Bicyclic analogues of D-myo-inositol 1,4,5-trisphosphate related to adenophostin a: Synthesis and biological activity

Article abstract of DOI:10.1021/jm0005499

The high affinity of adenophostin A for 1D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] receptors may be related to an alteration in the position of its 2′-phosphate group relative to the corresponding 1-phosphate group in Ins(1,4,5)P_3<

Full text of DOI:10.1021/jm0005499

2108
J . Med. Chem. 2001, 44, 2108-2117
Bicyclic An a logu es of D-m yo-In ositol 1,4,5-Tr isp h osp h a te Rela ted to
Ad en op h ostin A: Syn th esis a n d Biologica l Activity
Andrew M. Riley,† Vanessa Correa,‡ Mary F. Mahon,§ Colin W. Taylor,‡ and Barry V. L. Potter*,†
Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down,
Bath BA2 7AY, U.K., Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ , U.K.,
and Department of Chemistry, University of Bath, Claverton Down, Bath BA2 7AY, U.K.
Received December 27, 2000
The high affinity of adenophostin A for 1D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]
receptors may be related to an alteration in the position of its 2′-phosphate group relative to
the corresponding 1-phosphate group in Ins(1,4,5)P3. To investigate this possibility, two bicyclic
trisphosphates 9 and 10, designed to explore the effect of relocating the 1-phosphate group of
Ins(1,4,5)P3 using a novel fused-ring system, were synthesized from myo-inositol. Biological
evaluation of 9 and 10 at the Ins(1,4,5)P3 receptors of hepatocytes showed that both were
recognized by hepatic Ins(1,4,5)P3 receptors and both stimulated release of Ca2+ from
intracellular stores, but they had lower affinity than Ins(1,4,5)P3. This finding may be explained
by considering the three-dimensional structures of 9 and 10 in light of recent studies on the
conformation of adenophostin A.
In tr od u ction
(ribofuranosyl) ring. Removal of the adenine from 3 to
give 5 reduces affinity to about that of Ins(1,4,5)P3,6
Many extracellular stimuli activate the hydrolysis of
a phospholipid component of the plasma membrane,
phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2],
to generate 1D-myo-inositol 1,4,5-trisphosphate
[Ins(1,4,5)P3, 1, Figure 1].1 Ins(1,4,5)P3 acts as a second
messenger in most cells, where it binds to intracellular
Ins(1,4,5)P3 receptors [IP3R] to cause the opening of an
intrinsic Ca2+ channel through which Ca2+ leaks from
the lumen of the endoplasmic reticulum to the cytosol.
Each of the four subunits of each IP3R binds
Ins(1,4,5)P3 at a site formed by two distinct domains.
The site includes several positively charged Arg and Lys
residues which interact with the three phosphate groups
of Ins(1,4,5)P3.2 Structure-activity studies3 have shown
that all high-affinity agonists of IP3R contain groups
equivalent to the vicinal 4R,5R-trans-diequatorial
bisphosphate and adjacent 6-hydroxyl group of
Ins(1,4,5)P3, together with an appropriately positioned
nonvicinal phosphate group. Some variation in the
positioning of this third phosphate group is tolerated,
so that Ins(2,4,5)P3 (2), for example, is also recognized
by IP3Rs, although typically with some 25-fold lower
affinity than Ins(1,4,5)P3.4
In 1993, two new ligands of Ins(1,4,5)P3 receptors,
adenophostins A and B (3 and 4, Figure 1) were isolated
from culture broths of Penicillium brevicompactum and
found to be up to 100 times more potent than
Ins(1,4,5)P3 itself.5 In the adenophostins, the 3′′,4′′-
bisphosphate and 2′′-hydroxyl group of the glucopyra-
nosyl ring are thought to mimic the inositol 4,5-
bisphosphate and adjacent hydroxyl group of Ins(1,4,5)P3.
Intriguingly, the third phosphate group (analogous to
the 1-phosphate of Ins(1,4,5)P3) is located on a separate
* To whom correspondence should be addressed. Tel: +44 1225-
826639. Fax: +44 1225-826114. E-mail: B.V.L.Potter@bath.ac.uk.
† Department of Pharmacy and Pharmacology, University of Bath.
‡ University of Cambridge.
F igu r e 1. Structures of 1D-myo-inositol 1,4,5-trisphosphate
(1), 1D-myo-inositol 2,4,5-trisphosphate (2), adenophostins A
(3) and B (4), adenophostin analogues (5-8), and epimeric
bicyclic Ins(1,4,5)P3 analogues (9 and 10).
§ Department of Chemistry, University of Bath.
10.1021/jm0005499 CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/22/2001

Synthesis of Bicyclic Analogues of Ins(1,4,5)P3
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2109
Sch em e 1a
although further simplification by removal of the 4′-
hydroxymethyl group (6) has little further effect.6,7
However, opening of the five-membered ring, with a
consequent increase in the conformational mobility of
the nonvicinal phosphate, causes a greater reduction in
affinity. Thus, analogue 78,9 and its xylose-based equiva-
lent 810 were found to be 10 or more times weaker than
Ins(1,4,5)P3.
It has been suggested5,8 that, at the binding site of
the IP3R, the nonvicinal phosphate group in the adeno-
phostins may be held further away from the vicinal pair
than is the corresponding 1-phosphate of Ins(1,4,5)P3,
and that this modification may underlie the greater
affinity of the adenophostins for the IP3R. With this
explanation, the adenosine motif of 3 and 4 functions
simply to present the 2′-phosphate in a position that
modifies its interaction with one or more residues at the
receptor binding site. An alternative explanation for
the enhanced potency of the adenophostins is that
the adenine itself interacts with a region of the
Ins(1,4,5)P3 receptor close to the Ins(1,4,5)P3-binding
site.11,12 The two alternative explanations for adeno-
phostin activity (modified positioning of a phosphate
group versus a specific interaction of the adenine with
the receptor) are not necessarily mutually exclusive, and
an interplay between the two may be involved.
We sought to explore the consequences for biological
activity of relocating the 1-phosphate group of
Ins(1,4,5)P3 to a region of space further away from the
inositol ring by synthesising bicyclic analogues of
Ins(1,4,5)P3. Here we describe the synthesis and biologi-
cal evaluation of the first two such analogues 9 and 10.
These epimeric molecules, based on myo-inositol fused
to a seven-membered (1,4-dioxepane) ring, place their
nonvicinal phosphate groups in regions of space more
distant from the inositol ring than are normally acces-
sible to the 1-phosphate group of Ins(1,4,5)P3. They
retain the three-atom (O-C-C) linkage between the six-
membered ring and the nonvicinal phosphate charac-
teristic of the adenophostins and analogues 5-8, but
they lack the conformational freedom allowed by the
presence of the glycosidic linkage in these carbohydrate-
based compounds. A preliminary report of the synthesis
of 9 and 10 has appeared.13
a
Reagents and conditions: (i) butanedione, MeOH, CH(OMe)3,
(()-10-camphorsulfonic acid, reflux, 80%; (ii) (S)-(+)-acetylman-
delic acid, DCC, DMAP, CH2Cl2, -20 °C to room temperature, 34%
(13), 36% (14); (iii) NaOH, MeOH, reflux, 95%; (iv) CH2Cl2/
CF3COOH/H2O 25:24:1, 98%. Compounds (()-11 and (()-12 are
racemic
OH-group with (S)-(+)-acetylmandelic acid,17 we have
found that this method generally works well for myo-
inositols with this pattern of protection, giving diaste-
reoisomeric esters that are easily separated by flash
chromatography.18 An additional advantage of acetyl-
mandelic acid as a resolving agent is that both enanti-
omers are commercially available at similar cost and
in high optical purity. The resolution can initially be
carried out on a small scale, and once the absolute
configurations of the products are determined, the
appropriate enantiomer of acetylmandelic acid can be
chosen so as to optimize isolation of the required product
on scale-up.
Applying this method to (()-11, it was found that
regioselective esterification of (()-11 with (S)-(+)-acetyl-
mandelic acid gave diastereoisomeric acetylmandelate
esters which were readily separated by flash chroma-
tography followed by crystallization. The less polar ester
was particularly easy to isolate and was highly crystal-
line, while the more polar ester required more careful
chromatography and gave waxy, less satisfactory crys-
tals. Saponification of 13 and 14 gave the enantiomeric
diols (-)-12 and (+)-12, respectively. To determine the
absolute configurations, (-)-12 was converted into the
di-O-benzyl ether (-)-11 by treatment with TFA. The
tetrol (-)-11 had an optical rotation in agreement with
that reported for the known 1D-1,4-di-O-benzyl-myo-
inositol,18b identifying the more polar ester as 13 and
the more crystalline, less polar ester as the required 14.
Accordingly, (S)-(+)-acetylmandelic acid was used as
resolving agent in the full-scale route.
Resu lts a n d Discu ssion
The synthetic route to 9 and 10 begins with the
known DL-1,4-di-O-benzyl-myo-inositol [(()-11, Scheme
1], readily accessible on a 20 g scale without chroma-
tography from myo-inositol.14 This tetrol contains two
pairs of vicinal hydroxyl groups, one cis and one trans,
and it was found that the trans pair could be protected
selectively by using the butane diacetal (BDA) protect-
ing group. Thus, acid-catalyzed reaction of (()-11 with
tetramethoxybutane15 in refluxing methanol, in the
presence of trimethyl orthoformate, gave the crystalline
racemic diacetal (()-12. It was later found that identical
results could be achieved more cheaply and conveniently
using a modified procedure16 in which tetramethoxy-
butane is replaced with commercially available butane-
dione.
An optical resolution step was now used to isolate the
required enantiomer of diol 12. Following a report of
the optical resolution of (()-1,4,5,6-tetra-O-benzyl-myo-
inositol by regioselective esterification of the equatorial

2110 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Riley et al.
Sch em e 2a
a
Reagents and conditions: (i) NaH, 3-chloro-2-chloromethyl-1-propene, DMF, 84%; (ii) RuCl3, NaIO4, EtOAc/CH3CN/H2O, 82%; (iii)
NaBH4, MeOH, 0 °C to room temperature, 21% (17), 67% (18); (iv) CH2Cl2/CF3COOH/H2O 25:24:1, 83-84%; (v) (a) (BnO)2PNPri2, 1H-
tetrazole, CH2Cl2; (b) m-CPBA, -78 °C to room temperature, 82-90%; (vi) H2, 50 psi, Pd-C, MeOH, 89-90%.
Reaction of (+)-12 with sodium hydride and 3-chloro-
2-chloromethyl-1-propene in DMF (Scheme 2) gave the
alkene 15 in 84% yield. Flash dihydroxylation19 of the
alkene employing RuCl3/NaIO4 gave an inseparable 3:1
mixture of epimeric diols within 4 min. Prolongation of
the reaction time to 1.5 h gave the ketone 16, which
was isolated as a crystalline solid. Sodium borohydride
was chosen to reduce ketone 16, in the hope that both
epimeric alcohols 17 and 18 might be obtained. This
proved to be the case, and the reaction gave two alcohols
in a ratio of approximately 1:3. These alcohols were
separated by flash chromatography and isolated as
crystalline solids. A single-crystal X-ray study of the
more polar alcohol (major product) identified it as 18,
and thus the minor product was the epimer, 17. The
X-ray structure of 18 (Figure 2a) shows the relative
configuration at the C-2′ position, and the 1,4-dioxepane
ring is seen to take on a slightly twisted chair confor-
mation. The supramolecular structure is consolidated
by intermolecular hydrogen bonding, as shown in Figure
2b.
The BDA protecting groups were removed from 17
and 18 by treatment of each with TFA, giving the two
triols 19 and 20. Phosphitylation of 19 and of 20 using
bis(benzyloxy)(N,N-diisopropylamino)phosphine acti-
vated by 1H-tetrazole, followed by in situ oxidation of
phosphites with m-CPBA, gave the trisphosphate tri-
esters 21 and 22. While 22 was an oil, 21 was a
crystalline solid. Finally, deprotection of each epimer
by hydrogenolysis over Pd-C and purification of the
products by ion-exchange chromatography on Q-Sepha-
rose Fast Flow resin gave the pure epimeric trisphos-
phates 9 and 10 as their triethylammonium salts, which
were quantified by a modification of the Briggs total
phosphate assay.20
Ta ble 1. Binding of Ins(1,4,5)P3 (1), Adenophostin A (3), and
the Bicyclic Analogues 9 and 10 to Hepatic Ins(1,4,5)P3
Receptors
ratiob to
Ins(1,4,5)P3
Kd (nM)a
ha
n
Ins(1,4,5)P3 (1)
adenophostin A (3) 0.55 ( 0.12
9
4.56 ( 0.48
1
1.16 ( 0.19
1.17 ( 0.11
1.01 ( 0.07
1.12 ( 0.08
4
3
5
3
8.3 ( 2.0
-21 ( 6
-14 ( 2
98 ( 28
62 ( 4
10
a
Kds and Hill coefficients (h) were determined using equilibrium
competition binding with [3H]-Ins(1,4,5)P3. Results are shown as
means ( SEM for n independent experiments. Ratios greater
b
than 1 denote ligands with greater affinity than Ins(1,4,5)P3, and
ratios less than -1 denote ligands with lesser affinity than
Ins(1,4,5)P3.
hepatic Ins(1,4,5)P3 receptors was 8-fold greater than
that of Ins(1,4,5)P3, in line with previous results.11
However, analogues 9 and 10 had affinities 21- and 14-
fold lower than Ins(1,4,5)P3, respectively (Table 1).
These results were reflected in functional studies of
45Ca2+ release (Figure 3b). Maximal concentrations of
Ins(1,4,5)P3 adenophostin A and analogues 9 and 10
each released the same fraction (50-60%) of the intra-
cellular stores, but whereas adenophostin A was about
10-fold more potent than Ins(1,4,5)P3, analogues 9 and
10 were about 18- and 14-fold less potent, respectively
(Table 2). The rank order of potency of the ligands
[adenophostin > Ins(1,4,5)P3 > 10 > 9] was therefore
the same as that found with radioligand binding. In
summary, both bicyclic analogues had lower affinity for
hepatic IP3Rs than Ins(1,4,5)P3, and they appeared
closer to Ins(2,4,5)P3 in activity.
The simplest interpretation of these results is that
the 2′-phosphate groups in 9 and in 10 are unable to
interact effectively with the appropriate region of the
Ins(1,4,5)P3 receptor as a result of their three-dimen-
sional location and orientation at the receptor binding
site. This location will be influenced by the conformation
of the dioxepane rings in 9 and 10. In general, seven-
membered rings are flexible systems for which there
exist complex conformational equilibria between several
In equilibrium competition binding experiments using
[3H]-Ins(1,4,5)P3 and membranes prepared from rat
liver, adenophostin A, Ins(1,4,5)P3, and each of ana-
logues 9 and 10 completely displaced the specific [3H]-
Ins(1,4,5)P3 binding in a concentration-dependent man-
ner (Figure 3a). The affinity of adenophostin A for

Synthesis of Bicyclic Analogues of Ins(1,4,5)P3
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2111
F igu r e 3. Effects of 9 and 10 on equilibrium competition
3
binding of H-Ins(1,4,5)P3 to liver membranes (a) and 45Ca2+
release from the intracellular stores of permeabilized hepato-
3
cytes (b). (a) Specific H-Ins(1,4,5)P3 binding (%) is shown in
the presence of the indicated concentrations of adenophostin
A (filled squares), Ins(1,4,5)P3 (filled circles), 9 (filled triangles),
and 10 (open squares). (b) The amounts of 45Ca2+ released [%
of the Ins(1,4,5)P3-sensitive stores] by Ins(1,4,5)P3, 9, and 10
are shown [the symbols are the same as used in panel a]. For
both panels, results are means ( SEM of three to five
independent determinations.
Ta ble 2. Effects of Ins(1,4,5)P3 (1), Adenophostin A (3), and
the Bicyclic Analogues 9 and 10 on Ca2+ Release from the
Intracellular Stores of Permeabilized Hepatocytes
F igu r e 2. (a) ORTEX31 plot of 18. Ellipsoids are represented
at the 30% probability level. (b) Hydrogen-bonding interactions
in the supramolecular structure of 18.
maximal
EC50
ratiob to
Ins(1,4,5)P3
response
(nM)a
ha
(%)a
conformations of similar energies,21 although in the
bicyclic structures 9 and 10, the fusion of the 1,4-
dioxepane ring with myo-inositol greatly restricts the
number of possible conformations. Measurement of
Ins(1,4,5)P3 (1)
adenophostin A (3)
9
10
90 ( 7
9.2 ( 1.7
1650 ( 90
1230 ( 50
1
1.48 ( 0.21 59 ( 2
9.8 ( 2.0 2.99 ( 0.32 49 ( 3
-18 ( 2
-14 ( 1
2.61 ( 0.57 59 ( 1
2.9 ( 0.55 59 ( 1
1
vicinal coupling constants in the H NMR spectrum of
a
Concentration causing half the maximal effect (EC50), Hill
coefficient (h), and the fraction of the total Ca2+ stores released
by a maximal concentration of each agonist are shown (means (
SEM) for Ca2+ mobilization evoked by each of the ligands for three
the marginally more potent analogue 10, taken in D2O
buffered to physiological pH, confirmed that the con-
formation of the myo-inositol ring was close to a chair.
A
1H NMR-NOESY spectrum of 10 showed NOEs
b
independent experiments. Ratios greater than 1 denote ligands
with greater potency than Ins(1,4,5)P3 and ratios less than -1
denote ligands with lesser potency than Ins(1,4,5)P3.
between H-2 of the myo-inositol ring and one proton in
each pair of methylene protons at C-1′ and C-3′. This
supports the existence in solution of conformations of
type 10a (Figure 4a) in which the dioxepane ring adopts
a twist-chair conformation similar to that seen in the
X-ray structure of intermediate 18. The measured
vicinal coupling constants between H-2′ and the meth-
ylene protons on C-1′ and C-3′ in the dioxepane ring
were not wholly consistent with theoretical values
calculated22 for 10a alone, however, which suggested
that other conformations may be present in equilibrium
with 10a , leading to an averaging of measured J values
in the NMR spectrum. Molecular mechanics simulations
of 10 (see Experimental Section) supported this inter-
pretation; conformations of type 10a were predicted to
be the most stable, but variants with different puckers
of the dioexepane ring (Figure 4b) were also identified
with only slightly higher (1 to 2 kcal/mol) calculated
energies. Unfortunately, severe overlapping of signals
in the 1H NMR spectrum of 9 precluded detailed
analysis, but molecular modeling (not shown) as for 10
suggested that the 2′-phosphate group in 9 would be
constrained to a similar region of space as in 10 as a
result of the interplay between 2′-phosphate orientation

2112 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Riley et al.
F igu r e 4. (a) NOESY spectrum of 10 in D2O at pH 7.2 shows
NOEs between H-2 and two methylene protons in the 1,4-
dioxepane ring, indicating the presence of conformations of
type 10a . (b) Superimposed low-energy conformations of 10
as determined by molecular mechanics calculations (see
Experimental Section). For clarity, hydrogen atoms are not
shown.
and dioxepane ring-pucker. This finding would account
for the similar potencies of the two epimers.
F igu r e 5. Comparison of Ins(1,4,5)P3, adenophostin A and
bicyclic analogues. The shaded cones indicate regions of space
accessible to the nonvicinal phosphate group in each molecule.
Adenophostin A is shown in the conformation proposed by a
recent study.12 Analogues 9 and 10 are represented by a single
structure, shown in two binding orientations; dashed lines
indicate alternative dioxepane ring conformations.
While this work was in progress, a detailed confor-
mational study of adenophostin A using a combination
of NMR and molecular mechanics calculations was
reported.12 The authors proposed an active conformation
of adenophostin A in which the 2′-phosphate group is
held by the adenosine in a position close to the gluco-
pyranosyl ring in a slightly more extended position than
that of the 1-phosphate group in Ins(1,4,5)P3. Indepen-
dent evidence for this type of conformation of adeno-
phostin A in solution has recently emerged from a
combined NMR and potentiometric study by some of
us,23 which showed that, in solution, the protonation
state of the 2′-phosphate group in adenophostin A
influences the chemical shift of H-1′′, presumably via
an influence on the local electric field. There also
appears to be some pH-dependent interaction between
the 2′- and 3′′-phosphates of adenophostin A, analog-
ous to that between the 1- and 4-phosphates of
Ins(1,4,5)P3. (Indeed this study also found that, at
physiological pH, the ionization states of the three
phosphate groups in adenophostin A are remark-
ably similar to the corresponding phosphates in
Ins(1,4,5)P3, suggesting that differences in phosphate
ionization are unlikely to account for the greater potency
of adenophostin A).23 Thus, molecular mechanics simu-
lations (in vacuo) and studies of adenophostin A in
solution favor conformations for adenophostin A in
which the 2′-phosphate is held close to the glucopyra-
nosyl ring. If this proximity is retained in the receptor-
bound conformation of adenophostin A, the much lower
potency of 9 and 10 would be a result of their 2′-
phosphate groups being constrained to an overextended
position in either possible orientation of these molecules
at the IP3R binding site (Figure 5).
F igu r e 6. (a) Recently reported24 glucose-based bicyclic
analogues, spirophostins 23 and 24. (b) Acyclophostin25 (25)
and its xylose-based equivalent 26.26
These results do not necessarily imply that the goal
of constructing potent Ins(1,4,5)P3 analogues by confor-
mational restriction alone is unattainable, although it
is conceivable that adenophostin-like activity requires
exquisite precision in the location and orientation of the
2′-phosphate group at the receptor binding site. It has
been suggested, for example, that in the adenophostins
the adenine may finely tune this positioning by a subtle
effect on ribose conformation,12 hence the merely Ins-
(1,4,5)P3-like activity of analogues such as 5 and 6.
However, if the adenine itself is directly responsible for
the novel properties of the adenophostins, then confor-
mationally restricted analogues lacking the adenine (or
similar structure) may never exceed Ins(1,4,5)P3 in
potency. In support of a specific role for the adenine,
two recent reports25,26 have shown that its introduction
into analogues 7 and 8, to give 25 (“acyclophostin”) and
26, respectively (Figure 6b), can increase affinity for the
IP3R to Ins(1,4,5)P3-like levels, demonstrating that the
adenine can enhance binding to the IP3R, even in
combination with a flexible linkage to the nonvicinal
phosphate. The accumulating evidence therefore favors
the existence of a binding pocket for adenine, or for
similar aromatic/hydrophobic structures, close to the
Ins(1,4,5)P3 binding site. We are currently exploring this
possibility by investigating the effects of base-modified
analogues of adenophostin A, prepared by total synthe-
sis from D-xylose and D-glucose.27
During the preparation of this manuscript, a report
was published on the synthesis and evaluation of two
glucose-based bicyclic analogues (“spirophostins” 23 and
24, Figure 6a) designed as conformationally restricted
analogues of adenophostin A.24 These molecules con-
strain the nonvicinal phosphate group to a significantly
different region of space than in 9 and 10. However, the
spirophostins also bind to Ins(1,4,5)P3 receptors with
affinities lower than Ins(1,4,5)P3 (28-fold and 17-fold,
respectively, in bovine adrenal cortex membranes)
similar to those of 9 and 10 (21-fold and 14-fold lower
than Ins(1,4,5)P3, respectively, in hepatocytes).

Synthesis of Bicyclic Analogues of Ins(1,4,5)P3
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2113
using the TRIPOS force field and distance-dependent
a
Exp er im en ta l Section
dielectric function of ꢀ ) r, terminating at a gradient of 0.05
kcal mol-1 Å-1. Different conformations of the 1,4-dioxepane
ring in each structure were generated by 100 cycles of heating
to 1000 K for 2000 fs followed by exponential cooling to 0 K
over 10000 fs. During these cycles of simulated annealing, the
4,5-diequatorial bisphosphate conformation was retained by
constraining this region of the molecule as an aggregate. Each
cooled structure was then re-minimized as before. The protocol
was repeated three times, using different starting conforma-
tions of the dioxepane ring, to ensure that all stable conforma-
tions were located. Low-energy conformations identified in this
way were further investigated by systematically varying the
H2′-C2′-O2′-P2′ torsion in 10° steps, and re-minimizing after
each step.
[3H]-In s(1,4,5)P 3 Bin d in g to Hep a tic Mem br a n es. Rat
liver membranes were prepared from a liver perfused in situ
with cold saline, which was then excised and homogenized in
cold buffered sucrose (250 mM sucrose, 5 mM Hepes, 1 mM
EGTA, pH 7.4) using a Dounce homgenizer with 10 strokes of
a loose-fitting plunger and three with a tight plunger, before
filtration through gauze. After centrifugation (2500g, 10 min),
the pellet was resuspended and centrifuged (35000g, 30 min)
through a Percoll gradient (11.8%, Pharmacia). The mem-
branes were harvested, washed twice (48000g, 10 min) in
hypotonic medium (1 mM EGTA, 5 mM Hepes, pH 7.4), and
then resuspended in binding medium (50 mM Tris, 1 mM
EDTA, pH 8.3 at 4 °C) as previously described.11 For binding
experiments, membranes (0.1 mg protein/tube) in binding
medium (500 µL) were incubated with [3H]-Ins(1,4,5)P3 (1.3-
1.8 nM) and appropriate concentrations of competing ligand
for 5 min at 4 °C, before separation of bound from free ligand
by centrifugation (20000g, 5 min). After aspiration of the
supernatant, the pellets were resuspended in Ecoscint A liquid
scintillation cocktail (National Diagnostics, Atlanta, GA).
Equilibrium competition binding curves were fitted to logistic
equations using Kaleidagraph (Synergy Software), and the IC50
values were then used to calculate equilibrium dissociation
constants (Kd).
45Ca 2+ Relea se fr om P er m ea bilized Ra t Hep a tocytes.
The methods were similar to those reported previously.11
Briefly, permeabilized hepatocytes were loaded to steady state
(5 min at 37 °C) with 45Ca2+ in a cytosol-like medium (CLM:
140 mM KCl, 20 mM NaCl, 2 mM MgCl2, 1 mM EGTA, 300
µM CaCl2, 20 mM Pipes, pH 7.0) containing ATP (1.5 mM),
creatine phosphate (5 mM), creatine phosphokinase (5 units/
mL), and FCCP (10 µM). After 5 min, thapsigargin (1.25 µM)
was added to the cells to inhibit further Ca2+ uptake, 30 s later
the cells were added to appropriate concentrations of the
agonists, and after a further 60 s the Ca2+ contents of the
stores were determined by rapid filtration. Concentration-
response relationships were fitted to a four-parameter logistic
equation using Kaleidegraph software (Synergy Software, PA)
from which the maximal response, half-maximally effective
agonist concentration (EC50), and Hill slope (h) were
determined. All results are expressed as means ( SEM.
Ins(1,4,5)P3 was from American Radiolabeled Chemicals.
(2′RS,3′RS)-DL-1,4-Di-O-b en zyl-5,6-O-(2′,3′-d im et h oxy-
bu ta n e-2′,3′-d iyl)-m yo-in ositol [(()-12]. To a suspension of
DL-1,4-di-O-benzyl-myo-inositol14 [(()-11, 15.5 g, 43.0 mmol]
in MeOH (200 mL) and trimethyl orthoformate (20 mL) were
added camphorsulfonic acid (500 mg, 2.15 mmol) and butane-
dione (4.0 mL, 46 mmol). The mixture was heated at reflux
under N2 for 18 h. The resulting pink solution was allowed to
cool, and Et3N (1 mL) was added. The solution was stirred at
room temperature for a further 5 min, and the solvents were
removed by evaporation under reduced pressure to give a foam.
The foam was taken up in the minimum volume of CH2Cl2,
loaded onto a short column of silica gel, and eluted with EtOAc/
hexane (1:1) to give the racemic diol (()-12 (16.3 g, 34.3 mmol,
80% yield) as a white crystalline solid: mp 141-143 °C (from
EtOAc/hexane); 1H NMR (270 MHz, CDCl3) 1.36 (3 H, s, CH3),
1.37 (3 H, s, CH3), 2.62 (2 H, br s, D2O exch. OH-2 and OH-3),
3.30 (3 H, s, OCH3), 3.33 (3 H, s, OCH3), 3.43 (1 H, dd, J )
Thin-layer chromatography (TLC) was performed on pre-
coated plates (Merck TLC aluminum sheets silica 60F254) with
detection by UV light or with phosphomolybdic acid in
methanol followed by heating. Flash chromatography was
performed on silica gel (particle size 40-63 µm). NMR spectra
were recorded with a J EOL EX-270 or 400 spectrometer.
Chemical shifts are reported in parts per million (δ), and
signals are expressed as s (singlet), d (doublet), t (triplet), q
1
(quartet), m (multiplet), or br (broad). H NMR and 13C NMR
shifts are measured in ppm relative to internal tetramethyl-
1
silane (TMS). In the case of H NMR spectra taken in D2O,
acetone (δ 2.225 relative to TMS) was used as an internal
reference. 31P NMR shifts were measured in ppm relative to
external 85% phosphoric acid and are positive when downfield
from this reference. Coupling constants (J ) are given in Hz.
FAB mass spectra were recorded at the University of Bath
using m-nitrobenzyl alcohol as the matrix. Melting points
(uncorrected) were determined 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 triethylammonium bicarbonate (TEAB) as eluent.
Quantitative analysis of phosphate was performed using a
modification of the Briggs phosphate assay.20 Adenophostin
A was synthesized as previously reported.28
X-r a y Cr ysta llogr a p h y. A crystal of 18 having approxi-
mate dimensions 0.5 × 0.4 × 0.15 mm was used for data
collection. Crystal data: C29H38O9, M ) 530.59, orthorhombic,
a ) 10.163(3) Å, b ) 10.216(2) Å, c ) 26.818(8) Å, R ) 90°, â
) 90°, γ ) 90°, U ) 2784.4(13) Å3, space group P212121, Z ) 4,
Dc ) 1.266 g cm-3, µ(Mo-KR) ) 0.093 mm-1, F(000) ) 1136.
Crystallographic measurements were made at 293(2) K on a
CAD4 automatic four-circle diffractometer in the range 2.13
< θ < 22.93°. Data (2255 reflections) were corrected for Lorentz
and polarization but not for absorption. Early searches for
diffraction spots indicated that the sample chosen for this
determination (largest in batch) was not a strong diffractor,
possibly due to the smaller than desirable thickness of the
crystal. In an effort to counteract this, the scan parameters
for data collection were adjusted to increase the reliability of
the weaker data and thus maximize structural solution ability
using direct methods. In practice, the whole molecule was
evident in the first electron density map produced. Anisotropic
refinement of all atoms proceeded without any correlation
problems, although isotropic restraints were placed on carbons
1, 11, 14, and 20. The absolute configuration of the molecule
could not be determined reliably from a refined Flack param-
eter. This is probably due, in part, to the high proportion of
weak data present, reflected in an R(σ) value of 0.2796. In the
final least squares cycles, all atoms were allowed to vibrate
anisotropically. Hydrogen atoms were included at calculated
positions where relevant. Examination of the gross structure
revealed the presence of a hydrogen bonding feature along b.
Typically, the hydroxyl proton (H3) has possible contacts to
1
O2 and O4 of the molecule generated via the -x, -1/2 + y, /2
- z operation. The H3‚‚‚O4 distance is tenuous, but as the
hydrogens are included at calculated positions, this contact is
also illustrated on the packing diagram (Figure 2b) [O3‚‚‚O4,
3.23(1) Å; O3‚‚‚O2, 2.81(1) Å; H3‚‚‚O4, 2.5(1) Å; H3‚‚‚O2, 2.1-
(1) Å; O3-H3-O4, 144(9)°; O3-H3-O2, 147(9)°]. The solution
of the structure (SHELX-90)29 and refinement (SHELXL-97)30
converged to a conventional [i.e., based on 805 F2 data with
Fo > 4σ(Fo)] R1 ) 0.0824 and wR2 ) 0.1589. Goodness of fit )
0.785. The maximum and minimum residual densities were
0.257 and -0.221 e Å-3, respectively. The asymmetric unit
(shown in Figure 2a), along with the labeling scheme used,
was produced using ORTEX.31
Molecu la r Mod elin g. Modeling was carried out using the
Sybyl software package (v6.3, Tripos Associates) on a Silicon
Graphics workstation. The TRIPOS force field was used.
Charges were calculated using a semiempirical method (AM1)
for structures of 9 or 10 with fully-ionized phosphate groups,
and the resulting charged structures were energy-minimized

2114 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Riley et al.
9.9, 2.7, H-3), 3.48 (1 H, dd, J ) 9.2, 2.7, H-1), 3.64 (1 H, dd,
J ) 9.9, 9.7, H-5), 3.83 (1 H, t-like dd, J ) 9.5, 9.5, H-4 or
H-6), 4.15 (1 H, t-like dd, J ) 2.9, 2.9, H-2), 4.16 (1 H, t-like
dd, J ) 9.9, 9.9, H-4 or H-6), 4.70, 4.85 (2 H, ABq, J AB ) 11.9,
OCH2Ph), 4.71, 5.03 (2 H, ABq, J AB ) 11.2, OCH2Ph), 7.24-
7.38 (10 H, m, 2 × Ph); 13C NMR (CDCl3, 68 MHz) δ 17.74 (2
× CH3), 47.87 (2 × OCH3), 69.54, 70.40, 71.54, 71.81 (C-2, C-3,
C-5, and C-6), 76.63, 78.26 (C-1 and C-4), 73.21, 75.30 (OCH2-
Ph), 99.22 (2 × Cq BDA), 127.53, 127.68, 127.86, 128.31, 128.38
(Ph), 138.25, 138.64 (2 × C-1 of Ph); MS m/z (+ve ion FAB)
497 [(M + Na)+, 80%], 443 [(M - OCH3)+, 60%], 91 [(C7H7)+,
100%]; MS m/z (-ve ion FAB) 947 [(2 M - H)-, 20%], 627 [(M
+ NBA)-, 80%], 473 [(M - H)-, 100%]. Anal. (C26H34O8) C, H.
(2′R,3′R)-1D-3-O-[(S)-(+)-O-Acet ylm a n d elyl]-1,4-d i-O-
ben zyl-5,6-O-(2′,3′-d im eth oxybu ta n e-2′,3′-d iyl)-m yo-in osi-
tol (13) a n d (2′S,3′S)-1D-1-O-[(S)-(+)-O-Acetylm a n d elyl]-
3,6-d i-O-b en zyl-4,5-O-(2′,3′-d im et h oxyb u t a n e-2′,3′-d iyl)-
m yo-in ositol (14). To a stirred solution of (()-12 (11.0 g, 23.2
mmol), DCC (5.00 g, 24.2 mmol), and DMAP (50 mg) in dry
CH2Cl2 (100 mL) at -20 °C was added a solution of (S)-(+)-
acetylmandelic acid (5.00 g, 25.7 mmol) in dry CH2Cl2 (50 mL)
dropwise over 1 h. The cooling bath was removed, and stirring
was continued for 1 h, after which the suspension was filtered
through Celite to remove dicyclohexylurea. The filtrate was
concentrated by evaporation under reduced pressure, and the
residue was fractionated by flash chromatography (CHCl3/
acetone 30:1, then 10:1) on silica gel. The first fraction was
obtained as a white solid (7.22 g), and crystallization from
EtOAc/hexane gave pure 14 (5.39 g, 8.28 mmol, 36%, 72% of
this diastereoisomer). Further elution gave a second fraction
as an oil (6.63 g) slightly contaminated with 14. Crystallization
from EtOAc/hexane at -20 °C gave pure 13 as waxy crystals
(5.18 g, 7.96 mmol, 34%, 68% of this diastereoisomer).
MS m/z (-ve ion FAB); 803 [(M + NBA)-, 80%], 649 [(M -
H)-, 100%]. Anal. (C36H42O11) C, H.
(2′S,3′S)-1D-3,6-Di-O-b en zyl-4,5-O-(2′,3′-d im et h oxyb u -
ta n e-2′,3′-d iyl)-m yo-in ositol [(+)-12]. A solution of 14 (5.69
g, 8.74 mmol) in MeOH (150 mL) containing NaOH pellets (2.0
g, 50 mmol) was heated at reflux for 1 h. The solution was
allowed to cool and then neutralized by addition of solid CO2.
The solvents were removed by evaporation under reduced
pressure, and the residue was partitioned between CH2Cl2 and
water (100 mL of each). The organic layer was separated,
washed with water (2 × 100 mL), and dried (MgSO4). Evapo-
ration under reduced pressure gave the diol as a white
hygroscopic foam (3.94 g, 8.30 mmol, 95%); waxy crystals of
monohydrate (with difficulty) from Et2O/hexane at -20 °C:
[R]D24 ) + 69 (c ) 1, CHCl3); spectroscopic data were identical
to those obtained for (()-12. Anal. (C26H34O8‚H2O) C, H.
(2′R,3′R)-1D-1,4-Di-O-ben zyl-5,6-O-(2′,3′-d im eth oxybu -
ta n e-2′,3′-d iyl)-m yo-in ositol [(-)-12]. Saponification of 13
(5.0 g, 7.68 mmol) as described for 14 gave (-)-12 as a white
hygroscopic foam (3.46 g, 7.29 mmol, 95%); waxy crystals of
monohydrate (with difficulty) from Et2O/hexane at -20 °C:
26
[R]D ) -68 (c ) 1, CHCl3); spectroscopic data were identical
to those obtained for (+)-12. Anal. (C26H34O8‚H2O) C, H.
1D-1,4-Di-O-ben zyl-m yo-in ositol [(-)-11]. To a solution
of [(-)-12] (420 mg, 0.885 mmol) in CH2Cl2 (5 mL) was added
95% aqueous TFA (5 mL). The mixture was stirred at room
temperature for 30 min and then concentrated by evaporation
under reduced pressure to give (-)-11 as a white solid (312
mg, 0.866 mmol, 98%); crystals from EtOH: mp 172-173 °C,
Lit.18b mp 172-173 °C; [R]D ) -14 (c ) 1, MeOH), Lit.18b
20
[R]D20 ) - 16 (c ) 1, MeOH); NMR data were identical to those
previously reported.18b
(2′S,3′S)-1D-3,6-Di-O-ben zyl-4,5-(2′,3′-d im eth oxybu ta n e-
2′,3′-d iyl)-1,2-O-[(2′′-m eth ylen e)p r op a n e-1′′,3′′-d iyl]-m yo-
in ositol (15). To a solution of (+)-12 (3.94 g, 8.30 mmol) in
dry DMF (40 mL) was added NaH (1.30 g of a 60% dispersion
in oil, 32.5 mmol). The mixture was stirred at room temper-
ature for 30 min, and then 3-chloro-2-chloromethyl-1-propene
(2.0 mL, 2.16 g, 17.3 mmol) was added. After 1 h, TLC (ether)
showed that all the starting material (Rf 0.36) had been
consumed, with conversion into a major product at Rf 0.66.
Excess NaH was destroyed by cautious addition of MeOH, and
the solvents were removed by evaporation under reduced
pressure. The residue was partitioned between CH2Cl2 and
water (100 mL of each). The organic layer was washed with
water (2 × 100 mL), dried (MgSO4), and evaporated to give
an oil. Purification by flash chromatography (EtOAc/hexane
1:4) gave the alkene 15 as a colorless oil, later solidifying to a
brittle glass, which could not be recrystallized (3.67 g, 6.97
mmol, 84%): [R]D20 ) + 24 (c ) 1, CHCl3); 1H NMR (400 MHz,
CDCl3) 1.36 (3 H, s, CH3), 1.38 (3 H, s, CH3), 3.29 (3 H, s,
OCH3), 3.34 (3 H, s, OCH3), 3.43 (1 H, dd, J ) 10.1, 3.1, H-1
or H-3), 3.59 (1 H, dd, J ) 10.1, 2.1, H-1 or H-3), 3.66 (1 H,
t-like dd, J ) 9.5, 9.5, H-5), 3.85 (1 H, t-like dd, J ) 3.1, 2.1,
H-2); 3.99 (1 H, br d, J ) 13.4, H-1′′a or H-3′′a), 4.09-4.17 (3
H, m, H-4, H-6 and H-3′′a or H-1′′a), 4.18 (1 H, br d, J ) 13.4,
H-1′′b or H-3′′b), 4.47 (1 H, br d, J ) 14.3, H-3′′b or H-1′′b),
4.69, 4.82 (2 H, ABq, J AB ) 12.4, OCH2Ph), 4.78 (1 H, 0.5 of
ABq, J AB ) 11.6, OCH2Ph), 4.96-4.99 (2 H, m, CdCHaHb and
OCH2Ph), 5.06 (1 H, br s, CdCHaHb), 7.24-7.42 (10 H, m,
Ph); 13C NMR (100 MHz, CDCl3) 17.81 (2 × CH3), 47.88, 48.10
(2 × OCH3), 66.53 (C-1′′ or C-3′′), 69.17, 72.46 (2 × inositol
ring CH), 73.15, 73.57, 74.69 [2 × OCH2Ph and (C-1′′ or C-3′′)],
73.26, 75.88, 77.63, 80.39 (4 × inositol ring CH), 99.25, 99.41
(2 × Cq of BDA), 113.97 (dCH2), 127.58, 127.93, 128.25, 128.31
(10 × CH of Ph), 138.63, 138.93 (2 × C-1 of Ph), 146.96 (C-
2′′); MS m/z (+ve ion FAB) 495 [(M - CH3O)+, 70%], 266(90),
91 [(C7H7)+, 100%]. Anal. (C30H38O8) C, H.
Data for 13: mp 75-78 °C; Rf 0.24 (CHCl3/acetone 30:1);
[R]D ) -9.5 (c ) 2, CHCl3); 1H NMR (400 MHz, CDCl3) δ
26
1.31 (3 H, s, CH3), 1.34 (3 H, s, CH3), 2.18 [3 H, s, CH3C(O)],
2.67 (1 H, br s, D2O exch, OH-2), 3.15 (3 H, s, OCH3), 3.31 (3
H, s, OCH3), 3.54 (1 H, dd, J ) 10.3, 2.4, H-1), 3.62 (1 H, t-like
dd, J ) 10.3, 9.8, H-5), 3.99 (1 H, t-like dd J ) 9.8, 9.8, H-4),
4.07 (1 H, d, J ) 11.2, OCH2Ar), 4.16 (1 H, t-like dd, J ) 10.3,
9.8, H-6), 4.36 (1 H, br s, D2O exch gives t-like dd, J ) 2.4,
2.4, H-2), 4.55 (1 H, d, J ) 11.2, OCH2Ph), 4.69, 4.82 (2 H,
ABq, J ) 11.7, OCH2Ph), 5.99 [1 H, s, O2CCH(OAc)Ph], 6.94-
6.96 (2 H, m, Ph), 7.18-7.37 (11 H, m, Ph), 7.48-7.50 (2 H,
m, Ph); 13C NMR (CDCl3, 100 MHz) 17.67, 17.76 (2 × CH3),
20.67 [CH3C(O)], 47.82, 47.97 (2 × CH3O), 68.80, 69.13, 71.03
(C-2, C-5 and C-6), 73.39 (OCH2Ph), 74.87 (OCH2Ph), 74.82,
75.10 75.55, 76.52 [C-1, C-3, C-6 and O2CCH(OAc)Ph], 99.34
(2 × Cq of BDA), 127.21, 127.47, 127.69, 127.80, 127.96, 128.38,
128.84, 129.42 (15 × CH of Ph), 133.12 (C-1 of acetylmandelate
Ph), 138.06, 138.50 (2 × C-1 of Ph), 168.45 (s, CdO), 170.61
(s, CdO); MS m/z (+ve ion FAB); 673 [(M + Na)+, 50%], 619
[(M - CH3O)+, 70%], 91 [(C7H7)+, 100%]; MS m/z (-ve ion
FAB); 803 [(M + NBA)-, 70%], 649 [(M - H)-, 100%]. Anal.
(C36H42O11) C, H.
Data for 14: mp 132.5-134.5 °C; Rf 0.36 (CHCl3/acetone
30:1); [R]D26 ) +72 (c ) 2, CHCl3); 1H NMR (400 MHz, CDCl3)
1.34 (3 H, s, CH3), 1.35 (3 H, s, CH3), 2.19 [4 H, s, D2O exch.
gives 3 H, s, CH3C(O) and OH-2], 3.25 (3 H, s, OCH3), 3.30 (3
H, s, OCH3), 3.49 (1 H, dd, J ) 10.1, 2.7, H-3), 3.66 (1 H, t-like
dd, J ) 10.1, 9.8, H-5), 4.03 (1 H, t-like dd, J ) 9.8, 9.8, H-4
or H-6), 4.12-4.17 [2 H, m, H-2 and (H-4 or H-6)], 4.63-4.70
(2 H, m, 2 × 0.5 AB systems of OCH2Ph), 4.78 (1 H, dd, J )
10.1, 2.7, H-1), 4.78-4.87 (2 H, m, 2 × 0.5 AB systems of OCH2-
Ph), 5.95 [1 H, s, O2CCH(OAc)Ph], 7.24-7.32 (13 H, m, Ph),
7.43-7.45 (2 H, m, Ph); 13C NMR (CDCl3, 100 MHz) 17.72,
17.76 (2 × CH3), 20.68 [CH3C(O)], 47.88, 47.92 (2 × CH3O),
68.65, 69.07, 71.20 (C-2, C-4, and C-5), 73.41 (OCH2Ph), 74.78,
74.85, 75.43, 76.32 [C-1, C-3, C-6, and O2CCH(OAc)Ph], 75.16
(OCH2Ph), 99.34 (2 × BDA Cq), 127.39, 127.47, 127.61, 127.74,
127.94, 128.18, 128.35, 128.77, 129.22 (15 × CH of Ph), 133.37
(s, C-1 of acetylmandelate Ph), 138.11, 138.61 (2 × C-1 Ph),
168.09 (CdO), 170.79 (CdO); MS m/z (+ve ion FAB); 673 [(M
+ Na)+, 20%], 619 [(M - CH3O)+, 90%], 91 [(C7H7)+, 100%];
(2′S,3′S)-1D-3,6-Di-O-b en zyl-4,5-O-(2′,3′-d im et h oxyb u -
t a n e-2′,3′-d iyl)-1,2-O-[(2′′-oxo)p r op a n e-1′′,3′′-d iyl]-m yo-
in ositol (16). To a vigorously stirred solution of alkene 15
(3.67 g, 6.97 mmol) in EtOAc (20 mL) and CH3CN (20 mL)
was added NaIO4 (7.00 g, 32.7 mmol) followed by a solution of
RuCl3‚3H2O (40 mg in 20 mL of distilled water). The suspen-

Synthesis of Bicyclic Analogues of Ins(1,4,5)P3
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2115
Data for 18: mp 165-167 °C (from EtOAc/hexane); Rf 0.12
sion was stirred at room temperature for 1.5 h, after which
time TLC (EtOAc/hexane 1:2) showed complete conversion of
the alkene (Rf 0.40) into a slightly more polar product (Rf 0.34).
Water (200 mL) and Et2O (200 mL) were then added. The
organic layer was separated and washed with water (200 mL)
and a saturated aqueous solution of Na2S2O3 (100 mL). The
organic layer, which was now almost colorless, was dried
(MgSO4) and concentrated in vacuo to give the crude ketone
16 as a foam (3.02 g, 5.71 mmol, 82%) which was used in the
next step without further purification. For analytical purposes,
a small portion of this was purified by flash chromatography
(EtOAc/hexane 1:2) to give 16 as a white crystalline solid: mp
128-129 °C (from EtOAc/hexane); [R]D24 ) - 9 (c ) 1, CHCl3);
1H NMR (400 MHz, CDCl3) δ 1.37 (3 H, s, CH3), 1.40 (3 H, s,
CH3), 3.31 (3 H, s, OCH3), 3.34 (3 H, s, OCH3), 3.49 (1 H, dd,
J ) 10.5, 3.1, H-1 or H-3), 3.74 (1 H, t-like dd, J ) 9.8, 9.4,
H-5), 3.78 (1 H, dd, J ) 9.8, 2.7, H-1or H-3), 3.85 (1 H, d, J )
16.2, H-1′′a or H-3′′a), 3.86 (1 H, t-like dd, J ) 3.1, 2.7, H-2),
3.99 (1 H, d, J ) 17.4, H-3′′a or H-1′′a), 4.08 (1 H, t-like dd, J
) 10.2, 10.2, H-4 or H-6), 4.16 (1 H, d, J ) 16.2, H-1′′b or
H-3′′b), 4.17 (1 H, t-like dd, J ) 9.8, 9.4, H-4 or H-6), 4.38 (1
25
1
(CHCl3/acetone 10:1); [R]D ) + 29 (c ) 1, CHCl3); H NMR
(400 MHz, CDCl3) 1.36 (3 H, s, CH3), 1.38 (3 H, s, CH3), 2.58
(1 H, d, J ) 7.1, OH-2′′), 3.29 (3 H, s, OCH3), 3.32 (3 H, s,
OCH3), 3.40 (1 H, dd, J ) 10.3, 2.9, H-1 or H-3), 3.53 (1 H, dd,
J ) 12.7, 2.9, H-1′′a or H-3′′a), 3.59 (1 H, dd, J ) 12.7, 5.9,
H-3′′a or H-1′′a), 3.60-3.66 [2 H, m, H-5 and (H-1 or H-3)],
3.80 (1 H, br d, J ) 12.7, H-3′′b or H-1′′b), 3.92-3.97 (1 H, br
m, H-2′′), 3.98-4.10 [4 H, m, H-2, H-4, H-6 and (H-1′′b or
H-3′′b)], 4.69, 4.82 (2 H, ABq, J AB ) 12.2, CH2Ph), 4.78, 4.93
(2 H, ABq, J AB ) 11.7, CH2Ph), 7.24-7.42 (10 H, m, Ph); 13C
NMR (68 MHz, CDCl3) 17.80 (2 × CH3), 47.89, 48.10 (2 ×
OCH3), 64.79 (OCH2), 68.76, 71.29, 71.89 (3 × CH), 73.24
(OCH2), 73.95 (inositol ring CH), 74.88 (OCH2), 75.74 (CH),
77.63, 80.05 (2 × CH), 99.27, 99.34 (2 × BDA Cq), 127.58,
127.63, 127.99, 128.25, 128.35 (10 × CH of Ph), 138.52, 138.89
(2 × C-1 of Ph); MS m/z (+ve ion FAB); 553 [(M + Na)+, 40%],
529(20), 499 [(M - CH3O)+, 80%], 91 [(C7H7)+, 100%]; MS m/z
(-ve ion FAB); 683 [(M + NBA)-, 100%]. Anal. (C29H38O9) C,
H.
(2′S)-1D-3,6-Di-O-b en zyl-1,2-O-[(2′-h yd r oxy)p r op a n e-
1′,3′-d iyl]-m yo-in ositol (19). To a solution of the alcohol 17
(530 mg, 1.00 mmol) in CH2Cl2 (5 mL) at room temperature
was added 95% aqueous TFA (5 mL). The mixture was stirred
at room temperature for 15 min. The solvents were removed
by evaporation under reduced pressure, and the residue was
purified by flash chromatography (EtOAc/hexane 5:1) to give
the triol 19 as a hygroscopic white foam (346 mg, 0.83 mmol,
H, d, J ) 17.4, H-3′′b or H-1′′b), 4.67, 4.88 (2 H, ABq, J AB
)
12.5, OCH2Ph), 4.74, 5.04 (2 H, ABq, J AB ) 11.7, OCH2Ph),
7.27-7.38 (10 H, m, Ph); 13C NMR (68 MHz, CDCl3) 17.78 (2
× CH3), 47.97, 48.12 (2 × OCH3), 68.76 (inositol ring CH),
70.27 (C-1′′ or C-3′′), 71.88, 72.40 (2 × inositol ring CH), 73.56,
74.67 (OCH2Ph), 75.43 (inositol ring CH), 78.19 (C-1′′ or C-3′′),
78.35, 84.30 (2 × inositol ring CH), 99.27, 99.45 (2 × Cq of
BDA), 127.65, 127.83, 127.88, 128.01 (10 × CH of Ph), 138.34,
138.37 (2 × C-1 of Ph), 210.52 (C-2′′); MS m/z (+ve ion FAB);
551[(M + Na)+, 20%], 527(20), 497 [(M - CH3O)+, 75%],
266(40), 91 [(C7H7)+, 100%]; MS m/z (-ve ion FAB); 681
[(M + NBA)-, 100%], 527 [(M - H)-, 40%]. Anal. (C29H36O9)
C; H.
83%): [R]D ) - 3 (c ) 1, CHCl3); 1H NMR (400 MHz, CDCl3)
23
2.96 (1 H, d, J ) 10.7, OH-2′), 3.07 (1 H, br s, OH-4 or OH-5),
3.16 (1 H, br s, OH-4 or OH-5), 3.23-3.27 (2 H, m, H-3 and
(H-1′a or H-3′a)], 3.43 (1 H, t-like dd, J ) 9.3, 8.8, H-5), 3.59-
3.64 (2 H, m, H-2′ and H-6), 3.69 (1 H, dd, J ) 8.8, 3.4, H-1),
3.77-3.81 [2 H, m, H-2 and (H-3′a or H-1′a)], 3.85-3.92 [2 H,
m, H-4 and (H-3′b or H-1′b)], 4.04 (1 H, br d, J ) 12.7, H-1′b
or H-3′b), 4.62, 4.72 (2 H, ABq, J AB ) 11.7, CH2Ar), 4.78, 4.93
(2 H, ABq, J AB ) 11.2, CH2Ar), 7.25-7.39 (10 H, m, Ph); 13C
NMR (68 MHz, CDCl3) 67.56 (C-1′ or C-3′), 70.40, 71.15 (2 ×
CH), 72.93 (OCH2Ar), 73.86 (CH), 74.96, (OCH2Ph), 77.55 (d,
CH), 77.60 (C-1′ or C-3′), 78.14, 78.74, 81.04 (3 × CH), 127.83,
128.02, 128.14, 128.25, 128.44, 128.62 (10 × CH of Ph), 137.82
138.40 (2 × C-1 of Ph); MS m/z (+ve ion FAB); 417 [(M + H)+,
15%], 91 [(C7H7)+, 100%]; MS m/z (-ve ion FAB); 569 [(M +
NBA)-, 100%], 415 [(M - H)-, 60%]. Anal. (C23H28O7‚0.5H2O)
C, H.
(2′S,3′S,2′′R)-1D-3,6-Di-O-ben zyl-4,5-O-(2′,3′-d im eth oxy-
butane-2′,3′-diyl)-1,2-O-[(2′′-hydroxy)propane-1′′,3′′-diyl]-myo-
in osit ol (17) a n d (2′S,3′S,2′′S)-1D-3,6-Di-O-b en zyl-4,5-O-
(2′,3′-d im eth oxybu ta n e-2′,3′-d iyl)-1,2-O-[(2′′-h yd r oxy)p r o-
p a n e-1′′,3′′-d iyl]-m yo-in ositol (18). To a stirred solution of
ketone 16 (2.38 g, 4.50 mmol) in methanol (100 mL) at 0 °C
was added NaBH4 (800 mg, 21.1 mmol). The solution was
stirred at 0 °C for a further 5 min and then allowed to warm
to room temperature. TLC (CHCl3/acetone 10:1) showed total
conversion of the ketone (Rf 0.48) into a major product at Rf
0.12 and a minor product at Rf 0.20. Water (10 mL) was added,
and stirring was continued for a further 1 h. The solvents were
removed by evaporation under reduced pressure to give a solid
residue which was partitioned between CH2Cl2 and water (100
mL of each). The organic layer was removed, dried (MgSO4),
and evaporated under reduced pressure to give a white foam
which was fractionated by flash chromatography (CHCl3/
acetone 10:1) to give first the alcohol 17 (495 mg, 0.933 mmol,
21%). Second to elute was the epimeric alcohol 18 (1.60 g, 3.02
mmol, 67%).
(2′R)-1D-3,6-Di-O-b en zyl-1,2-O-[(2′-h yd r oxy)p r op a n e-
1′,3′-d iyl]-m yo-in ositol (20). The BDA group was removed
from 18 as described for the epimer 17. Purification by flash
chromatography (ethyl acetate) gave the triol 20 as a hygro-
23
scopic white foam in 84% yield: [R]D ) - 7 (c ) 1, CHCl3);
1H NMR (400 MHz, CDCl3) 2.52 (1 H, d, J ) 6.3 Hz, D2O exch.,
OH-2′), 2.74 (1 H, br s, D2O exch., OH-4 or OH-5), 2.80 (1 H,
br s, D2O exch., OH-4 or OH-5), 3.20 (1 H, dd, J ) 9.8, 2.9,
H-1 or H-3), 3.44 (1 H, dd, J ) 12.6, 3.5, H-1′a or H-3′a) 3.45
(1 H, t-like dd, J ) 9.2, 9.0, H-5), 3.66 (1 H, dd, J ) 12.7, 6.4,
H-3′a or H-1′a), 3.69 (1 H, dd, J ) 9.9, 2.7, H-1or H-3), 3.82-
3.89 (2 H, m, H-4 and H-6), 3.93 (1 H, br d, J ) 12.7, H-3′b or
H-1′b), 3.95-4.03 (1 H, m, C-2′-H), 4.08 (1 H, buried, H-2),
4.09 (1 H, dd, J ) 12.6, 5.9, H-1′b or H-3′b), 4.63, 4.68 (2 H,
ABq, J AB ) 11.7, CH2Ph), 4.79, 4.90 (2 H, ABq, J AB ) 11.2, CH2-
Ph), 7.26-7.41 (10 H, m, Ph); 13C NMR (68 MHz, CDCl3) 65.29
(C-1′ or C-3′), 70.90, 71.41 (2 × CH), 72.28 (OCH2Ph), 74.41
(CH), 74.45, 76.37 (2 × OCH2), 77.24, 77.41, 77.73, 78.10 (4 ×
CH), 127.75, 127.94, 128.07, 128.38, 128.57 (10 × CH of Ph),
137.61, 138.51 (2 × C-1 of Ph); MS m/z (+ve ion FAB); 838 [(2
M + H)+, 60%], 439 [(M + Na)+, 10%], 91 [(C7H7)+, 100%]; MS
m/z (-ve ion FAB); 569 [(M + NBA)-, 100%], 415[(M - H)-,
50%]. Anal. (C23H28O7‚0.5H2O) C, H.
Data for 17: mp 126-127 °C (from hexane); Rf 0.20 (CHCl3/
acetone 10:1); [R]D25 ) + 33 (c ) 1, CHCl3); 1H NMR (400 MHz,
CDCl3) 1.356 (3 H, s, CH3), 1.362 (3 H, s, CH3), 2.72 (1 H, d, J
) 10.7, OH-2′′), 3.29 (3 H, s, OCH3), 3.32 (3 H, s, OCH3), 3.37
(1 H, dd, J ) 12.7, 2.0, H-1′′a or H-3′′a), 3.45 (1 H, dd, J )
10.1, 3.4, H-1 or H-3), 3.57-3.64 [3 H, m, H-5, H-2′′ and (H-1
or H-3)], 3.73 (1 H, t-like dd, J ) 9.8, 9.8, H-4 or H-6), 3.78 (1
H, t-like dd, J ) 3.4, 3.4, H-2), 3.81-3.89 [2 H, m, (H-1′′a and
H-1′′b) or (H-3′′a and H-3′′b)], 4.07 (1 H, t-like dd, J ) 10.3,
H-4 or H-6), 4.07 (1 H, br d, J ) 12.7, H-1′′b or H-3′′b) 4.68,
4.89 (2 H, ABq, J ) 12.2, OCH2Ph), 4.83, 4.91 (2 H, ABq, J )
11.2 Hz, OCH2Ar), 7.25-7.42 (10 H, m, Ph); 13C NMR (100
MHz, CDCl3) 17.76, 17.81 (2 × CH3), 47.88, 48.08 (2 × OCH3),
67.14 (OCH2), 68.54, 70.39, 71.14 (3 × CH), 73.59, 75.26 (2 ×
OCH2), 75.51 (CH), 78.09 (OCH2), 78.47, 78.51, 79.44 (3 × CH),
99.21, 99.32 (2 × Cq of BDA), 127.56, 127.63, 127.69, 128.13,
128.20, 128.36 (10 × CH of Ph), 138.82, 138.62 (2 × C-1 of
Ph); MS m/z (+ve ion FAB); 553 [(M + Na)+, 60%], 529(50),
499 [(M - CH3O)+, 20%], 91 [(C7H7)+, 100%]; MS m/z (-ve ion
FAB); 683 [(M + NBA)-, 100%]. Anal. (C29H38O9) C, H.
(2′R)-1D-3,6-Di-O-ben zyl-4,5-bis-O-[d i(ben zyloxy)p h os-
p h or yl]-1,2-O-{2′-[d i(ben zyloxy)p h osp h or yloxy]p r op a n e-
1′,3′-d iyl}-m yo-in ositol (21). To a solution of bis(benzyloxy)-
diisopropylaminophosphine (870 mg, 2.52 mmol) in dry CH2Cl2
(5 mL) was added 1H-tetrazole (350 mg, 5.00 mmol). The
mixture was stirred at room temperature for 20 min, and then

2116 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Riley et al.
the triol 19 (175 mg, 0.420 mmol, previously dried in vacuo at
30 °C) was added. After 1 h at room temperature, the mixture
was cooled to -78 °C, m-CPBA (60%, 760 mg, 2.64 mmol) was
added, and the cooling bath was removed. The mixture was
allowed to reach room temperature and then diluted with CH2-
Cl2 (50 mL). The clear solution was washed with 10% Na2-
SO3, saturated NaHCO3, and brine (50 mL of each), dried
(MgSO4), and concentrated by evaporation under reduced
pressure. Purification of the residue by flash chromatography
(CHCl3/acetone 3:1) afforded 21 (412 mg, 0.344 mmol, 82%)
as a colorless oil which crystallized after a few days: mp 103-
concentrated by evaporation in vacuo, and MeOH was added
and evaporated repeatedly, eventually leaving the pure tri-
ethylammonium salt of 9 as a colorless glass (121 µmol, 90%):
[R]D ) -28 (c ) 1, MeOH); 1H NMR (400 MHz, 50 mM
21
phosphate buffer in D2O, pD 7.2) 3.76 (1 H, dd, J ) 9.8, 3.5,
H-1 or H-3), 3.77 (1 H, dd, J ) 13.3, 3.1, H-1′a or H-3′a), 3.82
(1 H, dd, J ) 9.4, 3.1 H-1 or H-3), 3.90-4.00 (2 H, m, H-5 and
H-6), 4.04-4.17 [5 H, m, H-2, H-4, (H2-3′b or H2-1′b) and (H-
3
1′b or H-3′b)], 4.27-4.33 (1 H, m, J HP 9.8, H-2′); 31P NMR
(162 MHz, 1H-coupled, D2O) -0.29 (1 P, d, 3J HP 9.4, P-2′), 0.44
(2 P, br m, P-4 and P-5); MS m/z (+ve ion FAB) 102 [Et3NH+,
100%]; MS m/z (-ve ion FAB) 475 [M-, 100%]. Accurate FAB-
calcd for C9H18O16P3-: 474.9808. Found: 474.9814.
19
1
105 °C (from ethanol); [R]D ) -14 (c ) 1, CHCl3); H NMR
(400 MHz CDCl3) 3.31 (1 H, br d, J ) 13.2, H-1′a or H-3′a),
3.50 (1 H, dd, J ) 9.3, 2.5 Hz, H-3), 3.61-3.66 [2 H, m, H-2
and (H-3′a or H-1′a], 3.73 (1 H, dd, J ) 9.2, 2.9 Hz, H-1), 3.94-
4.02 [2 H, m, C-6-H and (H-3′b or H-1′b)], 4.07 (1 H, br d, J )
13.2, H-1′b or H-3′b), 4.37-4.42 (1 H, m, H-2′), 4.46-4.53 (2
H, m, H-5 and 0.5 AB system of CH2Ph), 4.65-4.72 (3 H, m,
1.5 AB systems of CH2Ph), 4.80-5.06 (13 H, m, 6 × AB
systems of CH2Ph and H-4), 6.98-7.01 (2 H, m, Ph), 7.11-
7.34 (38 H, m, Ph); 13C NMR (100 MHz, CDCl3) δ 66.72
(broadened by J CP coupling, C-1′ or C-3′), 69.72-69.53 (over-
lapping signals with J CP couplings, 6 × POCH2Ph), 73.15,
74.07 (2 × OCH2Ph), 74.43 (J CP ) 3.7, C-1′ or C-3′), 76.74 (CH),
76.85 (J CP ) 5.5, C-2′), 77.36 (CH), 77.63 (with poorly resolved
J CP couplings, CH), 78.44, 78.77 (2 × CH), 78.91 (J CP ) 7.4,
5.5, CH), 127.12-128.66 (40 × CH of Ph), 135.50-136.15 (6
× C-1 of POCH2Ph with J CP couplings), 137.86, 138.68 (2 ×
(2′S)-1D-1,2-O-[(2′-P h osp h or yloxy)p r op a n e-1′,3′-d iyl]-
m yo-in ositol 4,5-bisp h osp h a te (10). Compound 22 (150 mg,
125 µmol) was deprotected as described for the epimer 22.
Purification by ion-exchange chromatography as before gave
the triethylammonium salt of 10 as a colorless glass (111 µmol,
22
1
89%): [R]D ) -24 (c ) 1.3, MeOH); H NMR (400 MHz, 50
mM phosphate buffer in D2O, pD 7.2) 3.53 (1 H, dd, J ) 12.5,
6.2, H-3′a), 3.75 (1 H, dd, J ) 9.8, 3.1 Hz, H-3), 3.81 (1 H, dd,
J ) 9.8, 2.7 H-1), 3.91 (1 H, dd, J ) 13.3, 7.0, H-1′a), 3.92-
4.05 (2 H, m, H-5 and H-6), 4.10 (1 H, dd, J ) 13.3, 2.0, H-1′b),
4.14 (1 H, q-like ddd, J ∼ 9, H-4), 4.19 (1 H, t-like dd, J ) 3.1,
2.7, H-2), 4.31 (1 H, dd, J ) 12.5, 6.6, H-3′b), 4.35-4.43 (1 H,
1
m H-2′); 31P NMR (162 MHz, H-coupled, D2O) -0.37 (1 P, d,
3J HP 9.5, P-2′), 0.47 (2 P, br m, P-4 and P-5); MS m/z (+ve ion
FAB) 102 [Et3NH+, 100%]; MS m/z (-ve ion FAB, relative
intensity); 950.5 (90), 474.9 [M-, 100%]. Accurate FAB- calcd
for C9H18O16P3-: 474.9808. Found: 474.9823.
1
C-1 of Ph); 31P NMR (162 MHz, H-coupled, CDCl3) -1.97 (1
P, apparent sextet, J ∼ 8), -1.65 (1 P, apparent sextet, J ∼
8), -1.36 (1 P, apparent sextet, J ∼ 8), MS m/z (+ve ion FAB);
1197 [(M + H)+, 90%], 91 [(C7H7)+, 100%]; MS m/z (-ve ion
FAB); 1349 [(M + NBA)-, 70%], 1242(20), 1105(40), 277
[(BnO)2P(O)O-, 100%]. Anal. (C65H67O16P3) C, H.
Ack n ow led gm en t. We thank the Wellcome Trust
for Programme Grant support (060554 to B.V.L.P. and
039662 to C.W.T.) and Professor A. Espinosa, University
of Granada, for useful discussions on the conformational
analysis of 1,4-dioxepane rings.
(2′S)-1D-3,6-Di-O-ben zyl-4,5-bis-O-[d i(ben zyloxy)p h os-
p h or yl]-1,2-O-{2′-[d i(ben zyloxy)p h osp h or yloxy]p r op a n e-
1′,3′-d iyl}-m yo-in ositol (22). Triol 20 (175 mg, 0.420 mmol)
was phosphorylated using the procedure described for 19.
Purification by flash chromatography (CHCl3/acetone 5:1)
Su p p or tin g In for m a tion Ava ila ble: NOESY spectrum
of 10 and X-ray structural data for 18. This material is
available free of charge via the Internet at http://pubs.acs.org.
20
afforded 22 as a colorless oil (449 mg, 0.375 mmol, 89%): [R]D
1
) -23 (c ) 1, CHCl3); H NMR (400 MHz, CDCl3) 3.24 (1 H,
Refer en ces
dd, J ) 12.7, 5.9, H-1′a or H-3′a), 3.42 (1 H, dd, J ) 9.8, 2.4
H-3), 3.58 (1 H, dd, J ) 13.2, 7.3, H-3′a or H-1′a), 3.69 (1 H,
dd, J ) 9.3, 2.9, H-1), 3.80-3.84 [2 H, m, H-2 and (H-3′b or
H-1′b)], 3.93 (1 H, t-like dd, J ) 9.3, 9.3 H-6), 4.11 (1 H, dd, J
) 12.7, 6.1, H-1′b or H-3′b), 4.42-4.53 (3 H, m, H-2′, H-5 and
0.5 AB system of OCH2Ph), 4.63 (1 H, d, J ) 12.2, 0.5 AB
system of OCH2Ph), 4.69-5.08 (15 H, m, AB systems of OCH2-
Ph and C-4-H), 7.01-7.04 (2 H, m, Ph), 7.11-7.36 (38 H, m,
Ph); 13C NMR (100 MHz, CDCl3) 65.40 (C-1′ or C-3′), 69.13,
69.39, 69.55 (signals with J CP couplings, 3 × POCH2Ph), 72.73,
73.61 (2 × OCH2Ph), 73.85 (3J CP 5.5, C-1′ or C-3′), 75.76 (J CP
) 5.6, C-2′), 76.41, 76.74, 77.25 (3 × CH), 77.38 (broadened
by J CP couplings CH), 77.61 (CH), 79.08 (J CP ) 5.5, 5.5, CH),
127.34-128.75 (40 × CH of Ph), 135.57, 135.97, 136.11 (6 ×
C-1 of POCH2Ph, with J CP couplings), 137.60, 138.42 (2 × C-1
of Ph); 31P NMR (162 MHz, 1H-decoupled, CDCl3,) -1.65 (1
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P), -1.79 (1 P), -2.01 (1 P) (overlapping sextets in H-coupled
spectrum); MS m/z (+ve ion FAB); 1197 [(M + H)+, 90%], 91
[(C7H7)+, 100%]; MS m/z (-ve ion FAB); 1349 [(M + NBA)-,
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