Novel 2-indolinone thiazole hybrids as sunitinib analogues: Design, synthesis, and potent VEGFR-2 inhibition with potential anti-renal cancer activity

Article abstract of DOI:10.1016/j.ejmech.2020.112752

Novel 2-indolinone thiazole hybrids were designed and synthesized as VEGFR-2 inhibitors based on sunitinib, an FDA-approved anticancer drug. The proposed structures of the prepared 2-indolinone thiazole hybrids were confirmed based on their spectral data and CHN analyses. The target compounds were screened in vitro for their anti-VEGFR-2 activity. All tested compounds exhibited a potent submicromolar inhibition of VEGFR-2 kinase with IC₅₀ values ranging from 0.067 to 0.422 μM, relative to sunitinib reference drug (IC₅₀ = 0.075 ± 0.002 μM). Compounds 5, 15a, 15b, 17, 19c displayed excellent VEGFR-2 inhibitory activity, comparable or nearly equipotent to sunitinib. Compound 13b stood out as the most potent against VEGFR-2 showing IC₅₀ value of 0.067 ± 0.002 μM, lower than that of sunitinib. In addition, the most potent derivatives were assessed for their anticancer activity against two renal cancer cell lines. Compound 13b (IC₅₀ = 3.9 ± 0.13 μM) was more potent than sunitinib (IC₅₀ = 4.93 ± 0.16 μM) against CAKI-1 cell line. Moreover, thiazole 15b displayed excellent anticancer activity against CAKI-1 cell line (IC₅₀ = 3.31 ± 0.11 μM), superior to that of sunitinib (IC₅₀ = 4.93 ± 0.16 μM). Thiazole 15b was also equipotent to sunitinib (IC₅₀ = 1.23 ± 0.04 μM) against A498 cell line. Besides, compound 15b revealed a safety profile much better than that of sunitinib against normal human renal cells. Furthermore, a docking study revealed a proper fitting of the most active compounds into the ATP binding site of VEGFR-2, rationalizing their potent anti-VEGFR-2 activity.

Full text of DOI:10.1016/j.ejmech.2020.112752

European Journal of Medicinal Chemistry 208 (2020) 112752
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Novel 2-indolinone thiazole hybrids as sunitinib analogues: Design,
synthesis, and potent VEGFR-2 inhibition with potential anti-renal
cancer activity
, b,
Huda K. Mahmoud a, Thoraya A. Farghaly a *, Hanan G. Abdulwahab c,
,
b
Nadia T. Al-Qurashi d, Mohamed R. Shaaban a
a Department of Chemistry, Faculty of Science, Cairo University, Giza, 12613, Egypt
b Department of Chemistry, Faculty of Applied Science, Umm Al-Qura University, Makkah Almukkarramah, Saudi Arabia
c Pharmaceutical Chemistry Department, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Egypt
d Department of Basic Science, University College in Adam, Umm Al-Qura University, Makkah Almukkarramah, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel 2-indolinone thiazole hybrids were designed and synthesized as VEGFR-2 inhibitors based on
sunitinib, an FDA-approved anticancer drug. The proposed structures of the prepared 2-indolinone
thiazole hybrids were confirmed based on their spectral data and CHN analyses. The target com-
pounds were screened in vitro for their anti-VEGFR-2 activity. All tested compounds exhibited a potent
Received 4 June 2020
Received in revised form
5 August 2020
Accepted 7 August 2020
Available online 21 August 2020
submicromolar inhibition of VEGFR-2 kinase with IC50 values ranging from 0.067 to 0.422
sunitinib reference drug (IC50 ¼ 0.075 0.002 M). Compounds 5, 15a, 15b, 17, 19c displayed excellent
VEGFR-2 inhibitory activity, comparable or nearly equipotent to sunitinib. Compound 13b stood out as
the most potent against VEGFR-2 showing IC50 value of 0.067 0.002 M, lower than that of sunitinib. In
addition, the most potent derivatives were assessed for their anticancer activity against two renal cancer
cell lines. Compound 13b (IC50 ¼ 3.9 0.13 M) was more potent than sunitinib (IC50 ¼ 4.93 0.16 M)
against CAKI-1 cell line. Moreover, thiazole 15b displayed excellent anticancer activity against CAKI-1 cell
line (IC50 ¼ 3.31 0.11 M), superior to that of sunitinib (IC50 ¼ 4.93 0.16 M). Thiazole 15b was also
equipotent to sunitinib (IC50 ¼ 1.23 0.04 M) against A498 cell line. Besides, compound 15b revealed a
mM, relative to
m
Keywords:
Indolinone
Thiazoles
Sunitinib
Anti-renal cancer activity
Docking study
m
m
m
m
m
m
safety profile much better than that of sunitinib against normal human renal cells. Furthermore, a
docking study revealed a proper fitting of the most active compounds into the ATP binding site of VEGFR-
2, rationalizing their potent anti-VEGFR-2 activity.
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
In many proteins, phosphorylation of their tyrosine moiety have
been achieved by catalysis of protein tyrosine kinases (PTKs). PTKs
Cancer is a worldwide health problem that leads to death in
both developing and developed countries [1]. Even though the
exploration for anticancer candidates started on the beginning of
the preceding century, there is a persistent need to develop new
anticancer agents to overcome the adverse effects and the resis-
tance development associated with most marketed anticancer
drugs [2]. Thus, extensive efforts have been devoted to the devel-
opment of new potent chemotherapeutic agents with higher
selectivity toward cancer cells [3].
take a pivotal part in proliferation, differentiation, and migration
signaling in the cell. It is well known that cancer development is
associated with abnormal activation or over-expression of PTKs
[4e6].
Vascular endothelial growth factor receptor-2 (VEGFR-2), a
transmembrane tyrosine kinase receptor, has been recognized as
the most critical factor in promoting angiogenesis [7e9]. Angio-
genesis is a complex process that involves the creation of new blood
vessels from the pre-existing vasculature in physiological and
pathophysiological conditions. Also, angiogenesis is considered as
one of the hallmarks of tumor growth, invasion and metastasis
[10e12]. Playing a central role in cancer pathophysiology, VEGFR-2
is well-established as the supreme significant target in anti-
* Corresponding author. Department of Chemistry, Faculty of Science, Cairo
University, Giza, 12613, Egypt.
E-mail
addresses:
thoraya-f@hotmail.com,
tamohamed@uqu.edu.sa
(T.A. Farghaly).
https://doi.org/10.1016/j.ejmech.2020.112752
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
H.K. Mahmoud et al. / European Journal of Medicinal Chemistry 208 (2020) 112752
angiogenesis therapy against cancer. In this context, several small
molecule VEGFR-2 inhibitors have been clinically approved or
tested for cancer treatment (Fig. 1) [13e15].
hydrophobic interactions with the hydrophobic pockets of the
VEGFR-2 active site e.g. vatalanib and sorafenib (Fig. 1) [31,32]. It
was also reported that the introduction of hydrophobic moieties to
the 2-indolinone core resulted in enhanced binding affinity and so
improved kinase inhibitory activity [33]. In this work, fluorine atom
in sunitinib was also replaced by its chlorine bioisostere. Addi-
tionally, simplification approach was also employed to obtain
compounds 3 and 5. Furthermore, the substituted pyrrole in suni-
tinib was replaced by substituted thiazole/thiazolidinone ring as
represented in target compounds 10a-c, 15a,b, 17 and 19a-c.
Compounds 13a-c were synthesized as open thiazolidinone ana-
logues with different substituents at para-position of phenyl ring.
Moreover, substituents of diverse size and electronic properties
were introduced to position 4 and 5 of thiazole ring in compounds
10a-c, 15a,b and 19a-c to explore their impact on VEGFR-2 inhibi-
tory activity. To study the effect of linker extension on anti-VEGFR-2
activity, the CH linker in sunitinib was also replaced by a hydrogen-
bond acceptor and relatively longer (¼NeN¼) linker in compounds
10e19.
Indolin-2-one scaffold attracted considerable attention towards
anticancer research, particularly the discovery of protein kinase
inhibitors [16,17]. Moreover, 2-indolinone derivatives were among
the initial structures recognized as kinase inhibitors and have been
thoroughly investigated for the inhibition of VEGFR (Fig. 1) [18].
Sunitinib (Fig. 1) is the first kinase inhibitor of the indolinone
category that reached the market. It is indicated for the treatment
of renal cell carcinoma and imatinib-resistant gastrointestinal
stromal tumor. Sunitinib binds to the kinase domain of VEGFR-2
and inhibits its activity [19].
On the other hand, thiazole is a fascinating heterocyclic core in
medicinal chemistry for the design and discovery of bioactive
compounds, particularly, anticancer agents [20e22]. Many thia-
zoles have been specially designed to target specific pathways that
are involved in cancer pathogenesis [20e22]. For example, the FDA-
approved thiazole-containing drugs, dabrafenib and dasatinib, are
selective tyrosine kinase inhibitors with very potent antitumor
activity (Fig. 2) [23,24].
Encouraged by these findings and in continuation of our
ongoing study project in designing and synthesis of novel biologi-
cally active heterocyclic compounds [25e30], novel 2-indolinone
thiazole hybrids were designed and synthesized as sunitinib ana-
logues with prospective VEGFR-2 inhibitory activity. The approach
utilized for designing our target candidates is illustrated in Fig. 3.
In the present work, while conserving the indolinone core of
sunitinib, a benzyl moiety was introduced to the nitrogen atom of
the indolinone core to improve binding affinity towards VEGFR-2
active site via hydrophobic interactions. Surveying literature
revealed that the improvement of the binding affinity towards
VEGFR-2 active site has been attained through the increase of the
All targeted products were screened for their in vitro VEGFR-2
inhibitory activity. Furthermore, the most potent derivatives were
further assessed in vitro for their anticancer activity against two
renal cancer cell lines. A docking study was also conducted to
predict the binding mode of the promising compounds within the
binding site of VEGFR-2 kinase.
2. Results and discussion
2.1. Chemistry
The versatile starting substrate 1-benzyl-5-chloroindoline-2,3-
dione (2) was prepared by treatment of 5-chloroisatin (1) with
benzyl chloride in DMF with stirring at room temperature using
Fig. 1. Clinically approved/tested VEGFR-2 inhibitors.

H.K. Mahmoud et al. / European Journal of Medicinal Chemistry 208 (2020) 112752
3
Fig. 2. Clinically approved thiazole-containing tyrosine kinase inhibitors.
Fig. 3. Design of target compounds based on Sunitinib VEGFR-2 inhibitor.

4
H.K. Mahmoud et al. / European Journal of Medicinal Chemistry 208 (2020) 112752
potassium carbonate as a catalyst following to reported procedure
[34] as shown in Scheme 1. The utility of the 1-benzyl-5-
chloroindoline-2,3-dione (2) in the synthesis of multifunctional
building blocks have been initiated by refluxing compound 2 and
thiosemicarbazide using ethanol containing HCl for 4 h as depicted
in Scheme 1 and reported in literature [34]. Condensation pro-
ceeded to afford the corresponding the thiosemicarbazone deriv-
ative 3 in 81% yield. On the other hand, a mixture of 1-benzyl-5-
chloroindoline-2,3-dione 2 and cyanoacetic acid hydrazide (4) in
ethanol/acetic acid solvent mixture was refluxed to give the amino
pyrazolone derivative 5 instead of the cyanoacetohydrazide deriv-
ative 6 as depicted in Scheme 1. The formation of 1H-pyr-
azolylidene derivatives 5 was confirmed by the extracted data from
1H NMR and IR as shown in Figs. 4 and 5. For detailed, the
derivatives 10a-c were characterized with the existence of only one
NH singlet signal at
d
¼ 11.2e13.19 ppm in addition to the aromatic
protons, moreover, the aliphatic region in the 1H NMR of derivatives
10b and 10c characterized with the appearance of two singlet
signals of each at
d
¼ 2.62 (CH3), 3.82 (OCH3), 2.37 (CH3) and 2.65
(CH3), respectively. The reaction of carbothioamide 3 with hydra-
zonoyl chlorides 7a-c, started with nucleophilic substitution of the
carbothioamide function in 3 through the sulfur atom reaction to
form the intermediates 8 as shown in Scheme 2. Cyclization of in-
termediate 8 with elimination of water molecule (H2O) through the
non-isolable intermediate 9 gave the final products 10a-c (Scheme
2).
Using the same reaction conditions, attempts to access the
thiazolones 12a-c, via one pot reaction of 2-indolinone-based thi-
osemicarbazone derivative 3 with C-ethoxycarbonyl-N-arylpropa-
nehydrazonoyl chlorides 11a-c in the presence of TEA (Scheme 3)
have been investigated. Condensation proceeded to afford the
corresponding amidrazone derivatives 13a-c rather than the ex-
pected thiazolone derivatives 12a-c. The molecular ion peak of all
mass spectra of derivatives 13a-c proved liberation of only HCl
molecule from the starting compounds 3 and 11. Furthermore, 1H
NMR spectra of all amidrazone derivatives 13a-c revealed the two
signals (t and q) for the CH3CH2 of the ester moiety near the regions
appearance of D2O-exchangeable broad signal at
d 4.42 ppm with
integration equivalent to NH2 group protons in 1H NMR confirmed
structure 5 rather than the acyclic isomer 6 as illustrated in Fig. 4.
Another evidence comes from its IR spectrum (Fig. 5) which free
from the cyano group (C^N) absorption at
n
z 2200 cmꢀ1. The
formation of compound 5 proceeded through the nucleophilic
condensation reaction with elimination of H2O from the active
methylene CH2 group of the cyano acid hydrazide 4 and the
carbonyl group of substituted isatin 2 followed by cyclization via
the nuecleophilic addition reaction (Mickle type addition reaction)
of the amino group to the C^N.
The targeted 2-indolinone incorporated arylazothiazole de-
rivatives 10a-c, were synthesized by condensation of the 2-
indolinone-based thiosemicarbazone derivative 3 with C-acetyl-
2-oxo-N-arylpropanehydrazonoyl chlorides 7a-c in dioxane and
using TEA as a basic catalyst (Scheme 2). The novel 2-indolinone-
thiazole hybrid derivatives 10a-c were obtained in 79e86% yield
under this applied reaction conditions. The structure of 2-
indolinone-thiazole hybrid derivatives 10a-c were confirmed by
their spectral and microanalyses. All 1H NMR spectra of the thiazole
d
1.35 and 4.40 ppm as well as the singlet signal of NH2 group at
3.60 ppm in addition to aliphatic and aromatic protons. Fig. 6
d
represents the 1H NMR of derivative 13c. Such data confirmed the
structure 13(I) rather than the isomer form 13(II) as illustrated in
Scheme 3.
Functionalized thiazole derivatives 15a,b have been accessed by
the reaction between 2-indolinone-based thiosemicarbazone de-
rivative 3 and
a-chloro-dicarbonyl compounds 14a,b in dioxane
and using triethylamine as a catalyst as depicted in Scheme 4. The
structure of the reaction products 15a,b has been elucidated by
spectral data evidences as well as elemental analyses. The IR
Scheme 1. Synthesis of the starting thiosemicarbazone derivative 3 and 3-(3-amino-5-oxo-1H-pyrazol-4(5H)-ylidene)-1-benzyl-5-chloroindolin-2-one (5).

H.K. Mahmoud et al. / European Journal of Medicinal Chemistry 208 (2020) 112752
5
Fig. 4. The 1H NMR spectrum of 3-(3-amino-5-oxo-1H-pyrazol-4(5H)-ylidene)-1-benzyl-5-chloroindolin-2-one (5).
Fig. 5. The IR spectrum of 3-(3-amino-5-oxo-1H-pyrazol-4(5H)-ylidene)-1-benzyl-5-chloroindolin-2-one (5).
spectrum of thiazoline derivative 15b (taken as an example) dis-
played a broad absorption band at 3448 cmꢀ1 corresponding to the
NH functions. 1H NMR spectrum of compound 15b revealed the
the form 19(I) in the solution state. This is due to the appearance of
two CH2 groups for the thiazole-CH2 and PhCH2 at 4.87 and
5.04 ppm. While, the IR spectra of derivatives 19a-c revealed the
broad band at 3417-3471 cm-1 indicated the presence of the isomer
19(II). For all thiazole derivatives 19aec, the mass spectrometry
results revealed the molecular ion peaks at the exact m/z values.
d
characteristic proton signals at
d 1.03 (t), 2.49 (s), 4.24 (q), 4.99 (s),
6.92e8.30 (m), 13.16 (s) ppm assigned to the CH3CH2, CH3 CH3CH2,
PhCH2, aromatic protons and NH groups, respectively.
Finally, 2-indolinone-based thiosemicarbazone derivative 3 re-
acts with ethyl chloroacetate (16) in fused sodium acetate/acetic
acid mixture to give the targeted thiazolone derivative 17 as shown
in Scheme 5. The structure elucidation of thiazolone derivative 17
has been achieved by spectral data and elemental analyses. In a
similar manner, reaction between 2-indolinone-based thio-
semicarbazone derivative 3 and phenacyl bromide derivatives 18a-
c in the presence of triethylamine afforded the corresponding
thiazole derivatives 19a-c as shown in Scheme 5. Such thiazole
derivatives 19 can present in one of the tautomeric structures 19(I)
or 19(II) (Scheme 5), the 1H NMR data of these derivatives assured
2.2. Biological activity
2.2.1. In vitro VEGFR-2 inhibition
In the present work, the title compounds were tested in vitro for
their ability to inhibit VEGFR-2 enzyme using sunitinib as a refer-
ence drug. The screening data are presented in Table 1.
As shown in Table 1, all tested compounds effectively inhibited
VEGFR-2 activity with IC50 values in the submicromolar level
ranging from 0.067 to 0.422
(IC50 ¼ 0.075 0.002 M). Compounds 5, 15a,b, 17, 19c displayed
mM, relative to sunitinib reference drug
m

6
H.K. Mahmoud et al. / European Journal of Medicinal Chemistry 208 (2020) 112752
Scheme 2. Reaction of 2-(1-benzyl-5-chloro-2-oxoindolin-3-ylidene)hydrazine-1-carbothioamide (3) with C-acetyl-2-oxo-N-arylpropanehydrazonoyl chlorides 7a-c.
excellent VEGFR-2 inhibitory activity (IC50 ¼ 0.084
0.002,
VEGFR-2 activity with IC50 values ranging from 0.067 to 0.133 mM.
0.092 0.003, 0.078 0.002 and 0.088 0.002 M, respectively),
m
Compound 13a; bearing 4-nitrophenyl moiety, was more potent
than its 4-bromophenyl analogue 13c. Among the tested com-
pounds, 4-tolyl hydrazono derivative 13b stood out as the most
active derivative against VEGFR-2 tyrosine kinase with IC50
comparable or nearly equipotent to sunitinib. Additionally, com-
pounds 13a and 13c exhibited high VEGFR-2 inhibitory activity
with IC50 ¼ 0.102
0.003 and 0.133
Furthermore, compounds 10c and 19b revealed half potency of
sunitinib against VEGFR-2 kinase with IC50 ¼ 0.153 0.004 and
0.004 mM, respectively.
value ¼ 0.067 0.002
mM, lower than that of sunitinib reference
drug. Besides, 13b was also two times more potent than its 4-tolyl
diazenylthiazole counterpart 10c.
0.158 0.004 mM, respectively. Equal VEGFR-2 inhibitory activity
was also detected for compounds 3 and 10b. Among the tested
compounds in this study, compound 13b emerged as the most
Noticeably, compounds 15a and 15b presented potent VEGFR-2
tyrosine kinase inhibitory activity comparable to that of sunitinib
with acetylthiazole 15a being more potent than its ethyl carbox-
ylate counterpart 15b. Moreover, thiazolidinone analogue 17
showed better anti-VEGFR-2 activity; almost equipotent to
sunitinib.
potent derivative with IC50 ¼ 0.067 0.002
mM, lower than that of
sunitinib reference drug. While, compounds 10a and 19a were the
least potent with IC50 ¼ 0.263
0.007 and 0.422
0.011 mM,
respectively.
Examining screening results revealed that simplification
approach was beneficial for VEGFR-2 inhibitory activity as indicated
by the nearly equipotent activity of pyrazole derivative 5 to suni-
tinib. Besides, the semicarbazone open analogue 3 displayed half
potency of sunitinb. It is worth mentioning that pyrazole-
containing VEGFR tyrosine kinase inhibitors such as axitinib and
pazopanib have been approved by FDA for the treatment of renal
cell carcinomas [35].
Concerning 4-arylthiazole derivatives 19a-c, the addition of
electron withdrawing chlorine atom to the para position of phenyl
ring in compound 19b resulted in more than two times increase in
VEGFR-2 inhibitory activity compared to the unsubstituted phenyl
derivative 19a. Further increase in potency (approximately five
times compared to 19a) was obtained upon the introduction of the
more electron withdrawing nitro group in compound 19c.
Generally, it could be concluded that the introduction of a large
bulky aryldiazenyl moiety to position 5 of thiazole ring is not
beneficial for VEGFR-2 kinase inhibitory activity as indicated by the
lower potency of compounds 10a-c comparing to their 5-acetyl 15a
and 5-ethyl carboxylate 15b analogues. Similarly, small methyl
group at position 4 of thiazole ring in compounds 15a and 15b is
more tolerated than relatively larger phenyl and chlorophenyl
moieties in compounds 19a and 19b, respectively.
Regarding 5-aryldiazenyl thiazole derivatives 10a-c, 4-
methyoxy (10b) and 4-methyl (10c) phenyl derivatives showed
significant VEGFR-2 inhibitory activity with IC50 ¼ 0.175 0.005
and 0.153 0.004 mM, respectively; much better than that of the
unsubstituted phenyl analogue 10a.
Comparing to thiazoles 10a-c, arylhydrazono derivatives 13a-c;
designed as open thiazolidinone analogues, displayed better anti-

H.K. Mahmoud et al. / European Journal of Medicinal Chemistry 208 (2020) 112752
7
Scheme 3. The reaction of 2-(1-benzyl-5-chloro-2-oxoindolin-3-ylidene)hydrazine-1-carbothioamide (3) with C-ethoxycarbonyl-N-arylpropanehydrazonoyl chlorides 11a-c.
2.2.2. In vitro anticancer activity
cancer cell line that was six folds more potent than sunitinib refer-
ence drug. Compound 5 also suppressed the growth of A498 renal
cancer cell line at one-digit micromolar IC50 value of 8.53 0.28 M.
Compound 13b, which was the most potent in anti-VEGFR-2
assay, showed cytotoxic activity against CAKI-1 cell line higher
than that of sunitinib. Besides, compound 13b efficiently inhibited
the growth of A498 cell line at low micromolar IC50 value of
As mentioned above, sunitinib is clinically used for the treat-
ment of renal cell carcinomas which are highly vascular tumors,
and therefore the effectiveness of sunitinib against renal cancer is
mainly attributed to the inhibition of VEGFR-2 [36]. With this in
mind, the most active compounds in anti-VEGFR-2 assay; 5, 13b,
15a, 15b, 17 and 19c were passed on to further evaluation of their
in vitro cytotoxic activity against two human renal cancer cell lines,
namely, CAKI-1 and A498 using MTT-based cytotoxicity assay [37].
Sunitinib was used as a reference drug (Table 2).
CAKI-1 and A498 cancer cell lines were obtained from American
Type Culture Collection. CAKI-1 is a widespread model of renal cell
carcinoma that is associated with high production of vascular
endothelial growth factor. In addition, A498 is a renal cancer cell
line belonging to NCI-60 panel and so is used extensively in renal
cancer research [38].
m
2.88 0.09 mM.
In addition, thiazole 15a effectively inhibited the growth of the
two tested cell lines; however A498 cell line was two times more
sensitive to thiazole 15a than CAKI-1 cell line.
Remarkably, it was observed that the replacement of acetyl
group in thiazole 15a by ethyl carboxylate moiety in compound 15b
led to more than four times increase in cytotoxic activity. Thiazole
15b revealed excellent anticancer activity against CAKI-1 renal
cancer cell line, superior to that of sunitinib. Moreover, thiazole 15b
emerged as the most potent compound against A498 renal cancer
cell line with cytotoxic activity equipotent to that of sunitinib.
On the contrary, the replacement of thiazole ring in compounds
15a and 15b by thiazolidinone ring in compound 17 resulted in a
marked decline in growth inhibitory activity against CAKI-1 cell
line. However, A498 cell line was much more sensitive to thiazo-
lidinone 17.
From the obtained results (Table 2), it can be observed that the
tested compounds displayed moderate to potent cytotoxicity
against CAKI-1 and A498 cell lines with IC50 values ranging from
two-digit micromolar to submicromolar concentrations compared
to sunitinib reference drug.
Interestingly, aminopyrazolone 5 with IC50 ¼ 0.82
0.03 mM
exhibited a highly potent cytotoxic activity against CAKI-1 renal

8
H.K. Mahmoud et al. / European Journal of Medicinal Chemistry 208 (2020) 112752
Fig. 6. The 1H NMR spectrum of compound 13c.
Scheme 4. Synthesis of thiazole derivatives 15a,b.
Finally, thiazole derivative 19c; bearing 4-nitrophenyl at posi-
2.2.4. Docking study
tion 4 of thiazole ring, demonstrated cytotoxic activity against
CAKI-1 cell line comparable to that of sunitinib with IC50 values of
5.78 0.19 and 4.93 0.16 mM, respectively. However, A498 cell line
In this investigation, a docking study of the most potent com-
pounds namely; 5, 13a, 15a, 15b, 17 and 19c into the ATP binding
site of VEGFR-2 kinase was performed using MOE 2014.0901. A
high-resolution VEGFR-2 co-crystallized with sunitinib (PDB:
4AGD) [39] was used for docking study. First, sunitinib was docked
into the active site of VEGFR-2 enzyme to validate docking pro-
cedure. Sunitinib reproduced a binding pose similar to that of the
co-crystallized ligand with docking score ¼ ꢀ-6.74 kcal/mol.
Sunitinib interacts by NH and CO of its indolinone scaffold with the
hinge region residues Glu917 and Cys919, respectively, in addition
to hydrophobic contacts with Leu840, Ala866, the gatekeeper
Val916, Phe918 of the hinge, and Leu1035.
Examining docking results (Figs. 7 and 8), it was found that the
analyzed compounds 5, 13a, 15a, 15b, 17, 19c were well-fitted into
the ATP binding site of VEGFR-2 with energy binding
scores ¼ ꢀ5.41, ꢀ6.10, ꢀ6.60, ꢀ6.70, ꢀ4.99, and ꢀ5.01 kcal/mol,
respectively. Moreover, the docked compounds demonstrated a
converged binding pattern similar to that of sunitinib, in which
hydrogen bonding interactions with the hinge region key residue
Cys919 were observed. It is worth mentioning that the azo linker in
was less sensitive to compound 19c.
Notably, it could be concluded that cyclized (thiazole) analogues
with relatively small substituents at position 4 and 5 of thiazole
ring are beneficial for both in vitro VEGFR-2 inhibition and anti-
cancer activity as indicated by the potent anti-VEGFR-2 activity and
superior cytotoxicity elicited by thiazole 15b.
2.2.3. In vitro cytotoxicity against normal human cells
The safety profile of the most effective analogue in this study;
15b was also assessed by testing its in vitro cytotoxicity against
RPTEC/TERT1 normal human cells, comparing to sutininb refer-
ence drug. RPTEC/TERT1 cells are human renal epithelial cells that
are employed as a model for basic kidney functions and are also
used to study nephrotoxicity [38]. It was found that compound
15b demonstrated a safety profile much better than that of suni-
tinib with IC50 values of 49.8
respectively.
2.79 and 16.9
0.95 mM,

H.K. Mahmoud et al. / European Journal of Medicinal Chemistry 208 (2020) 112752
9
Scheme 5. Synthesis of thiazole derivatives 17 and 19a-c.
Table 1
affinity of the inhibitors to the VEGFR-2 active site and so could
contribute to the high in vitro VEGFR-2 inhibitory activity displayed
by these compounds.
VEGFR-2 kinase inhibitory activity of the target compounds.
Comp. No.
IC50
(m
M)
Comp. No.
IC50 (mM)
As seen in Fig. 7A, compound 5 formed two hydrogen bonds
with Glu917 and Cys919 through the NH and carbonyl oxygen of its
pyrazolone ring, respectively. An intramolecular hydrogen bond
was also formed between pyrazole-NH2 and carbonyl of indolinone
core that could further help orient compound 5 within the enzyme
active site. Several literatures have described the positive impact of
intramolecular hydrogen bonding on ligand-receptor interaction
[40].
3
5
0.174 0.005
0.084 0.002
0.263 0.007
0.175 0.005
0.153 0.004
0.102 0.003
0.067 0.002
0.133 0.004
15a
15b
17
19a
19b
19c
Sunitinib
0.084 0.002
0.092 0.003
0.078 0.002
0.422 0.001
0.158 0.004
0.088 0.002
0.075 0.002
10a
10b
10c
13a
13b
13c
Additionally, as an H-bond acceptor, carbonyl oxygen of the
indolinone core in compound 13b formed two hydrogen bonds
with the hinge Phe918 and Cys919. 13b was further stabilized
within the active site of enzyme via an intramolecular hydrogen
bond between the azo linker and the tolylhydrazo NH, directing the
tolylhdrazo moiety toward the bulk solvent. In addition, benzyl
moiety was in a hydrophobic contact with Leu840, Val848 and
Phe1047 residues (Fig. 7B). This binding pose could explain the
superior anti-VEGFR-2 activity of 13b.
Considering the binding mode of acetyl thiazole 15a (Fig. 7C),
the azo linker nitrogen accepted one hydrogen bond from Cys919,
in addition to the formation of another hydrogen bond between the
thiazole ring NH and Glu917. Furthermore, methyl and acetyl
groups of thiazole ring were exposed tohydrophobic contacts with
gate keeper Val916, Leu840, Val848, Ala866, Val899, and Phe1047.
Focusing on the binding mode of thiazole 15b; a compound with
potent in vitro anti-VEGFR-2 activity and superior cytotoxicity,
thiazole NH and the azo linker N in compound 15b formed two
hydrogen bonds with the hinge Glu917 and Cys919, respectively.
Table 2
In vitro anticancer activity of compounds 5, 13b, 15a, 15b, 17 and 19c against CAKI-1
and A498 renal cancer cell lines.
Comp. No.
Cytotoxicity IC50 (mM)
CAKI-1
A498
5
0.82 0.03
3.9 0.13
12.8 0.42
3.31 0.11
35.6 1.16
5.78 0.19
4.93 0.16
8.53 0.28
2.88 0.09
6.27 0.2
1.23 0.04
3.45 0.11
24.7 0.8
1.25 0.04
13b
15a
15b
17
19c
Sunitinib
compounds 15a, 15b, 17, 19c acts as a hydrogen-bond acceptor
forming a hydrogen bond with Cys919. Additionally, as planned,
benzyl moiety in the docked compounds was fitted into a hydro-
phobic pocket lined with Leu840, Val848 and Phe1047 residues.
This hydrophobic interaction could probably improve the binding

10
H.K. Mahmoud et al. / European Journal of Medicinal Chemistry 208 (2020) 112752
Fig. 7. Compounds 5 (A), 13a (B), 15a (C), 17 (D), 19c (E) docked into the ATP binding site of VEGFR-2.
Fig. 8. Compound 15b docked into the ATP binding site of VEGFR-2; 2D (A), 3D (B).

H.K. Mahmoud et al. / European Journal of Medicinal Chemistry 208 (2020) 112752
11
Moreover, the ester carbonyl oxygen accepted a hydrogen bond
it in the biological evaluation.
from Cys1045 located in the back cleft of the enzyme. Hydrophobic
interaction was also observed between the benzyl moiety and
Leu840, Val848, and Phe1047 (Fig. 8).
4.2. Synthesis of 3-(3-amino-5-oxo-1H-pyrazol-4(5H)-ylidene)-1-
benzyl-5-chloroindolin-2-one (5)
On the other hand, thiazolidinone 17 and thiazole 19c revealed
almost the same binding mode within the active site of VEGFR-2 in
which the azo nitrogen and the carbonyl oxygen of indolinone core
formed two hydrogen bonds with Cys919 and Gly922, respectively.
Besides, benzene ring of indolinone core was exposed to hydro-
phobic residues; Val848, Ala866, Val918, Phe921, and Leu1035. As
well, the benzyl moiety participated in hydrophobic interactions
with Leu840, Val848, and Phe1047 (Fig. 7D and E).
Acidified ethanolic solution of 1-benzyl-5-chloroindoline-2,3-
dione and freshly prepared cyanoacetic acid hydrazide
(0.01 mol of each, 30 mL EtOH and 5 mL AcOH) was subjected to
homogenous heating for 5 min. After the solution was left in room
temperature to cool, it precipitated orange solid. The formed
colored solid was filtered, washed with CH3OH then crystallized
2
from
a mixture of ethanol/dioxane to afford pyrazolyl-5-
Observably, the ability of the docked compounds to bind to the
important amino acids within the enzyme active site could ratio-
nalize their potent anti-VEGFR-2 activity as indicated by their
docking pattern relative to that of sunitinib.
chloroindolin-2-one derivative 5 as orange solid, 72% yield, m.p.
178e180 ꢁC; IR (KBr): v 3201 (NH, NH2), 1720 (C]O), 1681 (C]O),
1612 (C]N), 1465, 1357, 1311, 1226, 1157, 1103, 1080, 1033,
1010 cmꢀ1
;
1H NMR (DMSO‑d6):
d
4.42 (br, 2H, NH2), 4.99 (s, 2H,
CH2), 7.05e7.63 (m, 8H, AreH), 12.49 (s, 1H, NH), 13C NMR
(DMSO‑d6): 42.3 (CH2), 110.5, 116.1, 120.4, 120.7, 123.2, 127.3, 127.4,
3. Conclusion
d
127.6,128.6,129.4,131.8,135.5,142.8,160.0 (C]O),160.2 (C]O). MS
m/z (%): 354 (Mþþ2, 14), 353 (Mþþ1, 9), 352 (Mþ, 38). Anal. Calcd
for C18H13ClN4O2 (352.77): C, 61.28; H, 3.71; N, 15.88. Found: C,
61.42; H, 3.69; N, 15.78%.
Herein, the synthesis of new 2-indolinone thiazole hybrids as
sunitinib analogues with potent anti-VEGFR-2 activity has been
described. All tested compounds effectively inhibited VEGFR-2 ac-
tivity with IC50 values in the submicromolar level. Compounds 5,
15a, 15b, 17, 19c displayed excellent VEGFR-2 inhibitory activity,
comparable or nearly equipotent to sunitinib. Compound 13b was
the most potent against VEGFR-2 showing IC50 value lower than
that of sunitinib reference drug. In addition, the most potent
compounds were estimated for their anticancer activity against two
renal cancer cell lines. The tested compounds exhibited moderate
to potent anticancer activity against CAKI-1 and A498 cell lines.
Compound 13b was more potent than sunitinib against CAKI-1 cell
line. Moreover, thiazole 15b was superior/equipotent to sunitinib
against CAKI-1/A498 cell lines, respectively. Besides, compound
15b demonstrated a safety profile much better than that of suni-
tinib against normal human renal cells. A docking study was also
conducted revealing the key interactions essential for VEGFR-2
inhibition.
4.3. Synthesis of compound 10a-c, 13a-c and 15a,b
Reaction of indolinone-thiosemicarbazone derivative 3 (0.34g,
1 mmol) with a-chlorocarbonyl compounds 7a-c, 11a-c or 14a,b
(1 mmol) in 15 mL of dioxane were proceeded under reflux in the
presence of 0.35 mL of triethylamine for 5 h. The progress of the
reactions were monitored through the TLC test. After the reactions
were completed, the solvent was then evaporated under vacuum
then washed with CH3OH. The colored solid produced was filtered
and the specific solvent used for each compound for crystallization
to give thiazolylhydrazonoindolin-2-one 10a-c, 13a-c and 15a,b.
4.3.1. 1-Benzyl-5-chloro-3-((4-methyl-5-(phenyldiazenyl)thiazol-
2(3H)-ylidene)hydrazono) -indolin-2-one (10a)
Red solid, 86% yield, m.p. 160e162 ꢁC (Dioxane); IR (KBr): v 3417
(NH), 1689 (C]O), 1612 (C]N), 1535, 1465, 1373, 1350, 1249, 1157,
4. Experimental
1103, 1033 cmꢀ1 1H NMR (DMSO‑d6):
; d 2.67 (s, 3H, CH3), 4.97 (s,
4.1. General methods
2H, CH2), 6.96e7.66 (m, 13H, AreH), 11.20 (s, 1H, NH), MS m/z (%):
489 (Mþþ2, 21), 488 (Mþþ1, 5), 487 (Mþ, 69). Anal. Calcd for
The melting points of all new 2-indolinone-based thiazole de-
rivatives were recorded using a SMP3 melting point apparatus (the
diameter of the glass capillaries is 0.5 mm). The 1430-Perkin-Elmer
infrared-spectrophotometer was utilized to record the IR-spectra
for all new 2-indolinone-based thiazole derivatives in the range
of wavenumber from 4000 cmꢀ1 to 200 cmꢀ1 as through the for-
mation of sample-KBr discs. The well-known Bruker Avance-300
instrument was utilized to record the H NMR-spectra for all new
2-indolinone-based thiazole derivatives at 300 MHz DMSO‑d6 so-
C25H19ClN6OS (486.98): C, 61.66; H, 3.93; N, 17.26. Found: C, 61.86;
H, 3.72; N, 17.45%.
4.3.2. 1-Benzyl-5-chloro-3-((5-((4-methoxyphenyl)diazenyl)-4-
methylthiazol-2(3H)-ylidene)hydrazono)indolin-2-one (10b)
Dark red solid, 84% yield, m.p. 198e200 ꢁC (EtOH/dioxane); IR
(KBr): v 3410 (NH), 1689 (C]O), 1587 (C]N), 1527, 1465, 1435,1357,
1311, 1249, 1149, 1103, 1033 cmꢀ1; 1H NMR (DMSO‑d6):
d 2.62 (s, 3H,
CH3), 3.82 (s, 3H, OCH3), 4.99 (s, 2H, CH2), 6.94e8.33 (m, 12H,
AreH), 13.19 (s, 1H, NH), MS m/z (%): 519 (Mþþ2, 28), 518 (Mþþ1,
11), 517 (Mþ, 85). Anal. Calcd for C26H21ClN6O2S (517.00): C, 60.40;
H, 4.09; N, 16.26. Found: C, 60.66; H, 4.18; N, 16.35%
lutions. Ppm and Hz characterize the chemical shifts (
coupling constants, respectively. To record the molecular weight of
all new 2-indolinone-based thiazole derivatives we used
d) and
a
Finnigan-MAT8222 spectrometer at 70 eV in Micro-analytical
center at Cairo University. Also, measuring of CHN elemental ana-
lyses were investigated on Elementar vario-LIII CeHeNeS analyzer.
The starting compounds 2,3 and hydrazonoyl chlorides 7 and 11
were prepared as illustrated in the literature reports [34,41]. The
purity of the newly synthesized compounds have been checked by
the TLC and further purification of the impure products have been
achieved by recrystallization using a proper solvent. The degree of
the purity have been tested through the TLC test again and can be
estimated from the results of the spectroscopic data. All the syn-
thesized compounds are characterized by high purity before using
4.3.3. 1-Benzyl-5-chloro-3-((4-methyl-5-(p-tolyldiazenyl)thiazol-
2(3H)-ylidene)-hydrazono) -indolin-2-one (10c)
Dark red solid, 79% yield, m.p. 214e216 ꢁC (Dioxane); IR (KBr): v
3417 (NH), 1681 (C]O), 1612 (C]N), 1535, 1465, 1381, 1357, 1311,
1157, 1103, 1080, 1033,1010 cmꢀ1; 1H NMR (DMSO‑d6):
d
2.37 (s, 3H,
CH3), 2.65 (s, 3H, CH3), 4.97 (s, 2H, CH2), 6.95e7.62 (m, 12H, AreH),
13.10 (s, 1H, NH), 13C NMR (DMSO‑d6):
14.2 (CH3), 18.9 (CH3), 42.1
d
(CH2), 114.2, 114.4, 115.5, 115.6, 115.9, 117.1, 118.0, 118.7, 122.8, 124.6,
129.3, 129.6, 129.8, 130.3, 133.3, 142.6, 144.8, 145.9, 163.1 (C]O). MS
m/z (%): 503 (Mþþ2, 8), 502 (Mþþ1, 3), 501 (Mþ, 30). Anal. Calcd for

12
H.K. Mahmoud et al. / European Journal of Medicinal Chemistry 208 (2020) 112752
C26H21ClN6OS (501.00): C, 62.33; H, 4.22; N, 16.77. Found: C, 62.56;
H, 4.34; N, 16.92%
4.4. Synthesis of 2-((1-benzyl-5-chloro-2-oxoindolin-3-ylidene)
hydrazono)thiazolidin-4-one (17)
4.3.4. N’-(1-benzyl-5-chloro-2-oxoindolin-3-ylidene)
carbamohydrazonic-2-ethoxy-N’-(4-nitrophenyl)-2-
Substitution and cyclization reaction of 1 mmol of each of
indolinone-thiosemicarbazone derivative 3 and ethylchloroacetate
16 was performed in 20 mL AcOH and 1 mmol of anhydrous sodium
acetate after refluxing for 4 h (monitored by TLC). The formed
precipitate (isolated after evaporation the acetic acid solvent under
vacuum) was washed with cold H2O, dried and recrystallized from
AcOH to give the titled thiazolone derivative 17 as orange solid,
with 72% yield, m.p. 240e242 ꢁC (EtOH); IR (KBr): v 3417 (NH), 1720
(C]O), 1620 (C]O), 1535 (C]N), 1465, 1381, 1342, 1242, 1180, 1111,
oxoacetohydrazonic thioanhydride (13a)
Orange solid, 76% yield, m.p. 160e162 ꢁC (EtOH); IR (KBr): v
3458 (br. NH2), 3340 (NH), 3070 (sp2 CH), 2924 (sp3 CH), 1720 (C]
O), 1681 (CO), 1589 (C]N), 1550, 1473, 1442, 1381, 1357, 1327, 1280,
1257, 1180, 1141, 1103, 1080, 1026 cmꢀ1; 1H NMR (DMSO‑d6):
d
1.33
(t, J ¼ 7 Hz, 3H, CH3), 4.38 (q, J ¼ 7 Hz, 2H, CH2), 3.57 (s, 2H, NH2),
4.98 (s, 2H, CH2), 7.01e7.73 (m, 12H, AreH), 12.42 (s, 1H, NH). 13
NMR (DMSO‑d6): 20.7 (CH3), 42.6 (CH2), 66.3 (CH2), 109.5, 122.6,
C
d
1033 cmꢀ1
CH2), 6.96e8.29 (m, 8H, AreH), 12.45 (s, 1H, NH). 13C NMR
(DMSO‑d6): 33.6 (CH2), 42.6 (CH2), 109.6, 116.6, 120.4, 120.8, 122.5,
; d 4.03 (s, 2H, CH2), 4.95 (s, 2H,
1H NMR (DMSO‑d6):
127.2, 127.6, 128.7, 128.9, 136.3, 144.2, 142.7, 142.8, 142.8, 142.9,
143.7, 143.9, 144.1, 144.5, 144.6, 144.7, 158.5 (C]O). MS m/z (%): 582
(Mþþ2, 11), 581 (Mþþ1, 9), 580 (Mþ, 42). Anal. Calcd for
d
127.4, 128.3, 129.8, 132.3, 136.1, 143.9, 146.7, 163.6 (C]O), 173.8 (C]
O). MS m/z (%): 386 (Mþþ2, 18), 385 (Mþþ1, 7), 384 (Mþ, 61). Anal.
Calcd for C18H13ClN4O2S (384.84): C, 56.18; H, 3.40; N, 14.56. Found:
C, 56.23; H, 3.31; N, 14.42%
C
26H22ClN7O5S (580.01): C, 53.84; H, 3.82; N,16.90. Found: C, 53.69;
H, 3.71; N, 16.83%.
4.3.5. N’-(1-Benzyl-5-chloro-2-oxoindolin-3-ylidene)
carbamohydrazonic-2-ethoxy-2-oxo-N’-(p-tolyl)acetohydrazonic
thioanhydride (13b)
4.5. Synthesis of 1-benzyl-5-chloro-3-((substitutedpnenylthiazol-
2(3H)-ylidene)hydrazono) -indolin-2-one (19a-c)
Orange solid, 85% yield, m.p. 230e232 ꢁC (EtOH/dioxane); IR
(KBr): v 3456 (br. NH2, NH), 1743 (C]O), 1705 (C]O), 1604 (C]N),
1535, 1489, 1465, 1381, 1350, 1288, 1180, 1095, 1056, 1026 cmꢀ1; 1H
Reaction of indolinone-thiosemicarbazone derivative 3 (0.34g,
1 mmol) with phenacyl bromide derivatives 18a-c (1 mmol) in
15 mL of dioxane were proceeded under reflux in the presence of
0.35 mL of trimethylamine for 5 h. The progress of the reactions
were monitored through the TLC test. After the reactions were
completed, the solvent was then evaporated under vacuum pres-
sure then washed with CH3OH. The colored solid produced was
filtered and recrystallized from the appropriate solvent to give
products 19a-c.
NMR (DMSO‑d6):
d
1.35 (t, J ¼ 8Hz, 3H, CH3), 2.43 (s, 3H, CH3), 3.57
(s, 2H, NH2), 4.42 (q, J ¼ 8Hz, 2H, CH2), 4.94 (s, 2H, CH2), 6.92e7.92
(m, 13H, AreH, NH), MS m/z (%): 551 (Mþþ2, 13), 550 (Mþþ1, 12),
549 (Mþ 89). Anal. Calcd for C27H25ClN6O3S (549.04): C, 59.06; H,
4.59; N, 15.31. Found: C, 59.15; H, 4.42; N, 15.52%
4.3.6. N’-(1-Benzyl-5-chloro-2-oxoindolin-3-ylidene)
carbamohydrazonic-N’-(4-bromo-phenyl)-2-ethoxy-2-
oxoacetohydrazonic thioanhydride (13c)
4.5.1. 1-Benzyl-5-chloro-3-((4-phenylthiazol-2(3H)-ylidene)
hydrazono)indolin-2-one (19a)
Orange solid, 87% yield, m.p. 220e222 ꢁC (EtOH); IR (KBr): v
3456 (br. NH2, NH), 1740 (C]O), 1705 (C]O), 1635 (C]N), 1527,
Orange solid, 92% yield, m.p. 200e202 ꢁC (EtOH); IR (KBr): v
3471 (NH), 1674 (C]O), 1612 (C]N), 1543, 1465, 1381, 1357, 1273,
1489, 1465, 1381, 1350, 1280, 1180, 1095, 1033 cmꢀ1 1H NMR
;
(DMSO‑d6):
d 1.36 (t, 3H, CH3), 3.57 (s, 2H, NH2), 4.40 (q, 2H, CH2),
1157, 1103, 1033 cmꢀ1; 1H NMR (DMSO‑d6):
d 4.95 (s, 2H, CH2), 5.04
4.96 (s, 2H, CH2), 6.95e7.93 (m, 13H, AreH, NH), MS m/z (%): 613.91
(Mþ). Anal. Calcd for C26H22BrClN6O3S (613.91): C, 50.87; H, 3.61; N,
13.69. Found: C, 50.99; H, 3.45; N, 13.82%
(s, 2H, CH2), 7.06e7.93 (m, 13H, AreH), MS m/z (%): 447 (Mþþ2, 7),
446 (Mþþ1, 3), 445 (Mþ, 26). Anal. Calcd for C24H17ClN4OS
(444.94): C, 64.79; H, 3.85; N, 12.59. Found: C, 64.57; H, 3.68; N,
12.49%
4.3.7. 3-((5-Acetyl-4-methylthiazol-2(3H)-ylidene)hydrazono)-1-
benzyl-5-chloroindolin-2-one (15a)
Orange solid, 91% yield, m.p. 176e178 ꢁC (EtOH); IR (KBr): v 3417
(NH), 1689 (C]O), 1620 (C]O), 1535 (C]N), 1495, 1381, 1350, 1311,
4.5.2. 1-Benzyl-5-chloro-3-((4-(4-chlorophenyl)thiazol-2(3H)-
ylidene)hydrazono)-indolin-2-one (19b)
Orange solid, 90% yield, m.p. 208e210 ꢁC (EtOH); v 3464 (NH),
1666 (CO), 1612 (C]N), 1550, 1465, 1380, 1357, 1250, 1165, 1103,
1273, 1203, 1157, 1103, 1080, 1033 cmꢀ1; 1H NMR (DMSO‑d6):
d
2.43
(s, 3H, CH3), 2.48 (s, 3H, CH3), 5.0 (s, 2H, CH2), 6.93e8.32 (m, 8H,
AreH), 13.20 (s, 1H, NH). 13C NMR (DMSO‑d6):
14.6 (CH3), 29.1
1033, 1010 cmꢀ1 1H NMR (DMSO‑d6):
; d 4.97 (s, 2H, CH2), 5.04 (s,
d
2H, CH2), 7.06e7.95 (m,12H, AreH), MS m/z (%): 483 (Mþþ4, 3), 481
(CH3), 42.4 (CH2), 110.3, 117.1, 120.1, 120.3, 122.1, 123.1, 126.8, 127.5,
128.5, 130.7,135.6, 136.5, 141.9, 142.7, 163.9 (C]O),188.9 (C]O). MS
m/z (%): 426 (Mþþ2, 8), 425 (Mþþ1, 5), 424 (Mþ, 26). Anal. Calcd for
(Mþþ2, 12), 480 (Mþþ1, 4), 479 (Mþ, 48). Anal. Calcd for
C24H16Cl2N4OS (479.38): C, 60.13; H, 3.36; N, 11.69 Found: C, 60.24;
H, 3.57; N, 11.46%
C
21H17ClN4O2S (424.90): C, 59.36; H, 4.03; N, 13.19. Found: C, 59.55;
H, 4.21; N, 13.32%
4.5.3. 1-Benzyl-5-chloro-3-((4-(4-nitrophenyl)thiazol-2(3H)-
ylidene)hydrazono)-indolin-2-one (19c)
4.3.8. Ethyl 2-((1-benzyl-5-chloro-2-oxoindolin-3-ylidene)
hydrazono)-4-methyl-2,3-dihydrothiazole-5-carboxylate (15b)
Orange solid, 74% yield, m.p. 198e200 ꢁC (EtOH/drops of
dioxane); IR (KBr): v 3448 (NH), 1697 (C]O), 1681 (C]O), 1643
(C]N), 1612, 1527, 1465, 1381, 1319, 1265, 1141, 1095, 1033 cmꢀ1; 1H
Orange solid, 93% yield, m.p. 260e262 ꢁC (EtOH); v 3417 (NH),
1681 (C]O), 1612 (C]N), 1558, 1504, 1465, 1385, 1342, 1273, 1157,
1103, 1080, 1033, 1002 cmꢀ1
;
1H NMR (DMSO‑d6):
d
4.93 (s, 2H,
CH2), 5.02 (s, 2H, CH2), 7.03e8.26 (m, 12H, AreH). 13C NMR
(DMSO‑d6): 42.6 (CH2), 107.1, 120.0, 121.9, 123.1, 125.6, 125.7, 127.4,
NMR (DMSO‑d6):
d
1.03 (t, J ¼ 8Hz, 3H, CH3), 2.49 (s, 3H, CH3), 4.24
d
(q, J ¼ 8Hz, 2H, CH2), 4.99 (s, 2H, CH2), 6.92e8.30 (m, 8H, AreH),
13.16 (s, 1H, NH), MS m/z (%): 456 (Mþþ2, 29), 455 (Mþþ1, 9), 454
(Mþ, 91). Anal. Calcd for C22H19ClN4O3S (454.93): C, 58.08; H, 4.21;
N, 12.32. Found: C, 58.25; H, 4.33; N, 12.46%
127.6, 128.0, 128.7, 128.9, 130.3, 132.0, 135.8, 139.0, 140.2, 142.5,
142.6, 161.1 (C]O). MS m/z (%): 492 (Mþþ2, 10), 490 (Mþ, 63). Anal.
Calcd for C24H16ClN5O3S (489.93): C, 58.84; H, 3.29; N, 14.29.
Found: C, 58.99; H, 3.35; N, 14.46%

H.K. Mahmoud et al. / European Journal of Medicinal Chemistry 208 (2020) 112752
13
10.1007/s10456-009-9160-6.
[14] R. Kerbel, J. Folkman, Clinical translation of angiogenesis inhibitors, Nat. Rev.
Canc. 2 (2002) 727e739, https://doi.org/10.1038/nrc905.
4.6. In vitro VEGFR-2 inhibition
The inhibition of VEGFR-2 enzyme was measured using VEGFR2
Kinase Assay Kit (BIOSCIENCE). The VEGFR2 Kinase Assay Kit mea-
sures VEGFR2 kinase activity for screening applications using Ki-
nase-Glo® MAX as a detection reagent [42]. Percent inhibition was
calculated by comparing test compounds to control. The inhibitory
concentration 50 was obtained from the concentration-inhibition
response curve (n ¼ 3) comparing to sunitinib reference drug.
[15] F. Wang, J. Molina, D. Satele, J. Yin, V.S. Lim, A.A. Adjei, A phase I study of the
vascular endothelial growth factor inhibitor vatalanib in combination with
pemetrexed disodium in patients with advanced solid tumors, Invest. N.
Drugs 37 (2019) 658e665, https://doi.org/10.1007/s10637-018-0690-x.
[16] A. Leoni, A. Locatelli, R. Morigi, M. Rambaldi, 2-Indolinone a Versatile Scaffold
for Treatment of Cancer: a Patent Review (2008-2014), Expert Opinion on
Therapeutic Patents, 2015, https://doi.org/10.1517/13543776.2016.1118059.
[17] M. Krug, A. Hilgeroth, Recent advances in the development of multi-kinase
inhibitors, Mini Rev. Med. Chem.
10.2174/138955708786369591.
8 (2008) 1312e1327, https://doi.org/
[18] C.R. Prakash, S. Raja, Indolinones as promising scaffold as kinase inhibitors: a
review, Mini Rev. Med. Chem. 12 (2012) 98e119, https://doi.org/10.2174/
138955712798995039.
4.7. In vitro cytotoxicity assay
[19] L.Q. Chow, S.G. Eckhardt, Sunitinib: from rational design to clinical efficacy,
J. Clin. Oncol. 25 (2007) 884e896, https://doi.org/10.1200/JCO.2006.06.3602.
[20] T.A. Farghaly, G.S. Masaret, Z.A. Muhamm, M.F. Harrasd, Discovery of thiazole-
based-chalcones and 4-hetarylthiazoles as potent anticancer agents: synthe-
sis, docking study and anticancer activity, Bioorg. Chem. 98 (2020) 103761,
https://doi.org/10.1016/j.bioorg.2020.103761.
[21] I. Althagafi, N.M. El-Metwaly, T.A. Farghaly, Characterization of new Pt(IV)e
thiazole complexes: analytical, spectral, molecular modeling and molecular
docking studies and applications in two opposing pathways, Appl. Organomet.
Chem. (2019) 5099, https://doi.org/10.1002/aoc.5099.
MTT assay [37] was utilized to evaluate the cytotoxic effect of
test compounds using in vitro MTT-based assay kit, Sigma. In brief,
A498, CAKI-1 and RPTEC/TERT1 cells; obtained from American Type
Culture Collection, were grown in DMEM supplemented with FBS,
penicillin and streptomycin and kept under 5% CO2 at 37 ꢁC. Cells
were washed with PBS, collected via trypsinization and were plated
and incubated under 5% CO2 at 37 ꢁC overnight. Cells were treated
with different concentrations of test compounds and were incu-
bated for 48 h at 37 ꢁC. MTT reagent was added and cells were
reincubated for 4 h at 37 ꢁC under dark condition. The absorbance
was measured at 590 nm by a plate reader and cell viability was
calculated. The inhibitory concentration 50 was obtained from the
concentration-inhibition response curve (n ¼ 3), comparing to
sunitinib.
[22] A. Leoni, A. Locatelli, R. Morigi, M. Rambaldi, Novel thiazole derivatives: a
patent review (2008-2012. Part 1), Expert Opin. Ther. Pat. 24 (2) (2014)
201e216, https://doi.org/10.1517/13543776.2014.858121.
ꢀ
[23] A. Puszkiel, G. Noe, A. Bellesoeur, et al., Clinical pharmacokinetics and phar-
macodynamics of dabrafenib, Clin. Pharmacokinet. 58 (4) (2019) 451e467,
https://doi.org/10.1007/s40262-018-0703-0.
[24] G.M. Keating, Dasatinib: a review in chronic myeloid leukaemia and Phþ
acute lymphoblastic leukaemia, Drugs 77 (1) (2017) 85e96, https://doi.org/
10.1007/s40265-016-0677-x.
[25] A.M. Gouda, H.A. El-Ghamry, T.M. Bawazeer, T.A. Farghaly, A.N. Abdalla,
A. Aslam, Antitumor activity of pyrrolizines and their Cu(II) complexes:
design, synthesis and cytotoxic screening with potential apoptosis-inducing
activity, Eur. J. Med. Chem. 145 (2018) 350e359, https://doi.org/10.1016/
j.ejmech.2018.01.009.
[26] D.H. Dawood, E.M.H. Abbas, T.A. Farghaly, M.M. Ali, M.F. Ibrahim, ZnO nano-
particles catalyst in synthesis of bioactive fused pyrimidines as anti-breast
cancer agents targeting VEGFR-2, Med. Chem. 15 (3) (2019) 277e286,
https://doi.org/10.2174/1573406414666180912113226.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
[27] Z.A. Muhammad, M.A.A. Radwan, T.A. Farghaly, H.M. Gaber, M.M. Elaasser,
Synthesis and antitumor activity of novel [1,2,4,5]-tetrazepino[6,7-b]indole
derivatives: marine natural product Hyrtioreticuline C and D analogues, Mini
Rev. Med. Chem. 19 (1) (2019) 79e86, https://doi.org/10.2174/
1389557518666180724094244.
[28] A.M.R. Alsaedi, T.A. Farghaly, M.R. Shaaban, Synthesis and antimicrobial
evaluation of novel pyrazolopyrimidines incorporated with mono- and
diphenylsulfonyl groups, Molecules 24 (2019) 4009, https://doi.org/10.3390/
molecules24214009.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112752.
References
[29] S.F. Mohamed, E.M.H. Abbas, H.S. Khalaf, T.A. Farghaly, D.N. Abd El-Shafy,
Triazolopyrimidines and thiazolopyrimidines: synthesis, anti-HSV-1, cyto-
toxicity and mechanism of action, Mini Rev. Med. Chem. 18 (2018) 794e802,
https://doi.org/10.2174/1389557518666171207161542.
[1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2020, CA Canc. J. Clin. 70
(2020) 7e30, https://doi.org/10.3322/caac.21590.
[2] V.T. DeVita, E. Chu, A history of cancer chemotherapy, Canc. Res. 68 (2008)
8643e8653, https://doi.org/10.1158/0008-5472.CAN-07-6611.
[30] T.A. Farghaly, M.A. Abdallah, G.S. Masaret, Z.A. Muhammad, New and efficient
approach for synthesis of novel bioactive [1,3,4]thiadiazoles incorporated
with 1,3-thiazole moiety, Eur. J. Med. Chem. (2015) 320e333, https://doi.org/
10.1016/j.ejmech.2015.05.009.
[31] R. Roskoski Jr., Classification of small molecule protein kinase inhibitors based
upon the structures of their drug-enzyme complexes, Pharmacol. Res. 103
(2016) 26e48, https://doi.org/10.1016/j.phrs.2015.10.021.
[32] W.M. Eldehna, S.M. Abou-Seri, A.M. El Kerdawy, R.R. Ayyad, A.M. Hamdy,
H.A. Ghabbour, M.M. Ali, D.A. Abou El Ella, Increasing the binding affinity of
VEGFR-2 inhibitors by extending their hydrophobic interaction with the
active site: design, synthesis and biological evaluation of 1-substituted-4-(4-
methoxybenzyl)phthalazine derivatives, Eur. J. Med. Chem. 113 (2016)
50e62, https://doi.org/10.1016/j.ejmech.2016.02.029.
[33] W.M. Eldehna, A.M. El Kerdawy, G.H. Al-Ansary, S.T. Al-Rashood, M.M. Ali,
A.E. Mahmoud, Type IIA - type IIB protein tyrosine kinase inhibitors hybrid-
ization as an efficient approach for potent multikinase inhibitor development:
design, synthesis, anti-proliferative activity, multikinase inhibitory activity
and molecular modeling of novel indolinone-based ureides and amides, Eur. J.
Med. Chem. 163 (2019) 37e53, https://doi.org/10.1016/j.ejmech.2018.11.061.
[34] S.S. Kari, V.S. Bahaduria, V. Rana, S. Kumar, P.G. Subbaro, U. Das, J. Balzarini,
E. De Clercq, J.R. Dimmock, 1-Arylmethyl-2,3-dioxo-2,3-dihydroindole thio-
semicarbazones as leads for developing cytotoxins and anticonvulsants,
J. Enzym. Inhib. Med. Chem. 24 (2) (2009) 537e544.
[3] V.V. Padma, An overview of targeted cancer therapy, Biomedicine 5 (2015)
1e6, https://doi.org/10.7603/s40681-015-0019-4.
[4] F. Broekman, E. Giovannetti, G.J. Peters, Tyrosine kinase inhibitors: multi-
targeted or single-targeted? World J. Clin. Oncol. 2 (2011) 80e93, https://
doi.org/10.5306/wjco.v2.i2.80.
[5] K. Parang, G. Sun, Protein kinase inhibitors drug discovery, in: S.C. Gad (Ed.),
Drug Discovery Handbook, John Wiley and Sons, Inc., 2005, pp. 1191e1257,
https://doi.org/10.1002/9780470571224.pse027.
[6] M. Pearson, C. García-Echeverría, D. Fabbro, Protein tyrosine kinases as targets
for cancer and other indications, in: D. Fabbro, F. Mc Cormick (Eds.), Protein
Tyrosine Kinases - from Inhibitors to Useful Drugs, Humana Press Inc., Totowa,
New Jersey, 2006, pp. 1e29, https://doi.org/10.1385/1-59259-962-1:001.
[7] P. Carmeliet, VEGF as a key mediator of angiogenesis in cancer, Oncology 69
(2005) 4e10, https://doi.org/10.1159/000088478.
[8] D.J. Hicklin, L.M. Ellis, Role of the vascular endothelial growth factor pathway
in tumor growth and angiogenesis, J. Clin. Oncol. 23 (2005) 1011e1027,
https://doi.org/10.1200/JCO.2005.06.081.
[9] Z.K. Otrock, J.A. Makarem, A.I. Shamseddine, Vascular endothelial growth
factor family of ligands and receptors: review, Blood Cell Mol. Dis. 38 (2007)
258e268, https://doi.org/10.1016/j.bcmd.2006.12.003.
[10] P. Carmeliet, R.K. Jain, Angiogenesis in cancer and other disease, Nature 407
(2000) 249e257, https://doi.org/10.1038/35025220.
[11] L.E. Benjamin, G. Bergers, Tumorigenesis and the angiogenic switch, Nat. Rev.
Canc. 3 (2003) 401e410, https://doi.org/10.1038/nrc1093.
[35] R. Roskoski Jr., Properties of FDA-approved small molecule protein kinase
inhibitors: a 2020 update, Pharmacol. Res. 152 (2020) 104609, https://doi.org/
10.1016/j.phrs.2019.104609.
[36] R. Roskoski Jr., Vascular endothelial growth factor (VEGF) signaling in tumor
progression, Crit. Rev. Oncol. Hematol. 62 (2007) 179e213, https://doi.org/
[12] R.S. Kerbel, Tumor angiogenesis: past, present and the near future, Carcino-
genesis 21 (2000) 505e515, https://doi.org/10.1093/carcin/21.3.505.
[13] K.J. Gotink, H.M.W. Verheul, Anti-angiogenic tyrosine kinase inhibitors:.what
is their mechanism of action? Angiogenesis 13 (2010) 1e14, https://doi.org/

14
H.K. Mahmoud et al. / European Journal of Medicinal Chemistry 208 (2020) 112752
[40] M.J.R. Yunta, It is important to compute intramolecular hydrogen bonding in
10.1016/j.critrevonc.2007.01.006.
[37] T. Mosmann, Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays, J. Immunol. Methods 65
(1983) 55e63, https://doi.org/10.1016/0022-1759(83)90303-4.
[38] K.K. Brodaczewska, C. Szczylik, M. Fiedorowicz, C. Porta, A.M. Czarnecka,
Choosing the right cell line for renal cell cancer research, Mol. Canc. 15 (1)
(2016) 83, https://doi.org/10.1186/s12943-016-0565-8.
[39] M. Mctigue, B.W. Murray, J.H. Chen, Y. Deng, J. Solowiej, R.S. Kania, Molecular
conformations, interactions, and properties associated with drug efficiency
and clinical performance among Vegfr tk inhibitors, Proc. Natl. Acad. Sci. U. S.
A. 109 (2012) 18281, https://doi.org/10.1073/pnas.1207759109.
drug design? Am. J. Model. Optim.
10.12691/ajmo-5-1-3.
[41] A.S. Shawali, H.A. Albar, Kinetics and mechanism of dehydrochlorination of N-
aryl-C-ethoxycarbonylformohydrazidoylchlorides, Can. J. Chem. 64 (1986)
871e875, https://doi.org/10.1139/v86-144.
[42] K. Sharma, P.S. Suresh, R. Mullangi, N.R. Srinivas, Quantitation of VEGFR2
(vascular endothelial growth factor receptor) inhibitors-review of assay
methodologies and perspectives, Biomed. Chromatogr. 29 (6) (2015)
803e834, https://doi.org/10.1002/bmc.3370.
5 (1) (2017) 24e57, https://doi.org/