Design, synthesis, molecular docking, anticancer evaluations, and in silico pharmacokinetic studies of novel 5-[(4-chloro/2,4-dichloro)benzylidene]thiazolidine-2,4-dione derivatives as VEGFR-2 inhibitors

Article abstract of DOI:10.1002/ardp.202000279

The anticancer activity of novel thiazolidine-2,4-diones was evaluated against HepG2, HCT-116, and MCF-7 cells. MCF-7 was the most sensitive cell line to the cytotoxicity of the new derivatives. In particular, compounds 18, 12, 17, and 16 were found to be the most potent derivatives over all the tested compounds against the cancer cell lines HepG2, HCT116, and MCF-7, with IC₅₀ = 9.16 ± 0.9, 8.98 ± 0.7, 5.49 ± 0.5 μM; 9.19 ± 0.5, 8.40 ± 0.7, 6.10 ± 0.4 μM; 10.78 ± 1.2, 8.87 ± 1.5, 7.08 ± 1.6 μM; and 10.87 ± 0.8, 9.05 ± 0.7, 7.32 ± 0.4 μM, respectively. Compounds 18 and 12 have nearly the same activities as sorafenib (IC₅₀ = 9.18 ± 0.6, 5.47 ± 0.3, and 7.26 ± 0.3 μM, respectively), against HepG2 cells, but slightly lower activity against HCT116 cells and slightly higher activity against the MCF-7 cancer cell line. Also, these compounds displayed lower activities than doxorubicin against HepG2 and HCT-116 cells but higher activity against MCF-7 cells (IC₅₀ = 7.94 ± 0.6, 8.07 ± 0.8, and 6.75 ± 0.4 μM, respectively). In contrast, compounds 17 and 16 exhibited lower activities than sorafenib against HepG2 and HCT116 cells, but nearly equipotent activity against the MCF-7 cancer cell line. Also, these compounds displayed lower activities than doxorubicin against the three cell lines. All the synthesized derivatives 7–18 were evaluated for their inhibitory activities against VEGFR-2. The tested compounds displayed high to medium inhibitory activity, with IC₅₀ values ranging from 0.17 ± 0.02 to 0.27 ± 0.03 μM. Compounds 18, 12, 17, and 16 potently inhibited VEGFR-2 at IC₅₀ values of 0.17 ± 0.02, 0.17 ± 0.02, 0.18 ± 0.02, and 0.18 ± 0.02 μM, respectively, which are nearly more than half of that of the IC₅₀ value for sorafenib (0.10 ± 0.02 μM).

Full text of DOI:10.1002/ardp.202000279

Received: 29 July 2020
Accepted: 16 September 2020
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DOI: 10.1002/ardp.202000279
F U L L P A P E R
Design, synthesis, molecular docking, anticancer evaluations,
and in silico pharmacokinetic studies of novel 5‐[(4‐chloro/
2,4‐dichloro)benzylidene]thiazolidine‐2,4‐dione derivatives
as VEGFR‐2 inhibitors
Khaled El‐Adl1,2
| Abdel‐Ghany A. El‐Helby1 | Helmy Sakr1 | Rezk R. Ayyad1
|
Hazem A. Mahdy1 | Mohamed Nasser1 | Hamada S. Abulkhair3,4
Sanadelaslam S. A. El‐Hddad1
|
1Pharmaceutical Medicinal Chemistry and
Drug Design Department, Faculty of Pharmacy
Abstract
(Boys), Al‐Azhar University, Cairo, Egypt
The anticancer activity of novel thiazolidine‐2,4‐diones was evaluated against HepG2,
HCT‐116, and MCF‐7 cells. MCF‐7 was the most sensitive cell line to the cytotoxicity of
the new derivatives. In particular, compounds 18, 12, 17, and 16 were found to be the
most potent derivatives over all the tested compounds against the cancer cell lines
HepG2, HCT116, and MCF‐7, with IC50 = 9.16 0.9, 8.98 0.7, 5.49 0.5 µM; 9.19 0.5,
8.40 0.7, 6.10 0.4 µM; 10.78 1.2, 8.87 1.5, 7.08 1.6 µM; and 10.87 0.8,
9.05 0.7, 7.32 0.4 µM, respectively. Compounds 18 and 12 have nearly the same ac-
tivities as sorafenib (IC50 = 9.18 0.6, 5.47 0.3, and 7.26 0.3 µM, respectively), against
HepG2 cells, but slightly lower activity against HCT116 cells and slightly higher activity
against the MCF‐7 cancer cell line. Also, these compounds displayed lower activities than
doxorubicin against HepG2 and HCT‐116 cells but higher activity against MCF‐7 cells
(IC50 = 7.94 0.6, 8.07 0.8, and 6.75 0.4 µM, respectively). In contrast, compounds
17 and 16 exhibited lower activities than sorafenib against HepG2 and HCT116 cells, but
nearly equipotent activity against the MCF‐7 cancer cell line. Also, these compounds
displayed lower activities than doxorubicin against the three cell lines. All the synthesized
derivatives 7–18 were evaluated for their inhibitory activities against VEGFR‐2. The
tested compounds displayed high to medium inhibitory activity, with IC50 values ranging
from 0.17 0.02 to 0.27 0.03 µM. Compounds 18, 12, 17, and 16 potently inhibited
VEGFR‐2 at IC50 values of 0.17 0.02, 0.17 0.02, 0.18 0.02, and 0.18 0.02 µM, re-
spectively, which are nearly more than half of that of the IC50 value for sorafenib
(0.10 0.02 µM).
2Pharmaceutical Chemistry Department,
Faculty of Pharmacy, Heliopolis University for
Sustainable Development, Cairo, Egypt
3Pharmaceutical Organic Chemistry
Department, Faculty of Pharmacy (Boys),
Al‐Azhar University, Cairo, Egypt
4Pharmaceutical Chemistry Department,
Faculty of Pharmacy, Horus University, New
Damietta, Egypt
Correspondence
Khaled El‐Adl, Pharmaceutical Medicinal
Chemistry and Drug Design Department,
Faculty of Pharmacy (Boys), Al‐Azhar
University, Nasr City 11884, Cairo, Egypt.
Email: eladlkhaled74@yahoo.com,
eladlkhaled74@azhar.edu.eg and
khaled.eladl@hu.edu.eg
K E Y W O R D S
anticancer agents, molecular docking, thiazolidine‐2‐4‐dione, VEGFR‐2 inhibitors
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Arch Pharm. 2020;e2000279.
wileyonlinelibrary.com/journal/ardp
© 2020 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.202000279

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1
INTRODUCTION
agents[13–20] and potent VEGFR‐2 inhibitors, for example, compound
II.[2,12] Owing to the importance of VEGFR‐2 in angiogenesis, this re-
ceptor is the most vital target in anti‐angiogenic therapy against cancer.
Numerous reports on VEGFR‐2 inhibitors, including the commercialized
sunitinib (III) (Figure 1), have been published.[21,22] Sorafenib (Nexavar®;
IV; (Figure 1) also is a potent VEGFR‐2 inhibitor and has been approved
as an anti‐angiogenic drug.[23–25] Our reported compounds V[19] and
VI[20] encourage us to synthesize new derivatives with the hydrophobic
electron‐withdrawing mono‐, chloro‐, and/or dichlorobenzylidene instead
of unsubstituted (V) or the hydrophobic electron‐donating methox-
ybenzylidenes (VI), respectively, to study the effect of these modifications
on the activity.
The developments in cancer therapies have focused on molecular targets,
particularly tumor angiogenesis.[1] Angiogenesis is the process by which
the existing vascular bed expands to form new blood vessels and is a
pivotal event in many physiological and pathological processes, including
tumor growth and metastasis.[2,3] Newly generated blood vessels supply
oxygen and essential nutrition, support tumor growth, and later aid in the
initiation of metastasis, which contributes to more than 90% of deaths in
various cancers.[4] The thiazolidine‐2,4‐diones (TZDs) have exhibited an-
titumor activity in a wide variety of experimental cancer models by af-
fecting cell cycle, induction of cell differentiation, and apoptosis as well as
by inhibiting tumor angiogenesis. TZD scaffold has been proposed for
hybridization with different bioactive moieties to have a different me-
chanism of action with a broad spectrum of activity against numerous
cancer cell lines.[5,6] Vascular endothelial growth factor (VEGF) is one of
the central regulators in angiogenesis.[7] Vascular endothelial growth
factor receptor‐2 (VEGFR‐2) is the receptor for VEGF and is the prime
mediator of VEGF‐induced pro‐angiogenesis signaling.[8] Binding of VEGF
to VEGFR‐2 leads to the dimerization of the receptors, activation of
tyrosine kinase, trans‐autophosphorylation, and initiation of the extra-
cellular signal‐regulated kinase.[9–11] Considering that angiogenesis is a
significant event in tumor development, blocking angiogenesis is one of
the most promising strategies to treat malignancies. A study reported by
Shah et al.,[12] showed that TZD derivative ciglitazone (I) (Figure 1) sig-
nificantly decreased the VEGF production in human granulose cells in an
in vitro model. Extensive studies were reported in the synthesis of several
5‐benzylidenethiazolidine‐2,4‐dione derivatives as potent anticancer
Study of the structure–activity relationships (SARs) and common
pharmacophoric features shared by sorafenib and various VEGFR‐2 in-
hibitors revealed that most VEGFR‐2 inhibitors shared four main features
as shown in Figure 2[26–28]: (a) the flat heteroaromatic ring system that
contains at least one N atom, (b) a central aryl ring (hydrophobic
spacer).[29] (c) A linker containing a functional group acting as pharma-
cophore (e.g., amino or urea) that possesses both H‐bond acceptor (HBA)
and donor (HBD) to bind with two crucial residues (Glu883 and
Asp1044). The NH motifs of the urea or amide moiety usually form one
hydrogen bond with Glu883, whereas the C═O motif forms another
hydrogen bond with Asp1044. (iv) The terminal hydrophobic moiety of
the inhibitors occupies the newly created allosteric hydrophobic pocket.
Thus, hydrophobic interactions are usually attained in this allosteric
binding region.[30] Furthermore, analysis of the X‐ray structure of various
inhibitors bound to VEGFR‐2 confirmed the sufficient space available for
various substituents around the terminal heteroaromatic ring.[31–33]
FIGURE 1 Reported vascular endothelial growth factor receptor‐2 inhibitors

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to ensure different electronic and lipophilic environments, which
could influence the activity of the target compounds. These mod-
ifications were performed to carry out further elaboration of the TZD
scaffolds and to explore a valuable SAR. The designed target 5‐([4‐
chloro/2,4‐dichloro]benzylidene)thiazolidine‐2,4‐dione
derivatives
were synthesized and evaluated as potential VEGFR‐2 inhibitory and
antitumor activities against three human tumor cell lines, namely,
hepatocellular carcinoma (HCC) type (HepG2), breast cancer
(Michigan Cancer Foundation‐7 [MCF‐7]), and human colorectal
carcinoma‐116 (HCT‐116).
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2
RESULTS AND DISCUSSION
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2.1
Rationale and structure‐based design
5‐([4‐Chloro/2,4‐dichloro]benzylidene)thiazolidine‐2,4‐dione deri-
vatives have the essential pharmacophoric features of VEGFR‐2
inhibitors[35–39] (Figure 3), which include the presence of five‐
membered hetero ring, TZD, substituted with 4‐chlorobenzylidene
and/or 2,4‐dichlorobenzylidene moieties, as hydrophobic portions,
forming 5‐(4‐chlorobenzylidene)thiazolidine‐2,4‐dione and 5‐(2,4‐
dichlorobenzylidene)thiazolidine‐2,4‐dione scaffolds linked to sub-
stituted hydrophobic phenyl tail through acetamide linkers con-
taining HBA‐HBD, which interacts as HBA through its C═O and as
H‐bond donor through its NH with the essential amino acid residues
Asp1044 and Glu883, respectively. Also, the substituted hydro-
phobic phenyl tails formed hydrophobic bonding interactions with
the hydrophobic pocket formed by Asp1044, Cys1043, Ile1042,
Hie1024, Leu1017, Val897, Leu887, Lys866, and Glu883. The hy-
drophobic phenyl tail was substituted with distal hydrophobic
moieties through amide linkers to increase the length of the
structure to enable the distal phenyl moieties to occupy new hy-
drophobic grooves formed by Arg1025, Ile1023, Cys1022, Leu1017,
Ile890, and Ile886. In addition, 4‐chlorobenzylidene and/or 2,4‐
dichlorobenzylidene moieties were designed to replace the pyridine
and 5‐fluoro‐2‐oxoindolin‐3‐ylidene moieties of the reference li-
gands sorafenib and sunitinib, respectively. Moreover, TZD was
designed to replace the central aryl and the five‐membered pyrrole
rings of the reference ligands sorafenib and sunitinib, respectively.
Furthermore, the hydrophobic 4‐chlorobenzylidene and/or 2,4‐
dichlorobenzylidene moieties occupied the hydrophobic groove
formed by Leu1033, Gly920, Lys918, Cys917, Phe916, Glu915,
Ala864, and Leu838. In contrast, the TZD moiety occupied the hy-
drophobic pocket formed by Cys1043, Leu1033, Val914, Val897,
and Lys866 (Figures 4 and 5).
FIGURE 2 The basic structural requirements for sorafenib and
sunitinib as reported VEGFR‐2 inhibitors. ATP, adenosine
triphosphate; HBA, H‐bond acceptor; HBD, H‐bond donor; VEGFR‐2,
vascular endothelial growth factor receptor‐2
Depending on ligand‐based drug design, particularly a molecular
hybridization approach that involves the coupling of two or more
groups with relevant biological properties,[34] molecular hybridiza-
tion of 5‐([4‐chloro/2,4‐dichloro]benzylidene)thiazolidine‐2,4‐diones
and other effective antitumor moieties were carried out in an at-
tempt to get new molecules with promising antitumor activities.
In continuation of our efforts to obtain new anticancer agents
targeting VEGFR‐2, the goal of our work was the synthesis of new
agents with the same essential pharmacophoric features of the
reported and clinically used VEGFR‐2 inhibitors (e.g., sorafenib).
The main core of our molecular design rationale comprised bioi-
sosteric modification strategies of VEGFR‐2 inhibitors at four
different positions (Figure 3).
Our target compounds were designed to have TZD spacers and
amide linkers having HBA‐HBD, the main pharmacophoric feature in
sorafenib hoping to obtain more potent VEGFR‐2 inhibitors. First, the
bioisosteric approach was adopted in the target 4‐chloro and/or 2,4‐
dichlorobenzylidene to replace the pyridine ring. The second strategy
is to use TZD to replace the central aryl ring of lead structure aiming
to increase VEGFR‐2 binding affinity. The third strategy is using
acetamide linkers containing HBA‐HBD functional groups that pos-
sess HBAs and/or HBDs. Also, the hydrophobic substituted phenyl
tail of the reported ligand sorafenib was substituted by other distal
hydrophobic moieties that occupied the newly formed hydrophobic
groove (Figure 4). Furthermore, the substitution pattern was selected
|
2.2
Chemistry
The synthetic strategy for the preparation of the target com-
pounds (7–18) is depicted in Scheme 1. The synthesis was in-
itiated by the cyclocondensation of thiourea with chloroacetic

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FIGURE 3 Structural similarities and pharmacophoric features of vascular endothelial growth factor receptor‐2 inhibitors and designed
compounds
acid to afford TZD (1),[40,41] which underwent further con-
densation reaction with the appropriate benzaldehyde, namely,
4‐chlorobenzaldehyde and/or 2,4‐dichlorobenzaldehyde, to af-
ford the corresponding 5‐(4‐chlorobenzylidene)thiazolidine‐2,4‐
hydroxide to afford the corresponding potassium salts (3a,b).
In contrast, chloroacyl chloride was reacted with 4‐aminobenzoic
acid (4) to obtain the corresponding chloroamide (5). The chlor-
oamide 5 was treated with ethyl chloroformate and then the
appropriate amine was added to afford the corresponding inter-
mediate (6a–f). The potassium salt (3a,b) was refluxed with the
dione
and
5‐(2,4‐dichlorobenzylidene)thiazolidine‐2,4‐dione
(2a,b), respectively, which was heated with alcoholic potassium

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FIGURE 4 Superimposition of compound
12 and sorafenib inside the binding pocket
of 1YWN
appropriate intermediate (6a–f) to obtain the corresponding de-
rivatives 7–18, respectively (Scheme 1).
concentration (IC50) values, which represented the compound con-
centrations required to produce a 50% inhibition of cell growth after
72 hr of incubation calculated from the concentration–inhibition re-
sponse curve and summarized in Table 1. From the obtained results, it
was explicated that most of the prepared compounds displayed excellent
to modest growth inhibitory activity against the tested cancer cell lines.
Investigations of the cytotoxic activity indicated that MCF‐7 was the
most sensitive cell line to the influence of the new derivatives. In parti-
cular, compounds 18, 12, 17, and 16 were found to be the most potent
derivatives in all the tested compounds against HepG2, HCT116, and
MCF‐7 cancer cell lines with IC50 = 9.16 0.9, 8.98 0.7, 5.49 0.5 µM;
|
2.3
Pharmacology/biology
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2.3.1
In vitro cytotoxic activity
Antiproliferative activity of the newly synthesized compounds 7–18 was
examined against three human tumor cell lines, namely hepatocellular
carcinoma (HepG2), colorectal carcinoma (HCT‐116), and breast cancer
(MCF‐7) using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium
bromide (MTT) colorimetric assay as described by Mosmann.[42–44] Sor-
afenib and doxorubicin were included in the experiments as reference
cytotoxic drugs. The results were expressed as growth inhibitory
9.19 0.5,
8.40 0.7,
6.10 0.4 µM;
10.78 1.2,
8.87 1.5,
7.08 1.6 µM; and 10.87 0.8, 9.05 0.7, 7.32 0.4 µM, respectively.
Compounds 18 and 12 have nearly the same activities as sorafenib
(IC50 = 9.18 0.6, 5.47 0.3, and 7.26 0.3 µM, respectively) against the
FIGURE 5 Superimposition of compound
18 and sorafenib inside the binding pocket
of 1YWN

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SCHEME 1 Synthetic route for the preparation of the target compounds 7–18
HepG2 cell line, but slightly lower activity against HCT116 and slightly
higher activity against MCF‐7 cancer cells, respectively. Also, these
compounds displayed lower activities than doxorubicin against the
HepG2 and HCT‐116 cell lines but higher activity against MCF‐7 cells,
respectively (IC50 = 7.94 0.6, 8.07 0.8, and 6.75 0.4 µM, respectively).
However, compounds 17 and 16 exhibited lower activities than sorafenib,
against HepG2 and HCT116 cells, but nearly equipotent activity against
the MCF‐7 cancer cell line, respectively. Also, these compounds displayed
lower activities than doxorubicin against the three cell lines.
Cytotoxicity evaluation against colorectal carcinoma (HCT‐116)
cell line discovered that compounds 10 and 15 displayed excellent
anticancer activities with IC50 = 9.35 1.0 and 10.11 0.7 µM, re-
spectively. Compounds 11, 8, 9, and 7 with IC50 = 12.71 1.2,
14.89 1.3, 15.03 1.5, and 20.66 1.6 µM displayed very good
cytotoxicity. In addition, compound 14 with IC50 = 23.66 1.9 µM
exhibited good cytotoxicity. While compound 13 with IC50 = 34.81
2.4 µM exhibited moderate cytotoxicity.
Cytotoxicity evaluation against the MCF‐7 cell line revealed that
compounds 10 and 15 displayed excellent anticancer activities with
IC50 = 7.41 1.4 and 8.21 0.4 µM, respectively. Compounds 11 and
14 with IC50 = 12.56 1.2 and 17.72 1.7 µM exhibited very good
cytotoxicity. Moreover, compounds 9, 13, and 8 with IC50 = 22.08
1.6, 24.11 2.3, and 28.12 1.9 µM exhibited good cytotoxicity.
With respect to the HepG2 hepatocellular carcinoma cell line,
compounds 8, 9, 10, 11, 14, and 15 displayed very good anticancer
activities with IC50 ranging from 11.04 1.5 to 19.43 1.8 µM, re-
spectively. Moreover, compounds 7 and 13 with IC50 = 21.34 2.1
and 29.37 2.1 µM displayed good cytotoxicity.

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TABLE 1 In vitro cytotoxic activities of
the newly synthesized compounds against
the HepG2, HCT‐116, and MCF‐7 cell lines
and VEGFR‐2 kinase assay
IC50 (µM)a
HepG2
Compound
HCT116
MCF‐7
VEGFR‐2
7
21.34 2.1
17.13 1.6
13.78 1.2
11.04 1.5
13.52 1.3
9.19 0.5
29.37 2.1
19.43 1.8
11.76 0.9
10.87 0.8
10.78 1.2
9.16 0.9
9.18 0.6
7.94 0.6
20.66 1.6
14.89 1.3
15.03 1.5
9.35 1.0
12.71 1.2
8.40 0.7
34.81 2.4
23.66 1.9
10.11 0.7
9.05 0.7
8.87 1.5
8.98 0.7
5.47 0.3
8.07 0.8
31.12 2.2
28.12 1.9
22.08 1.6
7.41 1.4
12.56 1.2
6.10 0.4
24.11 2.3
17.72 1.7
8.21 0.4
7.32 0.4
7.08 1.6
5.49 0.5
7.26 0.3
6.75 0.4
0.27 0.03
0.23 0.02
0.23 0.02
0.20 0.02
0.19 0.02
0.17 0.02
0.23 0.02
0.22 0.03
0.20 0.02
0.18 0.02
0.18 0.02
0.17 0.02
0.10 0.02
NTb
8
9
10
11
12
13
14
15
16
17
18
Sorafenib
Doxorubicin
aIC50 values are the mean SD of three separate experiments.
bNT: compounds not tested for their VEGFR‐2 inhibitory activity.
While compound 7 with IC50 = 31.12 2.2 µM exhibited moderate
with acetamide linkers, which interacts as HBAs through its carbonyl
group and as an H‐bond donor through its NH atom. These acetamide
linkers interact with the side chain NH of the essential amino acid residue
Asp1044 and carboxylate of the essential amino acid residue Glu883.
Also, hydrophobic interactions through the attached hydrophobic distal
moieties were recorded. The effect of replacement of pyridine and
5‐fluoro‐2‐oxoindolin‐3‐ylidene moieties of sorafenib and sunitinib,
respectively, by 4‐chlorobenzylidene and/or 2,4‐dichlorobenzylidene of
5‐([4‐chloro/2,4‐dichloro]benzylidene)thiazolidine‐2,4‐dione scaffold of
the synthesized compounds on the antitumor activities also was noticed.
This 5‐([4‐chloro/2,4‐dichloro]benzylidene moiety occupied the same
hydrophobic pocket which was occupied by the pyridine moiety of the
standard ligand. Moreover, the TZD was designed to replace the central
aryl and pyrrole rings of the reference ligands sorafenib and sunitinib,
respectively, and enables the target compounds to form new H‐bonds
through their 2‐carbonyl groups with the essential amino acid residue
Lys866. In contrast, different distal aliphatic and aromatic moieties were
introduced to the phenyl tail of the reference ligand with different lipo-
philicity and electronic nature to study their effects on the anticancer
activity. The presence of lipophilic distal moieties attached to the phenyl
ring through carboxamide linkers formed new hydrophobic and H‐
bonding interactions with the active site. The data obtained revealed that
the tested compounds displayed different levels of anticancer activity and
possessed a distinctive pattern of selectivity against the MCF‐7 cell lines.
Generally, the spacers, linkers (HBA‐HBD), lipophilicity, and electronic
nature exhibited an important role in anticancer activity. The acetamide
and carboxamide linkers were found to be responsible for high anticancer
activities. The 2,4‐dichlorobenzylidene moiety exhibited higher anticancer
activities than the 4‐chlorobenzylidene moiety, which may be due to the
cytotoxicity.
|
2.3.2
In vitro VEGFR‐2 kinase assay
All the synthesized derivatives 7–18 were evaluated for their in-
hibitory activities against VEGFR‐2 by using an antiphosphotyrosine
antibody with the AlphaScreen system (PerkinElmer). The results were
reported as a 50% inhibition concentration value (IC50) calculated
from the concentration–inhibition response curve and summarized in
Table 1. Sorafenib was used as a positive control in this assay. The
tested compounds displayed high to medium inhibitory activity with
IC50 values ranging from 0.17 0.02 to 0.27 0.03 µM. Among them,
compounds 18, 12, 17, and 16 potently inhibited VEGFR‐2 at IC50
values of 0.17 0.02, 0.17 0.02, 0.18 0.02, and 0.18 0.02 µM, re-
spectively, which are nearly more than the half of that of sorafenib
IC50 value (0.10 0.02 µM). Also, compounds 11, 10, and 15 possessed
very good VEGFR‐2 inhibition with IC50 values of 0.19 0.02,
0.20 0.02, and 0.20 0.02 µM, respectively, which are nearly half
that of sorafenib. Furthermore, compounds 14, 8, 9, 13, and 7 pos-
sessed good VEGFR‐2 inhibition with IC50 values = 0.22 0.03,
0.23 0.02, 0.23 0.02, 0.23 0.02, and 0.27 0.03 µM, respectively.
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2.4
SAR
The preliminary SAR study has focused on the effect of replacement of
the urea and carboxamide linkers of sorafenib and sunitinib, respectively,

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higher lipophilicity of the two lipophilic chloro atoms. Compounds with
distal phenyl moieties such as 10–12 and 16–18 showed higher activities
than those having distal aliphatic ones 7–9 and 13–15 against the three
HepG2, HCT116, and MCF‐7 cell lines. From the structure of the syn-
thesized derivatives and the data shown in Table 1, we can divide these
tested compounds into four groups. The first group has compounds 7–9
that contains 4‐chlorobenzylidene moiety. The aliphatic butyl distal
moiety 9 showed higher activities than the propyl 8 against both HepG2
and MCF‐7 cell lines, respectively, but it showed nearly the same activity
against HCT116 cells. Moreover, butyl 9 and propyl 8 derivatives ex-
hibited higher activities than ethyl 7 ones against the three HepG2,
HCT116, and MCF‐7 cell lines, respectively. The second group has
compounds 10–12 with 4‐chlorobenzylidene moiety also, the substituted
distal phenyl moiety with ethyl ester group as in compound 12 displayed
higher activities than that substituted with benzyl moiety 10 and methyl
group 11 against the three HepG2, HCT116, and MCF‐7 cell lines, re-
spectively. In the third group 13–15 with 2,4‐dichlorobenzylidene moiety,
the aliphatic butyl distal moiety 15 showed higher activities than the
propyl 14 and the ethyl 13 ones against the three HepG2, HCT116, and
MCF‐7 cell lines, respectively. In the fourth group 16–18 with 2,4‐
dichlorobenzylidene moiety also, the substituted distal phenyl moiety
with ethyl ester group as in compound 18 displayed higher activities than
that substituted with methyl group 17 against both HepG2 and MCF‐7
cell lines, respectively, but it showed nearly the same activity against
HCT116 cell line. Moreover, ethyl ester 18 and toluene derivative 17
derivatives exhibited higher activities than benzyl one 16 against the
three HepG2, HCT116, and MCF‐7 cell lines, respectively.
suggested that the absorption of a compound is more likely to be
better if the molecule achieves at least three out of four of the
following rules: (a) HBD groups ≤5, (b) HBA groups ≤10, (c) M.Wt.
<500, (d) logP <5. In this study, whereas the reference anticancer
agent doxorubicin violates three of Lipinski's rules, compounds 12,
16, 17, and 18 violated only two (M.Wt. and logP). All the highest
active derivatives have a number of HBA groups between five and
seven and only two HBDs, and these values agree with Lipinski's
rules. Also, the absorption, distribution, metabolism, excretion, and
toxicity (ADMET) profiles of the newly synthesized TZD deriva-
tives were preliminary assessed to analyze their potentials to build
up as good medication candidates. The prediction of ADMET
profiles was conducted with the aid of pkCSM descriptors algo-
rithm protocol.[46]
After assessing ADMET profiles of compounds 12, 16, 17, and 18
(Table 2), we can suggest that these derivatives have the advantage
of better intestinal absorption in humans than doxorubicin
(84.9–95.5, compared with 62.3 in the case of doxorubicin). This
preference may be attributed to the superior lipophilicity of our
designed ligands, which would make it easier to go along different
biological membranes.[47] Accordingly, they may have significantly
good bioavailability after oral administration. Studying the central
nervous system (CNS) permeability, TZD derivatives 16 and 17 de-
monstrated the best ability to penetrate the CNS (CNS permeability
values approx. −1.7), while doxorubicin is unable to penetrate (CNS
permeability < −4.0). It is also clear that cytochrome P3A4, the main
enzyme involved in drug metabolism, could be inhibited under the
effect of compounds 12, 16, 17, and 18, while doxorubicin could not.
This is also perhaps because of the higher lipophilicity of our new
ligands. Excretion was assessed in terms of the total clearance, which
is a significant parameter in deciding dose intervals. The obtained
data revealed that doxorubicin revealed the highest total clearance
value compared with other ligands. In contrast, new ligands showed
lower total clearance values (−0.592). Thus, doxorubicin could be
excreted quickly and accordingly require shorter dosing intervals.
Dissimilar to doxorubicin, new compounds exhibited slower clear-
ance rates, which means the preference of possible extended dosing
intervals of the novel thiazolidinediones. The last parameter ex-
amined in the ADMET profiles of our newly synthesized VEGFR‐2
inhibitors is the toxicity. As displayed in Table 2, doxorubicin and all
the new ligands except 18 shared the drawback of unwanted hepa-
totoxic effects. Gratifyingly, our designed compound 18 showed the
great advantage of no expected hepatotoxicity. In terms of the
maximum tolerated dose in humans, the new thiazolidinediones 17
and 18 showed more than eightfold of the reference compound
(≥0.700 compared with 0.081), which means the advantage of the
wide therapeutic index of the new derivative. Additionally, our de-
signed thiazolidinediones 12, 16, 17, and 18 revealed better toler-
ability (−0.30 to 0.70) compared with 0.08 for the reference
marketed anticancer agent. Finally, oral acute toxic doses of the new
compounds (LD50) are almost the same as that of the reference drug
(~2.35 for our new thiazolidinediones compared with 2.40 of
doxorubicin).
The data obtained from the VEGFR‐2 inhibition assay concluded
that, generally, compounds with 2,4‐dichlorobenzylidene moiety ex-
hibited higher VEGFR‐2 inhibition activities than those having
4‐chlorobenzylidene moiety. The distal phenyl moiety substituted
with ethyl ester groups either in 2,4‐dichlorobenzylidene or
4‐chlorobenzylidene derivatives as in compounds 18 and 12, respec-
tively, displayed the highest activities at the same IC50 value of
0.17 0.02 µM. Compounds with distal phenyl moieties such as 10–12
and 16–18 showed higher activities than those having distal aliphatic
ones 7–9 and 13–15, respectively. 2,4‐Dichlorobenzylidene with to-
luene 17 and/or benzylidene 16 distal moieties displayed the same
activity at IC50 value = 0.18 0.02 µM while toluene derivative 11 in
4‐chlorobenzylidene derivatives exhibited higher activity than the
benzylidene one 10. In 2,4‐dichlorobenzylidene derivatives, the aliphatic
butyl distal moiety 15 displayed higher activity than propyl 14 and ethyl
13 one, respectively. Furthermore, in 4‐chlorobenzylidene derivatives
the aliphatic butyl distal moiety 9 displayed the same activity as propyl
8, which was higher than that of ethyl 7 one.
|
2.5
Pharmacokinetic profiling study
In the present study, a computational study of the four most active
compounds (12, 16, 17, and 18) as representative TZDs was con-
ducted to determine the surface area and other physicochemical
properties according to the directions of Lipinski's rule.[45] Lipinski

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TABLE 2 ADMET profile of the four
most active compounds and doxorubicin
Parameter
12
16
17
18
Doxorubicin
Molecular properties
Molecular weight
LogP
564.019 526.401 540.428 598.464
543.525
5.444
5.9207
6.22912
6.0974
0.0013
Rotatable bonds
Acceptors
8
7
2
6
5
2
6
5
2
8
7
2
5
12
Donors
6
Surface area
232.707 214.640 221.005 243.010
222.081
Absorption
Water solubility
−4.376
0.462
84.982
−2.735
Yes
−3.489
0.659
95.074
−2.735
Yes
−3.494
0.56
−3.607
0.51
−2.915
0.457
62.372
−2.735
Yes
Caco‐2 permeability
Intestinal abs. (human)
Skin permeability
95.551
−2.735
Yes
90.603
−2.735
Yes
P‐glycoprotein substrate
P‐glycoprotein I inhibitor
P‐glycoprotein II inhibitor
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
No
Distribution
VDss (human)
0.051
0.035
0.09
0.124
0.12
0.174
0.108
1.647
0.215
Fraction unbound (human)
BBB permeability
CNS permeability
0.127
−1.726
−2.808
−1.503
−1.725
−1.527
−1.649
−1.911
−2.637
−1.379
−4.307
Metabolism
CYP2D6 substrate
CYP3A4 substrate
CYP1A2 inhibitor
CYP2C19 inhibitor
CYP2C9 inhibitor
CYP2D6 inhibitor
CYP3A4 inhibitor
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
No
No
Yes
No
No
No
No
No
No
No
No
Yes
Yes
No
Yes
Yes
No
Yes
Yes
Yes
Yes
Excretion
Total clearance
Renal OCT2 substrate
−0.530
−0.536
−0.592
−0.538
0.987
No
No
No
No
No
Toxicity
AMES toxicity
No
0.306
No
No
0.797
No
No
0.700
No
No
0.63
No
No
0.081
No
Max. tolerated dose (human)
hERG I inhibitor
hERG II inhibitor
Yes
Yes
Yes
Yes
Yes
Oral rat acute toxicity (LD50
Oral rat chronic toxicity (LOAEL)
Hepatotoxicity
)
2.272
1.78
Yes
2.21
1.278
Yes
2.236
1.167
Yes
2.308
0.774
No
2.408
3.339
Yes
Skin sensitization
No
No
No
No
No
Tetrahymena pyriformis toxicity
Minnow toxicity
0.292
0.524
0.319
1.702
0.322
1.57
0.298
1.583
0.285
4.412
Abbreviation: ADMET, absorption, distribution, metabolism, excretion, and toxicity.
|
2.6
Docking studies
VEGFR‐2, which reveals a large space bounded by a membrane‐
binding domain that serves as an entry channel for the substrate to
the active site (Figure 6). In addition, the affinity of any small mole-
cule can be considered as a unique tool in the field of drug design.
There is a relationship between the affinity of organic molecules and
free binding energy.[49–54] This relationship can contribute in the
prediction and interpretation of the activity of the organic
In the present work, all modeling experiments were performed using
Molsoft software. Each experiment used VEGFR‐2 downloaded from
the Brookhaven Protein Data Bank (PDB ID: 1YWN).[48]
The obtained results indicated that all studied ligands have si-
milar position and orientation inside the recognized binding site of

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compounds toward the specific target protein. The obtained results
of the free energy of binding (ΔG) explained that most of these
compounds had a good binding affinity toward the receptor and the
computed values reflected the overall trend (Table 3).
TABLE 3 The calculated ΔG (free energy of binding) and binding
affinities for the ligands (ΔG in kcal/mol)
Compound
ΔG (kcal/mol)
−84.62
Compound
ΔG (kcal/mol)
−92.92
7
14
The proposed binding mode of sorafenib revealed an affinity
value of −95.66 kcal/mol and four H‐bonds. The urea linker formed
one H‐bond with the key amino acid Glu883 (2.13 Å) through its NH
group and one H‐bond with Asp1044 (1.53 Å) through its carbonyl
group. The N‐methylpicolinamide moiety was stabilized by the for-
mation of two H‐bonds with Cys917, where the pyridine N atom
formed one H‐bond with the NH of Cys917 (2.46 Å) while its NH
group formed one H‐bond with the carbonyl of Cys917 (2.20 Å). The
N‐methylpicolinamide moiety occupied the hydrophobic groove
formed by Leu1033, Gly920, Lys918, Cys917, Phe916, Glu915,
Leu838, and Ala864. Moreover, the central phenyl ring occupied the
hydrophobic pocket formed by Cys1043, Leu1033, Val914, Val897,
and Lys866. Furthermore, the hydrophobic 3‐trifluromethyl‐4‐
chlorophenyl moiety attached to the urea linker occupied the hy-
drophobic pocket formed by Asp1044, Ile1042, His1024, Leu1017,
Ile890, and Leu887 (Figure 7). The urea linker played an important
role in the binding affinity toward the VEGFR‐2 enzyme, where it was
responsible for the higher binding affinity of sorafenib. These findings
encourage us to use different linkers resembling urea of sorafenib,
hoping to obtain potent VEGFR‐2 inhibitors.
8
−89.64
15
−96.25
9
−95.50
16
−96.37
10
11
12
13
−96.06
17
−98.76
−95.81
18
−101.17
−95.36
−101.14
−86.54
Sorafenib
Leu1033, Gly920, Lys918, Cys917, Phe916, Glu915, Ala864, and
Leu838. Moreover, the TZD moiety occupied the hydrophobic pocket
formed by Cys1043, Leu1033, Val914, Val897, and Lys866. The hy-
drophobic phenyl tail occupied the hydrophobic pocket formed by
Asp1044, Cys1043, Ile1042, Hie1024, Leu1017, Val897, Leu887,
Lys866, and Glu883. Furthermore, the distal phenyl ring occupied the
hydrophobic groove formed by Arg1025, Ile1023, Cys1022, Leu1017,
Ile890, and Ile886. In contrast, the distal ethyl group of the ester
moiety occupied the hydrophobic cleft formed by Cys1022, Lys1021,
Arg1020, and Hie889 (Figure 8). These interactions of compound 18
may explain its highest anticancer activity.
The proposed binding mode of compound 18 is virtually the same
as that of sorafenib, which revealed an affinity value of −101.17 kcal/
mol and six H‐bonds. The carbonyl group of the acetamide linker
formed one H‐bond with Asp1044 (2.36 Å), while its NH group formed
another H‐bond with Glu883 (1.76 Å). The carbonyl group at position‐
2 of TZD formed one H‐bond with Lys866 (2.71 Å). Moreover, the NH
group of the carboxamide linker formed one H‐bond with Asp1044
(2.61 Å). Furthermore, the carbonyl group of the distal ethyl ester
moiety formed two H‐bonds with Arg1025 (1.70 and 2.67 Å). The 2,4‐
dichlorophenyl moiety occupied the hydrophobic groove formed by
As planned, the proposed binding mode of compound 12 is vir-
tually the same as that of sorafenib, which revealed an affinity value
of −101.14 kcal/mol and five H‐bonds. The carbonyl group of the
acetamide linker formed one H‐bond with Asp1044 (2.42 Å) while its
NH group formed another H‐bond with Glu883 (1.74 Å). The car-
bonyl group at position‐2 of TZD formed one H‐bond with Lys866
(2.75 Å). Moreover, the NH group of the carboxamide linker formed
one H‐bond with Asp1044 (2.74 Å). Furthermore, the carbonyl group
of the distal ethyl ester moiety formed one H‐bond with Arg1025
FIGURE 6 Superimposition of some
docked compounds inside the binding pocket
of 1YWN

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FIGURE 7 Predicted binding mode for
sorafenib with 1WYN. H‐bonded atoms are
indicated by dotted lines
(1.92 Å). The 4‐chlorophenyl moiety occupied the hydrophobic
groove formed by Leu1033, Gly920, Lys918, Cys917, Phe916,
Glu915, Ala864, and Leu838. Moreover, the TZD moiety occupied
the hydrophobic pocket formed by Cys1043, Leu1033, Val914,
Val897, and Lys866. The hydrophobic phenyl tail occupied the hy-
drophobic pocket formed by Asp1044, Cys1043, Ile1042, Hie1024,
Leu1017, Val897, Leu887, Lys866, and Glu883. Furthermore, the
distal phenyl ring occupied the hydrophobic groove formed by
Arg1025, Ile1023, Cys1022, Leu1017, Ile890, and Ile886. In contrast,
the distal ethyl group of the ester moiety occupied the hydrophobic
cleft formed by Cys1022, Lys1021, Arg1020, and Hie889 (Figure 9).
These interactions of compound 12 may explain its high anticancer
activity.
H‐bonds. The carbonyl group of the acetamide linker formed one H‐bond
with Asp1044 (2.15 Å), while its NH group formed another H‐bond with
Glu883 (1.88 Å). The carbonyl group at position‐2 of TZD formed one
H‐bond with Lys866 (2.71 Å). Moreover, the NH group of the carbox-
amide linker formed one H‐bond with Asp1044 (2.75 Å). The 2,4‐
dichlorophenyl moiety occupied the hydrophobic groove formed by
Leu1033, Gly920, Lys918, Cys917, Phe916, Glu915, Ala864, and Leu838.
Moreover, the TZD moiety occupied the hydrophobic pocket formed by
Cys1043, Leu1033, Val914, Val897, and Lys866. The hydrophobic phenyl
tail occupied the hydrophobic pocket formed by Asp1044, Cys1043,
Ile1042, Hie1024, Leu1017, Val897, Leu887, Lys866, and Glu883. Fur-
thermore, the distal methyl phenyl moiety occupied the hydrophobic
groove formed by Arg1025, Ile1023, Cys1022, Leu1017, Ile890, and
Ile886 (Figure 10). These interactions of compound 17 may explain its
high anticancer activity.
The proposed binding mode of compound 17 is virtually the same as
that of 18, which revealed an affinity value of −98.76 kcal/mol and four
FIGURE 8 Predicted binding mode for
compound 18 with 1WYN

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EL‐ADL ET AL.
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FIGURE 9 Predicted binding mode for
compound 12 with 1WYN
The proposed binding mode of compound 16 is virtually the same
as that of sorafenib and 18, which revealed an affinity value of
−96.37 kcal/mol and four H‐bonds. The carbonyl group of the acetamide
linker formed one H‐bond with Asp1044 (2.22 Å), while its NH group
formed another H‐bond with Glu883 (1.95 Å). The carbonyl group at
position‐2 of TZD formed one H‐bond with Lys866 (2.60 Å). Moreover,
the NH group of the carboxamide linker formed one H‐bond with
Asp1044 (2.74 Å). The 2,4‐dichlorophenyl moiety occupied the hydro-
phobic groove formed by Leu1033, Gly920, Lys918, Cys917, Phe916,
Glu915, Ala864, and Leu838. Moreover, the TZD moiety occupied the
hydrophobic pocket formed by Cys1043, Leu1033, Val914, Val897, and
Lys866. The hydrophobic phenyl tail occupied the hydrophobic pocket
formed by Asp1044, Cys1043, Ile1042, Hie1024, Leu1017, Val897,
Leu887, Lys866, and Glu883. Furthermore, the distal benzyl moiety
occupied the hydrophobic groove formed by Arg1025, Ile1023,
Cys1022, Leu1017, Ile890, and Ile886 (Figure 11). These interactions of
compound 16 may explain its high anticancer activity.
From the obtained docking results (Table 1), we concluded that
the acetamide linker occupied the same groove occupied by the urea
linker of sorafenib and played the same role, which is essential for
higher affinity toward VEGFR‐2 enzyme. The lipophilicity of 2,
4‐dichlorobenzylidene and 4‐chlorobenzylidene moieties increased
the hydrophobic interactions and consequently, affinities toward the
VEGFR‐2 enzyme. The TZD enables the new compounds to form new
H‐bond through its carbonyl group at position‐2 with the essential
amino acid Asp1044. Elongation of the structure played an important
role in their VEGFR‐2 inhibitory activities. The hydrophobic distal
moiety and its linker which formed hydrogen bonding interactions
with the amino acid Arg1025 and/or Arg1044, respectively also in-
creased affinity toward VEGFR‐2 enzyme.
FIGURE 10 Predicted binding mode for
compound 17 with 1WYN

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EL‐ADL ET AL.
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FIGURE 11 Predicted binding mode for
compound 16 with 1WYN
|
3
CONCLUSION
performed to investigate the binding mode of the proposed compounds
with the VEGFR‐2 receptor. The data obtained from the docking studies
were highly correlated with that obtained from the biological screening.
The molecular design proved that the acetamide linker occupied the
same groove occupied by the urea linker of sorafenib and played the
same role, which is essential for a higher affinity toward VEGFR‐2
enzyme. The lipophilicity of 2,4‐dichlorobenzylidene and 4‐
chlorobenzylidene moieties increased the hydrophobic interactions
and, consequently, affinities toward the VEGFR‐2 enzyme. The TZD
enables the new compounds to form new H‐bond through its carbonyl
group at position‐2 with the essential amino acid Asp1044. Elongation
of the structure played an important role in their VEGFR‐2 inhibitory
activities. The hydrophobic distal moiety and its linker, which formed
hydrogen bonding interactions with the amino acid Arg1025 and/or
Arg1044, respectively increased the affinity toward the VEGFR‐2 en-
zyme. Furthermore, the ADMET profile was calculated for the four most
active compounds in comparison with doxorubicin as a reference drug.
Novel series of 5‐([4‐chloro/2,4‐chloro]benzylidene)thiazolidine‐2,4‐
dione derivatives 7–18 were designed, synthesized, and evaluated for
their anticancer activity against three human tumor cell lines hepato-
cellular carcinoma (HepG2), colorectal carcinoma (HCT‐116), and breast
cancer (MCF‐7) targeting VEGFR‐2 enzyme. All the tested compounds
showed variable anticancer activities. MCF‐7 was the most sensitive cell
line to the influence of the new derivatives. In particular, compounds 18,
12, 17, and 16 were found to be the most potent derivatives over all the
tested compounds against the HepG2, HCT116, and MCF‐7 cancer cell
lines with IC50 = 9.16 0.9, 8.98 0.7, 5.49 0.5 µM; 9.19 0.5,
8.40 0.7, 6.10 0.4 µM; 10.78 1.2, 8.87 1.5, 7.08 1.6 µM; and
10.87 0.8, 9.05 0.7, 7.32 0.4 µM, respectively. Compounds 18 and
12 have nearly the same activities as sorafenib (IC50 = 9.18 0.6,
5.47 0.3, and 7.26 0.3 µM, respectively) against HepG2 cells, but
slightly lower activity against HCT116 and slightly higher activity
against MCF‐7 cancer cell lines, respectively. Also, these compounds
displayed lower activities than doxorubicin against HepG2 and HCT‐
116 cell lines but higher activity against MCF‐7 cell line, respectively
(IC50 = 7.94 0.6, 8.07 0.8, and 6.75 0.4 µM, respectively). However,
compounds 17 and 16 exhibited lower activities than sorafenib, against
HepG2 and HCT116, but nearly equipotent activity against MCF‐7
cancer cell lines, respectively. Also, these compounds displayed lower
activities than doxorubicin against the three cell lines. All the synthe-
sized derivatives 7–18 were evaluated for their inhibitory activities
against VEGFR‐2. The tested compounds displayed high to medium
inhibitory activity with IC50 values ranging from 0.17 0.02 to
0.27 0.03 µM. Among them, compounds 18, 12, 17, and 16 potently
inhibited VEGFR‐2 at IC50 values of 0.17 0.02, 0.17 0.02, 0.18 0.02,
and 0.18 0.02 µM, respectively, which are nearly more than the half of
that of sorafenib IC50 value (0.10 0.02 µM). The molecular design was
|
4
EXPERIMENTAL
|
4.1
Chemistry
|
4.1.1
General
All melting points were carried out by open capillary method on a Gal-
lenkamp Melting Point Apparatus at Faculty of Pharmacy, Al‐Azhar
University and were uncorrected. The infrared spectra were recorded on
Pye Unicam SP1000 IR spectrophotometer at Microanalytical Unit, Fa-
culty of Pharmacy, Cairo University using the potassium bromide disc
technique. Proton magnetic resonance (1H NMR) spectra were recorded
on a Bruker 400 MHz NMR spectrophotometer at Microanalytical Unit,

14 of 17
EL‐ADL ET AL.
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Faculty of Pharmacy, Cairo University. 13C NMR spectra were recorded
on a Bruker 100 MHz NMR spectrophotometer at Microanalytical Unit,
Faculty of Pharmacy, Cairo University. Tetramethylsilane was used as
internal standard and chemical shifts were measured in δ scale (ppm). The
mass spectra were carried out on Direct Probe Controller Inlet part to
Single Quadropole Mass Analyzer in Thermo Scientific GCMS Model ISQ
LT using Thermo X‐Calibur software at the Regional Center for Mycology
and Biotechnology, Al‐Azhar University. Elemental analyses (C, H, N)
were performed on a CHN Analyzer at Regional Center for Mycology
and Biotechnology, Al‐Azhar University. All compounds were within 0.4
of the theoretical values. The reactions were monitored by thin‐layer
chromatography (TLC) using TLC sheets precoated with UV fluorescent
silica gel Merck 60 F254 plates and were visualized using a UV lamp and
different solvents as mobile phases.
(400 MHz, DMSO‐d6): 0.85 (t, 3H, –NHCH2CH2CH3), 1.46 (m, 2H,
–NHCH2CH2CH3), 3.17 (t, 2H, –CH2CH2CH3), 4.56 (s, 2H, –NCH2CO),
7.62 (m, 4H, Ar‐H of –C6H4Cl), 7.69 (d, 2H, Ar‐H H‐2 and H‐6 of
–NHC6H4), 7.82 (d, 2H, Ar‐H, H‐3 and H‐5 of –NHC6H4), 8.01 (s, 1H,
C═CH‐Ph), 8.36 (t, 1H, –NHCH2CH2CH3, D2O exchangeable), 10.63
(s, 1H, NH‐benzamide, D2O exchangeable); anal. calcd. for
C22H20ClN3O4S (457.93): C, 57.70; H, 4.40; N, 9.18. Found: C, 58.02; H,
4.73; N, 9.45.
N‐Butyl‐4‐{2‐[5‐(4‐chlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]
acetamido}benzamide (9)
Yield, 75%; m.p. 247–249°C; IRνmax (cm−1): 3,290, 3,120 (2 NH), 3,012
(CH aromatic), 2,908 (CH aliphatic), 1,743, 1,678, 1,635 (4C═O); 1H
NMR (400 MHz, DMSO‐d6): 0.92 (t, 3H, –CH2CH2CH2CH3), 1.28
(m, 2H, –CH2CH2CH2CH3), 1.46 (m, 2H, –CH2CH2CH2CH3), 3.23 (t,
2H, –CH2CH2CH2CH3), 4.55 (s, 2H, –NCH2CO), 7.63 (m, 4H, Ar‐H of
–C6H4Cl), 7.69 (d, 2H, Ar‐H, H‐2 and H‐6 of –NHC6H4), 7.81 (d, 2H,
Ar‐H, H‐3 and H‐5 of –NHC6H4), 8.01 (s, 1H, C═CH‐Ph), 8.34 (s, 1H,
–NHCH2CH3, D2O exchangeable), 10.64 (s, 1H, NH‐benzamide, D2O
exchangeable); anal. calcd. for C23H22ClN3O4S (471.96): C, 58.53; H,
4.70; N, 8.90. Found: C, 58.76; H, 4.89; N, 8.78.
The original spectra of the investigated compounds are provided
as Supporting Information. The InChI codes of the investigated
compounds, together with some biological activity data, are also
provided as Supporting Information.
TZD (1), 5‐(2,4‐dichlorobenzylidene)thiazolidine‐2,4‐dione,
and 5‐(4‐chlorobenzylidene)‐thiazolidine‐2,4‐dione (2a,b), the
corresponding potassium salts (3a,b), 4‐(2‐chloroacetamido)‐N‐
substitutedbenzamide derivative 6a–f were obtained according
to the reported procedures.[40,41,55–57]
N‐Benzyl‐4‐{2‐[5‐(4‐chlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]
acetamido}benzamide (10)
Yield, 60%; m.p. 260–261°C; IRνmax (cm−1): 3,282, 3,190 (2 NH), 3,039
(CH aromatic), 2,920 (CH aliphatic), 1,747, 1,681, 1,647 (4C═O); 1H NMR
(400 MHz, DMSO‐d6): 4.46 (s, 2H, –CH2Ph), 4.57 (s, 2H, –NCH2CO),
7.23–7.36 (m, 5H, Ar‐H, –C6H5), 7.55 (d, 2H, Ar‐H, H‐3 and H‐5 of
–C6H4Cl), 7.62 (d, 2H, Ar‐H, H‐2 and H‐6 of –C6H4Cl), 7.69 (d, 2H, Ar‐H,
H‐2 and H‐6 of –C6H4), 7.88 (d, 2H, Ar‐H, H‐3 and H‐5 of –C6H4), 8.02
(s, 1H, C═CH‐Ph), 8.96 (s, 1H, –NHCH2Ph, D2O exchangeable), 10.65
(s, 1H, –NH‐benzamide, D2O exchangeable); anal. calcd. for
C26H20ClN3O4S (505.97): C, 61.72; H, 3.98; N, 8.30. Found: C, 61.54; H,
4.07; N, 8.57.
|
4.1.2
General method for the synthesis of 4‐{2‐[5‐
(4‐chlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐
acetamido}‐N‐substitutedbenzamides 7–18
Equimolar quantities of the appropriate potassium salt 3a,b (0.01 mol)
and the appropriate 4‐(2‐chloroacetamido)‐N‐substitutedbenzamide
derivative 6a–f (0.01 mol) in dimethylformamide (DMF; 20 ml) were
heated on the water bath for 5 hr. The reaction mixture was poured
onto ice‐water (200 ml) and stirred for 30 min. The obtained solid was
filtered, washed with water, dried, and crystallized from ethanol to
give the corresponding target compounds, 7–18, respectively.
4‐{2‐[5‐(2,4‐Chlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]
acetamido}‐N‐(p‐tolyl)benzamide (11)
4‐{2‐[5‐(4‐Chlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]acetamido}‐
N‐ethylbenzamide (7)
Yield, 60%; m.p. 265–266°C; IRνmax (cm−1): 3,290, 3,190 (2 NH), 3,051
(CH aromatic), 2,935 (CH aliphatic), 1,743, 1,681, 1,635 (4C═O); 1H NMR
(400 MHz, DMSO‐d6): 2.28 (s, 3H, CH3), 4.59 (s, 2H, –NCH2CO), 7.14
(d, 2H, Ar‐H, H‐3 and H‐5 of –C6H4CH3), 7.61–7.71 (m, 8H, Ar‐H), 7.95
(d, 2H, Ar‐H, H‐3 and H‐5 of –C6H4CONH), 8.01 (s, 1H, C═CH‐Ph), 10.08
(s, 1H, NH, –NHC6H4CH3, D2O exchangeable), 10.71 (s, 1H, NH, –NH‐
benzamide, D2O exchangeable); anal. calcd. for C26H20ClN3O4S (505.97):
C, 61.72; H, 3.98; N, 8.30. Found: C, 61.50; H, 4.16; N, 8.37.
Yield, 85%; m.p. 242–244°C; IRνmax (cm−1): 3,240, 3,120 (2 NH), 3,012
(CH aromatic), 2,908 (CH aliphatic), 1,732, 1,685, 1,647 (4C═O); 1H NMR
(400 MHz, dimethyl sulfoxide [DMSO]‐d6): 1.11 (t, 3H, –NHCH2CH3),
3.27 (q, 2H, –NHCH2CH3), 4.56 (s, 2H, –NCH2CO), 7.64 (d, 2H, Ar‐H, H‐3,
H‐5 of –C6H4Cl), 7.69–7.81 (m, 4H, Ar‐H), 7.82 (d, 2H, Ar‐H, H‐3 and H‐5
of –NHC6H4), 8.01 (s, 1H, C═CH‐Ph), 8.36 (t, 1H, –NHCH2CH3, D2O
exchangeable), 10.63 (s, 1H, NH‐benzamide, D2O exchangeable); anal.
calcd. for C21H18ClN3O4S (443.90): C, 56.82; H, 4.09; N, 9.47. Found: C,
56.71; H, 4.23; N, 9.68.
Ethyl 4‐(4‐{2‐[5‐(4‐chlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]
acetamido}benzamido)benzoate (12)
Yield, 62%; m.p. 275–276°C; IRνmax (cm−1): 3,194, 3,109 (2 NH), 3,062
(CH aromatic), 2,939 (CH aliphatic), 1,739, 1,681, 1,631 (5C═O); 1H
NMR (400 MHz, DMSO‐d6): 1.30 (t, 3H, –COOCH2CH3), 4.27 (q, 2H,
–COOCH2CH3), 4.59 (s, 2H, –NCH2CO), 7.63 (d, 2H, Ar‐H, H‐3 and H‐5
of –C6H4Cl), 7.69 (d, 2H, Ar‐H, H‐2 and H‐6 of –C6H4Cl), 7.71 (d, 2H,
4‐{2‐[5‐(4‐Chlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]acetamido}‐
N‐propylbenzamide (8)
Yield, 85%; m.p. 245–247°C; IRνmax (cm−1): 3,282, 3,194 (2 NH), 3,059
(CH aromatic), 2,870 (CH aliphatic), 1,747, 1,678, 1,631 (4C═O); 1H NMR

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Ar‐H, H‐2 and H‐6 of –C6H4), 7.95–8.00 (m, 6H, Ar‐H), 8.02 (s, 1H,
C═CH‐Ph), 10.47 (s, 1H, NH, –C6H4CONH, D2O exchangeable); 13C
NMR (100 MHz, DMSO‐d6): 14.69, 44.66, 60.92, 118.95, 119.99 (2),
122.16, 124.92, 129.46 (2), 129.78, 129.99 (2), 130.51 (3), 132.22 (2),
142.02, 132.33 (2), 132.89, 135.94, 144.16, 164.78, 165.65, 165.85,
167.61; anal. calcd. for C28H22ClN3O6S (564.01): C, 59.63; H, 3.93; N,
7.45. Found: C, 59.89; H, 4.12; N, 7.53.
133.61, 133.68, 140.35, 141.42, 163.92, 164.55, 166.12; anal. calcd. for
26H19Cl2N3O4S (540.42): C, 57.79; H, 3.54; N, 7.78. Found: C, 58.02; H,
C
3.71; N, 7.94.
4‐{2‐[5‐(2,4‐Dichlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]
acetamido}‐N‐(p‐tolyl)benzamide (17)
Yield, 65%; m.p. 270–272°C; IR (KBr) νmax 3,309, 3,152, 3,063, 2,928,
1,728, 1,687, 1,666 cm−1 1H NMR (DMSO, 400 MHz): δ = 2.28 (s, 3H,
;
4‐{2‐[5‐(2,4‐Dichlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]
acetamido}‐N‐ethylbenzamide (13)
CH3), 4.58 (s, 2H, –NCH2CO), 7.12–7.16 (m, 4H, Ar‐H, –C6H4CH3),
7.62–7.67 (m, 4H, Ar‐H, –C6H4), 7.71 (m, 2H, Ar‐H, H‐5 of –C6H3 and
C═CH‐Ph), 7.95–7.99 (m, 2H, H‐3 and H‐6 of –C6H3), 10.08 (s, 1H, NH,
–NHC6H4CH3, D2O exchangeable), 10.71 (s, 1H, NH, –NHC6H4CO, D2O
exchangeable); anal. calcd. for C26H19Cl2N3O4S (540.42): C, 57.79; H,
3.54; N, 7.78. Found: C, 57.96; H, 3.48; N, 8.12.
Yield, 80%; m.p. 243–245°C; IR (KBr) νmax: 3,279, 3,066, 2,936, 1,751,
1,697 cm−1; 1H NMR (DMSO, 400 MHz): δ = 1.23 (t, 3H, –CH2CH3), 3.67
(q, 2H, –CH2CH3), 4.47 (s, 2H, –NCH2CO), 7.24 (d, 1H, Ar‐H, H‐5 of
–C6H3), 7.39–7.49 (m, 2H, Ar‐H, H‐2 and H‐6 of –C6H3), 7.54–7.65
(m, 2H, Ar‐H, H‐2 and H‐6 of –C6H4), 7.95–8.04 (m, 2H, Ar‐H, H‐3 and H‐
5 of –C6H4), 8.11 (s, 1H, C═CH‐Ph), 9.01 (s, 1H, –NHCH2CH3, D2O
exchangeable), 10.68 (s, 1H, NH‐C6H4, D2O exchangeable); 13C NMR
(100 MHz, DMSO‐d6): 14.18, 44.89, 46.73, 111.51, 115.41, 118.29,
123.70, 126.93 (2), 127.81, 128.61, 129.19 (2), 132.97, 136.06, 136.58,
140.05, 142.96, 161.46, 166.55, 167.37; anal. calcd. for C21H17Cl2N3O4S
(478.34): C, 52.73; H, 3.58; N, 8.78. Found: C, 52.59; H, 3.74; N, 9.07.
Ethyl 4‐(4‐{2‐[5‐(2,4‐dichlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]
acetamido}benzamido)benzoate (18)
Yield, 60%; m.p. 280–282°C; IR (KBr) νmax 3,233, 3,013, 2,939, 1,736,
;
1,686, 1,652 cm−1 13C NMR (DMSO, 100 MHz): δ = 14.70, 44.63, 60.91,
118.94, 119.98 (2), 121.40, 124.91, 129.46 (2), 129.77, 129.92 (2), 130.51
(3), 130.70 (2), 131.33, 133.32, 134.20, 142.04, 144.18, 164.83, 165.65,
165.76, 165.84, 167.61; electron ionization mass spectroscopy (EIMS)
m/z 602 [M++4] (0.88), 600 [M++2] (5.13), 598 [M+] (7.38), 414 (28.29),
120 (100), 80 (10.49); anal. calcd. for C28H21Cl2N3O6S (598.45): C, 56.20;
H, 3.54; N, 7.02. Found: C, 56.33; H, 3.70; N, 7.25.
4‐{2‐[5‐(2,4‐Dichlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]
acetamido}‐N‐propylbenzamide (14)
Yield, 80%; m.p. 246–248°C; IR (KBr) νmax 3,298, 3,059, 2,928,
1,740, 1,686, 1,670 cm−1; 1H NMR (DMSO, 400 MHz): δ = 0.89 (t, 3H,
–CH2CH2CH3), 1.50–1.56 (m, 2H, –CH2CH2CH3), 3.22 (t, 2H,
–CH2CH2CH3), 4.56 (s, 2H, –NCH2CO), 7.36 (d, 1H, Ar‐H, H‐5 of
–C6H3), 7.44–7.51 (m, 2H, Ar‐H, H‐3 and H‐6 of –C6H3), 7.53–7.59
(m, 2H, Ar‐H, H‐2 and H‐6 of –C6H4), 7.63–7.66 (m, 2H, Ar‐H, H‐3
and H‐5 of –C6H4), 7.82 (s, 1H, C═CH‐Ph), 8.37 (s, 1H,
–NHCH2CH2CH3, D2O exchangeable), 10.69 (s, 1H, NH‐C6H4, D2O
exchangeable); anal. calcd. for C22H19Cl2N3O4S (492.37): C, 53.67; H,
3.89; N, 8.53. Found: C, 53.49; H, 4.12; N, 8.70.
|
4.2
Biological assays
|
4.2.1
In vitro cytotoxic activity
Cancer cells from different cancer cell lines, such as hepatocellular
carcinoma (HepG2), breast cancer (MCF‐7), and colorectal carcinoma
(HCT‐116), were purchased from American Type Cell Culture Col-
lection (ATCC, Manassas) and grown on the appropriate growth
medium, Roswell Park Memorial Institute medium (RPMI 1640)
supplemented with 100 mg/ml of streptomycin, 100 units/ml of pe-
nicillin, and 10% of heat‐inactivated fetal bovine serum in a humidi-
fied, 5% (v/v) CO2 atmosphere at 37°C cytotoxicity assay by MTT.
Exponentially growing cells from different cancer cell lines were
trypsinized, counted, and seeded at the appropriate densities
(2,000–1,000 cells/0.33‐cm2 well) into 96‐well microtiter plates. Cells
were then incubated in a humidified atmosphere at 37°C for 24 hr.
Then, the cells were exposed to different concentrations of com-
pounds (0.1, 10, 100, and 1,000 µM) for 72 hr. Then, the viability of
treated cells was determined using MTT technique as follows. Cells
were incubated with 200 μl of 5% MTT solution/well (Sigma‐Aldrich,
MO) and were allowed to metabolize the dye into colored insoluble
formazan crystals for 2 hr. The remaining MTT solution was dis-
carded from the wells and the formazan crystals were dissolved in
200 µl/well acidified isopropanol for 30 min, covered with aluminum
foil and with continuous shaking using a MaxQ 2000 Plate Shaker
(Thermo Fisher Scientific Inc., MI) at room temperature. Absorbance
N‐Butyl‐4‐{2‐[5‐(2,4‐dichlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]
acetamido}benzamide (15)
Yield, 70%; m.p. 248–249°C; IR (KBr) νmax 3,267, 3,059, 2,932, 1,748,
;
1,693, 1,659 cm−1 1H NMR (DMSO, 400 MHz): δ = 0.88 (t, 3H,
–CH2CH2CH2CH3), 1.33 (m, 2H, –CH2CH2CH2CH3), 1.53 (m, 2H,
–CH2CH2CH2CH3), 3.24 (t, 2H, –CH2CH2CH2CH3), 4.59 (s, 2H,
–NCH2CO), 7.53 (m, 3H, Ar‐H, H‐3, H‐5 and H‐6 of –C6H3), 7.67 (m, 2H,
Ar‐H, H‐2 and H‐6 of –C6H4), 7.85 (m, 2H, Ar‐H, H‐3 and H‐5 of –C6H4),
7.97 (s, 1H, C═CH‐Ph), 8.40 (s, 1H, –NHCH2CH2CH3, D2O exchange-
able), 11.16 (s, 1H, NH‐C6H4, D2O exchangeable); anal. calcd. for
C23H21Cl2N3O4S (506.40): C, 54.55; H, 4.18; N, 8.30. Found: C, 54.83; H,
4.29; N, 8.59.
N‐Benzyl‐4‐{2‐[5‐(2,4‐dichlorobenzylidene)‐2,4‐dioxothiazolidin‐
3‐yl]acetamido}benzamide (16)
Yield, 65%; m.p. 262–264°C; IR (KBr) νmax 3,240, 3,055, 2,939, 1,739,
;
1,694 cm−1 13C NMR (DMSO, 100 MHz): δ = 42.99, 44.83, 118.86,
127.12 (3), 127.72 (4), 128.68 (5), 129.03, 129.35, 129.67, 133.50,

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EL‐ADL ET AL.
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was measured at 570 nm using a Stat FaxR 4200 Plate Reader
(Awareness Technology Inc., FL). The cell viability was expressed as a
percentage of control and the concentration that induces 50% of
maximum inhibition of cell proliferation (IC50) was determined using
Bank Directorate Manager, Ministry of Health, Cairo, Egypt for
helping in the pharmacological part.
CONFLICTS OF INTERESTS
GraphPad Prism version
CA).[42–44]
5
software (GraphPad Software Inc.,
The authors declare that there are no conflicts of interests.
ORCID
Khaled El‐Adl
http://orcid.org/0000-0002-8922-9770
|
4.2.2
In vitro VEGFR‐2 kinase assay
Hamada S. Abulkhair
http://orcid.org/0000-0001-6479-4573
The kinase activity of VEGFR‐2 was measured using an antipho-
sphotyrosine antibody with the AlphaScreen system (PerkinElmer) ac-
cording to the manufacturer's instructions.[58] Enzyme reactions were
performed in 50 mM Tris–HCl pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.01%
Tween‐20, and 2 mM dithiothreitol, containing 10 μM ATP, 0.1 μg/ml
biotinylated poly‐GluTyr (4:1), and 0.1 nM of VEGFR‐2 (Millipore, UK).
Before catalytic initiation with ATP, the tested compounds at final con-
centrations ranging from 0 to 300 μg/ml and enzyme were incubated for
5 min at room temperature. The reactions were quenched by the addition
of 25 μl of 100 mM EDTA, 10 μg/ml AlphaScreen streptavidin donor
beads and 10 μg/ml acceptor beads in 62.5 mM HEPES pH 7.4, 250 mM
NaCl, and 0.1% bovine serum albumin. The plate was incubated in the
dark overnight and then read by ELISA Reader (PerkinElmer). Wells
containing the substrate and the enzyme without compounds were used
as reaction control. Wells containing biotinylated poly‐GluTyr (4:1) and
enzyme without ATP were used as basal control. Percent inhibition was
calculated by the comparison of compounds treated to control incuba-
tions. The concentration of the test compound causing 50% inhibition
(IC50) was calculated from the concentration–inhibition response curve
(triplicate determinations) and the data were compared with sorafenib
(Sigma‐Aldrich) as standard VEGFR‐2 inhibitor.
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