Synthesis, anticancer activity and molecular docking studies on a series of heterocyclic trans-cyanocombretastatin analogues as antitubulin agents

Article abstract of DOI:10.1016/j.ejmech.2014.12.050

A series of heterocyclic combretastatin analogues have been synthesized and evaluated for their anticancer activity against a panel of 60 human cancer cell lines. The most potent compounds were two 3,4,5-trimethoxy phenyl analogues containing either an (Z

Full text of DOI:10.1016/j.ejmech.2014.12.050

European Journal of Medicinal Chemistry 92 (2015) 212e220
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, anticancer activity and molecular docking studies on a
series of heterocyclic trans-cyanocombretastatin analogues as
antitubulin agents
Narsimha Reddy Penthala a, Hongliang Zong b, Amit Ketkar c, Nikhil Reddy Madadi a,
,
*
Venumadav Janganati a, Robert L. Eoff c, Monica L. Guzman b, Peter A. Crooks a
a Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, USA
b Division of Hematology/Oncology, Department of Medicine, Weill Cornell Medical College, New York, NY 10021, USA
c Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of heterocyclic combretastatin analogues have been synthesized and evaluated for their anti-
cancer activity against a panel of 60 human cancer cell lines. The most potent compounds were two
3,4,5-trimethoxy phenyl analogues containing either an (Z)-indol-2-yl (8) or (Z)-benzo[b]furan-2-yl (12)
moiety; these compounds exhibited GI50 values of <10 nM against 74% and 70%, respectively, of the
human cancer cell lines in the 60-cell panel. Compounds 8, and 12 and two previously reported com-
pounds in the same structural class, i.e. 29 and 31, also showed potent anti-leukemic activity against
leukemia MV4-11 cell lines with LD50 values ¼ 44 nM, 47 nM, 18 nM, and 180 nM, respectively. From the
NCI anti-cancer screening results and the data from the in vitro toxicity screening on cultured AML cells,
seven compounds: 8, 12, 21, 23, 25, 29 and 31 were screened for their in vitro inhibitory activity on
tubulin polymerization in MV4-11 AML cells; at 50 nM, 8 and 29 inhibited polymerization of tubulin by
>50%. The binding modes of the three most active compounds (8, 12 and 29) to tubulin were also
investigated utilizing molecular docking studies. All three molecules were observed to bind in the same
Received 16 August 2014
Received in revised form
21 November 2014
Accepted 28 December 2014
Available online 29 December 2014
Keywords:
Trans-cyanocombretastatin analogues
Anti-cancer activity
Leukemia cell lines
Molecular docking
Tubulin binding
hydrophobic pocket at the interface of a- and b-tubulin that is occupied by colchicine, and were stabi-
lized by van der Waals’ interactions with surrounding tubulin residues. The results from the tubulin
polymerization and molecular docking studies indicate that compounds 8 and 29 are the most potent
anti-leukemic compounds in this structural class, and are considered lead compounds for further
development as anti-leukemic drugs.
© 2014 Published by Elsevier Masson SAS.
1. Introduction
Structurally related cyanocombretastatin analogues [8,9] (Fig. 1,
III) and similar analogues that incorporate different heterocyclic
A variety of natural products has been isolated from the bark of
the South African tree Combretum caffrum which includes the
combretastatins [1,2]. Combretastatins (Fig. 1, I and II) have been
shown to be cytotoxic, with combretastatin A-4 (CA-4, Fig. 1, I)
being the most potent [3,4]. CA-4 inhibits tubulin polymerization,
and competitively inhibits the binding of radiolabeled colchicines
to tubulin. CA-4 also exhibits potent cytotoxicity against a variety of
cancer cell lines, and has been shown to be active against multidrug
resistant (MDR) cancer cell lines [5e7].
moieties, such as indole, benzothiophene, quinoline and quinazo-
line have also been reported as cytotoxic compounds, and are
potent inhibitors of tubulin polymerization potencies comparable
to that of CA-4 [10e13]. In this respect, the 3,3-diarylacrylonitrile
analog CC-5079 has been reported as a novel synthetic tubulin
polymerization inhibitor with potential use in cancer chemo-
therapy [14] and 2,3-diarylacrylonitriles have emerged as impor-
tant synthons for the synthesis of a wide spectrum of biologically
active molecules [15]. Such compounds have been shown to
possess spasmolytic, estrogenic, hypotensive, antioxidant, tuber-
culostatic, antitrichomonal, and insecticidal properties [16].
Many natural medicinal agents, such as the combretastatins, the
* Corresponding author.
E-mail address: pacrooks@uams.edu (P.A. Crooks).
colchicines (Fig. 1, IV), and the podophyllotoxins, possess
a
http://dx.doi.org/10.1016/j.ejmech.2014.12.050
0223-5234/© 2014 Published by Elsevier Masson SAS.

N.R. Penthala et al. / European Journal of Medicinal Chemistry 92 (2015) 212e220
213
Fig. 2. Z-Benzo[b]thiophen-2-yl cyanocombretastatin analogues (29e31) [11].
Fig. 1. Chemical structures of potent antitubulin agents.
trimethoxyphenyl moiety in their structure. The cytotoxic proper-
ties of such compounds are believed to be related to their anti-
tubulin properties [17e21]. A large number of CA-4 derivatives
have been synthesized and evaluated for anti-tubulin activity
[22e29]. SAR studies have revealed that a 30,40,50-trimethox-
yphenyl moiety and a cis-configuration of the olefinic bond in these
compounds are essential structural elements for biological activity
[30,31]. The presence of the cis-ethylene bridge that connects the
two aryl rings (two planar rings tilted at 50e600 to each other
[1,31]) is believed to be the key structural factor that holds these
structural moieties at an appropriate distance apart, to maintain
the correct dihedral angle that maximizes interaction with the
colchicine binding site on tubulin protein [32]. We have recently
reported on the synthesis and anti-cancer activities of a series of
(Z)-quinolinylcyano- (V) and (E)- and (Z)-benzothiophene cyano-
combretastatin analogues (Fig. 1, VI) [11,12], and have determined
that such analogues (e.g. compound 29, Fig. 2) appear to overcome
cell-associated P-glycoprotein (P-gp)-mediated resistance in tumor
cells, since they are equipotent in inhibiting both OVCAR8 and NCI/
ADR-RES cell growth [11].
Previous studies on combretastatin analogues have shown that
replacement of the 3-hydroxy-4-methoxyphenyl moiety of CA-4
with heterocyclic groups such as quinoline, quinazoline and ben-
zothiophene results in improved anti-cancer activity [11,12,33]. In
the present work, we have synthesized a variety of heterocyclic (Z)-
cyanocombretastatin analogues that incorporate 2- and 3-indolyl,
2- and 3-benzofuranyl, 2-benzothiophenyl, and 2-benzothiazolyl
moieties as replacements for the 3-fluoro-4-methoxyphenyl
group in (Z)-cyano CA-4 [9] (Fig. 1, III).
Scheme 1. Synthesis of (Z)-indol-2-yl, (Z)-benzo[b]furan-2-yl, and (Z)-benzo[d] thia-
zol-2-yl acrylonitriles (8e17).
3-yl cyanocombretastatin analogues (25e27) were synthesized by
refluxing a variety of indole-3-carbaldehydes (18e19) or benzo[b]
furan-3-carbaldehyde (20) with appropriate phenylacetonitriles
(5e7) in 5% sodium methoxide/methanol (Scheme 2).
2.2. Biological evaluation
2.2.1. In vitro growth inhibition and cytotoxicity
All compounds were evaluated for their cytotoxic potency in a
preliminary screen against a panel of 60 human cancer cell lines
(NCI-60 panel) at a single analogue concentration (10ꢀ5 M). The 60
cell line panel is organized into subpanels representing leukemia,
non-small cell lung, colon, central nervous system, melanoma,
2. Results and discussion
2.1. Drug synthesis
(Z)-Indol-2-yl cyanocombretastatin analogues (8e11), (Z)-benzo
[b]furan-2-yl cyanocombretastatin analogues (12e14) and (Z)-
benzo[d]thiazol-2-yl cyanocombretastatin analogues (15e17) were
synthesized by refluxing the appropriate indole-2-carbaldehyde
(1), 4-cyanobenzyl substituted indole-2-carbaldehyde (2), benzo-
[b]furan-2-carbaldehyde (3) or benzo[d]thiazole-2-carbaldehyde
(4) with
a
variety of phenylacetonitriles, i.e., 3,4,5-
trimethoxyphenylacetonitrile (5), 3,4-dimethoxyphenyl acetoni-
trile (6), and 3,5-dimethoxyphenyl acetonitrile (7), in 5% sodium
methoxide/methanol (Scheme 1). Confirmation of the structure
and purity of these analogues was obtained from 1H NMR, 13C NMR
and high resolution mass spectroscopic analysis. The geometry of
the double bond (E- or Z-configuration) in these molecules was
established as the (Z)-isomer from single crystal X-ray crystallo-
graphic data [34,35].
Scheme 2. Synthesis of (Z)-indole-3-yl, and (Z)-benzo[b]furan-3-yl cyanocom-
bretastatins (21e27).
A second series of indole-3-yl (21e24), and (Z)-benzo[b]furan-

214
N.R. Penthala et al. / European Journal of Medicinal Chemistry 92 (2015) 212e220
Table 1
Growth inhibition (GI50
(12, 13), and (Z)-benzo[d]thiazol-2-yl (15 17) cyano combretastatin analogues.
Table 2
/m
M)a data for (Z)-indol-2-yl (8 11), (Z)-benzo[b]furan-2-yl
Growth inhibition (GI50
furan-3-yl (25) cyanocombretastatin analogues.
/m
M)a data for (Z)-indole-3-yl (21e24) and (Z)-benzo[b]
Panel/cell line
8
9
11
12
13
15
17
Panel/cell line
21
22
23
24
25
m
M
mM
m
M
m
M
m
M
m
M
m
M
m
M
m
M
mM
m
M
m
M
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
0.017 0.062
<0.01 0.034
<0.01 0.039
0.023 0.084
<0.01 0.151
<0.01 0.042
0.136
0.031 <0.01
0.039 <0.01
0.274
0.199
na
0.024 0.777 0.055 na
0.058
0.048
0.040
0.131
0.231
0.022
2.74
4.57
1.65
3.99
3.96
1.45
0.042
0.031
0.043
0.049
0.065
0.033
0.064
0.059
0.044
0.198
0.241
0.027
1.41
1.03
0.513
3.82
2.47
0.681
0.481 0.066 0.345
0.467 0.051 0.333
3.38
0.047 0.548 9.20
0.019 1.32
0.237 2.29
<0.01
0.470 0.107 0.402
Lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
Lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
<0.01 0.114
0.076 <0.01
0.520 0.379 na
0.078
0.596
0.082
1.01
0.194
0.133
nd
5.16
18.5
8.69
5.26
20.9
7.19
24.3
4.51
3.05
0.061
0.478
0.273
0.068
8.18
0.256
nd
0.039
0.027
0.086
0.485
0.133
0.083
0.170
0.101
0.044
0.037
0.086
0.665
na
1.16
0.307
na
0.059 na
na
na
<0.01 na
0.092 <0.01
0.077 <0.01
2.29
1.13
34.5
3.11
6.18
1.93
nd
0.844 4.11
3.25
2.29
3.35
0.534
6.98
3.02
6.20
4.58
19.1
0.839
0.261
<0.01 0.058
<0.01 0.399
<0.01 0.236
14.5
<0.01
0.246 <0.01
0.465 <0.01
0.093 <0.01
0.033 <0.01
2.84
>10.0
nd
0.735
<0.01 0.053
<0.01 0.049
0.472 0.336 2.86
0.438 0.022 na
0.041
0.037
0.016 0.232
0.018 0.366
<0.01 0.044
<0.01 0.048
<0.01 0.048
<0.01 0.076
<0.01 0.042
0.097 <0.01
0.404 0.036 0.574
0.474
0.107
0.048
0.051
0.038
0.043
0.047
5.31
12.6
35.5
3.70
3.32
4.04
3.53
0.047
0.207
0.042
0.053
0.038
0.040
0.044
0.051
0.119
0.045
0.068
0.038
0.044
0.046
0.449
4.13
0.265
0.01
2.33
0.297 8.39
0.047 <0.01
0.089 <0.01
0.048 <0.01
0.047 <0.01
0.048 <0.01
0.418 0.052 0.404
0.469 0.045 0.414
0.373 0.037 0.438
0.356 0.278 2.01
0.388 0.038 0.343
0.517
0.492
0.394
0.586
0.504
0.015 1.48
<0.01 0.036
<0.01 0.047
<0.01 0.189
<0.01 0.036
<0.01 0.075
0.086
0.247 2.05
1.03
3.09
2.54
18.9
4.92
8.42
24.2
6.04
4.97
0.078
0.045
0.052
0.062
0.034
0.045
2.14
4.79
0.037 <0.01
0.039 <0.01
0.085 <0.01
0.039 <0.01
0.044 <0.01
0.708 0.508 2.46
0.042
0.099
0.076
0.042
0.050
0.042
0.071
0.089
0.043
0.050
0.446
0.442
3.63
0.769
0.605
1.56
5.55
1.78
0.097 1.68
2.49
>10.0
0.145 2.13
U251
0.760 1.65
1.91
U251
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
<0.01 0.064
0.090 <0.01
0.930 0.073 1.84
0.629 0.515 0.699
0.437 0.193 1.21
0.229 0.021 0.199
0.487 0.287 0.958
0.075
12.5
0.053
0.024
na
0.090
0.042
11.3
5.32
9.91
3.75
3.95
4.70
6.25
4.27
11.9
6.98
0.078
nd
0.074
5.85
0.052
0.027
na
0.092
0.036
10.7
0.993
0.868
0.667
0.278
0.304
1.61
0.336
nd
0.519
8.28
nd
nd
59.9
<0.01 0.039
<0.01 0.021
<0.01 0.312
0.056 <0.01
0.024 <0.01
0.528 <0.01
0.161 <0.01
0.060 <0.01
0.062 63.2
0.058 <0.01
0.042
0.020
0.051
0.081
0.048
nd
na
<0.01 0.142
1.06 27.0
1.94
3.51
0.564 0.100 1.37
18.0 >100 na
0.557 0.052 0.755
1.81
1.98
<0.01 0.038
0.048
0.049
0.059
<0.01 0.482
<0.01 0.034
0.183
0.022 1.27
1.27
0.279 0.031 0.285
3.86 >10.0
0.706
0.417
0.033
0.338
10.6
0.121
0.039
0.051
21.0
5.68
22.1
>100
7.38
1.70
11.9
0.347
0.031
3.52
0.197
0.103
0.033
0.117
0.379
0.034
1.35
0.516
0.148
0.037
0.056
3.06
0.277
2.61
3.33
2.90
0.382
1.71
0.042 <0.01
15.2
0.08
0.735
0.471
0.672
0.259
0.039 7.19
0.042 5.23
0.347 4.85
0.438 3.13
<0.01 0.326
<0.01 0.040
<0.01 0.394
0.124 <0.01
0.073 <0.01
0.087 <0.01
3.98
0.317 0.059 1.47
2.49
5.84
0.626 1.79
<0.01 0.602
<0.01 0.040
<0.01 0.082
<0.01 0.103
<0.01 0.151
<0.01 0.779
<0.01 5.56
<0.01 0.071
0.137 <0.01
0.029 <0.01
1.06 2.44
0.090
0.035
1.76
0.058
0.036
0.699
11.1
14.1
3.36
23.7
4.34
4.48
8.10
13.2
14.3
0.077
0.035
nd
0.035
0.083
0.077
10.3
0.085
0.025
1.66
0.072
0.033
0.715
20.1
6.11
0.458
1.94
0.650
0.496
2.17
A498
ACHN
CAKI-1
RXF 393
SN12C
0.540 0.198 0.417
0.089 0.518
0.492 0.047 0.482
A498
ACHN
CAKI-1
RXF 393
SN12C
0.079
0.217 12.4
0.035 <0.01
0.055 <0.01
0.269 <0.01
1.30
0.148 0.551
0.956 0.515 4.68
TK-10
UO-31
20.1
0.201
73.6
2.29
2.28
3.79
0.063 2.78
0.083 0.679
TK-10
UO-31
2.10
2.89
1.41
0.663
1.26
Prostate cancer
PC-3
DU-145
Prostate cancer
PC-3
DU-145
<0.01 0.062
<0.01 0.206
0.105 <0.01
0.207 <0.01
2.41
1.30
0.341 2.74
0.333 1.07
0.054
0.132
7.85
22.6
0.075
0.130
0.054
0.153
1.84
1.88
Breast cancer
MCF7
MDA-MB-231/ATCC 0.014 0.291
HS 578T
BT-549
T-47D
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
0.016 0.289
0.039
0.294
0.092 <0.01
0.023 0.446 0.034 0.399
0.028 1.21 0.992 6.74
1.88
0.029 0.950 12.2
4.34 0.996 nd
0.034
0.245
0.131
1.94
0.058
0.041
3.10
7.18
12.9
5.38
6.83
na
0.041
0.644
0.611
0.049
nd
0.036
0.299
0.124
0.348
0.065
0.035
0.376
1.71
2.03
0.927
0.811
0.237
<0.01 0.133
<0.01 0.0978 0.109
1.29
2.01
3.45
1.18
2.50
0.573
95.2
0.041
T-47D
MDA-MB-468
MDA-MB-468
0.022 0.350
0.022 0.421 0.084 2.04
0.051
na: Not analyzed, nd: not determined, aGI50: 50% growth inhibition, concentration of
drug resulting in a 50% reduction in net cell growth as compared to cell numbers on
day 0.
na: Not analyzed, nd: not determined, aGI50: 50% growth inhibition, concentration of
drug resulting in a 50% reduction in net cell growth as compared to cell numbers on
day 0.

N.R. Penthala et al. / European Journal of Medicinal Chemistry 92 (2015) 212e220
215
ovary, renal, prostate, and breast cancer cell lines. The compounds
were considered for progression to full five-concentration assay if
they reduced the growth of cancer cells to 60% or more in at least
eight of the 60 cell lines screened. The single dose results are
expressed as the percent growth inhibition of treated cells at the
test concentration of 10ꢀ5 M following 48 h of incubation. From the
preliminary screens, compounds 8, 9, 11e13, 15, 17 and 21e25 were
selected as leads for more comprehensive studies designed to
determine GI50, TGI and LC50 values, which represent the molar
drug concentration required to cause 50% growth inhibition, total
growth inhibition, and the concentration that kills 50% of the cells,
respectively. The compounds were evaluated using five different
concentrations at 10-fold dilutions (10ꢀ4 M, 10ꢀ5 M, 10ꢀ6 M, 10ꢀ7 M
and 10ꢀ8 M) and incubations were carried out over 48 h exposure
to drug. Trans-cyanostilbenes analogues which contained the 3,4,5-
trimethoxyphenyl (8, 11, 12, 15, 21, 24 and 25) and dimethox-
yphenyl (9, 13, 17, 22 and 23) moieties exhibited low micromolar
level growth inhibition in subsequent five dose screening assays
against all 60 human cancer cell lines in the panel. The growth
inhibition results of the most potent of these compounds are pre-
sented in Tables 1 and 2. We hypothesized that the cytotoxic ac-
tivity of these novel analogues was likely due to their interaction
with the colchicine binding site on tubulin.
screened was 2.59
mM.
2.2.4. Benzo[d]thiazol-2-yl analogues
The substitution of a benzofuran-2-yl moiety in compound 12
for a benzo[d]thiazol-2-yl moiety afforded compound 15 [(Z)-3-
(benzo[d]thiazol-2-yl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile],
which exhibited potent growth inhibition against 94% of the cancer
cells in the 60-cell panel, with GI50 ranging from 0.021 to 12.2
The average GI50 value for this compound against all the cancer cell
lines screened was 0.93 M. This compound exhibited potent
growth inhibition against 73% of the cancer cell lines screened with
GI50 ¼ <1 M (Table 1) and exhibited potent growth inhibition
against MDA-MB-435 melanoma cancer cells with a GI50 of
0.021 M (Table 1).
mM.
m
m
m
Substitution of the 3,4,5-trimethoxyphenyl group in 15 for the
3,5-dimethoxyphenyl moiety afforded compound 17 [(Z)-3-(benzo
[d]thiazol-2-yl)-2-(3,5-dimethoxyphenyl)acrylonitrile],
exhibited potent growth inhibition against 94% of the cancer cells
in the 60-cell panel with GI50 ranging from 0.199 to 8.39 M. The
average GI50 value for this compound against all the cancer cells in
the panel was 1.87 M. This compound exhibited potent growth
inhibition against only 38% of the cancer cell lines screened with
GI50 ¼ <1 M (Table 2). This compound exhibited potent growth
inhibition against MDA-MB-435 melanoma cancer cells with a GI50
value of 0.199 M (Table 1).
which
m
m
m
2.2.2. Indol-2-yl analogues
m
Compound 8 [(Z)-3-(1H-indol-2-yl)-2-(3,4,5-trimethoxyphen-
yl)acrylonitrile] exhibited GI50 values ranging from <0.01
m
M to
2.2.5. Indol-3-yl analogues
1.16 M in 95% of the cancer cell lines screened, and showed potent
m
The indol-3-yl analog (21) of compound 8 exhibited good
growth inhibition in the 60-cell screen with GI50 values ranging
growth inhibition (GI50 ¼ <0.01
mM) in 74% of the cancer cells in the
60-cell panel (Table 1).
from 0.022
Compound 21 showed potent growth inhibition against SR leuke-
M), and exhibited <1 M growth inhibition
mM to 12.5 mM against all the cancer cell lines screened.
The 3,4-dimethoxyphenyl acrylonitrile analog of 8, compound 9,
which lacks the 5-methoxy group on the phenyl ring, also exhibited
potent growth inhibition against 93% of the cancer cell lines in the
mia cells (GI50 ¼ 0.022
m
m
against 83% of the cancer cell lines screened (Table 2).
panel, with GI50 values ranging from 0.021 to 0.779
m
M, and
The indol-3-yl analog (22) of compound 9 exhibited growth
inhibition against all the cancer cell lines screened with GI50 values
afforded an average GI50 value of 0.80 M against all the cancer cell
m
lines in the panel (Table 1). This compound exhibited potent
growth inhibition against MDA-MB-435 melanoma cancer cells
ranging from 1.45 mM to >100 mM (Table 2).
Replacement of the 3,4-dimethoxyphenyl group in compound
22 with a 3,5-dimethoxyphenyl moiety afforded compound 23 [(Z)-
3-(1H-indol-3-yl)-2-(3,5-dimethoxyphenyl)acrylonitrile], which
exhibited good growth inhibition against all the cells in the panel
with a GI50 ¼ 0.021
mM (Table 1).
The introduction of an N-(4-cyanobenzyl) group into the
structure of compound 8 afforded compound 11 [(Z)-4-((2-(2-
cyano-2-(3,4,5-trimethoxyphenyl)vinyl)-1H-indol-1-yl)-methyl)
with GI50 values ranging from 0.020
showed potent growth inhibition against melanoma MDA-MB-435
cancer cells with a GI50 value of 0.02 M. Compound 23 exhibited
<1 M growth inhibition against 85% of the cancer cell lines
screened (Table 2).
mM to 10.3 mM. This compound
benzonitrile], which exhibited GI50 values ranging from 0.024
to 0.672 M in 95% of the cancer cell lines screened and showed
potent growth inhibition properties in all five leukemia cell lines,
with GI50 values in the range 0.031e0.274 M. Compound 11 also
showed potent growth inhibition against the MDA-MB-435 mela-
noma cell line (GI50 ¼ 0.024 M), and exhibited growth inhibition <
M against 93% of the cancer cells in the 60-cell panel (Table 1).
mM
m
m
m
m
Replacement of the indol-3-yl group in compound 21 with a 5-
methoxyindol-3-yl moiety afforded compound 24 [(Z)-3-(5-
methoxy-1H-indol-3-yl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile],
which exhibited good growth inhibition against all the cancer cells
m
1
m
2.2.3. Benzofuran-2-yl analogues
Substitution of the 2-indolyl moiety in compound 8 for a
benzofuran-2-yl moiety afforded compound 12 [(Z)-3-(benzo-
in the panel, with GI50 values ranging from 0.025 to 20.1
compound exhibited potent growth inhibition against A498 renal
cancer cells with a GI50 value of 0.022 M. Compound 24 exhibited
<1 M growth inhibition against 88% of the cancer cell screened
mM. This
m
furan-2-yl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile],
exhibited good growth inhibition against all the cancer cells in the
60-cell panel with GI50 values ranging from <0.01 to 73.6 M. This
compound exhibited remarkably potent growth inhibition against
M (Table 1).
which
m
(Table 2).
m
2.2.6. Benzofuran-3-yl analogues
Replacement of the indol-3-yl group in compound 21 with a
benzofuran-3-yl moiety afforded compound 25 [(Z)-3-(benzo-
70% of the cancer cell lines screened with GI50 ¼ <0.01
m
Substitution of the 3,4,5-trimethoxyphenyl group in 12 for the
3,4-dimethoxyphenyl moiety afforded compound 13 [(Z)-3-
(benzofuran-2-yl)-2-(3,4-dimethoxyphenyl)acrylonitrile], which
exhibited good growth inhibition against 54% the cancer cells in the
furan-3-yl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile],
exhibited good growth inhibition against all the cancer cells in the
panel, with GI50 values ranging from 0.237 to 19.1 M. The average
GI50 value of this compound against all the cancer cell lines
screened was 1.99 M. This compound exhibited potent growth
inhibition against 52% of the cancer cell lines screened with GI50
values <1 M (Table 2).
which
m
panel with GI50 ranging from 0.229 to 0.996
exhibited potent growth inhibition of MDA-MB-435 melanoma
cancer cells with a GI50 value of 0.229 M (Table 1). The average
GI50 value of this compound against all the cancer cell lines
mM. This compound
m
m
m

216
N.R. Penthala et al. / European Journal of Medicinal Chemistry 92 (2015) 212e220
From structure-activity relationships (SAR) it is evident of that
the heterocyclic series of trans-cyano CA-4 analogues containing
benzo[b]thiophene-2-yl [11], indole-2-yl, benzofuran-2-yl and
benzothiazole-2-yl moieties exhibited more growth inhibitory ac-
tivity than the corresponding isomeric benzo[b]thiophene-3-yl,
indole-3-yl, benzofuran-3-yl analogues. Also, analogues containing
a 3,4,5-trimethoxyphenyl group generally exhibited better growth
inhibition properties than analogues containing
a
3,4-
dimethoxyphenyl or 3,5-dimethoxyphenyl moiety. Importantly,
introduction of a 2-benzo[b]thiophenyl or 2-indolyl heterocyclic
moiety in place of the 3-fluoro-4-methoxyphenyl moiety of trans-
cyano CA-4 [9] (Fig. 1, III) dramatically improved anti-cancer
activity.
2.3. Anti-leukemic activity
Fig. 3. Activity of compound 29 on 12 different leukemia cell lines.
Previously, we have reported on the synthesis of (Z)-benzo[b]
thiophen-2-yl acrylonitriles (29e31) (Fig. 2) [11] as potent anti-
cancer agents and we have evaluated the biological activity of
these compounds against PC3 prostate cancer cell lines [11].
Compound 29 was also screened for its in vitro inhibitory activity on
tubulin polymerization in these prostate cancer cells utilizing both
an immunofluorescence assay and by using antibody against
tubulin [11]. These benzo[b] thiophen-2-yl acrylonitrile com-
pounds were also able to overcome P-glycoprotein (P-gp)-mediated
resistance in PC3-DR prostate cancer cell lines and also exhibited a
concentration-dependent anti-tubulin interaction in both PC3 and
PC3-DR prostate cancer cells [11]. We have also reported that the
thiophen-2-yl analogue 29 exhibits more potent growth inhibition
than the isomeric thiophen-3-yl analogue 31 [11].
Fig. 4. Activity of compound 29 in a primary leukemia sample (blast crisis of chronic
myeloid leukemia) at 24 h, 48 h and 96 h time-points.
2.4. Tubulin polymerization inhibition
Based upon the 60-cell screening results from the leukemia cell
lines (Tables 1 and 2) and the data from the in vitro toxicity studies
on cultured AML cells (Table 3), seven compounds: 8, 12, 21, 23, 25,
29 and 31, were screened for their in vitro inhibitory activity on
tubulin polymerization utilizing both an immunofluorescence
assay and by using antibody against tubulin (a marker for dynamic
microtubules) (Fig. 5) [36].
MV4-11 cells were treated with the above seven compounds at
doses of 25, 50 and 100 nM for 2 h and cell-based tubulin depo-
lymerization assays performed. The polymerized tubulin in the
pellet (P) and unpolymerized tubulin in the supernatant (S) were
Compounds 29 and 30 both exhibited low nanomolar range
growth inhibition (GI50 < 10 nM) against all six leukemia cell lines
in the NCI 60 cell line panel [11]. These results indicate that (Z)-
benzo[b]thiophene analog 29 is
a potential anti-leukemic
compound.
In the current study, the novel cyanocombretastatin hetero-
aromatic analogues 8, 11e17, 21e25 and the previously reported
(Z)-benzo[b]thiophene CA-4 analogs 29e31 [11] were evaluated for
their anti-leukemic activity against the MV4-11 AML cell line
(Table 3). From these studies, analogues 8, 12 and 29 were deter-
mined to be the most active compounds against this leukemia cell
line.
Compound 29 was further screened against a panel of 12
different primary leukemia cell lines, and showed
a dose-
dependent toxicity against most of the cell lines tested (Fig. 3).
The average LD50 value for the 12 different leukemia cell lines was
132 nM (range: 18.0e271 nM). Specifically, MV4-11 cells exhibited
the most sensitivity to compound 29 (LD50 ¼ 18 nM), whereas THP-
1 and MLL-ENL cells were the most resistant cell line (LD50 ¼ 227.0
and 271 nM, respectively) (Fig. 3).
We also tested the activity of 29 against primary blast crisis
chronic myeloid leukemia (CML) cells and found that the response
to 29 was also time-dependent (Fig. 4.). We observed that at
96 h more than 50% of the cells were apoptotic/dead after exposure
to drug concentrations as low as 25 nM. We have also found that
the activity of 29 was mediated via the activation of caspase cas-
cades (MLG/HZ personal communication).
Fig. 5. Effect of trans-cyano CA-4 analogues 8, 12, 21, 23, 25, 29 and 31 to inhibit
tubulin polymerization in MV4-11 cells.
Table 3
Anti-leukemic activity (LD50) of the most potent compounds against the MV4-11 AML cell line.
Compound
LD50 M)
Compound
LD50 M)
8
11
0.369
22
12
0.047
23
0.375
13
>20
24
14
1.169
25
4.529
15
0.233
29
0.018
16
1.223
30
6.063
17
4.339
31
0.180
(
m
0.044
21
0.565
(
m
>20
0.467

N.R. Penthala et al. / European Journal of Medicinal Chemistry 92 (2015) 212e220
217
detected by immunoblotting using antibody against tubulin.
Compounds 8, 12, and 29 all demonstrated >50% inhibition of
tubulin polymerization at 50 nM concentration (Fig. 5).
b-tubulin. However, the heteroatom-ring in the two compounds is
rotated by 180ꢁ with respect to one another. The 3,4,5-
trimethoxyphenyl moiety is stabilized by Cys241, Leu242, Leu248,
Asp251, Lys254, Leu255, Met259, Val315 and Val318. Thus, the
binding modes of compounds 12 and 29 are exactly superimpos-
able on each other, except for their oppositely oriented hetero-
atoms (‘O’ in 12 and ‘S’ in 29). The orientation of compound 8, on
the other hand, is not superimposable on those of 12 and 29,
although 8 also occupies the same binding pocket at the interface of
2.5. Molecular docking
The binding modes of three of the most active compounds (8,12,
and 29) were determined at the colchicine binding site on tubulin
using in silico molecular docking protocols. Chemical structures of
the molecules were drawn using Marvin Sketch (ChemAxon).
a
- and
In summary, our molecular docking results were able to explain
the trend in potencies observed for the three chosen compounds.
This is further reflected in the free energy G) values
b-tubulin.
Atomic coordinates for the a,b-tubulin heterodimer were derived
from the PDB file 1SA0 of the crystal structure of tubulin-colchicine
complex. Input coordinate files for both the protein and the com-
pounds were generated using the Dock Prep module in the UCSF-
Chimera software.
(
D
of ꢀ8.12, ꢀ7.74 and ꢀ7.65, kcal/mol for compounds 29, 12 and 8,
respectively.
Docking was performed using SwissDock (http://www.
swissdock.ch/), which employs the EADock DSS algorithm to
generate binding modes [37], estimate CHARMM energies [38],
account for solvent effects using the FACTS implicit solvation model
[39], and rank binding modes with the most favorable energies.
The top binding poses for the above three compounds at the
colchicine-binding site of tubulin are shown in Fig. 6. The molecules
are structurally very similar, differing only in the nature of the
heterocyclic moiety in the molecule. All three of these heterocyclic
3. Conclusions
Novel heterocyclic cyanocombretastatin A-4 analogues have
been synthesized and evaluated for their anticancer activity against
a panel of 60 human cancer cell lines. Compounds containing a
trimethoxyphenyl moiety or a dimethoxyphenyl moiety showed
potent growth inhibition, with GI50 values generally <1 mM against
most of the cancer cell lines used, with 8 and 12 being the most
potent compounds. The novel cyanocombretastatin heteroaromatic
analogues 8,11e17, 21e25 and the previously reported (Z)-benzo[b]
thiophene CA-4 analogs 29e31 were evaluated for their anti-
leukemic activity against the MV4-11 AML cell line. Compounds
8, 12 and 29 also showed potent anti-leukemic activity against
leukemia MV4-11 cell lines (LD50 ¼ 44 nM, 47 nM, and 18 nM,
respectively). The most active compound from the series, 29, was
also screened against a variety of 12 different leukemia cell lines
and exhibited LD50 values <300 nM against all 12 leukemia cell
lines. Compounds 8, 12, 21, 23, 25, 29 and 31 were also screened for
their in vitro inhibitory activity on tubulin polymerization in MV4-
11 cells and compounds 8, 12 and 29 all demonstrated >50% inhi-
bition of tubulin polymerization at 50 nM concentrations. The
binding modes of the three most active compounds, 8, 12 and 29 at
the colchicine binding site on tubulin have been investigated uti-
lizing molecular docking studies are consistent with the rank po-
tency of these compounds as inhibitors of tubulin polymerization.
From the cell screening and molecular docking results, we consider
compounds 8 and 29 as lead compounds in the development of
new anticancer agents that target tubulin. Compounds 8 and 29 are
moieties are 10-p electron bicyclic aromatic ring systems. The
structural difference between the three compounds is due to the
nature of the heteroatoms in the fused 5-membered ring of the
heterocycle: i.e. indole (8), benzofuran (12), and benzothiophene
(29). Thus, an additional interest in carrying out the molecular
docking studies was to assess the effect of switching the hetero-
cyclic moiety in these compounds on tubulin binding characteris-
tics. None of the compounds make any polar contacts with tubulin
residues. Instead, all of them occupy the same hydrophobic pocket
at the
a,b-interface where colchicine binds, and are stabilized
through numerous van der Waals' interactions with residues from
both subunits. Compound 8 is stabilized through Van der Waal's
interactions with Gln11, Thr179 and and Val181 of
a-tubulin, and
Lys352, Val318, Cys241, Leu248, Lys254 of -tubulin (Fig. 6A). The
b
two compounds 12 and 29 show identical binding modes (Fig. 6B
and C) that involve interactions with the same set of residues. In
both cases, the ring containing the heteroatom (benzofuran in 12
and benzothiophene in 29) is stabilized through Van der Waal's
interactions with Asn101, Ser178, Thr179 and Val181 of
a-tubulin
(but not Gln11 as seen in compound 8), and Asn258 and Lys352 of
Fig. 6. Binding modes of A) compound 8; B) compound 12; C) compound 29; at the colchicine-binding site of tubulin. The inhibitors are shown as purple ball-and-sticks and the
tubulin residues are shown as orange (a-tubulin) and yellow (b-tubulin) sticks. (For interpretation of the references to color in this figure legend, the reader is referred to the web
version of this article.)

218
N.R. Penthala et al. / European Journal of Medicinal Chemistry 92 (2015) 212e220
considered lead compounds suitable for further development as
anti-leukemic drugs.
substituted phenylacetonitrile (1.1 mole) in 5% sodium methoxide/
methanol was heated under reflux for 3e6 h. The resulting solution
was cooled to room temperature and poured into ice-cold water to
afford a crude yellow solid. The solid was filtered off, washed with
water, and finally washed with cold methanol. The obtained crude
solid was recrystallized from methanol to afford the desired
condensation product as a pure yellow crystalline solid.
4. Experimental section
4.1. Chemistry
Melting points were recorded on a Kofler hot-stage apparatus
and are uncorrected. TLC controls were carried out on pre-coated
silica gel plates (F 254 Merck). 1H and 13C NMR spectra were
recorded on a Varian 400 MHz spectrometer equipped with a Linux
workstation running on vNMRj software. All the spectra were
phased, baseline was corrected where necessary, and solvent sig-
nals (CDCl3) were used as reference for both 1H and 13C spectra.
HRMS data was obtained on an Agilent 6210 LCTOF instrument
operated in multimode.
4.6.1. (Z)-3-(1H-indol-2-yl)-2-(3,4,5-trimethoxyphenyl)acrylon-
itrile (8)
mp: 147e148 ꢁC, 1H NMR (400 MHz, CDCl3):
d 3.83 (s, 6H), 3.93
(s, 3H), 6.73 (s, 2H), 6.77 (s, 1H), 7.08e7.25 (m, 4H), 7.57 (d,
J ¼ 8.0 Hz, 1H), 7.90 (brs, 1H, NH) ppm; 13C NMR (100 MHz, CDCl3):
d
56.23, 60.92, 102.78, 105.90, 111.54, 111.79, 112.73, 119.88, 120.97,
121.11, 121.48, 121.74, 127.37, 129.14, 130.67, 130.92, 132.26, 138.10,
138.99, 153.69 ppm. HRMS (ESI) m/z calcd for C20H19N2O3 [MþH]þ
335.1390; Found 335.1399.
4.2. Methodology for the in vitro 60 human cancer cell screen
4.6.2. (Z)-2-(3,4-Dimethoxyphenyl)-3-(1H-indol-2-yl)acryloni-trile
The methodology for the anti-cancer screening assay was car-
ried out as per the reported literature procedure [40], which is also
available at http://dtp.nci.nih.gov/branches/btb/ivclsp.html http://
dtp.nci.nih.gov/branches/btb/ivclsp.html.
(9)
mp: 170e172 ꢁC, 1H NMR (400 MHz, CDCl3):
d 3.92 (s, 3H), 3.97
(s, 3H), 6.90 (s, 1H), 6.92 (s, 1H), 7.11e7.16 (m, 1H), 7.22 (dd, J ¼ 2,
8.4 Hz, 1H), 7.29e7.41 (m 1H), 7.42e7.44 (m, 2H), 7.62 (d, J ¼ 8.4 Hz,
1H), 9.47 (brs, 1H, NH) ppm; 13C NMR (100 MHz, CDCl3):
d 56.04,
4.3. Methodology for anti-leukemic activity determination
56.06, 105.89, 108.14, 111.60, 112.11, 118.57, 119.98, 120.94, 121.49,
125.36, 126.34, 127.41, 129.56, 132.52, 137.98, 149.42, 149.95 ppm.
HRMS (ESI) m/z calcd for C19H17N2O2 [MþH]þ 305.1285; Found
305.1276.
MV4-11 cells were cultured in Iscove's Modified Dulbecco's
Media (IMDM) supplemented with 100 U/ml penicillin, 100 U/ml
streptomycin, and 10% fetal bovine serum (Life technologies). Cells
were seeded at 5 ꢂ105/ml, and treated with the test compounds. At
24 and 48 h post treatment, cells were stained with annexin V-FITC
4.6.3. (Z)-2-(3,5-Dimethoxyphenyl)-3-(1H-indol-2-yl)acrylonitrile
(10)
(BD Biosciences) and 1
mg/ml 7-AAD (Life Technologies). Percent
mp: 195e197 ꢁC, 1H NMR (400 MHz, CDCl3):
d 3.84 (s, 6H), 6.46
cell dead was determined by flow cytometry as the percent of
annexin Vþ cells. Data were analyzed using Flowjo 9.3.2 for Mac OS
X (TreeStar). The cell death was represented relative to vehicle
control (DMSO).
(t, J ¼ 2.0 Hz, 1H), 6.77 (d, J ¼ 2.4 Hz, 2H), 6.94 (d, J ¼ 2 Hz, 1H), 7.12
(t, J ¼ 8.4 Hz, 1H), 7.29e7.33 (m, 1H), 7.42 (d, J ¼ 8.8 Hz, 1H), 7.50 (s,
1H), 7.62 (d, J ¼ 7.6 Hz, 1H), 9.48 (brs, 1H, NH) ppm. 13C NMR
(100 MHz, CDCl3):
d 55.54, 100.96, 103.70, 105.84, 111.70, 113.06,
119.85, 121.03, 121.66, 125.69, 127.34, 131.62, 132.22, 135.45, 138.17,
4.4. Methodology for blast crisis chronic myeloid leukemia analyses
161.31 ppm. HRMS (ESI) m/z calcd for
305.1285; Found 305.1290.
C
19H17N2O2 [MþH]þ
Primary blast crisis chronic myeloid leukemia (CML) samples
were obtained with informed consent and IRB approval from Weill
Cornell Medical College. The cryopreserved primary samples were
thawed and cultured as previously described [41]. Cells were
treated with indicated doses of compound. At 48 h post treatment,
cells were stained with CD45-APC-H7 (BD Biosciences), followed by
annexin V-FITC and 7-AAD staining. Cell dead was determined by
flow cytometry. The percent cell dead of treated cells is represented
by percent of annexin Vþ blasts normalized to DMSO control.
4.6.4. (Z)-4-((2-(2-Cyano-2-(3,5-dimethoxyphenyl)vinyl)-1H-ind-
o1-yl)methyl)benzonitrile (11)
mp: 190e192 ꢁC, 1H NMR (400 MHz, CDCl3):
d 3.84 (s, 6H), 3.85
(s, 3H), 5.51 (s, 2H, eCH2), 6.64 (s, 2H), 7.11 (d, J ¼ 8 Hz, 2H),
7.18e7.29 (m, 4H), 7.58 (d, J ¼ 8.4 Hz, 2H), 7.74 (d, J ¼ 8 Hz, 1H), 7.79
(s, 1H) ppm; 13C NMR (100 MHz, CDCl3):
d 46.33, 56.27, 60.99,
103.25, 107.35, 107.33, 109.27, 110.92, 111.98, 118.07, 118.14, 121.44,
122.50, 125.11, 126.61, 127.58, 127.88, 129.72, 132.73, 132.88, 138.23,
142.59, 153.62 ppm. HRMS (ESI) m/z calcd for C28H24N3O3 [MþH]þ
450.1826; Found 450.1821.
4.5. Tubulin polymerization inhibition assay
MV4-11 cells were treated with indicated doses of compounds
for 2 h. Cells were then lysed in microtubule-stabilizing buffer
(100 mM Pipes, 1 mM EGTA, 1 mM MgSO4, 30% glycerol, 5% DMSO,
1 mM DTT, 0.02% NaN3, 0.125% NP-40, pH 6.9) at 37 ꢁC. Free tubulin
(supernatants, S) and polymerized tubulin (pellets, P) were exam-
ined by immunoblotting using tubulin antibody. Both microtubule-
stabilizing and microtubule-destabilizing drugs inhibit hypoxia-
inducible factor-1 alpha accumulation and activity by disrupting
microtubule function [42].
4.6.5. (Z)-3-(Benzofuran-2-yl)-2-(3,4,5-trimethoxyphenyl)acry-
lonitrile (12)
mp: 102e104 ꢁC, 1H NMR (400 MHz, CDCl3):
d 3.90 (s, 3H), 3.91
(s, 6H), 6.89 (s, 2H), 7.26e7.31 (m, 1H), 7.36e7.40 (m, 1H), 7.41 (s,
1H), 7.50 (s,1H), 7.53 (dd, J ¼ 1.2,10 Hz,1H), 7.63e7.65 (m, 1H) ppm;
13C NMR (100 MHz, CDCl3):
d 56.53, 61.25, 103.30, 110.89, 111.10,
111.71, 117.60, 122.16, 123.81, 126.94, 127.66, 128.29, 129.17, 139.50,
151.21, 153.68, 155.20 ppm. HRMS (ESI) m/z calcd for C20H18NO4
[MþH]þ 336.1230; Found 336.1224.
4.6. General synthetic procedure: synthesis of (Z)-heterocyclic
4.6.6. (Z)-3-(Benzofuran-2-yl)-2-(3,4-dimethoxyphenyl)acrylo-
cyanocombretastatins (8e17 and 21e27)
nitrile (13)
mp:111e113 ꢁC, 1H NMR (400 MHz, CDCl3):
d
3.91 (s, 3H), 3.95 (s,
A mixture of carbaldehyde (1.0 mole) and the appropriate
3H), 6.89 (d, J ¼ 8 Hz, 2H), 7.14 (d, J ¼ 2.4 Hz, 1H), 7.24e7.29 (m, 1H),

N.R. Penthala et al. / European Journal of Medicinal Chemistry 92 (2015) 212e220
219
7.34e7.38 (m, 2H), 7.45 (s, 1H), 7.51 (d, J ¼ 8.4 Hz, 1H), 7.60 (d,
4.6.13. (Z)-2-(3,5-Dimethoxyphenyl)-3-(1H-indol-3-yl)acrylo-
nitrile (23)
mp: 190e192 ꢁC, 1H NMR (400 MHz, CDCl3):
d 3.86 (s, 6H), 6.45
J ¼ 7.6 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3):
d 56.17, 108.50,
110.43, 110.87, 111.45, 111.64, 117.70, 119.33, 122.05, 123.72, 126.36,
126.45, 126.70, 128.40, 149.52, 150.56, 151.54, 155.18 ppm. HRMS
(ESI) m/z calcd for C19H16NO3 [MþH]þ 306.1125; Found 306.1131.
(s, 1H), 6.82 (s, 2H), 7.25e7.29 (m, 2H), 7.44 (d, J ¼ 8.0 Hz, 1H), 7.75
(d, J ¼ 7.2 Hz, 1H), 7.87 (s, 1H), 8.44 (s, 1H), 8.83 (brs, 1H, NH) ppm;
13C NMR (100 MHz, CDCl3):
d 55.52, 100.10, 103.67, 111.79, 118.00,
4.6.7. (Z)-3-(Benzofuran-2-yl)-2-(3,5-dimethoxyphenyl)acrylo-
119.95, 121.38, 123.50, 126.40, 127.28, 133.78, 135.45, 136.84,
nitrile (14)
161.21 ppm. HRMS (ESI) m/z calcd for
C
19H17N2O2 [MþH]þ
mp: 142e144 ꢁC, 1H NMR (400 MHz, CDCl3):
d
3.84 (s, 6H), 6.49
305.1285; Found 305.1297.
(s, 1H), 6.81 (d, J ¼ 2.0 Hz, 2H), 7.25 (t, J ¼ 7.6 Hz, 1H), 7.37 (t,
J ¼ 7.6 Hz,1H), 7.45 (s,1H), 7.49 (s, H), 7.53 (d, J ¼ 8.8 Hz,1H), 7.62 (d,
4.6.14. (Z)-3-(5-Methoxy-1H-indol-3-yl)-2-(3,4,5-trimethoxy-
J ¼ 8.0 Hz, 1H) ppm. 13C NMR (100 MHz, CDCl3):
d
55.54, 101.65,
phenyl)acrylonitrile (24)
104.08, 110.79, 111.36, 111.63, 117.41, 122.08, 123.66, 126.89, 128.15,
mp: 183e185 ꢁC, 1H NMR (400 MHz, CDCl3):
d 3.88 (s, 3H,
128.48, 135.43, 151.07, 155.22, 161.27 ppm. HRMS (ESI) m/z calcd for
C
eOCH3), 3.89 (s, 3H, eOCH3), 3.94 (s, 6H, eOCH3), 6.85 (s, 2H), 6.93
(d, J ¼ 8.8 Hz, 1H), 7.18 (s, 1H), 7.33 (d, J ¼ 8.4 Hz, 1H), 7.69 (s, 1H),
8.38 (s, 1H), 8.60 (brs, 1H, NH) ppm; 13C NMR (100 MHz, CDCl3):
19H16NO3 [MþH]þ 306.1125 Found 306.1126.
4.6.8. (Z)-3-(Benzo[d]thiazol-2-yl)-2-(3,4,5-trimethoxyphenyl)
d 55.98, 56.37, 61.00, 100.47, 102.95, 104.74, 111.72, 112.52, 113.21,
acrylonitrile (15)
120.02, 126.78, 127.93, 130.52, 130.80, 133.17, 153.60, 155.39 ppm.
HRMS (ESI) m/z calcd for C21H21N2O4 [MþH]þ 365.1496; Found
365.1502.
mp: 110e111 ꢁC, 1H NMR (400 MHz, CDCl3):
d 3.90 (s, 3H), 3.93
(s, 6H), 6.98 (s, 2H), 7.50e7.57 (m, 2H), 7.95e8.00 (m, 2H), 8.10 (d,
J ¼ 7.6 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3):
d 56.33, 61.0,
103.74, 110.0, 116.74, 116.97, 121.85, 123.88, 126.98, 127.05, 128.11,
133.82, 135.49, 140.37, 140.57, 152.51, 153.72, 161.33 ppm. HRMS
4.6.15. (Z)-3-(Benzofuran-3-yl)-2-(3,4,5-trimethoxyphenyl)ac-
rylonitrile (25)
(ESI) m/z calcd for
353.0968.
C
19H17N2O3S [MþH]þ 353.0960; Found
mp: 144e146 ꢁC, 1H NMR (400 MHz, CDCl3):
d 3.91 (s, 3H), 3.96
(s, 6H), 6.89 (s, 2H), 7.37e7.41 (m, 2H), 7.55 (s, 1H), 7.57 (d,
J ¼ 8.4 Hz, 1H), 7.72 (d, J ¼ 7.6 Hz, 1H), 8.67 (s, 1H) ppm; 13C NMR
4.6.9. (Z)-3-(Benzo[d]thiazol-2-yl)-2-(3,4-dimethoxyphenyl)ac-
(100 MHz, CDCl3): d 56.82, 61.47, 103.72, 112.10, 112.48, 116.53,
rylonitrile (16)
119.01, 119.37, 123.97, 125.99, 126.67, 129.83, 129.98, 139.70, 146.35,
154.14, 155.35 ppm. HRMS (ESI) m/z calcd for C20H18NO4 [MþH]þ
336.1236; Found 336.1218.
mp: 130e131 ꢁC, 1H NMR (400 MHz, CDCl3):
d 3.94 (s, 3H), 3.95
(s, 3H), 6.93 (d, J ¼ 8 Hz, 1H), 7.22 (s, 1H), 7.38 (d, J ¼ 8 Hz, 1H),
7.48e7.55 (m, 2H), 7.91e7.96 (m, 2H), 8.08 (d, J ¼ 8 Hz,1H) ppm; 13
C
NMR (100 MHz, CDCl3):
d
56.08, 56.09, 108.39, 111.34, 116.79, 117.19,
4.6.16. (Z)-3-(Benzofuran-3-yl)-2-(3,4-dimethoxyphenyl)acry-lo
120.47, 121.83, 123.59, 125.39, 126.88, 127.08, 131.90, 135.22, 149.58,
151.46, 152.02, 161.75 ppm. HRMS (ESI) m/z calcd for C18H15N2O2S
[MþH]þ 323.0854; Found 323.0842.
nitrile (26)
mp: 135e137 ꢁC, 1H NMR (400 MHz, CDCl3):
d 3.92 (s, 3H), 3.96
(s, 3H), 6.90 (d, J ¼ 8.8 Hz, 1H), 7.13 (d, J ¼ 2.4 Hz, 1H), 7.24 (dd,
J ¼ 2.4, 8.4 Hz,1H), 7.33e7.40 (m, 2H), 7.49 (s,1H), 7.54 (d, J ¼ 8.4 Hz,
4.6.10. (Z)-3-(Benzo[d]thiazol-2-yl)-2-(3,5-dimethoxyphenyl)-
1H), 8.62 (s, 1H) ppm; 13C NMR (100 MHz, CDCl3):
d 56.04, 56.08,
acrylonitrile (17)
108.64, 111.38, 111.48, 111.96, 116.18, 118.66, 118.82, 118.92, 123.42,
125.42, 126.32, 126.57, 128.11, 145.55, 149.38, 150.13, 154.84 ppm.
HRMS (ESI) m/z calcd for C19H16NO3 [MþH]þ 306.1125; Found
306.1126.
mp: 149e151 ꢁC, 1H NMR (400 MHz, CDCl3):
d 3.84 (s, 6H), 6.55
(s, 1H), 6.89 (s, 2H), 7.50e7.57 (m, 2H), 7.95e7.97 (d, J ¼ 8 Hz, 1H),
8.04 (s, 1H), 8.11 (d, J ¼ 8 Hz, 1H) ppm. 13C NMR (100 MHz, CDCl3):
d
55.60, 103.01, 104.55, 116.67, 117.32, 121.87, 123.86, 127.09, 127.14,
134.54, 134.58, 135.45, 152.15, 161.29, 161.37 ppm. HRMS (ESI) m/z
4.6.17. (Z)-3-(Benzofuran-3-yl)-2-(3,5-dimethoxyphenyl)acry lo
calcd for C18H15N2O2S [MþH]þ 323.0849; Found 323.0850.
nitrile (27)
mp:121e123 ꢁC, 1H NMR (400 MHz, CDCl3):
d 3.86 (s, 6H), 6.50
4.6.11. (Z)-3-(1H-Indol-3-yl)-2-(3,4,5-trimethoxyphenyl)acry-
(s, 1H), 6.81 (s, 2H), 7.34e7.42 (m, 2H), 7.56 (d, J ¼ 7.2 Hz, 1H), 7.62
(s, 1H), 7.69 (d, J ¼ 8 Hz, 1H), 8.68 (s, 1H); ppm; 13C NMR (100 MHz,
CDCl3): 55.54, 101.06, 104.11, 111.48, 111.98, 116.03, 118.50, 118.91,
123.57, 125.52, 126.24, 130.40, 135.65, 146.12, 154.85, 161.29 ppm.
HRMS (ESI) m/z calcd for C19H16NO3 [MþH]þ 306.1125; Found
306.1133.
lonitrile (21)
mp:178e180 ꢁC, 1H NMR (400 MHz, CDCl3):
d 3.89 (s, 3H), 3.95
(s, 6H), 6.87 (s, 2H), 7.24e7.31 (m, 2H), 7.45 (d, J ¼ 7.6 Hz, 1H), 7.77
(s, 1H), 7.78 (s, 1H), 8.42 (d, J ¼ 2.8 Hz, 1H), 8.94 (brs, 1H, NH) ppm;
13C NMR (100 MHz, CDCl3):
d 56.32, 61.02, 102.79, 104.99, 111.77,
111.88, 117.95, 120.01, 121.27, 123.46, 126.25, 127.22, 130.71, 133.13,
135.54, 138.24, 153.58 ppm. HRMS (ESI) m/z calcd for C20H19N2O3
[MþH]þ 335.1390; Found 335.1387.
Acknowledgments
We are grateful to NCI/NIH (Grant Number CA 140409) and to
the Arkansas Research Alliance (ARA) for financial support, M.L.G is
funded by the US National Institutes of Health (NIH) through the
NIH Director's New Innovator Award Program, 1 DP2 OD007399-01
and to the NCI Developmental Therapeutic Program (DTP) for
screening data.
4.6.12. (Z)-2-(3,4-Dimethoxyphenyl)-3-(1H-indol-3-yl)acrylonitrile
(22)
mp: 170e172 ꢁC, 1H NMR (400 MHz, CDCl3):
d 3.92 (s, 3H,
eOCH3), 3.97 (s, 3H, eOCH3), 6.91 (d, J ¼ 8.4 Hz, 1H), 7.15 (s, 1H),
7.24e7.21 (m, 3H), 7.44 (d, J ¼ 7.6 Hz, 1H), 7.75 (s, 1H), 7.78 (s, 1H),
8.40 (s, 1H), 8.75 (brs, 1H, NH) ppm; 13C NMR (100 MHz, CDCl3):
d
55.97, 56.12, 104.92, 108.44, 108.55, 111.47, 111.73, 111.89, 117.90,
Appendix A. Supplementary data
118.22, 120.14, 121.08, 121.24, 123.31, 125.90, 127.24, 127.83, 131.89,
135.53, 149.24 ppm. HRMS (ESI) m/z calcd for C19H17N2O2 [MþH]þ
305.1285; Found 305.1278.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.12.050.

220
N.R. Penthala et al. / European Journal of Medicinal Chemistry 92 (2015) 212e220
restricted naphthalene derivatives type isocombretastatin A-4 and isoerianin
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