Design and synthesis of novel tranilast analogs: Docking, antiproliferative evaluation and in-silico screening of TGFβR1 inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104368

The discovery of the antiproliferative potential of tranilast prompted additional studies directed at understanding the mechanisms of tranilast action. Its inhibitory effect on cell proliferation depends principally on the capacity of tranilast to interfere with transforming growth factor beta (TGFβR1) signaling. This work summarizes design, synthesis and biological evaluation of sixteen novel tranilast analogs on different tumors such as PC-3, HepG-2 and MCF-7 cell lines. The in vitro cytotoxicity was evaluated using MTT assay showed that, twelve compounds out of sixteen showed higher cytotoxic activities (IC₅₀’s 1.1–6.29 μM), than that of the reference standard, 5-FU (IC₅₀ 7.53 μM). The promising cytotoxic hits (4b, 7a, b and 14c-e), proved to be selective to cancer cells when their cytotoxicity's are examined on human normal cell line (WI-38). Then they are investigated for their possible mode of action as TGFβR1 inhibitors; remarkable inhibition of TGFβR1 by these hits was observed at the range of IC₅₀ 0.087–3.276 μM. The cell cycle analysis of the most potent TGFβR1 inhibitor, 4b revealed cell cycle arrest at G₂/M phase on prostate cancer cells. Additionally, it is clearly indicated apoptosis induction at Pre-G1 phase, this is substantiated by significant increase in the expression on the tumor suppressor gene, p53 and up regulation the level of apoptosis mediator, caspase-3. In addition, in silico study was performed for validating the physicochemical and ADME properties which revealed that, all compounds are orally bioavailable with no side effects complying with Lipinski rule. The proposed mode of action can be further explored on the light of molecular modeling simulation of the most potent compounds, 4b and 14e which were docked into the active sites of TGFβR1 to predict their affinities toward the receptor.

Full text of DOI:10.1016/j.bioorg.2020.104368

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Design and Synthesis of Novel Tranilast Analogs: Docking, Antiproliferative
Evaluation and In-silico Screening of TGFβR1 Inhibitors
Magda M.F. Ismail, Heba S.A. El-Zahabi, Rabab S. Ibrahim, Ahmed B. M.
Mehany
PII:
S0045-2068(20)31666-7
DOI:
https://doi.org/10.1016/j.bioorg.2020.104368
YBIOO 104368
Reference:
To appear in:
Bioorganic Chemistry
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21 April 2020
22 September 2020
8 October 2020
Please cite this article as: M.M.F. Ismail, H.S.A. El-Zahabi, R.S. Ibrahim, A. B. M. Mehany, Design and
Synthesis of Novel Tranilast Analogs: Docking, Antiproliferative Evaluation and In-silico Screening of TGFβR1
Inhibitors, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104368
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Design and Synthesis of Novel Tranilast Analogs:
Docking, Antiproliferative Evaluation and In-silico Screening of TGFβR1 Inhibitors
Magda M.F. Ismaila*, Heba S.A. El-Zahabia, Rabab S. Ibrahima, Ahmed B. M. Mehanyb
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University11754, Nasr City, Cairo, Egypt.
bDepartment of Zoology, Faculty of Science (Boys), Al-Azhar University11754, Nasr City, Cairo, Egypt.
ABSTRACT
The discovery of the antiproliferative potential of tranilast prompted additional studies directed at understanding the mechanisms
of tranilast action. Its inhibitory effect on cell proliferation depends principally on the capacity of tranilast to interfere with
transforming growth factor beta (TGFβR1) signaling. This work summarizes design, synthesis and biological evaluation of sixteen
novel tranilast analogs on different tumors such as PC-3, HepG-2 and MCF-7 cell lines. The in vitro cytotoxicity was evaluated
using MTT assay showed that, twelve compounds out of sixteen showed higher cytotoxic activities (IC50’s 1.1 -6.29 µM), than that
of the reference standard, 5-FU (IC50 7.53 µM). The promising cytotoxic hits (4b, 7a, b and 14c-e), proved to be selective to
cancer cells when their cytotoxicity's are examined on human normal cell line (WI-38). Then they are investigated for their possible
mode of action as TGFβR1 inhibitors; remarkable inhibition of TGFβR1 by these hits was observed at the range of IC50 0.087 –
3.276 μM. The cell cycle analysis of the most potent TGFβR1 inhibitor, 4b revealed cell cycle arrest at G2/M phase on prostate
cancer cells. Additionally, it is clearly indicated apoptosis induction at Pre-G1 phase, this is substantiated by significant increase
in the expression on the tumor suppressor gene, p53 and up regulation the level of apoptosis mediator, caspase-3. In addition, in
silico study was performed for validating the physicochemical and ADME properties which revealed that, all compounds are
orally bioavailable with no side effects complying with Lipinski rule. The proposed mode of action can be further explored on the
light of molecular modeling simulation of the most potent compounds, 4b and 14e which were docked into the active sites of
TGFβR1 to predict their affinities toward the receptor.
Keywords:
Synthesis, Anticancer activity, TGFβR1, Caspase-3, p-53, Molecular modeling
Corresponding author
Magda M. F. Ismail
Department of Pharmaceutical Chemistry,
Faculty of Pharmacy (Girls), Al-Azhar University,
Cairo 11754, Egypt
magdaismail@azhar.edu.eg
m.elalfy101@gmail.com
1

1. INTRODUCTION
Although, our understanding of mechanistic studies that lay behind cancer development and advancement has elevated, cancer
is still a significant health alarm in multiple advanced countries [1]. Cancer is a key factor of mortality far and wide; it roughly
causes 13% of all deaths every year. [2]On September 2018, WHO advertised that; 9.6 million persons died throughout the world;
in-depth, about 1 in 6 deaths is as a result of cancer. [3] There is a critical need for novel diagnostic and treatment options as well
as illumination of how cells gain the hallmarks, mandatory to become fully malignant. [1] Transforming growth factor-β (TGF-β)
superfamily is a leader in the regulation of a large number of physiological processes from development to pathogenesis including
cell proliferation, differentiation and apoptosis. [4-6] Three highly homologous isoforms of TGFβ exist in humans: TGFβ1, TGFβ2
and TGFβ3. [7] The most widely studied target in fighting cancer is the Transforming Growth Factor Receptor type I (TGFβR1),
a key player in cell proliferation; differentiation and apoptosis. [8] There are many pharmacological approaches to hinder TGFβR1
signaling, such as monoclonal antibodies, vaccines, antisense oligonucleotides, and small molecule inhibitors. [9] A new series of
small-molecule TGFβR1 inhibitors such as galunisertib, [10] SB-505124 [11] and tranilast (Rizaben®) [12] were discovered, (Fig.
1). Such small molecule inhibitors target the catalytic kinase domain of TGFβR1 and bind to the ATP-binding pocket of the
receptor to inhibit receptor auto-phosphorylation. This prevents initiation of the signaling cascade, and inhibits the growth and
proliferation of the cancer cells. [13] Galunisertib (LY2157299 monohydrate) is an oral small molecule inhibitor of the TGFβR I
kinase which particularly has negative impact on the phosphorylation of SMAD2, abrogating activation of TGFB signaling
pathway. [14] Besides, galunisertib has antitumor activity in animal models bearing tumors such as breast, colon, lung cancers,
and hepatocellular carcinoma, prostate cancer. [14] In 1982, tranilast (N-[3,4-dimethoxycinnamoyl]-anthranilic acid) has been
approved in Japan and South Korea as anti-allergic drug for treatment of bronchial asthma [15], with indications for keloids and
hypertrophic scar added in 1993. [16] The antitumor properties of tranilast were determined in the end of 1980s; where it was
emerged that tranilast displayed an inhibitory effect on TGF-β1 receptor. [16] Inhibitory activity of tranilast has been confirmed
on several cell lines of breast, pancreas, liver, prostate, glioma cancer, as well as gastric carcinoma and neurofibroma, [16] Tranilast
can also inhibit angiogenesis. [17]
N
N
HN
COOH
N
N
O
O
O
O
OCH3
OCH3
N
H
N
H2N
B
C
A
galunisertib
Tranilast
SB-505124
Fig. 1: Structures of small molecules act as TGFBR1 inhibitors
Tranilast was found to decrease the proliferation of fibroblasts from normal tissues and suppress the release of TGF-β from
fibroblasts. In addition, its antitumor activity has been reported in some cell lines such as prostate cancer cell lines (PC-3), breast
2

cancer cell lines like MCF-7, liver, lung and melanoma. Also, Yashiro et al [18] have described that, Tranilast decreases the
production of MMP-2 and TGF-β1 from fibroblasts, resulting in significant suppression of the invasion ability of gastric cancer
cells. It has also been shown to block cell cycle progression, inhibit migration and promote the apoptosis of cells. [19] Several
investigations have revealed that, cell cycle arrest is an important mechanism responsible for apoptosis-inducing ability of cancer
therapeutic agents in cancer cells. [20] Caspase-3 level and gene expression of p53 were known to be induced in programmed cell
death (apoptosis). [20] Structural features of tranilast covered various hydrophilic and hydrophobic points via two terminal
modifiable parts; anthranilate and aromatic moieties with central enone modifiable linker, (Fig. 2).
2
1
COOH
O
3
4
Anthranilate
modifiable moiety
Ar’
x
2
OCH3
OCH3
3
4
1
6
N
H
y
5
Ar
Aromatic
modifiable moiety
6
5
Enone
modifiable linker
Tranilast
The lead compound
Fig. 2: overview of the modifiable sites of the lead compound Tranilast
Herein, our strategy is directed toward the design and synthesis of four series of tranilast analogs acting as TGFβR1 inhibitors
and apoptosis inducers. Concerning series A, chain contraction strategy was applied where enaminone linker is replaced by
sulfonamide or amide as in compounds 3a-c and 4a, b respectively. Series B, bioisosterism approach was followed on the olefinic
linker -CH=CH- which was switched to -CH2-NH- in N-acetamido analogs, 5a-c. Series C was designed via variation of
substituent in the aromatic modifiable moiety as in chloroacrylamides, 6a-c, while series D concerned with applying ring fusion
and bioisosterism strategies on the anthranilate modifiable moiety as in acrylamides, 14a-e. These structural variations were
followed in an attempt to optimization of pharmacodynamic and pharmacokinetic properties of tranilast. Additionally, docking of
the most potent compounds into human TGF-β using Molsoft ICM 3.4-8c program was performed in order to predict the affinity
and orientation of the these compounds to the active site.
2. RESULTS AND DISCUSSION
2.1. Chemistry
Anthranilic acid represents a key starting molecule in many synthetic pathways. N-acetylation of anthranilic acid, 1 using
acetic anhydride afforded benzoxazinone intermediate which is immediately hydrolyzed with H2O to N-acetyl anthranilic acid, 2.
[20-24] N-Sulfonylation [25] of 2 with aryl sulfonyl chlorides in EtOH/TEA/reflux afforded the corresponding sulfonamides 3a-
c. Analogously, benzamide analogs, 4a, b were prepared using benzoyl chlorides in DMF/reflux, «Scheme 1». Their structures
were confirmed by elemental and spectral analysis. IR spectra of compounds 3a-c demonstrated stretching broad band at the range
2500-3400 cm−1 corresponding to OH group and bands at 1670 cm−1 and 1710 cm−1 for CON and COO groups respectively.1H
3

NMR spectrum of compound 3a as an example, revealed a singlet signal at δ 2.13 ppm for COCH3 protons. Two doublets recorded
at δ 7.37 ppm and 7.52 ppm disclosed to 4 aromatic protons of AB system with J value 8.00 Hz in addition to 4 aromatic protons
of anthranilic moiety. 13C-NMR (DMSO-d6) of compound 3a revealed signals at δ 19.3 (CH3), 138.7(C-SO2), 142.4 (C-N), 168
(C=O amide) and 169.4 (C=O of COOH) ppm in addition to other signals characterizing each structure. Mass spectrum of 3a
showed peaks at m/z 397 [M+, 1.96%] and 399 [M+2, 1.95%] in (1:1) ratio for Br isotope. On the other hand, IR spectra of
compounds 4a, b showed stretching broad band around 3367 cm−1 for OH group and bands at 1670 cm−1, 1700 cm−1 and 1710
cm−1 for three carbonyl groups.1H NMR spectrum of compound 4a revealed a singlet signal at δ 2.11 ppm for COCH3 protons and
two doublets for aromatic protons appeared at δ 8.05 ppm and 8.10 ppm with J value 8.00 Hz. 13C-NMR (DMSO-d6) δ (ppm)
showed signals at δ 20.2 (CH3), 137.3 (C-N), 166.8 (C-F), 172.1 (CONH) and 172.3 (COOH) (experimental section), «Scheme
1». Chloroacetylation of anthranilic acid, 1 with chloroacetyl chloride under reflux condition in benzene/ piperidine afforded 2-
chloroacetamidobenzoic acid, 5. [26, 27] Knoevenagel condensation [28-34] of the latter with different aldehydes or
acetophenones in ethanol/TEA yielded the corresponding 2-chloro-acrylamidobenzoic acids, 6a-c «Scheme 1». Their structures
were assigned based on spectral data. IR spectra showed broad bands at 3251-3400 cm−1 corresponding to OH and NH groups.
Also, bands appeared at 1661-1705 cm−1 for two carbonyl groups.1H NMR spectra of 6a-c exhibited a characteristic singlet signals
1
at the range of δ 7.53and δ 7.81 ppm for the olefinic proton, Cl-C=CH in compounds, 6a and 6b respectively. Also, H NMR
spectrum of 6c showed a characteristic singlet at δ 2.56 ppm corresponding to the methyl protons (Cl-C=C-CH3). 13C-NMR
(DMSO-d6) δ (ppm) displayed signals at δ 11.7 for CH3, also two signals were observed at 163.8 and 169.4 ppm corresponding
to the two carbonyl groups, (experimental section). Mass spectrum of 6c showed peaks at m/z: 349.14 (8.53, M+) and 351.35
(2.27, M+2) in 3:1 ratio for Cl isotope and base peak at 86.03 (100). Additionally, nucleophilic substitution reaction was performed
on compound 5 with different aryl amines in ethanol/TEA [35, 36] to give the corresponding derivatives, 7a-c, «scheme 1». The
structure of target compounds, 7a-c was proved by elemental and spectral analysis, (see experimental section).
4

COOH
O
COOH
O
O
S
N
N
O
X
O
X
3a-c
a: X=Br
b: X=F
c: X=Cl
4a,b
a: X=F
b: X=NO2
c
d
COOH
O
2
N
H
b
O
O
N
a
COOH
NH2
1
e
COOH
O
5
Cl
N
H
f
g
COOH
COOH
O
O
X
R2
H
N
N
H
N
H
Ar
Cl
6a-c
a; X=H, R1=OCH3, R2=H
R1
7a-c
a; Ar = 2-SH-ph
b; Ar = 2, 4-(CH3)2-ph
c; Ar = 4-pyridinyl
b; X=H, R1=Cl
c; X=CH3, R1=Cl,
R2=Cl
R2=H
Scheme 1: Reagents and conditions: (a) acetic anhydride, reflux 15 min, cooling; (b) H2O; (c) p-substituted sulfonyl chloride,
EtOH, TEA dps, reflux, 5-10 h.; (d) p-substituted benzoyl chloride, DMF, reflux, 6-8 h.; (e) ClCH2COCl, benzene, pip., reflux, 6
h.; (f) different aromatic aldehydes and acetophenone, EtOH, TEA, reflux, 8-12 h.; (g) different substituted amines, EtOH, TEA,
reflux, 8-12 h.
5

With the aim of preparing series of tranilast–based analogs bearing various bicyclic systems, nucleophilic substitution reaction
of 2-aminothiophene derivatives, 10 with the appropriate cinnamoyl chlorides, 13 in acetone / anhyd. K2CO3 /reflux [37] gave the
target compounds, 14a-e, «Scheme 2». The first intermediate, 2-aminothiophene derivatives, 10 were prepared following Gewald
reaction [38-43] via reacting equimolar amounts of alicyclic ketones e.g. cyclohexanone, 8a or cyclopentanone, 8b with the
appropriate active methylene reagent e.g. ethyl cyanoacetate, 9a or malononitrile, 9b and elemental sulfur in absolute ethanol /
morpholine. [44] The second intermediate, cinnamoyl chloride derivatives, 13 were prepared starting with Knoevenagel
condensation [32, 45 and 46] between substituted benzaldehydes, 11 and malonic acid in ethanol/ pip/reflux to produce the
corresponding cinnamic acids, 12. Then; chlorination of 12 using POCl3/ TEA, [47] afforded the corresponding cinnamoyl chloride
derivatives, 13. The IR spectra of 14a-e showed characteristic stretching bands at 3220 and 2200 and 1650 cm−1 corresponding to
NH, CN and CO groups respectively.1H NMR spectra of 14a-e exhibited two characteristic multiplet signals at δ 1.65 ppm and
2.49 ppm corresponding to the protons of cyclohexane. In case of cyclopentane; two multiplet signals appeared around δ 1.95 ppm
and 2.5 ppm. Generally, two more doublets appeared at δ 7.25 and 7.96 ppm for olefinic protons CH=CH with J constant 16 Hz
confirming tran (E) configuration (see experimental section).
R
O
R
a
NH2
CN
(n)
8
S
(n)
R
O
9
10
d
NH
X
(n)
S
O
O
14a-e
COOH
b
c
Cl
a: n= 1, R= COOC2H5, X= OH
b: n= 1, R= CN,
c: n= 1, R= CN,
d: n= 0, R= CN,
e: n= 0, R= CN,
X= OH
X= 2, 5 -(OCH3)2
X= OH
X
X
X
X= 2, 5 -(OCH3)2
12
11
13
Scheme 2: Reagents and conditions: (a) Elemental sulfur, morpholine , EtOH, reflux 4 h., cooling at 0 oC for 24 h; (b)
malonic acid, EtOH, piperidine, reflux 5 h.; (c) POCl3, dps TEA, reflux 20 mins; (d) acetone, anhydrous K2CO3, reflux, 6-8
h.
2.2. Biological results and discussion
2.2.1. In vitro anti-cancer activities
All the target compounds, 3a-c, 4a, b, 6a-c, 7a-c and 14a-e were tested for their anticancer activity using MTT assay [48]
against three cancer cell lines e.g. prostate cancer cell (PC-3), hepatocellular carcinoma (HepG-2) and human breast
adenocarcinoma (MCF-7) using 5-FU as a reference standard, (Table1). Obviously, the activity pattern of each compound
showed correlated results on the three cell lines. Conclusively, prostate cell line (PC-3) is the most sensitive one towards
our hits.
2.2.1.1 Antiprostate cancer activity (anti PC-3):
6

The antiproliferative profiles of the test compounds, 3a-c, 4a, b, 6a-c, 7a-c, and 14a-e showed variable activities compared
to the reference standard, 5-FU (IC50 7.53 μM), (Table 1). The highly potent area was covered by benzamido analog 4b,
chloroacrylamido derivative 6b, acetamido analogs 7a,b and acrylamides 14c-e showing IC50 values, 1.1, 4.0, 2.7, 3.4, 2.64, 3.4,
and 1.35 μM respectively. Compounds 4b and 14e were almost equipotent exerting seven folds the activity of 5-FU. Acetamido
derivative, 7a and acrylamide, 14c were almost equipotent eliciting 2.5 folds the activity of 5-FU. Furthermore, compounds 7b
and 14d displayed double the cytotoxicity of 5-FU. Another target compounds e.g. 3a-c, 4b and 14a, b showed better anticancer
activity than 5-FU, their IC50s ranges from 4 - 6.26 μM). The rest of compounds exhibited moderate to poor activity compared to
reference standard, (Table 1).
2.2.1.2. Antihepatic cancer activity (anti HepG-2):
The cytotoxicity of the target compounds on HepG-2 cancer cell line was parallel to the results on PC3 cell line. The most potent
derivatives were benzamido analog 4b, chloroacrylamido analog 6b, acetamido analogs 7a,b and acrylamides 14c-e having IC50s
1.5, 4.55, 3.2, 4.25, 2.85, 3.55, and 2.7 μM respectively compared to 5-FU (IC50 8.15 μM). The mentioned analogs showed two
to seven folds the activity of 5-FU against HepG-2 cancer cell line. Some derivatives were equipotent or slightly more potent
than 5-FU e.g. 3a-c and 14a, b and the rest of compounds demonstrated moderate to poor cytotoxicity, (Table 1).
2.2.1.3. Antibreast cancer activity (anti MCF-7):
The previous competitive test compounds e.g. 4b, 6b, 7a, b and 14c-e were also the key analogs in fighting MCF-7 cell line.
Among them, the benzamido analog, 4b showed three folds the activity (IC50 2.6 μM) of 5-FU (IC50 7.76 μM). Equipotent
acrylamides 14c, e (IC50 3.5, 3.2 μM) were twice times more active than 5-FU. Concerning 6b, 7a, b and 14d, they disposed 1.5
folds the activity of 5-FU where their IC50s are 4.79, 4.16, 4.85, and 4.92 μM respectively. The rest of the test compounds
exhibited moderate to poor activity compared to reference standard, (Table 1).
Table1
In vitro anti-proliferative activities towards PC-3, HepG2 and MCF7 cell lines.
IC50% (μM) ± S.E.
Compound No.
PC-3
HepG-2
MCF-7
3a
3b
3c
5.11 ± 0.72
5.94 ± 0.65
4.75 ± 0.53
8 ± 0.91
6.51 ± 0.84
6.42 ± 0.71
5.73 ± 0.68
9.7 ± 1.02
7.8 ± 0.87
8.8 ± 0.96
8.22 ± 0.97
11.28 ± 0.67
2.6 ± 0.42
4a
4b
6a
6b
6c
1.1 ± 0.32
29 ± 1.2
1.5 ± 0.52
31.42 ± 1.32
4.55 ± 0.32
18.07 ± 1.22
3.2 ± 0.42
33.16 ± 1.52
4.79 ± 0.19
23.62 ± 1.52
4.16 ± 0.56
4.85 ± 0.62
38.2 ± 1.72
8.27 ± 0.76
4 ± 0.52
16.45 ± 0.98
2.7 ± 0.81
3.4 ± 0.54
32.9 ± 1.32
6.29 ± 0.5
7a
7b
7c
4.25 ± 0.94
36.13 ± 1.92
8.3 ± 0.75
14a
7

14b
14c
5.2 ± 0.46
2.64 ± 0.21
3.4 ± 0.42
1.35 ± 0.04
7.53 ± 0.57
7.9 ± 0.67
2.85 ± 0.17
3.55 ± 0.28
2.7 ± 0.19
8.15 ± 0.61
7.5 ± 0.8
3.5 ± 0.26
4.92 ± 0.31
3.2 ± 0.28
7.76 ± 0.46
14d
14e
5-FU
2.2.1.4. The effect on normal cell line (anti-WI-38):
On the other hand, the safety profile of the promising compounds (4b, 7a, 7b, 14c- e) was further investigated via determination
of their cytotoxicity on human normal cell line (WI-38), where the recorded data showed high safety margin of them. Their
cytotoxic effect against cancer cell lines was several folds higher than that of normal cell line, (Table 2).
Table 2
Cytotoxicity of all compounds against the human non-cancerous cell line WI-38 cell.
Compound No.
IC50 (µM)a
WI-38 cell
234.43
4b
7a
278.81
7b
305.98
14c
14d
14e
259.90
236.30
185.20
2.2.1.5. Structural activity relationships
Structural profile of the target compounds (3a-c, 4a, b, 6a-c, 7a-c, and 14a-e) was based on the anticancer lead compound,
tranilast, considering three modifiable sites, anthranilate moiety, enone linker and aromatic moiety for tailoring tranilast analogs,
(Fig. 2). As previously mentioned, four main series, A-D were planned via drug rational approaches, (Fig.3). Regarding series A
(chain contraction), represented by 3a-c and 4a, b, benzamido analog 4b, was the most potent anticancer agent against prostate,
hepatic and breast cancer cell lines with IC50 1.1, 1.5 and 2.6 μM respectively. Replacement of p-NO2 by p-F as in 4a
dramatically decreased the activity against the tested cell lines; this could be attributed to the important effect of
hydrophilic/hydrophobic balance on the activity. Regarding sulfonamide analogs 3a-c, that illustrated switching between
different halogen atoms e.g. Br, F and Cl, the halogen replacement did not greatly affects the anticancer activity. Interestingly,
3b with NHSO2 group was more potent than 4a containing NHCO functionality against the three cancer cell lines. Series B
(bioisosterism) represented by compounds 7; It was found that 7a, b displayed remarkable anticancer activity against tested cell
lines. Noticeably, 7a (Ar= o-SHph) provided a slight increase in anticancer activity than its counterpart, 7b (Ar=o,p-(CH3)2ph).
Nevertheless, 7c (Ar=p-pyridyl) showed low the anticancer activity by almost eight folds compared to 5-FU. In case of series C,
enhanced cytotoxicity was recorded for 6b bearing o, p-diCl-ph moiety; when replacing it by e-donating group, p-OCH3-ph as in
6a, cytotoxicity is completely abolished. This anticancer profile reflected the vital role of e-withdrawing effect on the aromatic
modifiable moiety of compounds 6a-c, (Fig. 3).
8

In regard of series D (bioisosterism / ring fusion) included acrylamide derivatives, 14a-e, potential anticancer activity was
exhibited especially for compounds 14c-e. Generally, replacement of cyclohexan[b]thiophene moiety in 14b, c by
cyclopenta[b]thiophene moiety in 14d, e, enhanced the anticancer activity against the tested cell lines. Comparison of the
anticancer activity of cyclohexan[b]thiophene pairs 14b, c and cyclopenta[b]thiophene pairs 14d, e reflected the positive impact
of 2, 5-(OCH3)2 groups in 14 c, e on the anticancer activity rather than 4-OH group in their counterparts 14b, d, (Fig. 3). As well,
3-CN group in 14b showed higher cytotoxicity than 3-ester functionality in 14a. Collectively, cyclopenta[b]thiophene moiety, 3-
CN group and 2, 5-(OCH3)2 are preferred for anticancer activity of acrylamides.
chain contraction
Variation of substituent
COOH
COOH
Y
O
x
N
N
H
X
O
R
X= CO, SO2
X= Cl, Y= H, CH3
R
6a-c
3a-c& 4a,b
SERIES (C)
SERIES (A)
COOH
O
H
OCH3
OCH3
N
H
H
Tranilast
SERIES (D)
SERIES (B)
*Bioisosterism.
*Ring fusion
Bioisosterism
COOH
X
O
O
H
H
(n)
N
N
H
S
N
H
H
R
R
X= COOEt, CN
n = 0, 1
7a-c
14a-e
Fig. 3: Design of target compounds as tranilast analogs
2.2.3. TGFBR1 inhibition assay
The most potent hits 4b, 7a,b and 14c-e (IC50 0.087- 3.276 μM) were selected to explore their mechanism of action;
through evaluation of their inhibitory effect against TGFβR1. Results of TGFβR1 assay revealed that compounds 4b, 7a and
14e were the most potent TGFβR1 inhibitors showing IC50s in sub-micromolar range (IC50s 0.08 , 0.09, and 0.19 μM) respectively.
Compounds 4b and 14e are more potent TGFβR1 inhibitors than the reference standard galunisertib (IC50 0.17μM), while 7a
revealed equipotent inhibition to it, (Table 3).
9

Table 3
IC50 of the representative anticancer active compounds on TGFβR1 in PC-3 cells
Compound No.
IC50 (µM) a
TGFβR1
4b
0.087 ± 0.064
0.191 ± 0.013
3.276 ± 0.097
1.379 ± 1.42
2.114 ± 0.41
0.093 ± 0.16
0.170 ± 0.057
7a
7b
14c
14d
14e
Galunisertib
2.2.4. Cell cycle analysis
Cell cycle analysis of the most potent antiproliferative TGFβR1 inhibitor (4b) was examined [49] to clarify its possible apoptotic
inducing effect. Amongst the tested cancer cell lines, PC-3 exhibited the highest sensitivity towards 4b (IC50=1.1μM). It was
obvious from cell cycle analysis on PC-3 that, 4b induced a significant increase in the percentage of cells at Pre-G1phase by almost
12 folds and increased G2/M by 4 folds, compared to the control experiment, (Table 4). Such increase was accompanied by a
decrease in the percentage of cells at the Gᵒ/G1 and S-phases of the cell cycle. The Pre-G1 and G2/M phase results clearly indicated
that, compound 4b induces apoptosis at Pre-G1 phase and arrest the cell cycle at G2/M phase, (Figs. 4a-c).
Table 4
Results of cell cycle analysis in PC-3 cells expressed by (%) of cells in each phase when treated with compound 4b
Compound No.
%G0-G1
%S
%G2-M
%Pre-G1
4b / PC-3
26.36
56.39
15.43
32.44
42.37
9.86
15.84
1.31
Control/ PC-3
Fig. 4a: Cell cycle analysis and apoptosis effect in PC-3 cell line treated with compounds 4b
Fig. 4b: Cell cycle analysis of PC-3 cells treated with DMSO
Fig. 4c: Cell cycle analysis of PC-3 cells treated with compound 4b
10

2.2.5. Apoptosis detection studies
2.2.5.1. Annexin V-FITC apoptosis assay
In order to confirm the apoptosis induction of 4b, Annexin V binding studies by flow cytometer was carried out. [50] As
TGFBR1 inhibitors can induce cancer cell apoptosis, the apoptotic inducing effect of compound 4b against PC-3 cells was
evaluated via flow cytometer detection using Annexin V-FITC and propidium iodide (PI) double staining. The results revealed
that, compound 4b increased the early apoptosis ratio (lower right quadrant of the cytogram) from 0.63 to 6.69, and increased the
late apoptosis ratio (higher right quadrant of the cytogram) from 0.46 to 6.22 compared to the control experiment, (Table 5).
Comparative study for apoptosis illustrated high apoptotic effect of the target compound 4b; it was 12 folds as much as the control
experiment. These data suggest that, compound 4b triggered apoptosis via the programed cell death pathway rather than necrotic
pathway, (Figs. 5a-c).
Table 5
Percent of cell death induced by compound 4b on PC-3 cell.
Apoptosis %
Compound No.
Total
Necrosis %
Early
Late
11

4b / PC-3
15.84
1.31
6.69
0.63
6.22
0.46
2.93
0.22
Control/ PC-3
Fig. 5a: Effect of control on apoptosis of PC-3 cells
Fig. 5b: Effect of 4b on apoptosis of PC-3 cells.
The quadrants in the cytograms represent the following: Necrotic cells (higher left quadrant of the cytogram); late apoptotic cells (higher right quadrant
of the cytogram); Non-apoptotic and non-necrotic cells (living cells) (lower left quadrant of the cytogram); early apoptotic cells (lower right quadrant
of the cytogram).
Fig. 5c: Percent of cell death induced by compound 4b on PC-3 cells.
Fig. 5: Cell cycle analysis; a)-control PC-3, b) compound 4b by flow cytometer using PI staining method, c)% cell death induced by 4b
2.2.5.2. Effect on the level of active caspase-3
Activation of the caspase-3 pathway is a hallmark of apoptosis and can be used in cellular assays to quantify activators and
inhibitors of the “death cascade.” [51] The most active analog, 4b was evaluated for its effect on caspase-3 and the results revealed
12

that, 4b up regulated the caspase-3 level by almost 7 folds compared to the control, (Table 6). So, the recorded data proved the
apoptosis induction potential of this target compound, (Fig. 6).
2.2.5.3. Effect on the expression of p53 gene
P53 is the master guardian of the genome which mutated in over 50% of human cancers; its activity stops the formation of
tumors. The p53 protein is the product of p53 gene which acts as a regulator of transcription and mediates several biological
effects, such as growth arrest and apoptosis. It is believed that, tumor suppressor gene, p53 has the ability to induce apoptosis and
growth arrest may be as a result of its role in mastering the expression of a variety of target promoters such as p21, Bax, bcl-2,
CDK2 and PUMA α. [52] Results revealed that, 4b significantly increased the expression of p53 on PC-3 cells by almost 14 times
compared to the control and strongly shifted PC-3 cancer cells towards apoptosis, (Table 6, Fig. 6).
Table 6
The effect of compound 4b on the level of caspase-3 and gene expression of p53 gene.
Conc. (Pg./ml)
Compound No.
Caspase -3
p53
4b
362.8
51.39
259.6
19.22
Control
Fig. 6: The effect of 4b on the level of caspase-3 and p53.
2.3. Molecular Modeling
2.3.1. In-silico evaluation of physicochemical and ADME properties
Computational study of the synthesized compounds was performed to evaluate the physicochemical and ADME properties.
SwissADME [53] software was used to predict whether the compounds are likely to be bioactive according to some critical
parameters such as Lipinski and Veber rules. As delineated from Table 7; the physicochemical properties of all test compounds
have zero Lipinski’s violation. Also, all are in agreement with the parameters of veber rule [54], this indicates that these derivatives
have promising drug like properties. Relying on the topological polar surface area (TPSA), [55] all compounds exhibit
computational TPSA values between 54.02 and 120.50 Å2 and have good intestinal absorption. Besides, absorption (% ABS) was
estimated by using the equation % ABS = 109 – (0.345 x TPSA), [56] founding that, the calculated % ABS of all these hits ranged
13

between 67.42% and 90.36 %. The good ABS % demonstrated that, these hits may have the required cell membrane permeability
and bioavailability. It is apparent that, all the derivatives have high gastro-intestinal absorption. Along with, most of them have
no permeation to the blood brain barrier, which outweighed that, these systemically targeted molecules will have low to no CNS
side effects.
Table 7
Physicochemical properties based on Lipinski´s rule of five and number of rotatable bonds, the topological polar surface area (TPSA), %
ABS, GIT absorption and BBB permeability.
Cpd.
No.
HBD
HBA
M
logP
M.Wt
No. of
Lipinski´s
Violations
TPSA
%
ABS
GI
BBB
Rot.
Absorption Permeation
bonds
3a
3b
1
1
1
1
1
2
2
2
3
3
3
2
2
1
2
1
2
5
6
5
5
6
4
3
3
3
3
4
4
3
3
3
4
3
2.46
2.22
2.34
3.35
1.97
2.63
3.96
3.69
2.24
2.47
0.51
2.73
2.02
2.97
1.97
1.69
0.32
398.23
337.32
353.78
301.27
328.28
331.75
370.61
350.20
302.35
298.34
271.27
371.45
324.40
367.46
310.37
354.42
130.08
5
5
5
5
6
6
5
5
6
6
6
7
4
6
4
6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
100.13
100.13
100.13
74.68
74.45
74.45
74.45
83.23
67.42
82.90
86.09
86.09
68.55
81.94
77.49
73.16
74.03
82.84
74.03
74.64
86.33
high
high
high
high
high
high
high
high
high
high
high
high
high
high
high
high
high
No
No
3c
No
4a
Yes
No
4b
120.50
75.63
6a
Yes
Yes
Yes
No
6b
66.40
6c
66.40
7a
117.23
78.43
7b
Yes
No
7c
91.32
14a
14b
14c
14d
14e
5-FU
103.87
101.36
75.80
No
No
Yes
No
101.36
99.59
No
65.72
No
2.3.2. Molecular Docking studies
The transforming growth factor beta receptor type I (TGFBR1) crystal structure with naphthyridine inhibitor (ligand 460)
was obtained and downloaded from protein data bank, PDB file ID: 1VJY with resolution of 2.0Å and 303 amino acids
(http://www.pdb.org); it is considered as targets for docking simulations. We performed molecular docking studies in a trial to get
better understanding of the binding pattern of the new inhibitors, 4b, 14e and the lead compound, tranilast with TGFβR1 kinase
domain. We performed docking simulations using Molecular Operating Environment software 10.2010 (MOE), Chemical
Computing Group Inc., Montreal, Quebec, Canada. After download, refinement of TGFBR1 domain and removal of water chain,
then redocking of ligand 460 into the binding site to perform verification process. After that, the lead compound, tranilast and our
new hits, 4b and 14e are also docked to the same binding site of TGFβR1.
The docked model of the TGFβR1 inhibitor with ligand 460 showed docking score of -4.81 Kcal/mol and revealed the following
binding modes: hydrogen bonding interaction through its naphthyridine nitrogen atom with His283 (1.89 A0) and another H bonds
between pyrazolidine-H and Asp290 (2.27 A0), arene-H with Ile 211 amino acid. The obtained results are pictured in Fig. 7a, b.
14

The docked study of tranilast revealed better docking score (-5.72 Kcal/mol) than that with the ligand 460 and displayed hydrogen
bond (bond length 2.17 Å) between His283 and carbonyl-O. Also, hydrophobic and arene-H interactions were demonstrated in its
docking model between anthranilic ring with Leu340 and Val219. Beside, hydrophobic interaction was observed between
dimethoxy phenyl ring and Asp290, (Fig. 8a, b). On the other hand, the docked model of 4-nitrobenzamido derivative, 4b displayed
H bonding with the same amino acid His283 as ligand 460, through the carbonyl-O atom of COOH group, an additional H bond
(1.72 Å) with Ser287 through the oxygen atom of COCH3 group. In addition to, arene-H interaction between phenyl of anthranilic
acid moiety and Ile211 as long as other hydrophobic interactions with TGFβR1binding site through its phenyl moiety and
anthranilic ring with the important amino acids, Ile211, Gly286, Val219, Leu340 and Asp351. Compound 4b docking score energy
was -6.33 kcal/mol. These binding modes explained the enhanced activity of 4b, (Fig. 9a, b). Concerning compound 14e which is
acrylamide bearing 2-aminothiophen-3-carbonitrile fused with cyclopentane derivative, it revealed docking score energy (-6.92
kcal/mol). Also CN-N bonded to His283 via HBA (2.20 A0) and arene-H interaction of phenyl core and Lys132 together with
hydrophobic interactions with Ile211 Gly286, Val219, and Leu340. The obtained results are pictured in Figs. 10a, b. It worth to
mention that, overlay docking alignment of TGFβR1 inhibitors, 4b and 14e with the ligand 460 and tranilast showed good fitting
of all these compounds in the correct binding site of TGFβR1 as illustrated in Table 8 and Fig. 11.
Fig. 7: (a) 3D and (b) 2D binding interactions of ligand 460 with TGFβR1 binding site.
15

.
Fig. 8: (a) 3D and (b) 2D binding interactions of tranilast with TGFβR1 binding site.
Fig. 9: (a) 3D and (b) 2D binding interactions of compound 4b with TGFβR1 binding site.
16

Fig. 10: (a) 3D and (b) 2D binding interactions of compound 14e with TGFβR1 binding site.
Fig. 11: Overlay docking alignment of Ligand 460 (blue), tranilast (yellow), 4b (purple), and 14e (red) in the active site of TGFβR1 (PDB
code: 1VJY).
Table 8
The docking scores (energy) of ligand 460, tranilast, 4b and 14e in the binding pocket of TGFBR1 (PDB: 1VJY)
Cpd. No.
Docking score
(Kcal/mol)
No. of H-
bonds
Amino acid residues
(bond length A0)
Atoms of cpd.
Type of bonds
His283 (2.27);
Asp290 (1.89);
Ile211
Ile211
Leu340
N1-naphthyridin
N-pyrazole
H-bond (acceptor)
H-bond (donor)
Arene-H
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
Ligand
460
-4.81
2
N5-naphthyridin
Naphthyridinyl ring
Naphthyridinyl ring
Naphthyridinyl ring
Pyrazole ring
Val219
Asp290
His283 (2.17);
Leu340
VAI219
Leu340
Val219
Ile211
Carbonyl(amide)
Anthranilic ring
Anthranilic ring
Anthranilic ring
Anthranilic ring
Anthranilic ring
H-bond (acceptor)
Arene-H
Arene-H
Hydrophobic
Hydrophobic
Hydrophobic
-5.72
1
Tranilast
17

Aap290
Dimethoxy phenyl
Hydrophobic
His283 (1.64)
Ser287 (1.72);
Ile211
Ile211
Val219
Carbonyl(acetyl)
Carbonyl(acid)
Anthranilic ring
Anthranilic ring
Nitrophenyl
H-bond (acceptor)
H-bond (acceptor)
Arene-H
Hydrophobic
Hydrophobic
Hydrophobic
-6.33
-6.92
2
1
4b
Leu340
Nitrophenyl
His283 (2.20)
Lys232
Ile211
Val219
Leu340
Nitrile group
H-bond (acceptor)
Arene-H
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
14e
Dimethoxy phenyl
Cyclo pentane
Dimethoxy phenyl
Thiophene ring
Cyclo pentane
Asp290
3. CONCLUSION
In the process of anticancer drug discovery and to find new potential selective anticancer agents, we synthesized a novel
series of tranilast analogs. The in-vitro anticancer screening of sixteen new synthesized compounds was performed on PC-3, HepG-
2 and MCF-7 cell lines using 5-FU as reference drug. Twelve of the investigated compounds showed more potent cytotoxic activity
than 5-FU. Particularly, compound 4b displayed amazing anticancer activity 5-7 times that of reference drug against all the tested
cell lines. Six compounds, 4b, 7a, b and 14c-e, showed promising cytotoxicity and remarkable selectivity to cancer cells, were
further evaluated for their inhibitory activities against TGFβR1. The most active compound, 4b enhanced apoptosis and arrested
G2/M phase of cell cycle. Besides, it induced both caspase-3 level and expression of p53 gene of PC-3 cell. In silico study revealed
that all target compounds are in agreement with the parameters of Lipiniski's rule. Molecular docking of almost equipotent hits,
4b and 14e elicited higher affinities to TGFβR1’s binding site with good binding energies compared to ligand 460 and tranilast.
Therefore, compounds 4b and 14e can be considered as interesting candidates for further development of more potent anticancer
agents
4. EXPERIMENTAL PART
4.1. Chemistry
Melting points were measured in open capillary tubes using Griffin apparatus and are uncorrected. Elemental microanalyses
were carried out at the Regional Centre for Mycology and Biotechnology, Al-Azhar University. The IR spectra (KBr, cm-1) were
recorded using potassium bromide disc technique on a Schimadzu FT/IR 1650 (Perkin Elmer) at Faculty of Pharmacy, Cairo
University and Faculty of science, Al-Azhar University.1H NMR (400 MHz) and 13C NMR (100 MHz) spectra (δ, ppm) were
performed on Agilent Technologies NMR spectrophotometer at the Armed Forces Laboratories, faculty of pharmacy, Al-Mansoura
University and faculty of pharmacy, Ain shams university. DMSO-d6 was used as a solvent, and the chemical shifts were measured
in ppm, relative to TMS as an internal standard. As for the proton magnetic resonance, D2O was carried out for NH and OH
18

exchangeable protons. Mass spectra were recorded on a DI-50 unit of EI Shimadzu GC/ MS-QP 2010 plus Spectrometer (Japan)
or on single quadrpole EI mass Spectrometer ISQ LT (Thermo scientific) at the Regional Center for Mycology and Biotechnology,
Al-Azhar University. All reactions were monitored by TLC using pre-coated Aluminum sheets silica gel Merck 60 F254 and were
visualized by UV lamp.
4.1.1. 2-(N-acetyl-4-substituted benzensulfonyl) benzoic acids (3a-c)
To a solution of N-acetyl anthranilic acid (2) (0.01 mol) in ethanol (20 ml), p-substituted benzene sulfonyl chlorides (0.01 mol)
was added with few dps of TEA. The mixture was heated under reflux from 5-10 h. After completion of the reaction mixture, it
was concentrated and allowed to cool then poured into cold water. The precipitate was filtered and crystallized from ethanol to
give compounds 3a-c.
4.1.1.1. 2-(N-acetyl-4-bromobenzensulfonyl) benzoic acid (3a).
Brown powder; Yield (67%); m.p. 172-175oC; IR (KBr, cm-1): 3387 (br, OH), 1709, 1670 (C=O); 1HNMR (400 MHz, DMSO-
d6) δ(ppm): 2.13 (s, 3H, COCH3), 6.63 (t, Ar'-H,1H-3, J= 7.6 Hz), 6.83 (d, Ar'-H,1H-5, J= 8Hz), 7.29 (t, Ar'-H,1H-4, J= 8 Hz),
7.37 (d, Ar-H, 2H-3,5, J= 7.6 Hz), 7.52 (d, Ar-H, 2H-2,6 , J= 7.6 Hz), 7.73 (d, Ar'-H,1H-2, J=7.6 Hz), 11.1 (s, OH, D2O
exchangeable). 13CNMR (DMSO-d6) δ (ppm): 19.3 (CH3), 115.6, 121.5, 124.3, 126.3, 129.5, 130.5, 132, 134.2, 138.7(C-SO2) ,
142.4 (C-N) , 168 (C=O of amide), 169.4 (C=O of COOH). MS m/z (%): 397 (1.96, M+), 399 (1.95, M+2), 94.9 (100). Anal. Calcd
for: C15H12BrNO5S (397): C, 45.24; H, 3.04; N, 3.52; S, 8.05%, Found: C, 45.64; H, 3.54; N, 3.12; S, 8.39 %.
4.1.1.2. 2-(N-acetyl-4-flouro benzensulfonyl) benzoic acid (3b)
Buff powder; Yield (88%); m.p. 193-195oC; IR (KBr, cm-1): 3468 (br, OH), 1716, 1670 (C=O), 1HNMR (400 MHz, DMSO-d6) δ
(ppm): 2.30 (s, 3H, COCH3), 6.94 (t, Ar'-H, 1H-3, J= 6 Hz), 7.07 (d, Ar'-H, 1H-5, J= 7.2 Hz), 7.15 (t, Ar-H, 2H-3,5, J= 6.8 Hz),
7.43 (t, Ar'-H, 1H-4, J= 8Hz), 7.65 (t, Ar-H, 2H-2,6, J= 6.8 Hz), 7.84 (d, Ar'-H, 1H-2, J=8Hz), 11.1 (s, OH, D2O exchangeable).
13C-NMR (DMSO-d6) δ (ppm): 19.8 (CH3), 115.2, 116, 121.5, 124.5, 128.6, 130.5, 134.6, 135.3 (C-SO2), 142.4 (C-N) , 166 (C-
F),168 (C=O amide), 169 (C=O of COOH). MS m/z (%): 337.04 (3.96, M+), 94.9(100). Anal. Calcd for C15H12FNO5S (337.04):
C, 53.41; H, 3.59; N, 4.15; S, 9.51%, Found: C, 53.01; H, 3.83; N, 4.57; S, 9.15%.
4.1.1.3. 2-(N-acetyl-4-chlorobenzensulfonyl) benzoic acid (3c).
Brown powder; Yield (75%); m.p. 215-218oC; IR (KBr, cm-1): 3421 (br, OH), 1708, 1662 (C=O), 1HNMR (400 MHz, DMSO-d6)
δ (ppm): 2.2 (s, 3H, COCH3), 6.85 (t, Ar'-H, 1H-3, J= 7.6 Hz), 7.0 (d, Ar'-H, 1H-5, J= 7.6 Hz), 7.16 (t, Ar'-H, 1H-4 , J= 8.4 Hz),
7.39 (d, Ar-H, 2H-3,5, J= 8.4 Hz), 7.61 (d, Ar-H, 2H-2,6, J= 8.4 Hz), 7.81 (d, Ar'-H, 1H-2, J= 7.6 Hz), 11.3 (s, OH, D2O
exchangeable). 13C-NMR (DMSO-d6) δ (ppm): 19.2 (CH3), 116, 121.5, 124.2, 128.6, 129.2, 130.5, 134.2, 137.5 (C-Cl), 137.8 (C-
SO2), 142.4 (C-N), 168 (C=O amide), 169 (C=O of COOH). MS m/z (%): 353.01 (3.94, M+), 355 (1.5, M+2), 65.04 (100). Anal.
Calcd for: C15H12ClNO5S (353.01): C, 50.92; H, 3.42; N, 3.96; S, 9.06%, Found: C, 50.52; H, 3.76; N, 3.64; S, 9.43%.
4.1.2.2-(N-acetyl-4-substituted benzamido)benzoic acids (4a, b)
19

A solution N-acetyl anthranilic acid (2) (0.01 mol) and 4-Substituted benzoyl chlorides (0.01 mol) in dry DMF was heated under
reflux. The reaction was continued for 6-8 h., and cooled. The precipitate was filtered and crystallized from DMF to afford the
target compounds 4a, b.
4.1.2.1. 2-(N-acetyl-4-fluorobenzamido) benzoic acid (4a)
Pale yellow powder; Yield (67%); m.p. 160-162oC; IR (KBr, cm-1): 3367 (br, OH), 1693,1670 and 1654 (C=O); 1HNMR (400 Mz,
DMSO-d6) δ (ppm): 2.11 (s, 3H, CH3), 7.11 (t, Ar'-H, 1H-3, J= 6.8 Hz), 7.3 (t, Ar'-H, 1H-4, J= 6.8 Hz), 7.95 (d, 1 Ar'-H,1H-5,
J= 8 Hz ), 8.05 (d, Ar-H, 2H-3,5, J= 8 Hz), 8.1 (d, Ar-H, 2H-2,6, J= 8 Hz), 8.43 (d, Ar'-H,1H-2, J= 8Hz), 11.03 (s, OH, D2O
exchangeable). 13C-NMR(DMSO-d6) δ (ppm): 20.2 (CH3), 116, 121.5, 124.2, 129.1, 129.8, 130.5, 134.2, 137.3 (C-N), 166.8 (C-
F), 172.1 (CO of amide), 172.3 (C=O of COOH). MS m/z (%): 301 (6.96, M+), 302.19 (5, M+2), 76.04 (100). Anal. Calcd for:
C16H12FNO4 (301.01): C, 63.79; H, 4.01; N, 4.65 %, Found: C, 63.45; H, 4.39; N, 4.22 %.
4.1.2.2. 2-(N-acetyl-4-nitrobenzamido) benzoic acid (4b)
Pale yellow powder; Yield (54%); m.p. 224-227oC; IR (KBr, cm-1): 3367 (br, OH), 1710,1700 and 1685 (C=O); 1HNMR (400 Mz,
DMSO-d6) δ (ppm): 2.11(s, 3H, CH3), 7.11 (t, Ar'-H, 1H-3, J= 7.2 Hz), 7.54 (t, Ar'-H, 1H-4, J= 7.2 Hz), 7.94 (d, Ar'-H,1H-5, J =
8Hz ), 8.15 (d, Ar-H, 2H-3,5, J= 6.8 Hz), 8.3 (d, Ar-H, 2H-2,6, J= 6.8 Hz), 8.43 (d, Ar'-H,1H-2, J= 8Hz), 11.03 (s, OH, D2O
exchangeable). 13C-NMR (DMSO-d6) δ (ppm): 20.2 (CH3), 115.6, 121.2, 124.3, 128.4, 130.5, 134.2, 137.5 (C-N), 140.3, 151.5
(C-NO2), 172.1 (CO of amide), 172.3 (C=O of COOH). MS m/z (%): 328.43 (9.35, M+), 65.01 (100). Anal. Calcd for: C16H12N2O6
(328.43): C, 58.54; H, 3.68; N, 8.53 %, Found: C, 58.15; H, 3.32; N, 8.82 %.
4.1.3. 2-(2-Chloro-3-(aryl) acrylamido)benzoic acids (6a-c)
A mixture of equimolar amounts of 2-chloroacetamido benzoic acid (5) and the corresponding aldehydes or acetophenone, namely,
4-methoxy benzaldehyde, 2, 4-dichloro benzaldehyde or 4-chloro acetophenone was reacted in ethanol (20ml)/ TEA under reflux
for 8-12 h. The reaction poured into cold water. The precipitate was filtered and washed with water, then crystallized from ethanol
to give compounds 6a-c respectively.
4.1.3.1. 2-(2-Chloro-3-(4-methoxyphenyl) acrylamido) benzoic acid (6a)
o
1
Brown powder; Yield (65%); m.p. 168-170 C; IR (KBr, cm-1): 3251 (br, OH, NH), 1705, 1661 (C=O); HNMR (400 MHz,
DMSO-d6) δ (ppm): 3.88 (s, 3H, OCH3), 6.84 (d , Ar-H, 2H-3,5, J= 8 Hz), 7.13 (d, Ar H, 2H-2,6, J= 8 Hz), 7.19 (t, Ar'-H, 1H-3,
J= 12 Hz ), 7.53 (s, 1H, Cl-C=CH), 7.69 (t, Ar'-H, 1H-4, J=12 Hz), 7.85 (d, Ar'-H, 1H-5, J=12 Hz), 7.92 (d, Ar'-H,1H-2, J=12
Hz), 8.12 (s, NH, D2O exchangeable), 9.9 (s, OH, D2O exchangeable). 13C-NMR (DMSO-d6) δ (ppm): 56 (OCH3), 114.2, 115.6,
121.2, 124.3, 126.6 (C-Cl), 127.4, 127.5, 130.5, 134.2, 135.5 (C=C-Cl), 140.7(C-N), 159.9 (C-OCH3), 163.8 (CO of amide), 169.4
20

(C=O of COOH). MS m/z: 331.37 (23.43, M+), 333.3 (8.13, M+2), 72.48 (100). Anal. Calcd for: C17H14ClNO4 (331.37): C, 61.55;
H, 4.25; N, 4.22 %, Found: C, 61.12; H, 4.63; N, 4.56 %.
4.1.3.2. 2-(2-Chloro-3-(2,4-dichlorophenyl) acrylamido) benzoic acid (6b)
Dark brown powder; Yield (42%); m.p. 140-142oC; IR (KBr, cm-1): 3400 (br, OH, NH), 1705, 1695 (C=O); 1HNMR (400 MHz,
DMSO-d6) δ (ppm): 7.24-7.28 (m, Ar-H, 2H-5,6), 7.32-7.39 (m, Ar-H, 1H-3, Ar'-H, 1H-3), 7.64 (t, Ar'-H, 1H-4, J= 12 Hz), 7.81
(s, 1H, Cl-C=CH), 7.95 (s, NH, D2O exchangeable), 8.08 (d, Ar'-H, 1H-5, J=12 Hz), 8.66 (d, Ar'-H, 1H-2, J=12 Hz), 10.4 (s, OH,
D2O exchangeable). 13C-NMR (DMSO-d6) δ (ppm): 115.6, 121.2, 124.3, 126.6 (C-Cl), 127, 130.3, 130.5, 131.2, 132.6, 134.2,
134.9, 135.5 (C=C-Cl), 140.7(C-N), 163.8 (CO of amide), 169.4 (C=O of COOH). MS m/z: 369.27 (20.41, M+), 371.86 (6.5,
M+2), 373.01 (8.77, M+4), 374.95 (5.28, M+6), 85.91 (100). Anal. Calcd for: C16H10Cl3NO3 (369.27): C, 51.85; H, 2.72; N, 3.78
%, Found: C, 51.45; H, 2.35; N, 3.43 %.
3.1.3.3. 2-(2-Chloro-3-(4-chlorophenyl) but-2-enamido) benzoic acid (6c)
Dark brown powder; Yield (68%); m.p.230-233oC; IR (KBr, cm-1): 3349, 3271 (br, OH, NH), 1705, 1687 (C=O); 1HNMR (400
MHz, DMSO-d6) δ (ppm): 2.56 (s, 3H, CH3), 7.37 (d, Ar-H, 2H-2,6, J=12 Hz), 7.52 (t, Ar'-H, 1H-3, J= 8 Hz), 7.66 (d, Ar-H, 2H-
3,5, J= 12 Hz ), 7.77 (t, Ar'-H, 1H-4, J= 8 Hz), 7.99 (d, Ar'-H, 1H-5, J= 12 Hz), 8.12 (s, NH, D2O exchangeable), 8.42 (d, Ar'-H,
1H-2, J= 12 Hz),11.01 (s, OH, D2O exchangeable). 13C-NMR (DMSO-d6) δ (ppm): 11.7 (CH3), 115.6, 119.2 (C-Cl), 121.2, 124.3,
127.8, 128.8, 130.5, 133.5, 134.2, 140.7(C-N), 144.9 (C-CH3), 163.8 (CO of amide), 169.4 (C=O of COOH). MS m/z: 349.14
(8.53, M+), 351.35 (2.27, M+2), 86.03 (100). Anal. Calcd for C17H13Cl2NO3 (349.14): C, 58.31; H, 3.74; N, 4.00 %, Found: C,
58.65; H, 3.42; N, 4.32 %.
4.1.4.2-(2-(Aryl/ heteroarylamino) acetamido) benzoic acids (7a-c)
Equimolar amounts of 2-chloroacetamido benzoic acid (5) and different substituted amines, (2-amino thiophenol, 2,4-dimethyl
aniline and 4-amino pyridine) were refluxed in ethanol in presence of few drops of TEA for 8-12 h. The reaction mixture was
cooled poured into cold water. The precipitate was filtered, and crystalized from ethanol to afford the compounds 7a-c.
4.1.4.1. 2-(2-(2-Mercaptophenylamino) acetamido) benzoic acid (7a)
Yellowish green powder; Yield (67%); m.p. 145-147oC; IR (KBr, cm-1): 3448 (br, OH), 3352, 3309 (NH), 1710, 1662 (C=O);
1HNMR (400 Mz, DMSO-d6) δ (ppm):3.41(s, 1H, SH), 4.18-4.22 (m, 2H, CH2), 5.25 (s, NH, D2O exchangeable), 6.38-6.42 (m,
Ar-H, 2H-4,6 ), 6.47-6.53 (m, Ar-H, 2H-3,5), 7.06 (s, NH, D2O exchangeable), 6.92-6.97 (m, Ar'-H, 2H-3,5), 7.14 (t, Ar'-H, 1H-
4, J= 7.6 Hz), 7.29 (d, Ar'-H,1H-2, J= 7.2Hz),10.52 (s, OH, D2O exchangeable). 13C-NMR (DMSO-d6) δ (ppm): 54.8 (CH2), 113.4,
115.6, 116.3, 117.5, 121.5, 124, 126.4, 130.2, 130.5, 134.2, 143.3 (C-N), 144.5, 168.5 (CO amide), 169.4 (C=O of COOH). MS
m/z (%): 302 (44, M+), 64.09 (100). Anal. Calcd for: C15H14N2O3S (302.07): C, 59.59; H, 4.67; N, 9.27; S, 10.61 %, Found: C,
59.25; H, 4.33; N, 9.65; S, 10.28 %.
4.1.4.2. 2-(2-(2, 4-Dimethylphenylamino) acetamido)benzoic acid (7b)
21

Brown powder; Yield (53%); m.p. 223-225oC; IR (KBr, cm-1): 3447 (br, OH), 3381 (NH), 1685, 1605(C=O); 1HNMR (400 Mz,
DMSO-d6) δ (ppm): 2.2 (s, 3H, CH3), 2.3 (s, 3H, CH3), 4.49-4.51 (m, 2H, CH2, NH, D2O exchangeable ), 7.1 (d , Ar-H, 1H-6, J=
4 Hz), 7.22 (s, Ar-H, 1H-3), 7.24 (d, Ar-H, 1H-5, J= 4 Hz ), 7.52 (t, Ar'-H, 1H-3, J= 20 Hz), 7.78 (t, Ar'-H, 1H-4, J= 20 Hz), 8.09
(d, Ar'-H, 1H-5, J= 8 Hz), 8.21 (s, NH, D2O exchangeable), 8.33 (d, Ar'-H, 1H-2, J= 20 Hz), 11.36 (s, OH, D2O exchangeable).
13C-NMR (DMSO-d6) δ (ppm): 15.8, 24.6 (2CH3), 55.5(CH2) 113.3, 115.6, 116, 121.5, 124, 126.9, 130.5, 131.7, 134.2, 143.3 (C-
N), 143.5, 168.5 (CO amide), 169.4 (C=O of COOH).MS m/z (%): 298.21 (19.84, M+), 76.23(100). Anal. Calcd for C17H18N2O3
(298.21): C, 68.44, H, 6.08; N, 9.39%, Found: C, 68.17, H, 6.43; N, 9.62 %.
4.1.4.3. 2-(2-(Pyridin-4-ylamino) acetamido) benzoic acid (7c)
1
Buff powder; Yield (42%); m.p. 276-279oC; IR (KBr, cm-1): 3350 ( br, OH), 3200 (NH), 1670, 1651(C=O); HNMR (400 Mz,
DMSO-d6) δ (ppm): 4.31-4.34 (m, 2H, CH2, NH D2O exchangeable), 6.83 (d, Ar-H, 2H-2,6 of pyridine, J= 7.2 Hz), 6.94 (t, Ar'-
H, 1H-3, J= 8 Hz), 7.24 (t, Ar'-H, 1H-4, J= 8 Hz), 7.95 (d, Ar'-H, 1H-5, J= 8 Hz) , 8.12 (d, Ar-H, 2H-3,5 of pyridine, J= 7.6 Hz),
8.18 (s, NH, D2O exchangeable), 8.32 (d, Ar'-H, 1H-2, J= 7.6 Hz), 15.5 (s, OH, D2O exchangeable). 13C-NMR (DMSO-d6) δ
(ppm): 55 (CH2), 109.1, 115.6, 121.5, 124, 130.5, 134.2, 143.3 (C-N), 150.5, 155, 168.5 (CO amide), 169.4 (C=O of COOH). MS
m/z (%): 271.1(7.42, M+), 65.04 (100). Anal. Calcd for C14H13N3O3 (271.1): C, 61.99; H, 4.83; N, 15.49 %, Found: C, 61.55; H,
4.47; N, 15.17 %.
4.1.5. Acrylamides (14a-e)
Equimolar amounts of substituted amino thiophenes (10a-c) and substituted cinnamoyl chlorides (13a,b) namely: 4-
hydroxycinnamoyl or 2,5-dimethoxycinnamoyl chloride were refluxed in acetone in presence of anhydrous K2CO3 for 5-8 h. The
reaction medium was cooled and poured into acidified water. The precipitate was filtered, dried and crystalized from ethanol.
4.1.5.1. Ethyl-2-((E)-3-(4-hydroxyphenyl)acrylamido)-4,5,6,7tetrahydrobenzo[b]thiophene-3-carboxylate (14a)
o
1
Buff powder; Yield (79%); m.p. 116-118 C; IR (KBr, cm-1): 3404, 3298 (OH, NH), 1745, 1646 (C=O); HNMR (400 MHz,
DMSO-d6) δ (ppm): 1.24 (t, 3H, CH3, J= 8 Hz), 1.62-1.68 & 2.50-2.56 (m, 8H, CH2 of Cyclohexane), 4.14 (q, 2H, OCH2, J= 8
Hz), 5.2 (s, OH, D2O exchangeable). 6.84 (d , Ar-H, 2H-3,5, J= 8 Hz), 7.22 (d, olefinic, 1H-y, J= 16 Hz), 7.73 (d, Ar-H, 2H-2,6,
J= 8 Hz ), 7.96 (d, olefinic,1H-x, J= 16 Hz), 8.12 (s, NH, D2O exchangeable). 13C-NMR (DMSO-d6) δ (ppm): 15 (CH3), (20, 23.5,
25 cyclohexane), 61(OCH2), 97.6, 115.8, 119.1, 126.5, 127.4, 127.8, 144, 157.5 (C-OH) , 155, 160.4 (C=O of COOR),166.7 (CO
of amide), 176( C-S). MS m/z: 371.12 (21.53%, M+). Anal. Calcd for: C20H21NO4S (371.12): C, 64.67; H, 5.70; N, 3.77; S, 8.63
%, Found: C, 64.22; H, 5.35; N, 4.01; S, 8.43 %
4.1.5.2. (E)-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-(4-hydroxyphenyl)acrylamide (14b)
Buff powder; Yield (82%); m.p. 268-270 oC; IR (KBr, cm-1): 3322, 3257 (OH, NH), 2204(C≡N), 1633 (C=O); 1HNMR (400 MHz,
DMSO-d6) δ (ppm): 1.64-1.69 & 2.40-2.48 (m, 8H, CH2 of Cyclohexane), 4.99 (s, OH, D2O exchangeable). 6.88(d , Ar-H, 2H-
3,5, J= 8 Hz), 7.25 (d, olefinic, 1H-y, J= 16 Hz), 7.67 (d, Ar-H, 2H-2,6, J= 8 Hz ), 7.91 (d, olefinic,1H-x, J= 16 Hz), 8.1 (s, NH,
D2O exchangeable). 13C-NMR (DMSO-d6) δ (ppm): (19.4, 23, 23.5, 24.5 cyclohexane), 67.5(C-CN), 115.5 (C≡N), 115.8, 119.1,
126.5, 127, 127.8, 135, 144, 146.5(S-C-N), 157.5 (C-OH), 166.5 (CO of amide). MS m/z: 324.09 (M+). Anal. Calcd for:
C18H16N2O2S (324.09): C, 66.64; H, 4.97; N, 8.64; S, 9.88 %, Found: C, 66.23; H, 4.55; N, 8.87; S, 9.58 %
22

4.1.5.3. (E)-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-(2,5-dimethoxyphenyl)acrylamide (14c)
Brown powder; Yield (85%); m.p. 99-102 oC; IR (KBr, cm-1): 3220 (NH), 2203(C≡N), 1645 (C=O); 1HNMR (400 MHz, DMSO-
d6) δ (ppm): 1.64-1.69 & 2.44-2.51 (m, 8H, CH2 of Cyclohexane), 3.88 (s, 6H, 2OCH3), 6.76 (d , Ar-H, 1H-4, J= 7.6 Hz), 7.21 (d,
Ar-H, 1H-3, J= 8 Hz ), 7.33 (d, olefinic, 1H-y, J= 16 Hz), 7.43 (s, Ar-H, 1H-6), 8.01 (s, NH, D2O exchangeable), 8.03 (d,
olefinic,1H-x, J= 16 Hz). 13C-NMR (DMSO-d6) δ (ppm): (19.4, 23, 23.5, 24.5 cyclohexane), 55.9(, 56.5 (2OCH3), 68(C-
CN),111.2, 114, 115.5 (C≡N), 115.8, 117, 129, 135.5, 144, 146.5(S-C-N), 150, 153, 166.7 (CO amide). MS m/z: 368.45 (M+).
Anal. Calcd for: C20H20N2O3S (368.45): C, 65.20; H, 5.47; N, 7.60; S, 8.70 %, Found: C, 65.67; H, 5.12; N, 7.93; S, 8.32 %
4.1.5.4. (E)-N-(3-cyano-5,6-dihydro-4H-cyclopenta[b] thiophen-2-yl)-3-(4-hydroxyphenyl)acrylamide (14d)
o
1
Black powder; Yield (64%); m.p. 132-134 C; IR (KBr, cm-1): 3334, 3219 (OH, NH), 2193 (C≡N), 1650 (C=O); HNMR (400
MHz, DMSO-d6) δ (ppm): 1.92-1.96 & 2.43-2.50 (m, 6H, CH2 of Cyclopentane), 5.22 (s, OH, D2O exchangeable), 6.67 (d , Ar-
H, 2H-3,5, J= 8 Hz), 6.98 (d, olefinic, 1H-y, J= 16 Hz), 7.49 (d, Ar-H, 2H-2,6, J= 8 Hz ), 7.88 (d, olefinic,1H-x, J= 16 Hz), 8.1
(s, NH, D2O exchangeable). 13C-NMR (DMSO-d6) δ (ppm): (21.2, 25.5, 31.9 cyclopentane), 67.5(C-CN), 115.3 (C≡N), 115.8,
118.9, 127, 127.8, 129, 135.4, 144, 146.5(S-C-N), 157.7 (C-OH), 166.7 (CO of amide). MS m/z: 310.37 (M+). Anal. Calcd for:
C17H14N2O2S (310.37): C, 65.79; H, 4.55; N, 9.03; S, 10.33 %, Found: C, 65.79; H, 4.55; N, 9.03; S, 10.33 %
4.1.5.5. (E)-N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-3-(2,5-dimethoxyphenyl)acrylamide (14e)
1
Pale brown powder; Yield (55%); m.p. 264-266oC; IR (KBr, cm-1): 3170 (NH), 2201(C≡N), 1647 (C=O); HNMR (400 MHz,
DMSO-d6) δ (ppm): 1.88-1.96 & 2.38-2.46 (m, 6H, CH2 of Cyclopentane), 3.84 (s, 6H, 2OCH3), 6.64 (d , Ar-H, 1H-4, J= 7.6 Hz),
7.38 (d, Ar-H, 1H-3, J= 8 Hz ), 7.47 (d, olefinic, 1H-y, J= 16 Hz), 7.51 (s, Ar-H, 1H-6), 8.87 (d, olefinic,1H-x, J= 16 Hz), 7.99 (s,
NH, D2O exchangeable). 13C-NMR (DMSO-d6) δ (ppm): (21.2, 25.5, 31.9 cyclopentane), 55.9, 56.3 (2OCH3), 67.5(C-CN), 111.6,
114.5, 115.2, 115.3 (C≡N), 116, 118.9, 129, 135.4, 144, 146.8 (S-C-N), 150, 152.8 (2C-OCH3), 166.7 (CO of amide). MS m/z:
354.42 (M+). Anal. Calcd for: C19H18N2O3S (354.42): C, 64.39; H, 5.12; N, 7.90; S, 9.05 %, Found: C, 64.62; H, 5.45; N, 7.46; S,
9.55 %.
4.2. Biological activities.
4.2.1. In vitro anti-proliferative activities
Cancer cells from different cancer cell lines; Human prostate carcinoma cell lines (PC-3), hepatocellular carcinoma cell lines
(HepG-2) and human breast adenocarcinoma cell line (MCF-7) were obtained from VACSERA- Cell Culture Unit, Cairo, Egypt.
Also, the cytotoxic evaluation of all compounds against normal cells WI-38 was carried out to explore the toxicity and selectivity
of the tested compounds. For comparison, 5-FU was used as a standard reference drug. Anti-proliferative activities of the
synthesized compounds were carried out based on MTT assay. [48] Briefly, human cancer cell lines were dropped in 96-well plates
at a density of 3–8×103 cells/well. Next, the wells were incubated for 12 h in a 5% CO2 incubator at 37 °C. Then, for each well, the
growth medium was exchanged with 0.1 mL of fresh medium containing graded concentrations of the test compounds to be or
equal DMSO and incubated for two days. Then 10 μl MTT solutions (5μg/ml) was added to each well, and the cells were incubated
for additional 4 h. The crystals of MTT-formazan were dissolved in 100 μl of DMSO; the absorbance of each well was measured
23

at 490 nm using an automatic ELISA reader system (TECAN, CHE). The IC50 values were calculated using the nonlinear
regression fitting models (Graph Pad, Prism Version 5). The means of at least three separate experiments gave the reported results.
Statistical differences were analyzed according to one-way ANOVA test wherein the differences were considered to be significant
at p < 0.05.
4.2.2. Determination of TGFBR1 activity
The effects of the synthesized compounds on the activity of TGFβR1 were measured using Human TGF-beta1 ELISA (Enzyme-
Linked immunosorbent Assay) kit (Catalog #: ELH-TGFb1). The kit was activated by adding 0.1 ml 1 N HCI into 0.5 mL cell
culture supernatant. Incubated for 10 minutes at r.t., neutralized by adding 0.1 ml 1.2 N NaOH/0.5 M HEPES (PH=7.0~7.6), then
diluted with 1X Assay Diluent (Item E2) and assayed immediately according to the manufacturer's instructions. Shortly, the assay
was performed using 100 mL of the cells supernatant in each well, which were incubated for 2.5 h. at r.t, followed by adding 100
μl biotin antibody (incubated for 1 hr at r.t), 100 μl prepared Streptavidin solution (Incubate 45 minutes at r. t.) and 100 μl TMB
One-Step Substrate Reagent to each well (Incubate 30 minutes at r. t.). Finally, 50 μl Stop Solution was added to each well, the
optical density of each well was determined within 5 min using a microplate reader set at 450 nm.
4.2.3. Cell cycle analysis
PC-3 cells were seeded in a 6-well plate at concentration of 1 X 105 cells per well, then incubated for 24 h. The cells were treated
with (0.1% DMSO) vehicle or 0.36 μg/ml of compound 4b for 24 h. After that, the cells were harvested and fixed for 12 h using
ice-cold 70% ethanol at 4 oC. Removal of ethanol and washing cells with cold PBS was done. Then, the cells were incubated in
0.5 ml of PBS containing 1 mg/mL RNase at 37oC for 30 min. The cells were stained for 30 min with propidium iodide in the
dark. Afterwards, the flow cytometer was used to detect DNA contents. [49]
4.2.4. Apoptosis detection studies
4.2.4.1. Annexin-V assay
PC-3 cells were seeded in a 6-well plate (1X105cells/well), incubated for 24 h, and then treated with vehicle (0.1% DMSO) or 0.36
μg/ml of compound 4b for 24 h. Subsequently, the cells were harvested, washed using PBS and stained for 15 min at room
temperature in the dark using annexin V-FITC and PI in binding buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl2 at pH
7.4), then analyzed by the flow cytometer. [50]
4.2.4.2. Determination of the active caspase-3
To determine the effect of test compounds on apoptosis, the active caspase-3 level was measured by using Quantikine-Human
active Caspase-3 Immunoassay (R&D Systems, Inc. Minneapolis, USA) according to the manufacturer protocol. Briefly, after
washing the PC-3 cells with PBS, the cells were collected and lysed by adding it to the extraction buffer containing protease
inhibitors (1 mL per 1 x 107 cells.) then, the lysate was diluted immediately prior to the assay. At the end of the assay, the optical
density of each well was determined within 30 min using a microplate reader set at 450 nm.
4.2.4.3. Determination of the gene expression of some apoptosis key marker (p53)
24

The effect of compound 4b on the gene expression of apoptosis marker (p53) was measured using p53 in vitro Simple Step
ELISA® kit (Catalog #: ab171571). All materials and prepared reagents were kept at room temperature prior to use. Briefly, the
assay was performed using 50 μl samples and standards to appropriate wells, followed by adding 50 μL of the Antibody Cocktail.
The plate was sealed, covered and incubated for 1 h at room temperature on a plate shaker set to 400 rpm. After washing the PC-
3 cells with 3 x 350 μL 1X Wash Buffer PT, 100 μl TMB, One-Step Substrate Reagent was added to each well (Incubate 10
minutes at r. t. in the dark). Finally, 100 μl Stop Solution was added to each well and the optical density of each well was determined
within 5 min using a microplate reader set at 450 nm.
4.3. Molecular Modeling:
4.3.1. In silico physicochemical and ADME properties prediction
The molecular structures were converted into SMILES database using Chemdraw 12.0. Then, these SMILES were inserted
as input in SwissADME website to calculate the physicochemical descriptors, lipophilicity, pharmacokinetics properties, ADME
parameters, and medicinal chemistry friendliness.
4.3.2. Docking Studies
Crystallographic structures of TGFBR1 was retrieved from Protein Data Bank [PDB ID: 1VJY, with resolution of 2.0Å and 303
amino acids (http://www.pdb.org), and considered as targets for docking simulations. The docking analysis was performed using
MOE software to evaluate the free energies and binding mode of the designed molecules against TGFBR1. At first, the crystal
structures of TGFBR1was prepared by removing water molecules and retaining only one chain and its co-crystallized ligand,
naphthyridine. Then, the protein structure was protonated and the hydrogen atoms were hided. Next, the energy was minimized
and the binding pocket of the protein was defined. The 2D structures of the synthesized compounds and the co-crystallized ligand,
naphthyridine were sketched using ChemBioDraw Ultra 12.0 and saved as MDL-SD format. After that, the saved files were opened
using MOE, 3D structures were protonated and energy minimization was applied. Before docking the synthesized compounds,
validation of the docking protocol was carried out by running the simulation using the co-crystallized ligand and low RMSD
between docked and crystal conformations. The molecular docking of the synthesized compounds and the co-crystallized ligand
was performed using a default protocol. In each case, four docked structures were generated using genetic algorithm searches.
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27

Design and Synthesis of Novel Tranilast Analogs:
Docking, Antiproliferative Evaluation and In-silico Screening of TGFβR1
Inhibitors
Magda M.F. Ismaila*, Heba S.A. El-Zahabia, Rabab S. Ibrahima, Ahmed B. M. Mehanyb




PC-3
IC50=1.1 μm
HepG-2 IC50=1.5 μm
MCF-7 IC50=2.6 μm
TGFβR1 IC50=0.087 μm
COOH
4b
O
N
O
NO2
28

Research highlights






Novel series of tranilast analogs is synthesized as potential selective anticancer on PC-3, HepG-2 and MCF-7
cell lines using 5-FU as reference drug.
Twelve of the investigated compounds showed more potent cytotoxic agents than 5-FU, compound 4b
displayed amazing anticancer activity 5-7 times that of reference drug against all the tested cell lines.
Target compounds 4b, 7a and 14e were the most potent TGFβR1 inhibitors showing IC50s in sub-micromolar
range (IC50's 0.08, 0.09, and 0.19 μM).
4b enhanced apoptosis and arrested G2/M phase of cell cycle. Besides, it induced apoptosis via the increase of
both caspase-3 level and expression of p53 gene of PC-3 cell.
In silico study revealed that all target compounds are in agreement with the parameters of Lipiniski's rule and
Veber's violation.
Molecular docking of the most potent hits, 4b and 14e elicited higher affinities to their respective binding site
(TGFβR1) with good binding energies compared to ligand 460 and lead compound, tranilast.
29