Antioxidant-based lead discovery for cancer chemoprevention: the case of resveratrol

Article abstract of DOI:10.1021/jm8015415

Resveratrol is a well-known natural antioxidant and cancer chemopreventive agent that has attracted much nterest in the past decade. Resveratrol-directed compounds were synthesized, and their antioxidant effects gainst reactive oxygen species (ROS)-induced DNA damage, their prooxidant effects on DNA damage in he presence cupric ions, and their cytotoxic and apoptosis-inducing effects on human promyelocytic leukemia(HL-60) cells were investigated in vitro. It was found that the compounds bearing o-diphenoxyl groups exhibited remarkably higher activities in inhibiting ROS-induced DNA damage, accelerating DNA damage in the presence cupric ions, and inducing apoptosis of HL-60 cells compared with the ones bearing no such groups. The detail mechanism of the structure-activity relationship was also studied by the oxidative product analysis of resveratrol and its analogues with galvinoxyl radical or cupric ions and UV-visible spectra change in the presence cupric ions. This study reveals a good and interesting correlation between antioxidant and prooxidant activity, as well as cytotoxicity and apoptosis-inducing activity against HL-60 cells, and provides an idea for designing antioxidant-based cancer chemoprevention agents. ?2009 American Chemical Society.

Full text of DOI:10.1021/jm8015415

J. Med. Chem. 2009, 52, 1963–1974
1963
Antioxidant-Based Lead Discovery for Cancer Chemoprevention: The Case of Resveratrol
Yi-Ping Qian, Yu-Jun Cai,*,‡ Gui-Juan Fan, Qing-Yi Wei, Jie Yang, Li-Fang Zheng, Xiu-Zhuang Li, Jian-Guo Fang, and
Bo Zhou*
State Key Laboratory of Applied Organic Chemistry, Lanzhou UniVersity, Lanzhou, Gansu 730000, China
ReceiVed December 7, 2008
This paper was withdrawn on October 15, 2009 (J. Med. Chem. 2009, 52, 6504).
Resveratrol is a well-known natural antioxidant and cancer chemopreventive agent that has attracted much
interest in the past decade. Resveratrol-directed compounds were synthesized, and their antioxidant effects
against reactive oxygen species (ROS)-induced DNA damage, their prooxidant effects on DNA damage in
the presence cupric ions, and their cytotoxic and apoptosis-inducing effects on human promyelocytic leukemia
(HL-60) cells were investigated in vitro. It was found that the compounds bearing o-diphenoxyl groups
exhibited remarkably higher activities in inhibiting ROS-induced DNA damage, accelerating DNA damage
in the presence cupric ions, and inducing apoptosis of HL-60 cells compared with the ones bearing no such
groups. The detail mechanism of the structure-activity relationship was also studied by the oxidative product
analysis of resveratrol and its analogues with galvinoxyl radical or cupric ions and UV-visible spectra
change in the presence cupric ions. This study reveals a good and interesting correlation between antioxidant
and prooxidant activity, as well as cytotoxicity and apoptosis-inducing activity against HL-60 cells, and
provides an idea for designing antioxidant-based cancer chemoprevention agents.
Introduction
have attracted considerable attention.8 However, growing ex-
perimental evidence suggests that antioxidants present in food
as chemopreventive agents are independent of their ability to
scavenge reactive oxygen species (ROSa).9 Every antioxidant
is in fact a redox agent and thus might become a prooxidant to
accelerate DNA damage under special conditions. Actually,
resveratrol, as a prooxidant, could induce DNA damage in the
presence of cupric ions by the production of ROS,10 and its
prooxidant action is believed to play an important role in its
cancer chemopreventive and apoptosis-inducing properties, as
ROS can mediate apoptotic DNA fragmentation.6,10d,11
Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a phytoalexin
found in a wide variety of dietary sources including grapes,
plums, and peanuts.1 It is also present in red wine at high levels
(0.1-14.3 mg/L)1b and has been suggested to be linked to the
low incidence of heart diseases in some regions of France, the
so-called “French paradox”; i.e., despite high fat intake,
mortality from coronary heart disease is lower because of the
regular drinking of wine.2 Recently, Sinclair’s group and others
reported that resveratrol could activate sirtuin deacetylases and
extend the life span of yeast and flies,3 implicating its potential
as an antiaging agent in treating age-related human diseases.
More recently, Baur et al. showed that this compound could
improve the health of middle-aged mice on a high-calorie diet
and significantly increase their survival.4 One of the most
striking biological activities of resveratrol intensely investigated
during the past years has been its cancer chemopreventive or
anticancer properties.5 These properties were first appreciated
when Pezzuto and colleagues demonstrated the ability of
resveratrol to inhibit carcinogenesis at multiple stages.5a Since
then, resveratrol has been becoming a synonym of naturally
occurring molecules possessing chemopreventive activities and
research on the compound has been gathering momentum. This
has resulted in a flurry of papers reporting that resveratrol can
activate various signal pathways to inhibit tumor cells growth
and directly induce an apoptotic event, such as activation of
caspases, p53, and Bax and inhibition of Bcl2 and NF-κB
(reviewed in refs 5b-e).
The structure simplicity and low toxicity of resveratrol offer
the promise for designing new chemopreventive agents from
an antioxidant/prooxidant point of view. In our ongoing research
project on bioantioxidants, we previously found that simple
structural modification of resveratrol could significantly enhance
its antioxidative activity against free radical induced lipid
peroxidation in micelles, rat liver microsomes, and low-density
lipoprotein.12 Therefore, it is desirable to see if the same
structure-activity relationship (SAR) is also valid in inhibiting
ROS-induced DNA damage, accelerating DNA damage in the
presence of cupric ions, and inducing apoptosis of human
leukemia (HL-60) cells, with emphasis placed on the detail
mechanistic study of the SAR. Resveratrol-directed compounds
studied were 3,4-dihydroxy-trans-stilbene (3,4-DHS), 3,4,4′-
trihydroxy-trans-stilbene (3,4,4′-THS), 3,4,5-trihydroxy-trans-
stilbene (3,4,5-THS), 2,4-dihydroxy-trans-stilbene (2,4-DHS),
The cancer chemoprevention effect elicited by resveratrol can
be traced back to its antioxidant activity,1a,6 since free-radical-
mediated oxidative damage of DNA might play a causative role
in cancer.7 Therefore, the antioxidant activities of resveratrol
a Abbreviations: AAPH, 2,2′-azobis(2-amidinopropane hydrochloride);
3,4-DHS, 3,4-dihydroxy-trans-stilbene; 3,4,4′-THS, 3,4,4′-trihydroxy-trans-
stilbene; 3,4,5-THS, 3,4,5-trihydroxy-trans-stilbene; 2,4-DHS, 2,4-dihy-
droxy-trans-stilbene; 3,5-DHS, 3,5-dihydroxy-trans-stilbene; 3,5,4′-TMS,
3,5,4′-trimethoxy-trans-stilbene; ROHs, resveratrol and its analogues;
EDTA, ethylenediaminetetraacetic acid; EB, ethidium bromide; HL-60 cells,
human leukemia cells; HPLC, high performance liquid chromatography;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ROS,
reactive oxygen species; PBS, phosphate-buffered saline; PI, propidium
iodide; SDS, sodium dodecyl sulfate; SAR, structure-activity relationship.
* To whom correspondence should be addressed. For Y.-J.C.: phone,
(585) 276-9843; fax, (585) 276-9830; e-mail, yujun_cai@urmc.rochester.edu.
For B.Z.: phone, +86-931-8912500; fax, +86-931-8915557; e-mail,
bozhou@
lzu.edu.cn.
‡
Present address: Aab Cardiovascular Research Institute, University of
Rochester School of Medicine and Dentistry, Rochester, NY.
10.1021/jm8015415 CCC: $40.75
2009 American Chemical Society
Published on Web 03/09/2009

1964 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Qian et al.
Chart 1
Moreover, nuclear DNA was isolated from thymocytes treated
with 50 µΜ H2O2 for 6 h in the presence or absence of ROHs
and subjected to agarose gel electrophoresis (Figure 2C). The
addition of H2O2 resulted in the cleavage of DNA into
discontinuous mono- and oligonucleosomal size fragments that
formed a typical DNA ladder, a biochemical hallmark of
apoptosis (lane 2 in Figure 2C). It is clearly seen that 100 µΜ
3,4-DHS, 3,4,4′-THS, and 3,4,5-THS could significantly reduce
the formation of DNA ladder (lanes 3, 4, and 9 in Figure 2C).
Resveratrol and 3,5,4′-TMS showed a similar but somewhat
weaker effect (lanes 5 and 8 in Figure 2C). The SAR obtained
from this experiment is similar to that obtained from the strand
breakage of DNA by gel electrophoresis mentioned above.
Protective Effects of ROHs against H2O2-Induced DNA
Damage in Human Peripheral Blood Lymphocytes. Further-
more, we also investigated the inhibitory effects of ROHs on
H2O2-induced DNA damage in human peripheral blood lym-
phocytes using the single-cell alkaline electrophoresis (comet)
assay.15 It was found that human lymphocytes showed increased
DNA damage after treated with H2O2, and the percentage of
DNA damage was about 95% in the presence of 50 µΜ H2O2
(parts A and B of Figure 3). DNA damage was inhibited by
3,4-DHS in a concentration-dependent manner (parts C and D
of Figure 3). Figure 3E showed the protective effects of 12.5
µΜ ROHs against H2O2-induced DNA damage. All of these
analogues prevented the DNA damage of human lymphocytes
treated with H2O2 in the order of 3,4-DHS ∼ 3,4,4′-THS > 3,4,5-
THS > resveratrol > 2,4-DHS > 3,5-DHS > 3,5,4′-TMS (Figure
3E).
3,5-dihydroxy-trans-stilbene (3,5-DHS) and 3,5,4′-trimethoxy-
trans-stilbene (3,5,4′-TMS) (Chart 1).
Results
Antioxidant Effects of Resveratrol and Its Analogues
(ROHs). Inhibition of AAPH-Initiated DNA Strand Break-
age by ROHs. The single strand breakage in supercoiled
plasmid DNA leads to the formation of open circular form,
whereas the formation of the full length linear form of the
plasmid is indicative of a double-strand breakage.13 Since 2,2′-
azobis(2-amidinopropane hydrochloride) (AAPH) is water-
soluble and the rate of free radical generation from AAPH can
be easily controlled and measured, it has been extensively used
as a free radical initiator for biological studies. Thermal
decomposition of AAPH at physiological temperature generates
alkyl radicals that can react with oxygen and give alkylperoxyl
radicals. Alkylperoxyl radicals then attack plasmid DNA and
lead to DNA strand breakage. AAPH-induced plasmid pBR322
DNA strand breakage and its inhibition by ROHs were assessed
by agarose gel electrophoresis analysis (parts A-C of Figure
1). As shown in Figure 1A, the supercoiled DNA was gradually
converted into the circular and linear forms with an increase of
concentration of AAPH. The inhibition effect produced by
resveratrol depended on the concentration of resveratrol, as
illustrated in Figure 1B. With the increase of the concentration
of resveratrol, its inhibition effect became clearer and the content
of supercoiled DNA increased. The inhibition effects of res-
veratrol analogues also depended on the specific compound used,
as exemplified in Figure 1C. Resveratrol analogues (10 µM)
exhibited significant inhibition effects on the DNA breakage
excepting 3,5,4′-TMS. On the basis of the percentage of intact
supercoiled DNA, the activity for the inhibition of DNA strand
breakage followed the sequence of 3,4-DHS ∼ 3,4,4′-THS >
resveratrol > 3,4,5-THS > 2,4-DHS > 3,5-DHS > 3,5,4′-TMS
(Figure 1D).
Oxidative Products of Resveratrol and 3,4-DHS in the
Presence of Galvinoxyl Radical. Galvinoxyl radical is a
relatively stable oxygen radical and has been widely used for
evaluating antioxidant activities. To investigate the antioxidation
mechanism of ROHs, we isolated and identified the reaction
products of resveratrol and 3,4-DHS oxidized by galvinoxyl
radical in ethanol at room temperature. The major products
(Figure 4) were dihydrofuran dimer in the case of resveratrol
and a dioxane-like dimer in the case of 3,4-DHS by character-
izing with HRMS (ESI) and 1D and 2D NMR (see Supporting
Information). More than eight values for coupling constant (3J)
between H-7 and H-8 in the dimers suggested a predominant
pseudo-trans-axial arrangement for these two aliphatic protons.
Furthermore, enantiomeric composition of the dimers was
evaluated by high performance liquid chromatography (HPLC)
analysis on a chiral column and it was found that the dimer
was a racemic mixture. It is suggested from the oxidative
products that the hydroxyl group at the 4-position is much easier
to subject to oxidation than other hydroxyl groups, and the
dioxane-like dimer is formed via an o-quinone intermediate (vide
infra). The oxidative product analysis also suggests the impor-
tance of o-dihydroxyl groups and 4-hydroxyl groups.
Protective Effects of ROHs against H2O2-Induced Thy-
mocytes Death. Thymocytes from BALB/c mice were exposed
to H2O2 for 24 h, and its viability was assessed by the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
colorimetric assay.14 Upon addition of H2O2 to the cells, its
viability was decreased in a dose-dependent manner (Figure 2A).
Figure 2B showed the effects of increasing concentration of
ROHs in preventing H2O2-induced thymocytes death. It is seen
from the figure that the compounds bearing o-diphenoxyl groups
(3,4-DHS, 3,4,5-THS, and 3,4,4′-THS) exhibit remarkably
higher activities in preventing the cells death than resveratrol.
However, other analogues were ineffective in this experimental
condition.
Prooxidant Activities Analysis of ROHs. Calf Thymus
DNA Damage Induced by ROHs in the Presence of Cu(II).
The calf thymus DNA damage by ROHs in combination with
Cu(II) was assessed by fluorometric analysis with the ethidium
bromide (EB) binding assay16 (Figure 5). It was found that
neither resveratrol analogues nor Cu(II) alone caused detectable
DNA damage (data not shown). However, in the presence of
250 µM Cu(II) the calf thymus DNA was remarkably damaged
by 3,4-DHS, 3,4,4′-THS, and 3,4,5-THS in a concentration-
dependent manner, and to a lesser extent, by resveratrol and
2,4-DHS (Figure 5). 3,5-DHS and 3,5,4′-TMS were inactive
(data not shown). It is worth noting that the compounds bearing
o-dihydroxyl groups (3,4-DHS, 3,4,4′-THS, and 3,4,5-THS) are

ResVeratrol-Based Lead DiscoVery
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1965
Figure 1. Agarose gel electrophoresis pattern of pBR322 DNA strand breakage induced by AAPH and inhibited by ROHs. Supercoiled plasmid
DNA (100 ng) was incubated with AAPH and/or ROHs in phosphate-buffered saline (PBS, pH 7.4) at 37 °C for 60 min. (A) DNA strand breakage
induced by the indicated concentration of AAPH: (lane 1) control; (lanes 2-7) 1.25, 2.5, 5, 10, 20, and 40 mM AAPH, respectively. (B) Inhibitory
effect of resveratrol on AAPH (10 mM) induced DNA strand breakage: (lane 1) control; (lanes 2-7) 0, 2.5, 5, 10, 20, and 40 µM resveratrol,
respectively. (C) Inhibitory effects of ROHs (10 µM) on AAPH (10 mM) induced DNA strand breakage: (lane 1) control; (lane 2) 10 mM AAPH
alone; (lanes 3-9) 3,4,4′-THS, 3,4-DHS, resveratrol, 2,4-DHS, 3,5-DHS, 3,5,4′-TMS, and 3,4,5-THS, respectively. (D) Quantitative analysis of
protective effects of ROHs (10 µM) against AAPH (10 mM)-induced DNA strand breakage. DNA damage is represented by the percentage of
supercoiled DNA to native DNA. Experimental details are described in the section Material and Methods. Values represent the mean ( SD
(n ) 3).
Figure 2. (A) Effect of H2O2 on cell viability. Viable thymocytes were determined using MTT reduction assay as described in the section Materials
and Methods and expressed as a percentage of the untreated control cell samples. Samples were incubated for 24 h in the presence or absence of
H2O2 (10-50 µM). Values are the mean ( SD (n ) 4). (B) Inhibitory effects of ROHs on H2O2-induced thymocyte death. Thymocytes were
preincubated with resveratrol or its analogues for 30 min, and 50 µM H2O2 was then added to the medium. After incubation for 24 h, cell viability
was determined using MTT reduction assay. Values are the mean ( SD (n ) 4). (C) Inhibitory effects of ROHs on DNA fragmentation induced
by H2O2 in thymocytes. Thymocytes were preincubated with resveratrol or its analogues (100 µM) for 30 min, and 50 µM H2O2 was then added
to the medium. After incubation for 6 h, nuclear DNA was extracted from thymocytes and analyzed by agarose gel electrophoresis: (lane M) DNA
marker; (lane 1) control; (lane 2) 50 µM H2O2 alone; (lanes 3-9) 3,4,4′-THS, 3,4-THS, resveratrol, 2,4-DHS, 3,5-DHS, 3,5,4′-TMS, 3,4,5-THS,
respectively, in the presence of 50 µM H2O2.
most active, followed by compounds bearing 4-hydroxyl groups
(resveratrol and 2,4-DHS), while those bearing no such groups
(3,5-DHS and 3,5,4′-TMS) are inactive.
too fast to make the new peak corresponding to the formation
of the o-semiquinone anion or o-quinone observable (Figure
6C). In the case of resveratrol, its decay was significantly slower
than that of 3,4-DHS and a similar bathochromic shift and a
new peak of o-semiquinone anion or o-quinone were not
observed (inset in Figure 6A). There were no clear spectral
changes for 3,5-DHS and 3,5,4′-TMS in the presence of Cu(II).
The decay rate of ROHs and/or ROH-Cu(II) chelate complexes
follows the sequence of 3,4,5-THS > 3,4,4′-THS > 3,4-DHS >
resveratrol > 3,5-DHS and 3,5,4′-TMS. This sequence correlates
well with the activity sequence of the DNA damage mentioned
above.
The formation of 3,4-DHS-Cu(II) chelate was confirmed by
the reaction with ethylenediaminetetraacetic acid (EDTA), a
well-known chelating agent for metal ions (Figure 7). EDTA
was added after Cu(II) reacted with 3,4-DHS for 10 min. Upon
addition of EDTA, the red-shifted bands (342 nm) returned to
its initial position with a decrease in absorbance, but the new
produced band (459 nm) did not show any change. If EDTA
was added together with Cu(II), the spectrum of 3,4-DHS (line
1) did not show any change. These results indicate unambigu-
UV-Visible Spectral Changes of ROHs in the Presence
of Cu(II). In order to clarify the mechanism of the DNA
damage, the UV-visible absorption changes of ROHs in the
presence of Cu(II) were examined. The addition of Cu(II) to
3,4-DHS resulted in a rapid disappearance of the maximal
absorption at 317 nm accompanied by its bathochromic shift to
342 nm (Figure 6A). This peak (342 nm) is characteristic of
the formation of a chelate complex of 3,4-DHS with Cu(II). Its
intensity decreased with an increase of time, and a new peak
appeared at 459 nm because of the formation of the o-
semiquinone anions or o-quinone (vide infra). The isosbestic
point at 385 nm suggested a direct transition from one form
(3,4-DHS-Cu(II) chelate) to another one (o-semiquinone anion).
Similar slight bathochromic shift from 329 to 334 nm and
appearance of a new peak at 439 nm, as well as one isobestic
point, were observed in the case of 3,4,4′-THS (Figure 6B),
but its decay was faster than that of 3,4-DHS. The decay of
3,4,5-THS was the fastest one among these compounds. It was

1966 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Qian et al.
Figure 3. Representative single-cell alkaline electrophoresis patterns of human peripheral blood lymphocytes. Human lymphocytes in PBS were
pretreated with 12.5 µM ROHs analogues, and 50 µM H2O2 was then added to the medium. After incubation for 10 min, DNA damage of individual
lymphocytes was estimated by comet assay as described in the section Materials and Methods. (A) Untreated control. (B) H2O2 alone. (C) H2O2 +
3,4-DHS. (D) Protection of the indicated concentrations of 3,4-DHS from H2O2-induced DNA damage in human peripheral blood lymphocytes.
Values are the mean ( SD (n ) 3). (E) Protection of ROHs from H2O2-induced DNA damage in human peripheral blood lymphocytes. Values are
the mean ( SD (n ) 3).
oxygen in the reaction of 3,4-DHS with Cu(II) was studied by
mixing 3,4-DHS with Cu(II) under an inert atmosphere. It can
be seen from line 4 in the inset of Figure 6 that under an inert
atmosphere, although the 3,4-DHS-Cu(II) chelate still formed,
the band at 459 nm did not appear. That is, Cu(II) could not
induce the reaction of 3,4-DHS in the absence of O2.
Oxidative Products of Resveratrol and 3,4-DHS in the
Presence of Cu(II). To investigate the prooxidation mechanism
of ROHs, we also isolated and identified the oxidative products
Figure 4. Oxidation products of resveratrol (A) and 3,4-DHS (B) in
of resveratrol and 3,4-DHS in the presence of Cu(II) in
the presence of galvinoxyl radical.
acetonitrile at room temperature. The dimer products in the
presence of Cu(II) were the same as that in the presence of
galvinoxyl radical. Other ROHs bearing no 4-hydroxyl group
(3,5-DHS and 3,5,4′-TMS) gave no corresponding dimers. The
result also indicates unambiguously that o-quinone may be the
reaction intermediate in the case of 3,4-DHS.
Apoptosis-Inducing Activities of ROHs for HL-60
Cells. The antiproliferative effects of ROHs on HL-60 cells were
assessed by the colorimetric MTT assay,14 and results are
demonstrated in Figure 8A. It is seen from the figure that ROHs
exhibit dose-dependent inhibitory effects on HL-60 cell prolif-
eration. The cell viability was reduced to 70%, 54%, and 38%
after treatment with 10, 15, and 20 µM, respectively, in the
case of 3,4,5-THS for 48 h. Other analogues showed similar
effects, but to a lesser extent, on suppression of the cell viability
as exemplified in Figure 8A. The IC50 values for all of these
compounds are shown in Figure 8B. It indicates that 3,4-DHS,
3,4,4′-THS, and 3,4,5-THS exhibited higher cytotoxicity than
resveratrol and other analogues.
Figure 5. Extent of calf thymus DNA damages induced by ROHs in
the presence of Cu(II). DNA (80 µg) was incubated at 37 °C for 60
min with different concentrations of ROHs in the presence of 250 µM
Cu(II) in PBS buffer (pH 7.4) under air.
It is now well recognized that apoptosis is implicated for
multistage carcinogenesis and removal of damaged precancerous
cells via apoptosis provides an important strategy for the
treatment of cancer.17 Apoptosis can be characterized by the
cleavage of DNA into discontinuous mono- and oligo-nucleo-
ously that 3,4-DHS can chelate with Cu(II) as a bidentate ligand,
hence facilitating intramolecular electron transfer to form the
stable o-semiquinone anions (vide infra). The participation of

ResVeratrol-Based Lead DiscoVery
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1967
Figure 6. Absorption spectral changes of ROHs (50 µM) in the absence (dash line) and in the presence of 25 µM Cu(II) in PBS buffer (pH 7.4)
under air: (A) 3,4-DHS (inset, resveratrol) (interval, 3 min); (B) 3,4,4′-THS (interval, 1 min); (C) 3,4,5-THS (interval, 1 min). Arrows show the
time-related absorbance changes.
a lower incidence of various cancers.21 From this has developed
the idea that antioxidants in these foods are the effective cancer
chemopreventive agents.21 In the past year of the past century
the Chemoprevention Working Group to the American Associa-
tion for Cancer Research22 published its report “Prevention of
Cancer in the Next Millennium”, demonstrating that chemo-
prevention by using antioxidants has become a viable alternative
means in cancer control. On the other hand, however, there is
no doubt that free radical biology and medicine and antioxidant
therapy are still in their infancy. Many mechanisms on the action
of antioxidants in cancer chemoprevention are still unclear,
which are called “antioxidant conundrum”,23 and many ques-
tions are still under debate.24 The cancer chemopreventive
properties of antioxidants are generally believed to be due to
their ability to scavenge endogenous ROS. However, just as
Figure 7. Absorption spectral changes of 3,4-DHS: (trace 1) 3,4-DHS
(50 µM); (trace 2) 3,4-DHS (50 µM) plus Cu(II) (25 µM) for 10 min
ROS has pros and cons for human health, an antioxidant can
switch to a prooxidant under certain conditions and the
prooxidant action might not always be adverse for humans. As
a matter of fact, cancer cell apoptosis induced by polyphenolic
antioxidant are considered to be mediated by ROS,25 and these
antioxidants are also nontoxic to the normal cells.26 It is indeed
quite puzzling as to how these antioxidants can differentiate
between “normal” versus “abnormal tumor” cells in terms of
signaling, gene expression, and pharmacological effects. The
present work focused on the antioxidant and prooxidant activity
and the apoptosis-inducing activity against HL-60 cells of
resveratrol analogues with the different structure features that
enable us to deduce a clear picture of the structure determinants
for the activity and a structure-activity relationship. Further-
more, an interesting correlation among the antioxidant activity,
prooxidant activity, cytotoxicity, and apoptosis-inducing activity
has emerged. The present paper is intended to provide informa-
tion for understanding the chemopreventive mechanism of
polyphenolic antioxidants and an idea for designing antioxidant-
based cancer chemoprevention agents.
under air; (trace 3) same as 2 but adding EDTA (75 µM) under air;
(trace 4) same as 2 but recorded under argon atmosphere.
somal size fragments that form a typical DNA ladder during
gel electrophoresis.18 Therefore, nuclear DNA was isolated from
treated as well as untreated HL-60 cells and subjected to agarose
gel electrophoresis. As illustrated in Figure 9, electrophoresis
of DNA extracted from HL-60 cells treated with ROHs at certain
concentrations for 48 h exhibited a progressive increase of the
nonrandom fragmentation of the DNA into a ladder of 180-200
bp nucleosomes.
In order to quantify the apoptotic activity of ROHs flow
cytometric analysis was performed to detect the apoptotic cells,
i.e., cells with sub-G1 content of DNA.19 HL-60 cells were
treated with ROHs at certain concentrations for 48 h, then fixed
and stained with propidium iodide (PI) for flow cytometric
analysis. The results are shown in the DNA frequency distribu-
tion histograms (Figure 10), in which the M1 marker shows
the sub-G1 cell fraction that corresponds to the percentage of
apoptotic cells. It is seen from the figure that these compounds
at certain concentrations exhibit significant apoptotic activity
with the sub-G1 cell fraction ranging from 17.6% to 56.15%.
Especially, 50 µM 3,4,5-THS shows very high apoptotic activity
(Figure 10). Again, the molecules bearing the o-dihydroxyl
functionality exhibited remarkably higher activity than resvera-
trol and molecules bearing no such functionality.
Structure-Activity Relationship and Mechanism for
Antioxidant Reaction. Resveratrol and its analogues were found
to be able to protect DNA from AAPH-induced strand breakage
as illustrated in Figure 1. Similarly, we found that these
compounds could inhibit hydrogen peroxide induced apoptotic
death via oxidative stress in thymocytes (Figure 2). Furthermore,
it was found from Figure 3 that these compounds could inhibit
hydrogen peroxide induced DNA damage of human lympho-
cytes. It is worth noting that the activities of 3,4-DHS, 3,4,5-
THS, and 3,4,4′-THS, that is, the molecules bearing o-dihy-
droxyl groups, are appreciably higher than those of resveratrol
and molecules bearing no such groups in the above-mentioned
experimental systems. This can be understood because the
o-hydroxylphenoxyl radical, the oxidation intermediate for these
three more active species, is more stable because of the
Discussion
Chemoprevention is an active cancer preventive strategy to
intervene in the progress of carcinogenesis, using naturally
occurring or synthetic substances.20 There is considerable
evidence that ROS or free radical-induced oxidative DNA
damage contributes to human tumorigenesis7 and that fruits and
vegetables contain substantial amounts of various natural
compounds with antioxidant properties that are associated with

1968 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Qian et al.
Figure 8. (A) Inhibitory effects of ROHs on HL-60 cell viability. HL-60 cells were treated with tested compounds for 48 h, and the percent
viability was determined using the MTT method as described in the section Materials and Methods. Values are the mean ( SD of four independent
experiments. (B) In vitro cytotoxicity of ROHs for HL-60 cells. Cytotoxicity is expressed as IC50, the concentration of the compound to cause 50%
inhibition of the cell viability. Data are expressed as the mean ( SD of four determinations.
Figure 9. DNA fragmentation of HL-60 cells by ROHs for 48 h. The
nuclear DNA was extracted from treated cells and analyzed by
conventional agarose gel electrophoresis followed by ethidium bromide
staining as described in the section Materials and Methods: (lane M)
DNA molecular weight markers; (lane 1) control (cells treated with
medium); (lane 2) treated with 3,4-DHS (50 µM); (lane 3) treated with
3,4,4′-THS (50 µM); (lane 4) treated with resveratrol (150 µM); (lane
5) treated with 2,4,-DHS (200 µM); (lane 6) treated with 3,5-DHS (200
µM); (lane 7) treated with 3,5,4′-TMS (50 µM); (lane 8) treated with
3,4,5-THS (50 µM); (lane 9) treated with VP-16 (25 µM).
intramolecular hydrogen bonding interaction, as evidenced
recently from experiments by spectrophotometric measurement27
and theoretical calculations.28 The theoretical calculation showed
that the hydrogen bond in the o-OH phenoxyl radical is
approximately 4 kcal/mol stronger than that in the parent
catechol and that the bond dissociation energy (BDE) of catechol
is 9.1 kcal/mol lower than that of phenol and 8.8 kcal/mol lower
than that of resorcinol.28 In addition, it should be easier to further
oxidize the o-OH phenoxyl radical and/or o-semiquinone anion
to form the final o-quinone (Scheme 1) as evidenced from the
Figure 10. Flow cytometric analysis for apoptosis of HL-60 cells.
Cells were treated with ROHs with certain concentration for 48 h, then
formation of the dioxane-like dimer of 3,4-DHS (Figure 4) in
the presence of galvinoxyl radical in ethanol at room temper-
washed and harvested. DNA fragmentation was determined by flow
ature. Obviously, this dimer is the [4 + 2] Diels-Alder adduct
of the o-quinone intermediate and another molecule of 3,4-DHS.
Sugumaran reported that the formation of 1,2-dehydro-N-
acetyldopamine quinone is favorable in acidic media. But the
quinone is highly unstable in the neutral media.29 Davies also
pointed out that caffeoquinone is unstable at temperatures as
low as -20 °C.30 The 3,4-DHS o-quinone may be unstable
under our experimental conditions (pH 7.4), but it can be trapped
by 3,4-DHS via the Diels-Alder reaction, similar to the
formation of R-tocopherol dimer from the Ag2O oxidation of
R-tocopherol.31
cytometry after PI staining. The M1 marker shows the sub-G1 cell
fraction that corresponds to the apoptotic cell: (A) control (cells treated
with medium); (B) treated with 3,4-DHS (50 µM); (C) treated with
3,4,4′-THS (50 µM); (D) treated with 3,4,5-THS (50 µM); (E) treated
with resveratrol (150 µM); (F) treated with 3,5-DHS (200 µM); (G)
treated with 2,4,-DHS (200 µM); (H) treated with 3,5,4′-TMS (50 µM).
with lower oxidation potentials and reversible cyclic voltam-
mograms, that is, 3,4-DHS, 3,4,4′-THS, and 3,4,5-THS (0.36,
0.34, and 0.23 V vs SCE, respectively), exhibit higher activity,
while molecules with higher oxidation potentials and irreversible
cyclic voltammograms, that is, 3,5-DHS and resveratrol (0.79
and 0.67 V vs SCE, respectively), are less active.12a These facts
It is worth noting that the antioxidant activity is correlated
with the electrochemical behavior of the molecule. Molecules

ResVeratrol-Based Lead DiscoVery
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1969
Scheme 1. Antioxidative Mechanism of 3,4-DHSa
polyphenols rather than their antioxidant activity that may play
a critical role in their anticancer and apoptosis-inducing proper-
ties, as ROS can mediate apoptotic DNA fragmentation.25
Recently, Hadi and co-workers proposed a mechanism for the
cytotoxic action of plant polyphenols against cancer cells that
involves mobilization of endogenous copper and the consequent
prooxidant action.10d,11b Indeed such a common mechanism
better explains the anticancer effects of polyphenols with diverse
chemical structures. Therefore, it is of important to see how an
antioxidant can switch to a prooxidant and its reaction mechanism.
It is seen from Figure 5 that ROHs can show prooxidative
action to induce the calf thymus DNA damage in the presence
of Cu(II). It is clearly seen that the compounds bearing
o-dihydroxyl groups (3,4-DHS, 3,4,4′-THS, and 3,4,5-THS) are
the most active ones in inducing DNA damage, followed by
compounds bearing 4-hydroxyl groups (2,4-DHS and resvera-
trol), while those bearing no such groups (3,5-DHS and 3,5,4′-
TMS) are inactive. This activity sequence is similar to that
observed in the inhibition of DNA damage of ROHs.
The high activity of 3,4-DHS, 3,4,4′-THS, and 3,4,5-THS is
obviously due to their o-dihydroxyl groups, which can chelate
with Cu(II) to form the ArOH-Cu(II) complex, as evidenced
from Figures 6. The ArOH-Cu(II) complex can undergo
intramolecular electron transfer to form semiquinone anion and
Cu(I). It has been pointed out that the protonated phenolic group
is not a particularly good ligand for metal cations, but once
deprotonated, an oxygen center is generated that possesses a
high charge density, the so-called “hard” ligand.36 Although the
pKa1 value of catechol is 9.25,37 the hydroxyl proton could
dissociate at much lower pH values, e.g., 5.0-8.0 in the presence
of Cu(II).36 Therefore, 3,4-DHS should dissociate to form a
phenoxide, which chelates Cu(II) as a bidentate ligand and
undergoes intramolecular electron transfer to form o-OH phe-
noxyl radicals. The acidity dissociation constant of the latter is
much lower (pKa1 ) 4.1)38 than that of catechol (pKa1 ) 9.25).37
Thus, the o-OH phenoxyl radical should easily dissociate to form
the o-semiquinone anion, which chelates with Cu(I). The new
peak appearing at 459 nm in the case of 3,4-DHS demonstrates
the formation of the o-semiquinone anion or o-quinone (Figure
6A). This is similar to the previous observation of the absorbance
of o-semiquinone anion at 440 nm appearing during the
oxidation of resveratrol analogues bearing o-dihydroxyl
groups,8b of chlorogenic acid quinone at 408 nm,39 and of 1,2-
dehyo-N-acetyldopamine quinone at 390 nm.29 In addition, the
o-semiquinone anion should be more easily further oxidized to
form the final products o-quinone as evidenced from the dimer
product of 3,4-DHS in the presence of Cu(II) (Scheme 3).
a X• denotes free radical.
suggest that electron-transfer antioxidation might take place
simultaneously with the direct hydrogen-abstraction reaction,
as exemplified in Scheme 1. It is well-known that the phenoxide
(ArO-) undergoes electron transfer oxidation more easily to
produce the relatively stable phenoxyl radical (ArO•) in alkaline
media. Resveratrol, with a pKa1 of 8.8,32 partially dissociates
under our experimental conditions (pH 7.4); this makes the
electron-transfer reaction feasible. Cooperation between hydrogen-
abstraction and electron-transfer processes in an antioxidation
reaction by phenolic antioxidants has recently been discussed
theoretically.28 It was also proved by the oxidative product of
resveratrol that the hydroxyl group at the 4′-position is much
easier to subject to oxidation than other hydroxyl groups.
Therefore, the oxidation reactions take place at the 4′-OH
position in the case of resveratrol, resulting in the formation of
phenoxyl radicals or semiquinone (“A”, “B”, and “C”). Suc-
cessively, the coupling of one radical “B” and one radical “C”,
followed by tautomeric rearrangement and intramolecular nu-
cleophilic attack to the intermediate quinone, gave the dihy-
drofuran dimer as shown in Scheme 2.
Structure-Activity Relationship and Mechanism for
Prooxidant Reaction. Resveatrol is recognized as a naturally
occurring polyphenol antioxidant but also acts as a prooxidant,
inducing DNA damage in the presence of Cu(II).10 Copper is
an important metal ion present in chromatin and is closely
associated with DNA bases, in particular, guanine.33 It is also
one of the most redox active metal ions present in cells.34
Furthermore, evidence suggests that the concentration of copper
is greatly increased in various malignancies.11b,35 For example,
it was shown that copper concentrations in serum and cells of
leukemic patients (328 µg/mL and 52 µg/1010 cells in serum
and cells, respectively) were significantly higher than those of
health donors (114 µg/mL and 15 µg/1010 cells in serum and
cells, respectively).35 Copper ions from chromatin can be
mobilized by metal-chelating agents, leading to internucleosomal
DNA breakage, a property that is the hallmark of a cell
undergoing apoptosis.11b Much evidence suggests that antioxi-
dant properties of polyphenolic compounds may not fully
account for their chemopreventive effects.11b Therefore, it has
been proposed that it is the prooxidant action of plant-derived
The fact that Cu(II) could not effectively oxidize 3,4-DHS
to o-semiquinone anion intermediates in the absence of oxygen
(Figure 7) demonstrates unambiguously the involvement of O2
in the process. It is well-known that phenolics and Cu(II)-
mediated DNA damage is due to the involvement of ROS and
Cu(I) in the process.10 Therefore, possible mechanisms of DNA
damage induced by 3,4-DHS and resveratrol in the presence of
Cu(II) can be clarified as shown in Schemes 3 and 4,
respectively. The initial electron transfer oxidation of 3,4-DHS
or resveratrol with O2 generates the corresponding semiquinone
and superoxide radical anion. The semiquinone in the case of
3,4-DHS undergoes second electron transfer with O2 to form
o-quinone and the superoxide radical anion and subsequent
Diels-Alder reaction with another 3,4-DHS molecule leading
to dioxane-like dimer as the ultimate product as shown in
Scheme 3. In comparison, the semiquinone in the case of
resveratrol undergoes a radical coupling and intramolecular

1970 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Scheme 2. Antioxidative Mechanism of Resveratrola
Qian et al.
a X• denotes free radical.
Scheme 3. Prooxidative Mechanism of 3,4-DHS
Scheme 4. Prooxidative Mechanism of Resveratrol
lines.41 Of interest, intriguing results were recently described
that treatment of resveratrol-resistant Jurkat cells with 3,4,5-
THS rapidly induced extensive apoptosis.42
nucleophilic attack, resulting in the formation of the dihydro-
furan dimer as shown in Scheme 4. The superoxide radical anion
reacts with Cu(I), giving hydrogen peroxide which is readily
converted by a Fenton-type reaction to a hydroxyl radical to
induce the oxidative DNA damage. In addition, o-quinones have
also been suggested to be involved in DNA damage by forming
covalent adducts with DNA.40
Correlations among Antioxidant, Prooxidant, Cytotoxic,
and Apoptosis-Inducing Activities. The present work dem-
onstrates that resveratrol analogues bearing o-dihydroxyl groups
(3,4-DHS, 3,4,4′-THS, and 3,4,5-THS) exhibit signicantly higher
antioxidant and prooxidant activities than resveratrol and other
analogues bearing no such a groups. It is worth noting that these
compounds exhibit enhanced cytotoxicity (Figure 8) and apo-
ptosis-inducing activity (Figures 9 and 10) on HL-60 cells. It
was also recently reported that 3,4-DHS, 3,4,4′-THS, and 3,4,5-
THS had lower ApoEC50 values than resveratrol and other
analogues against human leukemia (HL-60 and Jurkat) cell
Comparison of the antioxidant activities and prooxidant
activities, cytotoxicities, and apoptosis-inducing activities against
HL-60 cells, as well as the oxidation potentials of those
molecules, reveals a very interesting correlation between these
activities and a very clear structure-activity relationship. That
is, the antioxidant activities of these molecules correlate well
with their prooxidant activities in the presence of Cu(II) and
with their cytotoxicities and apoptosis-inducing activities against
HL-60 cells. These activities also correlate well with the
oxidation potentials of the molecules, i.e., the lower is the
oxidation potential, the higher is the activity. The latter
correlation suggests clearly that electron transfer plays a critical
role in the antioxidative and prooxidative reactions, apoptosis-
inducing events against HL-60 cells of resveratrol analogues.
The finding appears to corroborate the observation from other

ResVeratrol-Based Lead DiscoVery
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1971
Scheme 5. Possible Mechanism for Chemopreventive Effects of
Phenolic Antioxidant
damage induced by polyphenolic antioxidants in the presence
of Cu(II) may be an important pathway through which preneo-
platic cells and neoplastic cells can be killed while normal cells
survived (Scheme 5).
It is also understood that 3,4-DHS, 3,4,4′-THS, and 3,4,5-
THS with low oxidation potentials (strong electron-transfer
capacity) and high antioxidant and prooxidant activities exhibit
high cytotoxic and apoptosis-inducing activities against HL-60
cells. In addition, the oxidative products of the compounds
bearing o-dihydroxyl groups (3,4-DHS, 3,4,4′-THS, and 3,4,5-
THS), i.e., o-dihydroxyphenoxyl radical and o-quinone, may
be also responsible for the apoptosis-inducing activities. It has
been reported that the o-quinone products from oxidation of
catecholic estrogen40a and dopamine40b are responsible for the
observed apoptotic effects of these chemicals in mutagenic and
neuroblastoma cells. Furthermore, their oxidative oligomer
products of resveratrol might also be responsible for the
cytotoxic and apoptosis-inducing activities against cancer cells.46
authors that electron-transfer capacity of catechin derivatives
correlate well with arrest activity of the cell cycle and apoptosis-
inducing activity in HT29 cells.43 The electron-transfer capacity
of exogenous plant phenolics and its influence on the delicate
balance between the antioxidant and prooxidant events govern-
ing cell functions may help to explain the putative cancer
chemopreventive properties of resveratrol analogues.
Conclusions
Resveratrol-directed compounds such as 3,4-DHS, 3,4,4′-
THS, and 3,4,5-THS bearing o-dihydroxyl groups exhibit
remarkably higher activity in the experiments of inhibiting ROS-
induced DNA damage and accelerating DNA damage in the
presence of copper than the ones bearing no such groups, and
the correlations among the antioxidant activities and prooxidant
activities, cytotoxicities, and apoptosis-inducing activities against
HL-60 cells are obtained. Although the precise mechanism is
still unclear, these results support the free radical theory of
cancer and give us useful information for antioxidant and
chemoprevention drug design.
How polyphenolic antioxidant mediates their cancer chemo-
prevention effects from the view of free radical biology is not
fully understood. Antioxidants counteract ROS production and
inhibit the latter-induced oxidative DNA damage and therefore
reduce the risk of cancer. On the other hand, growing experi-
mental evidence suggests that polyphenolic antioxidant-mediated
production of ROS (prooxidant action) may be responsible for
the ability to induce apoptosis of cancer cells.25 Therefore two
main questions rise from this paradox: (i) “When” will the
antioxidant and prooxidant activities of polyphenolics take place
to show efficacy? (ii) “Which” antioxidant and prooxidant
activities of polyphenolics are responsible for the ability of
cancer chemopreventive properties? Antioxidants such as res-
veratrol were shown to induce apoptoic cell death in HL-60
cell lines but not in normal peripheral blood lymphocytes.26b If
so, how can these antioxidants differentiate between “normal”
versus “abnormal tumor” cells? The present work (correlation
and structure-activity relationship), combined with the observa-
tion from other authors, can supply an explanation to this
paradox and these questions. As a matter of fact, antioxidants
induce a multitude of effects that depend on the cell type
(“normal” and cancer cells), cellular condition (normal, stressed,
or malignant), and concentration, and it can have opposing
activities. In the case of normal cells with low levels of copper,
antioxidants can counteract excess ROS production (oxidative
stress) and inhibit ROS-induced oxidative DNA damage,
retaining the delicate redox balance, and therefore reduce the
risk of cancer (Scheme 5). Compared with normal cells,
preneoplastic cells and neoplastic cells have been shown to
contain elevated levels of copper35 and may be more sensitive
to electron transfer with polyphenolic antioxidants to generate
ROS (strong prooxidant properties), resulting in strong but
“good” oxidative stress and DNA damage. It was also reported
that copper could enhance cytotoxic and apoptosis-inducing
activities of phenolic antioxidants against human leukemia and
breast cancer cells.44 Recently, Levine and co-workers demon-
strated that ascorbate (a famous antioxidant) at pharmacologic
concentrations was a prooxidant, generating hydrogen peroxide
dependent cytotoxicity toward a variety of cancer cells in vitro
without adversely affecting normal cells.45 Therefore, DNA
Materials and Methods
Materials. Resveratrol and its analogues 3,4-DHS, 3,4,4′-THS,
3,4,5-THS, 2,4-DHS, 3,5-DHS, and 3,5,4′-TMS were synthesized
as previously described.47 This method produced exclusively the
trans-isomer. Their structures were fully identified using 1H NMR
and EI-MS (see Supporting Information), and the data were
consistent with those reported in the literature.47 The purity (g95%)
of each compound was checked using a HPLC on a Waters 600
instrument with a photodiode array detector (see Supporting
Information).
Galvinoxyl radical, AAPH, EB, pBR322 DNA, calf thymus
DNA, agarose, low-melting point agarose, PI, RNase, MTT, and
sodium dodecyl sulfate (SDS) were purchased from Aldrich-Sigma.
RPMI medium 1640 was from GIBCO. All other chemicals were
of the highest quality available.
Assay for Oxidative DNA Strand Breakage Induced by
AAPH. The inhibition of AAPH-induced DNA strand breakage
by ROHs was assessed by measuring the conversion of the
supercoiled pBR322 plasmid DNA to open circular and linear forms
by gel electrophoresis.13a pBR322 DNA (0.1 µg) was incubated
with the indicated concentration of resveratrol, its analogues, and
AAPH in phosphate-buffered saline (PBS) at pH 7.4 and 37 °C for
1 h. After incubation, the samples were mixed with 5 µL of gel
loading buffer (0.13% bromophenol blue and 30% (w/v) sucrosel)
and immediately loaded in 1% agarose gels containing 40 mM Tris,
20 mM sodium acetate, and 2 mM EDTA and subjected to
electrophoresis in a horizontal slab gel apparatus in Tris/acetate/
EDTA gel buffer for 1 h. The gels were stained with 0.6 µg/mL
ethidium bromide for 30 min, followed by destaining in water for
30 min, and photographed under a transilluminator with UV light.
Gel_Pro Analyzer (version 3.0 from Media Cybernetics) was used
to quantify the density of supercoiled DNA form.
Thymocytes Preparation. Thymocytes were prepared as de-
scribed previously.48 Briefly, whole thymuses from young healthy
BALB/c mice were finely minced and pressed through stainless

1972 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Qian et al.
steel sieves in cold RPMI-1640 under aseptic conditions. After
centrifugation at 500g for 10 min, the cells were suspended in
RPMI-1640 medium supplemented with 10% fetal bovine serum,
penicillin (100 U/mL), and streptomycin (100 U/mL).
The slides were viewed with fluorescence microscope with
camera attachment. The representative views of slides were
photographed, and 50 randomly selected cell nucleoids were
scored on a slide and gave an overall comet score (A), in which
the tail is longer than half of length of head. The percentage
DNA damage was calculated from the ratio of the measurements
as follows: (A/50) × 100. The scoring method is consistent with
computer imaging analysis.50
Assessment of Thymocyte Viability. The thymocyte viability
was assessed by the MTT colorimetric assay which is based on
the reduction of MTT by the mitochondrial succinate dehydrogenase
of intact cells to a purple formazan product.14 Briefly, 100 µL
aliquots of thymocytes containing 5 × 105 cells/mL were added to
each well of a 96-well flat microtiter plate and incubated with
various amounts of hydrogen peroxides and/or ROHs dissolved in
0.1% dimethyl sulfoxide (DMSO). Six replicate wells were used
at each point in the experiments. After 24 h of incubation at 37
°C, MTT solution (5 mg/mL in PBS) was added and incubated for
another 4 h at 37 °C in a 5% CO2 incubator. The resulting MTT-
formazan product was dissolved by the same volume of lysis buffer
(10% SDS and 0.1 M HCl), and the incubation was continued
overnight at 37 °C. The amount of formazan was determined by
measuring the absorbance at 570 nm using a Bio-Rad 550 ELISA
microplate reader.
Analysis of DNA Fragmentation of Thymocytes by Agarose
Gel Electrophoresis. DNA fragmentation was assessed by agarose
gel electrophoresis as reported previously.18 Thymocytes were
suspended at a density of 1 × 105 cells/mL in RAPI 1640 medium
and incubated with 100 µM ROHs and 50 µM hydrogen peroxides
for 6 h. The cells were harvested by centrifugation at 1000g for 10
min, then washed with PBS and incubated with lysis buffer (50
mM Tris-HCl, pH 8.0, 10 mM NaCl, 10 mM EDTA, 0.5% SDS,
and 0.5 mg/mL proteinase K) at 37 °C for 2 h. The DNA was
extracted successively with phenol/chloroform (1:1, v/v) and
chloroform/isoamyl alcohol (24:1, v/v) and precipitated overnight
in -20 °C with ethanol containing 0.3 M sodium acetate (pH 5.2),
then centrifuged at 13000g at -4 °C for 15 min. The pellets were
dried in the air and resuspended in TE buffer (10 mM Tris-HCl,
pH 8.0, 1 mM EDTA). DNase-free RNase (100 µg/mL) was added
to each sample and incubated at 37 °C for 1 h. The DNA samples
were mixed with loading buffer and subjected to electrophoresis
on 1.5% agarose gel at 5 V/cm and visualized under UV light after
staining with ethidium bromide. The occurrence of apoptosis was
indicated by the appearance of a ladder of oligonucleosomal DNA
fragments that were approximately 180-200 bp multiples.
Isolation of Human Peripheral Blood Lymphocytes. Human
lymphocytes were isolated from fresh blood obtained from healthy
volunteers as described previously.49 Briefly, human peripheral
blood was collected and immediately mixed with heparin to avoid
coagulation and then diluted with PBS, layered onto an equal
volume of LymphoPrep LSM, and centrifuged at 700g for 30 min.
The lymphocytes were removed into a fresh tube and washed once
with PBS and then suspended in PBS for comet assay.
Assay for Cellular DNA Damage by Single-Cell Alkaline
Electrophoresis (Comet Assay). Cellular DNA damage was
assessed by comet assay.15 Normal melting point agarose at 0.5%
in PBS was dropped onto a microscope glass slides, and a
coverslip was applied immediately to cover it. The human
lymphocytes (2 × 106 cells) treated with hydrogen peroxide and/
or ROHs were washed with PBS and then mixed thoroughly
with 1% low melting point agarose dissolved in PBS at 39 °C.
Next, the mixture was transferred onto the microscope slide that
had been precoated with normal melting point agarose and a
glass coverslip was applied on the top of the liquid agarose to
spread the agarose across the surface of the slides, which was
placed on top of ice for 10 min. After removing the coverslips,
the slides were immersed slowly in ice-cold alkaline cell lysis
solution (2.5 M NaCl, 0.1 M EDTA, 1% N-lauroylsarcosine,
1% Triton X-100, 10% DMSO, 10 mM Tris, pH 10) and allowed
to stay for 1 h. Slides then were denatured in an eletrophoresis
tank containing 0.3 M NaOH/1 mM EDTA for 20 min. Next,
electrophoresis was carried out at 25 V/300 mA for 20 min.
Afterward, the slides were gently immersed in neutralization
buffer (0.4 mM Tris-HCl, pH 7.5) for 5 min after applying
ethidium bromide solution to stain and covering with coverslips.
Oxidation Product Analysis of Resveratrol or 3,4-DHS in
the Presence of Galvinoxyl Radical in Ethanol. The 60 mg of
resveratrol (0.26 mmol) and 220 mg of galvinoxyl (0.52 mmol)
were mixed in 30 mL of ethanol and stirred for 8 h. The products
were purified by silica gel chromatography and eluted with
chloroform-methanol (5:1, v:v) to afford 24 mg of dimer,
recovering unreacted resveratrol (29 mg) with the conversion, and
yields of 78% and 41%, respectively. The dehydrodimer of
1
resveratrol: H NMR (400 MHz, (CD3)2CO, δ): 4.62 (d, J ) 8.0
Hz, 1H, H-8), 5.48 (d, J ) 8.0 Hz, 1H, H-7), 6.19 (d, J ) 2.0 Hz,
2H, H-10 and H-14), 6.25 (d, J ) 2.0 Hz, 2H, H-12), 6.53 (d, J )
2.0 Hz, 2H, H-10′ and H-14′), 6.84 (d, J ) 8.0 Hz, 2H, H-3 and
H-5), 6.88 (d, J ) 8.0 Hz, 1H, H-3′), 6.92 and 7.08 (AB system,
d, J ) 16.0 Hz, 2H, H-7′ and H-8′), 7.24 (d, J ) 8.0 Hz, 2H, H-2
and H-6), 7.25 (d, J ) 8.0 Hz, 1H, H-2′), 7.45 (d, J ) 2.0 Hz, 1H,
H-6′). 13C NMR (100 MHz, (CD3)2CO, δ): 57.7 (1C, C-8), 94.2
(1C, C-7), 102.4 (1C, C-12), 102.7 (1C, C-12′), 105.7 (2C, C-10′
and C-14′), 107.4 (2C, C-10′ and C-14′), 110.1 (1C, C-3′), 116.1
(2C, C-3 and C-5), 123.9 (1C, C-2′), 127.2 (1C, C-7′), 128.5 (3C,
C-6′, C-2 and C-6), 129.1 (1C, C-8′), 131.7 (1C, C-1), 132.1 (1C,
C-5′), 132.5 (1C, C-1′), 140.7 (1C, C-9′), 145.1(1C, C-9), 158.4
(1C, C-4), 159.5 (2C, C-11′ and C-13′), 159.7 (2C, C-11 and C-13),
160.5 (1C, C-4′). HRMS (ESI): calcd m/z 455.1489, found m/z
455.1481 [M + H]+, error ) 1.8 ppm.
The 54 mg of 3,4-DHS (0.25 mmol) and 107 mg of galvinoxyl
radical (0.25 mmol) were mixed in 30 mL ethanol and stirred for
3 h. The products were purified by silica gel chromatography and
eluted with chloroform-methanol (10:1, v:v) to afford 47 mg of
1
dimer with an isolated yield of 87%. The dimer of 3,4-DHS: H
NMR (400 MHz, (CD3)2CO, δ): 4.92 (d, J ) 8 Hz, 1H, H-7), 5.05
(d, J ) 8, 2 Hz, 1H, H-8), 6.51 (d, J ) 8 Hz, 1H, H-6), 6.68 (d, J
) 8 Hz, 1H, H-5), 6.77 (d, J ) 8 Hz, 1H, H-2), 6.97 (d, J ) 8 Hz,
1H, H-3′), 7.13 (d, J ) 16 Hz, 1H, H-7′), 7.17 (d, 1H, J ) 8 Hz,
H-2′), 7.19 (d, J ) 16 Hz, 1H, H-8′), 7.23 (m, 2H, H-11 and H-13),
7.24 (d, J ) 7.8 Hz, 1H, H-12′), 7.25 (m, 1H, H-12), 7.26 (m, 2H,
H-10 and H-14), 7.27 (d, J ) 2.4 Hz, 1H, H-6′), 7.35 (d, J ) 7.8
Hz, 2H, H-11′ and H-13′), 7.57 (d, J ) 7.8 Hz, 2H, H-10′ and
H-14′). 13C NMR (100 MHz, (CD3)2CO, δ): 81.8 (1C, C-7), 81.3
(1C, C-8), 115.4 (1C, C-2), 115.5 (1C, C-3′), 115.6 (1C, C-6′),
117.9 (1C, C-5), 120.6 (1C, C-6), 120.9 (1C, C-2′), 127.1 (2C, C-10′
and C-14′), 127.9 (1C, C-7′), 128.1 (2C, C-1 and C-8′), 128.8 (2C,
C-10 and C-14), 128.9 (2C, C-11 and C-13), 129.2 (1C, C-12),
129.4 (1C, C-12′), 129.5 (2C, C-11′ and C-13′), 132.1 (1C, C-1′),
137.7 (1C, C-9), 138.6 (1C, C-9′), 144.8 (1C, C-4′), 145.2 (1C,
C-5′), 145.6 (1C, C-3), 146.2 (1C, C-4). HRMS (ESI): calcd m/z
423.1591, found m/z 423.1597 [M + H]+, error ) 1.4 ppm.
Determination of Calf Thymus DNA Damage. Calf thymus
DNA damage was determined by using the EB-binding assay.16
A total 3 mL of PBS (pH 7.4) containing calf thymus (80 µg/
mL) with different concentrations of ROHs and CuSO4 (250 µM)
was incubated at 37 °C for 1 h under aerobic conditions. After
incubation, 50 µL of 0.75 mg/mL EB was added and the
fluorescence was measured using a Shimadzu RF-540 spectrof-
luorimeter with excitation at 510 nm and emission at 590 nm.
A solution containing all reagents and DNA except ROHs was
used as the control for 100% fluorescence, and zero fluorescence
was assessed in a solution identical to the control except DNA.
The loss of the fluorescence was used as a measure of DNA
damage.
UV-Visible Spectral Measurement. UV-visible spectra were
measured at room temperature with a Hitachi 557 spectropho-
tometer. PBS containing 50 µM ROH was kept at room

ResVeratrol-Based Lead DiscoVery
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1973
(4) Baur, J. A.; Pearson, K. J.; Price, N. L.; Jamieson, H. A.; Lerin, C.;
Kalra, A.; Prabhu, V. V.; Allard, J. S.; Lopez-Lluch, G.; Lewis, K.;
Pistell, P. J.; Poosala, S.; Becker, K. G.; Boss, O.; Gwinn, D.; Wang,
M.; Ramaswamy, S.; Fishbein, K. W.; Spencer, R. G.; Lakatta, E. G.;
Le Couteur, D.; Shaw, R. J.; Navas, P.; Puigserver, P.; Ingram, D. K.;
de Cabo, R.; Sinclair, D. A. Resveratrol improves health and survival
of mice on a high-calorie diet. Nature 2006, 444, 337–342.
temperature, and the spectral tracing was started by addition of
25 µM CuSO4.
Oxidation Product Analysis of Resveratrol or 3,4-DHS in
the Presence of Cu(II). A solution of resveratrol or 3,4-DHS
(0.5mmol) and anhydrous copper sulfate (96 mg, 0.55mmol) were
mixed in anhydrous acetonitrile (50 mL) and stirred for 24 h at
room temperature, as monitored by silica gel (petroleum ether/
acetone, 20/1). The dimers of resveratrol and 3,4-DHS were isolated
by silica gel chromatography with isolated yields of 15.7 and 22%,
respectively. Their structures were characterized with HRMS (ESI)
and 1D and 2D NMR.
Cell Culture. HL-60 cell lines were originally obtained from
Shanghai Institute of Biochemistry and Cell Biology, Chinese
Academy of Sciences. The cells were grown in RPMI-1640 medium
supplemented with 10% (v/v) heat inactivated fetal bovine serum,
penicillin (100 U/mL), streptomycin (100 U/mL), and 2 mM of
glutamine. The cell cultures were maintained at 37 °C in a
humidified CO2 (5%) incubator. Exponentially growing cells were
used throughout.
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activity of resveratrol: a natural product derived from grapes. Science
1997, 275, 218–220. (b) Aggarwal, B. B.; Bhardwaj, A.; Aggarwal,
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Assessment of HL-60 Cell Viability. The HL-60 cell viability
was also assessed using the MTT colorimetric assay,14 and the
experimental procedure was the similar to that in the section of
assessment of thymocytes viability with different cell concentration
(5 × 104 cells/mL) and incubation time (48 h).
´
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Analysis of DNA Fragmentation of HL-60 Cells by
Agarose Gel Electrophoresis. DNA fragmentation of HL-60 cells
was also assessed by agarose gel electrophoresis, and the experi-
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of DNA fragmentation of thymocytes by agarose gel electrophoresis
with different incubation time (48 h), centrifugation rate (500g for
10 min), and lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA,
0.5% SDS, and 1 mg/mL proteinase K).
Dermination of Apoptosis Analysis by Flow Cytometry. Flow
cytometric DNA analysis was performed by an available method51
with minor modifications. Briefly, an amount of 10 mL of
exponentially growing HL-60 cells at a density of 1 × 105 cells/
mL was untreated or treated with various concentrations of ROHs.
The cells were then prepared as a single cell suspension in 200 µL
of PBS, fixed with 2 mL of ice-cold 70% ethanol, and maintained
at 4 °C overnight. The cells were harvested by 500g centrifugation
for 10 min, resuspended in 500 µL of PBS with 0.1% Triton-X100
and DNase-free RNase (100 µg/mL), incubated at 37 °C for 30
min, and stained with 50 mg/L PI in the dark at 4 °C for 30 min.
The red fluorescence of individual cells was determined with a flow
cytometer (FACS Calibur, Becton-Dickinson, CA). All data were
analyzed by CellQuest, version 3.3, software.
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Acknowledgment. This work was funded by the National
Natural Science Foundation of China (Grant Nos. 20502010
and 20621091) and the 111 Project and Program for New
Century Excellent Talents in University (Grant NCET-06-0906).
Supporting Information Available: Experimental details for
the synthesis and characterization of all compounds; HPLC, HRMS,
and NMR data. This material is available free of charge via the
Internet at http://pubs.acs.org.
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