Design, synthesis, molecular docking and cytotoxic evaluation of novel 2-furybenzimidazoles as VEGFR-2 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.04.068

Inhibition of angiogenesis through inhibition of vascular endothelial growth factor receptor 2 (VEGFR-2) has been applied in cancer therapy because of its important role in promoting cancer growth and metastasis. In the presented study, a series of benzimidazol-furan hybrids was designed and synthesized through facile synthetic pathways. Evaluation of the synthesized compounds for their in?vitro cytotoxic activity against breast (MCF-7) and hepatocellular (HepG2) carcinoma cell lines was performed. Two of the synthesized conjugates, 10b and 15, showed potent antiproliferative properties against MCF-7 cell line (IC₅₀?=?21.25, 21.35?μM, respectively) in comparison to tamoxifen (IC₅₀?=?21.57?μM). Additionally, compounds 10a, 10b, 15 and 17 showed promising potency (IC₅₀?=?25.95, 22.58, 26.94 and 31.06?μM, respectively) against liver carcinoma cell line HepG2 in contrast to cisplatin (IC₅₀?=?31.16?μM). Moreover, in?vitro evaluation of the synthesized compounds for their effect on the level of VEGFR-2 in MCF-7?cell line showed their potent inhibitory activity relative to control untreated cells. Four compounds 10a, 10b, 14 and 15 showed 92–96% reduction in VEGFR-2 level, compared with tamoxifen and sorafenib which showed inhibition percentage of 98% and 95.75%, respectively. Compound 10a was found to have promising VEGFR-2 inhibitory activity (IC₅₀?=?0.64?μM) in comparison to sorafenib (IC₅₀?=?0.1?μM). Molecular docking was performed to study the binding pattern of the newly synthesized compounds with VEGFR-2 active site. Molecular docking attributed their good VEGFR-2 inhibitory activity to their hydrogen bonding interaction with the key amino acids in VEGFR-2 active site, Glu885 and Asp1046, and their hydrophobic interaction by their 2-furylbenzimidazole moiety with the allosteric hydrophobic back pocket in a type III inhibitors-like binding mode. The binding interaction is augmented by a ring substituent with long chain extension at position 1 of the benzimidazole due to its hydrophobic interaction with the hydrophobic side chains of the amino acids at the interface between the ATP binding site and the allosteric back pocket. Structure-activity relationship (SAR) was inferred for future optimization based on the performed biological and docking studies.

Full text of DOI:10.1016/j.ejmech.2017.04.068

Accepted Manuscript
Design, synthesis, molecular docking and cytotoxic evaluation of novel 2-
furybenzimidazoles as VEGFR-2 inhibitors
Mona A. Abdullaziz, Heba T. Abdel-Mohsen, Ahmed M. El Kerdawy, Fatma A.F.
Ragab, Mamdouh M. Ali, Sherifa M. Abu-bakr, Adel S. Girgis, Hoda I. El Diwani
PII:
S0223-5234(17)30342-2
10.1016/j.ejmech.2017.04.068
EJMECH 9419
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 January 2017
Revised Date: 28 March 2017
Accepted Date: 20 April 2017
Please cite this article as: M.A. Abdullaziz, H.T. Abdel-Mohsen, A.M. El Kerdawy, F.A.F. Ragab, M.M.
Ali, S.M. Abu-bakr, A.S. Girgis, H.I. El Diwani, Design, synthesis, molecular docking and cytotoxic
evaluation of novel 2-furybenzimidazoles as VEGFR-2 inhibitors, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.04.068.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, Synthesis, Molecular Docking and Cytotoxic Evaluation of
Novel 2-Furybenzimidazoles as VEGFR-2 Inhibitors
a
a
b,c
b
Mona A. Abdullaziz, Heba T. Abdel-Mohsen,* Ahmed M. El Kerdawy, Fatma A. F. Ragab,
d
a
e
a
Mamdouh M. Ali, Sherifa M. Abu-bakr, Adel S. Girgis, Hoda I. El Diwani
a
Department of Chemistry of Natural and Microbial Products, Division of Pharmaceutical and Drug Industries, National
Research Centre, Dokki, Cairo, Egypt
b
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-AiniStreet, Cairo, P.O. Box
1
1562, Egypt.
c
Molecular Modeling Unit, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo, P.O. Box 11562, Egypt.
d
Department of Biochemistry, Division of Genetic Engineering and Biotechnology, National Research Centre, Cairo,
Egypt
e
Department of Pesticide Chemistry, Division of Chemical Industries Research, National Research Centre
*
Corresponding author: Email address: hebabdelmohsen@gmail.com
Abstract
Inhibition of angiogenesis through inhibition of vascular endothelial growth factor receptor 2
VEGFR-2) has been applied in cancer therapy because of its important role in promoting cancer
(
growth and metastasis. In the presented study, a series of benzimidazol-furan hybrids was designed
and synthesized through facile synthetic pathways. Evaluation of the synthesized compounds for their
in vitro cytotoxic activity against breast (MCF-7) and hepatocellular (HepG2) carcinoma cell lines
was performed. Two of the synthesized conjugates, 10b and 15, showed potent antiproliferative
properties against MCF7 cell line (IC = 21.25, 21.35 µM, respectively) in comparison to tamoxifen
5
0
(
IC50 = 21.57 µM). Additionally, compounds 10a, 10b, 15 and 17 showed promising potency (IC50
5.95, 22.58, 26.94 and 31.06 µM, respectively) against liver carcinoma cell line HepG2 in contrast
to cisplatin (IC = 31.16 µM). Moreover, in vitro evaluation of the synthesized compounds for their
=
2
5
0
effect on the level of VEGFR-2 in MCF-7 cell line showed their potent inhibitory activity relative to
control untreated cells. Four compounds 10a, 10b, 14 and 15 showed 92-96% reduction in VEGFR-2
level, compared with tamoxifen and sorafenib which showed inhibition percentage of 98% and
9
5.75%, respectively. Compound 10a was found to have promising VEGFR-2 inhibitory activity
(
IC = 0.64 µM) in comparison to sorafenib (IC = 0.1 µM). Molecular docking was performed to
5
0
50
study the binding pattern of the newly synthesized compounds with VEGFR-2 active site. Molecular
docking attributed their good VEGFR-2 inhibitory activity to their hydrogen bonding interaction with
the key amino acids in VEGFR-2 active site, Glu885 and Asp1046, and their hydrophobic interaction
by their 2-furylbenzimidazole moiety with the allosteric hydrophobic back pocket in a type III
1

ACCEPTED MANUSCRIPT
inhibitors-like binding mode. The binding interaction is augmented by a ring substituent with long
chain extension at position 1 of the benzimidazole due to its hydrophobic interaction with the
hydrophobic side chains of the amino acids at the interface between the ATP binding site and the
allosteric back pocket. Structure-activity relationship (SAR) was inferred for future optimization
based on the performed biological and docking studies.
Key words: Angiogenesis; VEGFR-2; 2-Furylbenzimidazole; MCF-7; HepG-2
1
. Introduction
Cancer is becoming one of the most serious health problems in the world. Moreover, adverse effects
of classical non-selective chemotherapies and resistance development for the existing anticancer
drugs make the search for more selective new anticancer agents an urgent research point. However,
designing a molecule that can selectively inhibit the proliferation of abnormal cells only with little or
no effect on normal cells is a difficult task [1].
Angiogenesis, the formation of new blood vessels from pre-existing ones, is a normal physiological
process taking place during embryogenesis, inflammation and wound healing. On the other hand,
pathological angiogenesis is associated with many disease conditions such as cancer, where new
blood vessels infiltrate tumor masses supplying them with oxygen and nutrients to enhance tumor
growth and metastasis [2-4]. Hence, blocking angiogenesis could be a strategy to hinder tumor
growth. Unlike classical chemotherapy, which causes severe adverse effects and leads to drug
resistance with long-term treatment, angiogenesis inhibition could be more effective in preventing
tumor progression and well-tolerated [2-5].
Numerous growth factors are involved in angiogenesis. One of the most known angiogenic molecules
is the vascular endothelial growth factor (VEGF) family members which are pivotal stimuli of
physiological as well as pathological angiogenesis [4]. Vascular endothelial growth factor (VEGF)
regulates blood and lymphatic vessel development and homeostasis [5,6]. VEGF ligands bind in an
overlapping pattern to three different, but structurally related, VEGF-receptor tyrosine kinases
(VEGFR-TK) which are VEGFR-1 (FLT1), VEGFR-2 (KDR/FLK1) and VEGFR-3 (FLT4) [7].
2

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VEGFR-2, a type III transmembrane receptor tyrosine kinase, is vital for angiogenesis [8]. Several
complex mechanisms are involved in the regulation of VEGFR-2 levels [9]. Hence, blocking of
VEGFR-2 or down regulation of its signaling became a main approach for the discovery of new
drugs for many human angiogenesis-dependent malignancies. To date, a humanized anti-VEGF
monoclonal antibody (bevacizumab) and several small molecule VEGFR-2 kinase inhibitors
(sorafenib, regorafenib, sunitinib, vandetanib, pazopanib, axitinib and cabozantinib) have been
approved as anti-angiogenic drugs [10-13]. However, some adverse effects have been observed
during their clinical use, such as bleeding complications, indicating that development of safer
VEGFR-2 inhibitors is still an active field of research.
As a member of the protein tyrosine kinase family, VEGFR-2 intracellular domain (catalytic domain)
has the conservative bi-lobed structure observed in all protein kinases (C-terminal lobe and N-
terminal lobe). The catalytic cleft is formed between these two lobes and consists of residues
contributed by both lobes [14]. The kinase domain has an ATP binding cleft located between these
two lobes. The C-terminal lobe contains an activation loop (A-loop) marked by a conserved triad
aspartate-phenylalanine-glycine (DFG) motif located at its beginning [15]. The A-loop can adopt
numerous conformations and according to the 3D orientation of the DFG motif, the protein kinase
structure can be found in the active or inactive conformation [16,17]. In the active state (DFG-in), the
A-loop is positioned away from the catalytic center (open conformation) exposing the residues
responsible for the protein substrate binding, simultaneously, it directs the catalytic aspartic acid into
the ATP binding pocket. Upon DFG motif flipping into an inactive (DFG-out) conformation, the A-
loop adapts a closed conformation leaving behind a hydrophobic back pocket nearby the ATP-
binding cleft [16]. This hydrophobic pocket is important for the binding of a group of the tyrosine
kinase inhibitors [16].
Generally, VEGFR-2 inhibitors are classified according to whether they competitively bind to the
ATP binding pocket in the active “DFG-in” conformation (type I) or the inactive “DFG-out”
conformation (type II), or noncompetitively via binding outside the ATP binding pocket in the
allosteric hydrophobic back pocket (type III) [15-18]. Type I inhibitors act on the active “DFG-in”
conformation via hydrogen bonding with the hinge region amino acid Cys919 and hydrophobic
interactions in and around the adenine region [17]. Type II inhibitors such as sorafenib occupy the
ATP binding site and extend over the gatekeeper Val916 into the adjacent allosteric hydrophobic
3

ACCEPTED MANUSCRIPT
back pocket in the inactive “DFG-out”conformation [17-20]
. Type III inhibitors bind to the inactive
“DFG-out”conformation beyond the gatekeeper Val916 exclusively to the less conservative allosteric
hydrophobic back pocket outside the ATP binding pocket locking VEGFR-2 in the inactive “DFG-
out”conformation. Therefore, they are expected to have superior selectivity profiles and offer new
opportunities for scaffold development [17,20].
Over the last few years, a wide range of VEGFR-2 inhibitors have been designed and synthesized
[21-23]. For example, 2-furylbenzimidazoles were reported as promising anti-angiogenic agents
through VEGFR-2 inhibition. 2-(5-Methylfuran-2-yl)-1H-benzimidazole (NP-184) was reported as a
potent anti-angiogenic agent. It was discovered to reduce the angiogenesis in vivo in material plug
assay model [24,25]. PJ-6 was also found to inhibit endothelial cells proliferation and migration when
evaluated for its anti-angiogenic activity. Also, PJ-8, 5-benzoyl-2-(5-methyl-2-furyl)-1H-
benzimidazole significantly inhibited VEGFR-2 and suppressed tumor-induced angiogenesis in vivo
(
Figure 1) [26].
The present work is a continuation of our effort to design novel anti-angiogenic 2-
furylbenzimidazoles [27,28]. This pursue is inspired by our development of the effective 2-
furylbenzimidazole derivatives A and B (Figure 1) which have structural resemblance to NP-184
[24,25], Pj-6 and Pj-8 [26]. Compounds A and B showed more potent antitumor properties against
breast carcinoma cell line MCF-7 than tamoxifen as well as potent anti-angiogenic activity against
VEGFR-2 [27,28].
4

ACCEPTED MANUSCRIPT
O
O
N
N
N
N
H
O
Me
N
O
Me
N
H
O
Me
NP184
Pj-6
Pj-8
N
N
N
N
O
O
O
NH
N
H C
3
CH3
A
B
Figure 1. Anti-angiogenicbenzimidazole based-compounds
The VEGFR-2 inhibitory activity of this series is assumed to be mediated through accommodation of
the benzimidazole ring into the allosteric hydrophobic back pocket of the VEGFR-2 in the inactive
“DFG-out” conformation [27] as was inferred from X-ray crystallography and molecular docking
studies [27,29]. Increasing the hydrophobicity of the benzimidazole ring for instance by addition of
trifluoromethyl substitution leads to increase in the VEGFR-2 inhibitory activity as it increases the
hydrophobic interaction with the hydrophobic pocket [29]. Further interactions may also take place
with the side chain carboxylate of Glu885 of the αC helix and/or with Asp1046 in the conserved DFG
motif through hydrogen bonding with the appropriate moieties in the inhibitors [27,29].
In the current work, starting from the reported inhibitor NP184 in figure 1, new analogues were
designed (Figure 2), such that modification on the benzimidazole ring was carried out through the
introduction of various hydrophobic groups such as alkyl, phenyl and heterocyclic ring systems
through a spacer or without a spacer in position 1. Also, substitution at position 5 of the furyl moiety
was carried out with a methylene morpholino group. The aim of this suggested modification was to
increase the hydrophobic nature of the newly synthesized derivatives in order to increase their
binding affinity to VEGFR-2 through increasing the hydrophobic interaction with its allosteric
hydrophobic back pocket.
5

ACCEPTED MANUSCRIPT
N
N
O
CH3
H or H C N
O
2
H
NP184
CH R (R = -CN, -COOH, -COOMe, -COOEt, -CONH , -CONHNH )
2
2
2
COR (R = alkyl, heterocycle)
N
(CH2)n
(n = 0, 1; X = CH , N, O, S)
2
X
R
Phenyl,
Het.
(
R = -H, -CN, -CONH2)
Figure 2. Schematic representation showing the designing strategy of 2-(2-furyl)-1H-benzimidazoles
The designed novel 2-furylbenzimidazole derivatives were synthesized and their antitumor activity
against MCF7 (breast) and HepG2 (hepatocellular) carcinoma cell lines was evaluated. Moreover, in
vitro evaluation of the synthesized compounds for their effect on the level of VEGFR-2 in MCF-7
cell line was carried out.
Molecular docking studies were performed to find out the possible binding mode of the newly
synthesized compounds with VEGFR-2, and to study their interaction with the receptor hot spots
(key amino acids) with the aim of explaining their VEGFR-2 inhibitory activity.
2
. Results and Discussion
.1. Chemistry
2
2
1
-Furylbenzimidazoles 4a,b were synthesized by the reaction of furfural bisulfite adducts 2a,b with
,2-phenylenediamine (3) under reflux [28,30,31]. Treatment of the benzimidazoles 4a,b with either
ethyl or methyl bromoacetate afforded the N-alkylated products 5a,b or 6a,b, respectively [27].
Hydrolysis of the formed methyl esters 6a,b to the corresponding carboxylic acids 7a,b was achieved
by refluxing in methanol under basic conditions. Reaction of the esters 5a,b with hydrazine hydrate
afforded the 1H-benzo[d]imidazol-1-yl-acetohydrazides 8a,b. Cyclization of the acid hydrazides 8a,b
to the corresponding oxadiazoles 9a,b was achieved by their reaction with formic acid or
trichloroacetic acid in the presence of POCl , respectively. The acid hydrazides 8a,b were also
3
6

ACCEPTED MANUSCRIPT
condensed with 2-chlorobenzaldehyde under basic conditions to afford the Schiff bases 10a,b,
respectively (Scheme 1).
N
N
(i)
OH
H
(ii)
(iii)
R
CHO
R
C
N
O
O
O
3
R
N
H
O
NaO S
R
3
1
O
4
2
O
6
(iv)
(v)
N
N
O
R
N
O
N
O
R
O
HOOC
5
7
(vi)
N
N
N
O
N
N
R
(viii)
(vii)
Cl
H
N
N
O
O
R
R
N
O
H
N
N
N
H N
2
10
O
O
R1
8
1
9
9
a, R = CCl
3
1
b, R = H
1
1
a, 2a, 4a, 5a, 6a, 7a, 8a, 9a, 10a; R = H
b, 2b, 4b, 5b, 6b, 7b, 8b, 9b, 10b; R = CH3
Reagents and conditions: (i) Na S O , H O, methanol, stirring, r.t., 15 min; (ii) 1,2-phenylenediamine, DMF,
2
2
5
2
reflux, 4 h; (iii) BrCH COOMe, anhydrous K CO , acetone, reflux, 8 h; (iv) BrCH COOEt, anhydrous K CO ,
2
2
3
2
2
3
acetone, reflux, 8 h; (v) methanol, anhydrous K CO , reflux, 4 h; (vi) NH NH .H O, methanol, reflux, 4 h; (vii)
2
3
2
2
2
2
R COOH, POCl , reflux, 20 h; (viii) 2-Chlorobenzaldehyde, 10% NaOH, ethanol, stirring, r.t., 4 h.
3
Scheme 1.Synthesis of benzimidazole-furan hybrids 4-10
Cyanomethylation of 4a by chloroacetonitrile in the presence of anhydrous potassium carbonate in
dry acetone gave the benzimidazole derivative 11. Subsequently, 11 was reacted with hydrazine
hydrate to give the acetimidohydrazide derivative 12. The thiadiazole derivative 13 was obtained by a
cyclization reaction of 12 with carbon disulphide in the presence of potassium hydroxide. Alkylation
of 13 with ethyl bromoacetate, in acetone containing anhydrous potassium carbonate, gave ethyl-2-
((5-((2-(furan-2-yl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3,4-thiadiazol-2-ylthio)acetate (14) which
7

ACCEPTED MANUSCRIPT
was converted to the corresponding hydrazide 15 by refluxing with hydrazine hydrate in methanol
(Scheme 2).
N
N
N
N
N
(
i)
(ii)
(iii)
N
H
O
O
O
H
N
4a
NC
H N
2
NH
1
1
1
2
N
N
N
N
N
N
(iv)
(v)
O
O
O
S
S
S
EtO
H NHN
2
N
N
N
S
S
S
N
N
N
H
O
O
1
3
14
15
Reagents and conditions: (i) ClCH CN, anhydrous K CO , acetone, reflux, 8 h; (ii)
2
2
3
NH NH .H O, methanol, reflux, 4 h; (iii) CS , KOH, ethanol, reflux, 10 h; (iv)
2
2
2
2
BrCH COOEt, anhydrous K CO , acetone, reflux, 4 h; (v) NH NH .H O, methanol, reflux,
2
2
3
2
2
2
4
h.
Scheme 2. Synthesis of benzimidazole-furan hybrids 11-15
2
-(2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)-2-(4-oxo-3-phenylthiazolidin-2-ylidene)acetonitrile
(16) was obtained through reaction of the cyanomethyl derivative 11 with phenylisothiocyanate
followed by cyclization using ethyl bromoacetate. Formation of the thiazolidine-4 one derivative 17
was performed by the reaction of 11 with thioglycolic acid. Hydrolysis of 11 to the corresponding
amide was achieved by refluxing with piperidine in ethanol. Knoevenagel condensation of 18 with
benzaldehyde or 2-chlorobenzaldehyde under basic condition yielded the corresponding ylidenes
1
9a,b, respectively (Scheme 3).
8

ACCEPTED MANUSCRIPT
N
N
N
N
(iv)
N
N
(i), (ii)
O
O
O
Ph
CN
N
H N
2
NC
11
O
S
O
18
16
(iii)
(v)
N
N
N
N
O
O
Ar
HN
H N
2
O
S
O
1
7
1
9a Ar = Ph
9b Ar = 2-Cl-Ph
1
Reagents and conditions: (i) Phenyl isothiocyanate, KOH, DMF, r.t., 30 min; (ii)
BrCH COOEt, stirring, 2 h; (iii) SHCH COOH, glacial acetic acid, ethanol,
2
2
reflux, 8 h; (iv) Ethanol, piperidine, reflux, 8 h; (v) RCHO, 10 % KOH solution,
ethanol, stirring, r.t., 4 h.
Scheme 3. Synthesis of benzimidazole-furan hybrids 16-19
Aminoalkylation of the furan ring of 4a using morpholine and paraformaldehyde was achieved
giving 20. 2-Chloro-1-(2-(furan-2-yl)-1H-benzo[d]imidazol-1-yl)ethan-1-one (21) was synthesized
by substitution reaction of the imidazolyl NH of 4a with chloroacetyl chloride. Compound 21 was
reacted with thiosemicarbazide or guanidine hydrochloride to afford the corresponding cyclized
products 22 and 23, respectively (Scheme 4).
9

ACCEPTED MANUSCRIPT
N
N
N
N
(
ii)
(iv)
N
H
O
N
O
O
O
N
4
a
21
SH
Cl
HN
N
(i)
2
2
(iii)
N
O
N
N
N
H
O
20
N
O
N
NH
HN
23
Reagents and conditions: (i) Morpholine, paraformaldehyde, HCl, methanol, reflux, 2 h; (ii) Chloroacetyl
chloride, anhydrous K CO , acetone, stirring, r.t., 8 h; (ii) Thiosemicarbazide, anhydrous K CO , acetone,
2
3
2
3
stirring, r.t., 8 h; (iv) Guanidine.HCl, K CO anhydrous, acetone, stirring, r.t., 8 h.
2
3
Scheme 4. Synthesis of benzimidazole-furan hybrids 20-23
Dimerization of 5-methyl-2-furyl-benzimidazole (4b) was achieved by its reaction with 0.5
equivalent diethyl carbonate or diethyl oxalate under acidic conditions to give the bis-
benzimidazoles24 and 25, respectively (Scheme 5).
N
N
N
N
O
N
O
O
(ii)
(i)
O
O
O
N
H
O
N
N
2
N
O
4
b
N
4
25
Reagents and conditions: (i) Diethyl carbonate, ethanol, conc. HCl, stirring, r.t., 5 h;
(
ii) Diethyl oxalate, ethanol, conc. HCl, stirring, r.t., 5 h.
Scheme 5. Synthesis of bisbenzimidazoles 24, 25
2
.2. Biological Studies
.2.1. In vitro anti-proliferative activity
2
The synthesized benzimidazoles were evaluated for their in vitro cytotoxic activity against human
breast cancer cell line MCF-7 and liver carcinoma cell line HepG-2 using SRB assay [32,33]. The
IC values of the tested compounds as well as the positive controls are summarized in Table 1.
5
0
1
0

ACCEPTED MANUSCRIPT
From the data obtained (Table 1), it is obvious that compounds 10b and 15 exhibited promising
activities (IC = 21.25 and 21.35µM, respectively) against breast carcinoma cell line MCF7 with in
5
0
comparison to tamoxifen (IC = 21.57µM). Additionally, compounds 10a, 14 and 17 (IC = 26.05,
5
0
50
2
8.50 and 29.13 µM, respectively) showed mild activities against MCF-7. Compounds 10a, 10b, 15
and 17 showed higher potency (IC = 25.95, 22.58, 26.94 and 31.06 µM, respectively) than that of
5
0
cisplatin (IC50 = 31.16 µM) against liver carcinoma cell line HepG2.
2
.2.2. In vitro effect of the synthesized compounds on the level of VEGFR-2 in MCF-7 cell line
Vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor (VEGFR-
) stimulate angiogenesis in tumor tissues thus promoting their nourishment and progression [34,35].
2
Recent studies demonstrated that VEGFR-2 is up-regulated in MCF-7 cell line [36]. The effect of the
newly synthesized molecules as well as tamoxifen, which was previously reported to have anti-
angiogenic effect through inhibition of VEGFR-2 [37,38], on the level of human vascular endothelial
growth factor receptor (VEGFR-2) was determined utilizing breast cancer cell line MCF-7 obtained
from the American Type Culture Collection (Rockville, MD, USA). This biological study was
undertaken utilizing double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to
assay the level of human VEGFR-2 in treated human breast cancer cell line MCF-7 samples as
compared to the level in the untreated cells. The observed results were depicted in Table 1. Most of
the tested compounds showed considerable inhibitory activity. Four compounds 10a,b, 14, and 15
showed 92-96% reduction in VEGFR-2 level in contrast to tamoxifen and sorafenib which showed
inhibition percentage of 98% and 95.75%, respectively.
Table 1. In vitro cytotoxic activity of the synthesized benzimidazoles against human hepatocellular
HepG2 and breast cancer MCF7cell lines as well as their effect on VEGFR-2 level in MCF-7 cell
line.
a
Compd.
HepG-2, IC50
MCF-7,
VEGFR-2
level (ng/ml)
2761.61
VEGFR-2
a
c
(
µ
g/ml, M)
µ
IC , (
µg/ml,
µM)
inhibition (%)
5
0
4a
4b
5a
5b
18.913, 102.53
20.300, 102.54
>50,>185.19
>50,>176.06
19.458,105.64
14.000, 55.55
15.200, 57.29
17.800, 63.53
43
39
57
25
2980.74
2210.12
3520.40
1
1

ACCEPTED MANUSCRIPT
6
6
7
7
8
8
9
9
a
b
a
b
a
b
a
b
21.087, 67.29
20.653, 80.60
23.510, 87.04
18.913, 63.08
35.610, 139.06
20.200, 79.22
22.400, 68.27
>50,>177.31
>50,>130.89
9.860, 26.05
8.330, 21.25
19.524, 88.13
17.391, 53.13
21.304, 52.61
11.413, 28.50
8.293, 21.35
21.848, 55.87
9.390, 29.13
28.355, 117.61
12.568, 38.62
14.575, 40.04
15.435, 55.46
19.633, 60.89
22.510, 60.52
24.524, 93.02
24.200, 58.34
29.510, 65.56
8.000, 21.57
4.33±0.60
1926.33
61
38
61
20
19
24
---
---
94
96
39
27
14
92
96
55
86
13
80
57
58
40
40
22
9
>50, >185.19
35.963, 148.54
>50, >195.31
>50, >195.31
>50, >185.19
37.000, 132.14
32.610, 85.57
9.680, 25.95
8.93, 22.58
3010.51
4200.63
3896.30
3942.11
3530.55
---
---
10a
290.20
10b
190.80
1
1
2
3
4
5
6
7
8
19.524, 88.13
19.022, 74.52
15.000, 47.56
24.458, 62.07
10.400, 26.94
15.611, 39.60
9.230, 31.06
37.568, 157.34
20.611, 63.04
16.304, 44.82
36.200, 127.9
19.633, 60.89
18.153, 62.05
16.630, 63.54
>50, >118.48
>50, >111.11
----
2987.11
3540.22
4195.46
362.80
1
1
1
1
1
1
1
210.60
1480.33
535.90
1926.33
961.85
19a
19b
1358.42
1329.61
2910.19
2910.61
3790.38
4420.65
4002.00
121.51
2
0
1
2
3
4
5
2
2
2
2
2
17
98
95.75
---
0
tamoxifen
Sorafenib
cisplatin
----
206.33±22.10
---
9.348, 31.16
---
---
DMSO
---
4855.00
a
The concentration required to produce 50% inhibition of cell growth compared to control experiments;
Data were expressed as mean of four independent experiments; Percentage changes as compared with
b
c
control untreated cells.
1
2

ACCEPTED MANUSCRIPT
2
.2.3. In vitro effect of the synthesized compounds on VEGFR-2
2
-Furylbenzimidazoles 10a, 10b, 15, which showed high % of inhibition on the level of
VEGFR-2 in MCF-7 cell line, were further measured for their ability to inhibit human
VEGFR-2 in vitro using Enzyme-Linked Immunosorbent Assay. IC50 of the tested
compounds were depicted in table 2. The results of the tested compounds were compared with
sorafenib as a positive control. From the observed results, it was found that 10a showed
promising IC against VEGFR-2 (IC = 0.64 µM) in comparison to sorafenib (IC = 0.1
5
0
50
50
µM). Compounds 10b and 15 showed moderate activity, IC50 = 1.26 and 1.41 µM,
respectively.
Table 2. IC (
µ
M) of selected compounds on VEGFR-2
Compound IC (
5
0
a
µ
M) against VEGFR-2
5
0
1
1
1
0a
0b
5
0.64
1.26
1
.41
.1
Sorafenib
a
0
The concentration required to produce 50% inhibition of cell growth
compared to control experiments.
2
.3. Molecular docking study and structure activity relationship (SAR)
Molecular docking was carried out with the aim of explaining the promising VEGFR-2 inhibitory
activity of the newly synthesized compounds through investigating their binding mode and their
interaction with the key amino acids (hot spots) in the active site of the VEGFR-2.
Several crystal structures are available in the protein data bank for VEGFR-2 [16], for this work we
selected (PDB ID: 4ASD) [39] which has VEGFR-2 in the inactive “DFG-out” conformation co-
crystallized with sorafenib as type II inhibitor. First, validation of the molecular docking protocol was
performed by re-docking of the co-crystallized ligand (sorafenib) in the VEGFR-2 active site. The re-
docking validation step reproduced the experimental binding pattern of the co-crystallized ligand
efficiently indicating the suitability of the used protocol for the planned docking study as
demonstrated by the small RMSD of 0.47Å between the docked pose and the co-crystallized ligand
(energy score (S) = −15.19 kcal/mol) and by the capability of the docking pose to reproduce all the
1
3

ACCEPTED MANUSCRIPT
key interactions accomplished by the co-crystallized ligand with the key amino acids (hot spots) in
the active site (Glu885, Cys919 and Asp1046) (Figure 3)
(
A)
(B)
Figure 3. (A) Superimposition of the docking pose (red) and the co-crystallized (blue)of sorafenib in the
VEGFR-2 active site with RMSD of 0.47Å. (B) 2D interaction diagram showing sorafenib docking pose
interactions with the key amino acids (hot spots) in the VEGFR-2 active site.(Distances in Å)
Table 3. Docking energy scores (S) in kcal/mol for the newly synthesized compounds and the
reference compounds in VEGFR-2 active site.
Energy score (S)
Compound
kcal/mol
4a
4b
5a
5b
6a
6b
7a
7b
8a
8b
9a
-9.02
-9.17
-10.19
-9.96
-9.42
-9.90
-9.67
-10.37
-9.17
-9.43
-10.30
14

ACCEPTED MANUSCRIPT
9
b
-11.84
-12.34
-13.44
-10.63
-9.83
1
0a
0b
1
1
1
2
3
4
5
6
7
8
1
1
1
1
1
1
1
-10.39
-12.38
-12.54
-13.79
-10.94
-9.15
1
9a
9b
-12.29
-13.25
-11.22
-9.80
1
2
0
1
2
3
4
5
2
2
2
2
2
-11.28
-10.35
-13.60
-14.07
-12.05
−15.19
Tamoxifen
Sorafenib
For compounds 4a, 4b, 11 and 20, the molecular docking study showed a predicted binding pattern
involving the fitting of the 2-furylbenzimidazole moiety in the middle of the active site at the
interface between the ATP binding site and the allosteric hydrophobic back pocket. Interaction takes
place through hydrogen bonding with the side chain carboxylate of Glu885 of the αC helix and with
1
5

ACCEPTED MANUSCRIPT
Asp1046 in the conserved DFG motif (Figure 4). As an exception, compound 11 interacts only with
Asp1046 as it has substituted nitrogen at position 1 and not NH as the rest of these compounds.
(A)
(B)
Figure 4. 2D diagram (A) and 3D representation (B) of compound 4b showing its interaction with the
VEGFR-2 receptor active site (Distances in Å).
Apart from compounds 4a, 4b, 11 and 20, the general predicted binding pattern of the newly
synthesized compounds according to the performed docking study involves the accommodation of
the 2-furylbenzimidazole moiety in the allosteric hydrophobic back pocket in line with the reported
binding mode (Figures 5-7) [27,28]. This binding pattern accomplishes a hydrophobic interaction
between the hydrophobic 2-furylbenzimidazole moiety and the hydrophobic back pocket which is
lined with the hydrophobic side chains of Ile888, Leu889, Ile892, Val898, Val899, Leu1019 and
Ile1044 amino acids achieving the promising VEGFR-2 inhibitory activity of the newly synthesized
compounds.
In addition to the 2-furylbenzimidazole hydrophobic interaction, most of the compounds performed
further interactions with the VEGFR-2 binding site through hydrogen bonding with the side chain
carboxylate of Glu885 of the αC helix and/or with Asp1046 in the conserved DFG motif (Figures 5-
7). This type of binding resembles VEGFR-2 type III inhibitors binding mode. (For further details see
supporting information).
16

ACCEPTED MANUSCRIPT
Compounds 10a and 10b showed both the 2-furylbenzimidazole hydrophobic interaction and the
hydrogen bonding interactions with the side chain carboxylate of Glu885 and with DFT motif
Asp1046 in the active site (figures 5 and 6). Moreover, this binding pattern allows 10a and 10b to
accommodate their o-chlorophenyl moiety in the vicinity of the hydrophobic side chains of Val848,
Lys868, Leu889, Val916 and Phe1047 amino acids to be involved in a hydrophobic interaction with
these hydrophobic side chains maximizing the binding interaction of both compounds with the
VEGFR-2 active site. This binding pattern of compounds 10a and 10b ensures that they have the best
VEGFR-2 inhibitory activity among the newly synthesized compounds (Table 3).
In compound 10b, the presence of a 5-methyl substitution on the furan ring increases the hydrophobic
interaction with the allosteric hydrophobic back pocket and so increases the binding interaction as
can be noticed in its docking score (−13.44 kcal/mol) as compared to that of its analogue 10a (−12.34
kcal/mol) (Table 3). This superiority is reflected in the effect on VEGFR-2 level as shown in table 1
(VEGFR-2 inhibition % of 98% in 10b vs. 96% in 10a).
(A)
(B)
Figure 5. 2D diagram (A) and 3D representation (B) of compound 10a showing its interaction with the
VEGFR-2 receptor active site. (Distances in Å)
17

ACCEPTED MANUSCRIPT
(A)
(B)
Figure 6. 2D diagram (A) and 3D representation (B) of compound 10b showing its interaction with the
VEGFR-2 receptor active site. (Distances in Å)
Increasing the size and the hydrophobic nature of the substituent at position 1 of the benzimidazole
ring increases the binding interaction with VEGFR-2 active site as indicated by the better docking
score of the compounds with cyclic ring substituent as compared to those without ring substituent
(e.g. compound 17 vs. 18 with docking score −10.94 vs.−9.15 kcal/mol, respectively and VEGFR-2
inhibition% of 86% vs. 13%, respectively. Also, the binding gets stronger when the ring substituent is
extended with a long chain, for example, compounds 14 and 15 with ring substitution (thiadiazole
moiety) at position 1 of the benzimidazole ring, extended with ethyl propionate moiety in compound
14 or with acid hydrazide moiety in compound 15, showed better binding interaction with the active
site as indicated in their docking scores (Table 3) compared to that of compound 13 which has no
chain extension (−12.38 and−12.54, respectively vs. −10.39 kcal/mol). This higher binding affinity is
reflected in their VEGFR-2 inhibitory activity (VEGFR-2 inhibition% = 92% and 96% vs.14% (table
1). Figure 7 shows the binding pattern of compound 15.
18

ACCEPTED MANUSCRIPT
(A)
(B)
Figure 7. 2D diagram (A) and 3D representation (B) of compound 15 showing its interaction with the
VEGFR-2 receptor active site. (Distances in Å)
In summary, for the 2-furyl-bezimidazoles to achieve high binding affinity (a) 5-Methyl substituent
on the furan ring is advantageous to increase the hydrophobic interaction with the allosteric
hydrophobic back pocket of VEGFR-2 active site. (b) Suitable hydrogen bond donor and acceptor to
interact with Glu885 and Asp1046, respectively. (c) Ring substituent with long chain extension at
position 1 of the benzimidazole for hydrophobic binding interaction with the hydrophobic side chains
of Val848, Lys868, Leu889, Val916 and Phe1047 amino acids at the interface between the ATP
binding site and the allosteric back pocket.
3
2
1
. Conclusion
-Furylbenzimidazoles are promising VEGFR-2 inhibitors, from the synthesized series, compounds
0a, 10b, 14 and 15 showed potent reductions in VEGFR-2 level in MCF-7 cell line in comparison to
tamoxifen and sorafenib. Compound 10a was found to have promising VEGFR-2 inhibitory activity
IC = 0.64 µM) in comparison to sorafenib (IC = 0.1 µM). Molecular docking attributed their high
(
5
0
50
potency to their type III inhibitors-like binding mode. They bind to the VEGFR-2 active site through
hydrogen bonding interaction with the key amino acids in VEGFR-2 active site, Glu885
andAsp1046, and their hydrophobic interaction by their 2-furyl-benzimidazole moiety with the
allosteric hydrophobic back pocket which is lined with the hydrophobic side chains of Ile888,
Leu889, Ile892, Val898, Val899, Leu1019 and Ile1044 amino acids. The binding interaction is
19

ACCEPTED MANUSCRIPT
augmented by the presence of a ring substituent with long chain extension at position 1 of the
benzimidazole due to its hydrophobic interaction with the hydrophobic side chains of Val848,
Lys868, Leu889, Val916 and Phe1047 amino acids at the interface between the ATP binding site and
the allosteric back pocket.
4
. Experimental
All chemicals were purchased from commercial suppliers. Analytical thin layer chromatography
TLC) was performed on precoated silica gel 60 F245aluminium plates (Merck) with visualization
(
under UV light. Melting points were determined on a Stuart SMP30 melting point apparatus with
open capillary tubes and are uncorrected. Elemental analysis and spectral data of the compounds were
performed in the Micro analytical labs, National Research Centre and Micro Analytical Laboratory
-1
Center, Faculty of Science, Cairo University, Cairo, Egypt. IR spectra (4000–400cm ) were recorded
using KBr pellets in a Jasco FT/IR 300E Fourier transform infrared spectrophotometer on a Perkin
1
13
Elmer FT-IR 1650(spectrophotometer). HNMR and CNMR spectra were recorded at 500 (125)
MHz, 400 (100) MHz and 300 (75) MHz on Jeol EX-, Bruker and Murcury instruments, respectively
using DMSO-d as a solvent. Coupling constants are reported in Hertz (Hz).Low resolution electron
6
impact mass spectra (EI-LRMS) were recorded at 70 eV on a Finnigan SSQ 7000 instrument. The
intensities are reported as percentages relative to the base peak (I = 100%).Chemical shifts are given
in parts per million (ppm) relative to TMS as internal standards
.
4
4
.1. Chemistry
.1.1. General procedure for the synthesis of 4a,b [28,31]
A solution of furfural (1a) (4.51 g, 47 mmol) or 5-methylfurfural (1b) (5.17 g, 47 mmol) in methanol
75 ml) was stirred for 10 min followed by addition of saturated solution of Na S O (10 ml). The
(
2
2
5
mixture was stirred at r.t. for 15 min followed by cooling in deep fridge overnight. Precipitated
furfural bisulfite adduct 2a,b was filtered and dried. A solution of 1,2-phenylenediamine (3) (4.10 g,
3
8 mmol), and the furfural bisulfite adduct (7.60 g, 38 mmol) or 5-methylfurfural bisulfite
adduct(8.13 g, 38 mmol) in DMF (40 ml) and the mixture was stirred under reflux for 4 h. The
reaction mixture was poured onto ice / water (200 ml) to give the corresponding crude product which
was collected by filtration and further purified by recrystalization from methanol.
2
0

ACCEPTED MANUSCRIPT
4
.1.1.1 2-(Furan-2-yl)-1H-benzo[d]imidazole (4a)[28,31]
Buff microcrystals; yield 80% (5.59 g); mp 285-287 °C (lit.mp:285-287 °C); R = 0.57 (petroleum
f
1
ether /EtOAc = 2:1); IR (KBr):
ṽ
3442, 3059, 1628, 1520; H-NMR (DMSO-d , 500 MHz):δH 6.73
6
(dd, J = 3.3, 1.8 Hz, 1H), 7.18-7.23 (m, 3H), 7.53-7.58 (m, 2H), 7.93 (dd,J = 2.7 Hz, 1.8 Hz, 1H),
1
3
1
1
7
2.88 (br s, 1H); C-NMR (DMSO-d , 125 MHz): δC 111.1, 112.9, 115.7, 115.8, 122.7, 123.4,
6
23.5, 144.2, 145.4, 145.6, 146.1; Anal. Calcd for C11
1.69; H, 4.11; N, 15.45.
8 2
H N O: C, 71.73; H, 4.38; N, 15.21. Found: C,
4
.1.1.2. 2-(5-Methylfuran-2-yl)-1H-benzo[d]imidazole (4b) [28]
Buff microcrystals; yield 85% (6.35 g); mp 272-274 °C (lit.mp: 275-277 °C); R = 0.71 (petroleum
f
1
ether /EtOAc = 2:1); IR (KBr):
ṽ
3423, 3050, 2950, 1631, 1505; H-NMR (DMSO-d
6
, 300 MHz):
δ
H
2
1
.39 (s, 3H), 6.32 (d, J = 2.7 Hz, 1H), 7.08 (d, J = 3.0 Hz, 1H), 7.15-7.19 (m, 2H), 7.54-7.56 (m, 2H),
1
3
3.14 (br s, 1H); C-NMR (DMSO-d , 75 MHz): δC 13.5, 108.6, 111.6, 122.1, 143.9, 144.1, 153.8;
6
10 2
Anal. Calcd for C12H N O: C, 72.71; H, 5.08; N, 14.13. Found: C, 72.55; H, 4.87; N, 14.48.
4
.1.2. General procedure for the synthesis of 5a,b [28]
Ethyl bromoacetate (1.08 g, 6.5 mmol) was added dropwise to a suspension of 4a (1.19 g, 6.5 mmol)
or 4b (1.29 g, 6.5 mmol) and anhydrous K CO (0.90 g, 6.5 mmol) in dry acetone (20 ml). The
2
3
mixture was stirred under reflux for 8 h. The mixture was then poured onto ice / water (100 ml), and
the resulting precipitate was collected by filtration and recrystallized from ethanol to give the
corresponding derivative 5a or 5b.
4
.1.2.1. Ethyl 2-(2-(furan-2-yl)-1H-benzo[d]imidazol-1-yl)acetate (5a) [28]
f
Gray microcrystals; yield 82% (1.43 g); mp151-153 °C (lit.mp: 150-152 °C); R = 0.62 (petroleum
1
ether /EtOAc = 1:1); H-NMR (DMSO-d , 400 MHz): δH 1.19 (t, J = 7.0 Hz, 3H), 4.17 (q, J = 7.0
6
Hz, 2H), 5.45 (s, 2H, CH
2
), 6.74 (t, J = 2.8 Hz, 1H), 7.21-7.29 (m, 3H), 7.67-7.69 (m, 2H), 7.80-7.94
1
3
(
m, 1H); C-NMR (DMSO-d , 100 MHz): δC 14.5, 44.6, 61.7, 110.8, 112.6, 112.8, 119.4, 123.0,
6
1
23.3, 136.3, 142.8, 144.3, 145.6, 169.6; Anal. Calcd for C H N O : C, 66.66; H, 5.22; N, 10.36.
15 14 2 3
Found: C, 66.35; H, 5.50; N, 10.12.
4
.1.2.2. Ethyl 2-(2-(5-methylfuran-2-yl)-1H-benzo[d]imidazol-1-yl)acetate (5b)[28]
2
1

ACCEPTED MANUSCRIPT
f
Gray microcrystals; yield 78% (1.44 g); mp 90-92 °C (lit.mp 88-90 °C); R = 0.50 (petroleum ether
1
/
EtOAc 1:1); H-NMR (DMSO-d , 300 MHz): δH 1.19 (t, J = 7.0 Hz, 3H), 2.35 (s, 3H), 4.18 (q, J =
6
7
.0 Hz, 2H), 5.41 (s, 2H), 6.35 (t, J = 2.8 Hz, 1H), 7.21 (t, J = 2.6 Hz, 1H), 7.27-7.29 (m, 2H), 7.67-
1
3
7
.69 (m, 2H); C-NMR (DMSO-d
6 C
, 75 MHz): δ 13.2, 14.0, 46.0, 52.3, 108.4, 110.1, 113.4, 118.7,
1
22.4, 135.8, 142.3, 143.3, 154.0, 169.0; Anal. Calcd for C H N O : C, 67.59; H, 5.67; N, 9.85.
1
6
16
2
3
Found: C, 67.81; H, 6.01; N, 10.10.
4
.1.3. General procedure for the synthesis of 6a,b
Methyl bromoacetate (0.99 g, 6.5 mmol) was added dropwise to a suspension of 4a (1.19 g, 6.5
mmol) or 4b (1.29 g, 6.5 mmol) and anhydrous K CO (0.90 g, 6.5 mmol) in dry acetone (20 ml).
2
3
The mixture was stirred under reflux for 8 h. The mixture was then poured onto ice / water (100 ml),
and the resulting precipitate was collected by filtration and recrystallized from ethanol to give 6a or
6
4
b.
.1.3.1. Methyl 2-(2-(furan-2-yl)-1H-benzo[d]imidazol-1-yl)acetate (6a)
f
Buff microcrystals; yield 79% (1.30 g); mp 90-92 °C; R = 0.65 (petroleum ether /EtOAc 1:1); IR
1
(
KBr):
ṽ
3046, 2954, 1745, 1612, 1509; H-NMR (DMSO-d , 300 MHz): δH 3.71 (s, 3H), 5.45 (s,
6
2
H), 6.74 (t, J = 2.4 Hz, 1H), 7.19 (dd, J = 3.3, 1.8 Hz, 1H), 7.26-7.28 (m, 2H), 7.64-7.68 (m, 2H),
1
3
7
.91-7.94 (m, 1H); C-NMR (DMSO-d
6 C
, 75 MHz): δ 46.4, 53.0, 110.9, 112.7, 113.0, 119.5, 123.1,
1
23.4, 136.4, 142.9, 144.4, 145.6, 169.5;Anal. Calcd for: C H N O : C, 65.62; H, 4.72; N, 10.93.
1
4
12
2
3
Found: C, 65.38; H, 4.56; N, 11.28.
4
.1.3.2. Methyl 2-(2-(5-methylfuran-2-yl)-1H-benzo[d]imidazol-1-yl)acetate (6b)
f
Buff microcrystals; yield 75% (1.32 g); mp 110-112 °C; R = 0.65 (petroleum ether /EtOAc 1:1); IR
1
(
KBr)
ṽ
3041, 2953, 1752, 1611, 1559; H-NMR (DMSO-d , 300 MHz): δH 2.37 (s, 3H), 3.73 (s,
6
3
2
1
6
H), 5.41 (s, 2H), 6.35 (d, J = 4.2 Hz, 1H), 7.19 (d, J = 3.3 Hz, 1H), 7.24-7.27 (m, 2H), 7.63-7.66 (m,
1
3
H); C-NMR (DMSO-d , 75 MHz): δC 13.2, 45.9, 52.3, 108.4, 110.1, 113.4, 118.7, 122.4, 135.7,
6
42.4, 143.9, 154.1, 169.0;Anal. Calcd for: C H N O : C, 66.66; H, 5.22; N, 10.36. Found: C,
1
5
14
2
3
6.42; H, 4.99; N, 10.11.
4
.1.4. General procedure for the synthesis of 7a,b
2
2

ACCEPTED MANUSCRIPT
2 3
A suspension of 6a (0.51 g, 2 mmol) or 6b (0.54 g, 2 mmol) and anhydrous K CO (0.28 g, 2 mmol)
in methanol (30 ml) was refluxed for 4 h. The mixture was filtered, the filterate was collected and
evaporated under vacuum and the precipitated product was collected, washed and recrysallized from
ethanol to give 7a and 7b, respectively.
4
.1.4.1. 2-(2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)acetic acid (7a)
Colorless microcrystals; yield 75% (0.36 g); mp 278-280 °C; R = 0.58 (petroleum ether / EtOAc
f
1
1
2
1
1
6
:1); IR (KBr)
ṽ
3430, 3010, 2923,1704, 1621, 1558; H-NMR (DMSO-d , 400 MHz): δH 5.34 (s,
6
H), 6.75 (t, J = 2.6 Hz, 1H), 7.20-7.30 (m, 3H), 7.65-7.96 (m, 2H), 7.95-7.96 (m, 1H), 13.29 (br s,
1
3
H); C-NMR (DMSO-d , 100 MHz): δC 46.0, 110.3, 112.1, 112.3, 118.9, 122.4, 122.8, 135.9, 142.3,
6
43.9, 144.9, 145.2, 169.6; Anal. Calcd for C13
4.25; H, 4.40; N, 11.15.
10 2 3
H N O : C, 64.46; H, 4.16; N, 11.56. Found: C,
4
.1.4.2. 2-(2-(5-Methylfuran-2-yl)-1H-benzo[d]imidazol-1-yl)acetic acid (7b)
Buff microcrystals; yield 78% (0.39 g); mp 258-260 °C; R = 0.71 (petroleum ether / EtOAc = 1:1);
f
1
IR (KBr)
ṽ
3424, 3009, 2956,1706, 1616, 1561; H-NMR (DMSO-d
6
, 300 MHz):
δ
H
2.36 (s, 3H), 5.26
1
3
(
s, 2H), 6.34 (d, J = 2.1 Hz, 1H), 7.06 (d, J = 3 Hz, 1H), 7.23-7.26 (m, 2H), 7.59-7.66 (m, 2H); C-
NMR (DMSO-d , 75 MHz): δC 13.3, 46.2, 108.3, 110.1, 113.3, 118.6, 122.2, 122.4, 135.9, 142.3,
6
1
6
43.4, 144.1, 153.8, 169.7; Anal. Calcd for C14
5.45; H, 4.48; N, 10.68.
12 2 3
H N O : C, 65.62; H, 4.72; N, 10.93. Found: C,
4
.1.5. General procedure for the synthesis of 8a, b [28]
Hydrazine hydrate (1.00 g, 20 mmol) was added dropwise to a solution of 5a (2.70 g, 10 mmol) or 5b
2.85 g, 10 mmol) in methanol (15 ml). The reaction mixture was refluxed for 4 h. After cooling to
(
r.t., the reaction mixture was concentrated by evaporation under reduced pressure. The crude product
was filtered, washed with small portions of cold ethanol and cold water repeatedly. Further
crystallization from ethanol afforded 8a and 8b, respectively.
4
.1.5.1. 2-(2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)acetohydrazide (8a) [28]
Buff microcrystals; yield 88% (2.25 g); mp 239-241 °C (lit.mp 240-242 °C); R = 0.70 (petroleum
f
1
ether /EtOAc = 1:1); IR (KBr):
ṽ
3325, 3254, 3131, 1650, 1611, 1510; H-NMR (DMSO-d
6
, 300
2
3

ACCEPTED MANUSCRIPT
MHz):
δ
H
4.32 (br s, 2H),5.12 (s, 2H), 6.72 (t, J = 3.0 Hz, 1H), 7.16 (dd, J = 3.6, 1.8 Hz,1H), 7.2-7.23
1
3
(
m, 2H), 7.49-7.62 (m, 2H), 7.91-7.92 (m, 1H), 9.49 (br s, 1H); C-NMR (DMSO-d , 75 MHz): δC
6
4
6.11, 110.7, 112.9, 119.3, 122.5, 123.1, 136.6, 142.9, 144.7, 145.2, 145.8, 166.7, 170.8; Anal. Calcd
12 4 2
for C13H N O : C, 60.93; H, 4.72; N, 21.86. Found: C, 60.67; H, 5.0; N, 21.55.
4
.1.5.2. 2-(2-(5-Methylfuran-2-yl)-1H-benzo[d]imidazol-1-yl)acetohydrazide (8b) [28]
Buff microcrystals; yield 90% (2.43 g); mp 244-246 °C (lit.mp 246-248 °C); R = 0.57 (petroleum
f
1
ether / EtOAc = 1:1); IR (KBr)
MHz):
.20-7.25 (m, 2H), 7.57-7.60 (m, 2H); Anal. Calcd for C H N O : C, 62.21; H, 5.22; N, 20.73.
ṽ
3348, 3252, 3131, 1660, 1612, 1510; H-NMR (DMSO-d , 300
6
H
δ 2.35 (s, 3H), 4.30 (br s, 2H),5.00 (s, 2H), 6.30 (d, J = 3.0 Hz, 1H), 7.00 (d, J = 3.0 Hz,1H),
7
1
4
14
4
2
Found: C, 62.45; H, 5.47; N, 21.00.
4
.1.6. 1-((5-(Trichloromethyl)-1,3,4-oxadiazol-2-yl)methyl)-2-(furan-2-yl)-1H-benzo[d]imid-azole
(9a)
A solution of 8a (2.50 g, 10 mmol) and trichloroacetic acid (1.60 g, 10 mmol) in phosphorus
oxychloride (15 ml) was refluxed for 20 h, followed by cooling to r.t. The mixture was poured onto
ice / water (50 ml) to give the precipitated crude product which was collected by filtration. Further
purification by crystallization from methanol afforded 9a as buff microcrystals; yield 65% (2.47 g);
1
mp 270-272 °C; R
f
= 0.67 (petroleum ether / EtOAc = 2:1); IR (KBr),
ṽ
3081, 2930, 1632, 1527; H-
NMR (DMSO-d , 400 MHz): δH 5.42 (s, 2H), 6.81 (t, J = 3.2 Hz, 1H), 7.34-7.39 (m, 3H), 7.69-7.79
6
1
3
(
m, 2H), 8.06 (dd, J = 3.3, 1.8 Hz, 1H); C-NMR (DMSO-d
16.4, 117.9, 125.5, 125.7, 134.6, 135.2, 140.7, 142.2, 148.3, 161.5, 165.6; Anal. Calcd for
C H Cl N O : C, 64.96; H, 2.36; N, 14.60. Found: C, 64.69; H, 2.70; N, 14.29.
6 C
, 100 MHz): δ 47.0, 91.5, 112.4, 113.6,
1
15
9
3
4
2
4
.1.7. 1-((1,3,4-Oxadiazol-2-yl)methyl)-2-(5-methylfuran-2-yl)-1H-benzo[d]imidazole (9b)
A solution of 8b (2.70 g, 10 mmol) and formic acid (0.50 g, 10 mmol) in phosphorus oxychloride (15
ml) was refluxed for 20 h, followed by cooling to r.t. The mixture was poured onto ice / water (50
ml) to give the precipitated crude product which was collected by filtration and further purified by
recrystalization from methanol to afford 9b as buff microcrystals; yield 70% (1.96 g); mp 260-262
1
°
C; R = 0.54 (petroleum ether /EtOAc = 2:1); IR (KBr)
ṽ
3060, 2930, 1618, 1514; H-NMR (DMSO-
f
6 H
d , 300 MHz): δ 2.39 (s, 3H), 5.12 (s, 2H), 6.33 (d, J = 2.4 Hz, 1H), 7.04 (d, J = 3.3 Hz, 1H), 7.21-
2
4

ACCEPTED MANUSCRIPT
1
3
7
1
6
.26 (m, 3H), 7.56-7.63 (m, 2H); C-NMR (DMSO-d
6 C
, 75 MHz): δ 12.4,45.6, 108.2, 110.3, 113.4,
18.6, 122.1, 122.3, 136.1, 134.6, 142.4, 143.1, 144.3, 153.9, 166.3; Anal. Calcd for C H N O : C,
1
5
12
4
2
4.28; H, 4.32; N, 19.99; Cl, 27.73. Found: C, 64.53; H, 4.01; N, 19.65; Cl, 27.55.
4
.1.8. General procedure for the synthesis of 10a,b
A solution of 8a (2.56 g, 10 mmol) or 8b (2.70 g, 10 mmol), 2-chlorobenzaldehyde (1.40 g, 10
mmol) and 10% NaOH (1 ml) in ethanol (20 ml) was stirred at r.t. for 4 h. The mixture was kept at
r.t. overnight and the formed crude product was collected by filtration and further purified by
recrystalization from methanol to give 10a and 10b, respectively.
4
.1.8.1.1-(1-(2-Chlorophenyl)-3-(2-(furan-2-yl)-1H-benzo[d]imidazol-1-yl)prop-1-en-2-
yl)hydrazine (10a)
Brown microcrystals; yield 80% (3.02 g); mp 220-222 °C; R = 0.50 (petroleum ether / EtOAc = 2:1);
f
1
IR (KBr):
ṽ
3407, 2995, 1650, 1610, 1509; H-NMR (DMSO-d
6
, 300 MHz):
δ
H
5.83 (s, 2H), 6.80 (t,
J = 2.5 Hz, 1H), 7.33-7.39 (m, 1H), 7.45-7.49 (m, 3H), 7.53-7.59 (m, 2H), 7.74-7.80 (m, 3H), 8.00-
1
3
8
4
1
9
6 C
.01 (m,1H), 8.46 (d, J = 7.8, 1H), 8.98 (s, 1H), 9.81 (br s, 1H ); C-NMR (DMSO-d , 75 MHz): δ
4.4, 110.9, 113.0, 113.9, 119.3, 123.8, 124.1, 128.0, 128.8, 130.2, 130.5, 131.2, 133.7, 134.8, 136.5,
44.4, 144.6, 146.2, 150.2, 161.7; Anal. Calcd for C H ClN O : C, 63.41; H, 3.99; N, 14.79; Cl,
2
0
15
4
2
.36. Found: C, 63.23; H, 4.25; N, 15.01; Cl, 9.16.
4
.1.8.2. 1-(1-(2-Chlorophenyl)-3-(2-(5-methylfuran-2-yl)-1H-benzo[d]imidazol-1-yl)prop-1-en-2-
yl)hydrazine (10b)
Buff microcrystals; yield 85% (3.33 g); mp 230-232 °C; R = 0.70 (petroleum ether / EtOAc = 2:1);
f
1
IR (KBr):
ṽ
3396, 3029, 2900, 1694, 1630, 1513; H-NMR (DMSO-d
6
, 300 MHz):
δ
H
2.4 (s, 3H),
5
.98 (s, 2H), 6.50 (d, J = 2.7 Hz, 1H), 7.33-7.39 (m, 3H), 7.45-7.49 (m, 3H), 7.53-7.59 (m, 2H), 8.12-
1
3
8
6 C
.15 (m,1H), 8.60 (s, 1H), 12.3 (br s, 1H); C-NMR (DMSO-d , 75 MHz): δ 13.3, 45.8, 108.5,
1
10.5, 113.7, 118.2, 122.5, 127.1, 127.6, 129.9, 131.4, 133.0, 136.1, 140.2, 143.2, 141.4, 143.8,
54.2, 168.7; Anal. Calcd for C H ClN O : C, 64.21; H, 4.36; N, 14.26; Cl, 9.36. Found: C, 63.95;
1
2
1
17
4
2
H, 4.19; N, 14.56; Cl, 9.19.
4
.1.9. 2-(2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)acetonitrile (11)
2
5

ACCEPTED MANUSCRIPT
Chloroacetonitrile (4.20 g, 56 mmol) was added dropwise to a suspension of 4a (10.30 g, 56 mmol)
and anhydrous K CO (7.73 g, 56 mmol) in acetone (30 ml). The mixture was refluxed for 8 h,
2
3
followed by addition of ice / water (200 ml). The precipitated crude product was filtered and further
purified by recrystallization from methanol to give 11 as gray microcrystals; yield 87% (10.81 g);
mp140-142 °C; R = (methylene chloride / petroleum ether = 2:1); IR (KBr)
ṽ 3052, 2917, 2194,
f
1
1
3
611, 1511; H-NMR (DMSO-d
6
, 500 MHz):
δ
H
5.80 (s, 2H), 6.79 (t, J = 3.3 Hz, 1H), 7.29-7.35 (m,
1
3
H), 7.52 (d, J = 7.6 Hz, 1H), 7.77 (d, J = 7.6 Hz, 1H), 8.04 (dd, J = 3.3, J = 1.5 Hz, 1H); C-NMR
(
DMSO-d , 125 MHz): δC34.0, 110.8, 113.0, 113.9, 116.7, 120.0, 123.9, 124.1, 135.3, 142.9, 143.6,
6
1
1
44.6, 146.2; Anal. Calcd for C13
9.13.
9 3
H N O: C, 69.95; H, 4.06; N, 18.82. Found C, 69.74; H, 3.75; N,
4
.1.10. 2-(2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)acetimidohydrazide (12)
Hydrazine hydrate (1.00 g, 20 mmol) was added dropwise to a solution of 11 (2.23 g, 10 mmol) in
methanol (15 ml). The reaction mixture was refluxed for 4 h. After cooling to r.t., the reaction
mixture was concentrated to one third by evaporation of the solvent under reduced pressure. The
crude product was collected by filtration, washed with small portions of cold ethanol and cold water
repeatedly then left to dry. The crude product was further purified by recrystallization from ethanol to
give 12 as buff microcrystals; yield 88% (2.24 g); mp 240-242 °C; R = 0.65 (petroleum ether /
f
1
EtOAc 1:1);IR (KBr)
ṽ
3325, 3254, 3131, 2930, 1611, 1510; H-NMR (DMSO-d
6
, 300 MHz):
δ
H
4
.59 (br s, 2H), 5.04 (s, 2H, CH ), 5.44 (br s, 1H), 6.69 (t, J = 1.8 Hz, 1H), 7.16-7.27 (m, 3H), 7.46-
2
1
3
7
.4 8 (m, 2H), 7.90 (dd, J = 2.1, 1.2 Hz, 1H); C-NMR (DMSO-d
6 C
, 75 MHz): δ 46.2, 111.4, 112.6,
1
13.0, 119.4, 122.6, 123.0, 136.7, 143.0, 143.8, 144.8, 145.5; Anal. Calcd for C H N O: C, 61.17;
1
3
13
5
H, 5.13; N, 27.43. Found: C, 61.45; H, 5.52; N, 27.15.
4
.1.11. 5-((-2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3,4-thiadiazole-2(3H)-thione (13)
Carbon disulfide (0.75 ml, 10 mmol) was added dropwise to an ice/cooled solution of acid hydrazide
2 (1.28 g, 5 mmol) and KOH (0.66 g, 5 mmol) in ethanol (10 ml). The reaction mixture was stirred
1
at r.t. for 2 h. After dilution with ethanol, the formed precipitate was filtered and washed twice with
ether. A solution of the formed intermediate (1.47 g, 4 mmol) in 10% KOH (10 ml) was refluxed for
1
0 h, followed by cooling to r.t. and acidification with conc. HCl (2 ml). The crude product was
filtered, washed with water, dried and crystallized from DMF to afford 13 as buff microcrystals; yield
2
6

ACCEPTED MANUSCRIPT
7
2
2
f
4% (1.16 g); mp 260-262 °C; R = 0.68 (petroleum ether /EtOAc = 1:1); IR (KBr) ṽ 3434, 3010,
1
944, 1610, 1510, 1260, 648; H-NMR (DMSO-d , 300 MHz): δH 5.97 (s, 2H, CH ), 6.75 (dd, J =
6
2
1
3
.1, 1.8 Hz, 1H), 7.28-7.34 (m, 3H), 7.68-7.70 (m, 2H), 7.99-8.00 (m, 1H), 14.20 (br s, 1H); C-
, 75 MHz): 42.4, 111.2, 113.0, 114.0, 119.5, 123.8, 124.0, 135.6, 142.2, 143.7,
44.5, 146.1, 159.2, 189.1; Anal. Calcd for C H N OS : C, 53.49; H, 3.21; N, 17.82; S, 20.40.
NMR (DMSO-d
6
δ
C
1
1
4
10
4
2
Found: C, 53.83; H, 3.52; N, 17.53; S, 20.18.
4
.1.12.Ethyl-2-((5-((2-(furan-2-yl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3,4-thiadiazol-2-
yl)thio)acetate (14)
A suspension of 13 (0.63 g, 2 mmol) and anhydrous K CO (0.28 g, 2 mmol) in dry acetone (30 ml)
2
3
was stirred at r.t. for 0.5 h, followed by addition of ethyl bromoacetate (0.33 g, 2 mmol). The reaction
mixture was refluxed for 4 h. After cooling to r.t., ice / water (100 ml) was added and the precipitated
product was filtered and recrysallized from ethanol to give 14 as buff microcrystals; yield 74% (0.59
f
g); mp 279-281 °C; R = 0.58 (petroleum ether / EtOAc = 1:3); IR (KBr) ṽ 3114, 2973, 1735, 1613,
1
1
2
3
1
510; H-NMR (DMSO-d , 300 MHz): δH 1.07 (t, J = 7.0 Hz, 3H), 4.06 (q, J = 7.0 Hz, 2H), 4.18 (s,
6
H), 6.21 (s, 2H), 6.75 (dd, J = 3.3, 1.5 Hz, 1H), 7.26-7.33 (m, 3H), 7.68-7.69 (m, 2H), 7.96 (dd, J =
1
3
.3, 1.8 Hz, 1H); C-NMR (DMSO-d , 75 MHz): δC 13.8, 35.1, 42.9, 61.3, 110.5, 112.3, 113.0,
6
19.2, 123.0, 123.2, 135.1, 142.5, 143.3, 144.4, 145.2, 165.7, 165.9, 167.7; Anal. Calcd for
18 16 4 3 2
C H N O S : C, 53.98; H, 4.03; N, 13.99; S, 16.01. Found: C, 53.64; H, 4.24; N, 13.65; S, 16.35.
4
.1.13.2-((5-((2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3,4-thiadiazol-2-
yl)thio)acetohydrazide (15)
A solution of 14 (4.00 g, 10 mmol) and hydrazine hydrate (1.00 g, 20 mmol) in methanol (15 ml) was
refluxed for 4 h, followed by cooling to r.t. Excess solvent was evaporated under vacuum and the
crude product was separated by filtration. The product was purified by recrystallization from ethanol
to give 15 as buff microcrystals; yield 85% (3.30 g); mp 250-252 °C; R
f
= 0.65 (petroleum ether /
1
EtOAc = 1:1); IR (KBr)
ṽ
3481, 3268, 3051, 2951, 1751, 1629, 1565, 660; H-NMR (DMSO-d , 400
6
MHz): δH 4.34 (s, 2H), 4.69 (br s, 2H), 5.14 (s, 2H), 6.74 (t, J = 2.5 Hz, 1H), 7.18 (dd, J = 3.3, 1.7
Hz,1H), 7.24-7.29 (m, 2H), 7.52 (d, J = 8.5 Hz, 1H), 7.54 (d, J = 8.6 Hz, 1H), 7.93-7.95 (m, 1H),
1
3
9
.53 (br s, 1H); C-NMR (DMSO-d , 100 MHz): δC 43.3, 43.6, 111.2, 112.8, 113.5, 119.7, 123.3,
6
2
7

ACCEPTED MANUSCRIPT
1
4
35.6, 143.0, 143.8, 144.9, 145.7, 153.6, 155.4, 166.1, 169.9; Anal. Calcd for
9.73; H, 3.65; N, 21.75; S, 16.59. Found: C, 49.55; H, 3.86; N, 21.93; S, 16.71.
16 14 6 2 2
C H N O S : C,
4
.1.14. 2-(2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)-2-(4-oxo-3-phenylthiazolidin-2-
ylidene)acetonitrile (16)
A solution of compound 11 (0.89 g, 4 mmol) and KOH (0.22 g, 4 mmol) in DMF (10 ml) was stirred
for 10 min, followed by addition of phenyl isothiocyanate (0.54 ml, 4 mmol) and the mixture was
stirred for additional 30 min. Ethyl bromoacetate (0.66 g, 4 mmol) was added and stirring was
continued for 2 h. The mixture was poured onto ice / water (200 ml) and the precipitated crude
product was collected by filtration. Further purification by recrystallization from methanol afforded
1
2
2
7
1
6
f
6 as buff microcrystals; yield 82% (1.30 g); mp 240-242 °C; R = 0.47 (petroleum ether / EtOAc =
1
:1); IR (KBr)
ṽ
3049, 2930, 2197, 1664, 1636, 1511; H-NMR (DMSO-d , 300 MHz):δH4.16 (s,
6
H), 6.86-6.89 (m, 2H), 7.08-7.13 (m, 2H), 7.31-7.36 (m, 3H), 7.39-7.40 (m, 2H), 7.40-7.52 (m, 2H),
1
3
.53-7.55 (m, 1H); C-NMR (DMSO-d
6 C
, 75 MHz): δ 32.8, 120.6, 124.2, 128.4, 128.9, 129.2, 135.3,
48.1, 155.8, 171.6; Anal. Calcd for C H N O S: C, 66.32; H, 3.54; N, 14.06; S, 8.05. Found: C,
2
2
14
4
2
6.02; H, 3.81; N, 13.76;S, 8.33.
4
.1.15. 2-((2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)methylene)thiazolidin-4-one (17)
Thioglycolic acid (2.73 g, 30 mmol) was added dropwiseto a solution of 11 (6.69 g, 30 mmol) in
ethanol (30 ml). The mixture was stirred under reflux for 8 h. The reaction mixture was poured onto
ice / water and the precipitated crude product was collected by filtration. Further purification by
recrystallization from ethanol afforded 17 as gray microcrystals; yield 74% (6.62 g); mp192-194 °C;
1
Rf = 0.62 (petroleum ether / EtOAc = 1:2); IR (KBr)
DMSO-d , 400 MHz):
ṽ
3289, 3096, 2922, 1705, 1644, 1508; H-NMR
(
6
δ
H
3.58 (s, 1H),5.50 (d, 2H), 6.75 (dd, J = 3.3, 1.5 Hz, 1H), 7.20-7.32 (m,
1
3
3
1
H), 7.66-7.70 (m, 2H), 7.63 (s, 1H), 8.00 (m,1H); C-NMR (DMSO-d , 75MHz): δC 46.0, 71.5,
6
10.3, 112.1, 112.5, 118.9, 122.7, 131.2, 135.9, 142.3, 144.3, 145.2, 169.6; Anal. Calcd for
C H N O S: C, 60.59; H, 3.73; N, 14.13; S, 10.78. Found: C, 60.19; H, 3.43; N, 14.43; S, 10.65.
15
11
3
2
4
.1.16. 2-(2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)acetamide (18)
Piperidine (2.55 g, 30 mmol) was added dropwise to a solution of 11 (6.69 g, 30 mmol) in ethanol
30 ml). The mixture was stirred under reflux for 8 h, the reaction mixture was poured onto ice /
(
2
8