1442
K. Fukuhara et al. / Bioorg. Med. Chem. 14 (2006) 1437–1443
of causing chromosomal aberrations and gene muta-
tions in cultured cells. The oxidative DNA damage
and/or alkylation of DNA that is responsible for the risk
of developing cancer are also induced by catechol estro-
gen metabolites. In fact, the catechol structure, which
can cause genotoxicity, is capable of inducing DNA
strand scission and the oxidation of DNA bases in the
presence of Cu(II). Recently, we reported the genotoxi-
city of 1, which induced micronucleus, sister chromatid
exchange, and S phase arrest.23 Among the many types
of hydroxylated stilbenes, 4-hydroxystilbene most effec-
tively caused genotoxic effects. Therefore, the finding
that the 4-hydroxylstilbene structure is responsible for
various biological activities, especially DNA damage
leading to genotoxicity, might be important for under-
standing the toxicity of polyphenols that do not have
a catechol structure.
odylate buffer (pH 7.1)/acetonitrile mixed solvent (1:1
v/v) was prepared and subjected to spectral analysis.
The binding constant between 1 and Cu(II) was ob-
tained according to the method described by Itoh
et al.25
4.4. Fluorescence measurements
Fluorescence excitation and emission spectra were
recorded on a Shimadzu RF-5300PC. A solution in a fi-
nal volume of 1 mL, which consisted of 20 lM of sample
and 0–100 lM calf thymus DNA in 10 mM sodium cac-
odylate buffer (pH 7.1) and DMF (10% by volume), was
used for fluorescence-quenching experiments. The exci-
tation wavelengths used were 255 nm for 1, 3, 6, and
7, and 260 nm for 2, 4, 5, and 1H2, and emissions were
recorded in the range of 300–500 nm. For all experi-
ments, the sample temperature was maintained at
37 ꢁC. The quenching data were analyzed by the
Stern–Volmer equation:26
4. Experimental
4.1. Materials
F 0=F ¼ 1 þ Ksv½Qꢁ;
where [Q] is the molar concentration of the calf thymus
DNA, F0 and F are the fluorescence intensities in the ab-
sence and in the presence of the calf thymus DNA [Q],
respectively, and Ksv is the Stern–Volmer quenching
constant.
Resveratrol 1 and calf thymus DNA were purchased
from Sigma (St. Louis, MO). Supercoiled plasmid
pBR322DNA was purchased from Nippon Gene (To-
kyo, Japan). Analogues of 1; 3,5,30-trihydroxy- (2),
3,5-dihydroxy- (3), 3,40-dihydroxy- (4), 3,30-dihydroxy-
(5), 3-hydroxy- (6), 4-hydroxy (7), 3,4,30,50-tetrahydr-
oxy- (8), and 3,4,30-trihydroxy-trans-stilbene (9), as
shown in Figure 1, were synthesized as previously
reported.24 Saturated form of 1 (1H2) was synthesized
by hydrogenation of 1 using 10% Pd/C as catalyst.
Yield: 98%. 1H NMR(acetone-d6): d 2.73 (m, 4H),
6.19 (d, 1H, J = 2.0 Hz), 6.22 (d, 1H, J = 2.0 Hz),
6.74 (d, 2H, J = 8.4 Hz), 7.03 (d, 2H, J = 8.4 Hz). All
other chemicals and solvents were of reagent grade or
better.
4.5. ESR analysis
ESR spectra were recorded at room temperature on a
JES-FE 2XG spectrometer (JEOL Co. Ltd., Tokyo,
Japan). The sample containing 1 mM CuCl2, 2 mM
NP of calf thymus DNA, and 1 mM of chemical in
50 mM phosphate buffer (pH 7.2) and acetonitrile
(5% by volume) was introduced into a quartz flat cell
and incubated at room temperature for 30 min. The
ESR spectrum was then recorded. The spectrometer
settings were modulation frequency, 100 kHz; modu-
lation amplitude, 10 G; and microwave power,
16 mW.
4.2. DNA-cleaving activity
DNA strand breakage was measured in terms of the
conversion of supercoiled pBR322 plasmid DNA to
the open circular and linear forms. Reactions were car-
ried out in 20 lL (total volume) of 50 mM Na cacodyl-
ate buffer (2.5% DMF), pH 7.2, containing 45 lM bp
pBR322 DNA, 10 lM CuCl2, and 100 lM of each stil-
bene derivative. The reaction mixtures were incubated
at 37 ꢁC for 1 h and then treated with 5 lL of loading
buffer (100 mM TBE buffer, pH 8.3, containing 30%
glycerol, 0.1% bromophenol blue) and applied to 1%
agarose gel. Horizontal gel electrophoresis was carried
out in 50 mM TBE buffer, pH 8.3. The gels were stained
with ethidium bromide (1 lg mLꢀ1) for 30 min, destain-
ed in water for 30 min, and photographed with UV
translumination.
4.6. Theoretical calculations
Density functional calculations were performed with
Gaussian03 (Revision C.02, Gaussian, Inc.) using the
unrestricted B3LYP functional for the open shell mole-
cule on an 8-processor QuantumCubeTM developed by
Parallel Quantum Solutions.
Acknowledgments
This work was supported partly by a grant (MF-16)
from the Organization for Pharmaceutical Safety and
Research of Japan, a grant from the Ministry of Health,
Labour and Welfare, a Grant-in-Aid for Research of
Health Sciences focusing on Drug Innovation
(KH51058) from the Japan Health Sciences Foundation,
and partly by Grants-in-Aid for Scientific Research (B)
(No. 17390033) and for Young Scientist (B) (No.
17790044) from the Ministry of Education, Culture,
Sports, Science and Technology, Japan.
4.3. UV–visible spectra measurements
UV–visible spectra were measured at 37 ꢁC with a
Hewlett Packard 8452A Diode Array Spectrophotome-
ter. A solution in a final volume of 1 mL consisted of
20 lM of sample and 0–100 lM CuCl2 in sodium cac-