Benzoxazole/benzothiazole-derived VEGFR-2 inhibitors: Design, synthesis, molecular docking, and anticancer evaluations

Article abstract of DOI:10.1002/ardp.201900178

A novel series of benzoxazole/benzothiazole derivatives 4a–c–11a–e were designed, synthesized, and evaluated for anticancer activity against HepG2, HCT-116, and MCF-7 cells. HCT-116 was the most sensitive cell line to the influence of the new derivatives. In particular, compound 4c was found to be the most potent derivative against HepG2, HCT-116, and MCF-7 cells, with IC₅₀ values = 9.45 ± 0.8, 5.76 ± 0.4, and 7.36 ± 0.5 μM, respectively. Compounds 4b, 9f, and 9c showed the highest anticancer activities against HepG2 cells with IC₅₀ values of 9.97 ± 0.8, 9.99 ± 0.8, and 11.02 ± 1.0 μM, respectively, HCT-116 cells with IC₅₀ values of 6.99 ± 0.5, 7.44 ± 0.4, and 8.15 ± 0.8 μM, respectively, and MCF-7 cells with IC₅₀ values of 7.89 ± 0.7, 8.24 ± 0.7, and 9.32 ± 0.7 μM, respectively, in comparison with sorafenib as reference drug with IC₅₀ values of 9.18 ± 0.6, 5.47 ± 0.3, and 7.26 ± 0.3 μM, respectively. The most active compounds 4a–c, 9b,c,e,f,h, and 11c,e were further evaluated for their VEGFR-2 inhibition. Compounds 4c and 4b potently inhibited VEGFR-2 at IC₅₀ values of 0.12 ± 0.01 and 0.13 ± 0.02 μM, respectively, which are nearly equipotent to the sorafenib IC₅₀ value (0.10 ± 0.02 μM). Furthermore, molecular docking studies were performed for all synthesized compounds to assess their binding pattern and affinity toward the VEGFR-2 active site.

Full text of DOI:10.1002/ardp.201900178

Received: 20 June 2019
Revised: 29 August 2019
Accepted: 1 September 2019
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DOI: 10.1002/ardp.201900178
F U L L P A P E R
Benzoxazole/benzothiazole‐derived VEGFR‐2 inhibitors:
Design, synthesis, molecular docking, and anticancer
evaluations
1
1
1
Abdel‐Ghany A. El‐Helby
Ahmed A. Al‐Karmalawy
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|
Helmy Sakr
|
Ibrahim H. Eissa
|
1
1,2
Khaled El‐Adl
1
Department of Pharmaceutical Chemistry,
Abstract
Faculty of Pharmacy, Al‐Azhar University,
Cairo, Egypt
A novel series of benzoxazole/benzothiazole derivatives 4a–c–11a–e were designed,
synthesized, and evaluated for anticancer activity against HepG2, HCT‐116, and
MCF‐7 cells. HCT‐116 was the most sensitive cell line to the influence of the new
derivatives. In particular, compound 4c was found to be the most potent derivative
against HepG2, HCT‐116, and MCF‐7 cells, with IC50 values = 9.45 ± 0.8, 5.76 ± 0.4,
and 7.36 ± 0.5 µM, respectively. Compounds 4b, 9f, and 9c showed the highest
anticancer activities against HepG2 cells with IC50 values of 9.97 ± 0.8, 9.99 ± 0.8, and
2
Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Heliopolis University for
Sustainable Development, Cairo, Egypt
Correspondence
Khaled El‐Adl and Ibrahim H. Eissa,
Pharmaceutical Chemistry Department,
Faculty of Pharmacy, Al‐Azhar University, 1
Al‐Mukhayam Al‐Daem Street, 6th District,
Nassr City, Cairo 00202‐2262‐5796, Egypt.
Email: eladlkhaled74@yahoo.com;
eladlkhaled74@azhar.edu.eg (K. E‐A.) and
ibrahimeissa@azhar.edu.eg (I. H. E.)
1
1.02 ± 1.0 µM, respectively, HCT‐116 cells with IC50 values of 6.99 ± 0.5, 7.44 ± 0.4,
and 8.15 ± 0.8 µM, respectively, and MCF‐7 cells with IC50 values of 7.89 ± 0.7,
.24 ± 0.7, and 9.32 ± 0.7 µM, respectively, in comparison with sorafenib as reference
8
drug with IC50 values of 9.18 ± 0.6, 5.47 ± 0.3, and 7.26 ± 0.3 µM, respectively. The
most active compounds 4a–c, 9b,c,e,f,h, and 11c,e were further evaluated for their
VEGFR‐2 inhibition. Compounds 4c and 4b potently inhibited VEGFR‐2 at IC50 values
of 0.12 ± 0.01 and 0.13 ± 0.02 µM, respectively, which are nearly equipotent to the
sorafenib IC50 value (0.10 ± 0.02 µM). Furthermore, molecular docking studies were
performed for all synthesized compounds to assess their binding pattern and affinity
toward the VEGFR‐2 active site.
K E Y W O R D S
anticancer agents, benzothiazole, benzoxazole, molecular docking, VEGFR‐2 inhibitors
1
|
INTRODUCTION
The VEGF signaling pathway plays fundamental roles in regulat-
ing tumor angiogenesis. VEGF, as a therapeutic target, has been
[
10]
Extensive studies have been reported on the synthesis of several
validated in various types of human cancers.
VEGFR‐2 represents
[1–5]
benzoxazole derivatives as promising anticancer agents
as potent
a major target within angiogenesis‐related kinases and hence is
vascular endothelial growth factor receptor‐2 (VEGFR‐2)
considered the most important transducer of VEGF‐dependent
[
4,5]
[11]
inhibitors,
for example, compound
I
(Figure 1). Moreover,
angiogenesis.
Thus, inhibition of the VEGF/VEGFR signaling
benzothiazole derivatives have attracted considerable attention as
pathway is regarded as an attractive therapeutic target for the
[12–15]
[
5–7]
promising and effective anticancer agents
targeting the VEGFR‐2
inhibition of tumor angiogenesis and subsequent tumor growth.
[
5,8,9]
®
enzyme.
It was suggested that benzothiazoles act as competitive
Sorafenib (Nexavar) is a potent VEGFR‐2 inhibitor and has been
[5]
[16–18]
inhibitors at the ATP‐binding site of tyrosine kinases, for example,
compound II (Figure 1).
approved as an antiangiogenic drug.
Studies on the
structure–activity relationships (SAR) and common pharmacophoric
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II
I
FIGURE 1 Reported benzoxazole and benzothiazole derivatives as VEGFR‐2 inhibitors
features shared by sorafenib and various VEGFR‐2 inhibitors
revealed that most VEGFR‐2 inhibitors shared four main features,
backbones of many bioactive compounds that show potential activities
[
27,28]
as VEGFR inhibitors. In addition, several 1,3,4‐thiadiazoles,
[
19–21]
[29]
[30]
as shown in Figure 2:
(a) The core structure of most inhibitors
thiazolidine‐2,4‐dione
have been reported to possess anticancer activities.
Depending on the ligand‐based drug design, particularly
molecular hybridization approach that involves the coupling of two
and 6‐phenyl‐5‐cyanothiouracil
moieties
consists of a flat heteroaromatic ring system that contains at least
one N atom, which occupies the catalytic adenosine triphosphate
a
(ATP)‐binding domain. (b) A central aryl ring (hydrophobic spacer),
[31]
occupying the linker region between the ATP‐binding domain and the
or more groups with relevant biological properties,
molecular
[22]
DFG domain of the enzyme.
(c) A linker containing a functional
hybridization of benzoxazole and/or benzothiazole and other
effective antitumor moieties were carried out in an attempt to get
new molecules with promising antitumor activities.
group acting as a pharmacophore (e.g., amino or urea) that possesses
both a H‐bond acceptor (HBA) and a donor (HBD) 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. (d) The terminal hydrophobic moiety
of the inhibitors occupies the newly created allosteric hydrophobic
pocket revealed when the phenylalanine residue of the DFG loop
flips out of its lipophilic pocket‐defining DFG‐out or inactive
conformation. Thus, hydrophobic interactions are usually attained
In continuation of our efforts to obtain new anticancer agents
targeting VEGFR‐2, the goal of our work was the synthesis of new
agents with the same essential pharmacophoric features of the
reported and clinically used VEGFR‐2 inhibitors (e.g., sorafenib). The
main core of our molecular design rationale comprised bioisosteric
modification strategies of VEGFR‐2 inhibitors at four different
positions (Figure 3).
Our target compounds were designed to have different spacers
and different cyclic linkers with HBA–HBD, the main pharmaco-
phoric feature in sorafenib, with the hope to obtain more potent
VEGFR‐2 inhibitors. First, a bioisosteric approach was adopted in
the target benzoxazole and/or benzothiazole to replace the
pyridine ring. The second strategy was to use acetamide, 2‐
sulfanylacetamide and/or 3‐sulfanylpropanamide to replace the
central aryl ring of the lead structure to increase the flexibility
with the aim to increase the VEGFR‐2 binding affinity. The third
strategy is using cyclic HBA–HBD linkers containing functional
groups that possess H‐bond acceptors and/or donors, such as
1,3,4‐thiadiazoles in compounds 4a–c, thiazolidine‐2,4‐dione in
compounds 9a–j, and 6‐phenyl‐5‐cyanothiouracil in compounds
[23]
in this allosteric binding region.
Furthermore, analysis of the X‐ray
structure of various inhibitors bound to VEGFR‐2 confirmed the
sufficient space available for various substituents around the
[24–26]
terminal heteroaromatic ring.
Benzoxazole and benzothiazole nuclei are privileged scaffolds that
form the most promising class of heterocycles that are well‐tolerated in
humans and possess antitumor activity. Moreover, they are the
aryl
ring
1
1a–e. Also, the hydrophobic substituted phenyl tail of the
reported ligand was substituted by other groups. Furthermore,
the substitution pattern was selected to ensure different electro-
nic and lipophilic environments, which could influence the activity
of the target compounds. On the contrary, the linkers were
designed in a different way; they constituted a part of the rigid
ring structures to study the effect of free‐rotated NHCONH of
sorafenib and the rigid ring structure linkers on SAR. These
modifications were performed to carry out further elaboration of
the benzoxazole and/or benzothiazole scaffolds and to explore a
valuable SAR. The designed target benzoxazole/benzothiazole
derivatives were synthesized and evaluated for their potential
VEGFR‐2 inhibitory and antitumor activities against three human
tumor cell lines: hepatocellular carcinoma (HCC) type (HepG2),
breast cancer (Michigan Cancer Foundation‐7 [MCF‐7]), and
human colorectal carcinoma‐116 (HCT‐116).
FIGURE 2 The basic structural requirements for sorafenib as
reported VEGFR‐2 inhibitor. ATP, adenosine triphosphate; HBA,
H‐bond acceptor; HBD, H‐bond donor; VEGFR‐2, vascular
endothelial growth factor receptor‐2

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aryl
ring
Sorafenib
FIGURE 3 Structural similarities and pharmacophoric features of VEGFR‐2 inhibitors and the designed compounds. HBA, H‐bond acceptor;
HBD, H‐bond donor; VEGFR‐2, vascular endothelial growth factor receptor‐2
2
2
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RESULTS AND DISCUSSION
decreased the flexibility of the linkers and the lack of the carbonyl
of the urea of sorafenib decreased the binding affinity where it did
not form H‐bond with the essential amino acid Glutamate883. This
may explain the importance of flexible free‐rotated linkers.
.1
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Rationale and structure‐based design
Benzoxazole/benzothiazole derivatives impart essential pharmaco-
[32–35]
phoric features to VEGFR‐2 inhibitors
(Figure 3), which include
the presence of five‐membered hetero rings, oxazole and/or thiazole,
fused with a benzene ring, as a hydrophobic portion, forming an
aromatic system represented by benzoxazole/benzothiazole rings
linked to an (un)substituted hydrophobic distal phenyl ring through
different spacers and cyclic linkers (HBA–HBD), which serve as
H‐bond acceptors through their two Ns with the essential amino acid
residue Aspartate1044, also through hydrophobic interaction with its
2
.2 Chemistry
|
The synthetic strategy for preparation of the target compounds
(4–11) is depicted in Schemes 1–3. The synthesis was initiated by
cyclocondensation of thiosemicarbazide with benzoic acid to
[
36,37]
afford 5‐phenyl‐1,3,4‐thiadiazol‐2‐amine,
which reacted with
the appropriate chloroacyl chloride to afford the corresponding
chloroamide (1a,b). On the contrary, 2‐amino‐4‐substituted phenol
was reacted with carbon disulphide in the presence of alcoholic
potassium hydroxide to provide the corresponding 2‐mercapto-
benzoxazole derivatives (2a,b), respectively, which were treated
with alcoholic potassium hydroxide to afford the corresponding
potassium salts (3a,b). The appropriate potassium salt (3a,b) was
refluxed with the appropriate chloroamide (1a,b) to afford the
corresponding acid amide derivatives (4a–c; Scheme 1). Chlor-
oacetic acid was refluxed with thiourea to afford thiazolidinedione
(un)substituted hydrophobic phenyl ring with the hydrophobic
pocket lined with the hydrophobic side chains of Alanine864,
Valine865, Lysine866, Valine897, Valine914, and Leucine1033. In
addition, oxazole and/or other moieties were designed to replace the
pyridine moiety of the reference ligand sorafenib. Moreover, the
hydrophobic benzoxazole/benzothiazole rings occupied the hydro-
phobic groove formed by Arginine1025, Histidine1024, Isoleu-
cine1023, Cysteine1022, Leucine1017, Isoleucine890, Histidine889,
and Isoleucine886. On the contrary, the cyclic rigid structure

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R = H,
R = H,
n = 1
n = 2
R = CH3, n = 2
SCHEME 1 Synthetic route for the preparation of the target compounds 4a–c
(
5) which underwent reaction with alcoholic potassium hydroxide
The obtained results indicated that all studied ligands have
similar position and orientation inside the putative binding site of
VEGFR‐2, which reveals a large space bounded by a membrane‐
binding domain which serves as entry channel for the substrate to
the active site (Figure 4). In addition, the affinity of any small
molecule can be considered as a unique tool in the field of drug
design. There is a relationship between the affinity of organic
to produce the corresponding potassium salt (6). 2‐Aminoben-
zothiazole was reacted with chloroacetyl chloride to obtain the
corresponding chloroamide (7), which was refluxed with the
obtained potassium salt (6) to get the corresponding hybrid
benzothiazole thiazolidinedione derivative (8). The formed hybrid
molecule (8) underwent further condensation reaction with the
appropriate benzaldehyde, namely, benzaldehyde, 2‐chlorobenzal-
dehyde, 4‐chlorobenzaldehyde, 2,6‐dichlorobenzaldehyde, 4‐fluor-
obenzaldehyde, 4‐methylbenzaldehyde, 2‐methoxybenzaldehyde,
[
39–42]
molecules and the free energy of binding.
This relationship
can contribute to 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 1).
4
‐methoxybenzaldehyde, 4‐dimethylaminobenzaldehyde, and/or
‐nitrobenzaldehyde to obtain the corresponding 5‐arylidine‐2,4‐
3
thiazolidinone derivatives by Knoevenagel condensation reaction
(
9a–j), respectively (Scheme 2). Cyclocondensation of ethyl
cyanoacetate, thiourea, and the appropriate benzaldehyde,
namely, benzaldehyde, 4‐fluorobenzaldehyde, 4‐methylbenzalde-
hyde, 4‐hydroxybenzaldehyde, and/or 4‐methoxybenzaldehyde
afforded the corresponding 6‐phenyl‐5‐cyanothiouracil deriva-
tives (10a–e) which underwent reaction with the chloroamide (7)
to produce the corresponding 1,6‐dihydropyrimidine derivatives
The proposed binding mode of sorafenib revealed an affinity
value of −95.36 kcal/mol and four H‐bonds. The urea linker formed
one H‐bond with the key amino acid Glutamate883 (2.13 Å) through
its NH group and one H‐bond with Aspartate1044 (1.65 Å) through
its carbonyl group. The central phenyl ring occupied the hydrophobic
pocket formed by Glutamate883, Isoleucine886, Leucine887, Iso-
leucine1042, Cysteine1043, and Aspartate1044. Moreover, the distal
hydrophobic 3‐trifluoromethyl‐4‐chlorophenyl moiety attached to
the urea linker occupied the hydrophobic pocket formed by
Cysteine1043, Leucine1033, Valine897, Valine914, Alanine864, Va-
line865, and Lysine866. Furthermore, the N‐methylpicolinamide
moiety occupied the hydrophobic groove formed by Arginine1025,
(
11a–e), respectively (Scheme 3).
2
.3 Docking studies
|
In the present work, all modeling experiments were performed using
Molsoft software. Each experiment used VEGFR‐2 downloaded from
[38]
the Brookhaven Protein Databank (PDB ID 1YWN).
Histidine1024,
Isoleucine1023,
Cysteine1022,
Leucine1017,

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R = H
R = 2-Cl
R = 4-Cl
R = 2,6-(Cl)2
R = 4-F
R = 4-CH3
R = 2-OCH3
R = 4-OCH3
R = 4-N(CH3)2
R = 3-NO2
SCHEME 2 Synthetic route for the preparation of the target compounds 8–j and 9a–j
Isoleucine890, Histidine889, and Isoleucine886, whereas its carbonyl
As planned, the proposed binding mode of compound 4c is
virtually the same as that of sorafenib which revealed affinity nearly
the same as that of sorafenib with a value of −94.71 kcal/mol and
four H‐bonds. The 1,3,4‐thiadiazole linker was stabilized by the
formation of three H‐bonds with Aspartate1044 (1.43, 2.30, and
was stabilized by the formation of two H‐bonds with Arginine1025
(1.90 and 2.12 Å; Figure 5). 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. This finding
encourages us to use different cyclic linkers resembling urea of
sorafenib, hoping to obtain potent VEGFR‐2 inhibitors.
2.88 Å). The SCH
2 2
CH CONH spacer formed one H‐bond with
Aspartate1044 (2.58 Å) through its carbonyl group. It also occupied
R = H
R = F
R = CH3
R = OH
R = OCH3
SCHEME 3 Synthetic route for the preparation of the target compounds 11a–e

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FIGURE 4 Superimposition of some
docked compounds inside the binding
pocket of 1YWN
the hydrophobic pocket formed by Glutamate883, Isoleucine886,
Leucine887, Leucine1017, Histidine1024, Isoleucine1042, Cy-
steine1043, and Aspartate1044. The distal phenyl moiety occupied
the hydrophobic pocket formed by Leucine1033, Valine897, Va-
line914, Glutamate915, Phenylalanine916, Cysteine917, Alanine864,
Valine865, Lysine866, and Glutamate883. Furthermore, the benzox-
azole moiety occupied the hydrophobic groove formed by Argi-
nine1025, Histidine1024, Isoleucine1023, Cysteine1022, Isoleu-
cine890, Histidine889, and Isoleucine886 (Figure 6). These interac-
tions of compound 4c may explain the highest anticancer activity.
The proposed binding mode of compound 4b is virtually the same
as that of 4c which revealed an affinity value of −92.39 kcal/mol and
four H‐bonds. The 1,3,4‐thiadiazole linker was stabilized by the
formation of three H‐bonds with Aspartate1044 (1.57, 2.68, and
Alanine864, Valine865, Lysine866, and Glutamate883. Furthermore,
the benzoxazole moiety occupied the hydrophobic groove formed by
Arginine1025, Histidine1024, Isoleucine1023, Cysteine1022, Isoleu-
cine890, Histidine889, and Isoleucine886 (Figure 7). These interac-
tions of compound 4b may explain the higher anticancer activity.
From the obtained docking results (Table 1), we concluded that
free‐rotated urea linker is essential for higher affinity towards
VEGFR‐2 enzyme than the cyclic linkers. The longer SCH
spacer showed higher binding affinities than the SCH
2
CH
2
CONH
2
CONH one.
The 1,3,4‐thiadiazoles exhibited higher affinities than thiazolidine‐
2,4‐dione and 1,6‐dihydropyrimidine, respectively. Also, lipophilicity
played an important role in their VEGFR‐2 inhibitory activities which
may be due to higher hydrophobic interactions.
2 2
2.79 Å). The SCH CH CONH spacer formed one H‐bond with
Aspartate1044 (1.87 Å) through its carbonyl group. It also occupied
the hydrophobic pocket formed by Glutamate883, Isoleucine886,
Leucine887, Leucine1017, Histidine1024, Isoleucine1042, Cy-
steine1043, and Aspartate1044. The distal phenyl moiety occupied
the hydrophobic pocket formed by Leucine1033, Valine897,
|
In vitro cytotoxic activity
2
.4
Antiproliferative activity of the newly synthesized benzoxazole/
benzothiazole derivatives 4a–c–11a–e was examined against three
human tumor cell lines namely, HepG2, MCF‐7, and HCT‐116 using 3‐
(
4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) col-
Valine914,
Glutamate915,
Phenylalanine916,
Cysteine917,
[43–45]
orimetric assay as described by Mosmann.
Sorafenib was
included in the experiments as a reference cytotoxic drug. The results
were expressed as growth inhibitory concentration (IC50) values,
which represent the compound concentrations required to produce a
TABLE 1 The calculated ΔG (free energy of binding) and binding
affinities for the ligands (ΔG in kcal/mol)
Compound
ΔG (kcal/mol)
−82.78
−92.39
−94.71
−71.37
−74.07
−82.90
−86.03
−82.12
−85.83
−89.58
Compound
ΔG (kcal/mol)
−77.30
−84.22
−80.98
−74.32
−74.89
−80.56
−85.52
−79.89
−82.59
−95.36
5
0% inhibition of cell growth after 72 hr of incubation calculated from
4
4
4
8
9
9
9
9
9
9
a
b
c
9g
the concentration–inhibition response curve and summarized in
Table 2. From the obtained results, it was explicated that most of
the prepared compounds displayed an excellent to modest growth
inhibitory activity against the tested cancer cell lines. Investigations of
the cytotoxic activity against HCT‐116 and MCF‐7 indicated that they
were more sensitive cell lines to the influence of the new derivatives,
respectively. In particular, compound 4c was found to be the most
potent derivative overall the tested compounds against HepG2, HCT‐
9h
9i
9j
a
b
c
d
e
f
11a
11b
11c
11d
11e
Sorafenib
1
16, and MCF‐7 cancer cell lines with IC50 = 9.45 ± 0.8, 5.76 ± 0.4, and
7
.36 ± 0.5 µM, respectively. It has nearly the same activity as sorafenib
against the three cell lines (IC50 = 9.18 ± 0.6, 5.47 ± 0.3, and

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FIGURE 5 Predicted binding mode for
sorafenib with 1WYN. H‐bonded atoms are
indicated by dotted lines
FIGURE 6 Predicted binding mode for
4c with 1WYN
FIGURE 7 Predicted binding mode for
b with 1WYN
4

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TABLE 2 In vitro cytotoxic activities of the newly synthesized compounds against HepG2, MCF‐7, and HCT‐116 cell lines and VEGFR‐2
kinase assay
a
a
IC50 (µM)
HepG2
IC50 (µM)
HepG2
Compd
HCT‐116
MCF‐7
VEGFR‐2
0.22 ± 0.02
0.13 ± 0.02
0.12 ± 0.01
NT
Compd
9g
HCT‐116
MCF‐7
VEGFR‐2
NT
4
4
4
8
9
9
9
9
9
9
a
b
c
15.88 ± 1.2
9.97 ± 0.8
9.45 ± 0.8
34.55 ± 3.1
25.65 ± 2.3
16.32 ± 1.4
11.02 ± 1.0
19.09 ± 1.7
11.67 ± 0.9
9.99 ± 0.8
12.45 ± 1.2
6.99 ± 0.5
5.76 ± 0.4
32.77 ± 3.2
25.59 ± 2.3
15.43 ± 1.4
8.15 ± 0.8
21.12 ± 1.7
8.88 ± 0.8
7.44 ± 0.4
12.65 ± 1.1
7.89 ± 0.7
7.36 ± 0.5
34.13 ± 3.2
26.08 ± 2.3
14.55 ± 1.4
9.32 ± 0.7
17.23 ± 1.8
9.94 ± 0.6
8.24 ± 0.7
24.74 ± 2.4
14.06 ± 1.1
23.24 ± 1.9
25.88 ± 2.3
25.15 ± 2.1
23.78 ± 1.9
12.12 ± 1.1
23.97 ± 2.1
17.99 ± 1.6
9.18 ± 0.6
24.77 ± 2.4
11.16 ± 1.1
22.11 ± 2.0
24.87 ± 2.3
22.66 ± 2.2
23.33 ± 2.0
12.14 ± 1.1
23.89 ± 2.2
16.22 ± 1.4
5.47 ± 0.3
24.99 ± 2.3
12.17 ± 1.2
22.15 ± 2.2
24.44 ± 2.3
24.54 ± 2.3
22.44 ± 2.1
10.34 ± 1.0
23.65 ± 2.3
14.97 ± 1.3
7.26 ± 0.3
9h
0.21 ± 0.02
NT
9i
9j
NT
a
b
c
d
e
f
NT
11a
11b
11c
NT
0.25 ± 0.02
0.16 ± 0.02
NT
NT
0.19 ± 0.02
NT
11d
11e
Sorafenib
0.19 ± 0.01
0.15 ± 0.02
0.30 ± 0.03
0.10 ± 0.02
Abbreviations: HCT‐116, human colorectal carcinoma‐116; HepG2, hepatocellular carcinoma (HCC) type; MCF‐7, Michigan Cancer Foundation‐7; NT,
compounds not tested for their VEGFR‐2 inhibitory activity; SD, standard deviation; VEGFR‐2, vascular endothelial growth factor receptor‐2.
a
IC50 values are the mean ± SD of three separate experiments.
7
.26 ± 0.3 µM, respectively). With respect to the HepG2 hepatocellular
VEGFR‐2 by using an antiphosphotyrosine antibody with the Alpha
Screen system (PerkinElmer). The results were reported as a 50%
inhibition concentration value (IC50) calculated from the concentra-
tion–inhibition response curve and summarized in Table 2. Sorafenib
was used as a positive control in this assay. The tested compounds
displayed high to good inhibitory activity with IC50 values ranging
from 0.12 ± 0.01 to 0.30 ± 0.03 µM. Among them, compounds 4c and
4b potently inhibited VEGFR‐2 at IC50 values of 0.12 ± 0.01 and
0.13 ± 0.02 µM, respectively, which are nearly equipotent as sorafe-
nib IC50 value (0.10 ± 0.02 µM). Also, compounds 4b, 9f, 9c, 9e, and
11c possessed good VEGFR‐2 inhibition with IC50 values of
0.13 ± 0.02, 0.15 ± 0.02, 0.16 ± 0.02, 0.19 ± 0.01, and 0.19 ± 0.02 µM,
respectively; whereas compounds 9h, 4a, 9b, and 11c possessed
moderate VEGFR‐2 inhibition with IC50 values of 0.21 ± 0.02,
0.22 ± 0.02, 0.25 ± 0.02, and 0.30 ± 0.03 µM, respectively.
carcinoma cell line, compounds 4b, 9f, 9c, 9e, and 11c displayed the
highest anticancer activities with (IC50 = 9.97 ± 0.8, 9.99 ± 0.8,
11.02 ± 1.0, 11.67 ± 0.9, and 12.12 ± 1.1 µM, respectively). Compounds
4a, 9b,d,h, and 11e, with IC50 ranging from 14.06 ± 1.1 to
19.09 ± 1.7 µM, displayed good cytotoxicity. Compounds 9a,g,i,j and
11a,b,d, with IC50 ranging from 23.24 ± 1.9 to 25.88 ± 2.3 µM
exhibited moderate cytotoxicity; whereas compound 8 with IC50
4.55 ± 3.1 µM exhibited the lowest cytotoxicity.
Cytotoxicity evaluation against colorectal carcinoma (HCT‐116)
=
3
cell line discovered that compounds 4b, 9f, 9c, 9e, 9h, 11c, and 4a
displayed the highest anticancer activities with IC50 = 6.99 ± 0.5,
7
1
.44 ± 0.4, 8.15 ± 0.8, 8.88 ± 0.8, 11.16 ± 1.1, 12.14 ± 1.1, and
2.45 ± 1.2 µM, respectively. Compounds 9b,d and 11e, with IC50
ranging from 15.43 ± 1.4 to 21.12 ± 1.7 µM, displayed good cytotoxi-
city. Compounds 9a,g,i,j and 11a,b,d, with IC50 ranging from
2
2.11 ± 2.0 to 25.59 ± 2.3 µM, exhibited moderate cytotoxicity;
2
.6 Structure–activity relationship (SAR)
|
whereas compound 8 with IC50 = 32.77 ± 3.2 µM exhibited the lowest
cytotoxicity.
The preliminary SAR study has focused on the effect of replacement
of the free‐rotated urea linker of sorafenib with different cyclic
linkers which interact as H‐bond acceptors through their N‐atoms as
in compounds 4a–c or their carbonyl groups as in compounds 9a–j or
as H‐bond donors through their NH atoms and as H‐bond acceptors
through their carbonyl groups as in compounds 11a–c. These cyclic
linkers interact with the side‐chain carboxylate of the essential amino
acid residue Aspartate1044. Also, hydrophobic interactions occur
through the attached (un)substituted hydrophobic moieties. The
effect of replacement of pyridine moiety of sorafenib by the
benzoxazole/benzothiazole scaffolds of the synthesized compounds
on the antitumor activities also was noticed. The benzoxazole/
benzothiazole scaffolds occupied the same hydrophobic pocket as
occupied by the pyridine moiety of the standard ligand. On the
contrary, different hydrophobic groups were introduced instead of
Cytotoxicity evaluation against MCF‐7 cell line revealed that
compounds 4b, 9f, 9c, 9e, and 11c displayed the highest anticancer
activities (with IC50 = 7.89 ± 0.7, 8.24 ± 0.7, 9.32 ± 0.7, 9.94 ± 0.6, and
10.34 ± 1.0 µM, respectively). Compounds 4a, 9b,d,h, and 11e (with
IC50 ranging from 12.17 ± 1.2 to 14.97 ± 1.3 µM) displayed good
cytotoxicity. Compounds 9a,g,b,i,j and 11a,b,d (with IC50 ranging
from 22.15 ± 2.2 to 26.08 ± 2.3 µM) exhibited moderate cytotoxicity;
whereas compound 8 with IC50 = 34.13 ± 3.2 µM exhibited the lowest
cytotoxicity.
2
.5
|
In vitro VEGFR‐2 kinase assay
The most active antiproliferative derivatives 4a–c, 9b,c,e,f,h, and
1c,e were selected to evaluate their inhibitory activities against
1

EL‐HELBY ET AL.
9 of 15
|
the phenyl moiety of the reference ligand. Moreover, different
substitutions were introduced to the phenyl group with different
lipophilicity and electronic nature to study their effect in the
anticancer activity. 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
and MCF‐7 cell lines, respectively. Generally, the spacers, linkers
variable anticancer activities. Novel series of benzoxazole/benzothia-
zole derivatives 4a–c–11a–e were designed, synthesized and
evaluated for their anticancer activity against three human tumor
cell lines HepG2, HCT‐116, and MCF‐7 targeting VEGFR‐2 enzyme.
HCT‐116 and MCF‐7 were the most sensitive cell lines to the
influence of the new derivatives. In particular, compound 4c was
found to be the most potent derivative against HepG2, HCT‐116, and
MCF‐7 with IC50 = 9.45 ± 0.8, 5.76 ± 0.4, and 7.36 ± 0.5 µM, respec-
tively. Compounds 4b, 9f, and 9c showed the highest anticancer
activities against HepG2 with IC50 of 9.97 ± 0.8, 9.99 ± 0.8, and
11.02 ± 1.0 µM, respectively; HCT‐116 with IC50 of 6.99 ± 0.5,
7.44 ± 0.4, and 8.15 ± 0.8 µM, respectively; and MCF‐7 with IC50 of
7.89 ± 0.7, 8.24 ± 0.7, and 9.32 ± 0.7 µM, respectively, in comparison
with sorafenib as a reference drug with IC50 of 9.18 ± 0.6, 5.47 ± 0.3,
and 7.26 ± 0.3 µM, respectively. The most active compounds 4a–c,
9b,c,e,f,h, and 11c,e were further evaluated for their VEGFR‐2
inhibition. Compounds 4c and 4b potently inhibited VEGFR‐2 at IC50
values of 0.12 ± 0.01 and 0.13 ± 0.02 µM, respectively, which are
nearly equipotent as sorafenib IC50 value (0.10 ± 0.02 µM).
The obtained results showed that the most active 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.
(HBA–HBD), lipophilicity, and electronic nature exhibited an im-
portant role in anticancer activity. The 1,3,4‐thiadiazoles linkers of
compounds 4a–c were found to be responsible for the higher
anticancer activity than that of thiazolidine‐2,4‐dione as in com-
pounds 9a–j and 6‐phenyl‐5‐cyanothiouracil as in compounds 11a–e,
respectively. The SCH
resulted in higher activities than the SCH
1a–e and CH CONH, respectively. The spacer SCH
compounds 4a–c showed higher activities than the retroarrangement
HNCOCH S as in compounds 11a–e. From the structure of the
2
CH
2
CONH spacers as in compounds 4b,c
CONH one as in 4a or
CONH as in
2
1
2
2
2
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 the 5‐methylbenzoxazole derivatives for
example compound 4c showed higher activities than the unsubsti-
tuted ones, for example, compounds 4b and 4a, respectively. In the
second group 9a–j, the distal phenyl group played an important role
in an activity where, the substituted phenyl group as in compounds
9b–j exhibited higher activities than the unsubstituted one 9a. The 4‐
substituted phenyl group, for example, 9c and 9h exhibited higher
activities than the 2‐substituted, for example, 9b and 9g and/or 3‐
substituted ones as 9j, respectively. The lipophilic electron‐donating
methyl group at position 4 as in compounds 9f displayed higher
anticancer activity than the electron‐withdrawing Cl group as in
compound 9e and F group as in 9c, respectively. The more lipophilic
4
|
EXPERIMENTAL
|
Chemistry
4.1
4
.1.1 | General
All melting points were carried out by open capillary method on a
Gallenkamp melting point apparatus at Faculty of Pharmacy of Al‐
Azhar University and were uncorrected. The infrared spectra were
recorded on Pye Unicam SP 1000 IR (infrared) spectrophotometer at
Pharmaceutical Analytical Unit, Faculty of Pharmacy, Al‐Azhar
University using potassium bromide disc technique. Proton magnetic
3
substituents at position 4 as CH , Cl, and F as in compounds 9a–c
exhibited higher activities than the lower lipophilic substituents as
methoxy and dimethylamino groups as in 9h and 9i, respectively. The
monosubstituted derivatives as in compounds 9c and 9b showed
higher activities than the disubstituted one as in compound 9d. In the
third group 11a–e, the substituted distal phenyl group as in
compounds 11b–e exhibited higher activities than the unsubstituted
one 11a. The lipophilic electron‐donating methyl and methoxy groups
as in compounds 11c and 11e exhibited higher anticancer activities
1
resonance H‐NMR spectra were recorded on a Bruker 400 MHZ‐
NMR spectrometer at Faculty of Sciences, Cairo University, Cairo,
1
3
Egypt. C‐NMR spectra were recorded on an Agilent 400 MHZ‐
NMR spectrometer at Chemical Laboratory, Ministry of Defense,
Cairo. TMS (tetramethylsilane) was used as internal standard and
chemical shifts were measured in δ scale (ppm). The mass spectra
were carried out on Direct Probe Controller Inlet part to Single
Quadropole mass analyzer in Thermo Scientific GCMS model ISQ LT
using Thermo X‐Calibur software at the Regional Center for
Mycology and Biotechnology, Al‐Azhar University. Elemental ana-
lyses (C, H, and N) were performed on a CHN analyzer at Regional
Center for Mycology and Biotechnology, Al‐Azhar University. All
compounds were within ±0.4 of the theoretical values. The reactions
were monitored by thin‐layer chromatography (TLC) using TLC
sheets precoated with UV fluorescent silica gel Merck 60 F254 plates
and were visualized using UV lamp and different solvents as mobile
phases.
than the electron‐withdrawing
F group as in compound 11b,
respectively. The lipophilic electron‐donating methyl and methoxy
groups as in compounds 11c and 11e exhibited higher anticancer
activities than the hydrophilic electron‐donating OH group as in
compound 11d, respectively.
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 fitted with that obtained
from the biological screening. All the tested compounds showed

10 of 15
EL‐HELBY ET AL.
|
2
‐Chloro‐N‐(5‐phenyl‐1,3,4‐thiadiazol‐2‐yl)acetamide (1a) 3‐
56.53; H, 3.69; N, 14.65; S, 16.77. Found: C, 56.45; H, 3.63; N, 15.02;
S, 16.74.
chloro‐N‐(5‐phenyl‐1,3,4‐thiadiazol‐2‐yl)propanamide (1b), 2‐mer-
captobenzoxazole (2a), 2‐mercapto‐5‐methylbenzoxazole (2b), and
their corresponding potassium salts (3a,b), thiazolidine‐2,4‐dione (5),
its potassium salt (6), N‐(benzothiazol‐2‐yl)‐2‐chloroacetamide (7),
and 2‐mercapto‐6‐oxo‐4‐(4‐(un)substituted phenyl)‐1,6‐dihydropyri-
midine‐5‐carbonitrile (10) were obtained according to the reported
3‐((5‐Methylbenzoxazol‐2‐yl)thio)‐N‐(5‐phenyl‐1,3,4‐thiadiazol‐2‐yl)‐
propanamide (4c)
−1
Yield, 79%; m.p. 273–275°C; IRνmax (cm ): 3,164 (NH), 3,090
1
[
35,36]
(CH aromatic), 2,908 (CH aliphatic), and 1,689 (C═O); H‐NMR
procedures.
(
400 MHz, DMSO‐d
CH CO), 4.53 (t, J = 6.8 Hz; 2H, –SCH
H‐6 of benzoxazole), 6.55 (dd, 1H, J = 8, 8 Hz; Ar‐H, H‐4 of phenyl),
.11 (dd, 2H, J = 7.6, 8 Hz; Ar‐H, H‐3 and H‐5 of phenyl), 7.37 (s, 1H;
6
): 2.36 (s, 3H, CH
3
), 3.07 (t, J = 6.8 Hz; 2H,
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting Information.
–
2
2
), 6.48 (d, 1H, J = 8.4 Hz; Ar‐H,
7
Ar‐H, H‐4 of benzoxazole), 7.53 (d, 1H, J = 8.4 Hz; Ar‐H, H‐7 of
benzoxazole), 7.94 (d, 2H, J = 7.6 Hz; Ar‐H, H‐2 and H‐6 of phenyl),
4
2
.1.2 | General method for the synthesis of
‐(benzoxazol‐2‐ylthio)‐N‐(5‐phenyl‐1,3,4‐thiadizol‐2‐
yl)acetamide (4a) and N‐(5‐phenyl‐1,3,4‐thiadizol‐2‐
yl)‐3‐[5‐(un)substituted benzoxazol‐2‐ylthio]‐
propanamides (4b,c)
and 12.90 (s, 1H, NH;
(m.w. 396.48): C, 57.56; H, 4.07; N, 14.13; S, 16.17.
Found: C, 57.12; H, 4.32; N, 14.01; S, 16.46.
2
D O exchangeable); Anal. calcd. for
19 16 4 2 2
C H N O S
To a mixture of the potassium salts 3a and/or 3b (0.002 mol) in dry
DMF (50 ml), the appropriate chloroamide derivatives (1a,b;
4
.1.3 | Synthesis of N‐(benzothiazol‐2‐yl)‐2‐(2,4‐
0.002 mol) were added. The reaction mixture was heated using a
dioxothiazolidin‐3‐yl)acetamide (8)
water bath for 12 hr. After cooling to room temperature, reaction
mixture was poured onto crushed ice. The precipitated solids were
collected by filtration, dried, and crystallized from ethanol to give the
target compounds 4a–c.
A mixture of the potassium salt of thiazolidin‐2,4‐dione (6; 1.55 g,
0.01 mol) and N‐(benzothiazol‐2‐yl)‐2‐chloroacetamide (7; 2.26 g,
0.01 mol) in dry DMF (20 ml) was heated on a water bath for 20 hr.
After cooling, the reaction mixture was poured on crushed ice. The
formed precipitate was filtered, dried, and crystallized from ethanol
affording the desired product (8; 2.63 g) as a beige solid.
2
‐(Benzoxazol‐2‐ylthio)‐N‐(5‐phenyl‐1,3,4‐thiadiazol‐2‐yl)acetamide
(4a)
−1
−1
Yield, 85%; m.p. 171–173°C; IRνmax (cm ): 3,328 (NH), 3,090 (CH
Yield, 81%; m.p. 176–178°C; IRνmax (cm ): 3,171 (NH), 3,037 (CH
1
1
aromatic), 2,991 (CH aliphatic), and 1,674 (C═O); H‐NMR (400 MHz,
aromatic), 2,915 (CH aliphatic), and 1,690 (C═O); H‐NMR (400 MHz,
DMSO‐d
6
): 2.65 (s, 2H, CH
Hz; Ar‐H, H‐5 of benzothiazole), 7.42 (dd, 1H, J = 8, 7.6 Hz; Ar‐H, H‐
of benzothiazole), 7.70 (d, 1H, J = 7.6 Hz; Ar‐H, H‐7 of benzothia-
2 2
S), 4.30 (s, 2H, CH N), 7.26 (dd, 1H, J = 8,
dimethyl sulfoxide [DMSO]‐d
Hz; Ar‐H, H‐6 of benzoxazole), 7.49 (dd, 1H, J = 8.8, 7 Hz; Ar‐H, H‐5
of benzoxazole), 7.55 (dd, 1H, J = 8.6, 8.6 Hz; Ar‐H, H‐4 of phenyl),
.60 (dd, 2H, J = 8.6, 9.6 Hz; Ar‐H, H‐3 and H‐5 of phenyl), 7.89 (d, 1H,
6 2
): 4.51 (s, 2H, CH ), 7.28 (dd, 1H, J = 8,
7
6
7
zole), 7.91 (d, 1H, J = 7 Hz; Ar‐H, H‐4 of benzothiazole), and 12.30 (s,
7
+
1
H, NH; D
2
O exchangeable); MS (m/z): 307 (M , 4.03%), 177
J = 8.8 Hz; Ar‐H, H‐4 of benzoxazole), 7.91 (d, 1H, J = 8 Hz; Ar‐H, H‐7
(
38.30%), 150 (100%, base peak), 135 (27.49%), and 69 (30.44%);
Anal. calcd. for C12 (307.34): C, 46.90; H, 2.95; N, 13.67; S,
0.86. Found: C, 46.47; H, 3.18; N, 13.37; S, 21.27.
of benzoxazole), 7.95 (d, 2H, J = 9.6 Hz; Ar‐H, H‐2 and H‐6 of phenyl),
+
9 3 3 2
H N O S
and 13.11 (s, 1H, NH; D
2
O exchangeable); MS (m/z): 368 (M , 100%,
2
base peak), 295 (29.10%), 204 (7.50%), 121 (14.78%), and 77
15.65%); Anal. calcd. for C17 (m.w. 368.43): C, 55.42; H,
.28; N, 15.21; S, 17.40. Found: C, 55.71; H, 3.25; N, 15.11; S, 17.48.
(
12 4 2 2
H N O S
3
4
.1.4 | General method for the synthesis of
N‐(benzothiazol‐2‐yl)‐2‐[5‐(un)substituted benzylidene‐
,4‐dioxothiazolidin‐3‐yl]acetamide (9a–j)
3
‐(Benzoxazol‐2‐ylthio)‐N‐(5‐phenyl‐1,3,4‐thiadiazol‐2‐yl)propana-
2
mide (4b)
−
1
Yield, 76%; m.p. 215–217°C; IRνmax (cm ): 3,168 (NH), 3,038 (CH
A mixture of N‐(benzothiazol‐2‐yl)‐2‐(2,4‐dioxothiazolidin‐3‐yl)acet-
amide 8 (0.307 g, 0.001 mol) and the appropriate benzaldehyde
(0.001 mol) namely benzaldehyde, 2‐chlorobenzaldehyde, 4‐chloro-
benzaldehyde, 2,6‐dichlorobenzaldehyde, 4‐fluorobenzaldehyde,
4‐methylbenzaldehyde, 2‐methoxybenzaldehyde, 4‐methoxybenzal-
dehyde, 4‐N,N‐dimethylaminobenzaldehyde, and/or 3‐nitrobenzalde-
hyde in the presence of catalytic quantity of piperidine was refluxed
in dry toluene for 8 hr. The reaction mixture was cooled to 25°C to
obtain a solid product. The product was crystallized from ethanol to
give the corresponding target compounds 9a–j, respectively.
1
aromatic), 2,905 (CH aliphatic), and 1,688 (C═O); H‐NMR (400 MHz,
DMSO‐d
SCH ), 7.30 (dd, 1H, J = 7.6, 7.2 Hz; Ar‐H, H‐6 of benzoxazole), 7.37
dd, 1H, J = 7.6, 8 Hz; Ar‐H, H‐5 of benzoxazole), 7.52 (dd, 1H, J = 7.6,
.6 Hz; Ar‐H, H‐4 of phenyl), 7.56 (dd, 2H, J = 6.6, 7.6 Hz; Ar‐H, H‐3
and H‐5 of phenyl), 7.65 (d, 1H, J = 8 Hz; Ar‐H, H‐4 of benzoxazole),
.93 (d, 1H, J = 7.2 Hz; Ar‐H, H‐7 of benzoxazole), 7.95 (d, 2H,
J = 6.6 Hz; Ar‐H, H‐2 and H‐6 of phenyl), and 12.82 (s, 1H, NH; D
exchangeable); Anal. calcd. for C18 (m.w. 382.46): C,
6 2
): 3.10 (t, 2H, J = 6.8 Hz, –CH CO), 4.51 (t, 2H, J = 6.8 Hz,
–
2
(
7
7
2
O
14 4 2 2
H N O S

EL‐HELBY ET AL.
11 of 15
|
N‐(Benzothiazol‐2‐yl)‐2‐(5‐benzylidene‐2,4‐dioxothiazolidin‐3‐yl)‐
acetamide (9a)
J = 7.6, 7.6 Hz; Ar‐H, H‐5 of benzothiazole), 7.42 (dd, 1H, J = 7.6,
8 Hz; Ar‐H, H‐6 of benzothiazole), 7.48 (dd, 1H, J = 8, 8 Hz; Ar‐H,
H‐4 of phenyl), 7.60 (d, 2H, J = 8 Hz; Ar‐H, H‐3 and H‐5 of phenyl),
7.75 (d, 1H, J = 8 Hz; Ar‐H, H‐7 of benzothiazole), 7.93 (s, 1H,
−
1
Yield, 90%; m.p. 232–233°C; IRνmax (cm ): 3,199 (NH), 3,080 (CH
1
aromatic), 2,999 (CH aliphatic), and 1,698 (C═O); H‐NMR
(
400 MHz, DMSO‐d
6
): 4.71 (s, 2H, CH
2
), 7.31 (dd, 1H, J = 8,
C═CH–ph), 7.97 (d, 1H, J = 7.6 Hz; Ar‐H, H‐4 of benzothiazole),
+
8
Hz; Ar‐H, H‐4 of phenyl), 7.44 (dd, 1H, J = 7.6, 7.6 Hz; Ar‐H, H‐5
and 12.85 (s, 1H, NH; D
2
O exchangeable); MS (m/z): 468 (M +4,
+
+
of benzothiazole), 7.51 (dd, 1H, J = 7.6, 8 Hz; Ar‐H, H‐6 of
benzothiazole), 7.57 (dd, 2H, J = 8, 7.4 Hz; Ar‐H, H‐3 and H‐5 of
phenyl), 7.67 (d, 2H, J = 7.4 Hz; Ar‐H, H‐2 and H‐6 of phenyl), 7.77
5.11%), 466 (M +2, 11.69%), 464 (M , 32.51%), 463 (100%, base
peak), 286 (38.23%), 177 (30.88%), and 56 (11.97%); Anal. calcd.
2 3 3 2
for C19H11Cl N O S (m.w. 464.34): C, 49.15; H, 2.39; N, 9.05; S,
(
d, 1H, J = 8 Hz; Ar‐H, H‐7 of benzothiazole), 7.98 (s, 1H,
C═CH–ph), 8.01 (d, 1H, J = 7.6 Hz; Ar‐H, H‐4 of benzothiazole),
and 12.87 (s, 1H, NH; D O exchangeable); Anal. calcd. for
(m.w. 395.04): C, 57.71; H, 3.31; N, 10.63; S,
6.21. Found: C, 57.90; H, 3.27; N, 10.53; S, 15.94.
13.81. Found: C, 48.75; H, 2.51; N, 9.06; S, 13.54.
2
N‐(Benzothiazol‐2‐yl)‐2‐(5‐(4‐fluorobenzylidene)‐2,4‐dioxothiazoli-
din‐3‐yl)acetamide (9e)
19 13 3 3 2
C H N O S
−
1
1
Yield, 85%; m.p. 237–239°C; IRνmax (cm ): 3,229 (NH), 3,076 (CH
1
aromatic), 2,984 (CH aliphatic), and 1,679 (C═O); H‐NMR
N‐(Benzothiazol‐2‐yl)‐2‐(5‐(2‐chlorobenzylidene)‐2,4‐dioxothiazoli-
din‐3‐yl)acetamide (9b)
6 2
(400 MHz, DMSO‐d ): 4.68 (s, 2H, CH ), 7.28 (dd, 1H, J = 8,
7.6 Hz; Ar‐H, H‐5 of benzothiazole), 7.37 (d, 2H, J = 8.4 Hz; Ar‐H,
H‐3 and H‐5 of phenyl), 7.42 (dd, 1H, J = 8, 8 Hz; Ar‐H, H‐6 of
benzothiazole), 7.71 (d, 2H, J = 8.4 Hz; Ar‐H, H‐2 and H‐6 of
phenyl), 7.74 (d, 1H, J = 8 Hz; Ar‐H, H‐7 of benzothiazole), 7.96 (d,
1H, J = 7.6 Hz; Ar‐H, H‐4 of benzothiazole), 8.01 (s, 1H,
−
1
Yield, 87%; m.p. 195–197°C; IRνmax (cm ): 3,197 (NH), 3,065 (CH
1
aromatic), 2,992 (CH aliphatic), and 1,698 (C═O); H‐NMR
(
400 MHz, DMSO‐d
Hz; Ar‐H, H‐5 of benzothiazol), 7.42 (dd, 1H, J = 7.4, 8.4 Hz; Ar‐H,
H‐6 of benzothiazole), 7.49 (d, 1H, J = 7 Hz; Ar‐H, H‐6 of phenyl),
.53 (dd, 1H, J = 7, 7.6 Hz; Ar‐H, H‐5 of phenyl), 7.62 (d, 1H,
6 2
): 4.69 (s, 2H, CH ), 7.29 (dd, 1H, J = 7.4,
8
2
C═CH–ph), and 12.84 (s, 1H, NH; D O exchangeable); MS (m/z):
+
+
7
415 (M +2, 14.51%), 413 (M , 98%), 236 (20.14%), 177 (100%,
base peak), 152 (55.65%), and 56 (28.81%); Anal. calcd. for
J = 8.4 Hz; Ar‐H, H‐7 of benzothiazole), 7.75 (dd, 1H, J = 7.6, 8 Hz;
Ar‐H, H‐4 of phenyl), 7.90 (s, 1H, C═CH–ph), 7.96 (d, 1H, J = 8 Hz;
Ar‐H, H‐4 of benzothiazole), 8.08 (d, 1H, J = 8 Hz; Ar‐H, H‐3 of
19 3 3 2
C H12FN O S (m.w. 413.44): C, 55.20; H, 2.93; N, 10.16; S,
15.51. Found: C, 55.34; H, 2.87; N, 9.90; S, 15.52.
2
phenyl), and 12.85 (s, 1H, NH; D O exchangeable); MS (m/z): 432
+
+
(
(
M +2, 12.36%), 430 (M , 27.63%), 429 (100%, base peak), 252
4.04%), 150 (22.86%), and 56 (15.13%); Anal. calcd. for
N‐(Benzothiazol‐2‐yl)‐2‐(5‐(4‐methylbenzylidene)‐2,4‐dioxothiazoli-
din‐3‐yl)acetamide (9f)
−
1
C
19
H
12ClN
3
O
3
S
2
(m.w. 429.89): C, 53.09; H, 2.81; N, 9.77; S,
Yield, 92%; m.p. 250–252°C; IRνmax (cm ): 3,208 (NH), 3,067 (CH
1
1
4.92. Found: C, 53.23; H, 2.74; N, 9.65; S, 14.98.
aromatic), 2,999 (CH aliphatic), 1,739, 1,698, and 1,659 (3C═O); H‐
6 3 2
NMR (400 MHz, DMSO‐d ): 2.36 (s, 3H, CH ), 4.67 (s, 2H, CH ), 7.29
N‐(Benzothiazol‐2‐yl)‐2‐(5‐(4‐chlorobenzylidene)‐2,4‐dioxothiazoli-
din‐3‐yl)acetamide (9c)
(dd, 1H, J = 8, 8 Hz; Ar‐H, H‐5 of benzothiazole), 7.35 (d, 2H, J = 8 Hz;
Ar‐H, H‐3 and H‐5 of phenyl), 7.42 (dd, 1H, J = 7.6, 8 Hz; Ar‐H, H‐6 of
benzothiazole), 7.54 (d, 2H, J = 8 Hz; Ar‐H, H‐2 and H‐6 of phenyl),
7.75 (d, 1H, J = 7.6 Hz; Ar‐H, H‐7 of benzothiazole), 7.95 (s, 1H,
C═CH–ph), 7.96 (d, 1H, J = 8 Hz; Ar‐H, H‐4 of benzothiazole), and
−1
Yield, 81%; m.p. 182–184°C; IRνmax (cm ): 3,215 (NH), 3,045
1
(
(
CH aromatic), 2,976 (CH aliphatic), and 1,678 (C═O); H‐NMR
6 2
400 MHz, DMSO‐d ): 4.68 (s, 2H, CH ), 7.28 (dd, 1H, J = 8, 8.4 Hz;
Ar‐H, H‐5 of benzothiazole), 7.41 (dd, 1H, J = 7.6, 8 Hz; Ar‐H, H‐6 of
benzothiazole), 7.60 (d, 2H, J = 8.8 Hz; Ar‐H, H‐3 and H‐5 of phenyl),
2 15 3 3 2
12.83 (s, 1H, NH; D O exchangeable); Anal. calcd. for C20H N O S
(m.w. 409.48): C, 58.66; H, 3.69; N, 10.26; S, 15.66. Found: C, 58.37;
H, 3.64; N, 10.38; S, 15.83.
7.66 (d, 2H, J = 8.8 Hz; Ar‐H, H‐2 and H‐6 of phenyl), 7.74 (d, 1H,
J = 7.6 Hz; Ar‐H, H‐7 of benzothiazole), 7.96 (d, 1H, J = 8.4 Hz; Ar‐H,
H‐4 of benzothiazole), 7.99 (s, 1H, C═CH–ph), and 12.83 (s, 1H, NH;
N‐(Benzothiazol‐2‐yl)‐2‐(5‐(2‐methoxybenzylidene)‐2,4‐dioxothiazo-
lidin‐3‐yl)acetamide (9g)
+
+
D
2
O exchangeable); MS (m/z): 431.89 (M +2, 2.72%), 429.91 (M ,
.47%), 251.84 (46.03%), 176.86 (100%, base peak), 149.93 (44.78%),
and 55.98 (26.90%); Anal. calcd. for C19 12ClN (m.w. 429.89):
−
1
6
Yield, 80%; m.p. 277–279°C; IRνmax (cm ): 3,280 (NH), 3,075 (CH
1
H
3
O
S
3 2
aromatic), 2,940 (CH aliphatic), and 1,681 (C═O); H‐NMR (400 MHz,
C, 53.09; H, 2.81; N, 9.77; S, 14.92. Found: C, 53.32; H, 2.94; N, 9.98;
S, 15.04.
6 3 2
DMSO‐d ): 3.89 (s, 3H, OCH ), 4.67 (s, 2H, CH ), 7.09 (dd, 1H, J = 7.6,
8 Hz; Ar‐H, H‐5 of phenyl), 7.16 (d, 1H, J = 8.4 Hz; Ar‐H, H‐3 of
phenyl), 7.29 (dd, 1H, J = 8.4, 8 Hz; Ar‐H, H‐4 of phenyl), 7.40 (dd, 1H,
J = 8, 8.4 Hz; Ar‐H, H‐5 of benzothiazole), 7.45 (dd, 1H, J = 7.6, 8.4 Hz;
Ar‐H, H‐6 of benzothiazole), 7.51 (d, 1 H, J = 7.6 Hz; Ar‐H, H‐6 of
phenyl), 7.75 (d, 1H, J = 7.6 Hz; Ar‐H, H‐7 of benzothiazole), 7.96 (d,
N‐(Benzothiazol‐2‐yl)‐2‐(5‐(2,6‐dichlorobenzylidene)‐2,4‐dioxothia-
zolidin‐3‐yl)acetamide (9d)
−
1
Yield, 80%; m.p. 190–192°C; IRνmax (cm ): 3,215 (NH), 3,090 (CH
1
aromatic), 2,935 (CH aliphatic), 1,743, 1,665, 1,659 (2C═O); H‐
1H, J = 8 Hz; Ar‐H, H‐4 of benzothiazole), 8.11 (s, 1H, C═CH–ph), and
+
NMR (400 MHz, DMSO‐d
6
): 4.69 (s, 2H, CH
2
), 7.29 (dd, 1H,
12.83 (s, 1H, NH; D
2
O exchangeable); MS (m/z): 425.89 (M , 10.31%),

12 of 15
EL‐HELBY ET AL.
|
4
24.90 (38.13%), 247.88 (15.07%), 176.89 (100%, base peak), 120.92
18.11%), and 76.93 (3.17%); Anal. calcd. for C20 15ClN (m.w.
25.48): C, 56.46; H, 3.55; N, 9.88; S, 15.07. Found: C, 56.31; H, 3.68;
N, 10.12; S, 15.21.
4.1.5 | General method for the synthesis of
(
H
3
O S
4 2
N‐(benzothiazol‐2‐yl)‐2‐((5‐cyano‐6‐oxo‐4‐(4‐(un)‐
substituted phenyl)‐1,6‐dihydropyrimidin‐2‐yl)thio)‐
acetamide (11a–e)
4
A mixture of N‐(benzothiazol‐2‐yl)‐2‐chloroacetamide (7; 0.23 g,
N‐(Benzothiazol‐2‐yl)‐2‐(5‐(4‐methoxybenzylidene)‐2,4‐dioxothiazo-
lidin‐3‐yl)acetamide (9h)
0
.001 mol) and the appropriate 2‐mercaptodihydropyrimidine
derivatives (10a–e; 0.001 mol) in the presence of anhydrous
potassium carbonate (0.14 g, 0.001 mol) was refluxed in dry
acetone (50 ml) for 8 hr. The reaction mixture was filtered while
hot and the residue was washed with hot acetone. The filtrate was
concentrated, cooled and poured onto ice water; the precipitated
solid product was filtered, washed with water, dried, and
recrystallized from ethanol to afford the corresponding target
compounds (11a–e), respectively.
−1
Yield, 85%; m.p. 270–272°C; IRνmax (cm ): 3,210 (NH), 3,059 (CH
1
aromatic), 2,937 (CH aliphatic), 1,683 (C═O), and 1,583 (C═N); H‐
NMR (400 MHz, DMSO‐d
6 3 2
): 3.81 (s, 3H, OCH ), 4.67 (s, 2H, CH ),
7.29 (dd, 1H, J = 7.2, 8 Hz; Ar‐H, H‐5 of benzothiazole), 7.42 (dd, 1H,
J = 7.2, 8.4 Hz; Ar‐H, H‐6 of benzothiazole), 7.53 (d, 2H, J = 8.8 Hz; Ar‐
H, H‐3 and H‐5 of phenyl), 7.61 (d, 2H, J = 8.8 Hz; Ar‐H, H‐2 and H‐6
of phenyl), 7.74 (d, 1H, J = 8.4 Hz; Ar‐H, H‐7 of benzothiazole), 7.94 (s,
1
H, C═CH–ph), 7.96 (d, 1H, J = 8 Hz; Ar‐H, H‐4 of benzothiazole), and
1
3
1
2.83 (s, 1H, NH; D
): δ = 44.01, 55.94, 115.36, 118.01, 120.73, 121.21, 122.29, 124.29,
25.74, 126.72, 132.29 (2C), 132.87 (2C), 134.30, 161.44, 161.83,
65.74, 167.63, and 168.41; Anal. calcd. for C20 15ClN (m.w.
25.48): C, 56.46; H, 3.55; N, 9.88; S, 15.07. Found: C, 56.06; H, 3.46;
2
O exchangeable); C‐NMR (100 MHz, DMSO‐
d
1
1
4
6
N‐(Benzothiazol‐2‐yl)‐2‐((5‐cyano‐6‐oxo‐4‐phenyl‐1,6‐dihydropyri-
midin‐2‐yl)thio)acetamide (11a)
H
3
O
S
4 2
−1
Yield, 60%; m.p. 217–219°C; IRνmax (cm ): 3,355, 3,186 (2 NH),
3
,072 (CH aromatic), 2,980 (CH aliphatic), 2,226 (C≡N), and 1,667
N, 10.43; S, 15.34.
1
(C═O); H‐NMR (400 MHz, DMSO‐d
6
): 4.24 (s, 1H, CH
2
), 7.19 (dd,
1H, J = 8.8 Hz; Ar‐H, H‐4 of phenyl), 7.28 (dd, 2H, J = 8, 7.6 Hz; Ar‐H,
N‐(Benzothiazol‐2‐yl)‐2‐(5‐(4‐(dimethylamino)benzylidene)‐2,4‐diox-
othiazolidin‐3‐yl)acetamide (9i)
H‐3 and H‐5 of phenyl), 7.35 (dd, 1H, J = 7.6, 7.4 Hz; Ar‐H, H‐5 of
benzothiazole), 7.42 (dd, 1H, J = 7.6, 10 Hz; Ar‐H, H‐6 of benzothia-
zole), 7.72 (d, 2H, J = 7.6 Hz; Ar‐H, H‐2 and H‐6 of phenyl), 7.74 (d,
−1
Yield, 85%; m.p. 240–242°C; IRνmax (cm ): 3,299 (NH), 3,045 (CH
1
aromatic), 2,905 (CH aliphatic), and 1,673 (C═O); H‐NMR (400 MHz,
1H, J = 10 Hz; Ar‐H, H‐7 of benzothiazole), 7.95 (d, 1H, J = 7.4 Hz; Ar‐
6 3 2 2
DMSO‐d ): 3.04 (s, 6H, N(CH ) ), 4.67 (s, 2H, CH ), 6.83 (d, 2H,
H, H‐4 of benzothiazole), 12.65 (s, 1H, NHCOCH
able), and 13.82 (s, 1H, OH of pyrimidinone; D
NMR (100 MHz, DMSO‐d
2
; D
2
O exchange-
J = 8.8 Hz; Ar‐H, H‐3 and H‐5 of phenyl), 7.31 (dd, 1H, J = 7.6, 8 Hz;
Ar‐H, H‐5 of benzothiazole), 7.41 (dd, 1H, J = 7.6, 8.4 Hz; Ar‐H, H‐6 of
benzothiazole), 7.49 (d, 2H, J = 8.8 Hz; Ar‐H, H‐2 and H‐6 of phenyl),
13
2
O exchangeable); C‐
6
): δ = 34.90, 92.31, 117.74, 121.03 (2C),
122.19 (2C), 124.05, 126.64, 128.57 (2C), 128.86 (2C), 131.27,
7.77 (d, 1H, J = 8.4 Hz; Ar‐H, H‐7 of benzothiazole), 7.86 (s, 1H,
131.92, 136.37, 149.02, 158.27, 167.56, and 167.95; MS (m/z): 421
C═CH–ph), 7.99 (d, 1H, J = 8 Hz; Ar‐H, H‐4 of benzothiazole), and
+
(
M , 8.37%), 339 (54.38%), 313 (100%, base peak), 269 (45.42%), and
1
3
1
d
2.85 (s, 1H, NH; D
2
O exchangeable); C‐NMR (100 MHz, DMSO‐
6
13 5 2 2
9 (18.95%); Anal. calcd. for C20H N O S (m.w. 419.48): C, 57.27;
6
): δ = 39.34, 43.88, 111.42, 112.50 (2C), 112.57, 113.10, 120.03
H, 3.12; N, 16.70; S, 15.29. Found: C, 57.01; H, 3.30; N, 16.60;
S, 15.41.
(
(
2C), 122.28, 124.27, 126.71, 132.56, 132.97 (2C), 135.28, 152.23
2C), 165.80, and 167.81; Anal. calcd. for C21 (m.w.
38.52): C, 57.52; H, 4.14; N, 12.78; S, 14.62. Found: C, 57.27; H,
.27; N, 12.62; S, 14.27.
18 4 3 2
H N O S
4
4
N‐(Benzothiazol‐2‐yl)‐2‐((5‐cyano‐4‐(4‐fluorophenyl)‐6‐oxo‐1,6‐di-
hydropyrimidin‐2‐yl)thio)acetamide (11b)
−
1
N‐(Benzothiazol‐2‐yl)‐2‐(5‐(3‐nitrobenzylidene)‐2,4‐dioxothiazolidin‐
‐yl)acetamide (9j)
Yield, 70%; m.p. 275–277°C; IRνmax (cm ): 3,360, 3,196 (2 NH),
3
3,068 (CH aromatic), 2,924 (CH aliphatic), 2,220 (C≡N), and 1,666
−
1
1
Yield, 89%; m.p. 207–209°C; IRνmax (cm ): 3,196 (NH), 3,069 (CH
(C═O); H‐NMR (400 MHz, DMSO‐d
6
): 4.28 (s, 1H, CH
2
), 7.02 (d,
1
aromatic), 2,988 (CH aliphatic), and 1,694 (C═O); H‐NMR
2H, J = 8 Hz; Ar‐H, H‐3 and H‐5 of phenyl), 7.28 (dd, 1H, J = 8,
7.2 Hz; Ar‐H, H‐5 of benzothiazole), 7.42 (dd, 1H, J = 7.6, 8 Hz; Ar‐
H, H‐6 of benzothiazole), 7.75 (d, 2H, J = 8 Hz; Ar‐H, H‐2 and H‐6
of phenyl), 7.82 (d, 1H, J = 7.6 Hz; Ar‐H, H‐7 of benzothiazole), 7.94
(d, 1H, J = 7.2 Hz; Ar‐H, H‐4 of benzothiazole), 12.67 (s, 1H,
(
400 MHz, DMSO‐d
6 2
): 4.70 (s, 2H, CH ), 7.29 (dd, 1H, J = 7.6,
8
Hz; Ar‐H, H‐5 of benzothiazole), 7.42 (dd, 1H, J = 7.4, 8 Hz; Ar‐
H, H‐6 of benzothiazole), 7.75 (d, 1H, J = 8 Hz; Ar‐H, H‐6 of
phenyl), 7.82 (dd, 1H, J = 8, 8 Hz; Ar‐H, H‐5 of phenyl), 7.97 (d, 1H,
J = 7.4 Hz; Ar‐H, H‐7 of benzothiazole), 8.06 (d, 1H, J = 8 Hz; Ar‐H,
H‐4 of phenyl), 8.17 (s, 1H, C═CH–ph), 8.31 (d, 1H, J = 7.6 Hz; Ar‐
H, H‐4 of benzothiazole), 8.52 (s, 1H; Ar‐H, H‐2 of phenyl), and
NHCOCH
2
;
D
2
O
exchangeable), and 13.96 (s, 1H, OH of
+
pyrimidinone; D
2
O exchangeable); MS (m/z): 439 (M +2, 1.39%),
+
437 (M , 4.15%), 329 (59.53%), 287 (100%, base peak), 69
1
2.85 (s, 1H, NH;
(m.w. 440.45): C, 51.81; H, 2.75; N, 12.72; S,
4.56. Found: C, 51.99; H, 2.65; N, 12.40; S, 14.61.
D
2
O
exchangeable); Anal. calcd. for
5 2 2
(57.34%), and 45 (26.38%); Anal. calcd. for C20H12FN O S (m.w.
C
19
H
12
N
4
O
5
S
2
437.47): C, 54.91; H, 2.77; N, 16.01; S, 14.66. Found: C, 54.79; H,
2.88; N, 15.84; S, 14.68.
1

EL‐HELBY ET AL.
13 of 15
|
N‐(Benzothiazol‐2‐yl)‐2‐((5‐cyano‐6‐oxo‐4‐(p‐tolyl)‐1,6‐dihydropyri-
midin‐2‐yl)thio)acetamide (11c)
protein–ligand interactions. It predicts how small flexible molecule
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 Databank (http://www.rcsb.org/pdb/explore/explore.do?
structureId=1YWN). 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 drugs
vatalanib and sorafenib were used as reference ligands.
−1
Yield, 55%; m.p. 224–226°C; IRνmax (cm ): 3,238, 3,183 (2 NH), 3,053
1
(
CH aromatic), 2,914 (CH aliphatic), 2,221 (C≡N), and 1,664 (C═O); H‐
6 3 2
NMR (400 MHz, DMSO‐d ): 2.13 (s, 3H, CH ), 4.32 (s, 1H, CH ), 6.97 (d,
2H, J = 8 Hz; Ar‐H, H‐3 and H‐5 of phenyl), 7.31 (dd, 1H, J = 7.6, 7.4 Hz;
Ar‐H, H‐5 of benzothiazole), 7.46 (dd, 1H, J = 7.6, 8 Hz; Ar‐H, H‐6 of
benzothiazole), 7.67 (d, 2H, J = 8 Hz; Ar‐H, H‐2 and H‐6 of phenyl), 7.79
(
d, 1H, J = 8 Hz; Ar‐H, H‐7 of benzothiazole), 7.96 (d, 1H, J = 7.4 Hz; Ar‐
H, H‐4 of benzothiazole), 12.69 (s, 1H, NHCOCH ; D O exchangeable),
and 13.91 (s, 1H, OH of pyrimidinone; D O exchangeable); C‐NMR
100 MHz, DMSO‐d ): δ = 21.27, 35.02, 93.03, 110.00, 116.40, 120.98,
22.19, 124.09, 126.65, 129.01 (2C), 129.26 (2C), 131.94, 132.63,
42.17, 149.00, 158.27, 165.69, 167.23, and 167.53; Anal. calcd. for
(m.w. 433.50): C, 58.18; H, 3.49; N, 16.16; S, 14.79.
2
2
13
2
(
6
4
.3 In vitro cytotoxic activity
|
1
1
The cytotoxicity assays were performed at the Pharmacology and
Toxicology Department, Faculty of Pharmacy, Al‐Azhar University,
Cairo, Egypt. Cancer cells from different cancer cell lines HepG2,
MCF‐7, and HCT‐116, were purchased from American Type Cell
Culture Collection (ATCC; Manassas) and grown on the appropriate
growth medium Roswell Park Memorial Institute medium (RPMI
1640) supplemented with 100 mg/ml of streptomycin, 100 units/ml
of penicillin, and 10% of heat‐inactivated fetal bovine serum in a
21 15 5 2 2
C H N O S
Found: C, 58.08; H, 3.82; N, 16.01; S, 14.69.
N‐(Benzothiazol‐2‐yl)‐2‐((5‐cyano‐4‐(4‐hydroxyphenyl)‐6‐oxo‐1,6‐di-
hydropyrimidin‐2‐yl)thio)acetamide (11d)
−1
Yield, 55%; m.p. 209–211°C; IRνmax (cm ): 3,432 (OH), 3,190 (NH),
3
,050 (CH aromatic), 2,993 (CH aliphatic), 2,248 (C≡N), and 1,660
1
(C═O); H‐NMR (400 MHz, DMSO‐d
6
): 5.15 (s, 1H, CH
2
), 7.09 (d, 2H,
humidified, 5% (v/v) CO
2
atmosphere at 37°C. Cytotoxicity assay was
J = 8 Hz; Ar‐H, H‐3 and H‐5 of phenyl), 7.36 (dd, 1H, J = 7.6, 8 Hz; Ar‐
H, H‐5 of benzothiazole), 7.48 (dd, 1H, J = 7.6, 8 Hz; Ar‐H, H‐6 of
benzothiazole), 7.89 (d, 2H, J = 8 Hz; Ar‐H, H‐2 and H‐6 of phenyl),
performed by using MTT.
The exponentially growing cells from different cancer cell lines
were trypsinized, counted, and seeded at the appropriate densities
2
7
.91 (d, 1H, J = 8 Hz; Ar‐H, H‐7 of benzothiazole), 8.04 (d, 1H,
J = 8 Hz; Ar‐H, H‐4 of benzothiazole), 9.47 (s, 1H, ph–OH; D
exchangeable), 12.94 (s, 1H, NHCOCH ; D O exchangeable), and
3.22 (s, 1H, OH of pyrimidinone; D O exchangeable); Anal. calcd. for
(m.w. 435.48): C, 55.16; H, 3.01; N, 16.08; S, 14.72.
(2,000–1,000 cells/0.33 cm well) into 96‐well microtiter plates. Cells
2
O
then were incubated in a humidified atmosphere at 37°C for 24 hr.
Then, cells were exposed to different concentrations of compounds
(0.1, 10, 100, and 1,000 µM) for 72 hr. Then the 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
2
2
1
2
20 13 5 3 2
C H N O S
Found: C, 55.20; H, 3.31; N, 15.80; S, 14.82.
(Sigma‐Aldrich, MO) and were allowed to metabolize the dye into
N‐(Benzothiazol‐2‐yl)‐2‐((5‐cyano‐4‐(4‐methoxyphenyl)‐6‐oxo‐1,6‐
dihydropyrimidin‐2‐yl)thio)acetamide (11e)
colored‐insoluble formazan crystals for 2 hr. The remaining MTT
solution was discarded from the wells and the formazan crystals
were dissolved in a 200‐µl/well acidified isopropanol for 30 min,
covered with aluminum foil and with continuous shaking using a
MaxQ 2000 plate shaker (Thermo Fisher Scientific Inc, MI) at room
temperature. Absorbance was measured at 570 nm using a Stat FaxR
4200 plate reader (Awareness Technology, Inc., FL). The cell viability
was expressed as a percentage of control and the concentration that
induces 50% of maximum inhibition of cell proliferation (IC50) was
determined using GraphPad Prism version 5 software (GraphPad
−1
Yield, 65%; m.p. 244–246°C; IRνmax (cm ): 3,344, 3,205 (2 NH),
3
,075 (CH aromatic), 2,991 (CH aliphatic), 2,233 (C≡N), and 1,663
1
(C═O); H‐NMR (400 MHz, DMSO‐d
6
): 3.57 (s, 3H, OCH
3
), 4.27 (s,
2
1H, CH ), 6.67 (d, 2H, J = 7.6 Hz; Ar‐H, H‐3 and H‐5 of phenyl), 7.28
(dd, 1H, J = 7.6, 7.6 Hz; Ar‐H, H‐5 of benzothiazole), 7.42 (dd, 1H,
J = 7.6, 8.4 Hz; Ar‐H, H‐6 of benzothiazole), 7.76 (d, 2H, J = 7.6 Hz; Ar‐
H, H‐2 and H‐6 of phenyl), 7.78 (d, 1H, J = 8.4 Hz; Ar‐H, H‐7 of
benzothiazole), 7.95 (d, 1H, J = 7.6 Hz; Ar‐H, H‐4 of benzothiazole),
[43–45]
1
2.69 (s, 1H, NHCOCH
pyrimidinone; D O exchangeable); Anal. calcd. for C21
m.w. 449.50): C, 56.11; H, 3.36; N, 15.58; S, 14.26. Found: C, 56.03;
H, 3.62; N, 15.65; S, 14.46.
2
; D
2
O exchangeable), and 13.77 (s, 1H, OH of
Software Inc., CA).
2
15 5 3 2
H N O S
(
4
.4
|
In vitro VEGFR‐2 kinase assay
The kinase activity of VEGFR‐2 was carried out in the
Pharmacology and Toxicology Department, Faculty of Pharmacy,
Al‐Azhar University, Cairo, Egypt, and measured by use of an
antiphosphotyrosine antibody with the Alpha Screen system
4
.2 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
[
15]
(PerkinElmer) according to manufacturer’s instructions.
zyme reactions were performed in 50 mM Tris‐HCl pH 7.5, 5 mM
MnCl , 5 mM MgCl , 0.01% Tween‐20 and 2 mM dithiothreitol,
En-
2
2

14 of 15
EL‐HELBY ET AL.
|
containing 10 μM ATP, 0.1 μg/ml biotinylated poly‐GluTyr (4:1)
and 0.1 nM of VEGFR‐2 (Millipore, UK). Before the catalytic
initiation with ATP, the tested compounds at final concentrations
ranging from 0 to 300 μg/ml and enzyme were incubated for
[6] S. Sulthana, P. Pandian, J. Drug Deliv. Ther. 2019, 9, 505. https://doi.
org/10.22270/jddt.v9i1‐s.2358
[
7] D. Osmaniye, S. Levent, A. B. Karaduman, S. Ilgın, Y. Özkay,
Z. A. Kaplancıklı, Molecules 2018, 23, 1054. https://doi.org/10.
3
390/molecules23051054
5
min at room temperature. The reactions were quenched by the
addition of 25 μl of 100 mM ethylenediaminetetraacetic acid,
0 μg/ml Alpha Screen streptavidin donor beads, and 10 μg/ml
[8] E. A. Abd El‐Meguid, Int. J. Pharm. Technol. 2015, 7, 10040.
[
9] Y. Oguro, D. R. Cary, N. Miyamoto, M. Tawada, H. Iwata, H. Miki, A.
Hori, S. Imamura, Bioorg. Med. Chem. 2013, 21, 4714.
1
[
10] G. Niu, X. Chen, Curr. Drug Targets 2010, 11, 1000.
11] K. Holmes, O. L. Roberts, A. M. Thomas, M. J. Cross, Cell. Signal. 2007,
19, 2003.
acceptor beads in 62.5 mM 4‐(2‐hydroxyethyl)‐1‐piperazineetha-
nesulfonic acid pH 7.4, 250 mM NaCl, and 0.1% bovine serum
albumin. The plate was incubated in the dark overnight and then
read by ELISA Reader (PerkinElmer). Wells containing the
substrate and the enzyme without compounds were used as
reaction control. Wells containing biotinylated poly‐GluTyr (4:1)
and enzyme without ATP were used as basal control. Percent
inhibition was calculated by the comparison of compounds
treated to control 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 standard VEGFR‐2 inhibitor.
[
[
12] S. Tugues, S. Koch, L. Gualandi, X. Li, L. Claesson‐Welsh, Mol. Aspects
Med. 2011, 32, 88.
[
13] W. M. Eldehna, H. S. Ibrahim, H. A. Abdel‐Aziz, N. N. Farrag,
M. M. Youssef, Eur. J. Med. Chem. 2015, 89, 549.
[14] W. M. Eldehna, S. M. Abou‐Seri, A. M. El Kerdawy, R. R. Ayyad, H. A.
Ghabbour, M. M. Ali, D. A. Abou El Ella, Eur. J. Med. Chem. 2016, 113,
5
0. https://doi.org/10.1016/j.ejmech.2016.02.029
[
15] S. M. Abou‐Seri, W. M. Eldehna, M. M. Ali, D. A. Abou El Ella, Eur. J.
Med. Chem. 2016, 107, 165. https://doi.org/10.1016/j.ejmech.2015.
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