Novel 2-cyanoacrylamido-4,5,6,7-tetrahydrobenzo[b]thiophene derivatives as potent anticancer agents

Article abstract of DOI:10.1002/ardp.202000069

Ethyl 2-acrylamido-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate as well as its corresponding bis-derivatives, 5–10, with aliphatic linkers were synthesized, fully characterized, and tested as novel anticancer agents. The targeted compounds, 5–10, wer

Full text of DOI:10.1002/ardp.202000069

Received: 6 March 2020
Revised: 18 June 2020
Accepted: 22 June 2020
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DOI: 10.1002/ardp.202000069
F U L L P A P E R
Novel 2‐cyanoacrylamido‐4,5,6,7‐tetrahydrobenzo[b]thio-
phene derivatives as potent anticancer agents
Farid M. Sroor1
Khaled Mahmoud3 | Ahmed H. M. Elwahy4
| Mohamad M. Aboelenin2 | Karima F. Mahrous2
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| Ismail A. Abdelhamid4
1Organometallic and Organometalloid
Chemistry Department, National Research
Abstract
Centre, Cairo, Egypt
Ethyl 2‐acrylamido‐4,5,6,7‐tetrahydrobenzo[b]thiophene‐3‐carboxylate as well
as its corresponding bis‐derivatives, 5–10, with aliphatic linkers were
synthesized, fully characterized, and tested as novel anticancer agents. The targeted
compounds, 5–10, were obtained by the Knoevenagel condensation reactions of
bis‐o‐ or ‐p‐aldehyde with a molar ratio of ethyl 2‐(2‐cyanoacetamido)‐4,5,6,7‐
tetrahydrobenzo[b]thiophene‐3‐carboxylate of 2 in the presence of piperidine in
excellent yields (93–98%). The in vitro anticancer activities of the prepared com-
pounds were evaluated against HepG2, MCF‐7, HCT‐116, and BJ1 cells. Compounds
7 and 9 emerged as the most promising compounds, with IC50 values of 13.5 and
32.2 µg/ml, respectively, against HepG2 cells, compared with the reference drug
doxorubicin (IC50: 21.6 µg/ml). Real‐time reverse‐transcription polymerase chain
reaction was used to measure the changes in expression levels of the COL10A1 and
COL11A1, ESR1, and ERBB2, or AXIN1 and CDKN2A genes within the treated cells,
as genetic markers for colon, breast, or liver cancers, respectively. Treatment of the
colon cancer cells with compounds 5, 9, and 10, or breast and liver cancers cells with
compounds 7, 8, 9, and 10 downregulated the expression of the investigated tumor
markers. The DNA damage values (depending on comet and DNA fragmentation
assays) increased significantly upon treatment of colon cancer cells with compounds
5, 9, and 10, and breast and liver cells with compounds 8, 9, and 10. The
structure–activity relationship suggested that the increase of the chain of the alkyl
linker increases the anticancer activity and the compounds with bis‐cyanoacrylamide
moieties are more active than those with one cyanoacrylamide moiety.
2Cell Biology Department, National Research
Centre, Dokki, Giza, Egypt
3Pharmacognosy Department, National
Research Centre, Dokki, Egypt
4Chemistry Department, Faculty of Science,
Cairo University, Giza, Egypt
Correspondence
Farid M. Sroor, Organometallic and
Organometalloid Chemistry Department,
National Research Centre, Cairo 12622, Egypt.
Email: faridsroor@gmx.de and
fm.sroor@nrc.sci.eg (F. M. S.)
Ahmed H. M. Elwahy and Ismail A. Abdelhamid,
Department of Chemistry, Faculty of Science,
Cairo University, Giza 12613, Egypt.
Email: aelwahy@yahoo.com (A. H. M. E.) and
ismail_shafy@yahoo.com (I. A. A.)
K E Y W O R D S
2‐cyanoacrylamide linked to 4,5,6,7‐tetrahydrobenzo[b]thiophene, alkyl linkers, anticancer,
bis(aromatic aldehydes), DNA damage, DNA fragmentation, gene expression
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1
INTRODUCTION
against cancer, which is the hot topic in the research area nowadays.
Thiophene and their fused derivatives are substantial building blocks and
Cancer is one of the main causes of major morbidity and mortality
worldwide, leading to a high percentage of deaths annually, as reported
by the World Health Organization (WHO).[1] Therefore, it is of prime
importance to develop novel and potent anticancer agents to fight
synthons in synthetic chemistry (Figure 1). The chemistry of these
molecules is of increasing interest, as they seem to be promising phar-
macological compounds with anticancer,[2‐4] anti‐inflammatory,[5,6] anti-
bacterial (I),[7‐9] antiproliferative,[10] antitubercular,[11] and antiviral
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Arch Pharm. 2020;e2000069.
wileyonlinelibrary.com/journal/ardp
© 2020 Deutsche Pharmazeutische Gesellschaft
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FIGURE 1 Examples of bioactive compounds containing the thiophene moiety and bioactive bis‐heterocycles with ether linkers
properties.[12,13] Moreover, the acrylamide derivatives have attracted
special attention due to their diverse biological and pharmacological
applications such as anticancer (II),[14‐16] anti‐inflammatory (III),[17,18]
antidiabetic,[19] antifungal,[20,21] and antimicrobial (IV) applications.[9,22,23]
In 2016, Aguiar et al.[24] reported a series of 2‐aminothiophene
derivatives, V, revealing their antiproliferative activity (Figure 1). The
activity of these compounds has been evaluated against HeLa, PANC‐1,
and 3T3 cells, which were exposed to the compounds at concentrations
of 5, 10, 25, or 50 µM for 24 or 48 hr. It was noted that nontumor
fibroblast cells (3T3) were protected by treatment with the synthesized
2‐aminothiophene derivatives and a pronounced proliferative effect was
observed, particularly after 48 hr, suggesting greater neoplastic anti-
proliferative selectivity. Likewise, in the same period, the reference drug
doxorubicin was more toxic than the selected 2‐aminothiophene deri-
vatives, which displayed a cell proliferation effect of <50%.[24]
of novel compounds with different applications such as redox
flow battery,[25] biofunctional dynamic covalent polymer,[26]
allosteric effectors of hemoglobin,[27] fluorescence‐based assay,[28]
in Groebke–Blackburn–Bienayme/Ugi reactions,[29] electrical
conductivity,[30] macrocyclization,[31] breaking and mending of the
porphyrin,[32] and a broad spectrum of biological activities.[16,23]
It is expected that the combination of tetrahydrobenzo[b]thio-
phene, acrylamide, and the alkyl linkers will increase the biological
profile of the targeted compounds. It is worth to mention that the
hydrophilic region in the targeted structures is homogeneously inside
the cell and represents a suitable probe for viscosity measurements
in the cytoplasm, whereas the hydrophobic region behaves as a
membrane layer to some extent. However, the hydrophobic region
also crosses the cell membrane and likely binds to intracellular pro-
teins,[33] as explained in Figure 2.
However, a few studies have been performed on the use
of bis‐benzaldehydes as a starting material for the preparation
In our endeavor toward the development and discovery of novel
potent anticancer agents and in continuation of our previous work to
FIGURE 2 The design concept used for the synthesis of the title compounds

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SCHEME 1 The synthesis of the bis(benzaldehydes) 3a–f
prepare biologically active organic and organometallic compounds,[34‐43]
we decided to design and prepare a novel series of bis‐(ethyl‐2‐(2‐
cyanoacrylamido)‐4,5,6,7‐tetrahydrobenzo[b]thiophene‐3‐carboxylates)
utilizing the appropriate bis‐aldehydes bearing aliphatic chain linkers as
precursors (Figure 2).
7‐tetrahydrobenzo[b]thiophene‐3‐carboxylate 4 in ethanol in the
presence of a few drops of piperidine at reflux for 30 min, condensa-
tion occurred from both sides with the two aldehydic groups and
resulted in the formation of bis‐ethyl 2‐(2‐cyanoacrylamido)‐4,5,6,7‐
tetrahydrobenzo[b]thiophene‐3‐carboxylate derivatives 5, 7, and 9
respectively (Schemes 2 and 3). However, the reaction of bis(benzal-
dehydes) 3b, 3d, and 3f with two equivalents of compound 4 afforded
the respective ethyl 2‐{2‐cyano‐3‐[4‐(4‐formylphenoxy)alkoxy]phenyl}
acrylamido‐4,5,6,7‐tetrahydrobenzo[b]thiophene‐3‐carboxylate deri-
vatives 6, 8, and 10 in which condensation occurred from one side only
(Schemes 2 and 3).
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2
RESULTS AND DISCUSSION
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2.1
Chemistry
The key starting materials, bis(benzaldehydes) 3a–f, were prepared, as
reported by our group, through the reaction of the potassium salt of
the appropriate hydroxybenzaldehydes 1a,b with the corresponding
dibromo compound 2 in dimethylformamide at reflux, as described
in Scheme 1.[44‐47] When the bis(benzaldehydes) 3a, 3c, and 3e
reacted with two equivalents of ethyl 2‐(2‐cyanoacetamido)‐4,5,6,
The chemical structures of the novel compounds 5–10 were
proved spectroscopically on the basis of the elemental analysis and
spectral data. The infrared (IR) spectrum of compound 5 as a
representative example indicated the presence of NH group at
3,410 cm−1 and the carbonyl band at 1,662 cm−1. The mass spectrum of
compound 5 revealed a molecular ion peak as a base peak at m/z 818.
SCHEME 2 The synthesis of compounds 5–10

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CN
CN
EtO2C
O
O
S
O
S
EtO2C
CO2Et
NH
O
OHC
O
HN
N
H
S
NC
O
O
5
6
(96%)
(93%)
CN
CN
O
S
O
EtO2C
O
EtO2C
CO2Et
OHC
NH
O
O
HN
N
H
S
S
NC
O
O
7
8
(95%)
(98%)
CN
CN
EtO2C
O
O
S
EtO2C
O
S
CO2Et
NH
O
OHC
O
HN
N
H
S
NC
O
O
9
10
(98%)
(97%)
SCHEME 3 Structures of the prepared derivatives 5–10
The 1H nuclear magnetic resonance (NMR) spectrum indicated the
ester group as multiplets at 1.30 and a quartet at 4.20 ppm. It showed
the methylene linkage as multiplets at 4.57 ppm. It also revealed a
singlet signal at 8.74 for the vinyl H3 proton. The amide NH group
resonated at δ 11.68 ppm. All other signals appeared at their expected
positions. On the contrary, the IR spectrum of compound 8 indicated
the presence of NH group at 3,421 cm−1 and the carbonyl band at
1,667 cm−1. The mass spectrum of compound 8 revealed a molecular
ion peak as a base peak at m/z 558. The 1H NMR spectrum indicated
one ester group as multiplets at 1.34 and multiplets at 4.30 ppm. It also
revealed a singlet signal integrated by one proton at 8.37 for the vinyl
H3 proton. It featured one CHO group at 9.87 ppm. The amide NH
group resonated at δ 11.95 ppm. All other signals appeared at their
expected positions. Unfortunately, the 13C NMR spectra of all the
compounds, 5–10, have not been assigned due to the poor solubility of
these compounds in most of the deuterated solvents.
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2.2
Anticancer activity
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2.2.1
Primary screening
Compounds 5–10 were screened against three human cancer cell
lines, namely Caucasian breast adenocarcinoma (MCF7), hepatocel-
lular carcinoma (HEPG2), and colon cell line (HCT116), at 100 µg/ml,
and their results were compared with the normal skin fibroblast cell
line (BJ1). The results revealed that compounds 6, 7, 8, 9, and 10
exhibited more than 75% mortality against the HEPG2 cell line,
TABLE 2 IC50 values (mM) for promising compounds
TABLE 1 % Mortality of cancer and normal cell lines at 100 μg/ml
Compounds
HEPG2
83.27
109.80
38.65
94.30
15.93
103.09
0.037
0%
HCT116
95.30
–
MCF7
BJ1
Compounds
HEPG2
68.2
76.2
100
HCT116
42.5
42.6
54.3
11.3
65.6
64.6
100
MCF7
47.6
49.5
49.5
42.6
52.6
51.8
100
BJ1
55.3
35.4
51.4
10.5
5.3
5
–
106.96
5
6
–
–
6
7
106.21
–
–
112.12
7
8
–
–
8
80.1
100
9
87.40
–
104.80
174.15
0.045
0%
–
9
10
–
10
75.3
100
22.4
100
0%
DOX
0.065
0%
0.057
0%
Positive control
Negative control
Negative control
0%
0%
0%
Abbreviation: DOX, doxorubicin.

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Secondary screening
whereas compounds 7, 9, and 10 exhibited more than 50% mortality
against HCT116. However, only two compounds, 9 and 10, exhibited
50% mortality against MCF7, as shown in Table 1. So these promising
compounds were subjected to secondary screening to calculate their
IC50 values and selectivity index.
2.2.2
Concerning IC50 values, the most promising compounds for HEPG2
were compounds 9 and 7, with IC50 values 15.93 and 38.65 mM,
respectively. Compound 9 was found to be more promising than 7,
FIGURE 3 Alterations in the gene expression level of (a) COL10A1 and (b) COL11A1 genes in colon cancer cell line; (c) ESR1 and (d) ERBB2
genes in breast cancer cell line; and (e) AXIN1 and (f) CDKN2A genes in liver cancer cell line due to the treatment with the target molecules.
Mean values with different superscript letters (a, b, c, and d) were significantly different (p < .05). Control (−ve): untreated and control (+ve):
doxorubicin

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FIGURE 4 The visual score of normal DNA (Class 0) and damaged DNA (Classes 1, 2, and 3) using the comet assay in the investigated cell
lines. Class 0, no tail; 1, tail length < diameter of nucleus; 2, tail length between 1× and 2× the diameter of nucleus; and 3, tail
length > 2× the diameter of nucleus
where its selectivity index was higher than that of compound 9 and
the values of IC50 ranged from 83.27 to 109.8 mM. However, in the
case of MCF7 and HCT116 cell lines, compound 9 is still a promising
compound, compared with others, but with a lower activity than for
the HEPG2 cell line, as depicted in Table 2. This result elucidates that
compound 9 exhibits selectivity against the HEPG2 cell line. Although
the activity of compound 9 is lower than the chosen medicine, it has a
higher selectivity index.
of the COL11A1 gene was decreased significantly in compounds 5‐ and
10‐treated cells, compared with compound 9‐treated cells.
Moreover, the expression levels of estrogen receptors (ESR1)
and Erb‐B2 receptor tyrosine kinase 2 (ERBB2) genes were used as
breast cancer markers, whereas the expression of AXIN1 and
CDKN2A genes was used as a liver cancer marker. The expression of
all the investigated breast and liver cancer markers in all treated and
positive control cells was significantly (p < .05) lower than their
expression in the negative control cells (Figure 3c–f). The expression
of all the studied breast or liver cancer gene markers in the negative
control cells was higher by around twofold than the positive control
cell lines. Although the treatment with compound 7 decreased the
expression level of the investigated breast and liver cancer markers,
their expression was higher than in the positive controls.
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2.2.3
Gene expression analysis
The gene expression analysis of colon cancer markers was carried out
using two colon cancer‐related genes, namely collagen type
X
α1 (COL10A1) and collagen type XI α1 (COL11A1). The results
(Figure 3a,b) revealed that the COL10A1 and COL11A1 genes were
found to be significantly overexpressed (p < .05) by about twofold in the
negative control colon cancer cells, compared with the positive control
(doxorubicin‐treated). However, the expression values of these genes
were decreased significantly in cells treated with compounds 5, 9, and
10, compared with negative control cell lines, but they did not differ
significantly from the positive control. However, the expression value of
the COL10A1 gene was decreased significantly in compound 5‐treated
cells, compared with compound 9‐treated cells. The expression value
Interestingly, the effects of the treatments with compounds 7, 8,
9, and 10 on the expression of the breast cancer markers ESR1 and
ERBB2, compared with liver cancer markers AXIN1 and CDKN2A,
respectively, had several similarities. In the cells that were treated
with compounds 8, 9, and 10, the expression of ESR1 (in breast
cancer cells) and AXIN1 (in liver cancer cells) was observed to be
downregulated, with a significant similarity with the positive control
cells. Additionally, compound 10‐treated cells had a significant lower
expression values for ESR1 and AXIN1 genes, compared with com-
pounds 7‐ and 9‐treated cells (Figure 3c,e).
TABLE 3 The visual score of DNA damage in control and treated colon tumor cell lines
No. of cells
No. of cells within each class
Treatment
No. of samples
Analyzed
300
Comets
35
0
1
2
3
DNA damaged cells % (mean SEM)
11.67 0.76c
Control (−ve)d
Control (+ve)e
Compound 5
Compound 9
Compound 10
3
3
3
3
3
265
229
226
239
228
25
32
20
25
26
6
4
300
71
21
33
23
22
18
21
13
24
23.65 1.04ab
300
74
24.71 0.66a
300
61
20.33 0.72b
300
72
24.11 0.50a
Note: Mean values with different superscripts (a, b, and c) between locations in the same column are significantly different at p < .05.
Abbreviation: SEM, standard error of the mean.
dUntreated.
eDoxorubicin‐treated.

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TABLE 4 The visual score of DNA damage in control and treated breast tumor cell lines
No. of cells
No. of cells within each class
Treatment
No. of samples
Analyzed
300
Comets
26
0
1
2
3
DNA damaged cells % (mean SEM)
8.67 0.62c
Control (−ve)d
Control (+ve)e
Compound 7
Compound 8
Compound 9
Compound 10
3
3
3
3
3
3
274
227
247
223
231
217
19
33
21
23
27
34
5
2
300
73
22
20
35
22
27
18
12
19
20
22
24.33 0.76a
300
53
17.66 0.75b
300
77
25.67 0.53a
300
69
23.00 1.04a,b
300
83
27.69 0.78a
Note: Mean values with different superscripts (a, b, and c) between locations in the same column are significantly different at p < .05.
Abbreviation: SEM, standard error of the mean.
dUntreated.
eDoxorubicin‐treated.
However, in cells treated with compounds 8 and 9, the expres-
sion of ERBB2 (in breast cancer cells) and CDKN2A (in liver cancer
cells) was observed to be downregulated, without significant differ-
ences as compared with the positive control. Cells treated with
compound 10 had the significantly lowest ERBB2 and CDKN2A
expression values among all treatments, except cells treated with
compound 8 (Figure 3d,f).
leads to significant higher DNA damage values than treatment with
compound 9.
Moreover, the comet assay results for the treated breast and
liver cancer cell lines (Tables 4 and 5, respectively) showed that the
positive control and all treated cells had a significantly (p < .05)
higher DNA damage value than the negative control. The treatment
with compound 7 had a lower DNA damage value as compared with
the positive control and all treated cells, except those treated with
compound 9. However, there are no significant differences in the
DNA damage value among the positive control and cells treated with
compounds 8, 9, and 10.
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2.2.4
DNA damage using the comet assay
The DNA damage in colon, breast, and liver cancer cell lines was
assessed by comet assay based on visual scores (from 0 to 3), as shown
in Figure 4. In the colon cancer cell line (Table 3), the results revealed
that negative cancer cell exhibited significantly lower (p < .05) DNA
damage values, compared with the positive control (doxorubicin‐
treated cells). The DNA damage values were increased significantly in
cell lines treated with compounds 5, 9, and 10, compared with the
negative control. Moreover, there were no significant differences
between DNA damage values of cells treated with compounds 5, 9,
and 10 and the positive control. Treatment with compounds 5 and 10
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2.2.5
DNA fragmentation assay
The rate of DNA fragmentation determined in colon cancer cell lines
is summarized in Table 6 and Figure 5a. The results showed that the
rate of DNA fragmentation was increased significantly (p < .05) in
positive control (doxorubicin‐treated) cancer cell lines, in addition to
cell lines treated with compounds 5, 9, and 10, compared with
negative control cancer cell lines. Moreover, there were no
TABLE 5 The visual score of DNA damage in control and treated liver tumor cell lines
No. of cells
No. of cells within each class
Treatment
No. of samples
Analyzed
300
Comets
29
0
1
2
3
DNA damaged cells % (mean SEM)
9.68 0.61c
Control (−ve)d
Control (+ve)e
Compound 7
Compound 8
Compound 9
Compound 10
3
3
3
3
3
3
271
224
246
221
229
215
22
34
20
22
26
33
4
3
300
76
24
23
36
23
29
18
11
21
22
23
25.32 0.56a
300
54
18.00 0.72b
300
79
26.33 0.65a
300
71
23.67 0.94a,b
300
85
28.33 0.56a
Note: Mean values with different superscripts (a, b, and c) between locations in the same column are significantly different at p < .05.
Abbreviation: SEM, standard error of the mean.
dUntreated.
eDoxorubicin‐treated.

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TABLE 6 Detected DNA fragmentation in control and treated colon tumor cell lines
Treatment
DNA fragmentation % (mean SEM)
5.63 0.1c
Change
0
Inhibition
0
Control (‐ve)d
Control (+ve)e
Compound 5
Compound 9
Compound 10
21.72 1.3a,b
16.1
17.3
12.8
16.5
0
23.9 1.26a
7.45
−20.49
2.48
18.41 0.53b
22.13 2.11a
Note: Mean values with different superscripts (a, b, and c) between locations in the same column are significantly different at p < .05.
Abbreviation: SEM, standard error of the mean.
dUntreated.
eDoxorubicin‐treated.
FIGURE 5 Detection of DNA fragmentation using agarose gel in (a) control and treated colon cancer, (b) breast cancer, (c) and liver cancer
cell lines. In the three images, M, DNA marker; 1, control (−ve); 2, control (+ve). In image (a), 3, compound 9; 4, compound 5; and
5, compound 10. In images (b) and (c), 3, compound 9; 4, compound 7; 5, compound 10; and 6, compound 8
significant differences between positive control cancer cell lines and
cell lines treated with compounds 5, 9, and 10, but the DNA damage
values in cell lines treated with compounds 5 and 10 were sig-
nificantly higher than those treated with compound 9.
The percentage of fragmented DNA in the positive control and breast
and liver cancer cells treated with compounds 7, 8, 9, and 10 was
significantly high (p < .05), compared with the negative control cells. No
significant differences were detected between the DNA damage value
of cells treated with compounds 8, 9, and 10 and positive control cells,
but the positive control cells and cells treated with compounds 8 and 10
had higher DNA damage values than those treated with compound 7.
Furthermore, the effect of the treatments on the percentage of
DNA fragmentation in the breast and liver cancer cell lines was
investigated (Tables 7 and 8, respectively, and Figure 5b,c, respectively).
TABLE 7 Detected DNA fragmentation in control and treated breast tumor cell lines
Treatment
DNA fragmentation % (mean SEM)
6.4 0.08c
Change
0
Inhibition
0
Control (−ve)d
Control (+ve)e
Compound 7
Compound 8
Compound 9
Compound 10
21.8 1.1a,b
15.4
9.5
0
15.9 0.53b
−38.31
14.93
−8.44
28.57
24.1 2.11a
17.7
14.1
19.8
20.5 1.3a,b
26.2 1.26a
Note: Mean values with different superscripts (a, b, and c) between locations in the same column are significantly different at p < .05.
Abbreviation: SEM, standard error of the mean.
dUntreated.
eDoxorubicin‐treated.

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TABLE 8 Detected DNA fragmentation in control and treated liver tumor cell lines
Treatment
DNA fragmentation % (mean SEM)
5.9 0.06c
Change
0
Inhibition
0
Control (−ve)d
Control (+ve)e
Compound 7
Compound 8
Compound 9
Compound 10
20.3 0.09a,b
14.7
10.1
18.3
13.8
20.2
0
14.8 0.07b
−37.41
15.63
−9.33
29.67
23.4 1.41a
19.7 1.3a,b
25.6 1.52a
Note: Mean values with different superscripts (a, b, and c) between locations in the same column are significantly different at p < .05.
Abbreviation: SEM, standard error of the mean.
dUntreated.
eDoxorubicin‐treated.
established by the different spectral tools as well as the elemental
analyses. All the promising compounds (5, 9, 10 as anti‐colon cancer and
7, 8, 9, 10 as anti‐breast and anti‐liver cancer agents) reduced the
expression levels of each type of cancer genetic marker and induced
higher DNA damage levels, compared with the negative controls.
Moreover, the anticancer activities of these compounds are comparable
and in some cases superior to doxorubicin as a known anticancer drug.
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4
EXPERIMENTAL
|
4.1
Chemistry
|
4.1.1
General
FIGURE 6 Structure–activity relationship of the prepared
compounds 5–10
All reactions were carried out in aerobic conditions at room tem-
perature. Acetonitrile was distilled and kept under an inert atmo-
sphere. All glassware was oven‐dried at 120°C for at least 24 hr
before use. 4‐Tolylsulfonylisocyanate, 4‐tolylisocyanate, benzoyl
isocyanate, and ethyl isocyanate were purchased from Sigma‐Aldrich
and used as received. The starting materials, 3a–f, have been pre-
pared as described in the literature.[44‐47] All melting points were
uncorrected and measured using Electrothermal IA 9100 apparatus
(Shimadzu, Japan). The IR spectra were recorded as potassium bro-
mide pellets on a JASCO spectrophotometer between 4,000 and
|
2.3
Structure–activity relationship (SAR)
The preliminary SAR of anticancer activity of compounds 5–10 is
summarized and discussed in Figure 6. It is suggested that the linker
length and the position of the cyanoacrylamide on the benzene ring
were essential for the anticancer activity. The increase in the length
of the linker was found to increase the activity of the compounds.
Likewise, the activity increases in the compounds in which the cya-
noacrylamide moieties are in the ortho position with respect to the
alkoxy group and with bis‐cyanoacrylamide moieties (5, 7, and 9),
compared with those in which the groups are located in the para
position with one cyanoacrylamide unit (6, 8, and 10).
400 cm−1 1H NMR spectra (see the Supporting Information Data for
.
the original spectra) were recorded in deuterated dimethylsulfoxide
(DMSO)‐d6 on a Bruker spectrometer (400 MHz) at 25°C, but for
compound 5, it was recorded on a Varian spectrometer (300 MHz) at
30°C. The chemical shifts were expressed as part per million (δ
values, ppm) using the solvent (DMSO = 2.51, water signal at 3.34) as
the reference. Microanalyses were performed using Elementar Vario
Cube apparatus and the mass spectra were recorded using Shimadzu
Qp‐2010 plus, Micro Analytical Center, Cairo University.
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as Supporting Informa-
tion Data.
|
3
CONCLUSIONS
We could develop an efficient and simple method for the synthesis of
bis‐2‐cyanoacetamide derivatives linked to the 4,5,6,7‐tetrahydrobenz-
o[b]thiophene moiety. The structures of the novel compounds were

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4.1.2
General procedures for the synthesis of the
Diethyl 2,2′‐({3,3′‐[(butane‐1,4‐diylbis(oxy))bis(2,1‐phenylene)]bis-
(2‐cyanoacryloyl)}bis(azanediyl))bis(4,5,6,7‐tetrahydrobenzo[b]thio-
phene‐3‐carboxylate) (9)
bis(cyanoacrylamido) derivatives 5–10
Few drops of piperidine were added to a solution of ethyl 2‐(2‐
cyanoacetamido)‐4,5,6,7‐tetrahydrobenzo[b]thiophene‐3‐carboxylate
4 (2 mmol) and bis(benzaldehydes) (3a–f) (1 mmol) in ethanol (15 ml).
The reaction mixture was then heated at reflux for 30 min. The pre-
cipitate was filtered off and washed with hot ethanol (2 × 5 ml) and
dried under vacuum to afford the products 5–10 as yellow solids.
Yield 97%, Mp > 300°C. IR (KBr, cm−1): 3,398 (NH), 2,823 (CH), 1,656
(C═O); 1H NMR (400 MHz, DMSO‐d6): δ, ppm: 1.32 (m, 6H, CH3), 1.74
(m, 8H, CH2), 2.01 (m, 4H, CH2), 2.70 (m, 8H, CH2), 4.30 (m, 8H, CH2),
7.04–7.23 (m, 4H, Ar‐H), 7.64 (m, 2H, Ar‐H), 8.15 (m, 2H, Ar‐H), 8.69
(s, 2H, vinyl‐H), 10.34 (s, 2H, 2 NH). Mass spectroscopy (MS): m/z 847
(M+). Anal. calcd. for C46H46N4O8S2 (847.01): C, 65.23; H, 5.47;
N, 6.61; found: C, 65.42; H, 5.95; N, 6.83%.
Diethyl 2,2′‐({3,3′‐[(ethane‐1,2‐diylbis(oxy))bis(2,1‐phenylene)]bis(2‐
cyanoacryloyl)}bis(azanediyl))bis(4,5,6,7‐tetrahydrobenzo[b]thio-
phene‐3‐carboxylate) (5)
Yield 96%, Mp > 300°C. IR (KBr, cm−1): 3,410 (NH), 1,662 (C═O),
2,215 (CN). 1H NMR (300 MHz, DMSO‐d6): δ, ppm: 1.30 (m, 6H, CH3),
1.72 (m, 8H, CH2), 2.50–2.60 (m, 8H, CH2), 4.20 (q, J = 6.9 Hz, 4H,
CH2), 4.57 (m, 4H, 2OCH2), 7.30 (m, 4H, Ar‐H), 7.69 (m, 2H, Ar‐H),
8.28 (d, J = 7.8 Hz, 2H, Ar‐H), 8.74 (s, 2H, vinyl‐H), 11.68 (s, 2H, NH).
Anal. calcd. for C44H42N4O8S2 (818.96): C, 64.53; H, 5.17; N, 6.84;
found: C, 64.44; H, 5.26; N, 6.98%.
Ethyl 2‐(2‐cyano‐3‐{4‐[4‐(4‐formylphenoxy)butoxy]phenyl}acryl-
amido)‐4,5,6,7‐tetrahydrobenzo[b]thiophene‐3‐carboxylate (10)
Yield 98%, Mp > 300°C. IR (KBr, cm−1): 3,418 (NH), 2,875 (CH), 1,663
(C═O); 1H NMR (400 MHz, DMSO‐d6): δ, ppm: 1.30 (m, 3H, CH3),
1.75 (m, 4H, CH2), 2.27 (m, 4H, CH2), 2.68 (m, 4H, CH2), 4.30 (m, 6H,
CH2 + 2OCH2), 7.19 (m, 4H, Ar‐H), 7.89 (m, 2H, Ar‐H), 8.11 (m, 2H,
Ar‐H), 8.40 (s, 1H, vinyl‐H), 9.88 (s, 1H, CHO), 11.97 (s, 1H, NH). Anal.
calcd. for C32H32N2O6S (572.68): C, 67.12; H, 5.63; N, 4.89; found: C,
67.23; H, 5.78; N, 4.97%.
Ethyl 2‐(2‐cyano‐3‐{4‐[2‐(4‐formylphenoxy)ethoxy]phenyl}-
acrylamide)‐4,5,6,7‐tetrahydrobenzo[b]thiophene‐3‐carboxylate (6)
Yield 93%, Mp > 300°C. IR (KBr, cm−1): 3,420 (NH), 1,665 (C═O),
2,210 (CN). 1H NMR (400 MHz, DMSO‐d6): δ, ppm: 1.33 (m, 3H,
CH3), 1.73 (m, 4H, CH2), 2.72 (m, 4H, CH2), 4.34–4.51 (m, 6H,
2OCH2 + CH2CH3), 7.23 (m, 4H, Ar‐H), 7.89 (m, 2H, Ar‐H), 8.12
(m, Hz, 2H, Ar‐H), 8.41 (s, 1H, vinyl‐H), 9.88 (s, 1H, CHO), 11.88
(s, 1H, NH). MS: m/z 544 (M+). Anal. calcd. for C30H28N2O6S
(544.62): C, 66.16; H, 5.18; N, 5.14; found: C, 66.37; H, 5.31;
N, 5.32%.
|
4.2
Anticancer evaluation
|
4.2.1
Materials and methods
The MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bro-
mide) assay developed by Mosmann was modified by Miura and used
to determine the in vitro inhibitory effects of the test compounds on
cell growth. Briefly, a medium containing 10 × 103 cells (HEPG2,
HCT‐116, MCF‐7, and BJ1 cells) in a fresh complete growth medium
was seeded into each well of a 96‐well microplate, with the com-
pound solution added simultaneously to triplicate wells, before the
final volume was made up to 100 ml. The plate was incubated at 37°C
for 72 hr in a humidified atmosphere of 5% CO2 using a water‐
jacketed carbon dioxide incubator (TC2323; Sheldon, Cornelius, OR).
The medium was aspirated, fresh medium (without serum) was ad-
ded, and cells were incubated, either alone (negative control) or with
different concentrations of the sample, to give a final concentration
of 100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0.78 mg/ml. Cells were
suspended in an RPMI‐1640 medium for HEPG2, MCF‐7, and
HCT‐116, and DMEM‐F12 for BJ1, 1% antibiotic–antimycotic mix-
ture (10,000 U/ml potassium penicillin, 10,000 mg/ml streptomycin
sulfate, and 25 mg/ml amphotericin B) and 1% L‐glutamine in a
96‐well flat‐bottom microplate at 37°C under 5% CO2. After 48 hr of
incubation, the medium was aspirated; 200 ml of 10% sodium do-
decyl sulfate (SDS) in deionized water was added to each well and
further incubated overnight at 37°C under 5% CO2. Then, 200 ml of
10% SDS in deionized water was added to each well to stop the
reaction and to solubilize any MTT formazan that had formed, and
then it was incubated overnight at 37°C. Doxorubicin, which is a
known natural cytotoxic agent, was used as a positive control
Diethyl 2,2′‐({3,3′‐[(propane‐1,3‐diylbis(oxy))bis(2,1‐phenylene)]bis-
(2‐cyanoacryloyl)}bis(azanediyl))bis(4,5,6,7‐tetrahydrobenzo[b]thio-
phene‐3‐carboxylate) (7)
Yield 98%, Mp > 300°C. IR (KBr, cm−1): 3,383 (NH), 2,825 (CH), 1,653
(C═O); 1H NMR (400 MHz, DMSO‐d6): δ, ppm: 1.31 (m, 6H, CH3),
1.74 (m, 6H, CH2), 2.33 (m, 4H, CH2), 2.64 (m, 8H, CH2), 4.39 (m, 8H,
CH2 + 2OCH2), 7.14–7.26 (m, 4H, Ar‐H), 7.58 (m, 2H, Ar‐H), 8.10 (m,
2H, Ar‐H), 8.64 (m, 2H, vinyl‐H), 11.92 (s, 2H, NH). Anal. calcd. for
C45H44N4O8S2 (832.99): C, 64.89; H, 5.32; N, 6.73; found: C, 65.25;
H, 5.82; N, 6.25%.
Ethyl 2‐(2‐cyano‐3‐{4‐[3‐(4‐formylphenoxy)propoxy]phenyl}-
acrylamido)‐4,5,6,7‐tetrahydrobenzo[b]thiophene‐3‐carboxylate (8)
Yield 95%, Mp > 300°C. IR (KBr, cm−1): 3,421 (NH), 2,829 (CH), 1,667
(C═O); 1H NMR (400 MHz, DMSO‐d6): δ, ppm: 1.34 (m, 3H, CH3),
1.74 (m, 4H, CH2), 2.26 (m, 2H, CH2), 2.73 (m, 4H, CH2), 4.30 (m, 6H,
2OCH2 + CH2CH3), 7.18 (m, 4H, Ar‐H), 7.88 (m, 2H, Ar‐H), 8.08 (m,
2H, Ar‐H), 8.37 (s, 1H, vinyl‐H), 9.87 (s, 1H, CHO), 11.95 (s, 1H, NH).
Anal. calcd. for C31H30N2O6S (558.65): C, 66.65; H, 5.41; N, 5.01;
found: C, 66.82; H, 5.59; N, 5.14%.

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(100 mg/ml), which exhibited 100% lethality under the same condi-
tions. Also, 100 ml of 0.02 N HCl/50% N,N‐dimethylformamide, 20%
SDS was added to solubilize any MTT formazan that had formed. The
optical density of each well was measured at 575 nm (OD575) using a
microplate multiwell reader (model 3350; Bio‐Rad Laboratories Inc.,
Hercules, CA), and the inhibition of cell growth (%) was calculated as
(1 − T/C) × 100, where C is the mean OD575 of the control group and
T is that of the treated group. The IC50 value was determined from
the dose–response curve. Statistical significance was tested between
samples and negative control (cells with vehicle) using independent
t‐test by SPSS 11 program (Chicago, IL).
|
4.2.2
The gene expression analysis
RNA isolation
RNeasy Mini Kit (Qiagen, Hilden, Germany) was used to extract total
RNA from each type of treated and control cancer cell line following
the kit manual, and then a treatment was applied using RNAse‐free
DNAse (Invitrogen, Germany) to eliminate any DNA contamination.
Whereas the RNA integrity was checked by formaldehyde‐containing
agarose gel electrophoresis, its quantity and purity were assessed
photospectrometrically at 260 nm and by 260/280 nm ratio, re-
spectively. Then, the extracted RNA aliquots were stored at −80°C.
Reverse‐transcription (RT) reaction
The RT reactions were performed for the isolated messenger RNA
from the treated and control cancer cell lines using RevertAid™ First
Strand cDNA Synthesis Kit (Fermentas, Germany). The kit's manual
instructions for the reaction setup and incubation were followed for
a reaction with 20 µl total volume that contained oligo‐dT as a
primer and 5 µg of RNA. Afterward, the complementary DNA (cDNA)
samples were stored at −80°C.
Quantitative real‐time PCR (qPCR)
The expression levels of the studied genes were quantified within the
treated and control cancer cell lines using StepOne™ Real‐Time PCR
System (Thermo Fisher Scientific, Waltham, MA). Real‐time reverse‐
transcription polymerase chain reaction (qRT‐PCR) reactions were
performed with a total volume of 25 μl, consisting of 12.5 μl 1×
SYBR® Premix Ex TaqTM (TaKaRa, Biotech Co. Ltd.), 0.5 μl of forward
and reverse primer pairs (0.2 μM each) that are specific for each gene
(Table 9), 6.5 μl ddH2O, and 5 μl of cDNA reaction. qRT‐PCR program
was divided into three stages. The first stage occurred at 95.0°C for
3 min. The second stage consisted of 40 cycles in which each cycle
was divided into three steps: 95.0°C for 15 s, the annealing tem-
perature of each primer pair (Table 9) for 30 s and finally 72.0°C for
30 s. At the end, a gradient dissociation protocol (0.5°C every 30 s
from 65°C to 95°C) was used to examine the specificity of the qPCR
primers and the occurrence of primer dimers. Each trial involved
ddH2O as a control. The relative expression quantification of the
target gene to the reference gene β‐actin was determined by using
the 2−ΔΔC method.
t

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4.2.3
DNA damage using the comet assay
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Contreras‐Martel, A. Dessen, A. J. O'Neill, D. Mengin‐Lecreulx, D.
Blanot, S. Gobec, Eur. J. Med. Chem. 2013, 66, 32.
The neutral comet assay was performed according to the following
protocol[51] for the treated and control cancer cell lines. In brief, a
single‐cell suspension was produced by trypsinization of each cell line
plate. About 1.5 × 104 cells were fixed in 0.75% low‐melting tem-
perature agarose and placed on a precoated microscope slide, and
the cells were lysed using the lysis buffer (0.5% SDS, 30 mM ethy-
lenediaminetetraacetic acid [EDTA], pH 8.0) for 4 hr at 50°C. Then,
the slides were rinsed overnight at room temperature in Tris/borate/
EDTA buffer, pH 8.0, and the slides were subjected to electrophor-
esis at 0.6 V/cm for 25 min. Further, the slides were stained by
propidium iodide. In total, 300 cells were studied to detect the ratio
of cells with DNA damage. The cells were classified as follows: Class
0, no detectable DNA damage and no tail; Class 1, tail with a
length < the diameter of the nucleus; Class 2, tail with length
between 1× and 2× the nuclear diameter; and Class 3, tail longer than
2× the diameter of the nucleus.[52]
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4.2.4
DNA fragmentation assay
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The cancer cell lines were treated with the investigated compounds,
and then the cells were trypsinized and washed with Dulbecco's
phosphate‐buffered saline. The cells were treated for 30 min on ice
by a lysis buffer (10 mM Tris [pH 7.4], 150 mM NaCl, 5 mM EDTA,
and 0.5% Triton X‐100). The cell lysates were centrifuged for 20 min
at 10,000g. An equal volume of neutral phenol/chloroform/:isoamyl
alcohol mixture (25:24:1) was added to supernatant to extract the
fragmented DNA and 2.5 volume of ethanol was used to precipitate
the DNA. The pellet was dried and dissolved in TE buffer. The DNA
was examined by electrophoresis on 2% agarose gels containing
0.1 μg/ml ethidium bromide.
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CONFLICTS OF INTERESTS
The authors declare that there are no conflicts of interests.
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Bunz, C. Ben Mamoun, D. R. Voelker, J. Biol. Chem. 2018, 293,
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Farid M. Sroor
http://orcid.org/0000-0003-1283-2157
Ahmed H. M. Elwahy
Ismail A. Abdelhamid
http://orcid.org/0000-0002-3992-9488
http://orcid.org/0000-0003-1220-8370
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