Discovery of quinazolin-4-amines bearing benzimidazole fragments as dual inhibitors of c-Met and VEGFR-2

Article abstract of DOI:10.1016/j.bmc.2014.07.008

Both c-Met and VEGFR-2 are important targets for the treatment of cancers. In this study, a series of N-(2-phenyl-1H-benzo[d]imidazol-5-yl)quinazolin-4- amine derivatives were designed and identified as dual c-Met and VEGFR-2 inhibitors. Among these compounds bearing quinazoline and benzimidazole fragments, compound 7j exhibited the most potent inhibitory activity against c-Met and VEGFR-2 with IC₅₀ of 0.05 μM and 0.02 μM, respectively. It also showed the highest anticancer activity against the tested cancer cell lines with IC₅₀ of 1.5 μM against MCF-7 and 8.7 μM against Hep-G2. Docking simulation supported the initial pharmacophoric hypothesis and suggested a common mode of interaction at the ATP-binding site of c-Met and VEGFR-2, which demonstrates that compound 7j is a potential agent for cancer therapy deserving further researching.

Full text of DOI:10.1016/j.bmc.2014.07.008

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of quinazolin-4-amines bearing benzimidazole fragments
as dual inhibitors of c-Met and VEGFR-2
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:
Both c-Met and VEGFR-2 are important targets for the treatment of cancers. In this study, a series of N-(2-
phenyl-1H-benzo[d]imidazol-5-yl)quinazolin-4-amine derivatives were designed and identified as dual
c-Met and VEGFR-2 inhibitors. Among these compounds bearing quinazoline and benzimidazole frag-
ments, compound 7j exhibited the most potent inhibitory activity against c-Met and VEGFR-2 with
Received 14 May 2014
Revised 2 July 2014
Accepted 4 July 2014
Available online xxxx
IC50 of 0.05
l
M and 0.02
lM, respectively. It also showed the highest anticancer activity against the
tested cancer cell lines with IC50 of 1.5
l
M against MCF-7 and 8.7 M against Hep-G2. Docking simulation
l
Keywords:
Quinazoline
Benzimidazole
Dual inhibitor
c-Met
supported the initial pharmacophoric hypothesis and suggested a common mode of interaction at the
ATP-binding site of c-Met and VEGFR-2, which demonstrates that compound 7j is a potential agent for
cancer therapy deserving further researching.
Ó 2014 Elsevier Ltd. All rights reserved.
VEGFR-2
1. Introduction
inhibition of both c-Met and VEGFR signaling represents a promis-
ing approach to cancer treatment.
The receptor tyrosine kinase c-Met and its natural ligand hepa-
tocyte growth factor (HGF) are involved in cell proliferation, migra-
tion, invasion, and survival and are essential for normal embryonic
development and wound healing.1,2 However, dysregulation of
c-Met/HGF pathway can lead to tumorigenesis and metastasis.3
Aberrant expression of c-Met/HGF axis has been identified in a
wide range of human malignancies such as bladder, breast, colo-
rectal, gastric, and lung cancers.4–8
VEGFR-2 (KDR), a member of vascular endothelial growth factor
receptors (VEGFRs), also belongs to receptor tyrosine kinase family.
Its activation by vascular endothelial growth factor (VEGF) initiates
downstream signaling, ultimately leading to angiogenesis, vascular
permeability enhancement, tumor proliferation, and tumor migra-
tion.9–12
In recent years, some c-Met/VEGFR-2 dual inhibitors have been
reported or have entered clinical trials.16–22 Most of these tyrosine
kinase inhibitors contain various heterocyclic scaffolds. In an
attempt to pursue new antitumor agents potently inhibited both
c-Met and VEGFR-2, a series of new quinazolin-4-amine deriva-
tives containing benzimidazole moieties were synthesized and
their inhibitory activities against c-Met and VEGFR-2 and two can-
cer cell lines were evaluated. In addition, the structure–activity
relationships and possible enzyme binding modes were also
illustrated.
2. Results and discussion
2.1. Chemistry
c-Met has been shown to collaborate synergistically with VEG-
FR-2, resulting in promoting development of angiogenesis and pro-
gression of various human cancers.10,13,14 Therefore, molecules
that potently inhibit both c-Met and VEGFR-2 may have advantage
over either c-Met-selective or VEGFR-2-selective inhibitor since
they can target multiple signaling pathways involved in tumor
angiogenesis, proliferation, and metastasis.15 Thus, the combined
The synthesis route of the title N-(2-phenyl-1H-benzo[d]imida-
zol-5-yl)quinazolin-4-amine derivatives is outlined in Scheme 1.
The preparation of these quinazolin-4-amines containing benz-
imidazole moiety started from a series of commercially available
substituted benzoic acids 2a–2u. Condensation of 2a–2u with
4-nitro-o-phenylenediamine (1) in polyphosphoric acid (PPA) at
120–150 °C for 5 h gave the 5-nitro-benzimidazole intermediates
3a–3u. Hydrogenation of 5-nitro-benzimidazole intermediates
3a–3u with Pd/C/H2 under normal pressure at room temperature
for 5 h or with Fe/AcOH/EtOH under reflux for 2 h provided the
⇑
Corresponding authors. Tel./fax: +86 25 83322067.
E-mail addresses: shilei@cpu.edu.cn (L. Shi), xyg@cpu.edu.cn (Y.-G. Xu).
Both authors contributed equally to the work.
http://dx.doi.org/10.1016/j.bmc.2014.07.008
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Shi, L.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.07.008

2
L. Shi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
O2N
NH2
NH2
H2N
b or c
COOH
O2N
N
a
N
+
R
N
H
R
R
N
H
3(a-u)
2(a-u)
1
4(a-u)
Cl
OH
d
N
N
N
N
6
5
2
3
,
4
7
and
R for
,
N
e
a
b
h
o
: R = 2-CF3
: R = H
: R = 2-F
: R = 3-F
R
N
H
HN
N
i
p
: R = 2-CH3
: R = 3-CF3
c: R = 3-CH3
j: R = 4-F
q: R = 4-CF3
N
d
e
k
r
: R = 2,6-di-Cl
: R = 4-CH3
: R = 2-Cl
l
s
: R = 2-OCH3
: R = 3-Cl
: R = 3,4-di-Cl
t: R = 3,5-di-Cl
f: R = 2-OH
m: R = 4-Cl
7(a-u)
g
n
u
: R = 2-Br, 5-F
: R = 4-CH2CH3
: R = 4-Br
Scheme 1. General procedure for the synthesis of N-(2-phenyl-1H-benzo[d]imidazol-6-yl)quinazolin-4-amine derivatives. Reagents and conditions: (a) PPA, 120–150 °C, 5 h;
(b) Pd/C, H2, CH3OH, rt, 5 h; (c) Fe, AcOH, EtOH, reflux, 2 h; (d) POCl3, 100 °C, 4 h; (e) isopropanol, reflux, 5 h.
5-amino-benzimidazole intermediates 4a–4u. Condensation of 4a–
4u with 4-chloroquinazoline (6), which was prepared by quinazo-
lin-4-ol (5) in POCl3 at 100 °C for 4 h, in isopropanol under reflux
for 5 h afforded the benzimidazole quinazolin-4-amines 7a–7u.
compound Golvatinib. Among these compounds, compound 7j
showed the most potent activities with IC50 of 0.02 M and
0.05 M against VEGFR-2 and c-Met, respectively, which is compa-
l
l
rable to Golvatinib (IC50 = 0.02
lM against c-Met and 0.04 lM
against VEGFR-2).
2.2. Biological evaluation
Structure–activity relationships (SARs) were inferred from data
of enzymatic experiments reported in Table 1. The type, number
and position of substituents on the phenyl ring linking to the benz-
imidazole moiety played important roles in the enzymatic activi-
ties. The inhibitory activities against c-Met and VEGFR-2 of
compounds 7a–7u with different single para-substituents
increased in the following order: -F (7j)>-Cl (7m)>-Br (7n)>-H
2.2.1. c-Met and VEGFR-2 inhibitory assay
All the synthesized quinazolin-4-amine derivatives containing
benzimidazole moiety 7a–7u were assayed with the enzymatic
activities against c-Met and VEGFR-2. The results were summa-
rized in Table 1. Also included was the activity of reference
Table 1
Enzymatic and cellular results for benzimidazole quinolin-4-amines
N
N
H
R
HN
N
N
Compd
R
Enzymatic inhibition (IC50
,
lM)
Proliferative inhibition (IC50
, lM)
c-Met
VEGFR-2
Hep-G2
MCF-7
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
H
1.34
>10
8.80
4.54
>10
0.83
9.35
6.57
4.76
>10
21.8
>100
85.7
36.8
>100
>100
80.3
64.8
37.6
8.7
73.1
31.6
15.8
24.7
>100
92.6
78.4
>100
19.5
28.4
68.3
65.5
9.6
2-CH3
3-CH3
4-CH3
2-OCH3
2-OH
4-CH2CH3
2-F
3-F
4-F
2-Cl
3-Cl
70.5
65.2
17.3
78.5
64.6
54.0
30.5
12.6
1.5
>10
>10
7.30
7.48
1.50
0.05
>10
4.24
0.15
0.56
>10
>10
>10
>10
1.53
3.58
7.85
0.02
8.69
6.35
0.82
0.02
8.70
1.58
0.09
0.38
>10
>10
8.85
>10
0.76
1.84
8.52
0.04
28.3
14.4
3.6
4-Cl
4-Br
8.3
2-CF3
3-CF3
4-CF3
2,6-Di-Cl
3,4-Di-Cl
3,5-Di-Cl
2-Br, 5-F
86.5
52.3
35.0
74.7
9.0
7.5
25.8
49.6
7s
7t
7u
Golvatinib
Please cite this article in press as: Shi, L.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.07.008

L. Shi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
Figure 1. (A) 2D molecular docking modeling of compound 7j with c-Met. The hydrogen bond is displayed as green dotted lines. The
shown as yellow line. (B) 3D model of the interaction between compound 7j and c-Met ATP binding site.
p–
p
interactions and
p–
r
interaction are
(7a)>-CH3 (7d)>-CH2CH3 (7g)>-CF3 (7q). The above results
indicated that introduction of electron-withdrawing small substi-
tuent is favorable for the activities while introduction of elec-
tron-donating substituent or strongly electron-withdrawing bulk
substituent causes obvious decrease of activities. This discipline
was also observed in ortho-substituted and meta-substituted
compounds.
compound 7t with two chloro substituents at two meta positions
displayed weaker activity (IC50 = 3.58 against c-Met and
1.84 M against VEGFR-2), however, introduction of two chloro
substituents to two ortho positions caused the lost of activity (com-
pound 7r: IC50 >10 M against c-Met and VEGFR-2). It also demon-
l
M
l
l
strated that introduction of certain substituents at para position is
more favorable.
Compounds 7h, 7i and 7j with fluoro substituent at different
position showed different inhibitory activity against c-Met and
VEGFR-2. Compound 7j with para-fluoro substituent showed more
2.2.2. Antiproliferation assay
All the synthesized compounds 7a–7u were evaluated for their
anticancer activity against MCF-7 (human breast cancer) and Hep-
G2 (human liver cancer) cell lines by MTT assay. The results were
also summarized in Table 1. It was clear that almost all the tested
compounds showed better activities against MCF-7 than against
Hep-G2. Among the tested compounds, compound 7j with the
most potent c-Met/VEGFR-2 inhibitory activities also showed the
potent activity (IC50 = 0.05
VEGFR-2) than compound 7i with meta-fluoro substituent
(IC50 = 1.50 M against c-Met and 0.82 M against VEGFR-2). The
activity of the latter was better than compound 7h with ortho-
chloro substituent (IC50 = 7.48 M against c-Met and 6.35
lM against c-Met and 0.02 lM against
l
l
l
lM
against VEGFR-2). The result suggested that substituents in
different positions led to different inhibitory activities (para-
> meta- > ortho-). This rule was also found in other single substi-
tuted compounds. Comparing the activities of compounds 7r, 7s
and 7t, we found that compound 7s with two chloro substituents
at para and meta positions exhibited the most potent activity
most potent anticancer activities with the IC50 value of 1.5
and 8.7 M against MCF-7 and HepG-2, respectively, which is bet-
ter than Golvatinib.
lM
l
The SARs analysis result of antiproliferation activities of the
tested compounds were consistent with that of their inhibitory
activities against c-Met and VEGFR-2, which suggested that the
(IC50 = 1.53 lM against c-Met and 0.76 lM against VEGFR-2), and
Please cite this article in press as: Shi, L.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.07.008

4
L. Shi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 2. (A) 2D molecular docking modeling of compound 7j with VEGFR-2. The hydrogen bonds are displayed as green or blue dotted lines. The
p–p interaction and
p–cation interactions are shown as yellow line. (B) 3D model of the interaction between compound 7j and VEGFR-2 ATP binding site.
potent anticancer activities of the synthesized compounds were
likely related to their dual c-Met/VEGFR-2 inhibitory activities.
(O. . .H–N: 4.0 Å) and CYS 919 (F. . .H–N: 3.9 Å), respectively. The
–cation interactions are not only formed between the quinazoline
ring and HIS1026 but also formed between the benzimidazole ring
p
2.3. Molecular docking studies
and LYS868. Additionally, the
the phenyl ring and PHE918.
p–p interaction is forged between
In order to better understand the interaction between com-
pounds and kinases, molecular docking studies on the potent com-
pound 7j were performed using the Discovery Studio 3.1/CDOCKER
protocol.
The nice binding model of compound 7j with c-Met and VEGFR-
2 is consistent with kinase assay data, which indicates that com-
pound 7j is a potent dual c-Met/VEGFR-2 inhibitor.
Figure 1 demonstrates the compound 7j docking into the bind-
ing site of c-Met kinase (PDB: 3CD8).23 In this binding model, the
protonated nitrogen atom of benzimidazole ring in 7j forms a
3. Conclusions
A series of quinazolin-4-amine derivatives containing benz-
imidazole moiety have been synthesized and discovered as dual
inhibitors against c-Met and VEGFR-2 that displayed good antipro-
liferation activities against two tumor cell lines (MCF-7 and Hep-
G2). Compound 7j exhibited the most potent inhibitory activity
hydrogen bond with ILE1084 (O. . .H–N: 3.8 Å). The
tions are formed between the benzimidazole ring and TYR1159.
Besides, interactions are also formed between the quinazoline
ring and TYR1230. In addition, the interaction is forged
between the imidazole ring and ILE1084.
p–p interac-
p–p
p–r
against c-Met and VEGFR-2 with IC50 of 0.05 lM and 0.02 lM,
respectively and also showed the greatest inhibitory activities
against human breast cancer cell MCF-7 and human liver cancer
The binding model of compound 7j into the ATP-binding cavity
of VEGFR-2 kinase (PDB: 2QU5)24 is depicted in Figure 2. In this
binding model, the protonated nitrogen atom between quinazoline
ring and benzimidazole ring and the fluorine atom of the phenyl
ring in 7j form hydrogen bonding interactions with GLU885
cell Hep-G2 with IC50 of 1.5
lM and 8.7 lM, respectively. Molecu-
lar docking of the most potent inhibitor 7j into ATP binding site of
Please cite this article in press as: Shi, L.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.07.008

L. Shi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
c-Met and VEGFR-2 was performed and the result suggested that
4.1.1.6.
2-(5-Nitro-1H-benzo[d]imidazol-2-yl)phenol
(3f).
compound 7j could bind well with the active site of c-Met and
VEGFR-2. The above results demonstrated that compound 7j could
be a potential anticancer agent.
Dark brown powder, yield: 37%, mp: 305–307 °C. 1H NMR
(300 MHz; DMSO-d6): 6.94–7.04 (m, 2H); 7.35 (t, J = 7.23 Hz, 1H);
7.71 (d, J = 8.8 Hz, 1H); 8.05 (dd, J = 8.8, 2.1 Hz, 1H); 8.26 (d,
J = 7.0 Hz, 1H); 8.47 (d, J = 1.95 Hz, 1H). MS (ESI+) m/z 256.1
(M+H)+. Anal. Calcd for C13H9N3O3: C, 61.18; H, 3.55; N, 16.46.
Found: C, 61.35; H, 3.51; N, 16.50.
4. Experimental section
4.1. Chemistry
4.1.1.7. 2-(4-Ethylphenyl)-5-nitro-1H-benzo[d]imidazole (3g).
Orange powder, yield: 90%, mp: 84–86 °C. 1H NMR (300 MHz;
DMSO-d6): 1.22 (t, J = 7.5 Hz, 3H); 2.68 (d, J = 7.5 Hz, 2H); 7.42 (d,
J = 8.1 Hz, 2H); 7.72 (d, J = 9.0 Hz, 1H); 8.08–8.13 (m, 3H); 8.42 (s,
1H); 13.48 (s, 1H). MS (ESI+) m/z 268.1 (M+H)+. Anal. Calcd for
C15H13N3O2: C, 67.40; H, 4.90; N, 15.72. Found: C, 67.52; H, 4.86;
N, 15.68.
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 Series LC/MSD Trap SL Mass
spectrometer., and 1H NMR spectra 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
CHN-O-Rapid instrument.
4.1.1.8. 2-(2-Fluorophenyl)-5-nitro-1H-benzo[d]imidazole (3h).
Orange powder, yield: 26%, mp: 171–173 °C. 1H NMR (300 MHz;
DMSO-d6): 7.41–7.52 (m, 2H); 7.60–7.68 (m, 1H); 7.81 (d,
J = 8.7 Hz, 1H); 8.15 (dd, J = 8.7, 1.8 Hz, 1H); 8.27 (t, J = 7.2 Hz,
1H); 8.52 (s, 1H); 13.21 (s, 1H). MS (ESI+) m/z 258.1 (M+H)+. Anal.
Calcd for C13H8FN3O2: C, 60.70; H, 3.13; N, 16.34. Found: C, 60.78;
H, 3.12; N, 16.30.
4.1.1. General procedure for the preparation of the 5-nitro-2-
phenyl-1H-benzo[d]imidazole derivatives 3a–3u
A mixture of 4-nitro-o-phenylenediamine (3.22 g, 21 mmol)
and substituted benzoic acid 2a–2u (20 mmol) in PPA (40 mL)
was stirred at 120–150 °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 3a–3u.
4.1.1.9. 2-(3-Fluorophenyl)-5-nitro-1H-benzo[d]imidazole (3i).
Pale yellow powder, yield: 42%, mp: 205–207 °C. 1H NMR
(300 MHz; DMSO-d6): 7.38–7.43 (m, 1H); 7.63–7.76 (m, 2H);
7.95–8.14 (m, 3H); 8.48 (s, 1H); 13.66 (s, 1H). MS (ESI+) m/z
258.1 (M+H)+. Anal. Calcd for C13H8FN3O2: C, 60.70; H, 3.13; N,
16.34. Found: C, 60.55; H, 3.15; N, 16.39.
4.1.1.1. 5-Nitro-2-phenyl-1H-benzo[d]imidazole (3a).
Pale
orange–red powder, yield: 88%, mp: 206–208 °C. 1H NMR
(300 MHz; DMSO-d6): 7.48–8.04 (m, 4H); 8.04–8.69 (m, 4H);
13.64 (s, 1H). MS (ESI +) m/z 294.1 (M+H)+. Anal. Calcd for
4.1.1.10.
(3j).
2-(4-Fluorophenyl)-5-nitro-1H-benzo[d]imidazole
Golden powder, mp: yield: 96%, mp: 258–260 °C. 1H
C13H9N3O2: C, 65.27; H, 3.79; N, 17.56. Found: C, 65.32; H, 3.82;
NMR (300 MHz; DMSO-d6): 7.44 (t, J = 9.0 Hz, 2H); 7.75 (d,
J = 8.7 Hz, 1H); 8.11 (dd, J = 9.0, 2.1 Hz, 1H); 8.23–8.27 (m, 2H);
8.45 (s, 1H); 13.59 (s, 1H). MS (ESI+) m/z 258.1 (M+H)+. Anal. Calcd
for C13H8FN3O2: C, 60.70; H, 3.13; N, 16.34. Found: C, 60.84; H,
3.16; N, 16.38.
N, 17.53.
4.1.1.2. 5-Nitro-2-o-tolyl-1H-benzo[d]imidazole (3b).
Yel-
low powder, yield: 86%, mp: 146–148 °C. 1H NMR (300 MHz;
DMSO-d6): 2.58 (s, 3H); 7.46 (s, 3H); 7.77–7.85 (m, 2H); 8.16 (s,
1H); 8.40–8.59 (m, 1H); 13.37 (s, 1H). MS (ESI+) m/z 254.1
(M+H)+. Anal. Calcd for C14H11N3O2: C, 66.40; H, 4.38; N, 16.59.
Found: C, 66.34; H, 4.41; N, 16.64.
4.1.1.11. 2-(2-Chlorophenyl)-5-nitro-1H-benzo[d]imidazole (3k).
Brown powder, yield: 16%, mp: 168–170 °C. 1H NMR (300 MHz;
DMSO-d6): 7.52–7.62 (m, 2H); 7.67–7.70 (m, 1H); 7.80 (d,
J = 9.0 Hz, 1H); 7.94 (dd, J = 7.2, 2.1 Hz, 1H); 8.15 (dd, J = 8.7,
2.1 Hz, 1H); 8.53 (s, 1H); 13.42 (s, 1H). MS (ESI+) m/z 274.1
(M+H)+. Anal. Calcd for C13H8ClN3O2: C, 57.05; H, 2.95; N, 15.35.
Found: C, 57.22; H, 2.97; N, 15.41.
4.1.1.3. 5-Nitro-2-m-tolyl-1H-benzo[d]imidazole (3c).
Yel-
low powder, yield: 99%, mp: 108–110 °C. 1H NMR (300 MHz;
DMSO-d6): 2.48 (s, 3H); 7.38–7.52 (m, 2H); 7.76 (d, J = 8.5 Hz,
1H); 8.00–8.15 (m, 3H); 8.47 (s, 1H); 13.58 (s, 1H). MS (ESI+) m/z
254.2 (M+H)+. Anal. Calcd for C14H11N3O2: C, 66.40; H, 4.38; N,
16.59. Found: C, 66.45; H, 4.42; N, 16.53.
4.1.1.12. 2-(3-Chlorophenyl)-5-nitro-1H-benzo[d]imidazole (3l).
Orange powder, yield: 83%, mp: 220–222 °C. 1H NMR (300 MHz;
DMSO-d6): 7.61 (d, J = 5.1 Hz, 2H); 7.76 (d, J = 9.0 Hz, 1H); 8.10–
8.17 (m, 2H); 8.22 (d, J = 0.9 Hz, 1H); 8.46 (d, J = 1.8 Hz, 1H);
13.69 (s, 1H). MS (ESI+) m/z 274.0 (M+H)+. Anal. Calcd for
4.1.1.4. 5-Nitro-2-p-tolyl-1H-benzo[d]imidazole (3d).
Yel-
low powder, yield: 81%, mp: 219–221 °C. 1H NMR (300 MHz;
DMSO-d6): 2.32 (s, 3H); 7.29 (d, J = 7.9 Hz, 2H); 7.61 (d, J = 8.8 Hz,
1H); 7.98 (dd, J = 8.8, 2.2 Hz, 1H); 8.09 (d, J = 8.1 Hz, 2H); 8.35 (d,
J = 2.1 Hz, 1H). MS (ESI+) m/z 254.1 (M+H)+. Anal. Calcd for
C13H8ClN3O2: C, 57.05; H, 2.95; N, 15.35. Found: C, 56.88;
H, 3.01; N, 15.43.
C
14H11N3O2: C, 66.40; H, 4.38; N, 16.59. Found: C, 66.52; H, 4.35;
4.1.1.13.
(3m).
2-(4-Chlorophenyl)-5-nitro-1H-benzo[d]imidazole
Yellow green powder, yield: 40%, mp: 301–303 °C. 1H
N, 16.65.
NMR (300 MHz; DMSO-d6): 7.66–7.77 (m, 3H); 8.13 (dd, J = 9.0,
2.1 Hz, 1H); 8.21 (d, J = 8.7 Hz, 2H); 8.49 (s, 1H); 13.66 (s, 1H).
MS (ESI+) m/z 274.0 (M+H)+. Anal. Calcd for C13H8ClN3O2: C,
57.05; H, 2.95; N, 15.35. Found: C, 57.19; H, 2.92; N, 15.29.
4.1.1.5. 2-(2-Methoxyphenyl)-5-nitro-1H-benzo[d]imidazole
(3e).
Brown powder, yield: 97%, mp: 208–209 °C. 1H NMR
(300 MHz; DMSO-d6): 4.08 (s, 3H); 7.17 (t, J = 7.4 Hz, 1H); 7.30
(d, J = 8.3 Hz, 1H); 7.57 (t, J = 7.4 Hz, 1H); 7.81 (d, J = 8.9 Hz, 1H);
8.13 (d, J = 8.7 Hz, 1H); 8.39 (d, J = 7.4 Hz, 1H); 8.54 (s, 1H); 12.67
(s, 1H). MS (ESI+) m/z 270.1 (M+H)+. Anal. Calcd for C14H11N3O3:
C, 62.45; H, 4.12; N, 15.61. Found: C, 62.66; H, 4.14; N, 15.67.
4.1.1.14. 2-(4-Bromophenyl)-5-nitro-1H-benzo[d]imidazole (3n).
Yellow powder, yield: 78%, mp: 296–298 °C. 1H NMR (300 MHz;
DMSO-d6): 7.77–7.85 (m, 3H); 8.13–8.18 (m, 3H); 8.50 (s, 1H). MS
Please cite this article in press as: Shi, L.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.07.008

6
L. Shi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
(ESI+) m/z 318.0 (M+H)+. 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: 292–293 °C. 1H NMR (300 MHz; DMSO-
d6): 3.17 (s, 2H); 6.54 (d, J = 8.5 Hz, 1H); 6.69 (s, 1H); 7.28 (d,
J = 8.5 Hz, 1H); 7.41 (t, J = 7.0 Hz, 1H); 7.50 (t, J = 7.6 Hz, 2H); 8.07
(d, J = 7.8 Hz, 2H). MS (ESI+) m/z 210.1 (M+H)+. Anal. Calcd for
4.1.1.15.
5-Nitro-2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]
imidazole (3o).
Pale orange powder, yield: 84%, mp: 190–
191 °C. 1H NMR (300 MHz; DMSO-d6): 7.80–7.91 (m, 4H); 7.98
(d, J = 7.5 Hz, 1H); 8.17 (d, J = 8.1 Hz, 1H); 8.54 (s, 1H), 13.52 (s,
1H). MS (ESI+) m/z 308.2 (M+H)+. Anal. Calcd for C14H8F3N3O2: C,
54.73; H, 2.62; N, 13.68. Found: C, 54.78; H, 2.64; N, 13.65.
C13H11N3: C, 74.62; H, 5.30; N, 20.08. Found: C, 74.48; H, 5.26; N,
20.14.
4.1.2.2.
2-o-Tolyl-1H-benzo[d]imidazol-5-amine
(4b).
Brown powder, yield: 64.28%, mp: 63–64 °C. 1H NMR (300 MHz;
CDCl3): 2.45 (s, 3H); 3.44 (s, 2H); 6.54 (t, J = 8.5 Hz, 1H); 7.08–
7.46 (m, 6H). MS (ESI+) m/z 224.1 (M+H)+. Anal. Calcd for
4.1.1.16.
5-Nitro-2-(3-(trifluoromethyl)phenyl)-1H-benzo[d]
imidazole (3p).
Brown crystal, yield: 56%, mp: 229–230 °C.
1H NMR (300 MHz; DMSO-d6): 7.76–7.93 (m, 3H); 8.12 (dd,
J = 9.0, 2.1 Hz, 1H); 8.47–8.52 (m, 3H); 13.79 (s, 1H). MS (ESI+)
m/z 308.1 (M+H)+. Anal. Calcd for C14H8F3N3O2: C, 54.73; H, 2.62;
N, 13.68. Found: C, 54.86; H, 2.59; N, 13.72.
C14H13N3: C, 75.31; H, 5.87; N, 18.82. Found: C, 75.52; H, 5.91; N,
18.77.
4.1.2.3. 2-m-Tolyl-1H-benzo[d]imidazol-5-amine (4c).
Yel-
low powder, yield: 82.33%, mp: 231–233 °C. 1H NMR (300 MHz;
CDCl3): 2.36 (s, 3H); 6.69 (dd, J = 8.7, 2.1 Hz, 1H); 6.85 (d,
J = 1.8 Hz, 1H); 7.22 (d, J = 7.8 Hz, 1H); 7.32 (t, J = 7.5 Hz, 1H);
7.48 (d, J = 8.4 Hz, 1H); 7.79 (d, J = 7.8 Hz, 1H); 7.90 (s, 1H). MS
(ESI+) m/z 224.1 (M+H)+. 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.
imidazole (3q).
5-Nitro-2-(4-(trifluoromethyl)phenyl)-1H-benzo[d]
Orange powder, yield: 21%, mp: 273–
274 °C. 1H NMR (300 MHz; DMSO-d6): 7.77 (d, J = 8.7 Hz, 1H);
7.94 (d, J = 8.7 Hz, 2H); 8.12 (dd, J = 9.0, 2.4 Hz, 1H); 8.37 (d,
J = 8.1 Hz, 2H); 8.47 (d, J = 1.8 Hz, 1H); 13.78 (s, 1H). MS (ESI+)
m/z 308.1 (M+H)+. 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 powder, yield: 89.4%, mp: 112–114 °C. 1H NMR
(300 MHz; CDCl3): 2.40 (s, 3H); 6.67 (dd, J = 8.7, 2.1 Hz, 1H); 6.84
(s, 1H); 7.88 (t, J = 1.8 Hz, 2H); 7.27 (s, 1H); 7.46 (d, J = 8.5 Hz,
1H); 7.88 (t, J = 1.8 Hz, 2H). MS (ESI+) m/z 224.1 (M+H)+. 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).
NMR (300 MHz; DMSO-d6): 7.60–7.71 (m, 3H); 7.83 (d, J = 9.0 Hz,
1H); 8.17 (dd, J = 9.0, 2.1 Hz, 1H); 8.58 (s, 1H); 13.65 (s, 1H). MS
(ESI+) m/z 307.1 (M+H)+. Anal. Calcd for C13H7Cl2N3O2: C, 50.67;
H, 2.29; N, 13.64. Found: C, 50.80; H, 2.31; N, 13.68.
Pale orange powder, yield: 80%, mp: 210–212 °C. 1H
4.1.2.5.
(4e).
2-(2-Methoxyphenyl)-1H-benzo[d]imidazol-5-amine
Brown powder, yield: 97%, mp: 193–194 °C. 1H NMR
4.1.1.19. 2-(3,4-Dichlorophenyl)-5-nitro-1H-benzo[d]imidazole
(3s).
NMR (300 MHz; DMSO-d6): 7.75 (d, J = 8.4 Hz, 1H); 7.82–7.88 (m,
1H); 8.03–8.15 (m, 2H); 8.37 (d, J = 2.1 Hz, 1H); 8.45 (s, 1H); 13.67
(s, 1H). MS (ESI+) m/z 307.1 (M+H)+. Anal. Calcd for C13H7Cl2N3O2: C,
50.67; H, 2.29; N, 13.64. Found: C, 50.82; H, 2.28; N, 13.60.
(300 MHz; DMSO-d6): 3.99 (s, 3H); 6.54 (dd, J = 8.5, 1.6 Hz, 1H);
6.75 (s, 1H); 7.06 (t, J = 7.5 Hz, 1H); 7.18 (d, J = 8.3 Hz, 1H); 7.28
(d, J = 8.5 Hz, 1H); 7.37–7.42 (m, 1H); 8.24 (d, J = 7.7 Hz, 1H). MS
(ESI+) m/z 240.1 (M+H)+. Anal. Calcd for C14H13N3O: C, 70.28; H,
5.48; N, 17.56. Found: C, 70.46; H, 5.50; N, 17.51.
Pale orange powder, yield: 42%, mp: 232–235 °C. 1H
4.1.2.6.
2-(5-Amino-1H-benzo[d]imidazol-2-yl)phenol
(4f).
4.1.1.20. 2-(3,5-Dichlorophenyl)-5-nitro-1H-benzo[d]imidazole
(3t).
NMR (300 MHz; DMSO-d6): 7.77–7.82 (m, 2H); 8.14 (dd, J = 9.0,
2.1 Hz, 1H); 8.20 (d, J = 2.1 Hz, 2H); 8.49 (d, J = 2.1 Hz, 1H); 13.77
(s, 1H). MS (ESI+) m/z 307.0 (M+H)+. Anal. Calcd for C13H7Cl2N3O2:
C, 50.67; H, 2.29; N, 13.64. Found: C, 50.53; H, 2.26; N, 13.57.
Yellow powder, yield: 46%, mp: 250–251 °C. 1H NMR (300 MHz;
DMSO-d6): 6.61 (d, J = 8.3 Hz, 1H); 6.74 (s, 1H); 6.97 (t, J = 8.4 Hz,
2H); 7.28–7.36 (m, 2H); 7.93 (d, J = 7.7 Hz, 1H); 12.63 (s, 1H);
13.22 (s, 1H). MS (ESI+) m/z 226.1 (M+H)+. Anal. Calcd for
Pale orange powder, yield: 12%, mp: 290–292 °C. 1H
C13H11N3O: C, 69.32; H, 4.92; N, 18.66. Found: C, 69.16; H, 4.95;
N, 18.72.
4.1.1.21.
imidazole (3u).
2-(2-Bromo-5-fluorophenyl)-5-nitro-1H-benzo[d]
Brown powder, mp: yield: 66%, mp: 218–
4.1.2.7. 2-(4-Ethylphenyl)-1H-benzo[d]imidazol-5-amine (4g).
Orange powder, yield: 71%, mp: 102–104 °C. 1H NMR (300 MHz;
CDCl3): 1.26 (t, J = 7.5 Hz, 3H); 2.67–2.71 (m, 2H); 5.30 (d,
J = 0.5 Hz, 1H); 6.67 (dd, J = 8.5, 2.0 Hz, 1H); 6.84 (s, 1H); 7.28 (s,
1H); 7.45 (d, J = 8.0 Hz, 1H); 7.92 (d, J = 8.0 Hz, 2H). MS (ESI+) m/z
238.1 (M+H)+. Anal. Calcd for C15H15N3: C, 75.92; H, 6.37; N,
17.71. Found: C, 80.18; H, 6.33; N, 17.66.
220 °C. 1H NMR (300 MHz; DMSO-d6): 7.43 (dd, J = 8.4, 3.0 Hz,
1H); 7.70–7.92 (m, 3H); 8.18 (d, J = 9.0 Hz, 1H); 8.60 (s, 1H);
13.55 (s, 1H). MS (ESI+) m/z 336.0 (M+H)+. Anal. Calcd for
C13H7BrFN3O2: C, 46.45; H, 2.10; N, 12.50. Found: C, 46.53;
H, 2.11; N, 12.55.
4.1.2. General procedure for the preparation of the 2-phenyl-
1H-benzo[d]imidazol-5-amine derivatives 4a–4u
4.1.2.8.
(4h).
2-(2-Fluorophenyl)-1H-benzo[d]imidazol-5-amine
White powder, yield: 61.9%, mp: 214–215 °C. 1H NMR
Method A: A suspension of 3 (5.0 mmol, R = CH3, OCH3, OH,
CH2CH3, CF3) and 10% Pd/C (0.13 g) in methanol (30 mL) was
hydrogenated under normal pressure for 5 h at room temperature.
Filtration and evaporation gave corresponding compound 4.
Method B: Compound 3 (5.0 mmol, R = 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. Filtra-
tion and evaporation gave corresponding compound 4.
(300 MHz; CDCl3): 6.72 (dd, J = 8.4, 2.1 Hz, 1H); 6.88 (s, 1H);
7.16–7.23 (m, 1H); 7.30–7.43 (m, 2H); 7.51 (d, J = 6.6 Hz, 1H);
8.46 (dd, J = 7.8, 1.8 Hz, 1H). MS (ESI+) m/z 228.1 (M+H)+. Anal.
Calcd for C13H10FN3: C, 68.71; H, 4.44; N, 18.49. Found: C, 68.56;
H, 4.43; N, 18.45.
4.1.2.9. 2-(3-Fluorophenyl)-1H-benzo[d]imidazol-5-amine (4i).
Pale orange powder, yield: 76%, mp: 245–246 °C. 1H NMR
(300 MHz; DMSO-d6): 4.98 (s, 2H); 6.54 (d, J = 8.4 Hz, 1H); 6.67
Please cite this article in press as: Shi, L.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.07.008

L. Shi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
(s, 1H); 7.19–7.30 (m, 2H); 7.49–7.56 (m, 1H); 7.81–7.91 (m, 2H);
4.1.2.18. 2-(2,6-Dichlorophenyl)-1H-benzo[d]imidazol-5-amine
(4r).
Brown crystal, yield: 64.25%, mp: 137–139 °C. 1H NMR
12.32 (s, 1H). MS (ESI+) m/z 228.1 (M+H)+. Anal. Calcd for
C
13H10FN3: C, 68.71; H, 4.44; N, 18.49. Found: C, 68.89; H, 4.46;
(300 MHz; DMSO-d6): 6.56 (dd, J = 8.7, 2.1 Hz, 1H); 6.67 (s, 1H);
7.28 (d, J = 8.4 Hz, 1H); 7.51–7.63 (m, 3H). MS (ESI+) m/z 278.0
(M+H)+. Anal. Calcd for C13H9Cl2N3: C, 56.14; H, 3.26; N, 15.11.
Found: C, 56.28; H, 3.23; N, 15.15.
N, 18.44.
4.1.2.10.
(4j).
2-(4-Fluorophenyl)-1H-benzo[d]imidazol-5-amine
Orange crystal, yield: 73.86%, mp: 68–69 °C. 1H NMR
(300 MHz; CDCl3): 3.50 (s, 2H); 6.66 (d, J = 8.4 Hz, 1H); 6.80 (s,
1H); 7.06 (t, J = 8.4 Hz, 2H); 7.42 (d, J = 8.4 Hz, 1H); 9.97 (m, 2H).
MS (ESI+) m/z 228.1 (M+H)+. 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).
(300 MHz; DMSO-d6): 6.57 (d, J = 8.5 Hz, 1H); 6.68 (s, 1H); 7.31
(d, J = 8.5 Hz, 1H); 7.77 (d, J = 8.4 Hz, 1H); 8.03 (d, J = 8.4 Hz, 1H);
8.27 (s, 1H). MS (ESI+) m/z 278.1 (M+H)+. Anal. Calcd for C13H9Cl2N3:
C, 56.14; H, 3.26; N, 15.11. Found: C, 56.32; H, 3.24; N, 15.15.
Orange crystal, yield: 71%, mp: 100–102 °C. 1H NMR
4.1.2.11.
(4k).
2-(2-Chlorophenyl)-1H-benzo[d]imidazol-5-amine
Brown powder, yield: 76.58%, mp: 87–89 °C. 1H NMR
(500 MHz; CDCl3): 6.73 (dd, J = 8.0, 2.0 Hz, 1H); 6.89 (s, 1H);
7.33–7.41 (m, 2H); 7.47 (dd, J = 8.0, 1.5 Hz, 1H); 7.52 (d,
J = 8.0 Hz, 1H); 8.39 (dd, J = 7.5, 1.5 Hz, 1H). MS (ESI+) m/z 244.1
(M+H)+. Anal. Calcd for C13H10ClN3: C, 64.07; H, 4.14; N, 17.24.
Found: C, 64.25; H, 4.16; N, 17.26.
4.1.2.20. 2-(3,5-Dichlorophenyl)-1H-benzo[d]imidazol-5-amine
(4t).
(300 MHz; DMSO-d6): 6.57 (d, J = 8.4 Hz, 1H); 6.60 (s, 1H); 7.30
(d, J = 8.4 Hz, 1H); 7.62 (s, 1H); 8.06 (s, 2H); 12.50 (s, 1H). MS
(ESI+) m/z 278.0 (M+H)+. Anal. Calcd for C13H9Cl2N3: C, 56.14; H,
3.26; N, 15.11. Found: C, 56.30; H, 3.29; N, 15.08.
Brown powder, yield = 51%, mp: 241–243 °C. 1H NMR
4.1.2.12.
(4l).
2-(3-Chlorophenyl)-1H-benzo[d]imidazol-5-amine
Orange powder, yield: 38%, mp: 72–74 °C. 1H NMR
4.1.2.21. 2-(2-Bromo-5-fluorophenyl)-1H-benzo[d]imidazol-5-
amine (4u).
Yellow powder, yield: 92%, mp: 96–97 °C. 1H
(300 MHz; CDCl3): 6.69 (dd, J = 8.7, 2.1 Hz, 1H); 6.82 (d,
J = 1.8 Hz, 1H); 7.33–7.35 (m, 2H); 7.47 (d, J = 8.4 Hz, 1H); 7.84–
7.88 (m, 1H); 7.97–7.99 (m, 1H). MS (ESI+) m/z 244.1 (M+H)+. Anal.
Calcd for C13H10ClN3: C, 64.07; H, 4.14; N, 17.24. Found: C, 63.88;
H, 4.17; N, 17.29.
NMR (300 MHz; CDCl3): 6.74 (dd, J = 8.4, 2.1 Hz, 1H); 6.86 (d,
J = 4.5 Hz, 1H); 7.01 (dd, J = 8.7, 3.0 Hz, 1H); 7.54–7.64 (m, 2H);
8.08 (dd, J = 9.9, 3.3 Hz, 1H). MS (ESI+) m/z 306.0 (M+H)+. Anal.
Calcd for C13H9FBrN3: C, 51.00; H, 2.96; N, 13.73. Found: C,
51.15; H, 2.99; N, 13.70.
4.1.2.13.
(4m).
2-(4-Chlorophenyl)-1H-benzo[d]imidazol-5-amine
Brown powder, yield: 57%, mp: 88–90 °C. 1H NMR
4.1.3. Synthesis of 4-chloroquinazoline (6)
(300 MHz; CDCl3): 2.17 (s, 2H); 6.63 (dd, J = 8.7, 2.1 Hz, 1H); 6.73
(d, J = 2.1 Hz, 1H); 7.31 (dd, J = 6.9, 2.4 Hz, 2H); 7.37 (d, J = 8.7 Hz,
1H); 8.85 (dd; J = 5.4, 1.8 Hz, 2H). MS (ESI+) m/z 244.2 (M+H)+. Anal.
Calcd for C13H10ClN3: C, 64.07; H, 4.14; N, 17.24. Found: C, 64.23;
H, 4.12; N, 17.28.
A suspension of quinazolin-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 resi-
due. The pH was adjusted to
6 with ammonia to allow
precipitation. The filter cake was washed with water and dried to
give 4-chloroquinazoline (6). White powder, yield: 92%, mp: 93–
94 °C. 1H NMR (300 MHz; CDCl3): 7.69 (t, J = 7.5 Hz, 1H); 7.90–
8.04 (m, 2H); 8.22 (d, J = 8.3 Hz, 1H); 8.99 (s, 1H). MS (ESI+) m/z
165.0 (M+H)+. Anal. Calcd for C8H5ClN2: C, 53.38; H, 3.06; N,
17.02. Found: C, 53.54; H, 3.02; N, 17.08.
4.1.2.14.
(4n).
2-(4-Bromophenyl)-1H-benzo[d]imidazol-5-amine
Orange powder, yield: 64.45%, mp: 104–106 °C. 1H
NMR (300 MHz; DMSO-d6): 6.54 (d, J = 8.4 Hz, 1H); 6.70 (s, 1H);
7.25 (d, J = 8.4 Hz, 1H); 7.64 (d, J = 8.4 Hz, 2H); 8.06 (d, J = 8.4 Hz,
2H). MS (ESI+) m/z 288.0 (M+H)+. Anal. Calcd for C13H10BrN3: C,
54.19; H, 3.50; N, 14.58. Found: C, 54.34; H, 3.51; N, 14.62.
4.1.4. General procedure for the preparation of the quinolin-4-
amine derivatives 7a–7u
4.1.2.15. 2-(2-(Trifluoromethyl)phenyl)-1H-benzo[d]imidazol-
A mixture of 4-chloroquinazoline (6, 5 mmol) and appropriate
substituted anilines (4a–4u, 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 7a–7u.
5-amine (4o).
Brown powder, yield: 93%, mp: 153–155 °C.
1H NMR (300 MHz; DMSO-d6): 5.12 (s, 2H); 6.50 (d, J = 7.4 Hz,
1H); 6.80 (s, 1H); 7.45 (d, J = 8.4 Hz, 1H); 7.61–7.66 (m, 2H);
8.54–8.71 (m, 2H). MS (ESI+) m/z 278.2 (M+H)+. Anal. Calcd for
C14H10F3N3: C, 60.65; H, 3.64; N, 15.16. Found: C, 60.58; H, 3.69;
N, 15.20.
4.1.4.1.
amine (7a).
N-(2-Phenyl-1H-benzo[d]imidazol-6-yl)quinazolin-4-
Yellow powder, yield: 92%, mp: 267–269 °C. 1H
4.1.2.16. 2-(3-(Trifluoromethyl)phenyl)-1H-benzo[d]imidazol-
5-amine (4p).
Brown crystal, yield: 76%, mp: 93–95 °C. 1H
NMR (300 MHz; DMSO-d6): 7.55–7.65 (m, 4H); 7.73 (d, J = 8.7 Hz,
1H); 7.83 (t, J = 8.1 Hz, 1H); 7.94 (d, J = 7.8 Hz, 1H); 8.06 (t,
J = 7.5 Hz, 1H); 8.13 (d, J = 1.8 Hz, 1H); 8.26 (dd, J = 7.8, 1.5 Hz,
2H); 8.86–8.90 (m, 2H); 11.50 (s, 1H). MS (ESI+) m/z 338.1
(M+H)+. Anal. Calcd for C21H15N5: C, 74.76; H, 4.48; N, 20.76.
Found: C, 74.62; H, 4.51; N, 20.80.
NMR (300 MHz; DMSO-d6): 5.02 (s, 2H); 6.58 (d, J = 8.4 Hz, 1H);
6.70 (s, 1H); 7.33 (d, J = 8.3 Hz, 1H); 7.71–7.76 (m, 2H); 8.35–
8.41 (m, 2H). MS (ESI+) m/z 278.1 (M+H)+. Anal. Calcd for
C14H10F3N3: C, 60.65; H, 3.64; N, 15.16. Found: C, 60.47; H, 3.61;
N, 15.22.
4.1.2.17. 2-(4-(Trifluoromethyl)phenyl)-1H-benzo[d]imidazol-
4.1.4.2.
amine (7b).
N-(2-o-Tolyl-1H-benzo[d]imidazol-6-yl)quinazolin-4-
Yellow powder, yield: 95%, mp: 303–304 °C. 1H
5-amine (4q).
White powder, yield: 82%, mp: 208–209 °C.
1H NMR (300 MHz; DMSO-d6): 5.09 (s, 2H); 6.57 (dd, J = 8.5,
1.6 Hz, 1H); 6.67 (s, 1H); 7.35(d, J = 8.5 Hz, 1H); 7.86 (d,
J = 8.1 Hz, 2H); 8.26 (d, J = 8.1 Hz, 2H); 12.51 (s, 1H). MS (ESI+)
m/z 278.1 (M+H)+. Anal. Calcd for C14H10F3N3: C, 60.65; H, 3.64;
N, 15.16. Found: C, 60.83; H, 3.67; N, 15.13.
NMR (300 MHz; DMSO-d6): 2.62 (s, 3H); 7.39–7.50 (m, 3H); 7.64
(dd, J = 8.7, 1.8 Hz, 1H); 7.74–7.86 (m, 3H); 7.92 (d, J = 8.4 Hz,
1H); 8.06 (d, J = 7.2 Hz, 1H); 8.15 (s, 1H); 8.84–8.87 (m, 2H);
11.40 (s, 1H). MS (ESI+) m/z 352.2 (M+H)+. Anal. Calcd for
Please cite this article in press as: Shi, L.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.07.008

8
L. Shi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
C22H17N5: C, 75.19; H, 4.88; N, 19.93. Found: C, 75.42; H, 4.85; N,
19.85.
4.1.4.10. N-(2-(4-Fluorophenyl)-1H-benzo[d]imidazol-6-yl)qui-
nazolin-4-amine (7j). Yellow powder, yield: 65%, mp: 297–
299 °C. 1H NMR (300 MHz; DMSO-d6): 7.42 (t, J = 8.8 Hz, 2H);
7.56 (dd, J = 8.7, 1.7 Hz, 1H); 7.67 (d, J = 8.6 Hz, 1H); 7.77 (t,
J = 7.9 Hz, 1H); 7.86 (d, J = 8.1 Hz, 1H); 8.00 (t, J = 7.3 Hz, 1H);
8.12 (s, 1H); 8.23–8.28 (m, 2H); 8.76–8.78 (m, 2H); 10.96 (s, 1H).
MS (ESI+) m/z 356.1 (M+H)+. Anal. Calcd for C21H14FN5: C, 70.98;
H, 3.97; N, 19.71. Found: C, 80.26; H, 3.93; N, 19.78.
4.1.4.3. N-(2-m-Tolyl-1H-benzo[d]imidazol-6-yl)quinazolin-4-
amine (7c).
Yellow powder, yield: 82%, mp: 285–287 °C. 1H
NMR (300 MHz; DMSO-d6): 2.45 (s, 3H); 7.42 (d, J = 7.4 Hz, 1H);
7.51 (t, J = 7.6 Hz, 1H); 7.67 (d, J = 8.6 Hz, 1H); 7.77 (d, J = 8.6 Hz,
1H); 7.86 (t, J = 7.6 Hz, 1H); 7.97 (d, J = 8.1 Hz, 1H); 8.07–8.14 (m,
4H); 8.91 (d, J = 8.5 Hz, 2H); 11.59 (s, 1H). MS (ESI+) m/z 352.3
(M+H)+. Anal. Calcd for C22H17N5: C, 75.19; H, 4.88; N, 19.93.
Found: C, 75.35; H, 4.92; N, 20.01.
4.1.4.11. N-(2-(2-Chlorophenyl)-1H-benzo[d]imidazol-6-yl)qui-
nazolin-4-amine (7k).
Yellow crystal, yield: 44%, mp: 289–
290 °C. 1H NMR (500 MHz; DMSO-d6): 7.52–7.61 (m, 3H); 7.67
(dd, J = 7.8, 1.9 Hz, 1H); 7.75 (d, J = 8.6 Hz, 1H); 7.85 (t, J = 7.6 Hz,
1H); 7.92–7.96 (m, 2H); 8.08 (t, J = 7.7 Hz, 2H); 8.88–8.92
(m, 2H); 11.65 (s, 1H). MS (ESI+) m/z 372.1 (M+H)+. Anal. Calcd
for C21H14ClN5: C, 67.83; H, 3.80; N, 18.84. Found: C, 67.98; H,
3.81; N, 18.87.
4.1.4.4.
amine (7d).
N-(2-p-Tolyl-1H-benzo[d]imidazol-6-yl)quinazolin-4-
Yellow powder, yield: 89%, mp: 238–240 °C. 1H
NMR (300 MHz; DMSO-d6): 2.42 (s, 3H); 7.43 (d, J = 8.0 Hz, 2H);
7.64–7.75 (m, 2H); 7.84 (t, J = 7.6 Hz, 1H); 7.94 (d, J = 8.2 Hz, 1H);
8.04–8.18 (m, 4H); 8.72–8.98 (m, 2H); 11.39 (s, 1H). MS (ESI+)
m/z 352.1 (M+H)+. Anal. Calcd for C22H17N5: C, 75.19; H, 4.88; N,
19.93. Found: C, 75.02; H, 4.91; N, 19.98.
4.1.4.12. N-(2-(3-Chlorophenyl)-1H-benzo[d]imidazol-6-yl)qui-
nazolin-4-amine (7l).
Yellow powder, yield: 79%, mp: 327–
329 °C. 1H NMR (500 MHz; DMSO-d6): 7.59–7.62 (m, 3H); 7.73
(d, J = 8.6 Hz, 1H); 7.85 (t, J = 7.8 Hz, 1H); 7.97 (d, J = 8.2 Hz, 1H);
8.06–8.11 (m, 2H); 8.21–8.23 (m, 1H); 8.31 (s, 1H); 8.89–8.96
(m, 2H); 11.75 (s, 1H). MS (ESI+) m/z 372.1 (M+H)+. Anal. Calcd for
4.1.4.5. N-(2-(2-Methoxyphenyl)-1H-benzo[d]imidazol-6-yl)qui-
nazolin-4-amine (7e).
Brown powder, yield: 71%, mp: 349–
350 °C. 1H NMR (300 MHz; DMSO-d6): 4.07 (s, 3H); 7.20 (t,
J = 7.4 Hz, 1H); 7.33 (d, J = 8.3 Hz, 1H); 7.57–7.68 (m, 2H); 7.77–
7.83 (m, 2H); 7.91 (d, J = 7.8 Hz, 1H); 8.00–8.06 (m, 1H); 8.27 (s,
1H); 8.33 (d, J = 7.5 Hz, 1H); 8.81 (s, 2H); 11.10 (s, 1H). MS (ESI+)
m/z 368.0 (M+H)+. Anal. Calcd for C22H17N5O: C, 71.92; H, 4.66;
N, 19.06. Found: C, 72.12; H, 4.63; N, 19.14.
C21H14ClN5: C, 67.83; H, 3.80; N, 18.84. Found: C, 68.02; H, 3.83; N,
18.92.
4.1.4.13. N-(2-(4-Chlorophenyl)-1H-benzo[d]imidazol-6-yl)qui-
nazolin-4-amine (7m).
Yellow powder, yield: 59%, mp:
337–339 °C. 1H NMR (300 MHz; DMSO-d6): 7.59–7.74 (m, 4H);
7.84 (t, J = 7.2 Hz, 1H); 7.96 (d, J = 8.3 Hz, 1H); 8.07 (t, J = 7.2 Hz,
2H); 8.27 (d, J = 8.5 Hz, 2H); 8.88–8.94 (m, 2H); 11.6 9(s, 1H). MS
(ESI+) m/z 372.2 (M+H)+. Anal. Calcd for C21H14ClN5: C, 67.83; H,
3.80; N, 18.84. Found: C, 67.65; H, 3.78; N, 18.78.
4.1.4.6.
yl)phenol (7f).
2-(6-(Quinazolin-4-ylamino)-1H-benzo[d]imidazol-2-
Yellow powder, mp: yield: 53%, mp: 341–
343 °C. 1H NMR (300 MHz; DMSO-d6): 7.01–7.11 (m, 2H); 7.42 (t,
J = 7.4 Hz, 1H); 7.65 (d, J = 8.6 Hz, 1H); 7.76–7.86 (m, 2H); 7.94
(d, J = 8.2 Hz, 1H); 8.07 (t, J = 7.6 Hz, 1H); 8.14 (d, J = 7.8 Hz, 2H);
8.88 (d, J = 8.3 Hz, 2H); 11.53 (s, 1H). MS (ESI+) m/z 354.3 (M+H)+.
Anal. Calcd for C21H15N5O: C, 71.38; H, 4.28; N, 19.82. Found: C,
71.25; H, 4.32; N, 19.88.
4.1.4.14. N-(2-(4-Bromophenyl)-1H-benzo[d]imidazol-6-yl)qui-
nazolin-4-amine (7n).
Yellow powder, yield: 78%, mp: 353–
355 °C. 1H NMR (300 MHz; DMSO-d6): 7.59 (dd, J = 8.7, 1.8 Hz,
1H); 7.70 (d, J = 8.7 Hz, 1H); 7.77–7.85 (m, 3H); 7.95(d, J = 8.7 Hz,
1H); 8.06 (t, J = 7.5 Hz, 2H); 8.18 (d, J = 8.4 Hz, 2H); 8.84–8.92 (m,
2H); 11.49 (s, 1H). MS (ESI+) m/z 416.0 (M+H)+. Anal. Calcd for
4.1.4.7. N-(2-(4-Ethylphenyl)-1H-benzo[d]imidazol-6-yl)quinaz-
olin-4-amine (7g).
Yellow powder, yield: 68%, mp: 307–
310 °C. 1H NMR (500 MHz; DMSO-d6): 1.23 (t, J = 7.6 Hz, 3H);
2.69 (q, J = 7.3 Hz, 2H); 7.44 (d, J = 8.2 Hz, 2H); 7.63 (dd, J = 8.6,
1.7 Hz, 1H); 7.72 (d, J = 8.7 Hz, 1H); 7.81 (t. J = 7.3 Hz, 1H); 7.92
(d, J = 8.4 Hz, 1H); 8.04 (t, J = 7.4 Hz, 1H); 8.17 (d, J = 8.3 Hz, 3H);
8.84 (d, J = 6.9 Hz, 2H); 11.33 (s, 1H). MS (ESI+) m/z 366.2 (M+H)+.
Anal. Calcd for C23H19N5: C, 75.59; H, 5.24; N, 19.16. Found: C,
75.64; H, 5.21; N, 19.20.
C21H14BrN5: C, 60.59; H, 3.39; N, 16.82. Found: C, 60.76; H, 3.41;
N, 16.86.
4.1.4.15. N-(2-(2-(Trifluoromethyl)phenyl)-1H-benzo[d]imida-
zol-6-yl)quinazolin-4-amine (7o).
Brown powder, yield:
54%, mp: 309–311 °C. 1H NMR (300 MHz; DMSO-d6): 7.59 (d,
J = 8.5 Hz, 1H); 7.71–7.85 (m, 5H); 7.94–8.09 (m, 4H); 8.89 (s,
1H); 8.91 (d, J = 8.2 Hz, 1H); 11.87 (s, 1H). MS (ESI+) m/z 406.1
(M+H)+. Anal. Calcd for C22H14F3N5: C, 65.18; H, 3.48; F, 14.06; N,
17.28. Found: C, 65.03; H, 3.50; N, 14.02.
4.1.4.8. N-(2-(2-Fluorophenyl)-1H-benzo[d]imidazol-6-yl)qui-
nazolin-4-amine (7h).
Yellow powder, yield: 64%, mp: 301–
304 °C. 1H NMR (300 MHz; DMSO-d6): 7.39–7.50 (m, 2H); 7.60
(d, J = 8.5 Hz, 2H); 7.75 (d, J = 8.6 Hz, 1H); 7.84 (t, J = 7.6 Hz, 1H);
7.97 (d, J = 8.2 Hz, 1H); 8.05–8.09 (m, 2H); 8.26 (t, J = 7.6 Hz, 1H);
8.88 (s, 1H); 8.95 (d, J = 8.3 Hz, 1H); 11.73 (s, 1H). MS (ESI+) m/z
356.1 (M+H)+. Anal. Calcd for C21H14FN5: C, 70.98; H, 3.97; N,
19.71. Found: C, 70.76; H, 3.95; N, 19.75.
4.1.4.16. N-(2-(3-(Trifluoromethyl)phenyl)-1H-benzo[d]imida-
zol-6-yl)quinazolin-4-amine (7p).
Yellow powder, yield:
55%, mp: 343–345 °C. 1H NMR (300 MHz; DMSO-d6): 7.60 (dd,
J = 8.6, 1.3 Hz, 1H); 7.75 (d, J = 8.6 Hz, 1H); 7.80–7.90 (m, 3H);
7.97 (d, J = 8.3 Hz, 1H); 8.08–8.12 (m, 2H); 8.54–8.59 (m, 2H);
8.90–8.94 (m, 2H); 11.75 (s, 1H). MS (ESI+) m/z 406.1 (M+H)+. Anal.
Calcd for C22H14F3N5: C, 65.18; H, 3.48; F, 14.06; N, 17.28. Found: C,
65.35; H, 3.51; N, 14.11.
4.1.4.9. N-(2-(3-Fluorophenyl)-1H-benzo[d]imidazol-6-yl)qui-
nazolin-4-amine (7i).
Green powder, yield: 77%, mp: 297–
299 °C. 1H NMR (300 MHz; DMSO-d6): 7.35 (t, J = 8.0 Hz, 1H);
7.57–7.65 (m, 2H); 7.70 (d, J = 8.6 Hz, 1H); 7.81 (t, J = 7.9 Hz, 1H);
7.92 (d, J = 8.3 Hz, 1H); 8.02–8.10 (m, 4H); 8.84–8.88 (m, 2H);
11.39 (s, 1H). MS (ESI+) m/z 356.1 (M+H)+. Anal. Calcd for
4.1.4.17. N-(2-(4-(Trifluoromethyl)phenyl)-1H-benzo[d]imida-
zol-6-yl)quinazolin-4-amine (7q).
Yellow powder, yield:
64%, mp: 334–337 °C. 1H NMR (300 MHz; DMSO-d6): 7.60 (d,
J = 8.0 Hz, 1H); 7.72 (d, J = 8.5 Hz, 1H); 7.82 (d, J = 7.1 Hz, 1H);
7.90–7.97 (m, 3H); 8.03–8.10 (m, 2H); 8.44 (d, J = 7.6 Hz, 2H);
C21H14FN5: C, 70.98; H, 3.97; N, 19.71. Found: C, 70.82; H, 4.00;
N, 19.63.
Please cite this article in press as: Shi, L.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.07.008

L. Shi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
8.87 (s, 1H); 8.94 (d, J = 7.9 Hz, 2H); 11.64 (s, 1H). MS (ESI+) m/z
406.1 (M+H)+. Anal. Calcd for C22H14F3N5: C, 65.18; H, 3.48; F,
14.06; N, 17.28. Found: C, 65.42; H, 3.46; N, 14.10.
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 tempera-
ture. Primary antibody [phosphorylated tyrosine monoclonal anti-
body [(pTyr-100), 1:1000 in PBS/T with 1% bovine 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, phos-
phorylation 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 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.
4.1.4.18.
yl)quinazolin-4-amine (7r).
N-(2-(2,6-Dichlorophenyl)-1H-benzo[d]imidazol-6-
Yellow powder, yield: 75%, mp:
336–339 °C. 1H NMR (300 MHz; DMSO-d6): 7.55–7.75 (m, 5H);
7.85 (t, J = 7.7 Hz, 1H); 7.98–8.12 (m, 3H); 8.89 (s, 1H); 9.01 (d,
J = 8.3 Hz, 1H); 11.93 (s, 1H). MS (ESI+) m/z 406.1 (M+H)+. Anal.
Calcd for C21H13Cl2N5: C, 62.08; H, 3.23; N, 17.24. Found: C,
62.31; H, 3.24; N, 17.29.
4.1.4.19.
yl)quinazolin-4-amine (7s).
N-(2-(3,4-Dichlorophenyl)-1H-benzo[d]imidazol-6-
Brown powder, yield: 81%, mp:
327–329 °C. 1H NMR (300 MHz; DMSO-d6): 7.58 (dd, J = 8.7,
1.5 Hz, 1H); 7.70 (d, J = 8.6 Hz, 1H); 7.80–7.84 (m, 2H); 7.95 (d,
J = 8.2 Hz, 1H); 8.03–8.06 (m, 2H); 8.20 (dd, J = 8.4, 1.6 Hz, 1H);
8.45–8.46 (d, J = 1.6 Hz, 2H); 8.86–8.93 (m, 2H); 11.60 (s, 1H). MS
(ESI+) m/z 406.0 (M+H)+. Anal. Calcd for C21H13Cl2N5: C, 62.08; H,
3.23; N, 17.24. Found: C, 61.85; H, 3.25; N, 17.27.
4.4. Molecular docking
Molecular docking of compound 7j into the three dimensional
X-ray structure of c-Met (PDB code: 3CD8) and VEGFR-2 (PDB
code: 2QU5) was carried out using the Discovery Studio (version
3.1) as implemented through the graphical user interface Discov-
ery Studio CDOCKER protocol.
4.1.4.20.
yl)quinazolin-4-amine (7t).
N-(2-(3,5-Dichlorophenyl)-1H-benzo[d]imidazol-6-
Brown powder, yield: 65%, mp:
315–317 °C. 1H NMR (300 MHz; DMSO-d6): 7.66–7.92 (m, 4H);
7.95–8.15 (m, 2H); 8.18–8.31 (m, 3H); 8.85 (d, J = 8.2 Hz, 1H);
8.94 (d, J = 8.4 Hz, 1H); 11.88 (s, 1H). MS (ESI+) m/z 406.0 (M+H)+.
Anal. Calcd for C21H13Cl2N5: C, 62.08; H, 3.23; N, 17.24. Found: C,
62.28; H, 3.21; N, 17.20.
The three-dimensional structure of the compound 7j was con-
structed using ChemBio 3D Ultra 11.0 software [Chemical Struc-
ture Drawing Standard; Cambridge Soft corporation, USA (2008)],
then it was energetically minimized by using MMFF94 with 5000
iterations and minimum RMS gradient of 0.10. The crystal struc-
tures 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 3CD8 or 2QU5 was defined as a
receptor and the site sphere was selected based on ATP binding site
of 3CD8 or 2QU5. Compound 7j were placed during the molecular
docking procedure. Types of interactions of the docked protein
with ligand were analyzed after the end of molecular docking.
4.1.4.21. N-(2-(2-Bromo-5-fluorophenyl)-1H-benzo[d]imidazol-
6-yl)quinazolin-4-amine (7u).
Yellow powder, yield: 73%,
mp: 328–331 °C. 1H NMR (300 MHz; DMSO-d6): 7.39 (dd, J = 8.5,
2.8 Hz, 1H); 7.59 (d, J = 8.8 Hz, 1H); 7.70–7.76 (m, 2H); 7.81–7.90
(m, 2H); 7.98 (d, J = 8.1 Hz, 1H); 8.06–8.11 (m, 2H); 8.89 (s, 1H);
8.99 (d, J = 8.2 Hz, 1H); 11.84 (s, 1H). MS (ESI+) m/z 434.0 (M+H)+.
Anal. Calcd for C21H13BrFN5: C, 58.08; H, 3.02; N, 16.13. Found: C,
58.32; H, 3.04; N, 16.18.
4.2. Cell proliferative assay
Acknowledgements
The antiproliferative activities of the prepared N-(2-phenyl-1H-
benzo[d]imidazol-5-yl)quinazolin-4-amine derivatives against
Hep-G2 and MCF-7 cells were evaluated by MTT assay.25 Briefly, tar-
get tumor cells were grown to log phase in RPMI 1640 medium sup-
plemented with 10% fetal bovine serum. After diluting to
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).
2 Â 104 cells mLÀ1 with the complete medium, 100
lL of the
obtained 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.
References
1. Comoglio, P. M.; Giordano, S.; Trusolino, L. Nat. Rev. Drug Disc. 2008, 7, 504.
2. Trusolino, L.; Comoglio, P. M. Nat. Rev. Cancer 2002, 2, 289.
3. Birchmeier, C.; Birchmeier, W.; Gherardi, E.; Vande Woude, G. F. Nature Rev.
Mol. Cell Biol. 2003, 4, 915.
4. Cheng, H.-L.; Trink, B.; Tzai, T.-S.; Liu, H.-S.; Chan, S.-H.; Ho, C.-L.; Sidransky, D.;
Chow, N.-H. J. Clin. Oncol. 2002, 20, 1544.
After 48 h exposure period, 40
of MTT was added to each well. Four hours later, 100
l
L of PBS containing 2.5 mg mLÀ1
L extraction
l
solution (10% SDS-5% isobutyl alcohol-0.010 M HCl) was added.
After an overnight incubation at 37 °C, the optical density was mea-
sured at a wavelength of 570 nm on an ELISA microplate reader.
5. Drebber, U.; Baldus, S. E.; Nolden, B.; Grass, G.; Bollschweiler, E.; Dienes, H. P.;
Hölscher, A. H.; Mönig, S. P. Oncol. Rep. 2008, 19, 1477.
6. Parr, C.; Watkins, G.; Mansel, R. E.; Jiang, W. G. Clin. Cancer Res. 2004, 10, 202.
7. Herynk, M. H.; Stoeltzing, O.; Reinmuth, N.; Parikh, N. U.; Abounader, R.;
Laterra, J.; Radinsky, R.; Ellis, L. M.; Gallick, G. E. Cancer Res. 2003, 63(11), 2990.
8. Maulik, G.; Kijima, T.; Ma, P. C.; Ghosh, S. K.; Lin, J.; Shapiro, G. I.; Schaefer, E.;
Tibaldi, E.; Johnson, B. E.; Salgia, R. Clin. Cancer Res. 2002, 8, 620.
9. Ferrara, N. Nat. Rev. Cancer 2002, 2, 795.
10. Ferrara, N.; Gerber, H. P.; LeCouter, J. Nat. Med. 2003, 9, 669.
11. Olsson, A. K.; Dimberg, A.; Kreuger, J.; Claesson-Welsh, L. Nat. Rev. Mol. Cell Biol.
2006, 7, 359.
12. Roy, H.; Bhardwaj, S.; Ylä-Herttuala, S. FEBS Lett. 2006, 580, 2879.
13. You, W.-K.; Sennino, B.; Williamson, C. W.; Falcón, B.; Hashizume, H.; Yao, L.-
C.; Aftab, D. T.; McDonald, D. M. Cancer Res. 2011, 71, 4758.
14. Sennino, B.; Ishiguro-Oonuma, T.; Wei, Y.; Naylor, R. M.; Williamson, C. W.;
Bhagwandin, V.; Tabruyn, S. P.; You, W.-K.; Chapman, H. A.; Christensen, J. G.;
Aftab, D. T.; McDonald, D. M. Cancer Discov. 2012, 2, 270.
4.3. In vitro kinase assay
In vitro kinase inhibitory ability was determined using HTScan
VEGF Receptor 2 Kinase Assay Kit and HTScan MET Kinase Assay
Kit (purchased from Cell Signaling Technology, Inc.) by colorimet-
ric ELISA assay according to the manufacturer’s instructions.26
Briefly, reaction cocktail containing recombinant human VEGFR-2
or Met kinase was incubated with various concentrations of tested
compounds or DMSO (0.1%) for 5 min at room temperature, and
then ATP/substrate peptide cocktail was added to the pre-
Please cite this article in press as: Shi, L.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.07.008

10
L. Shi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
15. Bilanges, B.; Torbett, N.; Vanhaesebroeck, B. Nat. Chem. Biol. 2008, 4, 648.
16. Kurzrock, R.; Sherman, S. I.; Ball, D. W.; Forastiere, A. A.; Cohen, R. B.; Mehra, R.;
Pfister, D. G.; Cohen, E. E. W.; Janisch, L.; Nauling, F.; Hong, D. S.; Ng, C. S.; Ye, L.;
Gagel, R. F.; Frye, J.; Müller, T.; Ratain, M. J.; Salgia, R. J. Clin. Oncol. 2011, 29,
2660.
17. Claridge, S.; Raeppel, F.; Granger, M.-C.; Berstein, N.; Saavedra, O.; Zhan, L.;
Llewellyn, D.; Wahhab, A.; Vaisburg, A. Bioorg. Med. Chem. Lett. 2008, 18, 2793.
18. Raeppel, S.; Claridge, S.; Saavedra, O.; Gaudette, F.; Zhan, L.; Mannion, M.; Zhou,
N.; Vaisburg, A. Bioorg. Med. Chem. Lett. 2009, 19, 1323.
22. Matsumoto, S.; Miyamoto, N.; Hirayama, T.; Oki, H.; Okada, K.; Tawada, M.;
Iwata, H.; Nakamura, K.; Yamasaki, S.; Miki, H.; Hori, A.; Imamura, S. Bioorg.
Med. Chem. Lett. 2013, 21, 7686.
23. Albrecht, B. K.; Harmange, J. C.; Bauer, D.; Berry, L.; Bode, C.; Boezio, A. A.; Chen,
A.; Choquette, D.; Dussault, I.; Fridrich, C.; Hirai, S.; Hoffman, D.; Larrow, J. F.;
Kaplan-Lefko, P.; Lin, J.; Lohman, J.; Long, A. M.; Moriguchi, J.; O’Connor, A.;
Potashman, M. H.; Reese, M.; Rex, K.; Siegmund, A.; Shah, K.; Shimanovich, R.;
Springer, S. K.; Teffera, Y.; Yang, Y.; Zhang, Y.; Bellon, S. F. J. Med. Chem. 2008,
51, 2879.
19. Nakagawa, T.; Tohyama, O.; Yamaguchi, A.; Matsushima, T.; Takahashi, K.;
Funasaka, S.; Shirotori, S.; Asada, M.; Obaishi, H. Cancer Sci. 2010, 101, 210.
20. Yakes, F. M.; Chen, J.; Tan, J.; Yamaguchi, K.; Shi, Y.; Yu, P.; Qian, F.; Chu, F.;
Bentzien, F.; Cancilla, B.; Orf, J.; You, A.; Laird, A. D.; Engst, S.; Lee, L.; Lesch, J.;
Chou, Y. C.; Joly, A. H. Mol. Cancer Ther. 2011, 10, 2298.
24. Potashman, M. H.; Bready, J.; Coxon, A.; Demelfi, T. M.; Dipietro, L.; Doerr, N.;
Elbaum, D.; Estrada, J.; Gallant, P.; Germain, J.; Gu, Y.; Harmange, J. C.;
Kaufman, S. A.; Kendall, R.; Kim, J. L.; Kumar, G. N.; Long, A. M.; Neervannan, S.;
Patel, V. F.; Polverino, A.; Rose, P.; Plas, S. V.; Whittington, D.; Zanon, R.; Zhao,
H. J. Med. Chem. 2007, 50, 4351.
21. Qian, F.; Engst, S.; Yamaguchi, K.; Yu, P.; Won, K. A.; Mock, L.; Lou, T.; Tan, J.; Li,
C.; Tam, D.; Lougheed, J.; Yakes, F. M.; Bentzien, F.; Xu, W.; Zaks, T.; Wooster,
R.; Greshock, J.; Joly, A. H. Cancer Res. 2009, 69, 8009.
25. Xu, Y.-Y.; Li, S.-N.; Yu, G.-J.; Hu, Q.-H.; Li, H.-Q. Bioorg. Med. Chem. 2013, 21, 6084.
26. Lee, J.-S.; Kang, Y.; Kim, J. T.; Thapa, D.; Lee, E.-S.; Kim, J.-A. Eur. J. Pharmacol.
2012, 677, 22.
Please cite this article in press as: Shi, L.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.07.008