5-aryl-1-arylideneamino-1h-imidazole-2(3h)-thiones: Synthesis and in vitro anticancer evaluation

Article abstract of DOI:10.3390/molecules26061706

A novel series of N-1 arylidene amino imidazole-2-thiones were synthesized, identified using IR,¹H-NMR, and¹³C-NMR spectral data. Cytotoxic effect of the prepared compounds was carried out utilizing three cancer cell lines; MCF-7 breast cancer, HepG2 liver cancer, and HCT-116 colon cancer cell lines. Imidazole derivative 5 was the most potent of all against three cell lines. DNA flow cytometric analysis showed that, imidazoles 4d and 5 exhibit pre-G1 apoptosis and cell cycle arrest at G2/M phase. The results of the VEGFR-2 and B-Raf kinase inhibition assay revealed that compounds 4d and 5 displayed good inhibitory activity compared with reference drug erlotinib.

Full text of DOI:10.3390/molecules26061706

molecules
Article
5-Aryl-1-Arylideneamino-1H-Imidazole-2(3H)-Thiones:
Synthesis and In Vitro Anticancer Evaluation
Ali H. Abu Almaaty 1 , Eslam E. M. Toson 2, El-Sherbiny H. El-Sayed 2, Mohamed A. M. Tantawy 3,
Eman Fayad 4 , Ola A. Abu Ali 5 and Islam Zaki 6,
*
1
Zoology Department, Faculty of Science, Port Said University, Port Said 42526, Egypt;
ali_zoology_2010@yahoo.com
2
3
4
5
Chemistry Department, Faculty of Science, Port Said University, Port Said 42526, Egypt;
eslamatwa2010@gmail.com (E.E.M.T.); saeed201691@yahoo.com (E.-S.H.E.-S.)
Medical Research Division, National Research Centre, Dokki, Giza 12511, Egypt;
mohamed_tantawy@daad-alumni.de
Biotechnology Department, Faculty of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia;
e.esmail@tu.edu.sa
Chemistry Department, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia;
O.abuali@tu.edu.sa
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Port Said University, Port Said 42526, Egypt
Correspondence: eslam.zaki@pharm.psu.edu.eg; Tel.: +20-1153436140
6
*
Abstract: A novel series of N-1 arylidene amino imidazole-2-thiones were synthesized, identified
using IR, 1H-NMR, and 13C-NMR spectral data. Cytotoxic effect of the prepared compounds was
carried out utilizing three cancer cell lines; MCF-7 breast cancer, HepG2 liver cancer, and HCT-116
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
colon cancer cell lines. Imidazole derivative
flow cytometric analysis showed that, imidazoles 4d and
5
was the most potent of all against three cell lines. DNA
exhibit pre-G1 apoptosis and cell cycle
5
Citation: Abu Almaaty, A.H.; Toson,
E.E.M.; El-Sayed, E.-S.H.; Tantawy,
M.A.M.; Fayad, E.; Abu Ali, O.A.;
Zaki, I. 5-Aryl-1-Arylideneamino-1H-
Imidazole-2(3H)-Thiones: Synthesis
and In Vitro Anticancer Evaluation.
Molecules 2021, 26, 1706. https://
doi.org/10.3390/molecules26061706
arrest at G2/M phase. The results of the VEGFR-2 and B-Raf kinase inhibition assay revealed that
compounds 4d and 5 displayed good inhibitory activity compared with reference drug erlotinib.
Keywords: imidazole; synthesis; cytotoxicity; apoptosis; cell cycle analysis; Annexin V; VEGFR-2
1. Introduction
Academic Editor: René Csuk
Cancer is the pathological uncontrolled proliferation of the abnormal cells that divide
rapidly [
forms in each body area [2,3
synthesize new biologically active molecules which might be useful in a cancer treatment.
Anticancer activity of the azole derivatives is most extensively studied and some of
1
]. Moreover, cancer can involve any tissue of the body and have many different
Received: 8 February 2021
Accepted: 15 March 2021
Published: 18 March 2021
]. Therefore, there is a constant and growing interest to
them are in clinical practice as antitumor active agents [
class of heterocyclic molecules and imidazole derivatives are reported to have anticancer
activity [ ]. Misonidazole displayed superior antiproliferative activity against a panel
of cancer cell lines [ ]. Tiazofurin II possesses good anticancer activity [10]. Lepidiline A
4,5]. Imidazoles are an important
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
6–
8
I
9
and B are imidazole compounds which exhibit cytotoxicity against various types of human
cancer cell lines at micromolar concentration [11]. Imidazole derivatives were found to
exert their anticancer activity by acting mainly as antiangiogenic agents, inhibitors of B-Raf
kinase, and as p38 MAP kinase inhibitors [12
potent antitumor activity against the MCF-7 cell line with an IC50 value of 3.26
compared with SOR (IC50 = 1.12 M) [15]. Additionally, compound IV has been reported
as a vascular endothelium growth factor (VEGFR-2) inhibitor and apoptotic inducer [15].
Moreover, it has been reported that imidazole molecule inhibits the kinase activity of Raf
–
14]. For example, compound III displayed
Copyright:
© 2021 by the authors.
µM, as
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
µ
V
in low nanomolecular concentrations [16]. Furthermore, compound VI was identified as a
submicromolar inhibitor of B-Raf kinase [17] (Figure 1).
Molecules 2021, 26, 1706. https://doi.org/10.3390/molecules26061706
https://www.mdpi.com/journal/molecules

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Based on the aforementioned aspects, the present study is concerned with the synthesis
of novel imidazole molecules bearing arylidene amino substituents at the N-1 position. It
is worthy to note that imidazole fragments are decorated at the C-5 position with phenyl
groups carrying chloro or methoxy substituents to show the impact on anticancer activity.
Finally, full details about the synthesis and evaluation of the antitumor activity in vitro are
reported herein.
Figure 1. Imidazole derivatives as anticancer agents.
2. Result and Discussion
2.1. Chemistry
2-Arylhydrazinecarbothioamides 3a
–e were prepared in high yield according to the
reported procedure [18 19] through condensation of aromatic aldehydes (namely, benzalde-
,
hyde, 2-hydroxybenzaldehyde, 5-bromo-2-hydroxybenzaldehyde, 4-methoxybenzaldehyde
4-hydroxy-3-methoxybenzaldehyde, and cinnamaldehyde) with thiosemicarbazide in re-
fluxing ethanol containing catalytic amounts of acetic acid. The structure of 3a
confirmed using different spectroscopic techniques. The 1H-NMR spectra revealed the
presence of a signal for a azomethine group (CH=N) at 7.92–8.38 ppm. In addition
to new signals attributed to NH and NH2 groups. Carbon signals observed in 13C-
NMR spectra at 140.10–145.19 and 178.01–178.10 ppm assigned to carbons of azome-
–e was
δ
δ
thine (CH=N) and thiocarbonyl (C=S) groups, respectively, confirmed formation of 2-
arylhydrazinecarbothioamides 3a–e.
Furthermore, the novel imidazole derivatives 4a
–e
and
5 were synthesized by cyclo-
condensation of 2-arylhydrazinecarbothioamides 3a
–e
and substituted phenacylbromide
in absolute ethanol in the presence of fused sodium acetate under reflux temperature
(Scheme 1). The structure of novel imidazoles 4a–e was characterized using different
spectroscopic methods. The 1H-NMR spectra revealed the presence of additional aromatic
signals attributed to phenyl ring B protons.
Compound 4b, as a representative example, was established chemically via an acety-
lation reaction with acetic anhydride, which gave 2-((3-acetyl-5-(4-chlorophenyl)-2-thioxo-
2,3-dihydro-1H-imidazol-1-ylimino)methyl)phenyl acetate (
spectrum of compound showed the absence NH signals, and OH groups with the ap-
pearance of two new singlet signals at 2.08, 2.51, and 2.54 ppm due to the protons of
two methyl function of acetyl groups. The 13C-NMR spectrum of compound
showed
the presence of four new carbon signals at 20.02, 22.95, 169.50, and 172.13 related to two
acetyl groups (CH3CO) which supported the formation of compound 6.
6
), Scheme 1. The 1H-NMR
6
δ
6
δ

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Scheme 1. Synthesis of imidazole-2-thione derivatives 4a–f, 5 and 6. Reagents and reaction condition: (i) EtOH, AcOH,
reflux; (ii) 4-chlorophenacylbromide, NaOAc, EtOH; (iii) 4-methoxyphenacylbromide, NaOAc, EtOH; (iv) Ac2O, reflux.
2.2. In Vitro Antitumor Evaluation
2.2.1. In Vitro Cytotoxic Activity against Three Cancer Cell Lines
The effect of imidazole molecules 4a–e and 5 on the viability of three cancer cell
lines were studied using the MTT assay. The cytotoxicity was assessed using Docetaxel
(DOC) as a standard antitumor drug. The three cancer cell lines were MCF-7 (breast
cancer cell line), colon carcinoma (HCT-116), and HepG2 (hepatocellular carcinoma cell
line). Treatment of MCF-7, HCT-116, and HepG2 cell lines with different concentrations
of imidazole derivatives revealed moderate to good antitumor activities as concluded
from their IC50 values shown in Table 1. Compound 1-(2-hydroxybenzylideneamino)-5-(4-
methoxyphenyl)-1H-imidazole-2(3H)-thione (
5) was the most potent cytotoxic compound
against all of the tested tumor cell lines with IC500 s < 5
µM. Moreover, the safety of

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compounds 4d and
5
was assessed against a normal breast cell line (MCF-10A) using the
MTT assay. The results revealed weak cytotoxic effect towards MCF-10A and compound
4d showed less cytotoxic effect than standard drug DOC.
Table 1. In vitro IC50 values (µM) of imidazole derivatives 4a–e and 5 over MCF-7, HepG2, HCT-116,
and MCF-10A cell lines. Data are expressed as the mean ± three experiments.
IC50 (µM)
Comp No.
MCF-7
HepG2
HCT-116
MCF-10A
4a
4b
4c
4d
4e
>50
61.81 ± 4.1
20.56 ± 1.4
23.53 ± 1.6
9.962 ± 0.7
28.95 ± 1.9
3.988 ± 0.3
3.99 ± 0.3
13.96 ± 0.9
NT
NT
NT
24.94 ± 1.6
43.95 ± 2.9
7.967 ± 0.5
13.15 ± 0.9
1.071 ± 0.1
11.09 ± 0.7
>50
11.36 ± 0.8
4.086 ± 0.3
17.01 ± 1.1
1.301 ± 0.1
8.128 ± 0.5
44.06 ± 0.23
NT
5
27.81 ± 0.31
33.17 ± 0.19
DOC
NT; not tested.
2.2.2. Cell Cycle Analysis
The most active compounds, 4d and
5 on MCF-7 were studied using DNA flow
cytometric analysis. Treatment of MCF-7 cells with IC50 concentration dose value of
compounds 4d and resulted in a significant alteration in cell cycle profile. There was a
significant increase in the percentage of cell population at the G2/M phase from (8.79%)
control to 35.55% and 25.63% upon treatment with compounds 4d and , respectively.
Additionally, a significant increase in the percentage of cells at pre-G1 phase from 1.61%
(control) to 22.36% and 22.81% upon treatment with compounds 4d and , respectively.
Therefore, it can be concluded that compounds 4d and inhibit the cell proliferation of
MCF-7 cells via cell cycle arrest at the G2/M phase and induction of apoptosis (Figure 2).
5
5
5
5
Figure 2.
) A graphical representation of the percentage of pre-G1 phase induced by compounds 4d and
IC50 (µM). (C) Cell cycle analysis of compounds 4d and 5 against MCF-7 at their IC50 (µM).
(
A
) A graphical representation of cell cycle analysis of compounds 4d and
5
against MCF-7 at their IC50
(µM).
(
B
5
against MCF-7 at their

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2.2.3. Annexin V-FITC/PI Apoptosis Induction Analysis
Several imidazole molecules have been reported in the literature as apoptosis inducers
in several tumor cell lines [20]. To evaluate the mode of cell death induced by compounds
4d and
5, Annexin V/FITC and propidium iodide (PI) assays were performed. MCF-7
cells were treated with compounds 4d and
5
at their IC50 concentration dose value for
48 h. The results showed that the selected candidates induce apoptosis of MCF-7 cells
at both early and later stages. As shown in Figure 3, the percent of early apoptosis was
increased from 0.43% (control) to 6.12% and 2.66% for compounds 4d and
5, respectively.
Additionally, the percent of late apoptosis was increased from 0.23% (control) to 10.23%
and 13.64% upon exposure to compounds 4d and
the antiproliferative activity of compounds 4d and
activity in MCF-7 cells.
5
5
, respectively. The results proved that
is attributed to its apoptosis inducing
Figure 3. (A) A graphical representation of apoptosis induction analysis of compounds 4d and 5 against MCF-7 at their IC50
(µM). (B) Apoptosis induction analysis of compounds 4d and 5 against MCF-7 at their IC50 (µM).
2.2.4. VEGFR-2 Kinase Inhibitory Activity
To investigate the possible molecular mechanism underlying the potent anticancer
activity of compounds 4d and . Their inhibitory activity against VEGFR-2 kinase was
determined using an ELISA assay. The results in (Figure 4) showed that the selected
imidazole molecules 4d and elicited potent activity against VEGFR-2 kinase with IC50
values 247.81 and 82.09 ng/mL, respectively, compared with erlotinib as the reference
5
5
drug. According to these results, imidazole molecules 4d and
5 possess good inhibition of
VEGFR-2 kinase activity.
2.2.5. B-Raf Ihibitory Activity
Raf is a protein kinase that initiates a cascade of other protein kinases by acting on
the protein kinases MEK-1 and MEK-2 [21]. Therefore, constitutive activation of this
signaling pathway is observed in variety of cancers. In the present assay, the activity
potential of compounds 4d and 5
was evaluated against B-RafV600E using an ELISA assay.

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The results showed that compounds 4d and
5
revealed good inhibitory activities against
B-RafV600E with IC50 values of 13.05 and 2.38
µg/mL, respectively, compared with the
value of 0.98 µg/mL for erlotinib (Figure 5).
Figure 4. A graphical representation of the IC50 values (ng/mL) of compounds 4d
, 5, and erlotinib
for the VEGFR-2 enzyme. Data are expressed as the mean (n = 3 experiments) SEM and statistical
±
comparisons were carried out using one-way analysis of variance (ANOVA) followed by Tukey’s
multiple comparisons test (**, p < 0.05).
Figure 5. A graphical representation of the IC50 values (
µg/mL) of compounds 4d, 5, and erlotinib
for the B-Raf enzyme. Data are expressed as mean (n = 3 experiments)
comparisons were carried out using one-way analysis of variance (ANOVA) followed by Tukey’s
±
SEM and statistical
multiple comparisons test (***, p < 0.005).
2.2.6. Molecular Docking Study
Compounds 4d and
5 were docked into the VEGFR-2 crystal structure (PDB code:
3U6J) [22]. Compound 4d interacted with the active site of 3U6J by hydrogen bonding with
the amino acid Lys 868 by its methoxy group. In addition, the hydrophobicity induced
by compound 4d resulted in a docking score of
compound interacted through hydrogen bonding with the amino acids Lys 868 and Ile
1025 by its methoxy and hydroxy groups, respectively, and its hydrophobicity resulted
in docking score of 21.58 kcal/mol (Figure 6). The interaction result is consistent with
the obtained IC50 values against the VEGFR-2 enzyme. Accordingly, from the obtained
−
20.69 kcal/mol. On the other hand,
5
−
results, compound
inhibitory activity.
5 was more potent than compound 4d and with higher VEGFR-2

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Figure 6.
representation of compound 4d docking inside the active site of 3U6J. (
compound docking inside the active site of 3U6J. ( ) 3D representation of compound 5
(
A
) 2D representation of compound 4d docking inside the active site of 3U6J. (
) 2D representation of
docking
B) 3D
C
5
D
inside the active site of 3U6J.
3. Material and Methods
3.1. General
1H and 13C-NMR spectra were measured with a Bruker 400 DRX, Avance NMR
spectrometer (Chichago, Elk Grove Village, USA) using the DMSO-d6 solvent. The IR data
were obtained with a shimadzu 470 spectrometer (Kyoto, Kyoto, Japan). Melting points of
the prepared derivatives were measured with an electro thermal melting point apparatus
and were not corrected. The microanalyses were implemented on a Perkin Elmer CHN
elemental analyzer (Haan, Germany). All chemicals were purchased from Aldrich chemical
company and all reagents were of analytical grade. The prepared imidazole molecules
were soluble in DMSO and not soluble in water at any concentration.
3.2. General Procedure for the Synthesis of 2-Arylhydrazinecarbothioamide (3a–d)
To a mixture of aromatic aldehyde (0.01 mol) and thiosemicarbazide
2 in absolute
ethanol (50 mL) and a few drops of glacial acetic acid were added. The reaction mixture was
heated under reflux for 4 h. After cooling, the separated solid was collected by filtration,
dried, and crystallized from ethanol to give 3a–d.
3.2.1. (E)-2-Benzylidenehydrazinecarbothioamide (3a)
◦
Pale yellow crystals, yield 83%. m.p. 188–190 C. IR (KBr)
ν
max: 3418, 3262, 3154 (NH),
3072 (arom.CH), 1591 (C=N), 1536, 1451 (C=C), 1292 (C=S) cm–1. H-NMR (DMSO-d6)
δ:
1
7.51–7.35 (m, 3H, arom. CH), 7.81 (dd, J = 6.4, 3.0 Hz, 2H, arom. CH), 8.03 (s, 1H, NH), 8.06
(s, 1H, CH=N), 8.24 (s, 1H, NH), 11.47 (s, 1H, NH) ppm. 13C-NMR (DMSO-d6)
δ: 125.52,
127.40, 129.32, 139.34 (C- aromatic), 145.19 (CH=N), 178.10 (C=S) ppm.

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3.2.2. (E)-2-(2-Hydroxybenzylidene)hydrazinecarbothioamide (3b)
◦
Yellow crystals, yield 81%, m.p. 210–212 C. IR (KBr)
ν
max: 3444 (OH), 3319, 3140
(NH), 3032 (arom.CH), 1613 (C=N), 1539, 1491 (C=C), 1368 (C=S), 1113 (C–O) cm−1. H-
1
1
NMR (DMSO-d6)
δ
: H-NMR (DMSO-d6)
δ
: 6.80 (t, J = 8.4 Hz, 1H, arom.CH), 6.86 (d,
J = 8.4 Hz, 1H, arom. CH), 7.20 (t, J = 8.5 Hz, 1H, arom.CH), 7.91 (s, 2H, arom.CH and NH),
8.15 (s, 1H, CH=N), 8.38 (s, 1H, NH), 9.90 (br. s, 1H, OH), 11.38 (s, 1H, NH) ppm. 13C-NMR
(DMSO-d6) δ: 116.50, 119.75, 120.80, 127.21, 131.57 (C-aromatic), 140.10 (CH=N), 156.87
(C–O), 178.10 (C=S) ppm.
3.2.3. (E)-2-(4-Methoxybenzylidene)hydrazinecarbothioamide (3c)
Yellow crystals, yield 81%, m.p. 192–194 ◦C. IR (KBr)
ν
max: 3404, 3290, 3154 (NH),
: 3.78 (s, 3H, OCH3),
1604 (C=N), 1536, 1511 (C=C), 1361 (C=S) cm−1. H-NMR (DMSO-d6)
δ
1
6.96 (d, J = 8.6 Hz, 2H, arom.CH), 7.74 (d, J = 8.6 Hz, 2H, arom.CH), 7.95 (s, 1H, NH), 8.02
(s, 1H, CH=N), 8.16, 11.37 (s, 2H, NH2) ppm. 13C-NMR (DMSO-d6)
δ
: 55.73 (OCH3), 114.60,
127.17, 129.38 (C-aromatic), 142.72 (CH=N), 161.12 (C–O), 178.01 (C=S) ppm.
3.2.4. (E)-2-((E)-3-Phenylallylidene)hydrazinecarbothioamide (3d)
◦
Pale yellow crystals, yield 79 %, m.p. 134–136 C. IR
ν
max: 3418, 3262, 3154 (NH),
3027 (arom.CH), 1591 (C=N), 1536, 1451 (C=C), 1371 (C=S) cm–1. H-NMR (DMSO-d6)
δ:
1
6.88 (dd, J = 16.1, 9.2 Hz, 1H olefinic CH), 7.03 (d, J = 16.1 Hz, 1H, olefinic CH), 7.45–7.34
(m, 3H, arom.CH), 7.57 (d, J = 7.2 Hz, 2H, arom.CH), 7.68 (s, 1H, NH), 7.92 (d, J = 9.2 Hz,
1H, CH=N), 8.22, 11.44 (s, 2H, NH2) ppm. 13C-NMR (DMSO-d6)
δ: 119.12, 125.52, 127.40,
129.32, 136.32, 139.34 (C-aromatic and olefinic), 145.19 (CH=N), 178.10 (C=S) ppm.
3.3. General Procedure for the Synthesis of Imidazole Derivatives 4a–e and 5
A suspension of thiosemicarbazone derivative 3a–e (0.01 mol) and freshly prepared
substituted phenacyl bromide (0.01 mol) in 30 mL of absolute ethanol and freshly fused
sodium acetate (0.03 mol) was refluxed for 6 h. After concentration and cooling, the
solid that formed was collected and crystallized from proper solvent to give the target
imidazole compound.
3.3.1. (E)-1-(Benzylideneamino)-5-(4-chlorophenyl)-1H-imidazole-2(3H)-thione (4a)
Pale yellow crystals, yield 78%, m.p. 121–123 ◦C crystallized from dioxane; IR (KBr)
1
ν
max: 3403 (NH), 3109 (arom.CH), 1604 (C=N), 1566, 1512 (C=C), 1255 (C=S) cm−1. H-
NMR (DMSO-d6)
(d, J = 7.1 Hz, 2H, arom.CH), 7.85 (d, J = 8.5 Hz, 2H, arom.CH), 8.04 (s, 1H, CH=N),
12.12 (s, 1H, NH) ppm. 13C-NMR (DMSO-d6)
: 104.93 (C4 imidazole), 126.71 (C2,6
δ: 7.32 (s, 1H, H-4 of imidazole ring), 7.48–7.38 (m, 5H, arom.CH), 7.64
δ
chlorophenyl), 127.67 (C3,5 chlorophenyl), 129.10 (C3,5 phenyl), 129.33 (C2,6 phenyl), 129.87
(C4 imidazole), 132.45 (C4 phenyl), 133.84 (C1 chlorophenyl), 134.68 (C4 chlorophenyl),
142.02 (C4 phenyl), 149.75 (C=N), 178.27 (C=S) ppm. Anal. Calcd for C16H12ClN3S (313.80).
Calculated: C, 61.24; H, 3.85; N, 13.39. Found: C, 61.12; H, 3.61; N, 13.48.
3.3.2. (E)-5-(4-Chlorophenyl)-1-(2-hydroxybenzylideneamino)-1H-imidazole-2(3H)-thione (4b
)
Yellow crystals, yield 79 %, m.p. 128–130 ◦C; crystallized from ethanol/ H2O (3:1); IR
(KBr)
ν
max: 3442 (OH), 3315 (NH), 3039 (arom.CH), 1614 (C=N), 1578. 1540 (C=C), 1267
(C=S), 1092 (C–O) cm–1. H-NMR (DMSO-d6)
δ: 6.95–6.88 (m, 2H, arom.CH), 7.29–7.18
1
(m, 2H, arom.CH), 7.40 (s, 1H, H-4 of imidazole ring), 7.48 (d, J = 8.5 Hz, 2H, arom.CH),
7.88 (d, J = 8.5 Hz, 2H, arom.CH), 8.35 (s, 1H, CH= N), 10.14 (s, 1H, OH), 12.18 (s, 1H, NH)
ppm. 13C-NMR (DMSO-d6)
δ: 104.64 (C4 imidazole), 116.61 (C3 hydroxyphenyl), 119.08
(C1 hydroxyphenyl), 120.83 (C5 hydroxyphenyl), 127.71 (C2,6 chlorophenyl), 129.12 (C3,5
chlorophenyl), 131.06 (C6 hydroxyphenyl), 131.55 (C5 imidazole), 132.43 (C1 chlorophenyl),
133.93 (C4 hydroxyphenyl), 139.98 (C4 chlorophenyl), 149.76 (C=N), 156.41 (C2 hydrox-
yphenyl), 178.08 (C=S) ppm. Anal. Calcd for C16H12ClN3OS (329.80). Calculated: C, 58.27;
H, 3.67; N, 12.74. Found: C, 58.41; H, 3.89; N, 12.49.

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3.3.3. (E)-1-(5-Bromo-2-hydroxybenzylideneamino)-5-(4-chlorophenyl)-1H-imidazole-
2(3H)-thione (4c)
Yellow crystals, yield 78%, m.p. 191–193 ◦C; crystallized from ethanol/ H2O (3:1); IR
(KBr)
ν
max: 3454 (OH), 3252 (NH), 3059 (arom.CH), 1592 (C=N), 1548, 1477 (C=C), 1263
(C=S), 1086 (C–O) cm−1. H-NMR (DMSO-d6)
δ
: 6.89 (d, J = 8.7 Hz, 1H, arom.CH), 7.35
1
(d, J = 8.7 Hz, 1H, arom.CH), 7.41 (s, 1H, H-4 of imidazole ring), 7.47 (d, J = 8.5 Hz, 2H,
arom.CH), 7.77 (s, 1H, arom.CH), 7.88 (d, J = 8.5 Hz, 2H, arom.CH), 8.27 (s, 1H, CH=N),
10.41 (s, 1H, OH), 12.29 (s, 1H, NH) ppm. 13C-NMR (DMSO-d6)
δ: 104.93 (C4 imida-
zole), 111.29 (C5 bromohydroxyphenyl), 118.87 (C3 bromohydroxyphenyl), 123.19 (C1
bromohydroxyphenyl), 127.70 (C2,6 chlorophenyl), 129.11 (C3,5 chlorophenyl), 132.45 (C5
imidazole and C6 bromohydroxyphenyl), 133.15 (C1,4 chlorophenyl), 137.50 (C4 bromohy-
droxyphenyl), 149.85 (C=N), 155.47 (C2 bromohydroxyphenyl), 178.28 (C=S) ppm. Anal.
Calcd for C16H11BrClN3OS (408.70). Calculated: C, 47.02; H, 2.71; N, 10.28. Found: C,
46.89; H, 2.57; N, 10.43.
3.3.4. (E)-5-(4-Chlorophenyl)-1-(4-methoxybenzylideneamino)-1H-imidazole-2(3H)-thione (4d
)
◦
Yellow crystals, yield 81%, m.p. 151–153 C; crystallized from ethanol/H2O (3:1);
IR (KBr) max: 3422 (NH), 3033 (arom.CH), 1575 (C=N), 1477, 1426 (C=C), 1352 (C=S),
1092 (C–O) cm–1. 1H-NMR (DMSO-d6)
: 3.78 (s, 3H, OCH3), 6.98 (d, J = 8.8 Hz, 2H,
ν
δ
arom.CH), 7.34 (s, 1H, H-4 of imidazole ring), 7.44 (d, J = 8.6 Hz, 2H, arom.CH), 7.59
(d, J = 8.8 Hz, 2H, arom.CH), 7.86 (d, J = 8.6 Hz, 2H, arom.CH), 7.99 (s, 1H, C=N), 12.04
(s, 1H, NH) ppm. 13C-NMR (DMSO-d6)
δ: 55.71 (OCH3), 104.621 (C4 imidazole), 114.80
(C3,5 methoxyphenyl), 127.43 (C1 methoxyphenyl), 127.66 (C2,6 chlorophenyl), 128.29
(C3,5 chlorophenyl), 129.07 (C2,6 methoxyphenyl), 132.34 (C5 imidazole), 134.05 (C1
chlorophenyl), 141.88 (C4 chlorophenyl), 142.68 (CH=N), 160.72 (C4 methoxyphenyl),
178.25 (C=S) ppm. Anal. Calcd for C17H14ClN3OS (343.83). Calculated: C, 59.38; H, 4.10;
N, 12.22. Found: C, 59.29; H, 3.89; N, 12.06.
3.3.5. 5-(4-Chlorophenyl)-1-((E)-((E)-3-phenylallylidene)amino)-1H-imidazole-2(3H)-thione (4e
)
Pale yellow crystals, yield 78%, m.p. 139–141 ◦C crystallized from dioxane; IR (KBr)
1
ν
max: 3499 (NH), 3059 (arom.CH), 1683 (C=N), 1565, 1480 (C=C), 1259 (C=S) cm−1. H-
NMR (DMSO-d6) δ: 6.94 (s, 1H, H-4 of imidazole ring), 7.02–7.04 (m, 1H, olefinic CH),
7.30 (d, J = 7.3 Hz, 1H, olefinic CH), 7.38–7.32 (m, 3H, arom.CH), 7.46 (d, J = 8.6 Hz,
2H, arom.CH), 7.58 (d, 2H, arom.CH), 7.88 (d, J = 10.1, 3.3 Hz, 2H, arom.CH), 7.93 (d,
J = 4.1 Hz, 1H, CH=N), 12.16 (s, 1H, NH) ppm. 13C-NMR (DMSO-d6)
δ: 104.81 (C4
imidazole), 125.63 (C2 allylidene), 127.36 (C2,6 chlorophenyl), 127.68 (C2,6 phenyl), 129.08
(C3,5 phenyl), 129.23 (C3,5 chlorophenyl), 130.49 (C4 phenyl), 132.45 (C5 imidazole), 133.98
(C1 chlorophenyl), 136.60 (C3 allylidene), 137.22 (C4 chlorophenyl), 144.59 (C1 phenyl),
149.80 (C=N), 178.16 (C=S) ppm. Anal. Calcd for C18H14ClN3S (339.84). Calculated: C,
63.62; H, 4.15; N, 12.36. Found: C, 63.53; H, 4.01; N, 12.12.
3.3.6. (E)-1-(2-Hydroxybenzylideneamino)-5-(4-methoxyphenyl)-1H-imidazole-2(3H)-thione (
5)
Yellow crystals, yield 76.40%, m.p. IR (KBr) max: 3442 (OH), 3319 (NH), 3032
ν
(arom.CH), 2986 (aliph.CH), 1613 (C=N), 1540, 1489 (C=C), 1271 (C=S), 1203, 1056 (C–
1
O) cm−1. H-NMR (DMSO-d6) δ: 3.89 (s, 3H, OCH3), 6.92–6.80 (m, 3H, arom.CH), 7.17 (d,
J = 8.8 Hz, 1H, arom.CH), 7.22 (d, J = 8.8 Hz, 1H, arom.CH), 7.31 (s, 1H, H-4 of imidazole
ring), 7.64 (d, J = 6.7 Hz, 1H, arom.CH), 7.94 (d, J = 6.3 Hz, 2H, arom.CH), 8.39 (s, 1H,
CH=N), 9.91 (s, 1H, OH), 11.41 (s, 1H, NH) ppm. 13C-NMR (DMSO-d6)
δ: 56.76 (OCH3),
102.99 (C4 imidazole), 111.25 (C3 hydroxyphenyl), 113.20 (C1 hydroxyphenyl), 116.47 (C5
hydroxyphenyl), 119.72 (C3,5 methoxyphenyl), 127.17 (C1 methoxyphenyl), 130.41 (C6
hydroxyphenyl), 131.05 (C5 imidazole), 131.55 (C2,6 methoxyphenyl), 139.97 (C4 hydrox-
yphenyl), 155.26 (C=N), 156.41 (C2 hydroxyphenyl), 156.86 (C4 methoxyphenyl), 178.06
(C=S) ppm. Anal. Calcd for C17H15N3O2S (325.38). Calculated: C, 62.75; H, 4.65; N, 12.91.
Found: C, 62.51; H, 4.78; N, 13.08.

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3.4. General Procedure for the Synthesis (E)-2-((3-Acetyl-5-(4-chlorophenyl)-2-thioxo-2,3-dihydro-
1H-imidazol-1-ylimino)methyl)phenyl Acetate (6)
A solution of compound 4b (0.01 mol) in acetic anhydride (20 mL) was heated under
reflux for 2 h, then cooled down and decanted into water. The reaction mixture was left for
24 h, and the precipitate formed was filtered off, washed with water and dried. Finally, the
product was crystallized from benzene to give 6.
Colorless crystals, yield 63%, m.p. 115–117 ◦C. IR (KBr)
ν
max: 3090 (arom.CH), 2934
(aliph.CH), 1766, 1696 (C=O), 1606 (C=N), 1521, 1484 (C=C), 1274 (C=S) cm−1. H-NMR
1
(DMSO-d6) δ: 2.08 (s, 3H, COCH3), 2.54 (s, 3H, COCH3), 7.29 (d, J = 8.1 Hz, 1H, arom.CH),
7.44 (t, J = 7.5 Hz, 1H, arom.CH), 7.52 (d, J = 8.5 Hz, 2H, arom.CH), 7.60 (t, 1H, arom.CH),
7.93 (d, J = 8.5 Hz, 2H, arom.CH), 8.10 (d, 1H, arom.CH), 8.15 (s, 1H, H-4 of imidazole
ring), 8.99 (s, 1H, CH=N) ppm. 13C-NMR (DMSO-d6)
δ: 20.02 (CH3CO), 22.95 (CH3CO),
114.59 (C4 imidazole), 124.04 (C3 acetoxyphenyl), 126.18 (C1 acetoxyphenyl), 127.10 (C5 ace-
toxyphenyl), 127.38 (C5 imidazole), 127.85 (C2,6 chlorophenyl), 129.34 (C3,5 chlorophenyl),
132.84 (C6 acetoxyphenyl), 133.05 (C4 acetoxyphenyl), 148.60 (C1 chlorophenyl), 148.80
(C4 chlorophenyl), 150.36 (C=N), 156.28 (C2 acetoxyphenyl), 169.50 (C=O), 172.13 (C=O),
172.53 (C=S) ppm. Anal. Calcd for C20H16ClN3O3S (413.88). Calculated: C, 58.04; H, 3.90;
N, 10.15. Found: C, 58.22; H, 4.07; N, 9.93.
3.5. Biological Studies
3.5.1. Antitumor Activity against Three Cancer Cell Lines
Antitumor activity of the newly prepared imidazole compounds 4a–e and 5 were
carried out against MCF-7, HepG2, HCT-116, and MCF-10A using the MTT assay method.
◦
Cells at a density of 1
×
104 were seeded in 96-well plates at 37 C for 48 h under 5%
CO2. After incubation, the cells were treated with different concentrations of the prepared
molecules and incubated for 24 h. MTT dye was added at the end of 24 h of drug treatment
◦
and incubated for 4 h at 37 C. Next, 100
µL of dimethyl sulfoxide was added to each well to
dissolve the purple formazan formed. The color intensity of the formazan product, which
represents the growth condition of the cells, is quantified using an ELISA plate reader at
570 nm. The experimental conditions were carried out with at least three replicates and the
experiments were repeated at least three times.
3.5.2. Cell Cycle Analysis of Compounds 4d and 5
MCF-7 cells (2
×
105/well) were harvested and washed twice in PBS. After that, cells
◦
were incubated at 37 C and 5% CO2. The medium was replaced with DMSO (1% v/v)
containing the tested compounds, then incubated for 48 h, washed twice in PBS, fixed
with 70% ethanol, rinsed again with PBS, and then stained with DNA fluorochrome PI for
15 min at 37 ◦C. DNA content was analyzed by flow cytometry on a FACS Calibur flow
cytometer (Becton and Dickinson, Heidelberg, Germany).
3.5.3. Annexin V FITC/PI Apoptosis Detection Staining Assay
Apoptosis in MCF-7 cells was investigated using fluorescent Annexin V-FITC/PI
detection kit by flow cytometry assay. Briefly, MCF-7 cells (2
×
105) after incubation for
12 h. Cells were treated with compounds 4d and at their IC50 concentration for 48 h, then
5
the cells were harvested and stained with Annexin V-FITC/PI dye for 15 min in the dark
at 37 ◦C. Flow cytometry analyses were performed using a FACS Calibur flow cytometer
(Becton and Dickinson, Heidelberg, Germany).
3.5.4. In Vitro VEGFR-2 Kinase Assay
VEGFR-2 inhibitory activity for compounds 4d, 5, and erlotinib was evaluated using
human VEGFR-2 ELISA kits according to the manufacturer’s instructions. A total of 100
µL
of standard solution and tested molecules were pipetted into the 96-wells 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 unbound biotinylated

Molecules 2021, 26, 1706
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antibody, 100
washing, 100
µ
L of conjugated streptavidin solution was pipetted into the well◦s. After
µL of TMB substrate solution was added to the wells for 30 min at 37 C. Stop
solution (50
450 nm.
µL) was added, and the intensity of the color was measured immediately at
3.5.5. In Vitro B-Raf Kinase Assay
RAF inhibitory activity for compounds 4d, 5, and erlotinib was evaluated using human
B-RAF ELISA kits according to the manufacturer’s instructions. Next, 100 µL of standard
solution and tested molecules were pipetted into the 96-wells and B-RAF was bound to the
wells by the immobilized antibody. The wells were washed and biotinylated antihuman
B-Raf antibody was added. After washing away unbound biotinylated antibody, 100 µL
of conjugated streptavidin solution was pipetted into the wells. After washing, 100
µ
L of
TMB substrate solution was added to the wells for 30 min at 37 ◦C. Stop solution (50
was added, and the intensity of the color is measured immediately at 450 nm.
µL)
3.6. Molecular Docking Study
A molecular docking study was performed using the MOE software program (MOE
2009.10). The VEGFR-2 crystal structure (PDB code: 3U6J) was obtained from a protein
data bank (Supplementary Materials).
4. Conclusions
In conclusion, a novel series of imidazoles bearing arylidene amino substituents at
the N-1 position were synthesized in order to investigate their anticancer activity. The
structure of the prepared imidazole derivatives was confirmed utilizing spectral data such
as IR, 1H-NMR, and 13C-NMR along with elemental analyses. The prepared imidazoles
molecules were screened for their cytotoxic activity against three cancer cell lines namely,
MCF-7 breast cancer, HepG2 liver cancer, and HCT-116 colon cancer cell lines. Compound
5
was the most potent of all against the three cell lines. DNA flow cytometric analysis
showed that, compounds 4d and exhibit pre-G1 apoptosis and cell cycle arrest at the
G2/M phase. The results of the VEGFR-2 and B-Raf kinase inhibition assays revealed that
compounds 4d and displayed good inhibitory activity compared with the reference drug
erlotinib. Finally, imidazole compounds 4d and could serve as lead chemical entities for
5
5
5
further structural optimization as anticancer agents.
Supplementary Materials: Figure S1: 1H-NMR spectrum of compound 3a, Figure S2: 13C-NMR
spectrum of compound 3a, Figure S3: 1H-NMR spectrum of compound 3b, Figure S4: 13C-NMR
spectrum of compound 3b, Figure S5: 1H-NMR spectrum of compound 3c, Figure S6: 13C-NMR
spectrum of compound 3c, Figure S7: 1H-NMR spectrum of compound 3d, Figure S8: 13C-NMR
1
spectrum of compound 3d, Figure S9: H-NMR spectrum of compound 4a, Figure S10: 13C-NMR
1
spectrum of compound 4a, Figure S11: H-NMR spectrum of compound 4b, Figure S12: 13C-NMR
1
spectrum of compound 4b, Figure S13: H-NMR spectrum of compound 4c, Figure S14: 13C-NMR
1
spectrum of compound 4c, Figure S15: H-NMR spectrum of compound 4d, Figure S16: 13C-NMR
1
spectrum of compound 4d, Figure S17: H-NMR spectrum of compound 4e, Figure S18: 13C-NMR
1
spectrum of compound 4e, Figure S19: H-NMR spectrum of compound
5
6
, Figure S20: 13C-NMR
, Figure S22: 13C-NMR
spectrum of compound
5
, Figure S21: 1H-NMR spectrum of compound
spectrum of compound 6.
Author Contributions: Conceptualization, A.H.A.A., E.-S.H.E.-S., M.A.M.T. and I.Z.; methodology,
E.E.M.T., M.A.M.T. and A.H.A.A.; data curation, E.F., O.A.A.A., A.H.A.A. and I.Z.; software, I.Z.,
A.H.A.A., E.F. and O.A.A.A.; resources, I.Z., A.H.A.A., E.F., O.A.A.A. and E.E.M.T.; supervision,
A.H.A.A., E.-S.H.E.-S., M.A.M.T.; funding acquisition, E.F. and O.A.A.A.; original draft preparation,
A.H.A.A., I.Z., E.F. and O.A.A.A.; Writing, review, and editing, all authors. All authors have read
and agreed to the published version of the manuscript.
Funding: Taif University Researchers Supporting Project number (TURSP-2020/220), Taif University,
Taif, Saudi Arabia.

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Data Availability Statement: MDPI Research Data Policies” at https://www.mdpi.com/ethics.
Acknowledgments: Taif University Researchers Supporting Project number (TURSP-2020/220), Taif
University, Taif, Saudi Arabia.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds not available from the authors.
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