Design, synthesis, and biological evaluation of 1-(5-(benzylthio)-1,3,4-thiadiazol-2-yl)-3-phenylurea derivatives as anticancer agents

Article abstract of DOI:10.1007/s00044-020-02616-2

A novel series of 1-(5-(benzylthio)-1,3,4-thiadiazol-2-yl)-3-phenylurea derivatives (5a–l) were designed and synthesized as sorafenib analogs. The in vitro cytotoxicity effects of synthesized compounds were evaluated against four different human cancer cells including MCF-7, HepG2, A549, and HeLa cell lines. The biological results showed that most of the compounds significantly prevented the proliferation of tested cancer cells. In particular, 2-F, 4-Cl, and 2,6-diF substituted derivatives (5d, 5g, and 5k) showed promising activities, especially against Hela cancer cells (IC₅₀ = 0.37, 0.73 and 0.95 μM, respectively) which were significantly more potent than sorafenib as the reference drug (IC₅₀ = 7.91 μM). Flow cytometry analysis revealed that the prototype compounds (5d, 5g, and 5k) significantly induced apoptotic cell death in HeLa cancer cells and blocked the cell cycle at the sub-G1 phase. Moreover, in silico docking study confirmed the binding of the prototype compound to the active site of VEGFR-2.

Full text of DOI:10.1007/s00044-020-02616-2

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02616-2
ORIGINAL RESEARCH
Design, synthesis, and biological evaluation of
1
-(5-(benzylthio)-1,3,4-thiadiazol-2-yl)-3-phenylurea derivatives
as anticancer agents
Ayoub Aghcheli1 Mahsa Toolabi Adileh Ayati Setareh Moghimi Loghman Firoozpour
2 ●
3 ●
3 ●
3 ●
●
4 ●
4 ●
5 ●
4 ●
1,3
Tayebeh Oghabi Bakhshaiesh Elahe Nazeri Maryam Norouzbahari Rezvan Esmaeili Alireza Foroumadi
Received: 18 May 2020 / Accepted: 13 August 2020
©
Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
A novel series of 1-(5-(benzylthio)-1,3,4-thiadiazol-2-yl)-3-phenylurea derivatives (5a–l) were designed and synthesized as
sorafenib analogs. The in vitro cytotoxicity effects of synthesized compounds were evaluated against four different human
cancer cells including MCF-7, HepG2, A549, and HeLa cell lines. The biological results showed that most of the compounds
significantly prevented the proliferation of tested cancer cells. In particular, 2-F, 4-Cl, and 2,6-diF substituted derivatives
(
5d, 5g, and 5k) showed promising activities, especially against Hela cancer cells (IC = 0.37, 0.73 and 0.95 µM,
50
respectively) which were significantly more potent than sorafenib as the reference drug (IC = 7.91 µM). Flow cytometry
5
0
analysis revealed that the prototype compounds (5d, 5g, and 5k) significantly induced apoptotic cell death in HeLa cancer
cells and blocked the cell cycle at the sub-G1 phase. Moreover, in silico docking study confirmed the binding of the
prototype compound to the active site of VEGFR-2.
●
●
●
Keywords 1,3,4-Thiadiazole Anticancer Sorafenib Cytotoxic activity
Introduction
Cancer is a serious risk of human health, caused by dys-
regulation of normal cell proliferation, currently known as
the second leading cause of death in the world (Elmetwally
et al. 2019; Elzahabi et al. 2018). Despite the rapid progress
made in diagnosis and treatment, cancer therapy represents
one of the greatest medical challenges. Multiple therapeutic
strategies including surgery, radiotherapy, and chemother-
apy have been used in cancer treatment, amongst che-
motherapeutic agents are known as the basic approach
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02616-2) contains supplementary
material, which is available to authorized users.
*
*
Rezvan Esmaeili
esmaeili.rezvan@gmail.com
Alireza Foroumadi
aforoumadi@yahoo.com
(Ayati et al. 2015, 2019). However, due to the low efficacy
1
2
3
and high toxicity of the available anticancer drugs, there is a
pressing need to discover and develop new scaffolds as
anticancer agents (Zheng et al. 2010).
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran
University of Medical Sciences, Tehran, Iran
Department of Medicinal Chemistry, School of Pharmacy, Ahvaz
Jundishapur University of Medical Sciences, Ahvaz, Iran
Thiadiazole is a prevalent 5-membered heterocyclic
system containing two nitrogen atoms and one sulfur atom.
This heterocyclic core exists in numerous biologically
active natural products and commercial drugs. The strong
aromaticity, lack of toxicity, and great in vivo stability are
advantageous features of this core made it prevalent in
bioactive compounds (Dawood and Farghaly 2017; Kush-
waha et al. 2012; Siddiquia et al. 2009). This heterocyclic
Drug Design and Development Research Center, The Institute of
Pharmaceutical Sciences (TIPS), Tehran University of Medical
Sciences, Tehran, Iran
4
5
Genetics Department, Breast Cancer Research Center, Motamed
Cancer Institute, ACECR, Tehran, Iran
Faculty of Medicine, Eastern Mediterranean University,
Famagusta, TRNC, via Mersin 10, Turkey

Medicinal Chemistry Research
ring exists in four isomeric structures including 1,2,3-thia-
diazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thia-
diazole. 1,3,4-Thiadiazole is considered as an important
class of multi-targeted scaffold which has been investigated
more than other isomers. 1,3,4-Thiadiazole-containing
compounds have shown a broad range of biological activ-
ities due to their mesoionic nature and liposolubility which
are attributed to the presence of sulfur atom. The good
liposolubility of the sulfur atom in this heterocyclic system
has a positive effect on the biological activity and phar-
macokinetic properties of thiadiazole-containing com-
pounds which make them capable to easily cross through
the cell membranes (Haider et al. 2015; Hu et al. 2014; Jain
et al. 2013; Shawali 2014). The recent reports have declared
the applications of 1,3,4-thiadiazole core in the structure of
novel therapeutic agents with antiviral (Shafique et al.
cancers such as metastatic breast, advanced gastric, thyroid,
non-small cell lung cancer, pancreatic, prostate, bladder,
metastatic colorectal, ovarian, brain, and leukemia (Kudo
2012; Wang et al. 2019; Wilhelm et al. 2006). The multiple
molecular targets of sorafenib make it a good candidate
against angiogenesis and tumorigenesis, however, its low
oral bioavailability and side reaction effects greatly limit its
clinical application (Liu et al. 2016). Regorafenib, the fluoro
analog of sorafenib, is a novel oral multikinase inhibitor
(Fig. 1), with potent antitumor effects in preclinical and
clinical trials. Regorafenib has been approved by the FDA
in 2017 for the treatment of hepatocellular carcinoma cancer
(HCC) with less severe side effects than those of sorafenib
(Liu et al. 2019).
Considering the promising anticancer effects of sorafenib
and in continuation of our recent studies on the synthesis of
anticancer agents (Akrami et al. 2017; Ayati et al. 2018;
Mahdavi et al. 2017; Rahmani-Nezhad et al. 2014; Toolabi
et al. 2020), herein, we describe the design, synthesis and
antiproliferative activities of the novel series of sorafenib
analogs, 1-(5-(benzylthio)-1,3,4-thiadiazol-2-yl)-3-pheny-
lurea derivatives (5a–l) (Fig. 1). The newly synthesized
compounds are designed by the attachment of 5-benzylthio-
1,3,4-thiadiazole to urea instead of 4-phenoxypyridine
moiety in the structure of sorafenib (Fig. 1). The in vitro
antiproliferative activities of synthesized compounds were
investigated against four different cancer cell lines. The
flow cytometry analysis was also performed to investigate
the mode of cell death caused by the most potent
compounds.
2
018), antimicrobial (Badr and Barwa 2011; Chandrakantha
et al. 2014), antioxidant (Kamboj et al. 2019), anti-
inflammatory (Gomha et al. 2017), antileishmanial
(
Almandil et al. 2019; Mojallal-Tabatabaei et al. 2019), and
antidepressant activities (Can et al. 2018). Moreover, a
number of studies have shown that 1,3,4-thiadiazole-con-
taining compounds showed remarkable anticancer activity
(
Akhtar et al. 2017). As an example, the benzylthio-1,3,4-
thiadiazol containing compound 6 showed significant
cytotoxicity against MV4-11 and HCT116 cancer cells (Li
et al. 2012).
Diarylurea derivatives are reported as potential anti-
proliferative agents against a large number of cancer cells
(
Choi et al. 2011; Jin et al. 2010; Khan et al. 2014; Li et al.
2
4
012; Li et al. 2019). Sorafenib, previously known as BAY
3-9006 (Fig. 1), with diarylurea moiety, is a potent
anticancer drug which extensively used in clinical targeted
therapy as a multi-target kinase inhibitor. Sorafenib is an
oral molecular agent approved by the U.S. Food and Drug
Administration (FDA) for the treatment of advanced renal
cell carcinoma in December 2005. Currently, sorafenib is
subjected to clinical trials for the treatment of some types of
Experimental
General chemistry
All commercially available reagents and solvents, used
without further purification, were purchased from Merck
Fig. 1 Structures of sorafenib
and the target compounds 5a–l

Medicinal Chemistry Research
−
1
and Sigma-Aldrich companies. Bruker FT-500 MHz spec-
m.p. 243 °C; IR (KBr, cm ): 3379 (N–H), 3192 (N–H),
1
trometer was used to confirm the structure of the target
1727 (C=O), 1598, 1539, 1321. H NMR (DMSO-d6,
1
13
compounds by H- and C-NMR in DMSO-d or CDCl as
500 MHz): 4.45 (s, 2H, CH ), 7.27 (t, 1H, J = 7.0 Hz, H-4),
6
3
2
the solvent. Chemical shifts reported in δ parts per million
7.33 (t, 2H, J = 7.2 Hz, H-3 and H-5), 7.40 (d, 2H, J =
7.5 Hz, H-2 and H-6), 7.64 (d, 1H, J = 9.0 Hz, H-5′),
7.73–7.74 (m, 1H, H-6′), 8.10 (s, 1H, H-2′), 9.65 (brs, 1H,
1
(
ppm) downfield from tetramethylsilane (TMS) for H- and
C-NMR and as the internal standard. F-NMR spectra
1
3
19
1
3
were also recorded with Bruker FT-300 MHz, using CFCl3
as internal standard. All the reactions were monitored
through thin-layer chromatography (TLC) on TLC plastic
sheets silica gel 60 F254. Elemental analysis was recorded
by a Perkin Elmer 2400 (automatic elemental analyzer).
Melting points were determined with a Kofler hot-plate
microscope apparatus and are uncorrected. IR spectroscopy
was performed using a Nicolet FT-IR Magna 550 spectro-
graph (KBr disks).
NH), 11.31 (brs, 1H, NH).
C NMR (DMSO-d6,
125 MHz): 37.7, 117.7 (q, J = 4.4 Hz), 122.7 (q, J =
241.9 Hz), 124.0, 125.1 (q, J = 4.4 Hz), 126.5, 127.1 (q,
J = 30.2 Hz), 127.7, 128.0, 128.7 (2C), 129.1 (2C), 132.2,
1
9
136.9, 138.5, 185.1. F NMR (DMSO-d , 282 MHz):
6
−61.45. Anal. Calcd for C H ClF N OS : C, 45.90; H,
1
7
12
3
4
2
2.72; N, 12.59; Found: C, 45.73; H, 2.54; N, 12.87. ESI-MS
+
m/z: 445.1 [M + H] .
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(5-((3-methylben-
General procedure for the synthesis of 2-amino-5-thiol-
zyl)thio)-1,3,4-thiadiazol-2-yl)urea (5b) White solid, Yield:
−
1
1
,3,4-thiadiazole (2)
75%; m.p. 226 °C; IR (KBr, cm ): 3373 (N–H), 3218
1
(
N–H), 1720 (C=O), 1595, 1547, 1317. H NMR (DMSO-
Carbon disulfide (2 mL) was added dropwise to a mixture of
thiosemicarbazide (1) (10 mmol) and sodium carbonate
d , 500 MHz): 4.42 (s, 2H, CH ), 7.35–7.37 (m, 3H, H-4,
6
2
H-5 and H-6), 7.49 (s, 1H, H-2), 7.65 (d, 1H, J = 8.7 Hz, H-
5′), 7.74–7.75 (m, 1H, H-6′), 8.11 (s, 1H, H-2′), 9.67 (brs,
(
9 mmol) in absolute ethanol (7 mL). The reaction mixture
1
3
was heated under reflux for 8 h. Upon completion, the
solvent was evaporated and the crude was dissolved in HCl
solution (6 M), the resulting solid was filtered and washed
with water.
1H, NH), 11.47 (brs, 1H, NH). C R (DMSO-d6,
125 MHz): 21.8, 38.5, 118.4 (q, J = 4.6 Hz), 123.5 (q, J =
236.7 Hz), 124.7, 126.4 (q, J = 4.6 Hz), 127.0, 127.7 (q,
J = 30.8 Hz), 129.2, 129.3, 130.5, 131.4, 133.0, 135.1,
1
9
137.4, 138.6, 139.2, 186.1.
F NMR (DMSO-d6,
General procedure for the synthesis of 2-amino-5-
282 MHz): −61.47. Anal. Calcd for C H ClF N OS : C,
1
8
14
3
4
2
(
benzylthio)-1,3,4-thiadiazole (3a–l)
47.11; H, 3.08; N, 12.21; Found: C, 47.28; H, 3.32; N,
+
12.02. ESI-MS m/z: 459.2 [M + H] .
To the chilled solution of 2-amino-5-thiol-1,3,4-thiadiazole
2) (7.5 mmol), KOH (8 mmol), in water (0.5 mL), and
ethanol (5 mL), benzyl bromide/chloride derivatives
7.9 mmol) were added slowly. Then, the reaction mixture
was stirred for 30 min. at room temperature. After the
consumption of the reagents, the mixture was diluted with
cold water. The obtained solid was filtered and washed with
water and then crystallized from ethanol.
(
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(5-((4-methoxy-
benzyl)thio)-1,3,4-thiadiazol-2-yl)urea (5c) White solid,
−
1
(
Yield: 72%; m.p. 232 °C; IR (KBr, cm ): 3368 (N–H),
1
3204 (N–H), 1726 (C=O), 1599, 1551, 1324. H NMR
(DMSO-d , 500 MHz): 3.72 (s, 3H, OCH ),4.40 (s, 2H,
6
3
CH ), 6.88 (d, 2H, J = 8.4 Hz, H-3 and H-5), 7.32 (d, 2H,
2
J = 8.4 Hz, H-2 and H-6), 7.65 (d, 1H, J = 9.0 Hz, H-5′),
7
.71–7.72 (m, 1H, H-6′), 8.09 (s, 1H, H-2′), 9.60 (brs, 1H,
1
3
General procedure for the synthesis of 1-(5-(benzylthio)-
NH), 11.40 (brs, 1H, NH).
C NMR (DMSO-d6,
1
,3,4-thiadiazol-2-yl)-3-(4-chloro-3-(trifluoromethyl)phenyl)
125 MHz): 36.7, 54.5, 113.4 (2C), 116.9 (q, J = 4.1 Hz),
121.8 (q, J = 248.2 Hz), 123.2, 124.4, 126.0 (q, J = 4.2 Hz),
urea derivatives (5a–l)
1
28.1 (q, J = 30.0 Hz), 128.9, 129.6 (2C), 130.6, 131.5,
1
9
A mixture of 1-chloro-4-isocyanato-2-(trifluoromethyl)
benzene (4) (0.3 mmol) in dry CH Cl (1 mL) was added to
132.4, 135.5, 185.8. F NMR (DMSO-d , 282 MHz):
6
−61.48. Anal. Calcd for C H ClF N O S : C, 45.52; H,
2
2
18 14
3
4
2 2
the solution of 2-amino-5-(benzylthio)-1,3,4-thiadiazole
3a–l) (0.45 mmol) in dry CH Cl (1 mL) and the reaction
2.97; N, 11.80; Found: C, 45.34; H, 2.68; N, 11.97. ESI-MS
m/z: 475.4 [M + H] .
+
(
2
2
mixture was stirred overnight. After the consumption of the
starting materials, the obtained solid was filtered and
washed with CH Cl .
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(5-((2-fluoroben-
zyl)thio)-1,3,4-thiadiazol-2-yl)urea (5d) White solid, Yield:
2
2
−
1
7
9%; m.p. 238 °C; IR (KBr, cm ): 3367 (N–H), 3259
1
1
-(5-(Benzylthio)-1,3,4-thiadiazol-2-yl)-3-(4-chloro-3-(tri-
(N–H), 1727 (C=O), 1604, 1551, 1321. H NMR (DMSO-
d , 500 MHz): 4.45 (s, 2H, CH ), 7.16 (t, 1H, J = 7.4 Hz,
fluoromethyl)phenyl)urea (5a) White solid, Yield: 75%;
6
2

Medicinal Chemistry Research
H-5), 7.20 (t, 1H, J = 9.1 Hz, H-6), 7.32–7.36 (m, 1H, H-4),
(s, 1H, H-2′), 9.63 (brs, 1H, NH), 11.39 (brs, 1H, NH). 13C
7
.44 (t, 1H, J = 7.5 Hz, H-3), 7.64 (d, 1H, J = 8.7 Hz, H-5′),
NMR (DMSO-d , 125 MHz): 36.8, 117.4 (q, J = 4.2 Hz),
6
7
.73–7.75 (m, 1H, H-6′), 8.09 (s, 1H, H-2′), 9.58 (brs, 1H,
123.6, 123.8, 124.7 (q, J = 250.5 Hz), 124.8 (q, J = 4.3 Hz),
126.9 (q, J = 33.1 Hz), 128.5 (2C), 130.9 (2C), 132.2,
134.3, 136.1, 138.2, 138.3, 185.2. F NMR (DMSO-d6,
13
NH), 11.41 (brs, 1H, NH).
1
4
C NMR (DMSO-d6,
25 MHz): 31.4, 115.4 (d, J = 21.0 Hz), 117.5 (q, J =
.2 Hz), 122.6 (q, J = 245.3 Hz), 123.8, 124.5, 126.0 (q,
1
9
282 MHz): −61.47. Anal. Calcd for C H Cl F N OS : C,
1
7
11
2
3
4
2
J = 4.7 Hz), 126.7 (q, J = 30.2 Hz), 129.9 (d, J = 8.2 Hz),
31.3, 132.0, 134.5 (d, J = 7.9 Hz), 135.5, 138.2, 155.6 (d,
42.60; H, 2.31; N, 11.69; Found: C, 42.83; H, 2.17; N,
11.52. ESI-MS m/z: 478.9 [M + H] .
+
1
19
J = 20.9 Hz), 160.3 (d, J = 255.0 Hz), 185.1. F NMR
DMSO-d , 282 MHz): −114.08, −61.42. Anal. Calcd for
(
1-(5-((4-Bromobenzyl)thio)-1,3,4-thiadiazol-2-yl)-3-(4-
6
C H ClF N OS : C, 44.11; H, 2.40; N, 12.10; Found: C,
chloro-3-(trifluoromethyl)phenyl)urea (5h) White solid,
1
7
11
4
4
2
+
−1
4
4.29; H, 2.24; N, 12.43. ESI-MS m/z: 463.2 [M + H] .
Yield: 77%; m.p. 228 °C; IR (KBr, cm ): 3370 (N–H),
1
3
257 (N–H), 1725 (C=O), 1598, 1550, 1320. H NMR
1
-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(5-((4-fluoroben-
(DMSO-d , 500 MHz): 4.43 (s, 2H, CH ), 7.35 (d, 2H, J =
6
2
zyl)thio)-1,3,4-thiadiazol-2-yl)urea (5e) White solid, Yield:
7.8 Hz, H-3 and H-5), 7.52 (d, 2H, J = 7.8 Hz, H-2 and H-
6), 7.64 (d, 1H, J = 8.6 Hz, H-5′), 7.78–7.80 (m, 1H, H-6′),
8.16 (s, 1H, H-2′), 10.62 (brs, 1H, NH). C NMR (DMSO-
−
1
7
5%; m.p. 235 °C; IR (KBr, cm ): 3365 (N–H), 3207
1
13
(
N–H), 1737 (C=O). 1592, 1526, 1321. H NMR (DMSO-
d , 500 MHz): 4.37 (s, 2H, CH ), 7.09 (t, 2H, J = 8.7 Hz, H-
d6, 125 MHz): 37.4, 117.4 (q, J = 4.9 Hz), 122.9 (q, J =
253.9 Hz), 123.6, 126.3 (q, J = 5.1 Hz), 127.3 (q, J =
6
2
3
1
and H-5), 7.25 (t, 2H, J = 8.7 Hz, H-2 and H-6), 7.63 (d,
H, J = 8.8 Hz, H-5′), 7.72–7.75 (m, 1H, H-6′), 8.12 (s, 1H,
31.2 Hz), 129.6 (2C), 130.2 (2C), 131.6, 131.8, 132.6,
13
19
H-2′), 9.57 (brs, 1H, NH), 11.38 (brs, 1H, NH). C NMR
133.7, 134.7, 135.5, 185.4.
F NMR (DMSO-d6,
(
DMSO-d , 125 MHz): 35.4, 116.3, 116.8 (q, J = 4.1 Hz),
282 MHz): −61.47. Anal. Calcd for C H BrClF N OS :
6
17 11 3 4 2
1
1
1
2
−
4
1
20.5, 122.6 (q, J = 252.1 Hz), 124.7, 126.4 (q, J = 4.0 Hz),
27.4 (q, J = 31.2 Hz), 129.8 (d, 2 C, J = 8.1 Hz), 130.9,
31.7 (d, 2C, J = 24.1 Hz), 132.4, 158.8, 162.2 (d, J =
C, 38.98; H, 2.12; N, 10.70; Found: C, 38.72; H, 2.41; N,
10.57. ESI-MS m/z: 523.2 [M + H] .
+
19
55.0 Hz), 185.2.
F
NMR (DMSO-d6, 282 MHz):
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(5-((2-nitrobenzyl)
thio)-1,3,4-thiadiazol-2-yl)urea (5i) White solid, Yield:
114.05, −61.45. Anal. Calcd for C H ClF N OS : C,
17
11
4
4
2
−
1
4.11; H, 2.40; N, 12.10; Found: C, 44.01; H, 2.71; N,
80%; m.p. 219 °C; IR (KBr, cm ): 3371 (N–H), 3364
+
2.32. ESI-MS m/z: 463.5 [M + H] .
(N–H), 1723 (C=O), 1600, 1551, 1528 (N–O), 1340 (N–O),
1
1
321. H NMR (DMSO-d , 500 MHz): 4.72 (s, 2H, CH ),
6
2
1
-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(5-((3-chloroben-
7.57 (t, 1H, J = 7.5 Hz, H-4), 7.63–7.65 (m, 2H, H-5 and H-
6), 7.69 (d, 1H, J = 7.4 Hz, H-5′), 7.72–7.74 (m, 1H, H-6′),
zyl)thio)-1,3,4-thiadiazol-2-yl)urea (5f) White solid, Yield:
8
−
1
0%; m.p. 216 °C; IR (KBr, cm ): 3368 (N–H), 3207
8.06–8.08 (m, 2H, H-3 and H-2′), 9.62 (brs, 1H, NH), 11.41
1
13
(
N–H), 1722 (C=O), 1598, 1545, 1320. H NMR (DMSO-
(brs, 1H, NH). C NMR (DMSO-d , 125 MHz): 34.8, 117.3
6
d , 500 MHz): 4.47 (s, 2H, CH ), 7.09 (d, 1H, J = 6.9 Hz,
(q, J = 4.4 Hz), 122.4 (q, J = 247.4 Hz), 123.6, 125.0, 126.5
(q, J = 3.9 Hz), 126.7, 127.6 (q, J = 30.0 Hz), 129.1, 131.9,
6
2
H-4), 7.18–7.22 (m, 3H, H-2, H-5 and H-6), 7.65 (d, 1H,
J = 8.7 Hz, H-5′), 7.74–7.76 (m, 1H, H-6′), 8.11 (s, 1H, H-
1
9
132.1, 132.2, 133.7, 134.9, 138.0, 147.8, 184.9. F NMR
(DMSO-d6, 282 MHz): −61.47. Anal. Calcd for
C H ClF N O S : C, 41.68; H, 2.26; N, 14.30; Found: C,
2
′), 9.67 (brs, 1H, NH), 11.44 (brs, 1H, NH).). 13C NMR
(
(
DMSO-d , 125 MHz): 36.8, 117.6 (q, J = 4.4 Hz), 122.4
q, J = 246.5 Hz), 123.92, 126.8 (q, J = 4.1 Hz), 127.0 (q,
6
17 11
3
5
3 2
+
41.35; H, 2.41; N, 14.47. ESI-MS m/z: 490.0 [M + H] .
J = 30.3 Hz), 127.6, 127.8, 128.9, 130.5, 132.2, 133.1,
1
9
1
2
4
1
34.7, 135.4, 136.4, 139.7, 185.3. F NMR (DMSO-d6,
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(5-((3-nitrobenzyl)
thio)-1,3,4-thiadiazol-2-yl)urea (5j) White solid, Yield:
85%; m.p. 219 °C; IR (KBr, cm ): 3374 (N–H), 3218
82 MHz): −61.47. Anal. Calcd for C H Cl F N OS : C,
1
7
11
2
3
4
2
−
1
2.60; H, 2.31; N, 11.69; Found: C, 42.43; H, 2.52; N,
+
1.82. ESI-MS m/z: 479.1 [M + H] .
(N–H), 1722 (C=O), 1599, 1551, 1528 (N–O), 1348
1
(
N–O), 1317. H NMR (DMSO-d , 500 MHz): 4.74 (s, 2H,
6
1
-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(5-((4-chloroben-
CH ), 7.61–7.65 (m, 2H, H-5 and H-6), 7.70–7.71 (m, 1H,
2
zyl)thio)-1,3,4-thiadiazol-2-yl)urea (5g) White solid, Yield:
H-4), 7.86 (d, 1H, J = 7.6 Hz, H-5′), 8.07 (s, 1H, H-2), 8.12
−
1
7
5%; m.p. 235 °C; IR (KBr, cm ): 3371 (N–H), 3217
(d, 1H, J = 7.9 Hz, H-6′), 8.29 (s, 1H, H-2′), 9.56 (brs, 1H,
1
13
(
N–H), 1725 (C=O), 1598, 1549, 1320. H NMR (DMSO-
NH), 11.36 (s, 1H, NH). C NMR (DMSO-d , 125 MHz):
6
d , 500 MHz): 4.45 (s, 2H, CH ), 7.38 (d, 2H, J = 8.4 Hz,
37.9, 117.8 (q, J = 4.2 Hz), 122.9 (q, J = 245.6 Hz), 124.0,
126.3, 127.0 (q, J = 4.1 Hz), 127.6 (q, J = 30.0 Hz), 128.5,
128.7, 129.8, 132.3, 134.2, 135.0, 136.7, 138.0, 138.6,
6
2
H-3 and H-5), 7.42 (d, 2H, J = 8.4 Hz, H-2 and H-6), 7.64
d, 1H, J = 9.0 Hz, H-5′), 7.72–7.74 (m, 1H, H-6′), 8.09
(

Medicinal Chemistry Research
1
83.6. 19F NMR (DMSO-d , 282 MHz): −61.50. Anal.
DMSO. The fresh medium containing 0.5 mg/ml of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) was added to each well and the optical density was
measured at 570 nm with DMSO as blank using a Bio-Rad
microplate reader (Model 680). The negative control was
0.1% DMSO and the positive control was sorafenib. The
IC50 values, the compounds, concentration required to
inhibit cell proliferation by 50%, were depicted as the mean
± SE from the dose–response curves of at least three inde-
pendent experiments.
6
Calcd for C H ClF N O S : C, 41.68; H, 2.26; N, 14.30;
Found: C, 41.84; H, 2.42; N, 14.12. ESI-MS m/z: 490.1 [M
+
1
7
11
3
5
3 2
+
H] .
1
-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(5-((2,6-difluoro-
benzyl)thio)-1,3,4-thiadiazol-2-yl)urea (5k) White solid,
Yield: 74%; m.p. 234 °C; IR (KBr, cm ): 3367 (N–H),
3
−
1
1
259 (N–H), 1726 (C=O), 1600, 1552, 1322. H NMR
(
DMSO-d , 500 MHz): 4.40 (s, 2H, CH ), 7.12 (t, 2H, J =
6
2
7
.9 Hz, H-3 and H-5), 7.39–7.44 (m, 1H, H-4), 7.65 (d, 1H,
J = 8.7 Hz, H-5′), 7.78–7.80 (m, 1H, H-6′), 8.13 (s, 1H, H-
Annexin V-FITC apoptosis assay
2
2
4
4
1
9
′), 9.8 (brs, 1H, NH). 13C NMR (DMSO-d , 125 MHz):
6
6.6, 112.1 (dd, 2C, J = 20.5, 4.5 Hz), 118.0 (q, J =
.4 Hz), 123.0 (q, J = 236.8 Hz), 124.3, 126.7 (q, J =
.9 Hz), 128.3 (q, J = 29.1 Hz), 130.9 (t, J = 10.1 Hz),
32.2, 132.5, 133.2, 134.9, 138.8, 161.0 (dd, 2C, J = 236.3,
Annexin V-FITC/PI (Propidium iodide) dual staining assay
was used to quantify the percentage of cells undergoing
apoptosis in the presence or absence of the most active
compounds. The results were compared with sorafenib as
1
9
.6 Hz), 182.9. F NMR (DMSO-d , 282 MHz): −114.10
tested at IC concentration. HeLa cancer cells were seeded
6
50
6
(
2F), −61.46. Anal. Calcd for C H ClF N OS : C, 42.46;
(1 × 10 ) into 6-well plates and incubated overnight at 37 °C
17
10
5
4
2
H, 2.10; N, 11.65; Found: C, 42.27; H, 2.34; N, 11.79. ESI-
MS m/z: 480.9 [M + H] .
under 5% CO then treated with IC concentrations of
2
50
+
compounds 5d, 5g, and 5k for 48 h more. The treated cells
were trypsinized, washed with PBS twice and centrifuged at
1200 rpm. After that, 500 µL of binding buffer and 5 µL of
Annexin V-FITC and PI were added to the suspended cells
and incubated for 15 min at room temperature in dark. The
percentage of apoptotic cell death was analyzed using a
flow cytometer (FACS Calibur Bectone-Dickinson) (Huang
et al. 2013).
1
-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(5-((2,4-dichloro-
benzyl)thio)-1,3,4-thiadiazol-2-yl)urea (5l) White solid,
−
1
Yield: 75%; m.p. 227 °C; IR (KBr, cm ): 3370 (N–H),
1
3
217 (N–H), 1726 (C=O), 1597, 1550, 1320. H NMR
(
DMSO-d , 500 MHz): 4.50 (s, 2H, CH ), 7.41 (d, 1H, J =
6
2
8.2 Hz, H-6), 7.51 (d, 1H, J = 8.3 Hz, H-5), 7.64–7.67 (m,
2H, H-3 and H-5′), 7.73–7.84 (m, 1H, H-6′), 8.09 (s, 1H, H-
2
′), 9.57 (brs, 1H, NH), 11.44 (s, 1H, NH). 13C NMR
Cell cycle assessment
(
DMSO-d , 125 MHz): 35.2, 117.5 (q, J = 4.4 Hz), 122.6
6
(
q, J = 250.9 Hz), 123.7, 123.8, 125.9 (q, J = 4.7 Hz), 126.7
The cell cycle distributions in HeLa cancer cells treated by
the most potent compounds (5d and 5g) were analyzed by
flow cytometry analysis using PI staining assay. The HeLa
cells were treated with IC50 concentrations of test com-
pounds (5d and 5g) for 48 h. Then the cells were trypsi-
nized, washed with PBS and centrifuged at 1000 rpm for
(
q, J = 30.0 Hz), 127.5, 129.0, 132.1, 132.5, 133.2, 133.5,
19
1
2
3
1
34.2, 136.1, 138.2, 185.1.
F NMR (DMSO-d6,
82 MHz): −61.45. Anal. Calcd for C H Cl F N OS : C,
17
10
3
3
4
2
9.74; H, 1.96; N, 10.91; Found: C, 39.58; H, 1.72; N,
+
1.28. ESI-MS m/z: 513.1 [M + H] .
5
min. The obtained HeLa cells were incubated with PBS
MTT assay
and fixed with 70% cold ethanol at −20 °C. The fixed cells
were further washed with PBS and treated sequentially with
RNase A (0.1 mg/ml, incubated for 30 min) and PI (50 mg/
mL, incubated for further 15 min). Novocyte flow cytometer
(ACEA Biosciences) was used to calculate the cell cycle
distribution, analyzed by NovoExpress 1.1.0 software
(Kamal et al. 2015).
Cytotoxic activity of synthesized compounds (5a–l) was
evaluated against four different tumor cells including breast
carcinoma (MCF-7), human liver cancer (HepG2), human
lung cancer (A549), and human cervical cancer (HeLa)
using MTT assay. The cancer cells, purchased from the
National Cell Bank of Iran (Pastor Institute, Tehran, Iran),
were grown in RPMI-1640 medium and DMEM with 10%
FBS (Gibco, Milano, Italy). Each well of a 96-well micro-
titer plate, containing 190 µl of complete medium and a
suspension of 8000 cells, incubated at 37 °C in a humidified
Molecular docking section
Docking analysis of compound 5d was performed using
Autodock 4.2.1. The co-crystal structures of VEGFR-2 and
sorafenib with 2.03 Å resolution (PDB entry code: 4ASD)
was used. The docking procedure was performed as pre-
viously conducted (Toolabi et al. 2020). Docking results
5
% CO incubator overnight. The cells were further incu-
2
bated for 48 h more after treatment with different con-
centrations of target compounds prepared by dissolving in

Medicinal Chemistry Research
including the 3D and 2D diagram interactions were viewed
by Discovery Studio visualizer 4.5.
better activities than sorafenib against MCF-7 and µHepG2
cancer cells. Compounds 5b, 5d, 5g, and 5k exhibited
significant cytotoxic effects toward all the tested cell lines
with promising results against HeLa cancer cells. These
derivatives showed IC50 values of 0.94, 0.37, 0.73, and
Results and discussion
0.95 µM, respectively, against HeLa cell line which were
Chemistry
about 10–21 times more potent than sorafenib.
The general structure of 5-benzylthio-2-phenyl urea-
1,3,4-thiadiazol has been varied by different R substitutions
attachment on the benzylthio warhead, which significantly
influenced on the anticancer effects of target compounds.
The unsubstituted derivative 5a showed better results than
sorafenib against all the tested cell lines with IC50s ranging
from 1.28 to 6.88 µM. The effect of substitutions on the
phenyl ring was depended on the type and the position of
substituents. The introduction of strong electron with-
drawing groups like fluoro, bromo and nitro at C-4, C-2 and
C-3 of benzylthio warhead, (5e, 5h–5j) dramatically
reduced the antiproliferative activities of compounds
against A549 cell line, while, the promising results against
HeLa cancer cells were observed for the 2-F, 2,6-diF and 4-
Cl derivatives (5d, 5k, and 5g). These derivatives had sig-
nificant cytotoxic effects against MCF-7 and HepG2 cells.
The nitro substituted derivatives (5i and 5j) were found to
be less potent compounds against A549 cell line with
moderate effects against other tested cancer cells. Moreover,
4-F derivative, 5e, showed the least activity against MCF-7
and HepG2 cell lines. Attachment of the electron donation
group (CH and OCH ) to the C-3 and C-4 position of
The synthetic route of the target compounds 5a–l is outlined
in Scheme 1. The compound 2, 2-amino-5-thiol-1,3,4-
thiadiazole, was prepared from the condensation reaction
between thiosemicarbazide (1) and carbon disulfide in
ethanol. In the next step, compound 2 was reacted with
different benzyl bromide/chloride derivatives in the pre-
sence of KOH solution to afford the appropriate 2-amino-5-
benzylthio-1,3,4-thiadiazoles (3a–l). Finally, with com-
pounds 3a–l in hands, the reaction with 1-chloro-4-iso-
cyanato-2-(trifluoromethyl)
dichloromethane furnished final derivatives 5a–l in good
yields.
benzene
(4)
in
dry
Antiproliferative activity
The in vitro antiproliferative activity of synthesized com-
pounds (5a–l) was investigated against four different cancer
cell lines including MCF-7, HepG2, A549, and HeLa, using
MTT assay. The results were presented as IC50 values and
compared with sorafenib as the reference drug (Table 1).
Based on the observed results, all the target compounds
have better anticancer activity against HeLa cancer cells
than the activity of sorafenib. The weakest cytotoxic
activity was observed toward A549 cell line.
As depicted in Table 1, the majority of compounds
exhibited good to excellent cytotoxic activity, displaying
IC50 values less than sorafenib. The reference drug sor-
afenib showed IC50 values ranging from 7.06 to 12.91 µM
against tested cell lines. All compounds except 5e showed
3
3
phenyl ring improved the cytotoxic effects of compounds
against all the tested cell lines with IC50s ranging from 0.94
to 7.04 µM. 4-Methoxy derivative, 5c, showed the highest
inhibitory activity against A549 cell line (IC = 4.44 µM)
5
0
which was about 3 times more potent than sorafenib as the
standard drug (IC = 12.25 µM). The best antiproliferative
50
effect of compound 5b with 3-methyl substitution was seen
against HeLa cancer cell with IC = 0.94 µM. As
5
0
Scheme 1 Synthesis of
compounds 5a–l. Reagents and
conditions: a (i) CS2, Na2CO3,
EtOH, reflux, 8 h; (ii) H2O, HCl;
b (i) H2O, KOH, r.t., 15 min.;
(
ii) EtOH, benzyl bromide/
chloride derivatives, r.t.,
0 min.; c 1-chloro-4-
3
isocyanato-2-(trifluoromethyl)
benzene, dry CH2Cl2, r.t.,
overnight

Medicinal Chemistry Research
Table 1 Antiproliferative activity of the target compounds 5a–l presented as IC50 (μM)a
Compound
R
MCF7
HepG2
A549
HeLa
5
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
H
1.52 ± 0.10
2.25 ± 0.12
3.14 ± 0.41
2.10 ± 0.34
16.82 ± 0.80
3.79 ± 0.21
2.46 ± 0.75
6.17 ± 0.51
5.24 ± 0.83
2.63 ± 0.22
3.94 ± 0.11
3.54 ± 0.25
7.06 ± 0.50
4.20 ± 0.24
4.03 ± 0.32
4.30 ± 0.11
6.14 ± 0.20
32.17 ± 0.77
6.94 ± 0.52
2.57 ± 0.79
6.47 ± 0.41
6.43 ± 0.40
1.72 ± 0.54
2.68 ± 0.41
2.21 ± 0.72
12.91 ± 0.47
6.88 ± 0.71
7.06 ± 0.57
4.44 ± 0.23
7.25 ± 0.15
25.15 ± 0.91
12.25 ± 0.73
7.58 ± 0.48
15.89 ± 0.71
15.32 ± 0.40
67.51 ± 1.14
5.82 ± 0.21
8.26 ± 0.45
12.25 ± 0.42
1.28 ± 0.34
0.94 ± 0.12
3.82 ± 0.27
0.37 ± 0.11
7.05 ± 0.54
3.98 ± 0.70
0.73 ± 0.37
7.71 ± 0.41
5.91 ± 0.43
2.02 ± 0.81
0.95 ± 0.48
2.32 ± 0.57
7.91 ± 0.31
3-CH3
4-OCH3
2-F
4-F
3-Cl
g
h
i
4-Cl
4-Br
2-NO2
3-NO2
2,6-diF
2,4-diCl
–
j
k
l
Sorafenib
aValues were the means of three replicates±standard deviation (SD)
evidenced from IC50 values, the best results were observed
from 2-F, 2,6-diF and 4-Cl derivatives 5d, 5g and 5k which
were significantly more potent than sorafenib against all
four tested cell lines.
2.8% of early apoptosis, respectively. These results con-
firmed that compounds 5d, 5g, and 5k significantly induced
early apoptosis to the HeLa cancer cells compared to sor-
afenib as the reference drug.
Determination of inducing apoptosis by the most
potent compound
Cell cycle arrest
The inhibition of cancer cells proliferation at specific check-
points was investigated by cell cycle analysis in HeLa cancer
cells (Kamal et al. 2015). In this regard, the HeLa cells were
treated with the most potent compounds 5d and 5g at the IC50
concentrations for 48 h. Then, the treated cells were harvested
and stained with PI and analyzed by flow cytometry. The
intensity of fluorescent emission from PI bounded to DNA
could visualize the proportion of DNA content. Non-treated
HeLa cells were used as control. As shown in Figs. 3, 4,
treatment of HeLa cells with 5d and 5g at IC concentrations,
To determine the mode of cell death by the most potent
compounds (5d, 5g, and 5k), the flow cytometry analysis
was performed on HeLa cells using Annexin V-FITC/PI
(
Propidium iodide) dual staining assay (Huang et al. 2013).
Non-treated cells were used as negative control and sor-
afenib at its IC50 concentration was used as the positive
control. The cells were treated with the IC50 concentrations
of the most potent compounds (5d, 5g, and 5k) for 48 h.
Then, the cells were harvested and stained with FITC
Annexin-V and PI solutions, respectively. The percentage
of apoptotic cells was determined by flow cytometry ana-
lysis. As shown in Fig. 2, the treatment of HeLa cells with
compounds 5d, 5g, and 5k led to the late apoptotic cell
death by 14.3%, 17.7%, and 15.9%, respectively, while, the
percentage of late apoptotic death by the negative and
positive control were 2.98% and 60.1%, respectively. These
compounds (5d, 5g, and 5k) induced early apoptosis to the
HeLa cells by 33.2%, 34.1%, and 25.8%, respectively,
whereas non-treated cells and sorafenib showed 3.9% and
5
0
increased the percentage of sub-G1 phase cells from 7.1% as
the control group to 11.9% and 9.2%, respectively. These
results confirmed that compounds 5d and 5g significantly
caused arrest in HeLa cancer cells.
Docking study
Docking study was conducted for the most potent compound
5d to manifest the binding mode of this compound in the
active site of VEGFR-2, using Autodock 4.2.1 software. The

Medicinal Chemistry Research
Fig. 2 Flow cytometry analysis of HeLa cells treated with synthetic
compounds 5d, 5g, and 5k. a Non-treated cells as the negative control
group; b treated with sorafenib as the positive control; c treated with
5d at IC50 concentration; d treated with 5g at IC50 concentration;
e treated with 5k at IC50 concentration
Fig. 3 Cell cycle analysis of HeLa cells by flow cytometry analysis. a Non-treated cells as the control group; b treated with 5d at IC50
concentration; c treated with 5g at IC50 concentration
results were visualized by Discovery Studio software. As
shown in Fig. 5, compound 5d formed a U shape config-
uration and interacted with Glu 885 and Asp 1046 residues
through hydrogen bonds with a bonding distance of 1.66 and
Val 898, respectively. To summarize, according to the
docking study, the results are consistent with the biological
evaluations.
2.28 Å, respectively. In general, the diarylurea and thiadiazole
moieties of this compound enhanced the complex stability
due to crucial hydrogen bonds interaction with the aspartate
and glutamate residues of the active site. Moreover, the
benzyl moiety of this compound extended to the hydrophobic
pocket of enzyme and made Van der Waals interactions with
Phe 1047, Val 916, and Leu 840 and also made π-sigma
and π-alkyl interactions with Val 848 and Ala 866. The
lipophilic trifluoromethyl group and chlorine atom of
phenyl ring formed alkyl interactions with Leu 1019 and
Conclusion
Encouraged by the broad-spectrum anticancer activities of
sorafenib, we extended the biological investigations to a
series of novel 1-(5-(benzylthio)-1,3,4-thiadiazol-2-yl)-3-
phenylureas (5a–l) as sorafenib analogs against four human
cancer cell lines including MCF-7, HepG2, A549, and
HeLa. Based on the cellular assays, most of the compounds
showed significant cytotoxic activity against all tested cell

Medicinal Chemistry Research
respectively). Furthermore, the flow cytometry analysis of
the most potent compounds was confirmed that 5d, 5g, and
5k significantly induced apoptotic cell death in HeLa cancer
cells and blocked the proliferation of HeLa cells at the sub-
G1 phase. The obtained results were in good agreement
with docking results which showed suitable interaction
between the most active compound 5d in the active site of
VEGFR-2. We investigated that this heterocyclic system
may undergo further modifications to find more potent and
selective anticancer agents.
Acknowledgements This work was supported and funded by National
Institute for Medical Research Development (NMIAD) Grant no.
Fig. 4 Quantitative analysis of cell cycle distributions; (1) Non-treated
cells as the control group; (2) treated with 5d at IC50 concentration; (3)
treated with 5g at IC50 concentration
9
63529.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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