Design, synthesis, molecular docking and anticancer evaluations of 5-benzylidenethiazolidine-2,4-dione derivatives targeting VEGFR-2 enzyme

Article abstract of DOI:10.1016/j.bioorg.2020.104059

Novel series of 5-benzylidenethiazolidine-2,4-dione derivatives 4_a-c-8_a-f were designed, synthesized and evaluated for anticancer activity against HepG2, HCT-116 and MCF-7 cell lines. MCF-7 was the most sensitive cell line to the influence of the new derivatives. In particular, compound 8_f was found to be the most potent derivative overall the tested compounds against the three HepG2, HCT116 and MCF-7 cancer cell lines with IC₅₀ = 11.19 ± 0.8, 8.99 ± 0.7 and 7.10 ± 0.4 μM respectively. Compound 8_f exhibited lower activity than sorafenib, (IC₅₀ = 9.18 ± 0.6, 8.37 ± 0.7 and 5.10 ± 0.4 μM respectively), against HepG2 and HCT116 but exhibited nearly the same activity against MCF-7 cancer cell lines respectively. Also, this compound displayed lower activity than doxorubicin, (IC₅₀ = 7.94 ± 0.6, 8.07 ± 0.8 and 6.75 ± 0.4 μM respectively), against HepG2 and HCT116 but nearly the same activity against MCF-7cell lines respectively. The most active derivatives 6_c,d,f,g and 8_a-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 compounds 8_a-f. Among them, compounds 8_f was found to be the most potent derivative that inhibited VEGFR-2 at IC₅₀ value of 0.22 ± 0.02 μM, which is nearly the half as that of sorafenib IC₅₀ value (0.10 ± 0.02 μM). Furthermore, molecular design was performed to investigate their binding mode and affinities towards VEGFR-2 receptor. The data obtained from docking studies were highly correlated with that obtained from the biological screening.

Full text of DOI:10.1016/j.bioorg.2020.104059

Journal Pre-proofs
Design, synthesis, molecular docking and anticancer evaluations of 5-benzyli-
denethiazolidine-2,4-dione derivatives targeting VEGFR-2 enzyme
Khaled El-Adl, Abdel-Ghany A. El-Helby, Helmy Sakr, Ibrahim H. Eissa,
Sanadelaslam SA El-Hddad, Fatma M. I. A. Shoman
PII:
S0045-2068(20)31356-0
DOI:
https://doi.org/10.1016/j.bioorg.2020.104059
YBIOO 104059
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
19 April 2020
30 May 2020
26 June 2020
Please cite this article as: K. El-Adl, A.A. El-Helby, H. Sakr, I.H. Eissa, S. SA El-Hddad, F. M. I. A. Shoman,
Design, synthesis, molecular docking and anticancer evaluations of 5-benzylidenethiazolidine-2,4-dione
derivatives targeting VEGFR-2 enzyme, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.
2020.104059
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Design, synthesis, molecular docking and anticancer evaluations of 5-
benzylidenethiazolidine-2,4-dione derivatives targeting VEGFR-2 enzyme
Khaled El-Adla,b,*, Abdel-Ghany A. El-Helbya, Helmy Sakra, Ibrahim H. Eissaa, Sanadelaslam SA El-Hddada, Fatma
M. I. A. Shomanc
a Pharmaceutical Chemistry Department, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt
b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Heliopolis University for Sustainable Development,
Cairo, Egypt
c MD in Clinical Pathology, Blood bank specialist, Blood bank directorate manager, Ministry of Health, Cairo, Egypt
Corresponding Authors:
Khaled El-Adl, eladlkhaled74@yahoo.com; eladlkhaled74@azhar.edu.eg; khaled.eladl@hu.edu.eg
Abstract
Novel series of 5-benzylidenethiazolidine-2,4-dione derivatives 4a-c-8a-f were designed, synthesized and evaluated for
anticancer activity against HepG2, HCT-116 and MCF-7 cell lines. MCF-7 was the most sensitive cell line to the
influence of the new derivatives. In particular, compound 8f was found to be the most potent derivative overall the
tested compounds against the three HepG2, HCT116 and MCF-7 cancer cell lines with IC50 = 11.19±0.8, 8.99±0.7 and
7.10±0.4 µM respectively. Compound 8f exhibited lower activity than sorafenib, (IC50 = 9.18±0.6, 8.37±0.7 and
5.10±0.4 µM respectively), against HepG2 and HCT116 but exhibited nearly the same activity against MCF-7 cancer
cell lines respectively. Also, this compound displayed lower activity than doxorubicin, (IC50 = 7.94±0.6, 8.07±0.8 and
6.75±0.4 µM respectively), against HepG2 and HCT116 but nearly the same activity against MCF-7cell lines
respectively. The most active derivatives 6c,d,f,g and 8a-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
compounds 8a-f. Among them, compounds 8f was found to be the most potent derivative that inhibited VEGFR-2 at IC50
value of 0.22±0.02 µM, which is nearly the half as that of sorafenib IC50 value (0.10 ± 0.02 µM). Furthermore, molecular
design was performed to investigate their binding mode and affinities towards VEGFR-2 receptor. The data obtained
from docking studies were highly correlated with that obtained from the biological screening.
Keywords: Thiazolidine-2,4-dione; Molecular docking; VEGFR-2 inhibitors; Anticancer agents.
1

1. Introduction
The thiazolidine-2,4-diones (TZDs) as heterocyclic compounds, have been reported to be a potential scaffold
that play an important role in cancer therapy. TZDs were reported to exhibit anticancer 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 [1-3]. Tumor angiogenesis is a crucial hallmark for the growth and metastasis of
malignant tumor [4].
Angiogenesis, the formation of new blood vessels sprouting from the pre-existing vasculature, is essential for
the rapid expansion of a tumor mass and is a critical process for the formation and metastases [3]. Targeting angiogenic
regulatory factors will be promising for cancer-treatment [5]. The vascular endothelial growth factor (VEGF) is a
positive regulator with its distinct specificity for vascular endothelial cells. The biological action of VEGF is mediated
by three structurally related receptors, VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1) and VEGFR-3[6]. VEGFR-2, a
tyrosine kinase subtype, is the major regulator of VEGF-driven responses in endothelial cells and can mediate
proliferation, differentiation, and microvascular permeability. Moreover, it has proven to be a prerequisite signal
transducer in both physiologic and pathologic angiogenesis [7, 8]. VEGFR-2 is over- expressed in several malignancies,
including hepatocellular carcinoma, breast, colorectal, ovarian and thyroid cancer, melanoma and medulloblastoma [9-
11]. Therefore, VEGFR-2 has been identified as an excellent remedial target for the production of novel anticancer
agents [12]. Hence, inhibition of angiogenesis via blocking VEGFR-2 pathway has attracted widespread interest as an
approach to anti-cancer therapy [13]. In addition, inhibition of VEGF signaling can also change or destroy tumor
vessels. VEGFR-2 inhibitors, as TKIs, prevent the vital processes of angiogenesis and lymph angiogenesis. Recently,
it was reported that a number of compounds have been confirmed as potent inhibitors of VEGFR-2 in vitro or possessed
antiangiogenic activity and achieved clinical success in the treatment of cancer [14].
In addition, several TZD derivatives, were reported to be potent anticancer agents and inhibitors of angiogenesis
targeting VEGFR-2 [3,15-18] via decreasing the VEGF production in an in vitro model e.g. ciglitazone I [19] compound
(II) [3]. Due to the importat role of VEGFR-2 in angiogenesis, this receptor is the most vital target in anti-angiogenic
therapy against cancer. Several potent VEGFR-2 inhibitors have been developed and approved as antiangiogenic drugs
for treatment of various cancers, e.g. sunitinib (III) [20, 21] and Sorafenib (Nexavar)®(IV) [22-24] (Figure 1).
S
NH
O
O
N
O
O
S
NH
S
O
O
O
N
H
Ciglitazone (I)
Compound (II)
CF3
H
N
H
N
Cl
F
O
O
N
N
O
N
H
O
HN
H
N
N
O
H
H3C
Sorafenib (IV)
Sunitinib (III)
2

Figure 1: Reported VEGFR-2 inhibitors.
In continuation of our previous efforts in the field of design and synthesis of new anticancer agents, especially
VEGFR-2 inhibitors [25-29], a new series of thiazolidine-2,4-dione derivatives were designed and synthesized on the
basis of the main pharmacophoric features of VEGFR-2 inhibitors.
A study of the structure-activity relationships (SAR) and binding pattern of sorafenib and various VEGFR-2
inhibitors revealed that they shared four main features (as shown in Figure 2) [30, 31]: (i) The core structure of most
inhibitors consists of a flat hetero aromatic ring system which occupied the catalytic adenosine triphosphate (ATP)-
binding domain [31]. (ii) A central aryl ring (hydrophobic spacer), occupying the linker region between the ATP-
binding domain and the DFG domain of the enzyme [32]. (iii) A linker containing a functional group acting as
pharmacophore (e.g. amino or urea) that possesses both H-bond acceptor (HBA) and donor (HBD) in order to bind with
two crucial residues (Glu883 and Asp1044) 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 Glu883,
whereas the C=O motif forms another hydrogen bond with Asp1044 [33]. (iv) The terminal hydrophobic moiety that
occupies the newly created allosteric hydrophobic pocket via various hydrophobic interactions [34].
Hetero
aromatic
ring
Central Aryl
Ring
Hydrophobic
tail
HBA-HBD
H
H
CF3
Cl
N
N
N
H
O
N
O
H3C
O
Sorafenib
F
O
N
N
H
HN
N
H
O
Sunitinib
Interact with
Glu883& Asp1044
in DFG domain
Occupy
linker
region
Occupy the
allosteric
binding region
ATP binding
domain
Figure 2: The basic structural requirements for sorafenib and sunitinib as reported VEGFR-2 inhibitors.
3

The goal of our work is to synthesize new 5-benzylidenethiazolidine-2,4-dione derivatives with the same
essential pharmacophoric features of the reported and clinically used VEGFR-2 inhibitors, in addition, to being
molecularly hybridized with effective antitumor moieties in an attempt to get more potent antitumor molecules. The
main core of our molecular design rationale was carried out by bioisosteric modification strategies of VEGFR-2
inhibitors (sorafenib & sunitinib) at four different positions (Figure 3).
H
H
CF3
Cl
N
N
N
H
O
N
O
H3C
O
Sorafenib
F
O
N
N
Sunitinib
H
HN
N
O
H
phenyl
group
Acetamide
moieties
(Un)substituted aromatic
or aliphatic groups
Thiazolidine-2,4-diones
O
R
R
O
O
R
R1
O
O
N
S
N
H
N
S
R
Distal moiety
O
Distal moiety
O
4a-c
6a-h
O
R2
R1
N
H
O
O
O
N
H
N
H
N
S
O
O
O
8a-c
N
N
S
H
O
8d-f
Figure 3: Structural similarities and pharmacophoric features of VEGFR-2 inhibitors and designed compounds
Firstly, bioisosteric approach was adopted in the target benzylidene to replace pyridine ring. The second strategy
is to use thiazolidine-2,4-dione to replace the central aryl ring of lead structure aiming to increase VEGFR-2 binding
affinity. The third strategy is using ester and/or acetamide linkers containing HBA-HBD functional groups that possess
H-bond acceptors and/or donors. In addition, the hydrophobic substituted phenyl tail of the reported ligand sorafenib
was replaced by other distal different hydrophobic moieties including (substituted) phenyl, aromatic heterocyclic or
4

aliphatic structures. The substitution pattern of these hydrophobic moieties was selected to ensure different electronic
and lipophilic environments, which could influence the activity of the target compounds. Moreover, the hydrophobic
moieties in several compounds (e.g. compounds 8a-f , Scheme 2) were further modified to carry additional HBA-HBD
groups and terminated different hydrophobic moieties that will be result in various extra binding interactions within the
back pocket leading to different inhibitory activity.
In general, the designed compounds were synthesized and evaluated for their in vitro antiproliferative activities
against three human tumor cell lines, namely, hepatocellular carcinoma (HCC) type (HepG2), breast cancer (michigan
cancer foundation-7 (MCF-7)) and human colorectal carcinoma-116 (HCT-116). The results prompted further
examinations to reach a deep insight into the mechanism of action of the synthesized compounds. Molecular docking
studies were conducted to understand the expected binding interactions of the target compounds with VEGFR-2 active
sites. Moreover, the most active cytotoxic compounds that showed promising IC50 values against three cancer cell lines,
were subjected to further investigation for their kinase inhibitory activities against VEGFR-2.
1.1.Rationale and structure-based design
5-Benzylidenethiazolidine-2,4-dione derivatives have the essential pharmacophoric features of VEGFR-2
inhibitors (Figure 3) which include: the presence of five membered hetero ring, thiazolidine-2,4-dione, substituted with
benzylidene moiety, forming 5-benzylidenethiazolidine-2,4-dione scaffold liked to (un)substituted hydrophobic tail
through ester and/or acetamide linkers. Benzylidene moiety (as hydrophobic portion), 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 benzylidene moiety occupied the
hydrophobic groove formed by Leucine1033, Glycine920, Lysine918, Cysteine917, Phenylalanine916, Glutamate915,
Alanine864 and Leucine838. On the other hand, the thiazolidine-2,4-dione moiety occupied the hydrophobic pocket
formed by Cysteine1043, Leucine1033, Valine914, Valine897 and Lysine866 (Figure 4).
The ester and/or acetamide linkers interact as H-bond acceptor through its C=O and as H-bond donor through
its NH with the essential amino acid residues Aspartate1044 and Glutamate883, respectively. Also the terminal
(un)substituted hydrophobic phenyl tail occupied the hydrophobic pocket formed by Aspartate1044, Cysteine1043,
Isoleucine1042, Histidine1024, Leucine1017, Valine897, Leucine887, Lysine866 and Glutamate883. Moreover, the
distal hydrophobic moieties as in compounds 8a-f attached to acetamide linker in order to increase the length of the
structure to enable these distal moieties to occupy new hydrophobic grooves formed by Arginine1025, Isoleucine1023,
Cysteine1022, Leucine1017, Isoleucine890 and Isoleucine886 and other one formed by Cysteine1022, Lysine1021,
Arginine1020 and Histidine889 e.g. 8f (Figure 4).
5

Figure 4: Superimposition of compound 8f and sorafenib inside the binding pocket of 1YWN.
2. Results and discussion
2.1. Chemistry
The synthetic strategy for preparation of the target compounds (4-8) is depicted in Schemes 1-2. Synthesis was initiated
by cyclocondensation of thiourea with chloroacetic acid to afford thiazolidine-2,4-dione (1) [26,35] which underwent
further condensation reaction with benzaldehyde in glacial acetic acid via Knoevenagel condensation [36,37] to afford
5-benzylidenethiazolidine-2,4-dione (2). Subsequent treatment of benzylidene derivative 2 with alcoholic potassium
hydroxide (KOH) afforded the corresponding potassium salt (3). The potassium salt (3) was heated with the appropriate
chloroester in dry dimethylformamide (DMF) to afford the corresponding ester derivatives (4a-c). The structures of (4a-c)
were confirmed by spectroscopic infrared (IR) and Proton nuclear magnetic resonance (1 H NMR) as well as elemental
analysis data. The IR spectra revealed the presence of carbonyl absorption bands at a range of 1682 -1685 cm-1 and
1740-1744 cm-1. 1H NMR spectra showed a new singlet signal for benzylidene methine proton (C═CHPh) in the range
of δ 7.99–8.02 ppm. In addition, 1H NMR spectra of (4a,b) showed the singlet signals of the aliphatic -NCH2 protons at
1
δ 4.53 and 4.51 ppm respectively for the introduced -NCH2 group. While, H NMR spectrum of 4c revealed the
presence the quartet signal of the aliphatic –NCH proton at δ 5.10-5.16 ppm. On the other hand, 2-Chloro-N-
arylacetamide (5a-h) were prepared according to the directions of Jacobs and Heidelberger [38] by slow addition of
chloroacetyl chloride to a solution of the appropriate aromatic amine to obtain the corresponding chloroamides (5a-h).
The reaction of potassium salt (3) with 2-chloro-N-arylacetamide (5a-i) in dry DMF yielded the corresponding acid
amide derivatives (6a-h) (Scheme1). The IR spectra of 6a-h revealed the presence of the carbonyl absorption band at a
1
range of 1636-1751 cm-1, NH band at a range of 3202-3329 cm-1. In addition; the H NMR spectra showed singlet
signals of the aliphatic -NCH2 protons at a δ range of 4.47-4.62 ppm, singlet signal for benzylidene methine proton
(C═CHPh) in the range of δ 7.79–8.59 ppm and presence of D2O exchangeable NH-amide protons at a δ range of 10.30
-12.54 ppm.
6

The chloroamide 5f was treated with ethyl chloroformate (ECF) in the presence of triethylamine (TEA) and then the
appropriate amine was added to afford the corresponding intermediate (7a-f). The potassium salt (3) was refluxed with
the appropriate intermediate (7a-f) to obtain the corresponding derivatives (8a-f), respectively (Scheme2). The IR spectra
of (8a-f) revealed the presence of the carbonyl absorption band at a range of 1647-1694 cm-1 and 1739-1748 cm-1, NH
bands at a range of 3144-3329cm-1 1H NMR spectra showed the presence of two singlet signals at ranges δ 8.34-10.47
and 10.24-10.97 ppm corresponding to two NH groups.
O
O
O
O
S
a
+
NH
O
NH
O
Cl
OH
H2N
NH2
S
S
b
(2)
(1)
c
O
R2
Cl
R2
O
N
O
O
O
N
H
N
H
NK
O
(5a-h)
S
S
e
(6a-h)
(3)
O
R2
d
Cl
R1
6a
H
O
R
4-CH3
6b
4-OCH3
R1
6c
6d
6e
6f
O
O
O
4-COCH3
2-COOH
4-COOH
N
S
R
O
(4a-c)
R
R1
4-COOC2H5
4-NO2
6g
6h
4a
4b
H
H
CH3
C2H5
C2H5
4c
CH3
a) HCl / / 2-3 h; b) Sodium acetate / acetic acid glacial;

c) KOH / Ethanol; d) DMF
e) DMF
7

Scheme 1: Synthetic route for preparation of the target compounds 4a-c-6a-h
O
NH-R2
O
OH
O
NK
a
S
O
(3)
HN
HN
Cl
Cl
O
(5f)
O
(7a-f
)
R2
b
8a
8b
C2H5
C3H7
C4H9
8c
O
O
8d
NH-R2
O
S
N
NH
8e
8f
O
O
O
(8a-f
)
a) ECF / TEA / DMF /NH2-R2; b) DMF
Scheme 2: Synthetic route for preparation of the target compounds 8a-f
2.2. Docking studies
All modeling experiments were carried out using Molsoft software. Each experiment used VEGFR-2
downloaded from the Brookhaven Protein Databank (PDB ID 1YWN) [39].
The obtained results indicated that all studied ligands have similar position and orientation inside the recognized
binding site of VEGFR-2 that reveals a large space bounded by a membrane-binding domain, which serves as entry
channel for substrate to the active site (Figure 5). The obtained results of the free energy of binding (∆G) explained
that most of these compounds had good binding affinity toward the receptor and the computed values reflected the
overall trend (Table 1).
8

Figure 5: Superimposition of some docked compounds inside the binding pocket of 1YWN.
The proposed binding mode of sorafenib revealed affinity value of -95.66 kcal/mol and 4 H-bonds. The urea
linker formed one H-bond with the key amino acid Glutamate883 (2.13 Å) through its NH group and one H-bond with
Aspartate1044 (1.53 Å) through its carbonyl group. The N-methylpicolinamide moiety was stabilized by formation of
2 H bonds with Cysteine917 where, the pyridine N atom formed 1H-bond with the NH of Cysteine917 (2.46 Å) while
its NH group formed 1H-bond with the carbonyl of Cysteine917 (2.20 Å). The N-methylpicolinamide moiety occupied
the hydrophobic groove formed by Leucine1033, Glycine920, Lysine918, Cysteine917, Phenylalanine916,
Glutamate915, Leucine838 and Alanine864. Moreover, the central phenyl ring occupied the hydrophobic pocket formed
by Cysteine1043, Leucine1033, Valine914, Valine897 and Lysine866. Furthermore, the hydrophobic 3-trifluromethyl-
4-chlorophenyl moiety attached to the urea linker occupied the hydrophobic pocket formed by Aspartate1044,
Isoleucine1042, Histidine1024, Leucine1017, Isoleucine890 and Leucine887 (Figure 6). The urea linker played an
important role in the binding affinity towards VEGFR-2 enzyme, where it was responsible for the higher binding affinity
of sorafenib. These findings encourage us to use different linker resembling urea of sorafenib hoping to obtain potent
VEGFR-2 inhibitors.
9

Figure 6: Predicted binding mode for sorafenib with 1WYN. H-bonded atoms are indicated by dotted lines.
The proposed binding mode of compound 8f is virtually the same as that of sorafenib which revealed affinity
value of -97.60 kcal/mol and 6 H-bonds. The carbonyl group of the acetamide linker formed 1 H-bond with
Aspartate1044 (2.42 Å) while its NH group formed another H bond with Glutamate883 (1.75 Å). The carbonyl group
at position-2 of thiazolidine-2,4-dione formed 1 H-bond with Lysine866 (2.67 Å). Moreover, the NH group of the
carboxamide linker formed 1 H-bond with Aspartate1044 (2.58 Å). Furthermore, the carbonyl group of the distal ethyl
ester moiety formed 2 H-bonds with Arginine1025 (1.66 Å and 2.59 Å). The 5-phenyl moiety occupied the hydrophobic
groove formed by Leucine1033, Glycine920, Lysine918, Cysteine917, Phenylalanine916, Glutamate915, Alanine864
and Leucine838. Moreover, the thiazolidine-2,4-dione moiety occupied the hydrophobic pocket formed by
Cysteine1043, Leucine1033, Valine914, Valine897 and Lysine866. The hydrophobic phenyl tail occupied the
hydrophobic pocket formed by Aspartate1044, Cysteine1043, Isoleucine1042, Histidine1024, Leucine1017, Valine897,
Leucine887, Lysine866 and Glutamate883. Furthermore, the distal phenyl ring occupied the hydrophobic groove
formed by Arginine1025, Isoleucine1023, Cysteine1022, Leucine1017, Isoleucine890 and Isoleucine886. On the other
hand the distal ethyl group of the ester moiety occupied the hydrophobic cleft formed by Cysteine1022, Lysine1021,
Arginine1020 and Histidine889 (Figure 7). These interactions of compound 8f may explain the highest anticancer
activity.
10

Figure 7: Predicted binding mode for 8f with 1WYN.
The proposed binding mode of compound 8e is virtually the same as that of 8f which revealed affinity value of -
93.51 kcal/mol and 4 H-bonds. The carbonyl group of the acetamide linker formed 1 H-bond with Aspartate1044 (2.29
Å) while its NH group formed another H bond with Glutamate883 (2.06 Å). The carbonyl group at position-2 of
thiazolidine-2,4-dione formed 1 H-bond with Lysine866 (2.58 Å). Moreover, the NH group of the carboxamide linker
formed 1 H-bond with Aspartate1044 (2.66 Å). The 2,4-dichlorophenyl moiety occupied the hydrophobic groove
formed by Leucine1033, Glycine920, Lysine918, Cysteine917, Phenylalanine916, Glutamate915, Alanine864 and
Leucine838. Moreover, the thiazolidine-2,4-dione moiety occupied the hydrophobic pocket formed by Cysteine1043,
Leucine1033, Valine914, Valine897 and Lysine866. The hydrophobic phenyl tail occupied the hydrophobic pocket
formed by Aspartate1044, Cysteine1043, Isoleucine1042, Histidine1024, Leucine1017, Valine897, Leucine887,
Lysine866 and Glutamate883. Furthermore, the distal methyl phenyl moiety occupied the hydrophobic groove formed
by Arginine1025, Isoleucine1023, Cysteine1022, Leucine1017, Isoleucine890 and Isoleucine886 (Figure 8). These
interactions of compound 8e may explain the high anticancer activity.
11

Figure 8: Predicted binding mode for 8e with 1WYN.
The proposed binding mode of compound 8d is virtually the same as that of sorafenib and 8f which revealed
affinity value of -93.44 kcal/mol and 4 H-bonds. The carbonyl group of the acetamide linker formed 1 H-bond with
Aspartate1044 (2.30 Å) while its NH group formed another H bond with Glutamate883 (1.80 Å). The carbonyl group
at position-2 of thiazolidine-2,4-dione formed 1 H-bond with Lysine866 (2.77 Å). Moreover, the NH group of the
carboxamide linker formed 1 H-bond with Aspartate1044 (2.87 Å). The 2,4-dichlorophenyl moiety occupied the
hydrophobic groove formed by Leucine1033, Glycine920, Lysine918, Cysteine917, Phenylalanine916, Glutamate915,
Alanine864 and Leucine838. Moreover, the thiazolidine-2,4-dione moiety occupied the hydrophobic pocket formed by
Cysteine1043, Leucine1033, Valine914, Valine897 and Lysine866. The hydrophobic phenyl tail occupied the
hydrophobic pocket formed by Aspartate1044, Cysteine1043, Isoleucine1042, Histidine1024, Leucine1017, Valine897,
Leucine887, Lysine866 and Glutamate883. Furthermore, the distal benzyl moiety occupied the hydrophobic groove
formed by Arginine1025, Isoleucine1023, Cysteine1022, Leucine1017, Isoleucine890 and Isoleucine886 (Figure 9).
These interactions of compound 8d may explain the high anticancer activity.
12

Figure 9: Predicted binding mode for 8d with 1WYN.
From the obtained docking results (Table 1), we concluded that, the acetamide linker occupied the same groove
occupied by urea linker of sorafenib and played the same role which is essential for higher affinity towards VEGFR-2
enzyme. The benzylidene increased hydrophobic interactions and consequently affinities towards VEGFR-2 enzyme.
The thiazolidine-2,4-dione enables the new compounds to form new H-bond through its carbonyl group at position-2
with the amino acid Lysine866. Elongation of the structure played an important role in their VEGFR-2 inhibitory
activities. The hydrophobic distal moiety and its linker as in compounds 8a-f formed hydrogen bonding and hydrophobic
interactions which increased affinity towards VEGFR-2 enzyme. On the other hand, these modifications compensated
hydrogen bonding and hydrophobic interactions of N-methylpicolinamide moiety of sorafenib.
Table 1: The calculated free energy of binding (∆G in Kcal/mole) for the ligands
Compound
∆G [kcal mol-1]
-55.41
Compound
∆G [kcal mol-1]
-82.65
4a
4b
4c
6a
6b
6c
6d
6e
6f
6g
6h
8a
8b
8c
8d
8e
8f
-58.75
-58.87
-64.54
-70.26
-70.98
-73.08
-69.96
-74.83
-65.64
-83.49
-86.61
-90.86
-93.44
-93.51
-97.60
-95.66
Sorafenib
2.3. In vitro cytotoxic activity
Anti-proliferative activity of the newly synthesized 5-benzylidenethiazolidine-2,4-dionederivatives 4a-c-8a-f was
examined against three human tumor cell lines namely, hepatocellular carcinoma (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 [40, 41]. Sorafenib and doxorubicin were included in the experiments as
reference cytotoxic drugs. The results were expressed as growth inhibitory concentration (IC50) values and summarized
13

in Table 2. From the obtained results, it was explicated that most of the prepared compounds displayed excellent to
modest growth inhibitory activity against the tested cancer cell lines. In general, investigations of the cytotoxic activity
indicated that MCF-7 was the most sensitive cell line to the influence of the new derivatives respectively. In particular,
compound 8f was found to be the most potent derivative overall the tested compounds against the three HepG2, HCT116
and MCF-7 cancer cell lines with IC50 = 11.19±0.8, 8.99±0.7 and 7.10±0.4 µM respectively. Compound 8f exhibited
lower activity than sorafenib, (IC50 = 9.18±0.6, 8.37±0.7 and 5.10±0.4 µM respectively), against HepG2 and HCT116
but exhibited nearly the same activity against MCF-7 cancer cell lines respectively. Also, this compound displayed
lower activity than doxorubicin, (IC50 = 7.94±0.6, 8.07±0.8 and 6.75±0.4 µM respectively), against HepG2 and HCT116
but nearly the same activity against MCF-7cell lines respectively. With respect to the HepG2 hepatocellular carcinoma
cell line, compounds 8e, 8d and 8c displayed the highest anticancer activities with (IC50 = 15.04±1.5, 17.08±1.6 and
21.75±2.1 µM, respectively). Compounds 8b, 8a and 6g, with IC50 = 29.12±2.5, 31.59±2.7 and 35.05±2.5 µM
respectively, displayed good cytotoxicity. Compounds 6b, 6c, 6d and 6f with (IC50 ranging from 51.22±3.6 to 72.72±4.6
µM) exhibited moderate cytotoxicity. While, compound 4a, 4b, 4c, 6a, 6e and 6h with IC50 ranging from 82.37±5.2 to
98.40±5.1 µM displayed the lowest cytotoxicity.
Cytotoxicity evaluation against colorectal carcinoma (HCT-116) cell line, discovered that compounds 8e, 8c, 8d and 8b
displayed the highest anticancer activities with (IC50 = 11.35±1.0, 18.35±1.8, 19.44±1.6 and 24.38±1.9 µM,
respectively). Compounds 8a, 6g and 6f with IC50 = 30.28±2.2, 36.18±1.8 and 39.45±3.1 µM respectively, displayed
good cytotoxicity. Compounds 6b, 6c, 6d and 6e with (IC50 ranging from 64.34±3.3 to 74.82±4.6 µM) exhibited moderate
cytotoxicity. While, compound 4a, 4b, 4c, 6a and 6h with IC50 ranging from 81.87±3.5 to >100 µM displayed the lowest
cytotoxicity.
Cytotoxicity evaluation against MCF-7 cell line, revealed that compounds 8e, 8d, 8b and 8c displayed the highest
anticancer activities with (IC50 = 12.41±1.4, 14.33±1.3, 19.61±1.3 and 25.12±1.9 µM, respectively). Compounds 8a, 6g
and 6f with IC50 = 28.07±3.1, 39.88±2.5 and 47.82±3.5 µM respectively, displayed good cytotoxicity. Compounds 6b,
6c, 6d and 6e with (IC50 ranging from 55.34±3.7 to 69.06±3.8 µM) exhibited moderate cytotoxicity. While, compound
4a, 4b, 4c, 6a and 6h with IC50 ranging from 80.71±5.2 to 93.43±5.3 µM displayed the lowest cytotoxicity.
Table 2: In vitro cytotoxic activities of the newly synthesized compounds against HepG2, HCT-116 and MCF-7cell
lines and VEGFR-2 kinase assay.
Compound
IC50 (µM)a
HepG2
HCT116
MCF-7
VEGFR-2
4a
4b
4c
6a
6b
6c
98.40±5.1
>100
93.43±5.3
NT
95.99±5.1
95.40±5.1
94.22±5.2
72.72±4.6
56.80±4.0
>100
91.67±5.3
83.91±4.9
80.71±5.2
68.65±3.8
58.96±4.8
NT
NT
>100
81.87±3.5
64.34±3.3
74.82±4.6
NT
NT
0.74±0.06
14

6d
6e
6f
54.16±3.3
82.37±5.2
51.22±3.6
35.05±2.5
68.34±4.5
73.55±2.5
39.45±3.1
36.18±1.8
55.34±3.7
69.06±3.8
47.82±3.5
39.88±2.5
0.72±0.06
NT
0.71±0.06
0.68±0.05
6g
6h
83.89±3.9
89.66±4.1
81.28±4.5
NT
8a
8b
8c
8d
8e
8f
31.59±2.7
29.12±2.5
21.75±2.1
17.08±1.6
15.04±1.5
11.19±0.8
30.28±2.2
24.38±1.9
18.35±1.8
19.44±1.6
11.35±1.0
8.99±0.7
28.07±3.1
19.61±1.3
25.12±1.9
14.33±1.3
12.41±1.4
7.10±0.4
0.68±0.05
0.68±0.05
0.40±0.03
0.28±0.02
0.25±0.02
0.22±0.02
Sorafenib
9.18±0.6
5.47±0.3
8.07±0.8
7.26±0.3
6.75±0.4
0.10 ± 0.02
bNT
Doxorubicin
7.94 ± 0.6
a IC50 values are the mean ± S.D. of three separate experiments.
bNT: Compounds not tested for their VEGFR-2 inhibitory activity.
2.4. In vitro VEGFR-2 kinase assay
Furthermore, the most active cytotoxic compounds 6c,d,f,g and 8a-f were selected to evaluate their inhibitory
activities against VEGFR-2 by using an anti-phosphotyrosine antibody with the Alpha Screen system (PerkinElmer,
USA). The results were reported as a 50% inhibition concentration value (IC50) calculated from the concentration-
inhibition response curve and summarized in Table 2. Sorafenib was used as positive control in this assay. The tested
compounds displayed high to medium inhibitory activity with IC50 values ranging from 0.22±0.02 to 0.74±0.05 µM.
Among them, compounds 8f was found to be the most potent derivative that inhibited VEGFR-2 at IC50 value of
0.22±0.02 µM, which is nearly the half as that of sorafenib IC50 value (0.10 ± 0.02 µM). Compounds 8e, 8d and 8c
exhibited very good activity with IC50 values of 0.25±0.02, 0.28±0.02 and 0.40±0.03 µM respectively. Also, compounds
8a, 8b and 6g possessed good VEGFR-2 inhibition with the same IC50 value of 0.68±0.05 µM. Compounds 6f, 6d and 6c
displayed the lowest VEGFR-2 inhibition with IC50 values = 0.71±0.06, 0.72±0.06 and 0.74±0.06 µM, respectively.
2.5. Structure Activity Relationship (SAR)
The preliminary SAR study has focused on the effect of replacement of the urea and carboxamide linkers of
sorafenib and sunitinib respectively with acetamide linkers which interacting as H-bond acceptors through its carbonyl
group and as H-bond donor through its NH atom. These acetamide linkers interacting with the side chain NH of the
essential amino acid residue Aspartate1044 and carboxylate of the essential amino acid residue Glutamate883. Also
hydrophobic interactions through the attached (un)substituted hydrophobic distal moieties. The effect of replacement
15

of pyridine and 5-fluoro-2-oxoindolin-3-ylidene moieties of sorafenib and sunitinib respectively by the benzylidene of
5-benzylidenethiazolidine-2,4-dione scaffold of the synthesized compounds on the antitumor activities also was
noticed. These benzylidene moiety occupied the same hydrophobic pocket which occupied by the pyridine moiety of
the standard ligand. Moreover, the thiazolidine-2,4-dione was designed to replace the central aryl and pyrrole rings of
the reference ligands sorafenib and sunitinib respectively and enables the target compounds to form new H-bonds
through their 2-carbonyl groups with the essential amino acid residue Lysine866. On the other hand different distal
aliphatic and aromatic moieties were introduced to the phenyl tail of the reference ligand with different lipophilicity
and electronic nature in order to study their effects on the anticancer activity. The presence of lipophilic distal moieties
attached to the phenyl ring through carboxamide linker as in compounds 8a-f formed new hydrophobic and H-bonding
interactions with the active site. The data obtained revealed that, the tested compounds displayed different levels of
anticancer activity and possessed a distinctive pattern of selectivity against the MCF-7 cell lines. Generally, the spacers,
linkers (HBA-HBD), lipophilicity and electronic nature exhibited an important role in anticancer activity. The presence
of acetamide and carboxamide linkers of compounds 8a-f was found to be responsible for their highest anticancer activity
than the other compounds that 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 three groups. The first group is compounds 4a-c
where ethyl isopropionate ester derivative 4c showed higher activities than the ethyl acetate 4b and the methyl one 4a
respectively. The second group is compounds 6a-f, the terminal phenyl tail was unsubstituted as in compound 6a, 2-
substituted with hydrophilic, electron withdrawing carboxylic group as in compound 6e and 4-substituted with different
hydrophobic, hydrophilic, electron donating and/or electron withdrawing groups. Generally, the substituted phenyl tail
as in compounds 6b-h showed higher anticancer activities than the unsubstituted one 6a against the three HepG2,
HCT116 and MCF-7 cell lines respectively except in 4-nitro derivative 6h which exhibited lower activity than
compound 6a against HCT116 and MCF-7 cell lines respectively. The 4-substituted phenyl tail with ethyl ester moiety
exhibited the highest anticancer activities within this group against the three HepG2, HCT116 and MCF-7 cell lines
respectively. The 4-substituted derivatives with hydrophilic electron withdrawing carboxylic group as in compound 6f
exhibited higher activities than that substituted with hydrophobic electron withdrawing acetyl group as in compound 6d
against the three HepG2, HCT116 and MCF-7 cell lines. The 4-substituted derivatives with carboxylic group 6f
exhibited higher activities than that at 2-position which indicated that the 4-position is critical for activity. While, 6d
with hydrophobic electron withdrawing acetyl group showed higher activities than that substituted with hydrophobic
electron donating methoxy group as in compound 6c against the three HepG2, HCT116 and MCF-7 cell lines. On the
other hand, the hydrophobic electron donating 4-methoxy derivative 6c showed higher activities than the hydrophobic
electron donating 4-methyl 6b one against both HepG2, and MCF-7 but exhibited lower activity against HCT116 cell
lines. In the third group 8a-f, generally, the substituted terminal phenyl group with aromatic substituents as in compounds
8e-f exhibited higher activities than that substituted with aliphatic ones 8a-c against the three HepG2, HCT116 and MCF-7
cell lines. The substituted distal phenyl moiety with ethyl ester group as in compound 8f displayed higher activities than
that substituted with methyl 8e and benzyl 8d against the three HepG2, HCT116 and MCF-7 cell lines respectively. In
addition, the aliphatic butyl distal moiety 8c showed higher activities than the propyl 8b and the ethyl 8a ones against
the three HepG2, HCT116 and MCF-7 cell lines respectively.
3. Conclusion
16

In summary, seventeen new 5-benzylidenethiazolidine-2,4-dione based derivatives have been designed,
synthesized and evaluated for their anticancer activities against three human tumor cell lines hepatocellular carcinoma
(HepG2), colorectal carcinoma (HCT-116) and breast cancer (MCF-7) targeting VEGFR-2 enzyme. All the tested
compounds showed variable anticancer activities. The molecular design was performed to investigate the binding mode
of the proposed compounds with VEGFR-2 receptor. The acetamide linker played the same role of urea linker of
sorafenib which is essential for higher affinity towards VEGFR-2 enzyme. The benzylidene moiety increased
hydrophobic interactions and the thiazolidine-2,4-dione enables the new compounds to form new H-bond through its
carbonyl group at position-2 with the amino acid Lysine866. Elongation of the structure exhibited higher VEGFR-2
inhibitory activities where the hydrophobic distal moiety and its linker as in compounds 8a-f formed hydrogen bonding
and hydrophobic interactions which increased affinity towards VEGFR-2 enzyme. On the other hand, these
modifications compensated hydrogen bonding and hydrophobic interactions of N-methylpicolinamide moiety of
sorafenib. 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. MCF-7 was the most sensitive cell line to
the influence of the new derivatives. In particular, compound 8f was found to be the most potent derivative overall the
tested compounds against the three HepG2, HCT116 and MCF-7 cancer cell lines with IC50 = 11.19±0.8, 8.99±0.7 and
7.10±0.4 µM respectively. Compound 8f exhibited lower activity than sorafenib, (IC50 = 9.18±0.6, 8.37±0.7 and
5.10±0.4 µM respectively), against HepG2 and HCT116 but exhibited nearly the same activity against MCF-7 cancer
cell lines respectively. Also, this compound displayed lower activity than doxorubicin, (IC50 = 7.94±0.6, 8.07±0.8 and
6.75±0.4 µM respectively), against HepG2 and HCT116 but nearly the same activity against MCF-7cell lines
respectively. The most active derivatives 6c,d,f,g and 8a-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
compounds 8a-f. Among them, compounds 8f was found to be the most potent derivative that inhibited VEGFR-2 at IC50
value of 0.22±0.02 µM, which is nearly the half as that of sorafenib IC50 value (0.10 ± 0.02 µM). Compounds 8e, 8d
and 8c exhibited very good activity with IC50 values of 0.25±0.02, 0.28±0.02 and 0.40±0.03 µM respectively. Also,
compounds 8a, 8b and 6g possessed good VEGFR-2 inhibition with the same IC50 value of 0.68±0.05 µM. Compounds
6f, 6d and 6c displayed the lowest VEGFR-2 inhibition with IC50 values = 0.71±0.06, 0.72±0.06 and 0.74±0.06 µM,
respectively. 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 inhibitors with higher
anticancer analogs.
4. Experimental
4.1.
Chemistry
4.1.1. General
All melting points were carried out by open capillary method on a Gallen kamp Melting point apparatus at
faculty of pharmacy Al-Azhar University and were uncorrected. The infrared spectra were recorded on pye Unicam SP
1000 IR spectrophotometer at Microanalytical Unit, Faculty of Pharmacy, Cairo University using potassium bromide
disc technique. Proton magnetic resonance 1HNMR spectra were recorded on a Bruker 400 Megahertz-nuclear magnetic
17

resonance (400 MHZ-NMR) spectrophotometer at Microanalytical Unit, Faculty of pharmacy, Cairo University.
Carbon-13 (C13) nuclear magnetic resonance (13CNMR) spectra were recorded on a Bruker 100 Megahertz-nuclear
magnetic resonance (100 MHZ-NMR) spectrophotometer at Microanalytical Unit, Faculty of pharmacy, Cairo
University. Tetramethylsilane (TMS) was used as internal standard and chemical shifts were measured in  scale one
part per million (ppm). The mass spectra were carried out on Direct Probe Controller Inlet part to Single Quadropole
mass analyzer in Thermo Scientific Gas chromatography–mass spectrometry (GCMS) model ISQ LT using Thermo X-
Calibur software at the Mycology and Biotechnology Regional Center, Al-Azhar University. Elemental analyses (C, H,
N) were performed on a carbon hydrogen and nitrogen (CHN) analyzer at Mycology and Biotechnology Regional
Center, Al-Azhar University. All compounds were within ±0.4 of the theoretical values. The reactions were monitored
by thin-layer chromatography (TLC) using TLC sheets precoated with UV fluorescent silica gel Merck 60 F254 plates
and were visualized using ultraviolet (UV) lamp and different solvents as mobile phases.
Compounds, thiazolidine-2,4-dione (1) [26,35], 5-benzylidenethiazolidine-2,4-dione (2) [42,43], potassium
salt of 5-benzylidenethiazolidine-2,4-dione (3) [43], N-aryl-2-chloroacetamide derivative 5a-h [44-47] and 4-(2-
chloroacetamido)-N-(substituted)benzamide derivatives 7a-f [48] were obtained according to the reported procedures.
4.1.2. General procedure for synthesis of target compounds (4a-c)
Equimolar quantities of the potassium salt 3 (2.43 g, 0.01 mol) and the appropriate chloroester derivative (0.01
mol) namely; methyl chloroacetate, ethyl chloroacetate and ethyl 2-chloropropanoate in DMF (20 mL) was heated on
water bath for 5 h. The reaction mixture was poured onto ice-water (200 mL) and stirred for 30 min. The obtained solid
was filtered and crystallized from ethanol to give the target compounds 4a-c, respectively.
4.1.2.1. Methyl 2-(5-benzylidene-2,4-dioxothiazolidin-3-yl)acetate (4a)
Yield, 90%; m.p. 120-2oC; IRνmax (cm-1): 3063 (CH aromatic), 2955 (CH aliphatic), 1740, 1682 (3C=O amide); 1HNMR
(400 MHz, DMSO-d6): 3.73 (s, 3H, -OCH3), 4.53 (s, 2H, CH2), 7.52-7.59 (m, 3H, J = 22 Hz, Ar-H, H-3, H-4 & H-5 of
phenyl), 7.66-7.68 (d, 2H, J = 22 Hz, Ar-H, H-2 & H-6 of phenyl), 8.02 (s, 1H, C=CH-Ph); 13CNMR (100 MHz,
DMSO-d6): 42.57, 53.16, 120.99, 129.91 (2), 130.74 (2), 131.43, 133.19, 134.64, 165.38, 167.33, 167.68; Anal. Calcd.
for C13H11NO4S (277.29): C, 56.31; H, 4.00; N, 5.05. Found: C, 56.03; H, 4.17; N, 5.28.
4.1.2.2. Ethyl 2-(5-benzylidene-2,4-dioxothiazolidin-3-yl)acetate (4b)
Yield, 85%; m.p. 110-2oC; IRνmax (cm-1): 3052 (CH aromatic), 2994 (CH aliphatic), 1740, 1685 (3C=O amide); 1HNMR
(400 MHz, DMSO-d6): 1.21-1.24 (t, 3H, J = 20 Hz, -OCH2CH3,), 4.15-4.21 (q, 2H, J = 21 Hz, -OCH2CH3), 4.51 (s,
2H, CH2), 7.51-7.59 (m, 3H, Ar-H, H-3, H-4 & H-5 of phenyl), 7.65-7.67 (d, 2H, Ar-H, H-2 & H-6 of phenyl), 8.01 (s,
1H, C=CH-Ph); Anal. Calcd. for C14H13NO4S ( 291.32): C, 57.72; H, 4.50; N, 4.81. Found: C, 57.98; H, 4.66; N, 4.94.
4.1.2.3. Ethyl 2-(5-benzylidene-2,4-dioxothiazolidin-3-yl)propanoate (4c)
Yield, 75%; m.p. 127-9oC; IRνmax (cm-1): 3090 (CH aromatic), 2912 (CH aliphatic), 1744, 1682 (3C=O amide); 1HNMR
(400 MHz, DMSO-d6): 1.14-1.19 (t, 3H, -OCH2CH3), 1.51-1.53 (d, 3H, -CHCH3), 4.13-4.17 (q, 2H, -OCH2CH3,), 5.10-
5.16 (q, 1H, -CHCH3), 7.50-7.58 (m, 3H, J = 20 Hz, Ar-H, H-3, H-4 & H-5 of phenyl), 7.64-7.66 (d, 2H, J = 20 Hz,
Ar-H, H-2 & H-6 of phenyl), 7.99 (s, 1H, C=CH-Ph); 13CNMR (100 MHz, DMSO-d6): 14.40 (2), 50.76, 61.94, 120.95,
129.90 (2), 130.71 (2), 131.37, 133.25, 134.48, 165.23, 167.12, 168.96; Anal. Calcd. for C15H15NO4S (305.35): C,
59.00; H, 4.95; N, 4.59; Found: C, 58.79; H, 5.12; N, 4.67.
18

4.1.3. General procedure for synthesis of target compounds (6a-h)
Equimolar quantities of the potassium salt 3 (2.43 g, 0.01 mol) and the appropriate N-aryl-2-chloroacetamide
derivative 5a-h (0.01 mol) in dry DMF (20 mL) was heated on a water bath for 3 h in the presence of catalytic amount
of potassium iodide (KI). After cooling to room temperature, reaction mixture was poured on to crushed ice. The
precipitated solids were filtered, dried and crystalized from ethanol to give the corresponding target compounds 6a-h
respectively.
4.1.3.1. 2-(5-Benzylidene-2,4-dioxothiazolidin-3-yl)-N-phenylacetamide (6a)
Yield, 60%; m.p. 150-2oC; IRνmax (cm-1): 3275 (NH), 3059 (CH aromatic), 2924 (CH aliphatic), 1694, 1657, 1636
(3C=O); 1HNMR (400 MHz, DMSO-d6): 4.53 (s, 2H, -CH2), 7.07-7.11 (m, 2H, Ar-H, H-4 of -NHC6H5 and H-4 of -
C6H5), 7.31-7.35 (m, 2H, Ar-H, H-3 & H-5 of -NHC6H5), 7.53-7.56 (m, 2H, Ar-H, H-3 & H-5 of -C6H5), 7.58-7.60
(m, 2H, Ar-H, H-2 & H-6 of -NHC6H5), 7.67-7.69 (m, 2H, Ar-H, H-2 & H-6 of -C6H5), 8.01 (s, 1H, -C=CH-Ph), 10.41
(s, 1H, NH, D2O exchangeable); Anal. Calcd. for C18H14N2O3S (338.38): C, 63.89; H, 4.17; N, 8.28. Found: C, 64.11;
H, 4.08; N, 8.59.
4.1.3.2. 2-(5-Benzylidene-2,4-dioxothiazolidin-3-yl)-N-(p-tolyl)acetamide (6b)
Yield, 70%; m.p. 154-6oC; IRνmax (cm-1): 3202 (NH), 3067 (CH aromatic), 2932 (CH aliphatic), 1697 (3C=O); 1HNMR
(400 MHz, DMSO-d6): 2.26 (s, 3H, -CH3), 4.51 (s, 2H, -CH2), 7.12-7-14 (d, 2H, Ar-H, H-3 & H-5 of -C6H4-CH3),
7.43-7.45 (d, 2H, Ar-H, H-2 & H-6 of -C6H4-CH3), 7.52-7.59 (m, 3H, Ar-H, H-3, H-4 & H-5 of -C6H5), 7.67-7.69 (d,
2H, Ar-H, H-2 & H-6 of -C6H5), 8.01 (s, 1H, -C=CH-Ph), 10.31 (s, 1H, NH, D2O exchangeable); 13CNMR (100 MHz,
DMSO-d6): 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): 352 (M+, 5.62 %), 203 (2.02 %), 107 (51.27 %), 72 (100 %, base beak); Anal. Calcd.
for C19H16N2O3S (352.41): C, 64.76; H, 4.58; N, 7.95. Found: C, 64.93; H, 4.75; N, 8.17.
4.1.3.3. 2-(5-Benzylidene-2,4-dioxothiazolidin-3-yl)-N-(p-methoxyphenyl)acetamide (6c)
Yield, 65%; m.p. 157-9oC; IRνmax (cm-1): 3329 (NH), 3063 (CH aromatic), 2928 (CH aliphatic), 1701, 1639 (3C=O);
1HNMR (400 MHz, DMSO-d6): 3.85 (s, 3H, OCH3), 4.62 (s, 2H, CH2), 7.09-7.16 (m, 4H, Ar-H, H-2, H-3,H-5 & H-
6 of -C6H4(OCH3)), 7.25-7.26 (m, 1H, Ar-H, H-4 of -C6H5), 7.49-7.54 (m, 2H, Ar-H, H-3 & H-5 of -C6H5), 7.55-7.64
(m, 2H, Ar-H, H-2 & H-6 of -C6H5), 7.96 (s, 1H, C=CH-Ph), 12.54 (s, 1H, NH, D2O exchangeable); 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.
4.1.3.4. N-(4-Acetylphenyl)-2-(5-benzylidene-2,4-dioxothiazolidin-3-yl)acetamide (6d)
Yield, 60%; m.p. 153-5oC; IRνmax (cm-1): 3305 (NH), 3032 (CH aromatic), 2924 (CH aliphatic), 1743, 1685, 1659
(4C=O); 1HNMR (400 MHz, DMSO-d6): 2.51 (s, 3H, COCH3), 4.50 (s, 2H, CH2), 7.12-7.13 (m, 5H, Ar-H of phenyl
ring), 7.43-7.46 (m, 2H, Ar-H, H-2 & H-6 of Acetophenone), 7.62-7.65 (m, 2H, Ar-H, H-3 & H-5 of Acetophenone),
7.96 (s, 1H, C=CH-Ph), 10.30 (s, 1H, NH, D2O exchangeable); MS (m/z): 381 (M++1, 17.26 %), 380 (M+, 50.58 %),
294 (100 %, base beak), 165 (24.80 %), 71 (49.68 %); Anal. Calcd. for C20H16N2O4S (380.42): C, 63.15; H, 4.24; N,
7.36. Found: C, 62.89; H, 4.63; N, 7.72.
4.1.3.5. 2-(2-(5-Benzylidene-2,4-dioxothiazolidin-3-yl)acetamido)benzoic acid (6e)
19

Yield, 65%; m.p. 180-2oC; IRνmax (cm-1): 3414 (OH), 3237 (NH), 3032 (CH aromatic), 2921 (CH aliphatic), 1728, 1697
(4C=O); 1HNMR (400 MHz, DMSO-d6): 4.61 (s, 2H, CH2), 7.36-7.41 (m, H, Ar-H, H-4 of C6H4-COOH), 7.51-7.65
(m, 3H, Ar-H, H-3, H-4 & H-5 of phenyl), 7.69-7.71 (m, 3H, Ar-H, H-2 & H-6 of phenyl and H-5 of C6H4-COOH),
7.82-7.87 (m, 2H, Ar-H, H-3 & H-6 of C6H4-COOH), 8.02 (s, 1H, C=CH-Ph), 10.67 (s, 1H, NH, D2O exchangeable),
12.20 (s, 1H, -OH, D2O exchangeable); MS (m/z): 382 (M+, 15.75 %), 214 (10.28 %), 133 (100 %, base beak), 71
(15.25 %); Anal. Calcd. for C19H14N2O5S (382.39): C, 59.68; H, 3.69; N, 7.33. Found: 59.86; H, 3.80; N, 7.21.
4.1.3.6. 4-(2-(5-Benzylidene-2,4-dioxothiazolidin-3-yl)acetamido)benzoic acid (6f)
Yield, 70%; m.p. 197-9oC; IRνmax (cm-1): 3414 (OH), 3233 (NH), 3032 (CH aromatic), 2928 (CH aliphatic), 1730, 1697
(4C=O); 1HNMR (400 MHz, DMSO-d6): 4.56 (s, 2H, CH2), 7.15 (m, H, Ar-H, H-4 of C6H5), 7.57 (m, 6H, Ar-H, H-
2, H-3, H-5 & H-6 of C6H5 & H-2 & H-6 of C6H4-COOH), 7.76 (m, 2H, Ar-H, H-3 & H-5 of C6H4-COOH), 7.99 (s,
1H, C=CH-Ph), 10.37 (s, 1H, NH, D2O exchangeable), 12.00 (s, 1H, -OH, D2O exchangeable; MS (m/z): 382 (M+,
22.75 %), 235 (63.87 %), 79 (100 %, base beak), 71 (20.30 %); Anal. Calcd. for C19H14N2O5S (382.39): C, 59.68; H,
3.69; N, 7.33. Found: 59.40; H, 3.47; N, 6.99.
4.1.3.7. Ethyl 4-(2-(5-benzylidene-2,4-dioxothiazolidin-3-yl)acetamido)benzoate (6g)
Yield, 82%; m.p. 155-7oC; IRνmax (cm-1): 3294 (NH), 3032 (CH aromatic), 2924 (CH aliphatic), 1751, 1694, 1670
(4C=O); 1HNMR (400 MHz, DMSO-d6): 1.13 (t, 3H, -OCH2CH3), 3.24 (q, 2H, -OCH2CH3), 4.55 (s, 2H, -NCH2CO),
7.39 (m, 3H, Ar-H, H-3, H-4 & H-5 of -C6H5), 7.63 (m, 2H, Ar-H, H-2 & H-6 of -C6H5), 7.82 (d, 2H, Ar-H, H-2 &
H-6 of -C6H4CO), 7.94 (d, 2H, Ar-H, H-3 & H-5 of -C6H4CO), 8.59 (s, 1H, C=CH-Ph), 10.67 (s, 1H, NH, D2O
exchangeable); Anal. Calcd. for C21H18N2O5S (410.44): C, 61.45; H, 4.42; N, 6.83. Found: C, 61.24; H, 4.23; N, 6.96.
4.1.3.8. 2-(5-Benzylidene-2,4-dioxothiazolidin-3-yl)-N-(4-nitrophenyl)acetamide (6h)
Yield, 55%; m.p. 157-9oC; IRνmax (cm-1): 3298 (NH), 3190 (CH aromatic), 2920 (CH aliphatic), 1705, 1694, 1682
(3C=O); 1HNMR (400 MHz, DMSO-d6): 4.47 (s, 2H, CH2), 7.14-7.17 (m, 1H, Ar-H, H-4 of pyridine), 7.38-7.42 (m,
1H, Ar-H, H-4 of -C6H5), 7.50-7.55 (m, 2H, Ar-H, H-2 & H-6 of -C6H5), 7.63-7.66 (m, 2H, Ar-H, H-3 & H-5 of -C6H5),
7.79 (s, 1H, C=CH-Ph), 7.80-7.82 (m, 1H, Ar-H, H-5 of pyridine), 7.95-8.00 (m, 1H, Ar-H, H-6 of pyridine), 8.32-
8.36 (d, 1H, Ar-H, H-3 of pyridine), 11.02 (s, 1H, NH, D2O exchangeable); MS (m/z): 340 (M++1, 19.33 %), 339 (M+,
87.24 %), 296 (5.87), 134 (100 %, base beak), 78 (26.77 %); Anal. Calcd. for C17H13N3O3S (339.37): C, 60.17; H, 3.86;
N, 12.38. Found: C, 59.89; H, 4.02; N, 12.54.
4.1.4. General procedure for synthesis of target compounds (8a-f)
Equimolar quantities of the potassium salt 3 (2.43 g, 0.01 mol) and the appropriate 4-(2-chloroacetamido)-N-
substitutedbenzamide derivative 7a-f (0.01 mol) in DMF (20 mL) was heated on water bath for 5 h. The reaction mixture
was poured onto ice-water (200 mL) and stirred for 30 min. The obtained solid was filtered, washed with water, dried
and crystallized from ethanol to give the corresponding target compounds, 8a-f respectively.
4.1.4.1. 4-(2-(5-Benzylidene-2,4-dioxothiazolidin-3-yl)acetamido)-N-ethylbenzamide (8a)
Yield, 85%; m.p. 242-3oC; IRνmax (cm-1): 3279 (2NH), 3055 (CH aromatic), 2932 (CH aliphatic), 1743, 1685 (4C=O);
1HNMR (400 MHz, DMSO-d6): 1.19 (t, 3H, CH2CH3), 3.28 (q, 2H, -CH2CH3), 4.61 (s, 2H, -NCH2CO), 7.36-7.71 (m,
20

5H, Ar-H of -C6H5), 7.87-7.98 (m, 4H, Ar-H of -C6H4), 8.08 (s, 1H, C=CH-Ph), 8.48 (s, 1H, -NHCH2CH3, D2O
exchangeable), 10.97 (s, 1H, NH-C6H4, D2O exchangeable); 411 (M++2, 5.96 %), MS (m/z): 410 (M++1, 6.78 %), 409
(M+, 27.79 %), 372 (43.77 %), 121 (55.18 %), 78 (100 %, base beak); Anal. Calcd. for C21H19N3O4S (409.46): C, 61.60;
H, 4.68; N, 10.26. Found: C, 61.83; H, 4.90; N, 10.13.
4.1.4.2. 4-(2-(5-Benzylidene-2,4-dioxothiazolidin-3-yl)acetamido)-N-propylbenzamide (8b)
Yield, 85%; m.p. 245-7oC; IRνmax (cm-1): 3329, 3283 (2NH), 3060 (CH aromatic), 2932 (CH aliphatic), 1748, 1680,
1678 (4C=O); 1HNMR (400 MHz, DMSO-d6): 0.90 (t, 3H, -CH2CH2CH3), 1.51 (m, 2H, -CH2CH2CH3), 3.21 (t, 2H, -
CH2CH2CH3), 4.56 (s, 2H, -NCH2CO), 7.52-7.58 (m, 3H, Ar-H, H-3, H-4 & H-5 of -C6H5), 7.60-7.62 (d, 2H, Ar-H,
H-2 & H-6 of -C6H5), 7.65-7.69 (d, 2H, Ar-H, H-2 & H-6 of -C6H4), 7.82-7.84 (d, 2H, Ar-H, H-3 & H-5 of -C6H4),
8.02 (s 1H, C=CH-Ph), 8.37 (s, 1H, -NHCH2CH2CH3, D2O exchangeable), 10.64 (s, 1H, NH- C6H4, D2O
exchangeable); 13CNMR (100 MHz, DMSO-d6): 34.66, 38.47, 119.23 (2), 121.84, 127.14 (3), 129.86 (3), 130.59 (3),
131.11, 133.29, 133.43, 138.84, 142.31, 165.94, 167.62, 169.42; Anal. Calcd. for C22H21N3O4S (423.49): C, 62.40; H,
5.00; N, 9.92. Found: C, 62.67; H, 4.89; N, 10.16.
4.1.4.3. 4-(2-(5-Benzylidene-2,4-dioxothiazolidin-3-yl)acetamido)-N-butylbenzamide (8c)
Yield, 75%; m.p. 247-9oC; IRνmax (cm-1): 3329, 3233 (2NH), 3066 (CH aromatic), 2943 (CH aliphatic), 1740, 1694,
1675 (4C=O); 1HNMR (400 MHz, DMSO-d6): 0.89 (t, 3H, -CH2CH2CH2CH3), 1.34 (m, 2H, -CH2CH2CH2CH3), 1.51
(m, 2H, -CH2CH2CH2CH3), 3.24 (t, 2H, -CH2CH2CH2CH3), 4.59 (s, 2H, -NCH2CO), 7.41-7.56 (m, 3H, Ar-H, H-3,
H-4 & H-5 of -C6H5), 7.64 (d, 2H, Ar-H, H-2 & H-6 of -C6H5), 7.68 (d, 2H, Ar-H, H-2 & H-6 of -C6H4), 7.81 (d, 2H,
Ar-H, H-3 & H-5 of -C6H4), 8.02 (s, 1H, C=CH-Ph), 8.34 (s, 1H, -NHCH2CH2CH3, D2O exchangeable), 10.74 (s, 1H,
NH-C6H4, D2O exchangeable); MS (m/z): 439 (M++2, 4.88 %), 438 (M++1, 14.27 %), 437 (M+, 24.35 %), 364 (13.81
%), 120 (100 %, base beak), 76 (17.84 %); Anal. Calcd. for C23H23N3O4S (437.51): C, 63.14; H, 5.30; N, 9.60. Found:
C, 63.40; H, 5.56; N, 9.87.
4.1.4.4. N-Benzyl-4-(2-(5-benzylidene-2,4-dioxothiazolidin-3-yl)acetamido)benzamide (8d)
Yield, 60%; m.p. 257-9oC; IRνmax (cm-1): 3267 (2NH), 3060 (CH aromatic), 2935 (CH aliphatic), 1747, 1694, 1659
1
(4C=O); HNMR (400 MHz, DMSO-d6): 4.11 (s, 2H, -CH2Ph), 4.59 (s, 2H, -NCH2CO), 7.14-7.18 (m, 5H, Ar-H, -
CH2C6H5), 7.45-7.53 (m, 1H, Ar-H, H-4 of -C6H5), 7.62-7.68 (m, 2H, Ar-H, H-3 & H-5 of -C6H5), 7.71-7.75 (m, 2H,
Ar-H, H-2 & H-6 of -C6H5), 7.93-7.96 (m, 2H, Ar-H, H-3 & H-5 of -C6H5), 7.98-8.02 (m, 2H, Ar-H, H-2 & H-6 of -
C6H5), 8.05 (s, 1H, C=CH-Ph), 10.10 (s, 1H, -NHCH2Ph, D2O exchangeable), 10.56 (s, 1H, NH-CO, D2O
exchangeable); Anal. Calcd. for C26H21N3O4S (471.53): C, 66.23; H, 4.49; N, 8.91. Found: C, 66.45; H, 4.68; N, 8.82.
4.1.4.5. 4-(2-(5-Benzylidene-2,4-dioxothiazolidin-3-yl)acetamido)-N-(p-tolyl)benzamide (8e)
Yield, 60%; m.p. 263-5oC; IRνmax (cm-1): 3144 (2NH), 3040 (CH aromatic), 2889 (CH aliphatic), 1739, 1693 (4C=O);
1HNMR (400 MHz, DMSO-d6): 2.30 (s, 3H, CH3), 4.10 (s, 2H, -NCH2CO), 7.14-7.18 (m, 3H, Ar-H, H-4 of C6H5,
H-3 & H-5 of -C6H4CH3), 7.32-7.39 (m, 2H, Ar-H, H-2 & H-6 of -C6H4CH3), 7.46-7.60 (m, 2H, Ar-H, H-3, & H-5 of
-C6H5), 6.65-7.72 (m, 4H, Ar-H, of -C6H4CO), 7.74-7.95 (m, 2H, Ar-H, H-2 & H-6 of -C6H5), 8.02 (s, 1H, C=CH-Ph),
10.04 (s, 1H, NH, -NHC6H4CH3, D2O exchangeable), 10.24 (s, 1H, NH, -NHC6H4CO, D2O exchangeable); Anal.
Calcd. for C26H21N3O4S (471.53): C, 66.23; H, 4.49; N, 8.91. Found: C, 66.38; H, 4.70; N, 9.07.
4.1.4.6. Ethyl 4-(4-(2-(5-benzylidene-2,4-dioxothiazolidin-3-yl)acetamido)benzamido)benzoate (8f)
21

Yield, 60%; m.p. 272-3oC; IRνmax (cm-1): 3283 (2NH), 3040 (CH aromatic), 2943 (CH aliphatic), 1748, 1690, 1681,
1
1647 (5C=O); HNMR (400 MHz, DMSO-d6): 1.32 (t, 3H, -OCH2CH3), 4.31 (q, 2H, -OCH2CH3), 4.59 (s, 2H, -
NCH2CO), 7.53-7.60 (m, 3H, Ar-H, H-3, H-4 & H-5 of -C6H5), 7.67-7.69 (m, 2H, Ar-H, H-2 & H-6 of -C6H5), 7.72-
7.74 (m, 2H, Ar-H, H-2 & H-6 of -C6H4CONH), 7.85 (s, 1H, C=CH-Ph), 7.95-8.02 (m, 2H, Ar-H, H-3 & H-5 of -
C6H4CONH), 8.27-8.29 (m, 2H, Ar-H, H-3 & H-5 of -C6H4COO ), 8.52-8.53 ( (m, 2H, Ar-H, H-2 & H-6 of -C6H4COO),
10.47 (s, 1H, NH, -C6H4CONH, D2O exchangeable), 10.75 (s, 1H, NH, -NHC6H4COO, D2O exchangeable); Anal.
Calcd. for C28H23N3O6S (529.57): C, 63.51; H, 4.38; N, 7.93. Found: C, 63.80; H, 4.50; N, 8.12.
5.1.Docking studies
In the present work, all the target compounds were subjected to docking study to explore their binding mode
towards VEGFR-2 enzyme. All modeling experiments were performed using molsoft program, which provides a unique
set of tools for the modeling of protein / ligandinteractions. It predicts how small flexible molecule such as substrates
or drug candidates bind to a protein of known 3D structure represented by grid interaction potentials
(http://www.molsoft.com/icm_pro.html). Each experiment used the biological target VEGFR-2 downloaded from the
Brookhaven Protein Databank (http://www.rcsb.org/pdb/explore/ explore.do?structureId=1YWN). In order to qualify
the docking results in terms of accuracy of the predicted binding conformations in comparison with the experimental
procedure, the reported VEGFR-2 inhibitor drug sorafenib was used as reference ligand.
5.2.In vitro cytotoxic activity
The cytotoxicity assays were performed at Pharmacology & Toxicology Department, Faculty of pharmacy, Al-
Azhar University, Cairo, Egypt. Cancer cells from different cancer cell lines hepatocellular carcinoma (HepG2), breast
cancer (MCF-7) and colorectal carcinoma (HCT-116), were purchased from American type Cell Culture collection
(ATCC, Manassas, USA) 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 bovine serum in a humidified, 5% (v/v) CO2 atmosphere at 37 ºC Cytotoxicity assay by 3-[4,5-dimethylthiazole-
2-yl]-2,5-diphenyltetrazolium bromide (MTT).
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 then were incubated in a
humidified atmosphere at 37˚C for 24 hours. Then, cells were exposed to different concentrations of compounds (0.1,
10, 100 and 1000 µM) for 72 hours. Then the viability of treated cells was determined using MTT technique as follow.
Media were removed; cells were incubated with 200 μl of 5% MTT solution/well (Sigma Aldrich, MO) and were
allowed to metabolize the dye into colored-insoluble formazan crystals for 2 hours. The remaining MTT solution were
discarded from the wells and the formazan crystals were dissolved in 200 µl/well acidified isopropanol for 30 min,
covered with aluminum foil and with continuous shaking using a MaxQ 2000 plate shaker (Thermo Fisher Scientific
Inc, MI) at room temperature. The colorimetric assay was measured and recorded at absorbance of 570 nm using a Stat
FaxR 4200 plate reader (Awareness Technology, Inc., FL). The cell viability were expressed as percentage of control
and the concentration that induces 50% of maximum inhibition of cell proliferation (IC50) were determined using Graph
Pad Prism version 5 software (Graph Pad software Inc, CA) [40, 41, 49].
22

5.3.In vitro VEGFR-2 kinase assay
The kinase activity of VEGFR-2 was carried out by Dr. Fatma M. I. A. Shoman, MD in Clinical Pathology,
Blood bank specialist, Blood bank directorate manager, Ministry of Health, Cairo, Egypt, and measured by use of an
anti-phosphotyrosine antibody with the Alpha Screen system (PerkinElmer, USA) according to manufacturer’s
instructions [50]. Enzyme reactions were performed in 50 mM Tris–HCl pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.01%
Tween-20 and 2 mM DTT, containing 10 μM ATP, 0.1 μg/mL biotinylated poly-GluTyr (4:1) and 0.1 nM of VEGFR-2
(Millipore, UK). Prior to catalytic initiation with ATP, the tested compounds at final concentrations ranging from 0-
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 Alpha Screen streptavidine donor beads and 10 μg/mL acceptor beads in 62.5 mM
HEPES pH 7.4, 250 mM NaCl, and 0.1% BSA. Plate was incubated in the dark overnight and then read by ELISA
Reader (PerkinElmer, USA). Wells containing the substrate and the enzyme without compounds were used as reaction
control. Wells containing biotinylated poly-GluTyr (4:1) and enzyme without ATP were used as basal control. Percent
inhibition was calculated by the comparison of compounds treated to control incubations. The concentration of the test
compound causing 50% inhibition (IC50) was calculated from the concentration–inhibition response curve (triplicate
determinations) and the data were compared with Sorafenib (Sigma-Aldrich, USA) as standard VEGFR-2 inhibitor.
Acknowledgments: The authors extend their appreciation and thanking to Dr. Tamer Abdel-Ghany, Pharmacology
& Toxicology Department, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt for helping in the pharmacological
part.
Conflict of interest: No
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► Seventeen compounds of Novel thiazolidin2,4-diones were designed and synthesized.
► In vitro cytotoxic activities were evaluated against three human cell lines HepG2, HCT-116 and
MCF-7
► In vitro VEGFR-2 kinase assay was carried out for the most potent 10 compounds.
► Molecular docking studies were carried out.
26