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. Author manuscript; available in PMC: 2016 Mar 6.
Published in final edited form as: Eur J Med Chem. 2014 Dec 29;92:212–220. doi: 10.1016/j.ejmech.2014.12.050

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,*
PMCID: PMC4336621  NIHMSID: NIHMS653620  PMID: 25557492

Abstract

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)-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 compounds 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 = 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 hydrophobic pocket at the interface of α- and β-tubulin that is occupied by colchicine, and were stablized 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.

Keywords: Trans-cyanocombretastatin analogues, Anti-cancer activity, Leukemia cell lines, Molecular docking, Tubulin binding

1. Introduction

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 [5–7].

Fig 1.

Fig 1

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Chemical structures of potent antitubulin agents

Structurally related cyanocombretastatin analogues [8, 9] (Fig. 1, III) and similar analogues that incorporate different heterocyclic moieties, such as indole, benzothiophene, quinoline and quinazoline have also been reported as cytotoxic compounds, and are potent inhibitors of tubulin polymerization potencies comparable to that of CA-4 [10–13]. 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 chemotherapy [14] and 2,3-diarylacrylonitriles have emerged as important synthons for the synthesis of a wide spectrum of biologically active molecules [15]. Such compounds have been shown to possess spasmolytic, estrogenic, hypotensive, antioxidant, tuberculostatic, antitrichomonal, and insecticidal properties [16].

Many natural medicinal agents, such as the combretastatins, the colchicines (Fig. 1, IV), and the podophyllotoxins, possess a trimethoxyphenyl moiety in their structure. The cytotoxic properties of such compounds are believed to be related to their anti-tubulin properties [17–21]. A large number of CA-4 derivatives have been synthesized and evaluated for anti-tubulin activity [22–29]. SAR studies have revealed that a 3′,4′,5′-trimethoxyphenyl 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 50–60° 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 cyanocombretastatin 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].

Fig 2.

Fig 2

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Z-Benzo[b]thiophen-2-yl cyanocombretastatin analogues (29–31) [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 benzothiophene 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).

2. Results and discussion

2.1. Drug Synthesis

(Z)-Indol-2-yl cyanocombretastatin analogues (8–11), (Z)-benzo[b]furan-2-yl cyanocombretastatin analogues (12–14) and (Z)-benzo[d]thiazol-2-yl cyanocombretastatin analogues (15–17) 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 acetonitrile (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 crystallographic data [34, 35].

Scheme 1.

Scheme 1

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Synthesis of (Z)-indol-2-yl, (Z)-benzo[b]furan-2-yl, and (Z)-benzo[d] thiazol-2-yl acrylonitriles (8–17)

A second series of indole-3-yl (21–24), and (Z)-benzo[b]furan-3-yl cyanocombretastatin analogues (25–27) were synthesized by refluxing a variety of indole-3-carbaldehydes (18–19) or benzo[b]furan-3-carbaldehyde (20) with appropriate phenylacetonitriles (5–7) in 5% sodium methoxide/methanol (Scheme 2).

Scheme 2.

Scheme 2

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Synthesis of (Z)--indole-3-yl, and (Z)--benzo[b]furan-3-yl cyanocombretastatins (21–27)

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 (10−5 M). The 60 cell line panel is organized into subpanels representing leukemia, non-small cell lung, colon, central nervous system, melanoma, 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 10−5 M following 48 h of incubation. From the preliminary screens, compounds 8, 9, 11–13, 15, 17 and 21–25 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 (10−4M, 10−5 M, 10−6 M, 10−7 M and 10−8 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 dimethoxyphenyl (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 presented in Tables 1 and 2. We hypothesized that the cytotoxic activity of these novel analogues was likely due to their interaction with the colchicine binding site on tubulin.

Table 1.

Growth inhibition (GI50/μM)a data for (Z)-indol-2-yl (8, 11), (Z)-benzo[b]furan-2-yl (12, 13), and (Z)-benzo[d]thiazol-2-yl (15, 17) cyano combretastatin analogues

Panel/cell line 8 9 11 12 13 15 17
μM μM μM μM μM μM μM
Leukemia
CCRF-CEM 0.017 0.062 0.136 0.024 0.777 0.055 na
HL-60(TB) <0.01 0.034 0.031 <0.01 0.481 0.066 0.345
K-562 <0.01 0.039 0.039 <0.01 0.467 0.051 0.333
MOLT-4 0.023 0.084 0.274 0.047 0.548 9.20 3.38
RPMI-8226 <0.01 0.151 0.199 0.019 1.32 0.237 2.29
SR <0.01 0.042 na <0.01 0.470 0.107 0.402
Lung Cancer
A549/ATCC <0.01 0.114 0.076 <0.01 0.520 0.379 na
EKVX 1.16 0.307 na 0.059 na na na
HOP-62 <0.01 na 0.092 <0.01 2.29 1.93 3.35
HOP-92 <0.01 0.058 0.077 <0.01 1.13 nd 0.534
NCI-H226 <0.01 0.399 14.5 <0.01 34.5 0.844 4.11
NCI-H23 <0.01 0.236 0.246 <0.01 3.11 3.25 2.84
NCI-H322M nd 0.735 0.465 <0.01 6.18 2.29 >10.0
NCI-H460 <0.01 0.053 0.093 <0.01 0.472 0.336 2.86
NCI-H522 <0.01 0.049 0.033 <0.01 0.438 0.022 na
Colon Cancer
COLO 205 0.016 0.232 0.097 <0.01 0.404 0.036 0.574
HCC-2998 0.018 0.366 0.265 0.01 2.33 0.297 8.39
HCT-116 <0.01 0.044 0.047 <0.01 0.418 0.052 0.404
HCT-15 <0.01 0.048 0.089 <0.01 0.469 0.045 0.414
HT29 <0.01 0.048 0.048 <0.01 0.373 0.037 0.438
KM12 <0.01 0.076 0.047 <0.01 0.356 0.278 2.01
SW-620 <0.01 0.042 0.048 <0.01 0.388 0.038 0.343
CNS Cancer
SF-268 0.015 1.48 0.086 0.247 2.05 1.03 3.09
SF-295 <0.01 0.036 0.037 <0.01 0.708 0.508 2.46
SF-539 <0.01 0.047 0.039 <0.01 1.56 0.097 1.68
SNB-19 <0.01 0.189 0.085 <0.01 5.55 2.49 >10.0
SNB-75 <0.01 0.036 0.039 <0.01 1.78 0.145 2.13
U251 <0.01 0.075 0.044 <0.01 0.760 1.65 1.91
Melanoma
LOX IMVI <0.01 0.064 0.090 <0.01 0.930 0.073 1.84
MALME-3M 8.28 nd nd 59.9 0.629 0.515 0.699
M14 <0.01 0.039 0.056 <0.01 0.437 0.193 1.21
MDA-MB-435 <0.01 0.021 0.024 <0.01 0.229 0.021 0.199
SK-MEL-2 <0.01 0.312 0.528 <0.01 0.487 0.287 0.958
SK-MEL-28 na 1.94 0.161 <0.01 3.51 1.81 1.98
SK-MEL-5 <0.01 0.142 0.060 <0.01 0.564 0.100 1.37
UACC-257 1.06 27.0 0.062 63.2 18.0 >100 na
UACC-62 <0.01 0.038 0.058 <0.01 0.557 0.052 0.755
Ovarian Cancer
IGROV1 <0.01 0.482 0.183 0.022 1.27 1.27 0.706
OVCAR-3 <0.01 0.034 0.042 <0.01 0.279 0.031 0.285
OVCAR-4 15.2 0.735 0.672 0.039 7.19 3.86 >10.0
OVCAR-5 0.08 0.471 0.259 0.042 5.23 0.347 4.85
OVCAR-8 <0.01 0.326 0.124 <0.01 3.98 0.438 3.13
NCI/ADR-RES <0.01 0.040 0.073 <0.01 0.317 0.059 1.47
SK-OV-3 <0.01 0.394 0.087 <0.01 2.49 0.626 1.79
Renal Cancer
786-0 <0.01 0.602 0.137 <0.01 5.84 1.06 2.44
A498 <0.01 0.040 0.029 <0.01 0.540 0.198 0.417
ACHN <0.01 0.082 0.079 0.217 12.4 0.089 0.518
CAKI-1 <0.01 0.103 0.035 <0.01 0.492 0.047 0.482
RXF 393 <0.01 0.151 0.055 <0.01 1.30 0.148 0.551
SN12C <0.01 0.779 0.269 <0.01 0.956 0.515 4.68
TK-10 <0.01 5.56 20.1 73.6 2.28 0.063 2.78
UO-31 <0.01 0.071 0.201 2.29 3.79 0.083 0.679
Prostate Cancer
PC-3 <0.01 0.062 0.105 <0.01 2.41 0.341 2.74
DU-145 <0.01 0.206 0.207 <0.01 1.30 0.333 1.07
Breast Cancer
MCF7 0.016 0.289 0.039 0.023 0.446 0.034 0.399
MDA-MB-231/ATCC 0.014 0.291 0.294 0.028 1.21 0.992 6.74
HS 578T <0.01 0.133 0.092 <0.01 1.88 1.29 2.01
BT-549 <0.01 0.0978 0.109 0.029 0.950 12.2 3.45
T-47D 2.50 0.573 95.2 4.34 0.996 nd 1.18
MDA-MB-468 0.022 0.350 0.041 0.022 0.421 0.084 2.04
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na: Not analyzed, nd: not determined,

a

GI50: 50% growth inhibition, concentration of drug resulting in a 50% reduction in net cell growth as compared to cell numbers on day 0.

Table 2.

Growth inhibition (GI50/μM)a data for (Z)-indole-3-yl (21–24) and (Z)-benzo[b]furan-3-yl (25) cyanocombretastatin analogues

Panel/cell line 21 22 23 24 25
μM μM μM μM μM
Leukemia
CCRF-CEM 0.058 2.74 0.042 0.064 1.41
HL-60(TB) 0.048 4.57 0.031 0.059 1.03
K-562 0.040 1.65 0.043 0.044 0.513
MOLT-4 0.131 3.99 0.049 0.198 3.82
RPMI-8226 0.231 3.96 0.065 0.241 2.47
SR 0.022 1.45 0.033 0.027 0.681
Lung Cancer
A549/ATCC 0.078 5.16 0.061 0.086 0.665
EKVX 0.596 18.5 0.478 0.485 na
HOP-62 0.082 8.69 0.273 0.133 6.98
HOP-92 1.01 5.26 0.068 0.083 3.02
NCI-H226 0.194 20.9 8.18 0.170 6.20
NCI-H23 0.133 7.19 0.256 0.101 4.58
NCI-H322M nd 24.3 nd 0.044 19.1
NCI-H460 0.041 4.51 0.039 0.037 0.839
NCI-H522 0.037 3.05 0.027 0.086 0.261
Colon Cancer
COLO 205 0.474 5.31 0.047 0.051 0.449
HCC-2998 0.107 12.6 0.207 0.119 4.13
HCT-116 0.048 35.5 0.042 0.045 0.517
HCT-15 0.051 3.70 0.053 0.068 0.492
HT29 0.038 3.32 0.038 0.038 0.394
KM12 0.043 4.04 0.040 0.044 0.586
SW-620 0.047 3.53 0.044 0.046 0.504
CNS Cancer
SF-268 2.54 18.9 0.078 2.14 4.79
SF-295 0.042 4.92 0.045 0.042 0.446
SF-539 0.099 8.42 0.052 0.071 0.442
SNB-19 0.076 24.2 0.062 0.089 3.63
SNB-75 0.042 6.04 0.034 0.043 0.769
U251 0.050 4.97 0.045 0.050 0.605
Melanoma
LOX IMVI 0.075 5.32 0.078 0.074 0.993
MALME-3M 12.5 9.91 nd 5.85 0.868
M14 0.053 3.75 0.042 0.052 0.667
MDA-MB-435 0.024 3.95 0.020 0.027 0.278
SK-MEL-2 na 4.70 0.051 na 0.304
SK-MEL-28 0.090 6.25 0.081 0.092 1.61
SK-MEL-5 0.042 4.27 0.048 0.036 0.336
UACC-257 11.3 11.9 nd 10.7 nd
UACC-62 0.048 6.98 0.049 0.059 0.519
Ovarian Cancer
IGROV1 0.417 21.0 0.347 0.379 3.06
OVCAR-3 0.033 5.68 0.031 0.034 0.277
OVCAR-4 0.338 22.1 3.52 1.35 2.61
OVCAR-5 10.6 >100 0.197 0.516 3.33
OVCAR-8 0.121 7.38 0.103 0.148 2.90
NCI/ADR-RES 0.039 1.70 0.033 0.037 0.382
SK-OV-3 0.051 11.9 0.117 0.056 1.71
Renal Cancer
786-0 0.090 14.1 0.077 0.085 6.11
A498 0.035 3.36 0.035 0.025 0.458
ACHN 1.76 23.7 nd 1.66 1.94
CAKI-1 0.058 4.34 0.035 0.072 0.650
RXF 393 0.036 4.48 0.083 0.033 0.496
SN12C 0.699 8.10 0.077 0.715 2.17
TK-10 11.1 13.2 10.3 20.1 2.10
UO-31 1.41 14.3 0.663 1.26 2.89
Prostate Cancer
PC-3 0.054 7.85 0.075 0.054 1.84
DU-145 0.132 22.6 0.130 0.153 1.88
Breast Cancer
MCF7 0.034 3.10 0.041 0.036 0.376
MDA-MB-231/ATCC 0.245 7.18 0.644 0.299 1.71
HS 578T 0.131 12.9 0.611 0.124 2.03
BT-549 1.94 5.38 0.049 0.348 0.927
T-47D 0.058 6.83 nd 0.065 0.811
MDA-MB-468 0.041 na 0.051 0.035 0.237
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na: Not analyzed, nd: not determined,

a

GI50: 50% growth inhibition, concentration of drug resulting in a 50% reduction in net cell growth as compared to cell numbers on day 0.

2.2.2 Indol-2-yl Analogues

Compound 8 [(Z)-3-(1H-indol-2-yl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile] exhibited GI50 values ranging from < 0.01 μM to 1.16 μM in 95% of the cancer cell lines screened, and showed potent growth inhibition (GI50 = < 0.01 μM) in 74% of the cancer cells in the 60-cell panel (Table 1).

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 panel, with GI50 values ranging from 0.021 to 0.779 μM, and afforded an average GI50 value of 0.80 μM against all the cancer cell lines in the panel (Table 1). This compound exhibited potent growth inhibition against MDA-MB-435 melanoma cancer cells with a GI50 = 0.021 μM (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) benzonitrile], which exhibited GI50 values ranging from 0.024 μM to 0.672 μM 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 0.031–0.274 μM. Compound 11 also showed potent growth inhibition against the MDA-MB-435 melanoma cell line (GI50 = 0.024 μM), and exhibited growth inhibition < 1 μM against 93 % of the cancer cells in the 60-cell panel (Table 1).

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-furan-2-yl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile], which exhibited good growth inhibition against all the cancer cells in the 60-cell panel with GI50 values ranging from < 0.01 to 73.6 μM. This compound exhibited remarkably potent growth inhibition against 70% of the cancer cell lines screened with GI50 = < 0.01 μM (Table 1).

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 panel with GI50 ranging from 0.229 to 0.996 μM. This compound exhibited potent growth inhibition of MDA-MB-435 melanoma cancer cells with a GI50 value of 0.229 μM (Table 1). The average GI50 value of this compound against all the cancer cell lines screened was 2.59 μM.

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 μM. The average GI50 value for this compound against all the cancer cell lines screened was 0.93 μM. This compound exhibited potent growth inhibition against 73% of the cancer cell lines screened with GI50 = <1 μM (Table 1) and exhibited potent growth inhibition against MDA-MB-435 melanoma cancer cells with a GI50 of 0.021 μM (Table 1).

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], which exhibited potent growth inhibition against 94% of the cancer cells in the 60-cell panel with GI50 ranging from 0.199 to 8.39 μM. The average GI50 value for this compound against all the cancer cells in the panel was 1.87 μM. This compound exhibited potent growth inhibition against only 38% of the cancer cell lines screened with GI50 = <1 μM (Table 2). This compound exhibited potent growth inhibition against MDA-MB-435 melanoma cancer cells with a GI50 value of 0.199 μM (Table 1).

Indol-3-yl Analogues

The indol-3-yl analog (21) of compound 8 exhibited good growth inhibition in the 60-cell screen with GI50 values ranging from 0.022 μM to 12.5 μM against all the cancer cell lines screened. Compound 21 showed potent growth inhibition against SR leukemia cells (GI50 = 0.022 μM), and exhibited < 1 μM growth inhibition against 83 % of the cancer cell lines screened (Table 2).

The indol-3-yl analog (22) of compound 9 exhibited growth inhibition against all the cancer cell lines screened with GI50 values ranging from 1.45 μM to > 100 μM (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 GI50 values ranging from 0.020 μM to 10.3 μM. This compound showed potent growth inhibition against melanoma MDA-MB-435 cancer cells with a GI50 value of 0.02 μM. Compound 23 exhibited < 1 μM growth inhibition against 85 % of the cancer cell lines screened (Table 2).

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 in the panel, with GI50 values ranging from 0.025 to 20.1 μM. This compound exhibited potent growth inhibition against A498 renal cancer cells with a GI50 value of 0.022 μM. Compound 24 exhibited < 1 μM growth inhibition against 88 % of the cancer cell screened (Table 2).

2.2.5. 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-(benzofuran-3-yl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile], which exhibited good growth inhibition against all the cancer cells in the panel, with GI50 values ranging from 0.237 to 19.1 μM. The average GI50 value of this compound against all the cancer cell lines screened was 1.99 μM. This compound exhibited potent growth inhibition against 52% of the cancer cell lines screened with GI50 values < 1 μM (Table 2).

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 activity 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

Previously, we have reported on the synthesis of (Z)-benzo[b]thiophen-2-yl acrylonitriles (29–31) (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 compounds 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].

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 heteroaromatic analogues 8, 11–17, 21–25 and the previously reported (Z)-benzo[b]thiophene CA-4 analogs 29–31 [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 determined to be the most active compounds against this leukemia cell line.

Table 3.

Anti-leukemic activity (LD50) of the most potent compounds against the MV4-11 AML cell line

Compound
8 11 12 13 14 15 16 17
LD50 (μM) 0.044 0.369 0.047 >20 1.169 0.233 1.223 4.339
Compound
21 22 23 24 25 29 30 31
LD50 (μM) 0.565 >20 0.375 0.467 4.529 0.018 6.063 0.180
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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: 18.0–271 nM). Specifically, MV4-11 cells exhibited the most sensitivity to compound 29 (LD50 = 18 nM), whereas THP-1 and MLL-ENL cells were the most resistant cell line (LD50 = 227.0 and 271 nM, respectively) (Fig. 3).

Fig 3.

Fig 3

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Activity of compound 29 on 12 different leukemia cell lines

We also tested the activity of 29 against a 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 hours 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 cascades (MLG/HZ personal communication).

Fig 4.

Fig 4

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Activity of compound 29 in a primary leukemia sample (blast crisis of chronic myeloid leukemia) at 24h, 48h 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].

Fig 5.

Fig 5

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Effect of trans-cyano CA-4 analogues 8, 12, 21, 23, 25, 29 and 31 to inhibit tubulin polymerization in MV4-11 cells.

MV4-11 cells were treated with the above seven compounds at doses of 25, 50 and 100 nM for 2 hours and cell-based tubulin depolymerization assays performed. The polymerized tubulin in the pellet (P) and unpolymerized tubulin in the supernatant (S) were 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).

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). Atomic coordinates for the α,β-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 compounds were generated using the Dock Prep module in the UCSF-Chimera software.

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 Figure 6. The molecules are structurally very similar, differing only in the nature of the heterocyclic moiety in the molecule. All three of these heterocyclic moieties are 10-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 heterocyclic moiety in these compounds on tubulin binding characteristics. None of the compounds make any polar contacts with tubulin residues. Instead, all of them occupy the same hydrophobic pocket at the α,β-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 Gln 11, Thr179 and and Val181 of α-tubulin, and Lys352, Val318, Cys241, Leu248, Lys254 of β-tubulin (Fig. 6A). The two compounds 12 and 29 show identical binding modes (Fig. 6B and 6C) 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 α-tubulin (but not Gln11 as seen in compound 8), and Asn258 and Lys352 of β-tubulin. However, the heteroatom-ring in the two compounds is rotated by 180° 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 superimposable on each other, except for their oppositely oriented heteroatoms (‘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 α- and β-tubulin.

Fig 6.

Fig 6

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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 (α-tubulin) and yellow (β-tubulin) sticks.

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 of −8.12, −7.74 and −7.65, kcal/mol for compounds 29, 12 and 8, respectively.

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 μM against most of the cancer cell lines used, with 8 and 12 being the most potent compounds. The novel cyanocombretastatin heteroaromatic analogues 8, 11–17, 21–25 and the previously reported (Z)-benzo[b]thiophene CA-4 analogs 29–31 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 = 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% inhibition 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 utilizing molecular docking studies are consistent with the rank potency 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 considered lead compounds suitable for further development as anti-leukemic drugs.

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 signals (CDCl3) were used as reference for both 1H and 13C spectra. HRMS data was obtained on an Agilent 6210 LCTOF instrument operated in multimode.

4.2. Methodology for the in vitro 60 human cancer cell screen

The methodology for the anti-cancer screening assay was carried out as per the reported literature procedure [40], which is also available at http://dtp.nci.nih.gov/branches/btb/ivclsp.html.

4.3. Methodology for anti-leukemic activity determination

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 × 105/ml, and treated with the test compounds. At 24 and 48 h post treatment, cells were stained with annexin V-FITC (BD Biosciences) and 1 μg/ml 7-AAD (Life Technologies). Percent cell dead was determined by flow cytometry as the percent of annexin V+ cells. Data were analyzed using Flowjo 9.3.2 for Mac OS X (TreeStar). The cell death was represented relative to vehicle control (DMSO).

4.4. Methodology for blast crisis chronic myeloid leukemia analyses

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 V+ blasts normalized to DMSO control.

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 examined 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. General synthetic procedure: synthesis of (Z)-heterocyclic cyanocombretastatins (8–17 and 21–27)

A mixture of carbaldehyde (1.0 mole) and the appropriate substituted phenylacetonitrile (1.1 mole) in 5% sodium methoxide/methanol was heated under reflux for 3 to 6 hrs. 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.

4.6.1.(Z)-3-(1H-indol-2-yl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile (8)

mp: 147–148 °C, 1H NMR (400 MHz, CDCl3): δ 3.83 (s, 6H), 3.93 (s, 3H), 6.73 (s, 2H), 6.77 (s, 1H), 7.08–7.25 (m, 4H), 7.57 (d, J = 8.0 Hz, 1H), 7.90 (brs, 1H, NH) ppm; 13C NMR (100 MHz, CDCl3): δ 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 [M+H]+ 335.1390; Found 335.1399.

4.6.2. (Z)-2-(3,4-Dimethoxyphenyl)-3-(1H-indol-2-yl)acrylonitrile (9)

mp: 170–172 °C, 1H NMR (400 MHz, CDCl3): δ 3.92 (s, 3H), 3.97 (s, 3H), 6.90 (s, 1H), 6.92 (s, 1H), 7.11–7.16 (m, 1H), 7.22 (dd, J = 2, 8.4 Hz, 1H), 7.29–7.41 (m 1H), 7.42–7.44 (m, 2H), 7.62 (d, J = 8.4 Hz, 1H), 9.47 (brs, 1H, NH) ppm; 13C NMR (100 MHz, CDCl3): δ 56.04, 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 [M+H]+ 305.1285; Found 305.1276.

4.6.3. (Z)-2-(3,5-Dimethoxyphenyl)-3-(1H-indol-2-yl)acrylonitrile (10)

mp: 195–197 °C, 1H NMR (400 MHz, CDCl3): δ 3.84 (s, 6H), 6.46 (t, J = 2.0 Hz, 1H), 6.77 (d, J = 2.4 Hz, 2H), 6.94 (d, J = 2 Hz, 1H), 7.12 (t, J = 8.4 Hz, 1H), 7.29–7.33 (m, 1H), 7.42 (d, J = 8.8 Hz, 1H), 7.50 (s, 1H), 7.62 (d, J = 7.6 Hz, 1H), 9.48 (brs, 1H, NH) ppm. 13C NMR (100 MHz, CDCl3): δ 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, 161.31 ppm. HRMS (ESI) m/z calcd for C19H17N2O2 [M+H]+ 305.1285; Found 305.1290.

4.6.4. (Z)-4-((2-(2-Cyano-2-(3,5-dimethoxyphenyl)vinyl)-1H-ind- o1-yl)methyl)benzonitrile (11)

mp: 190–192 °C, 1H NMR (400 MHz, CDCl3): δ 3.84 (s, 6H), 3.85 (s, 3H), 5.51 (s, 2H, -CH2), 6.64 (s, 2H), 7.11 (d, J = 8 Hz, 2H), 7.18–7.29 (m, 4H), 7.58 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 8 Hz, 1H), 7.79 (s, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ 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 [M+H]+ 450.1826; Found 450.1821.

4.6.5. (Z)-3-(Benzofuran-2-yl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile (12)

mp: 102–104°C, 1H NMR (400 MHz, CDCl3): δ 3.90 (s, 3H), 3.91 (s, 6H), 6.89 (s, 2H), 7.26–7.31 (m, 1H), 7.36–7.40 (m, 1H), 7.41 (s, 1H), 7.50 (s, 1H), 7.53 (dd, J = 1.2, 10 Hz, 1H), 7.63–7.65 (m, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ 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 [M+H]+ 336.1230; Found 336.1224.

4.6.6. (Z)-3-(Benzofuran-2-yl)-2-(3,4-dimethoxyphenyl)acrylonitrile (13)

mp:111–113 °C, 1H NMR (400 MHz, CDCl3): δ 3.91 (s, 3H), 3.95 (s, 3H), 6.89 (d, J = 8 Hz, 2H), 7.14 (d, J = 2.4 Hz, 1H), 7.24–7.29 (m, 1H), 7.34–7.38 (m, 2H), 7.45 (s, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 7.6 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ 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 [M+H]+ 306.1125; Found 306.1131.

4.6.7. (Z)-3-(Benzofuran-2-yl)-2-(3,5-dimethoxyphenyl)acrylonitrile (14)

mp: 142–144 °C, 1H NMR (400 MHz, CDCl3): δ 3.84 (s, 6H), 6.49 (s, 1H), 6.81 (d, J = 2.0 Hz, 2H), 7.25 (t, J = 7.6 Hz, 1H), 7.37 (t, J = 7.6 Hz, 1H), 7.45 (s, 1H), 7.49 (s, H), 7.53 (d, J = 8.8 Hz, 1H), 7.62 (d, J = 8.0 Hz, 1H) ppm. 13C NMR (100 MHz, CDCl3): δ 55.54, 101.65, 104.08, 110.79, 111.36, 111.63, 117.41, 122.08, 123.66, 126.89, 128.15, 128.48, 135.43, 151.07, 155.22, 161.27 ppm. HRMS (ESI) m/z calcd for C19H16NO3 [M+H]+ 306.1125 Found 306.1126.

4.6.8. (Z)-3-(Benzo[d]thiazol-2-yl)-2-(3,4,5-trimethoxyphenyl) acrylonitrile (15)

mp: 110–111°C, 1H NMR (400 MHz, CDCl3): δ 3.90 (s, 3H), 3.93 (s, 6H), 6.98 (s, 2H), 7.50–7.57 (m, 2H), 7.95–8.00 (m, 2H), 8.10 (d, J = 7.6 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ 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 (ESI) m/z calcd for C19H17N2O3S [M+H]+ 353.0960; Found 353.0968.

4.6.9. (Z)-3-(Benzo[d]thiazol-2-yl)-2-(3,4-dimethoxyphenyl)acrylonitrile (16)

mp: 130–131°C, 1H NMR (400 MHz, CDCl3): δ 3.94 (s, 3H), 3.95 (s, 3H), 6.93 (d, J = 8 Hz, 1H), 7.22 (s, 1H), 7.38 (d, J = 8 Hz, 1H), 7.48–7.55 (m, 2H), 7.91–7.96 (m, 2H), 8.08 (d, J = 8 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ 56.08, 56.09, 108.39, 111.34, 116.79, 117.19, 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 [M+H]+ 323.0854; Found 323.0842.

4.6.10. (Z)-3-(Benzo[d]thiazol-2-yl)-2-(3,5-dimethoxyphenyl)-acrylonitrile (17)

mp: 149–151 °C, 1H NMR (400 MHz, CDCl3): δ 3.84 (s, 6H), 6.55 (s, 1 H), 6.89 (s, 2H), 7.50–7.57 (m, 2H), 7.95–7.97 (d, J = 8Hz, 1H), 8.04 (s, 1H), 8.11 (d, J = 8Hz, 1H) ppm. 13C NMR (100 MHz, CDCl3): δ 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 calcd for C18H15N2O2S [M+H]+ 323.0849; Found 323.0850.

4.6.11. (Z)-3-(1H-Indol-3-yl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile (21)

mp:178–180 °C, 1H NMR (400 MHz, CDCl3): δ 3.89 (s, 3H), 3.95 (s, 6H), 6.87 (s, 2H), 7.24–7.31 (m, 2H), 7.45 (d, J = 7.6 Hz, 1H), 7.77 (s, 1H), 7.78 (s, 1H), 8.42 (d, J = 2.8 Hz, 1H), 8.94 (brs, 1H, NH) ppm; 13C NMR (100 MHz, CDCl3): δ 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 [M+H]+ 335.1390; Found 335.1387.

4.6.12. (Z)-2-(3,4-Dimethoxyphenyl)-3-(1H-indol-3-yl)acrylonitrile (22)

mp: 170–172 °C, 1H NMR (400 MHz, CDCl3): δ 3.92 (s, 3H, -OCH3), 3.97 (s, 3H, -OCH3), 6.91 (d, J = 8.4 Hz, 1H), 7.15 (s, 1H), 7.24-7.21 (m, 3H), 7.44 (d, J = 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): δ 55.97, 56.12, 104.92, 108.44, 108.55, 111.47, 111.73, 111.89, 117.90, 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 [M+H]+ 305.1285; Found 305.1278.

4.6.13. (Z)-2-(3,5-Dimethoxyphenyl)-3-(1H-indol-3-yl)acrylonitrile (23)

mp: 190–192 °C, 1H NMR (400 MHz, CDCl3): δ 3.86 (s, 6H), 6.45 (s, 1H), 6.82 (s, 2H), 7.25–7.29 (m, 2H), 7.44 (d, J = 8.0 Hz, 1H), 7.75 (d, J = 7.2 Hz, 1H), 7.87 (s, 1H), 8.44 (s, 1H), 8.83 (brs, 1H, NH) ppm; 13C NMR (100 MHz, CDCl3): δ 55.52, 100.10, 103.67, 111.79, 118.00, 119.95, 121.38, 123.50, 126.40, 127.28, 133.78, 135.45, 136.84, 161.21 ppm. HRMS (ESI) m/z calcd for C19H17N2O2 [M+H]+ 305.1285; Found 305.1297.

4.6.14. (Z)-3-(5-Methoxy-1H-indol-3-yl)-2-(3,4,5-trimethoxy- phenyl)acrylonitrile (24)

mp: 183–185 °C, 1H NMR (400 MHz, CDCl3): δ 3.88 (s, 3H, -OCH3), 3.89 (s, 3H, -OCH3), 3.94 (s, 6H, -OCH3), 6.85 (s, 2H), 6.93 (d, J = 8.8 Hz, 1H), 7.18 (s, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.69 (s, 1H), 8.38 (s, 1H), 8.60 (brs, 1H, NH) ppm; 13C NMR (100 MHz, CDCl3): δ 55.98, 56.37, 61.00, 100.47, 102.95, 104.74, 111.72, 112.52, 113.21, 120.02, 126.78, 127.93, 130.52, 130.80, 133.17, 153.60, 155.39 ppm. HRMS (ESI) m/z calcd for C21H21N2O4 [M+H]+ 365.1496; Found 365.1502.

4.6.15. (Z)-3-(Benzofuran-3-yl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile (25)

mp: 144–146 °C, 1H NMR (400 MHz, CDCl3): δ 3.91 (s, 3H), 3.96 (s, 6H), 6.89 (s, 2H), 7.37–7.41 (m, 2H), 7.55 (s, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.72 (d, J = 7.6 Hz, 1H), 8.67 (s, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ 56.82, 61.47, 103.72, 112.10, 112.48, 116.53, 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 [M+H]+ 336.1236; Found 336.1218.

4.6.16. (Z)-3-(Benzofuran-3-yl)-2-(3,4-dimethoxyphenyl)acrylonitrile (26)

mp: 135–137 °C, 1H NMR (400 MHz, CDCl3): δ 3.92 (s, 3H), 3.96 (s, 3H), 6.90 (d, J = 8.8 Hz, 1H), 7.13 (d, J = 2.4 Hz, 1H), 7.24 (dd, J = 2.4, 8.4 Hz, 1H), 7.33–7.40 (m, 2H), 7.49 (s, 1H), 7.54 (d, J = 8.4 Hz, 1H), 8.62 (s, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ 56.04, 56.08, 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 [M+H]+ 306.1125; Found 306.1126.

4.6.17. (Z)-3-(Benzofuran-3-yl)-2-(3,5-dimethoxyphenyl)acrylonitrile (27)

mp:121–123 °C, 1H NMR (400 MHz, CDCl3): δ 3.86 (s, 6H), 6.50 (s, 1H), 6.81 (s, 2H), 7.34–7.42 (m, 2H), 7.56 (d, J = 7.2Hz, 1H), 7.62 (s, 1H), 7.69 (d, J = 8Hz, 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 [M+H]+ 306.1125; Found 306.1133.

Supplementary Material

supplement

Highlights.

  • Synthesis of novel a series of heterocyclic trans cyano CA-4 analogs

  • Analogs evaluated for anti-cancer activity against 60 human cancer cell lines

  • Analogs evaluated against a variety of primary leukemia cell lines

  • Binding modes at the colchicine binding site on tubulin have been determined

  • Benzothiophen-2-yl and indol-2-yl analogs are the most potent anticancer agents

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.

Footnotes

Supplementary Material

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Notes and references

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