Design, synthesis, docking, ADMET profile, and anticancer evaluations of novel thiazolidine-2,4-dione derivatives as VEGFR-2 inhibitors

Article abstract of DOI:10.1002/ardp.202000491

The anticancer activity of novel thiazolidine-2,4-diones was evaluated against HepG2, HCT-116, and MCF-7 cells. Among the tested cancer cell lines, HCT-116 was the most sensitive one to the cytotoxic effect of the new derivatives. In particular, compounds 18, 11, and 10 were found to be the most potent derivatives among all the tested compounds against the HepG2, HCT-116, and MCF-7 cancer cell lines, with IC₅₀ values ranging from 38.76 to 53.99 μM. The most active antiproliferative derivatives (7–14 and 15–19) were subjected to further biological studies to evaluate their inhibitory potentials against VEGFR-2. The tested compounds displayed a good-to-medium inhibitory activity, with IC₅₀ values ranging from 0.26 to 0.72 μM. Among them, compounds 18, 11, and 10 potently inhibited VEGFR-2 at IC₅₀ values in the range of 0.26–0.29 μM, which are nearly three times that of the sorafenib IC₅₀ value (0.10 μM). Although our derivatives showed lower activities than the reference drug, they could be useful as a template for future design, optimization, adaptation, and investigation to produce more potent and selective VEGFR-2 inhibitors with higher anticancer analogs. The ADMET profile showed that compounds 18, 11, and 10 do not violate any of Lipinski's rules and have a comparable intestinal absorptivity in humans. Also, the new derivatives could not inhibit cytochrome P3A4. Unlike sorafenib and doxorubicin, compounds 18, 11, and 10 are expected to have prolonged dosing intervals. Moreover, compounds 10 and 18 displayed a wide therapeutic index and higher selectivity against cancer cells as compared with their cytotoxicity against normal cells.

Full text of DOI:10.1002/ardp.202000491

Received: 28 December 2020
DOI: 10.1002/ardp.202000491
Revised: 23 February 2021
Accepted: 26 February 2021
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F U L L P A P E R
Design, synthesis, docking, ADMET profile, and anticancer
evaluations of novel thiazolidine‐2,4‐dione derivatives as
VEGFR‐2 inhibitors
Khaled El‐Adl1,2
| Helmy Sakr1 | Sanadelaslam S. A. El‐Hddad1
|
Abdel‐Ghany A. El‐Helby1 | Mohamed Nasser1 | Hamada S. Abulkhair3,4
1Pharmaceutical Medicinal Chemistry and
Drug Design Department, Faculty of
Pharmacy (Boys), Al‐Azhar University, Nasr
City, Cairo, Egypt
Abstract
The anticancer activity of novel thiazolidine‐2,4‐diones was evaluated against
HepG2, HCT‐116, and MCF‐7 cells. Among the tested cancer cell lines, HCT‐116
was the most sensitive one to the cytotoxic effect of the new derivatives. In
particular, compounds 18, 11, and 10 were found to be the most potent
derivatives among all the tested compounds against the HepG2, HCT‐116, and
MCF‐7 cancer cell lines, with IC50 values ranging from 38.76 to 53.99 µM. The
most active antiproliferative derivatives (7–14 and 15–19) were subjected
to further biological studies to evaluate their inhibitory potentials against
VEGFR‐2. The tested compounds displayed a good‐to‐medium inhibitory activity,
with IC50 values ranging from 0.26 to 0.72 µM. Among them, compounds 18, 11,
and 10 potently inhibited VEGFR‐2 at IC50 values in the range of 0.26–0.29 µM,
which are nearly three times that of the sorafenib IC50 value (0.10 µM). Although
our derivatives showed lower activities than the reference drug, they could be
useful as a template for future design, optimization, adaptation, and investigation
to produce more potent and selective VEGFR‐2 inhibitors with higher anticancer
analogs. The ADMET profile showed that compounds 18, 11, and 10 do not
violate any of Lipinski's rules and have a comparable intestinal absorptivity in
humans. Also, the new derivatives could not inhibit cytochrome P3A4. Unlike
sorafenib and doxorubicin, compounds 18, 11, and 10 are expected to have
prolonged dosing intervals. Moreover, compounds 10 and 18 displayed a wide
therapeutic index and higher selectivity against cancer cells as compared with
their cytotoxicity against normal cells.
2Pharmaceutical Chemistry Department,
Faculty of Pharmacy, Heliopolis University for
Sustainable Development, El‐Salam City,
Cairo, Egypt
3Pharmaceutical Organic Chemistry
Department, Faculty of Pharmacy (Boys),
Al‐Azhar University, Nasr City, 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 and
eladlkhaled74@azhar.edu.eg or
khaled.eladl@hu.edu.eg
K E Y W O R D S
anticancer agents, HCT‐116, HepG2, MCF‐7, molecular docking, thiazolidine‐2,4‐dione,
VEGFR‐2 inhibitors
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Arch Pharm. 2021;e2000491.
wileyonlinelibrary.com/journal/ardp © 2021 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.202000491

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EL‐ADL ET AL.
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1
INTRODUCTION
The molecular hybridization of the thiazolidine‐2,4‐diones (TZDs)
scaffold with different bioactive moieties has recently been pro-
posed to have a different mechanism of action with a broad spec-
trum of activities against numerous cancer cell lines. TZDs have
exhibited an antitumor activity in a wide variety of experimental
cancer models by affecting cell cycle, induction of cell differentia-
tion, and apoptosis as well as by inhibiting tumor angiogenesis.[1]
Angiogenesis, the process of formation of new blood vessels from
the existing vasculature, is a fundamental event in tumor growth
and metastasis.[2] Angiogenesis is one of the main molecular targets
in recent developments in cancer therapies.[3] Newly generated
blood vessels supply oxygen and essential nutrition, support tumor
growth, and later aid in the initiation of metastasis, which con-
tributes to more than 90% of deaths in various cancers.[4] Vascular
endothelial growth factor (VEGF) is one of the central regulators in
angiogenesis.[5] VEGFR‐2 is the major mediator of VEGF‐induced
pro‐angiogenesis signaling.[6] The binding of VEGF to VEGFR2 leads
to activation of tyrosine kinase, trans‐autophosphorylation, and
initiation of the extracellular signal‐regulated kinase.[7,8] Blockade
of angiogenesis is one of the most promising strategies to treat
malignancies. Shah et al.[9] reported that TZD derivative ciglitazone
(I) (Figure 1) significantly decreased the VEGF production in human
granulosa cells in an in vitro model. Extensive studies were re-
ported on the synthesis of several 5‐benzylidenethiazolidine‐2,4‐
dione derivatives as potent anticancer agents[10–13] and potent
VEGFR‐2 inhibitors, for example, compound II.[9] Numerous reports
on VEGFR‐2 inhibitors, including the commercialized sunitinib (III)
(Figure 1), have been published.[14,15] Sorafenib (Nexavar)® (IV)
(Figure 1) is also a potent VEGFR‐2 inhibitor and has been approved
as an antiangiogenic drug.[16,17]
FIGURE 2 The basic structural requirements for sorafenib and
sunitinib as reported VEGFR‐2 inhibitors. ATP, adenosine
triphosphate; HBA, hydrogen‐bond acceptor; HBD, hydrogen‐bond
donor; VEGFR, vascular endothelial growth factor
(Michigan Cancer Foundation‐7 [MCF‐7]).[23,24] VEGFR‐2 inhibitors
were reported to impair the in vitro endothelial differentiation and
to promote angiogenesis, suggesting a VEGFR‐2‐mediated mechan-
ism of antiproliferative activity in human colorectal carcinoma.[21]
The overexpression of VEGFR‐2 receptors in breast cancer has been
verified as a sponsor in the resistance of such cancer against the
chemotherapeutic effect of tamoxifen.[23]
VEGFR‐2 was reported to be markedly overexpressed in HCC
(HepG2) cells.[18,19] Blockade of VEGFR‐2 signaling revealed a no-
ticeable inhibition of both the growth and metastasis of hepatocel-
lular carcinoma (HCC).[19,20] Also, VEGFR‐2 regulates endothelial
differentiation and is essential to cell survival of both the human
colorectal carcinoma (HCT‐116)[21,22] and breast cancer cells
A study of the structure–activity relationships (SAR) and common
pharmacophoric features shared by sorafenib and various VEGFR‐2
inhibitors revealed that most VEGFR‐2 inhibitors shared four main
FIGURE 1 Reported vascular endothelial growth factor‐2 inhibitors

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features, as shown in Figure 2.[25,26] The first feature is the flat het-
eroaromatic ring system that contains at least one N‐atom as the core
structure of most inhibitors, which occupied the catalytic adenosine
triphosphate (ATP)‐binding domain. The second feature is a hydro-
phobic spacer (central aryl ring), occupying the linker region between
the ATP‐binding domain and the DFG domain of the enzyme.[27] The
third one is a linker containing a functional group acting as a phar-
macophore (e.g., amino or urea) that possesses both H‐bond acceptor
(HBA) and donor (HBD) to bind with two crucial residues (Glu885 and/
or Asp1046) in the DFG (Asp–Phe–Gly) motif, an essential tripeptide
sequence in the active kinase domain. The NH motifs of the urea or
amide moiety usually form one hydrogen bond with Glu885, whereas
the C═O motif forms another hydrogen bond with Asp1046. Finally,
the fourth feature, the terminal hydrophobic moiety of the inhibitors
occupies the newly created allosteric hydrophobic pocket. Thus, hy-
drophobic interactions are usually attained in this allosteric binding
region.[28] The [4‐chloro‐3‐(trifluoromethyl)phenyl]urea and N‐[2‐
(diethylamino)ethyl]carboxamide tails of sorafenib and sunitinib, re-
spectively, were located at the solvent region of the VEGFR‐2 receptor.
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.[29,30]
Depending on ligand‐based drug design, especially a molecular
hybridization approach that involves the coupling of two or more
groups with relevant biological properties,[31–33] molecular hy-
bridization of 5‐[(4‐chloro/2,4‐dichloro)benzylidene]thiazolidine‐2,
4‐diones and other effective antitumor moieties was carried out in
an attempt to obtain new molecules with potent antitumor activity.
In view of the abovementioned findings and based on our con-
tinuous efforts to develop new anticancer agents,[70,34–39] especially
VEGFR‐2 inhibitors,[12,13,40–44] the goal of our work was to synthe-
size 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 bioisos-
teric modification strategies of VEGFR‐2 inhibitors at four different
positions (Figure 3).
Our target compounds were designed to have different spacers
and linkers while retaining the main pharmacophoric features of
sorafenib, hoping to obtain more potent VEGFR‐2 inhibitors. First, a
bioisosteric approach was adopted in the target 4‐chloro and/or 2,4‐
dichlorobenzylidenes to replace the hydrophobic group‐substituted
phenyl tail of the reported ligand sorafenib. The second strategy is to
use TZD to replace the central aryl ring of the lead structure, aiming
to increase the VEGFR‐2 binding affinity. The third strategy is using
FIGURE 3 Structural similarities and
pharmacophoric features of VEGFR‐2
inhibitors and designed compounds.
VEGFR, vascular endothelial growth
factor‐2

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ester and/or acetamide linkers that contain HBA–HBD functional
groups. In addition, the pyridine ring and indolin‐2‐one of sorafenib
and sunitinib, respectively, were replaced by their bioisosteres,
heteroaromatic, and/or substituted aromatic moieties. Lastly, the
substitution pattern was selected to ensure different electronic and
lipophilic environments that could influence the activity of the target
compounds. These modifications were performed to carry out
further elaboration of the TZD scaffolds and to explore a valuable
SAR. The designed target derivatives were synthesized and eval-
uated as potential VEGFR‐2 inhibitors and antitumor agents against
three human tumor cell lines, namely HCC type (HepG2), breast
cancer (MCF‐7), and human colorectal carcinoma‐116 (HCT‐116).
5‐fluoro‐2‐oxoindolin‐3‐ylidene moieties of the reference ligands sor-
afenib and sunitinib, respectively. In addition, 4‐chlorobenzylidene and
2,4‐dichlorobenzylidine moieties were designed to replace the hydro-
phobic tails of the reference ligands sorafenib and sunitinib. Moreover,
TZD was designed to replace the central aryl and pyrrole ring of the
reference ligands. Furthermore, the hydrophobic 4‐chlorobenzylidene
and/or 2,4‐dichlorobenzylidene moieties occupied the hydrophobic
groove formed by Asp1046, Cys1045, Hie1026, Ile892, Ile888, and
Glu885 (Figures 4 and 5).
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2.2
Chemistry
The adopted synthetic strategy for the preparation of the target
compounds (4–19) is depicted in Scheme 1. The synthesis was in-
itiated by cyclocondensation of thiourea with chloroacetic acid to
afford TZD (1),[12,13] which underwent further condensation reac-
tion with 4‐chlorobenzaldehyde and/or 2,4‐dichlorobenzaldehyde
to afford 5‐(4‐chlorobenzylidene)thiazolidine‐2,4‐dione and/or
5‐(2,4‐dichlorobenzylidene)thiazolidine‐2,4‐dione (2a,b), respec-
tively. Heating the latter benzylidines with an alcoholic solution of
potassium hydroxide afforded the corresponding potassium salts
(3a,b). The ester derivatives (4–6) were readily obtained by reacting
the potassium salt 3a under reflux with the appropriate
α‐chloroester derivative.[49–54] However, α‐chloroacetyl chloride
was allowed to react with a variety of aromatic amines to obtain the
corresponding α‐chloro‐N‐arylamides.[35,55] Treating the potassium
salt of 3a with the freshly prepared α‐chloro‐N‐arylamides
furnished the corresponding amide derivatives 7–14.[44,56–58]
Alternatively, the potassium salt 3b was refluxed with the same set
of α‐chloro‐N‐arylamides derivatives to afford our target amide
derivatives 15–19.
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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[15,44–48] (Figure 3), which include the following: the pre-
sence of five‐membered heterocyclic rings, 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. These scaffolds are linked to (un)substituted hydro-
phobic moieties through ester and/or acetamide linkers containing
HBA–HBD, which interact as an HBA through their C═O and as
HBD through their NH atom with the essential amino acid residues
Asp1046 and/or Glu885. Also, the heteroaromatic pyridine or
thiazole and substituted phenyl rings with hydrophilic carboxylic
and/or carboxamide groups were designed to replace the pyridine and
FIGURE 4 Superimposition of compound 18 and sorafenib inside the binding pocket of 3B8Q

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FIGURE 5 Superimposition of compound 11 and sorafenib inside the binding pocket of 3B8Q
O
O
O
NH
Cl
ii)
OH
i)
O
NH
O
S
S
S
Ar
2a-b
iii)
H2N
NH2
1
O
Ar
NK
S
O
3a-b
v); 3a
iv); 3a
vi); 3b
Cl
Cl
Cl
Cl
O
O
N
O
R
O
O
O
X
S
S
N
R2
S
Ar
N
O
N
H
N
H
R1
O
O
O
4-6
15-19
7-14
7: Ar = C6H5
4: R1 = H, R2 = CH3
5: R1 = H, R2 = C2H5
15: X = C, R = H
16: X = C, R = CH3
17: X = C, R = OCH3
18: X = C, R = COOH
19: X = N, R = H
11: Ar = 4-NH2COC6H4
12: Ar = 4-NO2C6H4
13: Ar = Pyridin-2-yl
14: Ar = Thiazol-2-yl
8: Ar = 4-CH3C6H4
9: Ar = 4-COCH3C6H4
10: Ar = 4-COOHC6H4
6: R1 = CH3, R2 = C2H5
SCHEME 1 Synthetic route for the preparation of the target compounds 4–19. Reagents and conditions: (i) HCl, 90°C, 3 h, 90%;
(ii) ArCHO/AcOH/AcONa, 90°C, 3 h, 74–80%; (iii) KOH/C2H5OH, 90°C, 1h, 95%; (iv) ClCH(R1)COOR2/DMF/K2CO3, water bath, 5 h,
81–90%; (v) ClH2CONH–Ar/DMF/K2CO3, water bath, 4 h, 55–92%; (vi) ClCH2CONH–Ar/DMF/K2CO3, water bath, 4 h, 60–70%.
DMF, dimethylformamide

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The process of chemical reactions was monitored by the thin‐
layer chromatography (TLC) approach. The structure and purity of
each new derivative were confirmed on the basis of spectral data and
elemental analysis. In all cases, a new typical ester and amide car-
bonyl stretching bands between 1681 and 1693 cm−1 were observed
in the infrared (IR) spectra. These findings confirm the tethering of
the ester and acetanilide fragments with the TZD nucleus. The
amidic NH group revealed a D2O‐exchangeable signal at the range of
10.32–11.05 ppm in 1H nuclear magnetic resonance (NMR) spectra
of compounds 7–14 and 15–19. Also, IR spectra of these amides
showed stretching bands in the range of 3232 and 3282 cm−1, sig-
nifying the secondary amide NH functionality. The most principal
feature in the IR and 1H NMR spectra of the ester derivatives 4–6
was the appearance of a distinctive carbonyl ester stretching band
around 1790 cm−1 and the protons of added alkyl ester moieties with
their relevant signals between 1.22 and 4.53 ppm.
TABLE 1 In vitro cytotoxic activities of the newly synthesized
compounds against HepG2, HCT‐116, and MCF‐7 cell lines and
VEGFR‐2 kinase assay
IC50 (µM)a
Compound
HepG2
HCT‐116
MCF‐7
VEGFR‐2
NTb
4
>100
>100
>100
5
>100
91.67 5.3
83.91 4.9
50.42 3.5
57.56 3.0
44.34 3.3
46.26 4.7
48.34 4.5
69.87 4.1
54.50 2.8
63.85 2.5
74.79 4.3
62.09 4.8
43.66 4.5
43.54 4.4
65.02 4.2
5.47 0.3
8.07 0.8
>100
NTb
6
>100
>100
NTb
7
94.35 5.2
82.69 4.8
53.75 3.9
42.34 3.2
40.26 3.3
72.72 4.6
64.19 4.2
92.35 5.2
71.02 3.8
65.98 4.8
52.86 3.7
38.76 2.9
70.11 6.1
9.18 0.6
7.94 0.6
90.27 5.2
77.36 4.9
58.65 3.8
53.99 3.7
52.34 3.7
71.28 4.5
75.85 4.9
89.06 3.8
59.34 3.9
49.87 4.6
43.20 4.5
50.13 3.5
55.38 4.1
7.26 0.3
6.75 0.4
0.67 0.03
0.61 0.03
0.34 0.06
0.29 0.03
0.28 0.03
0.68 0.03
0.63 0.05
0.72 0.03
0.62 0.02
0.49 0.04
0.32 0.02
0.26 0.03
0.58 0.05
0.10 0.02
NTb
8
9
10
11
12
13
14
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2.3
In vitro cytotoxic activity
15
The antiproliferative activity of the newly synthesized TZD derivatives
4–19 was examined against three human tumor cell lines, namely HCC
(HepG2), colorectal carcinoma (HCT‐116), and breast cancer (MCF‐7),
using 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide
(MTT) colorimetric assay, as described by Mosmann.[59] 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 h of incubation,
calculated from the concentration–inhibition response curve and
summarized in Table 1. From the obtained results, it was explicated
that most of the prepared compounds displayed a good‐to‐low growth
inhibitory activity against the tested cancer cell lines. Investigations of
the cytotoxic activity indicated that HCT‐116 was the cell line most
sensitive to the influence of the new derivatives. In particular, com-
pounds 18, 11, and 10 were found to be the most potent derivatives
among all the tested compounds against HepG2, HCT‐116, and MCF‐
7 cancer cell lines, with IC50 = 38.76 2.9, 43.54 4.4, 50.13 3.5 µM;
40.26 3.3, 48.34 4.5, 52.34 3.7 µM; and 42.34 3.2, 46.26 4.7,
53.99 3.7 µM, respectively.
16
17
18
19
Sorafenib
Doxorubicin
aIC50 values are the mean SD of three separate experiments.
bNT: Compounds not tested for their VEGFR‐2 inhibitory activity.
91.67 5.3 µM, respectively, exhibited the lowest cytotoxicity. How-
ever, compound 4 with IC50 >100 showed no cytotoxic activity.
Cytotoxicity evaluation against MCF‐7 cell line revealed that
compounds 17 and 16 displayed good anticancer activities with
IC50 = 43.20 4.5 and 49.87 4.6 µM, respectively. Compounds 19, 9,
15, 12, and 13 with IC50 = 55.38 4.1, 58.65 3.8, 59.34 3.9,
71.28 4.5, and 75.85 4.9 µM, respectively, displayed modest cyto-
toxicity. Compounds 8, 14, and 7 with IC50 = 77.36 4.9, 89.06 3.8,
and 90.27 5.2 µM, respectively, exhibited the lowest cytotoxicity.
However, compounds 4, 5, and 6 with IC50 >100 showed no cytotoxic
activity.
With respect to the HepG2 HCC cell line, compounds 17, 9, 13,
16, 19, 15, and 12 displayed modest anticancer activities, with IC50
=
52.86 3.7, 53.75 3.9, 64.19 4.2, 65.98 4.8, 70.11 6.1, 71.02
3.8, and 72.72 4.6 µM, respectively. Compounds 7, 8, and 14 with
IC50 values ranging from 82.69 4.8 to 94.35 5.2 µM exhibited
the lowest cytotoxicity. However, compounds 4, 5, and 6 with IC50
>100 showed no cytotoxic activity.
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2.4
In vitro VEGFR‐2 kinase assay
The newly synthesized 5‐(4‐chlorobenzylidene)thiazolidine‐2,4‐dione
derivatives 7–14 and 5‐(2,4‐dichlorobenzylidene)‐thiazolidine‐2,4‐
dione derivatives 15–19 were evaluated for their inhibitory activities
against VEGFR‐2 by using an anti‐phosphotyrosine 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.
Cytotoxicity evaluation against colorectal carcinoma (HCT‐116)
cell line revealed that compounds 17, 9, and 7 displayed good antic-
ancer activities with IC50 = 43.66 4.5, 44.34 3.3, and 50.42 3.5 µM,
respectively. Compounds 8, 12, 13, 14, 15, 16, and 19 displayed
modest anticancer activities with IC50 values ranging from 54.50 2.8
to 74.79 4.3 µM. Compounds 6 and 5 with IC50 = 83.91 4.9 and

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Sorafenib was used as a positive control in this assay. The tested
compounds displayed a good‐to‐medium inhibitory activity with IC50
values ranging from 0.26 0.03 to 0.72 0.03 µM. Among them,
compounds 18, 11, and 10 potently inhibited VEGFR‐2 at IC50 values
of 0.26 0.03, 0.28 0.03, and 0.29 0.03 µM, respectively, which
are nearly more than one‐third of that of sorafenib IC50 value
(0.10 0.02 µM). Also, compounds 17, 9, and 16 possessed good
VEGFR‐2 inhibition with IC50 values of 0.32 0.02, 0.34 0.06, and
0.49 0.04 µM, respectively. However, compounds 7, 8, 12, 13, 14,
15, and 19 exhibited moderate VEGFR‐2 inhibition with IC50 values
ranging from 0.58 0.05 to 0.72 0.03 µM.
hydrophilic electron‐withdrawing substituents, for example, 11, 10,
and 9, showed higher activities than that with the hydrophobic
electron‐donating one, 8, and the unsubstituted one, 7, against the
cell lines HepG2, HCT‐116, and MCF‐7. Compound 11 with car-
boxamide substitution exhibited higher activities than 10 with car-
boxylic and 9 with acetyl substitution against both HepG2 and MCF‐
7 cell lines, respectively, but it displayed a lower activity against
HCT‐116 cell line. The electron‐withdrawing nitro derivative 12
displayed higher activities than that with the electron‐donating
methyl one, 8, and the unsubstituted one, 7, against both HepG2 and
MCF‐7 cell lines, respectively, but it displayed a lower activity
against HCT‐116 cell line. Furthermore, the terminal pyridine tail, as
in compound 13, exhibited higher activities than thiazole, as in
compound 14, and the phenyl tail 7 against both HepG2 and MCF‐7
cell lines, respectively, but it displayed a lower activity against HCT‐
116 cell line.
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2.5
SAR
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 of our compounds, which inter-
act as HBAs through their carbonyl group and as HBD through their
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, our derivatives formed
hydrophobic interactions through the attached hydrophobic moi-
eties. The effect of replacement of pyridine and 5‐fluoro‐2‐
oxoindolin‐3‐ylidene moieties of sorafenib and sunitinib, respec-
tively, with the 4‐chlorobenzylidene and/or 2,4‐dichlorobenzylidene
on the antitumor activities also was noticed. These 5‐(4‐chloro/2,4‐
dichloro)benzylidene moieties occupied the same hydrophobic
pocket that was occupied by the pyridine moiety of the standard
ligand. Moreover, the TZD was designed to replace the central aryl
and pyrrole ring of the reference ligands sorafenib and sunitinib,
respectively, and enable the target compounds to form new H‐bonds
through their 2‐carbonyl groups with the essential amino acid re-
sidue Lys866. The data obtained revealed that the tested compounds
displayed different levels of anticancer activity and possessed a
distinctive pattern of selectivity against the HCT‐116 cell lines.
Generally, the spacers, linkers (HBA–HBD), lipophilicity, and elec-
tronic nature of substituents exhibited an important role in antic-
ancer activity. The 2,4‐dichlorobenzylidene moiety exhibited higher
anticancer activities than the 4‐chlorobenzylidene moiety, which may
be attributed to the higher lipophilicity of two lipophilic chloro
atoms.
In the third group, 15–19, the terminal phenyl tails were re-
placed by another heteroaromatic pyridine ring, as in compound 19.
In the other derivatives, they were unsubstituted, as in compound
15, and 4‐substituted with different hydrophobic, hydrophilic,
electron‐donating, and/or electron‐withdrawing groups. Generally,
compounds with
a hydrophilic electron‐withdrawing carboxylic
group, for example, 18, have higher activities than those with hy-
drophobic electron‐donating methoxy group, for example, 17, and
the hydrophobic electron‐donating methyl group, 16, against both
HepG2 and HCT‐116, respectively. However, the more hydrophobic
electron‐donating methoxy group, for example, 17, and the hydro-
phobic electron‐donating methyl group, 16, exhibited higher activ-
ities than the hydrophilic electron‐withdrawing carboxylic group, for
example, 18, against MCF‐7 cell line, respectively. Compounds with
substitutions at the phenyl tail, for example, 18, 17, and 16, displayed
higher activities than the unsubstituted one, for example, 15, against
HepG2, HCT‐116, and MCF‐7 cell lines. Furthermore, the terminal
pyridine tail, as in compound 19, exhibited higher activities than the
phenyl tail 15 against HepG2, HCT‐116, and MCF‐7 cell lines,
respectively.
Moreover, the dichloro derivatives with unsubstituted phenyl
group 15 and with 4‐methylphenyl group 16 exhibited higher an-
ticancer activities than the monochloro derivatives 7 and 8 against
both HepG2 and MCF‐7 cell lines, respectively, but they displayed
lower activity against HCT‐116 cell line, respectively. Furthermore,
the dichloro derivative with 4‐carboxylicphenyl group 18 exhibited
higher anticancer activities than the monochloro derivative 10
against HepG2, HCT‐116, and MCF‐7 cell lines, respectively.
In addition, the amide derivatives 7–14 in series 2 displayed
higher activities than the ester derivatives 4–6 in series 1 against
HepG2, HCT‐116, and MCF‐7 cell lines.
From the structure of the synthesized derivatives and the data
shown in Table 1, we can divide these tested compounds into three
groups. The first group consists of compounds 4–6, where ethyl
propionate ester derivative 6 showed higher activities than the ethyl
acetate 5 and the methyl one 4. In the second group, 7–14, the
terminal phenyl tails were replaced by another heteroaromatic pyr-
idine ring, as in compound 13, and/or thiazole ring, as in compound
14. In the other derivatives, terminal phenyl tails were replaced with
an (un)substituted phenyl, as in compound 7, or para‐substituted
with different hydrophobic, hydrophilic, electron‐donating, and/or
electron‐withdrawing groups. Generally, compounds with more
The data obtained from VEGFR‐2 inhibition assay concluded
that generally compounds with 2,4‐dichlorobenzylidene moiety, for
example, 15, 16, and 18, exhibited higher VEGFR‐2 inhibition activities
than that with 4‐chlorobenzylidene moiety, for example, 7, 8, and 10,
respectively. The phenyl tail substituted with hydrophilic electron‐
withdrawing carboxylic and aminocarbonyl groups, either in

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EL‐ADL ET AL.
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2,4‐dichlorobenzylidene and/or 4‐chlorobenzylidene derivatives, as in
compounds 18, 11, and 10 respectively, displayed the highest activ-
ities at IC50 values of 0.26 0.03, 0.28 0.03, and 0.29 0.03 µM,
respectively. In 4‐chlorobenzylidene derivatives, compounds with
acetyl substituent 9 showed higher activities than that with methyl 8,
pyridine 13, unsubstituted one 7, nitro substituent 12, and thiazole
hetero ring 14, respectively. Furthermore, in 2,4‐dichlorobenzylidene
derivatives, compounds with methoxy substituent, for example, 17,
displayed a higher activity than methyl 16, heteroaromatic pyridine
19, and the unsubstituted phenyl 15, respectively.
clearance values as compared with new ligands. Thus, sorafenib and
doxorubicin could be excreted faster, and are consequently expected
to have shorter dosing intervals. Unlike sorafenib and doxorubicin,
compounds 18, 11, and 10 displayed a slower clearance rate, which
reflects the preference of possible prolonged dosing intervals. The
last studied parameter in the ADMET profiles of our synthesized
VEGFR‐2 inhibitors is toxicity. As presented in Table 2, sorafenib,
doxorubicin, and all the new compounds shared the disadvantage of
undesirable hepatotoxic effects. Unlike the carboxamide derivative
11, sorafenib and doxorubicin, 10, and 18 showed the preference of
higher maximum tolerated dose, which means the advantage of the
narrow therapeutic index of the former and the wide therapeutic
index of 10 and 18. The oral acute toxic doses of the new compounds
(LD50) are roughly the same as for the reference drugs (~2.50 for
new thiazolidine‐diones compared with 2.50 of sorafenib and 2.40 of
doxorubicin). Finally, the lower Minnow toxicity values of the new
derivatives as compared with doxorubicin reflect the higher se-
lectivity of 18, 11, and 10 against cancer cells over their cytotoxicity
against normal cells.
|
2.6
Absorption, distribution, metabolism,
excretion, and toxicity (ADMET) profiling study
In this study, an in silico analysis of the three most active compounds
(18, 11, and 10) was conducted to evaluate their physicochemical
properties and the suggested ADMET profiles. This study was con-
ducted with the aid of pkCSM descriptors algorithm protocol[60] and
according to the directions of Lipinski's rule.[61] A compound is ex-
pected to have good absorption properties if the molecule conforms
to at least three of Lipinski's rules: (i) HBD groups ≤5; (ii) HBA groups
≤10; (iii) molecular weight <500; (iv) log P < 5. In the present work,
reference anticancer agents sorafenib and doxorubicin were found to
violate three and one of Lipinski's rules, respectively. Gratifyingly,
new compounds constructed in this study do not violate any of
Lipinski's rules. Whereas the molecular weight of doxorubicin ex-
ceeded the specified limit by 43 atomic mass units (amu), our newly
designed ligands were below the limit by more than 50 amu. Also,
sorafenib, the standard VEGFR‐2 degrader, violated the optimum
log P value, whereas new ligands did not. All the three active deri-
vatives have five HBA groups and a number of HBD groups between
2 and 3, and these values are in line with Lipinski's rules. ADMET
profiles of these three new TZDs were tentatively evaluated to
examine their potentials of building up as good drug candidates.
From the obtained results (Table 2), we can conclude that these
three derivatives have an intestinal absorptivity in humans (58.6–77.9)
that is comparable to that of sorafenib (62.3) and better than the
intestinal absorptivity of doxorubicin (62.3–84.7). This preferred
property would make the new compounds easier to go along various
biological membranes.[62] Therefore, they may have a considerable
good bioavailability after oral administration. Regarding the central
nervous system (CNS) permeability, our synthesized thiazolidine-
diones 18, 11, and 10 demonstrated an equivalent ability as sorafenib
to penetrate the CNS (CNS permeability values approximately −2.2),
whereas the standard anticancer agent doxorubicin was unable to
penetrate (CNS permeability < −4.0).
|
2.7
Docking studies
In the present work, all modeling experiments were performed using
Molsoft software. Each experiment used VEGFR‐2 downloaded from
the Brookhaven Protein Databank (PDB ID: 3B8Q).[63]
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 substrate to
the active site (Figure 6). In addition, the affinity of any small mo-
lecule 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.[49,64–66] This relationship 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 affinity toward the receptor, and the
computed values reflected the overall trend (Table 3).
The proposed binding mode of sorafenib revealed an affinity value
of −94.12 kcal/mol and four H‐bonds. The N‐methylpicolinamide
moiety was stabilized by the formation of two H‐bonds with the es-
sential amino acid Cys919, where the pyridine N atom formed one
H‐bond with the NH of Cys919 (2.94 Å), whereas its NH group formed
one H‐bond with the carbonyl of Cys919 (2.07 Å). The urea linker
formed one H‐bond with the key amino acid Glu885 (2.40 Å) through
its NH group and one H‐bond with Asp1046 (2.08 Å) through its
carbonyl group. The N‐methylpicolinamide moiety occupied the hy-
drophobic ATP‐binding pocket formed by Leu1035, Lys920, Cys919,
Phe918, Glu917, Val848, and Leu840. Moreover, the central phenyl
ring occupied the hydrophobic pocket formed by Cys1045, Leu1035,
Thr916, Lys868, and Val848. Furthermore, the hydrophobic
3‐trifluromethyl‐4‐chlorophenyl moiety attached to the urea linker
It is also obvious that cytochrome P3A4, the major enzyme in-
cluded in the metabolism of drug, could be inhibited by the effect of
sorafenib, whereas doxorubicin and new ligands could not inhibit it.
Excretion was predicted on the basis of the total clearance, which is a
considerable parameter in determining dose intervals. Tabulated
data revealed that sorafenib and doxorubicin revealed higher total

EL‐ADL ET AL.
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|
TABLE 2 ADMET profile of the three most active compounds, sorafenib, and doxorubicin
Parameters
10
11
18
Sorafenib
Doxorubicin
Molecular properties
Molecular weight
Log P
416.842
3.7132
5
415.858
3.1139
5
451.287
4.3666
5
464.831
5.5497
4
543.525
0.0013
12
H‐acceptors
H‐donors
2
2
2
3
6
Surface area
168.524
169.070
178.827
185.111
222.081
Absorption
Water solubility
Caco2 permeability
Intestinal abs. in human
Skin permeability
P‐glycoprotein substrate
P‐glycoprotein I inhibitor
Distribution
−4.219
1.016
58.617
−2.735
Yes
−4.51
0.975
77.939
−2.854
Yes
−4.312
0.786
59.796
−2.735
Yes
−4.888
0.613
84.731
−2.776
Yes
−2.915
0.457
62.372
−2.735
Yes
No
Yes
No
Yes
No
VDss (human)
−1.247
−1.321
−2.315
−0.246
−1.129
−2.412
−1.173
−1.472
−2.197
−0.233
−1.682
−1.995
1.647
BBB permeability
CNS permeability
Metabolism
−1.379
−4.307
CYP3A4 substrate
CYP1A2 inhibitor
CYP3A4 inhibitor
Excretion
No
No
No
No
No
No
No
No
No
Yes
Yes
Yes
No
No
No
Total clearance
Renal OCT2 substrate
Toxicity
−0.279
−0.488
−0.29
−0.212
0.987
No
No
No
No
No
Max. tolerated dose (human)
0.022
2.595
Yes
−0.389
2.312
Yes
0.135
2.684
Yes
0.677
2.595
Yes
0.081
2.408
Yes
Oral rat acute toxicity (LD50
)
Hepatotoxicity
Skin sensitization
No
No
No
No
No
Minnow toxicity
1.022
1.153
0.343
−0.421
4.412
Abbreviations: BBB, blood–brain barrier; CNS, central nervous system; VDss, steady‐state volume of distribution.
occupied the hydrophobic pocket formed by Asp1046, Cys1045,
Hie1026, Ile892, Ile888, and Glu885 (Figure 7). 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.
These findings encourage us to use acetamide linkers resembling urea
of sorafenib, hoping to obtain potent VEGFR‐2 inhibitors.
the acetamide linker formed one H‐bond with Thr916 (2.46 Å). More-
over, the carbonyl group at position 2 of TZD formed one H‐bond with
Asp1044 (2.58 Å). The 2,4‐dichlorophenyl moiety occupied the hydro-
phobic groove formed by Asp1046, Cys1045, Hie1026, Ile892, Ile888,
and Glu885. Moreover, the TZD moiety occupied the hydrophobic
pocket formed by Asp1046, Cys1045, Glu885, and Lys868. The
4‐carboxylicphenyl moiety occupied the hydrophobic ATP‐binding
pocket formed by Leu1035, Lys920, Cys919, Phe918, Glu917,
Thr916, Val848, and Leu840 (Figure 8).
As planned, the proposed binding mode of compound 18 is virtually
the same as that of sorafenib, which revealed an affinity value of
−94.02 kcal/mol and four H‐bonds. The carboxylic group was stabilized
by the formation of two H‐bonds with Cys919, the essential amino acid
in ATP‐binding site, with distances of 1.35 and 2.72 Å. The NH group of
The proposed binding mode of compound 11 is virtually the same
as that of sorafenib and 18, which revealed an affinity value of

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EL‐ADL ET AL.
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FIGURE 6 Superimposition of some docked compounds inside the binding pocket of 3B8Q
−82.30 kcal/mol and four H‐bonds. The carboxamide group was stabi-
lized by the formation of two H‐bonds with the essential amino acid
Cys919 (1.37 and 2.09 Å). The carbonyl group of the acetamide linker
formed one H‐bond with Asp1044 (2.96 Å). Moreover, the carbonyl
group at position 2 of TZD formed another H‐bond with Asp1044
(1.68 Å). The 4‐chlorophenyl moiety occupied the hydrophobic groove
formed by Asp1046, Cys1045, Hie1026, Ile892, Ile888, and Glu885.
Moreover, the TZD moiety occupied the hydrophobic pocket formed by
Asp1046, Cys1045, Glu885, and Lys868. The 4‐carboxamidephenyl
moiety occupied the hydrophobic ATP‐binding pocket formed by
Leu1035, Lys920, Cys919, Phe918, Glu917, Thr916, Val848, and Leu840
(Figure 9).
by the formation of two H‐bonds with the essential amino acid Cys919
(1.65 and 1.91 Å). The carbonyl group of the acetamide linker formed
one H‐bond with Lys868 (2.24 Å). Moreover, the carbonyl group
at position 2 of TZD formed one H‐bond with Asp1044 (1.46 Å). The
4‐chlorophenyl moiety occupied the hydrophobic groove formed by
Asp1046, Cys1045, Hie1026, Ile892, Ile888, and Glu885. Moreover, the
TZD moiety occupied the hydrophobic pocket formed by Asp1046,
Cys1045, Glu885, and Lys868. The 4‐carboxylicphenyl moiety occupied
the hydrophobic ATP‐binding pocket formed by Leu1035, Lys920,
Cys919, Phe918, Glu917, Thr916, Val848, and Leu840 (Figure 10).
The proposed binding mode of compound 17 is virtually the
same as that of sorafenib and 18, which revealed an affinity value of
−79.09 kcal/mol and three H‐bonds. The methoxy group formed one
H‐bond through its oxygen atom with the essential amino acid Cys919
(2.03 Å). The NH group of the acetamide linker formed one H‐bond with
Thr916 (2.72 Å). Moreover, the carbonyl group at position 2 of TZD
formed one H‐bond with Asp1044 (2.58 Å). The 2,4‐dichlorophenyl
moiety occupied the hydrophobic groove formed by Asp1046, Cys1045,
Hie1026, Ile892, Ile888, and Glu885. Moreover, the TZD moiety
occupied the hydrophobic pocket formed by Asp1046, Cys1045,
Glu885, and Lys868. The 4‐methoxyphenyl moiety occupied the
hydrophobic ATP‐binding pocket formed by Leu1035, Lys920, Cys919,
Phe918, Glu917, Thr916, Val848, and Leu840 (Figure 11).
The proposed binding mode of compound 10 is virtually the same
as that of sorafenib and 18, which revealed an affinity value of
−80.54 kcal/mol and four H‐bonds. The carboxylic group was stabilized
TABLE 3 The calculated ΔG (free energy of binding) and binding
affinities for the ligands
Compound
ΔG (kcal/mol)
−57.63
−60.27
−60.49
−62.99
−69.89
−73.76
−80.54
−82.30
−62.53
Compound
ΔG (kcal/mol)
−64.34
4
13
5
14
−58.39
6
15
−68.24
From the obtained docking results (Table 3), we concluded that
the acetamide linker played an important role in increasing the
affinity toward the VEGFR‐2 enzyme. The lipophilicity of the 2,4‐
dichlorobenzylidene and 4‐chlorobenzylidene moieties played an
important role in the hydrophobic interactions and, consequently, in
increasing affinities toward VEGFR‐2 enzyme. The TZD enables the
new compounds to form H‐bonds through its carbonyl group at position
2 with the amino acid residue Asp1046. Furthermore, the heteroaro-
matic rings and/or the substituted phenyl groups with hydrophilic
7
16
−72.54
8
17
−79.09
9
18
−94.02
10
11
12
19
−67.84
Sorafenib
−94.12

EL‐ADL ET AL.
11 of 18
|
FIGURE 7 The predicted binding mode of sorafenib with VEGFR‐2. H‐bonded atoms are indicated by dotted lines
moieties such as carboxylic and or carboxamide groups are necessary
for activity, because the NH groups and/or O atoms as the HBD–HBA
motif contribute hydrogen‐bonding interactions with carbonyl and NH
groups of the essential amino acid Cys919 in the adenine‐binding site of
the receptor.
with that obtained from the biological screening. All the tested
compounds showed variable anticancer activities. novel
A
series of 5‐(4‐chlorobenzylidene)‐thiazolidine‐2,4‐dione deriva-
tives 4–14 and 5‐(2,4‐dichlorobenzylidene)thiazolidine‐2,4‐dione
derivatives 15–19 was designed, synthesized, and evaluated
for their anticancer activity against three human tumor cell
lines, HCC (HepG2), colorectal carcinoma (HCT‐116), and breast
cancer (MCF‐7) cell lines, targeting VEGFR‐2 enzyme. All the
tested compounds showed variable anticancer activities. HCT‐116
was the most sensitive cell line to the influence of the new deri-
vatives. In particular, compounds 18, 11, and 10 were found to be
the most potent derivatives among all the tested compounds
|
3
CONCLUSION
The molecular design was performed to investigate the binding
mode of the proposed compounds with VEGFR‐2 receptor. The
data obtained from the docking studies were highly correlated
FIGURE 8 The predicted binding mode of 18 with 3B8Q

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EL‐ADL ET AL.
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FIGURE 9 The predicted binding mode of 11 with 3B8Q
against HepG2, HCT‐116, and MCF‐7 cancer cell lines. The most
active antiproliferative derivatives 7–19 were selected to evalu-
ate their inhibitory activities against VEGFR‐2. The tested com-
pounds displayed good‐to‐moderate inhibitory activities. Among
them, compounds 18, 11, and 10 were found to be the most potent
VEGFR‐2 inhibitors. Also, compounds 17, 9, and 16 possessed
good VEGFR‐2 inhibition. However, other compounds possessed
moderate VEGFR‐2 inhibition effects. Furthermore, ADMET pro-
file was calculated for the three most active compounds, 18, 11,
and 10, as compared with sorafenib and doxorubicin as reference
drugs. Compounds 18, 11, and 10 did not violate any of Lipinski's
rules and had an intestinal absorptivity in humans (58.6–77.9) that
is comparable to that of sorafenib (62.3) and better than the in-
testinal absorptivity of doxorubicin (62.3–84.7). Also, the new
derivatives could not inhibit cytochrome P3A4. Unlike sorafenib
and doxorubicin, compounds 18, 11, and 10 displayed a slower
clearance rate, which reflects the preference of possible pro-
longed dosing intervals. Moreover, compounds 10 and 18
displayed a wide therapeutic index. Finally, the lower Minnow
toxicity values of the new derivatives as compared with doxor-
ubicin reflect the higher selectivity of 18, 11, and 10 against
cancer cells over their cytotoxicity against normal cells.
FIGURE 10 The predicted binding mode of 10 with 3B8Q

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|
FIGURE 11 The predicted binding mode of 17 with 3B8Q
|
4
EXPERIMENTAL
Chemistry
potassium salts (3a,b), and N‐aryl‐2‐chloroacetamide derivatives were
obtained according to the reported procedure.[12,13]
|
4.1
|
|
General method for the synthesis of alkyl
4.1.1
General
4.1.2
2‐[5‐(4‐chlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐
acetate (4 and/or 5) and ethyl 2‐[5‐(4‐
chlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐
propanoate (6)
All melting points were determined by the open capillary method on
a Gallenkamp melting point apparatus at the Faculty of Pharmacy,
Al‐Azhar University, and were uncorrected. The IR spectra were
recorded on a Pye Unicam SP 1000 IR spectrophotometer at
Microanalytical Unit, Faculty of Pharmacy, Cairo University, using
the potassium bromide disc technique. 1H NMR spectra were
recorded on a Bruker 400 MHz‐NMR spectrophotometer at the
Microanalytical Unit, Faculty of Pharmacy, Cairo University. 13C
NMR spectra were recorded on a Bruker 100 MHz‐NMR spectro-
photometer at the Microanalytical Unit, Faculty of Pharmacy, Cairo
University. Tetramethylsilane was used as the internal standard and
chemical shifts were measured in δ scale (ppm). The mass spectra
were carried out on the direct probe controller inlet part of a 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 within 0.4 of the theoretical values. The reactions
were monitored by TLC using TLC sheets precoated with UV fluor-
escent silica gel Merck 60 F254 plates and were visualized using
a UV lamp and different solvents as mobile phases.
Equimolar quantities of the potassium salt 3a (2.76 g, 0.01 mol) and
the appropriate α‐chloroester derivative (0.01 mol), namely methyl
chloroacetate, ethyl chloroacetate, and ethyl 2‐chloropropanoate, in
dimethylformamide (DMF) (20 ml) were heated on a water bath for
5 h in the presence of K2CO3 (1.38 g, 0.01 mol). The reaction mixture
was poured into ice water (200 ml) and stirred for 30 min. The ob-
tained solid was filtered and crystallized from ethanol to give the
target compounds 4–6, respectively.
Methyl 2‐[5‐(4‐chlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐
acetate (4)
Yield, 90%; m.p. 110–112°C; IRνmax (cm−1): 3089 (CH aromatic),
2948 (CH aliphatic), 1741, 1681 (3C═O); 1H NMR (400 MHz,
dimethyl sulfoxide [DMSO]‐d6): 3.69 (s, 3H, OCH3), 4.53 (s, 2H, CH2),
7.64 (d, 2H, Ar–H, H‐3 and H‐5 of –C6H4, J = 8.8), 7.70 (d, 2H, Ar–H,
H‐2, and H‐6 of –C6H4, J = 8.4), 8.03 (s, 1H, C═CH–Ph); anal. calcd.
for C13H10ClNO4S (311.74): C, 50.09; H, 3.23; N, 4.49. Found: C,
50.21; H, 3.40; N, 4.26.
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.
Ethyl 2‐[5‐(4‐chlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐
acetate (5)
Yield, 85%; m.p. 125–126°C; IRνmax (cm−1): 3090 (CH aromatic),
2951 (CH aliphatic), 1743, 1681 (3C═O); 1H NMR (400 MHz, DMSO‐
d6): 1.22 (t, 3H, –OCH2CH3, J = 6.0), 4.17 (q, 2H, –OCH2CH3, J = 6.8),
TZD (1), 5‐(4‐chlorobenzylidene)thiazolidine‐2,4‐dione and 5‐(2,4‐
dichlorobenzylidene)thiazolidine‐2,4‐dione (2a,b), the corresponding

14 of 18
EL‐ADL ET AL.
|
4.51 (s, 2H, CH2), 7.63 (d, 2H, Ar–H, H‐3, and H‐5 of –C6H4, J = 7.2),
7.69 (d, 2H, Ar–H, H‐2, and H‐6 of –C6H4, J = 8.8), 8.01 (s, 1H,
C═H–Ph); anal. calcd. for C14H12ClNO4S (325.76): C, 51.62; H, 3.71;
N, 4.30. Found: C, 51.88; H, 3.84; N, 4.57.
J = 8.0), 8.01 (s, 1H, C═CH–Ph), 10.32 (s, 1H, NH, D2O exchange-
able); anal. calcd. for C19H15ClN2O3S (386.85): C, 58.99; H, 3.91; N,
7.24. Found: C, 59.17; H, 4.06; N, 7.40.
N‐(4‐Acetylphenyl)‐2‐[5‐(4‐chlorobenzylidene)‐2,4‐dioxothiazolidin‐
3‐yl]acetamide (9)
Ethyl 2‐[5‐(4‐chlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐
propanoate (6)
Yield, 60%; m.p. 153–155°C; IRνmax (cm−1): 3277 (NH), 3062 (CH
aromatic), 2855 (CH aliphatic), 1755, 1693 (4C═O); 1H NMR
(400 MHz, DMSO‐d6): 2.52 (s, 3H, COCH3), 4.58 (s, 2H, CH2), 7.56 (d,
2H, Ar–H, H‐3, and H‐5 of –C6H4Cl), 7.62 (d, 2H, Ar–H, H‐2, and H‐6
of –C6H4Cl), 7.71 (d, 2H, Ar–H, H‐2, and H‐6 of –C6H4COCH3), 7.96
(d, 2H, Ar–H, H‐3, and H‐5 of –C6H4COCH3), 7.98 (s, 1H, C═CH–Ph),
10.45 (s, 1H, NH, D2O exchangeable); 13C NMR (100 MHz, DMSO‐d6):
34.58, 38.52, 118.91 (2), 122.63, 129.93 (3), 130.77 (2), 131.94,
132.21 (3), 132.35, 135.69, 143.18, 165.83, 167.36, 169.30; anal.
calcd. for C20H15ClN2O4S (414.86): C, 57.90; H, 3.64; N, 6.75. Found:
C, 58.16; H, 3.75; N, 6.93.
Yield, 81%; m.p. 133–135°C; IRνmax (cm−1): 3090 (CH aromatic),
2912 (CH aliphatic), 1735, 1693 (3C═O); 1H NMR (400 MHz, DMSO‐
d6): 1.16 (t, 3H, –OCH2CH3, J = 7.2), 1.52 (d, 3H, –CHCH3, J = 7.2),
4.14 (q, 2H, –OCH2CH3, J = 7.6), 5.11 (q, 1H, –CHCH3, J = 7.6), 7.63
(d, 2H, Ar–H, H‐3, and H‐5 of –C6H4, J = 8.8), 7.68 (d, 2H, Ar–H, H‐2,
and H‐6 of –C6H4, J = 8.4), 7.98 (s, 1H, C═CH–Ph); mass spectro-
scopy (MS) (m/z): 342.08 (M++2, 1.51%), 341.07 (M++1, 7.30%),
339.96 (M+, 19.33%), 338.98 (87.24%), 133.97 (100%, base beak),
120.92 (35.83%); anal. calcd. for C15H14ClNO4S (339.79): C, 53.02;
H, 4.15; N, 4.12. Found: C, 53.29; H, 4.37; N, 4.25.
4‐{2‐[5‐(4‐Chlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]acetamido}‐
benzoic acid (10)
|
4.1.3
General method for the synthesis of 2‐[5‐(4‐
chlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐N‐
[(un)substituted aryl]acetamide (7–14)
Yield, 73%; m.p. 167–169°C; IRνmax (cm−1): 3417 (OH), 3232 (NH),
3012 (CH aromatic), 2839 (CH aliphatic), 1735, 1685 (4C═O); 1H
NMR (400 MHz, DMSO‐d6): δ = 4.72 (s, 2H, CH2), 7.07 (s, 1H,
C═CH–Ph), 7.42–7.67 (m, 2H, Ar–H, H‐2, and H‐6 of –C6H4–Cl),
7.66–7.73 (m, 2H, H‐3, and H‐5 of C6H4–Cl), 7.75–7.88 (m, 2H, Ar–H,
H‐2, and H‐6 of C6H4–COOH), 7.90–8.26 (m, 2H, Ar–H, H‐3, and H‐5
of C6H4–COOH), 11.31 (s, 1H, NH, D2O exchangeable), 12.53 (s, 1H,
–OH, D2O exchangeable); 13C NMR (100 MHz, DMSO‐d6): 36.42,
125.55, 129.80 (3), 130.34 (2), 132.02 (3), 132.56 (2), 135.29 (2),
168.56 (2), 173.57, 174.34; MS (m/z): 416 (M+, 15.75%), 246
(10.34%), 78 (100%, base beak), 71 (23.12%); anal. calcd. for
Equimolar quantities of the potassium salt 3a (2.76 g, 0.01 mol) and
the appropriate N‐aryl‐2‐chloroacetamide derivative (0.01 mol) in
dry DMF (20 ml) were heated on a water bath for 4 h in the presence
of K2CO3 (1.38 g, 0.01 mol). After cooling to room temperature, the
reaction mixture was poured over crushed ice. The precipitated
solids were filtered, dried, and crystallized from ethanol to give
the corresponding target compounds 7–14, respectively.
2‐[5‐(4‐Chlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐N‐
phenylacetamide (7)
C19H13ClN2O5S (416.83): C, 54.75; H, 3.14; N, 6.72. Found: 54.97; H,
3.42; N, 6.83.
Yield, 70%; m.p. 150–151°C; IRνmax (cm−1): 3275 (NH), 3059 (CH
aromatic), 2924 (CH aliphatic), 1694, 1657, 1636 (3C═O); 1H NMR
(400 MHz, DMSO‐d6): 4.53 (s, 2H, –CH2), 7.08 (m, 1H, Ar–H, H‐4 of
–C6H5), 7.35 (m, 2H, Ar–H, H‐3, and H‐5 of –C6H5), 7.55 (m, 4H,
Ar–H, H–4, and H‐6 of –C6H5 and Ar–H, H‐3, and H‐5 of C6H4Cl),
7.68 (m, 2H, Ar–H, H‐3, and H‐6 of C6H4Cl), 8.01 (s, 1H, C═CH–Ph),
10.41 (s, 1H, NH, D2O exchangeable); 13C NMR (100 MHz, DMSO‐
d6): 44.55, 119.64 (2), 122.21, 124.21, 129.36 (2), 129.97 (2), 132.23
(2), 132.31, 132.78, 135.91, 138.83, 164.19, 165.67, 167.36; anal.
calcd. for C18H13ClN2O3S (372.83): C, 57.99; H, 3.51; N, 7.51. Found:
C, 58.26; H, 3.68; N, 7.75.
4‐{2‐[5‐(4‐Chlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]acetamido}‐
benzamide (11)
Yield, 92%; m.p. 190–192°C; IRνmax (cm−1): 3282 (NH), 3066 (CH aro-
matic), 2843 (CH aliphatic), 1739, 1681 (4C═O); 1H NMR (400 MHz,
DMSO‐d6): 3.98 (s, 2H, CH2), 7.25 (s, 2H, NH2) (D2O exchangeable),
7.63–7.65 (m, 4H, Ar–H, H‐2, H‐3, H‐5, and H‐6 of –C6H4Cl), 7.72–7.75
(m, 4H, Ar–H, H‐2, H‐3, H‐5, and H‐6 of benzamide), 7.94 (s, 1H, C═CH‐
Ph), 10.40 (s, H, NH) (D2O exchangeable); 13C NMR (100 MHz, DMSO‐
d6): 44.57, 119.59 (2), 122.18, 129.09, 129.67 (2), 130.22 (2), 130.35 (3),
131.17, 133.04, 136.47, 140.54, 152.92, 164.62, 166.46; MS (m/z):
416.98 (M++2, 9.67%), 415 (M+, 25.44%), 338.06 (100%, base beak),
305.65 (58.57%), 236.23 (43.73%), 71.19 (15.14%); anal. calcd. for
2‐[5‐(4‐Chlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐N‐(p‐tolyl)‐
acetamide (8)
C19H14ClN3O4S (415.85): C, 54.88; H, 3.39; N, 10.10. Found: C, 54.71;
Yield, 70%; m.p. 155–157°C; IRνmax (cm−1): 3278 (NH), 3066 (CH
aromatic), 2850 (CH aliphatic), 1751, 1693 (3C═O); 1H NMR
(400 MHz, DMSO‐d6): 2.26 (s, 3H, –CH3), 4.49 (s, 2H, –CH2), 7.12 (d,
2H, Ar–H, H‐3, and H‐5 of –C6H4–CH3, J = 8.0), 7.42 (d, 2H, Ar–H,
H‐2, and H‐6 of –C6H4–CH3, J = 6.4), 7.52 (d, 2H, Ar–H, H‐3, and H‐5
of –C6H4–Cl, J = 6.0), 7.69 (d, 2H, Ar–H, H‐2, and H‐6 of –C6H4Cl,
H, 3.70; N, 10.34.
2‐[5‐(4‐Chlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐N‐(4‐
nitrophenyl)acetamide (12)
Yield, 68%; m.p. 180–182°C; IRνmax (cm−1): 3259 (NH), 3065 (CH
aromatic), 2852 (CH aliphatic), 1754, 1693 (3C═O); 1H NMR

EL‐ADL ET AL.
15 of 18
|
(400 MHz, DMSO‐d6): 4.61 (s, 2H, CH2), 7.64 (d, 2H, Ar–H, H‐3, and
H‐5 of –C6H4Cl, J = 6.8), 7.70 (d, 2H, Ar–H, H‐2, and H‐6 of –C6H4Cl,
J = 8.4), 7.82 (d, 2H, Ar–H, H‐2, and H‐6 of –C6H4–NO2, J = 10.0),
8.00 (s, 1H, C═CH–Ph), 8.25 (d, 2H, Ar–H, H‐3, and H‐5 of
–C6H4–NO2, J = 8.0), 11.04 (s, 1H, NH, D2O exchangeable); anal.
calcd. for C18H12ClN3O5S (417.82): C, 51.74; H, 2.90; N, 10.06.
Found: C, 51.91; H, 3.11; N, 10.35.
and H‐5 of –C6H5), 7.55 (d, 1H, Ar–H, H‐5 of –C6H3(Cl2), J = 9.6),
7.62–7.64 (m, 2H, Ar–H, H‐2, and H‐6 of –C6H5), 7.69–7.71 (m, 2H,
Ar–H, H‐3, and H‐6 of –C6H3(Cl2)), 8.00 (s, 1H, C═CH–Ph), 10.41 (s,
1H, NH, D2O exchangeable); MS (m/z): 409.86 (M++2, 21.87%),
407.90 (M++1, 34.66%), 407.22 (M+, 32.75%), 405.89 (61.28%),
373.03 (65.48%), 370.87 (100%, base beak), 93.27 (32.13%), 77.07
(50.54%); anal. calcd. for C18H12Cl2N2O3S (407.27): C, 53.09; H,
2.97; N, 6.88. Found: C, 53.13; H, 2.67; N, 6.79.
2‐[5‐(4‐Chlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐N‐(pyridin‐2‐
yl)acetamide (13)
2‐[5‐(2,4‐Dichlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐N‐
(p‐tolyl)acetamide (16)
Yield, 68%; m.p. 182–184°C; IRνmax (cm−1): 3151 (NH), 3051 (CH
aromatic), 2765 (CH aliphatic), 1755, 1678 (3C═O); 1H NMR
(400 MHz, DMSO‐d6): 4.61 (s, 2H, CH2), 7.15 (m, 1H, Ar–H, H‐4 of
pyridine), 7.53 (d, 2H, Ar–H, H‐3, and H‐5 of –C6H4Cl, J = 8.8), 7.65
(d, 2H, Ar–H, H‐2, and H‐6 of –C6H4Cl, J = 10.8), 7.82 (m, 1H, Ar–H,
H‐5 of pyridine), 7.98 (s, 1H, C═CH–Ph), 8.00 (d, 1H, Ar–H, H‐6 of
pyridine, J = 9.6), 8.36 (d, 1H, Ar–H, H‐3 of pyridine, J = 8.4), 11.02 (s,
1H, –NH, D2O exchangeable); 13C NMR (100 MHz, DMSO‐d6): 55.95,
113.96, 115.37, 120.32, 125.79, 132.04, 132.51, 132.86, 132.96,
134.10, 138.89, 148.59, 161.79, 161.91, 165.24, 165.87, 168.67;
anal. calcd. for C17H12ClN3O3S (373.81): C, 54.62; H, 3.24; N, 11.24.
Found: C, 54.85; H, 3.45; N, 11.47.
Yield, 70%; m.p. 175–176°C; IR (KBr) νmax 3202, 3067, 2932, 1697
;
cm−1 1H NMR (400 MHz, DMSO‐d6): δ = 2.26 (s, 3H, –CH3), 4.50 (s,
2H, –CH2), 7.13 (d, 2H, Ar–H, H‐3, and H‐5 of –C6H4–CH3, J = 6.0),
7.44 (d, 2H, Ar–H, H‐2, and H‐6 of –C6H4–CH3, J = 6.8), 7.62–7.70
(m, 3H, Ar–H, H‐3, H‐5, and H‐6 of –C6H3(Cl2)), 8.01 (s, 1H,
–C═CH–Ph), 10.33 (s, 1H, NH, D2O exchangeable); 13C NMR
(100 MHz, DMSO‐d6): δ = 20.90, 44.48, 119.65 (2), 121.49, 129.72
(2), 129.90 (2), 130.67 (2), 131.29, 133.14, 133.34, 134.05, 136.36,
163.97, 165.78, 167.60; MS (m/z): 423.88 (M++2, 11.97%), 422.94
(M++1, 10.89%), 421.90 (M+, 32.71%), 419.91 (47.52%), 373.84
(1.25%), 106.99 (51.27%), 77.04 (52.96%), 72.04 (100%, base beak);
anal. calcd. for C19H14Cl2N2O3S (421.29): C, 54.17; H, 3.35; N, 6.65.
Found: C, 53.96; H, 3.45; N, 6.87.
2‐[5‐(4‐Chlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐N‐(thiazol‐2‐
yl)acetamide (14)
Yield, 55%; m.p. 185–187°C; IRνmax (cm−1): 3275 (NH), 3086 (CH
aromatic), 2850 (CH aliphatic), 1747, 1693 (3C═O); 1H NMR
(400 MHz, DMSO‐d6): 4.25 (s, 2H, –CH2), 7.34 (d, 1H, Ar–H, H‐5 of
thiazol), 7.62 (d, 2H, Ar–H, H‐3, and H‐5 of –C6H5Cl, J = 7.6), 7.69 (d,
2H, Ar–H, H‐2, and H‐6 of –C6H5Cl, J = 8.0), 7.74 (d, 1H, Ar–H, H‐4 of
thiazol), 7.97 (s, 1H, –C═CH–Ph), 11.05 (s, 1H, NH, D2O exchange-
able); MS (m/z): 381.44 (M++2, 18.26%), 380.77 (M++1, 50.58%),
330.37 (79.85%), 293.81 (100%, base beak), 239.02 (83.26%), 71.49
(49.68%); anal. calcd. for C15H10ClN3O3S2 (379.83): C, 47.43; H,
2.65; N, 11.06. Found: C, 47.69; H, 2.91; N, 11.28.
2‐[5‐(2,4‐Dichlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐N‐(4‐
methoxyphenyl)acetamide (17)
Yield, 65%; m.p. 172–174°C; IR (KBr) νmax 3333, 3036, 2924,
;
1705, 1663 cm−1 1H NMR (400 MHz, DMSO‐d6): δ = 3.81 (s, 3H,
OCH3), 3.83 (s, 2H, CH2), 7.08–7.10 (m, 4H, Ar–H, H‐2, H‐3, H‐5,
and H‐6 of –C6H4(OCH3)), 7.64–7.66 (m, 4H, Ar–H, 3H of
–C6H3(Cl2) and 1H, C═CH–Ph), 11.34 (s, 1H, NH, D2O ex-
changeable); 13C NMR (100 MHz, DMSO‐d6): δ = 34.58, 38.52,
118.73 (2), 121.86, 129.86 (3), 130.59 (4), 131.10, 133.27,
133.44, 142.35, 165.94, 167.61, 169.14; MS (m/z): 439 (M++3,
4.88%), 438 (M++2, 14.27%), 437 (M++1, 24.35%), 436 (M+,
15.82%), 120 (100%, base beak), 91 (38.97%); anal. calcd. for
|
4.1.4
General procedure for the synthesis of
C19H14Cl2N2O4S (436.01): C, 52.19; H, 3.23; N, 6.41. Found:
target compounds (15–19)
52.06; H, 3.49; N, 6.21.
Equimolar quantities of the potassium salt 3b (3.12 g, 0.01 mol) and
the appropriate N‐aryl‐2‐chloroacetamide derivative (0.01 mol) in
dry DMF (20 ml) were heated on a water bath for 4 h in the presence
of K2CO3 (1.38 g, 0.01 mol). After cooling to room temperature,
the reaction mixture was poured over crushed ice. The precipitated
solids were filtered, dried, and crystallized from ethanol to give the
corresponding target compounds 15–19, respectively.
4‐{2‐[5‐(2,4‐Dichlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐
acetamido}benzoic acid (18)
Yield, 70%; m.p. 176–178°C; IR (KBr) νmax 3414, 3233, 3032, 2928,
;
1730, 1697 cm−1 1H NMR (400 MHz, DMSO‐d6): δ = 4.54 (s, 2H,
CH2), 7.15 (m, H, Ar–H, H‐5 of –C6H3(Cl2)), 7.65–7.71 (m, 4H, Ar–H,
H‐3, and H‐6 of –C6H3(Cl2) and H‐2 and H‐6 of C6H4–COOH),
7.78–7.84 (m, 2H, Ar–H, H‐3, and H‐5 of C6H4–COOH), 8.02 (s, 1H,
C═CH–Ph), 10.67 (s, 1H, NH, D2O exchangeable), 12.20 (s, 1H, –OH,
D2O exchangeable); 13C NMR (100 MHz, DMSO‐d6): 44.43, 119.73,
119.96, 120.24, 127.05 (2), 130.53 (2), 130.59, 130.76, 134.12,
136.61, 141.69, 144.26, 144.44, 164.01, 164.86, 165.84, 167.64; MS
(m/z): 454.01 (M++3, 21.09%), 453.09 (M++2, 23.89%), 450.75 (M+,
29.61%), 292.20 (100%, base beak), 259.20 (82.11%), 71.36
2‐[5‐(2,4‐Dichlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐N‐
phenylacetamide (15)
Yield, 60%; m.p. 170–171°C; IR (KBr) νmax 3294, 3063, 2932, 1701,
;
1667 cm−1 1H NMR (400 MHz, DMSO‐d6): δ = 4.52 (s, 2H, –CH2),
7.07–7.10 (m, 1H, Ar–H, H‐4 of –C6H5), 7.30–7.35 (m, 2H, Ar–H, H‐3,

16 of 18
EL‐ADL ET AL.
|
(37.04%); anal. calcd. for C19H12Cl2N2O5S (451.27): C, 50.57; H,
2.68; N, 6.21. Found: C, 50.28; H, 3.04; N, 6.25.
24 h. Then, cells were exposed to different concentrations of
compounds (0.1, 10, 100, and 1000 µM) for 72 h. Next, the viability
of treated cells was determined using the MTT technique[59,67] as
follows: Cells were incubated with 200 μl of MTT solution/well
(0.5 mg/ml) (Sigma‐Aldrich) and were allowed to metabolize the
dye into colored, insoluble formazan crystals for 2 h. The formazan
crystals were dissolved in 200 µl/well acidified isopropanol for
30 min and covered with aluminum foil, with continuous shaking
using a MaxQ 2000 plate shaker (Thermo Fisher Scientific Inc.), at
room temperature. Absorbance was measured at 570 nm using a
Stat Fax® 4200 Plate Reader (Awareness Technology Inc.). The cell
viability was expressed as a percentage of control, and the con-
centration that induces 50% of maximum inhibition of cell pro-
liferation (IC50) was determined using GraphPad Prism version
5 software (GraphPad Software Inc.).[68]
2‐[5‐(2,4‐Dichlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐yl]‐N‐(pyridin‐
2‐yl)acetamide (19)
Yield, 65%; m.p. 177–179°C; IR (KBr) νmax 3275, 3059, 2924, 1701,
;
1666, 1659 cm−1 1H NMR (400 MHz, DMSO‐d6): δ = 4.60 (s, 2H,
CH2), 7.15 (m, 1H, Ar–H, H‐4 of pyridine), 7.63 (d, 1H, Ar–H, H‐5 of
–C6H5(Cl2), J = 10.4), 7.69–7.72 (m, 2H, Ar–H, H‐3, and H‐6 of
–C6H5(Cl2)), 7.78 (s, 1H, C═CH–Ph), 7.97–8.01 (m, 2H, Ar–H, H‐5,
and H‐6 of pyridine), 8.36 (m, 1H, Ar–H, H‐3 of pyridine), 10.99 (s,
1H, NH, D2O exchangeable); MS (m/z): 409.98 (M++2, 6.78%), 408.95
(M++1, 27.79%), 407.98 (M+, 9.57%), 406.97 (44.69%), 371.98
(43.77%), 120.97 (55.18%), 77.98 (100%, base beak); anal. calcd. for
C
17H11Cl2N3O3S (408.25): C, 50.01; H, 2.72; N, 10.29. Found: C,
49.65; H, 2.34; N, 10.42.
|
4.4
In vitro VEGFR‐2 kinase assay
|
4.2
Docking studies
The kinase activity of VEGFR‐2 was carried out in Pharmacology
& Toxicology Department, Faculty of Pharmacy, Al‐Azhar
In the present work, 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 Molsoft
program, which provides a unique set of tools for the modeling of
protein/ligand interactions. It predicts how small flexible molecules
such as substrates or drug candidates bind to a protein of 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 in comparison with
the experimental procedure, the reported VEGFR‐2 inhibitor drugs
vatalanib and sorafenib were used as reference ligands.
University, Cairo, Egypt, and it was measured by use of an anti‐
phosphotyrosine antibody with the AlphaScreen system according
to manufacturer's instructions.[69,70] Enzyme reactions were per-
formed in 50 mM Tris‐HCl pH 7.5, 5 mM MnCl2, 5 mM MgCl2,
0.01% Tween‐20, and 2 mM DTT, containing 10 μM ATP, 0.1 μg/ml
biotinylated poly‐GluTyr (4:1), and 0.1 nM of VEGFR‐2 (Millipore).
Before catalytic initiation with ATP, the tested compounds at final
concentrations 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 streptavidine 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 the
reaction control. Wells containing biotinylated poly‐GluTyr (4:1)
and enzyme without ATP were used as the basal control. Percent
inhibition was calculated by the comparison of compounds
subjected to control incubations. The concentration of the test
compound causing 50% inhibition (IC50) was calculated from the
concentration–inhibition response curve (triplicate determina-
tions), and the data were compared with sorafenib (Sigma‐Aldrich)
as the standard VEGFR‐2 inhibitor.
|
4.3
In vitro cytotoxic activity
The cytotoxicity assays were performed at the Pharmacology &
Toxicology Department, Faculty of Pharmacy, Al‐Azhar University,
Cairo, Egypt. Cancer cells from different cancer cell lines,
HCC (HepG2), breast cancer (MCF‐7), and colorectal carcinoma
(HCT‐116), were purchased from American Type Cell Culture
Collection (ATCC) and grown in the appropriate growth medium,
Roswell Park Memorial Institute medium (RPMI‐1640), supple-
mented with 100 mg/ml of streptomycin, 100 units/ml of peni-
ACKNOWLEDGMENTS
cillin, and 10% of heat‐inactivated fetal bovine serum in
a
The authors extend their appreciation and gratitude to Asst. Prof.
humidified, 5% (v/v) CO2 atmosphere at 37°C. Cytotoxicity was
determined by MTT assay.
Tamer Abdel‐Ghany, Pharmacology & Toxicology Department,
Faculty of Pharmacy, Al‐Azhar University, Cairo, Egypt, for helping in
Exponentially growing cells from different cancer cell lines
were trypsinized, counted, and seeded at the appropriate densities
(2000–1000 cells/0.33 cm2 well) into 96‐well microtiter plates.
Cells were then incubated in a humidified atmosphere at 37°C for
the pharmacological part.
CONFLICTS OF INTERESTS
The authors declare that there are no conflicts of interests.

EL‐ADL ET AL.
17 of 18
|
ORCID
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Bioorg. Med. Chem. 2015, 23, 6497. https://doi.org/10.1016/j.bmc.
2015.08.010
Khaled El‐Adl
http://orcid.org/0000-0002-8922-9770
Hamada S. Abulkhair
https://orcid.org/0000-0001-6479-4573
[28] J. Dietrich, C. Hulme, L. H. Hurley, Bioorg. Med. Chem. 2010, 18,
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How to cite this article: K. El‐Adl, H. Sakr, S. S. A. El‐Hddad,
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