Effect of methyl substitution on the antioxidative property and genotoxicity of resveratrol

Article abstract of DOI:10.1021/tx7003008

Resveratrol (trans-3,4′,5-trihydroxystilbene) is a natural phytoalexin with various biological activities including inhibition of lipid peroxidation and free radical scavenging properties. In addition to its beneficial effects, resveratrol also has significant genotoxicity that leads to a high frequency of chromosome aberration together with micronucleus and sister chromatid exchanges. To enhance the radical scavenging activities and to reduce the genotoxicity of resveratrol, we designed 4′-methyl resveratrol analogues where a methyl group was introduced at the ortho position relative to the 4′-hydroxy group, which is responsible for both antioxidative activities and genotoxicity of resveratrol. These synthesized methyl analogues of resveratrol showed increased antioxidative activities against galvinoxyl radical as an oxyl radical species. Furthermore, the methyl analogues also surprisingly showed reduced in vitro genotoxicities, suggesting that methyl substitution may improve resveratrol efficacy.

Full text of DOI:10.1021/tx7003008

282
Chem. Res. Toxicol. 2008, 21, 282–287
Communication
Effect of Methyl Substitution on the Antioxidative Property and
Genotoxicity of Resveratrol
Kiyoshi Fukuhara,*,† Ikuo Nakanishi,‡,§ Atsuko Matsuoka,4 Tomohiro Matsumura,
Sachiko Honda,# Mikiko Hayashi,# Toshihiko Ozawa,‡, Naoki Miyata,O Shinichi Saito,
Nobuo Ikota,‡ and Haruhiro Okuda†
DiVision of Organic Chemistry, National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Redox
Regulation Research Group, Research Center for Radiation Safety, National Institute of Radiological Sciences,
Inage-ku, Chiba 263-8555, Graduate School of Engineering, Osaka UniVersity, SORST, Japan Science and
Technology Agency, Suita, Osaka 565-0871, DiVision of Medical DeVices, National Institute of Health Sciences,
Setagaya-ku, 158-8501 Tokyo, Faculty of Science, Tokyo UniVersity of Science, Shinjuku-ku, Tokyo 162-8601,
Department of Cancer Research, Toyama Institute of Health, 17-1 Nakataikouyama, Kosugi-machi, Imizu-gun,
Toyama 939-0363, New Industry Creation Hatchery Center, Yokohama College of Pharmacy, Tozuka-ku,
Yokohama, Kanagama 245-0066, and Graduate School of Pharmaceutical Sciences, Nagoya City UniVersity,
Mizuho-ku, Nagoya, Aichi 467-8603, Japan
ReceiVed August 21, 2007
Resveratrol (trans-3,4′,5-trihydroxystilbene) is a natural phytoalexin with various biological activities
including inhibition of lipid peroxidation and free radical scavenging properties. In addition to its beneficial
effects, resveratrol also has significant genotoxicity that leads to a high frequency of chromosome aberration
together with micronucleus and sister chromatid exchanges. To enhance the radical scavenging activities
and to reduce the genotoxicity of resveratrol, we designed 4′-methyl resveratrol analogues where a methyl
group was introduced at the ortho position relative to the 4′-hydroxy group, which is responsible for
both antioxidative activities and genotoxicity of resveratrol. These synthesized methyl analogues of
resveratrol showed increased antioxidative activities against galvinoxyl radical as an oxyl radical species.
Furthermore, the methyl analogues also surprisingly showed reduced in vitro genotoxicities, suggesting
that methyl substitution may improve resveratrol efficacy.
Besides, the 4′-hydroxyl group has been primarily responsible
for the copper binding property (7). In addition to its
beneficial effects, resveratrol is also reported to be genotoxic,
inducing a high frequency of chromosomal aberrations (CA),
micronucleus, and sister chromatid exchanges (SCE) in vitro
(8). Structure–activity relationship studies of resveratrol
analogues revealed that the 4′-hydroxyl group, besides being
essential for antioxidative activity, is also responsible for the
in vitro cytogenetic activity of resveratrol (7, 9, 10). In this
regard, our challenge is to create novel resveratrol analogues
that not only exert enhanced antioxidative abilities but also
have reduced in vitro genotoxicity. Such analogues could lead
to the development of new drugs against various diseases,
particularly those related to oxidative stress. In our attempt
to design new resveratrol analogues, we focused on the
methyl groups of the tocopherol due to their proven anti-
oxidative effects on the aromatic ring (11). In particular,
methyl groups at the ortho position to the hydroxyl group
contribute to delocalization of the unpaired electron of the
corresponding phenoxyl radical, which is generated in the
reaction with radical species, due to hyperconjugation. In this
study, we describe resveratrol analogues where methyl groups
are introduced into the ortho position of the 4′-hydroxyl
group. Their designs are relatively simple, but in comparison
Introduction
Resveratrol (3,4′,5-trihydroxy-trans-stilbene) is a natural
phytoalexin present in grapes and wine that has been shown
to play an essential role in the prevention of several human
pathological processes including inflammation (1), athero-
sclerosis (2), and carcinogenesis (3). The cancer preventive
activity of resveratrol is linked to its ability to eliminate free
radicals and to reduce oxidative and mutagenic stress. Lipid
peroxidation is one of the basic mechanisms of cell and tissue
damage leading to various diseases. So far, the protective
effect toward lipid peroxidation has proved to be a typical
antioxidative event of resveratrol (4). It has been demon-
strated that resveratrol suppresses lipid peroxidation by both
scavenging of free radicals and chelation of copper (5).
Among three hydroxyl groups in resveratrol, the 4′-hydroxyl
group is essential for radical scavenging activities (6).
* To whom correspondence should be addressed. Tel: +81-3-3700-1141.
Fax: +81-3-3707-6950. E-mail: fukuhara@nihs.go.jp.
† Division of Organic Chemistry, National Institute of Health Sciences.
‡ National Institute of Radiological Sciences.
§ Osaka University.
4 Division of Medical Devices, National Institute of Health Sciences.
Tokyo University of Science.
# Toyama Institute of Health.
Yokohama College of Pharmacy.
O Nagoya City University.
10.1021/tx7003008 CCC: $40.75
2008 American Chemical Society
Published on Web 01/05/2008

Communications
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 283
Scheme 1. Structure of Resveratrol and Methyl Derivatives
g, 30 mmol) in anhydrous diethyl ether (100 mL) over the period
of 30 min. The reaction mixture was stirred for 6 h. Excess
lithium aluminum hydride was decomposed by successive
dropwise addition of 2-propanol and 10% potassium hydroxide.
The organic layer was then passed through a Celite filter pad,
and the filtrate was dried over anhydrous Na2SO4. The solvent
was evaporated under vacuum to afford 6.09 g of 10 (91% yield)
as a white powder. 1H NMR (400 MHz, DMSO-d6): δ 2.06 (3H,
s), 4.43 (2H, d, J ) 5.6 Hz), 5.08 (4H, s), 5.15 (1H, t, J ) 5.6
Hz), 6.69 (2H, s), 7.40 (10H, m). MS (EI m/z) 334 [M+].
3,5-Bis(benzyloxy)-4-methylbenzyl bromide (11b). Phosphorus
tribromide (2.71 g, 10 mmol) was added to a stirred solution of 10
(3.34 g, 10 mmol) in dry CH2Cl2 (50 mL) at 0 °C under argon.
The stirring was continued for 2 h at 0 °C and at room temperature
for 2 h. The reaction mixture was poured onto ice water and
extracted with diethyl ether (3 × 100 mL). The ether layers were
combined, washed with brine, and dried over anhydrous Na2SO4.
The solvent was evaporated under vacuum, and the residue was
purified by silica gel column chromatography (n-hexane:CH2Cl2
) 1:1) to afford 10.44 g of 11b (3.62 g, 91% yield) as white needles.
1H NMR (400 MHz, DMSO-d6): δ 2.06 (3H, s), 4.64 (2H, s), 5.09
(4H, s), 6.85 (2H, s), 7.40 (10H, m). MS (EI m/z) 397 [M+].
Diethyl 3,5-dibenzyloxybenzyl phosphonate (5b). Triethyl
phosphite (3.0 g, 18 mmol) was added to 11b (4.77 g, 12 mmol)
containing a catalytic amount of tetrabutylammonium iodide,
and the resulting mixture was heated at 120 °C for 12 h. Excess
triethyl phosphite was removed by evaporater at 50 °C to afford
5b (5.18 g, 95% yield) as a light yellow oil. 1H NMR (400 MHz,
DMSO-d6): δ 1.24 (6H, t, J ) 7.2 Hz), 2.05 (3H, s), 3.86 (4H,
quint, J ) 7.2 Hz), 5.06 (4H, s), 6.66 (2H, s), 7.40 (12H, m).
MS (EI m/z) 454 [M+].
with resveratrol, their antioxidative abilities are significantly
increased, and surprisingly, in vitro genotoxicities are also
decreased.
Experimental Procedures
General. The reagents and solvents used were of commercial
origin (Wako Chemicals, Tokyo Chemical Industry, Sigma, and
1
Aldrich) and were employed without further purification. The H
NMR spectra and 13C NMR spectra were recorded with a Varian
AS 400 Mercury spectrometer. Chemical shifts are expressed in
ppm downfield shift from TMS (δ scale). Low- and high-resolution
mass spectra were obtained on a JEOL MS700 mass spectrometer.
The progress of all reactions was monitored by thin-layer chroma-
tography on silica gel 60 F254 (0.25 mm, Merck). Column
chromatography was performed on silica gel 60 (0.063–0.200 mm,
Merck). The purity of all synthetic compounds was approximately
1
4-Benzyloxy-3-methylbenzaldehyde (6a). To a well-stirred
solution of 4-hydroxy-3-methylbenzaldehyde (12) (3.40 g, 25
mmol) in DMF (100 mL), potassium carbonate (11.05 g, 80
mmol) was added under argon. After 1 h, benzyl bromide (10.68
g, 2.5 equiv, 62.5 mmol) was added dropwise over a period of
30 min and the reaction was allowed to proceed for 14 h. The
reaction mixture was poured on ice water (200 mL) and extracted
with CH2Cl2 (3 × 200 mL). The organic fractions were combined
and washed with water and brine and dried over anhydrous
Na2SO4. The residue was purified by silica gel column chro-
matography (n-hexane:ethyl acetate, 2:1) to afford 4.98 g of 6a
>98% (based on H NMR spectra).
Diethyl 3,5-bis(benzyloxy)benzylphosphonate (5a). Triethyl
phosphite (2.49 g, 15mmol) was added to a mixture of 3-benzyloxy-
4-methylbenzylbromide (11a) (3.83 g, 10 mmol) and tetrabuty-
lammonium iodide (73 mg, 0.2 mmol), and the resulting mixture
was heated for 8 h. Excess triethyl phosphite was removed by
heating for 3 h at 60 °C under vacuum to yield 4.32 g of 5a (98%
yield) as a light yellow oil. 1H NMR (400 MHz, DMSO-d6): δ
1.21 (t, 6H), 3.90 (q, 4H), 5.09 (s, 4H), 6.55 (d, 2H), 6.57 (s, 1H),
7.4 (m, 12H). MS (EI m/z) 440 [M+].
Methyl 3,5-dihydroxy-4-methylbenzoate (8). To a solution of
3,5-dihydroxy-4-methylbenzoic acid (10.08 g, 60 mmol) in
methanol (150 mL), sulfuric acid (3.0 mL) was added. The resulting
mixture was stirred for 18 h and poured into ice water (200 mL),
and then, the product was extracted with ethyl acetate (3 × 300
mL). The organic layer was washed with brine, dried over
anhydrous Na2SO4, and evaporated. The residue was purified by
silica gel column chromatography (n-hexane:ethyl acetate ) 1:1)
to afford 9.40 g of 8 (86% yield) as a colorless oil. 1H NMR (400
MHz, DMSO-d6): δ 1.96 (3H, s), 3.75 (3H, s), 6.91 (2H, s), 9.28
(2H, br). MS (EI m/z) 182 [M+].
Methyl 3,5-bis(benzyloxy)-4-methylbenzoate (9). To a well-
stirred solution of 8 (6.64 g, 36.5 mmol) in DMF (100 mL),
potassium carbonate (23.49 g, 170 mmol) was added under argon.
After 1 h, benzyl bromide (15.6 g, 2.5 equiv, 91.3 mmol) was added
dropwise over a period of 30 min and the reaction was allowed to
proceed for 16 h. The reaction mixture was poured into ice water
(200 mL) and extracted with CH2Cl2 (3 × 200 mL). The organic
fractions were combined and washed with water and brine and then
dried over anhydrous Na2SO4. The residue was purified by silica
gel column chromatography (n-hexane:ethyl acetate ) 2:1) to afford
10.44 g of 9 (79% yield) as a white powder. 1H NMR (400 MHz,
DMSO-d6): δ 2.15 (3H, s), 3.82 (3H, s), 5.19 (4H, s), 7.27 (2H, s),
7.40 (10H, m). MS (EI m/z) 362 [M+].
1
(88% yield) as a colorless oil. H NMR (400 MHz, DMSO-d6):
δ 2.15 (3H, s), 3.82 (3H, s), 5.19 (4H, s), 7.27 (2H, s), 7.40
(10H, m). MS (EI m/z) 226 [M+].
General Procedure for the Preparation of Stilbenes (13).
Sodium hydride (0.2 g, 4 mmol) was added to a well-stirred
suspension of the phosphate esters 5a,b (2 mmol) in dry THF(10
mL) at -8 °C under argon. After 30 min, the aldehydes 6a-c
(2 mmol) in dry THF (15 mL) were added dropwise, and the
reaction mixture was allowed to stir at room temperature for
16 h. The mixture was then cooled to 0 °C, and the excess
sodium hydride was quenched with water (20 mL). The reaction
mixture was then poured on ice, followed by addition of 2 M
HCl (5 mL), and the products were extracted with ethyl acetate
(2 × 100 mL). The organic layers were combined and were
washed with brine. The ethyl acetate layer was dried over
anhydrous Na2SO4 and evaporated. The purification of the crude
product was performed by silica gel chromatography (n-hexane:
CH2Cl2 ) 2:1).
(E)-1-(Benzyloxy)-4-[3,5-bis(benzyloxy)styryl]-2-methylben-
1
zene (13a). From 5a and 6a. Yield, 62.2%. H NMR (400 MHz,
DMSO-d6): δ 2.21 (3H, s), 5.10 (4H, s), 5.13 (2H, s), 6.54 (1H,
dd, J ) 2.0, 2.0 Hz), 6.82 (2H, d, J ) 2.0 Hz), 6.98 (1H, d, J )
16.0 Hz), 7.01 (1H, d, J ) 8.4 Hz), 7.16 (1H, d, J ) 16.0 Hz),
7.40 (17H, m). MS (EI m/z) 512 [M+].
3,5-Bis(benzyloxy)-4-methylbenzyl alcohol (10). A solution of
9 (7.25 g, 20.0 mmol) in anhydrous diethyl ether (50 mL) was
added to a cold suspension of lithium aluminum hydride (1.14

284 Chem. Res. Toxicol., Vol. 21, No. 2, 2008
Scheme 2. Synthesis of Methyl Derivatives of Resveratrola
Communications
a (a) H2SO4, MeOH, 25 °C (18 h). (b) C6H6CH2Br, K2CO3, DMF, 25 °C (16 h). (c) LiAlH4, Et2O, 25 °C (6 h). (d) PBr3, CH2Cl2, 25 °C (2 h). (e) P(OEt)3,
(n-Bu)4N+I-, 120 °C (12 h). (f) C6H6CH2Br, K2CO3, DMF, 25 °C (14 h). (g) NaH, THF, 25 °C (16 h). (h) AlCl3, (CH3)2NC6H6, CH2Cl2, 0 °C (16 h).
(E)-2-(Benzyloxy)-5-[3,5-bis(benzyloxy)styryl]-1,3-dimethylben-
zene (13b). From 5a and 4-benzyloxy-3,5-dimethylbenzaldehyde
(6b). Yield, 78.9%. H NMR (400 MHz, DMSO-d6): δ 2.25 (6H,
s), 4.80 (2H, s), 5.12 (4H, s), 6.57 (1H, dd, J ) 2.0, 2.0 Hz), 6.85
(2H, d, J ) 2.0 Hz), 7.04 (1H, d, J ) 16.4 Hz), 7.17 (1H, d, J )
16.4 Hz), 7.28 (2H, s), 7.40 (15H, m). MS (EI m/z) 526 [M+].
General Procedure for the Cleavage of the Benxyloxy Groups to
Afford Methyl Resveratrols (1–4 and 4-Methylresveratrol). To a well-
stirred solution of stilbene 13 (1 mmol) in dry CH2Cl2 (10 mL),
N,N-dimethylaniline (3 mmol) was added under argon atmo-
sphere at 0 °C. After 5 min, anhydrous AlCl3 (4 mmol) was
added to the reaction mixture. After 16 h, the reaction mixture
was quenched with water at 0 °C. The reaction mixture was
poured into a 1.0 M solution of HCl (20 mL). The resulting
mixture was extracted with ethyl acetate (2 × 100 mL), and
combined extracts were washed with brine (2 × 50 mL). The
ethyl acetate layer was dried over anhydrous Na2SO4 and
evaporated. The purification of the crude product was done by
thin-layer chromatography under argon atmosphere (silica gel
60, 2 mm coated silica plate, CH2Cl2:MeOH ) 10:1).
(E)-5-(4-Hydroxy-3-methylstyryl)benzene-1,3-diol (1). From
13a. Yield, 50.4%; mp 217–219 °C. 1H NMR (400 MHz, DMSO-
d6): δ 2.12 (3H, s), 6.09 (1H, dd, J ) 2.0, 2.0 Hz), 6.35 (2H, d,
J ) 2.0 Hz), 6.74 (1H, d, J ) 8.0 Hz), 6.78 (1H, d, J ) 16.4
Hz), 6.87 (1H, d, J ) 16.4 Hz), 7.18 (1H, dd, J ) 2.0, 8.0 Hz),
7.29 (1H, d, J ) 2.0 Hz), 9.18 (2H, s), 9.44 (1H, s). 13C NMR
(100 MHz, DMSO-d6): δ 160.2, 153.1, 140.3, 131.5, 128.6,
1
(E)-1,3-Bis(benzyloxy)-5-[4-(benzyloxy)styryl]-2-methylben-
zene (13c). From 5b and 4-(benzyloxy)benzaldehyde (6c). Yield,
75.7%. 1H NMR (400 MHz, DMSO-d6): δ 2.08 (3H, s), 5.11 (2H,
s), 5.15 (4H, s), 6.95 (2H, s), 7.01 (1H, d, J ) 16.0 Hz), 7.19 (1H,
d, J ) 16.0 Hz), 7.40 (19H, m). MS (EI m/z) 512 [M+].
(E)-1,3-Bis(benzyloxy)-5-[4-(benzyloxy)-3-methylstyryl]-2-meth-
1
ylbenzene (13d). From 5b and 6a. Yield, 62.4%. H NMR (400
MHz, DMSO-d6): δ 2.07 (3H, s), 2.21 (3H, s), 5.13 (2H, s), 5.15
(4H, s), 6.94 (2H, s), 7.01 (1H, d, J ) 16.0 Hz), 7.15 (1H, d, J )
16.0 Hz), 7.40 (18H, m). MS (EI m/z) 526 [M+].
(E)-1,3-Bis(benzyloxy)-5-[4-(benzyloxy)-3,5-dimethylstyryl]-2-me-
1
thylbenzene (13e). From 5b and 6b. Yield, 67.9%. H NMR (400
MHz, DMSO-d6): δ 2.09 (3H, s), 2.26 (6H, s), 4.81 (2H, s), 5.17
(4H, s), 6.97 (2H, s), 7.06 (1H, d, J ) 16.0 Hz), 7.17 (1H, d, J )
16.0 Hz), 7.28 (2H, s), 7.40 (15H, m). MS (EI m/z) 540 [M+].

Communications
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 285
127.7, 126.9, 123.6, 115.1, 104.7, 102.5, 19.8. MS (EI m/z) 242
[M+]. HRMS (EI m/z) calcd for C15H14O3 [M]+, 242.0943;
found, 242.0944.
(E)-5-(4-Hydroxy-3,5-dimethylstyryl)benzene-1,3-diol (2). From
13b. Yield, 68.6%; mp 205–208 °C. 1H NMR (400 MHz,
DMSO-d6): δ 2.16 (6H, s), 6.09 (1H, dd, J ) 2.0, 2.0 Hz), 6.35
(2H, d, J ) 2.0 Hz), 6.74 (1H, d, J ) 8.0 Hz), 6.78 (1H, d, J )
16.0 Hz), 6.84 (1H, d, J ) 16.0 Hz), 7.13 (2H, s), 8.37 (1H, s),
9.19 (2H, s). 13C NMR (100 MHz, DMSO-d6): δ 160.4, 152.0,
138.8, 135.5, 129.6, 125.3, 122.2, 114.3, 105.1, 102.9, 16.5. MS
(EI m/z) 256 [M+]. HRMS (EI m/z) calcd for C16H16O3 [M]+,
256.10995; found, 256.10997.
(E)-5-(4-Hydroxystyryl)-2-methylbenzene-1,3-diol (4-Methylres-
veratrol). From 13c. Yield, 59.8%; mp 239–242 °C. 1H NMR (400
MHz, DMSO-d6): δ 1.91 (3H, s), 6.43 (2H, s), 6.72 (2H, d, J )
8.8 Hz), 6.76 (2H, d, J ) 16.4 Hz), 6.79 (1H, d, J ) 16.4 Hz),
7.36 (1H, d, J ) 8.8 Hz), 9.07 (2H, s), 9.51(1H, s). 13C NMR (100
MHz, DMSO-d6): δ 160.6, 158.7, 138.1, 131.5, 130.2, 128.5, 114.7,
110.5, 105.5, 9.8. MS (EI m/z) 242 [M+]. HRMS (EI m/z) calcd
for C15H14O3 [M]+, 242.0943; found, 242.0943.
Figure 1. (a) ESR spectrum of phenoxyl radical generated in the
reaction of 4 (5.0 × 10-4 M) with G• (5.0 × 10-5 M) in deaerated
MeCN at 298 K. (b) The computer simulation spectrum.
Table 1. Rate Constants (kHT) of Hydrogen Transfer from
Resveratrol and Methyl Analogues to G•
(E)-5-(4-Hydroxy-3-methylstyryl)-2-methylbenzene-1,3-diol (3).
compd
kHT (M-1 s-1
)
compd
kHT (M-1 s-1
)
1
From 13d. Yield, 54.7%; mp 228–229 °C. H NMR (400 MHz,
DMSO-d6): δ 1.91 (3H, s), 2.11 (3H, s), 6.42 (2H, s), 6.72 (1H, d,
J ) 8.4 Hz), 6.74 (1H, d, J ) 16.4 Hz), 6.75 (1H, d, J ) 16.4 Hz),
7.16 (1H, dd, J ) 2.0, 8.0 Hz), 7.28 (1H, d, J ) 2.0 Hz), 9.08 (2H,
s), 9.40 (1H, s). 13C NMR (100 MHz, DMSO-d6): δ 161.6, 154.5,
138.8, 132.3, 129.8, 128.8, 127.0, 123.2, 116.9, 108.3, 104.1, 19.6,
9.4. MS (EI m/z) 256 [M+]. HRMS (EI m/z) calcd for C16H16O3
[M]+, 256.10995; found, 256.10996.
resveratrol
1
2
4.1
57.4
167
4-methylresveratrol
3
4
24.6
156
254
(E)-5-(4-Hydroxy-3,5-dimethylstyryl)-2-methylbenzene-1,3-diol (4).
1
From 13e. Yield, 73.0%; mp 205–208 °C. H NMR (400 MHz,
DMSO-d6): δ 1.91 (3H, s), 2.16 (6H, s), 6.43 (2H, s), 6.71 (1H, d,
J ) 16.4 Hz), 6.76 (1H, d, J ) 16.4 Hz), 7.12 (2H, s), 8.33 (1H,
s), 9.09 (2H, s). 13C NMR (100 MHz, DMSO-d6): δ 162.1, 158.2,
139.8, 132.6, 130.0, 128.4, 114.4, 109.5, 106.6, 9.8, -1.6. MS (EI
m/z) 270 [M+]. HRMS (EI m/z) calcd for C16H16O3 [M]+,
270.1256; found, 270.1259.
Spectral and Kinetic Measurements. Typically, an aliquot of
methyl resveratrol (1.0 × 10-2 M) in deaerated MeCN was added
to a quartz cuvette (10 mm i.d.) that contained galvinoxyl radical
(G•) (2.5 × 10-6 M) in deaerated MeCN (3.0 mL). This led to a
hydrogen transfer reaction from methyl resveratrol to G•. Changes
in the UV–vis spectrum associated with this reaction were
monitored using a Hewlett-Packard 8453 photodiode array spec-
trophotometer. The reaction rates were determined by following
the change in absorbance at 428 nm due to G• (ꢀ ) 1.43 × 105
M-1 cm-1). Pseudofirst-order rate constants (kobs) were determined
by a least-squares curve fitting using an Apple Macintosh personal
computer. The first-order plots of ln(A - A∞) vs time (A and A∞
are denoted to the absorbance at the reaction time and the final
absorbance, respectively) were linear until three or more half-lives,
with the correlation coefficient F > 0.999.
ESR Measurement. An aliquot of deaerated MeCN solution of
G• (1.0 × 10-3 M) was added to a LABOTEC LLC-04B ESR
sample tube containing deaerated MeCN solution of 4 (1.0 × 10-3
M), and ESR spectra of phenoxyl radical 4• produced in the reaction
between 4 and G• were taken on a JEOL X-band spectrometer (JES-
FA100). The ESR spectrum was recorded under nonsaturating
microwave power conditions. The magnitude of modulation was
chosen to optimize the resolution and the signal-to-noise (S/N) ratio
of the observed spectra. The g value and hyperfine coupling
constants were calibrated with a Mn2+ marker. Computer simulation
of the ESR spectra was carried out using Calleo ESR Version 1.2
program (Calleo Scientific Publisher) on an Apple Macintosh
personal computer.
Figure 2. Chromosome aberrations induced in vitro by resveratrol and
its methyl analogues. CHL cells were treated with the chemicals for
48 h.
122) in 5% CO2 in air at 37 °C. The doubling time was around
13 h, and the modal chromosome number was 25. Cells were seeded
at 1.5 × 105/plate (60 mm in diameter) and incubated at 37 °C for
17 h. They were then treated with a test sample for 48 h, and
colcemid (0.2 µg/mL) was added for the final 2 h. Chromosome
preparations were made as follows: Cells were trypsinized and
incubated in hypotonic KCl solution for 15 min and fixed three
times with ice-cold fixative (glacial acetic acid:methanol, 1:3). Two
drops of the fixed cell suspension were spread on a clean glass
slide, air-dried, and stained with Giemsa solution. All slides were
coded, and the number of cells with structural CAs was counted
for 100 well-spread metaphases with a modal chromosome number
of 25 ( 2.
Results and Discussion
Scheme 1 shows the structures of resveratrol and the designed
methyl derivatives. Because the 4′-hydroxyl group is essential
for both the antioxidative activity and the genotoxicity, res-
veratrol analogues were designed to introduce methyl groups
ortho to the 4′-hydroxyl group. Methyl groups were also
introduced at the 4-position para to the 4′-hydroxyl group. These
In Vitro CA Test. CHL cells were established from the lung of
a female newborn Chinese hamster by Koyama et al. (12) and
cloned by Ishidate and Odashima (13). They were maintained in
Eagle’s minimum essential medium (MEM; Gibco 11095-080)
supplemented with 10% heat-inactivated fetal bovine serum and
penicillin (100 U/mL)-streptomycin (100 µg/mL) (Gibco 15140-

286 Chem. Res. Toxicol., Vol. 21, No. 2, 2008
Communications
designed methyl derivatives (1 and 2) of resveratrol, 4-meth-
ylresveratrol, and its methyl derivatives (3 and 4) were
synthesized by means of Wittig–Horner reactions between
appropriate dibenzyloxybenzylphosphonic acid diethylesters
(5a,b) and benzyloxybenzaldehydes (6a-c), followed by depro-
tection using AlCl3 and N,N-dimethylaniline as outlined in
Scheme 2. Trans geometries of these compounds were confirmed
by their coupling constants (16.0–16.4 Hz) for the olefinic
proton.
In conclusion, we have described the synthesis, antioxi-
dative ability, and in vitro genotoxicity of resveratrol
analogues with methyl groups ortho to the 4′-hydroxyl group.
We demonstrated enhanced antioxidative activity coupled
with reduced genotoxicity, rendering the methyl analogues
1–4 potentially valuable for the development of drugs
effective for various types of diseases caused by oxidative
stress. The genotoxicity of resveratrol has been attributed to
the scavenging of tyrosyl free radicals in the R2 subunit of
ribonucleotide reductase that catalyzes the rate-limiting step
of de novo DNA synthesis (14). We previously reported that
the 4′-hydroxyl group is responsible for scavenging tyrosyl
radicals, which cause SCE and CA (15). Therefore, it is
possible that the lower CA frequency for 1–4 as compared
to resveratrol could be explained by the steric hindrance of
the o-methyl group with respect to the radical scavenging
reaction between the 4′-hydroxyl group and the tyrosyl
radical. On the other hand, comparison of resveratrol and its
o-methyl analogue (1 and 2) to the 4-methyl analogues (4-
methyresveratrol, 3 and 4), which have increased CA, shows
a potential functional relationship between structure and
enhanced radical scavenging activity. That is, slight increas-
ing CA frequency in the corresponding 4-methyl analogues
may be attributed to their enhanced radical scavenging
activities that are responsible for theinhibition of ribonucle-
otide reductase. Further detailed insight and in vivo studies
to fully exploit these potential benefits are currently underway.
The radical scavenging activities of resveratrol and its
analogues were evaluated by the hydrogen transfer reaction
using galvinoxyl radical (G•) as an oxyl radical species. The
hydrogen abstraction from resveratrols by G• in deaerated
acetonitrile was monitored by the decrease of absorbance at 428
nm due to G• that obeyed pseudofirst-order kinetics, when the
concentration of resveratrols was maintained at more than a 10-
fold excess of the G• concentration. The second-order rate
constant (kHT) for hydrogen abstraction was determined from
the linear plot of pseudofirst-order rate constant vs the resveratrol
concentration. As shown in Table 1, 3′-methylresveratrol (1),
where one methyl group was introduced at the ortho position
relative to the 4′-hydroxyl group, showed a significantly
increased radical scavenging activity ascompared to resveratrol.
A greater kHT value was also obtained in compound 2, which
has methyl groups at both positions ortho to the 4′-hydroxyl
group. In comparison to resveratrol, a 6-fold greater kHT value
was observed with 4-methylresveratrol, indicating that the
4-methyl group also affects the radical scavenging activities of
the 4′-hydroxyl group. Similar to the methyl analogues (1 and
2) of resveratrol, the kHT value of 4-methylresveratrol was
increased by the introduction of methyl ortho to the 4′-hydroxyl
group. Among resveratrol and its derivatives, compound 4 had
the strongest antioxidative activity with a 60-fold greater kHT
value than that of resveratrol.
Acknowledgment. This work was supported by a Grant from
the Ministry of Health, Labour and Welfare, and by Grant-in-
Aids for Scientific Research (B) (No. 17390033) and for Young
Scientists (B) (No. 17790044) from the Ministry of Education,
Culture, Sports, Science and Technology, Japan.
The strong antioxidative activity of tocopherol is attributed
to the delocalization of phenoxyl radical, which is generated in
the reaction with radical species, due to hyperconjugation with
the o-methyl group (11). Therefore, to verify that the o-methyl
group of 4 also contributes to delocalization of the unpaired
electron in the corresponding phenoxyl radical, the ESR
spectrum was measured for a solution containing 4 and G•
(Figure 1a). The observed ESR signals were characterized as
the phenoxyl radical derived from 4 by computer simulation
with the hyperfine splitting (hfs) values [aCH 3′(3H), aCH 5′(3H)
) 0.141 mT, aHR(1H) ) 0.601 mT] as shown in Figure 1b.
We clearly demonstrated that the delocalization of the unpaired
electron to the o-methyl groups by hyperconjugation results in
the stronger antioxidative ability of o-methyl derivatives as
compared to resveratrol.
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