5-(4-Methoxybenzylidene)thiazolidine-2,4-dione-derived VEGFR-2 inhibitors: Design, synthesis, molecular docking, and anticancer evaluations

Article abstract of DOI:10.1002/ardp.202000079

A novel series of 5-(4-methoxybenzylidene)thiazolidine-2,4-dione derivatives, 5a–g and 7a–f, was designed, synthesized, and evaluated for their anticancer activity against HepG2, HCT116, and MCF-7 cells. HepG2 and HCT116 were the most sensitive cell lines to the influence of the new derivatives. In particular, compounds 7f, 7e, 7d, and 7c were found to be the most potent derivatives of all the tested compounds against the HepG2, HCT116, and MCF-7 cancer cell lines. Compound 7f (IC₅₀ = 6.19 ± 0.5, 5.47 ± 0.3, and 7.26 ± 0.3 μM, respectively) exhibited a higher activity than sorafenib (IC₅₀ = 9.18 ± 0.6, 8.37 ± 0.7, and 5.10 ± 0.4 μM, respectively) against HepG2 and MCF-7, cells but a lower activity against HCT116 cancer cells, respectively. Also, this compound displayed a higher activity than doxorubicin (IC₅₀ = 7.94 ± 0.6, 8.07 ± 0.8, and 6.75 ± 0.4 μM, respectively) against HepG2 and MCF-7 cells, but nearly the same activity against HCT116 cells, respectively. All derivatives, 5a–g and 7a–f, were evaluated for their inhibitory activities against vascular endothelial growth factor receptor-2 (VEGFR-2). Among them, compound 7f was found to be the most potent derivative that inhibited VEGFR-2 at an IC₅₀ value of 0.12 ± 0.02 μM, which is nearly the same as that of sorafenib (IC₅₀ = 0.10 ± 0.02 μM). Compounds 7e, 7d, 7c, and 7b exhibited the highest activity, with IC₅₀ values of 0.13 ± 0.02, 0.14 ± 0.02, 0.14 ± 0.02, and 0.18 ± 0.03 μM, respectively, which are more than the half of that of sorafenib. Furthermore, molecular docking was performed to investigate their binding mode and affinities toward the VEGFR-2 receptor. The data obtained from the docking studies highly correlated with those obtained from the biological screening.

Full text of DOI:10.1002/ardp.202000079

Received: 17 March 2020
Revised: 11 May 2020
Accepted: 14 May 2020
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DOI: 10.1002/ardp.202000079
F U L L P A P E R
5‐(4‐Methoxybenzylidene)thiazolidine‐2,4‐dione‐derived
VEGFR‐2 inhibitors: Design, synthesis, molecular docking,
and anticancer evaluations
Khaled El‐Adl1,2
| Helmy Sakr1 | Mohamed Nasser1 | Mohamed Alswah3
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Fatma M. A. Shoman4
1Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Al‐Azhar University,
Cairo, Egypt
Abstract
A novel series of 5‐(4‐methoxybenzylidene)thiazolidine‐2,4‐dione derivatives, 5a–g and
7a–f, was designed, synthesized, and evaluated for their anticancer activity against
HepG2, HCT116, and MCF‐7 cells. HepG2 and HCT116 were the most sensitive cell
lines to the influence of the new derivatives. In particular, compounds 7f, 7e, 7d, and 7c
were found to be the most potent derivatives of all the tested compounds against the
HepG2, HCT116, and MCF‐7 cancer cell lines. Compound 7f (IC50 = 6.19 0.5,
5.47 0.3, and 7.26 0.3 µM, respectively) exhibited a higher activity than sorafenib
(IC50 = 9.18 0.6, 8.37 0.7, and 5.10 0.4 µM, respectively) against HepG2 and
MCF‐7, cells but a lower activity against HCT116 cancer cells, respectively. Also, this
compound displayed a higher activity than doxorubicin (IC50 = 7.94 0.6, 8.07 0.8,
and 6.75 0.4 µM, respectively) against HepG2 and MCF‐7 cells, but nearly the same
activity against HCT116 cells, respectively. All derivatives, 5a–g and 7a–f, were eval-
uated for their inhibitory activities against vascular endothelial growth factor receptor‐
2 (VEGFR‐2). Among them, compound 7f was found to be the most potent derivative
that inhibited VEGFR‐2 at an IC50 value of 0.12 0.02 µM, which is nearly the same as
that of sorafenib (IC50 = 0.10 0.02 µM). Compounds 7e, 7d, 7c, and 7b exhibited the
highest activity, with IC50 values of 0.13 0.02, 0.14 0.02, 0.14 0.02, and
0.18 0.03 µM, respectively, which are more than the half of that of sorafenib. Fur-
thermore, molecular docking was performed to investigate their binding mode and
affinities toward the VEGFR‐2 receptor. The data obtained from the docking studies
highly correlated with those obtained from the biological screening.
2Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Heliopolis University for
Sustainable Development, Cairo, Egypt
3Pharmaceutical Organic Chemistry
Department, Faculty of Pharmacy,
Al‐Azhar University, Cairo, Egypt
4Department of Clinical Pathology, Blood Bank
Specialist, Blood Bank Directorate, Ministry of
Health, Al‐Azhar University, Cairo, Egypt
Correspondence
Khaled El‐Adl, Pharmaceutical Chemistry
Department, Faculty of Pharmacy, Al‐Azhar
University, 1 Al‐Mukhayam Al‐Daem Str.,
6th District, Nassr City, Cairo 11765, 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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1
INTRODUCTION
generated blood vessels supply oxygen and essential nutrition, sup-
port tumor growth, and later, aid in the initiation of metastasis, which
Recent developments in cancer therapies have focused on molecular
targets, particularly tumor angiogenesis.[1] Angiogenesis is the pro-
cess by which the existing vascular bed expands to form new blood
vessels and it is a pivotal event in many physiological and patholo-
gical processes, including tumor growth and metastasis.[2,3] Newly
contributes to more than 90% of deaths in various cancers.[4]
Vascular endothelial growth factor (VEGF) is one of the central
regulators in angiogenesis.[5] Vascular endothelial growth factor
receptor‐2 (VEGFR‐2) is the receptor for VEGF and is the prime
mediator of VEGF‐induced proangiogenesis signaling.[6] Binding of
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VEGF to VEGFR‐2 leads to dimerization of the receptors, activation
of tyrosine kinase, trans‐autophosphorylation, and initiation of the
extracellular signal‐regulated kinase.[7–9] Considering that angiogen-
esis is a significant event in tumor development, blocking angiogen-
esis is one of the most promising strategies to treat malignancies. The
thiazolidine‐2,4‐diones (TZDs) scaffold has recently been proposed
for hybridization with different bioactive moieties to have different
mechanisms of action with a broad spectrum of activity against nu-
merous cancer cell lines. TZDs have exhibited antitumor activity in a
wide variety of experimental cancer models by affecting cell cycle,
induction of cell differentiation, and apoptosis, as well as by inhibiting
tumor angiogenesis.[10,11] A study reported by Shah et al.[12] showed
that TZD derivative ciglitazone (I) (Figure 1) significantly 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
agents[13–18] and potent vascular endothelial growth factor receptor‐
2 (VEGFR‐2) inhibitors, for example, compound II.[2,12] Owing to the
importance of VEGFR‐2 in angiogenesis, this receptor is the most
vital target in antiangiogenic therapy against cancer. Numerous re-
ports on VEGFR‐2 inhibitors, including the commercialized sunitinib
(III) (Figure 1), have been published.[19,20] Sorafenib (IV) (Nexavar®;
Figure 1) also is a potent VEGFR‐2 inhibitor and has been approved
as an antiangiogenic drug.[21–23]
A study of the structure–activity relationships (SAR) and com-
mon pharmacophoric features shared by sorafenib and various
VEGFR‐2 inhibitors revealed that most VEGFR‐2 inhibitors shared
four main features, as shown in Figure 2[24–26]: First, the core
structure of most inhibitors consists of a flat heteroaromatic ring
system that contains at least one N‐atom, which occupies the cata-
lytic adenosine triphosphate (ATP)‐binding domain. The second fea-
ture is the presence of a central aryl ring (hydrophobic spacer),
occupying the linker region between the ATP‐binding domain and the
DFG domain of the enzyme.[27] The third feature is the presence of a
linker containing a functional group acting as a pharmacophore (e.g.,
amino or urea) that possesses both H‐bond acceptor (HBA) and do-
nor (HBD) to bind with two crucial residues (Glu883 and Asp1044) in
the DFG (Asp–Phe–Gly) motif, an essential tripeptide sequence in the
FIGURE 2 The basic structural requirements for sorafenib and
sunitinib as reported VEGFR‐2 inhibitors. ATP, adenosine
triphosphate; VEGFR‐2, vascular endothelial growth factor
receptor‐2
active kinase domain. 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. Fourth, the
terminal hydrophobic moiety of the inhibitors occupies the newly
created allosteric hydrophobic pocket, which is revealed when the
phenylalanine residue of the DFG loop flips out of its lipophilic
pocket, defining the DFG‐out or inactive conformation. Thus, hy-
drophobic interactions are usually attained in this allosteric
I
II
FIGURE 1 Reported vascular endothelial
growth factor receptor‐2 inhibitors
IV
III

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binding region.[28] Furthermore, the 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.[29–31]
linkers containing HBA–HBD, which interacted as HBA through its
C═O and as HBD through its NH with the essential amino acid re-
sidues Asp1044 and Glu883, respectively. Also, its (un)substituted
hydrophobic phenyl tail occupied the hydrophobic pocket formed by
Cys1043, Leu1033, Val914, Val897, and Lys866. The hydrophobic
phenyl tail was substituted with distal hydrophobic moieties in some
derivatives such as in compounds 7a–f through an acetamide linker
to increase the length of the structure to enable the distal moiety to
occupy new hydrophobic grooves formed by Leu1033, Cys917,
Phe916, Leu838, and Ala864, and other one formed by Gly920,
Phe919, Lys918, Phe916, and Leu838, for example, compound 7f
(Figure 4). In addition, 4‐methoxybenzylidene moiety was designed to
replace the pyridine and 5‐fluoro‐2‐oxoindolin‐3‐ylidene moieties of
the reference ligands sorafenib and sunitinib, respectively. Moreover,
thiazolidine‐2,4‐dione 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‐methoxybenzylidene moiety occupied the hydrophobic groove
formed by Arg1025, His1024, Ile1023, Cys1022, Leu1017, Ile890,
and Ile886. On the contrary, the thiazolidine‐2,4‐dione moiety oc-
cupied the hydrophobic pocket formed by Asp1044, Ile890, Leu887,
Ile886, and Glu883 (Figure 4).
Depending on the ligand‐based drug design, particularly a mo-
lecular hybridization approach that involves the coupling of two or
more groups with relevant biological properties,[32] molecular hy-
bridization of 5‐(4‐methoxybenzylidene)thiazolidine‐2,4‐dione and
other effective antitumor moieties was carried out in an attempt to
obtain 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 to synthesize new
agents with the same essential pharmacophoric features of the re-
ported and clinically used VEGFR‐2 inhibitors (e.g., sorafenib). The
main core of our molecular design rationale comprised bioisosteric
modification strategies of VEGFR‐2 inhibitors at four different po-
sitions (Figure 3).
Our target compounds were designed to have different spacers
and different linkers with HBA–HBD, the main pharmacophoric
feature in sorafenib, hoping to obtain more potent VEGFR‐2 in-
hibitors. First, a bioisosteric approach was adopted for the target
4‐methoxybenzylidene to replace the pyridine ring. The second
strategy was to use thiazolidine‐2,4‐dione to replace the central aryl
ring of the lead structure, aiming to increase the VEGFR‐2 binding
affinity. The third strategy was using ester and/or acetamide linkers
containing HBA–HBD functional groups that possess HBAs and/or
HBDs. Also, the hydrophobic substituted phenyl tail of the reported
ligand sorafenib was substituted by other distal hydrophobic moi-
eties, which occupied new hydrophobic grooves, as in compounds
7a–f (Figure 4). Furthermore, the substitution pattern was selected
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
thiazolidine‐2,4‐dione scaffolds and to explore a valuable SAR. The
designed target, 5‐(4‐methoxybenzylidene)thiazolidine‐2,4‐dione de-
rivatives, was synthesized and evaluated for potential VEGFR‐2 in-
hibitory and antitumor activities against three human tumor cell
lines, namely hepatocellular carcinoma (HepG2), breast cancer (Mi-
chigan Cancer Foundation‐7, MCF‐7), and human colorectal
carcinoma‐116 (HCT116).
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2.2
Chemistry
The synthetic strategy for the preparation of the target compounds
(5–7) is depicted in Schemes 1 and 2. The synthesis was initiated
by cyclocondensation of thiourea with chloroacetic acid to
afford thiazolidine‐2,4‐dione (1),[39,40] which underwent further
condensation reaction with 4‐methoxybenzaldehyde to afford
5‐(4‐methoxybenzylidene)‐thiazolidine‐2,4‐dione (2), which was he-
ated with alcoholic potassium hydroxide to afford the corresponding
potassium salt (3). On the contrary, chloroacyl chloride was reacted
with the appropriate aromatic amine to obtain the corresponding
chloroamides (4a–g). The potassium salt (3) was refluxed with the
appropriate chloroamide (4a–g) to afford the corresponding acid
amide derivatives (5a–g; Scheme 1). The chloroamide 4c was treated
with ethyl chloroformate and then the appropriate amine was added
to afford the corresponding intermediates (6a–f). The potassium salt
(3) was refluxed with the appropriate intermediates (6a–f) to obtain
the corresponding derivatives (7a–f; Scheme 2).
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2
RESULTS AND DISCUSSION
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2.1
Rationale and structure‐based design
2.3
Docking studies
5‐(4‐Methoxybenzylidene)thiazolidine‐2,4‐dione derivatives have the
essential pharmacophoric features of VEGFR‐2 inhibitors[33–38]
In the present study, all modeling experiments were performed using
Molsoft software. Each experiment used VEGFR‐2 downloaded from
the Brookhaven Protein Data Bank (PDB ID 1YWN).[41]
(Figure 3), which include the following: the presence of
a
five‐membered hetero ring, thiazolidine‐2,4‐dione, substituted with
4‐methoxybenzylidene moiety, as a hydrophobic portion, forming a
5‐(4‐methoxybenzylidene)‐thiazolidine‐2,4‐dione scaffold linked to
(un)substituted hydrophobic tail through ester and/or acetamide
The obtained results indicated that all studied ligands have a
similar position and orientation inside the recognized binding site
of VEGFR‐2, which reveals
a large space bounded by a
membrane‐binding domain that serves as an entry channel for the

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FIGURE 3 Structural similarities and pharmacophoric features of the vascular endothelial growth factor receptor‐2 inhibitors and the
designed compounds
substrate to the active site (Figure 5). In addition, the affinity of
any small molecule can be considered as a unique tool in the field
of drug design. There is a relationship between the affinity of
organic molecules and the free binding energy.[42–47] This re-
lationship can contribute to the prediction and interpretation of
the activity of the organic 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 af-
finity toward the receptor and the computed values reflected the
overall trend (Table 1).

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FIGURE 4 The superimposition of compound 7f and sorafenib inside the binding pocket of 1YWN
The proposed binding mode of sorafenib revealed an affinity
value of −95.36 kcal/mol and four H‐bonds. The urea linker formed
one H‐bond with the key amino acid Glu883 (1.84 Å) through its NH
group and one H‐bond with Asp1044 (2.00 Å) through its carbonyl
group. The N‐methylpicolinamide moiety was stabilized by the for-
mation of two H‐bonds, where the pyridine N atom formed one
H‐bond with Arg1025 (2.77 Å), whereas its NH group formed one
H‐bond with Ile1023 (2.45 Å). The N‐methylpicolinamide moiety oc-
cupied the hydrophobic groove formed by Arg1025, His1024,
Ile1023, Cys1022, Leu1017, Ile890, His889, and Ile886. Moreover,
the central phenyl ring occupied the hydrophobic pocket formed by
Glu883, Ile886, Leu887, Ile1042, Cys1043, and Asp1044. Further-
more, the distal hydrophobic 3‐trifluoromethyl‐4‐chlorophenyl moi-
ety attached to the urea linker occupied the hydrophobic pocket
formed by Cys1043, Leu1033, Val897, Val914, Ala864, Val865, and
Lys866 (Figure 6). The urea linker played an important role in the
binding affinity toward VEGFR‐2 enzyme, where it was responsible
for the higher binding affinity of sorafenib. This finding encourages us
to use different linkers resembling urea of sorafenib and increase the
hydrophobic moieties, hoping to obtain potent VEGFR‐2 inhibitors.
As planned, the proposed binding mode of compound 7f is vir-
tually the same as that of sorafenib, which revealed affinity more
Arg1025 (2.97 Å) and Ile1023 (1.64 Å). The 4‐methoxybenzylidene
moiety occupied the hydrophobic groove formed by Arg1025,
His1024, Ile1023, Cys1022, Leu1017, Ile890, and Ile886. Moreover,
the thiazolidine‐2,4‐dione moiety occupied the hydrophobic pocket
formed by Asp1044, Ile890, Leu887, Ile886, and Glu883. The central
phenyl ring occupied the hydrophobic pocket formed by Cys1043,
Leu1033, Val914, Val897, and Lys866. Furthermore, the distal phe-
nyl ring occupied the hydrophobic groove formed by Leu1033,
Cys917, Phe916, Leu838, and Ala864. On the contrary, the distal
ethyl group of the ester moiety occupied the hydrophobic cleft
formed by Gly920, Phe919, Lys918, Phe916, and Leu838 (Figure 7).
These interactions of compound 7f may explain the highest antic-
ancer activity.
The proposed binding mode of compound 7e is virtually the same
as that of 7f, which revealed an affinity value of −101.92 kcal/mol
and 6 H‐bonds. The carbonyl group of the acetamide linker formed
one H‐bond with Asp1044 (1.32 Å), whereas its NH group formed
another H‐bond with Glu883 (2.74 Å). The carbonyl group at
position‐2 of thiazolidine‐2,4‐dione formed one H‐bond with
Asp1044 (2.93 Å). Moreover, the NH group of the carboxamide linker
formed an H‐bond with Glu915 (2.82 Å). The oxygen atom of the
methoxy group was stabilized by the formation of two H‐bonds with
Arg1025 (2.99 Å) and Ile1023 (1.56 Å). The 4‐methoxybenzylidene
moiety occupied the hydrophobic groove formed by Arg1025,
His1024, Ile1023, Cys1022, Leu1017, Ile890, and Ile886. Moreover,
the thiazolidine‐2,4‐dione moiety occupied the hydrophobic pocket
formed by Asp1044, Ile890, Leu887, Ile886, and Glu883. The central
phenyl ring occupied the hydrophobic pocket formed by Cys1043,
Leu1033, Val914, Val897, and Lys866. Furthermore, the distal
4‐methylphenyl ring occupied the hydrophobic groove formed by
than that of sorafenib, with
a value of −103.50 kcal/mol and
six H‐bonds. The carbonyl group of the acetamide linker formed
one H‐bond with Asp1044 (1.74 Å), whereas its NH group formed
another H‐bond with Glu883 (1.79 Å). The carbonyl group at
position‐2 of thiazolidine‐2,4‐dione formed one H‐bond with
Asp1044 (2.92 Å). Moreover, the NH group of the carboxamide linker
formed one H‐bond with Glu915 (2.79 Å). The oxygen atom of the
methoxy group was stabilized by the formation of two H‐bonds with

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SCHEME 1 The synthetic route for the preparation of the target compounds 5a–g
Leu1033, Gly920, Lys918, Cys917, Phe916, Ala864, and Leu838
(Figure 8). These interactions of compound 7e may explain the high
anticancer activity.
Glu883. The central phenyl ring occupied the hydrophobic pocket
formed by Cys1043, Leu1033, Val914, Val897, and Lys866. Fur-
thermore, the distal benzyl moiety occupied the hydrophobic groove
formed by Leu1033, Gly920, Lys918, Cys917, Phe916, Ala864, and
Leu838 (Figure 9). These interactions of compound 7d may explain
the high anticancer activity.
The proposed binding mode of compound 7d is virtually the same
as that of sorafenib and 7f, which revealed an affinity value of
−97.08 kcal/mol and six H‐bonds. The carbonyl group of the acet-
amide linker formed one H‐bond with Asp1044 (1.31 Å), whereas its
NH group formed another H‐bond with Glu883 (2.78 Å). The
carbonyl group at position‐2 of thiazolidine‐2,4‐dione formed one
H‐bond with Asp1044 (2.96 Å). Moreover, the NH group of the car-
boxamide linker formed one H‐bond with Glu915 (2.87 Å). The oxy-
gen atom of the methoxy group was stabilized by the formation of
two H‐bonds with Arg1025 (2.80 Å) and Ile1023 (2.47 Å). The
4‐methoxybenzylidene moiety occupied the hydrophobic groove
formed by Arg1025, His1024, Ile1023, Cys1022, Leu1017, Ile890,
and Ile886. Moreover, the thiazolidine‐2,4‐dione moiety occupied the
hydrophobic pocket formed by Asp1044, Ile890, Leu887, Ile886, and
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 4‐methoxybenzylidene
moiety compensated the effect of N‐methylpicolinamide moiety of
sorafenib and increased hydrogen bonding and hydrophobic inter-
actions, and consequently affinities toward VEGFR‐2 enzyme. The
thiazolidine‐2,4‐dione enables the new compounds to form a new
H‐bond through its carbonyl group at position‐2 with the essential
amino acid Asp1044. The elongation of the structure played an im-
portant role in their VEGFR‐2 inhibitory activities. The hydrophobic

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6a–f
7a–f
SCHEME 2 The synthetic route for preparation of the target compounds 7a–f
FIGURE 5 The superimposition of some docked compounds inside the binding pocket of 1YWN

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TABLE 1 The calculated free energy of binding (ΔG in kcal/mol)
for the ligands
summarized in Table 2. From the obtained results, it was explicated
that most of the prepared compounds displayed an excellent‐to‐
modest growth inhibitory activity against the tested cancer cell lines.
Investigations of the cytotoxic activity indicated that HepG2 and
HCT116 were the most sensitive cell lines to the influence of the new
derivatives. In particular, compounds 7f, 7e, 7d, and 7c were found to
be the most potent derivatives among all the tested compounds
Compound
ΔG (kcal/mol)
−71.52
Compound
ΔG (kcal/mol)
−85.32
5a
5b
5c
5d
5e
5f
7a
−76.01
7b
−90.40
−74.14
7c
−96.76
−86.91
7d
−97.08
against HepG2, HCT116, and MCF‐7 cancer cell lines, with IC50 =
6.19 0.5, 7.89 0.6, 10.83 1.1, 13.78 1.2 µM, 8.37 0.7,
9.56 0.9, 13.12 1.2, 18.03 1.5 µM, and 5.10 0.4, 10.18 0.9,
18.10 1.5, 22.08 1.6 µM, respectively. Compound 7f (IC50 = 6.19
0.5, 5.47 0.3, and 7.26 0.3 µM, respectively) exhibited a higher
activity than sorafenib (IC50 = 9.18 0.6, 8.37 0.7, and
5.10 0.4 µM, respectively) against HepG2 and MCF‐7 cell lines, but
a lower activity against HCT116 cancer cell line. Also, this compound
displayed higher activity than doxorubicin (IC50 = 7.94 0.6,
8.07 0.8, and 6.75 0.4 µM, respectively) against HepG2 and MCF‐
7 cell lines, but nearly the same activity against HCT116 cell line.
With respect to the HepG2 hepatocellular carcinoma cell line, com-
pounds 7e, 7d, 7c, and 8b displayed the highest anticancer activities,
with IC50 = 7.89 0.6, 10.83 1.1, 13.78 1.2, and 19.35 1.8 µM,
respectively. Compounds 5c and 7a, with IC50 = 23.11 2.3 and
24.11 2.3 µM, respectively, displayed good cytotoxicity. Com-
pounds 5a–c and 5e–g, with IC50 ranging from 40.33 3.3 to
54.37 5.2 µM, exhibited modest cytotoxicity.
−72.67
7e
−101.92
−103.50
−95.36
−67.65
7f
5g
−65.25
Sorafenib
distal moiety and its linker, as in compounds 7a–f, which formed
hydrogen bonding interactions with the amino acid Glu915, in-
creased affinity toward the VEGFR‐2 enzyme.
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2.4
In vitro cytotoxic activity
The antiproliferative activity of the newly synthesized 5‐(4‐
methoxybenzylidene)thiazolidine‐2,4‐dione derivatives, 5a–g and
7a–f, was examined against three human tumor cell lines, namely
HepG2, HCT116, and MCF‐7, using a 3‐[4,5‐dimethylthiazol‐2‐yl]‐
2,5‐diphenyltetrazolium bromide (MTT) colorimetric assay, as de-
scribed by Mosmann.[48–50] Sorafenib and doxorubicin were included
in the experiments as reference cytotoxic drugs. The results were
expressed as growth inhibitory concentration (IC50) values, which
represent the compound concentrations required to produce a 50%
inhibition of cell growth after 72 hr of incubation. They were calcu-
lated from the concentration–inhibition response curve, and they are
The cytotoxicity evaluation against colorectal carcinoma (HCT116)
cell line discovered that compounds 7e, 7d, and 7c displayed the highest
anticancer activities, with IC50 = 9.56 0.9, 13.12 1.2, and
18.03 1.5 µM, respectively. Compounds 7b, 7a, and 5c, with IC50
=
27.49 1.9, 29.37 2.1, and 29.40 2.1 µM, respectively, displayed good
cytotoxicity. Compounds 5a–c and 5e–g, with IC50 ranging from
54.92 3.7 to 66.26 4.9 µM, exhibited modest cytotoxicity.
FIGURE 6 The predicted binding mode for sorafenib with 1WYN. H‐bonded atoms are indicated by dotted lines

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FIGURE 7 The predicted binding mode for 7f with 1WYN
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The cytotoxicity evaluation against MCF‐7 cell line revealed
that compounds 7e and 7d displayed the highest anticancer
activities, with IC50 = 10.18 0.9 and 18.10 1.5 µM, respec-
tively. Compounds 7c, 7b, 5c, and 7a, with IC50 = 22.08 1.6,
23.72 1.7, 34.12 2.4, and 34.81 2.4 µM, respectively,
displayed good cytotoxicity. Compounds 5a–c and 5e–g, with
IC50 ranging from 50.25 4.9 to 79.33 6.8 µM, exhibited modest
cytotoxicity.
2.5
In vitro VEGFR‐2 kinase assay
All derivatives, 5a–g and 7a–f, were evaluated for their inhibitory
activities against VEGFR‐2 by using an antiphosphotyrosine antibody
with the Alpha Screen system (PerkinElmer). The results were re-
ported as IC50 values calculated from the concentration–inhibition
response curve, and they are summarized in Table 2. Sorafenib was
used as a positive control in this assay. The tested compounds
FIGURE 8 The predicted binding mode for 7e with 1WYN

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FIGURE 9 The predicted binding mode for 7d with 1WYN
TABLE 2 In vitro cytotoxic activities of the newly synthesized compounds against HepG2, HCT116, and MCF‐7 cell lines and VEGFR‐2 kinase
assay
IC50 (µM)a
Compound
HepG2
HCT116
MCF‐7
VEGFR‐2
5a
45.40 3.70
41.46 3.40
40.33 3.30
23.11 2.30
42.66 4.50
47.77 4.90
54.37 5.20
24.11 2.30
19.35 1.80
13.78 1.20
10.83 1.10
7.89 0.60
6.19 0.50
9.18 0.60
7.94 0.60
54.92 3.70
64.12 5.20
66.22 5.30
29.40 2.10
66.26 4.90
57.09 4.90
63.65 4.50
29.37 2.10
27.49 1.90
18.03 1.50
13.12 1.20
9.56 0.90
8.37 0.70
5.47 0.30
8.07 0.80
65.48 4.30
50.25 4.90
52.58 3.70
34.12 2.40
58.78 5.00
65.98 5.40
79.33 6.80
34.81 2.40
23.72 1.70
22.08 1.60
18.10 1.50
10.18 0.90
5.10 0.40
7.26 0.30
6.75 0.40
0.55 0.05
0.31 0.03
0.32 0.03
0.22 0.02
0.41 0.04
0.59 0.05
0.64 0.03
0.22 0.02
0.18 0.03
0.14 0.02
0.14 0.02
0.13 0.02
0.12 0.02
0.10 0.02
NT
5b
5c
5d
5e
5f
5g
7a
7b
7c
7d
7e
7f
Sorafenib
Doxorubicin
Abbreviations: HCT116, human colorectal carcinoma‐116; MCF‐7, Michigan Cancer Foundation‐7; NT, compounds not tested for their VEGFR‐2
inhibitory activity; SD, standard deviation; VEGFR‐2, vascular endothelial growth factor receptor‐2.
aIC50 values are the mean SD of three separate experiments.

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displayed a high‐to‐medium inhibitory activity, with IC50 values
ranging from 0.12 0.02 to 0.64 0.03 µM. Among them, compound
7f was found to be the most potent derivative that inhibited
VEGFR‐2 at an IC50 value of 0.12 0.02 µM, which is nearly the same
as that of sorafenib with IC50 value of 0.10 0.02 µM. Compounds
7e, 7d, 7c, and 7b exhibited the highest activity, with IC50 values of
0.13 0.02, 0.14 0.02, 0.14 0.02, and 0.18 0.03 µM, respectively,
which are more than the half of that of sorafenib. Also, compounds
5d and 7a possessed a very good VEGFR‐2 inhibition with the same
IC50 values of 0.22 0.02. Compounds 5b, 5c, and 5e possessed a
good VEGFR‐2 inhibition, with IC50 values = 0.31 0.03, 0.32 0.03,
and 0.41 0.04 µM, respectively. However, compounds 5a, 5f, and 5g
possessed a moderate VEGFR‐2 inhibition, with IC50 values ranging
from 0.55 0.05 to 0.64 0.03 µM.
activity than the other compounds containing only acetamide linkers.
From the structure of the synthesized derivatives and the data
shown in (Table 2), we can divide these tested compounds into two
groups. The first group is compounds 5a–g, where the terminal
phenyl tails were replaced by another heteroaromatic pyridine ring,
as in compound 5f, and/or thiazole ring, as in compound 5g. In the
other derivatives 5a–e, the terminal phenyl tail was unsubstituted as
in compound 5a and 4‐substituted with different hydrophobic,
electron‐donating, and/or electron‐withdrawing groups. Generally,
the substituted terminal phenyl with hydrophobic distal ethyl ester
group 5d showed higher activities than the other substituted com-
pounds (e.g., 5b, 5c, and 5e–g) and unsubstituted one, 5a, against the
three HepG2, HCT116, and MCF‐7 cell lines. Compound 5b with
hydrophobic electron‐donating methyl group exhibited higher activ-
ities than compounds 5c and 5e with hydrophilic electron‐
withdrawing carboxylic and/or nitro groups against both HCT116
and MCF‐7 cell lines, but it showed a lower activity against HepG2
cell line. Although compounds 5c and 5e with hydrophilic electron‐
withdrawing carboxylic and/or nitro groups exhibited higher activ-
ities than the unsubstituted compound 5a against both HepG2 and
MCF‐7 cell lines, they showed lower activities against HCT116 cell
line. Compound 5d with hydrophobic ethyl ester moiety exhibited
higher activities than compounds 5c and 5e with hydrophilic car-
boxylic and/or nitro groups against the three HepG2, HCT116, and
MCF‐7 cell lines. Moreover, the terminal unsubstituted phenyl deri-
vative 5a exhibited higher activities than the pyridine, as in com-
pound 5f, and the thiazole tail, as in compound 6g, against the three
HepG2, HCT116, and MCF‐7 cell lines. In the second group, 7a–f,
generally, the substituted terminal phenyl group with aromatic sub-
stituents, as in compounds 7e–f, exhibited higher activities than that
substituted with aliphatic ones, 7a–c, against the three HepG2,
HCT116, and MCF‐7 cell lines. The substituted distal phenyl moiety
with ethyl ester group, as in compound 7f, displayed higher activities
than that substituted with methyl as in compound 7e against the
three HepG2, HCT116, and MCF‐7 cell lines. The distal toluene
moiety 7e exhibited higher activities than the distal benzyl one 7d
against the three cancer cell lines. In addition, the aliphatic butyl
distal moiety 7c showed higher activities than the propyl 7b and the
ethyl 7a ones against the three HepG2, HCT116, and MCF‐7 cell
lines.
|
2.6
Structure–activity relationship
The preliminary SAR study has focused on the effect of replacement
of the urea and carboxamide linkers of sorafenib and sunitinib, re-
spectively, with acetamide linkers, which interact as HBAs through
its carbonyl group and as HBD through its NH atom. These acet-
amide linkers interact with the side chain NH of the essential amino
acid residue Asp1044 and the carboxylate of the essential amino acid
residue Glu883. Also, hydrophobic interactions take place through
the attached (un)substituted hydrophobic distal moieties. The effect
of replacement of pyridine and 5‐fluoro‐2‐oxoindolin‐3‐ylidene
moieties of sorafenib and sunitinib, respectively, by the
4‐methoxybenzylidene of 5‐(4‐methoxybenzylidene)thiazolidine‐2,
4‐dione scaffold of the synthesized compounds on the antitumor
activities also was observed. This 4‐methoxybenzylidene moiety oc-
cupied the same hydrophobic pocket that was occupied by the pyr-
idine moiety of the standard ligand. Moreover, the oxygen atom of
the methoxy group was stabilized by the formation of two H‐bonds
with the same amino acid residues, Arg1025 and Ile1023, which
formed two H‐bonds with N‐methylpicolinamide moiety of sorafenib.
The thiazolidine‐2,4‐dione was designed to replace the central aryl
and pyrrole rings of the reference ligands sorafenib and sunitinib,
respectively, which enables the target compounds to form new
H‐bonds through their 2‐carbonyl groups with the essential amino
acid residue Asp1044. On the contrary, different distal aliphatic and
aromatic moieties were introduced to the phenyl tail of the reference
ligand with different lipophilicity and electronic nature to study their
effects on the anticancer activity. The presence of lipophilic distal
moieties attached to the phenyl ring through the carboxamide linker,
as in compounds 7a–f, formed new hydrophobic and H‐bonding in-
teractions 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 HepG2
cell lines. Generally, the spacers, linkers (HBA–HBD), lipophilicity,
and electronic nature exhibited an important role in anticancer ac-
tivity. The presence of acetamide and carboxamide linkers of com-
pounds 7a–f was found to be responsible for their highest anticancer
|
3
CONCLUSION
The molecular design was 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. All the tested compounds
showed variable anticancer activities.
A novel series of 5‐(4‐
methoxybenzylidene)thiazolidine‐2,4‐dione derivatives, 5a–g and
7a–f, was designed, synthesized, and evaluated for their anticancer
activity against three human tumor cell lines, HepG2, HCT116, and
MCF‐7, targeting VEGFR‐2 enzyme. HepG2 and HCT116 were the

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most sensitive cell lines to the influence of the new derivatives. In
particular, compounds 7f, 7e, 7d, and 7c were found to be the most
potent derivatives among all the tested compounds against HepG2,
HCT116, and MCF‐7 cancer cell lines, with IC50 = 6.19 0.5,
7.89 0.6, 10.83 1.1, 13.78 1.2 µM, 8.37 0.7, 9.56 0.9,
13.12 1.2, 18.03 1.5 µM, and 5.10 0.4, 10.18 0.9, 18.10 1.5,
22.08 1.6 µM, respectively. Compound 7f (IC50 = 6.19 0.5,
5.47 0.3, and 7.26 0.3 µM, respectively) exhibited a higher activity
than sorafenib (IC50 = 9.18 0.6, 8.37 0.7, and 5.10 0.4 µM, re-
spectively) against HepG2 and MCF‐7 cell lines, but a lower activity
against HCT116 cancer cell line. Also, this compound displayed
higher activity than doxorubicin (IC50 = 7.94 0.6, 8.07 0.8, and
6.75 0.4 µM, respectively) against HepG2 and MCF‐7cell lines, but
nearly the same activity against HCT116 cell line. All derivatives,
5a–g and 7a–f, were evaluated for their inhibitory activities against
VEGFR‐2. The elongation of the structures to have distal moieties
enhanced anticancer and VEGFR‐2 inhibitory activities, as in com-
pounds 7a–f. Among them, compounds 7f was found to be the most
potent derivative that inhibited VEGFR‐2 at an IC50 value of
0.12 0.02 µM, which is nearly similar to the IC50 value of sorafenib:
0.10 0.02 µM. Compounds 7e, 7d, 7c, and 7b exhibited high activity,
with IC50 values of 0.13 0.02, 0.14 0.02, 0.14 0.02, and
0.18 0.03 µM, respectively, which are more than the half of that of
sorafenib. The obtained results showed that our compounds could be
useful as a template for future design, optimization, adaptation, and
investigation to produce more potent and selective VEGFR‐2 in-
hibitors with higher anticancer analogs.
within 0.4 of the theoretical values. The reactions were monitored by
thin‐layer chromatography (TLC) using TLC sheets precoated with ul-
traviolet (UV) fluorescent silica gel Merck 60 F254 plates and were
visualized using a UV lamp and different solvents as mobile phases.
The original spectra are provided as Supporting Information.
Thiazolidine‐2,4‐dione (1), 5‐(4‐methoxybenzylidene)thiazolidine‐2,4‐
dione (2), the corresponding potassium salt (3), N‐aryl‐2‐chloroacetamide
derivatives, 4a–g, and 4‐(2‐chloroacetamido)‐N‐substitutedbenzamide
derivatives, 6a–f, were obtained according to the reported
procedures.[39,40,51–55]
|
4.1.2
General method for the synthesis of 2‐[5‐(4‐
methoxybenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐N‐
[(un)substituted aryl]acetamides 5a–g
Equimolar quantities of the potassium salt 3 (2.73 g, 0.01 mol) and
the appropriate N‐aryl‐2‐chloroacetamide derivatives 4a–g
(0.01 mol) in dry DMF (20 ml) were heated in a water bath for 3 hr
in the presence of a catalytic amount of KI. After cooling to room
temperature, the reaction mixture was poured over crushed ice. The
precipitated solids were filtered, dried, and crystalized from ethanol
to give the corresponding target compounds 5a–g.
2‐[5‐(4‐Methoxybenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐N‐
phenylacetamide (5a)
Yield, 68%; m.p. 147–149°C; IRνmax (cm−1): 3,329 (NH), 3,063 (CH
aromatic), 2,928 (CH aliphatic), 1,701, and 1,639 (3C═O); 1H‐NMR
(400 MHz, DMSO‐d6): 3.84 (s, 3H, OCH3), 4.52 (s, 2H, CH2),
7.06–7.10 (m, 1H, Ar‐H, H‐4 of –C6H5), 7.30–7.35 (m, 2H, Ar‐H, H‐3,
H‐5 of –C6H5), 7.57 (d, 2H, J = 7.60 Hz, Ar‐H, H‐3 & H‐5 of –C6H4),
7.64 (d, 2H, J = 7.60 Hz, Ar‐H, H‐2 & H‐6 of –C6H4), 7.70 (d, 2H, Ar‐H,
J = 9.60 Hz, H‐2 & H‐6 of –C6H5), 8.00 (s, 1H, C═CH–Ph), and 10.41
(s, 1H, NH, D2O exchangeable); 13C‐NMR (100 MHz, DMSO‐d6):
44.44, 56.01, 115.51 (2), 118.14, 119.62 (2), 124.17, 125.80, 129.35
(2), 132.86 (2), 134.08, 138.87, 161.79, 164.31, 165.90, and 167.69;
Anal. calcd. for C19H16N2O4S (368.08): C, 61.94; H, 4.38; N, 7.60.
Found: C, 62.12; H, 4.67; N, 7.24.
|
4
EXPERIMENTAL
|
4.1
Chemistry
|
4.1.1
General
All melting points were measured by the open capillary method on a
Gallenkamp melting point apparatus at the Faculty of Pharmacy,
Al‐Azhar University, and they were uncorrected. The infrared (IR)
spectra were recorded on pye Unicam SP 1000 IR spectrophotometer at
Microanalytical Unit, Faculty of Pharmacy, Cairo University, using po-
tassium bromide disc technique. Proton magnetic resonance 1H‐NMR
(nuclear magnetic resonance) spectra were recorded on a Bruker
400 MHz NMR spectrophotometer at Microanalytical Unit, 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 the δ scale (ppm).
The mass spectra were performed on Direct Probe Controller Inlet part
to Single Quadrupole 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
2‐[5‐(4‐Methoxybenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐N‐(p‐tolyl)‐
acetamide (5b)
Yield, 70%; m.p. 150–152°C; IRνmax (cm−1): 3,298 (NH), 3,059 (CH
aromatic), 2,835 (CH aliphatic), 1,739, and 1,670 (3C═O); 1H‐NMR
(400 MHz, DMSO‐d6): 2.25 (s, 3H, CH3), 3.84 (s, 3H, –OCH3), 4.49
(s, 2H, CH2), 7.12 (d, 2H, J = 8.60 Hz, Ar‐H, H‐3
–C6H4–CH3), 7.13 (d, 2H, J = 7.40 Hz, Ar‐H, H‐3
of –C6H4–OCH3), 7.42 (d, 2H, J = 8.60 Hz, Ar‐H, H‐2 & H‐6 of
–C6H4–CH3), 7.62 (d, 2H, J = 7.40 Hz, Ar‐H, H‐2 H‐6 of
&
H‐5 of
&
H‐5
&
–C6H4–OCH3), 7.94 (s, 1H, –C═CH–Ph), and 10.29 (s, 1H, NH, D2O
exchangeable); MS (m/z): 383 (M++1, 3.12%), 382 (M+, 10.49%),
370 (100%, base peak), 285 (12.25%), and 93 (32.13%); Anal. calcd.
for C20H18N2O4S (382.43): C, 62.81; H, 4.74; N, 7.33. Found:
C, 62.64; H, 4.85; N, 7.59.

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4‐{2‐[5‐(4‐Methoxybenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐
acetamido}benzoic acid (5c)
2‐[5‐(4‐Methoxybenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐N‐(thiazol‐2‐
yl)acetamide (5g)
Yield, 66%; m.p. 168–169°C; IRνmax (cm−1): 3,414 (OH), 3,237 (NH),
3,032 (CH aromatic), 2,921 (CH aliphatic), 1,728, and 1,697 (4C═O);
1H‐NMR (400 MHz, DMSO‐d6): 3.80 (s, 3H, –OCH3), 4.56 (s, 2H,
CH2), 7.15 (d, 2H, J = 8.00 Hz, Ar‐H, H‐3 & H‐5 of C6H4–OCH3), 7.57
(d, 2H, J = 8.00 Hz, Ar‐H, H‐2 & H‐6 of C6H4–OCH3), 7.63 (d, 2H,
J = 8.80 Hz, Ar‐H, H‐2 & H‐6 of C6H4–COOH), 7.76 (d, 2H, J =
8.80 Hz, Ar‐H, H‐3 & H‐5 of C6H4–COOH), 7.99 (s, 1H, C═CH–Ph),
10.37 (s, 1H, NH, D2O exchangeable), and 12.00 (s, 1H, –OH, D2O
exchangeable); Anal. calcd. for C20H16N2O6S (412.42): C, 58.25;
H, 3.91; N, 6.79. Found: 58.16; H, 3.75; N, 6.93.
Yield, 70%; m.p. 187–189°C; IRνmax (cm−1): 3,205 (NH), 3,078 (CH
aromatic), 2,850 (CH aliphatic), 1,735, and 1,681 (3C═O); 1H‐NMR
(400 MHz, DMSO‐d6): 3.84 (s, 3H, OCH3), 4.62 (s, 2H, CH2), 7.13 (d,
2H, J = 9.60 Hz, Ar‐H, H‐3 & H‐5 of –C6H4), 7.25 (d, 1H, J = 6.60 Hz,
Ar‐H, H‐5 of thiazole), 7.49 (d, 1H, J = 6.60 Hz, Ar‐H, H‐4 of thiazole),
7.63 (d, 2H, J = 9.60 Hz, Ar‐H, H‐2 & H‐6 of –C6H4), 7.95 (s, 1H,
C═CH–Ph), and 12.53 (s, 1H, NH, D2O exchangeable); Anal. calcd. for
C16H13N3O4S2 (375.42): C, 51.19; H, 3.49; N, 11.19. Found: C, 51.37;
H, 3.70; N, 11.05.
|
General method for the synthesis of
4‐{2‐[5‐(4‐methoxybenzylidene)‐2,4‐dioxothiazolidin‐
3‐yl]acetamido}‐N‐substituted benzamides 7a–f
Ethyl 4‐{2‐[5‐(4‐methoxybenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐
acetamido}benzoate (5d)
4.1.3
Yield, 76%; m.p. 166–168°C; IRνmax (cm−1): 3,294 (NH), 3,032 (CH
aromatic), 2,924 (CH aliphatic), 1,751, 1,694, and 1,670 (4C═O); 1H‐
NMR (400 MHz, DMSO‐d6): 1.13 (t, 3H, J = 10.00 Hz, –OCH2CH3),
3.23 (q, 2H, J = 10.00 Hz, –OCH2CH3), 3.82 (s, 3H, –OCH3), 4.55 (s,
2H, –NCH2CO), 7.39 (d, 2H, J = 7.40 Hz, Ar‐H, H‐3 & H‐5 of
Equimolar quantities of the potassium salt 3 (2.73 g, 0.01 mol) and
the appropriate 4‐(2‐chloroacetamido)‐N‐substitutedbenzamide de-
rivatives 6a–f (0.01 mol) in DMF (20 ml) were heated in a water bath
for 5 hr. The reaction mixture was poured over 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, 7a–f.
–C6H4–OCH3), 7.63 (d, 2H, J = 7.40 Hz, Ar‐H, H‐2
of –C6H4–OCH3), 7.82 (d, 2H, J = 7.20 Hz, Ar‐H, H‐2 & H‐6 of
–C6H4COO), 7.94 (d, 2H, J = 7.20 Hz, Ar‐H, H‐3 H‐5 of
& H‐6
&
–C6H4COO), 8.59 (s, 1H, C═CH–Ph), and 10.67 (s, 1H, NH, D2O
exchangeable); Anal. calcd. for C22H20N2O6S (440.47): C, 59.99; H,
4.58; N, 6.36. Found: C, 59.86; H, 4.80; N, 6.21.
N‐Ethyl‐4‐{2‐[5‐(4‐methoxybenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐
acetamido}benzamide (7a)
2‐[5‐(4‐Methoxybenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐N‐(4‐
nitrophenyl)acetamide (5e)
Yield, 80%; m.p. 244–245°C; IRνmax (cm−1): 3,282, 3,190 (2NH), 3,039
(CH aromatic), 2,939 (CH aliphatic), 1,751, 1,681, and 1,647 (4C═O);
1H‐NMR (400 MHz, DMSO‐d6): 1.11 (t, 3H, –NHCH2CH3), 3.23–3.31
(q, 2H, –NHCH2CH3), 3.85 (s, 3H, OCH3), 4.54 (s, 2H, –NCH2CO),
7.13 (d, 2H, J = 8.40 Hz, Ar‐H, H‐3 & H‐5 of –C6H4OCH3), 7.62 (d, 2H,
Ar‐H, J = 8.40 Hz, H‐2 & H‐6 of –C6H4OCH3), 7.66 (d, 2H, Ar‐H, H‐2 &
H‐6 of –C6H4CONH), 7.82 (d, 2H, Ar‐H, H‐3 & H‐5 of –C6H4CONH),
7.97 (s, 1H, C═CH–Ph), 8.37 (s, 1H, –NHCH2CH3, D2O exchange-
able), and 10.62 (s, 1H, NH‐benzamide, D2O exchangeable); Anal.
calcd. for C22H21N3O5S (439.49): C, 60.13; H, 4.82; N, 9.56. Found: C,
60.41; H, 4.98; N, 9.75.
Yield, 60%; m.p. 174–176°C; IRνmax (cm–1): 3,282 (NH), 3,066 (CH
aromatic), 2,843 (CH aliphatic), 1,739, and 1,681 (3C═O);
1H‐NMR (400 MHz, DMSO‐d6): 3.83 (s, 3H, OCH3), 4.36 (s, 2H,
CH2), 7.08–7.11 (m, 4H, Ar‐H, –C6H4–OCH3), 7.63–7.66 (m, 5H,
Ar‐H, 4H, of –C6H4–NO2 & 1H, C═CH–Ph), and 11.34 (s, 1H,
NH.D2O exchangeable); 13C‐NMR (100 MHz, DMSO‐d6): 40.61,
55.19 (2), 114.56 (3), 120.91, 129.97, 131.91 (3), 137.55 (2),
161.23 (2), 168.61 (2), and 170.17 (2); Anal. calcd. for
C19H15N3O6S (413.40): C, 55.20; H, 3.66; N, 10.16. Found:
C, 55.43; H, 3.85; N, 10.35.
4‐{2‐[5‐(4‐Methoxybenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐
acetamido}‐N‐propylbenzamide (7b)
2‐[5‐(4‐Methoxybenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐N‐(pyridin‐
2‐yl)acetamide (5f)
Yield, 80%; m.p. 245–247°C; IRνmax (cm−1): 3,271, 3,105 (2NH), 3,032
(CH aromatic), 2,947 (CH aliphatic), 1,747, 1,697, and 1,631 (4C═O);
1H‐NMR (400 MHz, DMSO‐d6): 0.88 (t, 3H, J = 6.80 Hz,
–NHCH2CH2CH3), 1.50–1.55 (m, 2H, –NHCH2CH2CH3), 3.21 (t, 2H,
J = 6.40 Hz, –CH2CH2CH3), 3.86 (s, 3H, –OCH3), 4.55 (s, 2H,
–NCH2CO), 7.13 (d, 2H, J = 7.60 Hz, Ar‐H, H‐3 & H‐5 of –C6H4OCH3),
7.62 (d, 2H, J = 7.60 Hz, Ar‐H, H‐2 & H‐6 of –C6H4OCH3), 7.65 (d, 2H,
Yield, 73%; m.p. 167–169°C; IRνmax (cm−1): 3,151 (NH), 3,051 (CH
aromatic), 2,765 (CH aliphatic), 1,755, 1,678, and 1,608 (3C═O); 1H‐
NMR (400 MHz, DMSO‐d6): 3.81 (s, 3H, OCH3), 4.60 (s, 2H, CH2),
7.15–7.17 (m, 1H, Ar‐H, H‐4 of pyridine), 7.62 (d, 2H, J = 7.20 Hz, Ar‐
H, H‐3 & H‐5 of –C6H4), 7.69 (d, 2H, J = 7.20 Hz, Ar‐H, H‐2 & H‐6 of
–C6H4), 7.82–7.84 (m, 1H, Ar‐H, H‐5 of pyridine), 7.96 (s, 1H,
C═CH–Ph), 8.01 (d, 1H, Ar‐H, H‐6 of pyridine), 8.35 (d, 1H, Ar‐H, H‐3
of pyridine), and 10.99 (s, 1H, –NH, D2O exchangeable); Anal. calcd.
for C18H15N3O4S (369.40): C, 58.53; H, 4.09; N, 11.38. Found: C,
58.26; H, 3.68; N, 11.75.
Ar‐H, J = 7.60 Hz, H‐2
J = 7.60 Hz, Ar‐H, H‐3
&
H‐6 of –C6H4CONH), 7.82 (d, 2H,
H‐5 of –C6H4CONH), 7.96 (s, 1H,
&
C═CH–Ph), 8.36 (s, 1H, –NHCH2CH2CH3, D2O exchangeable), and
10.62 (s, 1H, NH‐benzamide, D2O exchangeable); 13C‐NMR

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EL‐ADL ET AL.
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(100 MHz, DMSO‐d6): 11.94, 22.88, 41.41, 44.51, 56.02, 115.53 (2),
118.09, 118.83, 125.79, 128.58 (2), 130.19, 132.87 (2), 134.14 (2),
141.20, 161.81, 164.71, 165.88, 165.97, and 167.69; Anal. calcd. for
C23H23N3O5S (453.51): C, 60.91; H, 5.11; N, 9.27. Found: C, 61.18; H,
5.24; N, 9.41.
118.90, 120.85 (2), 125.77, 129.16 (2), 129.43 (2), 130.29, 132.88 (2),
132.96 (2), 134.18, 137.13, 141.65, 161.82, 164.83, 165.06, 165.88,
and 167.71; Anal. calcd. for C27H23N3O5S (501.56): C, 64.66; H, 4.62;
N, 8.38. Found: C, 64.42; H, 4.89; N, 8.51.
Ethyl 4‐(4‐{2‐[5‐(4‐methoxybenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]
acetamido}benzamido)benzoate (7f)
N‐Butyl‐4‐{2‐[5‐(4‐methoxybenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐
acetamido}benzamide (7c)
Yield, 60%; m.p. 260–262°C; IRνmax (cm−1): 3,190, 3,109 (2NH), 3,062
(CH aromatic), 2,939 (CH aliphatic), 1,743, 1,685, and 1,631 (5C═O);
1H‐NMR (400 MHz, DMSO‐d6): 1.32 (t, 3H, J = 9.20 Hz, –CH2CH3),
3.85 (s, 3H, –OCH3), 4.27–4.32 (q, 2H, J = 9.20 Hz, –CH2CH3), 4.57
(s, 2H, –NCH2CO), 7.13 (d, 2H, J = 8.00 Hz, Ar‐H, H‐3 & H‐5 of
Yield, 73%; m.p. 251–253°C; IRνmax (cm−1): 3,194, 3,109 (2NH), 3,055
(CH aromatic), 2,931(CH aliphatic), 1,732, 1,693, and 1,631 (4C═O); 1H‐
NMR (400 MHz, DMSO‐d6): 0.90 (t, 3H, –CH2CH2CH2CH3), 1.26–1.34
(m, 2H, CH2CH2CH2CH3), 1.51–1.53 (m, 2H, –CH2CH2CH2CH3), 3.25 (t,
2H, –CH2CH2CH2CH3), 3.84 (s, 3H, –OCH3), 4.56 (s, 2H, −NCH2CO),
6.93 (d, 2H, J = 7.60 Hz, Ar‐H, H‐3 & H‐5 of –C6H4OCH3), 7.16 (d, 2H,
J = 7.60 Hz, Ar‐H, H‐2 & H‐6 of –C6H4OCH3), 7.66 (d, 2H, J = 7.60 Hz,
Ar‐H, H‐2 & H‐6 of –C6H4CONH), 7.86 (d, 2H, J = 7.60 Hz, Ar‐H, H‐3 &
H‐5 of –C6H4CONH), 8.00 (s, 1H, C═CH–Ph), 8.35 (s, 1H,
–NHCH2CH2CH2CH3, D2O exchangeable), and 10.71 (s, 1H, NH‐
benzamide, D2O exchangeable); MS (m/z): 467 (M+, 18.39%), 305
(8.33%), 375 (100%, base peak), and 76 (16.15%); Anal. calcd. for
C24H25N3O5S (467.54): C, 61.66; H, 5.39; N, 8.99. Found: C, 61.51; H,
5.45; N, 8.85.
–C6H4OCH3), 7.63 (d, 2H, J = 8.00 Hz, Ar‐H, H‐2
&
H‐6
of –C6H4OCH3), 7.71 (d, 2H, J = 12.00 Hz, Ar‐H, H‐2 & H‐6 of
–C6H4CONH), 7.93–7.98 (m, 6H, Ar‐H, H‐3 & H‐5 of –C6H4CONH &
4H of –C6H4COOC2H5), 8.10 (s, 1H, C═CH–Ph), 10.47 (s, 1H, NH,
–C6H4CONH, D2O exchangeable), and 10.74 (s, 1H, NH,
–NHC6H4COOC2H5, D2O exchangeable); MS (m/z): 559 (M+, 7.24%),
360 (14.29%), 458 (100%, base peak), and 91 (32.04%); Anal. calcd.
for C29H25N3O7S (559.59): C, 62.25; H, 4.50; N, 7.51. Found: C,
62.09; H, 4.66; N, 7.68.
|
Docking studies
N‐Benzyl‐4‐{2‐[5‐(4‐methoxybenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]
acetamido}benzamide (7d)
4.2
Yield, 60%; m.p. 262–264°C; IRνmax (cm−1): 3,275, 3,109 (2NH), 3,032
(CH aromatic), 2,931 (CH aliphatic), 1,743, 1,693, and 1,631 (4C═O);
1H‐NMR (400 MHz, DMSO‐d6): 3.86 (s, 3H, –OCH3), 4.47 (s, 2H,
–CH2Ph), 4.54 (s, 2H, –NCH2CO), 7.16 (d, 2H, J = 7.40 Hz, Ar‐H, H‐3
& H‐5 of –C6H4OCH3), 7.26–7.33 (m, 5H, Ar‐H, –C6H5), 7.65 (d, 4H,
Ar‐H, H‐2 & H‐6 of –C6H4OCH3, J = 7.40 Hz & H‐2 & H‐6 of
–C6H4CONH, J = 8.80 Hz), 7.90 (d, 2H, J = 8.80 Hz, Ar‐H, H‐3 & H‐5
of –C6H4CONH), 7.98 (s, 1H, C═CH–Ph), 8.96 (s, 1H, –NHCH2Ph,
D2O exchangeable), and 10.66 (s, 1H, –NH‐benzamide, D2O ex-
changeable); Anal. calcd. for C27H23N3O5S (501.56): C, 64.66; H,
4.62; N, 8.38. Found: C, 64.56; H, 5.12; N, 8.84.
In the present study, all the target compounds were subjected to a
docking study to explore their binding mode toward VEGFR‐2 en-
zyme. All modeling experiments were performed using the Molsoft
program, which provides a unique set of tools for the modeling of
protein/ligand interactions. It predicts how small flexible molecule
such as substrates or drug candidates bind to a protein of a known
three‐dimensional structure represented by grid interaction poten-
tials (http://www.molsoft.com/icm_pro.html). Each experiment used
the biological target VEGFR‐2 downloaded from the Brookhaven
Protein Data Bank (http://www.rcsb.org/pdb/explore/explore.do?
structureId=1YWN). To qualify the docking results in terms of ac-
curacy of the predicted binding conformations, as compared with the
experimental procedure, the reported VEGFR‐2 inhibitor drugs, va-
talanib and sorafenib, were used as reference ligands.
4‐{2‐[5‐(4‐Methoxybenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐
acetamido}‐N‐(p‐tolyl)benzamide (7e)
Yield, 65%; m.p. 270–272°C; IRνmax (cm−1): 3,190, 3,116 (2NH), 3,039
(CH aromatic), 2,939 (CH aliphatic), 1,743, 1,681, and 1,647 (4C═O);
1H‐NMR (400 MHz, DMSO‐d6): 2.27 (s, 3H, CH3), 3.84 (s, 3H,
–OCH3), 4.58 (s, 2H, –NCH2CO), 7.12 (d, 2H, J = 8.00 Hz, Ar‐H, H‐3 &
H‐5 of –C6H4OCH3), 7.14 (d, J = 8.40 Hz, 2H, Ar‐H, H‐3 & H‐5 of
–C6H4CH3), 7.62 (d, 2H, J = 8.40 Hz, Ar‐H, H‐2 & H‐6 of –C6H4CH3),
7.67 (d, 2H, J = 8.00 Hz, Ar‐H, H‐2 & H‐6 of –C6H4OCH3), 7.71 (d, 2H,
|
4.3
In vitro cytotoxic activity
The cytotoxicity assays were performed at the Pharmacology and
Toxicology Department, Faculty of Pharmacy, Al‐Azhar Uni-
versity, Cairo, Egypt. Different cancer cell lines—HepG2, MCF‐7,
and HCT116—were purchased from American Type Culture
Collection (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 penicillin, and 10% of heat‐inactivated fetal
J = 8.00 Hz, Ar‐H, H‐2,
J = 8.00 Hz, Ar‐H, H‐3
&
&
H‐6 of –C6H4CONH), 7.95 (d, 2H,
H‐5 of –C6H4CONH), 7.98 (s, 1H,
C═CH–Ph), 10.08 (s, 1H, NH, –NHC6H4CH3, D2O exchangeable), and
10.70 (s, 1H, NH, –NH‐benzamide, D2O exchangeable); 13C‐NMR
(100 MHz, DMSO‐d6): 20.95, 44.53, 56.01, 115.51 (2), 118.07,

15 of 16
EL‐ADL ET AL.
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bovine serum in a humidified, 5% (v/v) CO2 atmosphere at 37°C.
Cytotoxicity assay by MTT.
ACKNOWLEDGMENTS
The authors extend their appreciation and gratitude to Dr. Tamer
Abdel‐Ghany, Pharmacology and Toxicology Department, Faculty of
Pharmacy, Al‐Azhar University, Cairo, Egypt, for helping in the
pharmacological part.
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 then were incubated in a humidified atmosphere at 37°C for
24 hr. Then, cells were exposed to different concentrations of
compounds (0.1, 10, 100, and 1,000 µM) for 72 hr. Then, the viabi-
lity of treated cells was determined using MTT technique as follows:
Media were removed, cells were incubated with 200 μl of 5% MTT
solution/well (Sigma‐Aldrich, MO), and they were allowed to me-
tabolize the dye into colored insoluble formazan crystals for 2 hr.
The remaining MTT solution was discarded from the wells; the
formazan crystals were dissolved in 200 µl/well acidified iso-
propanol for 30 min, covered with aluminum foil, and continuously
shaked using a MaxQ 2000 plate shaker (Thermo Fisher Scientific
Inc, MI) at room temperature. Absorbance was measured at 570 nm
using a Stat FaxR 4200 plate reader (Awareness Technology, Inc.,
FL). The cell viability was expressed as percentage of control, and
the concentration that induces 50% of maximum inhibition of cell
proliferation (IC50) was determined using GraphPad Prism version 5
software (GraphPad software Inc, CA).[48–50]
CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.
ORCID
Khaled El‐Adl
http://orcid.org/0000-0002-8922-9770
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