Synthesis and biological evaluation of novel 4,5-disubstituted 2H-1,2,3-triazoles as cis-constrained analogues of combretastatin A-4

Article abstract of DOI:10.1016/j.ejmech.2015.08.041

A series of combretastatin A-4 (CA-4) analogues have been prepared from (Z)-substituted diarylacrylonitriles (1a-1p) obtained in a two-step synthesis from appropriate arylaldehydes and acrylonitriles. The resulting 4,5-disubstituted 2H-1,2,3-triazoles wer

Full text of DOI:10.1016/j.ejmech.2015.08.041

Accepted Manuscript
Synthesis and biological evaluation of novel 4,5-disubstituted 2H-1,2,3-triazoles as
cis-constrained analogues of combretastatin A-4
Nikhil R. Madadi, Narsimha R. Penthala, Kevin Howk, Amit Ketkar, Robert L. Eoff,
Michael J. Borrelli, Peter A. Crooks
PII:
S0223-5234(15)30225-7
10.1016/j.ejmech.2015.08.041
EJMECH 8077
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 June 2015
Revised Date: 14 August 2015
Accepted Date: 22 August 2015
Please cite this article as: N.R. Madadi, N.R. Penthala, K. Howk, A. Ketkar, R.L. Eoff, M.J. Borrelli,
P.A. Crooks, Synthesis and biological evaluation of novel 4,5-disubstituted 2H-1,2,3-triazoles as cis-
constrained analogues of combretastatin A-4, European Journal of Medicinal Chemistry (2015), doi:
10.1016/j.ejmech.2015.08.041.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis and biological evaluation of novel
,5-disubstituted 2H-1,2,3-triazoles as cis-constrained
Leave this area blank for abstract info.
4
analogues of combretastatin A-4
a
a
ab
c
c
b
Nikhil R. Madadi , Narsimha R. Penthala , Kevin Howk , Amit Ketkar , Robert L. Eoff , Michael J. Borrelli
a*
and Peter A. Crooks
a
Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
Department of Radiology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, USA
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, U.S.A.
b
c
2
l: GI50 = < 10.0 nM (against 95% of NCI 60-cell panel)
1
2
2
2
2
d: Ar = 3,4,5-trimethoxyphenyl; Ar = 2-quinolyl; GI50 = < 10 nM (MDA-MB-435, melanoma)
1
2
e: Ar = 3,4,5-trimethoxyphenyl; Ar = 3-methoxy-4-hydtoxyphenyl; LD50 = 7.5 nM (9LSF rat gliosarcoma cells)
1
2
m: Ar = 4-methoxyphenyl; Ar = 3,5-dimethoxyphenyl; GI = < 10 nM (K-562, leukemia; HCT-116, HCT-15, colon cancer;
5
0
M14, MDA-MB-435, melanoma; NCI/ADR-RES ovarian cancer; A498, renal cancer)
1
2
2
h: Ar = 3,4,5-trimethoxyphenyl; Ar = 3,4-ethylenedioxyphenyl; GI50 = < 10 nM (against 36% of NCI 60-cell panel)

ACCEPTED MANUSCRIPT
European Journal of Medicinal Chemistry
journal homepage: www.elsevier.com
Original article
Synthesis and biological evaluation of novel 4,5-disubstituted 2H-1,2,3-triazoles
as cis-constrained analogues of combretastatin A-4
a
a
ab
c
c
Nikhil R. Madadi , Narsimha R. Penthala , Kevin Howk , Amit Ketkar , Robert L. Eoff , Michael J.
b
a*
Borrelli and Peter A. Crooks
Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, USA
Department of Radiology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, USA
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, U.S.A.
a
b
c
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A series of combretastatin A-4 (CA-4) analogues have been prepared from (Z)-substituted
diarylacrylonitriles (1a-1p) obtained in a two-step synthesis from appropriate arylaldehydes and
acrylonitriles. The resulting 4,5-disubstituted 2H-1,2,3-triazoles were evaluated for their anti-cancer
activities against a panel of 60 human cancer cell lines. The diarylacrylonitrile analogue 2l exhibited
the most potent anti-cancer activity in the screening studies, with GI50 values of <10 nM against
almost all the cell lines in the human cancer cell panel and TGI values of <10nM against cancer cell
lines SF-539, MDA-MB-435, OVCAR-3 and A498. Furthermore, in silico docking studies of
compounds 2l, 2e and 2h within the active site of tubulin were carried out in order to rationalize the
mechanism of anti-cancer properties of these compounds. From the in silico studies, compound 2e was
predicted to have better affinity for the colchicine binding site on tubulin compared to compounds 2l
and 2h. Analogue 2e was also evaluated for its anti-cancer activity by colony formation assay against
Available online
Keywords:
Novel 4,5-disubstituted 2H-1,2,3-triazoles
Anti-cancer activity
Gliosarcoma cells
9
LSF rat gliosarcoma cells and afforded an LD50 of 7.5 nM. A cell cycle redistribution assay using
In silico docking studies
Cell cycle redistribution assay
analogue 2e was conducted to further understand the mechanism of action of these CA-4 analogues.
From this study, analogues 2e and 2l were the most potent anti-cancer agents in this structural class,
and were considered lead compounds for further development as anti-cancer drugs.
2009 Elsevier Ltd. All rights reserve.
1
. Introduction
There are three major binding sites for tubulin; i.e. the vinca,
taxane and colchicine domains. Vinca alkaloids, such as
vincristine and vinblastine, bind to the vinca domain inhibiting
the assembly of microtubule structures and arresting mitosis [3].
Paclitaxel acts at the taxane domain stabilizing microtubules and
interfering with the normal breakdown of microtubules during
mitosis [4]. Our area of interest focused on the colchicine binding
site. Colchicine binds to tubulin and inhibits microtubule
polymerization. Anti-mitotic agents such as combretastatin A-4
Cancer is the second most life threatening disease after
cardiovascular disease, affecting more than six million people per
year worldwide. Although, significant research has done to date
to treat cancer, there is still a lack of effective chemotherapeutic
treatment to cure it completely with minimal side effects. Also,
considerable effort has been put into identifying molecules with
anti-cancer properties from both natural and synthetic sources.
More than 60% of the anti-cancer drugs currently available are
from natural sources [1]. The search for potent semi synthetically
derived anti-cancer agents from the parent natural products
continues to be an important part of drug discovery process.
Anti-mitotic agents are a major class of cytotoxic drugs for the
treatment of cancer and drugs that target microtubule/tubulin
dynamics are widely used in cancer chemotherapy [2].
(CA-4) bind at the colchicine domain of tubulin, and have
received much attention in recent years; CA-4P, the water soluble
phosphate salt of CA-4 is currently in phase III clinical trial for
anaplastic thyroid cancer, and is also in phase II trials for
polypoidal choroidal vasculopathy and neovascular age-related
macular degeneration [5, 6].
CA-4 is classified as a cis-stilbene originating from the South
African willow tree combretum caffrum. CA-4 functions as a
microtubule targeting agent interfering with microtubule
dynamics and perturbs the mitotic cycle [7]. When compared to
colchicine, the vascular disrupting effects of CA-4 are well below
the maximum tolerable dose with fewer side effects in vivo [8].
_
___________
*Corresponding author. Tel.: +1-501-686-6495; fax: +1-501-686-6057;
E-mail address: pacrooks@uams.edu (P. A. Crooks).

However, CA-4 suffers from stability issues because of its
had completely disappeared, cold water was added and the
mixture stirred over 10–15 min, during which the final product
precipitated out and was filtered off, washed with water and
dried. In cases where there was an absence of a precipitate, the
product was extracted into ethyl acetate, the organic extract
washed with copious amounts of water, and the resulting organic
liquor evaporated to dryness on a rotary evaporator. The residue
obtained was purified by flash column chromatography to afford
the corresponding triazole (Scheme 1) [17]. The structure and
ACCEPTED MANUSCRIPT
tendency to undergo cis-trans double bond isomerism in solution.
CA-4 is a cis-configured stilbene which is readily converted to
the thermodynamically more stable, but less potent trans-isomer
[
9]. Extensive studies have been conducted in attempts to
stabilize the cis configuration by replacing the olefinic double
bond with heterocyclic ring systems such as β-lactam, azetidone,
thiazole, tetrazole, imidazole, pyrazole, oxazolone, triazole,
furanone moieties [10-15].
1
13
purity of the triazole derivatives were verified by H, C-NMR
spectroscopy, high resolution mass spectroscopy and X-ray
crystallography [17-20].
Based on previous SAR studies on cis constrained CA-4
analogues, triazole analogues initially chosen for synthesis
contained the 3,4,5-trimethoxyphenyl moiety (Ring A) and a
variably substituted aryl or heteroaryl moiety (Ring B) [7, 21]. In
later structural modifications we introduced halogeno, nitro and
hydroxyl functionalities ring A.
Fig. 1. Structures of combretastatin A-4 (CA-4, A), colchicine (B), triazole
analogue (2e, C) and reported anti-tubulin compounds (D-I).
Scheme 1. Synthesis of 4,5-disubstituted 2H-1,2,3-triazoles; reagents and
3 4 2
conditions: (a) 5% NaOMe, MeOH, reflux. (b) NaN , NH Cl, DMF/H O.
In the work described herein, we report on the synthesis of
series of novel cis-constrained 4,5-disubstituted 2H-1,2,3-triazole
analogues of CA-4. The 2H-1,2,3-triazole ring system was
designed to not only halt cis-trans isomerization, but also
improve the drug likeness of the resulting CA-4 analogue, along
with providing scope for further structural diversification.
Evaluation of these novel triazole analogues of CA-4 against a
panel of 60 human tumor cell lines has been performed, along
with cell cycle redistribution assays. The molecular mechanism
responsible for the of the anti-cancer activity of the three most
potent molecules, 2e, 2h and 2l, has been investigated by
performing molecular docking studies with the target molecule,
tubulin.
2
.2. Biological Evaluation
.2.1. Anti-cancer activity against a panel of NCI 60 human
2
cancer cells
The sulforhodamine B (SRB) assay procedure described by
Rubinstein et al. was used to screen the CA-4 analogues 2a-2s
against a panel of 60 human tumor cell lines [22]. Growth
inhibitory or cytotoxic effects were measured by percentage
growth (PG), which is proportional to optical density (OD) [23,
2
4]. OD measurements of SRB-derived color prior to and 48 hrs
after exposure of cells to the test compound or vehicle control
were recorded. Ten compounds (2c-2e, 2g, 2j, 2l-2n, 2r, and 2s)
were initially identified as “hits” after screening at all the
analogues at 10 M concentration. These single concentration
results are presented in the supplementary data section. The
screening protocol utilized to identify a hit was 60% growth
-
5
2
. Results and discussion
2
.1. Drug synthesis
-
5
inhibition at 10 M in at least eight cell lines from the panel of
0 cell lines. Compounds that met these criteria were then
selected for a complete dose response study at five different
The general procedure for the synthesis of the 4,5-disubstituted
6
2
H-1,2,3-triazole CA-4 analogues is illustrated in Scheme 1. In
-
4
-5
-6
-7
-8
the first step, a series of (Z)-substituted diarylacrylonitrile
analogues were synthesized by reacting substituted benzyl
concentrations, viz. 10 M, 10 M, 10 M, 10 M and 10 M.
Six of the preliminary hits (2d, 2g, 2h, and 2l-2n) were evaluated
in the full dose-response studies had effective GI50 and TGI
(Total growth inhibition) values against a variety of human tumor
cell lines (Table 2). All the compounds have LD50 values >100
µM against most of the human cancer cell lines, indicating that
the compounds are anti-proliferative.
carbaldehydes
with
their
corresponding
substituted
phenylacetonitriles in 5% NaOMe in methanol. The reaction
mixture was stirred at room temperature for 2-3 hours, during
which time the desired product precipitated out of the solution.
The resulting product was then filtered, washed with water, and
dried to yield the final compound; yields ranged from 70-95 %
Analogue 2l had impressive GI50 values of less than 10 nM
against almost all of the 60 cancer cell lines in the panel, except
for melanoma cancer cell line UACC-62, and colon cancer cell
lines COLO 205 and HCC-2998. Compound 2l also showed
potent anti-proliferative activity with TGI values of <10 nM
against CNS cancer cell line SF-539, melanoma cell MDA-MB-
(
Scheme 1) [16].
In the second step, 4,5-disubstituted-2H-1,2,3-triazoles were
obtained by refluxing a mixture of the (Z)-2,3-diarylacrylonitrile
(1a-1s) from step 1 with NaN , and NH Cl in a mole ratio of
3 4
1
:3:3 in 10:1 volumes of DMF/H O for 5–12 h. The reaction
2
4
35, ovarian cancer cell line OVCAR-3, and renal cancer cell
mixture was monitored by TLC, and when the starting material
line A498.

Table 1.
Synthesized (Z)-2,3-diarylacrylonitriles and 4,5-disubstituted-2H-1,2,3-triazoles from their corresponding acetonitrile and aldehyde precursors.
ACCEPTED MANUSCRIPT
Acetonitrile
Aldehyde
(Z)-2,3-Diarylacetonitriles (1a-1s)
4,5-Disubstituted 2H-1,2,3-triazoles (2a-2s)
OCH
3
CN
CHO
OCH
3
CN
Cl
OCH3
OCH3
Cl
Cl
Cl
1
a
2a
OCH
3
CN
OCH
OCH
3
3
CHO
3
H CO
CN
OCH
3
3
3
3
H3CO
OCH3
3
H CO
H
H
H
H
3
CO
OCH
3
OCH3
OCH
3
1
b
2
b
H
3
CO
OCH
3
CN
OCH
3
CHO
CN
3CO
OCH
N
OCH
3
N
1
c
2c
CN
OCH3
OCH3
CHO
N
N
3CO
OCH
OCH3
CN
d
OCH
3
1
2
d
OCH
3
H
N
CN
CHO
OCH
3
3
N
N
H3CO
H3CO
OCH
CN
OCH3
OCH
OH
3
H CO
3CO
OCH3
OH
OCH
CN
3
OCH3
OH
1
e
2e
H
S
N
N
N
CHO
CN
Cl
F
Cl
S
S
F
Cl
F
1
f
2
f
H
N
CHO
Cl
CN
Cl
N
N
Cl
N
N
CN
Cl
N
Cl
Cl
1
g
2
g
OCH3
CN
OCH3
OCH3
O
CHO
O
O
H
3CO
OCH
3
O
CN
OCH
3
1
h
2
h
NO2
CN
CHO
H3CO
H3CO
CN
H3CO
OCH3
OCH3
OCH3
NO2
1
i
2
i

ACCEPTED MANUSCRIPT
H
N
CN
N
N
O
O
CHO
O
O
O
CN
O
1
j
2
j
Br
3
H CO
CN
CHO
H3CO
NC
Br
Br
Br
1
k
2
k
CN
H3CO
CHO
3
H CO
OCH
3
H
3
CO
OCH
3
H
3
CO
NC
OCH3
CN
OCH
3
1l
2
l
H3CO
CHO
OCH
3
H
3
CO
NC
H3CO
OCH3
OCH
3
1m
2
m
OCH
3
CN
OCH
OCH
3
3
O
O
CHO
O
O
H
H
H
3
CO
OCH
3
3
3
CN
OCH
3
1
n
2
n
OCH
3
H
N
CN
OCH3
N
CHO
N
Cl
OCH3
OCH
3
CN
Cl
3
CO
OCH
Cl
Cl
OCH3
Cl
OCH
3
Cl
H3CO
1
o
2o
H
N
OCH
3
CN
N
N
CHO
OCH
OCH
3
3
OCH3
OCH3
N
N
3
CO
OCH
N
CN
OCH
3
H3CO
1
p
2
p
OCH3
CN
CHO
OCH3
CN
H3CO
OCH3
O2N
NO
2
1
q
2
q
H
N
OCH3
N
N
CN
CHO
O
O
OCH3
O
O
O
OCH3
CN
O
H3CO
OCH3
H3CO
1
r
2
r
H
N
N
N
N
CN
N
O
O
CHO
O
O
O
O
CN
N
1
s
2
s

Table 2.
Growth inhibition (GI50/µM) data of 2d, 2m, 2l, 2n and 2g against a panel of
0 human cancer cells.
appeared to be the most sensitive to the growth inhibitory effects
of 2m, exhibiting a TGI value of 10 nM.
ACCEPTED MANUSCRIPT
a
6
2
d
2m
2l
2h
2n
2g
Analogue 2h also showed potential growth inhibitory
properties with GI50 <10 nM against leukemia cancer cell lines
K-562 and SR, non-small cell lung cancer cell line HOP-92,
colon cancer cell lines HCT-116, HCT-15, KM-12, and SW-620,
CNS cancer cell lines SF-295, SF-539, and SNB-75, melanoma
cancer lines M14, MDA-MB-435, and SK-MEL-2, ovarian
cancer cell lines NCI/ADR-RES, and SK-OV-3, renal cancer cell
lines A498, ACHN, CAKI-1, and UO-31, and breast cancer cell
line MCF-7.
Panel/cell line
(
µM)
(µM)
(µM)
(µM)
(µM)
(µM)
Leukemia
CCRF-CEM
0.037 0.032 <0.01 0.034
0.28
3.38
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
Lung Cancer
A549/ATCC
HOP-62
0.030 0.023 <0.01 0.019
0.026 <0.01 <0.01 <0.01
0.068 0.048 0.012 0.040
0.037 0.037 <0.01 0.033
0.021 <0.01 <0.01 <0.01
0.20
0.05
0.57
0.33
0.04
5.75
5.50
4.23
10.7
5.43
0.054 0.037 <0.01 0.023
0.047 0.025 <0.01 0.018
0.099 <0.01 <0.01 <0.01
0.065 0.038 <0.01 0.048
0.038 0.034 <0.01 0.025
0.29
0.34
0.12
0.77
0.34
4.85
5.32
3.28
9.13
3.67
From the above screening studies, analogue 2l was considered
to be a lead candidate, since it exhibited very promising GI50 and
TGI values against a wide variety of both hematological and
solid tumor cell types. However, in silico tubulin docking studies
carried out with the triazole analogues revealed slightly better
tubulin binding affinity for compound 2e compared to 2l.
Unfortunately, analogue 2e, which is the triazole-derived form of
the CA4 molecule, was not selected for the 60 human cancer cell
screen. We therefore evaluated 2e for its anti-cancer activity by
colony formation assay against 9LSF rat gliosarcoma cells.
HOP-92
NCI-H23
NCI-H460
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
0.228 0.149 0.029 0.027
0.230 0.046 0.024 0.039
0.040 <0.01 <0.01 <0.01
0.037 <0.01 <0.01 <0.01
0.214 0.048 <0.01 0.025
0.035 0.030 <0.01 <0.01
0.041 0.030 <0.01 <0.01
0.31
>100
0.18
0.13
0.39
0.07
0.11
2.89
9.16
2.89
2.93
4.56
6.87
4.40
KM12
SW-620
CNS Cancer
SF-268
>
100
0
.503 0.177 <0.01 0.069
7.36
2
.2.2 Colony formation assay results for 2e utilizing 9LSF rat
SF-295
SF-539
SNB-75
U251
Melanoma
LOX IMVI
0.014 0.011 <0.01 <0.01
0.020 0.014 <0.01 <0.01
0.016 0.014 <0.01 <0.01
0.037 0.038 <0.01 0.020
0.10
0.18
0.08
0.30
3.01
na
1.58
5.35
gliosarcoma cells
9LSF cells were acquired from the laboratory of Dennis Deen,
Ph.D. (Brain Tumor Research Center, University of California,
San Francisco). Cells were exposed to 2e at concentrations of 1
nM, 3 nM, 10 nM, 30 nM, 100 nM, 1 µM, or 10 µM for 24 hours
prior to seeding for the colony formation assay. Cells were then
0.065 0.076 <0.01 0.018
0.022 <0.01 <0.01 <0.01
<0.01 <0.01 <0.01 <0.01
0.027 0.056 <0.01 <0.01
>100
0.013 0.027 <0.01 <0.01
0.157 <0.01 nd >100
0.55
na
0.09
0.02
0.24
>100
>100
4.83
14.3
2.20
6.52
5.39
7.89
3.35
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-62
2
na
na
>100
trypsinized, counted, seeded into 25 cm flasks, and incubated at
3
7°C to form colonies [25]. All conditions were seeded in
triplicate. Flasks that were plated with less than 50,000 cells were
previously plated with 50,000 lethally irradiated A549 cells to
serve as feeders [26]. Flasks were removed from incubation when
colonies were large enough to count (>50 cells). Colonies were
then fixed and stained with crystal violet, rinsed, allowed to dry,
and counted. The protocol was run in triplicate, and results
averaged, with error representing SEM. The LD50 value for 2e
was determined to be 7.5 nM using SigmaPlot 11.
Ovarian Cancer
IGROV1
0
.065 0.051 <0.01 0.033
0.011 0.024 <0.01 <0.01
0.077 na <0.01 0.034
>100
5.94
OVCAR-3
OVCAR-4
NCI/ADR-RES
SK-OV-3
Renal Cancer
0.43
0.08
0.08
0.49
3.38
3.04
3.92
2.72
0.023 <0.01 <0.01 <0.01
0.075 0.046 <0.01 <0.01
7
86-0
0.043 0.015 <0.01 <0.01
0.033 <0.01 <0.01 0.010
0.081 0.145 <0.01 <0.01
0.043 0.050 <0.01 <0.01
0.092 0.020 <0.01 <0.01
0.62
0.34
0.71
0.32
0.66
2.75
3.18
1.75
8.42
6.84
A498
ACHN
CAKI-1
UO-31
Prostate Cancer
PC-3
0.046 0.042 <0.01 0.018
0.027 0.045 <0.01 0.025
0.26
0.35
4.50
1.41
DU-145
Breast Cancer
MCF7
0.027 0.025 <0.01 <0.01
0.08
0.53
2.58
5.89
MDA-MB-
0
.094 0.046 <0.01 0.044
2
31/ATCC
HS 578T
MDA-MB-468
na 0.041 <0.01 0.668
0.44
0.23
11.6
3.20
0.035 0.023 <0.01 0.015
a
na: Not analyzed, nd; not determined. GI50: 50% growth inhibition,
concentration of drug resulting in a 50% reduction in net cell growth as
compared to cell numbers on day 0.
Analogue 2m exhibited potent growth inhibitory activity with
GI50 <10nM against leukemia cancer cell lines K-562 and SR,
non-small cell lung cancer cell line HOP-92, colon cancer cell
lines HCT-116 and HCT-15, melanoma cancer lines M14 and
UACC-62, ovarian cancer cell line NCI/ADR-RES, and renal
cancer cell line A498. Melanoma cancer cell line MDA-MB-435
Fig. 2. Colony formation assay, 2e-treated 9LSF cells. Cells were exposed to
2
e for 24 hours. Curve was fitted as four-parameter logistic curve (f1 = min +
(max-min)/(1 + (x/EC50)^(-Hillslope))). Values represent mean ± SEM of 3
independent colony formation experiments.

2
.2.3 Cell cycle redistribution using 2e
3. In silico molecular docking studies
ACCEPTED MANUSCRIPT
Two analogues (2h and 2l) from the 60 human cancer cell
panel screens, and analogue 2e, GI50 <10 nM) were chosen for
molecular docking studies utilizing the available crystal structure
of tubulin, in order to understand the possible mechanism of their
action as inhibitors of tubulin polymerization. Atomic
coordinates for tubulin (PDB 1SA0) were downloaded from the
protein structure database (www.rcsb.org/pdb). 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 coordinate files were
prepared for docking using the Dock Prep module in the UCSF-
Chimera package ((http://www.cgl.ucsf.edu/chimera). Docking
was performed using SwissDock (http://www.swissdock.ch/),
based on the docking algorithm EADock DSS [27]. Docking was
performed using protocols established in our previous studies
9
LSF cells were exposed to 50 nM 2e or 50 nM colchicine
COL) for 24 hrs in vitro, along with controls. Cells were then
trypsinized, counted, rinsed with PBS, and fixed using ice-cold
(
6
7
0% EtOH in PBS at 10 cells/mL. Samples were stored at 4°C
6
prior to flow cytometry analysis; 10 cells from each condition
were then pelleted and rinsed thrice with PBS. Samples were
resuspended in 1 mL PBS, to which 1 µL propidium iodide (PI,
1
2.5 mg/mL in DMSO) was added. Samples were exposed to PI
for 2 minutes at room temperature, then immediately analyzed by
flow cytometry (Cell Lab Quanta SC, Beckman Coulter, Inc.,
Brea, CA.) The protocol was run in triplicate. Cell cycle
distributions were analyzed using FCS Express 3 (De Novo
Software, Glendale, CA).
[
16]. Use of the most exhaustive and unbiased option in
SwissDock ensured the sampling of the maximum number of
binding modes for each molecule. The best hits based on the
SwissDock FullFitness scoring function (FF) from three repeated
docking runs were considered further.
All three compounds docked almost exclusively to the
colchicine-binding pocket on the α,β-tubulin heterodimer. This
indicates that the mode of inhibition of tubulin polymerization by
these three molecules is similar to that of colchicine and CA-4,
and that the introduction of a triazole ring in place of the cis-
olefinic bond still retains potent tubulin binding properties.
Among the three molecules examined, 2e had the most number
of ‘outlier’ poses that docked in non-colchicine binding, surface-
exposed pockets. However, 86% of the docked poses were still
localized at the colchicine-pocket. In the case of 2h and 2l, this
number was 95% and 99%, respectively (Fig. 4).
Fig 3. Cell cycle redistribution effects of 2e on 9L cells. (A) Percentage of
cell population in G2/M phase in control cells versus those exposed to 50 µM
2
e or 50 µM COL for 24 hrs. (B) Representative distribution of control cells.
C) Representative distribution of 2e-treated cells. (D) Representative
distribution of COL-treated cells.
(
Fig. 4. Docking poses of molecules 2e, 2h and 2l bound to tubulin. Subunits α and β of the tubulin heterodimer are shown as gray and black cartoons
respectively. All the docking poses generated by Swissdock for molecules 2e, 2h and 2l are shown in panels A), B) and C) respectively. As seen clearly, majority
of the poses bind to the ‘colchicine-binding’ pocket located at the interface of the two tubulin subunits for all three molecules. 2e shows the most, while 2l shows
the least number of ‘outlier’ poses.
All three molecules were primarily stabilized through
several van der Waals’ contacts with residues from the α- and
β-subunits of tubulin. Compound 2e was found to interact with
higher than that for 2e. Both scores followed the order: 2e < 2h
< 2l.
Table 3.
1
3 β residues and 5 α residues of tubulin (Fig 5). In contrast,
SwissDock statistics for the docking runs with compounds 2e, 2h and 2l
both molecules 2h and 2l, besides sharing all the contact
residues of 2e, made additional contacts with Met259 and
Thr314 of the β- subunit of tubulin (Fig 5B). This is reflected
in the FF and ∆G scores of the three compounds (Table 3),
with the FF score and free energy values for 2l and 2h being
(scores shown are averages from 3 docking runs).
Comp
FF score (kcal/mol)
∆G (kcal/mol)
2
l
-4224.7
-4216.5
-4211.4
-8.4
-8.2
-7.9
2
h
2
e

5
.2. General Synthetic procedure for the synthesis of 4,5-
ACCEPTED MANUSCRIPT
disubstituted-2H-1,2,3-triazoles
In the first synthetic step, a series of (Z)-substituted
diarylacrylonitrile analogues were synthesized by reacting
substituted benzyl carbaldehydes with their corresponding
substituted phenylacetonitriles in 5% NaOMe in methanol. The
reaction mixture was stirred at room temperature for 2-3 hours
for the reaction to complete and the final product to crash out
of the solution. The precipitate is filtered, washed with water
and dried to yield the final compounds with yields ranging
from 70-95 % (Scheme 1) [16].
Fig. 5. Atomic contacts between tubulin and molecules 2e and 2l. A) 2e is
shown as orange sticks, while residues of the α and β subunit of tubulin are
in light and dark gray, respectively. B) 2l (green sticks) is shown bound at
the colchicine-binding pocket of tubulin. The color scheme is the same as
in A, while the residues of β-tubulin unique to 2l pocket are shown in
magenta.
In the second synthetic step the 4,5-disubstituted-2H-1,2,3-
triazoles were synthesized by refluxing a mixture of the (Z)-
2
,3-diarylacrylonitrile (1a-1s), NaN , and NH Cl in a mole
3 4
ratio of 1:3:3 in 10:1 volumes of DMF/H O for 5–12 h. The
2
reaction was monitored by TLC. When the starting material
had completely disappeared, cold water was added and the
mixture was stirred over 10–15 min, during which time the
final product precipitated out and was filtered off. In the
absence of a precipitate, the product was extracted into ethyl
acetate, the organic extract washed with copious amounts of
water, and the resulting organic liquor evaporated to dryness
on a rotavaporator. The residue obtained was purified by flash
column chromatography to afford the corresponding triazole
analogue of CA-4.
4
. Conclusions
A series of 4,5-disubstituted 2H-1,2,3-triazoles designed as
CA4 analogues were synthesized from corresponding (Z)-
substituted diarylacrylonitriles (1a-1s). The synthesis
represents a facile and efficient reaction procedure for the
preparation of 4,5-diaryl-2H-1,2,3-triazoles in modest to good
yields. The resulting 4,5-disubstituted 2H-1,2,3-triazoles were
evaluated for their anticancer properties against a panel of 60
human cancer cell lines. The diarylacrylonitrile analogue 2l
exhibited the most potent anticancer activity in the cancer cell
screening studies, with GI50 values of <10 nM against almost
all the cell lines in the panel. Analogue 2l also exhibited
promising TGI values of <10 nM against CNS cancer cell line
SF-539, melanoma cell MDA-MB-435, ovarian cancer cell
line OVCAR-3 and renal cancer cell line A498. Analogues 2m
and 2h exhibited GI50 values <10nM against leukemia cell
lines K-562, SR, non-small lung cancer cell line HOP-92,
colon cancer cell lines HCT-116, and HCT-15, melanoma
cancer lines M14, and UACC-62, ovarian cancer cell line
NCI/ADR-RES and renal cancer cell line A498 Analogue 2e
was evaluated for anti-cancer activity by colony formation
assay against 9LSF rat gliosarcoma cells, and afforded an LD50
value of 7.5 nM. A cell cycle redistribution assay with 2e was
also conducted to further understand the mechanism of action
of this CA-4 analogue. In silico docking studies with
compounds 2l, 2e and 2h within the active site of tubulin were
carried out in order to rationalize the anticancer properties of
these compounds. From these studies, compound 2e exhibited
higher affinity for the colchicine binding site on tubulin
compared to 2l and 2h. From this study, analogues 2e and 2l
were considered lead compounds in this new structural class,
and worthy of further development as anti-cancer agents.
In the second synthetic step the 4,5-disubstituted-2H-1,2,3-
triazoles were synthesized by refluxing a mixture of the (Z)-
2
,3-diarylacrylonitrile (1a-1s), NaN , and NH Cl in a mole
3 4
ratio of 1:3:3 in 10:1 volumes of DMF/H O for 5–12 h. The
2
reaction was monitored by TLC. When the starting material
had completely disappeared, cold water was added and the
mixture was stirred over 10–15 min, during which time the
final product precipitated out and was filtered off. In the
absence of a precipitate, the product was extracted into ethyl
acetate, the organic extract washed with copious amounts of
water, and the resulting organic liquor evaporated to dryness
on a rotavaporator. The residue obtained was purified by flash
column chromatography to afford the corresponding triazole
analogue of CA-4.
5
.3. Analytical data of 4,5-disubstituted-2H-1,2,3-triazoles
.3.1. 4-(3,4-dichlorophenyl)-5-(3,4-dimethoxyphenyl)-2H-
5
1
,2,3-triazole (2a)
1
H NMR (400 MHz, CDCl ): δ 3.82 (s, 3H, -OCH ), δ 3.93
3
3
(
s, 3H, -OCH ), 6.88 (d, J = 8 Hz, 1H, ArH), 7.06 (d, J = 12
3
Hz, 2H, ArH), 7.42 (s, 2H, ArH), 7.78 (s, 1H, ArH), 12.49 (bs,
13
1
1
1
H, NH) ppm. C NMR (100 MHz, CDCl ): δ 55.87, 55.96,
3
11.19, 121.31, 127.31, 129.80, 129.89, 130.54, 132.60,
32.86, 149.15, 149.73 ppm. HRMS (ESI): m/z calcd for
C H Cl N O [M+H] : 350.0463; found 350.0465.
+
5
.0 Experimental Section
16 14
2
3
2
5
.1. Chemistry
5.3.2. 4,5-bis(3,4,5-trimethoxyphenyl)-2H-1,2,3-triazole (2b)
1
TLC experiments were carried out on pre-coated silica gel
H NMR (400 MHz, CDCl ): δ 3.76 (s, 12H, -OCH ), 3.88
3
3
1
13
plates (F 254 Merck). H and C NMR spectra were recorded
on a Varian 400 MHz spectrometer equipped with a Linux
workstation running on vNMRj software. All spectra were
phased, baseline was corrected where necessary, and solvent
(s, 6H, -OCH ), 6.84 (s, 4H, ArH), 12.50 (bs, 1H, NH) ppm.
2
13
C NMR (100 MHz, CDCl ): δ 56.08, 60.96, 105.57, 125.58,
3
138.28, 153.27 ppm. HRMS (ESI): m/z calcd for C H N O
6
2
0
24
3
+
[M+H] : 402.1665; found 402.1668.
1
13
signals (CDCl ) were used as reference for both H and
C
3
spectra. HRMS data was obtained on an Agilent 6210 LCTOF
instrument operated in multimode.
5
.3.3.
4-(5-(3,4,5-trimethoxyphenyl)-2H-1,2,3-triazol-4-yl)
quinolone (2c)

1
H NMR (400 MHz, CDCl ): δ 3.47 (s, 6H, -OCH ), 3.81 (s,
5.3.9. 4-(4-nitrophenyl)-5-(3,4,5-trimethoxyphenyl)-2H-1,2,3-
3
ACCEPTED MANUSCRIPT
3
3
H, -OCH ), 6.65 (s, 2H, ArH), 7.50-7.52 (t, J = 8 Hz, 1H,
ArH), 7.55 (d, J = 4.4 Hz, 1H, ArH), 7.77 (t, J = 1.6 Hz, 1H,
ArH), 7.83 (d, J = 8 Hz, 1H, ArH), 8.28 (d, J = 8.8 Hz, 1H,
triazole (2i)
3
1
H NMR (400 MHz, DMSO-d ): δ 3.70-3.72 (d, 9H, -OCH ),
6
3
6.80 (s, 2H, ArH), 7.86 (s, 2H, ArH), 8.30 (d, J = 8.4 Hz, 2H,
13
13
ArH), 9.03 (d, J = 4.4 Hz, 1H, ArH) ppm. C NMR (100 MHz,
ArH) ppm. C NMR (100 MHz, DMSO-d ): 56.23, 61.06,
6
CDCl ): δ 55.70, 60.85, 104.47, 122.68, 122.76, 124.69,
105.75, 123.91, 128.76, 137.57, 138.73, 147.50, 153.62 ppm.
3
+
1
1
25.95, 126.66, 127.52, 129.42, 130.21, 138.21, 138.45,
HRMS (ESI): m/z calcd for C H N O [M+H] : 357.1199;
17
17
4
5
48.15, 149.63, 149.69, 153.24 ppm. HRMS (ESI): m/z calcd
found 357.1199.
+
for C H N O [M+H] : 363.1457; found 363.1460.
20
19
4
3
5
.3.10.
4-(benzo[d][1,3]dioxol-5-yl)-5-phenyl-2H-1,2,3-
5
.3.4.
2-(5-(3,4,5-trimethoxyphenyl)-2H-1,2,3-triazol-4-yl)
triazole (2j)
1
quinolone (2d)
H NMR (400 MHz, CDCl ): δ 6.00 (s, 2H, -CH ), 6.81 (d, J
3 2
= 8.8 Hz, 1H, ArH), 7.04 (d, J = 6.8 Hz, 2H, ArH), 7.39 (t, J =
5.2 Hz, 3H, ArH), 7.57 (m, J = 9.6 Hz, 1H, ArH) ppm. C
1
H NMR (400 MHz, CDCl ): δ 3.78 (s, 6H, -OCH ), 3.91 (s,
3
3
13
3
H, -OCH3), 7.08 (s, 2H, ArH), 7.56 (t, J = 14.8 Hz, 1H,
ArH), 7.72 (t, J = 14.8 Hz, 1H, ArH), 7.83 (d, J = 6.4 Hz, 2H,
ArH), 8.09 (d, J = 8.8 Hz, 1H, ArH), 8.17 (d, J = 8.4 Hz, 1H,
NMR (100 MHz, CDCl ): δ 101.43, 108.77, 108.91, 122.46,
3
123.96, 128.41, 128.73, 128.88, 130.25, 142.29, 148.01,
13
+
ArH) ppm. C NMR (100 MHz, CDCl ): δ 56.08, 60.92,
148.11 ppm. HRMS (ESI): m/z calcd for C H N O [M+H] :
3
15 12
3
2
1
1
06.21, 120.70, 125.57, 127.12, 127.56, 127.73, 127.68,
266.0930; found 266.0928.
29.04, 130.20, 136.83, 138.50, 147.76, 153.19 ppm. HRMS
+
(ESI): m/z calcd for C H N O [M+H] : 363.1457; found
5.3.11. 4-(3,5-dibromophenyl)-5-(2-methoxyphenyl)-2H-1,2,3-
20
19
4
3
3
63.1456.
triazole (2k)
1
H NMR (400 MHz, CDCl ): δ 3.76 (s, 3H, -OCH ), 7.04 (d,
3
3
5
.3.5.
2-methoxy-5-(5-(3,4,5-trimethoxyphenyl)-2H-1,2,3-
J = 4.8 Hz, 2H, ArH), 7.39-7.47 (m, 2H, ArH), 7.63 (d, J = 1.6
Hz, 1H, ArH), 7.71 (s, 2H, ArH), 12.2 (bs, 1H, NH) ppm. C
1
3
triazol-4-yl)phenol (2e)
1
H NMR (400 MHz, CDCl ): δ 3.73 (s, 6H, -OCH ), 3.88 (s,
NMR (100 MHz, CDCl ): δ 55.51, 111.54, 121.21, 122.83,
3
3
3
6
1
H, -OCH ), 6.82 (d, J = 2 Hz, 3H, ArH), 7.04 (d, J = 8.4 Hz,
H, ArH), 7.20 (s, 1H, ArH) ppm. C NMR (100 MHz,
129.17, 130.48, 131.29, 133.34, 133.42, 134.96, 156.51 ppm.
3
13
+
HRMS (ESI): m/z calcd for C H N O Br [M+H] : 407.9347;
15
12
3
2
CDCl ): δ 55.92, 56.98, 56.07, 60.91, 60.98, 105.23, 110.63,
found 407.9342.
3
1
1
3
14.72, 120.61, 123.01, 125.74, 138.09, 145.66, 147.10,
+
53.23 ppm. HRMS (ESI): m/z calcd for C H N O [M+H] :
5.3.12. 4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-2H-
18
20
3
5
58.1403; found 358.1408.
1,2,3-triazole (2l)
1
H NMR (400 MHz, CDCl ): δ 3.74 (s, 6H, -OCH ), 3.84 (s,
3
3
5
.3.6. 4-(4-chloro-3-fluorophenyl)-5-(thiophen-3-yl)-2H-1,2,3-
3H, -OCH ), 3.89 (s, 3H, -OCH ), 6.83 (s, 2H, ArH), 6.92 (d, J
3 3
triazole (2f)
= 8.4 Hz, 2H, ArH), 7.52 (d, J = 8.0 Hz, 2H, ArH), 12.2 (bs,
1
13
H NMR (400 MHz, CDCl ): δ 7.22 (d, J = 4.8 Hz, 1H,
1H, NH) ppm. C NMR (100 MHz, CDCl ): δ 55.27, 55.96,
3
3
ArH), 7.32 (d, J = 8.4 Hz, 1H, ArH), 7.38-7.41 (m, 3H, ArH),
60.90, 105.14, 105.18, 114.03, 125.86, 129.79, 138.07, 153.27,
13
+
7
.50 (d, J = 1.2 Hz, 1H, ArH), 10.5 (bs, 1H, NH) ppm.
C
159.97 ppm. HRMS (ESI): m/z calcd for C H N O [M+H] :
18
20
3
4
NMR (100 MHz, CDCl ): δ 116.17, 121.32, 124.56, 126.73,
342.1454; found 342.1448.
3
1
27.01, 129.50, 130.75, 138.32, 141.30, 156.84, 159.31 ppm.
+
HRMS (ESI): m/z calcd for C H N FSCl [M+H] : 280.0111;
5.3.13. 4-(3,5-dimethoxyphenyl)-5-(4-methoxyphenyl)-2H-
12
8
3
found 280.0118.
1,2,3-triazole (2m)
1
H NMR (400 MHz, DMSO-d ): δ 3.68 (s, 6H, -OCH ), 3.78
6
3
5
.3.7. 2-(5-(2,4-dichlorophenyl)-2H-1,2,3-triazol-4-yl)pyridine
(s, 3H, -OCH ), 6.5 (s, 1H, Ar-H), 6.64 (2H, Ar-H), 7.01 (d,
3
(
21g)
2H, J = 8 Hz, ArH), 7.43 (d, 2H, J = 8.8 Hz, Ar-H), HRMS
+
H NMR (400 MHz, CDCl ): δ 7.26 (s, 1H, ArH), 7.39 (d, J
(ESI): m/z calcd for C H N O [M+H] : 312.1348; found
3
17 18
3
3
=
8 Hz, 2H, ArH), 7.49-7.54 (m, 2H, ArH), 7.67(d, J = 6.8 Hz,
312.1344.
13
1
H, ArH), 8.63 (s, 1H, ArH) ppm. C NMR (100 MHz,
CDCl ): δ 121.68, 123.21, 127.27, 129.64, 132.76, 134.74,
5.3.14.
4-(benzo[d][1,3]dioxol-5-yl)-5-(3,4,5-trimethoxy
3
1
35.52, 136.94, 149.45 ppm. HRMS (ESI): m/z calcd for
phenyl)-2H-1,2,3-triazole (2n)
+
1
C H N Cl [M+H] : 291.0204; found 291.0201.
H NMR (400 MHz, CDCl ): δ 3.77 (s, 6H, -OCH ), 3.89 (s,
13
9
4
2
3
3
3
H, -OCH ), 4.26-4.29 (q, J = 12.4 Hz, 4H, ArH), 6.84 (s, 2H,
3
5
.3.8.
4-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-(3,4,5-
ArH), 6.88 (d, J = 8 Hz, 1H, ArH), 7.04-7.07 (dd, J = 2 Hz, 8
trimethoxy phenyl)-2H-1,2,3-triazole (2h)
Hz, 1H, ArH), 7.16 (d, J = 1.6 Hz, 1H, ArH), 7.26 (s, 1H, ArH)
1
13
H NMR (400 MHz, CDCl ): δ 3.77 (s, 6H, 2xOCH ), 3.89
ppm. C NMR (100 MHz, CDCl ): δ 55.99, 56.08, 60.90,
3
3
3
(
s, 3H, OCH ), 4.26-4.29 (m, 2xCH ), 6.84 (s, 2H, ArH), 6.88
60.99, 64.27, 64.47, 105.24, 105.28, 117.42, 121.80, 138.18,
3
2
(
d, 1H, J = 8 Hz, Ar-H), 7.04-7.07 (dd, 1H, J = 2 and 8 Hz, 1H,
143.59, 144.09, 153.26 ppm. HRMS (ESI): m/z calcd for
13
+
Ar-H), 7.15 (d, 1H, Ar-H), 12.2 (bs, 1H, NH) ppm. C NMR
100 MHz, CDCl ): δ 55.99, 60.99, 64.27, 105.24, 105.28,
C H N O [M+H] : 356.1246; found 356.1248.
18
18
3
5
(
3
1
17.42, 121.80, 138.18, 143.59, 144.09, 153.26 ppm. HRMS
5.3.15. 4-(3,4-dichlorophenyl)-5-(3,4,5-trimethoxyphenyl)-2H-
+
(ESI): m/z calcd for C H N O [M+H] : 370.1403; found
1,2,3-triazole (2o)
19
20
3
5
1
3
70.1398.
H NMR (400 MHz, CDCl ): δ 3.79 (s, 6H, -OCH ), 3.91 (s,
3
3
3
H, -OCH ), 6.77 (s, 2H, ArH), 7.46 (s, 2H, ArH), 7.82 (s, 1H,
3
13
ArH) ppm. C NMR (100 MHz, CDCl ): δ 56.14, 60.98,
3

1
1
05.49, 127.51, 129.92, 130.57, 132.51, 132.77, 138.46,
[2] M.A. Jordan, Mechanism of action of antitumor drugs that
interact with microtubules and tubulin, Curr. Med. Chem.: Anti-
Cancer Agents, 2 (2002) 1-17.
ACCEPTED MANUSCRIPT
53.47 ppm. HRMS (ESI): m/z calcd for C H C N O
3
17
16 l2
3
+
[
M+H] : 380.0569; found 380.0564.
[
3] J.A. Hadfield, S. Ducki, N. Hirst, A.T. McGown, Tubulin and
microtubules as targets for anticancer drugs, Prog. Cell. Cycle.
Res. 5 (2003) 309-325.
5
.3.16.
2-(5-(3,4,5-trimethoxyphenyl)-2H-1,2,3-triazol-4-
yl)pyridine (2p)
[
4] M.A. Jordan, L. Wilson, Microtubules as a target for
1
H NMR (400 MHz, CDCl ): δ 3.81 (s, 6H, -OCH ), 3.90 (s,
3
3
anticancer drugs, Nat. Rev. Cancer 4 (2004) 253-265.
[5] S.L. Young, D.J. Chaplin, Combretastatin-A4 phosphate:
background and current clinical status, Expert. Opin. Investig.
Drugs 13 (2004) 1171-1182.
3
7
H, -OCH ), 6.9 (s, 2H, ArH), 7.28 (d, J =12.8 Hz, 2H, ArH),
3
.70 (s, 2H, ArH), 8.67 (s, 1H, ArH) ppm. C NMR (100
13
MHz, CDCl ): δ 56.10, 60.94, 106.04, 123.22, 123.46, 125.66,
1
for C H N O [M+H] : 313.1301; found 313.1298.
3
[
6] M.M. Cooney, J. Ortiz, R.M. Bukowski, S.C. Remick, Novel
37.06, 138.37, 149.36, 153.26 ppm. HRMS (ESI): m/z calcd
+
vascular targeting/disrupting agents: combretastatin-A4 phosphate
and related compounds, Curr. Oncol. Rep. 7 (2005) 90-95.
16
17
4
3
[
7] G.C. Tron, T. Pirali, G. Sorba, F. Pagliai, S. Busacca, A.A.
5
.3.17. 4-(3,5-dimethoxyphenyl)-5-(4-nitrophenyl)-2H-1,2,3-
Genazzani, Medicinal chemistry of combretastatin-A4: present
and future directions, J. Med. Chem. 49 (2006) 3033-3044.
[8] G.M. Tozer, C. Kanthou, C.S. Parkins, S.A. Hill, The biology
of the combretastatins as tumour vascular targeting agents, Int. J.
Exp. Pathol. 83 (2002) 21-38.
triazole (2q)
1
H NMR (400 MHz, CDCl ): δ 3.74 (s, 6H, -OCH ), 6.51 (d,
3
3
J = 1.2 Hz, 1H, ArH), 6.62 (s, 2H, ArH), 7.8 (d, J = 8.4 Hz,
2
13
H, ArH), 8.19 (d, J = 8.4 Hz, 2H, Ar-H) ppm. C NMR (100
MHz, CDCl ): δ 55.43, 101.28, 106.53, 123.85, 128.67,
[9] H.P. Hsieh, J.P. Liou, N. Mahindroo, Pharmaceutical design of
anti-mitotic agents based on combretastatins, Curr. Pharm. Des. 11
(2005) 1655-1677.
3
1
37.08, 147.49, 161.13 ppm. HRMS (ESI): m/z calcd for
+
C H N O [M+H] : 327.1093; found 327.1084.
16
15
4
4
[
10] M. Carr, L.M. Greene, A.J.S. Knox, D.G. Lloyd, D.M.
Zisterer, M.J. Meegan, Lead identification of conformationally
restricted β-lactam type combretastatin analogues: Synthesis,
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5
2
.3.18. 4-(benzo[d][1,3]dioxol-5-yl)-5-(3,5-dimethoxyphenyl)-
H-1,2,3-triazole (2r)
1
H NMR (400 MHz, CDCl ): δ 3.75 (s, 6H, -OCH ), 5.99 (s,
3
3
2
6
1
5
1
H, ArH), 6.48 (s, 1H, ArH), 6.73(d, J = 2.4 Hz, 2H, ArH),
.82 (d, J = 8.8 Hz, 1H, ArH), 7.06 (d, J = 6.4 Hz, 2H, ArH),
13
2.1 (bs, 1H, NH) ppm. C NMR (100 MHz, CDCl ): δ 55.34,
3
5.51, 101.05, 101.25, 106.12, 108.51, 108.80, 122.38, 131.91,
6
92-702.
47.81, 147.98, 160.87 ppm. HRMS (ESI): m/z calcd for
+
[12] T.M. Beale, P.J. Bond, J.D. Brenton, D.S. Charnock-Jones,
S.V. Ley, R.M. Myers, Increased endothelial cell selectivity of
triazole-bridged dihalogenated A-ring analogues of combretastatin
A–1, Bioorg. Med. Chem. 20 (2012) 1749-1759.
C H N O [M+H] : 326.1141; found 326.1133.
17
16
3
4
5
.3.19.
2-(5-(benzo[d][1,3]dioxol-5-yl)-2H-1,2,3-triazol-4-
yl)pyridine (2s)
[
13] D.V. Demchuk, A.V. Samet, N.B. Chernysheva, V.I.
1
H NMR (400 MHz, CDCl ): δ 5.99 (s, 2H, -CH ), 6.83 (d, J
Ushkarov, G.A. Stashina, L.D. Konyushkin, M.M. Raihstat, S.I.
Firgang, A.A. Philchenkov, M.P. Zavelevich, L.M. Kuiava, V.F.
Chekhun, D.Y. Blokhin, A.S. Kiselyov, M.N. Semenova, V.V.
Semenov, Synthesis and antiproliferative activity of
3
2
=
8
8.4 Hz, 1H, ArH), 7.14 (d, J = 6 Hz, 2H, ArH), 7.27 (d, J =
.4 Hz, 1H, ArH), 7.70 (d, J = 7.2 Hz, 2H, ArH), 8.69 (s, 1H,
13
ArH) ppm. C NMR (100 MHz, CDCl ): δ 101.15, 101.25,
1
1
3
conformationally restricted
1,2,3-triazole
analogues
of
01.35, 108.52, 109.31, 109.34, 122.78, 123.30, 124.04,
combretastatins in the sea urchin embryo model and against
human cancer cell lines, Bioorg. Med. Chem. 22 (2014) 738-755.
37.02, 147.77, 148.06, 149.56 ppm. HRMS (ESI): m/z calcd
+
for C H N O [M+H] : 267.0882; found 267.0879.
14
11
4
2
[
14] R. Shirai, H. Takayama, A. Nishikawa, Y. Koiso, Y.
Hashimoto, Asymmetric synthesis of antimitotic
Acknowledgements
combretadioxolane with potent antitumor activity against multi-
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2000.
We are grateful for support from NCI/NIH Grants CA
1
40409 (to P.A.C.) and CA 183895 (to R.L.E.), the UAMS
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Translational Research Institute (TRI), grant UL1TR000039
through the NIH National Center for Research Resources and
National Center for Advancing Translational Sciences, the
UAMS Department of Radiology the Arkansas Research
Alliance (ARA), and to the NCI Developmental Therapeutic
Program (DTP) for screening data.
[
16] N.R. Penthala, H. Zong, A. Ketkar, N.R. Madadi, V.
Janganati, R.L. Eoff, M.L. Guzman, P.A. Crooks, Synthesis, anti-
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Appendix A. Supplementary data
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(
(
[
Z)-2,3-diaryl substituted acrylonitriles, Tetrahedron Lett. 55
2014) 4207-4211.
18] N.R. Madadi, N.R. Penthala, S. Bommagani, S. Parkin, P.A.
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/....
Crooks, Crystal structure of 4,5-bis-(3,4,5-tri-meth-oxy-phen-yl)-
H-1,2,3-triazole methanol monosolvate, Acta. Crystallogr. Sect.
E: Struct. Rep. Online 70 (2014) o1128-1129.
19] N.R. Penthala, N.R. Madadi, S. Bommagani, S. Parkin, P.A.
Crooks, Comparison of crystal structures of 4-(benzo[b]thio-phen-
-yl)-5-(3,4,5-trimethoxyphenyl)-2H-1,2,3-triazole and 4-(benzo
b]thiophen-2-yl)-2-methyl-5-(3,4,5-trimethoxyphenyl)-2H-1,2,3-
2
Notes and references
[
[
1] M. Gordaliza, Natural products as leads to anticancer drugs,
Clin .Transl. Oncol. 9 (2007) 767-776.
2
[

triaz ole, Acta. Crystallogr . Sect. E: Struct. Rep. Online 70 (2014)
ACCEPTED MANUSCRIPT
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92-395.
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20] N.R. Penthala, N.R. Madadi, V. Janganati, P.A. Crooks, L-
Proline catalyzed one-step synthesis of 4,5-diaryl-2-1,2,3-triazoles
from heteroaryl cyanostilbenes via [3+2] cycloaddition of azide,
Tetrahedron Lett. 55 (2014) 5562-5565.
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21] N.H. Nam, Combretastatin-A4 analogues as anti-mitotic anti-
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S. Tosini, P. Skehan, D.A. Scudiero, A. Monks, M.R. Boyd,
Comparison of in vitro anticancer-drug-screening data generated
with a tetrazolium assay versus a protein assay against a diverse
panel of human tumor cell lines, J. Natl. Cancer. Inst. 82 (1990)
1
113-1118.
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Synthesis and anti-proliferative activity of aromatic substituted 5-
(1-benzyl-1H-indol-3-yl) methylene)-1,3-dimethylpyri midine-2,
, 6 (1H, 3H, 5H)-trione analogs against human tumor cell lines,
Bioorg. Med. Chem. Lett. 24 (2014) 601-603.
24] N.R. Madadi, H. Zong, A. Ketkar, C. Zheng, N.R. Penthala,
[
(
4
[
V. Janganati, S. Bommagani, R.L. Eoff, M.L. Guzman, P.A.
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analogues as potent anti-cancer agents that target tubulin, Med.
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25] M.J. Borrelli, D.M. Stafford, C.M. Rausch, L.J. Bernock,
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26] M.J. Borrelli, L.L. Thompson, W.C. Dewey, Evidence that
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substance, Int. J. Hyperthermia 5 (1989) 99-103.
[
27] A. Grosdidier, V. Zoete, O. Michielin, SwissDock, a protein-
small molecule docking web service based on EADock DSS,
Nucleic Acids Res. 39 (2011) W270-277.

List of Captions:
ACCEPTED MANUSCRIPT
Fig. 1. Structures of combretastatin A-4 (CA-4, A), colchicine (B), triazole
analogue (2e, C) and reported anti-tubulin compounds (D-I).
Scheme 1. Synthesis of 4,5-disubstituted 2H-1,2,3-triazoles; reagents and
conditions: (a) 5% NaOMe, MeOH, reflux. (b) NaN
Table 1. Synthesized (Z)-2,3-diarylacrylonitriles and 4,5-disubstituted-
H-1,2,3-triazoles from their corresponding acetonitrile and aldehyde
precursors.
3 4 2
, NH Cl, DMF/H O.
2
a
Table 2. Growth inhibition (GI50/µM) data of 2d, 2m, 2l, 2n and 2g
against a panel of 60 human cancer cells.
Fig. 2. Colony formation assay, 2e-treated 9LSF cells. Cells were exposed
to 2e for 24 hours. Curve was fitted as four-parameter logistic curve (f1 =
min + (max-min)/(1 + (x/EC50)^(-Hillslope))). Values represent mean ±
SEM of 3 independent colony formation experiments.
Fig 3. Cell cycle redistribution effects of 2e on 9L cells. (A) Percentage of
cell population in G2/M phase in control cells versus those exposed to 50
µM 2e or 50 µM COL for 24 hrs. (B) Representative distribution of control
cells. (C) Representative distribution of 2e-treated cells. (D)
Representative distribution of COL-treated cells.
Fig. 4. Docking poses of molecules 2e, 2h and 2l bound to tubulin.
Subunits α and β of the tubulin heterodimer are shown as gray and black
cartoons respectively. All the docking poses generated by Swissdock for
molecules 2e, 2h and 2l are shown in panels A), B) and C) respectively.
As seen clearly, majority of the poses bind to the ‘colchicine-binding’
pocket located at the interface of the two tubulin subunits for all three
molecules. 2e shows the most, while 2l shows the least number of ‘outlier’
poses.
Table 3. SwissDock statistics for the docking runs with compounds 2e, 2h
and 2l (scores shown are averages from 3 docking runs).
Fig. 5. Atomic contacts between tubulin and molecules 2e and 2l. A) 2e is
shown as orange sticks, while residues of the α and β subunit of tubulin are
in light and dark gray, respectively. B) 2l (green sticks) is shown bound at
the colchicine-binding pocket of tubulin. The color scheme is the same as
in A, while the residues of β-tubulin unique to 2l pocket are shown in
magenta.

Table 1.
Synthesized (Z)-2,3-diarylacrylonitriles and 4,5-disubstituted-2H-1,2,3-triazoles from their corresponding acetonitrile and aldehyde precursors.
ACCEPTED MANUSCRIPT
Acetonitrile
Aldehyde
(Z)-2,3-Diarylacetonitriles (1a-1s)
4,5-Disubstituted 2H-1,2,3-triazoles (2a-2s)
OCH
3
CN
CHO
OCH
3
CN
Cl
OCH3
OCH3
Cl
Cl
Cl
1
a
2a
OCH
3
CN
OCH
OCH
3
3
CHO
3
H CO
CN
OCH
3
3
3
3
H3CO
OCH3
3
H CO
H
H
H
H
3
CO
OCH
3
OCH3
OCH
3
1
b
2
b
H
3
CO
OCH
3
CN
OCH
3
CHO
CN
3CO
OCH
N
OCH
3
N
1
c
2c
CN
OCH3
OCH3
CHO
N
N
3CO
OCH
OCH3
CN
d
OCH
3
1
2
d
OCH
3
H
N
CN
CHO
OCH
3
3
N
N
H3CO
H3CO
OCH
CN
OCH3
OCH
OH
3
H CO
3CO
OCH3
OH
OCH
CN
3
OCH3
OH
1
e
2e
H
S
N
N
N
CHO
CN
Cl
F
Cl
S
S
F
Cl
F
1
f
2
f
H
N
CHO
Cl
CN
Cl
N
N
Cl
N
N
CN
Cl
N
Cl
Cl
1
g
2
g
OCH3
CN
OCH3
OCH3
O
CHO
O
O
H
3CO
OCH
3
O
CN
OCH
3
1
h
2
h
NO2
CN
CHO
H3CO
H3CO
CN
H3CO
OCH3
OCH3
OCH3
NO2
1
i
2
i

ACCEPTED MANUSCRIPT
H
N
CN
N
N
O
O
CHO
O
O
O
CN
O
1
j
2
j
Br
3
H CO
CN
CHO
H3CO
NC
Br
Br
Br
1
k
2
k
CN
H3CO
CHO
3
H CO
OCH
3
H
3
CO
OCH
3
H
3
CO
NC
OCH3
CN
OCH
3
1l
2
l
H3CO
CHO
OCH
3
H
3
CO
NC
H3CO
OCH3
OCH
3
1m
2
m
OCH
3
CN
OCH
OCH
3
3
O
O
CHO
O
O
H
H
H
3
CO
OCH
3
3
3
CN
OCH
3
1
n
2
n
OCH
3
H
N
CN
OCH3
N
CHO
N
Cl
OCH3
OCH
3
CN
Cl
3
CO
OCH
Cl
Cl
OCH3
Cl
OCH
3
Cl
H3CO
1
o
2o
H
N
OCH
3
CN
N
N
CHO
OCH
OCH
3
3
OCH3
OCH3
N
N
3
CO
OCH
N
CN
OCH
3
H3CO
1
p
2
p
OCH3
CN
CHO
OCH3
CN
H3CO
OCH3
O2N
NO
2
1
q
2
q
H
N
OCH3
N
N
CN
CHO
O
O
OCH3
O
O
O
OCH3
CN
O
H3CO
OCH3
H3CO
1
r
2
r
H
N
N
N
N
CN
N
O
O
CHO
O
O
O
O
CN
N
1
s
2
s

ACCEPTED MANUSCRIPT
Table 2.
a
Growth inhibition (GI50/µM) data of 2d, 2m, 2l, 2n and 2g against a
panel of 60 human cancer cells.
2
d
2m
2l
2h
2n
2g
Panel/cell line
(
µM
)
(
µM)
(µM)
(µM)
(µM)
(µM)
Leukemia
CCRF-CEM
0.037
0.030
0.026
0.068
0.037
0.021
0.032
0.023
<0.01
0.048
0.037
<0.01
<0.01
<0.01
<0.01
0.012
<0.01
<0.01
0.034
0.019
<0.01
0.040
0.033
<0.01
0.28
0.20
0.05
0.57
0.33
0.04
3.38
5.75
5.50
4.23
10.7
5.43
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
Lung Cancer
A549/ATCC
HOP-62
0.054
0.047
0.099
0.065
0.038
0.037
0.025
<0.01
0.038
0.034
<0.01
<0.01
<0.01
<0.01
<0.01
0.023
0.018
<0.01
0.048
0.025
0.29
0.34
0.12
0.77
0.34
4.85
5.32
3.28
9.13
3.67
HOP-92
NCI-H23
NCI-H460
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
0.228
0.230
0.040
0.037
0.214
0.035
0.041
0.149
0.046
<0.01
<0.01
0.048
0.030
0.030
0.029
0.024
<0.01
<0.01
<0.01
<0.01
<0.01
0.027
0.039
<0.01
<0.01
0.025
<0.01
<0.01
0.31
>10
0.18
0.13
0.39
0.07
0.11
2.89
9.16
2.89
2.93
4.56
6.87
4.40
KM12
SW-620
CNS Cancer
SF-268
>
0
10
0
.503
0.177
<0.01
0.069
7.36
SF-295
SF-539
SNB-75
U251
0.014
0.020
0.016
0.037
0.011
0.014
0.014
0.038
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
0.020
0.10
0.18
0.08
0.30
3.01
na
1.58
5.35
Melanoma
LOX IMVI
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-62
0.065
0.022
<0.01
0.027
>100
0.013
0.157
0.076
<0.01
<0.01
0.056
na
<0.01
<0.01
<0.01
<0.01
na
0.018
<0.01
<0.01
<0.01
>100
<0.01
>100
0.55
na
0.09
0.02
0.24
>10
>10
4.83
14.3
2.20
6.52
5.39
7.89
3.35
0.027
<0.01
<0.01
nd
Ovarian Cancer
IGROV1
>10
0
.065
0.051
0.024
na
<0.01
0.046
<0.01
0.033
5.94
0
OVCAR-3
OVCAR-4
NCI/ADR-RES
SK-OV-3
0.011
0.077
0.023
0.075
<0.01
<0.01
<0.01
<0.01
<0.01
0.034
<0.01
<0.01
0.43
3.38
3.04
3.92
2.72
0.08
0.08
0.49
Renal Cancer
7
86-0
0.043
0.033
0.081
0.043
0.092
0.015
<0.01
0.145
0.050
0.020
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
0.010
<0.01
<0.01
<0.01
0.62
0.34
0.71
0.32
0.66
2.75
3.18
1.75
8.42
6.84
A498
ACHN
CAKI-1
UO-31
Prostate Cancer
PC-3
0.046
0.027
0.042
0.045
<0.01
<0.01
0.018
0.025
0.26
0.35
4.50
1.41
DU-145
Breast Cancer
MCF7
0.027
0.025
0.046
<0.01
<0.01
<0.01
<0.01
0.044
0.08
0.53
2.58
5.89
MDA-MB-
0
.094
2
31/ATCC
HS 578T
MDA-MB-468
na
0.035
0.041
0.023
0.668
0.015
0.44
0.23
11.6
3.20
<0.01
a
na: Not analyzed, nd; not determined. GI50: 50% growth inhibition,
concentration of drug resulting in a 50% reduction in net cell growth as
compared to cell numbers on day 0.

ACCEPTED MANUSCRIPT
Table 3.
SwissDock statistics for the docking runs with compounds 2e, 2h and 2l (scores shown are averages from 3 docking runs).
Comp
FF score (kcal/mol)
∆G (kcal/mol)
2
l
-4224.7
-4216.5
-4211.4
-8.4
-8.2
-7.9
2
h
2
e

ACCEPTED MANUSCRIPT
Highlights
•
•
•
•
•
A variety of novel 4,5-disubstituted 2H-1,2,3-triazoles were synthesized from there corresponding
(Z)-substituted diarylacrylonitriles.
The synthesized analogs were evaluated for their anti-cancer activity against NCI 60 human cancer
cell lines.
Conducted in silico docking studies to rationalize the anti-cancer properties of the synthesized
compounds.
Cell cycle redistribution assay was conducted to further understand the mechanism of action of the
synthesized CA-4 analogues.
One of the analogue 2e showed promising anticancer activity with an LD value of 7.5 nM against
5
0
9
LSF rat gliosarcoma cells.

ACCEPTED MANUSCRIPT
Supporting Information
Synthesis and biological evaluation of novel 4,5-disubstituted 2H-1,2,3-triazoles as cis-
constrained analogues of combretastatin A-4
a
a
ab
c
c
Nikhil R. Madadi , Narsimha R. Penthala , Kevin Howk , Amit Ketkar , Robert L. Eoff ,
b
a*
Michael J. Borrelli and Peter A. Crooks
Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, USA
Department of Radiology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, USA
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, U.S.A.
a
b
c
*
Corresponding author: Tel.: +1-501-686-6495; fax: +1-501-686-6057; E-mail: pacrooks@uams.edu.

ACCEPTED MANUSCRIPT
1
Representative H NMR spectra of compound 4-(3,4-dichlorophenyl)-5-(3,4-dimethoxy
phenyl)-2H-1,2,3-triazole (2a)
1
3
Representative C NMR spectra of compound 4-(3,4-dichlorophenyl)-5-(3,4-dimethoxy
phenyl)-2H-1,2,3-triazole (2a)

ACCEPTED MANUSCRIPT
Representative HRMS spectra of compound 4-(3,4-dichlorophenyl)-5-(3,4-dimethoxy phenyl)-
2
H-1,2,3-triazole (2a)

ACCEPTED MANUSCRIPT
1
Representative H NMR spectra of compound 4,5-bis(3,4,5-trimethoxyphenyl)-2H-1,2,3-
triazole (2b)
1
3
Representative CNMR spectra of compound 4,5-bis(3,4,5-trimethoxyphenyl)-2H-1,2,3-
triazole (2b)

ACCEPTED MANUSCRIPT
Representative HRMS spectra of compound 4,5-bis(3,4,5-trimethoxyphenyl)-2H-1,2,3-triazole
(2b)

ACCEPTED MANUSCRIPT
1
Representative H NMR spectra of compound 4-(5-(3,4,5-trimethoxyphenyl)-2H-1,2,3-triazol-
-yl)quinolone (2c)
4
1
3
Representative C NMR spectra of compound 4-(5-(3,4,5-trimethoxyphenyl)-2H-1,2,3-triazol-
-yl)quinolone (2c)
4

ACCEPTED MANUSCRIPT
Representative HRMS spectra of compound 4-(5-(3,4,5-trimethoxyphenyl)-2H-1,2,3-triazol-4-
yl)quinolone (2c)

ACCEPTED MANUSCRIPT
1
Representative H NMR spectra of compound 2-(5-(3,4,5-trimethoxyphenyl)-2H-1,2,3-triazol-
-yl)quinolone (2d)
4
1
3
Representative C NMR spectra of compound 2-(5-(3,4,5-trimethoxyphenyl)-2H-1,2,3-triazol-
-yl)quinolone (2d)
4

ACCEPTED MANUSCRIPT
Representative HRMS spectra of compound 2-(5-(3,4,5-trimethoxyphenyl)-2H-1,2,3-triazol-4-
yl)quinolone (2d)

ACCEPTED MANUSCRIPT
1
Representative H NMR spectra of compound 2-methoxy-5-(5-(3,4,5-trimethoxyphenyl)-2H-
,2,3-triazol-4-yl)phenol (2e)
1
1
3
Representative C NMR spectra of compound 2-methoxy-5-(5-(3,4,5-trimethoxyphenyl)-2H-
,2,3-triazol-4-yl)phenol (2e)
1

ACCEPTED MANUSCRIPT
Representative HRMS spectra of compound 2-methoxy-5-(5-(3,4,5-trimethoxyphenyl)-2H-
1
,2,3-triazol-4-yl)phenol (2e)

ACCEPTED MANUSCRIPT
1
Representative H NMR spectra of compound 4-(4-chloro-3-fluorophenyl)-5-(thiophen-3-yl)-
H-1,2,3-triazole (2f)
2
1
3
Representative C NMR spectra of compound 4-(4-chloro-3-fluorophenyl)-5-(thiophen-3-yl)-
H-1,2,3-triazole (2f)
2