Synthesis of potent Ins(1,4,5)P3 5-phosphatase inhibitors by modification of myo-inositol 1,3,4,6-tetrakisphosphate

Article abstract of DOI:10.1016/S0968-0896(03)00347-X

Three myo-inositol tetrakisphosphate analogues were synthesised based upon myo-inositol 1,3,4,6-tetrakisphosphate: 2,5-di-O-methyl myo-inositol-1,3,4,6-tetrakisphosphate 19 and its phosphorothioate derivative 22, together with myo-inositol 1,3,4,6 tetraki

Full text of DOI:10.1016/S0968-0896(03)00347-X

Bioorganic & Medicinal Chemistry 11 (2003) 4245–4253
Synthesis of Potent Ins(1,4,5)P3 5-Phosphatase Inhibitors by
Modification of myo-Inositol 1,3,4,6-Tetrakisphosphate
Stephen J. Mills and Barry V. L. Potter*
Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath,
Claverton Down, Bath BA2 7AY, UK
Received 4 April 2003; accepted 20 May 2003
Abstract—Three myo-inositol tetrakisphosphate analogues were synthesised based upon myo-inositol 1,3,4,6-tetrakisphosphate:
2,5-di-O-methyl myo-inositol-1,3,4,6-tetrakisphosphate 19 and its phosphorothioate derivative 22, together with myo-inositol
1,3,4,6 tetrakisphosphorothioate 25. These compounds were prepared by phosphitylating 2,5-di-O-methyl-myo-inositol and 2,5-di-
O-benzyl-myo-inositol followed by oxidation with t-butylhydroperoxide or sulfoxidation at room temperature using sulfur in a
mixed solvent of DMF and pyridine. Sulfoxidation was complete within 15 min; however, without DMF, the reaction was much
slower, and required overnight. When evaluated against Ins(1,4,5)P3 5-phosphatase, 3-kinase and for Ca2+ release at the
Ins(1,4,5)P3 receptor, only weak activity was observed for Ca2+ release. 22 and 25 are potent 5-phosphatase inhibitors and 25 is a
moderate inhibitor of 3-kinase. Thus, we have synthesised potent enzyme inhibitors, which do not mobilise Ca2+ and devised
conditions for quick, clean and inexpensive sulfoxidation of inositol polyphosphite intermediates.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
Ins(1,4,5)P3 receptor with bound Ins(1,4,5)P3 at a reso-
lution of 2.2 A has also been published recently.5 Data
from the crystallographic studies illustrate the inter-
actions of specific arginine and lysine residues with
the three phosphate groups of Ins(1,4,5)P3. Knowing
the critical interactions between Ins(1,4,5)P3 and its
receptor we should be able to design effective new
ligands.
d-myo-Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3 1] is the
hydrophilic second messenger derived from the enzy-
matic cleavage of the minor membrane lipid, phospha-
tidylinositol 4,5-bisphosphate, in response to a primary
ligand stimulation.1,2 Ins(1,4,5)P3 interacts specifically
with a tetrameric Ins(1,4,5)P3 receptor-operated Ca2+
channel to mobilise Ca2+ from non-mitochondrial
stores.1,2 Design of receptor agonists, antagonists and
enzyme inhibitors for inositol phosphates as biological
tools, has been a serious enterprise for synthetic che-
mists for about 15 years.3 A study by Jiang et al.4
revealed the first three-dimensional structure of the
type-1 Ins(1,4,5)P3 receptor using cryo-electron micro-
scopy. The structure of the Ins(1,4,5)P3 receptor was
resolved at 24 A in which the Ins(1,4,5)P3 receptor
channel takes the form of an uneven dumbbell with one
end significantly bigger than the other. The Ins(1,4,5)P3
receptor can be sub-divided into three regions: first, the
amino terminal Ins(1,4,5)P3 binding domain, second,
the central modulatory region and third, the carboxy
terminal channel region. The first X-ray crystal struc-
ture of the Ins(1,4,5)P3 binding domain of mouse type-1
Two enzymes, Ins(1,4,5)P3 3-kinase and 5-phosphatase
deactivate Ins(1,4,5)P3 and switch off the Ca2+ signal.6
5-Phosphatase (type I)7a metabolises Ins(1,4,5)P3 to give
myo-inositol 1,4-bisphosphate Ins(1,4)P2 2, which has
no Ca2+ releasing activity. The crystal structure of an
inositol polyphosphate 5-phosphatase domain of
synaptojanin (from Schizosaccharmyces pombe synap-
tojanin, type II), bound to Ins(1,4)P2 and Ca2+ at 1.8 A
resolution has been reported8 and reveals the interac-
tions between critical amino acid residues of the 5-
phosphatase and the product of dephosphorylation,
Ins(1,4)P2. A number of disease states associated with
malfunctioning of 5-phosphatase (group II) have been
discovered over the last decade. One of these is Lowe’s
oculocerebrorenal syndrome (OCRL), an X-linked dis-
order which affects the brain, eye lens and kidney
development, and its associated gene product bears a
71% similarity to group II 5-phosphatase.9 A deficiency
*Corresponding author. Tel.: +44-1225-386639; fax: +44-1225-
386114; e-mail: b.v.l.potter@bath.ac.uk
0968-0896/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00347-X

4246
S. J. Mills, B. V. L. Potter / Bioorg. Med. Chem. 11 (2003) 4245–4253
or over expression of 5-phosphatase and other related
enzymes from Ins(1,4,5)P3 pathway would result in
unbalanced signalling products, so the design of specific
inhibitors and activators to counteract these effects will
make attractive targets for drug design of the future.
Results and Discussion
The tetraols 13 and 16 were prepared from the same
intermediate 11 via dl-1,4-di-O-allyl-myo-inositol 9
(Scheme 1). Compound 9 was prepared by treating
compound 8 with 80% acetic acid at reflux temperature
for 30 min, after which the solvent was evaporated and
the remaining solid was recrystallised. It was envisaged
that selective p-methoxybenzylation at the 3- and 6-
positions would give a protected 1,3,4,6-intermediate
which could be alkylated at the 2- and 5-positions then
deprotected to provide the 1,3,4,6-tetrol. A mixture of
dibutyltin oxide and dl-1,4-di-O-allyl-myo-inositol 9
was heated in toluene under reflux to form the cis-2,3
and the trans-5,6-dibutylstannylene derivative. The
toluene was evaporated and DMF was added to the
syrup together with CsF and p-methoxybenzyl chlo-
ride and the reaction was then stirred overnight.
TLC showed two products, dl-1,4-di-O-allyl-3,5-di-
O-p-methoxybenzyl-myo-inositol 10 and dl-1,4-di-O-
3-Kinase phosphorylates Ins(1,4,5)P3 at the 3-hydroxyl
to give d-myo-inositol 1,3,4,5-tetrakisphosphate
[Ins(1,3,4,5)P4 3]. This compound is then dephos-
phorylated to give d-myo-inositol 1,3,4-trisphosphate
[Ins(1,3,4)P3 4] and further phosphorylation of com-
pound 4 provides higher inositol polyphosphates such
as
Ins(1,3,4,6)P4
5,
Ins(1,3,4,5,6)P5
6
and
Ins(1,2,3,4,5,6)P6 7 (Fig. 1). One such tetrakispho-
sphate, Ins(1,3,4,6)P4 5 can mobilise Ca2+ in Xenopus
oocytes10 and neuroblastoma cells11 albeit with a much
weaker potency than for Ins(1,4,5)P3. Ins(1,3,4,6)P4 is a
partial agonist at the Ins(1,4,5)P3 receptor of SH-SY5Y
human neuroblastoma cells11 releasing only 85% of the
total Ca2+ pool and is the only naturally occurring
inositol polyphosphate that apparently acts as a partial
agonist at the Ins(1,4,5)P3 receptor. Data from our pre-
vious studies12 illustrate that Ins(1,3,4,6)P4 is a good 5-
phosphatase inhibitor having a Ki value of 7.7 mM. We
predict from this work that the tetrakispho-
sphorothioate analogue 25 should give a 5-phosphatase
inhibitor of sub-micromolar potency.12 Thus, myo-ino-
sitol 1,3,4,6-tetrakisphosphorothioate makes an attrac-
tive target for a potent 5-phosphatase inhibitor. scyllo-
Inositol 1,2,4,5-tetrakisphosphorothioate, is a high
intrinsic activity partial agonist at the Ins(1,4,5)P3
receptor that has been synthesised and evaluated.13 It is
a potent inhibitor of 5-phosphatase (Ki 0.3 mM); how-
ever, its ability to release Ca2+ makes it less useful as a
5-phosphatase inhibitor and a tool for investigating
Ca2+ signalling. Generally, if a phosphate is replaced
with a phosphorothioate group in inositol phosphate
compounds, the potency of inhibition for 5-phosphatase
increases.3 We report here the synthesis of three poten-
tial 5-phosphatase inhibitors, based upon Ins(1,3,4,6)P4,
which are poorly recognised by 3-kinase and are devoid
of Ca2+ release. We also report a simple method for the
improved preparation of phosphorothioate derivatives by
sulfoxidation of a suitable P(III) intermediate.
Scheme 1. (a) 80% HOAc, reflux, 30 min; (b) Bu2Sn¼O, toluene,
reflux, 2.5 h, then DMF, CsF (5 equiv), p-methoxybenzyl chloride; (c)
NaH, DMF, MeI; (d) Pd/C (10%), reflux, EtOH, PTSA; (e) BnBr,
NaH, DMF; (f) KOtBu, DMF, 85 ꢀC, 2 h; (g) CH2Cl2–TFA (10:1).
All, allyl; Bn, benzyl; Me, methyl; PMB, p-methoxybenzyl; Prop, cis-
prop-1-enyl.
Figure 1.

S. J. Mills, B. V. L. Potter / Bioorg. Med. Chem. 11 (2003) 4245–4253
4247
allyl-3,6-di-O-p-methoxybenzyl-myo-inositol 11. Diol 11
was methylated with methyl iodide using sodium
hydride as the base in DMF to give dl-1,4-di-O-allyl-
3,6-di-O-p-methoxybenzyl-2,5-di-O-methyl-myo-inositol
12 and isolated as a crystalline solid. The allyl and p-
methoxybenzyl protective groups were then removed in
one step by refluxing compound 12 in a mixture of eth-
anol–water with palladium on carbon and a catalytic
amount of acid. The palladium on carbon was filtered
off over a pad of Celite, the solvents were evaporated
and the crude mixture was recrystallised from ethanol to
give 2,5-di-O-methyl-myo-inositol 13. We found that
compound 13 decomposed between 266 and 268 ꢀC
where previously, a melting point of 270 ꢀC was repor-
ted.14 Compound 13 was used to synthesise 2,5-di-O-
methyl-myo-inositol 1,3,4,6-tetrakisphosphate 19 and
the corresponding phosphorothioate analogue 22
(Schemes 2 and 3).
derivative 15, by using freshly sublimed potassium t-
butoxide in dry DMF at 85 ꢀC for 2 h, which rendered
the prop-1-enyl ether susceptible to acidic deprotection.
The Rf value of the 1,4-di-O-cis-prop-1-enyl derivative
15 was identical to that of compound 14, but treatment
of a small quantity of the mixture with acid gave a more
polar derivative indicated by TLC. The compound was
extracted from the mixture, purified by flash chromato-
graphy then recrystallised from hexane to give com-
1
pound 15 in 83% yield. The H NMR data showed the
presence of 1,4-di-O-cis-prop-1-enyl groups because the
coupling constants for the protons across the double bond
of CH3HC¼CH–O were 6.2 and 6.4 Hz, respectively,
whereas a trans-prop-1-enyl ether would be ca. 15 Hz. The
cis-prop-1-enyl ether and the p-methoxybenzyl group are
both acid sensitive, and treatment of the derivative with
10% TFA in dichloromethane cleaved both groups whilst
leaving the benzyl ethers at position 2 and 5 intact. The
only product detected by TLC resulted from deprotection
of the p-methoxybenzyl group, which stained purple in the
presence of phosphomolybdic acid (PMA). The solvents
were evaporated then co-evaporated with water and eth-
anol. A white solid precipitated from the solution, which
was filtered off and washed with water, acetone and finally
ether to remove any impurities then crystallised from
DMF-ethanol to give 2,5-di-O-benzyl-myo-inositol15 16.
2,5-Di-O-benzyl-myo-inositol 16 was prepared in three
steps from dl-1,4-di-O-allyl-3,6-di-O-p-methoxybenzyl-
myo-inositol 11. Compound 11 was benzylated with
benzyl bromide and sodium hydride in DMF. Workup
and purification gave the fully protected compound 14
as a crystalline solid in 92% yield. Several methods were
attempted to deprotect the allyl and p-methoxybenzyl
groups simultaneously, but none of these was satisfac-
tory. However, success was achieved when the allyl
groups were isomerised to the cis-prop-1-enyl ether
1
The H NMR spectrum of 2,5-di-O-benzyl-myo-inositol
16 showed a similar symmetrical pattern to 2,5-di-O-
methyl-myo-inositol and a mass spectrum of compound
Scheme 2. (a) (EtO)2P–Cl, diisopropylethylamine, DMF; (b) t-
BuOOH; (c) TMSBr, CH2Cl2, room temperature, 16 h, then purifica-
tion on Q-Sepharose Fast Flow ion exchange resin. Et, ethyl; Me,
methyl.
Scheme 3. (a) Bis(benzyloxy)diisopropylaminophosphine, 1H-tetra-
zole, DMF, 1 h; (b) S8, pyridine; (c) Na/NH3, then purification on Q-
Sepharose Fast Flow ion exchange resin. Bn, benzyl; Me, methyl.

4248
S. J. Mills, B. V. L. Potter / Bioorg. Med. Chem. 11 (2003) 4245–4253
5
16 could not be obtained due to its inherent insolubil-
ity. 2,5-Di-O-benzyl-myo-inositol 16 was used to
prepare myo-inositol 1,3,4,6-tetrakisphosphorothioate
[Ins(1,3,4,6)PS4 25].
spin coupling pattern to compound 17, where JPP=4.3
Hz. Sulfoxidation was carried out using sulfur in a
mixed solvent of pyridine–DMF, and stirred for 10 min.
The remaining sulfur was filtered off, the solvents were
evaporated in vacuo and the residue was purified by
flash chromatography to give the decabenzyl derivative
24 in 71% yield. This is an efficient method for the
synthesis of phosphorothioates from phosphites and the
reaction time of 10 min is much faster than previously
reported.17 Deprotection of compound 24 with sodium
in liquid ammonia followed by purification by ion
exchange chromatography gave Ins(1,3,4,6)PS4 25 in
46% yield.
2,5-Di-O-methyl-myo-inositol 13 was dissolved in dry
DMF and dry N,N-diisopropylethylamine. The mixture
was cooled, diethoxychlorophosphine added dropwise
and was stirred for 45 min to give the 1,3,4,6-tetraki-
sphosphite intermediate 17. Oxidation of the P(III)
intermediate was carried out using t-butylhydroper-
oxide to give compound 18. Purification by flash chro-
matography was unnecessary since aqueous work up
provided the pure compound as a syrup. The eight ethyl
groups were removed from the protected tetrakispho-
sphate using a two-step deprotection method. Com-
pound 18 was dissolved in dry dichloromethane and
bromotrimethylsilane was added to the mixture under
nitrogen and stirred overnight. In this reaction, the eight
ethyl groups of compound 18 were replaced by tri-
methylsilyl functions in quantitative yield by 31P NMR,
with the release of bromoethane as the by-product. The
solvents were evaporated and water was added to the
residue in order to hydrolyse the temporary tri-
methylsilyl moieties. The crude product was purified by
ion exchange chromatography on Q-Sepharose Fast
Flow using a gradient of 200–1000 mM TEAB buffer
and the compound eluted at ca. 500 mM buffer then
isolated as its glassy triethylammonium salt.
Biological evaluation was carried out for compounds
19, 22 and 25 for the inhibition of Ins(1,4,5)P3 5-phos-
phatase (from human erythrocyte ghosts), Ins(1,4,5)P3
3-kinase (rat brain supernatant) and for Ca2+ release at
the Ins(1,4,5)P3 receptor of electrically permeabilised
SH-SY5Y human neuroblastoma cells.12 All three com-
pounds were poor agonists at the Ins(1,4,5)P3 receptor
mobilising less than 15% of the maximum pool of Ca2+
at 30 mM. Only Ins(1,3,4,6)PS4 25 significantly inhibited
Ins(1,4,5)P3 3-kinase with a Ki value of 46 mM, com-
pared with >100 and 105 mM for compounds 19 and 22
respectively. However, these compounds were potent
inhibitors of Ins(1,4,5)P3 5-phosphatase, where com-
pound 19 had a Ki value of 15.9 mM, while the phos-
phorothioate derivatives 22 and 25 had Ki values of 1.4
and 1.9 mM, respectively. The phosphorothioate com-
pound 22 is 75-fold more potent for the inhibition of 5-
The phosphorothioate derivatives 22 and 25 were pre-
pared using a different P(III) reagent. A mixture of
bis(benzyloxy)diisopropylaminophosphine and 1H-tet-
razole in DMF was stirred for 1 h to give a tetrazolide
intermediate.16 The formation of the complex in DMF
was slower than in dichloromethane, with the appear-
ance of two peaks at d=+ 126.12 and d=+ 127.47
ppm in the 31P NMR spectrum. 2,5-Di-O-methyl-myo-
inositol 13 was then added to the tetrazolide inter-
mediate16 and the mixture was stirred for a further 2 h.
The 31P NMR spectrum at this stage showed two peaks
at d=+141.26 and d=+139.45 ppm, which indicated
the phosphitylated myo-inositol derivative intermediate
20. However, at high resolution only two doublets (cor-
responding to phosphites at positions C-1, C-6, C-3 and
C-4) were observed in the 31P NMR spectrum, as part
of an AB spin coupling pattern where 5JPP=3.9 Hz, due
to the symmetrical nature of the molecule, where C-
1=C-3 and C-4=C-6. Sulfoxidation of this inter-
mediate with sulfur in pyridine–DMF gave the pro-
tected intermediate 21 within 15 min. The sulfur was
filtered off and the solvent was carefully evaporated in
vacuo below 30 ꢀC. The protected phosphorothioate 21
was purified by flash chromatography and isolated in
82% yield to give a syrup. The octabenzyl derivative 21
was deprotected with sodium in liquid ammonia then
purified on Q-Sepharose Fast Flow ion exchange resin
to afford compound 22 (25% yield) which eluted at ca.
800 mM TEAB buffer and was characterised as a trie-
thylammonium salt (Scheme 3).
phosphatase over 3-kinase, and very poor for Ca2+
-
release. For comparison, Ins(1,3,4,6)P4 had a Ki value
of 7.7 mM for 5-phosphatase inhibition, a Ki value of
150 mM for 3-kinase and an EC50 value of 19.6 mM for
Ca2+ release. Thus, methylation of the free hydroxyl
groups and replacement with phosphorothioate moieties
delivered a potent 5-phosphatase inhibitor 22 which
did not release Ca2+ and only interacted weakly with
3-kinase.
In conclusion, we synthesised myo-inositol-2,5-di-O-
methyl 1,3,4,6-tetrakisphosphorothioate 22 and myo-
inositol 1,3,4,6-tetrakisphosphate. These were better
5-phosphatase inhibitors (Ki for compounds 22 and 25
1.4 and 1.9 mM) than their corresponding tetrakispho-
sphates [Ki for compound 19 and Ins(1,3,4,6)P4 was 15.9
and 7.7 mM]. However, changing the trisphosphate
motif to give a trisphosphorothioate increases the
potency of 5-phosphatase inhibition and is more sig-
nificant compared to the two tetrakisphosphorothioate
compounds 22 and 25.12,17 For example, l-chiro-
Ins(2,3,5)P3 has a Ki value of 7.7 mM [the same as
Ins(1,3,4,6)P4], whereas the corresponding trisphos-
phorothioate has a Ki value of 0.23 mM. Compared to
l-chiro-Ins(2,3,5)PS3, tetrakisphosphorothioate 25 is 8-
fold less potent for 5-phosphatase inhibition at 1.9
mM.17 From a previous study,18 Ins(1,3,4,5)P4 was
found to be the most potent 5-phosphatase inhibitor
with an IC50 value of 0.15 mM. This compound is a
better fit at the active site of 5-phosphatase compared to
the tetrakisphosphorothioates 22 and 25 although com-
pounds 22 and 25 are potent inhibitors of the enzyme.
Compound 16 was phosphitylated in a similar way to
compound 13. Intermediate 23 showed a similar AB

S. J. Mills, B. V. L. Potter / Bioorg. Med. Chem. 11 (2003) 4245–4253
4249
ꢀ
Sulfoxidation of phosphites is slow using a mixture of
sulfur in pyridine.17 However, the addition of DMF
speeds up the rate significantly. DMF presumably
accelerates the rate-determining step by increasing the
polarisation of charge between sulfur atoms via dipole–
dipole interactions between the solvent and the atoms in
the S8 ring. We have thus succesfully synthesised two
new tetrakisphosphorothioate compounds (22 and 25)
pound 9. Yield (10.92 g, 84%); mp 136–137 C (from
ethanol); (lit.22 137–139 ꢀC). 1H NMR (Me2SO-d6,
270 MHz) d 2.96 (1H, dd, J=2.4, 9.7 Hz, H-1), 3.01
(1H, dt, J=4.6, 8.8 Hz, H-5), 3.16–3.28 (2H, m, H-3
and H-4), 3.48 (1H, dt, J=4.8, 9.3 Hz, H-6), 3.85 (1H,
br s, H-2), 3.97–4.28 (4H, m, 2ꢂO–CH2CH¼CH2), 4.53
(1H, d, J=6.2 Hz, D2O ex, OH), 4.62 (1H, d, J=3.7
Hz, D2O ex, OH), 4.67 (1H, d, J=4.8 Hz, D2O ex, OH),
4.69 (1H, d, J 4.8 Hz, D2O ex, OH), 5.01–5.33 (4H, m,
2ꢂO–CH2CH¼CH2), 5.83–6.00 (2H, m, 2ꢂO–
CH2CH¼CH2).
and
a
tetrakisphosphate (19) based upon the
Ins(1,3,4,6)P4 structure and discussed their inhibition of
5-phosphatase. We have also developed a simple,
faster method for sulfoxidation of inositol polyphos-
phites than previously reported,17 and this is now our
method of choice for preparing inositol phosphor-
othioates. These compounds will be of use in investi-
gating the polyphosphoinositide pathway of signal
transduction.
DL-1,4-Di-O-allyl-3,6-di-O-p-methoxybenzyl-myo-inositol
(11) and DL-1,4-di-O-allyl-3,5-di-O-p-methoxybenzyl-
myo-inositol (10). A mixture of dl-1,4-di-O-allyl-myo-
inositol 9 (5.2 g, 20 mmol) and dibutyltin oxide (12.5 g,
50 mmol) was suspended in toluene (400 mL) then
heated under reflux for 2.5 h whilst removing water in a
Dean and Stark apparatus. The solution was cooled and
the toluene was evaporated under reduced pressure. The
crystalline tin complex was dried at 130 ꢀC under
reduced pressure for a further 0.5 h. DMF (150 mL) and
CsF (15.19 g, 100 mmol) were added to the tin complex
and the suspension was stirred vigorously under nitro-
gen. p-Methoxybenzyl chloride (12.53 g, 60 mmol) was
added dropwise and the reaction was monitored by
TLC (ether). After 24 h at room temperature, TLC
revealed two products, Rf=0.40 and 0.56. The DMF
was evaporated in vacuo and the residue was parti-
tioned between a saturated aqueous solution of sodium
hydrogen carbonate (300 mL) and dichloromethane
(300 mL). The solution was stirred for 30 min and the
precipitated tin derivatives were filtered off over a bed of
Celite and washed with dichloromethane (2ꢂ100 mL).
The organic layer was separated and washed with water,
saturated brine and water again (200 mL of each). The
dichloromethane solution was dried over magnesium
sulfate and evaporated to give an oil. The mixture was
purified by flash chromatography (ether–light petro-
leum, 2:1) to give the title compounds, 11, (2.41 g,
24%), mp 115–117 ꢀC (from ethyl acetate–hexane),
(anal. calcd for C28H36O8 C, 67.17; H, 7.25; found: C,
66.9; H, 7.28), and 10 (1.2 g, 12%), mp 94–95 ꢀC (from
ethyl acetate–hexane), (anal. calcd for C28H36O8 C,
Experimental
Thin-layer chromatography (TLC) was performed on
pre-coated plates (Merck TLC aluminium sheets silica
60 F254): the product was visualised by spraying with
phosphomolybdic acid in MeOH, followed by heating.
Flash chromatography refers to the procedure devel-
oped by Still and coworkers19 and was carried out on
sorbsil C60 silica gel. The NMR spectra for the nuclei
31P, 1H and 13C were recorded on a Jeol FX-90Q,
GX270, GX400 or GLI EX400 spectrometers. Chemical
shifts were measured in parts per million (ppm) relative
to tetramethylsilane (Me4Si) in CDCl3; deuterium oxide
(D2O) and dimethyl sulfoxide-d6 (Me2SO-d6) were also
used. The 31P NMR shifts were measured in ppm rela-
tive to external 85% phosphoric acid. Coupling con-
stants J, were measured in hertz (Hz). Melting points
(uncorrected) were determined using a Reichert-Jung
Thermo Galen Kofler block, using two glass plates.
Microanalysis was carried out by the University of Bath
Microanalysis Service. Mass spectra were recorded by
the University of Bath Mass Spectrometry Service using
+ve and ꢁve Fast Atom Bombardment (FAB) with 3-
nitrobenzylalcohol (NBA) as the matrix. Ion exchange
chromatography was performed on an LKB-Pharmacia
Medium Pressure Ion Exchange Chromatograph using
Q-Sepharose and gradients of triethylammonium bicar-
bonate (TEAB) as eluent. Fractions containing phos-
1
67.17; H, 7.25; found: C, 67.1; H, 7.20); (11) H NMR
(CDCl3, 270 MHz) d 2.62 (2H, br s, D2O ex, 2ꢂOH),
3.24 (1H, dd, J=2.6, 9.5 Hz, H-3 or H-1), 3.30 (1H, dd,
J=2.6, 9.5 Hz, H-3 or H-1), 3.41 (1H, t, J=9.3 Hz, H-5),
3.68 (1H, t, J=9.3 Hz, H-4 or H-6), 3.76 (1H, t, J=9.5
Hz, H-4 or H-6), 3.77 (3H, s, CH2PhOMe), 3.79 (3H, s,
CH2PhOMe), 4.04–4.42 (5H, m, 2ꢂO–CH2CH¼CH2
and H-2), 4.58–4.85 (4H, m, 2ꢂCH2PhOMe), 5.14–5.33
(4H, m, 2ꢂO–CH2CH¼CH2), 5.87–6.05 (2H, m, 2ꢂO–
CH2CH¼CH2), 6.84–6.90 (4H, m, 2ꢂCH2PhOMe),
7.24–7.31 (4H, m, 2ꢂCH2PhOMe); 13C NMR (CDCl3,
68 MHz) d 55.2, 71.61, 72.29, 74.30, 75.15, 67.75, 74.21,
79.20, 79.40, 80.21, 80.27, 113.80, 116.79, 117.38, 129.44,
129.64, 130.00, 134.67, 135.28, 159.32; (ꢁve ion FAB)
phate and phosphorothioate were assayed by
a
modification of the Briggs phosphate test.17 The phos-
phitylating reagent was prepared by adding benzyl
alcohol to N,N-diisopropylaminodichlorophosphine20
in the presence of triethylamine at ꢁ78 ꢀC. The resulting
product21 was then purified by flash chromatography
(hexane-triethylamine, 10:1) in 96% yield.
DL-1,4-Di-O-allyl-myo-inositol (9). dl-3,6-Di-O-allyl-
1,2:4,5-di-O-isopropylidene-myo-inositol 8 (17 g, 50
mmol) was dissolved in 80% HOAc (200 mL) and the
mixture was heated under reflux for 30 min. The mix-
ture was then cooled and the acetic acid was evaporated
in vacuo to give a solid then co-evaporated with water
(100 mL) to remove any acetic acid. The remaining solid
was recrystallised from ethanol to give the title com-
m/z
653
(M+NBA)ꢁ,
499
(MꢁH)ꢁ,
379
(MꢁCH2PhOMe)ꢁ; (10) 1H NMR (CDCl3, 270 MHz) d
2.48 (1H, br s, D2O ex, OH), 2.57 (1H, br s, D2O ex,
OH), 3.13 (1H, dd, J=2.75, 9.7 Hz, H-3 or H-1), 3.25
(1H, t, J=9.3 Hz, H-5), 3.33 (1H, dd, J=2.7, 9.5 Hz,

4250
S. J. Mills, B. V. L. Potter / Bioorg. Med. Chem. 11 (2003) 4245–4253
H-3 or H-1), 3.76 (1H, t, J=9.5 Hz, H-4 or H-6), 3.79
(3H, s, CH2PhOMe), 3.80 (3H, s, CH2PhOMe), 3.94
(1H, t, J=9.5 Hz, H-4 or H-6), 4.06–4.43 (5H, m,
2ꢂOCH2CH¼CH2 and H-2), 4.60–4.85 (4H, m,
2ꢂCH2PhOMe), 5.16–5.34 (4H, m, 2ꢂCH2CH¼CH2),
5.87–6.05 (2H, m, 2ꢂOCH2CH¼CH2), 6.85–6.90 (4H,
m, 2ꢂCH2PhOMe), 7.25–7.32 (4H, m, 2ꢂCH2PhOMe);
13C NMR (CDCl3, 68 MHz) d 55.23, 67.17, 71.26,
72.00, 72.42, 74.43, 75.08, 78.78, 79.46, 80.56, 82.41,
113.84, 116.57, 117.74, 129.48, 129.61, 130.06, 130.84,
134.50, 135.28, 159.32; (ꢁve ion FAB) m/z 653
(M+NBA)ꢁ, 499 (MꢁH)ꢁ, 460 (MꢁCH2CH¼CH2)ꢁ,
379 (MꢁCH2PhOMe)ꢁ.
D2O ex, 2ꢂOH); 13C NMR (Me2SO-d6, 68 MHz) d
51.29, 59.90, 72.16, 72.58, 83.48, 85.72.
DL-1,4-Di-O-allyl-2,5-di-O-benzyl-3,6-di-O-p-methoxy-
benzyl-myo-inositol (14). A mixture of dl-1,4-di-O-
allyl-3,6-di-O-p-methoxybenzyl-myo-inositol 11 (2.0 g,
4.0 mmol) and sodium hydride (480 mg, 20 mmol) was
stirred in dry DMF (20 mL). Benzyl bromide (1.19 mL,
10 mmol) was added dropwise to the stirred solution at
room temperature. After 2 h, TLC (ether–hexane, 2:1)
showed a new product, Rf=0.60. Methanol (5 mL), was
added to destroy the excess sodium hydride and the
solvent was evaporated in vacuo. The resulting syrup
was partitioned between water and ether, then washed
with brine and water (100 mL of each). The organic
layer was dried over magnesium sulfate and the solvent
was evaporated to give a syrup. Flash chromatography
(ether–hexane, 2:1) removed the remaining benzyl bro-
mide to give compound 14. Yield (2.94 g, 92%); mp 91–
92 ꢀC (from hexane); (anal. calcd for C42H48O8 C, 74.07;
DL-1,4-Di-O-allyl-3,6-di-O-p-methoxybenzyl-2,5-di-O-
methyl-myo-inositol (12). A mixture of sodium hydride
(0.48 g, 20 mmol) and dl-1,4-di-O-allyl-3,6-di-O-p-
methoxybenzyl-myo-inositol 11 (2.0 g, 4 mmol) was
stirred in DMF (50 mL) at room temperature. Methyl
iodide (1.25 mL, 20 mmol) was added dropwise and the
mixture was stirred for 3 h after which TLC (ether)
showed a new product, Rf=0.80. The excess sodium
hydride was destroyed with methanol (10 mL) and the
solvents were evaporated in vacuo. The product was
taken up in ether (100 mL) washed with water, brine
and water again (50 mL of each). The organic layer was
dried over magnesium sulfate, evaporated and purified
by flash chromatography (ether–hexane, 2:1) to give
compound 12. Yield (1.91 g, 90%); mp 93-94 ꢀC (from
hexane), (anal. calcd for C30H40O8 C, 68.15; H, 7.73;
found: C, 68.4; H, 7.73); 1H NMR (CDCl3, 270 MHz) d
3.04 (1H, t, J=9.25 Hz, H-5), 3.14 (1H, dd, J=2.4, 9.9
Hz, H-3 or H-1), 3.33 (1H, dd, J=2.2, 9.9 Hz, H-3 or
H-1), 3.60 (3H, s, Ins-OMe), 3.64 (3H, s, Ins-OMe), 3.66–
3.73 (3H, m, H-2, H-4, H-6), 3.78 (3H, s, CH2PhOMe),
3.79 (3H, s, CH2PhOMe), 4.04–4.39 (4H, m,
2ꢂOCH2CH¼CH2), 4.40–4.85 (4H, m, 2ꢂCH2PhOMe),
5.14–5.32 (4H, m, 2ꢂOCH2CH¼CH2), 5.85–6.07
(2H, m, 2ꢂOCH2CH¼CH2), 6.85–6.90 (4H, m,
2ꢂCH2PhOMe), 7.25–7.32 (4H, m, 2ꢂCH2PhOMe);
13C NMR (CDCl3, 68 MHz) d 55.10, 61.05, 61.20,
71.76, 72.58, 74.27, 75.33, 77.53, 80.04, 80.14, 81.21,
81.24, 85.36, 113.61, 116.34, 116.73, 129.15, 129.65,
130.40, 131.07, 134.44, 135.89, 159.04, 159.09; (ꢁve ion
FAB) m/z 681 (M+NBA)ꢁ, 513 (MꢁMe)ꢁ.
1
H, 7.11; found: C, 74.3; H, 7.04); H NMR (CDCl3,
270 MHz) d 3.21 (1H, dd, J=2.0, 9.9 Hz, H-3 or H-1),
3.27 (1H, dd, J=2.0, 9.9 Hz, H-3 or H-1), 3.37 (1H, t,
J=9.3, H-5), 3.77 (3H, s, CH2PhOMe), 3.79 (3H, s,
CH2PhOMe), 3.90 (1H, t, J=9.3 Hz, H-4 or H-6), 3.96
(1H, t, J=9.9 Hz, H-4 or H-6), 3.97 (1H, br s, H-2),
4.00–4.12 (2H, m, OCH2CH¼CH2), 4.27–4.43 (2H, m,
OCH2CH¼CH2), 4.53, 4.59 (2H, AB, J=11.4 Hz,
CH2PhOMe or CH2Ph), 4.71, 4.80 (2H, AB, J=10.3 Hz,
CH2PhOMe or CH2Ph), 4.84 (4H, br s, 2ꢂCH2PhOMe
or 2ꢂCH2Ph), 5.12–5.33 (4H, m, 2ꢂOCH2CH¼CH2),
5.84–6.03 (2H, m, 2ꢂOCH2CH¼CH2), 6.81 (2H, d,
J=8.6 Hz, CH2PhOMe), 6.86 (2H, d, J=8.6 Hz,
CH2PhOMe), 7.21–7.41 (14H, m, 2ꢂCH2PhOMe and
2ꢂCH2Ph); 13C NMR (CDCl3, 68 MHz) d 55.23, 71.58,
72.52, 73.95, 74.44, 74.53, 75.44, 75.86, 80.50, 80.66,
81.28, 81.44, 83.65, 113.71, 116.57, 127.24, 127.47,
127.76, 127.86, 128.08, 128.31, 129.09, 129.77, 130.61,
131.13, 134.99, 135.51, 138.95, 139.01, 158.12; (ꢁve ion
FAB) m/z 833 (M+NBA)ꢁ.
DL-2,5-Di-O-benzyl-3,6-di-O-p-methoxybenzyl-1,4-di-O-
cis-prop-1-enyl-myo-inositol (15). A mixture of dl-1,4-
di-O-allyl-2,5-di-O-benzyl-3,6-di-O-p-methoxybenzyl-myo-
inositol 14 (2.38 g, 3.38 mmol) and sublimed potassium
t-butoxide (1.57 g, 14 mmol) in dry DMF (40 mL) was
stirred at 85 ꢀC for 2 h under an atmosphere of nitrogen.
TLC (ether–hexane 2:1) showed one product Rf=0.60
which was the same as starting material. The solution
was then cooled, water (100 mL) was added and the
product extracted with dichloromethane (4ꢂ100 mL).
The organic layer was dried over magnesium sulfate,
filtered and purified by flash chromatography (ether–
hexane, 2:1) to give compound 15. Yield (1.90 g, 83%);
mp 108–110 ꢀC (from hexane); (anal. calcd for
C42H48O8 C, 74.07; H, 7.11; found: C, 74.3; H, 7.07); 1H
NMR (CDCl3, 270 MHz): d 1.64 (3H, dd, J=1.5, 7.0
Hz, OCH¼CHCH3), 1.66 (3H, dd, J=1.65, 7.1 Hz,
OCH¼CHCH3), 3.32 (1H, dd, J=2.6, 9.7 Hz, H-3 or
H-1), 3.42 (1H, t, J=9.3 Hz, H-5), 3.51 (1H, dd, J=2.2,
9.7 Hz, H-3 or H-1), 3.77 (3H, s, CH2PhOMe), 3.79
(3H, s, CH2PhOMe), 3.98 (1H, br s, H-2), 4.02 (1H, t,
J=9.5 Hz, H-4 or H-6), 4.14 (1H, t, J=9.9 Hz, H-4 or
2,5-Di-O-methyl-myo-inositol (13). A mixture of dl-1,4-
di-O-allyl-3,6-di-O-p-methoxybenzyl-2,5-di-O-methyl-myo-
inositol 12 (1.056 g, 2 mmol) and palladium on acti-
vated charcoal, (10% Fluka, 0.30 g) and toluene-p-sul-
fonic acid (0.10 g, 0.52 mmol) was dissolved in a mixed
solvent of ethanol (55 mL) and water (5 mL) then
refluxed for 24 h after which, TLC (chloroform–metha-
nol, 3:1) showed a single product Rf=0.2. The solution
was filtered through Celite and recrystallised from eth-
anol to give compound 13. Yield (0.321 g, 77%); mp
266–268 ꢀC (from ethanol); (lit.14 270 ꢀC); (anal. calcd
for C8H16O6 C, 46.13; H, 7.75; found: C, 46.1; H, 7.70);
1H NMR (Me2SO-d6, 270 MHz) d 2.65 (1H, t, J=9.0
Hz, H-5), 3.19 (2H, ddd, J=2.6, 5.3, 9.9 Hz, H-3 and
H-1), 3.35 (1H, br s, H-2), 3.36 (2H, dt, J=4.95, 10.1
Hz, H-4 and H-6), 3.44 (6H, s, 2ꢂOMe), 4.57 (2H, d,
J=5.1 Hz, D2O ex, 2ꢂOH), 4.67 (2H, d, J=4.8 Hz,

S. J. Mills, B. V. L. Potter / Bioorg. Med. Chem. 11 (2003) 4245–4253
4251
H-6), 4.35 (1H, dq, J=6.8 Hz, OCH¼CHCH3), 4.44
(1H, dq, J=6.8 Hz, OCH¼CHCH3), 4.51–4.82 (8H, m,
CH2PhOMe and CH2Ph), 6.08 (1H, dd, J=1.65, 6.2
Hz, OCH¼CHCH3), 6.26 (1H, dd, J=1.65, 6.4 Hz,
OCH¼CHCH3), 6.84–6.90 (4H, m, CH2PhOMe), 7.22–
7.41 (14H, m, CH2PhOMe and CH2Ph); 13C NMR
(CDCl3, 68 MHz) d 9.33, 9.40, 55.23, 72.29, 74.46,
75.30, 75.73, 75.99, 78.42, 80.36, 82.57, 82.92, 84.39,
98.14, 100.77, 113.71, 127.36, 127.59, 127.82, 128.11,
128.27, 129.24, 129.96, 130.35, 130.80, 138.59, 138.75,
145.75, 147.77, 159.18; (ꢁve ion FAB) m/z 833
(M+NBA)ꢁ, 559 (M–PMB)ꢁ.
Hz P(O)OCH2CH3); (+ve ion FAB) m/z 753 (M+H)+,
Accurate mass spectrum requires: (M+H)+=753.2182;
found: 753.2231.
2,5-Di-O-methyl-myo-inositol 1,3,4,6-tetrakisphosphate
(19).
2,5-Di-O-methyl-1,3,4,6-tetrakis(diethoxyphos-
pho)-myo-inositol 18 (0.25 g, 0.33 mmol) was dissolved
in dry dichloromethane (5 mL) and kept under a blan-
ket of nitrogen at room temperature. Trimethylsilyl
bromide (0.70 mL, 5.3 mmol) was added dropwise and
the solution was stirred for 16 h at room temperature.
Evaporation gave a syrupy residue which was dissolved
in water (5 mL) then stirred for 1 h to give compound
19 in quantitative yield by 31P NMR. A small portion
was purified by ion exchange chromatography, using a
buffer gradient of 200–1000 mM of TEAB at a flow rate
of 5 mL/min. Yield (43.5 mmol) which eluted at 500 mM
buffer; 1H NMR (D2O, 270 MHz) d 3.51 (3H, br s,
OMe), 3.57 (4H, br s, OMe and H-5), 3.97 (1H, br s, H-
2), 4.06 (2H, t, J=9.5 Hz, H-3 and H-1), 4.28 (2H, q,
J=9.4 Hz, H-4 and H-6); 13C NMR (D2O, 68 MHz) d
59.64, 61.39, 73.97, 75.66, 75.76, 76.21, 80.01, 81.60; 31P
NMR (D2O, 162 MHz) d 0.00 (1H, d, J=9.9 Hz, 2P),
ꢁ0.58 (1H, d, J=8.0 Hz, 2P); (ꢁve ion FAB) m/z 527
2,5-Di-O-benzyl-myo-inositol (16). 2,5-Di-O-benzyl-3,6-
di-O-p-methoxybenzyl-1,4-di-O-cis-prop-1-enyl-myo-
inositol 15 (0.42 g, 0.62 mmol) was stirred in a mixture
of dichloromethane–trifluoroacetic acid [20 mL, (10:1)]
at room temperature overnight. TLC (ether) showed the
presence of a p-methoxybenzyl derivative only, and the
orange-red solution was evaporated to dryness. The
mixture was co-evaporated with water then ethanol (10
mL of each) to remove traces of acid, after which a fine
white solid precipitated from the solution. The solid was
filtered off and washed successively with water, acetone
and finally ether (20 mL of each) to give 16. Yield (0.17
g, 76%); mp 271–273 ꢀC (from DMF–ethanol); (lit.15
270–272 ꢀC); (anal. calcd for C20H24O6 C, 66.67; H,
(MꢁH)ꢁ;
Accurate
mass
spectrum
requires:
(MꢁH)=526.9520; found: 526.9520.
1
6.67; found: C, 66.4; H, 6.64); H NMR (Me2SO-d6,
2,5-Di-O-methyl-1,3,4,6-tetrakis[di(benzyloxyphosphoro-
thio)]-myo-inositol (21). A mixture of bis(benzyloxy)-
diisopropylaminophosphine (0.69 g, 2 mmol) and 1H-
tetrazole (350 mg, 5 mmol) was stirred in DMF (5 mL)
for 1 h. 2,5-Di-O-methyl-myo-inositol 13 (0.052 g, 0.25
mmol) was added to the mixture and stirred for a fur-
ther 2 h. TLC (ether–petroleum ether, 2:1) showed a
major product Rf=1.00 for the phosphite. Sulfur (0.096
g, 3 mmol) and dry pyridine (2 mL) were added and the
mixture stirred for 15 min after which TLC (ether–pet-
roleum ether, 2:1) showed a new product Rf=0.60. The
excess sulfur was filtered off and the solvents were eva-
porated in vacuo to give a syrup. Compound 21 was
purified by flash chromatography (ether–petroleum
ether, 2:1) and isolated as a syrup. Yield (0.27 g, 82%);
(anal. calcd for C64H68O14P4S4 C, 58.53; H, 5.22; found:
270 MHz) d 3.03 (1H, t, J=9.2 Hz, H-5), 3.33 (2H, ddd,
J=2.6, 4.8, 9.7 Hz, H-3 and H-1), 3.60 (2H, dt, J=5.1,
9.3 Hz, H-4 and H-6), 3.73 (1H, t, J=2.75 Hz, H-2),
4.75 (2H, d, J=4.8 Hz, D2O ex, OH-1 and OH-3), 4.78
(4H, s, 2ꢂCH2Ph), 4.83 (2H, d, J=5.1 Hz D2O ex, OH-
4 and OH-6), 7.20–7.43 (10H, m, 2ꢂCH2Ph); 13C NMR
(Me2SO-d6, 68 MHz) d 73.75, 74.17, 72.19, 73.07, 81.83,
84.23, 127.01, 127.53, 127.92, 127.98, 139.92, 140.01.
2,5-Di-O-methyl-1,3,4,6-tetrakis(diethoxyphospho)-myo-
inositol (18). A mixture of 2,5-di-O-methyl-myo-inositol
13 (0.104 g, 0.5 mmol) and dry diisopropylethylamine
(0.7 mL, 4 mmol) was dissolved in dry DMF (5 mL) and
kept under nitrogen at ꢁ78 ꢀC. Diethoxychlorophos-
phine (0.58 mL, 4 mmol), (90–95%) was added drop-
wise to the stirred solution and left for 45 min. The
mixture was oxidised with t-butylhydroperoxide (1 mL,
7.3 mmol) at ꢁ78 ꢀC and stirred for a further 30 min at
room temperature. The DMF was evaporated in vacuo
and the remaining syrup was partitioned between water
(50 mL) and dichloromethane (50 mL). The organic
layer was washed with 10% sodium metabisulfite solu-
tion, brine (20 mL of each) and finally water (2ꢂ20
mL). The organic layer was dried over magnesium sul-
fate and evaporated to give compound 18 Rf=0.40
1
C, 58.3; H, 5.21); H NMR (CDCl3, 270 MHz) d 2.23
(1H, t, J=9.3 Hz, H-5), 3.31 (3H, s, OMe), 3.50 (3H, s,
OMe), 4.33 (2H, dt, 2.0, 9.9 Hz, H-3 and H-1), 4.61
(1H, br s, H-2), 5.00–5.14 (18H, m, 8ꢂP(S)O–CH2Ph,
H-4 and H-6), 7.20–7.37 (40H, m, 8ꢂP(S)O–CH2Ph);
13C NMR (CDCl3, 68 MHz) d 61.29, 61.33, 69.47,
70.09, 76.02, 77.00, 77.25, 79.10, 79.30, 80.66, 127.69,
127.82, 127.89, 127.95, 128.08, 128.24, 135.18, 135.28,
135.41, 135.48, 135.57, 135.67, 135.80; 31P (CDCl3,
162 MHz) d+69.11 (dtt, J=8.0, 9.9, 9.9 Hz,
8ꢂP(S)OCH2Ph),+67.09 (dtt, J=7.9, 9.9, 9.9 Hz,
P(S)OCH2Ph); (+ve ion FAB) m/z 1314 (M+H)+.
1
(chloroform–methanol, 3:1). Yield (0.286 g, 76%); H
NMR (CDCl3, 270 MHz):
d 1.26–1.39 (24H, m,
8ꢂP(O)OCH2CH3), 3.23 (1H, t, J=9.4 Hz, H-5), 3.57
(3H, s, OMe), 3.63 (3H, s, OMe), 4.12–4.27 (18H,
8ꢂP(O)OCH2CH3, H-3 and H-1), 4.40 (1H, br s, H-2),
4.74 (2H, q, J=9.5, H-4 and H-6); 13C NMR (CDCl3,
68 MHz) d 16.05, 16.15, 59.64, 61.78, 63.89, 63.99,
64.09, 64.22, 64.31, 64.44, 64.51, 75.70, 76.28, 76.41,
76.51, 78.00, 80.79; 31P NMR (CDCl3, 109 MHz) d
ꢁ2.63 (q, J=7.5 Hz, P(O)OCH2CH3), ꢁ3.15 (q, J=7.5
2,5-Di-O-methyl-myo-inositol 1,3,4,6-tetrakisphosphoro-
thioate (22). Ammonia (80 mL) was distilled into a
three-neck flask and small slithers of freshly cut sodium
metal (0.80 g, 34.8 mmol) were added until the solution
remained blue. The dry-ice condenser was moved across
to the reaction flask and ammonia (40 mL) was gently
transferred to the flask by heating. Small slithers of

4252
S. J. Mills, B. V. L. Potter / Bioorg. Med. Chem. 11 (2003) 4245–4253
sodium (0.40 g, 17.4 mmol) were added to the ammonia
until the colour remained blue. 2,5-Di-O-methyl-1,3,4,6-
tetrakis[di(benzylphosphorothio)]-myo-inositol 21 (0.10
g, 76 mmol) in dry dioxan (1 mL) was added to the
sodium in liquid ammonia. The reaction was left for 2
min and quenched with methanol (20 mL). The ammo-
nia was then evaporated in a stream of nitrogen. MilliQ
water was then added to the residue and evaporated to
dryness in vacuo. The deprotected phosphorothioate 22
was purified by ion exchange chromatography using a
buffer gradient of 0–1000 mmol and eluted at ca. 800
mmol. Yield (19.2 mmol, 25%); 1H NMR (D2O,
270 MHz) d 3.32 (1H, t, J=9.5 Hz, H-5), 3.60 (3H, s,
OMe), 3.64 (3H, s, OMe), 4.15 (1H, t, J=2.4 Hz, H-2),
4.23 (2H, ddd, J=2.3, 10.3, 10.3 Hz, H-3 and H-1), 4.60
(2H, q, J=10.0, H-4 and H-6); 13C NMR (D2O,
68 MHz) d 59.57, 61.65, 74.07, 76.44, 80.14, 82.02; 31P
NMR (D2O, 109 MHz) d+46.7 (d, J=10.1 Hz, 2P),+48.8
(d, J=10.1 Hz, 2P); (ꢁve ion FAB) m/z 591 (MꢁH)ꢁ;
Accurate mass spectrum requires: (MꢁH)ꢁ=590.8662;
found: 590.8608.
the sodium in liquid ammonia. The reaction was left for
2 min and quenched with methanol (20 mL). The
ammonia was evaporated in a stream of nitrogen and
MilliQ water was then added to the residue then evap-
orated to dryness. The deprotected phosphorothioate 25
was purified by ion exchange chromatography using a
buffer gradient of 0–1000 mmol and eluted at ca. 800
mmol. Yield (18.5 mmol, 46%); 1H NMR (D2O,
270 MHz) d 3.61 (1H, t, J=9.0 Hz, H-5), 4.16 (1H, dt,
J=2.95, 9.5 Hz, H-1 and H-3), 4.51 (2H, q, J=9.9, H-4
and H-6), 4.99 (1H, br s, H-2); 31P NMR (D2O,
109 MHz) d+42.4 (d, J=12.25 Hz, 2P),+45.09 (d,
J=9.8 Hz, 2P); (ꢁve ion FAB) m/z 563 (MꢁH)ꢁ;
accurate mass spectrum requires: (MꢁH)ꢁ=562.8295;
found: 562.8314.
Acknowledgements
We thank the Welcome Trust (Programme Grant no.
060554 to B.V.L.P.) for financial support.
2,5-Di-O-benzyl-1,3,4,6-tetrakis[di(benzyloxyphosphoro-
thio)]-myo-inositol (24). A mixture of bis(benzyloxy)-
diisopropylaminophosphine (0.69 g, 2 mmol) and 1H-
tetrazole (0.35 g, 5 mmol) in DMF (2 mL) was stirred
for 1 h. 2,5-Di-O-benzyl-myo-inositol 16 (0.90 g, 0.25
mmol) was added to the mixture and stirred for a fur-
ther 1 h. TLC (ether–petroleum ether, 1:2) showed a
major product Rf=1.00 for the phosphite intermediate.
Sulfur (0.096 g, 3 mmol) and dry pyridine (2 mL) were
added and stirred for 10 min after which, TLC (ether–
petroleum ether, 1:2) showed a new product Rf=0.40.
The excess sulfur was filtered off and the solvents were
evaporated in vacuo at ambient temperature. Com-
pound 24 was purified by flash chromatography (ether–
petroleum ether, 1:2) and isolated as a syrup. Yield
(0.26 g, 71%); (anal. calcd for C76H76O14P4S4 C, 62.29;
References and Notes
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1
H, 5.23; found: C, 62.5; H, 5.32); H NMR (CDCl3,
400 MHz) d 3.59 (1H, t, J=9.15 Hz, H-5), 4.42–4.48
(3H, m, H-1, H-2 and H-3), 4.78–5.07 (20H, m,
8ꢂP(S)O–CH2Ph and 2ꢂCH2Ph), 5.38 (2H, dt, J=9.15,
12.5 Hz, H-4 and H-6), 6.86–7.37 (50H, 8ꢂP(S)O–
CH2Ph and 2ꢂCH2Ph); 13C NMR (CDCl3, 68 MHz) d
69.99, 70.19, 70.25, 70.54, 70.74, 74.01, 75.83, 76.51,
76.93, 77.84, 79.30, 127.01, 127.27, 127.53, 127.66,
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128.93, 135.74, 135.87, 135.93, 136.06, 136.13, 136.23,
136.36, 136.49, 138.75, 138.85; 31P NMR (CDCl3,
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such regulates Ca2+ signalling. Type II 5-phosphatase7b
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