Synthesis and evaluation of a series of resveratrol analogues as potent anti-cancer agents that target tubulin

Article abstract of DOI:10.1039/c4md00478g

A series of novel diarylacrylonitrile and trans-stilbene analogues of resveratrol has been synthesized and evaluated for their anticancer activities against a panel of 60 human cancer cell lines. The diarylacrylonitrile analogues 3b and 4a exhibited the m

Full text of DOI:10.1039/c4md00478g

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Medicinal Chemistry Communications
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DOI: 10.1039/C4MD00478G
Synthesis and evaluation of a series of resveratrol analogues as potent anti-
cancer agents that target tubulin
Nikhil R. Madadi,1 Hongliang Zong,2 Amit Ketkar,3 Chen Zheng,1 Narsimha R. Penthala,1 Venumadhav Janganati,1
Shobanbabu Bommagani,1 Robert L. Eoff,3 Monica L. Guzman,2 Peter A. Crooks.1*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: xxxxx
A series of novel diarylacrylonitrile and trans-stilbene analogues of resveratrol has been synthesized and evaluated for their anticancer
activities against a panel of 60 human cancer cell lines. The diarylacrylonitrile analogues 3b and 4a exhibited the most potent anticancer
activity of all the analogues synthesized in this study, with GI50 values of < 10 nM against almost all the cell lines in the human cancer cell
panel. Compounds 3b and 4a were also screened against the acute myeloid leukemia (AML) cell line, MV4-11, and were found to have
potent cytotoxic properties that are likely mediated through inhibition of tubulin polymerization. Results from molecular docking studies
indicate a common binding site for 4a and 3b on the α,β-tubulin heterodimer, with a slightly more favorable binding for 3b compared to 4a;
this is consistent with the results from the microtubule assays, which demonstrate that 4a is more potent than 3b in inhibiting tubulin
polymerization in MV4-11 cells. Taken together, these data suggest that diarylacrylonitriles 3b and 4a may have potential as antitubulin
therapeutics for treatment of both solid and hematological tumors.
Introduction
Intriguing results have been reported with the O-methylated
Chemicals that perturb microtubule and tubulin dynamics are
widely used in cancer chemotherapy.1 Taxane, vinca and colchicine
domains on tubulin are the major binding sites for anti-cancer drugs
such as paclitaxel, docetaxel, cabazitazel, vincristine, vinblastine
and colchicine.2 The mode of action of these drugs is via inhibition
of tubulin polymerization or by preventing depolymerization of
tubulin, resulting in mitotic arrest. Lately, anti-mitotic agents that
bind to the colchicine domain on tubulin have received much
attention, and drugs such as combretastatin A-4 (CA-4) (Fig. 1;
structure A) and its analogues have been extensively studied for
their anti-cancer activity.3-6 CA-4, a cis-stilbene analogue, has been
isolated from the South African native willow tree, Combretum
caffrum, and is a potent anti-proliferative agent.7 CA-4 inhibits the
polymerization of tubulin by interacting with the colchicine binding
site on tubulin, resulting in mitotic arrest. CA-4 is also a promising
anti-angiogenic molecule, and its water soluble phosphate salt is
currently in phase III clinical trials for treatment of anaplastic
thyroid cancer.8
resveratrol analogue (E)-3,4,5,4’-tetramethoxystilbene (Fig. 1;
structure C). This compound was found to potently inhibit the
proliferation of a variety of cancer cells, including HeLa cervical
cancer cells, MCF-7 and MDA-MB-435/LCC6 breast cancer cells,
HepG2 hepatoma cells, LnCaP prostate cancer cells and HT-29
colon cancer cells.12 Recently, our laboratory has reported on some
novel O-methylated resveratrol substrates for human hepatic,
intestinal, and renal UDP-glucuronosyl transferases. These
compounds were found to have increased bioavailability via altered
conjugation, and were considered as alternative scaffolds for the
development of new bioactive resveratrol analogues.13
More recently, we have reported on
a series of (Z)-
benzothiophene acrylonitrile derivatives of resveratrol (Fig. 1;
structure D); these compounds are potent anti-cancer agents which
are not substrates for cellular P-glycoprotein.14c In this respect,
Ohsumi et al. have reported that (E)-substituted diarylacrylonitrile
analogues structurally related to the combretastatins (Fig. 1;
structure E) are also effective anti-cancer agents against murine
solid tumors.15
The structurally related trans-stilbene analogue, resveratrol is a
phytolexin first isolated from white hellebore (Veratrum
grandiflorum O. Loes) and from about 70 other plant species.9
Resveratrol has been reported as a potential chemotherapeutic
agent, due to its striking inhibitory effects on cellular events
associated with cancer initiation, promotion, and progression.10
Chen and co-workers have synthesized and evaluated a series of
methoxylated resveratrol derivatives for their anti-cancer properties
against three different human cancer cell lines, i.e. K562, HT29,
and HePG2. They reported that (E)-3,5,4’-trimethoxystilbene (Fig.
1; structure B) and (E)-3,4,5,4’-tetramethoxystilbene (Fig. 1;
structure C) were the most effective anti-cancer agents among the
analogues investigated, and showed improved cytotoxicity
compared to resveratrol itself.11
_____________________________________________________
1Department of Pharmaceutical Sciences, College of Pharmacy, and 3Department of
Biochemistry and Molecular Biology, College of Medicine, University of Arkansas for
Medical Sciences, Little Rock, AR 72205, USA; 2Division of Hematology and
Medical Oncology, Department of Medicine, Weill Medical College of Cornell
University, New York, NY.
*Corresponding author, E-mail: pacrooks@uams.edu; Fax: +1-501-686-6057; Tel:
+1-501-686-6495.
Fig. 1 Structures of combretastatin A-4 and resveratrol related anticancer
agents.

Medicinal Chemistry Communications
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DOI: 10.1039/C4MD00478G
In our continuing quest to improve the potencies of natural anti-
cancer molecules through chemical modification, we have now
synthesized a small library of trans-stilbenes and related (Z)-
diarylacrylonitrile analogues structurally related to resveratrol, and
have evaluated them against a panel of 60 human tumor cell lines
and against MV-411 acute myeloid leukemia (AML) cells for their
anti-cancer activity. In these molecules, the trans-stilbene structural
moiety present in resveratrol have been modified by introducing a
cyano group at one of the sp2 carbons of the stilbene double bond,
and a variety of different aromatic substituents have been
introduced into the phenyl rings to improve anticancer activity. We
also describe the tubulin binding properties of the most potent of
these novel trans-stilbene analogues in comparison to the tubulin
binding properties of the corresponding cis-stilbene analogue.
inhibited the growth of cancer cell lines HepG2, B16-F10, and
A549 with IC50 values of 0.2, 0.1, and 1.4 ꢀg/mL, respectively.16 In
view of this finding, and the potent anticancer activity of the aryl-
substituted acrylonitrile analogues of structure E (Fig. 1), a series
of hybrid resveratrol derivatives possessing a phenylacrylonitrile
moiety attached to the C2-position of the (E)-3,5,4’-
trimethoxystilbene scaffold were investigated. The synthetic
strategy for preparing such compounds is given in Scheme 3. The
synthetic procedure for synthesizing the key intermediate 5
(Scheme 3) involves O-methylation of the hydroxyl groups of
resveratrol with MeI/K2CO3 in acetone, to form (E)-1,3-dimethoxy-
5-(4-methoxystyryl)-benzene, followed by C2-formylation with a
0
slight molar excess of POCl3 in DMF at 0 C for 30 min, to yield
trans-2-formyl-3,4′,5-trimethoxystilbene (5).17,18
With intermediate 5 in hand, a novel series of (Z)-3-(2,4-
dimethoxy-6-(4-methoxystyryl)phenyl)-2-phenylacrylonitrile
analogues (7a-7g) could be synthesized by reacting intermediate 5
with appropriately substituted phenylacetonitriles (6a-6d, 1a-1c) in
5% sodium methoxide/methanol. The reaction mixture was stirred
at room temperature for 2-3 h to allow the reaction to go to
completion, during which time the desired product was
precipitated. The resulting solid was filtered off, washed with water
and dried to yield the desired compound in yields ranging from 55-
80% (Scheme 3). Confirmation of the structure and purity of these
analogues was obtained from 1H, 13C-NMR and mass spectroscopic
analysis. According to previous literature, base-catalyzed
condensation of aryl/heteroaryl aldehydes with aryl/heteroaryl
acetonitriles leads exclusively to the formation of the (Z)-
isomer.14a-14c We confirmed the (Z) configuration of analogues 7a-
7g by carrying out carbon-proton coupling experiments to
determine the magnitude of the coupling constant (JCH) of the CN
carbon doublet that results from coupling with the adjacent olefinic
proton.14b
Chemistry
A series of (Z)-substituted diarylacrylonitrile analogues (3a-3h)
were synthesized by reacting a variety of substituted benzyl
carbaldehydes (2a-2e) with an appropriately substituted
phenylacetonitrile (1a-1d) in 5% sodium methoxide/methanol. The
reaction mixture was stirred at room temperature for 2-3 h to allow
the reaction to go to completion, during which time the desired
product crashed out of the solution. The resulting precipitate was
filtered, washed with water and dried to yield the desired
compound in yields ranging from 70-95% (Scheme 1).
Scheme 1 Synthesis of (Z)-substituted diarylacrylonitrile analogues (3a-
3h).
(E)-Substituted diarylacrylonitrile analogues 4a and 4b were
obtained by refluxing a methanolic solution of the (Z)-isomers 3b
and 3c in under ultraviolet light at 254 nm for 24 h. The time
course of the reaction was monitored by GC-MS. Once the reaction
was complete, the solution was cooled to room temperature and the
resulting precipitate was filtered off to yield the (E)-substituted
diarylacrylonitrile analogues 4a and 4b (Scheme 2).
Scheme 3 Synthesis of (Z)-3-(2,4-dimethoxy-6-(4-methoxystyryl)-2-phe-
nylacrylonitrile analogues (7a-7g). (a) MeI, K2CO3, acetone; (b) POCl3,
DMF, 0 oC, 69% yield; (c) NaOMe, EtOH, 6h reflux.
Scheme 2 Synthesis of (E)-substituted diarylacrylonitrile analogues (4a and
4b).
Biological evaluation
In related studies, Ruan et al. have reported the antitumor
activity of resveratrol derivatives possessing a chalcone moiety
(Fig. 1; structure F); these analogues exhibited potent anti-
proliferative and antitubulin activities, and compound F (Fig. 1)
A In vitro growth inhibition studies
Primary in vitro screening of all the synthesized compounds was
carried out against a panel of 60 human tumor cell lines utilizing

Page 3 of 8
Medicinal Chemistry Communications
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DOI: 10.1039/C4MD00478G
the sulforhodamine B (SRB) assay procedure described by
average GI50 values of the E/Z pair of isomers 4b and 3c were
similar (177 nM and 223 nM, respectively; Table 1), while the
other pair of E/Z isomers 3b and 4a were both found to be the most
potent compounds in this series from the five dose study data, with
GI50 values of < 10 nM against almost all the NCI human cancer
cell lines examined. Importantly, compounds 3b and 4a were
significantly more effective against the growth of several cancer
cell lines when compared to CA4 (Table 1). These include non-
small cell lung cancer A549/ATCC, colon cancer HCC-2998,
ovarian cancer (IGROV1, IGROV4, SK-OV-3), renal cancer (786-
0, UO-31) cell lines (Table 1).
Rubinstein et al.19,20 Compounds 3a-3h were initially screened at
10-5 M concentration to determine growth inhibition properties.
Only compounds that showed more than 60% growth inhibition at
10-5 M in at least eight cell lines from the panel of 60 cell lines
were selected for a complete dose-response study with five
different concentrations, i.e. 10-4 M, 10-5 M, 10-6 M, 10-7 M and 10-
8 M.
In the (Z)-diarylacrylonitrile series of compounds (3a-3h), only
compounds 3a-3d were selected for full dose-response studies
(Table 1). When the 4-methoxyphenyl group on ring B of
compound 3a was replaced with a 3,5-dimethoxyphenyl moiety
(3d), the average GI50 activity declined from 18 nM to 13 µM.
Also, when the 3,4,5-trimethoxyphenyl group on ring A of
compound 3a was replaced with a 3,5-dimethoxyphenyl moiety
(3f) the average GI50 activity declined from 18 nM to 400 nM.
Interestingly, introduction of a 3-hydroxy group into ring B of
compound 3a to afford compound 3b improved the average GI50
value of 18 nM against all the cancer cell lines in the NCI panel to
<10 nM. Comparing the growth inhibition activities of 3d and 3f
with their more active counterparts 3a and 3b suggests that both the
3,4,5-trimethoxy group on ring A and the 4-methoxy-3-hydroxy
group on ring B contribute significantly to the anticancer activity of
the (Z)-diarylacrylonitrile analogues.
In the (Z)-3-(2,4-dimethoxy-6-(4-methoxystyryl)phenyl)-2-
phenylacrylonitrile analogue series (7a-7g), two compounds, 7b
and 7e, showed good anticancer activity in the single dose cancer
cell screen, and were subsequently evaluated in the five dose
testing paradigm. Both compounds were found to be effective
against four particular cancer cell lines: viz. SR, NCI H522, SF-539
and MDA-MB-435 with GI50 values less than 300 nM (Table 1)
.
However, the average GI50 value of compounds 7b and 7e against
all 60 cell lines was only 2.79 and 1.19 µM, respectively.
Compound 7e (GI50 = 0.91 µM) was found to be somewhat more
potent than the previously reported reseveratrol-chalcone molecule
F (Figure 1) (GI50 = 1.40 µM) against non-small lung cancer cell
line A549.16
Interestingly, Ohsumi et al. has previously reported that the (E)-
diarylacrylonitrile analogue 4a potently inhibits the proliferation of
colon 26 cancer cells with an IC50 of 23 nM.15 Thus, (E)-isomers 4a
and 4b (Scheme 3) were synthesized to compare their growth
inhibitory values with those of their Z-counterparts, 3b and 3c. The
Thus, compounds 3b and 4a are the most potent compounds of
all the resveratrol analogues synthesized in this study, with GI50
values of < 10 nM against almost all the cell lines in the human
cancer cell panel.
Table 1 Growth inhibition (GI50)a data for compounds 3a, 3d, 3f, 3c, 4b, 3b, 4a, 7b, 7e and CA4 against a panel of 60 human cancer cell lines
Panel/ Cell line
3a
3d
3f
3c
GI50
( µM )
4b
GI50
( µM )
3b
GI50
( µM )
4a
GI50
( µM )
7b
GI50
( µM )
7e
GI50
( µM )
CA4b
GI50
( µM )
GI50
(µM)
GI50
(µM)
GI50
(µM)
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
0.003
0.002
0.002
0.003
0.003
0.003
0.03
0.02
<0.01
0.03
0.03
<0.01
51.2
68.6
3.14
28.2
6.64
4.14
0.03
0.03
0.02
0.05
0.04
0.02
0.04
0.03
0.03
0.07
0.05
0.03
0.75
1.66
0.43
4.38
1.16
0.91
0.32
0.28
0.31
0.43
0.33
0.29
2.24
0.58
0.43
3.30
2.85
0.28
Non-Small Cell
Lung Cancer
A549
HOP-62
HOP-92
NCI-H23
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
0.01
0.03
0.02
0.05
<0.01
6.35
7.63
3.57
43.0
20.5
0.21
0.03
1.69
0.07
<0.01
0.25
0.03
0.86
0.08
0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
NA
<0.01
<0.01
<0.01
0.91
0.97
NA
NA
0.24
0.016
0.003
0.003
0.003
0.002
0.38
0.51
NA
0.53
0.09
3.56
2.79
NA
NA
0.25
0.01
0.04
5.16
97.0
6.05
4.47
4.24
4.97
5.17
1.24
1.81
0.04
0.04
0.57
0.02
0.04
1.26
0.17
0.03
0.03
0.62
0.04
0.04
2.99
0.02
<0.01
<0.01
3.18
0.30
<0.01
<0.01
<0.01
0.32
1.24
2.49
0.49
0.65
0.69
0.78
0.50
0.100
0.063
0.003
0.004
0.100
0.005
0.003
0.27
0.34
0.33
0.33
0.28
0.38
0.42
2.28
5.47
1.29
0.76
0.48
0.64
0.71
<0.01
<0.01
<0.01
<0.01
0.01
HT29
KM12
SW-620
CNS Cancer
<0.01
<0.01
<0.01
<0.01
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
Melanoma
0.04
<0.01
<0.01
0.06
<0.01
0.02
25.3
5.33
6.60
5.46
2.67
7.82
0.06
0.10
0.02
0.05
0.02
0.04
0.08
0.58
0.02
0.04
0.02
0.05
<0.01
0.05
<0.01
<0.01
<0.01
0.01
<0.01
0.01
<0.01
<0.01
<0.01
<0.01
4.71
0.36
0.27
>100
0.47
0.64
0.006
0.004
0.003
0.004
0.008
0.004
0.84
0.22
0.22
0.98
0.29
0.343
4.18
2.51
1.55
8.77
2.28
2.77
LOX IMVI
0.01
5.31
0.06
0.07
<0.01
<0.01
NA
NA
0.003

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DOI: 10.1039/C4MD00478G
Panel/ Cell line
3a
3d
3f
3c
GI50
( µM )
4b
GI50
( µM )
3b
GI50
( µM )
4a
GI50
( µM )
7b
GI50
( µM )
7e
GI50
( µM )
CA4b
GI50
( µM )
GI50
(µM)
GI50
(µM)
GI50
(µM)
0.58
0.28
0.17
0.88
0.49
0.27
0.40
M14
<0.01
<0.01
<0.01
1.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
0.003
NA
0.004
0.008
0.004
0.006
0.01
<0.01
0.08
NA
0.01
4.45
1.63
5.44
14.0
4.70
2.42
0.03
<0.01
NA
1.11
0.04
0.02
0.02
<0.01
NA
1.27
0.05
NA
1.14
0.28
2.13
4.22
2.44
NA
1.38
0.24
NA
0.75
2.28
0.70
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-62
<0.01
Ovarian Cancer
IGROV1
OVCAR-3
OVCAR-4
NCI/ADR-RES
SK-OV-3
0.02
<0.01
0.01
<0.01
<0.01
14.6
6.69
7.48
3.25
14.4
0.06
0.02
1.56
0.02
0.05
0.07
0.02
1.07
0.02
0.05
<0.01
<0.01
<0.01
<0.01
0.01
<0.01
<0.01
<0.01
<0.01
<0.01
2.35
0.63
0.50
0.65
1.35
0.015
0.002
0.014
NA
0.53
0.30
0.73
0.27
0.37
5.28
1.38
3.59
0.71
4.73
0.059
Renal Cancer
786-0
A498
ACHN
CAKI-1
0.02
<0.01
0.01
NA
<0.01
13.0
1.29
9.78
6.19
1.92
0.36
0.02
0.08
NA
0.24
0.02
0.09
0.42
0.09
<0.01
<0.01
<0.01
0.03
0.02
1.72
0.38
0.83
0.50
0.81
0.100
0.006
0.006
0.026
0.019
0.42
0.25
0.53
0.54
0.49
3.25
1.37
4.25
2.20
3.44
<0.01
<0.01
<0.01
<0.01
UO-31
Breast Cancer
0.06
<0.01
MCF7
MDA-MB-231/ATCC
HS 578T
<0.01
0.03
<0.01
0.02
4.35
4.83
16.7
3.44
0.03
0.04
0.03
0.04
0.03
0.05
0.03
0.04
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
0.39
2.78
0.68
1.72
NA
NA
NA
NA
0.31
0.43
0.31
0.29
0.72
2.83
1.34
1.70
MDA-MB-468
a
NA: Not analyzed, GI50: 50% growth inhibition, concentration of drug resulting in a 50% reduction in net protein increase compared with control
cells. bNCI data for CA4 NSC 613729.3
Fig. 2 Lead compounds 3b, 4a, 3c and 4b exhibit potent anti-leukemia activity against MV-411 cells. MV-411 cells were treated with the indicated
compounds for 24 and 48 h. Cell viability was determined by Annexin V staining. Percent viability was calculated as the percent of Annexin v-/7-AAD- cells
relative to control. N = 5; error bars represent the SD.

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cancer cell lines tested, these molecules were chosen for
molecular docking studies utilizing the available crystal structure
of tubulin, in order to identify their binding sites on this protein.
The crystal structure of the tubulin-colchicine complex was
chosen as the target for docking analysis (PDB ID 1SA0).
Colchicine was removed from the coordinate file and the
coordinates of only chains A and B, corresponding to a α,β-
tubulin heterodimer were used for the docking studies. Atomic
coordinates for all compounds were generated using
MarvinSketch (ChemAxon), and both the ligand and target
protein coordinates files were prepared for docking using the
Dock Prep module in the UCSF-Chimera package23. Compound
3d, which was found to be significantly less potent than either 4a
or 3b in our biological assays (GI50 > 1 ꢀM), was also evaluated.
B In vitro toxicity study on AML cells and tubulin activity
Compounds 3c, 4b, and 3b and 4a were found to be very
effective cytotoxic agents against the leukemia cell sub-panel in
the 60 cancer cell screen (Table 1). Notably, compounds 3b and
4a exhibited GI50 values of < 10 nM across all six leukemia cell
lines. We have also tested the cytotoxicity of these four lead
compounds against MV-411 AML cells (Fig. 2), and have
conducted tubulin binding assays on these compounds in the
same cell line (Fig. 3).
Docking
was
performed
using
SwissDock
(http://www.swissdock.ch/), based on the docking algorithm
EADock DSS.24,25
Fig. 4 Compounds 4a and 3b bound in the colchicine binding site on
tubulin. The most favored docked poses of 3b (magenta) and 4a (yellow)
are shown in panels A and B, respectively, as ball-and-stick models in the
binding site on the α,β-tubulin heterodimer. α-Tubulin residues are shown
as grey sticks, while the β-tubulin residues are cyan. All structural
representations shown in this, and subsequent figures, were generated
using PyMol (DeLano Scientific, San Carlos, CA).
Fig. 3 Microtubule depolymerization assays with lead compounds 3b, 3c,
4a and 4b. P = pellet, S = supernatant.
MV4-11 cells were treated with increasing concentrations of
the above four lead compounds for 24 and 48 hours. Figure 2
shows the dose-response curves for each of the four compounds
at both time points. We found that after 48 hours of drug
treatment 4a was the most potent anti-leukemic compound
causing 50 percent cell death at a concentration of 2.5 nM (Fig.
2a). Compound 3b exhibited an LD50 value of 38.6 nM (Fig. 2b),
and compounds 3c and 4b afforded LD50 values of 353 nM and
409 nM, respectively (Figs. 2c and 2d). These data suggest that
compounds 4a and 3b hold promise as potential treatments for
AML.
We also investigated whether the above four lead compounds
could interfere with microtubule polymerization utilizing an
immunoblot assay.21,22 MV4-11 cells were treated with three
concentrations (25, 50 and 100 nM) of 3b, 3c, 4a and 4b for 2
hours. Cell-based tubulin depolymerization assays were then
performed. The polymerized α-tubulin in the pellets (P) and
unpolymerized α-tubulin in the supernatants (S) were detected by
Western blotting using antibody against α-tubulin. The data
demonstrate that lead compounds 4a and 3b bind to tubulin
directly to inhibit polymerization. Consistent with the superior
anti-leukemic activity observed for 4a over 3b in MV4-11 cells,
4a demonstrated a more potent inhibition of MT polymerization
when compared to 3b (Fig. 3).
Fig. 5 Relative binding of compounds 3b, 4a and 3d in the colchicine
binding site on tubulin. The colchicine-binding pocket on the β-subunit of
tubulin is shown as a solid molecular surface, which accounts for most of
the van der Waals’ interactions with compounds 3b (magenta), 4a
(yellow) and 3d (cyan). The atoms of α-tubulin have been removed for
better clarity. The A-ring moiety of the (Z)-isomer 3b can be seen clearly
projecting out of the binding cavity. In the case of the (E)-isomer 4a, both
A and B rings are placed snugly into the cavity, forming a more extensive
interacting surface with the protein as compared to 3b. Compound 3d,
which is the least potent of the three compounds, is the least buried in the
binding pocket, compared to both 3b and 4a.
C In silico molecular docking studies
Since the (E) and (Z) isomers 4a and 3b were found to be the
most potent compounds (GI50 <10 nM) against almost all the

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Previously, we have successfully used this method for docking
small-molecule inhibitors to human Y-family polymerases.26,27
Several trial docking runs were carried out to test the validity of
our protocol. We consistently obtained a binding mode for
colchicine in our trial docking runs that is essentially identical to
what was reported in the actual crystal structure (data not shown).
Docking with 4a, 3b and 3d was performed using the most
exhaustive and unbiased option in SwissDock, in order to sample
the maximum number of binding modes. The best hits based on
the SwissDock FullFitness scoring function (FF) from three
repeated docking runs were considered further.
through interference with tubulin polymerization. Results from
molecular docking studies indicate a common binding site for 3b
and 4a on the α,β-tubulin heterodimer, with a slightly more
favorable binding for 4a compared to 3b, which is consistent with
the results from microtubule depolymerization assays, which
demonstrate that 4a is more potent than 3b in inhibiting tubulin
polymerization in MV4-11 cells. Taken together, these data
suggest that diarylacrylonitriles 3b and 4a may have potential as
therapeutics for treatment of both solid and hematological tumors.
Acknowledgments
The crystal structure of the tubulin-colchicine complex shows
a single colchicine ligand bound to the β-subunit at the interface
with the α-subunit of each tubulin α,β-heterodimer. Our docking
results indicated that both 3b and 4a bind to the colchicine site in
the tubulin heterodimer (Figure 4). In the crystal structure,
colchicine does not have polar contacts with any residues of
either the α or the β subunit of tubulin. Colchicine binds in the 4-
5 Å wide cavity of its binding site and interacts with the protein
through van der Waals’ interactions with the mostly non-polar
side chains and backbone atoms of tubulin.
The authors thank the NIH/National Cancer Institute
(CA140409 to PAC and CA183895 to RLE), and the Arkansas
Research Alliance for financial support; MLG is funded by the
US National Institutes of Health (NIH) through the NIH
Director's New Innovator Award Program, 1 DP2 OD007399-01.
We are also grateful to the NCI Developmental Therapeutic
Program (DTP) for anticancer screening data.
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Graphical Abstract
Synthesis and evaluation of a series of resveratrol analogues as potent anti-cancer agents that target tubulin
Nikhil R. Madadi, Hongliang Zong, Amit Ketkar, Chen Zheng, Narsimha R. Penthala, Venumadhav Janganati,
Shobanbabu Bommagani, Robert L. Eoff, Monica L. Guzman, Peter A. Crooks