); 5.7-4.5 (m,
24H, containing at 5.35 (AB, J AB ) 13.9, ∆δ ) 0.47, 2H, -O-
CH2-C6H4-), at 5.28 (AB, J AB ) 13.7, ∆δ ) 0.34, 2H, -O-
(()-1,4,5-Tr i-O-a llyl-6-a m in o-2,3-d i-O-ben zyl-6-d eoxy-
m yo-in ositol (30). Dried (5 × 10-3 mbar; 50 °C; 12 h) tin
chloride (2 equiv; 276 mg; 1.46 mmol) was dissolved in
anhydrous acetonitrile (7 mL). Thiophenol (8 equiv; 600 µL;
5.84 mmol) and triethylamine (6 equiv; 610 µL; 4.38 mmol)
were added to the solution. (()-1,4,5-Tri-O-allyl-6-azido-2,3-
di-O-benzyl-6-deoxy-myo-inositol (28) dissolved in anhy-
drous acetonitrile (2 × 2 mL) was injected into the mixture.
After 1 h at TA, the solvents were removed and the residue
was dissolved in CH2Cl2 (20 mL) and 2 N NaOH (20 mL). The
aqueous phase was washed with CH2Cl2 (2 × 10 mL). The
organic layers were dried over Na2SO4, filtered, and evaporated
to dryness. The crude product was used as such for the next
step. An aliquot was purified by column chromatography
(ether). Rf ) 0.13 (ether). Anal. (C29H37O5N) C, H, N. 1H NMR
(CDCl3): 7.5-7.2 (m, 10H, -(C6H5)2); 6.1-5.8 (m, 3H, -(O-
CH2-CHdCH2)3); 5.4-5.1 (m, 6H, -(O-CH2-CHdCH2)3); 4.83
(AB, J AB ) 12.1, ∆δ ) 0.05, 2H, -O-CH2-C6H5); 4.68 (AB,
J AB ) 11.7, ∆δ ) 0.07, 2H, -O-CH2-C6H5); 4.5-4.1 (m, 4H,
-(O-CH2-CHdCH2)2); 4.1-3.8 (m, 4H, containing at 4.03 (t,
J ) 2.4, 1H, H-2), at 3.89 (t, J ) 9.3, 1H, H-4) and -O-CH2-
CHdCH2); 3.39 (t, J ) 9.9, 1H, H-6); 3.31 (dd, J ) 9.7, J )
2.4, 1H, H-3); 3.08 (t, J ) 9.5, 1H, H-5); 3.00 (dd, J ) 10.2, J
) 2.2, 1H, H-1); 1.83 (s, 2H, exchangeable, -NH2).
CH2-C6H4-), at 5.20 (t, J ) 9.3, 1H, H-4), at 4.93 (AB, J AB
)
13.7, ∆δ ) 0.64, 2H -O-CH2-C6H4-), at 4.51 (s, 1H, H-2),
-(O-CH2-C6H5)3, -(O-CH2-C6H4)-3, H-1, H-5, H-6 and NH);
4.16 (AB, J AB ) 11.3, ∆δ ) 0.10, 2H, -O-CH2-C6H5); 3.75
(dd, J ) 9.7, J ) 2.0, 1H, H-3). 31P NMR{1H} (CDCl3): -0.12
(s, 1P); -2.47 (s, 1P); -2.59 (s, 1P). MS (FAB+) M 1039.2: m/z
1040.1 [M + H].
(()-1,4,5-Tr i-O-a llyl-6-a m in o-2,3-d i-O-ben zyl-6-N-ben -
zyloxyca r bon yl-6-d eoxy-m yo-in ositol (31). Crude (()-1,4,5-
tri-O-allyl-6-amino-2,3-di-O-benzyl-6-deoxy-myo-inositol (30)
was dissolved in dioxane (14 mL) and 2 N NaOH (4 equiv; 1.5
mL; 2.92 mmol). At 0 °C benzyl chloroformate (4 equiv; 420
µL; 2.92 mmol) was slowly added. After 2 h at room temper-
ature, the solvents were evaporated and the crude product was
purified via silica gel column chromatography (ether-hexane,
1:2). The expected compound 31 was obtained with 88% yield
(from azide 28) (390 mg). Rf ) 0.33 (ether-hexane, 1:1). Anal.
(()-6-Am in o-6-deoxy-myo-in ositol 1,4,5-Tr is(ph osph ate)
(8). (()-6-amino-2,3-di-O-benzyl-6-N-benzyloxycarbonyl-6-
deoxy-myo-inositol 1,4,5-tri-O-(orthoxylylene)phosphate (33)
(100 mg; 0.096 mmol) dissolved in a CH2Cl2-MeOH-H2O (1:
2: 1) mixture (16 mL) was hydrogenized (5 atm) in the
presence of 10% Pd/C (50 mg) for 12 h at 20 °C. The Pd/C was
filtered through a Celite pad and the filtrate was evaporated
to dryness. The residue was dissolved in 1 mL bidistilled water
and then cooled to 0 °C; cyclohexylamine (1 mL) was added.
The mixture was evaporated to dryness and dissolved again
in 0.5 mL of bidistilled water. The cyclohexylammonium salts
were precipitated by adding 40 mL of acetone. The salts were
filtered and precipitated again by 40 mL of acetone. After
filtration and drying 53 mg (89%) of cyclohexylammonium salts
of (()-6-amino-6-deoxy-myo-inositol 1,4,5-tris(phosphate) (8)
1
(C37H43O7N) C, H, N. H NMR (DMSO-d6): 7.5-7.1 (m, 15H,
-(C6H5)3); 6.1-5.8 (m, 3H, -(O-CH2-CHdCH2)3); 5.4-5.1 (m,
8H, -(O-CH2-CHdCH2)3 and -O-CH2-C6H5); 4.75 (s, 2H,
-O-CH2-C6H5); 4.64 (AB, J AB ) 12.1, ∆δ ) 0.07, 2H, -O-
CH2-C6H5); 4.3-3.9 (m, 7H, -(O-CH2-CHdCH2)3 and H-2);
3.77 (q, J ) J H6NH ) 10.2, 1H, H-6); 3.59 (t, J ) 9.3, 1H, H-4);
3.4-3.2 (m, 3H, H-3, H-1, NH); 3.19 (t, J ) 9.5, 1H, H-5). MS
(FAB+) M 613.3: m/z 614.2 [M + H].
1
were obtained. H NMR{31P} (D2O, pH ) 11.4): 4.39 (t, J )
2.6, 1H, H-2); 4.25 (t, J ) 9.4, 1H, H-4); 3.94 (dd, J ) 9.7, J )
2.6, 1H, H-1); 3.93 (t, J ) 9.1, 1H, H-5); 3.79 (dd, J ) 9.4, J )
2.6, 1H, H-3); 3.25 (t, J ) 9.9, 1H, H-6). 31P NMR{1H} (D2O,
pH ) 11.4): 5.38 (s, 1P, P-4); 4.36 (s, 1P, P-5); 4.05 (s, 1P,
P-1). MS (FAB-) M 419.1: m/z 418.1 [M - H].
(()-6-Am in o-2,3-d i-O-ben zyl-6-N-ben zyloxyca r bon yl-6-
d eoxy-m yo-in osit ol (32). (()-1,4,5-Tri-O-allyl-6-amino-2,3-
di-O-benzyl-6-N-benzyloxycarbonyl-6-deoxy-myo-inositol (31)
(270 mg; 0.451 mmol) was dissolved in anhydrous THF (12
mL); dried (5 × 10-3 mbar, 100 °C, 12 h) ZnCl2 (8.1 equiv; 500
mg; 3.65 mmol) was added. After 15 min stirring at room
temperature, Pd(PPh3)4 (0.66 equiv; 345 mg; 0.3 mmol) was
added and stirring was maintained for 10 min. Bu3SnH (12
equiv; 1.45 mL; 5.42 mmol) was slowly added. After 1 h, the
reaction mixture was diluted with AcOEt (30 mL) and H2O (6
mL) and acidified. The aqueous phase was extracted with
AcOEt (3 × 30 mL). The organic phase was washed with brine,
dried over Na2SO4, filtered, and evaporated to dryness. The
residue was dissolved in acetonitrile (30 mL) and hexane (60
mL). The mixture was stirred for 30 min. The acetonitrile
phase was recovered and evaporated to dryness. Silica gel
column chromatography (CH2Cl2-MeOH, 97:3) furnished 187
mg (86%) of triol 32. Rf ) 0.41 (CH2Cl2-MeOH 97:3). Anal.
(C28H31O7N) C, H, N. 1H NMR (CDCl3): 7.5-7.2 (m, 15H,
-(C6H5)3); 5.12 (s, 2H, -O-CH2-C6H5); 4.87 (AB, J AB ) 11.5,
∆δ ) 0.30, 2H, -O-CH2-C6H5); 4.69 (AB, J AB ) 11.7, ∆δ )
0.07, 2H, -O-CH2-C6H5); 4.07 (t, J ) 2.6, 1H, H-2); 4.03 (td,
J ) 9.3, J ) 1.8, 1H, H-4); 3.82 (td, J ) 10.3, J ) 6.6, 1H,
H-6); 3.5-3.3 (m, 3H, H-1, H-5 and NH); 3.28 (dd, J ) 9.7, J
) 2.0, 1H, H-3); 3.0-2.8 (m, 1H, exchangeable. OH-5); 2.76
(d, J ) 2.0, 1H, exchangeable, OH-4). MS (FAB+) M 493.2:
m/z 494.1 [M + H].
In s(1,4,5)P 3 Der iva tives Bin d in g Assa y. Bovine adrenal
cortex microsomes were prepared as previously described.46
Bovine adrenal microsomes were incubated for 30 min at 0
°C in a medium containing 25 mM Tris/HCl buffered at pH
8.5, 100 mM KCl, 20 mM NaCl, 5 mM KH2PO4, and 1 mM
EDTA in a final volume of 500 µL with the appropriate
concentration of [3H]Ins(1,4,5)P3 (0.9 nM), Ins(1,4,5)P3, and
Ins(1,4,5)P3 derivatives. Nonspecific binding was determined
in the presence of 1 µM Ins(1,4,5)P3. Incubations were termi-
nated by centrifugation at 15000g for 5 min at 0 °C. The
receptor-bound radioactivity was analyzed by liquid scintilla-
tion spectrometry.
Ack n ow led gm en t. We thank Dr. Natasha Paul for
her help in correcting the final manuscript and Mrs.
Elisabeth Krempp for judicious advice on the NMR
experiments.
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