Part I. Synthesis, biological evaluation and docking studies of new 2-furylbenzimidazoles as antiangiogenic agents

Article abstract of DOI:10.1016/j.ejmech.2014.01.063

2-(2-Furyl)-1H-benzimidazoles 3-11 were synthesized and tested for their in vitro VEGF inhibition in MCF-7 cancer cell line. Compound 5a was more potent than Tamoxifen, and compounds 3b, 5a, 5c, 6b, 7a and 10 showed promising potency. Furthermore, compounds (6b, 7a and 10) showed remarkable selective inhibition of COX-2 enzyme close to that of Celecoxcib. Additionally, docking studies were performed using AutoDock 4.2 into the VEGFR2 kinase. Significant correlation exists between the biological activity (IC₅₀ and %VEGF inhibition) against MCF-7 cell line and the molecular docking results (K_i and ΔG_b) with correlation coefficients (R²) of 0.5513 and 0.4623 respectively. Accordingly, most of the synthesized 2-(2-furyl)-1H-benzimidazoles showed strong antiangiogenic activity against VEGFR2 kinase.

Full text of DOI:10.1016/j.ejmech.2014.01.063

European Journal of Medicinal Chemistry xxx (2014) 1e13
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Part I. Synthesis, biological evaluation and docking studies of new
-furylbenzimidazoles as antiangiogenic agents
2
a
a
,
b
c
*
Ahmed Temirak , Yasser M. Shaker , Fatma A.F. Ragab , Mamdouh M. Ali ,
d,
e
a
Hamed I. Ali , Hoda I. El Diwani
Department of Chemistry of Natural and Microbial Products, Division of Pharmaceutical and Drug Industries, National Research Centre, Dokki, 12622
Cairo, Egypt
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Egypt
Department of Biochemistry, Division of Genetic Engineering and Biotechnology, National Research Centre, 12622 Cairo, Egypt
a
b
c
d
Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, TX, USA
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, Cairo, Egypt
e
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 September 2013
Received in revised form
2-(2-Furyl)-1H-benzimidazoles 3e11 were synthesized and tested for their in vitro VEGF inhibition in
MCF-7 cancer cell line. Compound 5a was more potent than Tamoxifen, and compounds 3b, 5a, 5c, 6b, 7a
and 10 showed promising potency. Furthermore, compounds (6b, 7a and 10) showed remarkable se-
lective inhibition of COX-2 enzyme close to that of Celecoxcib. Additionally, docking studies were per-
formed using AutoDock 4.2 into the VEGFR2 kinase. Significant correlation exists between the biological
24 January 2014
Accepted 31 January 2014
Available online xxx
activity (IC50 and %VEGF inhibition) against MCF-7 cell line and the molecular docking results (K
i
and
2
DG ) with correlation coefficients (R ) of 0.5513 and 0.4623 respectively. Accordingly, most of the syn-
b
Keywords:
thesized 2-(2-furyl)-1H-benzimidazoles showed strong antiangiogenic activity against VEGFR2 kinase.
2-(2-Furyl)-1H-benzimidazoles
Ó 2014 Elsevier Masson SAS. All rights reserved.
Angiogenesis
Vascular endothelial growth factor (VEGF)
VEGFR2 kinase
Cytotoxicity
1. Introduction
kinases (RTK). VEGFR2 is considered as important receptor medi-
ating of all the cellular responses to VEGF [10]. Binding of VEGF to
Angiogenesis, new blood vessels formation, plays a central role
in cancer cell survival, tumor growth which is considered as a
precondition of metastasis for all solid tumors [1e3]. This process
consists of the formation of new capillaries through splitting from
pre-existing blood vessels. There are several growth factors
involved in tumor angiogenesis which include vascular endothelial
growth factor (VEGF) [4], basic fibroblast growth factor (bFGF) [5],
epidermal growth factor (EGF) [6], platelet-derived growth factor
its family of receptors (VEGFR), is the key mediator that promotes
the proliferation and survival of endothelial cells and consequently
cancer progression [11]. Therefore, looking for an effective anti-
VEGF/VEGFR drug became the main interest for many research
groups aiming to discover a new cancer therapy via angiogenesis
inhibition.
Benzimidazole nucleus is of well known promising biological
activity [12,13]. A series of 2-heteroaryl benzimidazole derivatives
was discovered in literature as effective VEGF/VEGFR inhibitors for
antiangiogenic tumor therapy. Fig.1 includes selected 2-substituted
benzimidazole structures as angiogenesis inhibitors. 2-
Arylbenzimidazoles were reported as novel multi-target VEGFR2,
EGFR and PDGFR kinases inhibitors [14]. 4-Amino-N-[2-[4-[(4-
aminobenzoyl)amino]phenyl]-1H-benzimidazol-5-yl]benzamide 1
was discovered as a novel benzimidazole derivative with unique
antiangiogenic characteristics that inhibited the vascular endo-
thelial growth factor (VEGF) and basic fibroblast growth factor
(bFGF)-induced growth of various endothelial cells without a
cytotoxic phase [15]. A series of novel heterocyclic analogs 4-
(
PDGF) [7] and angiopoietin Ref. [8]. Indeed, vascular endothelial
growth factor (VEGF) is the most important signaling protein
among the other growth factors that plays a vital role in stimulation
of angiogenesis [9]. The VEGF family consists of six members of
proteins (VEGF-A, B, C, D, E and placenta growth factor). These
proteins can bind to their VEGF receptors (VEGFR1, VEGFR2 and
VEGFR3). These receptors are belonging to receptor tyrosine
*
Corresponding author.
E-mail addresses: yabdelrahman@yahoo.com, yassershaker1972@yahoo.com (Y.
M. Shaker).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.01.063
Please cite this article in press as: A. Temirak, et al., Part I. Synthesis, biological evaluation and docking studies of new 2-furylbenzimidazoles as
antiangiogenic agents, European Journal of Medicinal Chemistry (2014), http://dx.doi.org/10.1016/j.ejmech.2014.01.063

2
A. Temirak et al. / European Journal of Medicinal Chemistry xxx (2014) 1e13
H N
2
position 5 of the furyl ring at the right hand side (part A) and/or
H
N
modification of the benzimidazole ring at the left hand side (part B)
through the introduction of various heterocyclic ring systems at
position 1 (Fig. 2).
The synthesized compounds 3e11 (Scheme 1) were tested for
their antitumor activity against MCF-7 breast cancer cell lines and
their in vitro inhibitory action against VEGFR kinase enzyme using
double-antibody sandwich enzyme-linked immunosorbent assay
N
NH
O
O
N
H
NH2
1
X
N
N
(ELISA) to assay the level of human VEGF in breast cancer cell line
N
H
NH
N
H
NH
MCF-7. These antiangiogenic studies were achieved in comparison
to the reference drug, Tamoxifen which is reported as a potent in-
hibitor of angiogenesis [22].
N
O
2
X = NH or OH
3
2
Some recent researches have stated that there is a significant
overexpression of cyclooxygenase-2 enzyme (COX-2) and VEGF in
malignant tumors which contribute to an increase in angiogenesis
via the activation of p38 and JNK kinase pathways [23,24]. More-
over, immunohistochemical studies have shown that COX-2 is
strongly associated with increased angiogenesis and the increase in
VEGF production [25]. Thus, study of the COX-2 selectivity inhibi-
tion of some of the compounds exhibiting excellent inhibition
against human VEGF in human breast cancer cell line MCF-7
showed that they are highly potent and selective to COX-2 rather
than COX-1.
N
Y
X
N
O
N
H
N
H
O
CH3
X = S, Y = CH
4
5
(NP-184)
X = NMe, Y = CH
X = NMe, Y = N
O
N
N
N
O
CH3
N
H
O
CH3
O
Molecular docking studies of 2-(2-furyl)-1H-benzimidazole
derivatives against receptor tyrosine kinase VEGFR2 (PDB code:
3
EWH) [26] using AutoDock 4.2 and correlating the docking results
6
(PJ-8)
7 (PJ-6)
with the in vitro inhibition against VEGF in human breast cancer cell
line MCF-7 helped in deducing the structure activity relationship of
the synthesized compounds as antiangiogenic VEGR2 inhibitors.
Compound 5a was found to be more potent than Tamoxifen against
MCF-7 cell line as well as possessing better in vitro VEGF inhibition
in human breast cancer than Tamoxifen and can be used as lead
compound in the future.
Fig. 1. Compounds 1e7.
amino- or 4-hydroxy-3-benzimidazol-2-ylhydroquinolin-2-one 2
and 3-benzimidazol-2-yl-1H-indazole 3 was developed as potent
VEGFR, FGFR, and PDGFR receptor tyrosine kinases (RTK) family
inhibitors which represent an attractive therapeutic modality with
interesting physicochemical and pharmacokinetic properties
16,17]. The DNA binding oligomers, incorporating benzimidazole
ring substituted at position 2 with five membered heterocyclic ring
, was designed to target the hypoxia inducible factor (HIF-1
[
2. Results and discussion
4
a
)
2.1. Chemistry
binding site on the promoter of VEGF gene [18]. 2-(5-Methyl-2-
furyl)-1H-benzimidazole (NP-184) 5 was discovered as a novel
potential candidate for the treatment of angiogenesis related-
diseases [19,20]. It was reported that NP-184, which exhibited a
potent anti-thrombotic activity, inhibited several angiogenic func-
tions of human umbilical vascular endothelial cells (HUVEC),
including proliferation, migration and tube formation. In addition,
NP-184 was discovered to reduce the angiogenesis in vivo in ma-
terial plug assay model [19,20]. PJ-8, 5-benzoyl-2-(5-methyl-2-
furyl)-1H-benzimidazole, 6 may function as a novel drug candi-
date in anticancer therapy because PJ-8 significantly inhibited
VEGFeVEGFR2 and suppressed tumor-induced angiogenesis in vivo
The target compounds 3e11 were synthesized according to the
reaction sequences outlined in Scheme 1. 2-(Furan-2-yl)-1H-benzo
[d]imidazole 3a and its 5-methyl derivative 3b were prepared ac-
cording to the literature procedure [27e29] via the condensation of
1,2-phenylenediamine 1 with furfural 2a or 5-methylfurfural 2b in
presence of p-toluenesulfonic acid/DMF. Reaction of 3a,b with
2 3
various chloro compounds was achieved in K CO /acetone at room
temperature to form the N-substituted product 5aec. Under the
same reaction conditions, treatment of the 1H-benzimidazoles 3a,b
with ethyl bromoacetate achieved the N-alkylated products 4a,b,
which were refluxed with hydrazine hydrate in ethanol to obtain
the desired acid hydrazide compounds, 2-[2-(furan-2-yl)-1H-benzo
[d]imidazol-1-yl]acetohydrazide 6a and its 5-methyl derivative 6b
as the key compounds to form the desired target products 7e11
(Scheme 1).
[21]. PJ-6, 6-benzoyl-1-benzyl-2-(5-methyl-2-furyl) benzimid-
azole, 7, the close chemical analog of PJ-8, was also evaluated for its
antiangiogenic activity through inhibiting cell proliferation and
migration of endothelial cells. Indeed, PJ-8 was expected to be a
novel candidate as an antiangiogenic agent [21].
Therefore, looking for derivatives of 2-(2-furyl)-1H-benzimid-
azole through modification and functionalization of the structure is
an important point of research aiming to find analogs for the drug
candidates 2-(2-furyl)-1H-benzimidazoles NP-184, 5, and PJ-8, 6 for
angiogenesis inhibition. In an effort to discover more effective
VEGF/VEGFR inhibitors as antiangiogenic agents, we have designed,
synthesized and evaluated a series of benzimidazole compounds in
general and bearing 2-furyl substituent at the 2-position more
specifically for their antiangiogenic activity. Our synthetic strategy
of synthesizing new analogues was based through substitution at
N
N
H
O
A
B
Fig. 2. Schematic diagram of the substitution positions on the lead compound.
Please cite this article in press as: A. Temirak, et al., Part I. Synthesis, biological evaluation and docking studies of new 2-furylbenzimidazoles as
antiangiogenic agents, European Journal of Medicinal Chemistry (2014), http://dx.doi.org/10.1016/j.ejmech.2014.01.063

A. Temirak et al. / European Journal of Medicinal Chemistry xxx (2014) 1e13
3
NH2
NH2
N
N
O
(i)
(iii)
R
O
N
H
O
N
O
R1
H
R
R2
2
a: R= H
b: R= CH3
1
3a: R= H
b: R= CH3
compound
R1
R2
(
ii)
5a
H
C
H
2
N
O
S
N
O
5b
H
R
O
O
S
O
O
5
c
CH
3
4a: R= H
O
b: R= CH3
(
iv)
N
N
N
N
(
v)
(vii)
H
N
N
O
O
N
O
R
R
R
H
N
CH3
O
O
H N
2
HN
N
O
NH
O
H N
2
6a: R= H
b: R= CH3
O
compound
R
N
(
vi)
7
a
b
9a: R= H
b: R= CH3
N
7
CH2
N
O
R
(
viii)
(ix)
O
N
N
N
N
N
S
N
O
O
H
8
a: R= H
O
N
HN
b: R= CH3
H C
3
N
N
S
NH2
N
H
S
10
11
ꢀ
Scheme 1. Synthesis of compounds 3e11. Reagents and conditions: (i) p-Toluenesulfonic acid, DMF, 100 C; (ii) Ethyl 2-bromoacetate, K
2
CO
3
, acetone, R.T.; (iii) Chloro compounds,
ꢀ
K
2
CO
3
, acetone, R.T.; (iv) Hydrazine hydrate, ethanol, 90 C; (v) Primary amine, paraformaldehyde, ethanol, reflux; (vi) Carbon disulphide, KOH, ethanol, ice bath followed by reflux;
vii) Ethyl acetoacetate, KOH, ethanol, reflux; (viii) Hydrazine hydrate, ethanol, 90 C; (ix) Methyl iodide, NaH, DMF, reflux.
ꢀ
(
Aminoalkylation of the furan ring of 6a using 4-aminopyridine
and paraformaldehyde (Mannich reaction) was achieved to form
was further stabilized to its tautomer thione form 10 (Scheme 3)
[31]. S-Alkylation of the thiol form of 8a with methyl iodide in
sodium hydride and DMF at reflux temperature yielded 11. The
reaction of 6a,b with ethyl acetoacetate in potassium hydroxide and
ethanol achieved successfully compounds 9a,b. The mechanism of
this reaction involves nucleophilic attack of the hydrazides 6a,b to
the carbon atom of the C]O of the acetyl group of ethyl acetoa-
cetate to form the target compounds 9a,b [32].
7a while alkylation of 6a with benzylamine was performed to give
7b. Compounds containing the oxadiazole ring 8a,b, 5-[(2-(furan-
2-yl)-1H-benzo[d]imidazol-1-yl)methyl]-1,3,4-oxadiazole-2(3H)-
thione and the 5-methyl derivative were obtained from the cycli-
zation reaction of the hydrazides 6a,b with carbon disulphide in
potassium hydroxide and ethanol. The reaction mechanism
involved a nucleophilic attack of the hydrazides 6a,b to the carbon
atom of carbon disulfide to form the intermediate A. The enol form
of A (intermediate B) underwent ring closure via intramolecular
nucleophilic attack of the lone pair of electrons of the OH oxygen
atom on the carbon of C]S to achieve the compounds 8a,b
2.2. Bioactivity
2.2.1. Cytotoxicity against human breast cancer cell line MCF-7
Cytotoxicity of the synthesized compounds 3e11 was tested
using Skehan et al. method [33] in human breast cancer cell line
MCF-7. The Cytotoxicity results were compared to that of Tamox-
ifen. Compound 5a exhibited higher potency against breast cancer
(Scheme 2) [30].
Then, 8a reacted with hydrazine hydrate in refluxing ethanol at
ꢀ
9
0
C to form compound 10. The postulated reaction mechanism
-carbon atom
involved nucleophilic attack of the hydrazine on the
a
cell line MCF-7 with (IC50 ¼ 6.98
Tamoxifen whose (IC50 ¼ 8.00 g/ml), while compound 5c was
equipotent to Tamoxifen with (IC50 ¼ 8.63 g/ml). The rest of the
compounds were of promising activity against MCF-7 cell line with
(IC50: 14.00e22.40 g/ml) and their order of reactivity was 3b, 4a,
mg/ml) lower than that of
of the oxadiazole ring of 8a to give the intermediate C which un-
dergo ring opening to form D. Cyclization of D via further nucleo-
philic attack of the hydrazide on the carbon of C]S was performed
to form E followed by dehydration to achieve the thiol form F which
m
m
m
Please cite this article in press as: A. Temirak, et al., Part I. Synthesis, biological evaluation and docking studies of new 2-furylbenzimidazoles as
antiangiogenic agents, European Journal of Medicinal Chemistry (2014), http://dx.doi.org/10.1016/j.ejmech.2014.01.063

4
A. Temirak et al. / European Journal of Medicinal Chemistry xxx (2014) 1e13
N
N
N
N
N
N
KOH
EtOH
O
O
O
R
R
R
HO
HO
O
S
C S
K
K
S
S
N
S
S
NH
N
N
H
N
H
H N
2
A
B
6
a: R= H
b: R= CH
3
N
N
O
R
O
N
S
N
H
8
a: R= H
b: R= CH
3
Scheme 2. Synthesis mechanism of compounds 8a,b.
8
b, 8a, 9a, 4b, 7b, 11, 9b, 6a, 10, 5b, 3a, 7a and 6b in a descending
screening results (Table 1) showed that most of the tested com-
pounds showed potent inhibition against human VEGF in the tested
samples. Nearly six compounds (3b, 5a, 5c, 6b, 7a and 10) were
found to be potent and selective similar to the positive drug,
Tamoxifen (98%) with percentage of inhibition values 95, 98, 98, 86,
order as shown in (Table 1).
2
7
.2.2. In vitro VEGF inhibition in human breast cancer cell line MCF-
90, 91% respectively as compared with control untreated cells. From
Angiogenesis is essential for tumor progression because tumor
the foregoing result it is clear that these results were consistent
with cell cytotoxicity activity, compounds 5a and 5c were the most
potent compounds against breast cancer cell line MCF-7 and at the
same time possess the highest inhibition against the human VEGF
activity equal to that of Tamoxifen.
3
mass cannot grow bigger than 2 mm without the nourishment of
blood vessels. Moreover, vascularization is required for the process
of extravasation in metastasis [34]. Hence, establishment of
chemotherapeutic strategy by blocking angiogenesis attracts much
attention in recent years. Besides, alteration of the cellular adap-
tation to hypoxia is also fundamental in cancer treatment because
angiogenesis or other metabolic modifications will be stimulated to
maintain tumor cell survival [35]. Vascular endothelial growth
factor (VEGF) has been identified as the most important angiogenic
factor for tumor progression because it is released by a variety of
tumor cells and over expresses in different human cancers. Drugs
that can inhibit the production of VEGF or block its receptor
signaling show significant inhibition of tumor growth.
2.2.3. In vitro cyclooxygenase (COX) inhibition
Furthermore, we have studied the ability of some of the best
active compounds inhibiting human VEGF in breast cancer cell line
MCF-7 to inhibit ovine COX-1 and COX-2 using an enzyme immu-
noassay (EIA) kit. The efficacies of the tested compounds were
determined as the concentration causing 50% enzyme inhibition
(IC50) (Table 2). All the tested compounds have showed no inhibi-
This biological in vitro study was done using double-antibody
sandwich enzyme-linked immunosorbent assay (ELISA) to assay
the level of human VEGF in human breast cancer cell line MCF-7
samples as compared to the inhibition for the untreated cells. The
tion of COX-1 with IC50 > 50
COX-2 inhibitory activity was observed with compounds 6b, 7a and
10 with IC50 0.47, 0.36, 0.4 M respectively. The selectivity indices
(COX-1/COX-2) were calculated and compared with that of the
mM. Moreover, a reasonable in vitro
m
N
N
N
N
N
N
O
O
O
H N NH
H N NH
HO
2
2
O
H N NH2
2
O
N
S
N
N
N
S
H
N
H
C
N
S
H
8
a
H
D
N
N
N
N
N
O
N
O
- H O
2
O
H N
H N
H N
2
2
2
N
N
N
HO
HS
N
N
N
S
N
HS
N
N
H
H
10
F
E
Scheme 3. Synthesis mechanism of compound 10.
Please cite this article in press as: A. Temirak, et al., Part I. Synthesis, biological evaluation and docking studies of new 2-furylbenzimidazoles as
antiangiogenic agents, European Journal of Medicinal Chemistry (2014), http://dx.doi.org/10.1016/j.ejmech.2014.01.063

A. Temirak et al. / European Journal of Medicinal Chemistry xxx (2014) 1e13
5
Table 1
Effect of the synthesized compounds 3e11 on the MCF-7 cancer cell line (IC50) and the VEGF level (Pg/ml) in MCF-7 cancer cell line.
N
N
O
R1
R2
Compound
R1
R2
IC50
(
m
g/ml)a
VEGF (Pg/ml)b
3
3
a
b
H
CH
3
H
H
21.50
14.00
4460.90
280.24
(15%)
(95%)
O
4
a
H
15.20
17.80
2000.82
3760.70
(62%)
(28%)
C
H
O
O
2
O
4
b
CH
3
C
H
2
5
5
a
H
H
C
6.98
90.70
(98%)
(14%)
H
2
O
S
b
21.20
4514.23
O
O
S
5
6
6
7
7
8
8
9
c
CH
H
3
8.63
20.20
22.40
22.00
19.30
16.90
16.60
17.00
125.18
4010.45
760.40
(98%)
(24%)
(86%)
(90%)
(19%)
(41%)
(39%)
(38%)
O
O
NH
NH
NH
NH
2
2
2
2
a
b
a
b
a
b
a
C
H
N
H
2
O
CH
3
C
H
N
H
2
O
H
N
C
N
530.33
C
H
H
2
2
N
H
2
O
H
N
H
C
2
C
H
4280.32
3112.78
3200.80
3265.80
C
H
N
H
2
N
O
NH
C
H
H
2
S
N
O
NH
C
H
CH
H
3
2
S
O
N
O
O
C
H
N
H
2
CH3
CH3
O
O
O
N
9
1
b
0
CH
H
3
19.60
20.50
3978.67
480.45
(24%)
(91%)
C
H
N
H
2
N
NH
C
H
2
N
N
S
(
continued on next page)
H
2
Please cite this article in press as: A. Temirak, et al., Part I. Synthesis, biological evaluation and docking studies of new 2-furylbenzimidazoles as
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6
A. Temirak et al. / European Journal of Medicinal Chemistry xxx (2014) 1e13
Table 1 (continued )
Compound
R1
H
R2
IC50
(
m
g/ml)a
VEGF (Pg/ml)b
4420.70
N
O
NH
C
H
11
19.30
(16%)
2
S
DMSO
Tamoxifen
e
e
e
e
e
5250.00
110.75
e
8.00
(98%)
a
Data were expressed as mean of six independent experiments.
Values between brackets indicated percentage changes as compared with control untreated cells.
b
standard COX-2 selective inhibitor, Celecoxib whose IC50 values on
COX-1 and COX-2 were determined to be >50 and 0.28 mM.
AutoDock 4.2 under our experimental parameters seems to be ac-
curate, being of small RMSD values, to be of high resemblance to the
biological co-crystallization. Hence, docking result of the natively
embedded ligand of VEGFR2 exhibited reasonable well comparable
and well correlated to their biological methods.
The correlation between VEGF inhibition in MCF-7 cancer cell
line and selective COX-2 inhibition proves that the antiangiogenic
agents targeting VEGFR2 kinase are probably inhibiting COX-2
where there is a significant over expression of COX-2 and VEGF in
malignant tumors.
2.3.2. Docking study of the synthesized compounds
Docking of our new compounds into the VEGFR2 kinase indi-
2
.3. Molecular docking study
cated that compounds revealing the lowest binding free energies
are 5a,c, 7a,b, 8a and 9b. They exhibited ( ) being: e11.36, e
DG
b
Molecular docking is a frequently used tool in computer-aided
1
2.56, e11.56, e11.85, e11.53, and ꢁ11.38 kcal/mol, respectively
structure-based rational drug design. It evaluates how small mol-
ecules called (e.g. drug candidates) and the target macromolecule
ꢀ
with RMSD range of 3.79e7.81 A. Moreover, they bind into kinase
domain through up to two hydrogen bonds, involving their
moieties being: imidazole ]N, CONH, Ph-NH, Terminal NH
which bound to the commonly involved amino acids being: Glu885
C]O), Asp1046 (NH), Leu840 (C]O), Lys868 (NH), Asn923 (NH),
Cys1045 (SH), and Phe1047 (C]O) (as cited in Table 1 in the
Supplementary data).
(
receptor or enzyme) fit together. AutoDock Tools (ADT) is a pro-
gram package of automated docking tools that is available from
http://autodock.scripps.edu/. It is designed to predict how small
molecules bind to a target protein of known 3D-structure. Besides
generating binding energies in these docking studies, the position
of the ligand in the host’s binding site can be visualized. It can be
useful for developing better drug candidates and also for the un-
derstanding the nature of the binding.
(
As illustrated in Fig. 3, compound 5a possess a high potential
binding affinity (
b
DG : e11.66 kcal/mol) into the binding site of the
3
D macromolecule (PDB code: 3EWH). Its high affinity is presum-
ably attributed to its hydrogen bond formed between its imidazole
C]N moiety and Lys868 (NH) amino acid of the binding site.
In this study, seventeen compounds: 3a, 3b, 4a,b, 5aec, 6a,b,
7
a,b, 8a,b, 9a,b, 10 and 11 were docked into the target crystal
structure of human VEGFR2 tyrosine kinase domain (PDB code:
EWH) in complex with a pyridyl-pyrimidine benzimidazole in-
hibitor (K11).
Compound 10 has a reasonable potential binding affinity (
b
DG : e
3
1
0.82 kcal/mol) into the binding site, forming two hydrogen bonds
between its terminal NH and SH and Thr916 (OH). In addition, both
compounds interact hydrophobically with the surrounding Leu840,
Lys868, and Leu889 amino acids.
On applying the flexible docking into VEGFR2 kinase, the high
binding affinity was elucidated by lower binding free energy. As
shown in Fig. 4 and cited in (as cited in Table 1 enclosed in the
2
.3.1. Evaluation of docking performance and accuracy into VEGFR2
kinase
As cited in (Table 1 enclosed in the supplementary data), the
RMSD values for the redocked native PTK inhibitor (K11) was
ꢀ
1
.49 A, where it is found that the docked ligand was superimposed
with the originally embedded native ligand. Moreover, it exhibited
the lowest binding free energy (
b
Supplementary data), compound 6b (DG : e11.47 kcal/mol) has a
promising antiproliferative activity through its high PTK binding
affinity. Compound 6b bound into VEGFR2 kinase through one
hydrogen bond between its terminal NH and the HN of Lys868
amino acid whereas, compound 3a revealed high binding energy
(DG : e9.54 kcal/mol) i.e. low binding affinity. These results are
b
highly correlated to the biological results.
DG
b
) of ꢁ13.04 kcal/mol and
further three hydrogen bonds with Glu885 (NH) and Cys919 (NH),
in addition to non covalent molecular interactions with the resi-
dues Lys868 and Phe1047. As cited in literature [36] if the RMSD of
ꢀ
the best docked conformation of the native ligand is ꢂ2.0 A from
the experimental one, the used scoring function is successful.
In the analysis of docking results, a fair overall correlation exists
Therefore these results indicated that flexible docking involving
between the biological results (IC50
(mg/ml) against breast cancer
cell line) and their corresponding inhibition constants (K
i
) pre-
dicted by AutoDock into the corresponding VEGFR2 kinase. Where
Table 2
many compound, namely 4b, 5aec, 6a, 8a,b, 9a, 10 and 11 revealed
Data of the in vitro COX-1/COX-2 enzyme inhibition assay of the designed
compounds.
2
a reasonable correlation coefficient (R ) of 0.5513 as represented in
Fig. 5(A). Whereas, compounds 3a, 4b, 5aec, 6b, 7a, 8b, 9a, 10 and
Compound
Cox-1 IC50
(
m
M)a
Cox-2 IC50
(
m
M)a
SIb
2
1
1 revealed significant correlation coefficient (R ¼ 0.4623) be-
6
7
1
b
a
0
>50
>50
>50
>50
0.40
0.47
0.36
0.28
125.0
106.4
138.9
178.6
tween their biological results; %VEGF inhibition against breast
b
cancer cell line MCF-7 and AutoDock binding free energies (DG )
into VEGFR2 kinase, as can be seen in Fig. 5(B) compounds of better
binding affinity to VEGFR2 kinase are greatly inhibiting the VEGF
level in cancer tissues.
Accordingly, from the aforementioned correlation between the
biological activity and molecular docking results, we can conclude
Celecoxib
a
IC50 value is the compound concentration required to produce 50% inhibition of
COX-1 or COX-2 for means of two determinations and deviation from the mean is
<
10% of the mean value.
b
Selectivity index (SI) (COX-1 IC50/COX-2 IC50).
Please cite this article in press as: A. Temirak, et al., Part I. Synthesis, biological evaluation and docking studies of new 2-furylbenzimidazoles as
antiangiogenic agents, European Journal of Medicinal Chemistry (2014), http://dx.doi.org/10.1016/j.ejmech.2014.01.063

A. Temirak et al. / European Journal of Medicinal Chemistry xxx (2014) 1e13
7
Fig. 3. Comparative docking modes of compounds 5a (ball and stick, colored by element) and 10 (blue sticks) involving flexible docking into VEGFR2 kinase. Compounds 5a and 10
ꢀ
exhibited one and two hydrogen bonds, respectively; shown as green dashed lines. They are shown superimposed (RMSD: 3.79 and 3.55 A) onto the K111 ligand (yellow sticks). PTK
is shown as solid backbone ribbon for protein and its and the binding site is shown in solid surface view with labeled amino acids. (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)
that our compounds in this study are showing significant anti-
the aromatic ring at position 1 of benzimidazole moiety causing
angiogenic inhibition of VEGFR2 kinase.
non covalent molecular interactions (piecation interaction); (iii)
2
the CH linker in benzyl group provided flexibility for the aromatic
2
.4. Theoretical estimation of partition coefficient
group. The benzyl group in 5a has more impact on activity than the
methylbenzenesulfonyl group in 5c, due to the bulkiness of the
sulfonyl group. The effect of the methyl group on increasing activity
was clear, 5c being more potent than 5b.
Partition coefficient (log P) is a measure of the lipophilicity
(
octanol/water partition coefficient) of compounds. Acceptable log
P values are preferable from 1 to 2.5 [37]. We have measured the
theoretical log P using CS ChemProp in ChemDraw [38]. Most
compounds showed acceptable log P values as shown in (Table 3).
Substitution of the furyl ring by a methyl group at position 5 in
the compounds carrying benzyl or methylbenzenesulfonyl in po-
sition 1 of the benzimidazole moiety as in 3b, 5c and 6b resulted in
better biological activity and binding affinity with the VEGFR2 than
3a, 5b and 6a respectively. This emphasizes the important role of
the methyl group in increasing the antiangiogenic activity of the
compound by the possible hydrophobic contact with the active site.
Additionally, replacing the methyl group at position 5 of the furyl
ring with methylaminopyridine group as in 7a increases the ac-
tivity, while the substitution with methylbenzylamine as in 7b
demolished the activity of the compound. The pronounced activity
of 7a relative to 7b indicated the importance of pyridine ring which
exhibited H-bonding with the Asp1046 residue on VEGFR2.
3
. Structure activity relationship
The comparison of the biological activity along with the docking
affinities and interactions of the synthesized compounds gave more
insight into the structure activity relationship of 2-furyl benz-
imidazole derivatives as VEGFR2 kinase inhibitors.
The high activity of 5a and 5c may be attributed to: (i) the
hydrogen bond formed between the C]N of the imidazole ring and
Lys868 (NH) amino acid of the binding site; (ii) the role exerted by
Fig. 4. Comparative docking modes of compounds 3a (yellow sticks) and 6b (ball and stick, colored by element), involving flexible docking into VEGFR2 kinase. They exhibited one
hydrogen bond for each; shown as green dashed lines with Glu885 and Lys868, respectively. Compound 6b is shown deeply embedded within the groove of the binding site. PTK is
shown as solid backbone ribbon for protein and its and the binding site is shown in solid surface view with labeled amino acids. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: A. Temirak, et al., Part I. Synthesis, biological evaluation and docking studies of new 2-furylbenzimidazoles as
antiangiogenic agents, European Journal of Medicinal Chemistry (2014), http://dx.doi.org/10.1016/j.ejmech.2014.01.063

8
A. Temirak et al. / European Journal of Medicinal Chemistry xxx (2014) 1e13
d) By a triazole ring, carrying the electron donating nitrogen
atoms, at position 1 of benzimidazole as in 10.
4. Conclusion
Our main goal throughout this manuscript was the synthesis of
new (2-furyl)-1H-benzimidazole derivatives as antiangiogenic
agents working via inhibiting VEGF-VEGFR2 complex formation,
thus suppressing proliferation and survival of endothelial cells and
consequently preventing cancer progression.
Some analogues for the drug candidates NP-184 5 and PJ-8 6
have been synthesized showing potential inhibition of VEGF
induced angiogenesis. Scheme 1 outlined the synthetic steps of the
target compounds 3e11 and the existence of these compounds was
verified using spectral and microanalyses data.
The bioactivity of the compounds 3e11 showed that 6 com-
pounds (3b, 5a, 5c, 6b, 7a and 10) have shown promising cytotoxic
activity against breast cancer cell line MCF-7 with (IC50: 6.98e
2
2.40
cancer cell line with percentages of inhibition (86e98%) in com-
parison to the positive drug, Tamoxifen (IC50: 8.00 g/ml, %
mg/ml) and potential inhibition of human VEGF in MCF-7
m
inhibition ¼ 98%) as compared with control untreated cells.
Moreover, compounds 6b, 7a and 10 expressed selective inhibition
of COX-2 enzyme in comparison to COX-1 enzyme with selectivity
indices quite similar to that of Celecoxcib.
Furthermore, molecular docking studies were done using
AutoDock 4.2 in order to understand the binding modes and
inhibitory effect of the synthesized compounds 3e11 against the
receptor tyrosine kinase VEGFR2, Compounds 5a,c, 7a,b, 8a, and 9b
showed promising binding interactions with the receptor. Consid-
erable correlation exists between the biological activity results (IC50
Fig. 5. (A) The overall correlation between IC50 against breast cancer cell line and the
inhibition constants (K
kinase. (B) The correlation between %VEGF inhibition in breast cancer cell line MCF-7
and the binding free energies ( ) for compounds 3a, 4b, 5aec, 6b, 7a, 8b, 9a, 10, and
i
) for compounds 4b, 5aec, 6a, 8a,b, 9a, 10, and 11 into VEGFR2
DG
b
1
1 into VEGFR2 kinase.
and %VEGF inhibition) against MCF-7 cell line and the molecular
2
docking results (K
i
and
D
G
b
) with correlation coefficient (R ) of
Changing the substitution at position 1 from oxadiazole ring in
a to triazole ring with terminal NH group in 10 increased the
group
0.5513 and 0.4623 respectively. Synthesized analogues of 2-(2-
8
2
furyl)-1H-benzimidazole have shown potential inhibition of
VEGFR2 kinase which may argument their advantage as promising
antiangiogenic agents.
activity, due to the H-bonding of the electron donating NH
2
with Thr916. The furyl ring in the 2-furylbenzimdazole derivatives
could form noncovalent molecular hydrophobic interactions with
the Lys868 residue in VEGFR2 kinase active site quite similar to that
made by the naphthalene ring in the native ligand (K11). This could
explain the closefitting of the 2-furylbenzimdazoles to the VEGFR2
kinase.
In conclusion, optimizing the activity of 2-furylbenzimidazole
derivatives as anti VEGFR2 kinase could be through the following
substitutions:
4.1. Physical measurements
Microanalyses and spectral data of the compounds were per-
formed in the Micro analytical labs, National Research Centre and
Micro analytical Laboratory Center, Faculty of Science, Cairo Uni-
ꢁ1
versity, Cairo, Egypt. The IR spectra (4000e400 cm ) were recor-
ded using KBr pellets in a Jasco FT/IR 300E Fourier transform
infrared spectrophotometer on a Perkin Elmer FT-IR 1650 (spec-
trophotometer). The melting points were determined on a Stuart
a) By a methyl group at position 5 of the furan ring as in 3b and
5
c.
1
SMP30 Melting Point Apparatus and are uncorrected. The H and
b) By an aromatic ring with electronegative nitrogen as pyri-
13
C
NMR spectra were recorded using Joel ECA 500 MHz NMR
as a solvent. Chemical shifts
dine linked to position 5 of the furan ring through NHeCH
linker as in 7a.
c) By an aromatic group at position 1 of the benzimidazole
2
spectrophotometers using DMSO-d
6
are reported in parts per million (ppm) from the tetramethylsilane
resonance in the indicated solvent. Coupling constants are reported
in Hertz (Hz); spectral splitting partners are designed as follow:
singlet (s); doublet (d); triplet (t); multiplet (m). The mass spectra
were carried out using Finnigan mat SSQ 7000 (Thermo. Inst. Sys.
Inc., USA) spectroscopy at 70 ev. Reaction progress was monitored
using thin layer chromatography (TLC) analysis on Silica Gel 60
F254 plate (Merck).
moiety, linked by a flexible linker, as in compound 5a.
Table 3
Theoretical partition coefficient (log P) of the 2-furylbenzimidazole derivatives 3e11.
Compound Theoretical Compound Theoretical Compound Theoretical
log P
log P
log P
3
3
4
4
5
5
a
b
a
b
a
b
1.48
1.82
1.58
1.92
3.45
2.93
5c
6a
6b
7a
7b
8a
3.26
0.08
0.42
0.03
1.58
1.82
8b
9a
9b
10
11
2.15
0.84
1.18
2.08
2.65
4
.1.1. General procedure for the preparation of compounds (3a,b)
[29]
1,2-Phenylenediamine 1 (10.8 g, 0.1 mol) and (0.1 mol) of
furfural 2a or 5-methylfurfural 2b were mixed in DMF (10 ml), then
p-toluenesulfonic acid (3.4 g, 0.02 mol) was added, and the solution
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A. Temirak et al. / European Journal of Medicinal Chemistry xxx (2014) 1e13
9
ꢀ
was heated and stirred at 100 C for 2 h. When the reaction was
completed, the reaction mixture was cooled and added dropwise
4.1.7. General procedure for the preparation of compounds (5aec)
To a well stirred solution of benzimidazoles (3a or 3b) (0.01 mol)
and anhydrous K CO (1.06 g, 0.01 mol) in dry acetone, benzyl
chloride and benzenesulphonyl chloride (0.01 mol) were added
dropwise. The reaction mixture was stirred for 3 h at room tem-
perature. Excess acetone was evaporated under reduced pressure
and the reaction mixture was poured onto cold water and the
precipitate obtained was filtered, dried and recrystallized from
ethanol.
with vigorous stirring onto a mixture of anhydrous Na
.1 mol) and distilled H O (20 ml). The product was collected by
filtration, washed with H O and dried.
2
CO
3
(10.6 g,
2
3
0
2
2
4
.1.2. 2-(Furan-2-yl)-1H-benzo[d]imidazole (3a)
ꢀ
Yield ¼ 85% (15.63 g). M.p.: 285e287 C. TLC R
f
¼ 0.57 (petro-
: 6.69 (dd,
2-furyl), 7.16e7.17
benzimidazole), 7.49e7.51 (m, 2H, H benz-
þ H
imidazole), 7.90 (dd, J ¼ 3 Hz, 1H, H 2-furyl), 13.00 (s, 1H, NH, D
exchangeable). IR (cm ): 3435 (NH),1629 (C]N). MS: m/z 185, 14%
1
leum ether/ethyl acetate, 2:1). H NMR (DMSO-d
6
)
d
J ¼ 3 Hz, 1H, H
4
2-furyl), 7.15 (dd, J ¼ 3 Hz, 1H, H
3
4
.1.8. 1-Benzyl-2-(furan-2-yl)-1H-benzo[d]imidazole (5a)
(
m, 2H, H
5
þ H
6
4
7
ꢀ
Yield ¼ 85% (2.30 g). M.p.: 255e257 C. TLC R
f
d
¼ 0.67 (petroleum
: 5.78 (s, 2H, CH ),
2-furyl), 7.06e7.08 (m, 2H, H
þ H
benzimidazole), 7.14 (dd, J ¼ 3 Hz, 1H, H 2-furyl), 7.20-7.30 (m, 5H,
eH phenyl), 7.57e7.60 (m, 2H, H benzimidazole), 7.93
þ H
dd, J ¼ 3 Hz, 1H, H 2-furyl). C NMR (500 MHZ, DMSO-d ): 48.26,
11.66, 112.92, 114.71, 118.87, 122.64, 123.84, 124.08, 126.91, 128.11,
29.27, 132.21, 135.65, 137.14, 141.19, 143.61, 144.14, 146.22. IR
5
2
O
1
ꢁ1
ether/ethyl acetate, 2:1). H NMR (DMSO-d
6.70 (dd, J ¼ 3 Hz, 1H, H
6
)
2
þ
þ
þ
þ
4
5
6
(
(
1
M þ1); m/z 184, 100% (M ); m/z 183, 21% (M ꢁ1); m/z 156, 25%
3
M ꢁCO). Anal. Calcd for C11
8 2
H N O (184.06): C, 71.73; H, 4.38; N,
H
(
1
1
2
0
6
0
4
7
5.21. Found C, 71.65; H, 4.41; N, 15.25.
13
5
6
4
.1.3. 2-(5-Methylfuran-2-yl)-1H-benzo[d]imidazole (3b)
ꢀ
ꢁ1
þ
þ
Yield ¼ 80% (15.92 g). M.p.: 275e277 C. TLC R
f
)
¼ 0.49 (petro-
: 2.36 (s, 3H,
2-
benzimidazole), 7.49e7.50 (m,
(
cm ): 1607 (C]N). MS: m/z 375, 7% (M þ1); m/z 374, 39% (M );
1
leum ether/ethyl acetate, 2:1). H NMR (DMSO-d
CH
), 6.31 (d, J ¼ 3 Hz, 1H, H
furyl), 7.13e7.15 (m, 2H, H
H, H benzimidazole), 12.00 (s,1H, NH, D
þ H
6
d
þ
m/z 373, 10% (M ꢁ1); m/z 197, 93%. Anal. Calcd for C18
H
14
N
2
O
3
4
2-furyl), 7.05 (d, J ¼ 3 Hz, 1H, H
3
(274.11): C, 78.85; H, 5.19; N, 10.11. Found C, 78.83; H, 5.12; N, 10.20.
5
þ H
6
2
(
1
4
7
2
O exchangeable). IR
4
.1.9. 1-(Benzenesulfonyl)-2-(furan-2-yl)-1H-1,3-benzodiazole (5b)
ꢁ
1
þ
cm ): 3446 (NH), 1632 (C]N). MS: m/z 199, 5% (M þ1); m/z 198,
ꢀ
Yield ¼ 80% (2.60 g). M.p.: 112e114 C. TLC R
f
¼ 0.62 (petroleum
þ
þ
00% (M ); m/z 183, 30% (M ꢁCH
3
); m/z 169, 33%; m/z 155, 24%.
1
ether/ethyl acetate, 2:1). H NMR (DMSO-d
1
H
6
)
d
: 6.70 (dd, J ¼ 3 Hz,
Anal. Calcd for C12
H
10
N
2
O (198.08): C, 72.71; H, 5.08; N, 14.13.
H, H
4
2-furyl), 7.18 (dd, J ¼ 3 Hz, 1H, H
benzimidazole), 7.56e7.60 (m, 2H, H
ꢁ H phenyl), 8.06 (dd, J ¼ 3 Hz, 1H, H
3
2-furyl), 7.23e7.26 (m, 2H,
þ H benzimidazole),
2-
Found C, 72.77; H, 5.02; N, 14.16.
5
þ H
6
4
7
7.70e7.90 (m, 5H, H
2
0
6
0
5
ꢁ1
furyl). IR (cm ): 1615 (C]N), 1368 and 1160 (S]O). MS: m/z
4
.1.4. General procedure for the preparation of compounds (4a,b)
To a well stirred solution of benzimidazoles (3a or 3b) (0.01 mol)
and anhydrous K CO (1.06 g, 0.01 mol) in dry acetone, ethyl 2-
2 3
bromoacetate (1.1 ml, 0.01 mol) was added dropwise. The reac-
tion mixture was stirred for 3 h at room temperature. The reaction
mixture was poured onto cold water and the precipitate was
filtered, dried and recrystallized from ethanol.
þ
þ
þ
3
3
8
25, 6% (M þ1); m/z 324, 26% (M ); m/z 323, 13% (M ꢁ1); m/z 184,
3%. Anal. Calcd for C17 S (324.06): C, 62.97; H, 3.78; N,
12 2 3
H N O
.58; S, 9.82. Found C, 62.93; H, 3.72; N, 8.65; S, 9.87.
4.1.10. 1-(Benzenesulfonyl)-2-(5-methylfuran-2-yl)-1H-1,3-
benzodiazole (5c)
Yield ¼ 78% (2.63 g). M.p.: 166e168 C. TLC R
ꢀ
f
d
¼ 0.65 (petroleum
: 2.36 (s, 3H, CH ),
2-furyl), 7.27 (d, J ¼ 3.05 Hz, 1H, H 2-
þ H benzimidazole), 7.55e7.60 (m,
benzimidazole), 7.72e7.74 (m, 5H, H þ H þ H þ H
þ H
phenyl protons), 8.16e8.18 (m, 2H, H benzimidazole). C NMR
500 MHZ, DMSO-d ): 13.90, 108.97, 110.74, 114.42, 114.99, 117.65,
20.77, 126.05, 127.28, 128.25, 129.14, 130.47, 135.76, 137.90, 141.21,
1
ether/ethyl acetate, 2:1). H NMR (DMSO-d
.21 (d, J ¼ 3.05 Hz, 1H, H
furyl), 7.37e7.39 (m, 2H, H
H, H
6
)
3
4
.1.5. Ethyl 2-(2-(furan-2-yl)-1H-benzo[d]imidazol-1-yl)acetate
4a)
Yield ¼ 90% (2.43 g). M.p.: 150e152 C. TLC R
6
4
3
(
5
6
ꢀ
f
¼ 0.65 (petroleum
: 1.15 (t, J ¼ 2.3 Hz,
), 5.43 (s, 2H, CH ), 6.70
2-furyl), 7.17 (dd, J ¼ 3 Hz,1H, H 2-furyl), 7.23e
þ H benzimidazole), 7.62e7.63 (m, 2H, H þ H
benzimidazole), 7.90 (dd, J ¼ 3 Hz, 1H, H
2
4
2
0
3
0
4
0
5
0
0
6
1
ether/ethyl acetate, 2:1). H NMR (DMSO-d
H, CH CH CH
), 4.12 (q, J ¼ 2.3 Hz, 2H, CH
dd, J ¼ 3 Hz,1H, H
.24 (m, 2H, H
6
)
d
13
7
3
2
3
2
3
2
(
6
(
4
3
1
1
7
5
6
4
7
ꢁ1
42.39,144.05,155.87. IR(cm ): 1610 (C]N),1370 and 1154 (S]O).
ꢁ
1
5
2-furyl). IR (cm ): 1742
þ
þ
MS: m/z 340, 5% (M þ2); m/z 338, 80% (M ); m/z 197, 100%; m/z
83, 5%. Anal. Calcd for C18 S (338.07): C, 63.89; H, 4.17; N,
.28; S, 9.48. Found C, 63.80; H, 4.19; N, 8.34; S, 9.49.
þ
þ
(
C]O), 1610 (C]N). MS: m/z 271, 14% (M þ1); m/z 270, 84% (M );
1
8
14 2 3
H N O
þ
m/z 269, 3%; m/z 197, 100%, (M ꢁCOOC
2
H
5
). Anal. Calcd for
(270.10): C, 66.66; H, 5.22; N, 10.36. Found C, 66.58; H,
.24; N, 10.45.
C
5
15 14 2 3
H N O
4.1.11. General procedure for the preparation of compounds (6a,b)
To a solution of benzimidazoles (4a or 4b) (0.01 mol) in meth-
4
.1.6. Ethyl 2-(2-(5-methylfuran-2-yl)-1H-benzo[d]imidazol-1-yl)
acetate (4b)
Yield ¼ 85% (2.41 g). M.p.: 88e90 C. TLC R
anol (15 ml), hydrazine hydrate (0.02 mol) was added dropwise and
the reaction mixture was refluxed for 4 h. The reaction mixture was
cooled and the excess solvent was evaporated using reduced
pressure. The product separated was filtered, washed with small
portions of cold ethanol and cold water repeatedly and dried. The
product was purified by recrystallization from ethanol.
ꢀ
f
¼ 0.60 (petroleum
: 1.17 (t, J ¼ 2.3 Hz,
), 4.14 (q, J ¼ 2.3 Hz, 2H, CH CH ), 5.05
), 6.33 (d, J ¼ 3 Hz, 1H, H 2-furyl), 7.05 (d, J ¼ 3 Hz,1H, H
-furyl), 7.21e7.23 (m, 2H, H benzimidazole), 7.59e7.61
þ H
m, 2H, H benzimidazole). C NMR (500 MHZ, DMSO-d ):
þ H
3.83, 14.59, 47.00, 61.75, 108.99, 110.69, 113.96, 119.28, 122.96,
1
ether/ethyl acetate, 2:1). H NMR (DMSO-d
H, CH CH ), 2.33 (s, 3H, CH
s, 2H, CH
2
(
6
) d
3
(
2
3
3
2
3
2
4
3
5
6
13
4
7
6
4.1.12. 2-(2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)
1
acetohydrazide (6a)
ꢁ
1
ꢀ
123.14, 136.36, 142.90, 143.98, 144.52, 154.50, 169.07. IR (cm ):
Yield ¼ 88% (2.25 g). M.p.: 240e242 C. TLC R
f
d
¼ 0.70 (petroleum
: 4.31 (s, 2H, NH
), 6.70 (dd, J ¼ 3 Hz, 1H, H 2-
2-furyl), 7.22e7.23 (m, 2H,
benzimidazole), 7.49 (m, 1H, H benzimidazole), 7.62 (m,
þ
1
1
748 (C]O), 1616 (C]N). MS: m/z 285, 99% (M þ1); m/z 284, 93%
ether/ethyl acetate, 1:1). H NMR (DMSO-d
O exchangeable), 5.10 (s, 2H, CH
furyl), 7.14 (dd, J ¼ 3 Hz, 1H, H
þ H
6
)
2
,
þ
þ
þ
(
M ); m/z 256, 13% (M eC
CH COOC eCH ). Anal. Calcd for C16
H, 5.67; N, 9.85. Found C, 67.50; H, 5.60; N, 9.89.
2
H
5
); m/z 211, 81%; m/z 183, 25% (M e
D
2
2
4
2
2
H
5
3
16
H N
2
O
3
(284.12): C, 67.59;
3
H
5
6
4
Please cite this article in press as: A. Temirak, et al., Part I. Synthesis, biological evaluation and docking studies of new 2-furylbenzimidazoles as
antiangiogenic agents, European Journal of Medicinal Chemistry (2014), http://dx.doi.org/10.1016/j.ejmech.2014.01.063

10
A. Temirak et al. / European Journal of Medicinal Chemistry xxx (2014) 1e13
1
H, H
7
benzimidazole), 7.90 (dd, J ¼ 3 Hz, 1H, H
5
2-furyl), 11.00 (s,
10% KOH (10 ml) was added, then the reaction mixture was refluxed
for 10 h, cooled, acidified with conc. HCl. The resulting solid was
filtered, washed with water, dried and crystallized from DMF.
ꢁ1
1
H, NH, D
2
O exchangeable). IR (cm ): 3325 and 3245 (NH and
þ
NH
2
), 1650 (C]O), 1616 (C]N). MS: m/z 271, 5% (M þ1); m/z 270,
þ
þ
þ
100% (M ); m/z 253, 7% (M eNH
2
); m/z 211, 81% (M eCONHNH
2
);
þ
m/z 198, 30% (M eCH
2
CONHNH
2
). Anal. Calcd for C13
H
12
N
4
O
2
4.1.18. 5-((2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3,4-
(
2
256.10): C, 60.93; H, 4.72; N, 21.86. Found C, 60.98; H, 4.77; N,
1.84.
oxadiazole-2(3H)-thione (8a)
ꢀ
Yield ¼ 80% (1.20 g). M.p.: 260e262 C. TLC R
f
d
¼ 0.68 (petroleum
: 5.90 (s, 2H, CH ),
2-furyl), 7.26 (dd, J ¼ 3 Hz, 1H, H 2-furyl),
þ H benzimidazole), 7.65e7.67 (m, 2H,
benzimidazole), 7.95 (dd, J ¼ 3 Hz, 1H, H 2-furyl), 11.00 (s,
O exchangeable). C NMR (500 MHZ, DMSO-d ): 46.50,
111.10, 112.83, 113.77, 119.71, 123.61, 123.92, 135.85, 142.78, 144.05,
1
ether/ethyl acetate, 1:1). H NMR (DMSO-d
6.74 (dd, J ¼ 3 Hz, 1H, H
7.28e7.30 (m, 2H, H
þ H
1H, NH, D
6
)
2
4
.1.13. 2-(2-(5-Methylfuran-2-yl)-1H-benzo[d]imidazol-1-yl)
acetohydrazide (6b)
Yield ¼ 90% (2.43 g). M.p.: 246e248 C. TLC R
4
3
5
6
ꢀ
f
¼ 0.57 (petroleum
: 2.35 (s, 3H, CH ),
), 6.30 (d, J ¼ 3 Hz,
2-furyl), 7.20e7.25 (m, 2H,
benzimidazole), 7.57e7.60 (m, 2H, H benzimidazole).
þ H
), 1660 (C]O), 1612 (C]N).
H
4
7
5
1
13
ether/ethyl acetate, 1:1). H NMR (DMSO-d
6
)
d
3
2
6
4
1
H
.30 (s, 2H, D
2
O exchangeable), 5.00 (s, 2H, CH
2
ꢁ
1
H, H
4
2-furyl), 7.00 (d, J ¼ 3 Hz, 1H, H
3
144.74, 145.94, 159.85, 178.51. IR (cm ): 3425 (NH), 1612 (C]N),
þ
þ
þ
5
þ H
6
4
7
1120 (C]S). MS: m/z 300, 2% (M þ2); m/z 299, 18% (M þ1); m/z
ꢁ
1
þ
IR (cm ): 3348 and 3252 (NH and NH
2
298, 100% (M ); m/z 297, 21% (M ꢁ1). Anal. Calcd for C14
10 4 2
H N O S
þ
þ
þ
MS: m/z 271, 4% (M þ1); m/z 270, 100% (M ); m/z 253, 17% (M e
(298.05): C, 56.37; H, 3.38; N, 18.78; S, 10.75. Found C, 56.32; H,
3.46; N, 18.85; S, 10.78.
þ
þ
NH
CH
2
); m/z 211, 65% (M eCONHNH ); m/z 198, 42% (M e
14 4 2
CONHNH ). Anal. Calcd for C14H N O (270.11): C, 62.21; H,
2
2
2
5
.22; N, 20.73. Found C, 62.27; H, 5.27; N, 20.79.
4.1.19. 5-((2-(5-Methylfuran-2-yl)-1H-benzo[d]imidazol-1-yl)
methyl)-1,3,4-oxadiazole-2(3H)-thione (8b)
ꢀ
4
.1.14. General procedure for the preparation of compounds (7a,b)
To a solution of the acid hydrazide (6a or 6b) (5 mmol) in
Yield ¼ 84% (1.31 g). M.p.: 265e267 C. TLC R
f
d
¼ 0.68 (petroleum
: 2.34 (s, 3H, CH ),
), 6.34 (d, J ¼ 3.05 Hz, 1H, H 2-furyl), 7.05 (d,
2-furyl), 7.24e7.29 (m, 2H, H benzimid-
1
ether/ethyl acetate, 1:1). H NMR (DMSO-d
5.90 (s, 2H, CH
J ¼ 3.05 Hz, 1H, H
azole), 7.62e7.67 (m, 2H, H
6
)
3
methanol (20 ml) was stirred at room temperature with para-
formaldehyde (0.01 g), conc. hydrochloric acid (1 ml) and primary
amine (5 mmol) namely: 4-amino pyridine and benzylamine for
2
4
3
5
þ H
6
ꢁ
1
4
þ H
7
benzimidazole). IR (cm ): 3428
þ
1
h. The reaction mixture was refluxed for 2 h, filtered while hot,
(NH), 1611 (C]N), 1122 (C]S). MS: m/z 314, 3% (M þ2); m/z 312,
þ
and the filtrate was concentrated to one-third of its original vol-
ume. The residual liquid was cooled for 24 h. The product that
separated out was washed and recrystallized from methanol.
26% (M ); m/z 253, 44%; m/z 237, 93%; m/z 211, 44%. Anal. Calcd for
15 12 4 2
C H N O S (312.10): C, 57.68; H, 3.87; N, 17.94; S, 10.27. Found C,
57.74; H, 3.83; N, 17.99; S, 10.28.
4
.1.15. 2-(2-(5-((Pyridin-4-ylamino)methyl)furan-2-yl)-1H-benzo
4.1.20. General procedure for the preparation of compounds (9a,b)
A mixture of hydrazide (6a or 6b) (5 mmol) and ethyl acetoa-
cetate (2 ml) was refluxed for 5 h. The reaction mixture was diluted
with petroleum ether (60e80) and the resulting solid was filtered,
washed with water, dried and crystallized from acetic acid and the
reaction mixture was then cooled, acidified with conc. HCl. The
resulting solid was filtered, washed with water, dried and crystal-
lized from acetic acid.
[d]imidazol-1-yl)acetohydrazide (7a)
ꢀ
Yield ¼ 72% (1.30 g). M.p.: >300 C. TLC R
f
¼ 0.67 (petroleum
ether/ethyl acetate/ethanol, 1:1:1). H NMR (DMSO-d : 4.64 (s,
O exchangeable),
2-furyl), 7.05 (d, J ¼ 3 Hz, 1H, H 2-furyl),
þ H benzimidazole), 7.40e7.50 (m, 2H,
benzimidazole), 7.85e7.91 (m, 4H, pyridyl). IR (cm ): 3390
1
6
) d
2
6
H, CH
2 2 2 2
2-furyl), 5.70 (s, 2H, CH ), 5.90 (NH , D
.67 (d, J ¼ 3 Hz, 1H, H
4
3
7.14e7.15 (m, 2H, H
5
6
ꢁ
1
H
4
þ H
7
and 3173 (NH and NH
2
), 1680 (C]O),1610 (C]N). MS: m/z 362, 45%
þ
þ
(
M ); m/z 332, 56% (M eN
2
H
3
); m/z 197, 95%; m/z 184, 48%. Anal.
4.1.21. Ethyl 3-(2-(2-(furan-2-yl)-1H-benzo[d]imidazol-1-yl)
Calcd for C19
H
18
N
6
O
2
(362.15): C, 62.97; H, 5.01; N, 23.19. Found C,
acetoylimino)butanoate (9a)
ꢀ
6
3.05; H, 5.10; N, 23.24.
Yield ¼ 70% (1.30 g). M.p.: 145e147 C. TLC R
f
¼ 0.60 (petroleum
1
ether/ethyl acetate, 1:1). H NMR (DMSO-d
1.90 (t, 3H, CH ), 4.10 (s, 2H, CH CH ), 5.30 (s, 2H, CH
5.54 (s, 2H, CH
6
)
d
: 1.17 (t, 3H, CH
2
CH
3
),
),
4.1.16. 2-(2-(5-((Benzylamino)methyl)furan-2-yl)-1H-benzo[d]
3
2
3
2
COOC H
2
5
imidazol-1-yl)acetohydrazide (7b)
Yield ¼ 78% (1.46 g). M.p.: >300 C. TLC R
2
“benzimidazole attached”), 6.69 (dd, J ¼ 3 Hz, 1H,
2-furyl), 7.10 (dd, J ¼ 3 Hz, 1H, H 2-furyl), 7.14e7.28 (m, 2H,
þ H benzimidazole), 7.53e7.624 (m, 2H, H þ H benzimid-
2-furyl), 11.0 (s, 1H, NH, D
ꢀ
f
¼ 0.70 (petroleum
ether/ethyl acetate/ethanol, 1:1:1). H NMR (DMSO-d : 3.58 (s,
), 6.67 (d,
2-furyl), 7.22e7.25
phenyl),
þ H
phenyl), 10.80 (s, 1H,
O exchangeable). IR
H
H
4
3
1
6
)
d
5
6
4
7
2
H, CH
J ¼ 3 Hz, 1H, H
m, 2H, H
þ H
.40e7.42 (m, 2H, H
benzimidazole), 7.74e7.76 (m, 2H, H
NH, D O exchangeable), 11.30 (s, 1H, NH, D
2
benzylic), 3.97 (s, 2H, CH
2
2-furyl), 5.70 (s, 2H, CH
2
azole), 7.90 (dd, J ¼ 3 Hz, 1H, H
5
2
O
ꢁ1
4
2-furyl), 7.20 (d, J ¼ 3 Hz, 1H, H
3
exchangeable). IR (cm ): 3423 (NH), 1733 (C]O), 1685 (C]O),
þ
þ
(
7
5
6
benzimidazole), 7.36e7.38 (m, 1H, H
4
0
1615 (C]N). MS: m/z 324, 24% (M eOC
2
H
5
); m/z 323, 14% (M e
); m/z 184, 32% (2-
(368.15): C,
þ
0
þ H
0
phenyl), 7.45e7.47 (m, 2H, H
þ H
OC
H
ꢁ1); m/z 296, 29% (M eCOOC
2 5
H
3
5
4
7
2
5
0
0
furylbenzimidazole). Anal. Calcd for
C
H
N
O
2
2
6
19
20
4
2
2
61.95; H, 5.47; N, 15.21. Found C, 62.07; H, 5.56; N, 15.26.
ꢁ
1
(
cm ): 3408 and 3185 (NH and NH
2
), 1685 (C]O), 1612 (C]N).
þ
þ
MS: m/z 375, 36% (M ); m/z 344, 68% (M eN
2
H
3
); m/z 197, 100%;
4.1.22. Ethyl 3-(2-(2-(5-methylfuran-2-yl)-1H-benzo[d]imidazol-1-
m/z 184, 60%. Anal. Calcd for C21
N, 18.65. Found C, 67.24; H, 5.67; N, 18.68.
H
21
N
5
O
2
(375.17): C, 67.18; H, 5.64;
yl)acetoylimino)butanoate (9b)
ꢀ
Yield ¼ 82% (1.56 g). M.p.: 160e162 C. TLC R
f
¼ 0.65 (petroleum
: 1.16 (t, 3H, CH CH ),
2-furyl), 4.11 (s, 2H, CH CH ), 5.30
), 5.57 (s, 2H, CH “benzimidazole attached”),
6.30 (dd, J ¼ 3 Hz, 1H, H 2-furyl), 6.95 (d, J ¼ 3 Hz, 1H, H 2-furyl),
7.15e7.27 (m, 2H, H benzimidazole), 7.53e7.61 (m, 2H,
þ H
þ H benzimidazole), 10.80 (s, 1H, NH, D O exchangeable). IR
(cm ): 3420 (NH), 1730 (C]O), 1685 (C]O), 1617 (C]N). MS: m/z
1
ether/ethyl acetate, 1:1). H NMR (DMSO-d
1.90 (t, 3H, CH ), 2.30 (t, 3H, CH
(s, 2H, CH COOC
6
)
d
2
3
4.1.17. General procedure for the preparation of compounds (8a,b)
3
3
2
3
Carbon disulfide (0.5 ml) was added dropwise to an ice cooled
2
2
H
5
2
solution of KOH (0.2 g) in ethanol (10 ml) containing the acid hy-
drazide (6a or 6b) (5 mmol). The reaction mixture was stirred at
room temperature 2 h. After dilution with ethanol, the solid ob-
tained was washed twice with ether. To the solid obtained (0.2 g),
4
3
5
6
H
4
7
2
ꢁ
1
Please cite this article in press as: A. Temirak, et al., Part I. Synthesis, biological evaluation and docking studies of new 2-furylbenzimidazoles as
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A. Temirak et al. / European Journal of Medicinal Chemistry xxx (2014) 1e13
11
þ
þ
3
36, 70% (M eOC
m/z 210, 63%. Anal. Calcd for C20
N, 15.21. Found C, 62.03; H, 5.52; N, 15.27.
2
H
5
); m/z 335, 24% (M eOC
2
H
5
ꢁ1); m/z 242, 59%;
4.2.2. In vitro VEGF inhibition in human breast cancer cell line MCF-
7
H
22
N
4
O
2
(382.16): C, 61.95; H, 5.47;
The effect of tested compounds on the level of human vascular
endothelial growth factor (VEGF) was determined utilizing human
tumor cell lines including breast cancer cell line MCF-7 obtained
from the American Type Culture Collection (Rockville, MD, USA).
The tumor cells were maintained in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% heat inactivated fetal calf
4
.1.23. 5-((2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl) methyl)-4-
amino-4H-1,2,4-triazole-3-thiol (10)
To a solution of compound 8a (2 mmol) in methanol (5 ml),
hydrazine hydrate (2 mmol) was added and the reaction mixture
was refluxed for 4 h, cooled and the excess solvent was evaporated
using reduced pressure. The product separated was filtered,
serum (GIBCO), penicillin (100 U/ml) and streptomycin (100
m
g/ml)
ꢀ
at 37 C in humidified atmosphere containing 5% CO
concentration of 0.50 ꢃ 10 were grown in a 25 cm flask in 5 ml of
2
. Cells at a
6
2
washed and dried. The product was purified by recrystallizion from
complete culture medium.
ꢀ
ethanol. Yield ¼ 85% (0.53 g). M.p.: 277e279 C. TLC R
f
¼ 0.74
: 5.80 (s,
2-furyl), 7.18 (dd, J ¼ 3 Hz, 1H, H
þ H benzimidazole), 7.64e7.67 (m,
benzimidazole), 7.90 (dd, J ¼ 3 Hz,1H, H 2-furyl),13.50
O exchangeable). C NMR (500 MHZ, DMSO-d ):
8.60, 110.05, 112.69, 113.49, 119.51, 123.35, 123.68, 130.09, 142.68,
The cells in culture medium were treated with 20 ml of IC50
1
(
petroleum ether/ethyl acetate, 1:3). H NMR (DMSO-d
6
)
d
values of the compounds or the standard reference drug, Tamoxifen
ꢀ
2
2
2
H, CH
-furyl), 7.23e7.25 (m, 2H, H
H, H
þ H
2
), 6.68 (dd, J ¼ 3 Hz, 1H, H
4
3
dissolved in DMSO, then incubated for 24 h at 37 C, in a humidified
5
6
2
5% CO atmosphere. The cells were harvested and homogenates
were prepared in saline using a tight pestle homogenizer until
complete cell disruption.
4
7
5
13
(
s, 2H, NH
2
, D
2
6
4
The kit uses a double-antibody sandwich enzyme-linked
ꢁ
1
144.35, 144.97, 145.71, 149.08, 167.45. IR (cm ): 3423 and 3285
immunosorbent assay (ELISA) to assay the level of human VEGF
in samples. Add VEGF to monoclonal antibody enzyme well which
is pre-coated with human VEGF monoclonal antibody, incubation;
then, add VEGF antibodies labeled with biotin, and combined with
Streptavidin-HRP to form immune complex; then carry out incu-
bation and washing again to remove the uncombined enzyme.
Then add Chromogen solution A, B, the color of the liquid changes
into the blue, and at the effect of acid, the color finally becomes
yellow. The chroma of color and the concentration of the human
TRK of sample were positively correlated and the optical density
was determined at 450 nm. The level of human VEGF in samples
was calculated (ng/L) as duplicate determinations from the stan-
dard curve.
þ
(
NH, NH
2
), 1690 (C]O), 1615 (C]N). MS: m/z 314, 3% (M þ2); m/z
þ
þ
2
312, 49% (M ); m/z 297, 25% (M eNH ); m/z 184, 100%. Anal. Calcd
for C14
12 6 2
H N O S (312.08): C, 53.83; H, 3.87; N, 26.91; S,10.27. Found
C, 53.89; H, 3.94; N, 26.95; S, 10.29.
4.1.24. 2-(Furan-2-yl)-1-(5-(methylthio)-1,3,4-oxadiazol-2-yl)-1H-
benzo[d]imidazole (11)
To a solution of compound 8a (2 mmol) in dry DMF, NaH (0.10 g)
was stirred at room temperature for 0.5 h. Then, methyl iodide
(
2 mmol) was added and the reaction mixture was refluxed for 4 h.
After cooling the reaction mixture, it was poured onto cold water
and the precipitated product was filtered, washed and recrysallized
ꢀ
from ethanol. Yield ¼ 74% (0.47 g). M.p.: 278e281 C. TLC R
f
¼ 0.58
: 2.60 (s,
2-furyl), 7.19 (dd,
benzimidazole),
benzimidazole), 7.66 (dd, J ¼ 3 Hz, 1H, H
-furyl). 13C NMR (500 MHZ, DMSO-d
): 14.82, 50.24,110.08,112.78,
4.2.3. In vitro cyclooxygenase (COX) inhibition assay
1
(
petroleum ether/ethyl acetate, 1:3). H NMR (DMSO-d
H, CH ), 6.00 (s, 2H, CH
), 6.7 (dd, J ¼ 3 Hz, 1H, H
2-furyl), 7.25e7.27 (m, 2H, H
þ H
þ H
6
)
d
The ability of the test compounds to inhibit ovine COX-1 and
COX-2 was determined using an enzyme immunoassay (EIA) (kit
catalog number 560131, Cayman Chemical, Ann Arbor, MI, USA)
according to the manufacturer’s instructions. Cyclooxygenase cat-
alyzes the first step in the biosynthesis of arachidonic acid (AA) to
3
3
2
4
J ¼ 3 Hz, 1H, H
3
5
6
7
.45e7.47 (m, 2H, H
4
7
5
2
6
1
13.61, 119.74, 123.48, 123.82, 135.91, 142.94, 144.08, 144.84, 145.87,
2 2a 2
PGH . PGF , produced from PGH by reduction with stannous
ꢁ1
þ
1
63.87, 165.94. IR (cm ): 1618 (C]N). MS: m/z 314, 2% (M þ2); m/
chloride, is measured by enzyme immunoassay (ACEÔ competitive
EIA). Stock solutions of test compounds were dissolved in a mini-
mum volume of DMSO. Briefly, to a series of supplied reaction
þ
þ
þ
z 313, 9% (M þ1); m/z 312, 100% (M ); m/z 311, 33% (M ꢁ1). Anal.
Calcd for C15 S (312.09): C, 57.31; H, 4.49; N, 17.82; S, 10.20.
Found C, 57.37; H, 4.42; N, 17.89; S, 10.28.
14 4 2
H N O
buffer solutions (960
EDTA and 2 mM phenol) with either COX-1 or COX-2 (10
enzyme in the presence of heme (10 L) were added 10
various concentrations of test drug solutions (0, 0.01, 0.1, 1, 10 and
m
L, 0.1 M TriseHCl pH 8.0 containing 5 mM
L)
L of
m
m
m
4
4
.2. Bioactivity materials and methods
5
0
m
M in a final volume of 1 mL). These solutions were incubated
ꢀ
.2.1. Cytotoxicity against human breast cancer cell line MCF-7
for a period of 5 min at 37 C after which 10
mL of AA (100
mM)
The antitumor activity against MCF-7 was performed in the
solution were added and the COX reaction was stopped by the
addition of 50 L of 1 M HCl after 2 min. PGF , produced from PGH
National Cancer Institute, Cancer Biology Department, Cairo, Egypt.
The cytotoxicity of the synthesized compounds 3e11 was tested
using the method of Skehan et al. [33]. Cells were plated in 96-
multiwell plate (104 cells/well) for 24 h before treatment with
the compounds to allow attachment of cell to wall of the plate.
Different Concentrations of the compound under test (0.0, 5.0, 12.5,
m
2
a
2
by reduction with stannous chloride was measured by enzyme
immunoassay. This assay is based on the competition between PGs
and a PG-acetylcholinesterase conjugate (PG tracer) for a limited
amount of PG antiserum. The amount of PG tracer, that is, able to
bind to the PG antiserum is inversely proportional to the concen-
tration of PGs in the wells since the concentration of PG tracer is
held constant while the concentration of PGs varies. This antibodye
PG complex binds to a mouse anti-rabbit monoclonal antibody that
had been previously attached to the well. The plate is washed to
remove any unbound reagents and then Ellman’s reagent, which
contains the substrate to acetylcholine esterase, is added to the
well.
2
5.0 and 50 mg/ml) were added to the cell monolayer. Six wells
were prepared for each individual dose. Monolayer cells were
ꢀ
incubated with the compounds for 48 h at 37 C and in atmosphere
2
of 5% CO . After 48 h cells were fixed, washed and stained with
Sulforhodamine B stain. Excess stain was washed with acetic acid
and attached stain was recovered with Tris EDTA buffer. Color in-
tensity was measured in an ELISA reader. The relation between
surviving fraction and drug conc. is plotted to get the survival curve
of each tumor cell line after the specified compound.
The product of this enzymatic reaction produces a distinct yel-
low color that absorbs at 410 nm. The intensity of this color,
Please cite this article in press as: A. Temirak, et al., Part I. Synthesis, biological evaluation and docking studies of new 2-furylbenzimidazoles as
antiangiogenic agents, European Journal of Medicinal Chemistry (2014), http://dx.doi.org/10.1016/j.ejmech.2014.01.063

12
A. Temirak et al. / European Journal of Medicinal Chemistry xxx (2014) 1e13
determined spectrophotometrically, is proportional to the amount
of PG tracer bound to the well, which is inversely proportional to
the amount of PGs present in the well during the incubation:
the docked inhibitors within PTK receptor, and flexible residues:
Glu885, Cys919, and Asp1046. Once those parameters were set in
one file, AutoGrid calculates grid parameter files for each type of
atom within a given area.
Absorbance
a [Bound PG Tracer] a1/PGs. Percent inhibition was
calculated by the comparison of compound treated to various
control incubations. The concentration of the test compound
4
.3.4. Preparing the docking parameter file and running AutoDock4
The docking parameter file tells AutoDock which map files to
causing 50% inhibition (IC50
, mM) was calculated from the concen-
trationeinhibition response curve (duplicate determinations).
use, the ligand molecule to move, what its center and number of
torsions are, where to start the ligand, the flexible residues to move
where side chain motion in the receptor is to be modeled, which
docking algorithm to use and how many runs to do. It usually has
the file extension, “.dpf”. Four different docking algorithms are
currently available in AutoDock: SA, the original Monte Carlo
simulated annealing; GA, a traditional Darwinian genetic algo-
rithm; LS, local search; and GALS (Lamarckian genetic algorithm),
which is a hybrid genetic algorithm with local search. Each search
method has its own set of parameters, and these must be set before
running the docking experiment itself. These parameters include
what kind of random number generator to use, step sizes, etc. The
most important parameters affect how long each docking will run.
In GALS, the number of energy evaluations and the number of
generations affect how long a docking will run. ADT lets you change
all of these parameters, and others not mentioned here. In our
study, we used AutoDock 4.2 to check the binding conformations of
the synthesized compounds into the entitled VEGFR2 kinase more
accurately. Lamarckian genetic algorithm was used for all molecular
docking simulations. Population size of 300, mutation rate of 0.02,
and crossover rate of 0.8 were set as the parameters. Simulations
were performed using up to 2.5 million energy evaluations with a
maximum of 27,000 generations. Each simulation was performed
4
.3. Molecular docking
This protocol presents a detailed outline and advice for use of
AutoDock and its graphical interface, AutoDock Tools, to analyze
biomolecular complexes using computational docking. The first
step is to prepare the coordinate files for the docking molecule and
the target molecule. The second step is the calculation of the af-
finity grid for the target molecule. In the third step, the docking
molecule is docked with the affinity grid, and, finally, the results are
analyzed.
4
.3.1. Preparing the target macromolecules investigated
Our analysis to the crystal structures in the Protein Data Bank
PDB comprising benzimidazole containing ligand bounded to the
receptor tyrosine kinase VEGFR2 revealed 3 entries out of which we
have chosen the entry with the best resolution (PDB code: 3EWH)
ꢀ
[26] with resolution of 1.6 A. The native ligand N-[4-({3-[2-
(
methylamino)pyrimidin-4-Yl]pyridin-2-Yl}oxy) naphthalen-1-Yl]-
6
-(trifluoro-methy-l)-1H-benzimidazol-2-amine (K11) in the co-
crystallized structure downloaded from the PDB website exhibi-
ted H-bonds with the main residues in the VEGFR2: Glu885,
Cys919, Lys868, Phe1047 and Asp1046. This was retrieved from the
Protein Data Bank, http://www.rcsb.org/pdb/home/home.do. For
each docking target, crucial amino acids of the active site were
identified using data in pdbsum, http://www.ebi.ac.uk/pdbsum/.
10 times, yielding 10 docked conformations. The lowest energy
conformations were regarded as the binding conformations be-
tween the ligands and the proteins.
4
.3.5. Analyzing the docking results
Reading a docking log or a set of docking logs is the first step in
4
.3.2. Preparing a ligand file for AutoDock
The ligands are originally drawn with a widely used chemical
analyzing the results of docking experiments. During its automated
docking procedure, AutoDock outputs a detailed record to the
result file has the extension “.dlg”. The output includes many de-
tails about the docking which are output as AutoDock parses the
input files and reports what it finds. After completing the runs,
AutoDock begins an analysis phase and records details of that
process. At the very end, it reports a summary of the amount of
time taken and the words ’Successful Completion’. The key results
in a docking log are the docked structures found at the end of each
run, the energies of these docked structures and their similarities to
each other. The similarity of docked structures is measured by
computing the root-mean-square-deviation, RMSD, between the
coordinates of the atoms. The docking results consist of the PDBQT
of the Cartesian coordinates of the atoms in the docked molecule,
along with the state variables that describe this docked confor-
mation and position. As a result of AutoDock calculations we obtain
the output file with in our case ten conformers of the protein-ligand
complex with flexible residues and the ligand located within the
binding pocket. Each structure are scored and ranked by the pro-
gram by the calculated interaction energy.
structure drawing software. The three-dimensional structures of
the aforementioned compounds were constructed using Chem3D
Ultra 8.0 software [Chemical Structure Drawing Standard; Cam-
bridge Soft corporation, USA (2003)] to obtain standard 3D struc-
tures (PDB format), then they were energetically minimized by
using MOPAC with 100 iterations and minimum RMS gradient of
0
.10. It is recommended to confirm whether all hydrogen atoms are
in the file before working with ADT. After opening the ligand, it can
be visualized and ADT now automatically computes Gasteiger
charges (empirical atomic partial charges) and distinguishes be-
tween hybridization state and type of each atom. As a part of
preparation, the program determines rotatable bonds of the ligand
to be able to generate different conformers for the docking.
4.3.3. Setting the grid box, preparing the GRID parameter file,
running AutoGrid4, and preparation of the flexible residue file
The grid parameter file tells AutoGrid4 which receptor to
compute the potentials around, the types of maps to compute and
the location and extent of those maps. In general, one map is
calculated for each atom type in the ligand plus an electrostatics
map and a separate desolvation map. The types of maps depend on
the types of atoms in the ligand. Thus one way to specify the types
of maps is by choosing a ligand. The grid maps of 60 ꢃ 60 ꢃ 60 grid
points, centered on the ligands of the complex structures, were
Acknowledgment
The authors are grateful to the National Cancer Institute, Cancer
Biology Department, Cairo, Egypt, to perform the antitumor activity
against MCF-7 cancer cell lines. Deep thanks are expressed to the ‘
Science & Technology Development Fund’ for financially supporting
the manuscript through the fund of the STDF project No 1517.
ꢀ
used to cover the binding pockets. A spacing of 0.375 A was set
ꢀ
centered at 14.078, ꢁ5.106 and 8.682 A, respectively for VEGFR2
kinase (PDB code: 3EWH), that encompassed the active site where
the co-crystallized ligand (K11) was embedded, was used to guide
Please cite this article in press as: A. Temirak, et al., Part I. Synthesis, biological evaluation and docking studies of new 2-furylbenzimidazoles as
antiangiogenic agents, European Journal of Medicinal Chemistry (2014), http://dx.doi.org/10.1016/j.ejmech.2014.01.063

A. Temirak et al. / European Journal of Medicinal Chemistry xxx (2014) 1e13
13
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Please cite this article in press as: A. Temirak, et al., Part I. Synthesis, biological evaluation and docking studies of new 2-furylbenzimidazoles as
antiangiogenic agents, European Journal of Medicinal Chemistry (2014), http://dx.doi.org/10.1016/j.ejmech.2014.01.063