Synthesis of feruloyl-myo-insitol derivatives and their inhibitory effects on phorbol ester-induced superoxide generation and Esptein-Barr virus activation

Article abstract of DOI:10.1016/S0968-0896(02)00010-X

We prepared 14 feruloyl-myo-inositol derivatives, and evaluated the relationships between their stereostructure and inhibitory activity toward the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced superoxide (O₂^-) generation. And further, their suppressive effect on the TPA-induced Epstein-Barr virus (EBV) activation was examined in order to estimate their anti-carcinogenic potentials. Among the derivatives tested, 1,6-O-bis[3-(4′-hydroxy-3′-methoxyphenyl)-2-propenoyl]-myo- inositol (6b) showed an excellent suppressive activity on the O₂^- generation at a concentration of 20 μM. For the suppressive effects on the EBV activation, 2,4,6-O-tris[3-(4′-hydroxy-3′-methoxyphenyl)-2-propenoyl]-myo- inositol 1,3,5-orthoformate (9b) showed the highest activity at a concentration of 100 μM among the derivatives tested. These results suggest that the inhibitory potencies of feruloyl-myo-inositol derivatives depend on the stereostructure of molecules rather than the hydrophobicity of molecules.

Full text of DOI:10.1016/S0968-0896(02)00010-X

Bioorganic & Medicinal Chemistry 10 (2002) 1855–1863
Synthesis of Feruloyl-myo-insitol Derivatives and their Inhibitory
Effects on Phorbol Ester-Induced Superoxide Generation and
Esptein–Barr Virus Activation
Asao Hosoda,a Eisaku Nomura,a Akira Murakami,b Koichi Koshimizu,b
Hajime Ohigashi,c Kazuhiko Mizunod and Hisaji Taniguchia,*
aIndustrial Technology Center of Wakayama Prefecture, 60 Ogura, Wakayama 649-6261, Japan
bDepartment of Biotechnological Science, Faculty of Biology-Oriented Science and Technology, Kinki University, Iwade-Uchita,
Wakayama 649-6493, Japan
cDivision of Applied Science, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
dDepartment of Applied Chemistry, College of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai,
Osaka 599-8531, Japan
Received 14 November 2001; accepted 22 December 2001
Abstract—We prepared 14 feruloyl-myo-inositol derivatives, and evaluated the relationships between their stereostructure and
inhibitory activity toward the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced superoxide (Oꢀ2 ) generation. And further, their
suppressive effect on the TPA-induced Epstein–Barr virus (EBV) activation was examined in order to estimate their anti-carcino-
genic potentials. Among the derivatives tested, 1,6-O-bis[3-(40-hydroxy-30-methoxyphenyl)-2-propenoyl]-myo-inositol (6b) showed
an excellent suppressive activity on the Oꢀ2 generation at a concentration of 20 mM. For the suppressive effects on the EBV activa-
tion, 2,4,6-O-tris[3-(40-hydroxy-30-methoxyphenyl)-2-propenoyl]-myo-inositol 1,3,5-orthoformate (9b) showed the highest activity at
a concentration of 100 mM among the derivatives tested. These results suggest that the inhibitory potencies of feruloyl-myo-inositol
derivatives depend on the stereostructure of molecules rather than the hydrophobicity of molecules. # 2002 Elsevier Science Ltd.
All rights reserved.
Introduction
gical systems. It has become clear that d-myo-inositol-
1,4,5-trisphosphate acts as an intracellular second mes-
Ferulic acid (1a) is a sort of polyphenolic compound
occurring widely in the plant kingdom.1,2 Some of the
authors have recently developed a novel method for the
convenient preparation of 1a from the oily component
of rice bran at kilogram or ton order.3,4 Since then, the
acid 1a and the related compounds have attracted con-
siderable attention in the field of cancer chemopreventive
study. For example, a ferulic acid derivative, ethyl 3-(40-
geranyloxy-30-methoxyphenyl)-2-propenoate (EGMP,
1b),4ꢀ6 in which geranyl group is bonded to the phenolic
hydroxyl group of ethyl ferulate (1d), shows a suppres-
sive effect on the formation of colonic tumor marker in
rats.7,8
senger for calcium mobilization.9,10 myo-Inositol hexa-
phosphate (IP6) is shown to have an anticancer action in
a variety of experimental tumor models.11 Thus, we
expected that feruloyl-myo-inositols, consisting of the
acid 1a and myo-inositol moieties, might have some
biological activities, including anti-oxidation and anti-
carcinogenesis.
On the other hand, myo-inositol (2) is also obtained
from rice bran, and it plays an important role in biolo-
*Corresponding author. Tel.: +81-73-477-1271; fax: +81-73-477-
2880; e-mail: taniguti@wakayama-kg.go.jp
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00010-X

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A. Hosoda et al. / Bioorg. Med. Chem. 10 (2002) 1855–1863
In a preliminary paper, we reported the synthesis of
seven ester compounds consisting of 1a and 2 and their
inhibitory effects on generation of the tumor promoter
12-O-tetradecanoylphorbol-13-acetate (TPA)-induced
superoxide (Oꢀ2 ) in differentiated HL-60 cells, which
contain the NADPH oxidase system generating Oꢀ2 and
have been used as a cellular system to search for anti-
tumor promoters.12 In this study, we found that only
3,4,5,6-tetra-O-acetyl-1,2-O-bis[3-(40-acetoxy-30-methoxy-
phenyl)-2-prop-enoyl]-myo-inositol (Fig. 1, 5a), in
which two feruloyl moieties are introduced to the
hydroxyl groups bonded to the 1,2-vicinal carbons in
myo-inositol, showed an excellent inhibitory activity on
the assay system. This observation suggests that the
inhibitory effect of bisferuloyl-myo-inositol derivatives
depends on factors as follows: (1) The phenolic and
inositol hydroxyl groups in the compounds are pro-
tected by acetyl group or not; (2) hydrophobicity of the
molecules; (3) stereostructure of the molecules. Among
these factors, we have been greatly interested in the
relationships between the stereostructure of feruloyl-
myo-inositols and their inhibitory effects toward the
TPA-induced Oꢀ2 generation, because 3,4,5,6-tetra-O-
acetyl-1-O-[3-(40 -acetoxy-30 -methoxyphenyl)-2-prope-
noyl]-myo-inositol (Fig. 1, 3) and 1,2:4,5-di-O-cyclo-
hexylidene-3,6-O-bis[3-(40-acetoxy-30-methoxyphenyl)-2-
propenoyl]-myo-inositol (Fig. 1, 6a) showed no activity.
Then, we designed to prepare a number of feruloyl-myo-
inositol derivatives to discuss the relationships between
their stereostructure and biological activity.
And further, we classified these compounds into five
types of molecular architectures in order to discuss the
relationships between their stereostructure and biologi-
cal activity. The structures of these compounds are
shown in Figure 1 with the typical stereostructure of
the molecules. 3,4,5,6-Tetra-O-acetyl-myo-inositol (10),
3-(40-acetoxy-30-methoxyphenyl)-2-propenoyl chloride
(11) and 1,6-O-bis[3-(40-acetyloxy-30-methoxyphenyl)-2-
propenoyl]-3,4-O-(1,1,3,3-tetraisoprop-yldisiloxanedi-1,3-
yl)-myo-inositol (13) were synthesized according to lit-
eratures.15,16 Compounds 3, 5a, 5b, 6a, 6b, 7b, 7c, 9a,
and 9b were synthesized by the method described in
previous papers.12,17
The treatment of two equivalent of acetyl chloride
with 10 in the presence of a mixture of pyridine and
4-(dimethylamino)pyridine (DMAP) in dichloro-
methane at room temperature overnight afforded
1,3,4,5,6-penta-O-acetyl-myo-inositol 12 in a 79% yield
which has a hydroxyl group on C-2. The reaction of 12
with two equivalent of 11 in the presence of a mixture of
triethylamine (Et3N) and DMAP in dichloromethane at
room temperature overnight gave 4 in a 78% yield
(Scheme 1). The treatment of hydrazine monohydrate
with 5a in a mixture of chloroform and methanol at
room temperature for 5 h gave 5c in an 80% yield
(Scheme 2). Compound 7a was obtained by a treatment
of acetic anhydride with 7c at 90 ꢁC for 2h in pyridine in
an 82% yield (Scheme 2). Compounds 8a and 8b were
synthesized as shown in Scheme 3. The desilylation of
13 was carried out by using a mixture of tetra-
butylammonium fluoride and benzoic acid in tetra-
hydrofuran (THF) at ꢀ5 to 0 ꢁC for 6 h to afford 1,6-O-
bis[3-(40-acetoxy-30-methoxyphenyl)-2-propenoyl]-myo-
inositol (14) in a quantitative yield. The reaction of 14
with acetic anhydride in the presence of DMAP in pyr-
idine at room temperature for 2h yielded 8a in a 91%
yield. The treatment of hydrazine monohydrate with 14
in methanol at room temperature for 2h gave 8b in an
80% yield.
In the present study, we synthesized five newly novel
feruloyl-myo-inositol derivatives and investigated their
inhibitory activity and that of other nine feruloyl-myo-
inositols toward the TPA-induced Oꢀ2 generation.13 The
suppressive effect on the TPA-induced Epstein–Barr
virus (EBV) activation was also examined in order to
estimate their anti-carcinogenic potential.14
Results and Discussion
Preparation of feruloyl-myo-inositols
Biological Evaluation
We prepared 14 myo-inositol derivatives 3, 1,3,4,5,6-
penta-O-acetyl-2-O-[3-(40-acetoxy-30-methoxyphenyl)-2-
propenoyl]-myo-inositol (4), 5a, 1,2-O-bis[3-(40-hydroxy
-30-methoxyphenyl)-2-propenoyl]-myo-inositol (5b), 3,4,5,
6-tetra-O-acetyl-1,2-O-bis[3-(40-hydroxy-30-methoxyphen-
yl)-2-propenoyl]-myo-inositol (5c), 6a, 1,2:4,5-di-O-
cyclohexylidene-3,6-O-bis[3-(40 -hydoxy-30 -methoxy-
phenyl)-2-propenoyl]-myo-inositol (6b), 1,2,4,5-tetra-O-
acetyl-3,6-O-bis[3-(40-acetoxy-30-methoxyphenyl)-2-pro-
penoyl]-myo-inositol (7a), 3,6-O-bis[3-(40-hydroxy-30-
methoxyphenyl)-2-propenoyl]-myo-inositol (7b), 3,6-O-
bis[3-(40-acetoxy-30-methoxyphenyl)-2-propenoyl]-myo-
inositol (7c), 2,3,4,5-tetra-O-acetyl-1,6-O-bis[3-(40-acet-
oxy-30-methoxyphenyl)-2-propenoyl]-myo-inositol (8a),
1,6-O-bis[3-(40-hydroxy-30-methoxyphenyl)-2-propenoyl]-
myo-inositol (8b), 2,4,6-O-tris[3-(40-acetoxy-30-methoxy-
phen-yl)-2-propenoyl]-myo-inositol 1,3,5-orthoformate
(9a) and 2,4,6-O-tris[3-(40-hydroxy-30-methoxyphenyl)-
2-propeno-yl]-myo-inositol 1,3,5-orthoformate (9b).
The suppressive effects on the NADPH oxidase system
responsible for the TPA-induced Oꢀ2 generation were
examined using DMSO-differentiated HL-60 cells, a
neutrophil model. The results, observed at a concentra-
tion of 100 mM, are shown in Figure 2.
Among the bisferuloyl-myo-inositols having the stereo-
structure of type A, compound 5a exhibited a high sup-
pressive activity toward the Oꢀ2 generation (inhibitory
rate of 100%) with moderate cytotoxicity, while 5b
showed a low activity (inhibitory rate of 12%).
The structural difference between 5a and 5b is whether
the hydroxyl groups are protected or not. In addition,
the compound 5c, in which inositol hydroxyl groups are
protected but phenolic ones are not, showed no activity.
These results show that it should be necessary that the
phenolic hydroxyl groups are protected for activity-

A. Hosoda et al. / Bioorg. Med. Chem. 10 (2002) 1855–1863
1857
Figure 1. The structure of feruloyl-myo-inositols. Typical stereostructures of the molecules (space-filling model) are provided as the energy-mini-
mized structures.

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A. Hosoda et al. / Bioorg. Med. Chem. 10 (2002) 1855–1863
exhibition on this assay system. Moreover, mono-
feruloyl-myo-inositol derivatives 3 and 4 also exhibited
low suppressive activities toward the Oꢀ2 generation.
Therefore, the stereostructure of type A, in which the
feruloyl group bonded to 2-position of myo-inositol is
perpendicular to that bonded to 1-position, should be
important for the suppression activity.
The stereostructure of type C has two feruloyl moieties
which are introduced into the equatorial hydroxyl
groups bonded to the vicinal 1 and 6 carbons in myo-
inositol. Compound 8a showed no activity, but com-
pound 8b exhibited a high suppressive activity on the
Oꢀ2 generation (inhibitory rate of 100%) without nota-
ble cytotoxicity. The structural difference between 8a
and 8b is whether the hydroxyl groups are protected or
not. This observation suggests that the stereostructure
of type C should be important toward this assay system
and the hydroxyl groups play important roles on the
suppression of the Oꢀ2 generation.
On the other hand, compounds 6a, 6b, 7a, 7b and 7c
exhibited low activities (inhibitory rates of 0–30%).
From these observations, it became clear that the ste-
reostructure of type B, in which two feruloyl moieties
are introduced into the equatorial hydroxyl groups
bonded to the 3 and 6 carbons in myo-inositol, was
unimportant for this assay system. These results also
suggest that the inhibitory effects on the Oꢀ2 generation
depend on the stereostructure of molecules rather than
the hydrophobicity of molecules.
In addition, trisferuloyl-myo-inositol derivative 9b,
which is converted into an adamantane type, showed a
moderate suppressive activity (inhibitory rate 62%),
while 9a showed no activity on the Oꢀ2 generation. The
structural difference between 9a and 9b is whether the
hydroxyl groups are protected or not.
On the other hand, 1a, EGMP (1b), methyl ferulate (1c),
1d, O-acetyl ferulate (1e) and 2 showed low suppression
activities (inhibitory rates of 0–42%). These findings
show that it is important that both ferulic acid and myo-
inositol are converted into feruloyl-myo-inositol in
order to enhance their attributes.
Then, to clarify the suppressive potencies of 5a, 8b, and
9b, which showed high activities at a concentration of
100 mM, we examined their suppressive effects at a con-
centration of 20 mM (Fig. 3).
Compound 8b showed an excellent suppressive activity
toward the Oꢀ2 generation (inhibitory rate of 93%)
without marked cytotoxicity. On the other hand, com-
pound 5a showed weak (inhibitory rate of 9%) and 9b
moderate (inhibitory rate of 62%) activities at 20 mM.
Scheme 1. (a) CH3COCl, pyridine, DMAP, CH2Cl2, rt; (b) Et3N,
DMAP, CH2Cl2, rt.
These results suggest that the inhibitory potencies of
feruloyl-myo-inositol derivatives may depend on the
ꢁ
Scheme 2. (a) NH2NH2ꢂ H2O, CHCl3/MeOH (10:1), rt; (b)
(CH3CO)2O, pyridine, 90 ꢁC.
Scheme 3. (a) n-Bu4NF 3H2O, PhCO2H, THF, ꢀ5 to 0 C; (b)
.
.
(CH3CO)2O, DMAP, pyridine, rt; (c) NH2NH2 H2O, MeOH, rt.

A. Hosoda et al. / Bioorg. Med. Chem. 10 (2002) 1855–1863
1859
Figure 2. Suppressive effects of feruloyl-myo-inositol derivatives on TPA-induced Oꢀ2 generation in differentiated HL-60 cells. O2ꢀ generation was
induced by TPA (50 nM) and detected by cytochrome c. Sample concentration was 100 mM. Open and closed bars represent cell viability (%) and O2ꢀ
generation suppression (%), respectively. Data are shown as mean values from duplicate experiments.
stereostructure of molecules rather than the hydro-
phorbicity of molecules.
tion of hydroxyl groups by acetyl group may increase
the suppressive activity on this assay system.
The suppressive effects on the TPA-induced EBV acti-
vation in vitro were examined in order to estimate the
anti-carcinogenic potential. We selected 16 compounds
1a, 1b, 1c, 1d, 1e, 2, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b, 9a and
9b, and examined their suppressive effects at a con-
centration of 100 mM. The results were shown in Figure
4. The acid 1a was inactive at a concentration of 100 mM
and methyl ferulate (1c) and myo-inositol (2), which are
control compounds, showed low suppression (inhibitory
rates of 17–30%) on the EBV activation. On the other
hand, the compound 1b exhibited high suppression
(inhibitory rate of 78%) without notable cytotoxicity.
Among the compounds tested, the compound 9b
showed marked suppression on the EBV activation
(inhibitory rate of 100%) without notable cytotoxicity.
The compound 9b has three feruloyl groups and has a
unique structure in which two ferulic acids are facing
each other by using a myo-inositol 1,3,5-orthoformate
skeleton. In a previous paper, we have reported that
compound 9b shows high suppressive effect on the
17
cyclooxygenase-2promoter activity. Therefore, the
remarkable inhibitory effect on the EBV activation of 9b
should be attributed to that special structure.
As shown in Figure 4, most of bisferuloyl-myo-inositol
derivatives showed moderate suppression (inhibitory
rates of 30–58%), and it became clear that the protec-
Conclusion
We synthesized newly five feruloyl-myo-inositol deriva-
tives 4, 5c, 7a, 8a and 8b. We evaluated the relationships
between their stereostructure and inhibitory activity of
fourteen feruloyl-myo-inositol derivatives on the TPA-
induced superoxide generation. Among these com-
pounds, we found that a bisferuloyl-myo-inositol 8b in
which two feruloyl moieties are introduced into the
hydroxyl groups bonded to 1,6-vicinal carbons in myo-
inositol shows an excellent activity on the assay system.
These results suggest that the inhibitory potencies of
feruloyl-myo-inositol derivatives depend on the stereo-
structure of molecules rather than the hydrophorbicity
of molecules. The derivative 8b may warrant further
evaluation in other biological assay systems with respect
to its anti-oxidation and anti-carcinogenesis activities.
On the other hand, it should be noted that trisferuloyl-
myo-inositol derivative 9b showed a distinct inhibitory
activity toward the TPA-induced EBV activation. It is
Figure 3. Suppressive effects of feruloyl-myo-inositol derivatives 5a,
8b, and 9b at a concentration of 20 mM. Data are shown as mean
values from duplicate experiments.

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A. Hosoda et al. / Bioorg. Med. Chem. 10 (2002) 1855–1863
Figure 4. Suppressive effects of feruloyl-myo-inositol derivatives on TPA-linduced EBV activation in Raji cells. Cells were incubated with n-butyric
acid (3 mM), TPA (50 nM), and test compound (100 mM). EBV activation was measured by detecting early antigen (EA)-induction. Open and closed
bars represent cell viability (%) and EBV activation suppression (%), respectively. Data are shown as mean values from duplicate experiments.
suggested that the suppressive activity by compound 9b
also may be attributable to its special molecular struc-
ture in which two ferulic acids are facing each other by
the use of a myo-inositol 1,3,5-orthoformate skeleton.
These results also suggest that the inhibitory potencies
of feruloyl-myo-inositol derivatives depend on the ste-
reostructure of molecules rather than the hydro-
phorbicity of molecules.
in Hertz. FT-IR spectra were recorded on a Shimadzu
FTIR-8200D instrument using a diffuse reflectance cell.
Melting points were measured in capillary tubes with a
Yanagimoto micro melting point apparatus, and are
uncorrected. Elemental analyses were performed on a
Perkin–Elmer 2400II. Compound purity was checked by
TLC on Silica Gel 60 F254 (E. Merck) with detection by
charring with phosphomolybdic acid (10% in EtOH solu-
tion). Column chromatography was performed on Silica
Gel, Wakogel C-200 (Wako Pure Chemical Industry).
Ferulic acid and myo-inositol were provided by Tsuno
Food Industrial Co., Ltd. Ferulic acid was recrystallized
from ethanol. Other chemicals were commercial pro-
ducts and used without further purification. Solvents
were reagent grade and in most cases dried prior to use.
The recent progress of molecular biology makes it pos-
sible to diagnose the susceptibility of cancer. Therefore,
the cancer chemopreventive study and cancer chemo-
preventive agents will be increasingly attracted much
attention. Many natural products are reported as cancer
chemopreventive agents, however, it is difficult for these
cancer chemopreventive agents to be used practically,
because the amounts of these agents are very small in
plants. Therefore, the synthetic compounds are in great
demand for cancer chemopreventive agents.
1,3,4,5,6-Penta-O-acetyl-myo-inositol (12). To a solution
of 10 (3.48 g, 10 mmol) in 50 mL CH2Cl2 were added
successively pyridine (1.58 g, 20 mmol), a catalytic
amount of DMAP and acetyl chloride (1.57 g,
20 mmol). The mixture was stirred at room temperature
overnight, and the reaction was quenched by adding
water. The organic layer was washed successively with
saturated aqueous NaHCO3, and brine. After the
organic layer was dried over Na2SO4, the solvent was
removed under reduced pressure. The residue was chro-
matographed (silica gel, CHCl3) to give 12 (3.08 g,
79%). Recrystallization of 12 from ethanol gave color-
less prisms: mp 170–173 ꢁC; IR (KBr) n 3496, 2985,
2925, 1747, 1369, 1236, 1043 cmꢀ1; 1H NMR (CDCl3) d
2.01 (s, 9H, Ac), 2.10 (s, 6H, Ac), 2.61 (d, 1H,
J=2.6 Hz, OH), 4.33 (dd, 1H, J=2.4, J=2.6 Hz, H-2),
5.02(dd, 2H, J=2.4, 10.0 Hz, H-1, and H-3), 5.19 (t,
1H, J=10.0 Hz, H-5), 5.60 (t, 2H, J=10.0 Hz, H-4 and
Experimental
Computational methods
Energy minimization was carried out by a MM2force
field in the program CAChe WorkSystem released 3.8
(CAChe Scientific, Inc.) on a Machintosh computer.
Chemistry
1
The H NMR (400 MHz) spectra were recorded on a
Varian unity-plus 400 spectrometer using tetra-
methylsilane as internal standard. Chemical shifts are
reported in d (ppm) and coupling constants (J) are given

A. Hosoda et al. / Bioorg. Med. Chem. 10 (2002) 1855–1863
1861
H-6); 13C NMR (CDCl3) d 20.4, 20.5, 20.6, 68.4, 69.4,
70.7, 70.9, 169.6, 169.7, 169.8. Anal. calcd for
.
C16H22O11 0.5H2O: C, 48.12; H, 5.81. Found: C, 48.40;
H, 5.77.
zation from EtOH gave 7a as a white solid (0.128 g,
82%): mp 235–238 ꢁC; IR (KBr) n 3072, 2982, 2945,
2847, 1765, 1716, 1632, 1601, 1508, 1421, 1369, 1230,
1
1036, 984, 905, 837 cmꢀ1; H NMR (CDCl3) d 1.97 (s,
3H, Ac), 2.00 (s, 3H, Ac), 2.01 (s, 3H, Ac), 2.24 (s, 3H,
Ac), 2.33 (s, 6H, Ac), 3.88 (s, 6H, OCH3), 5.20–5.35 (m,
3H, CH), 5.62–5.74 (m, 3H, CH), 6.28 (d, 1H,
J=16.0 Hz, CH¼), 6.31 (d, 1H, J=16.0 Hz, CH¼),
7.00–7.12(m, 6H, ArH), 7.63 (d, 1H, J=16.0 Hz,
CH¼), 7.64 (d, 1H, J=16.0 Hz, CH¼); 13C NMR
(CDCl3) d 20.5, 20.6, 20.8, 56.0, 68.3, 68.7, 69.5, 69.6,
70.8, 111.3, 116.3, 116.4, 121.8, 123.3, 132.7, 132.8,
141.9, 145.9, 146.1, 151.5, 165.2, 165.4, 168.7, 169.5,
169.6, 169.7, 169.9. Anal. calcd for C38H40O18: C, 58.16;
H, 5.14. Found: C, 57.98; H, 5.13.
1,3,4,5,6-Penta-O-acetyl-2-O-[3-(40-acetoxy-30-methoxy-
phenyl)-2-propenoyl]-myo-inositol (4). To a solution of
12 (1.56 g, 4.0 mmol) in 50 mL CH2Cl2 were added suc-
cessively Et3N (0.814 g, 8.0 mmol), a catalytic amount of
DMAP and acid chloride 11 (2.04 g, 8.0 mmol). The
mixture was stirred at room temperature overnight. The
reaction was quenched by adding water, and the organic
layer was washed successively with saturated aqueous
NaHCO3 and brine. After the organic layer was dried
over Na2SO4, the solvent was removed under reduced
pressure. The residue was chromatographed (silica gel,
7:1 CHCl3/n-hexane) to give 4 (1.91 g, 78%) as a white
solid: mp 182–184 ꢁC; IR (KBr) n 2947, 2849, 1761,
1,6-O-Bis[3-(40-acetyloxy-30-methoxyphenyl)-2-propeno-
yl]-myo-inositol (14). The compound 13 (0.859 g,
1.0 mmol) was dissolved in THF (20 mL) and treated
with n-Bu4NFꢂ 3H2O (0.80 mg, 2.54 mmol) in the pre-
sence of benzoic acid (0.366 g, 3.0 mmol). After being
stirred at ꢀ5 to 0 ꢁC for 6 h, the reaction mixture was
diluted with ethyl acetate and successively washed with
brine, saturated aqueous NaHCO3, and brine. The
organic layer was dried over anhydrous Na2SO4. The
solvent was evaporated and the residue was chromato-
graphed (silica gel, CHCl3 then 1:1 ethyl acetate/EtOH,)
to give 14 (0.62g, 100%) as a white solid: mp 129–
132 ꢁC; IR (KBr) n 3350, 2941, 2843, 1765, 1716, 1636,
1732, 1637, 1601, 1508, 1226, 1151, 1045, 984, 908 cmꢀ1
;
1H NMR (CDCl3) d 2.00 (s, 6H, Ac), 2.03 (s, 6H, Ac),
2.04 (s, 3H, Ac), 2.34 (s, 3H, Ac), 3.94 (s, 3H, OCH3),
5.16 (dd, 2H, J=2.8, 10.0 Hz, H-1 and H-3), 5.22 (t, 1H,
J=10.0 Hz, H-5), 5.59 (t, 2H, J=10.0 Hz, H-4 and H-
6), 5.75 (t, 1H, J=2.8 Hz, H-2), 6.58 (d, 1H, J=15.6 Hz,
CH¼), 7.08–7.23 (m, 3H, ArH), 7.72 (d, 1H,
J=15.6 Hz, CH¼); 13C NMR (CDCl3) d 20.4, 20.6,
56.0, 68.2, 68.5, 69.5, 71.1, 110.9, 116.5, 122.1, 123.3,
132.8, 141.9, 146.1, 151.5, 165.6, 168.6, 169.5, 169.6,
169.7. Anal. calcd for C28H32O15: C, 55.26; H, 5.30.
Found: C, 54.98; H, 5.31.
1
1599, 1510, 1261, 1155, 1034, 1010, 904, 833 cmꢀ1; H
NMR (DMSO-d6) d 2.24 (s, 3H, Ac), 2.25 (s, 3H, Ac),
3.30–3.54 (m, 3H, CH), 3.79 (s, 6H, OCH3), 3.97 (m,
1H, H-2), 4.48 (d, 1H, J=5.6 Hz, OH), 4.87 (dd, 1H,
J=2.0, 10.0 Hz, H-1), 4.92 (d, 1H, J=4.8 Hz, OH), 5.19
(d, 1H, J=5.2Hz, OH), 5.28 (d, 1H, J=4.4 Hz, OH),
5.43 (t, 1H, J=10.0 Hz, H-6), 6.61 (d, 1H, J=16.0 Hz,
CH¼), 6.66 (d, 1H, J=16.0 Hz, CH¼), 7.07–7.47 (m,
6H, ArH), 7.56 (d, 1H, J=16.0 Hz, CH¼), 7.59 (d, 1H,
J=16.0 Hz, CH¼2); 13C NMR (DMSO-d6) d 20.3, 55.8,
55.9, 69.9, 71.0, 72.4, 72.5, 111.4, 111.5, 118.0, 118.5,
121.6, 121.8, 123.0, 123.1, 132.7, 132.9, 140.8, 141.0,
143.7, 144.3, 151.0, 151.1, 165.6, 168.3. Anal. calcd for
3,4,5,6-Tetra-O-acetyl-1, 2-O-bis[3-(40-hydroxy-30-meth-
oxyphenyl)-2-propenoyl]-myo-inositol (5c). The com-
pound 5a (0.39 g, 0.5 mmol) was dissolved in a mixture
of CHCl3 (10 mL) and MeOH (1 mL) and treated with
ꢂ
NH2NH2 H2O (48.5 mL, 1.0 mmol). After being stirred
at room temperature for 5 h, the reaction mixture was
diluted with CHCl3 and successively washed with satu-
rated aqueous KHSO4, saturated aqueous NaHCO3,
and brine. The organic layer was dried over anhydrous
Na2SO4. The solvent was evaporated and the residue
was recrystallized from EtOH/H2O to provide 5c as a
white solid (0.28 g, 80%): mp 121–124 ꢁC; IR (KBr) n
3430, 2943, 2843, 1755, 1724, 1631, 1590, 1516, 1431,
.
C30H32O14 H2O: C, 56.78; H, 5.40. Found: C, 56.98; H,
5.33.
1
1369, 1229, 1153, 1043, 982, 847, 820 cmꢀ1; H NMR
(DMSO-d6) d 1.92(s, 3H, Ac), 1.95 (s, 3H, Ac), 1.96 (s,
2,3,4,5-Tetra-O-acetyl-1,6-O-bis[3-(40-acetyloxy-30-meth-
oxyphenyl)-2-propenoyl]-myo-inositol (8a). To a solution
of 14 (0.312g, 0.507 mmol) in 2mL pyridine were added
acetic anhydride (1 mL, 10.0 mmol) and catalytic
amount of DMAP. The mixture was stirred for 2h at
room temperature. The mixture was poured on ice, and
resulting powder was collected. Recrystallization from
n-hexane/ethyl acetate gave 8a as a white solid (0.361 g,
91%): mp 118–121 ꢁC; IR (KBr) n 3074, 2943, 2843,
3H, Ac), 1.99 (s, 3H, Ac), 3.72(s, 3H, OCH ), 3.84 (s,
3
3H, OCH3), 5.40–5.69 (m, 6H, CH), 6.35 (d, 1H,
J=16.0 Hz), 6.70–7.45 (m, 8H, CH¼ and ArH), 7.61 (d,
1H, J=16.0 Hz), 9.67 (s, 1H, ArOH), 9.72(s, 1H,
ArOH); 13C NMR (DMSO-d6) d 20.3, 20.5, 55.8, 56.0,
67.7, 68.3, 68.7, 69.3, 69.6, 70.4, 111.1, 111.3, 112.9,
113.1, 115.6, 124.0, 124.5, 125.4, 125.5, 146.8, 147.4,
148.1, 148.2, 149.9, 150.0, 165.7, 166.2, 169.4, 169.6,
169.7. Anal. calcd for C34H36O16: C, 58.28; H, 5.18.
Found: C, 58.05; H, 5.19.
1
1759, 1724, 1635, 1601, 1369, 1155, 1036, 903 cmꢀ1; H
NMR (CDCl3) d 2.00 (s, 3H, Ac), 2.02 (s, 3H, Ac), 2.04
(s, 3H, Ac), 2.24 (s, 3H, Ac), 2.31 (s, 6H, Ac), 3.84 (s,
6H, OCH3), 5.18 (dd, 1H, J=2.8, 10.0 Hz, H-3), 5.30–
5.56 (m, 2H, H-1 and H-5), 5.57 (t, 1H, J=10.0 Hz, H-
4), 5.73–5.78 (m, 2H, H-2 and H-6), 6.25 (d, 1H,
J=16.0 Hz, CH¼), 6.28 (d, 1H, J=16.0 Hz, CH¼),
7.00–7.08 (m, 6H, ArH), 7.58 (d, 1H, J=16.0 Hz,
CH¼), 7.60 (d, 1H, J=16.0 Hz, CH¼); 13C NMR
1,2,4,5-Tetra-O-acetyl-3, 6-O-bis[3-(40-acetoxy-30-meth-
oxyphenyl)-2-propenoyl]-myo-inositol (7a). To a solution
of 7c (0.123 g, 0.2 mmol) in 2 mL pyridine was added
acetic anhydride (380 mL, 4.0 mmol). The mixture was
ꢁ
stirred for 2h at 90 C. The cooled mixture was poured
on ice, and resulting powder was collected. Recrystalli-

1862
A. Hosoda et al. / Bioorg. Med. Chem. 10 (2002) 1855–1863
(CDCl3) d 20.4, 20.5, 20.6, 20.8, 55.9, 68.3, 68.6, 68.8,
69.5, 70.8, 111.2, 116.3, 116.4, 121.8, 123.2, 123.3, 132.7,
132.8, 141.8, 145.9, 146.1, 149.5, 151.4, 165.2, 165.5,
168.6, 169.5, 169.7. Anal. calcd for C38H40O18: C, 58.16;
H, 5.14. Found: C, 58.03; H, 5.09.
(10,000 U/mL) and being placed on ice. After cen-
trifugation, the level of extracellular Oꢀ2 was measured
by the cytochrome c reduction method, in which
reduced cytochrome c was quantified by measuring the
visible absorption of the supernatant at 550 nm. Cells
treated with a compound, cytochrome c, and the vehicle
without TPA were used as a negative control, while cells
with the vehicle, but without the compound, cyto-
chrome c, or TPA were used as a positive control. Cells
treated with the vehicle, but without the compound,
cytochrome c, or the vehicle without TPA, were used as
a blank. Inhibitory rates were calculated by the follow-
ing formula: inhibitory rate (%)={1ꢀ[(test compound,
Abs550)ꢀ(negative, Abs550)/(positive, Abs550)ꢀ(blank,
Abs550)]}ꢃ100(%). Cell viability was determined by a
Trypan Blue dye exclusion test. Each experiment was
done independently in duplicate twice, and the data are
shown as mean values.
1,6-O-Bis[3-(40-hydroxy-30-methoxyphenyl)-2-propeno-yl]-
myo-inositol (8b). The compound 14 (0.247 g, 0.4 mmol)
was dissolved in methanol (5 mL) and treated with
ꢂ
NH2NH2 H2O (43 mL, 0.88 mmol). After being stirred
at room temperature for 2h, the reaction mixture was
diluted with ethyl acetate and successively washed with
saturated aqueous KHSO4 and brine. The organic layer
was dried over anhydrous Na2SO4. The solvent was
evaporated and the residue was recrystallized from
EtOH/H2O to provide 8b as white a solid (0.17 g, 80%):
mp 183–186 C; IR (KBr) n 3320, 2947, 2844, 1695, 1633,
1
1593, 1512, 1275, 1180, 1033, 1010, 845, 820 cmꢀ1; H
NMR (DMSO-d6) 3.30–3.60 (m, 3H, CH), 3.77 (s, 6H,
OCH3), 3.94 (m, 1H, H-2), 4.75 (d, 1H, J=4.8 Hz, OH),
4.80 (dd, 1H, J=2.4, 10.0 Hz, H-1), 4.86 (d, 1H,
J=4.4 Hz, OH), 5.11 (d, 1H, J=5.2Hz, OH), 5.20 (d,
1H, J=4.4 Hz, OH), 5.38 (t, 1H, J=10.0 Hz, H-6), 6.34
(d, 1H, J=16.0 Hz, CH¼), 6.39 (d, 1H, J=16.0 Hz,
CH¼), 6.70–7.24 (m, 6H, ArH), 7.46 (d, 1H,
J=16.0 Hz, CH¼), 7.48 (d, 1H, J=16.0 Hz, CH¼), 9.59
(m, 2H, ArOH); 13C NMR (CDCl3) d 55.8, 55.9, 70.1,
71.2, 72.3, 72.7, 111.1, 111.3, 114.5, 115.0, 115.6, 123.2,
123.5, 125.7, 125.8, 145.0, 145.6, 148.0, 148.1, 149.3,
EBV activation
An EBV activation inhibitory test was performed as
previously reported.14 Raji cells were incubated in 1 mL
of RPMI 1640 medium (supplemented with 10% FBS)
containing sodium n-butyric acid (BA) (3 mM), TPA
(50 nM), and the test compound (100 mM) at 37 ꢁC
under a 5% CO2 atmosphere for 48 h. EBV activation
was evaluated by the detection of EA, stained by an
indirect immunofluorescence method with high-titer
EA-positive sera from nasopharyngeal carcinoma
patients, followed by flourescein isothiocyanate (FITC)
labeled IgG. The rate of EA-induced cells was com-
pared with the rate obtained in a control experiment
using only BA, TPA, and DMSO [0.5% (v/ v)], in which
the rate of EA-induced cells was ordinarily around
40%. Inhibitory rates were calculated by the following
formula: inhibitory rate (%)={1ꢀ[(sample,%EA-posi-
tive cells)/(TPA+n-BA,%EA-positive cells)]}ꢃ100. Cell
viability (CV) was determined by a Trypan Blue dye
exclusion test. Each experiment was done in duplicate,
and the mean values are shown.
.
149.5, 166.3. Anal. calcd for C26H28O12 3H2O: C, 53.24;
H, 5.84. Found: C, 53.10; H, 5.71.
Chemicals and cells for bioassay
TPA was obtained from Research Biochemicals Inter-
national, Natick, MA. DMEM and RPMI 1640 media,
and fetal bovine serum (FBS) were purchased from
Gibco BRL, NY. FITC-labeled anti-human IgG was
obtained from Dako, (Glostrup, Denmark). Cyto-
chrome c was obtained from Sigma, MO. Other chemi-
cals were purchased from Wako Pure Chemical
Industries, Ltd., unless specified otherwise. Human B-
lymphoblastoid Raji cells and high-titer EBV-early
antigen (EA)-positive sera from naso-pharyngeal carci-
noma patients were kindly provided by. Dr. Ohsato
(Health Sciences University of Hokkaido, Japan).
Acknowledgements
This study was performed through support from the
Special Coordination Funds for Promoting Science and
Technology (Leading Research Utilizing Potential of
Regional Science and Technology) of the Ministry of
Education, Culture, Sports, Science and Technology of
the Japanese Government.
Oꢀ2 generation. Inhibitory tests of TPA-induced O2ꢀ
generation were performed as previously reported.13
HL-60 cells (5ꢃ105 cells/mL) were incubated in 1.25%
dimethylsulfoxide in RPMI medium (supplemented
with 10% FBS) for 6 days to induce differentiation into
granulocyte-like cells. Differentiated HL-60 cells, sus-
pended in 1 mL of Hank’s buffer, were treated with
100 mM (2 0mM) of each test compound (5 mL of stock
solution), or the vehicle. After preincubation at 37 ꢁC
for 15 min, the suspension was centrifuged and the
extracellular compounds were removed by washing with
1% bovine serum albumin (BSA) in Hank’s buffer. The
cells were then suspended in 1 mL of Hank’s buffer, and
incubated with 100 mM TPA or the vehicle and 1 mg/mL
cytochrome c at 37 ꢁC for 30 min. The reaction was ter-
minated by adding a superoxide dismutase solution
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