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Potent Inhibitory Effects of Resveratrol Derivatives on Progression of Prostate Cancer Cells.

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238
Arch. Pharm. Chem. Life Sci. 2006, 339, 238 – 241
Full Paper
Potent Inhibitory Effects of Resveratrol Derivatives on
Progression of Prostate Cancer Cells
Kyung Mi Yoo1, Sanghee Kim2, Bo Kyung Moon3, Sung Soo Kim1, Kyung Tack Kim1,
Sun Yeou Kim4 and Sang Yoon Choi1
1
Korea Food Research Institute, Songnam, Korea
Department of Pharmacy, Seoul National University, Seoul, Korea
3
Department of Food and Nutrition, Chung-Ang University, Seoul, Korea
4
Graduate School of East-West Medical Science, Kyunghee University, Seoul, Korea
2
Resveratrol has been identified as a potent anticancer agent in a variety of studies. In this study,
several resveratrol derivatives were synthesized and investigated in the search for an anticancer
agent with higher efficacy than resveratrol. During our examination of cancer cell lines, compounds C, F, and G evidenced higher inhibitory activity than resveratrol with regard to the
growth of PC-3 and LNCaP human prostate cancer cells. Moreover, four derivatives of resveratrol
evidenced potent growth inhibitory activity (IC50 0.01 l 0.04 lM) in LNCaP cells. The levels of
activity in these derivatives were 25 l 100 times stronger than that associated with resveratrol
(IC50 1.0 lM). Our results suggested that compounds C, D, F, and G might function as anticancer
agents on prostate tumors. This study also contains a discussion regarding the structure-activity
relationships of several resveratrol derivatives.
Keywords: Resveratrol derivative / Prostate cancer / PC-3 / LNCaP /
Received: October 24, 2005; accepted: January 27, 2006
DOI 10.1002/ardp.200500228
Introduction
Resveratrol is a polyphenol compound, which has been
detected primarily in grape skin and mori cortex [1, 2].
This compound exhibits antioxidative and cardioprotective properties and is, in part, the compound responsible
for the so-called “French Paradox” [3 – 5]. Particularly,
resveratrol has been extensively studied, and repeatedly
demonstrated to exert profound anticancer effects.
Resveratrol appears to exert potent effects on a number
of cancer cells, with properties involving inhibition of
initiation, promotion, and progression [6 – 8]. However,
although resveratrol clearly exerts an effective anticancer influence, recent experimental results have shown
Correspondence: Sang Yoon Choi, Korea Food Research Institute,
Strategic Program Division, San 46-1, Baekhyun-Dong, Bundang-Ku,
Songnam-Si, Kyunggi-Do, 463-746, South Korea.
E-mail: sychoi@kfri.re.kr
Fax: +82 31 780-9096
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2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
that the bioavailability of this compound is quite low [9,
10]. Resveratrol is rapidly removed in the animal body
with a half-life of approximately 5 min [10]. Therefore, in
order to develop a resveratrol-related anticancer drug, at
first it is necessary locating or synthesizing a potent antitumorigenic resveratrol derivative, in which the free phenolic groups are blocked, thus preventing the rapid biotransformation associated with resveratrol itself. Here,
we have conducted an investigation into the anti-progression activities of a series of resveratrol derivatives on a
prostate cancer cell line. In this assay, we employed the
cell lines most frequently used in prostate cancer studies
PC-3 and LNCaP cells. PC-3 cells are an androgen-insensitive human prostatic carcinoma cell line and LNCaP cells
are androgen-sensitive [11]. During this examination, we
noted the potent anti-progression activities of several
derivatives of resveratrol on the PC-3 and LNCaP cell
lines. These compounds all feature blocked free phenolic
groups, and also appear to exert more profound anti-progression effects than does resveratrol itself.
Arch. Pharm. Chem. Life Sci. 2006, 339, 238 – 241
Resveratrol Derivatives
239
Scheme 1. Synthesis of compound D and F.
Results and discussion
Preparation of resveratrol derivatives
In recent experiments, we have developed methods for
the preparation of resveratrol derivatives [12]. Five resveratrol derivatives were synthesized using the previously
reported methods, and two additional resveratrol derivatives were synthesized in this study using coupling
reagents (Scheme 1). These compounds have methoxy
groups and three types of connecting chains (trans-carbon double bond, nitrogen-carbon double bond, and
amide bond, respectively) between the phenyl rings. As
compared with the structure of resveratrol, the phenyl
ring of the derivatives harbors dimethoxy groups, rather
than dihydroxy groups, at its 3,5-positions. The other
phenyl ring harbors modified functional groups in positions 29 and 49.
Anti-progression effects on human prostate cancer
cell lines
In order to characterize the progression-inhibition
effects of these compounds on human prostate cancer
cells, we employed the PC-3 and LNCaP cell lines in our
experiments [13, 14]. Synthetic resveratrol derivatives
were administered to the cells, and the ensuing cytotoxicity was evaluated. As a result, compound C was shown to
exert the most profound degree of anti-progression
effects on the prostate cancer cells. Compound C manifested more profound anti-progression effects than did
resveratrol on both cancer cell lines, with an IC50 of
0.05 lM on PC-3 and an IC50 of 0.01 lM on LNCaP. The
anti-progression activity of compound C on the LNCaP
cells was 100 times as strong as that measured for resver-
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2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
atrol. Also, compounds G and F substantially inhibited
the progression of the PC-3 cells, and compounds D, G,
and F exerted potent cytotoxic effects on the LNCaP cells.
Structure-activity relationships
As is shown in Table 1, compound C harbors two methoxyl groups at positions 3 and 5, and also contains a
nitrogen at the 49 position. As compared with compound
A, the nitrogen may play an important role in these aforementioned anti-progression effects. This activity was
reduced when the nitrogen was replaced by a C-methoxy
group in case of compound A. In the connecting chains,
the carbon double bond (compound A) and the benzamide bond (compound E) were less active than the nitrogen-carbon double bond (compound G). On the basis of
these results, the development of more effective compounds, possibly including a compound harboring a
nitrogen-carbon double bond and a nitrogen in the 4'
position, will require more work in the future. The logPvalues for each of the compounds are shown in Table 2.
In this study, we identified several resveratrol derivatives having profound anti-progression effects on prostate cancer cells. Among these compounds, compound C
was shown to be the most effective of the agents on cancer cell lines. Compound C features blocked free phenolic
groups, and a higher degree of anti-progression activity
than has been associated with resveratrol. Our findings
appear to suggest that compound C may also function as
an anticancer agent. Furthermore, compound C may
prove useful in further studies in the search for a suitable
anti-prostate cancer agent. The development of more
effective agents derived from the most effective of the
compounds in this study (compound C) will require
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240
K. M. Yoo et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 238 – 241
Table 1. Anti-progression effects of resveratrol derivatives on PC-3 and LNCaP cells.
Stilbene analogues:
Compounds
A
B
C
Resveratrol
Substituent
PC-3
LNCaP
R1
R2
R3
R4
X
IC50
AC50
IC50
AC50
OCH3
OCH3
OCH3
OH
OCH3
OCH3
OCH3
OH
H
OCH3
H
H
OCH3
OCH3
–
OH
C
C
N
C
3.6 l 0.9
2.0 l 0.1
0.05 l 0.03
0.6 l 0.01
12.1 l 0.7
5.0 l 0.5
0.20 l 0.01
4.0 l 0.02
0.8 l 0.1
1.1 l 0.3
0.01 l 0.01
1.0 l 0.01
6.3 l 0.7
4.2 l 0.3
0.80 l 0.02
6.0 l 0.01
Amide analogues:
Compounds
D
E
F
Substituent
PC-3
AC50
LNCaP
R1
R2
R3
R4
X
IC50
IC50
AC50
H
H
OCH3
OCH3
OCH3
H
OCH3
OCH3
OCH3
H
OCH3
H
OCH3
OCH3
OCH3
43.0 l 0.09 120.0 l 19.2
0.04 l 0.01 0.01 l 0.01
35.0 l 5.4
99.0 l 10.4 29.0 l 4.4 101.0 l 11.3
0.09 l 0.04
0.41 l 0.05 0.04 l 0.01 0.30 l 0.01
Imine analogues:
Compound
G
PC-3
LNCaP
IC50
AC50
IC50
AC50
0.07 l 0.01
0.21 l 0.20
0.03 l 0.01
0.11 l 0.60
IC50 and AC50 were represented with lM Each value represents the mean l l S.E. of three independent experiments.
Table 2. LogP-value of each compound.
Experimental
Compounds
LogP-values
Materials
A
B
C
D
E
F
G
Resveratrol
3.85
3.72
2.63
3.06
2.46
2.33
2.46
3.06
The DMEM (growth medium), fetal bovine serum (FBS), and penicillin-streptomycin (PS) used in this study were purchased from
Gibco-BRL (USA). The trans-resveratrol was acquired from the
Sigma Co. (USA). All other chemicals and solvents were of analytical grade, and were used without further purification.
further experimentation, as well as in vivo investigation
into the anticancer effects of new anti-prostate cancer
agent candidates.
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2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Synthesis of resveratrol derivatives
Five different resveratrol derivatives were synthesized via previously reported methods [12]: 3,5,49-Trimethoxystilbene (A),
3,5,29,49-tetramethoxystilbene (B), 4-[2-(3,5-dimethoxyphenyl)vinyl]pyridine (C), N-(2,4-dimethoxyphenyl)-3,5-dimethoxy benzamide (E), (3,5-dimethoxybenzylidene)-(2,4-dimethoxyphenyl)amine (G).
www.archpharm.com
Arch. Pharm. Chem. Life Sci. 2006, 339, 238 – 241
The N-(4-dimethoxyphenyl)-3,5-dimethoxybenzamide was prepared as follows: To a mixture of 3,5-dimethoxybenzoic acid
(500 mg, 2.8 mmol) and trichloroacetonitrile (550 mL,
5.5 mmol) in CH2Cl2 (8.7 mL), PPh3 (1.44 g, 5.5 mmol) in CH2Cl2
(5 mL) was added in an argon atmosphere at room temperature.
After 4 h of stirring, the reaction mixture was treated with p-anisidine (338 mg, 2.8 mmol), and this mixture was stirred for a
further 12 h. The reaction mixture was then poured into water
and extracted with ethyl acetate. The extract was washed with
brine, dried over MgSO4, and concentrated in vacuo. The residue
was then purified via flash column chromatography on a silica
gel (eluent: CH2Cl2/MeOH = 40/1), ultimately yielding 443 mg
(56.2%) of compound D, appearing as a white solid.
N-(4-Methoxyphenyl)-3,5-dimethoxybenzamide (D);
white solid
1
H-NMR (CDCl3) d: 8.11 (s, 1H), 7.51 (d, 2H, J = 9.0 Hz), 6.94 (d, 2H, J
= 2.7 Hz), 6.84 (d, 2H, J = 9.0 Hz), 6.55 (t, 1H, J = 2.7 Hz), 3.78 (s,
3H), 3.76 (s, 6H); 13C-NMR (75 MHz, CDCl3) d: 165.39, 161.01,
156.63, 137.30, 130.95, 121.99, 114.23, 104.93, 103.66, 55.60,
55.49; MS (EI+) m/z: 287 [M+] (74), 165 (100), 137 (50), 107 (20).
N-(4-Methoxyphenyl)-2,5-dimethoxybenzamide was synthesized in accordance with the following methods: 2.5-Dimethoxylbenzoic acid (200 mg, 1.1 mmol) was added to a stirred solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (232 mg,
1.2 mmol) in anhydrous THF (11 mL). p-Anisidine (122 mg,
0.99 mmol) was then added. After 4 h of stirring, the reaction
mixture was poured into water and extracted with CH2Cl2. The
extract was washed in brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed (silica gel, eluent: CH2Cl2/MeOH = 30/1) and recrystallized (ethylacetate/heaxane), yielding 85 mg (30%) of compound F, appearing as a white
solid.
N-(4-Methoxy-phenyl)-2,5-dimethoxybenzamide (F);
white solid:
1
H-NMR (300 MHz, CDCl3) d: 7.85 (d, 1H, J = 2.7 Hz), 7.61 – 7.56 (m,
3H), 7.03 (Dd, 1H, J = 2.7 Hz; 2.7 Hz), 6.97 (d, 1H, 9.0 Hz), 6.91 (d,
2H, J = 9.0 Hz), 4.01 (s, 3H), 3.85 (s, 3H), 3.82 (s, 3H); 13C-NMR
(75 MHz, CDCl3) d: 162.69, 156.25, 154.08, 151.42, 131.55,
122.37, 121.93, 119.59, 115.57, 114.07, 113.19, 56.76, 55.76,
55.43; MS (EI+) m/z: 287 (M+] (31), 165 (100), 149 (9), 136 (8), 122
(22), 107 (25), 95 (19), 81 (33), 69 (66).
Biological assays
Cell culture
PC-3 and LNCaP, both of which are human prostate cancer cell
lines, were provided by the American Type Culture Collection
(Rockville, MD, USA). This cell line was cultured in DMEM supplemented with 10% heat-inactivated FBS, 4 mmol/L L-glutamine,
and antibiotics (40 g/L penicillin and 40,000 U/L streptomycin)
at 378C in a humidified atmosphere consisting of 5% CO2 in air.
This cell line was passaged twice a week.
Determination of anti-progression effects
In order to evaluate the effects of the resveratrol derivatives on
the v iability of the PC-3 and LNCaP cells, the cells were seeded at
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2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Resveratrol Derivatives
241
a density of 56103 cells per well, in 96-well plates containing
100 lL of culture medium per well. After 24 hours of cultivation,
the medium was exchanged with fresh medium supplemented
with resveratrol derivatives. The final THF concentration in the
culture medium was 0.575% (v/v), and the control culture was
treated only with THF (vehicle alone). After 72 h of cultivation,
cell viability was evaluated via MTT assay, and was expressed as
the percentage of the value of the control culture treated with
the vehicle alone (THF).
Statistical analysis
Data in this study are expressed as means l S.E. from three independent experiments. Statistical comparisons between different
treatments were conducted via Student's t-tests.
This study was supported by the Technology Development Program for
Agriculture and Forestry, Ministry of Agriculture and Forestry, Republic of Korea.
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