Synthesis and biological evaluation of L-α-Phosphatidyl-D-3-deoxy-3-heteromethyl-myo-inositols as phosphoinositide 3-kinase inhibitors

Article abstract of DOI:10.1016/S0968-0896(01)00232-2

We have synthesized a series of 3-deoxy-3-heteromethyl derivatives of L-α-phosphatidyl-D-myo-inositol as part of our effort to develop specific, reversible inhibitors of phosphoinositide (PI) 3-kinase. Among various derivatives examined, phosphatidyl-D-3-

Full text of DOI:10.1016/S0968-0896(01)00232-2

Bioorganic & Medicinal Chemistry 9 (2001) 3165–3172
Synthesis and Biological Evaluation of L-ꢀ-Phosphatidyl-D-3-
deoxy-3-heteromethyl-myo-inositols as Phosphoinositide
3-Kinase Inhibitors
Da-Sheng Wanga and Ching-Shih Chena,b,
*
aDivision of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA
bDivision of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
Received 1 February 2001; accepted 21 May 2001
Abstract—We have synthesized a series of 3-deoxy-3-heteromethyl derivatives of l-a-phosphatidyl-d-myo-inositol as part of our
effort to develop specific, reversible inhibitors of phosphoinositide (PI) 3-kinase. Among various derivatives examined, phosphati-
dyl-d-3-deoxy-3-aminomethyl-myo-inositol displays the highest potency in inhibiting PI 3-kinase both in vitro and in cells. It
effectively suppressed antigen-stimulated degranulation in mast cells (IC50, 17 mM), suggesting a potential application of this PI 3-
kinase inhibitor as a mast cell-stabilizing agent. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
LY294002 have received wide attention. These two PI 3-
kinase inhibitors are structurally and mechanistically
distinct molecules (Fig. 1).
The pivotal role of PI 3-kinase in the regulation of
diverse physiological functions has been well doc-
umented.1À3 Cellular responses that are mediated by PI
3-kinase include cell growth and differentiation,4,5 cell
survival,6 platelet aggregation,7,8 T-cell activation,6
mast cell degranulation,9 membrane trafficking,10 and
so forth. In stimulated cells, PI 3-kinase phosphorylates
the 3-OH of the inositol ring of phosphoinositides,
resulting in transient accumulations of phosphatidyli-
nositol 3,4,5-trisphosphate [PI(3,4,5)P3] and phosphati-
dylinositol 3,4-bisphosphate [PI(3,4)P2]. These lipid
second messengers act as both membrane anchors and
allosteric regulators, serving to recruit and activate down-
stream enzymes and their protein substrates. Through this
membrane recruitment process, PI 3-kinase behaves as a
molecular switch to regulate downstream signaling path-
ways that culminate in various physiological responses.
Concerning potential therapeutic applications, PI 3-
kinase can be targeted toblock undesired cell prolifera-
tion or activation under pathological conditions.
Wortmannin, a fungal metabolite, is a highly potent
inhibitor (IC50, 2 nM) that covalently modifies the Lys-
802 of the p110 catalytic subunit, thereby preventing the
12
binding of PI(4,5)P2 and ATP tothe enzyme. On the
other hand, LY294002 [2-(4-morpholinyl)-8-phenyl-4H-
1-benzopyran-4-one] inhibits PI 3-kinase (IC50, 1.4 mM)
by blocking the ATP-binding site of the enzyme.13 More
recently, Kozikowski and coworkers developed various
3-substituted derivatives of phosphatidylinositol that
acted as anti-proliferative agents against cancer cell
growth.11,14,15 In our laboratory, we have embarked on
In the literature, several types of PI 3-kinase inhibitors
have been reported,11 among which wortmannin and
*Corresponding author at present address: The Ohio State University,
College of Pharmacy, Division of Mecidinal Chemistry and Pharma-
cognosy, 500 West 12th Avenue, Columbus, OH 43210-1291, USA.
Tel.: +1-614-688-4008; fax: +1-859-257-2489; e-mail: chen.844@osu.
edu
Figure 1. Structures of wortmannin and LY294002.
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00232-2

3166
D.-S. Wang, C.-S. Chen / Bioorg. Med. Chem. 9 (2001) 3165–3172
ꢀ
the development of PI 3-kinase inhibitors as anti-allergic
agents in light of the involvement of this enzyme in mast
cell activation.16 Our data suggest that PI 3-kinase plays
a key role in mast cell degranulation by stimulating Ca2+
influx via a phosphatidylinositol 3,4,5-trisphosphate
[PI(3,4,5)P3]-sensitive Ca2+ entry mechanism.16 Here,
we report the synthesis of a series of the 3-deoxy-3-het-
eromethyl analogues of l-a-phosphatidyl-d-myo-inosi-
tol (1–6) (Fig. 2) and their activity against PI 3-kinase
both in vitro and in cells.
the presence of CCl4, CBr4, I2, and Zn(N3)2 Py2/DEAD
(diethylazodicarboxylate), respectively. In addition, 10
underwent O-benzylation to generate (À)-16. These 3-
heteromethyl analogues (11–16) were exposed to tri-
fluoroacetic acid (TFA) in CH2Cl2 toremove the
p-
methoxybenzyl group to give the key compounds (À)-17
to(+)- 22. Reaction of these intermediates with 1,2-
dipalmitoyl-sn-glycerol 3-(benzyl N,N-diisopropylphos-
phoramidite) (23) in the presence of 1H-tetrazole, fol-
lowed by m-chloroperoxybenzoic acid (m-CPBA), gave
the perbenzylated derivatives (+)-24 to(+)- 29 that
underwent hydrogenolysis to generate the target mole-
cules 1–6, with overall yields from (+)-7 ranging from
25 to35%.
Among these analogues, l-a-phosphatidyl-d-3-deoxy-3-
aminomethyl-myo-inositol (5) displayed the highest
potency in PI 3-kinase inhibition, and effectively inhib-
ited antigen-stimulated degranulation in mast cells, a
process leading to allergic and inflammatory responses.
PI 3-kinase inhibition
The potency of the 3-deoxy-3-heteromethyl derivatives
(1–6) in enzyme inhibition was examined by exposing
immunoprecipitated rat liver PI 3-kinase to PI(4,5)P2
and [g-32P]-ATP in the presence of individual
compounds at different concentrations. 32P-Labeled
PI(3,4,5)P3 derived from the enzymatic 3-O-phosphor-
ylation of PI(4,5)P2 was extracted and analyzed by thin
layer chromatography, followed by autoradiography.
Figure 4A depicts the effect of compounds 1–6 at 50 mM
o n 3[2P]PI(3,4,5)P3 production. Among these six inhibi-
tors, the 3-deoxy-3-aminomethy derivative (5) was most
potent (>98% inhibition), followed by the 3-deoxy-3-
hydroxymethyl counterpart (6) (85% inhibition),
whereas the 3-halomethyl derivatives (1–4) exhibited a
modest activity in blocking [32P]PI(3,4,5)P3 production
(55–65% inhibition).
Results and Discussion
Synthesis
The aforementioned d-3-deoxy-3-heteromethyl-myo-
inositol derivatives (1–6) were synthesized using (+)-7
as a precursor (Fig. 3), which was used as an inter-
mediate in our previous synthesis of PI(3)P.17
The efficient use of (+)-7 was made of by converting it
tothe ketnoe ( À)-8 via DMSO-Ac2O oxidation as
described,18 followed by the Wittig reaction to afford
the olefin (À)-9 by reacting with triphenylphosphonium
methylide in the presence of 1 molar equivalent of t-
1
butoxide. H NMR analysis of the protons at C-2 and
C-4 of both 8 and 9 showed no appreciable epimeriza-
tion during the alkene formation. Hydroboration of 9
with 9-BBN (9-borabicyclo[3,3,1]nonane) followed by
oxidation with alkaline H2O2 solution gave the sterically
more favorable isomer (À)-10 as a single product after
chromatography. The C-3 stereochemistry of 10 was
According to the dose–response curves (Fig. 4B), the
IC50 values of compounds 5 and 6 were 20Æ1 and
35Æ2 mM, respectively (n=3). It is worth noting that
both 5 and 6 were not substrates for PI 3-kinase. Our
preliminary kinetic data suggest that this inhibition was
attributable tocompetitive binding with the substrate to
the enzyme active site.
1
confirmed by comparing its H NMR signal (2.63–2.68,
m, 1H) with that reported for the C-3 protons in both R
and S configurations.15 Compound 10 served as a ver-
satile intermediate for the synthesis of various 3-hetero-
methyl derivatives. The 3-fluoromethyl counterpart
(+)-11 was obtained by reacting 10 with DAST (die-
thylaminosulfur trifluoride), while (+)-12 to( À)-15
were yielded by treating 10 with triphenylphosphine in
Effect of phosphatidyl-D-3-deoxy-3-aminomethyl-myo-in-
ositol (5) on IgE-induced degranulation in mast cells.
The effect of the synthetic PI 3-kinase inhibitor was
tested on the degranulation of mast cells. Upon activa-
tion by antigens, mast cells immediately extrude gran-
ule-associated mediators that induce immediate allergic
inflammation.19 Published data indicate that inhibition
of PI 3-kinase could effectively block the IgE-induced
secretion of inflammatory mediators,9,20 establishing a
link between PI 3-kinase and mast cell activation. The
inhibitory effect of 5 was assessed on antigen-induced
secretion of hexosaminidase in IgE-sensitized rat baso-
philic leukemia (RBL-2H3) cells, a tumor analogue of
rat mucosal mast cells.21 In this study, we chose
LY294002 as a control in lieu of wortmannin. Wortman-
nin has been shown to inhibit myosin light-chain kinase,
one of the key regulatory enzymes in mast cells.20
After sensitizing RBL-2H3 cells with dinitrophenol
(DNP)-specific monoclonal IgE, the cells were incubated
with LY294002 or compound 5 at various concentrations
Figure 2. Structures of phosphatidyl-d-3-deoxy-3-heteromethyl-myo-
inositols (1–6).

D.-S. Wang, C.-S. Chen / Bioorg. Med. Chem. 9 (2001) 3165–3172
3167
Figure 3. General scheme for the synthesis of phosphatidyl-d-3-deoxy-3-heteromethyl-myo-inositols (1–6).
at 37 ꢁC for 15 min. The cells were then stimulated with
the antigen DNP-human serum albumin (HAS) con-
jugates for 1 h, and the release of hexosaminidase into
medium was determined as an index of degranulation.
As shown in Figure 5, both LY294002 and 5 inhibited
hexosaminidase release in a dose-dependent manner.
The respective IC50 values were 1.8 and 17 mM, respec-
tively, which were in line with those for PI 3-kinase
inhibition in vitro, that is 1.4 and 20 mM. This observa-
tion suggests that both inhibitors could readily cross cell
membranes toaccess intracellular PI 3-kinase.
underlying the inhibitory effect of compound 5 o n PI 3-
kinase is different from that of wortmannin and
LY294002. Thus, it provides an additional tool to study
the physiological function of PI 3-kinase in vivo. Fur-
ther studies of the effect of compound 5 on the mast cell
physiology are currently under way in this laboratory.
Experimental
Material and methods
(+)-2,4,5,6-Tetra-O-benzyl-1-O-(p-methoxybenzyl)-myo-
inositol (7) was synthesized from (+)-1,2:5,6-dicyclo-
hexylidene-myo-inositol as previously described.17 1,2-
Di-O-palmitoyl-sn-glycerol 3-(benzyl N,N-diisopropyl-
phosphoramidite) (23) was prepared using a method
described by Dreef et al.22 DNP–HSA was purchased
Conclusion
The present study demonstrates the biochemical utility
of compound 5 as a PI 3-kinase inhibitor and mast cell-
stabilizing agent. It is noteworthy that the mechanism

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D.-S. Wang, C.-S. Chen / Bioorg. Med. Chem. 9 (2001) 3165–3172
from Sigma, and DNP-specific monoclonal IgE was a
kind gift from Dr. Henry Metzger (National Institutes
of Health). Monoclonal antibody against the p85 sub-
unit of PI 3-kinase was obtained from Transduction
Laboratory. [g-32P]ATP was purchased from NEN Life
Science Products. PI 3-kinase was prepared by immu-
noprecipitation from rat liver cytosol using anti-p85
antibody according to a reported procedure.23
(m, 10H), 6.83 (d, J=8.4 Hz, 2 H), 7.18–7.38 (m, 22H);
HR-MALDI (positive) calcd m/z for C42H42O7:
658.2931. Found 681.2828 (M+Na+).
(À)-2,4,5,6-Tetra-O-benzyl-1-O-(p-methoxybenzyl)-3-
deoxy-3-methylene-myo-inositol (9). A mixture of potas-
sium t-butoxide (135 mg, 1.2 mmol) and methyl-triphe-
nylphosphine bromide (643 mg, 1.8 mmol) in dry
toluene (4 mL) was stirred under reflux for 30 min (À)-8
(789 mg, 1.2 mmol) was added in one pot. The reaction
mixture was stirred at 100–110 ꢁC for 2 h, cooled to
room temperature, diluted with water, extracted with
ethyl acetate (3Â20 mL), dried, and concentrated. Col-
umn chromatography (ether–hexane 1:15) of the residue
gave 9 (syrup, 624 mg, 80%): [a]2D0 À4.9ꢁ (c 2, CHCl3);
1H NMR (CDCl3) d 3.35–3.42 (m, 2H), 3.80 (s, 3H),
4.04–4.12 (m, 2H), 4.23–4.30 (m, 2H), 4.50–4.54 (m, 3H),
4.65(q, J=11.4, 13.5 Hz, 2H), 4.82–4.97 (m, 4H), 5.02 (t,
J=1.5 Hz), 5.43 (t, J=1.5 Hz, 1H), 6.83 (d, J=8.7 Hz,
2H), 7.24 (d, J=8.7 Hz, 2H), 7.26–7.36 (m, 20H); HR-
MALDI (positive) calcd m/z for C43H46O7: 674.3244.
Found: 697.3141, (M+Na+), 713.2881 (M+K+).
(À)-2,4,5,6-Tetra-O-benzyl-1-O-(p-methoxybenzyl)-3-
deoxy-3-oxo-myo-inositol (8). A solution of (+)-7
(1016 mg, 1.5 mmol) in dry DMSO (10 mL) was stirred
with acetic anhydride (3.2 mL, 31.3 mmol) under argon
at 23 ꢁC overnight. The reaction mixture was stirred
with saturated NaHCO3 solution for 3 h, and extracted
with CH2Cl2 (3Â20 mL). The organic phase was washed
with brine, and concentrated. Column chromatography
(ether–hexane 1:10) of the residue gave 8 (amorphous,
912 mg, 90%): [a]2D0=À9.6ꢁ (c 2, CHCl3); 1H NMR
(CDCl3) d 3.43 (dd, J=3, 9.6 Hz, 1H), 3.49 (t, J=9 Hz,
1H), 3.73–3.94 (m, 5H), 4.26 (t, J=9 Hz, 1H), 4.42–4.90
(À)-2,4,5,6-Tetra-O-benzyl-1-O-(p-methoxybenzyl)-3-
deoxy-3-hydroxymethyl-myo-inositol (10). A solution of
(À)-9 (524 mg, 0.8 mmol) in dry benzene (2 mL) was
added 9-BBN (1.69 mL, 0.5 M solution in THF) at
room temperature, and stirred under argon overnight.
Methanol (0.51 mL), 2 N NaOH (0.17 mL), and H2O2
(0.34 mL) were added in tandem. The mixture was stir-
red at 55 ꢁC for 2 h, and concentrated. The residue was
taken up with ether, washed with brine, dried and con-
centrated. Column chromatography (ether–hexane 1:10)
of the residue gave (À)-10 (syrup, 384 mg, 71%) as the
single product: [a]2D0 À5.1ꢁ (c 3, CHCl3); 1H NMR
(CDCl3) d 1.91 (br s, 1H), 2.46–2.58 (m, 1H), 3.31 (dd,
J=4.5, 10.8 Hz, 1H), 3.41 (dd, J=3.0, 9.6 Hz, 1H), 3.61
(t, J=3.0 Hz, 1H), 3.72–3.84 (m, 5H), 3.95 (t, J=8.7 Hz,
1H), 4.03 (d, J=6.3 Hz, 1H), 4.47–4.92 (m, 10H), 6.84
(d, J=8.7 Hz, 2H), 7.24 (d, J=8.7 Hz, 2H), 7.25–7.33
(m, 20H); HR-MALDI (positive) calcd m/z for
Figure 4. (A) Effect of various phosphatidyl-d-3-deoxy-3-hetero-
methyl-myo-inositols (1–6) at 50 mM on PI 3-kinase. Lane A represents
the control in which only solvent carrier was added to the assay. The
autoradiogram is the representative of three independent experiments.
(B) Dose-dependent effect of compounds 5 and 6 on PI 3-kinase. Each
data point represents the means+SD (n=3).
Figure 5. Dose-dependent effect of phosphatidyl-d-3-deoxy-3-amino-
methyl-myo-inositol (5) and LY294002 on antigen-stimulated secre-
tion of b-hexosaminidase from RBL-2H3 mast cells.

D.-S. Wang, C.-S. Chen / Bioorg. Med. Chem. 9 (2001) 3165–3172
3169
C43H44O6 656.3138. Found 679.3036, (M+Na+),
695.2775 (M+K+).
(18.8 mg, 0.075 mmol), and imidazole (5.2 mg,
0.075 mmol) in toluene (2 mL) was stirred at 0 ꢁC, and I2
(21 mg, 0.079 mmol) was slowly added. After 45 min, the
solution was warmed to 70 ꢁC and stirred for another
hour, cooled to room temperature, and diluted with
ether (25 mL). The solution was washed, in tandem,
with 0.5% HCl solution, saturated NaHCO3 solution,
and brine, dried, and concentrated. Column chromato-
graphy ether–hexane 1:15) of the residue gave (+)-14
(syrup, 30 mg, 70%): [a]2D0 +7.7ꢁ (c 1, CHCl3); 1H NMR
(CDCl3) d 2.52–2.58 (m, 1H), 2.74 (t, J=11.7 Hz, 1H),
3.42–3.65 (m, 4H), 3.80 (s, 3H), 3.91 (t, J=9.6 Hz, 1H),
4.08 (t, J=3.3 Hz, 1H), 4.46–4.95 (m, 10H), 6.83 (d,
J=8.7 Hz, 2H), 7.25 (d, J=8.7 Hz, 2H), 7.29–7.35 (m,
20H); HR-MALDI (positive) calcd m/z for C43H45O6I:
784.2261. Found: 807.2159 (M+Na+), 823.1898
(M+K+).
(+)-2,4,5,6-Tetra-O-benzyl-1-O-(p-methoxybenzyl)-3-
deoxy-3-fluoromethyl-myo-inositol (11). A solution of
(À)-10 (37 mg, 0.055 mmol) in CH2Cl2 (1 mL) was
added dropwise to a stirred solution of DAST (7.55 mL,
60.4 mmol) in CH2Cl2 (1 mL) under Ar at 0 ꢁC within a
period of 1 h. The mixture was warmed up to 25 ꢁC,
stirred for another 1 h, and poured into ice-cold water.
The organic phase was washed with water, dried, and
concentrated. Column chromatography (ether–hexane
1:15) of the residue gave (+)-11 (syrup, 28 mg, 75%):
[a]D20 +8.3ꢁ (c 1, CHCl3); 1H NMR (CDCl3) d 2.63–2.68
(m, 1H), 3.38–3.50 (m, 2H), 3.68–3.73 (m, 1H), 3.79 (s,
3H), 3.83–3.92 (m, 3H), 3.99–4.04 (t, J=3.3 Hz, 1H),
4.30–4.69 (m, 10H), 6.80 (d, J=8.7 Hz, 2H), 7.22 (d,
J=8.7 Hz, 2H), 7.25–7.33 (m, 20H); HR-MALDI
(positive) calcd m/z for C43H45O6F: 676.3200. Found
699.3098 (M+Na+).
(À)-2,4,5,6-Tetra-O-benzyl-1-O-(p-methoxybenzyl)-3-
deoxy-3-azidomethyl-myo-inositol (15). A mixture of
(+)-10 (24 mg, 0.036 mmol), triphenylphosphine
(19 mg, 0.075 mmol) in toluene (2 mL) was stirred at
(+)-2,4,5,6-Tetra-O-benzyl-1-O-(p-methoxybenzyl)-3-
deoxy-3-chloromethyl-myo-inositol (12). A so lutio n o f 0 C, and Zn(N3)2 Py2 (8.4 mg, 0.027 mmol) followed by
ꢁ
ꢀ
(+)-10 (37 mg, 0.055 mmol) in dry pyridine (1 mL) was
treated with triphenylphosphine (28.8mg, 0.11mmol),
followed by CCl4 (5.3 mL, 0.055 mmol) in several portions
DEAD (12.6 mg, 0.072 mol) were added. The solution
was stirred at 80 ꢁC for 4 h, cooled to room temperature,
and concentrated. Column chromatography (ether–
hexane 1:15) of the residue afforded (À)-15 (syrup,
20 mg, 80%): [a]2D0 À10.2ꢁ (c 2, CHCl3); 1H NMR
(CDCl3) d 2.61–2.68 (m, 1H), 3.45–3.52 (m, 1H), 3.56–
3.61 (m, 1H), 3.65–3.71 (m, 1H), 3.80 (s, 3H), 3.85 (t,
J=2.7 Hz, 1H), 3.94–4.03 (m, 1H), 4.13–4.21 (m, 2H),
4.40–5.04 (m, 10H), 6.80 (d, J=8.4 Hz, 2H), 7.25–7.38
(m, 22H); HR-MALDI (positive) calcd m/z for
C43H45O6N3: 699.3308. Found: 722.3206 (M+Na+).
ꢁ
at 0 ꢁC. The resulting mixture was heated to70 C, stirred
for 1 h, cooled to room temperature, and diluted with ether
(25 mL). The solution was washed, in tandem, with 0.5%
HCl solution, saturated NaHCO3 solution, and brine,
dried and concentrated. Column chromatography (ether–
hexane 1:15) of the residue gave (+)-12 (syrup, 27 mg,
70%): [a]2D0 +4.7ꢁ (c 2, CHCl3); H NMR (CDCl3) d
1
2.52–2.58 (m, 1H), 3.26 (t, J=11.4 Hz,1H), 3.47–3.59
(m, 3H), 3.80 (s, 3H), 3.83–4.03 (m, 2H), 4.08 (t,
J=3.3 Hz, 1H), 4.43–4.97 (m, 10H), 6.84 (d, J=8.7 Hz,
2H), 7.25 (d, J=8.7 Hz, 2H), 7.29–7.35 (m, 20H); HR-
MALDI (positive) calcd m/z for C43H45O6Cl: 692.2905.
Found: 715.2802 (M+Na+), 731.2542 (M+K+).
(À)-2,4,5,6-Tetra-O-benzyl-1-O-(p-methoxybenzyl)-3-
deoxy-3-benzyloxy-methyl-myo-inositol (16). A solution
of (+)-10 (36 mg, 0.053 mmol) in DMF (1 mL) was
treated with NaH (3 mg, 85%, 0.11 mmol) at 0 ꢁC fo r
30 min, followed benzyl bromide (12 mL, 0.08 mmol).
The mixture was stirred 40 ꢁC for 1 h. Excess NaH was
destroyed by CH3OH. The solution was diluted with
ethyl acetate (20 mL), washed with water, dried, and
concentrated. Column chromatography (ether–hexane
1:25) of the residue afforded (À)-16 (syrup, 39 mg,
(+)-2,4,5,6-Tetra-O-benzyl-1-O-(p-methoxybenzyl)-3-
deoxy-3-bromomethyl-myo-inositol (13). A solution of
(+)-10 (37 mg, 0.055 mmol) in dry pyridine (1 mL) was
treated with triphenylphosphine (28.8 mg, 0.11 mmol),
followed by CBr4 (18.2 mg, 0.055 mmol) in several por-
tions at 0 ꢁC. The resulting mixture was heated to70 ꢁC
for 1 h, stirred for 1 h, cooled to room temperature, and
diluted with ether (25 mL). The solution was washed, in
tandem, with 0.5% HCl solution, saturated NaHCO3
solution, and brine, dried and concentrated. Column
chromatography (ether–hexane 1:15) of the residue gave
(+)-13 (syrup, 32 mg, 79%): [a]2D0 +6.8ꢁ (c 3, CHCl3);
1H NMR (CDCl3) d 2.52–2.60 (m, 1H), 3.07 (t,
J=11.7 Hz, 1H), 3.35–3.59 (m, 3H), 3.68–3.80 (m, 1H),
3.80 (s, 3H), 3.90–3.93 (m, 1H), 4.11 (t, J=3.3 Hz, 1H),
4.45–4.97 (m, 10H), 6.85 (d, J=8.7 Hz, 2H), 7.25 (d,
J=8.7 Hz, 2H), 7.26–7.35 (m, 2H); HR-MALDI (posi-
tive) calcd m/z for C43H45O6Br: 736.2400. Found:
759.2297 (M+Na+), 775.2037 (M+K+).
ꢁ
1
96%); [a]2D0 À13.0 (c 2, CHCl3); H NMR (CDCl3) d
2.34–2.41 (m, 1H), 3.01–3.09 (m, 1H), 3.44–3.69 (m,
3H), 3.80 (s, 3H), 3.89–3.97 (m, 2H), 4.02 (dd, J=4.2,
6 Hz, 1H), 4.43–4.90 (m, 12H), 6.85 (d, J=8.4 Hz, 2H),
7.24 (d, J=8.4 Hz, 2H), 7.25–7.36 (m, 25H); HR-
MALDI (positive) calcd m/z for C50H52O7: 764.3713.
Found: 787.3612 (M+Na+).
(À)-2,4,5,6-Tetra-O-benzyl-3-deoxy-3-fluoromethyl-myo-
inositol (17). Removal of the p-methoxybenzyl group of
(+)-11 (25 mg, 0.037 mmol) was achieved by exposing
toCH 2Cl2–TFA–CH3OH (3:1.5:0.5, 5 mL) with stirring
at 0 ꢁC for 1 h. The mixture was diluted with CH2Cl2
(20 mL), washed, in tandem, with saturated NaHCO3
solution and brine, dried and concentrated. Column
chromatography (ether–hexane 1:10) of the residue gave
(À)-17 (syrup, 18.5 mg, 90%); [a]2D0 À5.3ꢁ (c 1, CHCl3);
1H NMR (CDCl3) d 2.01 (br s, 1H), 2.64–2.68 (m, 1H),
(+)-2,4,5,6-Tetra-O-benzyl-1-O-(p-methoxybenzyl)-3-
deoxy-3-iodomethyl-myo-inositol (14). A mixture of
(+)-10 (37 mg, 0.055 mmol), triphenylphosphine

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D.-S. Wang, C.-S. Chen / Bioorg. Med. Chem. 9 (2001) 3165–3172
3.50–3.53 (m, 1H), 3.68–3.75 (m, 2H), 3.90 (d,
J=7.8 Hz, 1H), 4.02 (dd, J=5.1, 7.8 Hz, 1H), 4.30 (d,
J=3.9 Hz, 1H), 4.42–4.62 (m, 9H), 7.25–7.36 (m, 20H);
HR-MALDI (positive) calcd m/z for C35H37O5F:
556.2625. Found: 579.2523 (M+Na+).
(73 mg, 0.047 mmol) and 1H-tetrazole (10 mg,
0.063 mmol) in CH2Cl2 (2 mL) was stirred at 23 ꢁC
under argon for 30 min and (À)-17 (17 mg, 0.031 mmol)
was added. The mixture was kept under the same con-
ditions for another 12 h, cooled to À40 ꢁC, and then
treated with m-CPBA (19 mg, 57% purity, 0.064 mmol).
The reaction mixture was stirred at À40 ꢁC for 30 min,
allowed to attain room temperature for 1 h, diluted with
CH2Cl2 (20 mL), washed, in tandem, with 10%
NaHCO3 solution and brine, dried, and concentrated.
Column chromatography (ether–hexane 1:3) of the
residue gave (+)-24 (syrup, 33.6 mg, 85%); [a]2D0 +2.4ꢁ
(À)-2,4,5,6-Tetra-O-benzyl-3-deoxy-3-chloromethyl-myo-
inositol (18). Removal of the p-methoxybenzyl group of
(+)-12 (24 mg, 0.033 mmol) with TFA, as describꢁed for
17, gave (À)-18 (syrup, 18.5 mg, 92%); [a]2D0 À7.6 (c 1,
1
CHCl3); H NMR (CDCl3) d 2.14 (br s, 1H), 2.56–2.62
(m, 1H), 3.47 (t, J=10.8 Hz, 1H), 3.54–3.62 (m, 2H),
3.75 (t, J=8.1 Hz, 1H), 3.86–4.04 (m, 2H), 4.06 (t,
J=4.2 Hz, 1H), 4.45–5.01 (m, 8H), 7.26–7.35 (m, 20H);
HR-MALDI (positive) calcd m/z for C35H37O5Cl:
572.2330. Found: 595.2228, (M+Na+).
1
(c 1, CHCl3); H NMR (CDCl3) d 0.87 (t, J=7.2 Hz,
6H), 1.20–1.30 (m, 48H), 1.42–1.60 (m, 4H), 2.20–2.30
(m, 5H), 3.45–3.52 (m, 1H), 3.85–4.10 (m, 9H), 4.20–
4.25 (m, 1H), 4.47–4.93 (m, 10H), 4.96–5.05 (m, 1H),
7.24–7.34 (m, 25H); 31P NMR (CDCl3, H3PO4 as
external reference) d À1.42, À1.51; HR-MALDI (posi-
tive) calcd m/z for C77H111O12FP: 1277.7797. Found:
1300.6795 (M+Na+).
(À)-2,4,5,6-Tetra-O-benzyl-3-deoxy-3-bromomethyl-myo-
inositol (19). Removal of the p-methoxybenzyl group
(+)-13 (28 mg, 0.038 mmol) with TFA, as described for
17, gave (À)-19 (syrup, 21 mg, 90%); [a]2D0 À3.2ꢁ (c 1,
1
CHCl3); H NMR (CDCl3) d 2.50 (br s, 1H), 2.59–2.66
(+)-1-O-(1,2-Di-O-palmitoyl-sn-glycerol-3-benzoxypho-
sphoryl)-2,4,5,6-tetra-O-benzyl-3-deoxy-3-chloromethyl-
myo-inositol (25). Coupling of (À)-18 (18 mg,
0.031 mmol) with 23 (73 mg, 0.047 mmol), as described
for 24, yielded (+)-25 (syrup, 33 mg, 80%); [a]2D0 +10.0ꢁ
(m, 1H), 3.52–3.61 (m, 2H), 3.71–3.90 (m, 3H), 3.93–
3.98 (m, 1H), 4.05–4.10 (m, 1H), 4.53–5.01 (m, 8H),
7.25–7.36 (m, 20H); HR-MALDI (positive) calcd m/z
for C35H37O5Br: 618.1804. Found: 641.1702,
(M+Na+).
1
(c 2, CHCl3); H NMR (CDCl3) d 0.87 (t, J=7.2 Hz,
6H), 1.20–1.32 (m, 48H), 1.48–1.64 (m, 4H), 2.17–2.25
(m, 4H), 2.54–2.62 (m, 1H), 3.41 (t, J=11.4 Hz, 1H),
3.51–3.60 (m, 2H), 3.76–3.81 (m, 1H), 3.92–4.19 (m,
6H), 4.29 (t, J=7.8 Hz, 1H), 4.39–4.71 (m, 5H), 4.79–
5.08 (m, 6H), 7.25–7.36 (m, 25H); 31P NMR (CDCl3,
H3PO4 as external reference) d À1.12, À1.37; HR-
MALDI (positive) calcd m/z for C77H111O12ClP:
1293.7502. Found: 1316.3399 (M+Na+).
(À)-2,4,5,6-Tetra-O-benzyl-3-deoxy-3-iodomethyl-myo-
inositol (20). Removal of the p-methoxybenzyl group of
(+)-14 (27 mg, 0.034 mmol) with TFA, as described for
17, gave (À)-20 (syrup, 19 mg, 94%); [a]2D0 À8.7ꢁ (c 1,
1
CHCl3); H NMR (CDCl3) d 2.12 (br s, 1H), 2.52–2.57
(m, 1H), 3.56–3.66 (m, 3H), 3.69–3.86 (m, 3H), 4.03–
4.07 (m, 1H), 4.44–5.01 (m, 8H), 7.25–7.35 (m, 20H);
HR-MALDI (positive) calcd m/z for C35H37O5I:
664.6686. Found: 687.6583 (M+Na+).
(+)-1-O-(1,2-Di-O-palmitoyl-sn-glycerol-3-benzoxypho-
sphoryl)-2,4,5,6-tetra-O-benzyl-3-deoxy-3-bromomethyl-
myo-inositol (26). Coupling of (À)-19 (20 mg,
0.032 mmol) with 23 (73 mg, 0.047 mmol), as described
for 24, yielded (+)-26 (syrup, 35 mg, 81%); [a]2D0 +8.2ꢁ
(À)-2,4,5,6-Tetra-O-benzyl-3-deoxy-3-azidomethyl-myo-
inositol (21). Removal of the p-methoxybenzyl group of
(À)-15 (18 mg, 0.026 mmol) TFA, as described for 17,
gave (À)-21 (syrup, 14 mg, 94%); [a]2D0 À6.3ꢁ (c 1,
1
(c 1, CHCl3); H NMR (CDCl3) d 0.87 (t, J=7.2 Hz,
1
CHCl3); H NMR (CDCl3) d 2.42 (br s, 1H), 2.64–2.72
6H), 1.22–1.28 (m, 48H), 1.48–1.60 (m, 4H), 2.18–2.25
(m, 4H), 2.56–2.60 (m, 1H), 3.28 (t, J=9.9 Hz, 1H), 3.44–
3.56 (m, 2H), 3.65–3.77 (m, 2H), 3.96–4.30 (m, 6H), 4.40–
4.70 (m, 4H), 4.78–5.11 (m, 7H), 7.24–7.34 (m, 25H); 31P
NMR (CDCl3, H3PO4 as external reference) d À1.13,
À1.37; HR-MALDI (positive) calcd m/z for
C77H111O12BrP: 1337.6997. Found 1360.6894 (M+Na+).
(m, 1H), 3.54–3.65 (m, 1H), 3.72 (t, J=8.4 Hz, 1H), 3.85
(t, J=9.3 Hz, 1H), 3.91–3.97 (m, 1H), 4.07–4.23 (m,
3H), 4.38–4.59 (m, 4H), 4.69–4.99 (m, 4H), 7.25–7.33
(m, 20H); HR-MALDI (positive) calcd m/z for
C35H37O5N3: 579.2733. Found: 602.2630 (M+Na+).
(+)-2,4,5,6-Tetra-O-benzyl-3-deoxy-3-benzyloxymethyl-
myo-inositol (22). Removal of the p-methoxybenzyl
group of (À)-16 (35 mg, 0.046 mmol) TFA, as described
for 17, gave (+)-22 (syrup, 27 mg, 92%); [a]2D0 +6.0ꢁ (c
(+)-1-O-(1,2-Di-O-palmitoyl-sn-glycerol-3-benzoxypho-
sphoryl)-2,4,5,6-tetra-O-benzyl-3-deoxy-3-iodomethyl-
myo-inositol (27). Coupling of (À)-20 (18 mg,
0.027 mmol) with 23 (73 mg, 0.047 mmol), as described
for 24, yielded (+)-27 (syrup, 32 mg, 85%); [a]2D0 +8.7ꢁ
1
1, CHCl3); H NMR (CDCl3) d 2.17 (s, 1H), 2.59–2.68
(m, 1H), 3.34 (t, J=9.9 Hz, 1H), 3.44–3.71 (m, 4H), 3.94–
4.09 (m, 2H), 4.33–4.61 (m, 6H), 4.70–5.02 (m, 4H),
7.26–7.38 (m, 25H); HR-MALDI (positive) calcd m/z for
C42H44O6: 644.3138. Found: 667.3036 (M+Na+).
1
(c 2, CHCl3); H NMR (CDCl3) d 0.87 (t, J=7.2 Hz,
6H), 1.18–1.36 (m, 48H), 1.44–1.62 (m, 4H), 2.16–2.24
(m, 4H), 2.68–2.76 (m, 1H), 3.47–3.50 (m, 1H), 3.60–
3.68 (m, 2H), 3.92–4.31 (m, 8H), 4.47–5.18 (m, 11H),
7.25–7.30 (m, 25H); 31P NMR (CDCl3, H3PO4 as
external reference) d À1.12, À1.37; HR-MALDI (posi-
tive) calcd m/z for C77H111O12IP: 1385.6858. Found:
1408.6756 (M+Na+).
(+)-1-O-(1,2-Di-O-palmitoyl-sn-glycerol-3-benzoxypho-
sphoryl)-2,4,5,6-tetra-O-benzyl-3-deoxy-3-fluoromethyl-
myo-inositol (24). A solution of 1,2-di-O-palmitoyl-sn-
glycerol 3-(benzyl N,N-diisopropylphosphoramidite) 23

D.-S. Wang, C.-S. Chen / Bioorg. Med. Chem. 9 (2001) 3165–3172
3171
(+)-1-O-(1,2-Di-O-palmitoyl-sn-glycerol-3-benzoxypho-
sphoryl)-2,4,5,6-tetra-O-benzyl-3-deoxy-3-azidomethyl-
myo-inositol (28). Coupling of (À)-21 (13 mg,
0.023 mmol) with 23 (54 mg, 0.035 mmol), as described
for 24, yielded (+)-28 (24 mg, 83%); [a]2D0 +8.2ꢁ (c 1,
external reference) d À1.03; HR-MALDI (negative)
calcd m/z for C42H81O12BrP: 889.4629. Found: 807.5387
(MÀHBr).
L-ꢀ-Phosphatidyl-D-3-deoxy-3-iodomethyl-myo-inositol
(4). The perbenzylated derivative (+)-27 (30 mg,
0.022 mmol) was subjected to hydrogenolysis, as descri-
bed for 24, gave 4 (lyophilized powder, 19 mg, 94%);
1
CHCl3); H NMR (CDCl3) d 0.87 (t, J=7.2 Hz, 6H),
1.20–1.30 (m, 48H), 1.46–1.67 (m, 4H), 2.13–2.24 (m,
4H), 2.60–2.68 (m, 1H), 3.56–3.65 (m, 2H), 3.95–4.21
(m, 7H), 4.29–4.48 (m, 2H), 4.52–5.05 (m, 11H), 7.25–
7.38 (m, 25H); 31P NMR (CDCl3, H3PO4 as external
reference) d À0.57; HR-MALDI (positive) calcd m/z for
[a]2D0 +3.0ꢁ (c 1.0, CHCl3); H NMR (CDCl3) d 0.87 (t,
1
J=7.2 Hz, 6 H), 1.21–1.35 (m, 48H), 1.48–1.62 (m, 4H),
2.28–2.37 (m, 4H), 3.47–3.51 (m, 1H), 3.66–3.67 (m,
1H), 3.72–3.77 (m, 1H), 4.08–4.30 (m, 9H), 5.20–5.30
(m, 1H); 31P NMR (CDCl3, H3PO4 as external refer-
ence) d À0.86; HR-MALDI (negative) calcd m/z for
C42H81O12IP: 934.4432. Found: 807.5387 (MÀHI).
C77H111O12N3P:
(M+Na+).
1300.7905.
Found
1323.7802
(+)-1-O-(1,2-Di-O-palmitoyl-sn-glycerol-3-benzoxypho-
sphoryl)-2,4,5,6-tetra-O-benzyl-3-deoxy-3-benzyloxy-
methyl-myo-inositol (29). Coupling of (À)-22 (24 mg,
0.037 mmol), with 23 (87 mg, 0.056 mmol), as described
for 24, yielded (+)-29 (syrup, 41 mg, 81%); [a]2D0 +11.0ꢁ
L-ꢀ-Phosphatidyl-D-3-deoxy-3-aminomethyl-myo-inositol
(5). The perbenzylated derivative (+)-28 (22 mg,
0.017 mmol) was subjected to hydrogenolysis, as descri-
bed for 24, gave 5 (lyophilized powder, 13.2 mg, 92%);
1
(c 2, CHCl3); H NMR (CDCl3) d 0.87 (t, J=7.2 Hz,
[a]2D0 +3.8ꢁ (c 1, CHCl3); H NMR (CDCl3) d 0.87 (t,
1
6H), 1.19–1.36 (m, 48H), 1.45–1.62 (m, 4H), 2.17–2.38
(m, 4H), 2.58–2.65 (m, 1H), 3.46–3.65 (m, 3H), 3.98–
4.29 (m, 7H), 4.33–4.52 (m, 1H), 4.48–5.05 (m, 13H),
7.25–7.36 (m, 30H); 31P NMR (CDCl3, H3PO4 as
external reference) d À0.46; HR-MALDI (positive)
calcd m/z for C84H118O13P:1365.8310. Found:
1388.8207 (M+Na+).
J=7.2 Hz, 6H), 1.21–1.25 (m, 48H), 1.49–1.62 (m, 4H),
2.25–2.40 (m, 4H), 3.46–4.30 (m, 12H), 4.60–4.70 (m,
1H), 5.20–5.30 (m, 1H); 31P NMR (CDCl3, H3PO4 as
external reference) d À1.11; HR-MALDI (negative)
calcd m/z for C42H83O12NP: 824.5653. Found: 823.5575
(MÀH).
L-ꢀ-Phosphatidyl-D-3-deoxy-3-fluoromethyl-myo-inositol
(1). A suspension of 24 (32 mg, 0.025 mmol) and palla-
dium black (30 mg) in aqueous 80% ethanol (4 mL) was
shaken under H2 (55 psi) for 16 h, filtered and con-
centrated. The residual aqueous solution was lyophi-
lized tofurnish 1 (lyophilized powder, 20 mg, 97%);
L-ꢀ-Phosphatidyl-D-3-deoxy-3-hydroxymethyl-myo-inosi-
tol (6). The perbenzylated derivative (+)-29 (38 mg,
0.028 mmol) was subjected to hydrogenolysis, as descri-
bed for 24, gave 6 (lyophilized powder, 22 mg, 95%);
[a]2D0 +6.9ꢁ (c 1, CHCl3); H NMR (CDCl3) d 0.87 (t,
1
J=7.2 Hz, 6H), 1.25–1.37 (m, 48H), 1.50–1.67 (m, 4H),
2.17–2.38 (m, 4H), 3.45–4.51 (m, 1H), 3.60–4.48 (m,
11H), 5.20–5.30 (m, 1H); 31P NMR (CDCl3, H3PO4 as
external reference) d À0.99; HR-MALDI (negative)
calcd m/z for C42H82O13P: 825.5493. Found 824.5415
(MÀH), 848.5391, (M+Na+).
[a]2D0 +9.1ꢁ (c 1, CHCl3); H NMR (CDCl3) d 0.87 (t,
1
J=7.2 Hz, 6H), 1.20–1.32 (m, 48H), 1.49–1.64 (m, 4H),
2.23–2.40 (m, 4H), 3.44–3.60 (m, 1H), 3.72–3.73 (m,
1H), 3.95–4.29 (m, 10H), 5.08–5.30 (m, 1H); 31P NMR
(CDCl3, H3PO4 as external reference) d À1.01; HR-
MALDI (negative) calcd m/z for C42H81O12FP:
827.5450. Found: 807.5387 (MÀHF), 850.5347
(M+Na+).
PI 3-kinase assay
The enzyme assay was carried out using PI 3-kinase that
was immunoprecipitated with anti-p85 antibodies from
rat liver cytosol as described.23 In brief, PI(4,5)P2
(10 mg) and phosphatidylserine (40 mg) were suspended
in 100 mL of 30 mM Hepes, pH 7.5, containing 1 mM
EDTA and 1 mM EGTA, sonicated in a water bath-
type sonicator for 5 min, and mixed vigorously with a
vortex mixer before assays. The immunoprecipitated PI
3-kinase in 30 mM Hepes, pH 7.5, with appropriate
dilution (10 mL) was incubated with 80 mL of the same
buffer containing 125 mM ATP, 10 mCi [g-32P]ATP, and
6.25 mM MgCl2. The reaction was initiated by adding
10 mL of the phospholipid solution, incubated at 37 ꢁC
for 10 min, and stopped by adding 5 mL of 1 mM EDTA
and 25 mL of 5 M HCl, followed by 160 mL of CHCl3–
CH3OH (1:1). The phases were separated by cen-
trifugation at 6000g for 5 min. The organic layer was
dried by a stream of N2, spotted onto 1% oxalic acid-
treated TLC plates, and then developed with n-propra-
nol–2M acetic acid (65:35) overnight. After drying,
spots were located by autoradiography and compared
L-ꢀ-Phosphatidyl-D-3-deoxy-3-chloromethyl-myo-inositol
(2). The perbenzylated derivative (+)-25 (31 mg,
0.024 mmol) was subjected to hydrogenolysis, as descri-
bed for 24, gave 2 (lyophilized powder, 19 mg, 94%);
[a]2D0 +2.8ꢁ (c 2, CHCl3); H NMR (CDCl3) d 0.87 (t,
1
J=7.2 Hz, 6H), 1.21–1.35 (m, 48H), 1.49–1.64 (m, 4H),
2.28–2.37 (m, 4H), 3.40–4.45 (m, 12H), 5.20–5.30 (m,
1H); 31P NMR (CDCl3, H3PO4 as external reference) d
À1.22; HR-MALDI (negative) calcd m/z for
C42H81O12ClP: 843.5155. Found: 807.5387 (MÀHCl),
866.5053 (M+Na+).
L-ꢀ-Phosphatidyl-D-3-deoxy-3-bromomethyl-myo-inositol
(3). The perbenzylated derivative (+)-26 (33 mg,
0.025 mmol) was subjected to hydrogenolysis, as descri-
bed for 24, gave 3 (lyophilized powder, 21 mg, 96%);
[a]2D0 +6.3ꢁ (c 2, CHCl3); H NMR (CDCl3) d 0.87 (t,
1
J=7.2 Hz, 6H), 1.21–1.32 (m, 48H), 1.50–1.58 (m, 4H),
2.29–2.37 (m, 4H), 3.18–3.77 (m, 4H), 3.92–4.56 (m,
8H), 5.20–5.30 (m, 1H); 31P NMR (CDCl3, H3PO4 as

3172
D.-S. Wang, C.-S. Chen / Bioorg. Med. Chem. 9 (2001) 3165–3172
with standards. The autoradiograms were scanned by a
Photodyne image system and quantified using an NIH
imaging program (version 1.59).
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Hexosaminidase secretion assay
The release of mast cell mediators by exocytosis was
monitored by the b-hexosaminidase assay. RBL-2H3
cells were grown in 12-well plates and passively sensi-
tized with DNP-specific IgE. The IgE-sensitized cells
were washed twice with Tyrode’s buffer consisting of
10 mM Hepes, pH 7.4, 130 mM NaCl, 5 mM KCl,
1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, and
0.1% BSA. Secretion was initiated by exposing cells to
the antigen DNP-HSA (1 mg/mL). After 1 h, the reac-
tion was terminated by placing the plate on ice. The
enzyme activities of b-hexosaminidase in 50 mL o f
supernatants and attached cells solubilized with 0.5%
Triton X-100 were measured with 200 mL o f 1 mMp-
nitrophenyl N-acetyl-b-d-glucosaminide in 0.1 M
sodium citrate, pH 4.5, at 37 ꢁC for 1 h. The reaction
was stopped by the addition of 500 mL of 0.1 M
NaHCO3. The release of the product p-nitrophenol was
measured by monitoring the absorbance at 400 nm.
Percentage of degranulation was calculated by dividing
the absorbance of the supernatant over the combined
absorbance of the supernatant and cell lysate.
14. Kozikowski, A. P.; Kiddle, J. J.; Frew, T.; Berggren, M.;
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16. Ching, T. T.; Hsu, A. L.; Johnson, A. J.; Chen, C.-S. J.
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Acknowledgements
19. Metcalfe, D. D.; Baram, D.; Mekori, Y. A. Physiol. Rev.
1997, 77, 1033.
This work was supported by National Institutes of
Health grant GM53448. We are grateful toDr. Henry
Metzger (NIAMS/NIH) for providing DNP-specific
IgE.
20. Yano, H.; Agatsuma, T.; Nakanishi, S.; Saitoh, Y.; Fukui,
Y.; Nonomura, Y.; Matsuda, Y. Biochem. J. 1995, 312, 145.
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