Effects of 1,3-di-O-substituted-myo-inositol derivatives on the antiproliferation and caspase-3
activity of HCT-116 and HL-60 cells
Noriyuki Hatae,a,* Satoe Yamauchi,b Takafumi Saeki,b Ichiro Suzuki,a Tominari Choshi,c Satoshi
Hibino,c Chiaki Okada,a Minoru Hayashi,b Yutaka Watanabe,b Eiko Toyotaa,*
a
School of Pharmaceutical Sciences, Health Sciences University of Hokkaido; Ishikari-Tobetsu,
Hokkaido 061-0293, Japan:
b
Department of Materials Science and Biotechnology, Graduate
School of Science and Engineering, Ehime Universit; 3 Bunkyo-cho, Matsuyama 790-8577, Japan:
and c Graduate School of Pharmacy & Pharmaceutical Sciences, and Faculty of Pharmacy and
Pharmaceutical Sciences, Fukuyama University; Fukuyama, Hiroshima 729-0292, Japan.
Supporting Information
Materials and Experimental Procedures
S2-S6
Spectral Data for 1,3-di-O-substituted-myo-inositol derivativess
S7-S10
References
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S1
Materials
The myo-inositol derivatives were synthesized according to our previously described methods,
and their spectral data were identical to those reported previously.1,2
bromide
(MTT)
was
obtained
from
Sigma-Aldrich
Corp.
Thiazolyl blue tetrazolium
(MO,
USA).
Both
2-(4-iodophenyl)-3-4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1)
and 1-methox-5-methylphenazinium methylsulfate (1-methoxy PMS) were obtained from Dojindo
Laboratories (Kumamoto, Japan). The CellToxTM Green Cytotoxicity Assay kit was obtained from
Promega (WI, USA).
Fix Buffer I, Perm/Wash Buffer I, and fluorescein isothiocyanate
(FITC)-conjugated anti-active caspase-3 monoclonal antibody were obtained from BD Biosciences
Pharmingen (NJ, USA).
General
myo-Inositol (Tsuno Food Co., Ltd) was dried by heating at 200 °C for 12 h under reduced
pressure (0.5 mmHg).
Anhydrous N,N-dimethylacetamide (DMA) and dimethyl sulfoxide
(DMSO) were obtained by treating with BaO and CaH2 overnight, respectively, and sequent
distillation (about 70 °C/25 mmHg).
LiCl is so hydroscopic that it was weighed quickly in a
reaction vessel and dried by application of heat at about 400 °C under reduced pressure (0.5 mmHg)
for a few minutes.
Analytical thin-layer chromatography was performed with silica gel 60 F254
(Merck). Flash column chromatography was performed using silica gel (Fuji Silysia Chemical Ltd.
BW-300). All 1H-NMR spectra were recorded at 400 MHz or 270 MHz, and the 13C-NMR spectra
were recorded at 100 MHz, generally using CDCl3 as a solvent.
Multiplicity is indicated by one or
more of the following: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or br (broad).
Low and high resolution mass spectra were recorded on ESI-MS or FAB-MS, using m-nitrobenzyl
alcohol as a matrix.
The
synthesis
and
properties
of
1,3-di-O-substituted-myo-Inositols
containing
l-menthyloxycarbonyl (10), dibutyl phosphoryl (11), TIPS (13), or TBDMS (14), have never been
reported previously, and the experimental procedures used to synthesize them and their spectral data
were presented below.
1,3-Di-O-(l-menthyloxycarbonyl)-myo-inositol (10)
To a reaction flask containing LiCl (80 mg, 1.89 mmol) was added myo-inositol (100 mg, 0.55
mmol) and DMA (1 mL), and the mixture was heated at about 120˚C until the mixture became a
clear solution.
After addition of Et3N (391 mg, 3.89 mmol), the resultant solution was kept at 0 ˚C,
and then l-menthylchloroformate (364 mg, 1.67 mmol) and DMAP (13.6 mg, 0.11 mmol) were
added. The mixture was stirred at the same temperature for 8 h. H2O (about 0.1 mL) was added
and the mixture was stirred for 10 min, partitioned to AcOEt and H2O layers. The aqueous layer
S2
was extracted three times with AcOEt.
The combined extract was washed with H2O (x3) and brine,
dried over Na2SO4, filtered, and then evaporated. The resultant residue was purified by a flash
column chromatography (AcOEt/Hexane 3:2) to furnish white powder 10 (114 mg, 38%).
1
H-NMR (270 MHz, CDCl3)  0.55 (6H, d, J=6.9 Hz), 0.89 (6H, d, J=6.9 Hz), 0.92 (6H, d, J=6.9
Hz), 1.07 (4H, m), 1.42 (4H, m), 1.67 (4H, m), 1.94 (2H, m), 2.08 (2H, m), 3.51 (1H, t, J=9.4 Hz),
4.00 (2H, t, J=9.4 Hz), 4.36 (2H, m), 4.56 (2H, m), 4.68 (2H, m);
CD3OD/CDCl3
13
C-NMR (100 MHz,

1:0.1)
76.40 (2C), 79.32(2C); HRMS (ESI) Calcd for C28H48NaO10 [(M+Na)+]: 567.31452. Found:
567.31629.
1,3-Di-O-(dibutyl phosphoryl)-myo-inositol (11)
To a reaction flask containing LiCl (80 mg, 1.89 mmol) was added myo-inositol (100 mg, 0.55
mmol) and DMA (1 mL), and the mixture was heated at about 120 ˚C until the mixture became a
clear solution. After addition of dibutyltin chloride (203 mg, 0.67 mmol) and Et3N (225 mg, 2.22
mmol), the resultant solution was stirred at r.t. for 2 h, and then dibutoxyphosphoryl chloride (317
mg, 1.39 mmol) was added. The mixture was stirred at 0 °C for 24 h.
TMSCl (1.00 g, 9.20
mmol) and pyridine (2 mL) were added, and the resultant solution was stirred at r.t. for 16 h. H 2O
(about 0.1 mL) was added and the mixture was stirred for 10 min, partitioned to AcOEt and H2O
layers. The organic layer was washed with H2O (x5), saturated KHSO4 solution, H2O, and then
brine, dried over Na2SO4, filtered, and then evaporated. The resultant residue was dissolved in
CHCl3 (1.5 mL) and MeOH (3 mL), and CF3CO2H (380 mg, 3.33 mmol) were added. The
solution was stirred for 16 h at r.t., and all the volatile materials were distilled off under reduced
pressure (1.0 mmHg).
The resultant residue was purified by a flash column chromatography
(MeOH/CHCl3 1:8) to furnish white powder 11 (184 mg, 59%).
1
H-NMR (400 MHz, CDCl3) 
0.927 (12H, t, J=7.3 Hz), 1.40 (8H, m), 1.65 (8H, m), 3.40 (1H, t, J=9.3 Hz), 3.95 (2H, t, J=9.3 Hz),
4.08 (8H, m), 4.17 (2H, m), 4.77 (1H, m);
13
C-NMR (100 MHz, CDCl3)  13.5 (4C), 18.6 (4C),
32.1 (4C), 68.1 (4C), 70.3, 70.8 (2C), 74.2, 78.0 (2C); HRMS (FAB+, m-nitrobenzyl alcohol) Calcd
for C22H47O12P2 [(M+H)+]: 565.2543. Found: 565.2558.
1,3-Di-O-(triisopropylsilyl)-myo-inositol (13)
The suspension of myo-Inositol (100 mg, 0.55 mmol) in DMSO ( 2.5 mL) was heated at about 120
˚C until the suspension became a clear solution.
The solution was kept at 0 °C, and
triisopropylsilylchloride (335 mg, 1.74 mmol) and a catalytic amount of DMAP were added. The
resultant mixture was stirred at r.t. for 5 d. H2O (about 0.1 mL) was added and the mixture was
S3
stirred for 10 min, partitioned to AcOEt and H2O layers. The aqueous layer was extracted three
times with AcOEt.
The combined extract was washed with H2O (x2), 1N HCl solution, H2O,
saturated NaHCO3 solution, H2O, and brine, dried over Na2SO4, filtered, and then evaporated.
The resultant residue was purified by a flash column chromatography (AcOEt/Hexane 2:3) to
furnish white solid 13 (72 mg, 26%).
1
H-NMR (270 MHz, CDCl3)  1.06-1.17 (42H, m), 3.36 (1H,
t, J=9.2 Hz), 3.62 (2H, dd, J=2.7 and 9.2 Hz), 3.84 (2H, t, J=9.2 Hz), 4.05 (1H, t, J=2.7 Hz);
C-NMR (100 MHz, CDCl3)  12.46 (6C), 17.97 & 18.01 (12C), 73.10 (2C), 73.54 (2C), 73.72,
13
74.17; HRMS (ESI) Calcd for C24H52O6Si2Na [(M+Na)+]: 515.32001. Found: 515.32003.
1,3-Di-O-(tert-butyldimethylsilyl)-myo-inositol (14)
The suspension of myo-Inositol (200 mg, 1.11 mmol) in DMSO (5.0 mL) was heated at about 120
˚C until the suspension became a clear solution.
The solution was kept at 0 °C,
tert-butyldimethylsilylchloride (916 mg, 3.33 mmol) and pyridine (3.0 mL) were added.
The
resultant mixture was stirred at r.t. for 3 d. H2O (about 0.1 mL) was added and the mixture was
stirred for 10 min, partitioned to AcOEt and H2O layers. The aqueous layer was extracted three
times with AcOEt.
The combined extract was washed with H2O, 1N HCl solution, H2O, saturated
NaHCO3 solution, H2O, and brine, dried over Na2SO4, filtered, and then evaporated. The resultant
residue was purified by a flash column chromatography (AcOEt/Hexane 2:3) to furnish white
amorphous 14 (68 mg, 15%).
1
H-NMR (400 MHz, CDCl3)  0.11 (6H, s), 0.12 (6H, s), 0.91 (18H,
s), 3.31 (1H, t, J=9.2Hz), 3.46 (2H, dd, J=2.8 and 9.2 Hz), 3.76 (2H, t, J=9.2 Hz), 3.87 (1H, t, J=2.8
Hz);
C-NMR (100 MHz, CDCl3)  -4.76 (2C), -4.37 (2C), 18.20 (2C), 25.84 (6C), 72.71 (2C),
13
73.48 (2C), 73.78, 74.19; HRMS (ESI) Calcd for C18H40O6Si2Na [(M+Na)+]: 431.22611. Found:
431.22547.
Cell lines and cell cultures
For testing the antitumor cell activities of the synthesized molecules, two cancer cell lines were
used: HCT-116 cells (human colon cancer cells) and HL-60 cells (human promyelocytic leukemia
cells), which were purchased from the American Type Culture Collection (VA, USA).
The
HCT-116 cells were maintained in McCOY 5A medium supplemented with L-glutamine and 10%
heat inactivated (55 °C for 30 min) fetal bovine serum (FBS) at 37 °C in an atmosphere of 5% CO2.
The HL-60 cells were cultured in RPMI-1640 medium supplemented with L-glutamine and 10%
heat inactivated FBS at 37 °C in an atmosphere of 5% CO2.
Cell viability assays
The HCT-116 cell viability assay was carried out using the MTT based method described by
Mosmann.3
Briefly, cells were placed in 96-well flat-bottomed tissue culture plates at a density of
S4
6.0 x 103 cells per well in 100 L culture medium.
The cells were then incubated at 37 °C in an
atmosphere of 5% CO2 for 24 h to allow cells attachment onto the wells.
The cells were
subsequently treated with the indicated concentrations of the test agents in culture medium without
FBS.
Following a further 24 h incubation, 10 L of MTT (5 mg/mL in PBS buffer) was added per
well and the plates were incubated for 4 h to allow the MTT to be metabolized by cellular
mitochondrial dehydrogenases.
The excess MTT was aspirated, and the formazan crystals that had
formed were dissolved by the addition of 100 L of DMSO.
was read at 570 nm using a microplate reader.
The absorbance of purple formazan
The results following test agents exposure were
calculated as a percentage relative to untreated controls.
The HL-60 cell viability assay was carried out using the WST-1 based method described by
Ishiyama.4
The cells were seeded in 96-well flat-bottomed tissue culture plates at a density of 3.0
x 104 cells per well in 100 L of the FBS contained culture medium with the indicated
concentrations of the test agents.
Following a further 24 h incubation, 10 L of a mixture of
WST-1/1-methoxy PMS solution containing 5 mM WST-1 and 0.2 mM 1-methoxy PMS in 40 mM
HEPES-NaOH (pH 7.4) were added to each well and the plates were incubated for 3 h to allow the
WST-1 to be metabolized by cellular mitochondrial dehydrogenases.
formazan was read at 415 nm using a microplate reader.
The absorbance of yellow
The results following test agents
exposure were calculated as a percentage relative to untreated controls.
Cytotoxicity assay
HL-60 cells were seeded in 96-well flat-bottomed culture plates at a density of 1.0 x 104 cells per
well in 100 L of the FBS contained culture medium with test agents and CellTox TM Green Dye.
After the cells had been incubated for the indicated hours, the fluorescence of the wells were
measured at an excitation wavelength of 480 nm and an emission wavelength of 530 nm using a
microplate reader.
The results following test agents exposure were calculated as a percentage
relative to cytotoxicity controls.
Caspase-3 activation assay
FACS based analysis of caspase-3 activation was carried out adopting a method previously
reported.5
HL-60 cells were incubated with test agents in Krebs-Ringer’s HEPES buffer
containing 15 mM HEPES-NaOH (pH 7.4), 120 mM NaCl, 5 mM KCl, 0.7 mM MgSO4, 1.2 mM
CaCl2 and 1.8 g/L glucose for 4 h at 37 °C. After washing the cells with ice-cold PBS buffer, they
were fixed with Fixed Buffer I for 30 min on ice. The cells were then permeabilized and stained
with anti-active caspase-3 monoclonal antibody in Perm/Wash Buffer I at 37 °C in the dark for 30
min. The cells were then washed and diluted with staining buffer (2% FBS in PBS buffer), and
fluorescence measurements were obtained using a FACSCanto with BD Diva software (BD
S5
Biosciences).
Statistical analysis
Concentration-cell viability relationships were fitted to a four-parameter logistic equation using a
non-linear curve-fitting program, which derived the LC50 values for each test agent and cell type
(Kaleida-graph; Synergy Software, Reading, PA). Where appropriate, the results were expressed
as means ± sem values with n = 3 or higher for one of at least three similar experiments.
S6
1H-NMR
for 1,3-di-O-l-menthylocycarbonyl-myo-inositol (10)
13C-NMR
for 1,3-di-O-l-menthylocycarbonyl-myo-inositol (10)
S7
1H-NMR
for 1,3-di-O-dibutylphosphoryl-myo-inositol (11)
13C-NMR
for 1,3-di-O-dibutylphosphoryl-myo-inositol (11)
S8
1H-NMR
for 1,3-di-O-triisopropylsilyl-myo-inositol (13)
13C-NMR
for 1,3-di-O-triisopropylsilyl-myo-inositol (13)
S9
1H-NMR
for 1,3-di-O-tert-butyldimethylsilyl-myo-inositol (14)
13C-NMR
for 1,3-di-O-tert-butyldimethylsilyl-myo-inositol (14)
S10
References
1
S. Yamauchi, M. Hayashi, Y. Watanabe.
myo-Inositol by Dissolution Strategy.
2
One-Step Regioselective Functionalization of
Synlett 2009, 2287-2290.
Y. Watanabe, T. Uemura, S. Yamauchi, K. Tomita, T. Saeki, R. Ishida, M. Hayashi.
Regioselective functionalization of unprotected myo-inositol by electrophilic substitution.
TETRAHEDRON 2013, 69, 4657-4664.
3
T. Mosmann.
Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays.
4
J. Immunol. Methods 1983, 65, 55-63.
M. Ishiyama, M. Shiga, K. Sasamoto, M. Mizoguchi, P. He.
A New Sulfonated Tetrazolium
Salt That Produces a Highly Water-Soluble Formazan Dye.
Chem. Pharm. Bull. 1993, 41,
1118-1122 (1993).
5
N. Hatae, T. Nagayama, H. Esaki, E. Kujime, M. Minami, M. Ishikura, T. Choshi, S. Hibino, C.
Okada, E. Toyota, H. Nagasawa, T. Iwamura. Synthesis of 4-Arylpiperidin-4-ol Derivatives
of Loperamide as Agents with Potent Antiproliferative Effects against HCT-116 and HL-60
Cells.
Heterocycles 2014, 88, 663-673.
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