Synthesis and antitumor activity of inositol phosphotriester analogues

Article abstract of DOI:10.1039/c2ob00031h

Inositol phosphates, as important second messengers of signal transduction, regulate many biological functions. However, cell penetration and phospholipase stability could be two main issues faced by inositol phosphate analogues used as lead compounds for drug discovery. Inositol phosphotriester analogues could be more beneficial to diffuse across plasma membrane. In this paper, we describe the design and synthesis of a series of inositol phosphotriester analogues based on phosphatidylinositol, along with the initial antitumor activity analysis. Several compounds exhibited good cytotoxic activity against human cancer cell lines A549, HepG2, MDA-MB-231 and HeLa, especially compound 33 was cytotoxic against all the four cancer cell lines with good IC₅₀ values.

Full text of DOI:10.1039/c2ob00031h

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PAPER
Synthesis and antitumor activity of inositol phosphotriester analogues†
Fanbo Song,a Jing Zhang,b Yuefang Zhao,a Wenbin Chen,a Luyuan Lib and Zhen Xi*a
Received 5th January 2012, Accepted 23rd February 2012
DOI: 10.1039/c2ob00031h
Inositol phosphates, as important second messengers of signal transduction, regulate many biological
functions. However, cell penetration and phospholipase stability could be two main issues faced by
inositol phosphate analogues used as lead compounds for drug discovery. Inositol phosphotriester
analogues could be more beneficial to diffuse across plasma membrane. In this paper, we describe the
design and synthesis of a series of inositol phosphotriester analogues based on phosphatidylinositol,
along with the initial antitumor activity analysis. Several compounds exhibited good cytotoxic activity
against human cancer cell lines A549, HepG2, MDA-MB-231 and HeLa, especially compound 33 was
cytotoxic against all the four cancer cell lines with good IC50 values.
The signaling pathways regulated by inositol phosphates and
phosphatidylinositol phosphates have become important cancer
Introduction
Inositol phosphates, inositol pyrophosphates, and phosphatidyl-
inositol phosphates comprise an extremely diverse signaling
molecules family for which variations in biological properties
lead to independent regulation of numerous key cellular pro-
cesses.1 In particular, inositol 1,4,5-trisphosphate (InsP3), as ubi-
quitous intracellular second messenger, stimulates the release of
calcium ions. This signaling molecule is generated through the
hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2)
by the intracellular phosphatidylinositol-specific phospholipase
C (PI-PLC), which also produces another important signaling
lipid diacylglycerol (DAG). InsP3-mediated Ca2+ release is rela-
tive to a plethora of subsequent signaling events, including cell
proliferation, muscle contraction, apoptosis, and gene transcrip-
tion.2 Because of the biological importance as second messenger
in the intracellular signal transduction, a lot of InsP3 analogues
have been synthesized, such as deoxy analogues, fluorinated ana-
logues, ring-modified analogues, phosphorothioate analogues,
phosphonate analogues, bicyclic analogues, conformationally
restricted analogues and adenophostin mimic analogues, many
of which could behave as InsP3 receptor agonists or antagonists.3
Synthetic inositol-containing natural products and analogues are
invaluable for elucidating the complex biological activities. Fur-
thermore, they are useful for the development of new molecular
probes that can interfere with cellular processes and new drug
leads discovery based on novel therapeutic targets.
therapeutic targets. Significant evidence suggests that PI-PLC
could be target for the development of antitumor drugs. Hyper-
activation of PI-PLC exists in a number of human tumors.4 The
majority of human breast cancers have detectable PI-PLCγ
immunoreactive protein compared to benign breast tissue.5 Cyto-
solic PI-PLC activity was evidently increased in human non-
small cell lung cancers and renal cell cancers.6 In addition, the
cellular signaling pathway of phosphatidylinositol 3-kinase
(PI3K)/protein kinase B (AKT)/mammalian target of rapamycin
(mTOR), which regulates a number of important biological pro-
cesses such as cell growth, proliferation, survival, and apoptosis,
is commonly deregulated in many human cancers.7 Hyperactiva-
tion of the PI3K cascade, including phosphatase and tensin hom-
ologue (PTEN) deactivation and PI3K activation mutations,
exists in almost every form of human cancers, for example colon
cancers, breast cancers, melanomas, brain cancers, and gastric
cancers.8 Moreover, the activation of PI3K/AKT/mTOR signal-
ing pathway would result in the resistance of cancer cells to both
targeted anticancer therapies and conventional cytotoxic agents.9
Accordingly, considerable drug discovery efforts are ongoing,
targeting several components of the signaling cascade with
single or multiple target strategies.8f,h,10
To date, few studies have addressed the application of inositol
phosphate and phosphatidylinositol phosphate analogues as
inhibitors to show antitumor activity. Some analogues were
designed to repress the PI3K-dependent signaling pathway
(Fig. 1). Falasca group found Ins(1,3,4,5,6)P5 could inhibit the
growth of cancer cell by the competitive binding of the AKT
pleckstrin homology (PH) domain.11 They recently has demon-
strated that 2-O-Bn-Ins(1,3,4,5,6)P5 exhibits enhanced antitumor
activity.12 Perifosine, which has a similar structure to naturally
occurring phospholipids, was shown to inhibit the growth of
PC-3 prostate carcinoma cells, in which PTEN mutation
aState Key Laboratory of Elemento-Organic Chemistry and Department
of Chemical Biology, Nankai University, Tianjin, 300071, China.
E-mail: zhenxi@nankai.edu.cn; Fax: (+86)22-23504782; Tel: (+86)22-
23504782
bCollege of Pharmacy, Nankai University, Tianjin, 300071, China
†Electronic supplementary information (ESI) available: Copies of 31P
NMR, 1H NMR and 13C NMR spectra for all new synthetic compounds.
See DOI: 10.1039/c2ob00031h
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Fig. 2 The design concept of target molecules.
Fig. 1 Representative lipid-based antagonists of the PI3K pathway.
happened and the AKT pathway was highly activated.13 Kozi-
kowski’s group found several 3-modified phosphatidylinositol
analogues could block PI3K, AKT, and then repressed cancer
cell growth.14
Cell penetration and phospholipase stability could be two
main issues faced by inositol phosphate analogues used as lead
compounds for drug discovery. The intrinsic charge associated
with the phosphate groups of inositol phosphates renders these
molecules unable to passively diffuse across the lipophilic cell
membrane. If small molecules are to be truly useful as drug
leads, they have to be readily membrane permeant. As the devel-
opment is in its early stages, the structures of the analogues are
similar to the natural products and often contain polar groups.
Inositol phosphotriester analogues could be more beneficial to
diffuse across plasma membrane.15 However, little attention has
been paid to inositol phosphotriester analogues to study their
biological activities. In this paper, we reported the design and
synthesis of a series of inositol phosphotriester analogues based
on phosphatidylinositol, along with the initial antitumor activity
analysis.
Scheme 1 Synthesis of mono-hydroxyl inositol compounds. Reagents
and conditions: (a) HC(OEt)3, PTSA monohydrate, DMF, 110 °C, 28 h,
83%; (b) NaH, BnBr, DMF, 0 °C–RT, overnight, 48% for 2, 74% for 3,
68% for 4; (c) TBDMSCl, imidazole, DMF, RT, overnight, 90%; (d)
AlMe3, CH2Cl2, 0 °C–RT, 5 h, 68%; (e) DIBAL, CH2Cl2, 0 °C–RT, 4 h,
95%.
each hydroxyl group has its own steric configuration, the phos-
phorylation occurred at different positions of myo-inositol, may
lead to different stereo-configurations when interact with the
target. Because Ins(1,3,4,5,6)P5 and 2-O-Bn-Ins(1,3,4,5,6)P5
exhibited good antitumor activity, some analogues also con-
tained different numbers of benzyl groups or phosphotriester
groups to enhance their interactions with the target through
hydrogen bond or stacking.
The mono-hydroxyl compounds 4, 5, 6 and 7 were syn-
thesized using myo-inositol as starting material according to the
route shown in Scheme 1. Treatment of myo-inositol with triethyl
orthoformate in DMF provided the triol 1.16 Benzyl protection
of the 2-, 4-, or 6-hydroxyl groups, the mono-hydroxyl com-
pound 4, the diol 2 and the total protected compound 3 were
obtained respectively.16b The mono-hydroxyl compound 5 was
afforded by the silylation of 2 with tert-butyldimethylsilyl chlor-
ide.17 Selective ring-opening of 3 with trimethyl aluminium or
diisobutylaluminium hydride gave 6 and 7, respectively.18
Results and discussion
Synthesis
To try to improve the issues faced by inositol phosphate ana-
logues used as drug leads, a series of inositol phosphotriester
analogues of phosphatidylinositol were designed (Fig. 2). Firstly,
DAG, as a second messenger, which could active PKC, plays
important role in crucial biological function together with Ca2+,
and has contribution to the PI-PLC hydrolysis of phosphatidyl-
inositol, so the lipophilic diacylglycerol (DAG) group was
replaced with two decyl chains as phosphotriester to improve
cell penetration and PI-PLC stability. Secondly, the DAG group
is on the C1 position of myo-inositol in the natural phosphatidyl-
inositol phosphates, however, myo-inositol is a meso-compound,
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Scheme 4 Synthesis of 4(6)-phosphorylated analogues 18–23.
Reagents and conditions: (a) 1H-tetrazole, i-Pr2NP(OC10H21 2 (8),
)
CH2Cl2, RT, overnight, then m-CPBA, 0 °C–RT, 2 h, 60%; (b) HCl,
MeOH, RT-40 °C, 6 h, 95%; (c) Pd–C, ethanol, RT, overnight, 82% for
20, 91% for 22, 91% for 23, (d) TBAF, THF, RT, 30 min, 98%.
Scheme 2 Synthesis of 1(3)-phosphorylated analogues 9–12. Reagents
and conditions: (a) 1H-tetrazole, i-Pr2NP(OC10H21 2 (8), CH2Cl2, RT,
)
overnight, then m-CPBA, 0 °C–RT, 2 h, 72%; (b) HCl, MeOH, RT, 2 h,
99%; (c) Pd–C, ethanol, RT, overnight, 93%; (d) Bt2O, pyridine, RT,
overnight, 90%.
Scheme 5 Synthesis of 5-phosphorylated analogues 24–27. Reagents
and conditions: (a) 1H-tetrazole, i-Pr2NP(OC10H21)2 (8), CH2Cl2, RT,
overnight, then m-CPBA, 0 °C–RT, 2 h, 86%; (b) HCl, MeOH, RT, 1 h,
then reflux, 6 h, 80%; (c) Pd–C, ethanol, RT, overnight, 73% for 26,
90% for 27.
Scheme 3 Synthesis of 2-phosphorylated analogues 13–17. Reagents
and conditions: (a) 1H-tetrazole, i-Pr2NP(OC10H21)2 (8), CH2Cl2, RT,
overnight, then m-CPBA, 0 °C–RT, 2 h, 93%; (b) HCl, MeOH, RT–
35 °C, 6 h, 75%; (c) Pd–C, ethanol, RT, overnight, 80% for 15, 24% for
16, 99% for 17.
lung cancer is improving, survival of advanced NSCLC patients
has been greatly limited by standard methods such as chemother-
apy and radiotherapy.20 The 5-year survival rate of NSCLC is
still lacking in significant improvement. The major chemother-
apy agents towards NSCLC are still cisplatin or its derivatives
combined with other agents, which have apparent side effects in
clinical therapies. For that matter, there is an urgent need to
develop novel antitumor agents which can be successfully admi-
nistered to NSCLC patients. Therefore, having established the
synthesis of inositol phosphotriester analogues 9–27, we assayed
the inhibitory effects of these compounds against NSCLC cell
line A549 firstly by the MTT test with cisplatin as positive
control. The NSCLC cells were exposed to all the nineteen ana-
logues at 10 μg mL−1 (final concentration in the test well). A
compound was considered active if it reduced the growth of the
cell line to 60% or less. The biological activity assay showed
that analogues 14, 16, 17 and 21 were generally proved to be
more potent than others. Analogues 11, 15, 20, 26, which were
more similar to phosphatidylinositol in structure, exhibited little
inhibition activity. The similar results were obtained with
Phosphorylation of the mono-hydroxyl compounds 4, 5, 6, 7
with 8 afforded 9, 13, 18, 24 (Schemes 2–5), which were trans-
formed into 16, 17, 22, 27 employing catalytic hydrogenation.
Acidic hydrolysis of compounds 9, 13, 18, 24 provided 10, 14,
19 and 25. Subsequently, hydrogenolytic removal of benzyl
groups furnished 11, 15, 20 and 26. Treatment of compound 18
with tetrabutylammonium fluoride in THF to remove the tert-
butyldimethylsilyl group gave 21, which was converted to 23
using hydrogenolysis reaction. In order to enhance the ability to
diffuse across plasma membrane, the hydroxyl groups of com-
pound 11 were protected with butyryl to synthesize 12. Com-
pounds 14, 17 and 21 were further phosphorylated to provide
28–35 (Scheme 6).
Cytotoxicity evaluation
Non-small cell lung cancer (NSCLC) constitutes over 85% of
primary lung cancers.19 Although the treatment of advanced
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Table 1 Growth inhibition effects of inositol phosphotriester
analogues on cancer cell lines and normal breast epithelial cell line
MCF10A
IC50 (μM)a
MDA-
MB-231
Compound A549
HepG2
HeLa
MCF10A
14
16
17
21
30
31
33
16.6 0.2 13.4 0.6 22.2 2.5 28.4 1.0 >40.0
22.8 2.0 13.7 1.2 33.1 0.2 23.6 3.3 37.4 1.7
14.0 1.0 10.3 0.5 15.2 2.4 18.2 2.5 29.6 1.4
12.3 0.6 10.6 0.6 12.2 1.9 16.1 0.6 37.8 1.1
16.4 1.0 12.0 1.2 16.3 1.4 18.2 2.5 >40.0
11.9 1.5 8.5 0.1 21.9 2.4 15.4 0.1 34.9 0.8
6.6 0.1 5.8 0.2 6.2 0.2 5.9 0.2 15.0 1.5
Cisplatin 7.0 2.7 1.0 0.1 8.0 3.0 1.7 0.1 3.4 0.2
a IC50 values are the half-maximal inhibitory concentrations as measured
by MTT assay; data represent the mean value
Experimental section for details.
SD. See the
cisplatin. Removal of benzyl groups of 33 by catalytic hydrogen-
ation to generate 34, showed much less inhibition activity.
The seven inositol phosphotriester analogues, which showed
the highest potency against A549, were then screened for their
anti-cancer activity against the other three human cancer cell
lines (HepG2, MDA-MB-231 and HeLa) in order to determine
the selectivity between different cancer cell lines (Table 1). Com-
pound 14 exhibited better activities against HepG2 and A549
than MDA-MB-231 and HeLa. The cytotoxic activity of ana-
logues 16 and 31 were higher against HepG2 than those against
the other three cancer cell lines. Compounds 17, 21, 30 and 33
showed similar inhibition activities against all the four cancer
cell lines. The general toxicity of these compounds was exam-
ined on the non-cancerous cell line MCF10A, and found to be
less toxic than cisplatin (Table 1).
As initial research on the mechanism, these inositol phospho-
triester analogues were tested for the ability to inhibit p100/p85
PI3K using the method of competitive fluorescence polariz-
ation,21 but the inhibition activity was little. Actually, the bio-
logical result was unexpected and surprised for us. Analogues
11, 15, 20, 26, which were more similar to phosphatidylinositol,
exhibited little inhibition activity. But some analogues phos-
phorylated at 2- or 4(6)-position, which contain benzyl groups
and/or rigid structure, showed good antitumor activity. In
addition, the active compounds 14, 16, 21, 31 and 33 retained at
least one benzyl group, which could be unlikely to be removed
in vitro.15a But the benzyl groups were very important for the
antitumor activity, especially analogues 14, 21 and 33. Also, the
Falasca group has demonstrated that 2-O-Bn-Ins(1,3,4,5,6)P5
exhibits enhanced antitumor activity.12 Furthermore, the effect of
2-O-Bn-Ins(1,3,4,5,6)P5 was highly specific, as this compound
only inhibited PDK1 and to a lesser extent mTOR in a panel of
almost 60 kinases.12 However, the role of the benzyl group was
still not clear. Examination of the X-ray crystal structure of the
PH-domain of AKT, which binds to PtdIns(3,4,5)P3 and PtdIns
(3,4)P2, indicates that benzyl groups could not be tolerated in
the binding site, making it unlikely that these phosphotriester
analogues are acting as inhibitors of the AKT PH-domain.22
And, it could be difficult for these analogues to interact with a
highly charged phosphate-binding domain. Maybe an alternative
Scheme 6 Synthesis of further phosphorylated analogues 28–35.
Reagents and conditions: (a) 1H-tetrazole, i-Pr2NP(OC10H21 2 (8),
)
CH2Cl2, RT, overnight, then m-CPBA, 0 °C–RT, 2 h, 55% for 28, 39%
for 29; (b) 1H-tetrazole, i-Pr2NP(OCH3)2, CH2Cl2, RT, overnight,
then m-CPBA, 0 °C–RT, 2 h, 76% for 30, 80% for 31, 80% for 33; (c)
1H-tetrazole, i-Pr2NP(OBn)2, CH2Cl2, RT, overnight, then m-CPBA,
0 °C–RT, 2 h, 70% for 32, 70% for 35; (d) Pd/C, ethanol, RT, overnight,
99% for 34.
analogues 9, 13, 18, 24, which did not contain free hydroxyl
groups. On the other hand, the inhibition activity was related
with the phosphorylated position, 2-phosphorylated and 4(6)-
phosphorylated analogues represented better inhibition activity.
Analogues 14, 16, 17 and 21 were further tested in an eight-dose
mode to determine their potency. Their IC50 values were in the
10–25 μM range (Table 1).
The natural phosphatidylinositol phosphates contain one more
phosphate group at least to interact with the target. The hydroxyl
groups of 14, 17 and 21 were further converted to different phos-
photriester groups to improve the biological activity (Scheme 6).
Compounds 28 and 29, which contain didecyl phosphates,
showed little inhibition activity. Similar results were obtained
with analogues 32 and 35, which contain dibenzyl phosphates.
The analogues containing dimethyl phosphates still exhibited
antitumor activity, the IC50 values of 30 and 31 were equivalent
to 17 and 21. Moreover, the IC50 value of 33 advanced to
6.6 μM after phosphorylation of compound 14, comparable to
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( )-6-O-Benzyl-D-myo-inositol-1,3,5-O-orthoformate (2). Sodium
hydride (60.5% dispersion; 1.59 g, 40 mmol) was added in
batches to a stirred solution of myo-inositol orthoformate 1
(7.6 g, 40 mmol) and imidazole (100 mg) in anhydrous dimethyl
formamide (150 mL) under N2. The solution was stirred for
30 min, and then benzyl bromide (4.72 mL, 40 mmol) in anhy-
drous dimethyl formamide (10 mL) was added dropwise. After
stirring overnight, the reaction was quenched with water and the
solvent evaporated in vacuo. The residue dissolved in dichloro-
methane and water. The layers were separated, and the aqueous
layer was extracted with dichloromethane. The combined
organic layers were washed with brine, dried (sodium sulfate),
filtered, and concentrated under reduced pressure. Purification by
silica gel column chromatography, eluting with ethyl acetate and
petroleum ether (1 : 3), yielded 2 as a white solid (7.58 g, 68%):
antitumor mechanism underlying cellular response to these
agents exists. Our further work will focus on researching the
mechanism. This will be useful for deep understanding of the
signal transduction mechanism regulated by inositol phosphates
and anticancer drugs design based on inositol phosphates.
Conclusion
In this paper, we report the synthesis and biological activity of
several novel inositol phosphotriester analogues based on phos-
phatidylinositol. Seven compounds exhibited good cytotoxic
activity against human cancer cell lines A549, HepG2,
MDA-MB-231 and HeLa, especially compound 33 was cyto-
toxic against all the four cancer cell lines with good IC50 values.
Moreover, the IC50 values of 33 against A549 and
MDA-MB-231 were comparable to cisplatin. Further target
study of these analogues is being actively pursued to elucidate
the molecular basis of the drug–target interaction. To the best of
our knowledge, this work represents the first attempt to examine
the effects of inositol phosphotriester analogues on cancer cell
growth inhibition properties.
1
Rf 0.30 (ethyl acetate–petroleum ether 1 : 2); mp: 92–94 °C; H
NMR (400 MHz, CDCl3): δH = 3.18 (d, J = 12.0 Hz, 1 H), 3.75
(d, J = 10.0 Hz, 1 H), 4.09 (d, J = 11.6 Hz, 1 H), 4.21 (m, 1 H),
4.25 (m, 1 H), 4.29 (m, 1 H), 4.43–4.48 (m, 2 H), 4.67 (dd, J =
11.6, 16.8 Hz, 2 H), 5.44 (s, 1 H), 7.30–7.41 (m, 5 H); 13C
NMR (75.5 MHz, CDCl3): δC = 60.5, 67.2, 67.8, 72.1, 73.0,
74.1, 74.6, 102.6, 128.0, 128.7, 128.8, 135.8; HRMS m/z [M +
Na]+ calcd for C14H16O6Na 303.0839, found 303.0837.
Experimental section
meso-2,4,6-Tri-O-benzyl-D-myo-inositol-1,3,5-O-orthoformate (3).
Sodium hydride (60.5% dispersion; 2.54 g, 64 mmol) was added
in batches to a stirred solution of myo-inositol orthoformate 1
(3.45 g, 16 mmol) in anhydrous dimethyl formamide (70 mL)
under N2. The solution was stirred for 30 min, and then benzyl
bromide (8.5 mL, 72 mmol) in anhydrous dimethyl formamide
(10 mL) was added. After stirring overnight, the reaction was
quenched with water and the solvent evaporated in vacuo. The
residue dissolved in dichloromethane and water. The layers were
separated, and the aqueous layer was extracted with dichloro-
methane. The combined organic layers were washed with brine,
dried (sodium sulfate), filtered, and concentrated under reduced
pressure. Purification by silica gel column chromatography,
eluting with ethyl acetate and petroleum ether (1 : 8), yielded 3
as a white solid (5.4 g, 74%): Rf 0.50 (ethyl acetate–petroleum
ether 1 : 5); mp: 109–111 °C; 1H NMR (300 MHz, CDCl3): δH =
4.06 (m, 1 H), 4.30–4.32 (m, 2 H), 4.35 (t, J = 4.8 Hz, 2 H),
4.45 (m, 1 H), 4.55 (dd, J = 15.6, 72.0 Hz, 4 H), 4.65 (s, 2 H),
5.55 (s, 1 H), 7.21–7.40 (m, 15 H); 13C NMR (75.5 MHz,
CDCl3): δC = 67.1, 68.0, 70.4, 71.3, 71.4, 74.0, 103.1, 127.4,
127.6, 127.9, 128.2, 128.3, 137.5, 137.7; HRMS m/z [M + Na]+
calcd for C28H28O6Na 483.1778, found 483.1770.
Synthesis
General experimental procedures. Unless stated otherwise,
commercial reagents and solvents were used without further
purification. Reactions were monitored by thin-layer chromato-
graphy carried out on silica gel plates (GF254) using UV light as
the visualizing agent and phosphomolybdic acid and heat as
developing agents. Column chromatography was performed on
silica gel. All yields refer to isolated products. Melting points
(mp) were determined on a TaiKe X-4 melting point apparatus
and were uncorrected. 1H NMR, 13C NMR and 13P NMR
spectra were recorded on either 300 MHz or 400 MHz spec-
1
trometer. H NMR chemical shifts were reported in ppm relative
to tetramethylsilane (TMS, δ 0.00 ppm) or residual CHCl3 (δ
7.26 ppm) in CDCl3, 13C NMR chemical shifts were reported
using the central line of CDCl3 (δ 77.0 ppm) as the internal stan-
dard. In other solvents, the solvent itself was used as the refer-
ence. High resolution mass spectral analyses (HRMS) were
measured using ESI or MALDI ionization.
meso-D-myo-Inositol-1,3,5-O-orthoformate (1). A solution of
myo-inositol (18.0 g, 100 mmol) and triethyl orthoformate
(30 mL) in anhydrous dimethyl formamide (180 mL) in the pres-
ence of p-toluenesulfonic acid monohydrate (5.2 g) was heated
for 28 h at 110 °C under N2. After cooling, saturated aqueous
NaHCO3 solution (25 mL) was added, and stirring was contin-
ued for 30 min. The color of the solution changed from dark
brown to purple. The reaction mixture was evaporated to dryness
in vacuo. The resulting syrup was recrystallized from hot metha-
nol to yield 1 as a white solid (15.83 g, 83%): Rf 0.70 (dichloro-
methane–methanol 6 : 1); mp: 285 °C (dec); 1H NMR
(300 MHz, D2O): δH = 4.27–4.31 (m, 3 H), 4.36 (m, 1 H),
4.62–4.64 (m, 2 H), 5.65 (s, 1 H); 13C NMR (100.6 MHz, D2O):
δC = 59.6, 66.7, 69.3, 73.8, 102.1; HRMS m/z [M − H]− calcd
for C7H9O6 189.0405, found 189.0407.
meso-4,6-Di-O-benzyl-D-myo-inositol-1,3,5-O-orthoformate (4).
Sodium hydride (60.5% dispersion; 1.63 g, 40 mmol) was added
in batches to a stirred solution of myo-inositol orthoformate 1
(3.8 g, 20 mmol) and imidazole (40 mg) in anhydrous dimethyl
formamide (90 mL) under N2. The solution was stirred for
30 min, and then benzyl bromide (5.8 mL, 48 mmol) in anhy-
drous dimethyl formamide (10 mL) was added dropwise. After
stirring overnight, the reaction was quenched with water and the
solvent evaporated in vacuo. The residue dissolved in dichloro-
methane and water. The layers were separated, and the aqueous
layer was extracted with dichloromethane. The combined
organic layers were washed with brine, dried (sodium sulfate),
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filtered, and concentrated under reduced pressure. Purification by
silica gel column chromatography, eluting with ethyl acetate and
petroleum ether (1 : 3), yielded 4 as a colorless solid (3.56 g,
48%): Rf 0.20 (ethyl acetate–petroleum ether 1 : 3); mp:
under N2 at 0 °C. The mixture was warmed to room temperature,
stirred for 4 h, then poured into a solution of sodium potassium
tartrate (108 g) in water (180 mL) and saturated aqueous
ammonium chloride (144 mL), added ethyl acetate (430 mL),
and stirred for 1 h at room temperature. The product was
extracted into ethyl acetate, the combined organic layers were
dried (sodium sulfate), filtered, and concentrated under reduced
pressure. Purification by silica gel column chromatography,
eluting with ethyl acetate and petroleum ether (1 : 4), yielded 7
as a colorless oil (7.6 g, 92%): Rf 0.25 (ethyl acetate–petroleum
1 : 4); 1H NMR (300 MHz, CDCl3): δH = 4.08 (m, 1 H),
4.12–4.14 (m, 2 H), 4.41 (m, 1 H), 4.54–4.56 (m, 2 H),
4.63–4.77 (m, 7 H), 5.66 (d, J = 3.6 Hz, 1 H), 7.37–7.42 (m, 15
H); 13C NMR (75.5 MHz, CDCl3): δC = 69.5, 70.2, 70.8, 72.1,
72.7, 81.3, 85.7, 127.6, 127.8, 127.9, 128.0, 128.5, 128.6,
137.7, 138.0; HRMS m/z [M + Na]+ calcd for C28H30O6Na
485.1935, found 485.1936.
1
130–132 °C; H NMR (300 MHz, CDCl3): δH = 4.12–4.17 (m,
3 H), 4.29–4.31 (m, 2 H), 4.39 (m, 1 H), 4.55 (dd, J = 11.7, 13.5
Hz, 4 H), 5.40 (s, 1 H), 7.18–7.24 (m, 10 H);13C NMR
(75.5 MHz, CDCl3): δC = 61.4, 67.8, 71.7, 73.0, 73.8, 103.4,
127.7, 127.9, 128.5, 137.5; HRMS m/z [M + Na]+ calcd for
C21H22O6Na 393.1309, found 393.1303.
( )-2-O-tert-Butyldimethylsilyl-6-O-benzyl-myo-inositol-1,3,5-O-
orthoformate (5). Imidazole (4.62 g, 32.4 mmol) was added to a
stirred solution of 2 (7.6 g, 27 mmol) in anhydrous dimethyl for-
mamide (150 mL) under N2. Then tert-butyldimethylsilyl chlor-
ide (4.9 mL, 67.5 mmol) in anhydrous dimethyl formamide
(20 mL) was added dropwise under ice-water bath. After stirring
overnight at room temperature, the reaction was quenched with
water and the solvent evaporated in vacuo. The residue dissolved
in dichloromethane and water. The layers were separated, and
the aqueous layer was extracted with dichloromethane. The com-
bined organic layers were washed with brine, dried (sodium
sulfate), filtered, and concentrated under reduced pressure. Purifi-
cation by silica gel column chromatography, eluting with ethyl
acetate and petroleum ether (1 : 5), yielded 5 as a colorless oil
Didecyl N,N-diisopropylphosphoramidite (8). Dry diisopropy-
lamine (57 mL, 405.5 mmol) was added dropwise to a solution
of phosphorus trichloride (17 mL, 194.8 mmol) in dry THF
(200 mL) within one hour whilst stirring under N2 in ice-salt
bath. The resulting mixture was stirred at room temperature for
three hours more, then filtered. The precipitate was washed with
dry THF. After evaporation of THF, the yellow oil was distilled
under reduced pressure to give N,N-diisopropylphosphoramide
dichloridite (28.3 g, 71%): 31P NMR (162 MHz, CDCl3): δP =
1
(9.58 g, 90%): Rf 0.60 (ethyl acetate–petroleum ether 1 : 3); H
NMR (300 MHz, CDCl3): δH = 0.15 (s, 6 H), 0.95 (s, 9 H), 3.65
(d, J = 10.2 Hz, 1 H), 4.13–4.15 (m, 2 H), 4.26–4.27 (m, 2 H),
4.40–4.45 (m, 2 H), 4.66 (s, 2 H), 5.49 (s, 1 H), 7.26–7.40 (m, 5
H); 13C NMR (100.6 MHz, CDCl3): δC = −4.8, −4.7, 25.8,
60.8, 67.4, 68.2, 72.4, 73.0, 74.7, 74.8, 102.4, 127.9, 128.6,
128.8, 135.9; HRMS m/z [M + Na]+ calcd for C20H30O6SiNa
417.1704, found 417.1705.
1
169.6; H NMR (400 MHz, CDCl3): δH = 1.21 (d, J = 6.8 Hz,
12 H), 3.84–3.89 (m, 2 H).
Dry decanol (39 mL, 204.2 mmol) was added dropwise to a
solution of N,N-diisopropylphosphoramide dichloridite (20.9 g,
101.4 mmol) and dry triethylamine (30 mL, 207.4 mmol) in dry
THF (100 mL), within one hour whilst stirring under N2 in ice-
water bath. The resulting white slurry was stirred at room temp-
erature for three hours more, and then filtered. The precipitate
was washed with dry THF. After evaporation of THF, 8 was
obtained as a colorless oil (36.9 g, 99%): 31P NMR (162 MHz,
( )-2,4,6-Tri-O-benzyl-3,5-O-ethylidene-D-myo-inositol (6). AlMe3
(15 mL) in dry CH2Cl2 (10 mL) dropwise was added to a stirred
solution of 3 (5.4g, 11.8 mmol) in dry CH2Cl2 (60 mL) under
N2 at 0 °C. The mixture was warmed to room temperature,
stirred for 5 h, then poured into a solution of sodium potassium
tartrate (150 g) in water (300 mL) and saturated aqueous
ammonium chloride (300 mL), and stirred for 1 h at room temp-
erature. The product was extracted into CH2Cl2, the combined
organic layers were dried (sodium sulfate), filtered, and concen-
trated under reduced pressure. Purification by silica gel column
chromatography, eluting with ethyl acetate and petroleum ether
(1 : 9), yielded 6 as a colorless oil (3.8 g, 68%): Rf 0.20 (ethyl
1
CDCl3): δP = 145.0; H NMR (400 MHz, CDCl3): δH = 0.86 (t,
J = 7.2 Hz, 6 H), 1.15 (d, J = 6.8 Hz, 12 H), 1.24 (s, 28 H),
1.55–1.62 (m, 4 H), 3.51–3.64 (m, 6 H); 13C NMR (100.6 MHz,
CDCl3): δC = 14.1, 22.6, 24.5, 24.6, 25.9, 29.31, 29.35, 29.5,
29.6, 31.26, 31.33, 31.9, 42.6, 42.7, 63.3, 63.5; HRMS m/z
[M + H]+ calcd for C26H57NO2P 446.4121, found 446.4125.
( )-2,4,6-Tri-O-benzyl-3,5-O-ethylidene-D-myo-inositol-1-(didecyl
phosphate) (9). Didecyl N,N-diisopropylphosphoramidite (1.16 g,
2.6 mmol) was stirred with 1H-tetrazole (182 mg, 2.6 mmol) in
dry CH2Cl2 (25 mL) under N2 for 30 min at room temperature.
Compound 6 (612 mg, 1.3 mmol) dissolved in dry CH2Cl2
(25 mL) was added and the resulting mixture was stirred over-
night. The mixture was cooled to 0 °C, and 3-chloroperoxyben-
zoic acid (650 mg, 2.6 mmol) was added. The resulting mixture
was allowed to warm to room temperature and stirred for 2 h.
The 3-chloroperoxybenzoic acid was quenched with a 10%
aqueous solution of sodium hydrogen sulfite. The layers were
separated, and the aqueous layer was extracted with dichloro-
methane. The combined organic layers were washed with a satu-
rated aqueous solution of sodium hydrogen carbonate and brine,
dried (sodium sulfate), filtered, and concentrated under reduced
acetate–petroleum 1 : 8); 1H NMR (300 MHz, CDCl3): δH
=
1.23 (d, J = 4.8 Hz, 3 H), 3.21 (d, J = 6.6 Hz, 1 H), 3.81 (d, J =
6.2 Hz, 1 H), 3.97 (t, J = 3.7 Hz, 1 H), 4.15 (m, 1 H), 4.33 (m, 1
H), 4.39–4.44 (m, 3 H), 4.50 (d, J = 11.0 Hz, 1 H), 4.69 (d, J =
11.0 Hz, 1 H), 4.74–4.82 (m, 3 H), 5.26 (q, J = 4.9 Hz, 1 H),
7.25–7.39 (m, 15 H); 13C NMR (75.5 MHz, CDCl3): δC = 20.9,
68.3, 68.8, 71.1, 71.8, 71.9, 72.5, 72.7, 73.0, 82.3, 90.7, 127.4,
127.5, 127.8, 128.1, 128.2, 128.3, 128.4, 128.5, 137.2, 137.6,
138.4; HRMS m/z [M + Na]+ calcd for C29H32O6Na 499.2091,
found 499.2086.
( )-2,4,6-Tri-O-benzyl-1,3-O-methylidene-D-myo-inositol
(7).
DIBAL (36 mL) in dry CH2Cl2 (15 mL) was added dropwise to
a stirred solution of 3 (8.2 g, 17.8 mmol) in dry CH2Cl2 (75 mL)
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 3642–3654 | 3647

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pressure. Purification by silica gel column chromatography,
eluting with ethyl acetate and petroleum ether (1 : 10, then 1 : 8),
yielded 9 as a colorless oil (780 mg, 72%): Rf 0.30 (ethyl
(sodium sulfate), filtered, and concentrated under reduced
pressure. Purification by silica gel column chromatography,
eluting with ethyl acetate and petroleum ether (1 : 6, then 1 : 4),
yielded 12 as a colorless oil (120 mg, 90%): Rf 0.30 (ethyl
acetate–petroleum ether 1 : 4); 31P NMR (162 MHz, CDCl3): δP
= −1.7; 1H NMR (400 MHz, CDCl3): δH = 0.86–1.02 (m,
21 H), 1.25–1.32 (m, 28 H), 1.53–1.63 (m, 10 H), 1.66–1.74 (m,
4 H), 2.16–2.22 (m, 6 H), 2.26–2.35 (m, 2 H), 2.43 (t, J = 7.2
Hz, 2 H), 3.91–3.99 (m, 4 H), 4.58 (ddd, J = 3.2, 10.0, 12.4 Hz,
1 H), 5.02 (dd, J = 2.4 Hz, 10.4 Hz, 1 H), 5.17 (t, J = 9.6 Hz,
1 H), 5.50 (dd, J = 9.6, 18.8 Hz, 2 H), 5.73 (t, J = 2.8 Hz, 1 H);
13C NMR (100.6 MHz, CDCl3): δC = 13.4, 13.50, 13.55, 14.0,
17.9, 18.0, 18.1, 18.2, 18.5, 22.6, 25.2, 25.3, 29.0, 29.2, 29.4,
30.0, 30.1, 31.8, 35.6, 35.76, 35.79, 35.9, 68.1, 68.2, 68.4, 68.5,
68.6, 68.9, 69.80, 69.85, 70.2, 72.78, 72.83, 171.9, 172.0,
172.1, 172.3; HRMS m/z [M + Na]+ calcd for C46H83O14PNa
913.5413, found 913.5410.
acetate–petroleum 1 : 8); 31P NMR (162 MHz, CDCl3): δP
=
1
−1.4; H NMR (400 MHz, CDCl3): δH = 0.86–0.90 (m, 6 H),
1.19–1.23 (m, 31 H), 1.49–1.53 (m, 4 H), 3.88–4.00 (m, 5 H),
4.03 (m, 1 H), 4.30 (m, 1 H), 4.38–4.41 (m, 2 H), 4.48 (m, 1 H),
4.58 (m, 1 H), 4.65–4.69 (m, 2 H), 4.72–4.77 (m, 2 H), 5.17 (m,
1 H), 5.32 (m, 1 H), 7.28–7.37 (m, 15 H); 13C NMR
(100.6 MHz, CDCl3): δC = 14.0, 20.6, 22.5, 25.18, 25.22, 29.0,
29.2, 29.4, 30.0, 30.08, 30.14, 31.7, 67.57, 67.63, 67.7, 68.5,
71.3, 71.7, 72.9, 73.0, 74.7, 74.8, 79.47, 79.55, 90.8, 127.36,
127.44, 127.7, 127.8, 127.9, 128.0, 128.1, 128.3, 137.3, 137.9,
138.0; HRMS m/z [M + Na]+ calcd for C49H73O9PNa 859.4885,
found 859.4889.
( )-2,4,6-Tri-O-benzyl-D-myo-inositol-1-(didecyl phosphate) (10).
Concentrated hydrochloric acid (0.3 mL) was added to a stirred
solution of 9 (360 mg, 0.43 mmol) in methanol (25 mL). The
reaction mixture was stirred at room temperature for 2 h, concen-
trated under reduced pressure. Purification by silica gel column
chromatography, eluting with ethyl acetate and petroleum ether
(1 : 6, then 1 : 3), yielded 10 as a colorless oil (350 mg, 99%): Rf
0.20 (ethyl acetate–petroleum ether 1 : 3); 31P NMR (162 MHz,
CDCl3): δP = −1.3; 1H NMR (400 MHz, CDCl3): δH = 0.88 (t, J
= 6.8 Hz, 6 H), 1.23–1.25 (m, 28 H), 1.56–1.64 (m, 4 H),
3.52–3.58 (m, 2 H), 3.68 (m, 1 H), 3.89 (m, 1 H), 3.94–4.06 (m,
4 H), 4.26–4.31 (m, 2 H), 4.73–4.83 (m, 3 H), 4.87–4.92 (m, 3
H), 7.28–7.39 (m, 15 H); 13C NMR (100.6 MHz, CDCl3): δC =
14.0, 22.5, 25.2, 25.3, 29.0, 29.1, 29.4, 29.5, 30.1, 31.7, 67.9,
68.0, 71.6, 74.6, 74.7, 75.1, 75.3, 78.18, 79.22, 80.0, 81.2,
127.3, 127.4, 127.5, 127.6, 127.7, 127.9, 128.16, 128.19, 128.3,
138.4, 138.5, 138.6; HRMS m/z [M + Na]+ calcd for
C47H71O9PNa 833.4728, found 833.4732.
meso-4,6-Di-O-benzyl-1,3,5-O-orthoformate-D-myo-inositol-2-
(didecyl phosphate) (13). Didecyl N,N-diisopropylphosphorami-
dite (900 mg, 2 mmol) was stirred with 1H-tetrazole (140 mg,
2 mmol) in dry CH2Cl2 (40 mL) under N2 for 30 min at room
temperature. Compound 4 (370 mg, 1 mmol) was added and the
resulting mixture stirred overnight. The mixture was cooled to
0 °C, and 3-chloroperoxybenzoic acid (495 mg, 2 mmol) was
added. The resulting mixture was allowed to warm to room
temperature and stirred for 2 h. The 3-chloroperoxybenzoic acid
was quenched with a 10% aqueous solution of sodium hydrogen
sulfite. The layers were separated, and the aqueous layer was
extracted with dichloromethane. The combined organic layers
were washed with a saturated aqueous solution of sodium hydro-
gen carbonate and brine, dried (sodium sulfate), filtered, and
concentrated under reduced pressure. Purification by silica gel
column chromatography, eluting with ethyl acetate and pet-
roleum ether (1 : 10, then 1 : 6), yielded 13 as a colorless oil
(685 mg, 93%): Rf 0.20 (ethyl acetate–petroleum 1 : 6); 31P
NMR (162 MHz, CDCl3): δP = −1.3; 1H NMR (400 MHz,
CDCl3): δH = 0.88 (t, J = 7.0 Hz, 6 H), 1.25–1.33 (m, 28 H),
1.64–1.68 (m, 4 H), 4.05–4.09 (m, 4 H), 4.36–4.37 (m, 2 H),
4.44–4.46 (m, 3 H), 4.58–4.65 (m, 4 H), 4.95 (d, J = 7.2 Hz,
1 H), 5.50 (s, 1 H), 7.26–7.27 (m, 10 H); 13C NMR
(100.6 MHz, CDCl3): δC = 13.8, 22.4, 25.1, 28.8, 29.0, 29.2,
29.3, 29.9, 30.0, 31.6, 66.9, 67.0, 67.6, 67.8, 67.9, 70.7, 70.8,
71.2, 73.5, 102.9, 127.46, 127.55, 128.1, 137.1; HRMS m/z
[M + Na]+ calcd for C41H63O9PNa 753.4102, found 753.4098.
( )-D-myo-Inositol-1-(didecyl phosphate) (11). 5% palladium
black (300 mg) was added to a stirred solution of 10 (290 mg,
0.36 mmol) in ethanol (15 mL), and the flask was flushed three
times with a balloon of hydrogen. After the reaction mixture was
stirred overnight at room temperature, the catalyst was removed
by filtering through Celite. The combined organic layers were
concentrated under reduced pressure, yielded 11 as a white solid
(180 mg, 93%): mp: 187–189 °C; 31P NMR (162 MHz,
1
CD3OD): δP = −1.7; H NMR (400 MHz, CD3OD): δH = 0.89
(t, J = 6.4 Hz, 6 H), 1.29 (m, 28 H), 1.67–1.69 (m, 4 H), 3.17
(t, J = 9.6 Hz, 1 H), 3.35 (d, J = 9.6 Hz, 1 H), 3.64 (t, J =
9.6 Hz, 1 H), 3.78 (t, J = 9.6 Hz, 1 H), 4.05–4.14 (m, 6 H); 13C
NMR (100.6 MHz, CD3OD): δC = 14.5, 23.8, 26.6, 30.3, 30.5,
30.7, 31.3, 31.4, 33.1, 69.3, 69.4, 69.5, 69.6, 72.5, 72.6, 72.8,
72.9, 73.9, 76.2, 80.6, 80.7; HRMS m/z [M + Na]+ calcd for
C26H53O9PNa 563.3319, found 563.3325.
meso-4,6-Di-O-benzyl-D-myo-inositol-2-(didecyl phosphate) (14).
Concentrated hydrochloric acid (0.12 mL) was added to a stirred
solution of 13 (110 mg, 0.15 mmol) in methanol (12 mL). The
reaction mixture was stirred at room temperature for 1 h, and
then heated to 35 °C for 6 h, concentrated under reduced
pressure. Purification by silica gel column chromatography,
eluting with ethyl acetate and petroleum ether (1 : 2, then 1 : 1),
yielded 14 as a colorless oil (75 mg, 75%): Rf 0.20 (ethyl
acetate–petroleum ether 1 : 1); 31P NMR (162 MHz, CDCl3): δP
( )-2,3,4,5,6-Penta-O-butyryl-D-myo-inositol-1-(didecyl phos-
phate) (12). Compound 11 (83 mg, 0.15 mmol) was dissolved in
dry pyridine (6 mL), to the stirred solution was added DMAP
(18 mg, 0.15 mmol) and butyric anhydride (0.26 mL,
1.5 mmol). After stirring overnight at room temperature, the
reaction was quenched with water. The aqueous layer was
extracted with dichloromethane. The combined organic layers
were washed with dilute hydrochloric acid and brine, dried
1
= 0.9; H NMR(400 MHz, CDCl3): δH = 0.88 (t, J = 6.0 Hz,
6 H), 1.26–1.37 (m, 28 H), 1.65–1.72 (m, 4 H), 2.62 (m, 1 H),
3.28–3.35 (m, 2 H), 3.56–3.62 (m, 4 H), 3.91 (s, 1 H),
4.05–4.13 (m, 4 H), 4.76 (d, J = 7.2 Hz, 1 H), 4.88 (dd, J =
3648 | Org. Biomol. Chem., 2012, 10, 3642–3654
This journal is © The Royal Society of Chemistry 2012

View Article Online
11.2, 14.8 Hz, 4 H), 7.29–7.39 (m, 10 H); 13C NMR
(100.6 MHz, CDCl3): δC = 14.1, 22.7, 25.5, 29.2, 29.3, 29.6,
30.2, 30.3, 31.9, 68.66, 68.72, 70.92, 70.95, 74.7, 75.0, 80.0,
80.11, 81.14, 127.9, 128.1, 128.5, 138.6; HRMS m/z [M + Na]+
calcd for C40H65O9PNa 743.4258, found 743.4263.
( )-2-O-tert-Butyldimethylsilyl-6-O-benzyl-D-myo-inositol-1,3,5-
O-orthoformate-4-(didecyl phosphate) (18). Didecyl N,N-diiso-
propylphosphoramidite (900 mg, 2 mmol) was stirred with 1H-
tetrazole (140 mg, 2 mmol) in dry CH2Cl2 (40 mL) under N2 for
30 min at room temperature. Compound 5 (403 mg, 1 mmol)
was added and the resulting mixture stirred overnight. The
mixture was cooled to 0 °C, and 3-chloroperoxybenzoic acid
(504 mg, 2 mmol) was added. The resulting mixture was
allowed to warm to room temperature and stirred for 2 h. The 3-
chloroperoxybenzoic acid was quenched with a 10% aqueous
solution of sodium hydrogen sulfite. The layers were separated,
and the aqueous layer was extracted with dichloromethane. The
combined organic layers were washed with a saturated aqueous
solution of sodium hydrogen carbonate and brine, dried (sodium
sulfate), filtered, and concentrated under reduced pressure. Purifi-
cation by silica gel column chromatography, eluting with ethyl
acetate and petroleum ether (1 : 15, then 1 : 10), yielded 18 as a
colorless oil (450 mg, 60%): Rf 0.20 (ethyl acetate–petroleum
1 : 10); 31P NMR (162 MHz, CDCl3): δP = −1.7; 1H NMR
(400 MHz, CDCl3): δH = 0.15 (s, 6 H), 0.88 (t, J = 6.8 Hz, 6 H),
0.94 (s, 9 H), 1.24–1.26 (m, 28 H), 1.52–1.63 (m, 4 H),
3.87–3.99 (m, 4 H), 4.15 (m, 1 H), 4.21 (m, 1 H), 4.30–4.33 (m,
2 H), 4.54–4.72 (m, 3 H), 5.07 (m, 1 H), 5.52 (s, 1 H),
meso-D-myo-Inositol-2-(didecyl phosphate) (15). 5% palladium
black (200 mg) was added to a stirred solution of 14 (75 mg,
0.1 mmol) in ethanol (10 mL), and the flask was flushed three
times with a balloon of hydrogen. After the reaction mixture was
stirred overnight at room temperature, the catalyst was removed
by filtering through Celite. The combined organic layers were
concentrated under reduced pressure, yielded 15 as a white solid
(42 mg, 80%): mp: 188–190 °C; 31P NMR (162 MHz, CD3OD):
1
δP = −1.5; H NMR (400 MHz, CD3OD): δH = 0.91 (t, J = 6.4
Hz, 6 H), 1.32–1.42 (m, 28 H), 1.67–1.71 (m, 4 H), 3.22 (t, J =
8.8 Hz, 1 H), 3.50–3.60 (m, 4 H), 4.14–4.19 (m, 4 H), 4.71 (d,
J = 8.4 Hz, 1 H); 13C NMR (100.6 MHz, CD3OD): δC = 14.5,
23.8, 26.7, 30.4, 30.5, 30.76, 30.79, 31.37, 31.44, 33.1, 69.47,
69.54, 71.80, 71.83, 74.3, 76.4, 82.8, 82.9; HRMS m/z [M +
Na]+ calcd for C26H53O9PNa 563.3319, found 563.3310.
( )-4-O-Benzyl-1,3,5-O-orthoformate-D-myo-inositol-2-(didecyl
phosphate) (16). 5% palladium black (150 mg) was added to a
stirred solution of 13 (100 mg, 0.13 mmol) in ethanol (15 mL),
and the flask was flushed three times with a balloon of hydrogen.
After the reaction mixture was stirred overnight at room tempera-
ture, the catalyst was removed by filtering through Celite. The
combined organic layers were concentrated under reduced
pressure. Purification by silica gel column chromatography,
eluting with ethyl acetate and petroleum ether (1 : 4), yielded 16
as a colorless oil (20 mg, 24%): Rf 0.20 (ethyl acetate–petroleum
1 : 4); 31P NMR (162 MHz, CDCl3): δP = −1.2; 1H NMR
(400 MHz, CDCl3): δH = 0.87 (t, J = 6.8 Hz, 6 H), 1.25–1.38
(m, 28 H), 1.68–1.71 (m, 4 H), 4.06–4.12 (m, 4 H), 4.26 (m, 1
H), 4.36 (m, 1 H), 4.41 (m, 1 H), 4.47 (m, 1 H), 4.53 (m, 1 H),
4.68 (dd, J = 11.6, 52.4 Hz, 2 H), 4.84 (m, 1 H), 5.47 (s, 1 H),
7.32–7.40 (m, 5 H); 13C NMR (100.6 MHz, CDCl3): δC = 14.1,
22.6, 25.4, 29.1, 29.3, 29.5, 29.7, 30.15, 30.17, 30.21, 30.24,
31.8, 66.37, 66.41, 67.5, 68.1, 68.27, 68.33, 70.27, 70.30, 72.8,
72.97, 73.02, 73.9, 102.5, 128.3, 128.8, 128.9, 135.6; HRMS m/
z [M + Na]+ calcd for C34H57O9PNa 663.3632, found 663.3630.
7.28–7.35 (m, 5 H); 13C NMR (100.6 MHz, CDCl3): δC
=
−4.92, −4.88, 13.9, 18.2, 22.5, 25.17, 25.21, 25.7, 28.9, 29.1,
29.3, 29.9, 30.0, 30.1, 31.7, 60.8, 67.60, 67.64, 68.0, 68.09,
68.14, 70.87, 70.92, 71.4, 72.8, 73.7, 102.7, 127.1, 127.6,
128.2, 137.3; HRMS m/z [M + Na]+ calcd for C40H71O9PSiNa
777.4497, found 777.4497.
( )-6-O-Benzyl-D-myo-inositol-4-(didecyl phosphate) (19). Con-
centrated hydrochloric acid (0.16 mL) was added to a stirred sol-
ution of 18 (200 mg, 0.26 mmol) in methanol (15 mL). The
reaction mixture was stirred at room temperature for 1 h, then
heated to 40 °C for 6 h, concentrated under reduced pressure.
Purification by silica gel column chromatography, eluting with
ethyl acetate and petroleum ether (1 : 1, then 2 : 1), yielded 19 as
a colorless oil (160 mg, 95%): Rf 0.30 (ethyl acetate–petroleum
1
ether 2 : 1); 31P NMR (162 MHz, CDCl3): δP = 1.0; H NMR
(400 MHz, CDCl3): δH = 0.88 (t, J = 7.2 Hz, 6 H), 1.26–1.36
(m, 28 H), 1.65–1.72 (m, 4 H), 2.69 (m, 1 H), 3.25 (m, 1 H),
3.56–3.63 (m, 3 H), 3.72 (m, 1 H), 3.91 (s, 2 H), 4.09–4.17 (m,
4 H), 4.22 (m, 1 H), 4.44 (m, 1 H), 4.90 (dd, J = 11.2, 30.8 Hz,
2 H), 7.30–7.40 (m, 5 H); 13C NMR (100.6 MHz, CDCl3): δC =
14.1, 22.7, 25.4, 29.2, 29.3, 29.5, 30.1, 30.2, 31.9, 52.6, 68.6,
68.66, 68.71, 71.2, 71.8, 72.3, 73.62, 73.65, 75.1, 81.4, 81.66,
81.72, 127.8, 128.1, 128.5, 128.9, 131.1, 138.7; HRMS m/z
[M + Na]+ calcd for C33H59O9PNa 653.3789, found 653.3796.
meso-1,3,5-O-Orthoformate-D-myo-inositol-2-(didecyl
phos-
phate) (17). 5% palladium black (350 mg) was added to a stirred
solution of 13 (157 mg, 0.2 mmol) in ethanol (15 mL), and the
flask was flushed three times with a balloon of hydrogen. After
the reaction mixture was stirred overnight at room temperature,
the catalyst was removed by filtering through Celite. The com-
bined organic layers were concentrated under reduced pressure,
yielded 17 as a colorless oil (110 mg, 99%): Rf 0.20 (ethyl
( )-D-myo-Inositol-4-(didecyl phosphate) (20). 5% palladium
black (200 mg) was added to a stirred solution of 19 (100 mg,
0.16 mmol) in ethanol (15 mL), and the flask was flushed three
times with a balloon of hydrogen. After the reaction mixture was
stirred overnight at room temperature, the catalyst was removed
by filtering through Celite. The combined organic layers were
concentrated under reduced pressure, yielded 20 as a white solid
(60 mg, 82%): mp: 193–195 °C; 31P NMR (162 MHz, CD3OD):
acetate–petroleum 1 : 2); 31P NMR (162 MHz, CDCl3): δP
=
1
−1.8; H NMR (400 MHz, CDCl3): δH = 0.88 (t, J = 7.2 Hz,
6 H), 1.26–1.39 (m, 28 H), 1.66–1.73 (m, 4 H), 4.06–4.11 (m,
4 H), 4.30 (m, 1 H), 4.36–4.40 (m, 2 H), 4.56–4.58 (m, 2 H),
4.89 (m, 1 H), 5.47 (s, 1 H); 13C NMR (100.6 MHz, CDCl3): δC
= 14.1, 22.7, 25.4, 29.1, 29.3, 29.48, 29.52, 30.07, 30.14, 31.9,
67.2, 67.3, 67.9, 68.2, 68.7, 68.8, 72.8, 72.9, 102.4; HRMS m/z
[M + Na]+ calcd for C27H51O9PNa 573.3163, found 573.3160.
1
δP = −0.6; H NMR (400 MHz, CD3OD): δH = 0.85–0.91 (m,
6 H), 1.28–1.38 (m, 28 H), 1.60–1.72 (m, 4 H), 3.33–3.36 (m,
This journal is © The Royal Society of Chemistry 2012
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2 H), 3.52 (m, 1 H), 3.62 (m, 1 H), 3.94 (m, 1 H), 4.10–4.12 (m,
4 H), 4.37 (m, 1 H); 13C NMR (100.6 MHz, CD3OD): δC
14.5, 23.8, 26.7, 30.4, 30.5, 30.76, 30.78, 31.3, 31.4, 33.1,
69.36, 69.39, 69.42, 69.5, 72.28, 72.31, 73.1, 74.3, 75.0, 75.1,
83.3, 83.4; HRMS m/z [M + Na]+ calcd for C26H53O9PNa
563.3319, found 563.3323.
CDCl3): δC = 14.0, 22.6, 25.3, 29.0, 29.2, 29.4, 29.6, 30.08,
30.15, 30.2, 31.8, 60.1, 67.1, 68.65, 68.68, 68.8, 68.88, 68.94,
71.5, 71.6, 72.36, 72.44, 74.0, 102.8; HRMS m/z [M + Na]+
calcd for C27H51O9PNa 573.3163, found 573.3165.
=
meso-2,4,6-Tri-O-benzyl-1,3-O-methylidene-D-myo-inositol-5-
(didecyl phosphate) (24). Didecyl N,N-diisopropylphosphorami-
dite (360 mg, 0.75 mmol) was stirred with 1H-tetrazole (62 mg,
0.75 mmol) in dry CH2Cl2 (20 mL) under N2 for 30 min at
room temperature. Compound 7 (120 mg, 0.25 mmol) dissolved
in dry CH2Cl2 (10 mL) was added and the resulting mixture
stirred overnight. The mixture was cooled to 0 °C, and 3-chloro-
peroxybenzoic acid (190 mg, 0.75 mmol) was added. The result-
ing mixture was allowed to warm to room temperature and
stirred for 2 h. The 3-chloroperoxybenzoic acid was quenched
with a 10% aqueous solution of sodium hydrogen sulfite. The
layers were separated, and the aqueous layer was extracted with
dichloromethane. The combined organic layers were washed
with a saturated aqueous solution of sodium hydrogen carbonate
and brine, dried (sodium sulfate), filtered, and concentrated
under reduced pressure. Purification by silica gel column chrom-
atography, eluting with ethyl acetate and petroleum ether (1 : 15,
1 : 10, then 1 : 8), yielded 24 as a colorless oil (175 mg, 86%): Rf
0.20 (ethyl acetate–petroleum 1 : 8); 31P NMR (162 MHz,
( )-6-O-Benzyl-D-myo-inositol-1,3,5-O-orthoformate-4-(didecyl
phosphate) (21). TBAF (0.56 mL, 0.56 mmol) was added to
compound 18 (280 mg, 0.37 mmol) in dry THF (20 mL), after
the mixture was stirred for 30 min at room temperature,
quenched with water. The aqueous layer was extracted with
dichloromethane. The combined organic layers were washed
with brine, dried (sodium sulfate), filtered, and concentrated
under reduced pressure. Purification by silica gel column chrom-
atography, eluting with ethyl acetate and petroleum ether (1 : 4),
yielded 21 as a colorless oil (232 mg, 98%): Rf 0.20 (ethyl
acetate–petroleum 1 : 4); 31P NMR (162 MHz, CDCl3): δP
=
1
−1.7; H NMR (400 MHz, CDCl3): δH = 0.88 (t, J = 6.8 Hz, 6
H), 1.24–1.26 (m, 28 H), 1.53–1.64 (m, 4 H), 3.90–4.01 (m, 4
H), 4.14 (m, 1 H), 4.23 (m, 1 H), 4.29 (m, 1 H), 4.36 (m, 1 H),
4.55–4.72 (m, 3 H), 5.10 (m, 1 H), 5.47 (s, 1 H), 7.31–7.34 (m,
5 H); 13C NMR (100.6 MHz, CDCl3): δC = 14.0, 22.6, 25.3,
29.0, 29.2, 29.4, 30.0, 30.08, 30.14, 31.8, 60.5, 67.48, 67.52,
68.2, 68.29, 68.34, 70.45, 70.5, 71.54, 72.59, 72.65, 73.2,
103.0, 127.4, 127.8, 128.3, 137.2; HRMS m/z [M + Na]+ calcd
for C34H57O9PNa 663.3632, found 663.3635.
1
CDCl3): δP = −1.7; H NMR (400 MHz, CDCl3): δH = 0.88 (t,
J = 6.8 Hz, 6 H), 1.24–1.29 (m, 28 H), 1.58–1.65 (m, 4 H),
3.93–4.02 (m, 4 H), 4.07–4.08 (m, 2 H), 4.17 (m, 1 H),
4.33–4.35 (m, 2 H), 4.60–4.74 (m, 8 H), 5.45 (d, J = 4.4 Hz, 1
H), 7.26–7.33 (m, 15 H); 13C NMR (100.6 MHz, CDCl3): δC =
14.1, 22.6, 25.4, 29.1, 29.3, 29.5, 30.1, 30.2, 31.8, 67.8, 67.9,
69.9, 70.6, 70.9, 71.8, 73.67, 73.74, 80.49, 80.53, 85.2, 127.5,
127.6, 127.8, 128.3, 128.4, 137.61, 137.65; HRMS m/z
[M + Na]+ calcd for C48H71O9PNa 845.4728, found 845.4732.
( )-2-O-tert-Butyldimethylsilyl-D-myo-inositol-1,3,5-O-orthofor-
mate-4-(didecyl phosphate) (22). 5% palladium black (300 mg)
was added to a stirred solution of 18 (125 mg, 0.15 mmol) in
ethanol (15 mL), and the flask was flushed three times with a
balloon of hydrogen. After the reaction mixture was stirred over-
night at room temperature, the catalyst was removed by filtering
through Celite. The combined organic layers were concentrated
under reduced pressure, yielded 22 as a colorless oil (100 mg,
91%): Rf 0.20 (ethyl acetate–petroleum 1 : 5); 31P NMR
(162 MHz, CDCl3): δP = −2.1; 1H NMR (400 MHz, CDCl3): δH
= 0.15 (s, 6 H), 0.88 (t, J = 6.8 Hz, 6 H), 0.95 (s, 9 H), 1.26 (m,
28 H), 1.68–1.69 (m, 4 H), 4.05–4.10 (m, 4 H), 4.14 (m, 1 H),
4.19 (m, 1 H), 4.28 (m, 1 H), 4.52 (m, 1 H), 4.57 (m, 1 H), 5.03
meso-2,4,6-Tri-O-benzyl-D-myo-inositol-5-(didecyl phosphate)
(25). Concentrated hydrochloric acid (0.5 mL) was added to a
stirred solution of 24 (110 mg, 0.12 mmol) in methanol
(15 mL). The reaction mixture was stirred at room temperature
for 1 h, refluxed for 6 h, concentrated under reduced pressure.
Purification by silica gel column chromatography, eluting with
ethyl acetate and petroleum ether (1 : 2, then 2 : 3), yielded 25 as
a colorless oil (82 mg, 80%): Rf 0.15 (ethyl acetate–petroleum
(m, 1 H), 5.52 (s, 1 H); 13C NMR(100.6 MHz, CDCl3): δC
=
1
−4.8, −4.7, 14.0, 18.3, 22.6, 25.3, 25.8, 29.0, 29.2, 29.4, 30.1,
30.17, 30.24, 31.8, 60.46, 67.53, 68.8, 68.9, 68.95, 68.98,
72.08, 72.13, 72.77, 72.85, 74.3, 102.6; HRMS m/z [M + Na]+
calcd for C33H65O9PSiNa 687.4028, found 687.4032.
ether 2 : 3); 31P NMR (162 MHz, CDCl3): δP = −1.2; H NMR
(400 MHz, CDCl3): δH = 0.88 (t, J = 6.8 Hz, 6 H), 1.18–1.29
(m, 28 H), 1.47–1.50 (m, 4 H), 2.50–2.52 (m, 2 H), 3.50–3.54
(m, 2 H), 3.76–3.80 (m, 2 H), 3.86–3.90 (m, 3 H), 3.95–3.99
(m, 2 H), 4.37 (m, 1 H), 4.79 (s, 2 H), 4.83 (dd, J = 11.2, 60.0
Hz, 4 H), 7.25–7.44 (m, 15 H); 13C NMR (100.6 MHz, CDCl3):
δC = 14.1, 22.7, 25.4, 29.2, 29.3, 29.5, 30.2, 30.3, 31.9, 67.9,
68.0, 71.9, 74.5, 75.2, 79.2, 80.2, 80.3, 80.49, 80.52, 127.7,
127.8, 127.9, 128.0, 128.4, 128.5, 138.5, 138.6; HRMS m/z
[M + Na]+ calcd for C47H71O9PNa 833.4728, found 833.4730.
( )-D-myo-Inositol-1,3,5-O-orthoformate-4-(didecyl phosphate)
(23). 5% palladium black (350 mg) was added to a stirred sol-
ution of 21 (130 mg, 0.2 mmol) in ethanol (15 mL), and the
flask was flushed three times with a balloon of hydrogen. After
the reaction mixture was stirred overnight at room temperature,
the catalyst was removed by filtering through Celite. The com-
bined organic layers were concentrated under reduced pressure,
yielded 23 as a colorless oil (100 mg, 91%): Rf 0.50 (ethyl
meso-D-myo-Inositol-5-(didecyl phosphate) (26). 5% palladium
black (270 mg) was added to a stirred solution of 25 (70 mg,
0.09 mmol) in ethanol (10 mL), and the flask was flushed three
times with a balloon of hydrogen. After the reaction mixture was
stirred overnight at room temperature, the catalyst was removed
by filtering through Celite. The combined organic layers were
concentrated under reduced pressure, yielded 26 as a white solid
acetate–petroleum 1 : 1); 31P NMR (162 MHz, CDCl3): δP
=
1
−2.1; H NMR (400 MHz, CDCl3): δH = 0.88 (t, J = 6.8 Hz, 6
H), 1.26 (m, 28 H), 1.66–1.72 (m, 4 H), 4.06–4.10 (m, 4 H),
4.14 (m, 1 H), 4.22 (m, 1 H), 4.28 (m, 1 H), 4.56 (m, 1 H), 4.60
(m, 1 H), 5.07 (m, 1 H), 5.47 (s, 1 H); 13C NMR (100.6 MHz,
3650 | Org. Biomol. Chem., 2012, 10, 3642–3654
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(30 mg, 73%): mp: 206–208 °C; 31P NMR (162 MHz, CD3OD):
δP = −1.3; H NMR (400 MHz, CD3OD): δH = 0.90 (t, J = 6.4
colorless oil (30 mg, 39%): Rf 0.20 (ethyl acetate–petroleum
1
1
1 : 4); 31P NMR (162 MHz, CDCl3): δP = −1.7, −1.6; H NMR
Hz, 6 H), 1.30–1.40 (m, 28 H), 1.65–1.70 (m, 4 H), 3.36–3.39
(m, 2 H), 3.73–3.78 (m, 2 H), 3.96–4.02 (m, 2 H), 4.11–4.16
(m, 4 H); 13C NMR (100.6 MHz, CD3OD): δC = 14.5, 23.8,
26.7, 30.4, 30.5, 30.76, 30.78, 31.36, 31.43, 33.1, 69.37, 69.43,
73.0, 73.1, 73.2, 73.8, 84.46, 84.53; HRMS m/z [M + Na]+
calcd for C26H53O9PNa 563.3319, found 563.3322.
(400 MHz, CDCl3): δH = 0.80–0.90 (m, 18 H), 1.20–1.40 (m, 84
H), 1.67–1.69 (m, 12 H), 4.07–4.08 (m, 12 H), 4.50–4.55 (m, 3
H), 4.79 (m, 1 H), 5.10–5.13 (m, 2 H), 5.51 (s, 1 H); 13C NMR
(100.6 MHz, CDCl3): δC = 14.1, 22.6, 25.38, 25.42, 29.1, 29.2,
29.3, 29.5, 30.2, 30.26, 30.33, 31.9, 65.30, 65.34, 67.9, 67.99,
68.04, 68.2, 68.3, 68.49, 68.55, 70.6, 70.69, 70.74, 102.6;
HRMS m/z [M + Na]+ calcd for C67H133O15P3Na 1293.8749,
found 1293.8752.
meso-1,3-O-Methylidene-D-myo-inositol-5-(didecyl phosphate)
(27). 5% palladium black (450 mg) was added to a stirred sol-
ution of 24 (120 mg, 0.15 mmol) in ethanol (15 mL), and the
flask was flushed three times with a balloon of hydrogen. After
the reaction mixture was stirred overnight at room temperature,
the catalyst was removed by filtering through Celite. The com-
bined organic layers were concentrated under reduced pressure.
Purification by silica gel column chromatography, eluting with
ethyl acetate and petroleum ether (1 : 1, then 3 : 2), yielded 27 as
a colorless oil (72 mg, 90%): Rf 0.20 (ethyl acetate–petroleum
meso-1,3,5-O-Orthoformate-D-myo-inositol-4,6-bi(dimethyl phos-
phate)-2-(didecyl phosphate) (30). Dimethyl N,N-diisopropylpho-
sphoramidite (75 mg, 0.348 mmol) was stirred with 1H-tetrazole
(24 mg, 0.348 mmol) in dry CH2Cl2 (5 mL) under N2 for
30 min at room temperature. Compound 17 (48 mg,
0.087 mmol) dissolved in dry CH2Cl2 (5 mL) was added and the
resulting mixture stirred overnight. The mixture was cooled to
0 °C, and 3-chloroperoxybenzoic acid (88 mg, 0.348 mmol) was
added. The resulting mixture was allowed to warm to room
temperature and stirred for 2 h. The 3-chloroperoxybenzoic acid
was quenched with a 10% aqueous solution of sodium hydrogen
sulfite. The layers were separated, and the aqueous layer was
extracted with dichloromethane. The combined organic layers
were washed with a saturated aqueous solution of sodium hydro-
gen carbonate and brine, dried (sodium sulfate), filtered, and
concentrated under reduced pressure. Purification by silica gel
column chromatography, eluting with ethyl acetate and pet-
roleum ether (1 : 1), then ethyl acetate and petroleum ether and
methanol (1 : 1 : 0.06), yielded 30 as a colorless oil (51 mg,
76%): Rf 0.10 (ethyl acetate–petroleum–methanol 1 : 1 : 0.06);
31P NMR (162 MHz, CDCl3): δP = −1.5, 0.1; 1H NMR
(400 MHz, CDCl3): δH = 0.85 (t, J = 6.6 Hz, 6 H), 1.23–1.35
(m, 28 H), 1.64–1.71 (m, 4 H), 3.78 (s, 6 H), 3.81 (s, 6 H),
4.05–4.08 (m, 4 H), 4.46–4.50 (m, 2 H), 4.56 (m, 1 H), 4.78 (d,
J = 3.6 Hz, 1 H), 5.10–5.12 (m, 2 H), 5.50 (s, 1 H); 13C NMR
(100.6 MHz, CDCl3): δC = 14.0, 22.6, 25.3, 29.1, 29.2, 29.43,
29.46, 30.08, 30.15, 31.8, 54.78, 54.83, 54.84, 65.12, 65.16,
67.70, 67.75, 67.8, 68.3, 68.4, 70.2, 70.3, 70.49, 70.53, 70.6,
102.5; HRMS m/z [M + Na]+ calcd for C31H61O15P3Na
789.3115, found 789.3113.
1
ether 3 : 2); 31P NMR (162 MHz, CDCl3): δP = −0.2; H NMR
(400 MHz, CDCl3): δH = 0.87 (t, J = 6.8 Hz, 6 H), 1.25–1.34
(m, 28 H), 1.63–1.70 (m, 4 H), 2.80 (m, 1 H), 3.90 (s, 1 H),
4.04–4.09 (m, 5 H), 4.16–4.31 (m, 4 H), 4.62–4.72 (m, 2 H),
4.75 (d, J = 6.0 Hz, 1 H), 5.07 (d, J = 6.0 Hz, 1 H); 13C NMR
(100.6 MHz, CDCl3): δC = 14.0, 22.6, 25.4, 29.1, 29.3, 29.5,
30.08, 30.15, 31.8, 52.6, 62.0, 65.5, 68.7, 68.8, 73.09, 73.14,
83.0, 83.1, 85.1; HRMS m/z [M + Na]+ calcd for C27H53O9PNa
575.3319, found 575.3313.
( )-1,3,5-O-Orthoformate-D-myo-inositol-2,4-bi(didecyl phos-
phate) (28) and meso-1,3,5-O-orthoformate-D-myo-inositol-2,4,6-
tri(didecyl phosphate) (29). Didecyl N,N-diisopropylphosphora-
midite (116 mg, 0.24 mmol) was stirred with 1H-tetrazole
(19 mg, 0.24 mmol) in dry CH2Cl2 (5 mL) under N2 for 30 min
at room temperature. Compound 17 (33 mg, 0.06 mmol) dis-
solved in dry CH2Cl2 (5 mL) was added and the resulting
mixture stirred overnight. The mixture was cooled to 0 °C, and
3-chloroperoxybenzoic acid (64 mg, 0.24 mmol) was added. The
resulting mixture was allowed to warm to room temperature and
stirred for 2 h. The 3-chloroperoxybenzoic acid was quenched
with a 10% aqueous solution of sodium hydrogen sulfite. The
layers were separated, and the aqueous layer was extracted with
dichloromethane. The combined organic layers were washed
with a saturated aqueous solution of sodium hydrogen carbonate
and brine, dried (sodium sulfate), filtered, and concentrated
under reduced pressure. Purification by silica gel column chrom-
atography, eluting with ethyl acetate and petroleum ether (1 : 6,
1 : 4, 1 : 3, then 1 : 2), yielded 28 as a colorless oil (30 mg,
55%): Rf 0.10 (ethyl acetate–petroleum 1 : 4); 31P NMR
(162 MHz, CDCl3): δP = −2.2, −1.5; 1H NMR (400 MHz,
CDCl3): δH = 0.88 (t, J = 6.8 Hz, 12 H), 1.20–1.40 (m, 56 H),
1.68–1.70 (m, 8 H), 3.90 (m, 1 H), 4.08–4.09 (m, 8 H), 4.40 (m,
1 H), 4.46 (m, 1 H), 4.57–4.62 (m, 2 H), 4.87 (d, J = 3.2 Hz, 1
H), 5.06 (m, 1H), 5.50 (s, 1 H); 13C NMR (100.6 MHz, CDCl3):
δC = 14.1, 22.6, 25.36, 25.38, 29.10, 29.13, 29.3, 29.5, 30.11,
30.14, 30.17, 30.21, 31.8, 65.9, 66.0, 67.1, 68.2, 68.3, 68.82,
68.83, 68.9, 69.0, 70.69, 70.74, 70.78, 70.82, 71.36, 71.41,
72.20, 72.24, 76.7, 102.6; HRMS m/z [M + Na]+ calcd for
C47H92O12P2Na 933.5956, found 933.5945; yielded 29 as a
( )-6-O-Benzyl-D-myo-inositol-1,3,5-O-orthoformate-2-(dimethyl
phosphate)-4-(didecyl phosphate) (31). Dimethyl N,N-diisopro-
pylphosphoramidite (46 mg, 0.2 mmol) was stirred with 1H-tet-
razole (16 mg, 0.2 mmol) in dry CH2Cl2 (10 mL) under N2 for
30 min at room temperature. Compound 21 (68 mg, 0.1 mmol)
was added and the resulting mixture stirred overnight. The
mixture was cooled to 0 °C, and 3-chloroperoxybenzoic acid
(51 mg, 0.2 mmol) was added. The resulting mixture was
allowed to warm to room temperature and stirred for 2 h. The 3-
chloroperoxybenzoic acid was quenched with a 10% aqueous
solution of sodium hydrogen sulfite. The layers were separated,
and the aqueous layer was extracted with dichloromethane. The
combined organic layers were washed with a saturated aqueous
solution of sodium hydrogen carbonate and brine, dried (sodium
sulfate), filtered, and concentrated under reduced pressure. Purifi-
cation by silica gel column chromatography, eluting with ethyl
acetate and petroleum ether (1 : 4, 1 : 3, then 1;2), yielded 31 as
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 3642–3654 | 3651

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a colorless oil (60 mg, 80%): Rf 0.10 (ethyl acetate–petroleum
hydrogen carbonate and brine, dried (sodium sulfate), filtered,
and concentrated under reduced pressure. Purification by silica
gel column chromatography, eluting with ethyl acetate and pet-
roleum ether (1 : 1), then ethyl acetate and petroleum ether and
methanol (1 : 1 : 0.15), yielded 33 as a colorless oil (140 mg,
99%): Rf 0.20 (ethyl acetate–petroleum–methanol 1 : 1 : 0.15);
1
1 : 2); 31P NMR (162 MHz, CDCl3): δP = −1.8, 0.4; H NMR
(400 MHz, CDCl3): δH = 0.88 (t, J = 6.8 Hz, 6 H), 1.24–1.33
(m, 28 H), 1.54–1.65 (m, 4 H), 3.82 (s, 3 H), 3.84 (s, 3 H),
3.89–4.02 (m, 4 H), 4.38 (m,1 H), 4.46–4.50 (m, 2 H), 4.58 (m,
1 H), 4.64 (dd, J = 11.6, 29.6 Hz, 2 H), 4.88 (d, J = 6.8 Hz, 1
H), 5.10 (m, 1 H), 5.51 (s, 1 H), 7.29–7.35 (m, 5 H); 13C NMR
(100.6 MHz, CDCl3): δC = 14.0, 22.6, 25.31, 25.32, 29.1, 29.2,
29.43, 29.45, 30.06, 30.13, 30.2, 31.8, 54.5, 54.6, 66.60, 66.65,
67.64, 67.68, 68.3, 68.4, 68.5, 70.38, 70.43, 70.61, 70.65,
70.80, 70.84, 70.9, 71.6, 73.0, 102.8, 127.7, 128.0, 128.4,
136.9; HRMS m/z [M + Na]+ calcd for C36H62O12P2Na
771.3609, found 771.3612.
1
31P NMR (162 MHz, CDCl3): δP = −1.7, 0.9, 1.1; H NMR
(300 MHz, CDCl3): δH = 0.87 (t, J = 6.8 Hz, 6 H), 1.20–1.41
(m, 28 H), 1.68–1.77 (m, 4 H), 3.45 (s, 3 H), 3.49 (s, 3 H), 3.59
(s, 3 H), 3.63 (s, 3 H), 3.76 (s, 3 H), 3.79 (s, 3 H), 3.94 (t, J =
9.6 Hz, 2 H), 4.08–4.15 (m, 4 H), 4.38–4.51 (m, 3 H), 4.83 (s, 4
H), 5.13 (d, J = 0.9 Hz, 1 H), 7.21–7.45 (m, 10 H); 13C NMR
(75.5 MHz, CDCl3): δC = 13.9, 22.5, 25.4, 29.1, 29.2, 29.4,
30.1, 30.2, 31.7, 54.26, 54.34, 54.4, 54.5, 54.6, 68.1, 68.2, 74.7,
75.2, 76.7, 76.8, 77.7, 79.0, 79.1, 127.2, 127.9, 137.7; HRMS
m/z [M + Na]+ calcd for C46H80O18P4Na 1067.4187, found
1067.4179.
( )-6-O-Benzyl-D-myo-inositol-1,3,5-O-orthoformate-2-(dibenzyl
phosphate)-4-(didecyl phosphate) (32). Dibenzyl N,N-diisopro-
pylphosphoramidite (82 mg, 0.2 mmol) was stirred with 1H-tet-
razole (17 mg, 0.2 mmol) in dry CH2Cl2 (10 mL) under N2 for
30 min at room temperature. Compound 21 (64 mg, 0.1 mmol)
was added and the resulting mixture stirred overnight. The
mixture was cooled to 0 °C, and 3-chloroperoxybenzoic acid
(50 mg, 0.2 mmol) was added. The resulting mixture was
allowed to warm to room temperature and stirred for 2 h. The 3-
chloroperoxybenzoic acid was quenched with a 10% aqueous
solution of sodium hydrogen sulfite. The layers were separated,
and the aqueous layer was extracted with dichloromethane. The
combined organic layers were washed with a saturated aqueous
solution of sodium hydrogen carbonate and brine, dried (sodium
sulfate), filtered, and concentrated under reduced pressure. Purifi-
cation by silica gel column chromatography, eluting with ethyl
acetate and petroleum ether (1 : 6, 1 : 4, then 1 : 3), yielded 32 as
a colorless oil (63 mg, 70%): Rf 0.10 (ethyl acetate–petroleum
meso-D-myo-Inositol-1,3,5-tri(dimethyl phosphate)-2-(didecyl
phosphate) (34). 5% palladium black (180 mg) was added to a
stirred solution of 33 (65 mg, 0.06 mmol) in ethanol (10 mL),
and the flask was flushed three times with a balloon of hydrogen.
After the reaction mixture was stirred overnight at room tempera-
ture, the catalyst was removed by filtering through Celite. The
combined organic layers were concentrated under reduced
pressure, yielded 34 as a colorless oil (50 mg, 98%): Rf 0.20
(ethyl acetate–petroleum ether–methanol 1 : 1 : 0.2); 31P NMR
1
(162 MHz, CDCl3): δP = −1.9, 1.1, 1.6; H NMR (400 MHz,
CDCl3): δH = 0.87 (t, J = 6.4 Hz, 6 H), 1.22–1.38 (m, 28 H),
1.62–1.72 (m, 4 H), 3.83–3.88 (m, 18 H), 3.97–4.10 (m, 6 H),
4.49 (m, 1 H), 4.62 (t, J = 9.6 Hz, 2 H), 5.08 (d, J = 8.4 Hz, 1
H); 13C NMR (100.6 MHz, CDCl3): δC = 14.0, 22.6, 25.4, 29.1,
29.2, 29.5, 30.1, 30.2, 31.8, 54.7, 54.8, 54.96, 55.02, 55.04,
55.1, 68.0, 68.1, 70.8, 75.7, 81.4, 81.5; HRMS m/z [M + Na]+
calcd for C32H68O18P4Na 887.3248, found 887.3244.
1
1 : 3); 31P NMR (162 MHz, CDCl3): δP = −1.8, −1.7; H NMR
(400 MHz, CDCl3): δH = 0.88 (t, J = 6.8 Hz, 6 H), 1.22–1.30
(m, 28 H), 1.51–1.61 (m, 4 H), 3.83–3.98 (m, 4 H), 4.33 (m, 1
H), 4.38–4.42 (m, 2 H), 4.57 (m, 1 H), 4.58 (dd, J = 11.6, 27.6
Hz, 2 H), 4.90 (d, J = 3.2 Hz, 1 H), 5.08–5.11 (m, 5 H), 5.51 (s,
1 H), 7.28–7.33 (m, 15 H); 13C NMR (100.6 MHz, CDCl3): δC
= 14.0, 22.6, 25.3, 29.1, 29.2, 29.42, 29.45, 30.0, 30.1, 30.2,
31.8, 66.68, 66.73, 67.65, 67.68, 68.28, 68.34, 68.37, 68.43,
69.51, 69.54, 69.6, 70.38, 70.43, 70.5, 70.7, 70.8, 70.9, 71.5,
73.0, 102.8, 127.6, 127.90, 127.93, 128.4, 128.5, 135.41,
135.44, 135.49, 135.51, 136.9; HRMS m/z [M + Na]+ calcd for
C48H70O12P2Na 923.4235, found 923.4230.
meso-4,6-Di-O-benzyl-D-myo-inositol-1,3,5-tri(dibenzyl phos-
phate)-2-(didecyl phosphate) (35). Dibenzyl N,N-diisopropyl-
phosphoramidite (423 mg, 1.224 mmol) was stirred with 1H-
tetrazole (86 mg, 1.224 mmol) in dry CH2Cl2 (10 mL) under N2
for 30 min at room temperature. Compound 14 (98 mg,
0.136 mmol) dissolved in dry CH2Cl2 (5 mL) was added and the
resulting mixture stirred overnight. The mixture was cooled to
0 °C, and 3-chloroperoxybenzoic acid (346 mg, 1.224 mmol)
was added. The resulting mixture was allowed to warm to room
temperature and stirred for 2 h. The 3-chloroperoxybenzoic acid
was quenched with a 10% aqueous solution of sodium hydrogen
sulfite. The layers were separated, and the aqueous layer was
extracted with dichloromethane. The combined organic layers
were washed with a saturated aqueous solution of sodium hydro-
gen carbonate and brine, dried (sodium sulfate), filtered, and
concentrated under reduced pressure. Purification by silica gel
column chromatography, eluting with ethyl acetate and pet-
roleum ether (1 : 3, 1 : 2, then 1 : 1), yielded 35 as a colorless a
colorless oil (186 mg, 91%): Rf 0.30 (ethyl acetate–petroleum
meso-4,6-Di-O-benzyl-D-myo-inositol-1,3,5-tri(dimethyl phos-
phate)-2-(didecyl phosphate) (33). Dimethyl N,N-diisopropyl-
phosphoramidite (255 mg, 1.224 mmol) was stirred with 1H-
tetrazole (87 mg, 1.224 mmol) in dry CH2Cl2 (10 mL) under N2
for 30 min at room temperature. Compound 14 (98 mg,
0.136 mmol) dissolved in dry CH2Cl2 (5 mL) was added and the
resulting mixture stirred overnight. The mixture was cooled to
0 °C, and 3-chloroperoxybenzoic acid (335 mg, 1.224 mmol)
was added. The resulting mixture was allowed to warm to room
temperature and stirred for 2 h. The 3-chloroperoxybenzoic acid
was quenched with a 10% aqueous solution of sodium hydrogen
sulfite. The layers were separated, and the aqueous layer was
extracted with dichloromethane. The combined organic layers
were washed with a saturated aqueous solution of sodium
1
1 : 1); 31P NMR (162 MHz, CDCl3): δP = −1.7, −1.6, −1.4; H
NMR (400 MHz, CDCl3): δH = 0.87 (t, J = 6.8 Hz, 6 H),
1.22–1.32 (m, 28 H), 1.59–1.66 (m, 4 H), 3.99 (t, J = 9.6 Hz, 2
H), 4.05–4.13 (m, 4 H), 4.33–4.38 (m, 2 H), 4.46 (m, 1 H),
3652 | Org. Biomol. Chem., 2012, 10, 3642–3654
This journal is © The Royal Society of Chemistry 2012

View Article Online
2 M. J. Berridge, M. D. Bootman and H. L. Roderick, Nat. Rev. Mol. Cell
Biol., 2003, 4, 517.
4.60–4.66 (m, 2 H), 4.77–4.89 (m, 8 H), 4.95–5.00 (m, 6 H),
5.29 (d, J = 7.2 Hz, 1 H), 6.96–6.98 (m, 4 H), 7.11–7.12 (m, 4
H), 7.16–7.26 (m, 28 H), 7.37–7.39 (m, 4 H); 13C NMR
(75.5 MHz, CDCl3): δC = 14.0, 22.5, 25.4, 29.14, 29.19, 29.5,
30.1, 30.2, 31.8, 68.2, 68.3, 69.16, 69.23, 69.26, 69.33, 69.5,
69.6, 74.6, 75.2, 75.3, 77.7, 127.2, 127.4, 127.6, 127.8, 128.0,
128.1, 128.2, 128.28, 128.33, 135.5, 135.56, 135.60, 135.64,
135.70, 135.73, 137.7; HRMS m/z [M + Na]+ calcd for
C82H104O18P4Na 1523.6065, found 1523.6060.
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Antiproliferative assay
Non-small cell lung cancer cell line A549, liver cancer cell line
HepG2, breast cancer cell line MDA-MB-231, cervical carci-
noma cell line HeLa and normal breast epithelial cell line
MCF10A were obtained from ATCC (American type culture col-
lection) and were maintained in 5% CO2 at 37 °C. A549,
HepG2, MDA-MB-231 and HeLa cells were grown in Dulbec-
co’s modified Eagle’s medium (DMEM, Gibco) supplemented
with 10% fetal bovine serum (FBS, Omega Scientific) and 1%
penicillin/streptomycin (Omega Scientific). MCF10A cells were
grown in DMEM/F12 (Gibco) supplemented with 5% horse
serum, 20 ng mL−1 EGF (Peprotech), 0.5 μg mL−1 hydrocorti-
sone (Sigma), 100 ng mL−1 cholera toxin (Sigma), 10 μg mL−1
insulin (Sigma), 1% Pen/Strep (Invitrogen). Cisplatin (Sigma)
was dissolved in 1% NaCl (Sigma) solution (pH 7).
Approximately 1000 cells were seeded into individual wells
of 96-well tissue culture plates and incubated for 12 h, medium
was 0.2 mL per well. The compound was diluted to 10 μg mL−1
using DMEM (final concentration in the test well), to analyze
the inhibition effect on A549 roughly. After that, cells were
exposed to triplicates of different concentration solutions (from
0.39 to 50 μg mL−1) of test compounds to determine their
potency. The analyzed inhibitors were dissolved in DMSO reach-
ing a final DMSO concentration of 0.5%. Viability was normal-
ized to control cells which were treated with the vehicle, DMSO.
After 72 h incubation at 37 °C and 5% CO2, cell viability was
assessed by MTT assay. Cells were replenished with fresh
medium (0.1 mL per well) which contains 10% MTT. Culture
medium was removed after 4 h, and the formazan was dissolved
in DMSO (200 μL per well). Then OD570 were measured by
Plate Reader (BIORAD). The IC50 values were calculated by
Origin 6.0.
Acknowledgements
This work was financially supported by Ministry of Science and
Technology of China (2010CB126102, 2011BAE06B05,
2008DFA30770) and National Natural Science Foundation of
China (20932005, 20872067).
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