Synthesis of novel polyphenols consisted of ferulic and gallic acids, and their inhibitory effects on phorbol ester-induced Epstein-Barr virus activation and superoxide generation

Article abstract of DOI:10.1016/S0968-0896(01)00361-3

We prepared novel polyphenols which were esters composed of two naturally occurring products, ferulic and gallic acids, and investigated their inhibitory effects on 2-O-tetradecanoylphorbol- 3-acetate (TP )-induced Epstein Barr virus (EBV) activation and

Full text of DOI:10.1016/S0968-0896(01)00361-3

Bioorganic & Medicinal Chemistry 10 (2002) 1069–1075
Synthesis of Novel Polyphenols Consisted of Ferulic and Gallic
Acids, and Their Inhibitory Effects on Phorbol Ester-Induced
Epstein–Barr Virus Activation and Superoxide Generation
Eisaku Nomura,a,* Asao Hosoda,a Hideko Morishita,b Akira Murakami,c
Koichi Koshimizu,c Hajime Ohigashid and Hisaji Taniguchia,*
aIndustrial Technology Center of Wakayama Prefecture, 60 Ogura, Wakayama 649-6261, Japan
bFaculty of Education, Wakayama University, 930 Sakaedani, Wakayama 640-8510, Japan
cFaculty of Biology-Oriented Science and Technology, Kinki University, Iwade-Uchita, Wakayama 649-6493, Japan
dDivision of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
Received 31 August 2001; accepted 11 October 2001
Abstract—We prepared novel polyphenols which were esters composed of two naturally occurring products, ferulic and gallic acids,
and investigated their inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Epstein–Barr virus (EBV) activ-
ation and superoxide (Oꢀ2 ) generation. Most of these compounds exhibited significant EBV activation suppression at a concentra-
tion of 20 mM and in particular, the ester 5f having 2-metyl-1-butyl group showed high activity. The suppressive effects on O2ꢀ
generation were also observed in most of the esters. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
and mio-inositol exert the inhibitory effect on super-
oxide generation.10
Ferulic acid 1a is a phenolic compound and some
authors have recently developed a mass production
method of 1a from rice bran.1,2 The acid 1a is known to
form various derivatives having a variety of biological
functions by combining with other compounds. For
example, chlorogenic acids are natural products con-
taining ferulic acid skeleton and they have the potential
for anti-carcinogenesis.3,4 Although there are many
natural compounds which have biological activities, it is
difficult to utilize practically as chemopreventive agents
due to the presence of small quantities of these com-
pounds in plants. Therefore, interest in the design of the
synthetic compounds with enhanced chemopreventive
activities has intensified in recent years.5,6 The studies
along with this line were performed by some authors.
Alkyl ferulates have a high anti-carcinogenic potential.7
A ferulic acid derivative in which geranyl group is
attached to the phenolic hydroxyl group of ethyl fer-
ulate exhibits colon anti-carcinogenesis in rodent mod-
els.2,8,9 The ester compounds consisting of ferulic acid
We now prepared novel polyphenols which were com-
posed of two naturally occurring products, ferulic acid
and gallic acid, and assessed their anti-caricinogenic
potential. It was found that these polyphenols were
obtained in quantity and had higher activity as an anti-
carcinogen than the original phytochemicals. Gallic acid
2a is a component of plant tannin and exhibits a variety
of chemical and biological properties that include phar-
macological activity.11,12 In this paper, we describe the
condensation of the two molecules and investigations of
inhibitory effects of the conjugates on 12-O-tetra-
decanoylphorbol-13-acetate (TPA)-induced Epstein–
Barr virus (EBV) activation and superoxide generation
in vitro.13,14
*Corresponding author. Tel.: +81-73-477-1271; fax: +81-73-477-
2880; e-mail: nomura@wakayamakg.go.jp
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00361-3

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E. Nomura et al. / Bioorg. Med. Chem. 10 (2002) 1069–1075
Results and Discussion
O(10)ꢁ ꢁ ꢁ O(3) are 2.795(5) and 2.845(6) A, respectively.
The rest of the hydroxyl group of galloyl group forms a
hydrogen bond with the ester oxygen O(7) of neighbor-
ing molecule and the distance is 2.722(5) A.
Chemistry
Two types of esters can be prepared by the esterification
of ferulic acid with gallic acid. One is a family that gal-
loyl group introduced into the hydroxyl group of ferulic
acid. The other is a family that feruloyl group intro-
duced into the hydroxyl groups of gallic acid. The latter
family has five possible kinds of esters as feruloyl group
can be stayed in the three hydroxyl groups of gallic acid.
We chose compounds of the former family because it
was easy to prepare and structurally simple. Various
types of alkyl ferulates 1b were chosen to the condensa-
tion with gallic acid because some authors reported
previously that 1b were more effective chemopreventive
agents than 1a.7 The reaction was performed by the
reaction of alkyl ferulates with O-acetyl galloyl chloride,
followed by deacetylation as shown in Scheme 1. O-
acetyl gallic acid was prepared by the reaction of gallic
acid with acetic anhydride in aqueous NaOH in high
yield and then converted to the acid chloride using
thionyl chloride in a quantitative yield. The esterifica-
tion was performed in dichloromethane in the presence
of triethylamine at room temperature to afford corre-
sponding esters 5a–g in moderate or high yields (67–
86%). The removal of acetyl groups without cleaving
ester bond was carried out using hydrazine mono-
hydrate at room temperature to yield 6a–g. The com-
pounds which we prepared are listed in Table 1. The
esters 6a–g are target polyphenols to estimate biological
activities. The acetylated compounds 5a–g might exhibit
the biological activities because naturally occurring
product such as 10-actoxychavicol acetate showed the
inhibition of a tumor promotion.15 Therefore, we used
the esters 5a–g and 6a–g to assess their anti-caricino-
genic potential.
Biological activity
For the ester compounds prepared, we investigated their
suppressive effects on 12-O-tetradecanoylphorbol-13-
acetate (TPA)-induced EBV activation in vitro in order
to estimate their anti-carcinogenic potential. This assay
system was established for the evaluation of anti-tumor-
promoting activities in vitro.13,14 The results were
shown in Figure 2. The compounds 5a–g are OH-pro-
tected hydrophobic molecules and 6a–g are molecules
carrying three OH groups. As shown in Figure 2A, 1a
6
was inactive at a concentration of 100 mM and methyl
gallate 2b, which is a control compound, showed mod-
erate suppression (inhibitory rate of 64%) of the EBV
activation. The esters 5a and 6a showed marked sup-
pression of the EBV activation (inhibitory rates of
100%) without notable cytotoxicity. On the other hand,
while 5b–g and 6b–g exhibited high suppression of the
EBV
activation, substantial cytotoxicity
(cell
viability=CV<25%) to Raji cells was observed at a
concentration of 100 mM. Figure 2B shows the activities
for 5b–g and 6b–g at a concentration of 20 mM. All
compounds tested still exhibited high suppression (inhi-
bitory rates of 70–100%) and low cytotoxicity
(CV=60–81%) at this concentration. The suppressive
effects of alkyl ferulates 1b at a concentration of 20 mM
were previously reported.7 The ferulates 1b carrying
straight chains (carbon atom number: C5–C12) showed
the activation (inhibitory rates of 3.3–67.5%) and only
1b having 2-methyl-1-hexyl group exhibited high-sup-
pression (inhibitory rate of 100%). We then examined
the activities for 5a–g and 6a–g at a lower concentration
of 4 mM as shown in Figure 2C. The compounds 5a–b
and 6a–b showed weak suppressive activity (inhibitory
rates of 13–30%), and 5c–e and 6c–e exhibited low or
no activities at 4 mM. Moreover, 5f–g and 6f–g, having
branched alkyl groups, showed weak or high activity
(inhibitory rates of 10–83%) and it was found that 5f
and 6f were higher potent suppressor (inhibitory rates of
The ester 6f was recrystallized from methanol to give
prism crystals which included methanol molecules inside
the crystals. The crystal structure of 6f was determined
by a single crystal X-ray analysis (Fig. 1). Two metha-
nol molecules are captured in the crystal lattice and
form hydrogen bonds with two hydroxyl groups of
galloyl group. The distances of O(9)ꢁ ꢁ ꢁ O(2) and
Scheme 1.

E. Nomura et al. / Bioorg. Med. Chem. 10 (2002) 1069–1075
1071
62 and 83%, respectively) among the compounds eval-
uated.
molecules to incorporate into cells, which were demon-
strated by the cellular uptake experiments.
The suppressive effects on the NADPH oxidase system
responsible for TPA-induced Oꢀ2 generation were
examined using DMSO-differentiated HL-60 cells, a
neutrophil model. The suppressive effects for 5a–g and
6a–g at 20 mM are shown in Figure 3. The compounds
5a–c, 5f–g, 6a–c, and 6g exhibited high suppressive
activities on Oꢀ2 generation (inhibitory rates of 53–
100%), however, and 5d–e and 6d–f were less potent
(inhibitory rates of 13–40%). It was noteworthy that 5c
and 5f suppressed Oꢀ2 generations by 100% and 6f was
less active (inhibitory rates of 13%) in contrast to the
data shown in the EBV activation test. The structural
difference between 5f and 6f is whether phenolic
hydroxyl groups are protected or not.
Conclusion
The novel polyphenols consisting of two naturally
occurring compounds, ferulic and gallic acids, were
prepared. Most of these compounds exhibited their
inhibitory abilities on TPA-induced EBV activation and
Oꢀ2 generation in vitro. In particular, 5f could be the
most promising chemopreventive agents among the
compounds evaluated in the present study. Mass pro-
duction method of ferulic acid from rice bran was
developed in recent years1,2 and the production of gallic
acid was already on a commercial basis. Therefore, it is
valuable to obtain more effective anti-carcinogenic
agents as derivatives from natural compounds for prac-
tical use. While the present study provided the anti-car-
cinogenic capability of the conjugates in the first stage
In the EBV activation assay, an important structural
factor seems to be not the presence of the OH group but
the nature of alkyl chain. On the other hand, in attenu-
ating Oꢀ2 generation, the presence of the OH groups and
the nature of alkyl chain affect the activities. The OH
group and alkyl groups in bioactive phytochemicals are
supposed to be effective or ineffective depending on
assay system or permeability of cellular membrane. For
example, alkyl ferulates having a longer or branched
alkyl chain are highly active in above both assay sys-
tems.7 It was assumed that their potent suppressors of
Oꢀ2 generation were correlated to hydrophobicity of the
Table 1. The esters consisting of farulic and gallic acids
Compound
R1
R2
Yield (%)
5a
6a
5b
6b
5c
6c
5d
6d
5e
6e
5f
Acetyl
H
Acetyl
H
Acetyl
H
Acetyl
H
Acetyl
H
Acetyl
H
Acetyl
H
Ethyl
Ethyl
Butyl
Butyl
Hexyl
Hexyl
Decyl
Decyl
Undecyl
Undecyl
83
60
86
70
84
78
73
83
67
73
67
71
81
42
2-Methyl-1-butyl
2-Methyl-1-butyl
2-Ethyl-1-hexyl
2-Ethyl-1-hexyl
6f
5g
6g
Figure 2. Suppressive effects of the polyphenols on TPA-induced EBV
activation in Raji cells. Cells were incubated with n-BA (3 mM), TPA
(50 nM), and test compounds (0, 4 (panel C), 20 (panel B), or 100
(panel A) mM). EBV activation was measured by detecting EA-induc-
tion. Open and closed bars represent cell viability (%) and EBV acti-
vation suppression (%), respectively. Data are shown as mean values
from duplicate experiments.
Figure 1. ORTEP drawing of 6f.

1072
E. Nomura et al. / Bioorg. Med. Chem. 10 (2002) 1069–1075
of the assay systems, the studies of these compounds in
rodent model in the future will serve to elucidate the
efficacies.
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).
Experimental
General
EBV activation
An EBV activation inhibitory test was performed as
previously reported.16 Raji cells were incubated in 1mL
of RPMI 1640 medium (supplemented with 10% FBS)
containing sodium n-butyric acid (BA) (3 mM), TPA
(50 nM), and the test compound (4, 20, and 100 mM) at
37 ꢂC under a 5% CO2 atmosphere for 48 h. EBV acti-
vation 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)]}Â100. Cell
viability (CV) was determined by a Trypan Blue dye
exclusion test. Each experiment was done in duplicate,
and the mean values are shown.
Ferulic acid and alkyl ferulate were generously supplied
by Tsuno Rice Fine Chemicals Co., Ltd. (Wakayama,
Japan). Gallic acid and methyl gallate were kindly sup-
plied by Fuji Chemical Co., Ltd. (Wakayama, Japan).
Anhydrous CH2Cl2 was purchased from Kanto Chemi-
cal Co., Inc. (Tokyo, Japan) and used without further
purification. Other solvents and reagents were pur-
chased from Wako Pure Chemical Industries, Ltd.
(Osaka, Japan), and used without further purification.
Using acetic anhydride in aqueous NaOH was
performed acetylation for hydroxyl groups of gallic
acid. O-acetylgallic acid was converted to the acid chlo-
ride by using thionyl chloride in the presence of pyridine
and a small amount of DMF. Melting points were
determined by a Yanaco micro melting point apparatus
and are uncorrected. Elemental analyses were per-
1
formed on a Perkin–Elmer 2400II. H and 13C NMR
spectra were recorded on a Varian Unity-plus 400 spec-
trometer using a residual solvent as an internal stan-
dard. FT-IR spectra were obtained on a Shimadzu FT-
IR 8200D spectrometer using a diffuse reflectance cell.
Oꢀ2 generation
Inhibitory tests of TPA-induced Oꢀ2 generation were
performed as previously reported.13 HL-60 cells
(5Â105 cells/mL) were incubated in 1.3% dimethylsulf-
oxide in RPMI medium (supplemented with 10% FBS)
for 6 days to induce differentiation into granulocyte-
mimic cells. Differentiated HL-60 cells, suspended in
1mL of Hank’s buffer, were treated with 20 mM of each
test compound (5 mL of stock solution), or the vehicle.
After preincubation at 37 ꢂC 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 1mL of Hank’s buffer, and incubated with 100 mM
TPA or the vehicle and 1mg/mL cytochrome c at 37 ꢂC
for 30 min. The reaction was terminated by adding a
superoxide dismutase solution (10,000 U/mL) and being
placed on ice. After centrifugation, the level of extra-
cellular Oꢀ2 was measured by the cytochrome c reduc-
tion 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, cytochrome 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 following formula: inhibitory
rate (%)={1 ꢀ [(test compound, Abs550) ꢀ (negative,
Abs550)/(positive, Abs550) ꢀ (blank, Abs550)]} Â 100(%).
Cell viability was determined by a Trypan Blue dye
exclusion test. Each experiment was done independently
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
Figure 3. Suppressive effects of the polyphenols on TPA-induced O2ꢀ
generation in differentiated HL-60 cells. Oꢀ2 generation was induced by
TPA (50 nM) and detected by cytochrome c. Sample concentrations
were 0 or 20 mM). Open and closed bars represent cell viability (%)
and Oꢀ2 generation suppression (%), respectively. Data are shown as
mean values from duplicate experiments.

E. Nomura et al. / Bioorg. Med. Chem. 10 (2002) 1069–1075
1073
1
in duplicate twice, and the data are shown as mean
values.
1508 cmꢀ1; H NMR (CDCl3) d 0.86 (t, 3H, J=6.9 Hz,
CH3), 1.2–1.5 (m, 14H, CH2), 1.66–1.71 (m, 2H, CH2),
2.30 (s, 6H, Ac), 2.31(s, 3H, Ac), 3.82 (s, 3H, OCH ),
3
4.19 (t, 2H, J=6.8 Hz, OCH2), 6.39 (d, 1H, J=16.0 Hz,
¼CH), 7.10–7.13 (m, 3H, ArH), 7.64 (d, 1H,
J=16.0 Hz, ¼CH), 7.94 (s, 2H, ArH); 13C NMR
(CDCl3) d 14.1, 20.2, 20.6, 22.7, 26.0, 28.7, 29.3, 29.5,
32.0, 56.0, 64.8, 111.2, 118.6, 121.2, 123.0, 123.2, 127.3,
133.7, 139.2, 141.2, 143.5, 143.8, 151.4, 162.3, 166.4,
166.9, 167.6. Anal. calcd for C33H40O11: C, 64.69%; H,
6.55%; found: C, 64.31%; H, 6.55%.
General procedure of the reaction of alkyl ferulate with
3,4,5-triacetoxybenzoyl chloride
To a solution of 0.5 g of alkyl ferulate and triethylamine
(two times molar amounts of alkyl ferulate) in anhy-
drous CH2Cl2 (20 mL) was added 3,4,5-triacetoxy-
benzoyl chloride (two times molar amounts of alkyl
ferulate). The mixture was stirred for 4 h at rt under
nitrogen. Ice-water was added to the reaction mixture,
and the organic portion was extracted with CH2Cl2
(20 mL Â 3) and dried over MgSO4. After removal of
the solvent, the product was purified through SiO2 col-
umn (CHCl3) and recrystallzed from CHCl3/MeOH.
Undecyl 3-{4-(3,4,5-triacetyloxybenzoayloxy)-3-meth-oxy-
phenyl}-2-propenoate (5e). 0.59 g (67%); mp 97–99 ꢂC;
IR (KBr)? n 1774, 1746, 1715, 1636, 1601, 1508 cmꢀ1
;
1H NMR (CDCl3) d 0.86 (t, 3H, J=6.9 Hz, CH3), 1.2–
1.4 (m, 16H, CH2), 1.67–1.71 (m, 2H, CH2), 2.30 (s, 6H,
Ethyl
3-{4-(3,4,5-triactyloxybenzoayloxy)-3-methoxy
Ac), 2.31(s, 3H, Ac), 3.82 (s, 3H, OCH ), 4.19 (t, 2H,
3
phenyl}-2-propenoate (5a). 0.95 g (83%) of crystals; mp
148–149 ꢂC; IR (KBr) n 1782, 1747, 1713, 1641, 1599,
J=6.8 Hz, OCH2), 6.39 (d, 1H, J=15.9 Hz, ¼CH),
7.10–7.13 (m, 3H, ArH), 7.64 (d, 1H, J=15.9 Hz,
¼CH), 7.94 (s, 2H, ArH); 13C NMR (CDCl3) d 14.1,
20.2, 20.6, 22.7, 26.0, 28.7, 29.3, 29.3, 29.5, 29.6, 31.9,
55.9, 64.8, 111.2, 118.7, 121.2, 123.0, 123.2, 127.4, 133.7,
139.2, 141.2, 143.5, 143.8, 151.4, 162.3, 166.4, 166.9,
167.6. Anal. calcd for C34H42O11: C, 65.16%; H, 6.76%;
found: C, 64.71%; H, 6.68%.
1
1508 cmꢀ1; H NMR (CDCl3) d 1.33 (t, 3H, J=7.1Hz,
CH3) 2.30 (s, 6H, Ac), 2.31(s, 3H, Ac), 3.82 (s, 3H,
OCH3), 4.26 (q, 2H, J=7.1Hz, CH 2), 6.39 (d, 1H,
J=16.0 Hz,=CH), 7.11–7.13 (m, 3H, ArH), 7.64 (d,
1H,J=16.0 z,=CH), 7.94 (s, 2H, ArH); 13C NMR
(CDCl3) d 14.3, 20.2, 20.6, 55.9, 60.6, 111.2, 118.6, 121.2,
123, 123.2, 127.3, 133.7, 139.2, 141.2, 143.5, 143.8, 151.4,
162.3, 166.4, 166.8, 167.6. Anal. calcd for C25H24O11: C,
60.00%; H, 4.83%; found: C, 59.96%; H, 4.72%.
2-Methyl-1-butyl 3-{4-(3,4,5-triacetyloxybenzoayloxy)-
3-methoxyphenyl}-2-propenoate (5f). 0.69 g (67%); mp
140–142 ꢂC; IR (KBr) n 1784, 1753, 1699, 1636, 1601,
Butyl 3-{4-(3,4,5-triacetyloxybenzoayloxy)-3-methoxy-
phenyl}-2-propenoate (5b). 0.86 g (86%) of crystals; mp
143–144.5 ꢂC; IR (KBr) n 1784, 1753, 1701, 1638, 1601,
1508 cmꢀ1 1H NMR (CDCl3) d 0.91–0.97 (m, 6H,
;
CH3), 1.19–1.26 (m, 1H, CH2), 1.26–1.54 (m, 1H, CH2),
1.74–1.82 (m, 1H, CH), 2.30 (s, 6H, OAc), 2.31 (s, 3H,
OAc), 3.83 (s, 3H, OCH3), 3.98–4.11 (m, 2H, OCH2),
6.40 (d, 1H, J=15.9 Hz, ¼CH), 7.10–7.16 (m, 3H,
ArH), 7.64 (d, 1H, J=15.9 Hz, ¼CH), 7.94 (s, 2H,
ArH); 13C NMR (CDCl3) d 11.3, 16.5, 20.2, 20.6, 26.1,
34.2, 55.9, 69.3, 111.2, 118.7, 121.2, 123.0, 123.2, 127.3,
133.7, 139.2, 141.2, 143.5, 143.8, 151.4, 162.3, 166.4,
166.9, 167.6. Anal. calcd for C28H30O11: C, 61.99%; H,
5.57%; found: C, 61.41%; H, 5.45%.
1508 cmꢀ1
;
1H NMR (CDCl3)
d
0.95 (t, 3H,
J=7.3 Hz, CH3), 1.43 (m, 2H, CH2), 1.68 (m, 2H,
CH2), 2.30 (s, 6H, Ac), 2.31(s, 3H, Ac), 3.82 (s, 3H,
OCH3), 4.20 (t, 2H, J=6.7 Hz, OCH2), 6.39 (d, 1H,
J=15.9 Hz, ¼CH), 7.10–7.13 (m, 3H, ArH), 7.64 (d,
1H, J=15.9 Hz, ¼ CH), 7.94 (s, 2H, ArH); 13C NMR
(CDCl3) d 13.8, 19.2, 20.2, 20.6, 30.7, 55.9, 64.5, 111.2,
118.6, 121.2, 123.0, 123.2, 127.3, 133.7, 139.2, 141.2,
143.5, 143.8, 151.4, 162.3, 166.4, 167.0, 167.6. Anal.
.
calcd for C27H28O11 0.5H2O: C, 60.33%; H, 5.44%;
found: C, 60.00%; H, 5.10%.
2-Ethyl-1-hexyl 3-{4-(3,4,5-triacetyloxybenzoayloxy)-3-
methoxyphenyl}-2-propenoate (5g). 0.76 g (81%); mp
116–118 ꢂC; IR (KBr) n 1784, 1726, 1593, 1491 cmꢀ1. 1H
NMR (CDCl3) d 0.87–0.93 (m, 6H, CH3), 1.31–1.43 (m,
8H, CH2), 1.61–1.65 (m, 1H, CH), 2.30 (s, 6H, OAc),
Hexyl 3-{4-(3,4,5-triacetyloxybenzoayloxy)-3-methoxy-
phenyl}-2-propenoate (5c). 0.84 g (84%) of crystals; mp
144–145 ꢂC; IR (KBr) n 1798, 1774, 1740, 1713, 1639,
2.31(s, 3H, OAc), 3.83 (s, 3H, OCH ), 4.10–4.13 (m,
3
1599, 1515 cmꢀ1
;
1H NMR (CDCl3) d 0.89 (t, 3H,
2H, OCH2), 6.39 (d, 1H, J=15.9 Hz, ¼CH), 7.10–7.14
(m, 3H, ArH), 7.63 (d, 1H, J=15.9 Hz, ¼CH), 7.94 (s,
2H, ArH). 13C NMR (CDCl3) d 11.0, 14.1, 20.2, 20.6,
23.0, 23.8, 29.0, 30.4, 38.9, 55.9, 67.1, 111.2, 118.7,
121.2, 123.0, 123.2, 127.3, 133.7, 139.2, 141.2, 143.5,
143.7, 151.4, 162.3, 166.4, 167.0, 167.6. Anal. calcd for
C31H36O11: C, 63.69%; H, 6.21%; found: C, 63.48%; H,
6.12%.
J=7.1Hz, CH 3), 1.31–1.40 (m, 6H, CH2), 1.67–1.71 (m,
2H, CH2), 2.30 (s, 6H, Ac), 2.31(s, 3H, Ac), 3.82 (s, 3H,
OCH3), 4.19 (t, 2H, J=6.9 Hz, OCH2), 6.39 (d, 1H,
J=15.8 Hz, ¼CH), 7.10–7.13 (m, 3H, ArH), 7.64 (d,
1H, J=15.8 Hz, ¼CH), 7.94 (s, 2H, ArH); 13C NMR
(CDCl3) d 14.0, 20.2, 20.6, 22.5, 25.6, 28.7, 31.5, 55.9,
64.8, 111.2, 118.6, 121.2, 123.0, 123.2, 127.3, 133.7,
139.2, 141.1, 143.5, 143.8, 151.4, 162.3, 166.4, 166.9,
167.6. Anal. calcd for C29H32O11: C, 62.58%; H, 5.80%;
found: C, 62.71%; H, 5.72%.
General procedure of the reaction of deacetylation
To a solution of 0.5 g of the esters 5 in 20 mL acetoni-
trile was added hydrazine monohydrate (three times
molar amounts of alkyl ferulate). The mixture was stir-
red for 15 min at rt. Acetic acid was added to the mix-
Decyl 3-{4-(3,4,5-triacetyloxybenzoayloxy)-3-methoxy-
phenyl}-2-propenoate (5d). 0.67 g (73%) of crystals; mp
118–120 ꢂC; IR (KBr) n 1778, 1740, 1717, 1639, 1601,

1074
E. Nomura et al. / Bioorg. Med. Chem. 10 (2002) 1069–1075
ture. The organic portion was extracted with ethyl ace-
tate (50 mL), washed with water (30 mL Â 2) and satd
aqueous NaCl (20 mL), and dried over MgSO4. The
solvent was evaporated to give a sold.
J=8.2 Hz, ArH), 7.54 (d, 1H, J=1.6 Hz, ArH), 7.64 (d,
1H,J=15.9 Hz, ¼CH), 9.3 (brs, 3H, OH); 13C NMR
(DMSO-d6) d 14.2, 22.3, 25.6, 28.4, 28.9, 29.2, 31.5,
56.2, 64.2, 109.4, 112.0, 118.0, 118.5, 121.9, 123.7, 133.1,
139.5, 141.6, 144.1, 145.9, 151.6, 164.1, 166.5. Anal.
.
Ethyl 3-{4-(3,4,5-trihydroxybenzoayloxy)-3-methoxy-
phenyl}-2-propenoate (6a). Recrystallized from MeOH
calcd for C27H34O8 H2O: C, 64.27%; H, 7.19%; found:
C, 63.98%; H, 7.12%.
to afford 0.224 g (60%) of crystals: mp 241–244 ꢂC; IR
1
(KBr) n 3477, 1736, 1666, 1612, 1537, 1508 cmꢀ1; H
Undecyl 3-{4-(3,4,5-trihydroxybenzoayloxy)-3-methoxy-
phenyl}-2-propenoate (6e). Recrystallized from MeOH/
CHCl3 to afford 0.29 g (73%) of crystals: mp 92–
94.5 ꢂC; IR (KBr) n 3659, 3558, 3518, 3269, 1728, 1684,
1634, 1601, 1512 cmꢀ1; 1H NMR (CDCl3) d 0.86 (t, 3H,
J=6.9 Hz, CH3), 1.1–1.4 (m, 16H, CH2), 1.67–1.71 (m,
2H, CH2), 3.76 (s, 3H, OCH3), 4.20 (t, 2H, J=6.8 Hz,
OCH2), 6.39 (d, 1H, J=15.9 Hz, ¼CH), 7.06–7.13 (m,
3H, ArH), 7.31(s, 2H, ArH), 7.65 (d, 1H, J=15.9 Hz,
¼CH); 13C NMR (CDCl3) d 14.1, 22.7, 26.0, 28.7, 29.27,
29.32, 29.5, 29.6, 31.9, 55.9, 65.1, 110.6, 111.4, 118.2,
120.4, 121.2 123.3, 133.3, 137.1, 141.7, 143.6, 144.3, 151.5,
NMR (DMSO-d6) d 1.26 (t, 3H, J=7.1Hz, CH 3), 3.80
(s, 3H, OCH3), 4.19 (q, 2H, J=7.1Hz, CH 2), 6.72 (d,
1H, J=16.0 Hz, ¼CH), 7.06 (s, 2H, ArH), 7.20 (d, 1H,
J=8.2 Hz, ArH), 7.32 (d, 1H, J=8.2 Hz, ArH), 7.54 (s,
1H, ArH), 7.65 (d, 1H, J=16.0 Hz, ¼CH), 9.3 (brs, 3H,
OH); 13C NMR (DMSO-d6) d 14.4, 56.2, 60.3, 109.4,
112.1, 118.0, 118.6, 121.9, 123.7, 133.1, 139.5, 141.6,
144.1, 146.0, 151.6, 164.1, 166.5. Anal. calcd for
C19H18O8: C, 60.96%; H, 4.85%; found: C, 60.74%; H,
4.80%.
.
Butyl
3-{4-(3,4,5-trihydroxybenzoayloxy)-3-methoxy-
164.5, 167.4. Anal. calcd for C28H36O8 H2O: C, 64.85%;
H, 7.39%; found: C, 64.82%; H, 7.33%.
phenyl}-2-propenoate (6b). Recrystallized from MeOH
to afford 0.28 g (70%) of crystals: mp 168–169 ꢂC; IR
(KBr) n 3476, 3310, 1724, 1684, 1635, 1610, 1601, 1541,
2-Methyl-1-butyl 3-{4-(3,4,5-trihydroxybenzoayloxy)-3-
methoxyphenyl}-2-propenoate (6f). Recrystallized from
MeOH/CHCl3 to afford 0.27 g (71%) of crystals: mp
150–153 ꢂC; IR (KBr) n 3310, 1724, 1682, 1636, 1601,
1514 cmꢀ1
;
1H NMR (DMSO-d6) d 0.91(t, 3H,
J=7.3 Hz, CH3), 1.33–1.43 (m, 2H, CH2), 1.59–1.66 (m,
2H, CH2), 3.80 (s, 3H, OCH3), 4.15 (t, 2H, J=6.7 Hz,
OCH2), 6.71(d, 1H, J=16.0 Hz, ¼CH), 7.06 (s, 2H,
ArH), 7.20 (d, 1H, J=8.4 Hz, ArH), 7.32 (dd, 1H,
J=8.4, 1.6 Hz, ArH), 7.54 (d, 1H, J=1.6 Hz, ArH), 7.65
(d, 1H, J=16.0 Hz, ¼CH), 9.3 (brs, 3H, OH); 13C NMR
(DMSO-d6) d 13.81, 18.9, 30.5, 56.2, 64.0, 109.4, 112.1,
118.0, 118.5, 121.9, 123.7, 133.1, 139.5, 141.6, 144.1,
146.0, 151.6, 164.1, 165.6. Anal. calcd for
1
1514 cmꢀ1; H NMR (DMSO-d6) d 0.87–0.94 (m, 6H,
CH3), 1.16–1.21 (m, 1H, CH2), 1.42–1.48 (m, 1H, CH2),
1.71–1.76 (m, 1H, CH), 3.80 (s, 3H, OCH3), 3.95–4.06
(m, 2H, OCH2), 6.73 (d, 1H, J=15.9 Hz, ¼CH), 7.06 (s,
2H, ArH), 7.20 (d, 1H, J=8.2 Hz, ArH), 7.32 (dd, 1H,
J=8.2, 1.8 Hz, ArH), 7.55 (d, 1H, J=1.8 Hz, ArH), 7.65
(d, 1H, J=15.9 Hz, ¼CH), 9.15 (brs, 1H, OH), 9.41
(brs, 2H, OH); 13C NMR (DMSO-d6) d 11.3, 16.4, 25.7,
33.9, 56.2, 68.6, 109.4, 112.0, 118.0, 118.5, 122.0, 123.7,
133.1, 139.5, 141.6, 144.1, 145.9, 151.6, 164.1, 166.6.
.
C21H22O8 H2O: C, 59.99%; H, 5.75%; found: C,
60.02%; H, 5.31%.
.
Hexyl
3-{4-(3,4,5-trihydroxybenzoayloxy)-3-methoxy-
Anal. calcd for C22H24O8 1.5H2O: C, 59.59%; H,
6.14%; found: C, 59.83%; H, 5.87%.
phenyl}-2-propenoate (6c). Recrystallized from MeOH
to afford 0.30 g (78%) of crystals: mp 138–140 ꢂC; IR
(KBr) n 3580, 3477, 3327, 1774, 1724, 1690, 1639, 1599,
2-Ethyl-1-hexyl 3-{4-(3,4,5-trihydroxybenzoayloxy)-3-
methoxyphenyl}-2-propenoate (6g). Recrystallized from
MeOH/H2O to afford 0.16 g (42%) of crystals: mp
130.5–132 ꢂC; IR (KBr) n 3320, 1726, 1688, 1600–1670,
1
1537, 1514 cmꢀ1; H NMR (DMSO-d6) d 0.87 (t, 3H,
J=6.9 Hz, CH3), 1.28–1.36 (m, 6H, CH2), 1.60–1.65 (m,
2H, CH2), 3.80 (s, 3H, OCH3), 4.14 (t, 2H, J=6.7 Hz,
OCH2), 6.72 (d, 1H, J=16.0 Hz, ¼CH), 7.06 (s, 2H,
ArH), 7.20 (d, 1H, J=8.1Hz, ArH), 7.32 (dd,
1H,J=8.1, 1.6 Hz, ArH), 7.54 (d, 1H, J=1.6 Hz, ArH),
7.65 (d, 1H, J=16.0 Hz, ¼CH), 9.15 (bs, 1H, OH), 9.41
(bs, 2H, OH); 13C NMR (DMSO-d6) d 14.1, 22.2, 25.3,
28.4, 31.1, 56.2, 64.2, 109.4, 112.0, 118.0, 118.5, 121.9,
123.7, 133.1, 139.5, 141.6, 144.1, 145.9, 151.6, 164.1,
166.6. Anal. calcd for C23H26O80.5H2O: C, 62.86%; H,
6.19%; found: C, 63.17%; H, 5.95%.
1
1514 cmꢀ1; H NMR (DMSO-d6) d 0.86–0.90 (m, 6H,
CH3), 1.29–1.39 (m, 8H, CH2), 1.58–1.63 (m, 1H, CH),
3.80 (s, 3H, OCH3), 4.06–4.10 (m, 2H, OCH2), 6.72 (d,
1H, J=15.9 Hz, ¼CH), 7.06 (s, 2H, ArH), 7.20 (d, 1H,
J=8.2 Hz, ArH), 7.32 (d, 1H, J=8.2 Hz, ArH), 7.55 (s,
1H, ArH), 7.64 (d, 1H, J=15.9 Hz, ¼CH), 9.15 (brs,
1H, OH), 9.41 (brs, 2H, OH); 13C NMR (DMSO-d6) d
11.0, 14.1, 22.6, 23.4, 28.6, 30.0, 38.5, 56.2, 66.3, 109.4,
112.0, 118.0, 118.5, 122.0, 123.7, 133.1, 139.5, 141.6,
144.1, 145.9, 151.6, 164.1, 166.6. Anal. calcd for
.
Decyl
3-{4-(3,4,5-trihydroxybenzoayloxy)-3-methoxy-
C25H30O8 H2O: C, 63.01%; H, 6.77%; found: C,
63.09%; H, 6.69%.
phenyl}-2-propenoate (6d). Recrystallized from MeOH/
CHCl3 to afford 0.33 g (83%) of crystals: mp 114–
115 ꢂC; IR (KBr) n 3490, 3305, 1726, 1690, 1634, 1601,
Single crystal X-ray diffraction for 6f
1
1551, 1514 cmꢀ1; H NMR (DMSO-d6) d 0.84 (t, 3H,
J=6.7 Hz, CH3), 1.2–1.4 (m, 14H, CH2), 1.61–1.65 (m,
2H, CH2), 3.80 (s, 3H, OCH3), 4.14 (t, 2H,J=6.6 Hz,
OCH2), 6.72 (d, 1H, J=15.9 Hz, ¼CH), 7.06 (s, 2H,
ArH), 7.20 (d, 1H, J=8.2 Hz, ArH), 7.32 (d, 1H,
The crystal mounted on a glass fiber. The single crystal
X-ray data was collected on a Rigaku R-AXIS RAPID
Imaging plate diffractometer. All calculations were per-
formed with the crystallographic software package teX-

E. Nomura et al. / Bioorg. Med. Chem. 10 (2002) 1069–1075
1075
san (Molecular Structure Corporation, 1985 and 1999).
The structure was solved by direct method (SHELXS-
97)17 and expanded using Fourier techniques (DIR-
DIF94).18 Non-hydrogen atoms were refined aniso-
tropically. Hydrogen atoms except OH groups were
included at calculated positions: Crystal data for
3. Huang, M. T.; Smart, R. C.; Wong, C. Q.; Conney, A. H.
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4. Mori, H.; Tanaka, T.; Shima, H.; Kuniyasu, T.; Takahashi,
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Nomura, E.; Hosoda, H.; Tsuno, T.; Maruta, Y.; Ohigashi,
H.; Koshimizu, K. Cancer Lett. 2000, 157, 77.
8. Tsuda, H.; Takasuka, N.; Toriyama, Y.; Nomura, E.;
Taniguchi, H. Anticancer Res. 1999, 19, 3779.
9. Han, B. M.; Park, C. B.; Takasuka, N.; Naito, A.; Sekine,
K.; Nomura, E.; Taniguchi, H.; Tsuno, T.; Tsuda, H. Jpn. J.
Cancer Res. 2001, 92, 404.
10. Hosoda, A.; Nomura, E.; Murakami, A.; Koshimizu, K.;
Ohigashi, H.; Mizuno, K.; Taniguchi, H. Bioorg. Med. Chem.
Lett. 2000, 19, 1439.
.
C22H24O8 2(CH4O)
6f:
M=480.51,
monoclinic,
a=7.257(1), b=8.996(2), c=38.848(9) A, b=92.341(9)ꢂ,
V=2534.2(9) A3, T=23 ꢂC, space group P21/c (no. 14),
Z=4, ꢀ(CuKa)=8.26 cmꢀ1, Dc=1.259 g cmꢀ3, 14,346
reflections measured, 4548 unique (Rint=0.029), Full-
matrix least-squares refinement was based on 2915
observed reflections [I>2.00s(I)] and 307 variable
parameters. R=0.074, Rw=0.124, GOF=1.08. Crystal-
lographic data (excluding structure factors) for the struc-
ture in this paper have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publica-
tion numbers CCDC 167664. Copies of the data can be
obtained, free of charge, on application to CCDC, 12
Union Road, Cambridge CB2 1EZ, UK (fax: +44-1223-
336033; e-mail: http:www.deposit@ccdc.cam.ac.uk).
11. Hemingway, R. W. Laks, P. E., Ed. Plantpolyphenols
Synthesis, Properties, Significance. Plenum Press: New York,
1992.
12. Wagnner, H., Farnsworth, N. R., Eds. Economic and
Medicinal Plant Research. Vol.5. Academic Press: New York,
1991.
13. Murakami, A.; Kuki, W.; Takahashi, Y.; Yonei, H.;
Nakamura, Y.; Ohto, Y.; Ohigashi, H.; Koshimizu, K. Jpn. J.
Cancer Res. 1997, 88, 443.
14. Murakami, A.; Nakamura, Y.; Koshimizu, K.; Ohigashi,
H. J. Agric. Food Chem. 1995, 43, 2779.
15. Murakami, A.; Ohura, S.; Nakamura, Y.; Koshimizu, K.;
Ohigashi, H. Oncology 1996, 53, 386.
Acknowledgements
This research was performed through the Special Coor-
dination 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 Japa-
nese Government.
16. Kim, O.-K.; Murakami, A.; Nakamura, Y.; Ohigashi, H.
Cancer Lett. 1998, 125, 199.
17. Sheldric, G. M. Program for the Solution of Crystal
Structure; University of Goettingen: Germany, 1997.
18. Beurskens, P. T.; Admiraal, G.; Beurskens, G.; Bos-
man, W. P.; de Gelder, R.; Israel, R.; Smits, J. M. M. The
DIRDIF-94 Program System. Technical Report. Crystal-
lography Laboratory; University of Nijmegen: The Nether-
lands, 1994.
References and Notes
1. Taniguchi, H.; Nomura, E.; Tsuno, T.; Minami, S.; Kato,
K.; Hayashi, C. JP-2095088.
2. Taniguchi, H.; Hosoda, A.; Tsuno, T.; Maruta, Y.;
Nomura, E. Anticancer Res. 1999, 19, 3779.