Multisubstituted indole-acrylonitrile hybrids as potential cytotoxic agents

Article abstract of DOI:10.1016/j.bmcl.2014.03.011

A series of multisubstituted indole-acrylonitrile hybrids were designed, synthesized and evaluated for their potential cytotoxic activities. The bio-evaluation results indicated that some of the target compounds (such as 3a, 3f, 3k, 3n) exhibited good to moderate cytotoxic effect on HepG2, BCG-823, BEL-7402, and HL-7702 cell lines. Especially, the compounds 3a and 3k also exhibited high cytotoxic activities (3a, 19.38 ± 3.38 μM; 3k, 15.43 ± 3.54 μM) against the BEL-7402 cell line resistant to Taxol (>25 μM) and 5-FU (>500 μM), which might be developed as novel lead scaffold for potential anticancer agents.

Full text of DOI:10.1016/j.bmcl.2014.03.011

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1907–1911
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Multisubstituted indole–acrylonitrile hybrids as potential cytotoxic
agents
⇑
⇑
Shaoyong Ke , Ziwen Yang, Zhigang Zhang, Ying Liang, Kaimei Wang, Manli Liu, Liqiao Shi
National Biopesticide Engineering Research Center, Hubei Academy of Agricultural Sciences, Wuhan 430064, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of multisubstituted indole–acrylonitrile hybrids were designed, synthesized and evaluated for
their potential cytotoxic activities. The bio-evaluation results indicated that some of the target com-
pounds (such as 3a, 3f, 3k, 3n) exhibited good to moderate cytotoxic effect on HepG2, BCG-823, BEL-
7402, and HL-7702 cell lines. Especially, the compounds 3a and 3k also exhibited high cytotoxic activities
Received 16 January 2014
Revised 3 March 2014
Accepted 5 March 2014
Available online 17 March 2014
(3a, 19.38 3.38
5-FU (>500
lM; 3k, 15.43 3.54 lM) against the BEL-7402 cell line resistant to Taxol (>25 lM) and
M), which might be developed as novel lead scaffold for potential anticancer agents.
l
Keywords:
Multisubstituted indole–acrylonitrile
Hybrids
Ó 2014 Elsevier Ltd. All rights reserved.
Synthesis
Cytotoxic activity
Nitrogen-containing heterocycles always display extensive bio-
activities, and which are also extremely versatile structural units
serving as important intermediates for the construction of active
molecules in drug design and agrochemical industry.1–3 Among
these heterocyclic building blocks, the myriad indole skeleton
has emerged as a most promising class of synthons for pharmaceu-
tical drugs and functional materials.4–7 Up to now, many heterocy-
clic compounds derived from indole have been identified as
potential antibacterial, anticancer, antiviral agents and protein ki-
nase inhibitors.8–11 In addition, some indole intermediates can also
be used as important synthons for further transformation to re-
lated fused-heterocycles or various indole alkaloids.
On the other hand, acrylonitrile units have also attracted con-
siderable attention for decades, and which are privileged scaffolds
because of their broad applications range from medicinal
agents,12–16 agrochemicals17–19 to functional materials.20–22 Re-
cently, many examples bearing this moiety have been reported
to confirm that the introduction of this pharmacophores can result
in highly potential activity as protein kinase inhibitors or antican-
cer agents. As shown in Figure 1, the compounds AG-49023 and AG-
1824 are potential EGFR inhibitors, the compound AG-102425 is an
IGF-1R autophosphorylation inhibitors, WP1066 is a novel inhibi-
tor of JAK2 and STAT3,26 WP1130 is a selective deubiquitinase
inhibitor.27 Meanwhile, the flexible properties and itself character-
istics of acrylonitrile group make it present important chemical
significance, and which is always the focus field to researchers in
materials due to its conjugation system.20–22
Thus, based on the aforementioned statements, this work fo-
cused on the design, convenient synthesis, and cytotoxic evaluation
of a series of multisubstituted indole–acrylonitrile derivatives
based on pharmacophores hybridization. We utilized indole scaf-
fold as key prototype structural unit and planed for the integration
of indole skeleton and acrylonitrile pharmacophores to the core
structure as shown in Figure 2, which might be developed as lead
compounds for high potential cytotoxic agents.
The general procedures for the preparation of novel
multisubstituted indole–acrylonitrile derivatives 3a–p are outlined
in Scheme 1.
The key building blocks N-substituted indoles 1b were obtained
by alkylation reaction of indole in the presence of Me2SO4/K2CO3,
and then the reactants 1a–c were conveniently transferred to the
corresponding 3-(1H-indol-3-yl)-3-oxopropanenitriles 2a–c via
classical Friedel–Crafts acylation reaction under the condition of
Ac2O/NCCH2COOH. The following Knoevenagel condensation reac-
tions of intermediates 2a–c were treated with various aldehydes
resulting in multisubstituted indole–acrylonitrile derivatives 3a–
p. All target compounds gave satisfactory chemical analyses, and
the general procedures and spectra data were described in Supple-
mentary data.
The newly prepared multisubstituted indole–acrylonitrile hy-
brids 3a–p were evaluated for their in vitro cytotoxic effects
against HepG2 (hepatocellular liver carcinoma), BCG-823 (gastric
cancer), BEL-7402 (hepatocellular carcinoma), and HL-7702 (nor-
mal liver cell) cell lines by the standard MTT (3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyl tetrazolium bromide) assay28 using 5-FU
(5-Fluorouracil) and Taxol as a positive control. The preliminary
results were summarized in Table 1.
⇑
Corresponding authors. Tel.: +86 2759101956 (S.K.).
E-mail addresses: keshaoyong@163.com (S. Ke), shiliqiao_75@163.com (L. Shi).
http://dx.doi.org/10.1016/j.bmcl.2014.03.011
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

1908
S. Ke et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1907–1911
O
N
N
HO
HO
HO
HO
Br
N
N
N
N
Br
N
H
H
N
H
N
HO
N
N
N
Br
O
O
AG-490 (Tyrphostin)
AG-18
AG-1024
WP1066
WP1130
Figure 1. Representative compounds containing acrylonitrile unit.
Generally, as shown in Table 1, the newly prepared indole–acry-
lonitrile hybrids (3a–p) displayed moderate to good inhibition
activities against the four human cancer cell lines. Notably, the
compounds 3a, 3f, 3k, and 3n exhibited significant inhibitory
activities (71.07–87.21%) against all four tested cell lines at
cancer cell lines. The results of preliminary cytotoxic assay indi-
cated this combination of indole core and acrylonitrile unit lead
to the high potential active scaffold, and which can be used as lead
compounds for optimization of anticancer agents.
Based on the preliminary results, most of the target compounds
displayed good inhibitory activities against the tested cell lines, so
in order to further investigate the potential activities, the IC50 val-
ues were further evaluated using the above cell-based method. The
inhibitory activities expressed as IC50 values for the target com-
pounds are presented in Table 2. The results further testify that
some of the designed indole–acrylonitrile hybrids 3a–p exhibited
higher inhibition activities compared to the commercial 5-FU un-
der the same conditions. As indicated in Table 2, compound 3a
showed the strongest inhibitory effect on all four cell lines, with
an IC50 values of 7.07 0.92 (HepG2), 4.92 2.46 (BCG-823),
40 lg/mL concentration. Also, it is interesting to note that com-
pound 3b and 3d presented selective cytotoxicity for a special hu-
man hepatocellular liver carcinoma cell, and the inhibitions against
HepG2 cell were up to 72.05% and 71.52%, respectively. However,
from the results indicated in Table 1, we also observed that the
intermediates 2a–c exhibited lower activities against the tested
N
N
O
Ar
R
19.38 3.38 (BEL-7402) and 18.76 5.84 (HL-7702)
lM, respec-
R
N
tively. Especially, we can find that the most potential compounds
3a, 3f, 3k and 3n also showed higher cytotoxic activities (3a,
Pharmacophores
Hybridization
H
N
H
19.38 3.38
28.12 5.13
l
M; 3f, 20.99 4.11 lM; 3k, 15.43 3.54 lM; 3n,
Figure 2. Design strategy of multisubstituted indole–acrylonitrile hybrids.
lM) against the BEL-7402 cell line resistant to Taxol
R1
N
O
R1
O
R1
Ac2O/NCCH2COOH
Cat. KOH
EtOH
CN
R3
N
70-80°C
R2
N
N
R2
R2
3a-p
1a-c
2a-c
1a R1 = R2 = H
2a R1 = R2 = H
1b R1 = H, R2 = Me
2b R1 = H, R2 = Me
2c R1 = CN, R2 = H
1c R1 = CN, R = H
2
Scheme 1. General synthetic route for multisubstituted indole–acrylonitrile derivatives.
Table 1
Cytotoxic activity (% cell growth inhibition) of the compounds 3a–p and 2a–c at 40
l
g/mL concentration against various human cancer cell lines
Growth-inhibitory properties (%)
Entry
Compd no.
HepG2a
BCG-823a
BEL-7402a
HL-7702a
1
2
3
4
5
6
7
8
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
2a
2b
2c
71.07 3.25
72.05 4.24
23.87 7.49
71.52 1.40
39.71 3.23
75.71 4.22
72.40 2.03
8.23 6.16
70.94 9.01
73.18 2.01
72.88 6.35
36.11 6.49
16.88 5.42
71.53 0.49
25.27 5.29
0.00 0.00
77.51 7.48
63.94 6.80
15.89 6.48
61.53 8.00
26.19 9.80
81.74 4.07
76.74 2.49
3.66 6.33
81.00 5.84
33.95 12.31
82.37 3.13
41.72 8.24
3.90 5.56
76.51 6.41
2.37 4.10
0.00 0.00
31.06 2.08
62.61 2.23
21.89 12.09
84.73 8.82
2.16 1.87
72.08 5.66
66.51 3.16
23.99 7.01
38.53 6.60
41.51 4.99
77.79 4.35
69.56 3.41
35.33 9.59
66.03 8.11
42.95 7.49
74.10 3.03
37.26 10.42
26.51 7.12
74.13 3.45
12.50 6.21
1.63 2.82
23.91 11.56
14.10 5.24
32.79 10.86
82.55 8.36
28.50 2.77
44.54 6.82
78.18 12.28
24.63 7.66
87.21 5.85
48.59 6.44
49.81 7.96
79.40 6.61
25.74 5.36
20.15 6.50
5.55 6.39
9
10
11
12
13
14
15
16
17
18
19
71.74 5.42
73.24 2.44
12.59 7.47
31.08 8.58
38.40 2.88
35.09 3.26
26.26 10.64
51.15 9.03
a
Abbreviations: HepG2—human hepatocellular liver carcinoma cell line; BCG-823—human gastric cancer cell line; BEL-7402—human hepatocellular carcinoma cell line;
HL-7702—human normal liver cell line.

S. Ke et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1907–1911
1909
Table 2
Cytotoxic activity of the compounds 3a–p and 2a–c against various cell lines
N
O
R1
R3
N
R2
3a-p
a
Entry
Compd no.
Substituents
R3
In vitro cytotoxicity IC50 (lM)
BCG-823b
R1
H
R2
HepG2b
BEL-7402b
HL-7702b
NH
1
2
3a
3b
Me
7.07 0.92
4.92 2.46
19.38 3.38
18.76 5.84
OMe
OMe
OMe
H
Me
18.61 7.18
90.93 1.60
>100
76.84 12.76
3
4
5
3c
3d
3e
H
H
H
Me
Me
Me
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
HO
N
24.38 5.57
>100
NC
NO2
Cl
6
3f
H
Me
15.79 5.19
12.33 1.29
20.99 4.11
15.58 0.87
N
N
HN
OMe
OMe
OMe
7
8
3g
3h
H
H
H
H
37.28 4.69
>100
56.89 2.49
>100
>100
>100
55.51 6.63
>100
9
3i
H
H
H
H
H
H
83.65 15.97
58.07 15.07
38.89 2.89
67.34 2.43
>100
72.19 9.02
>100
86.78 14.58
>100
HO
10
11
3j
NH
3k
17.04 4.18
15.43 3.54
40.50 8.04
12
13
3l
H
H
H
H
>100
>100
>100
>100
>100
>100
>100
>100
N
NO2
N
3m
N
S
HN
Cl
NO2
Cl
14
15
3n
3o
H
H
22.76 4.24
>100
24.10 8.26
>100
28.12 5.13
>100
15.84 4.69
>100
N
N
HN
OMe
OMe
CN
H
H
OMe
NH
16
17
3p
2a
CN
—
>100
>100
>200
>100
>200
>100
>200
44.54 14.67
(continued on next page)

1910
S. Ke et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1907–1911
Table 2 (continued)
a
Entry
Compd no.
Substituents
R3
In vitro cytotoxicity IC50 (lM)
BCG-823b
R1
R2
HepG2b
BEL-7402b
HL-7702b
18
19
20
21
2b
2c
—
—
—
—
31.80 9.09
>200
0.0064 0.0045
86.87 9.23
87.33 7.07
>200
0.0028 0.0003
67.65 16.91
>200
>200
>25
>200
>200
0.0518 0.0395
95.36 6.92
Taxolc
5-FUd
>500
a
IC50—compound concentration required to inhibit tumor cell proliferation by 50%.
Abbreviations: HepG2—human hepatocellular liver carcinoma cell line; BCG-823—human gastric cancer cell line; BEL-7402—human hepatocellular carcinoma cell line;
b
HL-7702—human normal liver cell line.
c
Taxol, used as a positive control.
5-Fluorouracil, used as a positive control.
d
Figure 3. Morphology image of BEL-7402 cells treated with the compounds 3a, 3f, 3k, 3n, or Taxol for 48 h (400Â). A is the control, the cells treated with DMSO 0.1% (v/v) as a
vehicle control. B–F are treated with the compounds 3a, 3f, 3k, 3n, or Taxol at concentration of 10 lg/mL, respectively.
(>25
l
M) and 5-FU (>500
l
M). The most potent compounds 3a and
agents. Bio-evaluation indicated that some of the newly synthe-
sized compounds exhibited good cytotoxic activities. Especially,
the most potent compounds 3a, 3f, 3k and 3n also showed higher
cytotoxic activities against the BEL-7402 cell line resistant to Taxol
3k all contain two indole cores in single molecule, which may have
appropriate hydrophobic–lipophilic interactions with proteins. In
contrast, compound 3p bearing two indole moieties almost lost
inhibitory activity due to its poor solubility. By comprehensive
consideration of the structure–activity relationships for these com-
pounds, we also can conclude that the compounds bearing indole
core (entries 1 and 11) or containing multisubstituted heterocycles
(entries 6 and 14) exhibited higher activities. These interesting re-
sults might be used to develop novel lead scaffolds for potential
anticancer agents.
Based on the aforementioned results, we can find that the com-
pounds 3a, 3f, 3k and 3n also showed higher cytotoxic activities
against the BEL-7402 cell line resistant to Taxol and 5-FU, which
are very interesting. Furthermore, during the experiments, signifi-
cant morphological changes of the cells have been observed under
an inverted microscope. So we present the selective morphological
image of the cells treated with compounds 3a, 3f, 3k and 3n at
and 5-FU, which is characterized by IC50 values in the low
lM
range. These interesting results might be used to develop novel
lead scaffold for potential anticancer agents.
Acknowledgments
We gratefully acknowledge the support of this work by the Pro-
jects in the National Science & Technology Pillar Program during
the Twelfth Five-year Plan Period (2011BAE06B004-02,
2011BAE06B004-15) and the partial support from Hubei Agricul-
tural Science Innovation Centre (2012-620-008-002).
Supplementary data
10 lg/mL for 48 h in the following pictures (Fig. 3). Contrast with
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.
2014.03.011.
the presented images in Figure 3, we found that the quantities of
the cells treated with compounds 3a, 3f, 3k and 3n were obviously
decreased greatly for BEL-7402 cell lines. On the other hand, signif-
icant volume shrink and changing to globular and fragment of
some BEL-7402 cells have also been observed. However, such mor-
phological changes were not apparent in the control cells (Fig. 3A)
and the cells treated with Taxol (Fig. 3F).
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hybrids have been designed and synthesized as potential cytotoxic

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