A Conformationally Restricted Cyclic Phosphate Analogue of Inositol Trisphosphate: Synthesis and Physicochemical Properties

Article abstract of DOI:10.1021/jo9714425

The design and total synthesis of DL-6-deoxy-6-(hydroxymethyl)-scyllo-inositol 1:7-cyclic 2,4-trisphosphate (4), a conformationally restricted cyclic phosphate analogue of the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃], is described. The protected inosose 2,4,6/3,5-pentahydroxy-3,5-bis-O-(p-methoxybenzyl)-2,4,6-O- methylidynecyclohexanone (7) was obtained from myoinositol orthoformate in two steps, and Wittig methylenation and then hydroboration-oxidation using 9-BBN-H/OH^-/H₂O₂ gave the axial hydroxymethyl derivative 9. A series of protection/ deprotection steps provided the diol 13, which was converted into two cyclic phosphate esters 14a and 14b, epimeric at phosphorus, by reaction with (benzyloxy)bis(N,N-diisopropylamino)phosphine/ 1H-tetrazole followed by m-CPBA. Two other hydroxyl groups were then exposed and phosphorylated, and total deprotection gave racemic 4. NMR studies confirmed that in 4 the phosphate group equivalent to the 4-phosphate of Ins(1,4,5)P₃ is held in the positive gauche orientation and that the inositol ring maintains a chair conformation from pH 2 to 12. Investigation of the acid-base properties of 4 using potentiometric and ³¹P NMR techniques showed that, over the physiological pH range, 4 behaves as a diprotic acid and that the ionization of the phosphate group equivalent to the 5-phosphate of Ins(1,4,5)P₃ is enhanced. In biological assays, 4 appears to behave as a weak full agonist at the platelet Ins(1,4,5)P₃ receptor, and the possible interpretation of this result is discussed.

Full text of DOI:10.1021/jo9714425

J . Org. Chem. 1998, 63, 295-305
295
A Con for m a tion a lly Restr icted Cyclic P h osp h a te An a logu e of
In ositol Tr isp h osp h a te: Syn th esis a n d P h ysicoch em ica l
P r op er ties
Andrew M. Riley,† Philippe Gue´dat,‡ Gilbert Schlewer,‡ Bernard Spiess,‡ and
Barry V. L. Potter*,†
Department of Medicinal Chemistry, School of Pharmacy and Pharmacology, University of Bath,
Claverton Down, Bath BA2 7AY, U.K., and Laboratoire de Pharmacochimie Mole´culaire,
Faculte´ de Pharmacie, Universite Louis Pasteur, Strasbourg, France
Received August 4, 1997X
The design and total synthesis of DL-6-deoxy-6-(hydroxymethyl)-scyllo-inositol 1:7-cyclic 2,4-
trisphosphate (4), a conformationally restricted cyclic phosphate analogue of the second messenger
inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], is described. The protected inosose 2,4,6/3,5-pentahy-
droxy-3,5-bis-O-(p-methoxybenzyl)-2,4,6-O-methylidynecyclohexanone (7) was obtained from myo-
inositol orthoformate in two steps, and Wittig methylenation and then hydroboration-oxidation
using 9-BBN-H/OH-/H2O2 gave the axial hydroxymethyl derivative 9. A series of protection/
deprotection steps provided the diol 13, which was converted into two cyclic phosphate esters 14a
and 14b, epimeric at phosphorus, by reaction with (benzyloxy)bis(N,N-diisopropylamino)phosphine
/1H-tetrazole followed by m-CPBA. Two other hydroxyl groups were then exposed and phospho-
rylated, and total deprotection gave racemic 4. NMR studies confirmed that in 4 the phosphate
group equivalent to the 4-phosphate of Ins(1,4,5)P3 is held in the positive gauche orientation and
that the inositol ring maintains a chair conformation from pH 2 to 12. Investigation of the acid-
base properties of 4 using potentiometric and 31P NMR techniques showed that, over the
physiological pH range, 4 behaves as a diprotic acid and that the ionization of the phosphate group
equivalent to the 5-phosphate of Ins(1,4,5)P3 is enhanced. In biological assays, 4 appears to behave
as a weak full agonist at the platelet Ins(1,4,5)P3 receptor, and the possible interpretation of this
result is discussed.
1. In tr od u ction
tors have contained a structure equivalent to the vicinal
trans-4,5-bisphosphate of Ins(1,4,5)P3, although isosteres
such as phosphorothioate5 may be tolerated to some
extent.
D-myo-Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3, 1] func-
tions as an intracellular signaling molecule by interacting
with a family of receptor-operated Ca2+ channels to
mobilize nonmitochondrial Ca2+ in many cell types.1
Ins(1,4,5)P3 receptors (IP3Rs) are now known to be
tetrameric with a central ion channel, and the four
subunits may be of different subtypes, allowing the
existence of heterotetramers.2 It is currently assumed
that each IP3R subunit has a positively charged pocket
containing basic amino acid residues that engage in ionic
interactions with the negative charges on the three
phosphate groups of the Ins(1,4,5)P3 molecule,3 although
no direct information is yet available on the three-
dimensional structure of the binding site, nor of the
receptor-bound conformation of Ins(1,4,5)P3. Many ana-
logues of Ins(1,4,5)P3 have been synthesized4 and to date
all molecules behaving as agonists at Ins(1,4,5)P 3 recep-
Previously, we demonstrated that linking of the vicinal
phosphates in Ins(1,4,5)P3 to give the 1-phosphate-4,5-
pyrophosphate analogue 2 abolishes Ca2+-releasing activ-
ity.6 Although 2 may be regarded as a conformationally
restricted analogue of Ins(1,4,5)P3, it differs from Ins-
(1,4,5)P3 in two important ways. First, the formation of
the cyclic pyrophosphate in 2 reduces the negative charge
on the equivalents of both the 4- and 5-phosphate groups
in Ins(1,4,5)P3. Physicochemical studies by some of us
have shown that the affinity of Ins(1,4,5)P3 for its
receptor is correlated with the degree of ionization of the
phosphate group at position 5, suggesting that binding
affinity will be maximized for a fully ionized 5-phos-
phate.7 Second, it is probable that 2 mimics a high-
energy conformation of Ins(1,4,5)P3, unlikely to exist in
appreciable amounts in solution or at the IP3R binding
site, due to electrostatic repulsion between the two vicinal
phosphates. The remaining pyrophosphate oxygens would
therefore be incorrectly placed to interact with the
appropriate basic amino acid residues in the Ins(1,4,5)-
P3 binding pocket. We therefore set out to design a
* Address correspondence to this author at School of Pharmacy and
Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY,
U.K. Tel: 1225-826639. Fax: 1225-826114. Email: B.V.L.Potter@
bath.ac.uk.
† University of Bath.
‡ Universite Louis Pasteur.
X Abstract published in Advance ACS Abstracts, December 15, 1997.
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34, 1933-1972.
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sis, Biochemistry and Therapeutic Potential; Reitz, A. B., Ed.; American
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(6) Noble, N. J .; Dubreuil, D.; Potter, B. V. L. Bioorg. Med. Chem.
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S0022-3263(97)01442-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/01/1998

296 J . Org. Chem., Vol. 63, No. 2, 1998
Riley et al.
Sch em e 1a
different conformationally restricted Ins(1,4,5)P3 ana-
logue that would mimimize these difficulties by con-
straining only the 4-phosphate group, preferably in an
orientation that would mimic a low-energy conformation
of the 4,5-bisphosphate in Ins(1,4,5)P3. It would be
interesting to see whether such an analogue could retain
some of the binding affinity of Ins(1,4,5)P3 or show novel
activity in its interaction with the Ins(1,4,5)P3 receptor
or Ins(1,4,5)P3-metabolizing enzymes.
a
Key: (a) NaH (2.1 equiv), PMBCl (2.0 equiv), DMF; (b) DMSO,
(COCl)2, CH2Cl2, -60 °C and then Et3N. PMB ) p-methoxybenzyl.
5J Ha-Hb ) 0.9 Hz.
synclinal were energetically more favorable than those
having the antiperiplanar conformation. This suggested
that the cyclohexane and dioxaphosphorinane rings of the
target molecule should be fused in a trans sense, to mimic
a low-energy conformation. Taken together then, all
these considerations suggested the initial target 4. Ret-
rosynthetic analysis of 4 led to a symmetrical precursor
which, in turn, could be synthesized from myo-inositol
orthoformate. This strategy would initially lead to
racemic 4 but should be capable of modification to give
optically pure material by resolution of an intermediate
should this prove necessary. We report here a synthetic
route to racemic 4 and an examination of its inframo-
lecular acid-base properties. Some of the synthetic work
has been presented in Communication form.15
To constrain the 4-phosphate group it would be neces-
sary to tether it to the carbon atom at position 3 of the
inositol ring, to give a cyclic phosphate. This would, in
turn, require the removal of the 3-hydroxyl group, and
its replacement with a methylene bridge. This strategy
is valid because it had previously been shown that
3-deoxy-Ins(1,4,5)P3 is highly active,8,9 and it is also
known that the Ins(1,4,5)P3 receptor can accommodate
slightly increased bulk around the 3-position.10 In con-
trast, steric bulk at position 6 is poorly tolerated,11 and
deletion of the 6-hydroxyl group leads to a 70-fold
reduction in activity,12 affirming the importance of
retaining the 6-hydroxyl group in the target structure.
We had previously shown that the scyllo analogue of Ins-
(1,4,5)P3 [scyllo-Ins(1,2,4)P3, 3]13 in which the relative
stereochemistry is all-trans, showed high potency, ap-
proaching that of Ins(1,4,5)P3 itself. The implication of
this, that it was not necessary to retain the myo-inositol
configuration in the target molecule, would greatly
simplify the synthetic strategy. This conjecture was later
borne out by the finding that the then newly discovered
adenophostins showed remarkably high potency at the
IP3R, while lacking an equivalent to the axial 2-hydroxyl
group of Ins(1,4,5)P3.14
2. Resu lts a n d Discu ssion
2.1 Syn th esis. Reaction of myo-inositol orthoformate
(5)16 with 2.1 equiv of p-methoxybenzyl chloride and 2.3
equiv of sodium hydride gave the symmetrical 4,6-
disubstituted alcohol 6 as the highly crystalline major
product, easily recognizable from the NMR spectra by its
plane of symmetry. With careful workup and chroma-
tography, it was possible to increase the yield of 6 to
around 40% (Scheme 1).
Swern oxidation of 6 using DMSO/oxalyl chloride was
expected to give the ketone 7. However, after aqueous
workup, an IR spectrum of the product showed two bands
at 3540 and 3440 cm-1, suggesting two alcohol groups,
together with a band at 1760 cm-1, corresponding to the
expected ketone. The 13C NMR spectrum of the product
showed the unusual feature of a quaternary carbon
resonating at δ 88.67, corresponding to C(OH)2, confirm-
ing that 7 had been partially converted into the gem-diol
7a . After the mixture of products was refluxed in
toluene, with azeotropic removal of water, pure ketone
7 was isolated in 92% yield. The highly crystalline gem-
diol 7a could also be isolated by allowing a solution of
the ketone in dioxane with a few drops of water added
to stand for a few days. The ease of hydration of ketone
7 may be attributable to strain effects in its rigid cagelike
structure, resulting in destabilization of sp2-hybridized
carbon relative to sp3, and since our preliminary report15
a similar phenomenon has been reported for a related
compound.17 Ketone 7 also reacted with methanol at
room temperature to give a single hemiketal.
Finally, our own molecular modeling investigations of
Ins(1,4,5)P3 showed that conformers of Ins(1,4,5)P3 in
which the P4-O4-C4-H4 torsion angle was positive and
(8) Seewald, M. J .; Aksoy, I. A.; Powis, G.; Fauq, A. H.; Kozikowski,
A. P. J . Chem. Soc., Chem. Commun. 1990, 1638-1639.
(9) Kozikowski, A. P.; Ognyanov, V. I.; Fauq, A. H.; Nahorski, S.
R.; Wilcox, R. A. J . Am. Chem. Soc. 1993, 115, 4429-4434.
(10) Liu, C.; Potter, B. V. L. Tetrahedron Lett. 1994, 35, 8457-8460;
Liu, C.; Potter, B. I. L. J . Org. Chem. 1997, 62, 8335-8340.
(11) Polokoff, M. A.; Bencen, G. H.; Vacca, J . P.; DeSolms, S. J .;
Young, S. D.; Huff, J . R. J . Biol. Chem. 1988, 263, 11922-11927.
(12) Safrany, S. T.; Wojcikiewicz, R. J . H.; Strupish, J .; Nahorski,
S. R.; Dubreuil, D.; Cleophax, J .; Gero, S. D.; Potter, B. V. L. FEBS
Lett. 1991, 278, 252-256.
(15) Riley, A. M.; Potter, B. V. L. J . Org. Chem. 1995, 60, 4970-
4971.
(16) Lee, H. W.; Kishi, Y. J . Org. Chem. 1985, 50, 4402-4404.
(17) Wu, Y.; Zhou, C.; Roberts, M. F. Biochemistry 1997, 36, 356-
363.
(13) Lampe, D.; Potter, B. V. L. Tetrahedron Lett. 1993, 34, 2365-
2368.
(14) Takahashi, M.; Tanzawa, K.; Takahashi, S. J . Biol. Chem. 1994,
269, 369-372.

Cyclic Phosphate Analog of Inositol Trisphosphate
J . Org. Chem., Vol. 63, No. 2, 1998 297
Sch em e 2a
Sch em e 3a
a
Key: (a) CH3PPh3Br, t-BuOK, THF, reflux; (b) (i) 9BBN-H,
THF, 50 °C, (ii) H2O2, OH-; (c) (i) 1 M HCl/MeOH 1:10, 50 °C, (ii)
NH3(aq); (d) C6H5CH(OMe)2, DMF, p-TsOH; (e) NaH, BnBr, DMF;
(f) 1 M HCl/THF/MeOH 1:5:5, reflux. PMB ) p-methoxybenzyl.
Bn ) benzyl. Asymmetrical compounds are racemic.
The next step was the conversion of ketone 7 into the
symmetrical alkene 8 by Wittig methylenation using
methylenetriphenylphosphorane (Scheme 2). In the first
attempts, using methyltriphenylphosphonium bromide,
potassium tert-butoxide as base and heating to 50 °C for
2 h, the yields were consistently low (30 to 50%). Further
investigations showed that the intermediate oxaphos-
phetane, visible in the 31P NMR spectrum as a single
resonance at δP -68.9, was unusually stable. A sample
of this material could be kept for several days at 4 °C
with only slight decomposition. When the reaction was
repeated, but with heating to reflux for 2 h after adding
ketone to Wittig reagent, 8 was obtained in 91% yield.
The alkene 8 was converted into alcohol 9 by hydrobo-
ration/oxidation using 9-BBN-H followed by alkaline
hydrogen peroxide solution, and a single product was
obtained in 97% yield after chromatography. The strat-
egy exploits the fact that one diastereotopic face of alkene
8 is exposed while the other is very hindered by the two
p-methoxybenzyl groups, allowing only one orientation
of approach by the bulky 9-BBN-H molecule. Hydrobo-
ration using borane-THF was less successful, giving
mainly 9, but accompanied by various other minor
products, which were not identified. It was possible to
deduce the stereochemistry of 9 as follows: It has been
a
Key (a) (i) BnOP(NPri2)2, 1H-tetrazole, (ii) m-CPBA, -78 °C;
(b) DDQ, CH2Cl2, H2O; (c) (i) (BnO)2PNPri2, 1H-tetrazole, (ii)
m-CPBA, -78 °C; (d) Na/liq NH3. PMB ) p-methoxybenzyl. Bn )
benzyl. All compounds are racemic.
necessary to add aqueous ammonia after the acid hy-
drolysis stage, to cleave a persistent formate ester
intermediate resulting from partial hydrolysis of the cage.
The H NMR spectrum of 10 showed only axial-axial J
couplings between ring protons, confirming the previ-
ously deduced stereochemistry of the precursor 9 beyond
doubt.
Two protection steps were now used to produce the
fully protected 12. The racemic benzylidene acetal 11
was prepared in 93% yield by the reaction of tetrol 10
with benzaldehyde dimethyl acetal in dry DMF with a
catalytic amount of PTSA at 70 °C. High yields and short
reaction times were made possible by continuous removal
of formed methanol using an air condenser attached to
a filter pump. Benzylation under standard conditions
then gave fully protected 12. This is a versatile inter-
mediate19 because either the benzylidene or p-methoxy-
benzyl groups can be removed chemoselectively or the
benzylidene ring can be reduced regioselectively in either
direction.
1
3
1
noted that the H NMR spectra of myo-inositol orthofor-
The benzylidene acetal was removed selectively by mild
acid hydrolysis, giving diol 13, which was reacted with
1.2 equiv of the bifunctional phosphitylating agent (ben-
zyloxy)bis(N,N-diisopropylamino)phosphine20 in the pres-
ence of 3 equiv of 1H-tetrazole to give two cyclic phosphite
triesters, epimeric at phosphorus as expected (Scheme
3). These intermediates were visible as two spots close
together on the TLC plate, and also as two distinct
signals at δP 125.0 and δP 130.4 in the 31P NMR spectrum.
These signals disappeared and were replaced by peaks
at δP -4.6 and -7.5 after addition of m-CPBA. It was
possible to separate the two epimeric cyclic phosphate
mate and many of its derivatives show a long-range five-
bond coupling (typically around 1 Hz) between the
methylidyne proton and the axial proton at C-2.18 Such
1
a coupling was observed in the H NMR spectrum of 6
(Scheme 1). Now the corresponding signal in the spec-
trum of the hydroboration/oxidation product was a sharp
singlet (δ 5.58). The fact that there was no coupling to
the equivalent proton of H-2 was evidence that this
proton was no longer axial and that the product therefore
had the desired structure 9.
Mild acid treatment removed the orthoformate ester
without significant loss of p-methoxybenzyl groups to give
the tetrol 10. To maximize the yield of 10, it was
(19) Riley, A. M.; Murphy, C. T.; Lindley, C. J .; Westwick, J .; Potter,
B. V. L. Bioorg. Med. Chem. Lett. 1996, 6, 2197-2200.
(20) Dreef, C. E.; Elie, C. J . J .; Hoogerhout, P.; van der Marel, G.
A.; van Boom, J . H. Tetrahedron Lett. 1988, 29, 6513-6516.
(18) Baudin, G.; Gla¨nzer, B. I.; Swaminathan, K. S.; Vasella, A. Helv.
Chim. Acta 1988, 71, 1367-1378.

298 J . Org. Chem., Vol. 63, No. 2, 1998
Riley et al.
triesters by column chromatography, and they were
isolated as crystalline solids.
The configurations at phosphorus of the two epimers
were determined by examination of their 31P and 1H NMR
3
chemical shifts, J HCOP coupling constants and PdO
stretching frequencies. Epimers 14a and 14b can be
regarded as esters of 2-oxo-1,3,2-dioxaphosphorinanes,
and studies have been published on simple related
compounds.21-23 In all previous studies on isomeric pairs
of phosphorinanes (see ref 23 and references therein) the
axially substituted epimer has an upfield 31P chemical
shift. This identifies the less polar epimer (δP -7.5) as
14a and the more polar isomer (δP -4.6) as the equato-
rially substituted 14b. The PdO stretching frequency
in the IR spectrum of 14b is 21 cm-1 lower than that for
14a (νPdO 1287 cm-1), confirming this assignment.
F igu r e 1. 162 MHz 31P NMR spectrum of 4 (1H-coupled, Na+
salt, D2O, pH ∼8).
The p-methoxybenzyl ethers of 14a and 14b were
cleaved with DDQ, giving the corresponding diols 15a
and 15b. TLCs taken during the reaction suggested that
a competing reaction was loss of the benzyl protecting
group on the cyclic phosphate, but the diols were isolated
phate groups at positions 2 and 4 appearing as doublets
3
with J HP approximately 7 Hz. The signal from the cyclic
phosphate phosphorus atom (δP -2.76) is also a doublet,
with a large coupling constant (3J P-Heq ) 22.5 Hz). The
1
in moderate yields. A closer examination of the H NMR
3
predicted J HP coupling constant for a dihedral angle of
spectra of 14a , 14b, 15a , and 15b revealed further
evidence for the assigned configurations at phosphorus.
In the equatorially protected epimers 14b and 15b, H-1
and H-7ax are deshielded, as expected for protons in a
2,4-diaxial relationship with a PdO group. These pro-
tons therefore resonate downfield of the corresponding
protons in 14a and 15a , while the H-7eq protons have
similar chemical shifts in all four molecules (approxi-
mately δ 4.4).
180° is approximately 23 Hz,25 and so we can conclude
that the angle HeqCOP is close to 180°. This suggests
that the dioxaphosphorinane ring adopts a chair confor-
mation as shown in Figure 1.
2.2 P h ysicoch em istr y. We now proceeded to inves-
tigate the physicochemical properties of 4 by the use of
potentiometric and NMR investigations. Previous
studies26-28 have shown drastic changes in the binding
profile and Ca2+ mobilization of Ins(1,4,5)P3 as well as
of other inositol phosphates as a function of pH. Such
alterations can be of biological significance since intra-
cellular pH varies in subcellular compartments following,
for instance, phospholipase C activation or trophic factor
action.29 Thus, a knowledge of the acid-base properties
of an inositol phosphate may contribute to a better
understanding of the events occurring at a molecular
level. These properties are usually expressed as overall
protonation or dissociation constants which characterize
a molecule as a whole, but a detailed investigation of the
ionization state of each individual phosphate group
provides a more accurate picture of the distribution of
charges in the molecule at any given pH. The protona-
tion process has therefore to be expressed in terms of
microprotonation equilibria, which are characterized by
their related microconstants. These equilibria are influ-
enced in a complex way by various aspects of the
molecular structure, such as the nature and orientation
of neighboring functional groups. We have previously
determined the microprotonation scheme for various bis
and tris phosphates7,30 and analyzed the protonation of
tetrakis phosphates31 at an “inframolecular level”, i.e.,
at a level referring to a unique donor site. The confor-
mationally restricted analogue 4 is unique, however, in
The diols 15a and 15b were next phosphitylated using
the monofunctional phosphitylating agent bis(benzyl-
oxy)(N,N-diisopropylamino)phosphine24 with 1H-tetra-
zole. This step involves the formation of a phosphite
triester at a position vicinal to an existing phosphate
triester, and in each case we were able to observe in the
5
31P NMR spectrum a J PP spin-spin coupling of 1.2 Hz
between the phosphorus atoms of neighboring phosphate
and phosphite groups. To the best of our knowledge, such
a long-range P(III)-P(V) coupling has not previously
been reported, although it is known in vicinal P(III)-
P(III) systems. Oxidation with m-CPBA and purification
gave crystalline 16a and 16b, neither of which showed
any phosphorus-phosphorus couplings.
The axially protected 16a was deprotected using
sodium in liquid ammonia, and purification by ion-
exchange chromatography on Q-Sepharose Fast Flow
gave the target compound 4 in 78% yield as the triethy-
1
lammonium salt. The structure was confirmed by H,
31P, and 13C NMR.
A
1H-1H COSY NMR spectrum
allowed all the proton resonances to be assigned un-
equivocally. The equatorially protected 16b was also
deblocked with equal success.
The proton-coupled 31P NMR spectrum of 4 (Figure 1)
shows clearly the two different classes of phosphate
esters and the expected heteronuclear spin couplings,
with the phosphorus atoms in the unconstrained phos-
(25) Lankhorst, P. P.; Haasnoot, C. A. G.; Erkelens, C.; Altona, C.
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982.

Cyclic Phosphate Analog of Inositol Trisphosphate
J . Org. Chem., Vol. 63, No. 2, 1998 299
F igu r e 2. Microprotonation states of 4.
F igu r e 3. Mean number of protons bound per molecule of 4
(pj) vs pH calculated from potentiometric (b) and 31P NMR (s)
measurements.
that it combines a cyclic phosphate diester and phosphate
monoesters together in the same molecule. Since the
ionization of the 5-phosphate group appears to be so
crucial in Ca2+ mobilizing activity, and the overall
ionization of the crucial 4,5-bisphosphate system is
clearly different than that of two individual phosphate
groups, we felt it of particular interest to study a system
where a weakly acidic function [represented by one pKa
of the 4-phosphate in Ins(1,4,5)P3] had effectively been
removed and the conformational access of this phosphate
group to its vicinal partner was highly restricted.
Ta ble 1a
y
log Ky ( σ
i
log ki ( σ
ii′
log kii′ ( σ
6.20 ( 0.02
1
6.70 ( 0.01
6.76 ( 0.03
5.60 ( 0.01
5.64 ( 0.04
4
6.10 ( 0.01
42
2
2
6.57 ( 0.01
24
5.73 ( 0.02
a
Logarithms of the macro- and microprotonation constants for
4 in 0.2 M KCl at 37 °C. The macroconstants given in italic were
determined from the 31P-NMR data. log ki and log kii′ represent
a general designation for, respectively, the logarithms of the first
and second stepwise microprotonation constants. The uncertain-
ties are estimates of the standard deviation as calculated by
Superquad, Hypnmr, and Enzfitter (Elsevier-Biosoft) for the
macro- and microconstants.
A sample of 4 (triethylammonium salt) was first
converted into the free acid and then titrated with
potassium hydroxide solution. The study was performed
in a 0.2 mol‚dm-3 KCl solution at 37 °C, conditions that
roughly mimic those encountered within the cell. When
the pH was decreased from 10 to 3, the fully deprotonated
4, bearing five negative charges, gained only two protons,
leading to a diprotonated species. Thus, in the pH range
considered, 4 behaves as a difunctional ligand character-
ized by the macroscopic stepwise protonation equilibria
calculated from both the potentiometric and NMR data
according to eqs 2 and 3, respectively.
CH - [H+] + [OH-]
pj )
(2)
(3)
CL
Hy-1L
(5-y+1)- + H+ a HyL(5-y)-
i)N
pj )
f
i,p
∑
and the related Ky constants, where y ) 1 and 2 stand
for the first and second step, respectively. The micro-
protonation diagram is depicted in Figure 2. Note that
the second protonation of the phosphates P2 and P4 and
the protonation of P1 occur below pH 3, well outside the
physiological range, and the related constants were
therefore not considered in this study.
In previous studies30 we have shown that, in a number
of favorable cases, the analytical concentration of the
coexisting microspecies can ultimately be obtained from
31P NMR spectroscopy carried out over a large pH range.
If the observed chemical shift for the phosphorus reso-
nance δoi bs depends on the electronic effects accompany-
ing the variations in protonation state, then the
protonated fraction fi,p of a phosphate group in position
i on the inositol ring can be calculated by eq 1,
i)1
In these equations, pj is the mean number of protons
bound per molecule of 4, while CH and CL correspond to
the analytical concentrations of the acid and 4, respec-
tively. Figure 3 shows the excellent superimposition of
both pj ) f(pH) curves for 4, thus allowing further
interpretation of the δiobs in terms of microprotonation
constants.
As shown earlier,7 fi,p can be expressed as a function
of the macro- and microprotonation constants and the
proton concentration. For instance, the fractions of
protonation of phosphate P2 (f2,p) are defined as
k24k2[H+]2 + k2[H+]
k24k2[H+]2 + (k2 + k4)[H+] + 1
f2,p
)
(4)
δoi bs - δi,d
δi,p - δi,d
The equation is solved by nonlinear regression, introduc-
ing K1 ) k2 + k4 obtained by potentiometry or NMR
experiments. It can be seen that the logarithms of the
macroprotonation constants (Table 1) determined by both
techniques are in good agreement.
fi,p
)
(1)
where δi,p and δi,d correspond respectively to the chemical
shifts of the protonated and deprotonated fractions of the
phosphates in position i. The previous condition can be
checked by the superimposition of the pj ) f(pH) curves
The 31P NMR titration curves and the corresponding
protonation fractions of 4 are shown in Figure 4. These
curves appear monophasic for P2 and P4, indicating the

300 J . Org. Chem., Vol. 63, No. 2, 1998
Riley et al.
F igu r e 5. Distribution curves of the protonated microspecies
of 4 in 0.2 M KCl at 37 °C plotted against pH.
Figure 5 shows the distribution of the various micro-
protonated species as a function of pH, providing a direct
observation of the protonation state of the P2 and P4
phosphate groups. It is noteworthy that, in the ligand
concentration used in this study, and at physiological pH
(7.5), phosphate P2 is only 10% protonated, whereas in
Ins(1,4,5)P3, the P5 phosphate (the equivalent of P2 in
4) is protonated to the extent of about 40%.7 As discussed
above, it has been shown that for Ins(1,4,5)P3 specific
binding to brain membrane receptors increases with pH
and especially with the ionization of the P5 phosphate.
Thus, the higher dissociation of the equivalent phosphate
group in 4 would therefore be expected to enhance
binding to the receptor at physiological pH.
F igu r e 4. Chemical shifts δ from a 31P NMR titration for 4
(A) and the corresponding protonation fraction curves fi,p (B)
as a function of pH in 0.2 M KCl at 37 °C (H2O-10%D2O).
The least-squares fit of fi,p vs pH according to type 4 equations
is shown in solid line.
1H NMR titration curves (Figure 6) were also obtained,
to follow possible conformational changes over the stud-
ied pH range. On going from the lowest to the highest
pH limits (2.0 < pH < 12) the H-1, H-2, H-3, and H-4
resonances are shifted upfield by about 0.15 ppm while
the rest of the signals are almost unaffected. As we
observed previously,30 the 1H resonances of the myo-
inositol ring may be influenced by the ionization of both
the distal and adjacent phosphates. In addition, the
relative independence of the two phosphate groups, while
the P1 chemical shifts do not change significantly,
affirming that the cyclic phosphate remains monocharged
over all the studied pH range. The determination of the
microconstants (Table 1) and the derived k2/k4 ) 2.95
ratio reveal that P2 is more basic than P4. This confirms
the importance of the presence of neighboring OH groups
which tend to decrease the basic character of a phosphate
either through an inductive field effect or via changing
the local dielectric constant of the solvent.7,30 Thus, the
higher basicity of phosphate P2 seems due to the presence
of a phosphate ester oxygen, rather than through the
stabilization of the negative charge of P1 which appears
not to be affected by the deprotonation of P2. Calculation
of the interactivity parameters allows the quantification
of the basicity changes at one site when the other site
takes up a proton. For 4 this parameter is defined as
∆log k2-4 ) log k2 - log k42 ) log k4 - log k24 ) 0.37. This
value is not too far from ∆log k1-4 ) 0.07 for Ins(1,4)P2
compared to ∆log k4-5 ) 2.12 for Ins(4,5)P2.7 Such a
result confirms that P2 and P4 tend to behave indepen-
dently during the protonation process.
3
coupling pattern as well as the J H-H coupling constants
of the ring protons remain unchanged from pH 2 to 12.
They undoubtedly correspond to an equatorial chair
conformation slightly distorted by the cyclization of the
P1 phosphate.
2.3 Con clu sion s. We have described the design and
synthesis of a novel conformationally restricted cyclic
phosphate analogue of Ins(1,4,5)P3. NMR studies con-
firm that in 4 the phosphate group corresponding to P4
of Ins(1,4,5)P3 is held in the positive gauche orientation
and the inositol ring maintains a chair conformation over
the entire pH range studied. One consequence of intro-
ducing the cyclic phosphate has been to simplify the
acid-base properties of the analogue so that 4 behaves
essentially as a diprotic acid under physiological condi-
tions, with the equivalent of P4 in Ins(1,4,5)P3 being
singly charged over the physiological pH range. Another
effect has been to enhance the acidity of the equivalent
of P5 in Ins(1,4,5)P3. Finally, the scyllo-inositol structure
of 4 appears to have slightly modified the behavior of the
equivalent of the 1-phosphate in Ins(1,4,5)P3.
It should also be noticed that the δoi bs ) f(pH) curves
of 4 appear very straightforward with regard to those of
Ins(1,4,5)P3.7 In particular, the unexpected initial down-
field shift of P1 (equivalent of P4 for 4) occurring for Ins-
(1,4,5)P3 with the addition of the first equivalent of
protons is not observed. This may be related to the scyllo-
inositol structure of 4 and the difference in the configu-
ration of one vicinal hydroxyl group in the two molecules.
So far, the biological properties of 4 are less clear.
When 4 was examined for Ca2+ mobilizing ability at
platelet Ins(1,4,5)P3 receptors using saponin-permeabi-
lized platelets loaded with 45Ca2+, it appeared to behave

Cyclic Phosphate Analog of Inositol Trisphosphate
J . Org. Chem., Vol. 63, No. 2, 1998 301
valid; the scyllo-inositol structure and added methylene
group are clearly no obstacle to potent activity.
In summary, we have designed an efficient synthetic
route to the first example of a conformationally restricted
inositol polyphosphate analogue which is, we believe, a
unique example of a molecule possessing both phosphate
monoester and six-membered cyclic phosphodiester groups.
Investigation of microprotonation equilibria has shown
clear differences in the analogue as compared to Ins-
(1,4,5)P3. While the biological activity of 4 may be
complex and is under current examination, it seems likely
that the approaches outlined here will find further
application in the phosphoinositide field among the
wealth of newly discovered inositol phosphates and
phospholipids.
3. Exp er im en ta l Section
Thin-layer chromatography (TLC) was performed on pre-
coated plates (Merck TLC aluminum sheets silica 60 F254) with
detection by UV light or with phosphomolybdic acid in
methanol followed by heating. Flash chromatography was
performed on silica gel (Sorbsil C60). Dichloromethane was
distilled over phosphorus pentoxide and stored in the presence
of 4 Å molecular sieves. DMF was purchased in anhydrous
form and stored over 4 Å sieves. THF was distilled from
sodium benzophenone ketyl under a nitrogen atmosphere. 31
P
NMR shifts were measured in ppm relative to external 85%
phosphoric acid and are positive when downfield from this
reference. FAB mass spectra were recorded at the EPSRC
Mass Spectrometry Service Centre, Swansea, and at the
University of Bath using m-nitrobenzyl alcohol as the matrix.
Microanalysis was carried out by the Microanalysis Service,
University of Bath. Melting points (uncorrected) were deter-
mined using a Reichert-J ung hot stage microscope apparatus.
Ion-exchange chromatography was performed on an LKB-
Pharmacia medium-pressure ion exchange chromatograph
using Q-Sepharose Fast Flow and gradients of triethylammo-
nium bicarbonate (TEAB) as eluent. Quantitative analysis of
phosphate was performed using a modification of the Briggs
phosphate assay.32,33
Potentiometric and NMR determinations were carried out
as previously described.7,30 The experiments were performed
in two steps in which the same initial solution of 4 of about 3
× 10-3 mol‚dm-3 was successively subjected to potentiometric
and 31P NMR or 1H NMR titrations. The processing of the
pH measurements allowed the total concentration of the ligand
and the acid as well as the macroscopic protonation constants
(by using SUPERQUAD34) to be determined. 31P NMR spectra
were recorded at 81.015 MHz on a Bruker AC 200 Fourier
transform spectrometer. Field-frequency lock was achieved
using 10% D2O. The HypNMR program35 was used to check
the potentiometrically determined protonation constants. The
1H NMR titration was performed on a Bruker 300 DPX
spectrometer on 0.45 mL of solution in D2O instead of 2 mL
for the 31P NMR titration. Phosphorus resonances of 4 were
assigned by performing phosphorus-proton and proton-
proton 2D correlation experiments at pH ) 6.8.
F igu r e 6. Chemical shifts δ from a 1H NMR titration for 4
as a function of pH in 0.2 M KCl at 37 °C (D2O). The H6
resonance appearing at about 2.3 ppm has been omitted.
as a full agonist, being able to release all of the Ins(1,4,5)-
P3 -sensitive Ca2+ pool, but was 40-70-fold weaker than
Ins(1,4,5)P3 itself. Assuming that only one enantiomer
of 4 is active, this would imply an approximately 30-fold
reduction in potency compared to Ins(1,4,5)P3. Binding
studies in rat cerebellar membranes showed that the
affinity of 4 for the cerebellar Ins(1,4,5)P3 receptor was
much reduced (200 to 300-fold) compared to Ins(1,4,5)-
P3. However, we have now shown that 6-deoxy-6-
(hydroxymethyl)-scyllo-inositol 1,2,4-trisphosphate [6-CH2-
OH-scyllo-Ins(1,2,4)P3, (17)], an analogue of 4 in which
the dioxaphosphorinane ring has been opened to give a
phosphate monoester with an adjacent hydroxymethyl
group, shows remarkably high activity in the same
assays, being equipotent to Ins(1,4,5)P3 itself, despite
being racemic.19
4,6-Bis-O-(p-m eth oxyben zyl)-1,3,5-O-m eth ylid yn e-m yo-
in ositol (6). To a solution of myo-inositol orthoformate16 (5)
(15.0 g, 78 mmol) in dry DMF (300 mL) at 0 °C was added
sodium hydride (7.2 g of a 60% dispersion in oil, 180 mmol).
The mixture was stirred for 20 min at 0 °C, and then
p-methoxybenzyl chloride (22.5 mL, 166 mmol) was added. The
mixture was stirred for a further 2 h at rt, after which TLC
While it is possible then that the cyclic phosphate of 4
might be able to mimic the 4-phosphate of Ins(1,4,5)P3,
it is difficult to be certain at this stage whether the
apparent behavior of 4 as weak full agonist might not
originate from partial enzymatic hydrolysis of 4 by
nonspecific phosphodiesterases to give 17 during the
biological assays. Because of the high activity of 17, this
is particularly difficult to assess. The Ins(1,4,5)P3-like
activity of 17 does, however, confirm that the assump-
tions discussed above regarding the design of 4 were
(32) Briggs, A. P. J . Biol. Chem. 1922, 53, 13-16.
(33) Lampe, D.; Liu, C.; Potter, B. V. L. J . Med. Chem. 1994, 37,
907-912.
(34) Gans, P.; Sabatini, A.; Vacca, A. J . Chem. Soc., Dalton Trans.
1985, 1195-1200.
(35) Frassineti, C.; Ghelli, S.; Gans, P.; Sabatini, A.; Moruzzi, M.
S.; Vacca, A. Anal. Biochem. 1995, 231, 374-382.

302 J . Org. Chem., Vol. 63, No. 2, 1998
Riley et al.
(dichloromethane/ethyl acetate 2:1) showed a major product
at Rf 0.49 and minor products (mono- and tri-O-p-methoxy-
benzylated material) at Rf 0.22 and 0.64. Water (10 mL) was
added carefully to quench the reaction. Solvents were evapo-
rated in vacuo, and the residue was partitioned between water
(200 mL) and dichloromethane (400 mL). The organic layer
was washed with brine (200 mL), dried (MgSO4), and evapo-
rated to give an oil which was purified by flash chromatogra-
phy (dichloromethane/ethyl acetate 5:1) giving, in order of
elution, the tri-O-p-methoxybenzylated ether, di-O-p-meth-
oxybenzylated material, and, finally, monosubstituted prod-
ucts. Recrystallization of the second fraction from ethyl
acetate/hexane gave 6 (13.5 g, 31.4 mmol, 40% yield): mp 120-
121 °C (from ethyl acetate/hexane); 1H NMR (CDCl3, 400 MHz)
δ 3.26 (1 H, d, J ) 11.3 Hz, D2O ex), 3.79 (6 H, s), 4.16 (1 H,
br d, J ) 11.2 Hz, D2O ex gives br s), 4.18-4.20 (2 H, m) 4.32
(2 H, t, J ) 3.4 Hz), 4.40 (1 H, tt, J ) 3.4 Hz, 1.8 Hz), 4.48,
4.56 (4 H, AB, J AB ) 11.0 Hz) 5.46 (1 H, d, J ) 0.9 Hz), 6.81
(4 H, d, J ) 8.9 Hz), 7.17 (4 H, d, J ) 8.6 Hz); 13C NMR (CDCl3,
67.8 MHz) δ 55.14, 61.12, 67.74, 71.21, 72.90, 73.38, 103.24,
113.75, 129.27, 129.53, 159.29; MS m/z (positive ion FAB, rel
intensity) 431 [(M + H)+, 12%], 309 [(M - C7H6OCH3)+, 15%],
121 [(C7H6OCH3)+, 100%]. Anal. Calcd for C23H26O8
(430.45): C, 64.18; H, 6.09. Found: C, 63.9; H, 6.05.
MHz) δ 55.25, 67.51, 71.42, 73.32, 73.56, 88.67, 102.30, 113.95,
128.74, 129.76, 159.58; MS m/z (positive ion FAB, relative
intensity) 447 [(M + H)+, 2%], 121 [(C7H6OCH3)+, 100%]. Anal.
Calcd for C23H26O9 (446.45): C, 61.88; H, 5.87. Found: C, 61.8;
H, 5.89.
2,4-Bis-O-(p -m e t h oxyb e n zyl)-6-m e t h yle n e -1,3,5-O-
m et h ylid yn ecycloh exa n e-1,3,5/2,4-p en t ol (8). Methyl-
triphenylphosphonium bromide (6.13 g, 17.2 mmol), previously
dried in vacuo at 70 °C, was suspended in dry THF (20 mL)
under N2 at 0 °C. Potassium tert-butoxide (16.3 mL of a 1 M
solution in THF, 16.3 mmol) was added. The resulting yellow
suspension was allowed to reach rt and was then stirred at rt
for 10 min. A solution of ketone 7 (3.50 g. 8.17 mmol) in dry
THF (30 mL) was added. [At this stage, a 31P NMR spectrum
of a sample taken from the yellow suspension showed the
presence of an oxaphosphetane intermediate (δP -68.9 ppm).
This signal could still be observed in the NMR sample after
several days at 4 °C.] The mixture was refluxed for 2 h, after
which time its color had darkened to orange and TLC (ethyl
acetate/hexane 1:1) showed the reaction to be complete, with
the product at Rf 0.52. The solvent was removed by evapora-
tion in vacuo, the residue was taken up in ether (100 mL),
and the solution was washed with brine (100 mL), dried
(MgSO4), and evaporated to give a clear brown oil. Purification
by flash chromatography (ethyl acetate/hexane 1:2) gave the
alkene 8 as a white crystalline solid (3.17 g, 7.42 mmol, 91%):
mp 95-97 °C (from ethanol); 1H NMR (CDCl3, 270 MHz) δ
3.80 (6 H, s), 4.23 (2 H, t, J ) 3.6 Hz), 4.31 (1 H, tt, J ) 3.6
Hz, 1.7 Hz), 4.40 (2 H, dd, J ) 3.6 Hz, 1.7 Hz), 4.54, 4.58 (4 H,
AB, J AB ) 11.8 Hz), 5.25 (2 H, s), 5.57 (1 H, s), 6.82-6.87 (4
H, m), 7.22-7.26 (4 H, m); 13C NMR (CDCl3, 67.8 MHz) δ
55.19, 68.94, 73.55, 74.26, 71.05, 103.73, 113.75, 129.43,
114.27, 129.82, 137.15, 159.32; MS m/z (positive ion FAB,
relative intensity) 427 [(M + H)+, 1%], 305 [(M - C7H6OCH3)+,
6%], 121 [(C7H6OCH3)+, 100%]. Anal. Calcd for C24H26O7
(426.47): C, 67.59; H, 6.15. Found: C, 67.3; H, 6.15.
2,4,6/3,5-P en t a h yd r oxy-3,5-b is-O-(p -m et h oxyb en zyl)-
2,4,6-O-m et h ylid yn ecycloh exa n on e (7). Dry dichloro-
methane (40 mL) was placed into a 250 mL flask under an
atmosphere of N2. A solution of oxalyl chloride in dry dichlo-
romethane (12.8 mL of a 2 M solution, 25.6 mmol) was added,
and the flask was cooled to -60 °C using a chloroform/solid
CO2 bath. A solution of anhydrous DMSO (3.6 mL, 51 mmol)
in dry dichloromethane (5 mL) was added dropwise over 5 min
(care! rapid evolution of gas) and stirring was continued for 5
min. A solution of 6 (10.0 g, 23.2 mmol) in dry dichlo-
romethane (30 mL) was added dropwise over 5 min and
stirring continued for an additional 20 min, maintaining a
temperature of -55 to -60 °C. Triethylamine (15 mL) was
added dropwise over 2 min, and the reaction was stirred for a
further 5 min before allowing it to warm to rt. The mixture
was stirred with water (100 mL) for 10 min, and then
dichloromethane (200 mL) was added. The organic layer was
separated, and the aqueous layer re-extracted with a further
200 mL of dichloromethane. The combined organic layers were
then washed successively with saturated NaCl, 1% HCl, water,
10% NaHCO3, and water (200 mL of each), dried (MgSO4), and
evaporated to give a white solid consisting of a mixture of
ketone 7 and its hydrated gem-diol (7a ). The mixture was
dissolved in toluene (300 mL) and refluxed with azeotropic
removal of water in a Dean-Stark apparatus for 3 h. The
toluene was removed by evaporation in vacuo, and the residue
was recrystallized from ethyl acetate/hexane to give ketone 7
(9.14 g, 21.3 mmol, 92%): mp 125-126 °C (from ethyl acetate/
(1,3,5/2,4,6)-6-(H yd r oxym et h yl)-1,3,5-O-m et h ylid yn e-
2,4-b is-O-(p -m et h oxyb en zyl)cycloh exa n e-1,2,3,4,5-p en -
tol (9). The alkene 8 (6.0 g, 14.0 mmol, previously dried at
60 °C in vacuo) was placed in a dry three-neck 250 mL flask,
and 9-BBN-H (60 mL of a 0.5M solution in THF, 30 mmol)
was added under an atmosphere of N2 at rt. The temperature
was increased to 50 °C, and the mixture was stirred under N2
for 2 h. The mixture was allowed to cool to room temperature
and then further cooled to 0 °C in an ice bath. Ethanol (20
mL), 6 M NaOH (5 mL), and 30% H2O2 (10 mL) were added
dropwise (care! exothermic reaction with rapid evolution of
gas), and then the temperature was increased to 50 °C. After
being stirred at 50 °C for 30 min, the mixture was cooled to
room temperature and the aqueous layer was saturated with
K2CO3. The organic layer was removed, dried (MgSO4), and
evaporated in vacuo to give a colorless oil which was purified
by column chromatography, giving 9 as a white solid (6.04 g,
13.6 mmol, 97%): mp 81-82 °C (from ethanol or ethyl acetate/
1
hexane); IR (KBr disk) νCdO 1760 cm-1; H NMR (CDCl3, 270
MHz) δ 3.78 (6 H, s), 4.38-4.42 (2 H, m), 4.47-4.56 (3 H, m),
4.52 (4 H, br s), 5.63 (1 H, s), 6.81 (4 H, d, J ) 8.2 Hz), 7.16 (4
H, d, J ) 8.4 Hz); 13C NMR (CDCl3, 67.8 MHz) δ 55.22, 68.88,
71.18, 76.37, 77.97, 102.64, 113.85, 128.90, 129.50, 159.47,
199.23; MS m/z (positive ion FAB, relative intensity) 447 [(M
+ H2O + H)+, 0.3%], 429 [(M + H)+, 1.2%], 121 [(C7H6OCH3)+,
100%]; MS m/z (negative ion FAB, relative intensity) 580 [(M
+ NBA - H)-, 75%], 427 [(M - H)-, 30%], 322 (80), 303 (100),
287 (60%). Anal. Calcd for C23H24O8 (428.44): C, 64.48; H,
5.65. Found: C, 64.6; H, 5.66.
1
hexane); H NMR (CDCl3, 270 MHz) δ 1.58 (1 H, t, J ) 5.9
Hz, D2O ex), 2.97 (1 H, br t, J ) 8.2 Hz), 3.80 (6 H, s), 4.08 (2
H, dd, J ) 8.2 Hz, 5.9 Hz, D2O ex gives d, J ) 8.2 Hz), 4.27 (2
H, br s), 4.36 (2 H, br s), 4.49, 4.59 (4 H, AB, J AB ) 10.8 Hz),
4.53 (1 H, partly obscured by AB system), 5.58 (1 H, s), 6.79-
6.82 (4 H, m), 7.15-7.26 (4 H, m); 13C NMR (CDCl3, 67.8 MHz)
δ 42.88, 55.17, 59.89, 68.50, 69.02, 73.06, 71.41, 103.84, 113.78,
129.29, 129.63, 159.26; MS m/z (positive ion FAB, relative
intensity) 445 [(M + H)+, 1.2%], 323 [(M - C7H6OCH3)+, 3.1%],
121 [(C7H6OCH3)+, 100%]; MS m/z (negative ion FAB, relative
intensity) 597 [(M + NBA)-, 100%], 323 [(M - C7H6OCH3)-,
90%]. Anal. Calcd for C24H28O8 (444.48): C, 64.85; H, 6.35.
Found: C, 65.1; H, 6.47.
2-C-H yd r oxy-4,6-b is-O-(p -m e t h oxyb e n zyl)-1,3,5-O-
m eth ylid yn e-m yo-in ositol (gem -d iol 7a ). The ketone 7 (400
mg, 0.924 mmol) was dissolved in dioxane (4 mL), and water
(0.4 mL) was added. The solution was left at room tempera-
ture for 3 days, and then water was added dropwise until
crystals began to appear. After another day at room temper-
ature, the crystals were filtered off and were found to consist
of pure gem-diol 7a (315 mg, 0.706 mmol, 76%): mp 129-131
(1,3,5/2,4,6)-6-(Hyd r oxym et h yl)-2,4-b is-O-(p-m et h oxy-
ben zyl)cycloh exa n e-1,2,3,4,5-p en tol (10). Alcohol 9 (2.32
g, 5. 22 mmol) was dissolved in methanol (100 mL) and heated
to 50 °C. Then 1 M HCl (10 mL) was added and the mixture
stirred at 50 °C for 30 min. Excess concentrated ammonia
solution was added and the mixture was allowed to cool.
Stirring was continued at room temperature for a further 30
min, and the solvents were removed by evaporation in vacuo.
1
°C (from ethyl acetate/hexane); H NMR (CDCl3, 270 MHz) δ
3.79 (6H, s), 3.82 (1H, s, D2O ex), 4.12-4.15 (2H, m), 4.41-
4.60 (7 H, m), 4.97 (1 H, s, D2O ex), 5.50 (1 H, s), 6.80 (4 H, d,
J ) 8.6 Hz), 7.12 (4 H, d, J ) 8.6 Hz); 13C NMR (CDCl3, 68

Cyclic Phosphate Analog of Inositol Trisphosphate
J . Org. Chem., Vol. 63, No. 2, 1998 303
1
The residue was extracted with hot ethyl acetate (2 × 100 mL),
and the combined extracts were evaporated in vacuo to give a
white solid which was purified by flash chromatography
(CHCl3/MeOH 100:0-50:50), giving tetrol 10 (1.98 g, 4.56
mmol, 87%): mp 136-137 °C (from ethyl acetate/hexane); 1H
NMR (d6-DMSO, 400 MHz, 1H-1H COSY) δ 1.25 (1 H, tt, J )
10.7 Hz, 2.1 Hz, C-6-H), 3.09 (2 H, t, J ) 9.3 Hz, 9.3 Hz, C-2-H
and C-4-H), 3.20 (1 H, td, J ) 9.2 Hz, 5.4 Hz, D2O ex gives t,
J ) 9.2 Hz, C-3-H), 3.29 (2 H, ddd, J ) 10.7 Hz, 9.3 Hz, 5.9
Hz, D2O ex gives dd, J ) 10.7 Hz, 9.3 Hz, C-1-H and C-5-H),
3.69 (2 H, dd, J ) 5.4 Hz, 2.1 Hz, D2O ex gives d, J ) 2.1 Hz,
CH2OH), 3.73 (6 H, s, 2 × OCH3), 4.27 (1 H, t, J ) 5.4 Hz,
D2O ex, CH2OH), 4.71 (4 H, s, CH2OPMB), 4.74 (2 H, d, J )
5.9 Hz, D2O ex, C-1-OH and C-5-OH), 4.92 (1 H, d, J ) 5.4
Hz, D2O ex, C-3-OH), 6.83-6.87 (4 H, m, C6H4OMe), 7.29-
7.36 (4 H, m, C6H4OMe); 13C NMR (d6-DMSO, 67.8 MHz) δ
48.13, 55.49, 57.54, 69.00, 73.87, 73.88, 85.90, 113.78, 129.80,
132.07, 158.91; MS m/z (positive ion FAB, relative intensity)
433 [(M - H)+, 2.0%], 313 [(M - C7H6OCH3)+, 7.0%], 121
[(C7H6OCH3)+, 100%]; MS m/z (negative ion FAB, relative
intensity) 587 [(M + NBA)-, 100%], 433 [(M - H)-, 100%],
313 [(M - C7H6OCH3)-, 20%]. Anal. Calcd for C23H30O8
(434.49): C, 63.58; H, 6.96. Found: C, 63.4; H, 6.94.
137 °C (from ethanol); H NMR (CDCl3, 400 MHz) δ 1.98 (1
H, qd, J ) 11 Hz, 4.4 Hz), 3.21 (1 H, dd, J ) 10.8 Hz, 9.3 Hz),
3.45-3.73 (5 H, m), 3.76 (3 H, s), 3.78 (3 H, s), 4.42 (1 H, dd,
J ) 11.2 Hz, 4.4 Hz), 4.51-4.97 (8 H, m), 5.48 (1 H, s), 6.77-
6.81 (4 H, m), 7.20-7.50 (19 H, m); 13C NMR (CDCl3, 100 MHz)
δ 39.69, 55.21, 69.31, 74.93, 75.20, 75.62, 76.06, 77.94, 80.03,
82.77, 83.19, 85.70, 100.95, 113.73, 113.84, 125.91, 127.60,
127.78, 127.96, 128.05, 128.18, 128.38, 128.51, 128.77, 129.59,
129.83, 130.58, 130.74, 138.00, 138.04, 138.64, 159.20, 159.23;
MS m/z (positive ion FAB, relative intensity) 703 [(M + H)+,
0.5%], 702 [M+, 0.7%], 611 [(M - C7H7)+, 0.3%], 581 [(M -
C7H6OCH3)+, 3.5%], 121 [(C7H6OCH3)+, 100%], 91 [(C7H7)+,
22%]; MS m/z (negative ion FAB, rel intensity) 855 [(M +
NBA)-, 100%], 611 [(M - C7H7)-, 40%], 581 [(M - C7H6OCH3)-,
20%]. Anal. Calcd for C44H46O8 (702.84): C, 75.19; H, 6.60.
Found: C, 75.2; H, 6.62.
DL-(1,3,5/2,4,6)-1,3-Di-O-ben zyl-6-(h yd r oxym eth yl)-2,4-
bis-O-(p-m eth oxyben zyl)cycloh exan e-1,2,3,4,5-pen tol (13).
Compound 12 (1.00 g, 1.42 mmol) was dissolved in a mixture
of THF (25 mL) and methanol (25 mL). Then 1 M HCl (5 mL)
was added and the solution was refluxed for 30 min, after
which TLC (ethyl acetate) showed that no starting material
remained. Excess NaHCO3 (1 g) was added, and the mixture
was allowed to cool to rt with stirring before the solvents were
removed by evaporation in vacuo. The residue was taken up
in dichloromethane (100 mL), washed with water (50 mL) and
brine (50 mL), dried (MgSO4), and evaporated in vacuo to give
a solid which was purified by flash chromatography, yielding
the diol 13 (720 mg, 1.17 mmol, 82%): mp 116-117.5 °C (from
ethanol); 1H NMR (CDCl3, 270 MHz) δ 1.65 (1 H, tdd, J ) 10
Hz, 4.5 Hz, 3.0 Hz), 2.27 (1 H, br t, J ) 5.1 Hz, 5.1 Hz, D2O
ex), 2.77 (1 H, d, J ) 1.5 Hz, D2O ex), 3.34-3.51 (4 H, m),
3.63 (1 H, t, J ) 9.3 Hz, 9.3 Hz), 3.73 (1 H, br m, D2O ex gives
dd, J ) 11.2 Hz, 4.5 Hz), 3.77 (3 H, s), 3.78 (3 H, s), 3.89 (1 H,
br m, D2O ex gives dd, J ) 11.2 Hz, 3.0 Hz), 4.59-4.97 (8 H,
m), 6.79-6.87 (4 H, m), 7.18-7.23 (4 H, m), 7.29-7.38 (10 H,
m); 13C NMR (CDCl3, 67.8 MHz) δ 46.07, 55.20, 60.22, 70.37,
77.52, 83.20, 84.80, 85.95, 75.10, 75.20, 75.43, 75.57, 113.80,
114.03, 127.63, 127.84, 127.99, 128.41, 129.37, 129.53, 130.49,
130.60, 138.14, 138.45, 159.14, 159.37; MS m/z (positive ion
FAB, rel intensity) 615 [(M + H)+, 0.5%], 614 [M+, 0.4%], 523
[(M - C7H7)+, 0.2%], 493 [(M - C7H6OCH3)+, 2.8%], 121 [(C7H6-
OCH3)+, 100%], 91 [(C7H7)+, 20%]; MS m/z (negative ion FAB,
rel intensity) 767 [(M + NBA)-, 60%], 613 [(M - H)-, 100%),
493 [(M - C7H6OCH3)-, 35%]. Anal. Calcd for C37H42O8
(614.74): C, 72.29; H, 6.89. Found: C, 72.3; H, 6.88.
DL-(1,3,5/2,4,6)-1,3-Di-O-b en zyl-5,7-O-(b en zyloxyp h os-
p h or yl)-6-(h yd r oxym eth yl)-2,4-bis-O-(p-m eth oxyben zyl)-
cycloh exan e-1,2,3,4,5-pen tol (Epim er s 14a an d 14b). (Ben-
zyloxy)bis(N,N-diisopropylamino)phosphine20 (285 mg, 0.84
mmol) was placed in a dry round-bottomed flask, and dry
dichloromethane (5 mL) was added, followed by 1H-tetrazole
(150 mg, 2.14 mmol). The suspension was stirred for 10 min
and then cooled to 0 °C. The diol 13 (430 mg, 0.70 mmol,
previously dried in vacuo at 60 °C) was added, and stirring
was continued at 0 °C for 2 h. 31P NMR spectroscopy now
showed signals at δP 125.0 and 130.4 ppm, corresponding to
the two cyclic phosphite triester invertomers. The mixture was
cooled to -78 °C, and m-CPBA (240 mg, 1.4 mmol) was added.
The clear solution was now allowed to warm to room temper-
ature and then diluted with ethyl acetate (50 mL), washed
with 10% Na2SO3, 1 M HCl, saturated NaHCO3 and brine (50
mL of each), dried (MgSO4), and evaporated in vacuo to give
a colorless oil. Purification by flash chromatography (ethyl
acetate/hexane 1:1) gave the two epimeric cyclic phosphate
triesters 14a , Rf 0.30 (246 mg, 0.32 mmol), and 14b, Rf 0.18
(214 mg, 0.28 mmol), corresponding to a total yield of 0.60
mmol (86% from 13).
DL-(1,3,5/2,4,6)-1,7-O-Ben zylid en e-6-(h yd r oxym eth yl)-
2,4-b is-O-(p -m et h oxyb en zyl)cycloh exa n e-1,2,3,4,5-p en -
tol (11). The tetrol 10 (2.00 g, 4.60 mmol) was dissolved in
dry DMF (10 mL) in a 100 mL round-bottomed flask.
A
catalytic amount of toluene-p-sulfonic acid (50 mg) was added,
followed by benzaldehyde dimethyl acetal (0.80 mL, 5.33
mmol). The flask was fitted with a 250 mm air condenser
connected to a filter pump, and the solution was stirred at 65-
75 °C under reduced pressure for 1 h, after which TLC (ethyl
acetate) showed the reaction to be complete. The solution was
cooled to room temperature and triethylamine (1 mL) added.
After the solution was stirred for 30 min at room temperature,
the solvents were removed by evaporation in vacuo. The
residue was taken up in dichloromethane (100 mL), washed
with brine (50 mL), dried (MgSO4), and evaporated in vacuo
to give a solid which was purified by column chromatography
(ethyl acetate/chloroform 1:1), giving the diol 11 as a white
solid (2.24 g, 4.29 mmol, 93%): mp 158-160 °C (from ethyl
1
acetate/hexane); H NMR (CDCl3, 270 MHz) δ 1.92 (1 H, qd,
J ) 11 Hz, 4.4 Hz), 2.36 (1 H, br s, D2O ex), 2.69 (1 H, br s,
D2O ex), 3.22 (1 H, br dd, D2O ex gives dd, J ) 11.0 Hz, 8.8
Hz), 3.33 (1 H, dd, J ) 8.8 Hz, 8.6 Hz), 3.50-3.66 (3 H, m),
3.68 (1 H, dd, J ) 11.1 Hz, 11.1 Hz), 3.78 (6 H, br s), 4.49 (1
H, dd, J ) 11.2 Hz, 4.4 Hz), 4.61, 4.97 (2 H, AB, J AB ) 11.2
Hz), 4.63, 4.96 (2 H, AB, J AB ) 11.0 Hz), 5.53 (1 H, s), 6.84-
6.92 (4 H, m), 7.24-7.32 (4 H, m), 7.34-7.54 (5 H, m); 13C NMR
(CDCl3, 67.8 MHz) δ 39.51, 55.27, 69.39, 69.41, 74.68, 80.03,
82.03, 84.33, 74.62, 74.75, 101.10, 113.97, 114.09, 125.98,
128.27, 128.87, 129.74, 129.87, 130.47, 130.62, 138.06, 159.42,
159.45; MS m/z (positive ion FAB, relative intensity) 523 [(M
+ H)+, 1.0%], 522 [M+, 2.0%], 401 [(M - C7H6OCH3)+, 2.0%],
121 [(C7H6OCH3)+, 100%]; MS m/z (negative ion FAB, rel
intensity) 1043 [(2M - H)-, 30%], 828 (10%), 688 (10%), 675
[(M + NBA)-, 100%], 521 [(M - H)-, 100%], 401 [(M -
C7H6OCH3)-, 40%]. Anal. Calcd for C30H34O8 (522.60): C,
68.95; H, 6.56. Found: C, 68.8; H, 6.53.
DL-(1,3,5/2,4,6)-1,3-Di-O-b e n zyl-5,7-O-b e n zylid e n e -6-
(h yd r oxym eth yl)-2,4-bis-O-(p-m eth oxyben zyl)cycloh ex-
a n e-1,2,3,4,5-p en tol (12). The diol 11 (1.00 g, 1.91 mmol)
was dissolved in dry DMF, and sodium hydride (250 mg of a
60% dispersion in oil, 6.25 mmol) was added. The suspension
was stirred for 20 min at room temperature, and then benzyl
bromide (0.50 mL, 4.60 mmol) was added and stirring was
continued for 2 h, after which TLC (ethyl acetate/hexane 1:1)
showed the reaction to be complete. Excess NaH was carefully
destroyed by dropwise addition of water, and the solvents were
removed by evaporation in vacuo. The residue (which had a
very low solubility in ether) was taken up in dichloromethane
(50 mL), washed with brine (2 × 50 mL), dried (MgSO4), and
evaporated in vacuo to give a white solid which was washed
with pentane and then recrystallized from hot ethanol, yielding
12 (1.26 g, 1.79 mmol, 94%) as colorless crystals: mp 135-
14a : mp 130-132 °C (from ethyl acetate/hexane); IR: (KBr
disk) νPdO 1287 cm-1; 1H NMR (CDCl3, 270 MHz) δ 2.19 (1 H,
qd, J ) 11 Hz, 4.5 Hz), 3.08 (1 H, dd, J ) 11.2 Hz, 9.2 Hz),
3.40 (1 H, t, J ) 9.3 Hz), 3.63 (1 H, t, J ) 9.4 Hz), 3.66 (1 H,
t, J ) 9.4 Hz), 3.76 (1 H, dd, J ) 11.3 Hz, 11.3 Hz), 3.768 (3
H, s), 3.773 (3 H, s), 4.04 (1 H, dd, J ) 11.2 Hz, 9.2 Hz), 4.41
(1 H, ddd, J ) 24.2 Hz, 11.4 Hz, 4.5 Hz), 4.41 (1 H, d, J ) 11.2

304 J . Org. Chem., Vol. 63, No. 2, 1998
Riley et al.
Hz, part of a broad AB system), 4.68-4.93 (7 H, m), 5.06, 5.11
(2 H, ABX, J AB ) 11.7 Hz, J HP ) 7.7 Hz, 7.7 Hz), 6.79-6.83 (4
H, m), 7.15-7.36 (19 H, m); 13C NMR (CDCl3, 68 MHz) δ 40.28
(3J CP ) 5.5 Hz), 55.20, 68.93 (2J CP ) 5.5 Hz), 70.26 (2J CP ) 7.7
Hz), 75.02, 75.59, 76.01, 76.29, 81.22 (J CP ) 7.7 Hz), 82.30 (J CP
) 2.2 Hz), 82.60 (J CP ) 7.7 Hz), 84.93, 113.77, 113.83, 127.58,
127.65, 127.97, 128.13, 128.38, 128.56, 128.67, 128.72, 129.50,
129.76, 130.15, 130.24, 135.44 (3J CP ) 7.7 Hz), 137.30, 138.25,
159.30; 31P NMR (CDCl3, 162 MHz) -7.49 (dt, 3J HP ) 24.2 Hz,
7.7 Hz, 7.7 Hz); MS m/z (positive ion FAB, relative intensity)
767 [(M + H)+, 1.2%], 675 [(M - C7H7)+, 1.2%], 645 [(M - C7H6-
OCH3)+, 1.4%], 121 [(C7H6OCH3)+, 100%], 91 [(C7H7)+, 28%];
MS m/z (negative ion FAB, rel intensity) 919 [(M + NBA)-,
80%], 765 [(M - H)-, 30%), 675 [(M - C7H7)-,100%], 645 [(M
- C7H6OCH3)-, 30%], 187 [C7H7OPO3H)-, 80%], 97 [(H2PO4)-,
45%]. Anal. Calcd for C44H47O10P (766.82): C, 68.92; H, 6.18.
Found: C, 69.1; H, 6.11.
187 [C7H7OPO3H)-, 40%], 97 [(H2PO4)-, 20%]. Anal. Calcd
for C28H31O8P (526.52): C, 63.87; H, 5.93. Found: C, 63.7; H,
6.00.
DL-(1,3,5/2,4,6)-1,3-Di-O-b en zyl-5,7-O-(b en zyloxyp h os-
p h or yl)-6-(h yd r oxym eth yl)cycloh exa n e-1,2,3,4,5-p en tol
(Ep im er 15b). The p-methoxybenzyl groups were removed
from 14b (260 mg, 0.34 mmol) using the same procedure as
for 14a. Purification by column chromatography (ethyl acetate/
dichloromethane 1:1) gave 15b (125 mg, 0.237 mmol, 70%):
mp 160-162 °C (from ethyl acetate/hexane); Rf 0.25 (ethyl
acetate/dichloromethane 1:1), Rf 0.15 (ethyl acetate/hexane
2:1); IR (KBr disk) νPdO 1263 cm-1; 1H NMR (CDCl3, 400 MHz)
δ 2.13 (1 H, qd, J ) 11 Hz, 4.9 Hz), 2.93 (1 H, d, J ) 2.4 Hz,
D2O ex), 3.13 (1 H, dd, J ) 10.7 Hz, 9.3 Hz), 3.25 (1 H, t, J )
9.3 Hz), 3.50 (1 H, br s, D2O ex), 3.63-3.69 (2 H, br m), 4.08
(1 H, td, J ) 11.2 Hz, J HP ) 4.4 Hz), 4.17 (1 H, dd, J ) 10.8
Hz, 10.7 Hz), 4.42 (1 H, ddd, J ) 20.0 Hz, 10.7 Hz, 4.9 Hz),
4.51-5.12 (6 H, AB systems), 7.25-7.40 (15 H, m); 13C NMR
(CDCl3, 100.4 MHz) δ 40.13 (3J CP ) 7.4 Hz), 70.16 (2J CP ) 7.4
Hz), 70.86 (2J CP ) 7.4 Hz), 74.81, 75.39 (J CP ) 7.3 Hz), 75.54,
76.56, 77.13, 80.08, 75.39 (J CP ) 5.5 Hz), 81.94, 128.32, 128.48,
128.55, 128.72, 128.87, 128.90, 129.05, 135.41 (3J CP ) 7.3 Hz),
137.90, 138.56; 31P NMR (CDCl3, 162 MHz) δ -4.43 (1P, br
14b: mp 101-102.5 °C (from ethanol); IR (KBr disk) νPdO
1
1266 cm-1; H NMR (CDCl3, 270 MHz) δ 2.17 (1 H, qd, J )
11.2 Hz, 4.8 Hz), 3.18 (1 H, dd, J ) 11.0 Hz, 9.0 Hz), 3.44 (1
H, t, 9.0 Hz), 3.51 (1 H, t, J ) 9.1 Hz), 3.60 (1 H, t, J ) 9.0
Hz), 3.76 (3 H, s), 3.78 (3 H, s), 4.03 (1 H, td, J ) 11.2 Hz, 3.9
Hz), 4.32 (1 H, br dd, J ) 11.2 Hz, 9.2 Hz), 4.39 (1 H, ddd, J
) 20.4 Hz, 11.2 Hz, 4.7 Hz), 4.45-4.90 (8 H, m), 5.08, 5.13 (2
H, ABX, J AB ) 11.9 Hz, J HP ) 9.9 Hz, 9.9 Hz), 6.78-6.84 (4 H,
m), 7.17-7.38 (19 H, m); 13C NMR (CDCl3, 68 MHz) δ 40.41
(3J CP ) 6.6 Hz), 55.19, 55.22, 69.47 (2J CP ) 5.5 Hz), 70.40 (2J CP
) 6.6 Hz), 75.12, 75.59, 76.06, 76.47, 81.14 (J CP ) 5.5 Hz),
82.29, 82.65 (J CP ) 7.7 Hz), 85.04, 113.65, 113.85, 127.63,
127.71, 127.86, 128.17, 128.26, 128.35, 128.62, 128.65, 129.50,
129.72, 130.18, 130.26, 135.45 (2J CP not readable), 137.28,
138.30, 159.22, 159.30; 31P NMR (CDCl3, 162 MHz) δ -4.56
3
dtd, J HP ) 20 Hz, 8 Hz, 4 Hz); MS m/z (positive ion FAB,
relative intensity) 527 [(M + H)+, 20%], 435 [(M - C7H7)+, 4%],
91 [(C7H7)+, 100%]; MS m/z (negative ion FAB, rel intensity)
1051 [(2M - H)-, 4%], 960 (5), 679 [(M + NBA)-, 40%], 525
[(M - H)-, 80%), 435 [(M - C7H7)-, 100%], 187 [C7H7OPO3H)-,
50%], 97 [(H2PO4)-, 22%]. Anal. Calcd for C28H31O8P
(526.52): C, 63.87; H, 5.93. Found: C, 63.6; H, 6.01.
DL-(1,3,5/2,4,6)-1,3-Di-O-b en zyl-5,7-O-(b en zyloxyp h os-
p h o r y l )-2 ,4 -b i s -O -[b i s (b e n z y l o x y )p h o s p h o r y l ]-6 -
(h yd r oxym et h yl)cycloh exa n e-1,2,3,4,5-p en t ol (E p im er
16a ). To a solution of bis(benzyloxy)(N,N-diisopropylamino-
)phosphine24 (345 mg, 1.00 mmol) in dry dichloromethane (3
mL) was added 1H-tetrazole (140 mg, 2.00 mmol). The
mixture was stirred at rt for 20 min, and then the diol 15a
(130 mg, 0.247 mmol) was added. Stirring was continued for
30 min, after which 31P NMR showed signals at δ 143 (1 P, s,
phosphite at C-4), 142 (1 P, d, 5J PP ) 1.2 Hz, phosphite at C-2),
3
(dtd, J HP ) 20.3 Hz, 9.9 Hz, 3.9 Hz); MS m/z (positive ion
FAB, relative intensity) 767 [(M +H)+, 2.0%], 675 [(M -
C7H7)+, 1.0%], 645 [(M - C7H6OCH3)+, 4.0%], 121 [(C7H6-
OCH3)+, 100%], 91 [(C7H7)+, 22%]; MS m/z (negative ion FAB,
relative intensity) 919 [(M + NBA)-, 20%], 765 [(M - H)-,
10%), 675 [(M - C7H7)-,100%], 645 [(M - C7H6OCH3)-, 12%],
187 [C7H7OPO3H)-, 70%], 97 [(H2PO4)-, 45%]. Anal. Calcd
for C44H47O10P (766.82): C, 68.92; H, 6.18. Found: C, 69.1;
H, 6.13.
5
-7.9 (1 P, d, J PP ) 1.2 Hz, cyclic phosphate). The mixture
was cooled to -78 °C, m-CPBA (345 mg, 2.00 mmol) was
added, and the cooling bath was removed. The mixture was
allowed to reach rt and then diluted with ethyl acetate (50
mL). The clear solution was washed with 10% Na2SO3, 1 M
HCl, saturated NaHCO3, and brine (50 mL of each), dried
(MgSO4), and evaporated in vacuo, giving a solid residue.
Purification by column chromatography (ethyl acetate/dichlo-
romethane 1:2) afforded 16a (192 mg, 0.183 mmol, 74%) as a
white solid: mp 171-172.5 °C (from ethanol); 1H NMR (CDCl3,
270 MHz) δ 2.20 (1 H, qd, J ) 11 Hz, 4.4 Hz), 3.21 (1 H, dd,
J ) 11.0 Hz, 9.0 Hz), 3.58 (1 H, t, J ) 9.2 Hz, 9.2 Hz), 3.68 (1
H, dd, J ) 11.2 Hz, 11.0 Hz), 4.10 (1 H, dd, J ) 11.2 Hz, 9.4
Hz), 4.32 (1 H, ddd, J ) 24.2 Hz, 11.0 Hz, 4.4 Hz), 4.33 (1 H,
d, J ) 11.4 Hz, part of a broad AB system), 4.50-4.68 (2 H,
m), 4.72-5.10 (13 H, m), 6.95-7.42 (35 H, m); 13C NMR
(CDCl3, 68 MHz) δ 39.0 (3J CP ) 5.5 Hz), 69.04 (2J CP ) 5.5 Hz),
69.40, 69.49, 69.60, 69.68, 74.16, 75.04, 78.20, 78.75, 79.38,
80.81, 127.36, 127.44, 127.84, 127.89, 128.09, 128.17, 128.23,
128.26, 128.36, 128.39, 128.51, 128.67, 128.77, 135.51, 135.63,
136.75, 137.54 (many signals showed J CP couplings that were
unreadable due to complex or overlapping multiplets); 31P
NMR (CDCl3, 162 MHz) δ -8.33 (1 P, dt, J ) 24.1 Hz, 7.6
Hz), -1.81, -1.65 (2 P, overlap to give m in 1H-coupled
spectrum); MS m/z (positive ion FAB, relative intensity) 1047
[(M + H)+, 5%], 181 (10), 91 [(C7H7)+, 100%]; MS m/z (negative
ion FAB, rel intensity) 1199 [(M + NBA)-, 8%], 1045 [(M -
H)-, 3%] 955 [(M - C7H7)-, 55%], 277 [(BnO)2PO2-, 100%],
187(38%), 97 [H2PO4-, 10%]. Anal. Calcd for C56H57O14P3
(1046.98): C, 64.24; H, 5.49. Found: C, 63.9; H, 5.46.
DL-(1,3,5/2,4,6)-1,3-Di-O-b en zyl-5,7-O-(b en zyloxyp h os-
p h or yl)-6-(h yd r oxym eth yl)cycloh exa n e-1,2,3,4,5-p en tol
(Ep im er 15a ). To a solution of 14a (300 mg, 0.39 mmol) in
dichloromethane (10 mL) were added water (1 mL) and DDQ
(355 mg, 1.56 mmol). The mixture was stirred at rt for 2.5 h,
after which TLC (ethyl acetate/ hexane 2:1) showed the
reaction to be complete. Dichloromethane (60 mL) was added,
and the organic layer was washed with a 10% Na2SO3 solution
(3 × 50 mL), a saturated NaHCO3 solution, and brine (50 mL
of each), dried (MgSO4), and evaporated in vacuo to give a
yellow oil which was purified by column chromatography (ethyl
acetate/pentane 3:2), giving the diol 15a (137 mg, 0.260 mmol,
66%): mp 145-149 °C (from ethyl acetate/ hexane); Rf 0.45
(ethyl acetate/dichloromethane 1:1), Rf 0.31 (ethyl acetate/
1
hexane 2:1); IR (KBr disk) νPdO 1280 cm-1; H NMR (CDCl3,
1
400 MHz, H-1H COSY) δ 2.07 (1 H, qd, J ) 11 Hz, 10.7 Hz,
4.4 Hz, C-6-H), 2.83 (1 H, d, J ) 1.96 Hz, D2O ex, C-2-OH),
3.07 (1 H, dd, J ) 10.7 Hz, 9.3 Hz, C-1-H), 3.20 (1 H, t, J )
9.3 Hz, C-3-H), 3.55 (1 H, d, J ) 2.9 Hz, D2O ex, C-4-OH),
3.63-3.70 (2 H, m, C-2-H and C-4-H), 3.81 (1 H, t, J ) 11.2
Hz, C-7-Hax), 3.86 (1 H, dd, J ) 10.7 Hz, 9.3 Hz, C-5-H), 4.43
(1 H, ddd, J ) 24.4 Hz, 11.2 Hz, 4.4 Hz, C-7-Heq), 4.48-5.13
(6 H, AB systems, CH2OBn), 7.25-7.38 (15 H, m); 13C NMR
(CDCl3, 100.4 MHz) δ 39.60 (3J CP ) 3.6 Hz), 69.15 (2J CP ) 5.5
Hz), 70.45 (2J CP ) 7.4 Hz), 74.35, 75.17, 74.70 (3J CP ) 7.4 Hz),
75.85, 76.69 (J CP unreadable), 80.90 (J CP ) 7.3 Hz), 81.32,
127.99, 128.06, 128.13, 128.28, 128.39, 128.52, 128.59, 128.72,
128.85, 135.28 (2J CP ) 7.3 Hz), 137.59, 138.17; 31P NMR
3
(CDCl3, 162 MHz) δ -7.27 (1 P, dt, J HP ) 24.2 Hz, 8.1 Hz);
DL-(1,3,5/2,4,6)-1,3-Di-O-b en zyl-5,7-O-(b en zyloxyp h os-
p h o r y l )-2 ,4 -b i s -O -[b i s (b e n z y l o x y )p h o s p h o r y l ]-6 -
(h yd r oxym et h yl)cycloh exa n e-1,2,3,4,5-p en t ol (E p im er
16b). Compound 15b (110 mg, 0.209 mmol) was phosphory-
lated using the procedure described for 15a . Purification by
MS m/z (positive ion FAB, relative intensity) 527 [(M + H)+,
22%], 435 [(M - C7H7)+, 3%], 391 (10), 181 (6), 91 [(C7H7)+,
100%]; MS m/z (negative ion FAB, relative intensity) 679 [(M
+ NBA)-, 90%], 525 [(M - H)-, 100%), 435 [(M - C7H7)-, 90%],

Cyclic Phosphate Analog of Inositol Trisphosphate
J . Org. Chem., Vol. 63, No. 2, 1998 305
flash chromatography (ethyl acetate/dichloromethane 1:3)
afforded 16b (190 mg, 0.182 mmol, 87%) as a white solid: mp
136-138 °C (from ethyl acetate/hexane); 1H NMR (CDCl3, 400
MHz) δ 2.24 (1 H, qd, J ) 11 Hz, 4.6 Hz), 3.33 (1 H, t, J ) 9.8
Hz, 9.8 Hz), 3.62 (1 H, dd, J ) 9.2 Hz, 8.9 Hz), 4.00 (1 H, br
t, J ) 11 Hz), 4.31 (1 H, ddd, J ) 21.4 Hz, 11.0 Hz, 4.6 Hz),
4.39 (1 H, d, J ) 11.3 Hz, part of a broad AB system), 4.44 (1
H, br dd, J ) 10.7 Hz, 10.1 Hz), 4.61 (1 H, td, J ) 9.2 Hz, 8.9
Hz), 4.67-5.27 (14 H, m), 6.99-7.40 (35 H, m); 13C NMR
(CDCl3, 100.4 MHz) δ 39.3 (3J CP ) 5.5 Hz), 68.88 (2J CP ) 3.7
Hz), 69.17 (2J CP ) 5.5 Hz), 69.54 (2J CP ) 5.5 Hz), 69.59 (2J CP
) 5.5 Hz), 70.82 (2J CP ) 7.4 Hz), 74.18, 74.34, 75.31, 77.14,
78.82, 79.44, 81.03, 127.27, 127.32, 127.72, 127.89, 128.00,
blue-black for 10 min. Either compound 16a or 16b (95 mg,
91 µmol) was dissolved in anhydrous dioxane (2 mL) and added
to the vigorously stirring sodium-liquid ammonia mixture.
After 60-90 s the reaction was quenched by careful addition
of methanol until the blue color disappeared, followed by
deionized water (10 mL). Ammonia and solvents were then
removed by evaporation in vacuo. The residue was dissolved
in deionized water (500 mL) and purified by ion-exchange
chromatography on Q Sepharose Fast Flow Resin, eluting with
a gradient of triethylammonium bicarbonate buffer (0 to 1 M),
pH 8.0. The glassy triethylammonium salt of 4 eluted between
500 and 650 mM TEAB and was quantified by phosphate assay
1
(yield 78-82%): 1H NMR (D2O, 400 MHz H-1H COSY, Na+
128.13, 128.22, 128.33, 128.42, 128.60, 128.71, 134.88 (3J CP
)
salt, pH 8) δ 1.94 (1 H, qd J ) 11 Hz, 4.4 Hz, C-6-H), 3.32 (1
H, dd, J ) 10.7 Hz, 9.3 Hz, C-5-H), 3.41 (1 H, dd, J ) 9.3 Hz,
8.8 Hz, C-3-H), 3.80 (1 H, td, J ) 8.8 Hz, 7.8 Hz, C-4-H), 3.85-
3.98 (3 H, m, C-1-H, C-2-H, C-7-Hax), 4.21 (1 H, ddd, J ) 22.9
Hz, 11.3 Hz, 4.4 Hz, C-7-Heq); 13C NMR (D2O, 100.4 MHz, Na+
salt, pH 8) δ 41.67 (3J CP ) 3.6 Hz, C-6), 69.15 (2J CP ) 5.4 Hz,
C-7), 70.58, 74.37 (J CP ) 3.7 Hz), 77.57 (J CP ) 5.5 Hz, 5.5 Hz),
78.14 (J CP ) 7.3 Hz, 5.5 Hz), 80.21 (J CP ) 5.5 Hz); 31P NMR
7.3 Hz), 135.54 (3J CP ) 7.3 Hz), 135.66, 135.73, 135.83, 136.76,
137.65 (many signals showed J CP couplings that were unread-
able due to complex or overlapping multiplets); 31P NMR
(CDCl3, 162 MHz) δ -5.28 (1 P, dtd, J ) 21.4 Hz, 7.6 Hz, 2
Hz), -1.58, -1.51 (2 P, overlap to give m in 1H-coupled
spectrum); MS m/z (positive ion FAB, relative intensity) 1047
[(M + H)+, 70%], 181 (12), 91 [(C7H7)+, 100%]; MS m/z
(negative ion FAB, relative intensity) 1212 (65), 1199 [(M +
NBA)-, 30%], 1046 (50), 955 [(M - C7H7)-, 100%], 277
[(C6H5O)2PO2-, 60%]. Anal. Calcd for C56H57O14P3 (1046.98):
C, 64.24; H, 5.49. Found: C, 64.4; H, 5.55.
1
(D2O, 162 MHz, H-coupled, Na+ salt, pH 8) δ -2.76 (1 P, d,
J HP ) 22.5 Hz, P1), 2.99 (1 P, d, J HP ) 7.1 Hz, P2), 3.96 (1 P,
d, J HP ) 6.8 Hz, P4); MS m/z (negative ion FAB, relative
intensity) 831 [ (2M - H)-, 20%], 415 [(M - H)-, 90%], 159-
(100), 97 [H2PO4-, 83%]; accurate mass negative ion FAB m/z
calcd for C7H14O14P3-, 414.960, found 414.962.
DL-(1,3,5/2,4,6)-6-(Hydr oxym eth yl)cycloh exan e-1,2,3,4,5-
p en t ol 1:7-Cyclic 2,4-Tr isp h osp h a t e [DL-6-d eoxy-6-(h y-
d r oxym et h yl)-scyllo-in osit ol 1:7-Cyclic 2,4-Tr isp h os-
p h a te] (4). Ammonia (∼100 mL) was condensed into a three-
necked flask at -78 °C. An excess of sodium was added to
dry the liquid ammonia, and the blue-black solution was
stirred at -78 °C for 30 min. A small volume of ammonia (∼30
mL) was then distilled into a second three-necked flask and
kept at -78 °C. Sodium was added until the solution remained
Ack n ow led gm en t. We thank the Wellcome Trust
(Program Grant 045491) for financial support and
Professor J . Westwick for preliminary biological evalu-
ation of 4.
J O9714425