Structural determinants of resveratrol for cell proliferation inhibition potency: Experimental and docking studies of new analogs

Article abstract of DOI:10.1016/j.ejmech.2010.03.024

Resveratrol is the subject of intense research because of the abundance of this compound in the human diet and as one of the most valuable natural chemopreventive agents. Further advances require new resveratrol analogs be used to identify the structural determinants of resveratrol for the inhibition potency of cell proliferation by comparing experimental and docking studies. Therefore, we synthesized new trans/(E)- and cis/(Z)-resveratrol - analogs not reported to date - by modifying the hydroxylation pattern of resveratrol and a double bond geometry. We included them in a larger panel of 14 molecules, including (Z)-3,5,4′-trimethoxystilbene, the most powerful molecule that is used as reference. Using a docking model complementary to experimental studies on the proliferation inhibition of the human colorectal tumor SW480 cell line, we show that methylation is the determinant substitution in inhibition efficacy, but only in molecules bearing a Z configuration. Most of the synthetic methylated derivatives (E or Z) stop mitosis at the M phase and lead to polyploid cells, while (E)-resveratrol inhibits cells at the S phase. Docking studies show that almost all of the docked structures of (Z)-polymethoxy isomers, but not most of the (E)-polymethoxy isomers substantially overlap the docked structure of combretastatin A-4, taken as reference ligand at the colchicine-tubulin binding site.

Full text of DOI:10.1016/j.ejmech.2010.03.024

European Journal of Medicinal Chemistry 45 (2010) 2972e2980
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structural determinants of resveratrol for cell proliferation inhibition potency:
Experimental and docking studies of new analogs
a
a
a
a
c
c
Frédéric Mazué , Didier Colin , Jessica Gobbo , Maria Wegner , Antonio Rescifina , Carmela Spatafora ,
b
a
b
c
a,
*
Dominique Fasseur , Dominique Delmas , Philippe Meunier , Corrado Tringali , Norbert Latruffe
INSERM U 866; University of Burgundy, Laboratory of Biochemistry of Metabolism and Nutrition, 6, Bd Gabriel, F-21000 Dijon, France
ICMUB-UMR CNRS 5260, University of Burgundy, Institute of Molecular Chemistry, Ave Savary, F-21000 Dijon, France
Università degli Studi di Catania, Dipartimento di Scienze Chimiche, Viale Andrea Doria 6, I-95125 Catania, Italy
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 November 2009
Received in revised form
Resveratrol is the subject of intense research because of the abundance of this compound in the human
diet and as one of the most valuable natural chemopreventive agents. Further advances require new
resveratrol analogs be used to identify the structural determinants of resveratrol for the inhibition
potency of cell proliferation by comparing experimental and docking studies. Therefore, we synthesized
new trans/(E)- and cis/(Z)-resveratrol e analogs not reported to date e by modifying the hydroxylation
pattern of resveratrol and a double bond geometry. We included them in a larger panel of 14 molecules,
15 March 2010
Accepted 16 March 2010
Available online 25 March 2010
0
including (Z)-3,5,4 -trimethoxystilbene, the most powerful molecule that is used as reference. Using
Keywords:
a docking model complementary to experimental studies on the proliferation inhibition of the human
colorectal tumor SW480 cell line, we show that methylation is the determinant substitution in inhibition
efficacy, but only in molecules bearing a Z configuration. Most of the synthetic methylated derivatives (E
or Z) stop mitosis at the M phase and lead to polyploid cells, while (E)-resveratrol inhibits cells at the S
phase. Docking studies show that almost all of the docked structures of (Z)-polymethoxy isomers, but not
most of the (E)-polymethoxy isomers substantially overlap the docked structure of combretastatin A-4,
taken as reference ligand at the colchicineetubulin binding site.
Resveratrol
Polymethoxy-stilbenes
Tubulin polymerization
Colon cancer
Docking studies
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
Nevertheless, various studies have documented that stilbenes
and flavonoids, despite efficient absorption by the organism,
Recent evidence suggests that the use of resveratrol, a well-
known polyphenol that is abundant in the human diet, in combi-
nationwith drugs, ionizing radiation, or cytokines can be effective in
unfortunately have low bioavailability, glucuronidation and sulfa-
tion being limiting factors [9e11]. We have recently developed
acetylated forms of resveratrol and oligomers, showing that acet-
ylation of resveratrol inhibits cancer cell proliferation in the same
manner as resveratrol [12e14]. In contrast, the isomerization of
molecules and the methylation of hydroxyl groups change the cell
molecular targets and are essential to strengthen the efficiency of
resveratrol derivatives for blocking the cell cycle [15e17], sug-
gesting that polymethoxy-stilbenes and related compounds are
a subgroup of resveratrol analogs showing promising antitumor
properties (see for review [18]). In addition, in vivo studies indicate
that polymethoxy-stilbenes undergo a different metabolic conver-
sion and have a higher bioavailability than resveratrol.
0
the sensitization to apoptosis. Natural trans-resveratrol [(E)-3,5,4 -
trihydroxystilbene] targets a wide variety of intracellular mecha-
nisms involved in the pathways leading to malignancy. In various in
vitro and in vivo models, this polyphenolic compound has proven to
be capable of retarding or preventing the various steps of carcino-
genesis [1]. This protective effect could be related to the ability of
resveratrol to arrest cell cycle progression [2,3] or to trigger tumor
cell death byapoptosis [4,5]. Recently, resveratrol has been shown to
behavealsoas a sensitizerof anticancerdrugs such as 5-fluoro-uracil
[6] and paclitaxel [7], radiation [8], and cytokines such as TRAIL [5].
While previous studies already reported the synthesis of poly-
0
methoxy-stilbene analogs [18], especially highlighting (Z)-3,5,4 -
trimethoxystilbene, which exhibits strong antiproliferative activity
by acting as an inhibitor of tubulin polymerization [19], the struc-
tureeactivity relationship remains unclear, for example, the methyl
*
Corresponding author.
E-mail address: latruffe@u-bourgogne.fr (N. Latruffe).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.03.024

F. Mazué et al. / European Journal of Medicinal Chemistry 45 (2010) 2972e2980
2973
position and number, double bond configuration, or the additional
hydroxyl group. The originality of our work is based on two strate-
gies: synthesis of three new resveratrol analogs as yet unpublished
depends on Z-stereochemistry. The presence of a supplementary
hydroxyl group decreases the efficiency of the antiproliferative
properties of resveratrol analogs. Interestingly, (Z)-isomer treat-
ments lead to a polyploidy phenomenon in colon cancer cells. Using
a computational docking approach, we show that Z-isomers, apart
from (Z)-resveratrol and (Z)-tetramethoxy-stilbene, can be incor-
porated into the colchicine site of tubulin. All (Z)-isomers substan-
tially overlap the docked structure of combretastatin A-4 (15), taken
as reference.
(
compounds 6, 10 and 14) and the use of docking modeling studies
and their comparison with experimental data from the human
colorectal tumor SW480 cell line to identify biological targets. For
a coherent approach to critical methylation resveratrol analogs and
for a better understanding, we tested a larger panel of molecules
0
from already published resveratrol analogs including (Z)-3,5,4 -tri-
methoxystilbene (compound 4), the most powerful molecule. Thus,
with apropersynthetic methodology, previouslyemployed for some
of the compounds reported here [20] and exemplified in Fig. 1,
a library of resveratrol analogs was obtained from (E)- and (Z)-
resveratrol (Fig.1, compounds 1 and 2). Both (E)- and (Z)-isomers for
2. Material and methods
2.1. General
0
The H and 13C NMR spectra were run on a Varian Unity Inova
1
each substrate were prepared. In the first group, the 3,5,4 -hydroxyl
groups of resveratrol were replaced with methoxy groups
spectrometer at 500 and 125 MHz, respectively, in CDCl or C
3
6 6
D
(
compounds 3 and 4) and a further hydroxyl group was inserted at
solutions with TMS as internal standard. Mass spectra were recor-
ded in ESI positive mode on a Micromass ZQ2000 spectrometer
(Waters). All reactions were monitored by TLC on commercially
available precoated plates (silica gel 60 F254) and the products
were visualized with cerium sulfate solution. A silica gel 60 was
employed for column chromatography. Resveratrol (1) was
purchased from Sigma; m-chloroperbenzoic acid (m-CPBA) 99%
assay was obtained by washing the commercial 77% material
(Aldrich) with a phosphate buffer at pH 7.5 and drying the residue
under reduced pressure.
position 2 (compounds 5 and 6); a second group was represented by
3
a hydroxyl group in C-2 (compounds 9 and 10); in a third group of
3
cally substituted (compounds 11 and 12), and also in this case the 2-
hydroxy analogs were prepared (compounds 13 and 14). We
established that the number of methyl groups of resveratrol is
crucial for determining the inhibitory properties of colorectal cancer
cell proliferation and in cell cycle arrest. The strongest effect
0
0
,5,3 ,5 -tetramethoxy-stilbenes (compounds 7 and 8) also bearing
0 0
,5,3 ,4 -tetramethoxy-stilbenes, the two rings were asymmetri-
Fig. 1. Structure of (E)/trans-resveratrol (A), (Z)/cis-resveratrol (B) related analogs and combretastatin A-4 (C).

2974
F. Mazué et al. / European Journal of Medicinal Chemistry 45 (2010) 2972e2980
The known compounds 3, 5, 7, 9, 11, and 13 were synthesized as
2.4. Biological methods
previously reported, based on an Arbuzov rearrangement followed
by the HornereEmmonseWadsworth reaction [20]. Compound 4
was prepared by the classic Wittig reaction: 4-methoxybenzyl-
triphosfonium chloride was reacted with 3,5-dimethox-
ybenzaldehyde and BuLi in THF to afford 4. Spectral data of the
known compounds are in perfect agreement with those obtained
previously [20]. Calculated log D values were obtained with ACD/
labs Log D program version 11.
2.4.1. Proliferation and cytotoxicity assays
The human colon carcinoma cell line SW480 was cultured in
RPMI-Medium with 10% fetal bovine serum and 1% antibiotics.
Proliferation inhibition assays were performed in 24-well plates in
triplicate, and each experiment was conducted three times. In all,
30,000 cells were seeded per well and after 24 h were treated with
media containing either 0.1% dimethylsulfoxide with (E)-resvera-
trol, or with resveratrol derivatives, or with 0.1% dimethylsulfoxide
as control. After 24, 48, and 72 h, adherent cells collected by
trypsinization and detached cells were labeled in 1 mL medium
2.2. Photo-isomerization
containing 1 mg/mL of propidium iodide. Cells were counted using
Irradiation experiments were conducted in a 200-mL quartz
a Cyflow green flow cytometer (Partec, Münster). Dead cells were
distinguished from viable cells by incorporation of propidium
iodide. Subsequently, 48h IC50 values were determined by per-
vessel using a Rayonet photochemical reactor equipped with
a variable number of “black light” phosphor lamps with emission in
the 310 to 390nm range and a maximum at 350 nm. The fluence
forming 1 nMe100
mM treatments and the IC50 values were
ꢀ2
rate at the irradiation position was measured to be 5 mW$cm . A
obtained after parametric regressions on the percentages of viable
cells versus the control.
ꢀ
4
2
ꢁ 10 M solution (200 mL) of each compound (1, 3, 7, and 11) in
ethanol was irradiated in the reactor for 10 min under nitrogen
bubbling. The irradiated solution was then reduced to a small
volume under vacuum and charged onto the appropriate silica gel
column to separate the (Z)-product from the residual (E)-isomer. All
photo-isomerizations were obtained with 78e82% conversion,
2.4.2. Cell cycle and DNA content analysis and microscopic
examination of cell nuclei morphology
See the techniques previously used in [12e14].
1
based on H NMR measurements. Spectral data of the 2, 4, 8, and 12
2.5. Computational docking studies
compounds obtained are in perfect agreement with those previ-
ously reported [17,21e23].
In view of the structural similarities of (Z)-polymethoxy stil-
benes with combretastatin, we also investigated the binding model
of all (E)- and (Z)-isomers 1e14 in comparison with combretastatin
A-4 to delineate their structureeactivity relationships (SARs). Thus,
given that the cytotoxicity mechanism of combretastatin and
related structures has been shown to involve the inhibition of
tubulin polymerization by binding tubulin at the colchicine binding
site [25], we conducted molecular docking simulations of all stil-
bene analogs into this pocket [26]. The reported 3D structure of the
tubulineDAMA-colchicineestathmin-like domain complex was
retrieved from the Protein Data Bank (http://www.rcsb.org/PDB;
code 1SAO), but it has a resolution of only 3.58 Å and therefore
requires considerable computational effort before models derived
from it can be considered “all-atom” [27]. Thus, the stathmin-like
domain, the C and D subunits, and the DAMA-colchicine were
removed from the model, the missing atoms on chain A residues
Gln 35, Asp 47, Thr 51, Glu 55, Thr 56, Glu 77, Arg 221, Gln 285, Arg
308, Ile 335, Lys 336, Lys 338, Arg 339, Gln 342, and chain B residues
His 37, Asn 59, Lys 124, Ser 126, Arg 215, Lys 218, Leu 219, Arg 322,
Lys 326, Lys 338, Arg 369, Lys 372, Asp 437 were added and locally
optimized. Then, using the MolProbity Web server [28], the
hydrogen atoms were added and the orientations of the hydroxyl
hydrogens from the Ser, Thr, and Tyr, the sulfhydryl orientations of
Cys, and the methyls of Met amino acids were optimized; at the
same time the positions of hydrogens on histidine, asparagine, and
glutamine residues were assigned, ensuring suitable ionization
states. Finally, the GasteigereMarsili charges [29] were assigned
and the whole protein, with the combretastatin A-4 positioned in
the place of the DAMA-colchicine ligand and aligned with the A-
ring, and the tree methoxyl groups were optimized to an energy
gradient of 0.005 kcal-Å/mol with amber 96 force field [30].
2.3. Chemical procedure of hydroxylation
The compounds 6, 10, and 14 were synthesized as follow:
a solution of m-CPBA in CH
a stirred solution of the substrate in CH
room temperature. The reaction mixtures were then washed with
a NaHSO solution and subsequently with saturated aqueous
NaHCO . The organic layer was dried (Na SO ), filtered, and
2
Cl
2
(0.150 mmol/mL) was added to
2
2
Cl (0.105 mmol/mL) at
3
3
2
4
concentrated in vacuo; the residues were submitted to flash-chro-
matography on a 3 ꢁ 25 cm silica gel column, eluted with EtOAc in
n-hexane (from 0% to 30%).
0
(
Z)-2-Hydroxy-3,5,4 -trimethoxystilbene (6): EI-MS m/z 285
ꢀ
1
0
[
M-H] ; H NMR (CDCl
3
):
d
3.53 (s, 3H, 4 -OCH
); 5.35 (s, 1H, OH); 6.33 (d, 1H, J ¼ 2.0 Hz,
); 6.61
); 6.75 (d, 2H, J ¼ 7.5 Hz, H-3 and H-5 ); 7.23 (d,
3
); 3.77 (s, 3H, 5-
OCH ); 3.86 (s, 3H, 3-OCH
3
3
H-4); 6.38 (d, 1H, J ¼ 2.0 Hz, H-6); 6.57 (d, 1H, J ¼ 12.0 Hz, H-
a
0
0
(
2
1
5
d,1H, J ¼ 12 Hz, H-
b
0
0
13
H, J ¼ 7.5 Hz, H-2 and H-6 ). C NMR (CDCl
3
): d 158.7, 152.4, 147.0,
37.6, 130.4, 128.6, 127.4, 123.5, 122.9, 114.2, 100.5, 98.8, 56.2, 55.9,
5.5.
Z)-2-Hydroxy-3,5,3 ,5 -tetramethoxystilbene (10): EI-MS m/z
0
0
(
ꢀ
1
3
3
6
15 [M-H] ; H NMR (CDCl
); 3.86 (s, 3H, 3-OCH
.33 (s, 1H, H-6); 6.37 (s, 1H, H-4); 6.47 (s, 2H, H-2 and H-6 ); 6.60
3
):
d
3.52 (s, 3H, 5-OCH
3
); 3.65 (s, 6H,
0
0
0
,5 -OCH
3
3
); 5.38 (s,1H, OH); 6.31 (s,1H, H-4 );
0
0
13
(
(
1
d, 1H, J ¼ 12.5 Hz, H-
CDCl ): 159.8, 153.9, 146.9, 139.2, 137.2, 131.0, 123.9, 122.5, 104.2,
00.0, 99.1, 56.1, 55.7, 55.4.
a
); 6.71 (d, 1H, J ¼ 12.5 Hz, H-
b). C NMR
3
d
0 0
Z)-2-Hydroxy-3,5,3 ,4 -tetramethoxystilbene (14): EI-MS m/z
(
ꢀ
1
0
0
3
); 3.63 (s, 3H, 3 -
3
15 [M-H] ; H NMR (CDCl
3
):
d
3.35 (s, 3H, 4 OCH
OCH ); 3.85 (s, 6H, 3,5-OCH
3
3
); 5.36 (s, 1H, OH); 6.35 (d, 1H,
J ¼ 2.5 Hz, H-4); 6.38 (d, 1H, J ¼ 2.5 Hz, H-6); 6.59 (bs, 2H, H-
a
and
3. Results
0
H-b
); 6.74 (d, 1H, J ¼ 8.5 Hz, H-3); 6.85 (d, 1H, J ¼ 8.5, H-2 ) over-
0
13
lapped with 6.74 (d, 1H, J ¼ 2 Hz, H-6 ). C NMR (CDCl
50.1, 149.8, 148.2, 137.6, 130.6, 129.9, 123.7, 122.1, 121.1, 111.7, 110.7,
04.3, 98.9, 56.2, 55.7, 55.5, 55.4.
The spectral data of already published resveratrol analogs are in
agreement with those reported in the literature [24].
3
):
d
151.9,
We compared the potency of resveratrol synthetic analogs
toward the human colorectal tumor cell line SW480 by comparing
two reference natural molecules, i.e., (E)-3,5,4 -Trihydroxystilbene
(1) and (Z)-3,5,4 -Trihydroxystilbene (2) (Fig. 1); the (E)- and (Z)-
isomers of the 3,5,4 -Trimethoxystilbene (3 and 4, respectively),
1
1
0
0
0

F. Mazué et al. / European Journal of Medicinal Chemistry 45 (2010) 2972e2980
2975
permethylated analogs of resveratrol, were also included in view of
their previously reported high antiproliferative activity.
3.1. Antiproliferative activity toward SW480 colon cancer cell line
Tocomparethe effectofresveratrolanalogsoncellgrowth, SW480
cells were treated with molecules from our library and the anti-
proliferative activity of compounds 1e14 was determined as IC50 (in
m
M)and is reported inTable 1. The most active compounds, inorder of
0
decreasing potency, were the following: (Z)-3,5,4 -Trimethox-
0 0
ystilbene (4), 0.3
10), 7 M > (Z)-3,5,3 ,4 -Tetramethoxystilbene (12), 9.5
Hydroxy-3,5,3 ,4 -Tetramethoxystilbene (14), 10
Tetramethoxystilbene (8), 13 M > (E)-2-Hydroxy-3,5,3 ,5 -Tetra-
methoxystilbene (9), 17 M > (E)-Resveratrol (1), 20 M.
mM > (Z)-2-Hydroxy-3,5,3 ,5 -Tetramethoxystilbene
0
0
(
m
mM > (Z)-2-
0
0
0 0
M > (Z)-3,5,3 ,5 -
m
0
0
m
m
m
When compound 11 was compared with 7 (which is only
different in the position of one methoxy group) it appeared to be
less active. The same tendency was observed between (E)-tetra-
methoxy derivatives, i.e., 13 compared with 9. For the derivatives
with trans configuration, too, a slightly higher effect was seen with
0
0
a methoxy group in another position (12 > 8, 10 > 14). (Z)-3,5,3 ,4 -
0
0
Tetramethoxystilbene (12) and (Z)-2-Hydroxy-3,5,3 ,4 -tetrame-
thoxystilbene (14), two (Z)-tetramethoxystilbenes, were potent but
they appeared to be less effective than the (Z)-trimethoxy analog 4
as a reference of the most potent resveratrol analog.
3.2. Cell cycle and DNA content
To further explore the mechanisms by which all these poly-
phenols exert their antiproliferative potencies, we studied their
effects on the cell cycle distribution of SW480 cells. As shown in the
top panel of Fig. 2A, percentages of cells were determined by
electronic gating on cell populations according to their DNA
content: the diploid cell cycle was divided into a 2n G
a S phase of DNA replication (2 < n < 4), and a 4n G
0
/G
1
phase,
2
/M phase.
Moreover, it should be noted that cancer cells, mainly colon cancer
cells, are usually unstable and display >4n DNA content associated
with polyploidy. Fig. 2A shows the flow cytometry diagrams
obtained after 48h treatments of SW480 cells with all the
compounds from our library at 30
used to evaluate (E)-resveratrol anticancer potencies in vitro. Only
compound 4, which is highly toxic at 30 M, was used at 1 M for
mM, a common concentration
m
m
comparison. The synthetic histograms of the cell distribution in the
different phases of the cell cycle are also shown in Fig. 2B.
Table 1
Potencies of resveratrol derivatives antiproliferative activities toward SW480 human
colorectal cells after 48 h of treatment.
Compound
Number
IC50 (mM)
A. IC50 of (E)-resveratrol derivatives
(
(
(
(
(
(
(
E)-Resveratrol
E)-3,5,4 -Trimethoxystilbene
1
3
5
7
9
20 ꢂ 3
54 ꢂ 8
43 ꢂ 6
48 ꢂ 7
17 ꢂ 2
100 ꢂ 15
42 ꢂ 3
0
0
E)-2-Hydroxy-3,5,4 -trimethoxystilbene
0
0
E)-3,5,3 ,5 -Tetramethoxystilbene
0
0
E)-2-Hydroxy-3,5,3 ,5 -tetramethoxystilbene
0
0
E)-3,5,3 ,4 -Tetramethoxystilbene
11
13
0
0
E)-2-Hydroxy-3,5,3 ,4 -tetramethoxystilbene
B. IC50 of (Z)-resveratrol derivatives
(
(
(
(
(
(
(
Z)-Resveratrol
Z)-3,5,4 -Trimethoxystilbene
2
4
6
90 ꢂ 12
0.3 ꢂ 0.04
18 ꢂ 2
13 ꢂ 2
7 ꢂ 2
Fig. 2. Effect of methoxyresveratrol analogs on SW480 cell cycle. Flow cytometry
analysis of SW480 cells distribution in the cell cycle after 48 h treatments by resver-
atrol analogues (number). Diagrams show cell numbers against DNA content
measurement by propidium iodide staining. Synthetic histograms of cell distribution
among their cell cycle. Data are means ꢂ SD of a representative experiment among
three independent. For experimental procedure, see material and methods.
0
0
Z)-2-Hydroxy-3,5,4 -trimethoxystilbene
0
0
Z)-3,5,3 ,5 -Tetramethoxystilbene
8
0
0
Z)-2-Hydroxy-3,5,3 ,5 -tetramethoxystilbene
10
12
14
0
0
Z)-3,5,3 ,4 -Tetramethoxystilbene
9.5 ꢂ 2
10 ꢂ 2
0
0
Z)-2-Hydroxy- 3,5,3 ,4 -tetramethoxystilbene

2976
F. Mazué et al. / European Journal of Medicinal Chemistry 45 (2010) 2972e2980
Three types of compounds can be reported, according to their
effects on cell cycle distribution: those that exert no effects and are
essentially (E)-isomers plus (Z)-resveratrol (compounds 2, 7, 9, 11,
We then used microscopic analysis to illustrate the accumula-
tion of polyploid cells after treatment with compound 4 (Fig. 3C).
Hoechst staining allowed us to confirm that this (Z)-isomer leads to
mitotic disturbances. Indeed, as compared to resveratrol treatment,
which induces nuclear swelling, compound 4 inhibits cytokinesis
resulting in endocycle and multinucleated cells (karyokinesis
inhibition would have produced giant nuclei).
1
1
0 1
3) with an average of 50% G /G cells, 15% S cells, 25% 4n cells, and
0% >4n cells. Another compound is the well-known (E)-resvera-
trol by itself, which induces a strong S-phase arrest and a small
increase of polyploidy. Finally, many compounds (especially (Z)-
isomers, compounds 3, 4, 5, 6, 8, 10, 12, 14) completely disturb the
diploid cell cycle, leading to the generation of polyploid cells, more
precisely of tetraploid cells because of the loss of 2n cells and of the
increase in 4n and 8n cells.
3.3. Binding model of (E)- and (Z)-stilbene isomers
Starting from the experimental data (presented in previous
sections), we looked for possible relationships between structures
of resveratrol analogs and their affinity to bind colchicine at the
tubulin-binding site, responsible for mitosis inhibition and poly-
ploidy, via a computational docking approach.
To have an overall view of the mechanisms involved in the
polyploidization induced by most of the (Z)-isomers, we performed
a time- and concentration-dependent analysis of the effects of (Z)-
0
3
,5,4 -trimethoxystilbene (4) as reference [19]. The antiproliferative
activity of this compound occurred in a time- and dose-dependent
manner as compared to (E)-resveratrol, and did not exert toxic
To validate the model, we docked combretastatin A-4 (Fig. 1C)
and some structurally diversified combretastatin-like analogs that
bind to colchicine at the tubulin binding site to compare their
experimentally obtained IC50 values with the calculated values. The
results obtained (not reported here; see Supplementary Data)
showed that the model is able to reproduce the IC50 experimental
values with a high precision that is, to our knowledge, the best
obtained to date by docking methodology. The model was applied
successively to compounds 1e14 and the results obtained, arranged
in order of decreasing calculated activity, are presented in Table 2.
Although almost all the (E)-isomers were less active than the
(Z)-isomers against the inhibition of SW480 cells, they were
effects at the highest concentration used (1
also noted that in contrast to (E)-resveratrol, there was no growth
resurgence after 72 h. Moreover, at 1 M), compound
M (IC50 ¼ 0.3
did not seem to be more potent than 0.2 M or 0.5 M. Thus, cell
cycle analyses after treatment with the same concentrations
showed comparable results: 0.5 M and 1.0 M treatments led to
exactly the same profile of cell cycle disruption with an increase of
n and >4n cells (Fig. 3B). Nevertheless, 0.2 M treatments exerted
mM) (Fig. 3A). It was
m
m
4
m
m
m
m
4
m
weaker effects than higher concentrations with regard to cell
distribution in the cell cycle.
Fig. 3. Inherent mechanisms of antiproliferative activity of compound 4. A. Proliferation of SW480 cells treated with resveratrol (compound 1) at 30
to 1.0
M during 24, 48 and 72 h. Data are means ꢂ SD of a representative experiment among three independent. For experimental procedure, see material and methods. B. Flow
cytometry analysis of SW480 cells distribution in the cell cycle after 48 h treatments by compound 1 and 4 at indicated concentrations. Diagrams show cell numbers against DNA
content measurement by propidium iodide staining. C. Microscopic analysis of SW480 cells left untreated (Ctl) or treated with 30 M resveratrol (1) or with 0.5 M of compound 4
mM and compound 4 from 0.2
m
m
m
for 48 h. Green fluorescence (Alexa 488) is due to actin immunostaining performed to see the shape of the cells. Blue fluorescence (Hoechst 33342) reveals nuclei. All the pictures
have been taken with the same conditions and magnification (100ꢁ). This figure represents an example of three independent experiments. [For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.]

F. Mazué et al. / European Journal of Medicinal Chemistry 45 (2010) 2972e2980
2977
Table 2
with our previous assumptions [15], i.e., that the mechanism of
the antiproliferative activity of resveratrol seems to be different
from that of polymethoxy-stilbenes, the latter probably being
related to inhibition of tubulin.
Calculated and experimental binding affinity to colchicine site of tubulin for
substituted stilbenes 1e14.
Ligand
D
G
Bind calcd. (kcal/mol)
IC50 calcd. (mM)
0
1
6
1
4
2
1
1
9
7
8
3
5
1
1
0
ꢀ7.87
ꢀ7.72
ꢀ7.53
ꢀ7.50
ꢀ7.27
ꢀ7.24
ꢀ7.14
ꢀ7.01
ꢀ7.01
ꢀ6.93
ꢀ6.82
ꢀ6.79
ꢀ6.72
ꢀ6.46
1.69
2.18
3.00
- Among (Z)-polymethoxy stilbenes, 3,5,4 -trimethoxystilbene
(
4) is confirmed to be the most active compound by far. This
result is also in perfect agreement with the data of Schneider
M versus SW480 cells
2
a
3.16
et al. [19], who reported an IC50 ¼ 0.23
m
4.66
4.90
5.80
7.23
7.23
8.27
9.96
10.48
11.79
18.30
for 4. This is partly in agreement with our docking results,
indicating that compound 4 integrated well within the tubulin
hydrophobic pocket but with a lower score than compounds 6,
4
10, and 12. Nevertheless, 6 was (relative to 4) scarcely hydro-
phobic (see calculated log D values) and this could explain the
difference between tubulin fitting and in vitro activity: scarcely
hydrophobic compounds (like resveratrol itself) could not
reach the hydrophobic pocket of tubulin, regardless of their
docking “score”. Compound 4 was also more hydrophobic than
1
3
a
An experimental value of 4.00 is reported. See Schneider et al. [19].
10 and 12, and this is probably an important factor to target the
tubulin site. Both compounds 6 and 10 were hydroxylated at C-
2 and this structural detail seems important for tubulin fitting,
but is probably an unfavorable factor in bioassays, perhaps
because of easier metabolic conversion or air oxidation. It is
known from the literature that 1 has a rapid metabolic
conversion [31]. Compound 12 is probably in an intermediate
situation, i.e., it has less tubulin affinity but it may be less
sensitive to degradation.
integrated into the colchicine site of tubulin. Moreover, compounds
, 7, and 9 showed a binding affinity comparable to that of
1
compounds 14 and 8; these data point out that the efficiency in the
interaction with the receptor site, alone, is not sufficient to eluci-
date the anticancer potency, and many other factors, such as
absorption, distribution, and metabolism, must be considered
especially important in this case. However, some conclusions can
be made from the docking results. All docked structures of (Z)-
isomers, with the exception of compound 2, which is moved down,
and compound 8, which is overturned, substantially overlap the
docked structure of combretastatin A-4, taken as reference
- Compounds 8 and 14 were in a similar range of activity, which
was partly confirmed by the docking data (Table 3). Analo-
gously, the group of (E)-polymethoxy stilbenes with lower
activity (3, 5, 7,11, 13; experimental IC50 values: 42e100
not fit well with the tubulin pocket (calculated IC50 values:
7e18 M).
mM) do
(Fig. 4A). Moreover, compounds 6 and 14 collocated the 3,5-
dimethoxy groups of the A-ring in the same position of the corre-
sponding combretastatin A-4 rings with the hydroxylic moiety,
which engages a hydrogen bond with Ser B132 (not shown);
conversely, compound 10, which possesses the best affinity, has
two m-methoxy groups in both the A- and B-rings so it could be
arranged into two possible orientations with respect to com-
bretastatin A-4. The best arrangement results from the B-ring
overlapping with the A-ring of compound 15, as shown in Fig. 4B. In
this position, compound 10 establishes six hydrogen bonds with
m
4. Discussion
We compared two series of molecules starting from (E)-
stereoisomer and its (Z)-counterpart. It must be noted that to date
the use of (Z)-resveratrol has not been reported. Some of the
compounds tested demonstrate much more potencies than the
natural parent molecule. Compared with (E)-resveratrol, which
leads to a cell growth arrest in S phase, the methylated derivatives
0
0
the tubulin receptor: 3 -methoxyl with the Cys B238 sulfhydryl, 5 -
methoxyl with the Ser A132 hydroxyl, 2-hydroxyl with both
carbonyl of Thr A133 and Asn B255, and 3-methoxyl with the Lys
A67 ammonium and with Ala A134 amidic hydrogen (Fig. 4B).
Finally, Fig. 4C shows how all (E)-isomers overlap compared with
combretastatin A-4. Also, in this case all compounds are sufficiently
aligned between them but not with combretastatin, with the
exception of compounds 3 and 5, which, possessing only one
methoxyl in the B-ring, prefer to interact with the hydrophobic
channel that joins the A and B subunits protruding to the A
subunits. Interestingly, the three compounds with the 2-hydroxyl
group are almost perfectly overlapping with the B-ring of the
reference compound combretastatin.
stop cell proliferation by inducing G
ploidization of the SW480 cell line. (E)-resveratrol derivatives also
induce cancer cell apoptosis since sub-G peaks were found during
2
/M failures and also a poly-
1
the flow cytometry analysis. Cell polyploidization, which can occur
naturally to repair damaged DNA, is induced by all of the methyl-
0
ated compounds. (Z)-3,5,4 -trimethoxystilbene (4) has been
described as an inhibitor of tubulin polymerization [31]. Inhibition
leads to mitosis defect and cytokinesis impairment. The destiny of
tetraploidy-induced cells is unknown; these cells could still
proliferate and become more resistant [32] or die by mitotic
catastrophe [33].
The study of synthetic (E)-resveratrol derivatives could offer
a wide range of compounds that are potentially more active than
(E)-resveratrol (about 66-fold for 4), but these molecules seem to
have a different way of delaying cancer cell growth.
The comparison between binding models of (E)- and (Z)-
resveratrol isomers with regard to tubulin and cell proliferation
inhibition potencies is presented in Table 3, with the following
results:
Our team previously tested similar resveratrol analogs on
various tumor cell lines: DU145 (androgen-nonresponsive human
prostate cancer), LNCaP (androgen-responsive human prostate
cancer tumor), M14 (human melanoma), and KB (human mouth
epidermoid carcinoma) [15]. In all cell lines, results indicate
-
Almost all the (Z)-polymethoxy stilbenes were more active
than the (E)-polymethoxy stilbenes. One exception may occur
for compound 9, but the difference is within the SD range of
compound 6. Conversely, (E)-resveratrol (1) is significantly
more active than (Z)-resveratrol (2) notwithstanding that both
showed a similar affinity for the tubulin pocket (similar IC50
values calculated from docking). These data are in agreement
0
a stronger effect of (E)-3,5,4 -trimethoxystilbene (3) than (E)-
resveratrol, especially toward DU145 cells. The corresponding (Z)-
trimethoxy analog 4 was very active toward the KB cell line but
with a poor effect on M14 cells.

2978
F. Mazué et al. / European Journal of Medicinal Chemistry 45 (2010) 2972e2980
Szekeres’s group [34] reported on the influence of several
E)-resveratrol analogs on HT29 human colon cancer cell
(
proliferation inhibition and apoptosis, and some results are
similar to those obtained by our group, i.e., the poor effect of
0
0
(
E)-3,5,3 ,4 -tetramethoxystilbene (11) and blockade on G
0
-G .
1
0
Other results are conflicting; a strong effect of (E)-3,5,4 -trime-
thoxystilbene (3) on HT29 cells and a weaker effect on SW480
cells (our data). More generally, methylated resveratrol analogs,
although nonantioxidant molecules, have a stronger effect than
the parent molecule. Indeed, they inhibit the human tumor
necrosis factor alpha-induced activation of transcription factor
nuclear factor kappa B [35].
In summary, while (E)-resveratrol is considered to be
a promising molecule for fighting cancer [36], synthetic resver-
atrol analogs could offer a wide range of compounds that are
potentially more active than (E)-resveratrol. These molecules
seem to have a different way of delaying cancer cell growth.
Resveratrol inhibits cells in S phase, while most of the other
synthetic derivatives stop mitosis or block it in an unknown
manner (7, 9, 11, 13). We can consider that these methylated
derivatives, which are prevented from any hydroxyl group-
conjugation dependency, would be less metabolized than
resveratrol and potentially more bioavailable. Indeed, we have
observed (unpublished results) limited metabolism of 4 after
incubation with the SW480 cell line. Moreover, recent results
from Lin and Ho [37,38] are in agreement with this statement,
0
since they reported that the pharmacokinetics of (E)-3,5,4 -tri-
0
0
methoxystilbene (3) and (E)-3,5,3 4 -tetramethoxystilbene (11) in
rat plasma are much slower than those of (E)-resveratrol, i.e.,
greater plasma exposure, longer elimination exposure, and lower
clearance. In addition, the stronger effect of (Z)-methoxy deriva-
tives with respect to their (E)-isomers is not related to the lack of
an antioxidative effect (disappearance of hydroxyl groups) but is
probably due to a steric-dependent mechanism leading to inter-
ference in different pathways as compared to the trans
derivatives.
We learned of the recent work of Li et al. [39], while our
work was just being completed and submitted. This work
reports the effect of a pentamethoxy resveratrol derivative and
the analysis of its apoptotic properties. Although from our
respective analogs the (Z)-methoxy derivatives have both
microtubule targets, our present paper does not have the same
objective since our goal was the analysis of a structureefunction
relationship using a large series of analog (E)- and (Z)-deriva-
tives, especially by combining experimental data and an original
docking approach. Moreover, in our cell model and molecules
we did not detect a strong apoptosis but a strong polyploidy
Fig. 4. Computational studies and binding model of (E)-and (Z)-stilbenes isomers. For part
A and C, the binding models for all stilbenes were constructed using the combretastatin
model [25] as a template and reference ligand in the binding site. Computational docking
was carried out applying the Lamarckian genetic algorithm (LGA) implemented in Auto-
Dock 4.0 [29]. For fine docking, we used the following parameters: grid spacing ¼ 0.261 Å,
number of runs ¼ 100, npts ¼ 50-60-50 centered on combretastatin A-4, ga_num_
evals ¼ 20,000,000, ga_pop_size ¼ 150, and ga_num_generations ¼ 27,000. A. Superpo-
sition of all (Z)-stilbene analogues docked at the colchicine-binding site. The docked
structure of combretastatin A-4, represented in orange (tube rendering), is inserted for
comparison. Only polar hydrogens are represented, for clarity. Ligands are rendered as
sticks with the subsequent colour code: 2 yellow, 4 red, 6 green, 8 blue, 10 brown, 12
magenta and 14 cyan.B. Moleculardockingresultofcompound10 at the colchicine-binding
site oftubulin.Only theaminoacidresidues within 4.5 Å around theinhibitorare shownfor
clarity. The ligand is represented with the carbon skeleton in brown with the only polar
hydrogen. Dotted lines represent hydrogen bonds between ligand and receptor. The
numerotation of amino acid residues is decremented by 3 unit for subunit A and 56 for
subunit B respect to the original PDB file due to the strong manipulation and multiple file
format conversions experienced. C. Superposition of all (E)-stilbene analogues docked at
the colchicine-binding site. The docked structure of combretastatin A-4, represented in
orange (tube rendering), is inserted for comparison. Only polar hydrogens are represented,
forclarity. Ligands are renderedas sticks with thesubsequentcolourcode: 1 yellow, 3 red, 5
green, 7 blue, 9 brown, 11 magenta and 13 cyan. [For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.]
(see Fig. 2).
With regard to the docking work, it is noteworthy that the
adopted procedure, consisting in the extrusion of colchicine from
the tubulin binding site, reconstruction of protein deficiencies
(lacking in amino acid residues, hydrogen bondings, hydrogen
orientations, etc.), restoring the appropriate ligand (com-
bretastatin A-4), molecular mechanic minimization of the whole
system, and, finally, submission to the AutoDock docking
program [40], allowed us to obtain a model that was able to
reproduce the IC50 experimental values of other combretastatin
analogs also reported in the literature with a high grade of
precision that is, to our knowledge, the best obtained to date
using docking methodology. Although the results obtained do
not always coincide with the antitumoral activity, this is probably
attributable to the ADME implications and not to the mechanism
of actions. Then the described procedure results in a new valid
approach for determining a potential tubulin ligand in the
colchicine-binding site.

F. Mazué et al. / European Journal of Medicinal Chemistry 45 (2010) 2972e2980
2979
Table 3
Appendix. Supplementary material
Ranked comparison between binding models of compounds 1e14 to tubulin, cell
proliferation inhibition potencies and log D values.
Supplementary material associated with this article can be
found in the on-line version, at doi:10.1016/j.ejmech.2010.03.024.
Compounda Calculated tubulin Experimental
Calculated log D
b
IC50 range values (m
M)c
IC50
(
mM)
1
6
1
4
2
1
1
9
7
8
3
5
1
1
0
1.69
2.18
3.00
3.16
4.66
4.90
5.80
7.23
7.23
8.27
9.96
10.48
11.79
18.30
5e9
3.88
3.56
3.98
4.32
3.02
3.02
3.23
3.88
4.64
4.64
4.31
3.56
3.98
3.23
References
16e20
7.5e11.5
0.26e0.34
78e102
17e23
8e12
15e19
41e55
11e15
46e62
37e49
85e115
39e45
2
[
1] M. Jang, L. Cai, G.O. Udeani, K.V. Slowing, C.F. Thomas, C.W. Beecher, et al.,
Cancer chemopreventive activity of resveratrol, a natural product derived
from grapes. Science 275 (1997) 218e220.
[
2] D. Delmas, P. Passilly-Degrace, B. Jannin, M.C. Malki, N. Latruffe, Resveratrol,
a chemopreventive agent, disrupts the cell cycle control of human SW480
colorectal tumor cells. Int. J. Mol. Med. 10 (2002) 193e199.
4
[3] D. Delmas, B. Jannin, M.C. Malki, N. Latruffe, Inhibitory effect of resveratrol on
the proliferation of human and rat hepatic derived cell lines. Oncol. Rep. 7
(2000) 847e852.
[4] D. Delmas, C. Rebe, S. Lacour, R. Filomenko, A. Athias, P. Gambert, et al.,
Resveratrol-induced apoptosis is associated with Fas redistribution in the rafts
and the formation of a death-inducing signaling complex in colon cancer cells.
J. Biol. Chem. 278 (2003) 1482e1490.
[5] D. Delmas, C. Rebe, O. Micheau, A. Athias, P. Gambert, S. Grazide, et al.,
Redistribution of CD95, DR4 and DR5 in rafts accounts for the synergistic
toxicity of resveratrol and death receptor ligands in colon carcinoma cells.
Oncogene 23 (2004) 8979e8986.
1
3
a
b
c
Compounds are listed in decreasing order of calculated tubulin IC50 values.
IC50 values calculated from docking and referred to tubulin inhibition.
Range values determined on the basis of SW480 cell growth IC50 values ꢂ SD.
[
6] S. Fulda, K.M. Debatin, Sensitization for anticancer drug-induced apoptosis by
the chemopreventive agent resveratrol. Oncogene 23 (2004) 6702e6711.
7] A.R. Jazirehi, B. Bonavida, Resveratrol modifies the expression of apoptotic
regulatory proteins and sensitizes non-Hodgkin’s lymphoma and multiple
myeloma cell lines to paclitaxel-induced apoptosis. Mol. Cancer Ther. 3 (2004)
5
. Conclusions
[
1
- The relatively weak effect of (E)-RSV is not exclusively attrib-
utable to its high metabolic rate and efflux, since the masking of
hydroxyl groups by methylation does not significantly improve
the resveratrol analog efficacy;
71e84.
[8] I. Zoberi, C.M. Bradbury, H.A. Curry, K.S. Bisht, P.C. Goswami, J.L. Roti Roti, et
al., Radiosensitizing and anti-proliferative effects of resveratrol in two human
cervical tumor cell lines. Cancer Lett. 175 (2002) 165e173.
2
- The Z configuration does not enhance resveratrol efficacy since its
potency is weaker than that of the isomer with the E configuration;
- In contrast to the preceding statement (#2), the Z configuration
associated with the substitution of hydroxyl groups by methoxyl
groups is crucial and leads to potent inhibition efficacy;
- Following the preceding statement (#3) the strong efficacy of
[
9] X. Xu, H.J. Wang, P.A. Murphy, L. Cook, S. Hendrich, Daidzein is a more
bioavailable soymilk isoflavone than is genistein in adult women. J. Nutr. 124
(1994) 825e832.
3
[
10] P.C. Hollman, J.M. van Trijp, M.N. Buysman, M.S. van der Gaag, M.J. Mengelers,
J.H. de Vries, et al., Relative bioavailability of the antioxidant flavonoid
quercetin from various foods in man. FEBS Lett. 418 (1997) 152e156.
[11] M.J. Lee, Z.Y. Wang, H. Li, L. Chen, Y. Sun, S. Gobbo, et al., Analysis of plasma
and urinary tea polyphenols in human subjects. Cancer Epidemiol. Biomarkers
Prev. 4 (1995) 393e399.
12] D. Colin, A. Gimazane, G. Lizard, J.C. Izard, E. Solary, N. Latruffe, et al., Effects of
resveratrol analogs on cell cycle progression, cell cycle associated proteins
and 5fluoro-uracil sensitivity in human derived colon cancer cells. Int. J.
Cancer 124 (2009) 2780e2788.
13] D. Colin, A. Lancon, D. Delmas, G. Lizard, J. Abrossinow, E. Kahn, et al., Anti-
proliferative activities of resveratrol and related compounds in human
hepatocyte derived HepG2 cells are associated with biochemical cell distur-
bance revealed by fluorescence analyses. Biochimie 90 (2008) 1674e1684.
14] A.K. Marel, G. Lizard, J.C. Izard, N. Latruffe, D. Delmas, Inhibitory effects of
trans-resveratrol analogs molecules on the proliferation and the cell cycle
progression of human colon tumoral cells. Mol. Nutr. Food Res. 52 (2008)
4
0
the (Z)-3,5,4 -trimethoxystilbene 4 would also be associated
with the molecule’s poor cell metabolism;
[
[
[
5
- The presence of more than three methoxyl groups on the (Z)-
resveratrol core structure does not enhance resveratrol analog
efficacy but rather gives it a weaker effect;
- The presence of a new hydroxyl group on tri-methylated (Z)-
resveratrol analogsdramatically decreases the inhibitory potency;
- Concerning the inhibitory mechanism, the presence of
methoxyl groups, whichever resveratrol isomer (E)-, (Z)- is
considered, induces cell polyploidy resulting from the blockade
of cell divisions at the mitosis level;
6
7
538e548.
[
15] V. Cardile, R. Chillemi, L. Lombardo, S. Sciuto, C. Spatafora, C. Tringali, Anti-
proliferative activity of methylated analogues of E- and Z-resveratrol. Z.
Naturforsch. C 62 (2007) 189e195.
16] M. Roberti, D. Pizzirani, D. Simoni, R. Rondanin, R. Baruchello, C. Bonora, et al.,
Synthesis and biological evaluation of resveratrol and analogues as apoptosis-
inducing agents. J. Med. Chem. 46 (2003) 3546e3554.
[17] G.R. Pettit, M.P. Grealish, M.K. Jung, E. Hamel, R.K. Pettit, J.C. Chapuis, et al.,
Antineoplastic agents. 465. Structural modification of resveratrol: sodium
resverastatin phosphate. J. Med. Chem. 45 (2002) 2534e2542.
18] R. Chillemi, S. Sciuto, C. Spatafora, C. Tringali, Anti-tumor properties of stil-
bene-based resveratrol analogues: recent results. Nat. Prod. Commun. 2
8
- The relationship between spatial conformations of resveratrol
analogs and their effect fits quite well with docking studies where
almost all of the docked structures of (Z)-polymethoxy isomers
overlap substantially with the docked structure of combretastatin
A-4 (15), taken as reference ligand, to bind at the colchici-
neetubulin binding site, while mostof the (E)-polymethoxy isomer
counterparts fit between them but not with combretastatin.
[
[
[
[
(2007) 1e15.
19] Y. Schneider, P. Chabert, J. Stutzmann, D. Coelho, A. Fougerousse, F. Gosse, et
Conflict of interest statement
The authors have no conflict of interest to declare.
0
al., Resveratrol analog (Z)-3,5,4 -trimethoxystilbene is a potent anti-mitotic
drug inhibiting tubulin polymerization. Int. J. Cancer 107 (2003) 189e196.
20] C. Spatafora, G. Basini, L. Baioni, F. Grasselli, A. Sofia, C. Tringali, Antiangiogenic
resveratrol analogues by mild m-CPBA aromatic hydroxylation of 3,5-dime-
thoxystilbenes. Nat. Prod. Commun. 4 (2009) 239e246.
[
21] M. Deak, H. Falk, On the chemistry of the resveratrol diastereomers. Monatsh.
Chem. 134 (2003) 883e888.
Acknowledgements
[22] Atsuya Momotake, Mayuko Ud, Tatsuo Arai, Photochemical cyclisation reac-
tion in cis-tetramethoxystilbene. J. Photochem. Photobiol. A Chem. 158 (2003)
This study was supported by BIVB, the Regional Council of
Burgundy, the Cancer League of Côte d’Or and Jura, INSERM U866,
CNRS, UMR 5260, MIUR, Ministero dell’Università e della Ricerca
7
e12.
[
[
23] M. Gao, M. Wang, K. Miller, G. Sledge, G. Hutchins, Q. Zhang, Synthesis of
radiolabelled stilbene derivative a new potential PET probe for arylhy-
drocarbon receptor in cancer. Bioorg. Med. Chem. Lett. 16 (2006) 5767e5772.
24] D. Koh, K.H. Park, J. Jung, Mok KH Yang, Y. Lim, Spectral assignments and
reference data NMR spectra of resveratrol derivatives complete assignment of
(
PRIN 2007, Rome, Italy), and Università degli Studi di Catania, (PRA,
Catania, Italy). We thank Mrs. Linda Northrup for the improvement
of English.
1
13
the H and C. Magn. Reson. Chem. 39 (2001) 768e770.

2980
F. Mazué et al. / European Journal of Medicinal Chemistry 45 (2010) 2972e2980
[
25] N.H. Nam, Combretastatin A-4 analogues as antimitotic antitumor agents.
Curr. Med. Chem. 10 (2003) 1697e1722.
26] R.B. Ravelli, B. Gigant, P.A. Curmi, I. Jourdain, S. Lachkar, A. Sobel, et al., Insight
into tubulin regulation from a complex with colchicine and a stathmin-like
domain. Nature 428 (2004) 198e202.
27] T.L. Nguyen, C. McGrath, A.R. Hermone, J.C. Burnett, D.W. Zaharevitz, B.W. Day,
et al., A common pharmacophore for a diverse set of colchicine site inhibitors
using a structure-based approach. J. Med. Chem. 48 (2005) 6107e6116.
28] S.C. Lovell, I.W. Davis, W.B. Arendall 3rd, P.I. de Bakker, J.M. Word, M.
G. Prisant, et al., Structure validation by Calpha geometry: phi, psi and Cbeta
deviation. Proteins 50 (2003) 437e450.
[34] P. Saiko, M. Pemberger, Z. Horvath, I. Savinc, M. Grusch, N. Handler, et al.,
Novel resveratrol analogs induce apoptosis and cause cell cycle arrest in HT29
human colon cancer cells: inhibition of ribonucleotide reductase activity.
Oncol. Rep. 19 (2008) 1621e1626.
[35] J.J. Heynekamp, W.M. Weber, L.A. Hunsaker, A.M. Gonzales, R.A. Orlando, L.
M. Deck, et al., Substituted trans-stilbenes, including analogues of the natural
product resveratrol, inhibit the human tumor necrosis factor alpha-induced
activation of transcription factor nuclear factor kappaB. J. Med. Chem. 49
(2006) 7182e7189.
[
[
[
[36] J.A. Baur, D.A. Sinclair, Therapeutic potential of resveratrol: the in vivo
evidence. Nat. Rev. Drug Discov. 5 (2006) 493e506.
[37] H.S. Lin, P.C. Ho, A rapid HPLC method for the quantification of 3,5,4 -trime-
0
[29] J. Gasteiger, M. Marsili, A new model for calculating atomic charges in
molecules. Tetrahedron Lett. 34 (1978) 3181e3184.
thoxy-trans-stilbene (TMS) in rat plasma and its application in pharmacoki-
netic study. J. Pharm. Biomed. Anal. 49 (2009) 387e392.
[
30] W. Cornell, P. Cieplak, C. Bayly, I. Gould, K. Merz Jr., D. Ferguson, et al., A
second generation force field for the simulation of proteins, nucleic acids, and
organic molecules. J. Am. Chem. Soc. 117 (1995) 3e13.
31] N. Seiler, Y. Schneider, F. Gosse, R. Schleiffer, F. Raul, Polyploidisation of meta-
static colon carcinoma cells by microtubule and tubulin interacting drugs: effect
on proteolytic activity and invasiveness. Int. J. Oncol. 25 (2004) 1039e1048.
32] J. Erenpreisa, M. Kalejs, M.S. Cragg, Mitotic catastrophe and endomitosis in
tumour cells: an evolutionary key to a molecular solution. Cell Biol. Int. 29
[38] H.S. Lin, C. Tringali, C. Spatafora, C. Wu, P.C. Ho, A simple and sensitive HPLC-UV
0
0
methodforthequantificationofpiceatannolanalog3,5,3 ,4 -tetramethoxy-trans-
stilbeneinratplasmaanditsapplicationforapre-clinicalpharmacokineticstudy.
J.Pharm.Biomed.Anal.51(2010)679e684.
[
[
[
[39] H. Li, W.K. Wu, Z. Zheng, C.T. Che, L. Yu, Z.J. Li, Y.C. Wu, K.W. Cheng, J. Yu, C.
0
0
0
H. Cho, M. Wang, 2,3 ,4,4 ,5 -Pentamethoxy-trans-stilbene,
a resveratrol
derivative, is a potent inducer of apoptosis in colon cancer cells via targeting
microtubules. Biochem. Pharmacol. 78 (2009) 1224e1232.
(
2005) 1012e1018.
33] S. Mansilla, W. Priebe, J. Portugal, Mitotic catastrophe results in cell death by
caspase-dependent and caspase-independent mechanisms. Cell Cycle
2006) 53e60.
[40] G. Morris, D. Goodsell, R. Halliday, R. Huey, W. Hart, R. Belew, et al., Auto-
mated docking using a Lamarckian genetic algorithm and an empirical
binding free energy function. J. Comput. Chem. 19 (1998) 1639e1662.
5
(