Design, synthesis, and cytotoxicity screening of new synthesized imidazolidine-2-thiones as VEGFR-2 enzyme inhibitors

Article abstract of DOI:10.1002/ardp.202000121

A series of imidazolin-2-thione derivatives was synthesized and structurally confirmed through the use of different spectroscopic techniques such as infrared, nuclear magnetic resonance, and mass spectrometry along with elemental analyses. The breast canc

Full text of DOI:10.1002/ardp.202000121

Received: 19 April 2020
Revised: 6 June 2020
Accepted: 6 July 2020
|
|
DOI: 10.1002/ardp.202000121
F U L L P A P E R
Design, synthesis, and cytotoxicity screening of new
synthesized imidazolidine‐2‐thiones as VEGFR‐2
enzyme inhibitors
1
2
2
Islam Zaki
| Heba M. M. Ramadan | El‐Sherbiny H. El‐Sayed
|
2
Mohamed Abd El‐Moneim
1
Pharmaceutical Organic Chemistry
Department, Faculty of Pharmacy, Port Said
University, Port Said, Egypt
Abstract
A series of imidazolin‐2‐thione derivatives was synthesized and structurally confirmed
through the use of different spectroscopic techniques such as infrared, nuclear mag-
netic resonance, and mass spectrometry along with elemental analyses. The breast
cancer cell line MCF‐7 was utilized in the evaluation of the cytotoxic activity of the
prepared molecules. The tested molecules 3 and 7 exhibited the best results on MCF‐7
cells, with mean IC50 values of 3.26 and 4.31 µM, respectively. The results of the
VEGFR‐2 assay indicated that compounds 3 and 7 displayed a good inhibition of the
VEGFR‐2 kinase enzyme. Additionally, DNA flow cytometry of compounds 3 and 7
showed cell cycle arrest at the G0/G1 phase, cell apoptosis, and marked DNA frag-
mentation in MCF‐7 cells. Finally, compounds 3 and 7 were proved to upregulate the
activation of effector caspase‐3/7, as presented by the caspase‐3/7 green flow
cytometry assay.
2
Chemistry Department, Faculty of Science,
Port Said University, Port Said, Egypt
Correspondence
Islam Zaki, Pharmaceutical Organic Chemistry
Department, Faculty of Pharmacy, Port Said
University, Port Said 42526, Egypt.
Email: Eslam.Zaki@pharm.psu.edu.eg
K E Y W O R D S
annexin‐V, caspase‐3/7, cell cycle analysis, imidazolidine, VEGFR‐2
1
| INTRODUCTION
receptor tyrosine kinases (RTK) and promotes proliferation and migra-
[11,12]
tion of lymphatic vessels.
Several literature reports indicate that
In recent years, target‐based chemotherapy has become an important
class of anticancer agents. The targeted anticancer molecules interfere
with specific cellular targets such as angiogenesis, tubulin, and DNA
the VEGFR‐2 tyrosine kinase overexpression is remarkable in the
angiogenesis of many types of human cancers including breast
[13]
carcinoma.
Thus, blockade of the VEGF signaling pathway through
[
1–4]
present in cancer cells and normal cells.
Angiogenesis, the formation
the inhibition of the RTK activity with small molecules is found to be an
attractive strategy for angiogenesis inhibition, and hence anticancer
drug development.
of new blood vessels from the existing ones, is a fundamental process in
[5,6]
the pathogenesis of various disorders including cancer.
Tumor an-
giogenesis is a prerequisite for tumor metastasis, and it is a crucial
Sorafenib (SOR; Figure 1) is a pan protein kinase (PK) inhibitor,
approved by the Food and Drug Administration for treating hepato-
cellular carcinoma. Similarly, axitinib and semaxanib (Figure 1) are
[7]
factor in cancer growth and progression. Accordingly, selective in-
hibition of tumor angiogenesis is considered one of the most effective
targets in treating cancer. The process of tumor angiogenesis is regu-
lated by a number of signaling proteins produced by human and tumor
cells, including vascular endothelial growth factor (VEGF), platelet‐
[14,15]
multikinase inhibitors.
Despite having different chemical struc-
tures, the mentioned VEGFR‐2 inhibiting drugs share similar binding
sites, as the molecular skeleton of the drugs is structurally hybrid from
head and tail parts. The head has been developed to act as a com-
petitive inhibitor with adenosine triphosphate and is composed of an
orthogonal aryl ring system. Meanwhile, the tail is formed from a
[8,9]
derived growth factor, and fibroblast growth factor.
Among these
proteins, VEGF is the pivotal regulator of physiological and pathological
[10]
angiogenesis. The binding of VEGF to its receptor, VEGFR‐2, triggers
Arch Pharm. 2020;e2000121.
wileyonlinelibrary.com/journal/ardp © 2020 Deutsche Pharmazeutische Gesellschaft
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FIGURE 1 Examples of antiangiogenic
compounds
Tail part
Head part
Tail part
Linker
Head part
Linker
Sorafenib (SOR), 1
Axitinib, 2
Semaxanib, 3
linker attached to hydrophobic aryl groups to increase binding affinity
of designing potent anticancer agents. Three structural models were
designed and synthesized. The model has the following structural out-
line: Starting with 3‐(cinnamylidene)aminoimidazolin‐2‐thione (2) as a
lead molecule, the first structural model involves N‐1 extension with
either a large hydrophobic group, ethyl 3‐(4‐aryl)‐2‐cyano‐acrylate, to
give compounds 3–5 or with a small group, acetyl group, to afford
[3,15]
for enzyme sites.
Furthermore, literature reports indicate that
thiazole derivative 4 (Figure 1) exhibited promising PK inhibition
[16]
activities as SOR.
From the above‐mentioned findings, herein, we reported the
synthesis of a new series of imidazolin‐2‐thione derivatives with the aim
R
FIGURE 2 The design strategy of the imidazolidin‐2‐thiones‐based compounds 2–8

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compound 6. Additionally, the second structural model is obtained by
C‐5 extension of the lead compound with 4‐methoxybenzylidene moiety
to obtain compound 7. Furthermore, the third structural model includes
core extension of compound 3 into imidazo[5,1‐b]‐1,3,4‐oxadiazole to
afford compound 8 (Figure 2).
(3‐phenylallylidene)amino‐2‐thioxo‐2,3‐dihydro‐1H‐imidazol‐1‐yl]‐3‐(4‐
[19]
methoxyphenyl)acrylate (5), respectively
(Scheme 2).
Moreover, compound 2 on reaction with acetic anhydride under
reflux afforded 1‐[4‐hydroxy‐3‐(3‐phenylallylidene)amino‐2‐thioxo‐
2,3‐dihydro‐1H‐imidazol‐1‐yl]ethanone (6), as shown in Scheme 3.
The Fourier transform infrared (FT‐IR) spectrum of compound 6 in-
−
1
dicated the presence of a new absorption band at 1,705 cm for
1
2
2
| RESULTS AND DISCUSSION
.1 | Chemistry
C═O (acetyl group). Furthermore, the H‐NMR spectrum of com-
pound 6 indicated that the appearance of new signals at δ 2.37 ppm
3
of methyl protons for the acetyl group (–COCH ) obviously con-
firmed the formation of compound 6. The remaining proton signal of
the hydroxyl group (–OH) at C‐4 of the imidazolidine ring was
observed in the expected region at δ 11.30–11.80 ppm.
Condensation of cinnamaldehyde with thiosemicarbazide in ethanol aims
at giving cinnamaldehyde thiosemicarbazone (1) as a key starting material
for the design and synthesis of 5‐hydroxy‐1‐(3‐phenylallylidene)amino‐
The structure of compound 2 presented in a keto form was
1H‐imidazole‐2(3H)‐thione (2) via cyclocondensation of thiosemicarba-
confirmed through the condensation of compound 2 with 4‐
zone molecule (1) with ethyl chloroacetate in ethanol in the presence of
methoxybenzaldehyde in the presence of piperidine as a catalyst, lead-
[
17]
fused sodium acetate under reflux, following the reported procedure.
ing to the formation of 5‐(4‐methoxybenzylidene)‐3‐[(3‐phenylallylidene)
[17]
The structure of imidazolin‐2‐thione (2) was modified via
replacement of proton at position‐1 with different substituents.
The hydrogen proton at N‐1 in the imidazolidine ring of com-
pound 2 is utilized in designing and synthesizing 1‐N‐[substituted‐3‐
amino]‐2‐thioxoimidazolidin‐4‐one (7) (Scheme 3).
Furthermore, heating of ethyl 2‐cyano‐3‐[4‐hydroxy‐3‐
(phenylallylidene)amino‐2‐thioxo‐2,3‐dihydro‐1H‐imidazol‐1‐yl]‐3‐(4‐
hydroxyphenyl)acrylate (3) with piperidine under fusion yielded ethyl
2‐cyano‐3‐(4‐hydroxyphenyl)‐3‐(2‐styryl‐5‐thioxoimidazo[5,1‐b]
[1,3,4]oxadiazol‐6(5H)‐yl)acrylate (8) (Scheme 4).
(
3‐phenylallylidene)amino]‐4‐hydroxyimidazolidine‐2‐thione deriva-
tives 3–6, as demonstrated in Scheme 1.
Treatment of 5‐hydroxy‐1‐(3‐phenylallylidene)amino‐1H‐imidazole‐
2
(3H)‐thione (2) with ethyl 3‐(4‐hydroxyphenyl)‐2‐cyano‐acrylate in
the presence of triethylamine (Et N), as base catalyst in ethanol under
reflux, afforded the corresponding ethyl 2‐cyano‐3‐[4‐hydroxy‐
‐(phenylallylidene)amino‐2‐thioxo‐2,3‐dihydro‐1H‐imidazol‐1‐yl]‐3‐(4‐
2.2 | Biological activity
3
3
2.2.1 | Cytotoxic activity against the breast cancer
cell line MCF‐7
[18]
hydroxyphenyl)acrylate (3).
Acetylation and alkylation of compound 3 with acetic anhydride and
methyl iodide resulted in the formation of ethyl 3‐(4‐acetoxy phenyl)‐
Compounds 2–8 were screened for their in vitro cytotoxic activity
against the breast cancer cell line (MCF‐7) through the use of the 3‐
(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT)
2
‐cyano‐3‐[4‐hydroxy‐3‐(3‐phenylallylidene)amino‐2‐thioxo‐2,3‐dihydro‐
H‐imidazol‐1‐yl]acrylate (4) and ethyl 2‐cyano‐3‐[4‐hydroxy‐3‐
1
(
i)
(
ii)
SCHEME 1 The synthesis of 1,3‐
disubstituted‐4‐hydroxyl‐imidazolidine‐2‐thiones
2
and 3. Reagents and reaction conditions: (i)
ClCH COOEt, NaOAc, (ii) ethyl 3‐(4‐
hydroxyphenyl)‐2‐cyano‐acrylate, Et
2
3
N

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SCHEME 2 The synthesis of the 1,3‐
disubstituted‐4‐hydroxyl‐imidazolidine‐2‐
thiones 4 and 5. Reagents and reaction
2 3 2 3
conditions: (iii) Ac O, (iv) CH I, K CO ,
acetone
(iii)
(iv)
SCHEME 3 The synthesis of 2‐
thioxoimidazolidine derivatives 6 and 7. Reagents
and reaction conditions: (v) Ac
CH OC H CHO, piperidine
2
O, (vi) 4‐
(
v)
3
6
4
(
vi)
[
20,21]
colorimetric assay.
SOR was utilized as a reference compound in
the imidazolin‐2‐thione ring with a large hydrophobic group has proved
to be the most cytotoxic molecule in comparison with the compound
containing a small group such as acetyl group. The second structural
variation was at C4 of the imidazolin‐2‐thione ring. The results indicated
that compound 7 revealed a good cytotoxic activity, with an IC50 value
of 4.31 µM. The third structural variation involves the core extension of
the imidazolin‐2‐thione ring into thioxoimidazo[5,1‐b][1,3,4]oxadiazole
ring to afford compound 8, which revealed a relatively poor cytotoxic
the present study. The results were reported as summarized in Table 1
and Figure 3. From the results in Table 1, it is noteworthy that
compound 3 demonstrated the most potent cytotoxic activity within the
tested molecules, with an IC50 value of 3.26 µM, as compared with SOR.
Structurally, the tested molecules comprise three structural variations
as follows: the first structural variation was at N‐1 of the imidazolin‐2‐
thione ring. The data obtained revealed that N‐1 structural extension of
(vii)
SCHEME 4 The synthesis of the fused
imidazolidine derivative 8. Reagents and
reaction conditions: (vii) piperidine, reflux

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TABLE 1 The in vitro cytotoxic activity of the synthesized
derivatives 2–8 against MCF‐7 and MCF‐10a cells
inhibition. Compound 3 exhibited a percentage inhibition of 81.23%,
whereas compound 7 displayed 63.42% VEGFR‐2 inhibition activity,
as presented in Figure 4a. Furthermore, the IC50 values for com-
pounds 3 and 7 against VEGFR‐2 enzyme inhibitory activity were
also investigated with five different concentrations. As summarized
in Figure 4b, compounds 3 and 7 presented IC50 values of 63.16 and
a
IC50 (µM)
Comp no.
MCF‐7
MCF‐10a
2
18.72 ± 0.54***
3.26 ± 0.12***
19.56 ± 0.47***
>50
NT
3
34.09 ± 0.17***
114.04 nM, respectively, in comparison with SOR, which presented a
4
NT
value of 36.22 nM. These results were in a correlation with those of
in vitro study on MCF‐7 cells, which might indicate the VEGFR‐2
kinase enzyme inhibitory activity.
5
NT
6
8.04 ± 0.32***
4.31 ± 0.09***
42.34 ± 0.81**
1.21 ± 0.09***
NT
7
76.17 ± 0.15***
NT
8
2.2.3 | Immunofluorescence staining of
VEGFR‐2/KDR
Sorafenib
83.72 ± 0.11***
Note: One‐way analysis of variance was applied to determine the
significance of the difference between tested compounds and untreated
The effect of compounds 3 and 7 on the VEGFR‐2/KDR expression in
MCF‐7 human breast cancer cells as compared with SOR was visualized
after treatment with selected candidates at their IC50 concentrations
for 24 hr, and it was then submitted to immunofluorescence labeling
and analysis under confocal microscope. VEGFR‐2 antibody was utilized
for visualizing the protein expression. The results in Figure 5 demon-
strate that in untreated control cells, VEGFR‐2 was observed, and after
treatment with selected molecules, there was notable demolishment of
cytoplasmic VEGFR‐2 staining. The results in this experiment confirmed
that VEGFR‐2 is inhibited by compounds 3 and 7 in MCF‐7 cells, which
is in line with the previously discussed data.
control (n = 3), followed by Tukey's multiple comparison test at p < .05.
,
*
* *** Indicate one way ANOVA for analysis of difference between tested
molecule and no treated control.
Abbreviation: NT, not tested.
a
IC50 values are the mean of three separate experiments.
activity, with an IC50 value of 27.34 µM. In addition, the cytotoxic effect
of compounds 3 and 7 was tested in vitro against the normal breast cell
line MCF‐10a. In the study, both the tested molecules exhibited a safe
mode of the cytotoxic effect, with IC50 values of 34.09 and 76.17 µM,
respectively. In conclusion, the most cytotoxic compounds are 3 and 7,
with IC50 values of 3.26 and 4.31 µM, respectively, against MCF‐7 cells
in comparison with a value of 1.21 µM for SOR.
2
.2.4 | The cell cycle analysis
2
.2.2 | VEGFR‐2 inhibitory activity
DNA flow cytometry assay
DNA‐flow cytometry assay after staining of the nuclei with propi-
dium iodide (PI) was carried out to investigate the effect of com-
pounds 3 and 7 on the cell cycle progression of MCF‐7 cells in
To investigate whether cytotoxicity is related to the VEGFR‐2 en-
zyme inhibition activity, compounds 3 and 7 were assessed for their
VEGFR‐2 inhibitory activity at their IC50 concentration dose value
with SOR as a positive control. The results were reported as sum-
marized in Figure 4. The results revealed a good inhibitory activity
against VEGFR‐2 enzyme, as concluded from their percentage
[22]
comparison with SOR as a reference drug.
As presented in
Figure 6, the obtained data demonstrated that the tested molecules 3
and 7 increased cell percentage at G1 phase by 1.5‐ and 1‐fold, re-
spectively, compared with the untreated control, which indicates cell
(a)
(b)
FIGURE 3 The cytotoxic activity of the synthesized compounds 2–8 and sorafenib (SOR) against the MCF‐7 breast cancer cell line

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(a)
(b)
FIGURE 4 (a) A graphical representation for % inhibition of vascular endothelial growth factor receptor 2 (VEGFR‐2) enzyme for compounds
, 7, and sorafenib (SOR) at their IC50 concentrations (µM). (b) A graphical representation for dose–response curve for the tested compounds, 3
and 7, and SOR at five different concentrations for determination of IC50 (nM) values for the VEGFR enzyme
3
cycle arrest at G1 phase. Meanwhile, SOR induced cell cycle arrest at
G1 phase (twofold) and reduced cell population at S phase in com-
parison with the untreated control groups. The data suggested that
the tested compounds result in significant cell cycle arrest at
G1 phase, together with apoptosis‐inducing activity, marked by an
increasing percentage of apoptotic cells population at G0 phase.
the untreated control, whereas SOR showed an increase in the per-
centage of early apoptosis by 14.39‐fold. Meanwhile, the percentage
of the cell population of the late apoptosis after treatment with
compounds 3 and 7 was increased by 67.50‐ and 24.59‐fold, re-
spectively, in comparison with untreated control. At the same time,
SOR induced the late cell apoptosis by 31.18‐fold. Finally, the results
confirm that the tested molecules led to the induction of apoptotic
cell death in MCF‐7 cells, and compound 3, in particular, induced
total cell apoptosis by one‐fold, compared with SOR, as shown in
Figure 7.
Annexin‐V‐fluorescein isothiocyanate (FITC)/PI apoptosis detection
staining assay
The degree of apoptosis in MCF‐7 cells was evaluated through
staining with fluorescent annexin‐V/PI and was analyzed using the
[23]
flow cytometry assay.
The nuclear condensation and DNA frag-
mentation were increased in cells treated with compounds 3 and 7 as
compared with untreated control cells. The percentage of cells of the
early apoptotic stage after treatment with compounds 3 and 7 was
increased by 25.55‐ and 12.59‐fold, respectively, in comparison with
2.2.5 | In vitro caspase‐3/7 green flow cytometry
assay
Activation of the terminal caspase‐3/7 has the main role in tumor cell
[24]
apoptosis and death.
The level of active caspase‐3/7 was mea-
sured in MCF‐7 cells for compounds 3 and 7 through the use of green
flow cytometry assay. The results in Figure 8 displayed that treat-
ment of MCF‐7 cells with the tested molecules resulted in an
increased percentage of the level of active caspase‐3/7 by 16.66‐ and
6.75‐fold when compared to the untreated control, whereas SOR
induced caspase‐3/7 activation by 11.63‐fold. This result indicates
that compounds 3 and 7 can improve caspase‐3/7 activation and
subsequently induce apoptosis.
2
.2.6 | Molecular docking study
Compound 3 and SOR were investigated for molecular docking into the
[25]
active site of VEGFR‐2 protein (PDB ID: 4ASD)
using Molecular
FIGURE 5 Immunofluorescence detection of vascular endothelial
growth factor receptor 2 (VEGFR‐2)/KDR in MCF‐7 cells after
treatment with 3, 7, and sorafenib (SOR) as compared with untreated
control
Operating Environment (MOE), with the aim of elucidating the binding
mode of these compounds with VEGFR‐2. The results of binding
interaction with VEGFR‐2 protein are summarized in Figure 9.

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(b)
(
a)
FIGURE 6 (a) A graphical representation of the cell cycle analysis of compounds 3 and 7 at their IC50 concentrations (µM) as compared with
sorafenib (SOR). (b) The cell cycle analysis of compounds 3 and 7 at their IC50 concentrations (µM) as compared with SOR
(b)
(
a)
FIGURE 7 (a) A graphical representation of the flow cytometric analysis of annexin‐V‐fluorescein isothiocyanate (FITC)/propidium iodide
PI) induced by compounds 3 and 7 at their IC50 concentrations (µM) as compared with sorafenib (SOR). (b) The flow cytometric analysis of
annexin‐V‐FITC/PI induced by compounds 3 and 7 at their IC50 concentrations (µM) as compared with SOR
(

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(b)
(
a)
µM
µM
µM
FIGURE 8 (a) A graphical representation of the effects of compounds 3 and 7, as compared with sorafenib (SOR), on the activation of
caspase‐3/7 using the green flow cytometry analysis. (b) The green flow cytometry analysis of active caspase‐3/7 induced by compounds 3 and 7
at their IC50 concentrations (µM) as compared with SOR
The docking results of compound 3 and SOR molecules reveal that they
exhibited energy scores of −13.05 and −13.63 kcal/mol, respectively.
After inspecting the docking results, it is observed that compound 3
interacted with different amino acids as follows: The OH group at the
imidazoline ring and the linker N were engaged with two hydrogen
bonds with Lys 868. The phenolic OH was able to form two hydrogen
bonds with Asp 814 and Arg 1027. As H‐bond acceptor, compound 3
interacted with its CN group with the amino acid His 1026.
FIGURE 9 Two‐dimensional interaction of compound 3 and ligand (sorafenib [SOR]) with the amino acids of the active site of vascular
endothelial growth factor receptor 2 (VEGFR‐2)

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3
| CONCLUSION
(m, 2H, arom. CH and NH), 7.54 (d, J = 7.4 Hz, 1H, arom. CH), 7.62
d, J = 8.1 Hz, 1H, arom. CH), 7.90 (d, J = 9.1 Hz, 1H, arom. CH),
8.11–8.30 (m, 1H, olefinic H), and 11.43 (s, 1H, OH) ppm. MS: m/z
(
In summary, three structural models of imidazolin‐2‐thione deriva-
tives were synthesized. Compounds 3 and 7 demonstrated the most
potent antitumor activity, with mean IC50 values of 3.26 and 4.31 µM,
respectively, against the breast cancer cell line MCF‐7. The results of
the VEGFR‐2 enzyme assay demonstrated that compounds 3 and 7
displayed a good inhibition of the VEGFR‐2 kinase enzyme. The DNA
flow cytometry analysis of compounds 3 and 7 showed that MCF‐7
cells exhibited cell cycle arrest at G0/G1 phase of the cell cycle
profile with apoptosis‐inducing activity, as indicated by an increased
percentage of cell population at G0 phase. Finally, compounds 3 and
+
+
(%) = 246 (M +1, 8.90) and 245 (M , 39.30). Anal. calcd. for
OS (245): C, 58.76; H, 4.52; N, 17.13. Found: C, 58.58; H,
12 11 3
C H N
4.27; N, 17.27.
Ethyl 2‐cyano‐3‐[4‐hydroxy‐3‐(phenylallylidene)amino‐2‐thioxo‐2,3‐
dihydro‐1H‐imidazol‐1‐yl]‐3‐(4‐hydroxyphenyl)acrylate (3)
A mixture of imidazolin‐2‐thione molecule 2 (0.01 mol) and ethyl
β‐(4‐hydroxyphenyl)‐α‐(cyano)acrylate (0.01 mol) in 30 ml absolute
3
ethanol in the presence of Et N (2 ml) was refluxed for 6 hr. The
7
showed an increased level of effector caspase‐3/7, as presented by
reaction mixture was then cooled, poured into a beaker containing
50 ml cold water, and then neutralized with few drops of dilute
hydrochloric acid (2%). The obtained product was crystallized from
absolute ethanol to give compound 3. Green crystals; yield 76%, m.p.
110–112°C. IR (KBr) ʋmax: 3,414–3,158 (br. OH), 2,231 (CN), 1,729
the caspase‐3/7 green flow cytometry assay. Accordingly, this novel
class of imidazolidine derivatives 3 and 7 emerged as a valuable lead
series that might be useful as antitumor agents, and hence promising
candidates for further in vivo evaluation.
(C═O), 1,712 (C═O), 1,646 (C═N), 1,589 (C═C), 1,250, 1,175, and
−
1
1
1
,086 (C–O) cm
.
H‐NMR (400 MHz, DMSO‐d
CH ), 3.88 (s, 1H, H‐5 of imidazolidine), 4.28
q, J = 7.1 Hz, 2H, OCH CH ), 6.87 (dd, J = 16.1, 9.2 Hz, 1H,
6
)
δ: 1.28
4
4
| EXPERIMENTAL
.1 | Chemistry
(t, J = 7.1 Hz, 3H, OCH
2
3
(
2
3
olefinic H), 6.95 (d, J = 8.7 Hz, 2H, arom. CH), 7.02 (d, J = 16.1 Hz, 1H,
olefinic H), 7.29–7.43 (m, 1H, arom. CH), 7.55–7.62 (m, 2H, arom.
CH), 7.63 (d, J = 7.1 Hz, 2H, arom. CH), 7.99 (d, J = 8.7 Hz, 2H, arom.
4
.1.1 | General
CH), 8.21–8.31 (m, 2H, olefinic H and OH), and 11.41 (s, 1H, OH)
13
1
13
H‐NMR (400 MHz) and
C‐NMR (101 MHz) spectra (see the
ppm. C‐NMR (101 MHz, DMSO‐d
6
) δ: 14.13 (OCH
2 3
CH ), 62.04
Supporting Information) were measured using a Bruker 400 DRX‐
Avance NMR spectrometer. The IR data were produced with a
Shimadzu 470 spectrometer. Mass spectra were obtained by using an
electron ionization (EI) mass spectrometer with a probe Agilent MSD
2 3
(OCH CH ), 79.23 (C2 ethyl acrylate), 97.09 (C5 imidazolidine),
116.45 (C3,5‐hydroxy phenyl), 116.55 (CN), 122.58 (C2 allylidene),
125.12 (C1 hydroxy phenyl), 126.98 (C2,6 phenyl), 127.42 (C4 phe-
nyl), 128.91 (C3,5 phenyl), 134.06 (C2,6 hydroxy phenyl), 135.93 (C3
allylidene), 138.92 (C1 phenyl), 141.36 (C4 imidazolidine), 144.76 (C1
allylidene), 154.77 (C3 ethyl acrylate), 162.68 (C4 hydroxy phenyl),
162.96 (C1 ethyl acrylate), and 177.69 (C═S) ppm. MS: m/z (%) = 461
5975 instrument at 70 eV. Melting points of crystalline molecules
were determined with an electrothermal melting point apparatus and
were uncorrected. The elemental analysis was performed on a
Perkin‐Elmer 2400 series II‐CHN elemental analyzer.
+
+
20 4 4
(M +1, 2.30) and 460 (M , 1.10). Anal. calcd. for C24H N O S (460):
The InChI codes of the investigated compounds, together with
C, 62.60; H, 4.38; N, 12.17. Found: C, 62.79; H, 4.23; N, 11.98.
some biological activity data, are provided as Supporting Information.
Ethyl 3‐(4‐acetoxyphenyl)‐2‐cyano‐3‐[4‐hydroxy‐3‐(3‐
phenylallylidene)amino‐2‐thioxo‐2,3‐dihydro‐1H‐imidazol‐1‐yl]
acrylate (4)
4
.1.2 | Synthesis
A solution of ester 3 (0.01 mol) in 20 ml of acetic anhydride was
refluxed for 2 hr. The reaction mixture was then cooled, poured into
ice‐cold water, and stirred. The reaction mixture was left for 24 hr.
The obtained product was filtered off and crystallized from absolute
ethanol to give compound 4. Yellow crystals; yield 63%, m.p.
120–122°C. IR (KBr) ʋmax = 3,212 (OH), 2,223 (CN), 1,757 (C═O),
1,634 (C═N), 1,608, 1,598 (C═C), 1,201, 1,186, and 1,013
5
‐Hydroxy‐1‐(3‐phenylallylidene)amino‐1H‐imidazole‐2(3H)‐
thione (2)
A mixture of thiosemicarbazone molecule 1 (0.01 mol) and ethyl
chloroacetate (0.01 mol) in 50 ml absolute ethanol in the presence of
anhydrous sodium acetate (0.03 mol) was refluxed for 4 hr. The re-
action mixture was then cooled, poured into cold water, and stirred.
The obtained solid was purified by crystallization from absolute
ethanol to give compound 2. Pale yellow crystals; yield 79%, m.p.
−
1 1
(C–O) cm
OCH CH
.
H‐NMR (400 MHz, DMSO‐d
), 2.31 (s, 3H, OCH ), 3.86 (s, 1H, H‐5 of imidazolidine),
CH ), 6.28 (dd, J = 15.8, 6.3 Hz, 1H,
6
) δ: 1.31 (t, J = 7.0 Hz, 3H,
2
3
3
1
1
91–193°C. IR (KBr) ʋmax: 3,225 (NH), 1,732 (C═O), 1,633 (C═N).
4.32 (q, J = 7.0 Hz, 2H, OCH
2
3
−1
1
,605, 1,588 (C═C), 1,093, and 1,056 (C–O) cm
) δ: 3.88 (s, 1H, H‐5 of imi-
dazolidine), 6.86 (dd, J = 16.1, 9.2 Hz, 1H, olefinic H), 7.01
d, J = 16.1 Hz, 1H, olefinic H), 7.07–7.16 (m, 1H, arom. CH), 7.36
.
H‐NMR
olefinic H), 6.37–6.55 (m, 2H, arom. CH and olefinic H), 7.33
(t, J = 7.3 Hz, 3H, arom. CH), 7.38 (d, J = 8.4 Hz, 2H, arom. CH), 7.45
(d, J = 7.3 Hz, 2H, arom. CH), 8.12 (d, J = 8.4 Hz, 2H olefinic H and
(400 MHz, dimethyl sulfoxide [DMSO]‐d
6
1
3
(
arom. CH), and 11.73 (s, 1H, OH) ppm.
C‐NMR (101 MHz,

10 of 12
ZAKI ET AL.
|
1
3
DMSO‐d
4.93 (C2 ethyl acrylate), 102.39 (C5 imidazolidine), 115.64 (CN), 122.96
C3,5 acetoxy phenyl), 126.28 (C2 allylidene), 126.77 (C2,6 phenyl),
28.18 (C4 phenyl), 128.70 (C3,5 phenyl), 129.00 (C1 acetoxy phenyl),
29.63 (C3 allylidene), 132.46 (C2,6 acetoxy phenyl), 135.45 (C1 phenyl),
46.32 (C4 imidazolidine), 154.01 (C1 allylidene), 154.14 (C4 acetoxy
6
) δ: 14.03 (–OCH
2
CH
3
), 20.93 (CH
3
CO–), 62.44 (–OCH
2
CH
3
),
6 3
(s, 1H, OH) ppm. C‐NMR (101 MHz, DMSO‐d ) δ: 21.95 (COCH ),
6
126.30 (C5 imidazolidine), 126.78 (C2,6 phenyl), 127.05 (C2 allyli-
dene), 128.21 (C4 phenyl), 128.73 (C3,5 phenyl), 128.92 (C3 allyli-
dene), 129.68 (C1 phenyl), 135.48 (C4 imidazolidine), 146.38 (C1
allylidene), 167.49 (C═O), and 169.44 (C═S) ppm. MS: m/z (%) = 289
(
1
1
+
+
1
13 3 2
(M +2, 63.30) and 287 (M , 6.60). Anal. calcd. for C14H N O S
phenyl), 161.82 (C3 ethyl acrylate), 167.42 (C1 ethyl acrylate), 168.84
(287): C, 58.52; H, 4.56; N, 14.62. Found: C, 58.22; H, 4.32; N, 14.81.
+
(
C═O of CH
unstable). Anal. calcd. for C26
Found: C, 61.95; H, 4.58; N, 11.32.
3
CO–), and 169.39 (C═S) ppm. MS: m/z (%) = 502 (M ,
H
22
N
4
O
5
S (502): C, 62.14; H, 4.41; N, 11.15.
5‐(4‐Methoxybenzylidene)‐3‐[(3‐phenylallylidene)amino]‐2‐
thioxoimidazolidin‐4‐one (7)
Imidazolin‐2‐thione 2 (0.01 mol), 4‐methoxybenzaldehyde (0.01 mol),
and piperidine (1 ml) were fused on a hot plate for 2–3 min, and then
ethanol (30 ml) was added. The reaction mixture was refluxed for
2 hr, then cooled, poured into water, and neutralized by few drops of
dilute hydrochloric acid (2%). The obtained product was filtered off,
washed with water, dried, and crystallized from benzene to give
compound 7. Orange crystals; yield 71%, m.p. 205–207°C. IR (KBr)
Ethyl 2‐cyano‐3‐[4‐hydroxy‐3‐(3‐phenylallylidene)amino‐2‐thioxo‐
,3‐dihydro‐1H‐imidazol‐1‐yl]‐3‐(4‐methoxyphenyl)acrylate (5)
2
A mixture of compound 3 (0.01 mol) and methyl iodide (0.01 mol) in
dry acetone (40 ml) in the presence of anhydrous potassium carbo-
nate (0.03 mol) was refluxed for 24 hr. Excess acetone was evapo-
rated under reduced pressure and cold water was added, which was
then stirred. The solid obtained was filtered off and crystallized from
absolute ethanol to afford compound 5. Yellow crystals; yield 71%,
m.p. 140–142°C. IR (KBr) ʋmax = 3,414 (OH), 2,215 (CN), 1,733
ʋmax: 3,215 (NH), 1,705 (C═O), 1,633 (C═N), 1,615, 1,605, 1,585
−
1 1
(C═C), 1,127, and 1,056 (C–O) cm
.
6
H‐NMR (400 MHz, DMSO‐d )
3
δ: 3.89 (s, 3H, OCH ), 6.86 (dd, J = 16.1, 9.2 Hz, 1H, olefinic H), 6.96
(
(
C═O), 1,643 (C═N), 1,602, 1,588 (C═C), 1,210, 1,177, and 1,086
(d, J = 8.7 Hz, 1H, olefinic H), 7.00 (s, 1H, olefinic H), 7.09–7.16 (m,
1H, arom. CH), 7.36–7.29 (m, 3H, arom. CH), 7.56 (d, J = 7.3 Hz, 1H,
arom. CH), 7.64 (d, J = 7.2 Hz, 2H, arom. CH), 7.89 (d, J = 9.2 Hz, 1H,
olefinic H), 8.18 (d, J = 9.0 Hz, 2H, arom. CH), and 11.42 (s, 1H,
−
1 1
C–O) cm
CH
.
H‐NMR (400 MHz, DMSO‐d
), 3.88 (s, 3H, OCH ), 3.89 (s, 1H, H‐5 of imidazolidine),
CH ), 6.87 (dd, J = 16.1, 9.2 Hz, 1H,
6
) δ: 1.30 (t, J = 7.1 Hz, 3H,
OCH
2
3
3
4
.30 (d, J = 7.1 Hz, 2H, OCH
2
3
1
3
olefinic H), 7.03 (d, J = 16.2 Hz, 1H, olefinic H), 7.17 (d, J = 9.0 Hz, 2H,
arom. CH), 7.38–7.41 (m, 2H, arom. CH), 7.56 (d, J = 7.3 Hz, 2H, arom.
CH), 7.90 (d, J = 9.2 Hz, 1H, arom. CH), 8.10 (d, J = 8.9 Hz, 2H, arom.
CH), 8.18 (d, J = 9.0 Hz, 1H, olefinic H), and 11.42 (s, 1H, OH) ppm.
6 3
NH) ppm. C‐NMR (101 MHz, DMSO‐d ) δ: 55.33 (OCH ), 114.20
(olefinic), 125.11 (C5 imidazolidine), 125.31 (C2 allylidene), 126.98
(C3,5 methoxy phenyl), 127.42 (C2,6 phenyl), 128.94 (C3,5 phenyl),
129.23 (C1 methoxy phenyl), 135.78 (C4 phenyl), 135.92 (C3 allyli-
dene), 138.91 (C1 phenyl), 141.36 (C2,6 methoxy phenyl), 144.75 (C1
allylidene), 158.82 (C4 methoxy phenyl), 160.71 (C═O of imidazoli-
1
3
6 2 3 3
C‐NMR (101 MHz, DMSO‐d ) δ: 14.08 (OCH CH ), 55.82 (OCH ),
6
2.12 (OCH CH ), 81.19 (C2 ethyl acrylate), 98.57 (C5 imidazoli-
2
3
+
dine), 115.03 (C3,5 methoxy phenyl), 116.31 (CN), 123.99 (C2 ally-
lidene), 125.07 (C1 methoxy phenyl), 126.95 (C2,6 phenyl), 127.39
dine), and 177.68 (C═S) ppm. MS: m/z (%) = 364 (M +1, 4.30) and 363
+
(M , 16.90). Anal. calcd for C20
17
H N
O
3 2
S (363): C, 66.10; H, 4.71; N,
(
(
C4 phenyl), 128.87 (C3,5 phenyl), 131.85 (C3 allylidene), 133.57
C2,6 methoxy phenyl), 135.90 (C1 phenyl), 138.87 (C4 imidazoli-
11.56. Found: C, 65.97; H, 4.38; N, 11.68.
dine), 144.72 (C1 allylidene), 154.53 (C3 ethyl acrylate), 162.40 (C4
Ethyl 2‐cyano‐3‐(4‐hydroxyphenyl)‐3‐(2‐styryl‐5‐thioxoimidazo[5,1‐
b][1,3,4]oxadiazol‐6(5H)‐yl)acrylate (8)
methoxy phenyl), 163.56 (C1 ethyl acrylate), and 177.66 (C═S) ppm.
+
+
MS: m/z (%)= 475 (M +1, 1.20) and 474 (M , 0.70). Anal. calcd. for
S (474): C, 63.28; H, 4.67; N, 11.81. Found: C, 63.07; H,
.48; N, 12.04.
Compound 3 (0.01 mol) in piperidine (1 ml) was fused on a hot plate
for 10–15 min, and then ethanol (30 ml) was added. The mixture was
refluxed for 2 hr, then cooled, poured into water, and neutralized by
few drops of dilute hydrochloric acid (2%). The solid product was
25 22 4 4
C H N O
4
1
‐[4‐Hydroxy‐3‐(3‐phenylallylidene)amino‐2‐thioxo‐2,3‐dihydro‐1H‐
collected by filtration and crystallized from a mixture of
imidazol‐1‐yl]ethanone (6)
ethanol–dioxane (2:1) to afford compound 8. Yellow crystals; yield
63%, m.p. 155–157°C, IR (KBr) ʋmax: 3,147–2,782 (br. OH), 2,220
(CN), 1,741 (C═O), 1,634 (C═N), 1,615, 1,582 (C═C), 1,208, 1,069,
A solution of compound 2 (0.01 mol) in 25 ml of acetic anhydride was
heated under reflux for 2 hr, which was then cooled, poured into ice‐
cold water, and stirred. The reaction mixture was left for 24 hr, and
the solid obtained was filtered off and crystallized from absolute
ethanol to give compound 6. Colorless crystals; yield 68%, m.p.
−
1
1
and 1,087 (C–O) cm
.
H‐NMR (400 MHz, DMSO‐d
), 4.28 (q, J = 7.0 Hz, 2H, OCH
6
) δ: 1.27 (t,
J = 7.0 Hz, 3H, OCH CH
2
3
2
CH ), 6.86 (d,
3
J = 8.1 Hz, 1H, olefinic H), 6.96 (d, J = 8.5 Hz, 1H, arom. CH),
7.42–7.54 (m, 5H, arom. CH and olefinic H), 8.01 (d, J = 8.5 Hz, 2H,
arom. CH), 8.33 (d, J = 8.1 Hz, 2H, arom. CH), 8.52 (s, 1H, olefinic H),
165–167°C. IR (KBr) ʋmax: 3,453 (br. OH), 1,703 (C═O), 1,632 (C═N),
−1
1
1
,605, 1,593 (C═O), 1,105, and 1,083 (C–O) cm
) δ: 2.37 (s, 3H, COCH ), 6.60 (dd, J = 15.6,
.0 Hz, 1H, olefinic H), 6.85–6.70 (m, 2H, arom. CH and H‐5 of imi-
.
H‐NMR
1
3
(400 MHz, DMSO‐d
6
3
and 10.89 (s, 1H, OH) ppm. C‐NMR (101 MHz, DMSO‐d
6
) δ: 14.31
6
(OCH CH ), 62.21 (OCH CH ), 97.23 (C2 ethyl acrylate), 116.49
2
3
2
3
dazolidine), 7.58 (d, J = 6.7 Hz, 1H, olefinic H), 7.71–7.60 (m, 3H,
arom. CH and olefinic H), 7.76 (d, J = 6.8 Hz, 2H, arom. CH), and 12.05
(CN), 116.67 (C3,5 hydroxy phenyl), 119.48 (C5 imidazolidine),
122.75 C1 (olefinic), 127.16 (C1 hydroxy phenyl), 127.88 (C2,6

ZAKI ET AL.
11 of 12
|
phenyl), 128.94 (C4 phenyl), 129.08 (C3,5 phenyl), 129.12 (C1 ole-
finic), 130.88 (C1 phenyl), 131.41 (C2 oxadiazole), 134.24 (C2,6 hy-
droxy phenyl), 154.96 (C4 imidazolidine), 156.47 (C3 ethyl acrylate),
5 min. After washing, cells were blocked with 4% goat serum at 37°C for
30 min and incubated with polyclonal rabbit antibodies against VEGFR‐
2/KDR (1:25) at 4°C overnight. After washing, slides were soaked in
FITC‐conjugated goat immunoglobulin G antibodies (1:1,200) at room
temperature for 1 hr. After washing, the slides were incubated with
acridine orange solution (100 µg/ml in phosphate‐buffered saline [PBS])
for 10 min. After washing, cells were visualized using a fluorescence
microscope (Axiostar plus; Zeiss, Göttingen, Germany) equipped with an
156.89 (C4 hydroxy phenyl), 162.04 (C1 ethyl acrylate), and 163.17
+
+
(
C═S) ppm. MS: m/z (%) = 459 (M +1, 2.60) and 458 (M , 2.10). Anal.
calcd for C24 S (458): C, 62.87; H, 3.96; N, 12.22. Found: C,
3.04; H, 4.10; N, 12.02.
18 4 4
H N O
6
[27,28]
image analyzer and digital camera (Power Shot; Canon).
4
.2 | Biological studies
4
.2.1 | Cell viability assay
4.2.4 | The cell cycle analysis
The antitumor effect of the newly prepared compounds was determined
against MCF‐7 (ATCC Cat. No. HTB‐22) using the MTT assay method.
Compounds 3, 7, and SOR were screened for their effects on the normal
breast cell line MCF‐10a (ATCC Cat. No. CRL‐10317). Cells at a density
DNA flow cytometry assay
DNA content in the cell cycle analysis was quantified with the help of
a FACS Calibur flow cytometer at 488 nm according to the manu-
5
facturer's instructions. Briefly, 2 × 10 cells/well were treated with
4
of 1 × 10 were seeded in a 96‐well plate at 37°C for 48 hr under 5%
selected molecules 3 and 7 at their IC50 concentrations for 24 hr.
After treatment, cells were washed two times and resuspended in
PBS. After washing, 0.7 ml absolute ethanol was then added, followed
by incubation at −20°C for 20 min. After washing, 500 μl RNase was
added, followed by incubation for 30 min. PI was then added,
followed by incubation for 30 min (light was avoided).
2
CO . After incubation, the cells were treated with different con-
centrations of the prepared molecules and incubated for 24 hr. MTT dye
was added after 24 hr of drug treatment and incubated for 4 hr at 37°C.
Also, 100 µl of dimethyl sulphoxide was added to each well to dissolve
the purple formazan formed. The color intensity of the formazan pro-
duct, which represents the growth condition of the cells, was quantified
using an enzyme‐linked immunosorbent assay (ELISA) plate reader at
Annexin‐V‐FITC/PI apoptosis detection staining solution
5
70 nm. The experiments were carried out with at least three replicates
Apoptosis detection in MCF‐7 cells was assessed using BioVision®
annexin‐V‐FITC apoptosis detection kit according to the manu-
facturer's instructions and quantified by a FACS Calibur flow cyt-
and they were repeated at least three times.
5
ometer at 488 nm. Briefly, 1–5 × 10 cells were collected by
4
.2.2 | VEGFR‐2 enzyme assay
centrifugation. Cells were treated with compounds 3 and 7 at their
IC50 concentrations for 24 hr and resuspended in 500 µl of binding
buffer. Annexin‐V‐FITC and PI were added. They were then in-
cubated at room temperature for 5 min in the dark. Annexin‐V‐FITC
binding was analyzed using a specific signal detector.
The VEGFR‐2 inhibitory activity for compounds 3, 7, and SOR was
evaluated using human VEGFR‐2 ELISA kits according to the manu-
facturer's instructions. A series of tested molecules and standard con-
centrations were pipetted into the 96‐well plate, and VEGFR‐2 was
bound to the wells by the immobilized antibody. The wells were washed
and biotinylated antihuman VEGFR‐2 antibody was added. After
washing away the unbound biotinylated antibody, 100 µl of conjugated
streptavidin solution was pipetted into the wells. After washing, 100 µl
of 3,3′,5,5′‐tetramethylbenzidine substrate solution was added to the
wells for 30 min at 37°C. Stop solution (50 µl) was added, and the op-
tical density of the color was measured immediately at 450 nm. The IC50
values against VEGFR‐2 were determined by the concentration that
4.2.5 | In vitro caspase‐3/7 green flow cytometry
assay
®
The activity of caspase‐3/7 was evaluated using CellEvent Caspase‐
3/7 green flow cytometry assay kit according to the manufacturer's
instructions and quantified by a FACS Calibur flow cytometer at
488 nm. Briefly, flow cytometric tubes, each containing 1 ml of no‐
treatment control and compounds 3 and 7‐treated MCF‐7 cells
[26]
induced 50% maximal percent activity.
5
7
(
1 × 10 –1 × 10 cells/ml), were prepared. Samples were combined
with the reaction buffer and incubated with a specific detection re-
agent for 25 min at 37°C. All experiments were done in triplicates.
4
.2.3 | Immunofluorescence staining of
VEGFR‐2/KDR
5
MCF‐7 cells (1 × 10 cells/well) were cultured on multi‐well confocal
adhesive slides for 24 hr. The next day, cells were treated with com-
pounds 3, 7, and SOR at their IC50 concentrations and incubated for
4.2.6 | Molecular docking study
The X‐ray crystallographic structure of VEGFR‐2 cocrystalized with
24 hr. The cells were fixed on slides by cold methanol at −20°C for
SOR as an inhibitor was downloaded from the Protein Data Bank

12 of 12
ZAKI ET AL.
|
(PDB code: 4ASD). All molecular docking calculations and docking
[13] X. Yuan, Q. Yang, T. Liu, K. Li, Y. Liu, C. Zhu, Z. Zhang, L. Li, C. Zhang,
M. Xie, J. Lin, J. Zhang, Y. Jin, Eur. J. Med. Chem. 2019, 179, 147.
studies were carried out using MOE (2009.10). The three‐
dimensional (3D) structures of the ligand were built using MOE and
were subjected to the following procedure: (a) 3D protonation of the
structure, (b) hiding the hydrogen atoms, and (c) selecting the least
energetic conformer. The enzyme was prepared for docking studies
by the following procedure: (a) Discarding water molecules, (b) pro-
tonating 3D protocol, (c) hiding the hydrogen atoms, (d) redocking of
the cocrystallized ligand (SOR) in the vicinity of the active site of the
receptor, and (e) predicting the ligand–receptor interactions at the
active site for compound 3.
[
14] R. J. Motzer, M. D. Michaelson, B. G. Redman, G. R. Hudes, G.
Wilding, R. A. Figlin, M. S. Ginsberg, S. T. Kim, C. M. Baum, S. E.
DePrimo, J. Z. Li, C. L. Bello, C. P. Theuer, D. J. George, B. I. Rini,
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15] T. A. Barnes, G. M. O'Kane, M. D. Vincent, N. B. Leighl, Front. Oncol.
2
017, 7, 113.
[
16] E. Perspicace, V. Jouan‐Hureaux, R. Ragno, F. Ballante, S. Sartini, C.
La Motta, F. Da Settimo, B. Chen, G. Kirsch, S. Schneider, B. Faivre, S.
Hesse, Eur. J. Med. Chem. 2013, 63, 765.
[
[
[
[
[
17] A. G. Eliwi, S. Abdulla, Z. Kedaer, A. J. Kh, M. Mubarak, M. Abbas, Al‐
Mustansiriyah J. Sci. 2018, 29, 79.
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CONFLICTS OF INTEREST
19] Y. W. Rizzk, I. M. El‐Deen, F. Z. Mohammed, M. S. Abdelhamid, A. I.
Khedr, Anti‐Cancer Agents Med. Chem. 2019, 19, 2010.
20] D. K. Maurya, N. Nandakumar, T. P. A. Devasagayam, J. Clin. Biochem.
Nutr. 2010, 48, 85.
The authors declare that there are no conflicts of interest.
ORCID
21] S. Ranganathan, D. Halagowder, N. D. Sivasithambaram, PLOS One
Islam Zaki
http://orcid.org/0000-0002-2026-7373
2
015, 10, e0141370.
El‐Sherbiny H. El‐Sayed
http://orcid.org/0000-0002-3848-4739
[22] I. Zaki, M. K. Abdelhameid, I. M. El‐Deen, A. H. A. Abdel Wahab, A. M.
Ashmawy, K. O. Mohamed, Eur. J. Med. Chem. 2018, 156, 563.
[
[
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23] K. O. Mohamed, I. Zaki, I. M. El‐Deen, M. K. Abdelhameid, Bioorg.
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
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