Quinone reductase induction activity of methoxylated analogues of resveratrol

Article abstract of DOI:10.1016/j.ejmech.2006.12.012

Agents that induce the activity of phase II enzymes play an important role in intervening with the carcinogenic process at the initiation stage. Resveratrol is well known for its chemopreventive activity against major stages of carcinogenesis. In this study, several methoxylated analogues of resveratrol were synthesized and evaluated for their ability to induce the activity of the phase II enzyme quinone reductase (QR). Methoxy groups serve to increase lipophilicity and improve metabolic stability. Compared to resveratrol, analogues with ortho-methoxy substituents were found to be more potent inducers of QR and to exert their activity in a qualitatively different manner. The greater induction activities associated with these stilbenoids serve as a useful starting point for the design of improved chemopreventive agents.

Full text of DOI:10.1016/j.ejmech.2006.12.012

European Journal of Medicinal Chemistry 42 (2007) 841e850
http://www.elsevier.com/locate/ejmech
Original article
Quinone reductase induction activity of methoxylated
analogues of resveratrol
*
W. Zhang, M.L. Go
Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore
Received 30 October 2006; received in revised form 28 November 2006; accepted 12 December 2006
Available online 9 January 2007
Abstract
Agents that induce the activity of phase II enzymes play an important role in intervening with the carcinogenic process at the initiation stage.
Resveratrol is well known for its chemopreventive activity against major stages of carcinogenesis. In this study, several methoxylated analogues
of resveratrol were synthesized and evaluated for their ability to induce the activity of the phase II enzyme quinone reductase (QR). Methoxy
groups serve to increase lipophilicity and improve metabolic stability. Compared to resveratrol, analogues with ortho-methoxy substituents were
found to be more potent inducers of QR and to exert their activity in a qualitatively different manner. The greater induction activities associated
with these stilbenoids serve as a useful starting point for the design of improved chemopreventive agents.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Methoxylated stilbenes; Resveratrol; Chemopreventive activity; Induction of quinone reductase
1. Introduction
potent as resveratrol in terms of antioxidant activity and anti-
carcinogenic activity in a mouse mammary organ culture
model [6]. Rhapontigenin (3,5,30-trihydroxy-40-methoxy-
trans-stilbene), a component of Korean rhubarb (Rheum undu-
latum), was an inhibitor of human CYP1A1 [7] while another
methoxy analogue (20,3,40,5-tetramethoxystilbene) was also
a CYP inhibitor but selective for the CYP1B1 subtype [8].
The methoxylation of hydroxyl groups results in an increase
in lipophilicity and a loss of hydrogen bond donor property.
These changes will influence bioavailability, susceptibility to
metabolism and possibly the pharmacological profile of the
resulting analogue. A case in point is 3,4,5,40-tetramethoxy-
The stilbene moiety is commonly encountered in natural
products and many members are associated with therapeuti-
cally important pharmacological properties [1]. Resveratrol
(E-3,40,5-trihydroxystilbene) is probably the best-known stil-
benoid. Its therapeutic potential is recognized in several areas
like the chemoprevention of cancer, cardiovascular diseases
and neurodegeneration [2,3]. Unfortunately, resveratrol is rap-
idly inactivated by phase II conjugation reactions [4] and it is
generally a weak growth inhibitor (IC50 40e200 mM) [5].
Novel stilbenoids based on the resveratrol structure may pro-
vide a solution to these limitations. Among naturally occurring
stilbenoids, the biological potential of methoxylated stilbenes
is particularly promising. For example, pterostilbene (trans-
3,5-dimethoxy-40-hydroxystilbene), found in grapes and heart-
wood extracts of Pterocarpus marsupium, was almost as
trans-stilbene,
a
synthetic methoxylated analogue of
resveratrol which had an improved pharmacokinetic profile,
selective growth inhibitory effect against cancer cells and
a unique mode of action compared to resveratrol [9].
Resveratrol exerts many effects on cellular events associ-
ated with cancer initiation, promotion and progression. It is
an antioxidant, antimutagen, induces apoptosis and moderates
the activity of drug metabolizing enzymes [10e12]. Resvera-
trol is a moderate inducer of the phase II enzyme quinone
* Corresponding author. Tel.: þ65 65162654; fax: þ65 67791554.
E-mail address: phagoml@nus.edu.sg (M.L. Go).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.12.012

842
W. Zhang, M.L. Go / European Journal of Medicinal Chemistry 42 (2007) 841e850
reductase (NAD(P)H:quinone reductase, QR), with a CD (con-
centration required to double the specific activity of QR) of
21 mM [10]. It is also a monofunctional inducer in that it in-
duces only phase II enzymes without affecting the activity
of phase I enzymes [10]. QR induction is widely used as a bio-
marker for evaluating chemopreventive potential of com-
pounds against cancer initiation [13].
In view of the biological potential of methoxylated stil-
benes and the shortcomings associated with resveratrol, it is
of interest to explore the potential of methoxylated derivatives
of resveratrol as chemopreventive agents. For this purpose,
a number of E and Z isomers of methoxylated stilbenes were
synthesized and evaluated for QR induction activity. As QR
also serves to maintain the antioxidant function of cells [14],
antioxidants are often associated with QR induction activity
[15], and the compounds were also screened for their antiox-
idant properties using an in vitro assay.
and Z isomers. These were separated by column chromatogra-
phy, with Z isomers obtained in higher yields. Stilbene 11
which has a 2-hydroxyl group on ring A was also synthesized
by this route as protection of the 2-hydroxyl group was not
necessary.
In the case of other stilbenes with hydroxyl groups on ring
A (12e14), the phenolic hydroxyl group was protected by
conversion to the t-butyldimethylsilyloxy ether before reacting
with 2 (Scheme 2). The silyloxystilbenes 19 and 20 were
obtained exclusively as Z isomers. Conversion to the E isomer
was effected by refluxing in heptane in the presence of a cata-
lytic amount of iodine. On the other hand, the siloxystilbene
21 was obtained as a mixture of E and Z isomers. The mixture
was separated by column chromatography, after which the si-
loxyl protecting function was removed by reaction with tetra-
butylammonium fluoride to give the final compounds (12e14).
Unlike the other members, stilbene 15 was obtained by
McMurry coupling of 2-methoxyaldehyde under Mukaiyama
conditions [16] (Scheme 3).
2. Chemistry
Table 1 lists the stilbenes (3e15) synthesized in this inves-
tigation. All the stilbenes have a 40-methoxy group on ring B,
with the exception of stilbene 15 which has a 20-methoxy
group on ring B. The substitution pattern on ring A is as fol-
lows: methoxy groups only (3e10, 15), hydroxyl groups only
(11e13) and one member (14) with both hydroxyl and me-
thoxy groups. Z and E isomers were prepared for all members,
except for stilbenes 9, 11 and 15 which were synthesized as E
isomers.
Stilbenes with 40-methoxy substituent (3e14) were
obtained by the Wittig reaction (Scheme 1). 4-Methoxyben-
zyltriphenylphosphonium bromide (2) was synthesized from
4-methoxybenzylalcohol by bromination, followed by reaction
with triphenylphosphine. The phosphonium salt 2 was reacted
with the methoxylated benzaldehyde in the presence of n-bu-
tyllithium as base to give the desired stilbene as a mixture of E
3. Biological screening
3.1. Antioxidant activity
Antioxidant activity was evaluated by the ABTS [2,20-
azino-3-ethylbenzo-thiazoline-6-sulfonic acid] radical cation
decolorisation assay [17]. This method is based on the ability
of a potential antioxidant to scavenge the stable radical cation
of ABTS (ABTSꢀþ). The nitrogen radical of this species im-
parted a deep purple color at 734 nm that was lost when
quenched by an antioxidant. The loss in absorbance of the
ABTS radical cation was monitored at different concentrations
of the test compound after a fixed time period of 5 min. A plot
of the reduction in absorbance against different concentrations
of the test compound gave a straight line, the gradient of which
was correlated to the antioxidant activity of the test compound.
Table 1
Structures of stilbenes investigated for chemopreventive activity
3
2
4
5
A
6
R
2'
3'
R'
B
6'
4'
5'
Compound
R
R0
Compound
R
R0
3Z, 3E
4Z, 4E
5Z, 5E
6Z, 6E
7Z, 7E
8Z, 8E
9E
2-OCH3
3-OCH3
4-OCH3
40-OCH3
40-OCH3
40-OCH3
40-OCH3
40-OCH3
40-OCH3
40-OCH3
40-OCH3
11E
2-OH
3-OH
40-OCH3
40-OCH3
40-OCH3
40-OCH3
20-OCH3
12Z, 12E
13Z, 13E
14Z, 14E
15E
3,5-OH
3-OH, 4-OCH3
2-OCH3
2,4-OCH3
3,4-OCH3
3,5-OCH3
2,6-OCH3
3,4,5-OCH3
Resveratrola
Pinosylvina
3,5-OH
3,5-OH
40-OH
H
10Z, 10E
a
Purchased from commercial sources.

W. Zhang, M.L. Go / European Journal of Medicinal Chemistry 42 (2007) 841e850
843
CH2PPh3+Br-
CH2Br
CH2OH
A
R
a
b
B
H3CO
OCH3
OCH3
OCH3
c
1
2
B
CHO
H3CO
A
R
R
9E: R = 2,6- (OCH3)2
10E,Z: R = 3,4,5-(OCH3)3
11E: R = 2-OH
3E,Z: R = 2-OCH3
4E,Z: R = 3-OCH3
5E,Z: R = 4-OCH3
6E,Z: R = 2,4-(OCH3)2
7E,Z: R = 3,4-(OCH3)2
8E,Z: R = 3,5-(OCH3)2
Scheme 1. Synthetic pathway for stilbenes 3e11. Reagents and conditions: (a) PBr3, CH2Cl2, 0 ꢁC; (b) PPh3, toluene, reflux; (c) n-BuLi, THF, ꢂ78 ꢁC. Only E
isomers of compounds 9 and 11 were synthesized.
R1
R2
R1
R3
R2
H3CO
R3
H3CO
12Z, 13Z
12E, 13E
12Z,E: R1= OH, R2=R3 =H
13Z,E: R1=R3= OH; R2 =H
d
d
R1
CHO
R2
R3
c
R1
b
R1
R3
R2
H3CO
R3
H3CO
R2
16, 17
19Z, 20Z
19E, 20E
a
CHO
R2
16, 19Z,19 E:R1= OSi(CH3)2C(CH3)3; R2=R3 =H
17, 20Z,20 E:R1=R3= OSi(CH3)2C(CH3)3; R2 =H
R3
R1
OSi(CH3)2C(CH3)3
OCH3
OH
OCH3
a
CHO
H3CO
H3CO
d
14Z
21Z
+
b
(H3C)3C(H3C)2SiO
(H3C)3C(H3C)2SiO
OH
OCH3
OCH3
OCH3
d
18
H3CO
H3CO
14E
21E
Scheme 2. Synthetic pathways for stilbenes 12e14. Reagents and conditions: (a) t-Bu(CH3)2SiCl, DIEA, THF; (b) n-BuLi, THF, ꢂ78 ꢁC, 4-methoxybenzyltriphe-
nylphosphonium bromide 3; (c) I2, heptane, 12 h reflux; (d) Bu4NF, THF. Compound 21 was obtained as a mixture of Z and E isomers. These were separated by
column chromatography and then individually reacted via (d).

844
W. Zhang, M.L. Go / European Journal of Medicinal Chemistry 42 (2007) 841e850
compounds were screened for QR induction at concentrations
that maintained cell viability at ꢃ55%.
OCH3
CHO
OCH3
The QR induction assay can also be used to determine if an
agent is monofunctional (induces phase II enzymes only) or
bifunctional (induces both phase I and II enzymes) in nature.
This is done by comparing the levels of induction in wild
type Hepa 1c1c7 cells with that observed in a mutant cell
line (c1) which lacks CYP1A1 dependent aryl hydrocarbon
hydrolase (AHH) activity. Monofunctional inducers induce
QR activity of wild type and mutant Hepa cell lines to similar
extents but bifunctional inducers induce QR activity of wild
type cells to a greater degree. This is because the mutant
cell line lacks an inducible phase I enzyme which is required
to convert the compound into an activated form for the induc-
tion of the phase II enzyme. As reported in the literature [20],
the induction properties of sulphoraphane and b-naphthofla-
vone were found in our assays to be monofunctional and
bifunctional, respectively, when tested on wild type and mu-
tant cell lines.
a
OCH3
15E
Scheme 3. Synthetic pathway for stilbene 15E. Reagents and conditions: (a)
TiCl4, zinc powder, THF, 12 h reflux.
The experiment was also carried out using a standard antiox-
idant, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-car-
boxylic acid). The quotient of the gradients of the lines
obtained with the test compound and Trolox gave the TEAC
(Trolox equivalent antioxidant capacity) of the test compound.
The TEAC indicates the number of times the test compound is
more effective than Trolox in quenching the ABTS radical cat-
ion under similar experimental conditions. TEAC of stilbenes
9E, 11E and 15E were not determined.
3.2. Microculture tetrazolium assay for determining cell
viability
4. Results and discussion
The assay is based on the ability of the mitochondrial en-
zyme succinate dehydrogenase of viable cells to reduce the
yellow tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyl-2H-tetrazolium bromide (MTT) to an insoluble purple
substance called formazan. The formation of formazan can be
quantified by spectrophotometry (lmax 590 nm) and afforded
a convenient and sensitive means of monitoring the effect of
test compounds on cell viability [18]. In this investigation,
the MTT assay was carried out on murine hepatoma cells
(Hepa 1c1c7). These cells were used in the QR induction assay
and the MTT assay served to determine the concentration of
test compound to be used in that assay. The concentration of
test compound should be one that did not adversely affect the
viability of the Hepa 1c1c cells (ꢃ55% viability at the test
concentration).
4.1. Antioxidant activity of stilbenes
The antioxidant activity of the stilbenes was assessed from
their TEAC values, which compares the radical scavenging
activity of the test compound to that of a standard antioxidant
(Trolox). Under the present experimental conditions, the
TEAC of resveratrol was found to be 2.6 (Table 2). Pinosylvin
(E-3,5-dihydroxystilbene) which does not have a 40-OH on
ring B had a lower TEAC value of 2.0. Interestingly, 13E
which has 40-methoxy on ring B but the same ring A substit-
uents (3,5-dihydroxyl) as resveratrol had a TEAC of 2.5.
Thus, the antioxidant activity of resveratrol owes little to the
presence of the 40-OH group on ring B, as this group can be
methoxylated or omitted with no loss of antioxidant activity.
In contrast, the dihydroxyl group on ring A of resveratrol con-
tributed significantly to the antioxidant activity. Starting from
13E which had almost the same TEAC as resveratrol, removal
of one OH group from ring A to give 12E caused a sharp drop
in TEAC (1.1). Replacing both OH groups with methoxy
3.3. Induction of QR activity
The murine hepatoma cell line Hepa 1c1c7 contains easily
measurable inducible QR. The induction of QR has been
shown to correlate with the elevation of other protective phase
2 enzymes. Thus it serves as a reliable biomarker for identify-
ing potential cancer chemopreventive agents [13]. Briefly, the
QR induction assay is based on the generation of NADPH
when glucose-6-phosphate was reduced by glucose-6-phos-
phate dehydrogenase in the presence of its cofactor NADP.
Once formed, NADPH served as an electron donor for the
QR mediated reduction of menadione to menadiol. Menadiol
reduced MTT to formazan which was measured at 590 nm.
Both NADP and menadione were regenerated during the cata-
lytic cycle and therefore need not be replenished during the
course of the assay. An inducer of QR increases the rate at
which menadiol is generated and this would in turn lead to
more formation of formazan. In this investigation, the
Table 2
Radical quenching ability of stilbenes measured in terms of TEAC values
Compounda
TEACb
12Z
12E
1.04 (0.05)
1.11 (0.03)
1.86 (0.03)
2.49 (0.06)
1.11 (0.02)
1.29 (0.06)
2.74 (0.09)
2.04 (0.06)
13Z
13E
14Z
14E
Resveratrol
Pinosylvin
a
Other stilbenes (3E, Ze8E, Z, 10E, Z, 12E, Ze14E, Z) had no radical
quenching properties in this assay. Compounds 9E, 11E and 15E were not
evaluated.
b
Trolox equivalent antioxidant capacity, given as mean (ꢄSD) of three
determinations.

W. Zhang, M.L. Go / European Journal of Medicinal Chemistry 42 (2007) 841e850
845
groups (8E) completely abolished radical scavenging activity.
When one of the two hydroxyl groups on ring A was methoxy-
lated to give 14E, TEAC was reduced to 1.3. Thus, antioxidant
activity depends on the presence of at least one OH group on
ring A. Stilbene 11E which has a 2-OH group on ring A
should also possess antioxidant activity but its TEAC was
not evaluated in this investigation. Should future investigations
show otherwise, this may be due to the anomalous effect of the
ortho-OH group (‘‘ortho-effect’’).
No antioxidant activity was observed when only methoxy
groups were present on ring A. In contrast, methoxy groups
on ring B did not adversely affect the antioxidant activity.
As for the antioxidant activity of Z and E isomers, a compari-
son of the TEAC values of three pairs of Z/E isomers (12, 13
and 14) showed that the E isomer had greater antioxidant
activity.
Of the stilbenes tested at 12.5 mM, only five compounds in-
duced QR to a greater extent than resveratrol, namely 3E, 4E,
9E, 14E and 15E (Table 3). Of these, the QR induction activ-
ity of 3E, 9E and 15E fell within a narrow range of 1.8e2.0
and were significantly greater than that of 4E, 14E and resver-
atrol tested at similar concentrations. Compounds 3E, 9E and
15E share in common the presence of ortho-methoxy substit-
uents on their stilbene framework. It is notable that the
regioisomers of 3E (2-OCH3), namely 4E (3-OCH3) and 5E
(4-OCH3), had significantly lower QR induction activities.
This serves to underscore the importance of the ortho-methoxy
substituent on ring A. A similar observation was made for 9E
(two ortho-methoxy groups on ring A) and 15E (ortho-me-
thoxy substituents on both rings A and B). The QR induction
activity of 3E was further determined at a lower concentration
of 6.3 mM and found to be 1.8, which still exceeded that of
resveratrol (1.1) tested at the same concentration. Although
these observations suggest that ortho-methoxy groups have
a special role in inducing QR activity, there are limitations
that the compounds had variable effects on cell viability at
the test concentration (12.5 mM) (Table 3). A better approach
might be to determine the CD values of the promising com-
pounds but this would still be difficult for 3E whose IC50
was estimated to be near 12.5 mM. Notwithstanding this limi-
tation, the association of ortho-methoxy groups with good QR
induction activity in stilbenes has not been observed before. It
is notable that none of these compounds have antioxidant
properties. Most inducers of phase II enzymes activate the an-
tioxidant response element (ARE) through Keap1 and Nrf2
[22]. ARE is activated by electrophiles and antioxidants, and
this may be the means by which resveratrol, a known antiox-
idant, induced phase II enzymes. The QR induction activities
of the methoxylated resveratrol analogues are likely to be
4.2. Induction of quinone reductase activity
Prior to carry out the assay on QR induction activity, the
viability of the test compounds was evaluated at various con-
centrations to identify suitable concentrations that maintained
viability of the Hepa 1c1c7 cells at 55% or more. Based on the
results, the stilbenes were evaluated at 12.5 mM, 5 or 1 mM.
Greater cytotoxicity was generally observed for the Z isomers.
The concentration of resveratrol required to double the spe-
cific activity of QR (CD value) was reported to vary between
21e25 mM [19,21]. In our study, resveratrol at 25 mM in-
creased quinone reductase activity by 1.5 times. The viability
of the cells exposed to 25 mM resveratrol was about 55%, and
as this might affect induction activity, resveratrol was tested at
a lower concentration of 12.5 mM. At this concentration, cell
viability rose to 80% but induction of QR was only 1.2-fold.
Table 3
Effect of test compounds on viability of Hepa 1c1c7 cells and QR induction activities
Compound
% Viabilitya
QR induction
of Hepa 1c1c cellsb
Compound
% Viabilitya
QR induction
of Hepa 1c1c cellsb
Wild type
Mutant (c1)
Wild type
Mutant (c1)
3E
3Z
4E
4Z
5E
5Z
6E
6Z
7E
7Z
8E
8Z
9E
a
58 (12.5 mM)c
56 (1 mM)
2.04 (0.07)c
<1.0
1.14 (0.06)
NDd
<1.0
ND
10E
10Z
86 (1 mM)
88 (1 mM)
<1.0
<1.0
ND
ND
90 (12.5 mM)
92 (1 mM)
1.44 (0.05)
<1.0
11E
12E
80 (12.5 mM)
81 (12.5 mM)
90 (5 mM)
1.13 (0.04)
1.08 (0.03)
<1.0
ND
ND
67 (12.5 mM)
64 (12.5 mM)
58 (12.5 mM)
85 (1 mM)
1.14 (0.04)
1.06 (0.04)
1.18 (0.05)
<1.0
ND
ND
12Z
13E
ND
ND
87 (12.5 mM)
96 (12.5 mM)
77 (12.5 mM)
75 (5 mM)
1.14 (0.05)
1.04 (0.04)
1.33 (0.05)
<1.0
ND
ND
13Z
14E
ND
ND
100 (1 mM)
86 (1 mM)
94 (1 mM)
<1.0
<1.0
ND
ND
14Z
15E
ND
82 (12.5 mM)
80 (12.5 mM)
61 (12.5 mM)
1.81 (0.02)
1.24 (0.03)
1.07 (0.04)
<1.0
1.16 (0.04)
ND
<1.0
<1.0
ND
ND
Resveratrol
Pinosylvin
82 (1 mM)
90 (12.5 mM)
1.92 (0.06)
1.05 (0.02)
Mean of three determinations on Hepa 1c1c7 cells by MTT assay. Values in bracket indicate concentration of compound tested.
b
QR induction activity (mean ꢄ SD, n ¼ 3) was determined on wild type Hepa 1c1c7 cells at the same concentration used to determine % viability. Compounds
with QR activity > 1.2 were also tested on the mutant Hepa 1c1c7c1 cell line. CD (concentration required to double QR activity) values of sulforaphane were 0.26
(ꢄ0.04) mM and 0.28 (ꢄ0.04) mM on Hepa 1c1c7 wild type and c1 mutant cells, respectively. CD value of b-napthoflavaone was 0.028 (ꢄ0.003) mM on Hepa
1c1c7 wild type cells. It had no activity on c1 mutant cells.
c
When tested at 6.3 mM, viability of Hepa 1c1c7 cells was 73%. QR induction at this concentration was 1.8.
ND ¼ not determined.
d

846
W. Zhang, M.L. Go / European Journal of Medicinal Chemistry 42 (2007) 841e850
mediated by mechanisms that do not involve their antioxidant
properties.
lacked the antioxidant activity of resveratrol and thus may
induce QR activity by a different mechanism. The methoxyla-
tion of hydroxyl groups impart greater lipophilicity and stabil-
ity against metabolic inactivation. Methoxylated analogues of
resveratrol that retain QR induction activity, as those identified
in this study, provide a useful starting point for the rational
design of chemopreventive agents with improved pharmacoki-
netic and pharmacodynamic properties.
The induction properties of the ortho-methoxylated stil-
benes 3E, 4E, 9E and 15E were further investigated to deter-
mine if they were mono or bifunctional inducers of
metabolizing enzymes. Bifunctional inducers induce activities
of both phase I and II enzymes, unlike monofunctional
inducers which elevate phase II enzymes selectively without
inducing phase I enzymes [22]. Resveratrol induced QR activ-
ity of the wild type Hepa 1c1c7 cells to the same extent as the
mutant c1 cells that lacked a phase I enzyme (Table 3). Thus it
is a monofunctional inducer and its ability to induce QR did
not depend on prior biotransformation to an active metabolite
by oxidative metabolism. In contrast, the methoxylated ana-
logs 3E, 4E, 9E and 15E were found to be bifunctional in-
ducers. Their bifunctional status raised the question as to the
likely active metabolite responsible for the induction activity.
Because these compounds have methoxyl groups, it is possible
that one or more of these groups are dealkylated by phase I en-
zymes to generate phenolic hydroxyl groups. This has been
observed for the antioxidant 2(3)-tert-butyl-4-hydroxyanisole
(BHA) whose induction activity was traced for conversion to
a 1,4-diphenol by O-dealkylases in the Hepa cells [23]. Taking
3E as an example, dealkylation of the ortho-methoxy group on
ring A would result in 11E, but since the latter had negligible
QR induction activity (Table 3), this was not a likely option.
Another possibility was the dealkylation of the 40-methoxy
of 3E to give an active metabolite (2-methoxy, 40-hydroxystil-
bene). This compound was not synthesized but a comparison
of the induction activities of resveratrol (1.2 at 12.5 mM) and
its 40-methoxylated analogue (13E, 1.1 at 12.5 mM) suggested
that the state of the 40-substituent was unlikely to affect QR
induction activity. Other options are possible such as hydrox-
ylation of the methoxylated analogues at either ring A or B.
Notwithstanding these possibilities, it is clear that ortho-
substituted methoxylated stilbenes induced QR activity to
a greater extent and work through different mechanisms com-
pared to resveratrol.
6. Experimental
6.1. Chemistry
Melting points were determined on a Buchi melting point
apparatus (B540) in open glass capillary tubes and were uncor-
1
rected. H and 13C NMR spectra were recorded on a Bruker
ACF (DPX 300 MHz) spectrometer. 1H and 13C Chemical
shifts were reported in d (ppm), relative to tetramethylsilane
as internal standard (for 1H NMR only) and obtained in
CDCl3 as solvent, unless otherwise stated. Thin layer chroma-
tography (TLC) was carried out on pre-coated plates (silica gel
60F254, Merck) and visualized with ultraviolet light. Flash col-
umn chromatography was performed with silica gel 60 (70e
230 mesh) with hexane/ethyl acetate as eluting solvent. The
purity of compounds 3e15 was assessed by reverse phase
high-pressure column chromatography. Determinations were
carried out on a Waters Delta 600 liquid chromatography
system, using Zorbax Eclipse XDB-C18 column
(4.6 mm ꢅ 150 mm, 5 mm) and UV detection (278 nm). The
isocratic mode was employed using two solvent systems
(methanolewater and acetonitrileewater in the ratio of 9:1).
The area under the main peak was determined and expressed
as a percentage of total peak area during a 15 min run. Com-
pounds 3e15 were purified until their chromatograms showed
a main peak with areas > 97% on both solvent systems.
6.1.1. 4-Methoxybenzyl bromide (1)
Phosphorus tribromide (3.1 ml) was slowly added to a solu-
tion of 4-methoxybenzylalcohol (12.79 ml) in dichlorome-
thane (150 ml) at 0 ꢁC, and stirring was continued for 12 h.
The reaction mixture was poured into aqueous sodium bicar-
bonate and extracted with dichloromethane. Removal of the
solvent in vacuo from the organic phase gave the product as
5. Conclusion
A library of Z and E methoxylated analogues of resveratrol
were synthesized and investigated for their chemopreventive
potential through QR induction and antioxidant assays. The in-
troduction of methoxy groups generally diminished QR induc-
tion except in compounds with ortho-methoxy substituents.
We found that the QR induction activities of these analogues
exceeded that of resveratrol. The best compound identified
in this study (3E) was 1.8 times more effective than resveratrol
as a QR inducer when tested at 6.3 mM. However, there was
a qualitative difference in the way these compounds induced
QR activity. Unlike resveratrol, the methoxylated analogues
were bifunctional inducers, that is, they required prior acti-
vation by phase I enzymes before they can induce phase II
enzymes. The nature of the active metabolite is yet to be de-
termined but it is quite clear that dealkylation of the methoxy
groups was not involved. The methoxylated analogues also
1
a clear oil (19.1 g, 95%). H NMR: d 7.3 (2H, d, J ¼ 8.67),
6.9 (2H, d, J ¼ 8.64), 4.5 (2H, s, CH2), 3.9 (3H, s, OCH3).
6.1.2. 4-Methoxybenzyltriphenylphosphonium
bromide (2)
Triphenylphosphine (25 g) was added to a solution of
bromide 1 (19.1 g) in toluene (250 ml). The mixture was
heated to reflux for 6 h and then cooled to room temperature.
The product was collected, recrystallized from ethanol and
obtained as a colorless solid (42.3 g, 96%), mp 234e235 ꢁC.
1H NMR: d 7.7 (15H, m), 7.0 (2H, d, J ¼ 8.70), 6.6 (2H, d,
J ¼ 8.70), 5.7 (2H, s), 3.7 (3H, s, OCH3).

W. Zhang, M.L. Go / European Journal of Medicinal Chemistry 42 (2007) 841e850
847
6.1.3. General procedure for the synthesis
of stilbenes (3e11)
(300 MHz): d 160.2, 158.8, 157.8, 131.2, 127.5, 126.9,
126.6, 121.2, 119.9, 114.0, 104.9, 98.5, 55.5, 55.4, 55.3.
n-Butyllithium (1.6 M in hexane, 1 mmol) was added to 4-
methoxybenzyltriphenylphosphonium bromide 2 (1.1 mmol)
in anhydrous tetrahydrofuran (30 ml) at ꢂ78 ꢁC, and the re-
sulting red solution was stirred under nitrogen for 30 min. A
solution of aldehyde (1 mmol) in anhydrous tetrahydrofuran
was added dropwise over 30 min and the mixture was stirred
for 16 h. The resulting suspension was poured into water
and extracted with ethyl acetate. The organic phase was
washed with brine, and removal of the solvent in vacuo gave
a mixture of cis and trans isomers. The two isomers were
separated by flash column chromatography (99:1 hexane/ethyl
acetate), with the Z isomer eluted first from the column
followed by the E isomer. The Z isomer was obtained as a clear
oil, in contrast to the E isomer which was obtained as a color-
less solid.
6.1.3.5. 3,4,40-Trimethoxystilbene (7). Yield 77%, 0.94 g; 7Z
[29] (0.66 g, oil): 1H NMR: d 7.21e7.26 (3H, m), 6.74e
6.84 (4H, m), 6.48 (1H, d, J ¼ 12.03 Hz), 6.43 (1H, d,
J ¼ 12.06 Hz), 3.87 (3H, s, OCH3), 3.79 (3H, s, OCH3), 3.65
(3H, s, OCH3). Compound 7E (0.28 g), mp 136e137 ꢁC, lit.
1
mp 136e138 ꢁC [30]. H NMR: d 7.44 (2H, d, J ¼ 8.67 Hz),
6.84e7.05 (7H, m), 3.95 (3H, s, OCH3), 3.90 (3H, s,
OCH3), 3.83 (3H, s, OCH3).
6.1.3.6. 3,40,5-Trimethoxystilbene (8). Yield 60%, 0.74 g; 8Z
[31] (0.55 g): 1H NMR: d 7.21 (2H, d, J ¼ 8.67 Hz), 6.77
(2H, d, J ¼ 8.67 Hz), 6.53 (1H, d, J ¼ 12.42 Hz), 6.44 (1H,
d, J ¼ 12.03 Hz), 6.43 (2H, d, J ¼ 2.25 Hz), 6.32 (1H, t,
J ¼ 2.25 Hz), 3.78 (3H, s, OCH3), 3.67 (6H, s, OCH3). Com-
pound 8E (0.19 g), mp 49e54 ꢁC, lit. mp 50 ꢁC [32]. 1H
6.1.3.1. 2,40-Dimethoxystilbene (3). Yield 60%, 0.72 g; 3Z
[24] (0.54 g): 1H NMR: d 7.22 (2H, d, J ¼ 7.2 Hz), 7.17
(2H, d, J ¼ 8.67), 6.90 (1H, d, J ¼ 8.3), 6.70e6.78 (3H, m),
6.57 (2H, s), 3.77 (3H, s, OCH3), 3.83 (3H, s, OCH3).
Compound 3E (0.18 g), mp 85e87 ꢁC, lit. 85e86 ꢁC [25].
1H NMR: d 7.57 (1H, d, J ¼ 7.53 Hz), 7.47 (2H, d,
J ¼ 8.67 Hz), 7.35 (1H, d, J ¼ 16.2 Hz), 7.21 (1H, d,
J ¼ 6.9 Hz), 7.06 (1H, d, J ¼ 16.5 Hz), 6.88e6.98 (4H, m),
3.83 (3H, s, OCH3), 3.89 (3H, s, OCH3).
NMR:
d
7.45 (2H, d, J ¼ 8.64 Hz), 7.04 (1H, d,
J ¼ 16.2 Hz), 6.902 (1H, d, J ¼ 16.56 Hz), 6.90 (2H, d,
J ¼ 9.06 Hz), 6.65 (2H, d, J ¼ 2.28 Hz), 6.38 (1H, t,
J ¼ 2.28 Hz), 3.83 (9H, s, OCH3).
6.1.3.7. 2,40,6-Trimethoxystilbene (9). Compound 9E (0.65 g,
48% yield), mp 79e80 ꢁC. 1H NMR: d 7.52 (1H, d,
J ¼ 16.56 Hz), 7.47 (2H, d, J ¼ 9.06 Hz), 7.31 (1H, d,
J ¼ 16.56 Hz), 7.14 (1H, t, J ¼ 8.28 Hz), 6.88 (2H, d,
J ¼ 8.64 Hz), 6.59 (2H, d, J ¼ 8.28 Hz), 3.89 (6H, s, OCH3),
3.82 (3H, s, OCH3). 13C NMR (300 MHz): d 158.8, 158.5,
132.1, 131.9, 127.6, 127.5, 117.8, 115.1, 113.9, 104.0, 55.8,
55.3.
6.1.3.2. 3,40-Dimethoxystilbene (4). Yield 72%, 0.86 g; 4Z
1
[24] (0.64 g, oil): H NMR: d 7.15 (1H, d, J ¼ 7.9 Hz), 7.2
(2H, d, J ¼ 8.67 Hz), 6.83e6.88 (2H, m), 6.73e6.79 (3H,
m), 6.54 (1H, d, J ¼ 12.42 Hz), 6.48 (1H, d, J ¼ 12.42 Hz),
3.79 (3H, s, OCH3), 3.69 (3H, s, OCH3). Compound 4E
6.1.3.8. 3,4,40,5-Tetramethoxystilbene (10). Yield 80%, 1.2 g;
1
1
(0.22 g), mp 106e109 ꢁC, lit. 107e108 ꢁC [26]. H NMR:
10Z [33] (0.8 g, oil): H NMR: d 7.24 (2H, d, J ¼ 8.67 Hz),
d 7.45 (d, J ¼ 8.67 Hz, 2H), 7.26 (t, J ¼ 8.3 Hz, 1H), 7.04e
7.10 (m, 3H), 6.89e6.97 (m, 3H), 6.80 (q, J ¼ 1.9 Hz, 1H),
3.85 (3H, s, OCH3), 3.83 (3H, s, OCH3).
6.79 (2H, d, J ¼ 8.67 Hz), 6.52 (1H, d, J ¼ 12.06 Hz), 6.50
(2H, s), 6.42 (1H, d, J ¼ 12.06 Hz), 3.84 (3H, s, OCH3),
3.79 (3H, s, OCH3), 3.69 (6H, s, OCH3). Compound 10E
(0.4 g), mp 158e159 ꢁC, lit. mp 157 ꢁC [34]. 1H NMR:
d 7.44 (2H, d, J ¼ 8.64 Hz), 6.98 (1H, d, J ¼ 16.2 Hz), 6.90
(2H, d, J ¼ 9.39 Hz), 6.89 (1H, d, J ¼ 16.2 Hz), 6.72 (2H, s),
3.92 (3H, s, OCH3), 3.87 (3H, s, OCH3), 3.84 (3H, s, OCH3).
6.1.3.3. 4,40-Dimethoxystilbene (5). Yield 72%, 0.99 g; 5Z
[27] (0.69 g, oil): 1H NMR: d 7.2 (4H, d, J ¼ 9 Hz), 6.77
(4H, d, J ¼ 9.0 Hz), 6.45 (s, 2H), 3.79 (6H, s, OCH3). Com-
1
pound 5E (0.3 g), mp 214e215 ꢁC, lit. 214e216 ꢁC [28]. H
NMR: d 7.43 (4H, d, J ¼ 9 Hz), 6.93 (s, 2H), 6.89 (4H, d,
6.1.3.9. 2-Hydroxy-40-methoxystilbene (11). Compound 11E
(0.023 g, 2% yield), mp 141e143 ꢁC, lit. mp 145e147 ꢁC
J ¼ 9.0 Hz), 3.83 (6H, s, OCH3).
1
[35]. H NMR (DMSO-d6): d 9.66 (1H, s, OH), 7.53 (1H, d,
6.1.3.4. 2,4,40-Trimethoxystilbene (6). Yield 84%, 1.03 g; 6Z
(0.7 g, oil): 1H NMR: d 7.1e7.2 (3H, m), 6.73 (2H, d,
J ¼ 8.6 Hz), 6.5 (2H, d, J ¼ 1.5 Hz), 6.46 (1H, d,
J ¼ 2.3 Hz), 6.33 (1H, q, J ¼ 2.6 Hz), 3.81 (3H, s, OCH3),
3.80 (3H, s, OCH3), 3.77 (3H, s, OCH3). 13C NMR
(300 MHz): d 160.2, 158.4, 158.3, 130.5, 130.2, 130.0,
128.6, 123.7, 119.1, 113.4, 104.3, 98.3, 55.5, 55.3, 55.1.
J ¼ 7.9 Hz), 7.49 (2H, d, J ¼ 8.67 Hz), 7.26 (1H, d,
J ¼ 16.6 Hz), 7.13 (1H, d, J ¼ 16.6 Hz), 7.04e7.09 (1H, m),
6.94 (2H, d, J ¼ 8.67 Hz), 6.78e6.86 (2H, m), 3.77 (3H, s,
OCH3).
6.1.4. General procedure for the protection of phenolic
groups with tert-butyldimethylsilyl chloride
1
Compound 6E (0.33 g), mp 86e88 ꢁC. H NMR: d 7.43e
To a solution of the hydroxybenzaldehyde (5 mmol) in
anhydrous tetrahydrofuran (30 ml) was added N,N-diisopropy-
lethylamine (DIEA, 1.29 g, 2 equiv). The solution was stirred
for 15 min, after which tert-butyldimethylsilyl chloride
(1.79 g, 2 equiv) was added, and the solution was stirred for
7.5 (3H, m), 7.25 (1H, d, J ¼ 16.2 Hz), 6.95 (1H, d,
J ¼ 16.6 Hz), 6.88 (2H, d, J ¼ 8.67 Hz), 6.52 (1H, q,
J ¼ 2.3 Hz), 6.47 (1H, d, J ¼ 2.3 Hz), 3.86 (3H, s, OCH3),
3.83 (3H, s, OCH3), 3.82 (3H, s, OCH3). 13C NMR

848
W. Zhang, M.L. Go / European Journal of Medicinal Chemistry 42 (2007) 841e850
12 h. The reaction mixture was poured into water and ex-
tracted with ethyl acetate. Removal of the solvent in vacuo
from the organic phase provided a clear oil. The oil was
separated by flash column chromatography (9:1 hexane/ethyl
acetate) to give the product as a clear oil.
(d, J ¼ 8.67 Hz, 2H), 6.75 (d, J ¼ 8.64 Hz, 2H), 6.5 (d,
J ¼ 12.42 Hz, 1H), 6.4 (d, J ¼ 12.06 Hz, 1H), 6.36 (d,
J ¼ 1.89 Hz, 2H), 6.19 (t, J ¼ 2.25 Hz, 1H), 3.77 (3H, s,
OCH3), 0.93 (18H, s, C(CH3)3), 0.097 (12H, s, Si(CH3)2).
1
Compound 20E: H NMR: d 7.44 (d, J ¼ 8.67 Hz, 2H),
6.97 (d, J ¼ 16.56 Hz, 1H), 6.89 (d, J ¼ 8.64 Hz, 2H), 6.83
(d, J ¼ 16.2 Hz, 1H), 6.6 (d, J ¼ 2.25 Hz, 2H), 6.24 (t,
J ¼ 2.28 Hz, 1H), 3.83 (3H, s, OCH3), 0.99 (18H, s,
C(CH3)3), 0.21 (12H, s, Si(CH3)2).
6.1.4.1. 3-(tert-Butyldimethylsilyloxy)benzaldehyde (16). Yield
1
70%, 0.93 g. H NMR: d 9.95 (1H, s, J ¼ 1 Hz, CHO), 7.48
(1H, t, J ¼ 1.14 Hz), 7.40 (1H, t, J ¼ 7.53 Hz), 7.33 (1H, t,
J ¼ 2.64 Hz), 7.10 (1H, q, J ¼ 1.14 Hz), 1.0 (9H, s,
C(CH3)3), 0.23 (6H, s, Si(CH3)2).
6.1.5.3. 3-(tert-Butyldimethylsilyloxy)-4,40-dimethoxystilbene
1
(21). Yield 71%, 1.32 g; 21Z (0.8 g): H NMR: d 7.19 (d,
6.1.4.2. 3,5-Di(tert-butyldimethylsilyloxy) benzaldehyde (17). Four
equivalents (2.58 g) of DIEA and 2 equiv (3.58 g) of tert-
butyldimethylsilyl chloride were reacted with 3,5-dihydroxyben-
J ¼ 8.67 Hz, 2H), 6.7e6.83 (m, 5H), 6.44 (d, J ¼ 12.06 Hz,
1
(9H, s, C(CH3)3), 0.06 (6H, s, Si(CH3)2). 21E: (0.52 g). H
NMR: d 7.42 (d, J ¼ 8.67 Hz, 1H), 7.03 (d, J ¼ 7.89 Hz,
2H), 6.81e6.9 (m, 5H), 3.83 (3H, s, OCH3), 3.82 (3H, s,
OCH3), 1.02 (9H, s, C(CH3)3), 0.18 (6H, s, Si(CH3)2).
1H), 6.39 (d, J ¼ 12.06 Hz, 1H), 3.78 (6H, s, OCH3), 0.93
1
zaldehyde (5 mmol) as described earlier. Yield 58%, 0.97 g. H
NMR:
d
9.86 (s, J ¼ 1H, CHO), 6.96 (d, J ¼
2.25 Hz, 2H), 6.59 (t, J ¼ 2.28 Hz, 1H), 0.99 (18H, s,
C(CH3)3), 0.22 (12H, s, Si(CH3)2).
6.1.6. General procedure for deprotection of
silyoxystilbenes (19e21)
6.1.4.3. 3-(tert-Butyldimethylsilyloxy)-4-methoxy benzaldehyde
(18). Two equivalents (1.29 g) of DIEA and 2 equiv (1.79 g)
of tert-butyldimethylsilyl chloride were reacted with 3-
hydroxy-4-methoxybenzaldehyde (5 mmol) as described ear-
Tetrabutylammonium fluoride was added to a solution of
the E or Z silyloxy-protected stilbene in anhydrous tetrahydro-
furan (20 ml). The pale yellow solution was stirred for 45 min,
poured into water, and extracted with dichloromethane, from
which removal of the solvent in vacuo provided a clear oil
in 80e90% yield. The oil was separated by gravity column
chromatography (9:1 hexane/ethyl acetate) to give the product.
1
lier. Yield 80%, 1.07 g. H NMR: d 9.8 (s, J ¼ 1H, CHO),
7.48 (q, J ¼ 1.86 Hz, 1H), 7.37 (d, J ¼ 1.89 Hz, 1H), 6.96 (d,
J ¼ 8.31 Hz, 2H), 3.9 (3H, s, OCH3), 1.0 (9H, s, C(CH3)3),
0.17 (6H, s, Si(CH3)2).
6.1.5. General procedure for the synthesis of
silyloxystilbenes (19e21)
6.1.6.1. 3-Hydroxy-40-methoxystilbene (12). Compound 12Z
1
(80% yield). H NMR: d 7.19 (d, J ¼ 8.67 Hz, 2H), 7.1 (d,
Equimolar quantities (5 mmol) of benzaldehyde (16, 17 or
18) and 4-methoxybenzyltriphenyl phosphonium bromide 2
were reacted in the presence of n-butyllithium and tetrahydrofu-
ran as solvent as described earlier. Stilbenes 19 and 20 were ob-
tained as Z isomers. The E isomer was obtained by refluxing
a solution of Z isomer (5 mmol) in heptane (20 ml) in the pres-
ence of a catalytic amount of iodine (one crystal) for 16 h. The
reaction mixture was diluted with 20 ml ether and washed
with saturated aqueous sodium bisulfite (40 ml) and brine
(2 ꢅ 10 ml). The organic layer was dried over anhydrous
MgSO4 and concentrated in vacuo to give the trans isomer in
90e92% yields. Stilbene 21 was obtained as a mixture of Z
and E isomers that were separated by column chromatography.
J ¼ 7.53 Hz, 1H), 6.84 (d, J ¼ 7.53 Hz, 1H), 6.73e6.78 (m,
3H), 6.68 (q, J ¼ 2.64 Hz, 1H), 6.52 (d, J ¼ 12.42 Hz, 1H),
6.44 (d, J ¼ 12.06 Hz, 1H), 3.78 (3H, s, OCH3). 13C NMR
(300 MHz): d 158.6, 155.4, 139.2, 130.2, 130.0, 129.7,
129.6, 128.4, 121.4, 115.4, 114.1, 113.6, 55.2.
Compound 12E (91% yield), mp 157e160 ꢁC, lit. mp
1
159e160 ꢁC [36]. H NMR (DMSO-d6): d 9.39 (1H, s, OH),
7.5 (d, J ¼ 8.64 Hz, 2H), 7.16 (d, J ¼ 7.5 Hz, 1H), 7.1 (d,
J ¼ 15.5 Hz, 1H), 7.0 (d, J ¼ 15.8 Hz, 1H), 6.93e7.0 (m,
4H), 6.65 (q, J ¼ 1.89 Hz, 1H), 3.34 (3H, s, OCH3).
6.1.6.2. 3,5-Dihydroxy-40-methoxystilbene (13). Compound
1
13Z [37] (94% yield). H NMR: d 7.2 (2H, d, J ¼ 8.67 Hz),
6.1.5.1. 3-(tert-Butyldimethylsilyloxy)-40-methoxystilbene (19).
Compound 19Z: clear oil, 76% yield. 1H NMR: d 7.18
(d, J ¼ 9.06 Hz, 2H), 7.09 (d, J ¼ 7.92 Hz, 2H), 6.82e6.99
(m, 3H), 6.75 (d, J ¼ 9.03 Hz, 1H), 6.52 (d, J ¼ 12.06 Hz,
1H), 6.45 (d, J ¼ 12.06 Hz, 1H), 3.78 (3H, s, OCH3), 0.93
(9H, s, C(CH3)3), 0.093 (6H, s, Si(CH3)2).
6.76 (2H, d, J ¼ 8.67 Hz), 6.5 (1H, d, J ¼ 12.03 Hz), 6.37
(1H, d, J ¼ 12.03 Hz), 6.31 (2H, d, J ¼ 2.25 Hz), 6.22 (1H, t,
J ¼ 2.25 Hz), 3.78 (3H, s, OCH3). 13E (82% yield), mp
164e167 ꢁC, lit. mp 158e166 ꢁC [38]. 1H NMR (DMSO-
d6): d 9.21 (2H, s, OH), 7.52 (2H, d, J ¼ 8.67 Hz), 6.86e
7.01 (4H, m), 6.4 (2H, d, J ¼ 1.89 Hz), 6.1 (1H, t,
J ¼ 3.69 Hz), 3.34 (3H, s, OCH3).
1
Compound 19E: H NMR: d 7.45 (d, J ¼ 8.67 Hz, 2H),
7.19 (d, J ¼ 15.8 Hz, 1H), 7.08 (d, J ¼ 15.8 Hz, 1H), 6.88e
6.99 (m, 5H), 6.72 (q, J ¼ 2.28 Hz, 1H), 3.83 (3H, s,
OCH3), 1 (9H, s, C(CH3)3), 0.22 (6H, s, Si(CH3)2).
6.1.6.3. 3-Hydroxy-4,40-dimethoxystilbene (14). Compound
14Z (94% yield), mp 77e79 ꢁC. 1H NMR: d 7.2 (2H, d,
J ¼ 8.67 Hz), 6.86 (1H, d, J ¼ 1.89 Hz), 6.7e6.79 (4H, m),
6.45 (1H, d, J ¼ 12.03 Hz), 6.39 (1H, d, J ¼ 12.06 Hz), 5.5
(1H, s, OH), 3.87 (3H, s, OCH3), 3.79 (3H, s, OCH3). 13C
6.1.5.2. 3,5-Di-(tert-butyldimethylsilyloxy)-40-methoxystilbene
1
(20). Compound 20Z: clear oil, 68% yield. H NMR: d 7.17

W. Zhang, M.L. Go / European Journal of Medicinal Chemistry 42 (2007) 841e850
849
NMR (300 MHz): d 158.6, 145.6, 145.2, 131.0, 130.1, 129.8,
128.8, 128.3, 120.9, 114.9, 113.6, 110.4, 55.9, 55.2.
Compound 14E [39] (92% yield), mp 192e193 ꢁC. 1H
NMR (DMSO-d6): d 8.95 (1H, s, OH), 7.49 (2H, d,
J ¼ 8.67 Hz), 7.0 (1H, d, J ¼ 1.86 Hz), 6.88e6.96 (6H, m),
3.78 (3H, s, OCH3), 3.77 (3H, s, OCH3).
formazan was observed in each well, the absorbance of which
was measured at 590 nm on a plate reader. Blank wells contai-
ned no cells and control wells contain Hepa 1c1c7 cells treated
with medium containing 0.5% DMSO but without the test
compound. QR induction activity of a compound tested at
a given concentration was determined from the ratio of absor-
bances obtained in the presence of test compound and the con-
trol well, after subtracting the absorbance of the blank well.
QR induction activity was also determined in the same manner
on a mutant Hepa 1c1c7 cell line (c1, ATCC CRL-2716) for
selected target compounds. In addition, the QR induction
activities of other compounds were concurrently determined:
resveratrol and sulforaphane (SigmaeAldrich Chemical Com-
pany), pinosylvin (Sequoia Research Products), b-naphthofla-
vone (Fluka Chemical Company). Experiments were carried
out at least three times on separate occasions.
6.1.7. E-2,20-dimethoxystilbene (15)
Two equivalents of titanium tetrachloride in dichlorome-
thane (1.5 M) was added dropwise to powdered zinc
(2.5 equiv) in a round bottom flask that was charged with N2
gas and cooled in an ice water bath [16]. About 50 ml of
THF was then added and the mixture was brought to reflux
for 2 h. It was then cooled to room temperature and a solution
of 2-methoxybenzaldehyde (5 mmol) in THF was added drop-
wise. The mixture was then refluxed for 12 h. The reaction
mixture was extracted with dichloromethane (3 times) and
the organic layer was washed with brine and dried over anhy-
drous MgSO4. The organic solvent was removed in vacuo and
the residue was purified by column chromatography (99%
hexane, 1% ethyl acetate). The product was obtained as a mix-
ture of Z and E isomers, and converted to the E isomer by
refluxing in heptane in the presence of iodine. Compound
15E was obtained in 10% yield, mp 135e136 ꢁC, lit. mp
6.3. Determination of antioxidant activity
The ABTS [2,20-azinobis-(3-ethylbenzothiazoline-6-sul-
fonic acid)] radical cation decolorisation assay was carried
out according to a reported method [17]. Briefly, a solution
of ABTS free radical (ABTSꢀþ) was prepared by reacting
ABTS (SigmaeAldrich) with potassium persulfate. It was
then diluted with PBS (pH 7.4) to give an absorbance of
0.700 (ꢄ0.02) at 734 nm, 25 ꢁC. The absorbance of the diluted
solution remained unchanged for up to 15 min. An aliquot of
the test compound prepared from a stock solution in ethanol
was added to the diluted ABTSꢀþ solution (1 ml) and the
decrease in absorbance was monitored at 1, 3 and 5 min.
The percentage reduction in absorbance at 5 min was plotted
as a function of concentration (2.5e25 mM) of test compound.
A straight line was obtained, the gradient of which reflected
the scavenging ability of the test compound. The experiment
was repeated with the known antioxidant Trolox (6-hydroxy-
2,5,7,8-tetramethylchroman-2-carboxylic acid, SigmaeAl-
drich Company). The ratio of the gradients of the lines
obtained from test compound and Trolox was used to deter-
mine the TEAC (Trolox equivalent antioxidant capacity) of
the test compound.
1
135e136 ꢁC [40]. H NMR: d 7.63e7.66 (2H, m), 7.47 (2H,
s), 7.2e7.23 (2H, m), 6.87e6.98 (4H, m), 3.88 (6H, s, OCH3).
6.2. Determination of quinone reductase activity
Activity of quinone reductase was determined in Hepa
1c1c7 murine hepatoma cells (American Type Culture Coll-
ection) grown in 96-well microtitre plates according to a
described procedure [19]. Briefly, cells (104 per well) were
grown for 24 h in a-minimal essential medium (without ribo-
nucleosides or deoxyribonucleosides) containing 10% (v/v) fe-
tal calf serum, 0.01% penicillin G, 0.15% sodium bicarbonate,
0.01% streptomycin sulfate in an humidified atmosphere of
5% CO2 at 37 ꢁC. The cells were then exposed to a known
concentration of the test compound (stock solution prepared
in DMSO) for 24 h. The final concentration of DMSO in
each well was kept at 0.5% v/v or lower. After this time, the
media was decanted and the cells lysed by a solution contain-
ing 0.8% w/v digitonin and 2 mM EDTA with agitation
(10 min). An aliquot (200 ml) of a solution (‘‘complete reac-
tion mixture’’) was then added to each well. This solution
was freshly prepared just prior to use and consisted of
7.5 ml 0.5 M Tris-Cl (pH ¼ 7.4), 100 mg bovine serum, 1 ml
1.5% Tween-20, 0.1 ml 7.5 mM FAD, 1 ml 150 mM
glucose-6-phosphate, 90 ml 50 mM NADP, 300 units yeast glu-
cose-6-phosphate dehydrogenase, 45 mg MTT and deionized
water to a final volume of 150 ml. Menadione (0.2 ml of
a 50 mM solution prepared in acetonitrile) was added just
before the mixture was dispensed into the wells. After addi-
tion, the plate was gently agitated for 5 min, after which the
reaction was quenched by adding a solution (50 ml) of
0.3 mM dicoumarol in 0.5% DMSO and 5 mM potassium
phosphate, pH ¼ 7.4. A blue color due to the formation of
TEAC ¼ GradientTest compound/GradientTrolox
.
Experiments were carried out at least three times, on sepa-
rate occasions.
6.4. Microculture tetrazolium assay for determining cell
viability
The cytotoxicity of stilbenes 3e15 was determined on
Hepa 1c1c7 cells using the microculture tetrazolium assay
[18]. Stock solutions of the test compounds were prepared in
DMSO and serially diluted to give final concentrations of
1e25 mM. Not more than 1% DMSO (final concentration)
was present in each well. The test compounds were incubated
with the cells for 24 h, after which MTT [3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide), SigmaeAl-
drich] was added for 3 h and the cells lysed to release the
formazan product. The latter was dissolved in DMSO

850
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(150 ml) and the absorbance was determined within 30 min at
590 nm on a microtitre plate reader. The absorbance values
from no less than three independent determinations were aver-
aged, adjusted by subtraction of blank values (wells without
cells) and expressed as a percentage of the average absorbance
of control wells (wells with untreated cells).
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Acknowledgements
Zhang Wei is supported by a research scholarship from the
National University of Singapore. This work is made possible
by Grant RP140000058112 to M.L. Go from NUS.
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