Chemical transformations of oxyresveratrol (trans-2,4,3′,5′-tetrahydroxystilbene) into a potent tyrosinase inhibitor and a strong cytotoxic agent

Article abstract of DOI:10.1016/j.bmcl.2006.08.018

From oxyresveratrol (trans-2,4,3′,5′-tetrahydroxystilbene 1), seven derivatives were prepared, including trans-2-methoxy-4,3′,5′-trihydroxystilbene (2), trans-2,3′-dimethoxy-4,5′-dihydroxystilbene (3), trans-4,3′-dimethoxy-2,5′-dihydroxystilbene (4), tran

Full text of DOI:10.1016/j.bmcl.2006.08.018

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5650–5653
Chemical transformations of oxyresveratrol
(trans-2,4,30,50-tetrahydroxystilbene) into a potent
tyrosinase inhibitor and a strong cytotoxic agent
Kittisak Likhitwitayawuid,a,* Acom Sornsute,a
Boonchoo Sritularaka and Poonsakdi Ploypradithb
aDepartment of Pharmacognosy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
bChulabhorn Research Institute, Vibhavadee-Rangsit Highway, Bangkok 10210, Thailand
Received 4 April 2006; revised 29 July 2006; accepted 2 August 2006
Available online 17 August 2006
Abstract—From oxyresveratrol (trans-2,4,30,50-tetrahydroxystilbene 1), seven derivatives were prepared, including trans-2-methoxy-
4,30,50-trihydroxystilbene (2), trans-2,30-dimethoxy-4,50-dihydroxystilbene (3), trans-4,30-dimethoxy-2,50-dihydroxystilbene (4),
trans-2,4,30,50-tetramethoxystilbene (5) and cis-2,4,30,50-tetramethoxystilbene (6), 2,4,30,50-tetrahydroxybibenzyl (7), and 2,4,30,50-
tetramethoxybibenzyl (8). The tetrahydroxybibenzyl 7, a hydrogenation product of 1, exhibited more potent tyrosinase inhibitory
activity than the parent compound, without cytotoxicity. A kinetic study revealed that 7 was a reversible and non-competitive inhib-
itor of mushroom tyrosinase with L-dopa as the substrate. Analysis of the Ki values indicated that 7 has a slightly higher affinity to
the enzyme than 1. Compound 6, a tetra-O-methylated analogue of 1 with cis-configuration, was deprived of inhibitory effect on the
enzyme tyrosinase, but showed very strong cytotoxicity against the human cancer cells KB, BC, and NCI-H187, with potency com-
parable to those of the anticancer agents ellipticine and doxorubicin. Data on the tyrosinase inhibitory activity and cytotoxicity of
1–8 indicated that O methylation on stilbene 1 destroyed anti-tyrosinase activity but generated cytotoxicity. Thus, facile prepara-
tions of a potent tyrosinase inhibitor (7) and a strong cytotoxic agent (6) from the natural product 1 were achieved through simple
chemical reactions.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Oxyresveratrol (trans-2,4,30,50-tetrahydroxystilbene, 1)
is a major constituent of the heartwood of Artocarpus
lakoocha Roxb.1,2 The compound is the active principle
of ‘Puag-Haad,’ a dried aqueous extract traditionally
used in Thailand as an anthelmintic.3 Recent studies
have revealed that 1 also possesses potent inhibitory
activity against tyrosinase,4,5 a key enzyme in the bio-
synthetic pathway of melanin pigments in both plants
and animals. Further investigations have shown that
the compound has strong skin depigmenting effects in
both animals and humans.6 As a strong tyrosinase
inhibitor, 1 has potential applications as a skin-whiten-
ing agent in cosmetic preparations or as an anti-brown-
ing agent for food products of plant origin. Other
interesting biological activities reported for 1 include
antiherpetic and anti-HIV,2 antiinflammatory,7 antioxi-
dative,8 and antiapoptotic and neuroprotective activi-
ties.9 Since 1 can be obtained in large amounts from
the widely available A. lakoocha,2–4 the compound ap-
pears to be an attractive target for chemical studies in
terms of structure–activity relationships. In the present
communication, we describe our efforts to prepare, from
1, analogues with higher potency and selectivity with
regard to their cytotoxic and anti-tyrosinase activities.
The reaction of 1 with CH3I (3 equiv, reflux, 24 h) gave
three partially methylated products, namely trans-2-
methoxy,4,30,50-trihydroxystilbene (2), trans-2,30-dime-
thoxy-4,50-dihydroxystilbene (3), and trans-4,30-dime-
thoxy-2,50-dihydroxystilbene (4). Full O-methylation of
1 was achieved when CH3I (6 equiv) was employed,
leading to the formation of trans-2,4,30,50-tetramethoxy-
stilbene (5). Attempts to prepare other mono-, di-, and
tri-methoxy derivatives of 1 using CH3I or (CH3)2SO4
in various conditions were not successful. When trans-
tetramethoxystilbene 5 was subjected to a photochemi-
cal reaction, the cis-isomer 6 was formed. Catalytic
Pd/C hydrogenation of 1 and 5 gave bibenzyl com-
pounds 7 and 8, respectively. The positions of the
Keywords: Stilbene; Tyrosinase inhibitor; Cytotoxicity; Oxyresveratrol.
*
Corresponding author. Fax: +662 2188357; e-mail: Kittisak.L@
chula.ac.th
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.018

K. Likhitwitayawuid et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5650–5653
5651
OH
(IC50 > 100 lM). The loss of activity in 2–6 and 8 was
caused by the disappearance of the 4-alkyl resorcinol-
like structure of ring A.12–14 Compound 7, a bibenzyl
derivative prepared from 1, showed more inhibitory
activity than did the parent compound, being about 8-
fold stronger in view of the IC50 value. Further analysis
of the data obtained for 7 indicated that this compound,
as well as 1 and kojic acid, inhibited the enzyme in a
dose-dependent manner (Fig. 2). The higher tyrosinase
inhibitory activity of 7, as compared with 1, was proba-
bly due to its bibenzyl structure which gave more flexi-
bility and thus allowed the phenolic groups to interact
with the enzyme more effectively. Kinetic studies were
then conducted on 7, in comparison with 1, with regard
to the ability to inhibit mushroom tyrosinase with L-do-
pa as the substrate. As summarized in Table 2, the Vmax
(DA490/min) for the dopa oxidase activity of the enzyme
was 2.0 · 10ꢀ1, and the Km value was 0.7 mM L-dopa.
It can be seen from the Lineweaver–Burk plot of 1/V
CH3I (3 eq)
OH
2
+
3
+ 4
OH
hν
1
CH3I (6 eq)
5
8
6
HO
H2/Pd
H2/Pd
7
OMe
OR3
3'
α
2
β
OR1
2
B
α
4
MeO
OR4
5'
5'
3'
β
A
MeO
OMe
R2O
4
6
OR3
3'
R1
R2
R3
R4
OR1
2
B
α
1
H
H
H
H
H
H
H
H
OR4
5'
β
A
2
3
4
5
Me
Me
H
4
R2O
Me
H
R1
H
R2
R3
R4
100
80
60
40
20
0
Me Me
H
7
8
H
H
H
Me
Me Me Me
Me
Me Me Me
Figure 1. Chemical transformations of 1 into 2–8.
methoxy groups in 2–4 were determined by NOESY
experiments. The cis-configuration of 6 was established
from the coupling constant between H-a and H-b
(J = 12.1 Hz) (Jab = 16.5 Hz in 5), and from the NOESY
effect observed between H-6 and H-20(60). These natural
and synthetic stilbenoids (1–8) (Fig. 1) were then evalu-
ated for tyrosinase inhibitory activity and cytotoxic
potential.10
For the evaluation of tyrosinase inhibitory activity, as-
says were performed with L-dopa as the substrate as pre-
viously described, using kojic acid, a well-known strong
tyrosinase inhibitor, as the positive control.4,11–13 Table
1 summarizes the percentages of inhibition and the IC50
values of 1–8, as compared with kojic acid. It should be
mentioned that oxyresveratrol (1) was earlier reported
to be a stronger tyrosinase inhibitor than kojic acid,5
and this was confirmed in this study, as reflected in
their respective IC50 values of 12.7 and 133.4 lM
(Table 1). For the O-methylated products (2–6 and8),
none of them showed significant inhibitory activity
0.1.1
1
10
100
1000
Sample (μM)
Figure 2. Dose-dependent inhibitory effects on mushroom tyrosinase
by oxyresveratrol (1), 2,4,30,50-tetrahydroxybibenzyl (7), and kojic
acid. Samples shown are oxyresveratrol (circle, d), 2,4,30,50-tetrahydr-
oxybibenzyl (triangle, m), and kojic acid (diamond, Ç).
Table 1. Tyrosinase inhibitory activity of 1–8
Compound
% inhibition (at 100 lM)
IC50 (lM)
2,4,30,50-Tetrahydroxystilbene (1)
trans-2-Methoxy-4,30,50-trihydroxystilbene (2)
trans-2,30-Dimethoxy-4,50-dihydroxystilbene (3)
trans-4,30-Dimethoxy-2,50-dihydroxystilbene (4)
trans-2,4,30,50-Tetramethoxystilbene (5)
cis-2,4,30,50-Tetramethoxystilbene (6)
2,4,30,50-Tetrahydroxybibenzyl (7)
2,4,30,50-Tetramethoxybibenzyl (8)
Kojic acid
83.5 0.9
4.8 1.6
<0 1.4
<0 3.6
<0 2.6
<0 1.9
96.3 0.6
2.3 3.2
41.1 1.4
12.7
>100
>100
>100
>100
>100
1.6
>100
133.4

5652
K. Likhitwitayawuid et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5650–5653
Table 2. Kinetic parameters of mushroom tyrosinase in the presence of
1 or 7
of our investigation, as reflected in the unchanged Km
value (0.7 mM L-dopa) in the presence of 1 (Table 2).
When the Ki values of 1 (2.2 · 10ꢀ5–2.8 · 10ꢀ5 M) and
7 (4.8 · 10ꢀ6–5.6 · 10ꢀ6 M) were compared (Table 2),
it was found that 7 had about 5-fold higher affinity to
the enzyme than 1. The higher potency of the former
is probably due to this kinetic property.
Inhibitor
Dose
(lM)
Km
Vmax
Ki
(M)
(DA490/min)
(M)
None
—
0.7 · 10ꢀ3
0.7 · 10ꢀ3
0.7 · 10ꢀ3
0.7 · 10ꢀ3
0.7 · 10ꢀ3
2.0 · 10ꢀ1
1.6 · 10ꢀ1
1.4 · 10ꢀ1
1.4 · 10ꢀ1
1.2 · 10ꢀ1
—
7
7
1
1
1.2
2.4
9.2
18.4
4.8 · 10ꢀ6
5.6 · 10ꢀ6
2.2 · 10ꢀ5
2.8 · 10ꢀ5
A study of the cytotoxicity of 1–8 was carried out using
a battery of cancer cell lines, including KB, BC, and
NCI-H187, with the anticancer agents ellipticine and
doxorubicin as positive controls.16,17 Table 3 illustrates
the IC50 values for these compounds. It is obvious that
while the parent compound 1 was not cytotoxic, all of
its O-methylated derivatives (2–6) demonstrated cyto-
toxic potential, ranging from weak to very strong activ-
ity. Thus, it appears that the introduction of methoxy
groups to the aromatic rings generated cytotoxicity. It
should be noted that the cis-polymethoxystilbene 6
was more potent than the trans-isomer 5. The bibenzyl
structures 7 and 8, however, were devoid of cytotoxicity.
Therefore, it could be deduced from these data that the
requirements for cytotoxicity of the stilbenoids were: (1)
polymethoxy groups, (2) unsaturation at a- and b-car-
bons, and (3) cis-configuration. In view of the IC50 val-
ue, 6 showed more cytotoxicity than the positive control
ellipticine in all cell lines. However, when compared with
doxorubicin, 6 was equally active in KB but less active
in BC and NCI-H187 cells. Structurally, stilbene 6 could
be considered, as related to combretastatins, a group of
natural products possessing strong anticancer potential,
which were first isolated from Combretum caffrum
(Combretaceae).18 Studies have shown that trans-poly-
methoxystilbenoids exert their cytotoxicity by inhibition
of cytochrome P450 1B1,19 whereas cis-analogues have
inhibitory effects on tubulin polymerization of cancer
cells.18 More studies on 6, such as in vitro assays in
other cancer cell lines and in vivo experiments in ani-
mals, as well as examinations of the mechanism of ac-
tion, are needed before the anticancer potential of this
compound can be fully determined.
values with different concentrations of L-dopa (Fig. 3)
that the presence of 7 at different concentrations (1.2
and 2.4 lM) did not affect the Km value (0.7 mM L-do-
pa) of the enzyme, but decreased the Vmax values to
1.6 · 10ꢀ1 and 1.4 · 10ꢀ1, respectively (Table 2). There-
fore, 7 was a non-competitive inhibitor of tyrosinase on
L-dopa with the Ki values of 4.8–5.6 lM (Table 2). A
previous kinetic study on mushroom tyrosinase with L-
dopa as the substrate indicated that 1 was a non-com-
petitive inhibitor.15 This is in agreement with the results
50
40
30
Α
20
10
-2
0
2
4
6
8
10
1/[L-DOPA], mM
Figure 3. Lineweaver–Burk plot of mushroom tyrosinase in the
presence of 2,4,30,50-tetrahydroxybibenzyl (7). Data were obtained as
mean values of 1/V, inverse of the increase of absorbance at the
wavelength 490 nm per min (DA490/min), with different concentrations
of L-dopa as substrate. Inhibitors of the enzyme were 2,4,30,50-
tetrahydroxybibenzyl (7) with 2.4 lM (triangle, .), 1.2 lM (circle, s),
and no 2,4,30,50-tetrahydroxybibenzyl (7) (circle, d).
In summary, this study showed that a more potent
tyrosinase inhibitor (7) can be obtained from oxyresve-
ratrol (1) through a single-step reduction reaction. The
bibenzyl structure 7 was a non-competitive inhibitor of
mushroom tyrosinase with higher affinity to the enzyme
Table 3. Cytotoxicity of 1–8
Compound
IC50 (lM)
KB
BC
NCI-H187
2,4,30,50-Tetrahydroxystilbene (1)
trans-2-Methoxy-4,30,50-trihydroxystilbene (2)
trans-2,30-Dimethoxy-4,50-dihydroxystilbene (3)
trans-4,30-Dimethoxy-2,50-dihydroxystilbene (4)
trans-2,4,30,50-Tetramethoxystilbene (5)
cis-2,4,30,50-Tetramethoxystilbene (6)
2,4,30,50-Tetrahydroxybibenzyl (7)
2,4,30,50-Tetramethoxybibenzyl (8)
Ellipticine
NAa
NA
5.5
NA
66.6
10.8
13.9
5.6
NA
NA
10.9
33.5
8.0
16.5
8.6
0.3
1.0
0.3
NA
NA
2.4
NA
NA
2.7
NA
NA
1.9
Doxorubicin
0.3
0.5
0.1
a NA, no activity, showing less than 50% inhibition at 20 lg/ml.

K. Likhitwitayawuid et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5650–5653
5653
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Acknowledgments
We are grateful to the Asahi Glass Foundation for par-
tial financial support (for the study of tyrosinase inhib-
itory activity). Thanks are due to the National Center
for Genetic Engineering and Biotechnology for the as-
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