Discovery of N-(2-phenyl-1H-benzo[d]imidazol-5-yl)quinolin-4-amine derivatives as novel VEGFR-2 kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.07.071

Inhibition of the VEGF signaling pathway has become a valuable approach in the treatment of cancers. In this work, a series of N-(2-phenyl-1H-benzo[d]imidazol-5-yl)quinolin-4-amine derivatives were designed and identified as potent inhibitors of VEGFR-2 (

Full text of DOI:10.1016/j.ejmech.2014.07.071

European Journal of Medicinal Chemistry 84 (2014) 698e707
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Discovery of N-(2-phenyl-1H-benzo[d]imidazol-5-yl)quinolin-4-
amine derivatives as novel VEGFR-2 kinase inhibitors
,
, *
Lei Shi a *, Ting-Ting Wu a, Zhi Wang a, Jia-Yu Xue b, Yun-Gen Xu a
a Jiangsu Key Laboratory of Drug Design and Optimization, Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, PR China
b Jiangsu Provincial Key Laboratory for Plant Ex Situ Conservation, Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014,
PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibition of the VEGF signaling pathway has become a valuable approach in the treatment of cancers. In
Received 3 April 2014
Received in revised form
16 July 2014
Accepted 19 July 2014
Available online 21 July 2014
this work,
a series of N-(2-phenyl-1H-benzo[d]imidazol-5-yl)quinolin-4-amine derivatives were
designed and identified as potent inhibitors of VEGFR-2 (KDR) kinase. These compounds with quinoline
scaffold and benzimidazole moiety were synthesized and their biological activities against VEGFR-2 and
two human cancer cell lines were evaluated. Among them, compound 7s exhibited the most potent
inhibitory activity against VEGFR-2 with IC50 of 0.03
tivity against the tested cancer cell lines with IC50 of 1.2
Docking simulation supported the initial pharmacophoric hypothesis and suggested a common mode of
interaction at the ATP-binding site of VEGFR-2, which demonstrates that compound 7s is a potential
agent for cancer therapy deserving further researching.
m
M and it also showed the highest anticancer ac-
M against MCF-7 and 13.3 M against Hep-G2.
m
m
Keywords:
VEGFR-2 kinase inhibitor
Antitumor
Quinolin
© 2014 Elsevier Masson SAS. All rights reserved.
Benzimidazole
1. Introduction
Inhibition of angiogenesis by blocking the VEGF signaling
pathway has become a valuable approach in the treatment of
Angiogenesis, the formation of new capillary blood vessels from
existing vasculature, is a normal physiological event that occurs
during embryogenesis, wound healing, and the menstrual cycle [1].
It also contributes to a number of pathological disorders such as
psoriasis, arthritis, endometriosis and diabetic retinopathy [2].
More importantly, it is required for the growth and metastasis of
solid tumors [3e5]. In fact, in order to grow beyond a critical size,
solid tumors need new blood capillaries to create their own
nutrient supply to remove metabolic waste and to facilitate
metastasis formation [6e8].
Among the many proangiogenic factors identified to date,
vascular endothelial growth factor (VEGF) has been identified as
the most common regulator of tumor angiogenesis and the
increased expression of VEGF has been implicated in tumor growth
and metastasis [9,10]. VEGF binds to VEGFR-2 (KDR), a receptor
tyrosine kinase (RTK), which upon dimerization by VEGF undergoes
autophosphorylation and initiates downstream signaling, ulti-
mately leading to angiogenesis, vascular permeability enhance-
ment, tumor proliferation, and tumor migration [11e14].
cancers [15]. Several successful strategies for the inhibition of
angiogenesis have been effectively demonstrated in preclinical and
clinical settings. These approaches include VEGF soluble decoy re-
ceptors, antibodies directed against VEGF, and small molecules that
inhibit KDR [16]. Bevacizumab, a monoclonal antibody to VEGF, has
been approved by the FDA for the treatment of metastatic colo-
rectal cancer and non-small-cell lung cancer [17,18]. In addition to
biological-based antiangiogenics, small-molecule tyrosine kinase
inhibitors of KDR, such as sorafenib [19], sunitinib [20], pazopanib
[21], vandetanib [22], axitinib [23] and cabozantinib [24] have been
approved for treatment of various types of cancers (Fig. 1).
Recently, a large number of VEGFR inhibitors have been re-
ported or have entered clinical trials [6,25e32]. Some compounds
containing quinoline or benzimidazole moiety exhibited potent
antitumor activities [33e41]. We have recently identified a series of
quinoline amides as effective inhibitors of KDR at the enzyme and
cellular levels [42]. In an attempt to pursue new potent antitumor
agents with KDR inhibitory activity, a series of new quinolin-4-
amine derivatives containing quinoline and benzimidazole moi-
eties were synthesized and their inhibitory activities against KDR
and two cancer cell lines were evaluated. In addition, the struc-
tureeactivity relationships and possible enzyme binding modes
were also illustrated.
* Corresponding authors.
E-mail addresses: shilei@cpu.edu.cn, shilei0828@gmail.com (L. Shi), xyg@cpu.
edu.cn (Y.-G. Xu).
http://dx.doi.org/10.1016/j.ejmech.2014.07.071
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

L. Shi et al. / European Journal of Medicinal Chemistry 84 (2014) 698e707
699
Fig. 1. VEGFR inhibitors approved for clinical use.
2. Results and discussion
substituent exhibited the weakest inhibitory activity with IC50 of
18.9 M, which was about one sixth of compound 7m. The above
m
2.1. Chemistry
results indicated that introduction of weakly electron-withdrawing
substituent is favorable for the activity while introduction of
strongly electron-withdrawing bulk substituent or strongly
electron-donating bulk substituent causes obvious decrease of ac-
tivity. This discipline was also observed in ortho-substituted and
meta-substituted compounds. Compounds 7k, 7l and 7m with
chloro substituent at different position showed distinct inhibitory
activity against MCF-7. Compound 7m with para-chloro substituent
The route that enabled the synthesis of the title N-(2-phenyl-
1H-benzo[d]imidazol-5-yl)quinolin-4-amine derivatives is out-
lined in Scheme 1. The synthesis of these benzimidazole quinolin-
4-amines started from
a series of commercially available
substituted benzoic acids 2ae2u. Condensation of 2ae2u with 4-
nitro-o-phenylenediamine (1) in polyphosphoric acid (PPA) at
120e150 ꢀC for 5 h gave the 5-nitro-benzimidazole intermediates
3ae3u. Hydrogenation of 5-nitro-benzimidazole intermediates
3ae3u with Pd/C/H2 under normal pressure at room temperature
for 5 h or Fe/AcOH/EtOH under reflux for 2 h provided the 5-amino-
benzimidazole intermediates 4ae4u. Condensation of 4ae4u with
4-chloroquinoline (6), which was prepared by quinolin-4-ol (5) in
POCl3 at 100 ꢀC for 4 h, in isopropanol under reflux for 5 h gave the
title benzimidazole quinolin-4-amines 7ae7u.
showed more potent activity (IC50 ¼ 3.2
m
M) than compound 7l
with meta-chloro substituent (IC50 ¼ 6.3
mM). The activity of the
latter was higher than compound 7k with ortho-chloro substituent
(IC50 ¼ 19.6
mM). The result suggested that substituents in different
positions led to different inhibitory activities (para- > meta-
> ortho-). This rule was also found in other single substituted
compounds. Comparing the activities of compounds 7r, 7s and 7t
against MCF-7, we found that compound 7s with two chloro sub-
stituents at para and meta positions exhibited the most potent
activity (IC50 ¼ 1.2
stituents at two meta positions displayed slightly weaker activity
(IC50 ¼ 2.3 M), however, introduction of two chloro substituents to
two ortho positions caused an obvious decrease of activity (com-
M). It also demonstrated that introduction of
certain substituents at para or meta position is more favorable than
at ortho position.
All the above SARs were also suitable for the inhibitory activities
of these compounds against Hep-G2, however, the inhibitory ac-
tivities against Hep-G2 were much weaker than that against MCF-7.
mM), and compound 7t with two chloro sub-
2.2. Biological evaluation
m
2.2.1. Antiproliferation assay
All the synthesized compounds 7ae7u were evaluated for their
anticancer activity against MCF-7 (human breast cancer) and Hep-
G2 (human liver cancer) cell lines by MTT assay. They were also
evaluated for cytotoxicity on a human normal liver cell line L02. The
results were summarized in Table 1. It was clear that all the tested
compounds showed better activities against MCF-7 than against
Hep-G2. It also indicated that these compounds exhibited weak
cytotoxic activity against L02. Among the tested compounds,
compound 7s showed the most potent activities with the IC50 value
pound 7r: IC50 ¼ 24.7
m
2.2.2. VEGFR-2 inhibitory assay
The VEGFR-2 kinase inhibitory potency of the synthesized qui-
nolin-4-amine derivatives containing benzimidazole moiety was
examined and the results were also summarized in Table 1. Most of
the tested compounds exhibited potent VEGFR-2 inhibitory activ-
ities. Among them, compound 7s displayed the most potent
of 1.2 mM and 13.3 mM against MCF-7 and HepG-2, respectively,
which was better than the positive control sunitinib.
Structureeactivity relationships (SARs) were inferred from data
of cell proliferative experiments reported in Table 1. The type,
number and position of substituents on the phenyl ring linking to
the benzimidazole moiety played important roles in the anti-
proliferation activities. The inhibitory activities against MCF-7 of
compounds 7ae7u with different para-substituents increased in
the following order: compound 7m with para-chloro substituent
and compound 7n with para-bromo substituent exhibited potent
inhibitory activity with IC50 value of 0.03 mM, which was similar to
the positive control sunitinib. The SARs analysis result of VEGFR-2
inhibitory activities of the tested compounds were consistent
with that of their anticancer activities, which suggested that the
potent anticancer activities of the synthesized compounds were
likely related to their VEGFR-2 inhibitory activities.
inhibitory activities with IC50 of 3.2 mM and 3.4 mM, respectively;
compound 7a with no substituent on para-position and compound
7j with para-fluoro substituent displayed slightly weaker inhibitory
2.3. Molecular docking studies
activities with IC50 of 4.6
7d with para-methyl and compound 7g with para-ethyl substituent
showed much weaker inhibitory activities with IC50 of 6.6 M and
8.0 M, respectively; compound 7q with para-trifluoromethyl
mM and 4.3 mM, respectively; compound
In order to better understand the interaction between the syn-
thesized compounds and VEGFR-2 kinase, molecular docking of the
potent compound 7s into the ATP binding site of VEGFR-2 kinase
m
m

700
L. Shi et al. / European Journal of Medicinal Chemistry 84 (2014) 698e707
Scheme 1. General procedure for the synthesis of N-(2-phenyl-1H-benzo[d]imidazol-5-yl)quinolin-4-amine derivatives. Reagents and conditions: (a) PPA, 120e150 ꢀC, 5 h; (b) Pd/C,
H2, CH3OH, r.t., 5 h; (c) Fe, AcOH, EtOH, reflux, 2 h; (d) POCl3, 100 ꢀC, 4 h; (e) isopropanol, reflux, 5 h.
(PDB: 2QU5) was performed using the Discovery Studio 3.1/
CDOCKER protocol. The binding model is depicted in Fig. 2. In this
binding model, compound 7s is nicely bound to the ATP-binding
suggested that compound 7s could bind well with the VEGFR-2
active site. The above results provided theoretical basis for
further structural optimization of benzimidazole quinolin-4-amine
derivatives as VEGFR-2 inhibitors and demonstrated that com-
pound 7s could be a potential anticancer agent.
cavity of VEGFR-2 via three hydrogen bonds, four
p-cation in-
teractions, two interactions and one interaction. The
p
ep
sep
nitrogen atom of quinoline forms a hydrogen bond (N/HeN:
2.24 Å, 128.79ꢀ) with the amino hydrogen atom of Cys919 and the
4. Experimental section
phenyl ring of quinoline forms a
with the phenyl ring of Phe918 and also forms a
(distance: 2.83 Å) with the 4-position carbon atom of Leu840 while
the pyridine ring of quinoline form another interaction (dis-
p
ep
interaction (distance: 5.28 Å)
sep
interaction
4.1. Chemistry
pep
Unless otherwise noted, all solvents and reagents were
commercially available and used without further purification.
Melting points (uncorrected) were determined on a RY-1 MP
apparatus. ESI-MS spectra were recorded on an Agilent/HP 1100
tance: 6.01 Å) with the phenyl ring of Phe918, which suggests that
the quinoline group plays an important role in the combination of
the receptor and ligand. In addition, the nitrogen atom of benz-
imidazole forms a hydrogen bond (N/HeN: 1.94 Å, 164.27ꢀ) with
the amino hydrogen atom of Asp1046 and the amino hydrogen
atom of benzimidazole forms another hydrogen bond (NeH/O:
2.15 Å, 134.48ꢀ) with the carbonyl oxygen atom of Glu885. Both the
Series LC/MSD Trap SL Mass spectrometer. 1H NMR spectra and 13
C
NMR were recorded on a Bruker AV-300 or AV-500 spectrometer at
25 ꢀC with TMS and solvent signals allotted as internal standards.
Chemical shifts were reported in ppm (d). Elemental analyses were
performed on a CHNeO-Rapid instrument.
imidazole ring and the phenyl ring of benzimidazole form p-cation
interactions (distance: 4.38 Å for imidazole ring and 4.85 Å for
phenyl ring) with the end amino cation of Lys868. The results
indicated that the introduction of benzimidazole group to the
quinoline group might reinforce the combination of compound 7s
and the receptor, which might enhance the binding affinity,
resulting in the increased anticancer activity of this compound.
Furthermore, the amino cation linking quinoline and benzimid-
4.1.1. General procedure for the preparation of the 5-nitro-2-
phenyl-1H-benzo[d]imidazole derivatives 3ae3u
A mixture of 4-nitro-o-phenylenediamine (3.22 g, 21 mmol) and
substituted benzoic acid 2ae2u (20 mmol) in PPA (40 mL) was
stirred at 120e150 ꢀC for 5 h. The reaction was quenched with
water and the pH was adjusted to 6 with saturated NaOH. The filter
cake was washed with water and recrystallized from ethyl acetate
to give corresponding compounds 3ae3u.
azole forms a
phenyl ring of Phe1047 while the phenyl ring with two chlorine
atoms linking to benzimidazole forms another -cation interaction
p-cation interaction (distance: 4.78 Å) with the
p
(distance: 5.32 Å) with the imidazole amino cation of His1026.
4.1.1.1. 5-Nitro-2-phenyl-1H-benzo[d]imidazole (3a). Pale orange-
red powder, yield: 88%, mp: 206e208 ꢀC. 1H NMR (300 MHz; d6-
DMSO): 7.48e8.04 (m, 4H); 8.04e8.69 (m, 4H); 13.64 (s, 1H). MS
(ESIþ) m/z 294.1 (MþH)þ. Anal. Calcd for C13H9N3O2: C, 65.27; H,
3.79; N, 17.56; Found: C, 65.32; H, 3.82; N, 17.53.
3. Conclusions
A series of quinolin-4-amine derivatives containing benzimid-
azole moiety have been synthesized and discovered as novel
VEGFR-2 inhibitors that displayed good antiproliferation activities
against two tumor cell lines (MCF-7 and Hep-G2) and weak cyto-
toxic activities against the human normal live cell line L02. Com-
pound 7s exhibited the most potent inhibitory activity against
VEGFR-2 with IC50 of 0.03 mM, which was equivalent to the com-
mercial VEGFR-2 inhibitor sunitinib and also showed the greatest
inhibitory activities against human breast cancer cell MCF-7 and
4.1.1.2. 5-Nitro-2-o-tolyl-1H-benzo[d]imidazole (3b). Yellow pow-
der, yield: 86%, mp: 146e148 ꢀC. 1H NMR (300 MHz; d6-DMSO):
2.58 (s, 3H); 7.46 (s, 3H); 7.77e7.85 (m, 2H); 8.16 (s, 1H); 8.40e8.59
(m, 1H); 13.37 (s, 1H). MS (ESIþ) m/z 254.1 (MþH)þ. Anal. Calcd for
C14H11N3O2: C, 66.40; H, 4.38; N, 16.59; Found: C, 66.34; H, 4.41; N,
16.64.
human liver cancer cell Hep-G2 with IC50 of 1.2
respectively. Molecular docking of the most potent inhibitor 7s into
ATP binding site of VEGFR-2 kinase was performed and the result
m
M and 13.3
m
M,
4.1.1.3. 5-Nitro-2-m-tolyl-1H-benzo[d]imidazole (3c). Yellow pow-
der, yield: 99%, mp: 108e110 ꢀC. 1H NMR (300 MHz; d6-DMSO):
2.48 (s, 3H); 7.38e7.52 (m, 2H); 7.76 (d, J ¼ 8.5 Hz, 1H); 8.00e8.15

L. Shi et al. / European Journal of Medicinal Chemistry 84 (2014) 698e707
701
Table 1
Enzymatic and cellular results for benzimidazole quinolin-4-amines.
4.1.1.8. 2-(2-Fluorophenyl)-5-nitro-1H-benzo[d]imidazole
(3h).
Orange powder, yield: 26%, mp: 171e173 ꢀC. 1H NMR (300 MHz; d6-
DMSO): 7.41e7.52 (m, 2H); 7.60e7.68 (m, 1H); 7.81 (d, J ¼ 8.7 Hz,
1H); 8.15 (dd, J ¼ 8.7, 1.8 Hz, 1H); 8.27 (t, J ¼ 7.2 Hz, 1H); 8.52 (s, 1H);
13.21 (s, 1H). MS (ESIþ) m/z 258.1 (MþH)þ. Anal. Calcd for
C13H8FN3O2: C, 60.70; H, 3.13; N, 16.34; Found: C, 60.78; H, 3.12; N,
16.30.
Compound
VEGFR-2
(IC50, mM)
Proliferative inhibition (IC50
,
m
M)
L02
No.
R
Hep-G2
MCF-7
4.1.1.9. 2-(3-Fluorophenyl)-5-nitro-1H-benzo[d]imidazole
(3i).
Pale yellow powder, yield: 42%, mp: 205e207 ꢀC. 1H NMR
(300 MHz; d6-DMSO): 7.38e7.43 (m, 1H); 7.63e7.76 (m, 2H);
7.95e8.14 (m, 3H); 8.48 (s, 1H); 13.66 (s, 1H). MS (ESIþ) m/z 258.1
(MþH)þ. Anal. Calcd for C13H8FN3O2: C, 60.70; H, 3.13; N, 16.34;
Found: C, 60.55; H, 3.15; N, 16.39.
7a
7b
7c
7d
7e
7f
7g
7h
7i
H
2.48
>10
6.80
4.65
>10
9.53
5.49
>10
3.52
1.36
>10
2.40
0.24
0.18
>10
>10
>10
8.35
0.03
0.08
7.82
0.04
116.0
>100
59.7
54.1
>100
52.5
63.3
>100
85.2
49.4
67.0
38.0
45.4
42.8
>100
>100
63.0
85.9
13.3
26.8
62.7
13.6
4.6
>100
>100
>100
>100
>100
>100
>100
>100
>100
54.0
2-CH3
3-CH3
4-CH3
2-OCH3
2-OH
4-CH2CH3
2-F
3-F
4-F
2-Cl
3-Cl
54.2
18.9
6.6
75.0
11.4
8.0
31.2
9.6
4.3
4.1.1.10. 2-(4-Fluorophenyl)-5-nitro-1H-benzo[d]imidazole
(3j).
Golden powder, mp: yield: 96%, mp: 258e260 ꢀC. 1H NMR
(300 MHz; d6-DMSO): 7.44 (t, J ¼ 9.0 Hz, 2H); 7.75 (d, J ¼ 8.7 Hz,
1H); 8.11 (dd, J ¼ 9.0, 2.1 Hz, 1H); 8.23e8.27 (m, 2H); 8.45 (s, 1H);
13.59 (s, 1H). MS (ESIþ) m/z 258.1 (MþH)þ. Anal. Calcd for
C13H8FN3O2: C, 60.70; H, 3.13; N, 16.34; Found: C, 60.84; H, 3.16; N,
16.38.
7j
7k
7l
19.6
6.3
3.2
>100
>100
65.8
7m
7n
7o
7p
7q
7r
7s
7t
4-Cl
4-Br
3.4
92.5
2-CF3
3-CF3
4-CF3
2,6-di-Cl
3,4-di-Cl
3,5-di-Cl
2-Br,5-F
51.1
42.8
18.9
24.7
1.2
2.3
20.4
10.5
>100
>100
>100
>100
59.5
82.3
>100
75.6
7u
sunitinib
(m, 3H); 8.47 (s, 1H); 13.58 (s, 1H). MS (ESIþ) m/z 254.2 (MþH)þ.
Anal. Calcd for C14H11N3O2: C, 66.40; H, 4.38; N, 16.59; Found: C,
66.45; H, 4.42; N, 16.53.
4.1.1.4. 5-Nitro-2-p-tolyl-1H-benzo[d]imidazole (3d). Yellow pow-
der, yield: 81%, mp: 219e221 ꢀC. 1H NMR (300 MHz; d6-DMSO):
2.32 (s, 3H); 7.29 (d, J ¼ 7.9 Hz, 2H); 7.61 (d, J ¼ 8.8 Hz, 1H); 7.98 (dd,
J ¼ 8.8, 2.2 Hz, 1H); 8.09 (d, J ¼ 8.1 Hz, 2H); 8.35 (d, J ¼ 2.1 Hz, 1H).
MS (ESIþ) m/z 254.1 (MþH)þ. Anal. Calcd for C14H11N3O2: C, 66.40;
H, 4.38; N, 16.59; Found: C, 66.52; H, 4.35; N, 16.65.
4.1.1.5. 2-(2-Methoxyphenyl)-5-nitro-1H-benzo[d]imidazole
(3e).
Brown powder, yield: 97%, mp: 208e209 ꢀC. 1H NMR (300 MHz; d6-
DMSO): 4.08 (s, 3H); 7.17 (t, J ¼ 7.4 Hz, 1H); 7.30 (d, J ¼ 8.3 Hz, 1H);
7.57 (t, J ¼ 7.4 Hz,1H); 7.81 (d, J ¼ 8.9 Hz,1H); 8.13 (d, J ¼ 8.7 Hz,1H);
8.39 (d, J ¼ 7.4 Hz, 1H); 8.54 (s, 1H); 12.67 (s, 1H). MS (ESIþ) m/z
270.1 (MþH)þ. Anal. Calcd for C14H11N3O3: C, 62.45; H, 4.12; N,
15.61; Found: C, 62.66; H, 4.14; N, 15.67.
4.1.1.6. 2-(5-Nitro-1H-benzo[d]imidazol-2-yl)phenol
(3f). Dark
brown powder, yield: 37%, mp: 305e307 ꢀC. 1H NMR (300 MHz; d6-
DMSO): 6.94e7.04 (m, 2H); 7.35 (t, J ¼ 7.23 Hz, 1H); 7.71 (d,
J ¼ 8.8 Hz, 1H); 8.05 (dd, J ¼ 8.8, 2.1 Hz, 1H); 8.26 (d, J ¼ 7.0 Hz, 1H);
8.47 (d, J ¼ 1.95 Hz, 1H). MS (ESIþ) m/z 256.1 (MþH)þ. Anal. Calcd
for C13H9N3O3: C, 61.18; H, 3.55; N, 16.46; Found: C, 61.35; H, 3.51;
N, 16.50.
4.1.1.7. 2-(4-Ethylphenyl)-5-nitro-1H-benzo[d]imidazole
(3g).
Orange powder, yield: 90%, mp: 84e86 ꢀC. 1H NMR (300 MHz; d6-
DMSO): 1.22 (t, J ¼ 7.5 Hz, 3H); 2.68 (d, J ¼ 7.5 Hz, 2H); 7.42 (d,
J ¼ 8.1 Hz, 2H); 7.72 (d, J ¼ 9.0 Hz, 1H); 8.08e8.13 (m, 3H); 8.42 (s,
1H); 13.48 (s, 1H). MS (ESIþ) m/z 268.1 (MþH)þ. Anal. Calcd for
Fig. 2. (A) 2D molecular docking modeling of compound 7s with VEGFR-2 kinase. The
hydrogen bonds are displayed as green or blue dotted lines. The
p-cation interactions,
pep
interactions and interaction are shown as yellow line. (B) 3D model of the
sep
interaction between compound 7s and VEGFR-2 kinase ATP binding site. (For inter-
pretation of the references to color in this figure legend, the reader is referred to the
web version of this article.)
C
15H13N3O2: C, 67.40; H, 4.90; N, 15.72; Found: C, 67.52; H, 4.86; N,
15.68.

702
L. Shi et al. / European Journal of Medicinal Chemistry 84 (2014) 698e707
4.1.1.11. 2-(2-Chlorophenyl)-5-nitro-1H-benzo[d]imidazole
(3k).
4.1.1.20. 2-(3,5-Dichlorophenyl)-5-nitro-1H-benzo[d]imidazole (3t).
Pale orange powder, yield: 12%, mp: 290e292 ꢀC. 1H NMR
(300 MHz; d6-DMSO): 7.77e7.82 (m, 2H); 8.14 (dd, J ¼ 9.0, 2.1 Hz,
1H); 8.20 (d, J ¼ 2.1 Hz, 2H); 8.49 (d, J ¼ 2.1 Hz,1H); 13.77 (s,1H). MS
(ESIþ) m/z 307.0 (MþH)þ. Anal. Calcd for C13H7Cl2N3O2: C, 50.67; H,
2.29; N, 13.64; Found: C, 50.53; H, 2.26; N, 13.57.
Brown powder, yield: 16%, mp: 168e170 ꢀC. 1H NMR (300 MHz; d6-
DMSO): 7.52e7.62 (m, 2H); 7.67e7.70 (m, 1H); 7.80 (d, J ¼ 9.0 Hz,
1H); 7.94 (dd, J ¼ 7.2, 2.1 Hz, 1H); 8.15 (dd, J ¼ 8.7, 2.1 Hz, 1H); 8.53
(s, 1H); 13.42 (s, 1H). MS (ESIþ) m/z 274.1 (MþH)þ. Anal. Calcd for
C
13H8ClN3O2: C, 57.05; H, 2.95; N, 15.35; Found: C, 57.22; H, 2.97; N,
15.41.
4.1.1.21. 2-(2-Bromo-5-fluorophenyl)-5-nitro-1H-benzo[d]imidazole
(3u). Brown powder, mp: yield: 66%, mp: 218e220 ꢀC. 1H NMR
(300 MHz; d6-DMSO): 7.43 (dd, J ¼ 8.4, 3.0 Hz, 1H); 7.70e7.92 (m,
3H); 8.18 (d, J ¼ 9.0 Hz, 1H); 8.60 (s, 1H); 13.55 (s, 1H). MS (ESIþ) m/z
336.0 (MþH)þ. Anal. Calcd for C13H7BrFN3O2: C, 46.45; H, 2.10; N,
12.50; Found: C, 46.53; H, 2.11; N, 12.55.
4.1.1.12. 2-(3-Chlorophenyl)-5-nitro-1H-benzo[d]imidazole
(3l).
Orange powder, yield: 83%, mp: 220e222 ꢀC. 1H NMR (300 MHz;
d6-DMSO): 7.61 (d, J ¼ 5.1 Hz, 2H); 7.76 (d, J ¼ 9.0 Hz, 1H); 8.10e8.17
(m, 2H); 8.22 (d, J ¼ 0.9 Hz, 1H); 8.46 (d, J ¼ 1.8 Hz, 1H); 13.69 (s,
1H). MS (ESIþ) m/z 274.0 (MþH)þ. Anal. Calcd for C13H8ClN3O2: C,
57.05; H, 2.95; N, 15.35; Found: C, 56.88; H, 3.01; N, 15.43.
4.1.2. General procedure for the preparation of the 2-phenyl-1H-
benzo[d]imidazol-5-amine derivatives 4ae4u
4.1.1.13. 2-(4-Chlorophenyl)-5-nitro-1H-benzo[d]imidazole
(3m).
Method A: A suspension of 3 (5.0 mmol, R ¼ CH3, OCH3, OH,
CH2CH3, CF3) and 10% Pd/C (0.13 g) in methanol (30 mL) was hy-
drogenated under normal pressure for 5 h at room temperature.
Filtration and evaporation gave corresponding compound 4.
Method B: Compound 3 (5.0 mmol, R ¼ F, Cl, Br) and iron (1.1 g,
20 mmol) were suspended in aqueous ethanol (120 mL, 70% v/v)
containing acetic acid (2 mL, 30 mmol) and heated at reflux for 2 h.
The reaction mixture was cooled to room temperature. Filtration
and evaporation gave corresponding compound 4.
Yellow green powder, yield: 40%, mp: 301e303 ꢀC. 1H NMR
(300 MHz; d6-DMSO): 7.66e7.77 (m, 3H); 8.13 (dd, J ¼ 9.0, 2.1 Hz,
1H); 8.21 (d, J ¼ 8.7 Hz, 2H); 8.49 (s,1H); 13.66 (s,1H). MS (ESIþ) m/z
274.0 (MþH)þ. Anal. Calcd for C13H8ClN3O2: C, 57.05; H, 2.95; N,
15.35; Found: C, 57.19; H, 2.92; N, 15.29.
4.1.1.14. 2-(4-Bromophenyl)-5-nitro-1H-benzo[d]imidazole
(3n).
Yellow powder, yield: 78%, mp: 296e298 ꢀC. 1H NMR (300 MHz; d6-
DMSO): 7.77e7.85 (m, 3H); 8.13e8.18 (m, 3H); 8.50 (s, 1H). MS
(ESIþ) m/z 318.0 (MþH)þ. Anal. Calcd for C13H8BrN3O2: C, 49.08; H,
2.53; N, 13.21; Found: C, 49.24; H, 2.52; N, 13.26.
4.1.2.1. 2-Phenyl-1H-benzo[d]imidazol-5-amine (4a). Pink crystal,
yield: 99%, mp: 292e293 ꢀC. 1H NMR (300 MHz; d6-DMSO): 3.17 (s,
2H); 6.54 (d, J ¼ 8.5 Hz,1H); 6.69 (s,1H); 7.28 (d, J ¼ 8.5 Hz,1H); 7.41
(t, J ¼ 7.0 Hz, 1H); 7.50 (t, J ¼ 7.6 Hz, 2H); 8.07 (d, J ¼ 7.8 Hz, 2H). MS
(ESIþ) m/z 210.1 (MþH)þ. Anal. Calcd for C13H11N3: C, 74.62; H, 5.30;
N, 20.08; Found: C, 74.48; H, 5.26; N, 20.14.
4.1.1.15. 5-Nitro-2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]imid-
azole (3o). Pale orange powder, yield: 84%, mp: 190e191 ꢀC. 1H
NMR (300 MHz; d6-DMSO): 7.80e7.91 (m, 4H); 7.98 (d, J ¼ 7.5 Hz,
1H); 8.17 (d, J ¼ 8.1 Hz, 1H); 8.54 (s, 1H), 13.52 (s, 1H). MS (ESIþ) m/z
308.2 (MþH)þ. Anal. Calcd for C14H8F3N3O2: C, 54.73; H, 2.62; N,
13.68; Found: C, 54.78; H, 2.64; N, 13.65.
4.1.2.2. 2-o-Tolyl-1H-benzo[d]imidazol-5-amine (4b). Brown pow-
der, yield: 64.28%, mp: 63e64 ꢀC. 1H NMR (300 MHz; CDCl3): 2.45
(s, 3H); 3.44 (s, 2H); 6.54 (t, J ¼ 8.5 Hz, 1H); 7.08e7.46 (m, 6H). MS
(ESIþ) m/z 224.1 (MþH)þ. Anal. Calcd for C14H13N3: C, 75.31; H,
5.87; N, 18.82; Found: C, 75.52; H, 5.91; N, 18.77.
4.1.1.16. 5-Nitro-2-(3-(trifluoromethyl)phenyl)-1H-benzo[d]imid-
azole (3p). Brown crystal, yield: 56%, mp: 229e230 ꢀC. 1H NMR
(300 MHz; d6-DMSO): 7.76e7.93 (m, 3H); 8.12 (dd, J ¼ 9.0, 2.1 Hz,
1H); 8.47e8.52 (m, 3H); 13.79 (s, 1H). MS (ESIþ) m/z 308.1 (MþH)þ.
Anal. Calcd for C14H8F3N3O2: C, 54.73; H, 2.62; N, 13.68; Found: C,
54.86; H, 2.59; N, 13.72.
4.1.2.3. 2-m-Tolyl-1H-benzo[d]imidazol-5-amine (4c). Yellow pow-
der, yield: 82.33%, mp: 231e233 ꢀC. 1H NMR (300 MHz; CDCl3): 2.36
(s, 3H); 6.69 (dd, J ¼ 8.7, 2.1 Hz, 1H); 6.85 (d, J ¼ 1.8 Hz, 1H); 7.22 (d,
J ¼ 7.8 Hz,1H); 7.32 (t, J ¼ 7.5 Hz,1H); 7.48 (d, J ¼ 8.4 Hz,1H); 7.79 (d,
J ¼ 7.8 Hz,1H); 7.90 (s,1H). MS (ESIþ) m/z 224.1 (MþH)þ. Anal. Calcd
for C14H13N3: C, 75.31; H, 5.87; N, 18.82; Found: C, 75.38; H, 5.84; N,
18.86.
4.1.1.17. 5-Nitro-2-(4-(trifluoromethyl)phenyl)-1H-benzo[d]imid-
azole (3q). Orange powder, yield: 21%, mp: 273e274 ꢀC. 1H NMR
(300 MHz; d6-DMSO): 7.77 (d, J ¼ 8.7 Hz, 1H); 7.94 (d, J ¼ 8.7 Hz,
2H); 8.12 (dd, J ¼ 9.0, 2.4 Hz, 1H); 8.37 (d, J ¼ 8.1 Hz, 2H); 8.47 (d,
J ¼ 1.8 Hz, 1H); 13.78 (s, 1H). MS (ESIþ) m/z 308.1 (MþH)þ. Anal.
Calcd for C14H8F3N3O2: C, 54.73; H, 2.62; N, 13.68; Found: C, 54.88;
H, 2.60; N, 13.64.
4.1.2.4. 2-p-Tolyl-1H-benzo[d]imidazol-5-amine (4d). Orange pow-
der, yield: 89.4%, mp: 112e114 ꢀC. 1H NMR (300 MHz; CDCl3): 2.40
(s, 3H); 6.67 (dd, J ¼ 8.7, 2.1 Hz, 1H); 6.84 (s, 1H); 7.88 (t, J ¼ 1.8 Hz,
2H); 7.27 (s, 1H); 7.46 (d, J ¼ 8.5 Hz, 1H); 7.88 (t, J ¼ 1.8 Hz, 2H). MS
(ESIþ) m/z 224.1 (MþH)þ. Anal. Calcd for C14H13N3: C, 75.31; H,
5.87; N, 18.82; Found: C, 75.05; H, 5.91; N, 18.79.
4.1.1.18. 2-(2,6-Dichlorophenyl)-5-nitro-1H-benzo[d]imidazole (3r).
Pale orange powder, yield: 80%, mp: 210e212 ꢀC. 1H NMR
(300 MHz; d6-DMSO): 7.60e7.71 (m, 3H); 7.83 (d, J ¼ 9.0 Hz, 1H);
8.17 (dd, J ¼ 9.0, 2.1 Hz,1H); 8.58 (s, 1H); 13.65 (s, 1H). MS (ESIþ) m/z
307.1 (MþH)þ. Anal. Calcd for C13H7Cl2N3O2: C, 50.67; H, 2.29; N,
13.64; Found: C, 50.80; H, 2.31; N, 13.68.
4.1.2.5. 2-(2-Methoxyphenyl)-1H-benzo[d]imidazol-5-amine
(4e).
Brown powder, yield: 97%, mp: 193e194 ꢀC. 1H NMR (300 MHz; d6-
DMSO): 3.99 (s, 3H); 6.54 (dd, J ¼ 8.5, 1.6 Hz, 1H); 6.75 (s, 1H); 7.06
(t, J ¼ 7.5 Hz, 1H); 7.18 (d, J ¼ 8.3 Hz, 1H); 7.28 (d, J ¼ 8.5 Hz, 1H);
7.37e7.42 (m, 1H); 8.24 (d, J ¼ 7.7 Hz, 1H). MS (ESIþ) m/z 240.1
(MþH)þ. Anal. Calcd for C14H13N3O: C, 70.28; H, 5.48; N, 17.56;
Found: C, 70.46; H, 5.50; N, 17.51.
4.1.1.19. 2-(3,4-Dichlorophenyl)-5-nitro-1H-benzo[d]imidazole (3s).
Pale orange powder, yield: 42%, mp: 232e235 ꢀC. 1H NMR
(300 MHz; d6-DMSO): 7.75 (d, J ¼ 8.4 Hz, 1H); 7.82e7.88 (m, 1H);
8.03e8.15 (m, 2H); 8.37 (d, J ¼ 2.1 Hz,1H); 8.45 (s, 1H); 13.67 (s, 1H).
MS (ESIþ) m/z 307.1 (MþH)þ. Anal. Calcd for C13H7Cl2N3O2: C,
50.67; H, 2.29; N, 13.64; Found: C, 50.82; H, 2.28; N, 13.60.
4.1.2.6. 2-(5-Amino-1H-benzo[d]imidazol-2-yl)phenol
(4f).
Yellow powder, yield: 46%, mp: 250e251 ꢀC. 1H NMR (300 MHz; d6-
DMSO): 6.61 (d, J ¼ 8.3 Hz, 1H); 6.74 (s, 1H); 6.97 (t, J ¼ 8.4 Hz, 2H);

L. Shi et al. / European Journal of Medicinal Chemistry 84 (2014) 698e707
703
7.28e7.36 (m, 2H); 7.93 (d, J ¼ 7.7 Hz, 1H); 12.63 (s, 1H); 13.22 (s,
1H). MS (ESIþ) m/z 226.1 (MþH)þ. Anal. Calcd for C13H11N3O: C,
69.32; H, 4.92; N, 18.66; Found: C, 69.16; H, 4.95; N, 18.72.
4.1.2.15. 2-(2-(Trifluoromethyl)phenyl)-1H-benzo[d]imidazol-5-
amine (4o). Brown powder, yield: 93%, mp: 153e155 ꢀC. 1H NMR
(300 MHz; d6-DMSO): 5.12 (s, 2H); 6.50 (d, J ¼ 7.4 Hz, 1H); 6.80 (s,
1H); 7.45 (d, J ¼ 8.4 Hz, 1H); 7.61e7.66 (m, 2H); 8.54e8.71 (m, 2H).
MS (ESIþ) m/z 278.2 (MþH)þ. Anal. Calcd for C14H10F3N3: C, 60.65;
H, 3.64; N, 15.16; Found: C, 60.58; H, 3.69; N, 15.20.
4.1.2.7. 2-(4-Ethylphenyl)-1H-benzo[d]imidazol-5-amine
(4g).
Orange powder, yield: 71%, mp: 102e104 ꢀC. 1H NMR (300 MHz;
CDCl3): 1.26 (t, J ¼ 7.5 Hz, 3H); 2.67e2.71 (m, 2H); 5.30 (d,
J ¼ 0.5 Hz, 1H); 6.67 (dd, J ¼ 8.5, 2.0 Hz, 1H); 6.84 (s, 1H); 7.28 (s,
1H); 7.45 (d, J ¼ 8.0 Hz, 1H); 7.92 (d, J ¼ 8.0 Hz, 2H). MS (ESIþ) m/z
238.1 (MþH)þ. Anal. Calcd for C15H15N3: C, 75.92; H, 6.37; N, 17.71;
Found: C, 80.18; H, 6.33; N, 17.66.
4.1.2.16. 2-(3-(Trifluoromethyl)phenyl)-1H-benzo[d]imidazol-5-
amine (4p). Brown crystal, yield: 76%, mp: 93e95 ꢀC. 1H NMR
(300 MHz; d6-DMSO): 5.02 (s, 2H); 6.58 (d, J ¼ 8.4 Hz, 1H); 6.70 (s,
1H); 7.33 (d, J ¼ 8.3 Hz, 1H); 7.71e7.76 (m, 2H); 8.35e8.41 (m, 2H).
MS (ESIþ) m/z 278.1 (MþH)þ. Anal. Calcd for C14H10F3N3: C, 60.65;
H, 3.64; N, 15.16; Found: C, 60.47; H, 3.61; N, 15.22.
4.1.2.8. 2-(2-Fluorophenyl)-1H-benzo[d]imidazol-5-amine
(4h).
White powder, yield: 61.9%, mp: 214e215 ꢀC. 1H NMR (300 MHz;
CDCl3): 6.72 (dd, J ¼ 8.4, 2.1 Hz, 1H); 6.88 (s, 1H); 7.16e7.23 (m, 1H);
7.30e7.43 (m, 2H); 7.51 (d, J ¼ 6.6 Hz, 1H); 8.46 (dd, J ¼ 7.8, 1.8 Hz,
1H). MS (ESIþ) m/z 228.1 (MþH)þ. Anal. Calcd for C13H10FN3: C,
68.71; H, 4.44; N, 18.49; Found: C, 68.56; H, 4.43; N, 18.45.
4.1.2.17. 2-(4-(Trifluoromethyl)phenyl)-1H-benzo[d]imidazol-5-
amine (4q). White powder, yield: 82%, mp: 208e209 ꢀC. 1H NMR
(300 MHz; d6-DMSO): 5.09 (s, 2H); 6.57 (dd, J ¼ 8.5,1.6 Hz,1H); 6.67
(s, 1H); 7.35 (d, J ¼ 8.5 Hz, 1H); 7.86 (d, J ¼ 8.1 Hz, 2H); 8.26 (d,
J ¼ 8.1 Hz, 2H); 12.51 (s, 1H). MS (ESIþ) m/z 278.1 (MþH)þ. Anal.
Calcd for C14H10F3N3: C, 60.65; H, 3.64; N, 15.16; Found: C, 60.83; H,
3.67; N, 15.13.
4.1.2.9. 2-(3-Fluorophenyl)-1H-benzo[d]imidazol-5-amine
(4i).
Pale orange powder, yield: 76%, mp: 245e246 ꢀC. 1H NMR
(300 MHz; d6-DMSO): 4.98 (s, 2H); 6.54 (d, J ¼ 8.4 Hz, 1H); 6.67 (s,
1H); 7.19e7.30 (m, 2H); 7.49e7.56 (m, 1H); 7.81e7.91 (m, 2H); 12.32
(s, 1H). MS (ESIþ) m/z 228.1 (MþH)þ. Anal. Calcd for C13H10FN3: C,
68.71; H, 4.44; N, 18.49; Found: C, 68.89; H, 4.46; N, 18.44.
4.1.2.18. 2-(2,6-Dichlorophenyl)-1H-benzo[d]imidazol-5-amine (4r).
Brown crystal, yield: 64.25%, mp: 137e139 ꢀC. 1H NMR (300 MHz;
d6-DMSO): 6.56 (dd, J ¼ 8.7, 2.1 Hz, 1H); 6.67 (s, 1H); 7.28 (d,
J ¼ 8.4 Hz, 1H); 7.51e7.63 (m, 3H). MS (ESIþ) m/z 278.0 (MþH)þ.
Anal. Calcd for C13H9Cl2N3: C, 56.14; H, 3.26; N, 15.11; Found: C,
56.28; H, 3.23; N, 15.15.
4.1.2.10. 2-(4-Fluorophenyl)-1H-benzo[d]imidazol-5-amine
(4j).
Orange crystal, yield: 73.86%, mp: 68e69 ꢀC. 1H NMR (300 MHz;
CDCl3): 3.50 (s, 2H); 6.66 (d, J ¼ 8.4 Hz, 1H); 6.80 (s, 1H); 7.06 (t,
J ¼ 8.4 Hz, 2H); 7.42 (d, J ¼ 8.4 Hz, 1H); 9.97 (m, 2H). MS (ESIþ) m/z
228.1 (MþH)þ. Anal. Calcd for C13H10FN3: C, 68.71; H, 4.44; N,18.49;
Found: C, 68.93; H, 4.41; N, 18.55.
4.1.2.19. 2-(3,4-Dichlorophenyl)-1H-benzo[d]imidazol-5-amine (4s).
Orange crystal, yield: 71%, mp: 100e102 ꢀC. 1H NMR (300 MHz; d6-
DMSO): 6.57 (d, J ¼ 8.5 Hz, 1H); 6.68 (s, 1H); 7.31 (d, J ¼ 8.5 Hz, 1H);
7.77 (d, J ¼ 8.4 Hz, 1H); 8.03 (d, J ¼ 8.4 Hz, 1H); 8.27 (s, 1H). MS
(ESIþ) m/z 278.1 (MþH)þ. Anal. Calcd for C13H9Cl2N3: C, 56.14; H,
3.26; N, 15.11; Found: C, 56.32; H, 3.24; N, 15.15.
4.1.2.11. 2-(2-Chlorophenyl)-1H-benzo[d]imidazol-5-amine
(4k).
Brown powder, yield: 76.58%, mp: 87e89 ꢀC. 1H NMR (500 MHz;
CDCl3): 6.73 (dd, J ¼ 8.0, 2.0 Hz, 1H); 6.89 (s, 1H); 7.33e7.41 (m, 2H);
7.47 (dd, J ¼ 8.0, 1.5 Hz, 1H); 7.52 (d, J ¼ 8.0 Hz, 1H); 8.39 (dd, J ¼ 7.5,
1.5 Hz, 1H). MS (ESIþ) m/z 244.1 (MþH)þ. Anal. Calcd for
4.1.2.20. 2-(3,5-Dichlorophenyl)-1H-benzo[d]imidazol-5-amine (4t).
Brown powder, yield ¼ 51%, mp: 241e243 ꢀC. 1H NMR (300 MHz;
d6-DMSO): 6.57 (d, J ¼ 8.4 Hz, 1H); 6.60 (s, 1H); 7.30 (d, J ¼ 8.4 Hz,
1H); 7.62 (s, 1H); 8.06 (s, 2H); 12.50 (s, 1H). MS (ESIþ) m/z 278.0
(MþH)þ. Anal. Calcd for C13H9Cl2N3: C, 56.14; H, 3.26; N, 15.11;
Found: C, 56.30; H, 3.29; N, 15.08.
C
13H10ClN3: C, 64.07; H, 4.14; N, 17.24; Found: C, 64.25; H, 4.16; N,
17.26.
4.1.2.12. 2-(3-Chlorophenyl)-1H-benzo[d]imidazol-5-amine
(4l).
Orange powder, yield: 38%, mp: 72e74 ꢀC. 1H NMR (300 MHz;
CDCl3): 6.69 (dd, J ¼ 8.7, 2.1 Hz, 1H); 6.82 (d, J ¼ 1.8 Hz, 1H);
7.33e7.35 (m, 2H); 7.47 (d, J ¼ 8.4 Hz, 1H); 7.84e7.88 (m, 1H);
7.97e7.99 (m, 1H). MS (ESIþ) m/z 244.1 (MþH)þ. Anal. Calcd for
4.1.2.21. 2-(2-Bromo-5-fluorophenyl)-1H-benzo[d]imidazol-5-amine
(4u). Yellow powder, yield: 92%, mp: 96e97 ꢀC. 1H NMR (300 MHz;
CDCl3): 6.74 (dd, J ¼ 8.4, 2.1 Hz, 1H); 6.86 (d, J ¼ 4.5 Hz, 1H); 7.01
(dd, J ¼ 8.7, 3.0 Hz, 1H); 7.54e7.64 (m, 2H); 8.08 (dd, J ¼ 9.9, 3.3 Hz,
1H). MS (ESIþ) m/z 306.0 (MþH)þ. Anal. Calcd for C13H9FBrN3: C,
51.00; H, 2.96; N, 13.73; Found: C, 51.15; H, 2.99; N, 13.70.
C
13H10ClN3: C, 64.07; H, 4.14; N, 17.24; Found: C, 63.88; H, 4.17; N,
17.29.
4.1.2.13. 2-(4-Chlorophenyl)-1H-benzo[d]imidazol-5-amine
(4m).
Brown powder, yield: 57%, mp: 88e90 ꢀC. 1H NMR (300 MHz;
CDCl3): 2.17 (s, 2H); 6.63 (dd, J ¼ 8.7, 2.1 Hz, 1H); 6.73 (d, J ¼ 2.1 Hz,
1H); 7.31 (dd, J ¼ 6.9, 2.4 Hz, 2H); 7.37 (d, J ¼ 8.7 Hz, 1H); 8.85 (dd;
J ¼ 5.4, 1.8 Hz, 2H). MS (ESIþ) m/z 244.2 (MþH)þ. Anal. Calcd for
C13H10ClN3: C, 64.07; H, 4.14; N, 17.24; Found: C, 64.23; H, 4.12; N,
17.28.
4.1.3. Synthesis of 4-chloroquinoline (6)
A suspension of quinolin-4-ol (5) (2 g) in POCl3 (30 mL) was
heated at 100 ꢀC for 4 h. After cooling, the mixture was concen-
trated under reduced pressure and the ice was added to the residue.
The pH was adjusted to 6 with ammonia to allow precipitation. The
filter cake was washed with water and dried to give 4-
chloroquinoline (6). White powder, yield: 77%, mp: 28e31 ꢀC. 1H
NMR (300 MHz; CDCl3): 7.41 (d, J ¼ 4.7 Hz, 1H); 7.54e7.60 (m, 1H);
7.69e7.74 (m, 1H); 8.09e8.17 (m, 2H); 8.73 (d, J ¼ 4.7 Hz, 1H). MS
(ESIþ) m/z 164.0 (MþH)þ. Anal. Calcd for C9H6ClN: C, 66.07; H, 3.70;
N, 8.56; Found: C, 66.18; H, 3.68; N, 8.52.
4.1.2.14. 2-(4-Bromophenyl)-1H-benzo[d]imidazol-5-amine
(4n).
Orange powder, yield: 64.45%, mp: 104e106 ꢀC. 1H NMR (300 MHz;
d6-DMSO): 6.54 (d, J ¼ 8.4 Hz, 1H); 6.70 (s, 1H); 7.25 (d, J ¼ 8.4 Hz,
1H); 7.64 (d, J ¼ 8.4 Hz, 2H); 8.06 (d, J ¼ 8.4 Hz, 2H). MS (ESIþ) m/z
288.0 (MþH)þ. Anal. Calcd for C13H10BrN3: C, 54.19; H, 3.50; N,
14.58; Found: C, 54.34; H, 3.51; N, 14.62.

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4.1.4. General procedure for the preparation of the N-phenyl-1H-
benzo[d]imidazol-5-yl)quinolin-4-amine derivatives 7ae7u
A mixture of 4-chloroquinoline (6, 5 mmol) and appropriate
substituted anilines (4ae4u, 6 mmol) in isopropanol (40 mL) was
stirred at reflux for 5 h. The reaction mixture was concentrated
under reduced pressure and the solid residue was purified by col-
umn chromatography on silica gel, eluting with CH2Cl2/CH3OH (10/
1) to furnish title compound 7ae7u.
J ¼ 7.8 Hz, 1H); 8.46 (d, J ¼ 6.6 Hz, 1H); 8.71 (d, J ¼ 8.4 Hz, 1H); 10.61
(s, 1H); 12.89 (s, 1H); 13.50 (s, 1H). 13C NMR (300 MHz; d6-DMSO):
100.4, 112.8, 116.1, 117.8, 121.9, 124.5, 129.2, 132.2, 135.0, 139.0, 147.2,
153.4, 155.6. MS (ESIþ) m/z 353.1 (MþH)þ. Anal. Calcd for
C22H16N4O: C, 74.98; H, 4.58; N, 15.90; Found: C, 74.75; H, 4.60; N,
15.95.
4.1.4.7. N-(2-(4-ethylphenyl)-1H-benzo[d]imidazol-6-yl)quinolin-4-
amine (7g). Yellow powder, yield: 10%, mp: 351e353 ꢀC. 1H NMR
(500 MHz; d6-DMSO): 1.23 (t, J ¼ 7.5 Hz, 3H); 2.69 (q, J ¼ 4.5 Hz,
2H); 6.76 (s,1H); 7.26 (d, J ¼ 8.5 Hz,1H); 7.40 (d, J ¼ 8.0 Hz, 2H); 7.76
(t, J ¼ 7.5 Hz, 3H); 7.97 (t, J ¼ 7.5 Hz,1H); 8.04 (d, J ¼ 8.5 Hz,1H); 8.14
(d, J ¼ 8.0 Hz, 2H); 8.44 (d, J ¼ 6.5 Hz, 1H); 8.77 (d, J ¼ 8.5 Hz, 1H);
10.76 (s, 1H); 13.24 (s, 1H). 13C NMR (300 MHz; d6-DMSO): 15.3,
28.0, 99.9, 117.6, 123.2, 126.1, 126.6, 127.4, 128.3, 132.5, 144.6, 146.0,
152.7. MS (ESIþ) m/z 365.2 (MþH)þ. Anal. Calcd for C24H20N4: C,
79.10; H, 5.53; N, 15.37; Found: C, 79.28; H, 5.54; N, 15.41.
4.1.4.1. N-(2-phenyl-1H-benzo[d]imidazol-6-yl)quinolin-4-amine
(7a). Yellow powder, yield: 47%, mp: 323e325 ꢀC. 1H NMR
(300 MHz; d6-DMSO): 6.77 (d, J ¼ 6.9 Hz, 1H); 7.31 (dd, J ¼ 8.7,
1.8 Hz, 1H); 7.51e7.62 (m, 3H); 7.73e7.84 (m, 3H); 8.01e8.11 (m,
2H); 8.25 (dd, J ¼ 8.1, 1.5 Hz, 2H); 8.47 (d, J ¼ 6.9 Hz, 1H); 8.85 (d,
J ¼ 8.7 Hz, 1H); 11.09 (s, 1H); 13.44 (s, 1H). 13C NMR (300 MHz; d6-
DMSO): 99.7, 117.0, 120.2, 123.6, 126.6, 136.9, 129.0, 129.8, 130.2,
133.7, 138.2, 142.5, 152.8, 155.6. MS (ESIþ) m/z 337.2 (MþH)þ. Anal.
Calcd for C22H16N4: C, 78.55; H, 4.79; N, 16.66; Found: C, 78.81; H,
4.81; N, 16.62.
4.1.4.8. N-(2-(2-fluorophenyl)-1H-benzo[d]imidazol-6-yl)quinolin-4-
amine (7h). Yellow powder, yield: 68%, mp: 337e339 ꢀC. 1H NMR
(300 MHz; d6-DMSO): 6.76 (s, 1H); 7.32 (dd, J ¼ 8.4, 1.8 Hz, 1H);
7.39e7.49 (m, 2H); 7.56e7.63 (m,1H); 7.77e7.82 (m, 3H); 7.99e8.09
(m, 2H); 8.23e8.29 (m, 1H); 8.45 (d, J ¼ 6.9 Hz, 1H); 8.84 (d,
J ¼ 8.4 Hz, 1H); 11.10 (s, 1H); 12.91 (s, 1H). 13C NMR (300 MHz; d6-
DMSO): 99.7, 116.4, 116.8, 120.1, 123.7, 125.1, 126.9, 130.2, 132.1,
133.7, 134.3, 138.2, 142.5, 155.7, 161.1. MS (ESIþ) m/z 355.1 (MþH)þ.
Anal. Calcd for C22H15FN4: C, 74.56; H, 4.27; N, 15.81; Found: C,
74.69; H, 4.30; N, 15.77.
4.1.4.2. N-(2-o-tolyl-1H-benzo[d]imidazol-6-yl)quinolin-4-amine
(7b). Bright yellow crystal, yield: 56%, mp: 218e220 ꢀC. 1H NMR
(300 MHz; CDCl3): 2.52 (s, 3H); 6.58 (d, J ¼ 6.4 Hz, 1H); 7.05e7.63
(m, 10H); 7.97e8.04 (m, 1H); 8.67 (d, J ¼ 8.2 Hz, 1H). 13C NMR
(300 MHz; d6-DMSO): 21.9, 100.6, 118.1, 122.0, 124.4, 126.8, 127.3,
130.3, 130.5, 132.1, 134.0, 137.9, 144.1, 154.2, 155.7. MS (ESIþ) m/z
351.1 (MþH)þ. Anal. Calcd for C23H18N4: C, 78.83; H, 5.18; N, 15.99;
Found: C, 79.02; H, 5.14; N, 16.03.
4.1.4.3. N-(2-m-tolyl-1H-benzo[d]imidazol-6-yl)quinolin-4-amine
(7c). Bright yellow crystal, yield: 75%, mp: 237e239 ꢀC. 1H NMR
(300 MHz; CDCl3): 2.43 (s, 3H); 6.76 (d, J ¼ 6.9 Hz, 1H); 7.28e7.36
(m, 2H); 7.46 (t, J ¼ 7.5 Hz, 1H); 7.71e7.83 (m, 3H); 8.00-8.12 (m,
4H); 8.46 (d, J ¼ 6.9 Hz, 1H); 8.86 (d, J ¼ 8.7 Hz, 1H); 11.09 (s, 1H);
13.04 (s, 1H). 13C NMR (300 MHz; d6-DMSO): 21.0, 99.7, 117.1, 120.6,
123.6, 123.8, 126.7, 127.1, 128.9, 129.7, 130.7, 133.5, 138.2, 142.8,
152.8, 155.2. MS (ESIþ) m/z 351.1 (MþH)þ. Anal. Calcd for C23H18N4:
C, 78.83; H, 5.18; N, 15.99; Found: C, 78.95; H, 5.21; N, 15.95.
4.1.4.9. N-(2-(3-fluorophenyl)-1H-benzo[d]imidazol-6-yl)quinolin-4-
amine (7i). Dark yellow powder, yield: 46%, mp: 342e343 ꢀC. 1H
NMR (300 MHz; d6-DMSO): 6.75 (d, J ¼ 6.6 Hz, 1H); 7.30e7.40 (m,
2H); 7.59e7.83 (m, 4H); 8.02e8.10 (m, 4H); 8.46 (d, J ¼ 7.2 Hz, 1H);
8.81 (d, J ¼ 8.4 Hz, 1H); 11.07 (s, 1H); 13.54 (s, 1H). 13C NMR
(300 MHz; d6-DMSO): 99.0, 113.1, 113.4, 116.7, 117.0, 120.1, 120.5,
122.7, 123.7, 126.8, 131.1, 132.0,133.7, 138.2,142.4,151.5, 155.5, 160.8,
164.0. MS (ESIþ) m/z 355.1 (MþH)þ. Anal. Calcd for C22H15FN4: C,
74.56; H, 4.27; N, 15.81; Found: C, 74.35; H, 4.29; N, 15.85.
4.1.4.4. N-(2-p-tolyl-1H-benzo[d]imidazol-6-yl)quinolin-4-amine
(7d). Yellow powder, yield: 73%, mp: 363e365 ꢀC. 1H NMR
(300 MHz; CDCl3): 2.41 (s, 3H); 6.76 (s, 1H); 7.29 (d, J ¼ 8.4 Hz, 1H);
7.4 (d, J ¼ 8.1 Hz, 2H); 7.74e7.85 (m, 3H); 8.01e8.06 (m, 2H); 8.12 (d,
J ¼ 7.8 Hz, 2H); 8.47 (d, J ¼ 6.9 Hz, 1H); 8.8 (d, J ¼ 8.4 Hz, 1H); 11.01
(s, 1H); 13.24 (s, 1H). 13C NMR (300 MHz; d6-DMSO): 21.0, 99.6,
117.0, 120.3, 123.6, 126.6, 126.8, 127.1, 129.5, 133.6, 138.4, 139.9,
142.5, 152.9, 155.5. MS (ESIþ) m/z 351.1 (MþH)þ. Anal. Calcd for
4.1.4.10. N-(2-(4-fluorophenyl)-1H-benzo[d]imidazol-6-yl)quinolin-
4-amine (7j). Bright yellow powder, yield: 62%, mp: 353e355 ꢀC.
1H NMR (300 MHz; d6-DMSO): 6.73 (s, 1H); 7.29e7.40 (m, 3H);
7.70e7.76 (m, 3H); 8.00e8.07 (m, 2H); 8.29e8.44 (m, 3H); 8.85 (s,
1H); 11.12 (s, 1H); 13.55 (s, 1H). 13C NMR (300 MHz; d6-DMSO): 99.7,
115.9, 116.2, 116.9, 120.1, 123.6, 126.4, 126.8, 129.0, 133.7, 138.2,
142.4, 151.9, 155.6. MS (ESIþ) m/z 355.1 (MþH)þ. Anal. Calcd for
C
22H15FN4: C, 74.56; H, 4.27; N, 15.81; Found: C, 74.37; H, 4.25; N,
C
23H18N4: C, 78.83; H, 5.18; N, 15.99; Found: C, 78.67; H, 5.22; N,
15.76.
16.04.
4.1.4.11. N-(2-(2-chlorophenyl)-1H-benzo[d]imidazol-6-yl)quinolin-
4-amine (7k). Brown crystal, yield: 56%, mp: 330e332 ꢀC. 1H NMR
(500 MHz; d6-DMSO): 6.76 (s, 1H); 7.32 (d, J ¼ 8.0 Hz,1H); 7.51e7.57
(m, 2H); 7.65e7.76 (m, 4H); 7.94e7.98 (m, 2H); 8.07 (d, J ¼ 8.5 Hz,
1H); 8.44 (d, J ¼ 7.0 Hz, 1H); 8.84 (d, J ¼ 8.5 Hz, 1H); 10.90 (s, 1H);
13.06 (s, 1H). 13C NMR (300 MHz; d6-DMSO): 99.8, 117.3, 121.4,
123.6, 126.4, 127.5, 129.6, 130.4, 131.4, 131.6, 132.0, 133.0, 143.5,
150.4, 154.7. MS (ESIþ) m/z 371.1 (MþH)þ. Anal. Calcd for
4.1.4.5. N-(2-(2-methoxyphenyl)-1H-benzo[d]imidazol-6-yl)quino-
lin-4-amine (7e). Dark brown crystal, yield: 78%, mp: 220e223 ꢀC.
1H NMR (300 MHz; d6-DMSO): 6.06 (s, 3H); 6.77 (d, J ¼ 6.9 Hz, 1H);
7.12e7.18 (m, 1H); 7.27e7.31 (m, 2H); 7.50e7.56 (m, 1H); 7.74e7.83
(m, 3H); 8.00e8.11 (m. 2H); 8.36 (dd, J ¼ 7.8, 1.5 Hz, 1H); 8.47 (d,
J ¼ 6.9 Hz,1H); 8.86 (d, J ¼ 8.4 Hz,1H); 11.10 (s,1H); 12.42 (s,1H). 13
C
NMR (300 MHz; d6-DMSO): 56.6, 100.7, 113.4, 118.0, 120.0, 124.3,
127.5, 132.8, 134.3, 144.0, 145.4, 158.7. MS (ESIþ) m/z 367.1 (MþH)þ.
Anal. Calcd for C23H18N4O: C, 75.39; H, 4.95; N, 15.29; Found: C,
75.48; H, 4.91; N, 15.34.
C
22H15ClN4: C, 71.25; H, 4.08; N, 15.11; Found: C, 71.48; H, 4.12; N,
15.15.
4.1.4.12. N-(2-(3-chlorophenyl)-1H-benzo[d]imidazol-6-yl)quinolin-
4-amine (7l). Yellow powder, yield: 54%, mp: 323e325 ꢀC. 1H NMR
(500 MHz; d6-DMSO): 6.77 (s, 1H); 7.32 (d, J ¼ 8.0 Hz, 1H);
7.57e7.63 (m, 2H); 7.77 (t, J ¼ 7.5 Hz, 3H); 7.99 (t, J ¼ 7.0 Hz, 1H);
8.09 (d, J ¼ 8.5 Hz, 1H); 8.24 (d, J ¼ 7.5 Hz, 1H); 8.32 (s, 1H); 8.46 (d,
4.1.4.6. 2-(6-(Quinolin-4-ylamino)-1H-benzo[d]imidazol-2-yl)phenol
(7f). Yellow crystal, mp: yield: 10%, mp: 241e243 ꢀC. 1H NMR
(300 MHz; d6-DMSO): 6.79 (s, 1H); 7.00e7.07 (m, 2H); 7.33e7.42
(m, 2H); 7.75 (t, J ¼ 6.9 Hz, 3H); 7.92e8.02 (m, 2H); 8.12 (d,

L. Shi et al. / European Journal of Medicinal Chemistry 84 (2014) 698e707
705
J ¼ 7.0 Hz, 1H); 8.85 (d, J ¼ 8.5 Hz, 1H); 10.93 (s, 1H); 13.67 (s, 1H).
13C NMR (300 MHz; d6-DMSO): 100.6, 121.8, 124.4, 126.0, 127.0,
127.4, 130.6, 131.8, 132.7, 134.1, 134.6, 144.0, 154.5, 163.4. MS (ESIþ)
m/z 371.1 (MþH)þ. Anal. Calcd for C22H15ClN4: C, 71.25; H, 4.08; N,
15.11; Found: C, 71.46; H, 4.05; N, 15.06.
(MþH)þ. Anal. Calcd for C22H14Cl2N4: C, 65.20; H, 3.48; N, 13.82;
Found: C, 65.38; H, 3.46; N, 13.85.
4.1.4.19. N-(2-(3,4-dichlorophenyl)-1H-benzo[d]imidazol-6-yl)qui-
nolin-4-amine (7s). Yellow powder, yield: 56%, mp: 247e249 ꢀC. 1H
NMR (300 MHz; d6-DMSO): 6.74 (s, 1H); 7.32 (d, J ¼ 8.1 Hz, 1H);
7.68e7.87 (m, 4H); 7.99e8.06 (m, 2H); 8.21 (dd, J ¼ 8.4, 1.8 Hz, 1H);
8.46e8.47 (m, 2H); 8.80 (d, J ¼ 8.7 Hz, 1H); 11.06 (s, 1H); 13.61 (s,
1H). 13C NMR (300 MHz; d6-DMSO): 101.6, 119.2, 121.8, 123.1, 124.7,
128.0, 129.2, 130.3, 131.5, 133.0, 134.1, 134.9, 135.8, 140.4, 144.0,
153.5, 158.5. MS (ESIþ) m/z 405.2 (MþH)þ. Anal. Calcd for
4.1.4.13. N-(2-(4-chlorophenyl)-1H-benzo[d]imidazol-6-yl)quinolin-
4-amine (7m). Yellow powder, yield: 55%, mp: 345e347 ꢀC. 1H
NMR (300 MHz; d6-DMSO): 6.76 (s, 1H); 7.32 (d, J ¼ 7.8 Hz; 1H);
7.65e7.83 (m, 5H); 8.00e8.10 (m, 2H); 8.26 (d, J ¼ 8.7 Hz, 2H); 8.47
(d, J ¼ 6.9 Hz,1H); 8.83 (d, J ¼ 8.7 Hz,1H); 11.06 (s,1H); 13.53 (s,1H).
13C NMR (300 MHz; d6-DMSO): 99.8,117.0,120.2,123.4,123.7,126.9,
128.4, 130.2, 132.6, 133.8, 134.9, 138.2, 142.5, 146.3, 151.8, 155.6. MS
(ESIþ) m/z 371.2 (MþH)þ. Anal. Calcd for C22H15ClN4: C, 71.25; H,
4.08; N, 15.11; Found: C, 71.07; H, 4.10; N, 15.14.
C22H14Cl2N4: C, 65.20; H, 3.48; N, 13.82; Found: C, 65.41; H, 3.45; N,
13.78.
4.1.4.20. N-(2-(3,5-dichlorophenyl)-1H-benzo[d]imidazol-6-yl)qui-
nolin-4-amine (7t). Dark yellow powder, yield: 18%, mp:
349e350 ꢀC. 1H NMR (300 MHz; d6-DMSO): 6.75 (s, 1H); 7.25e7.40
(m, 1H); 7.63e7.92 (m, 4H); 7.95e8.15 (m, 2H); 8.26 (s, 2H); 8.46 (d,
J ¼ 6.9 Hz, 1H); 8.80 (d, J ¼ 8.1 Hz, 1H); 11.05 (s, 1H); 13.65 (s, 1H).
13C NMR (300 MHz; d6-DMSO): 100.5, 114.9, 120.7, 122.6, 124.4,
125.8, 127.7, 129.9, 133.9, 134.4, 135.7, 142.6, 150.6, 161.1. MS (ESIþ)
m/z 405.1 (MþH)þ. Anal. Calcd for C22H14Cl2N4: C, 65.20; H, 3.48; N,
13.82; Found: C, 65.42; H, 3.52; N, 13.86.
4.1.4.14. N-(2-(4- bromophenyl)-1H-benzo[d]imidazol-6-yl)quinolin-
4-amine (7n). Pale yellow powder, yield: 62%, mp: 359e361 ꢀC. 1H
NMR (500 MHz; d6-DMSO): 6.76 (m, 1H); 7.31 (s, 1H); 7.66e7.80 (m,
5H); 8.02 (t, J ¼ 8.0 Hz, 1H); 8.11 (d, J ¼ 8.5 Hz, 1H); 8.21 (d,
J ¼ 8.0 Hz, 2H); 8.46 (d, J ¼ 6.5 Hz,1H); 8.86 (d, J ¼ 8.5 Hz, 1H); 11.06
(s, 1H); 13.62 (s, 1H). 13C NMR (300 MHz; d6-DMSO): 100.2, 116.5,
120.6, 123.7, 126.5, 128.5, 129.1, 131.9, 133.7, 135.0, 139.4, 144.2,
151.6, 155.4. MS (ESIþ) m/z 415.1 (MþH)þ. Anal. Calcd for
4.1.4.21. N-(2-(2-bromo-5-fluorophenyl)-1H-benzo[d]imidazol-6-yl)
quinolin-4-amine (7u). Brown crystal, yield: 40%, mp: 238e239 ꢀC.
1H NMR (300 MHz; d6-DMSO): 6.73 (s, 1H); 7.32e7.43 (m, 2H);
7.69e7.90 (m, 5H); 7.99e8.07 (m, 2H); 8.45 (d, J ¼ 6.9 Hz, 1H); 8.82
(d, J ¼ 8.4 Hz,1H); 10.95 (s,1H); 11.17 (s,1H). 13C NMR (300 MHz; d6-
DMSO): 100.4, 119.7, 120.0, 124.2, 127.2, 129.0, 132.7, 136.3, 139.6,
141.4, 151.6, 160.2. MS (ESIþ) m/z 433.0 (MþH)þ. Anal. Calcd for
C
22H15BrN4: C, 63.63; H, 3.64; N, 13.49; Found: C, 63.85; H, 3.60; N,
13.53.
4.1.4.15. N-(2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-6-
yl)quinolin-4-amine (7o). Dark yellow powder, yield: 34%, mp:
244e246 ꢀC. 1H NMR (300 MHz; d6-DMSO): 6.73e6.78 (m, 1H);
7.34 (s, 1H); 7.66e7.87 (m, 6H); 7.95e8.08 (m, 3H); 8.46 (d,
C22H14BrFN4: C, 60.99; H, 3.26; N, 12.93; Found: C, 61.21; H, 3.29; N,
J ¼ 6.9 Hz,1H); 8.83 (d, J ¼ 8.4 Hz,1H); 11.06 (s,1H); 13.15 (s,1H). 13
C
12.97.
NMR (300 MHz; d6-DMSO): 100.9, 120.5, 123.6, 126.7, 130.4, 132.2,
132.5, 133.6, 145.8, 149.2, 163.1. MS (ESIþ) m/z 405.1 (MþH)þ. Anal.
Calcd for C23H15F3N4: C, 68.31; H, 3.74; N, 13.85; Found: C, 68.16; H,
3.78; N, 13.81.
4.2. Cell proliferative assay
The antiproliferative activities of the prepared N-(2-phenyl-1H-
benzo[d]imidazol-5-yl)quinolin-4-amine derivatives against Hep-
G2, MCF-7 and L02 cells were evaluated by MTT assay. Briefly,
target tumor cells were grown to log phase in RPMI 1640 medium
supplemented with 10% fetal bovine serum. After diluting to
4.1.4.16. N-(2-(3-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-6-
yl)quinolin-4-amine (7p). Brown powder, yield: 60%, mp:
243e245 ꢀC. 1H NMR (300 MHz; d6-DMSO): 6.74e6.78 (m,1H); 7.33
(d, J ¼ 6.3 Hz, 1H); 7.69e7.87 (m, 5H); 7.99e8.05 (m, 2H); 8.45e8.57
(m, 3H); 8.82 (d, J ¼ 8.4 Hz, 1H); 11.05 (s, 1H); 13.68 (s, 1H). 13C NMR
(300 MHz; d6-DMSO): 100.6, 119.3, 123.7, 124.5, 126.4, 127.0, 130.5,
132.4, 133.8, 138.2, 143.8, 152.2, 162.5. MS (ESIþ) m/z 405.1 (MþH)þ.
Anal. Calcd for C23H15F3N4: C, 68.31; H, 3.74; N, 13.85; Found: C,
68.08; H, 3.72; N, 13.80.
2 ꢁ 104 cells mLꢂ1 with the complete medium, 100
mL of the ob-
tained cell suspension was added to each well of 96-well culture
plates. The subsequent incubation was permitted at 37 ꢀC, 5% CO2
atmosphere for 24 h before the antiproliferative assessments.
Tested samples at pre-set concentrations were added to 6 wells.
After 48 h exposure period, 40
m
L of PBS containing 2.5 mg mLꢂ1 of
L extraction
MTT was added to each well. Four hours later, 100
m
4.1.4.17. N-(2-(4-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-6-
yl)quinolin-4-amine (7q). Dark brown powder, yield: 21%, mp:
258e260 ꢀC. 1H NMR (300 MHz; d6-DMSO): 6.76e6.85 (m, 1H);
7.35 (d, J ¼ 7.5 Hz, 1H); 7.71e8.11 (m, 7H); 8.48 (d, J ¼ 7.4 Hz, 3H);
8.85 (d, J ¼ 8.3 Hz, 1H); 11.02 (s, 1H); 13.77 (s, 1H). 13C NMR
(300 MHz; d6-DMSO): 101.1, 120.2, 123.4, 125.9, 126.5, 127.2, 130.4,
133.7, 142.1, 154.6, 161.7. MS (ESIþ) m/z 405.3 (MþH)þ. Anal. Calcd
for C23H15F3N4: C, 68.31; H, 3.74; N, 13.85; Found: C, 68.55; H, 3.77;
N, 13.89.
solution (10% SDS-5% isobutyl alcohol-0.010 M HCl) was added.
After an overnight incubation at 37 ꢀC, the optical density was
measured at a wavelength of 570 nm on an ELISA microplate reader.
4.3. In vitro kinase assay
In vitro kinase assay was tested using HTScan VEGF Receptor 2
Kinase Assay Kit (purchased from Cell Signaling Technology, Inc.) by
colorimetric ELISA assay according to the instructions. Reaction
cocktail containing VEGFR2 was incubated with various concen-
trations of tested compounds or DMSO (0.1%) for 5 min at room
temperature, and then ATP/substrate peptide cocktail was added to
the pre-incubated reaction cocktail. After incubation at room
temperature for 30 min, the reaction was stopped and transferred
to a 96-well streptavidin-coated plate, and incubated for 1 h at
room temperature. Primary antibody [phosphorylated tyrosine
monoclonal antibody [(pTyr-100), 1:1000 in PBS/T with 1% bovine
4.1.4.18. N-(2-(2,6-dichlorophenyl)-1H-benzo[d]imidazol-6-yl)qui-
nolin-4-amine (7r). Pale yellow powder, yield: 52%, mp:
354e356 ꢀC. 1H NMR (300 MHz; d6-DMSO): 6.79 (s, 1H); 7.35 (d,
J ¼ 9.0 Hz, 1H); 7.60e7.85 (m, 6H); 8.03 (d, J ¼ 3.9 Hz, 2H); 8.47 (d,
J ¼ 6.9 Hz,1H); 8.80 (d, J ¼ 8.4 Hz,1H); 11.03 (s,1H); 13.19 (s,1H). 13
C
NMR (300 MHz; d6-DMSO): 100.6, 117.8, 121.0, 124.5, 127.7, 129.2,
133.4, 134.6, 135.8, 139.0, 143.4, 153.2, 156.5. MS (ESIþ) m/z 405.2

706
L. Shi et al. / European Journal of Medicinal Chemistry 84 (2014) 698e707
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serum albumin (BSA)] was added into per well until the wells were
washed thrice with PBS/T. After incubated at room temperature for
1 h, phosphorylation of the substrate was monitored with HRP-
labeled anti-mouse IgG antibody (1:500 in PBS/T with 1% BSA),
followed by a chromogenic reaction. Finally, the VEGFR2 kinase
assay was detected at 450 nm with microplate reader. The reaction
processed with only DMSO (0.1%) served as a vehicle control. The
results were expressed as percent kinase activity of the vehicle
control, and IC50 was defined as the compound concentration that
resulted in 50% inhibition of enzyme activity. The kinase assay was
performed thrice independently.
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€
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4.4. Molecular docking
Molecular docking of compound 7s into the three dimensional
X-ray structure of VEGFR-2 kinase (PDB code: 2QU5) was carried
out using the Discovery Stutio (version 3.1) as implemented
through the graphical user interface DiscoveryStudio CDOCKER
protocol.
The three-dimensional structure of the compound 7s was con-
structed using ChemBio 3D Ultra 11.0 software [Chemical Structure
Drawing Standard; Cambridge Soft corporation, USA (2008)], then
it was energetically minimized by using MMFF94 with 5000 iter-
ations and minimum RMS gradient of 0.10. The crystal structures of
VEGFR-2 kinase were retrieved from the RCSB Protein Data Bank
(http://www.rcsb.org/pdb/home/home.-do). All bound waters and
ligands were eliminated from the protein and the polar hydrogen
was added. The whole 2QU5 was defined as a receptor and the site
sphere was selected based on ATP binding site of 2QU5. Compound
7s were placed during the molecular docking procedure. Types of
interactions of the docked protein with ligand were analyzed after
the end of molecular docking.
Acknowledgments
This work was supported by National Natural Science Founda-
tion of China (Grant No. 21102182), Natural Science Foundation of
Jiangsu Province (Grant No. BK2012760) and Research Fund of
young scholars for the Doctoral Program of Higher Education of
China (Grant No. 20110096120008).
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