Synthesis and binding properties of cyclopentane analogues of myo-inositol 1,4,5-tris(phosphate)

Article abstract of DOI:10.1016/j.bmc.2004.06.005

Cyclopentanic analogues of myo-inositol 1,4,5-tris(phosphate) were synthesised starting from cyclopentadiene. The affinities of the trisphosphorylated derivatives for the Ins(1,4,5)P₃ receptors were equipotent to that of compound 4, showing that the relative orientation of the functional groups, particularly of the hydroxyl, is not of prime importance in this series. The ³¹P NMR titration curves show that the tris(phosphate) 5 behaves as the superimposition of an independent phosphate and a vicinal bis(phosphate).

Full text of DOI:10.1016/j.bmc.2004.06.005

Bioorganic & Medicinal Chemistry 12 (2004) 3995–4001
Synthesis and binding properties of cyclopentane analogues
of myo-inositol 1,4,5-tris(phosphate)
a
Marc-Antoine Moris, Annabelle Z. Caron, Gaetan Guillemette and Gilbert Schlewer
b
b
a,
*
ꢀ
aLaboratoire de Pharmacochimie de la Communication Cellulaire UMR 7081 du CNRS,
Facultꢀe de Pharmacie, 74, route du Rhin 67401 Illkirch, France
bDꢀepartement de Pharmacologie, Facultꢀe de Mꢀedecine, Universitꢀe de Sherbrooke,
3001, 12 avenue Nord Sherbrooke, Quebec, Canada J1H 5N4
Received 30 March 2004; accepted 2 June 2004
Abstract—Cyclopentanic analogues of myo-inositol 1,4,5-tris(phosphate) were synthesised starting from cyclopentadiene. The
affinities of the trisphosphorylated derivatives for the Ins(1,4,5)P3 receptors were equipotent to that of compound 4, showing that
the relative orientation of the functional groups, particularly of the hydroxyl, is not of prime importance in this series. The 31P NMR
titration curves show that the tris(phosphate) 5 behaves as the superimposition of an independent phosphate and a vicinal
bis(phosphate).
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
The structure–activity relationship developed around
this compound, especially towards the Ins(1,4,5)P3R,4–6
permitted Kozikowski to propose a pharmacophore
model showing the crucial role of the vicinal bis(phos-
phate) in positions 4 and 5, the amplifying effect of the
third phosphate in position 1, the major role played by
the hydroxyl group in position 6 and the slight impor-
tance of the two hydroxyls in positions 2 and 3 (Fig.
1).7;8
It is now well established that myo-inositol 1,4,5-
tris(phosphate) (Ins(1,4,5)P3) (1) acts as an intracellular
second messenger. The binding of Ins(1,4,5)P3 with its
receptors (Ins(1,4,5)P3R) induces the mobilisation of
calcium from intracellular stores.1–3
NH2
N
OH
OR
HO
O
N
HO
OPO3H2
OH
O
The adenophostines 2 and 3 extracted from Penicillium
brevicompactum are highly potent agonists of the
Ins(1,4,5)P3 receptors (10–30 times more potent than
Ins(1,4,5)P3).9–12 These molecules contain two sugar
backbones. Starting from the bisphosphorylated hexose
moiety it is easy to imagine new Ins(1,4,5)P3 analogues
and such analogues were recently published.13–16 Dre-
iding stereomodels show that the main functionalities of
Ins(1,4,5)P3 could be arranged around a cyclopentanic
ring. Potter et al. have reported cyclopentanic analogues
such as compound 4. The relative configuration of the
ring substituent strictly respect that of the parent
Ins(1,4,5)P3. This compound is a weak agonist for the
Ins(1,4,5)P3R (20% Caþþ mobilisation at 10ꢀ4 M) and
possesses a weak affinity for this receptor (three orders
of magnitude lower than the parent compound).17
H2O3PO
H2O3PO
O
N
N
HO
H2O3PO
OPO3H2
OPO3H2
2
3
R = H
R = Ac
1
OH
H2O3PO
OPO3H2
H2O3PO
OH
4
Keywords: myo-Inositol 1,4,5-tris(phosphate); Cyclopentane tris(phos-
phate); IP3 analogues; Binding; 31P NMR.
Using 31P NMR, it has been shown that the differ-
ent functions of Ins(1,4,5)P3 establish intramolecular
* Corresponding author. Tel.: +33-390-24-42-19; fax: +33-390-24-43-
10; e-mail: schlewer@pharma.u-strasbg.fr
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.06.005

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M.-A. Moris et al. / Bioorg. Med. Chem. 12 (2004) 3995–4001
Less important groups
OH
4
HO
H2O3PO
H2O3PO
2
6
3
OPO3H2
Amplifying effect
5
HO
1
Hydrogen bond donor
Crucial
Figure 1. Ins(1,4,5)P3 pharmacophore model proposed by Kozikowski.
cooperative effects especially between the phosphate
groups. The cooperative effect seemed to be relayed by
the neighbouring hydroxyls.18–23 The same studies car-
ried on compound 4 did not reveal the same type of
cooperation.24 The orientation of the functional groups
induced by the five member ring could disrupt the
interactions between the phosphate functions if the rel-
ative configuration of the Ins(1,4,5)P3 backbone is too
starkly transposed to the cyclopentanic ring. The inter-
functional cooperation could be restored by changing
the configuration of some of the backbone carbons.
remaining double bond using potassium permanga-
nate26 led to the intermediates 11–13. The isomer 12
proceeded by migration of the benzoate in position 4 to
the neighbouring cis hydroxy group. All the isomers
were obtained pure after column chromatography on
silicagel. The diols 11 and 13 were successively reacted
with dibutyltinoxide and benzyl bromide in the presence
of tetrabutyl ammonium bromide27 to yield the di-O-
benzoyl mono-O-benzyl cyclopentan tetraols 14, 15 and
16 (Scheme 1).
Hydrolysis of the benzoates 14 and 16 by means of
sodium hydroxide furnished the triols 17 and 18. These
hydroxyls were phosphorylated using the phosphite
method by means of N,N-diethylamino benzo-
dioxa(1,3,2) phosphepane and tetrazole followed by
m-chloroperbenzoic acid28 yielding the protected final
products 19 and 20. Total deprotection was obtained in
one step by hydrogenolysis using palladium on charcoal
as a catalyst.21 The expected compounds 5 and 6, as
racemates, were stabilised as cyclohexylammonium salts
to prevent phosphate migrations (Scheme 2).
We report here the synthesis and the properties of such
cyclopentanic analogues of Ins(1,4,5)P3. For these
derivatives (5, 6) we suppressed the hydroxyl in position
2, which did not seem important according to the
Kozikowski’s model.
H2O3PO
OPO3H2 H2O3PO
OPO3H2
4
1
1
4
2
3
2
3
H2O3PO
OH
H2O3PO
OH
5
6
3. Binding properties
For compound 5 the hydroxyl in position 3 corre-
sponding to the hydroxyl in position 6 on Ins(1,4,5)P3
was epimerised to see if this modification could restore
the cooperative effects observed for Ins(1,4,5)P3. For the
analogue 6 we wanted to keep the cis relative orientation
of the hydroxyl in position 3 and the phosphate in
position 2. In a first attempt, the products were prepared
as racemates. If their affinity justified it, the racemates
could be resolved to identify the more active enantio-
mer.
The affinity of the prepared compounds for the
Ins(1,4,5)P3 receptors was measured by binding on bovine
adrenal cortex microsomes.29 The results are reported as
Kd values (lM) in Table 1.
4. 31P NMR titrations
As examples, Figure 2 shows the 31P NMR titrations of
the tris(phosphate) 5 and the reference compound 1. As
for analogue 4, the titration curves of the tris(phos-
phate) 5 appear as a superimposition of an isolated
monophasic phosphate for phosphate 4 equivalent to
phosphate 1 of Ins(1,4,5)P3 and a vicinal bis(phosphate)
for the phosphates 1 and 2 equivalent to the phosphates
4 and 5 of the parent compound 1. The epimerisation of
hydroxyl 3 does not improve the cooperative intra-
molecular effects compared to compound 4. The same
behaviour of the phosphates was observed for the
tris(phosphate) 6 where the phosphates 1 and 2 were
epimerised compared to compound 4 leading to a cis
2. Synthesis
Our synthetic scheme started with cyclopentadiene (7),
which was treated with silver benzoate and iodine25
giving three dibenzoate isomers; the trans 3-cyclopent-
ene 1,2-dibenzoate (8), trans 2-cyclopentene 1,4-
dibenzoate (9) and the cis 2-cyclopentene 1,4-dibenzoate
(10) in a 15/20/20 proportion, respectively. The three
isomers were separated by crystallisation and column
chromatography. The cis dihydroxylation of the

M.-A. Moris et al. / Bioorg. Med. Chem. 12 (2004) 3995–4001
3997
OBz
BzO
OBz
c
OBz
BzO
HO
OBn
14
OBz
OBz
HO
OH
11
b
8
a
c
BzO
BzO
OBz
BzO
OH
a
BzO
9
7
HO
OBz
BnO
OH
15
12
13
a
BzO
OBz
OBz
b
BzO
OBz
c
HO
OH
10
HO
OBn
16
Scheme 1. Reagents and conditions: (a) BzOAg, I2, dry Et2O, reflux, yield 8–10 63%; (b) KMnO4, Me2CO, H2O, 0 ꢁC, yield 11 33%, 12 17%, 13 66%;
(c) Bu2SnO, Bu4NBr, CH3CN, MS 3 A, BnBr, reflux, yield 14 27%, 15 27%, 16 67%.
ꢁ
H2O3PO
OPO3H2
OH
HO
P
P
O
P
P
O
e
e
f
f
g
14
16
H2O3PO
OH
HO
OBn
P
O
OBn
17
18
19
20
5
6
H2O3PO
OPO3H2
O
O
HO
OH
g
H2O3PO
OH
P
O
O
OBn
HO
OBn
O
P
P
=
O
Scheme 2. Reagents and conditions: (e) NaOH 1 N, MeOH, THF, rt, yield 17 41%, 18 85%; (f) N,N-diethylamino benzodioxa(1,3,2) phosphepane,
1H-tetrazole, dry THF, )78 ꢁC, then rt overnight; then )78 ꢁC, m-CPBA, CH2Cl2, then rt, yield 19 60%, 20 55%; (g) H2, 5 atm, Pd/C 10%, MeOH,
CH2Cl2, H2O, AcOH, rt, overnight, then evaporation, H2O, cyclohexylamine, (Me)2CO, yield 5 45%, 6 57%.
compound and are of the same order of magnitude as
the cyclopentanic analogue 4 prepared by Potter et al.
One can observe that the epimerisation of the hydroxyl
as well as the inversion of the relative configuration of
the phosphate maintain about the same level of affinity.
The relative orientation of the functional groups does
not seem of prime importance in this series. The removal
of the hydroxyl in position 5 does not dramatically
reduce the affinity confirming Kozikowski’s pharmaco-
phore model. In terms of intramolecular cooperative
effects, the changes we made on the relative configura-
tions around the cyclopentanic ring do not induce sig-
nificant modifications on the NMR titrations compared
to the observations made for compound 4 and do not
correspond to the aspect observed for the parent
Ins(1,4,5)P3 (1). Five member ring contracted analogues
Table 1. Binding properties of the synthesised products and reference
compounds
Nr
1
4
5
6
Kd (lM)
0.025
>2517
100
70
system between the phosphate 2 and the hydroxyl group
in position 3 (titration curves not shown).
5. Discussion
Among the synthesised cyclopentanic analogues of
Ins(1,4,5)P3, the trisphosphorylated compounds show a
significant affinity for the Ins(1,4,5)P3 receptors. These
affinities remained, however, lower than the parent

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M.-A. Moris et al. / Bioorg. Med. Chem. 12 (2004) 3995–4001
6
P4
5
P5
OH
4
3
2
1
0
HO
OPO3H2
OH
P1
1
4
5
H2O3PO
OPO3H2
1
2
4
6
8
10
12
pH
6
5
4
3
2
1
0
P4
P2
P1
H2O3PO
OPO3H2
4
1
2
3
H2O3PO
OH
5
2
4
6
8
10
12
pH
Figure 2. 31P NMR titration analyses of compounds 5 and 1.
of Ins(1,4,5)P3 did not seem to lead to highly potent
analogues. As the observed affinities were relatively low,
it did not seem worth-while to define their agonist or
antagonist character.
solution of freshly distilled cyclopentadiene (1 equiv) in
dry ether (25 mL) was then slowly added; the exothermic
reaction mixture was warming to reflux and the addition
rate adjusted to maintain the reflux. After the complete
addition, the reflux was maintained by heating for an
additional 1 h. The cooled reaction mixture was filtered
and the organic layer washed with a saturated aqueous
solution of sodium hydrogenocarbonate (2 · 100 mL),
and brine (100 mL) and dried over magnesium sulfate.
The solvent volume was reduced to 50 mL and the
solution was allowed to slowly crystallise. The crystals
were filtered and recrystallised from ethanol giving 8.8 g
of trans-1,4-cyclohexene dibenzoate 9. (RF 0.38 heptane–
ethyl acetate 9/1); mp 122 ꢁC; 1H NMR (CDCl3): 2.54 (t,
J ¼ 5:1, 2H, CH2-5), 6.16 (td, J ¼ 5:1, J ¼ 0:9, 2H, H-1,
H-4), 6.33 (d, J ¼ 0:9, 2H, H-2, H-3), 7.4–8.1 (3m, 10H,
(C6H5)2). The filtrates were evaporated to dryness and
purified by column chromatography on silicagel (300 g)
eluted with a heptane–ether gradient starting from 9/1 to
2/1. An oily product possessing the same RF as the
crystals of 9 was isolated but corresponding to the cis-
More work is in progress to understand the SAR. The
recently published XR structures of the Ins(1,4,5)P3 R1
binding domain30;31 suggest the synthesis of new
Ins(1,4,5)P3 analogues.
6. Experimental part
Melting points were measured on a Mettler PF62
apparatus and are uncorrected. If not specified, NMR
spectra were recorded on Bruker Avance 300 spec-
trometer using the d scale; the CHCl3 residual signal was
fixed at 7.26 ppm. The abbreviations s, d, t, q, qu, m are
related to singlet, doublet, triplet, quadruplet, quin-
tuplet and multiplet, respectively. All the new com-
pounds gave satisfactory CHP analyses.
1
1,4-cyclohexene dibenzoate 10. H NMR (CDCl3): 2.11
6.1. Dibenzoates 8–1025
(dt, J ¼ 15:5, J ¼ 3:9, 1H, H-5b), 3.16 (dt, J ¼ 14:9,
J ¼ 7:4, 1H, H-5a), 5.92 (br dd, J ¼ 7:3, J ¼ 4:0, 2H, H-
1, H-4), 6.33 (br s, 2H, H-2, H-3), 7.2–8.3 (2m, 10H,
(C6H5)2). Finally a third isomer was isolated as an oil
corresponding to the trans-1,2-cylohexene dibenzoate 8
Sublimated iodine (29.0 g, 0.127 mol) was dropwise
added (25 min) to a suspension of silver benzoate32
(62.0 g, 0.27 mol) in dry ether (500 mL) with stirring. A

M.-A. Moris et al. / Bioorg. Med. Chem. 12 (2004) 3995–4001
3999
(RF 0.28 in heptane–ethyl acetate 9/1).1 H NMR
(CDCl3, 200 MHz): 2.4–2.6 (2m, 1H, H-5a), 3.22 (ddq,
J ¼ 18:1, J ¼ 7:1, J ¼ 1:9 1H, H-5b), 5.71 (dt, J ¼ 7:2,
J ¼ 3:5, 1H, H-1), 6.00 (dq, J ¼ 5:6, J ¼ 1:9, 1H, H-4),
6.1–6.2 (m, 2H, H-2, H-3), 7.4–8.2 (3m, 10H, (C6H5)2).
the diol 11 (12.5 g, 36.5 mmol) in acetonitrile (500 mL) in
ꢁ
a 1 L vessel equipped with a Soxhlet containing 3 A
molecular sieves. The mixture was refluxed for 3 h.
Benzyl bromide (29.5 g) was slowly added and the mix-
ture refluxed again for 2 days. Acetonitrile was evapo-
rated and CH2Cl2 (300 mL) and NaHCO3 (200 mL
saturated aqueous solution) were poured on the residue
and the mixture was stirred at room temperature for 1 h.
CH2Cl2 was recovered and the aqueous solution ex-
tracted twice with CH2Cl2 (100 mL). The organic layers
were dried over MgSO4, filtered and evaporated to
dryness giving a crude product, which was purified by
column chromatography on silicagel eluted with a
mixture of heptane–ether 7/3 leading to the expected
monobenzylated derivative 14 (5.2 g, 33%). 1H NMR
(CDCl3): 2:69 (AB part of an ABXY system, Dd ¼ 0:46,
JAB ¼ 14:7, JAX ¼ 4:2, JAY ¼ 7:3, JBX ¼ 8:2, JBY ¼ 5:9,
2H, CH2-5), 3.20 (d, J ¼ 4:9, 1H, exchangeable with
D2O, OH), 4.45 (t, J ¼ 4:5, 1H, H-2), 4.52 (q, J ¼ 4:5,
became t after exchange with D2O, 1H, H-3), 4:93 (AB,
Dd ¼ 0:12, JAB ¼ 11:6, 2H, CH2–C6H5), 5.63 (dt,
J ¼ 8:1, J ¼ 3:8, 1H, H-4), 5.85 (br q, J ¼ 5:4, 1H, H-1)
7.4–8.3 (m, 15H, (C6H5)3). The monobenzylated ana-
logue 15 was not obtained pure (4.0 g mixture).
6.2. 2b,3b-Dihydroxy cyclohexane 1b,4a-dibenzoate (11)
and 1b,3b-dihydroxy cyclohexane 2b,4a-dibenzoate (12)
Cyclopentene dibenzoate 9 (6.16 g, 20 mmol), was added
and stirred in a 1 L flask containing 500 mL acetone and
cooled to 0 ꢁC. KMnO4 (2.12 g, 20 mmol) dissolved in
water (150 mL) was added dropwise. The reaction mix-
ture was kept at 4 ꢁC overnight, filtered and the solid
washed with acetone (50 mL). The filtrate was evapo-
rated to dryness and dissolved in ether (200 mL). This
organic phase was successively washed with an aqueous
K2CO3 saturated solution (50 mL) and brine (50 mL)
and dried over MgSO4. The crude product obtained
after filtration and solvent evaporation was purified on a
silicagel column eluted with a gradient starting from
heptane–ether 85/15 to heptane–ether–ethyl acetate 5/5/
1. Cyclopentene dibenzoate 9 was recovered (1.4 g) fol-
lowed by the dihydroxylated compound 11 (1.4 g, 26%).
1
RF heptane–ethyl acetate 1/1: 0.52. H NMR (CDCl3):
3:70 (AB part of an ABXY system, Dd ¼ 0:32,
JAB ¼ 15:0, JAX ¼ 7:2, JAY ¼ 4:7, JBX ¼ 6:6, JBY ¼ 8:7,
2H, CH2-5), 3.10 (br d, J ¼ 3:0, exchangeable with D2O,
1H, OH), 3.72 (br d, J ¼ 3:0, exchangeable with D2O,
1H, OH), 4.24 (br q, J ¼ 3:0, became t after exchange
with D2O, 1H, CHOH), 4.45–4.55 (m, 1H, became t
after exchange with D2O, J ¼ 3:0, CHOH), 5.34 (dt,
J ¼ 8:7, J ¼ 4:4, 1H, H-4), 5.52 (td, J ¼ 7:2, J ¼ 4:4,
1H, H-1), 7.4–8.2 (m, 10H, (C6H5)2). The dihydroxyl-
ated compound 12 was eluted just after compound 11
(0.8 g, 15%). RF heptane–ethyl acetate 1/1: 0.44. 1H
NMR (CDCl3): 2:24 (AB part of an ABXY system,
Dd ¼ 0:42, JAB ¼ 15:0, JAX ¼ 6:2, JAY ¼ 5:3, JBX ¼ 8:4,
JBY ¼ 5:0, 2H, CH2-5), 4.40–4.55 (m, 2H, modified after
exchange with D2O, CHOH-1 and CHOH-3), 5.48 (t,
J ¼ 4:7, 1H, H-2), 5.75 (dt, J ¼ 8:1, J ¼ 5:3, 1H, H-4),
7.3–8.1 (m, 10H, (C6H5)2).
6.5. 2a-Benzyloxy-3-hydroxy cyclohexane 1b,4b-di-
benzoate (16)
Same procedure as for 14 starting from the diol 13
(3.5 g) and using dibutyltinoxide (3.02 g), tetrabutylam-
monium bromide (3.75 g), benzyl bromide (8.3 mL) and
acetonitrile (300 mL). Purification by column chroma-
tography on silicagel eluted with a mixture of heptane–
ethyl acetate 4/1 gave the expected monobenzylated
1
derivative 16 (3.28 g, 74%). H NMR (CDCl3): 1.96 (dt,
J ¼ 15:4, J ¼ 3:8, 1H, H-5b), 2.91 (d, J ¼ 5:6, 1H, be-
came s after exchange with D2O, 1H, OH), 3.04 (dt,
J ¼ 15:5, J ¼ 7:9, 1H, H-5a), 4.23 ( dd, J ¼ 4:3, J ¼ 4:1,
1H, H-2), 4.44 (q, J ¼ 5:3, became t after exchange with
D2O, 1H, H-3), 4:81 (AB system, Dd ¼ 0:12, JAB ¼ 11:7,
2H, CH2–C6H5), 5.30 (dt, J ¼ 7:5, J ¼ 4:1, 1H, H-4),
5.50 (dt, J ¼ 7:5, J ¼ 3:4, 1H, H-1), 7.2–8.1 (m, 15H,
(C6H5)3).
6.3. 2b,3b-Dihydroxy cyclohexane 1a,4a-dibenzoate (13)
Same procedure as for 11 starting from the cis di-
benzoate 10 (11.0 g) and using KMnO4 (3.79 g) led to
unreacted starting material (4.0 g) and the expected di-
hydroxylated derivative 13 (5.3 g, 43%). 1H NMR
(CDCl3): 2:60 (AB part of an ABX2 system, Dd ¼ 0:89,
JAB ¼ 15:3, JAX ¼ 3:6, JBX ¼ 7:8, 2H, CH2-5), 3.61 (br s,
2H, exchangeable with D2O, (OH)2), 4.39 (d, J ¼ 1:4,
2H, H-2 and H-3), 5.2–5.4 (m, 2H, H-1, H-4), 7.3–8.2
(m, 10H, (C6H5)2).
6.6. 2b-Benzyloxy-1b,3b,4a-trihydroxy cyclohexane (17)
Dibenzoate 14 (1.2 g) dissolved in methanol (25 mL) and
THF (25 mL) was stirred overnight at room temperature
in the presence of NaOH (20 mL, 1 N). The pH was then
adjusted to 6 by addition of HCl (1 N) and the mixture
was evaporated to dryness. The crude product was
purified on a silicagel column eluted with a mixture of
ethyl acetate–methanol 9/1 leading to the expected triol
1
17 (240 mg, 47%). H NMR (200 MHz, CDCl3): 2:02
(AB part of an ABXY system, Dd ¼ 0:43, JAB ¼ 14:7,
JAX ¼ JAY ¼ 5:6, JBX ¼ 7:8, JBY ¼ 2:7, 2H, CH2-5),
3.38 (br s, 3H, exchangeable with D2O, (OH)3), 3.97
(br s, 2H,), 4.23 (dd, J ¼ 4:3, J ¼ 4:1, 1H, H-2), 4.32
(br s, 2H,), 4.72 (s, 2H, CH2–C6H5), 7.2–7.7 (m, 5H,
C6H5).
6.4. 3b-Benzyloxy-2b-hydroxy cyclohexane 1b,4a-di-
benzoate (14)
Dibutyltinoxide (10.8 g, 1.2 equiv) and tetrabutylam-
monium bromide (11.6 g) were added to a solution of

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M.-A. Moris et al. / Bioorg. Med. Chem. 12 (2004) 3995–4001
6.7. 2a-Benzyloxy-1b,3a,4b-trihydroxy cyclohexane (18)
(16 mL), H2O (8 mL), CH2Cl2 (8 mL) and CH3CO2H
(1 mL). Pd/C 10% (200 mg) was added and the mixture
was hydrogenolysed under 5 atm in a Parr shaker for
10 h. After filtration, the solvents were evaporated in
vacuo, the residue redissolved in pure water (5 mL), and
cooled to 0 ꢁC. Cyclohexylamine (2 mL) was added and
the mixture was evaporated again to dryness in vacuo.
The residue was dissolved in pure water (1 mL) and
dropped in dry acetone (150 mL). The precipitate was
filtered and redissolved in water (1 mL) and precipitated
again in acetone (150 mL). The beige powder was fil-
tered and dried in vacuo leading to the expected product
as its pentacyclohexylammonuim salt (113 mg, 50%).
Same procedure as for 17 starting from dibenzoate 16
(1.6 g) leading to the triol 18 (690 mg, 84%). Mp 114.5 ꢁC
(dec). H NMR (200 MHz, CDCl3): 1.59 (dt, 1H, H-5),
1
2.48, dt, 1H, H-5), 2.64 (br s 3H, exchangeable with
D2O, (OH)3), 3.9–4.3 (m, 4H, H-1, H-2, H-3, H-4), 4:68
(AB system, Dd ¼ 0:06, JAB ¼ 11:5, 2H, CH2–C6H5-2),
7.2–7.5 (m, 5H, C6H5).
6.8. 2b-Benzyloxy-cyclohexyl 1b,3b,4a-tris(xylyl-phos-
phate) (19)
Mp (dec). Analyses calculated for (C5H7O13P3)6ꢀ
;
The triol 17 (240 mg, 1.08 mmol) was dissolved in dry
THF and stirred under inert argon atmosphere and
cooled to )78 ꢁC. 1H-tetrazole (925 mg, 13.2 mmol) was
added followed by N,N-diethylamino benzodioxa (1,3,2)
phosphepane (3.20 g, 6.6 mmol). The reaction mixture
was warmed up and kept at room temperature over-
night. Then the vessel was cooled again to )78 ꢁC and
dry m-CPBA (3.25 g) dissolved in dry CH2Cl2 (20 mL)
was added dropwise. After warming to room tempera-
ture, the mixture was stirred for an additional 30 min
and the solvents cautiously evaporated in vacuo. The
residue was dissolved again in CH2Cl2 (300 mL), which
was successively washed with Na2S2O3 (2 · 50 mL
aqueous saturated solution) and NaHCO3 (100 mL
aqueous saturated solution) and finally dried over
MgSO4. The crude product obtained after concentration
in vacuo was purified on a silicagel column eluted with a
mixture of CH2Cl2–CH3OH 99-1 to 80/20, giving the
expected tris(phosphate) 19 (502 mg, 60%). Mp 80 ꢁC.
Analyses calculated for C36H37O13P3; 1/2H2O (770.61):
C, 55.47; H, 4.91; P, 11.92; found: C, 55.24; H, 5.02; P,
11.46. 1H NMR (200 MHz, CDCl3): 2:60 (AB part of an
4C6H14Nþ; 2Naþ (814.74): C, 42.75; H, 7.7; P, 11.40; N,
6.72; found C, 42.33; H, 8.19; P, 10.78; N, 6.64. 1H
NMR (200 MHz, D2O, CD3OD 1/1 + COSY): 0.9–2.0
(6m, 51H, CH-5, 25 CH2 cyclohexyl), 2.1–2.3 (m, 1H,
H-5), 2.9–3.0 (m, 5H, (NH2CH)5), 4.09 (t, J ¼ 4:0, 1H,
H-3), 4.26 (dt, J ¼ 9:2, J ¼ 4:7, became t after {31P},
1H, H-2), 4.42–4.51 (m, became td after {31P}, J ¼ 7:4,
J ¼ 3:9, 1H, H-4), 4.51–4.61 (m, became td after {31P},
J ¼ 9:2, J ¼ 5:0, 1H, H-1); 13C NMR (75.48 MHz, D2O,
CD3OD 1/1 + HSQC): 24.15, 24.63, 30.74, 50.55 (cy-
clohexylammonium), 36.88 (C-5), 72.68 (C-3), 73.17
(C-2), 78.30 (C-4), 80.40 (C-1). {1H} 31P NMR
(80.96 MHz, H2O, D2O 95/5, KCl 2 M, pH 7.2): 2.97,
3.27, 4.08.
6.11. 3a-Hydroxy-cyclohexyl 1b,2a,4b-tris(phosphate) (6)
Same procedure as for 5 starting from the protected
compound 26 (710 mg, 1.00 mmol) gave tris(phosphate)
7 as its hexacyclohexylammonium salt (546 mg, 61%).
Mp (dec). Analyses calculated for (C5H7O13P3)6ꢀ
;
ABXY system, Dd ¼ 0:49, JAB ¼ 15:0, JAX ¼ 7:2, JAY
¼
6C6H14Nþ, 2H2O: C, 48.99; H, 9.53; N, 8.36; P, 9.25;
1
4:1, 2H, CH2-5), 4.28 (t, J ¼ 4:7, 1H, CH–OBn), 4.7–5.5
(m, 17H, (CH2C6H5)7 + (CH–OP)3), 7.0–7.6 (m, 17H,
C6H5 + (C6H4)3).
found: C, 48.68; H, 9.83; N, 7.99; P, 8.83. H NMR
(D2O + COSY): 0.9–2.0 (4m, 61H containing at 1.63
CH-5b as the A part of an ABXY system + 30 CH2
cyclohexyl), 2.97 (dt, J ¼ 15:0, J ¼ 6:8, 1H, H-5a), 2.9–
3.0 (m, 6H, (NH2CH)5), 4.03 (t, J ¼ 4:5, 1H, H-3), 4.25
(dt, J ¼ 7:4, J ¼ 4:7, became dd after {31P}, 1H, H-4),
4.35–4.50 (m, simplified after {31P}, 2H, H-2, H-1); {1H}
31P NMR (80.96 MHz, H2O, D2O 95/5, KCl 2 M,
pH 6.8): 2.54, 2.84, 3.23.
6.9. 3a-Benzyloxy-cyclohexyl 1b,2b4b-tris(xylyl-phos-
phate) (20)
Same procedure as for 19 starting from 2a-benzyloxy-
1b,3a,4b-trihydroxy
cyclohexane
(18)
(630 mg,
2.81 mmol) and using tetrazole (1.62 g, 8 equiv), N,N-
diethylamino benzodioxa(1,3,2) phosphepane (5.60 g,
6 equiv), m-CPBA (5.7 g), produced tris(phosphate) 20
(1.20 g) after purification on a silicagel column eluted
with a mixture of heptane–ethyl acetate 6/4. Mp 61 ꢁC.
1H NMR (CDCl3): 2.21 (dt, J ¼ 15:4, J ¼ 3:0, 1H, H-
5b), 2.92 (dt, J ¼ 15:6, J ¼ 7:8, 1H, H-5a), 4.30 (t, J ¼
3:1, 1H, H-3), 4:71 (AB system, Dd ¼ 0:03, JAB ¼ 11:6,
2H, CH2–C6H5), 4.8–5.4 (m, 15H, (CH2C6H4CH2)3, H-
1, H-2 and H-4), 7.1–7.4 (m, 17H, C6H5 and (C6H4)3).
6.12. 31P NMR titrations
The acid base properties of the synthesised products
were studied using cyclohexyl phosphate as internal pH
sensor. Thus, the tris(phosphate) (7.12 mg) and cyclo-
hexyl phosphate (1.5 equiv) cyclohexyl ammonium salts
were eluted through an Amberlist IRN 77 column in its
acidic form to regenerate the free acids as previously
reported.15 The lyophilised acids were dissolved in
0.5 mL D2O/H2O (5/95) 0.2 M in KCl. The initial pH
was measured and the solution introduced in a 5 mm
NMR tube at 26 ꢁC. Between each {1H}31P spectrum
acquisition, 10 lL NaOH (0.2 N) were added. The
internal pH was obtained using the cyclohexyl phos-
phate chemical shift, which was compared to this of a
6.10. 3b-Hydroxy-cyclohexyl 1a,2b,4b-tris(phosphate) (5)
The totally protected tris(phosphate) 19 (200 mg,
0.26 mmol) was dissolved in a mixture of CH3OH

M.-A. Moris et al. / Bioorg. Med. Chem. 12 (2004) 3995–4001
4001
8. Bonis, D.; Sezan, A.; Mauduit, P.; Cleophax, J.; Gero, S.
D.; Rossignol, B. Biochem. Biophys. Res. Commun. 1991,
175, 894–900.
previous titration of this compound. The final pH was
also measured using a classical pH electrode.
9. Takahashi, M.; Kagasaki, T.; Hosoya, T.; Takahashi, S.
J. Antibiot. (Tokyo) 1993, 46, 1643–1647.
10. Takahashi, M.; Tanzawa, K.; Takahashi, S. J. Biol. Chem.
1994, 269, 369–372.
11. Correa, V.; Riley, A. M.; Shuto, S.; Horne, G.; Nerou, E.
P.; Marwood, R. D.; Potter, B. V. L.; Taylor, C. W. Mol.
Pharmacol. 2001, 59, 1206–1215.
12. Murphy, C. T.; Riley, A. M.; Lindley, D. J.; Jenkins, D. J.;
Westwick, J.; Potter, B. V. L. Mol. Pharmacol. 1997, 52,
741–748.
13. Roussel, F.; Moitessier, N.; Hilly, M.; Chretien, F.;
Mauger, J. P.; Chapleur, Y. Bioorg. Med. Chem. 2002,
10, 759–768.
14. Beecroft, M. D.; Marchant, J. S.; Riley, A. M.; van
Straten, N. C. R.; van der Marel, G. A.; van Boom, J. H.;
Potter, B. V. L.; Taylor, C. W. Mol. Pharmacol. 1999, 1,
109–117.
15. Jenkins, D. J.; Potter, B. V. L. J. Chem. Soc., Chem.
Commun. 1995, 1169–1170.
6.13. Binding assay
Bovine adrenal cortices (dissected free of medullary
tissue) were homogenised with eight strokes of a Dounce
homogeniser (loose pestle) in a medium containing
110 mM KCl, 10 mM NaCl, 2 mM MgCl2, 25 mM Tris–
HCl pH 7.2, 5 mM KH2PO4, 1 mM dithiothreitol and
2 mM EGTA. After stirring for 5 min and centrifugation
at 500g for 15 min, the supernatant was centrifuged at
35,000g for 20 min. The pellet was resuspended in the
medium without EGTA, supplemented with glycerol
(1.4% w/v), at a concentration of 20–30 mg of protein/
mL. The microsomes were stored at )70 ꢁC. This
microsomal preparation has been successfully used since
1987.29
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 lL with
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 lM In-
s(1,4,5)P3. Incubations were terminated by centrifuga-
tion at 15,000g for 5 min at 0 ꢁC. The receptor-bound
radioactivity was analysed by liquid scintillation spec-
trometry.
16. Jenkins, D. J.; Potter, B. V. L. Carbohydr. Res. 1996, 287,
169–182.
17. Jenkins, D. J.; Potter, B. V. L. J. Chem. Soc., Perkin
Trans. 1 1998, 41–49.
18. Schmitt, L.; Schlewer, G.; Spiess, B. Biochim. Biophys.
Acta 1991, 139–140.
ꢀ
19. Guedat, P.; Poitras, M.; Spiess, B.; Guillemette, G.;
Schlewer, G. Bioorg. Med. Chem. Lett. 1996, 6, 1175–
1178.
20. Schmitt, L.; Bortmann, P.; Schlewer, G.; Spiess, B.
J. Chem. Soc., Perkin Trans. 1 1993, 2257–2263.
21. Ballereau, S.; Guedat, P.; Poirier, S. N.; Guillemette, G.;
Spiess, B.; Schlewer, G. J. Med. Chem. 1999, 42, 4824–
4835.
22. Felemez, M.; Ballereau, S.; Schlewer, G.; Spiess, B. New J.
Chem. 2000, 24, 631–638.
23. Felemez, M.; Bernard, P.; Schlewer, G.; Spiess, B. J. Am.
Chem. Soc. 2000, 122, 3156–3165.
24. Felemez, M.; Schlewer, G.; Jenkins, D. J.; Correa, V.;
Taylor, C. W.; Potter, B. V. L.; Spiess, B. Carbohydr. Res.
1999, 95–101.
Acknowledgements
We thank la Ligue Contre le Cancer for financial sup-
port and Mary H. Richards for proofreading the man-
uscript.
25. Gaoni, Y. Bull. Soc. Chim. Fr. 1950, 705–710.
26. Sable, H. Z.; Anderson, T.; Tolbert, B.; Posternak, T.
Helv. Chim. Acta 1963, 1157–1165.
References and notes
27. Nagashima, N.; Ohno, M. Chem. Lett. 1987, 141–144.
28. Perich, J. W.; Johns, R. B. Tetrahedron Lett. 1987, 28,
101–102.
29. Guillemette, G.; Balla, T.; Baukal, A. J.; Spat, A.; Catt, K.
J. J. Biol. Chem. 1987, 262, 1010–1015.
30. Serysheva, I. I.; Bare, D. J.; Ludtke, S. J.; Kettlun, C. S.;
Chiu, W.; Mignery, A. J. Biol. Chem. 2003, 278, 21319–
21322.
31. Bosanac, I.; Alattla, J. R.; Mal, T. K.; Chan, J.; Talarico,
S.; Tong, F. K.; Tong, K. I.; Yoshlkawa, F.; Furulchl, T.;
Iwal, M.; Michlkawa, T.; Mikoshiba, K.; Ikura, M.
Nature 2002, 420, 696–700.
1. Streb, H.; Irvine, R. F.; Berridge, M. J.; Schultz, I. Nature
1983, 312, 375–376.
2. Berridge, M. J.; Irvine, R. F. Nature 1984, 312, 315–321.
3. Berridge, M. J. Nature 1993, 361, 315–325.
4. Billington, D. C. Chem. Soc. Rev. 1989, 18, 83–122.
5. Billington, D. C. The Inositol Phosphates; VCH: Wein-
heim, 1993. ISBN 3-527-28152-5; p 153.
6. Potter, B. V. L.; Lampe, D. Angew. Chem., Int. Ed. Engl.
1995, 34, 1933–1972.
7. Kozikowski, A. P.; Ognyanov, V. I.; Fauq, A. H.;
Nahorski, S. R.; Wilcox, R. A. J. Am. Chem. Soc. 1993,
115, 4429–4434.
32. Neumann, M. S.; Beal, P. F. J. Am. Chem. Soc. 1950, 72,
5163–5165.