Synthesis and biological evaluation of 1-cyano-2-Amino-benzimidazole derivatives as a novel class of antitumor agents

Article abstract of DOI:10.1007/s00044-013-0888-6

A series of 1-cyano-2-Amino-benzimidazole derivatives were synthesized and evaluated for their cytotoxic activities in vitro against three human cancer cell lines (human lung carcinoma cell line: A549, human leukemia cell line: K562, and human prostate cancer cell line: PC-3). Most of these compounds showed potent activities against these tumor cell lines, especially against A549 and K562 cell lines. The preliminary structure-Activity relationships of 1-cyano-2-Amino-benzimidazole derivatives were also discussed. The cell cycle analysis was carried out in K562 cells and the results showed that compound 4d caused a marked increase of cells in G ₂/M phase.

Full text of DOI:10.1007/s00044-013-0888-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0888-6
ORIGINAL RESEARCH
Synthesis and biological evaluation of 1-cyano-2-amino-
benzimidazole derivatives as a novel class of antitumor agents
•
Zhang Hu Lili Ou Sidong Li Lei Yang
•
•
Received: 17 October 2013 / Accepted: 7 December 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of 1-cyano-2-amino-benzimidazole
derivatives were synthesized and evaluated for their cyto-
toxic activities in vitro against three human cancer cell
lines (human lung carcinoma cell line: A549, human leu-
kemia cell line: K562, and human prostate cancer cell line:
PC-3). Most of these compounds showed potent activities
against these tumor cell lines, especially against A549 and
K562 cell lines. The preliminary structure–activity rela-
tionships of 1-cyano-2-amino-benzimidazole derivatives
were also discussed. The cell cycle analysis was carried out
in K562 cells and the results showed that compound 4d
caused a marked increase of cells in G2/M phase.
still suffer from many toxic side effects of the drugs such as
bone marrow suppression, gastrointestinal tract lesions,
nausea, hair loss, drug resistance, and so on (Nussbaumer
et al., 2011). Therefore, the development of novel, effi-
cient, and less toxic anticancer agents is still of utmost
importance.
The benzimidazole nucleus is an important pharmaco-
phore and privileged structure in drug discovery. Benz-
imidazole derivatives have been found to possess a wide
variety of biological activities including antiviral (Mor-
ningstar et al., 2007), anti-inflammatory (Mader et al.,
2008), antihypertensive (Kaur et al., 2008; Kohara et al.,
1996), antioxidant (Kus¸ et al., 2008, 2009), antitubercular
(Camacho et al., 2011), antiulcer (Ku¨hler et al., 1998), and
antimicrobial (Ansari and Lal, 2009; El-masry et al., 2000).
Particularly noteworthy is that benzimidazole derivatives
have also been found to be of potent anticancer activities
(Hranjec et al., 2008a, b, 2010; Gellis et al., 2008; Thimme
Gowda et al., 2008; Thimme Gowda et al., 2009; Winfield
et al., 2008), and benzimidazole nucleus was considered to
be an essential part of many antineoplastic derivatives
(Badawey and Kappe, 1995). Bendamustine hydrochloride
containing alkylating group and benzimidazole component
has been approved by FDA to be used for the treatment of
patients with chronic lymphocytic leukemia (Cheson and
Rummel, 2009). Due to the significant medicinal impor-
tance, the synthesis and biological evaluation of benz-
imidazole derivatives have attracted considerable attention
in the recent years.
Keywords 1-Cyano-2-amino-benzimidazole derivatives ꢀ
Antitumor activity ꢀ Structure–activity relationships ꢀ
Cell cycle analysis
Introduction
Cancer is one of the major leading causes of death around
the world. Taking into account the existing cancer thera-
pies, chemotherapy has turned out to be one of the most
significant treatments in cancer management (Harrison
et al., 2009). Although the success of clinical practice in
identifying a large number of potent chemotherapeutic
anticancer agents has been significant, clinical treatments
Z. Hu (&) ꢀ S. Li ꢀ L. Yang
The benzimidazole compounds with cyano substituent
in general strongly enhance the cytotoxic activity. Some
cyano-substituted benzimidazole compounds were found to
possess cytotoxic activity on several human cancer cell
lines (Hranjec et al., 2010, 2008a, b). Earlier, we reported
that the intramolecular C–N coupling reactions of various
Department of Chemistry, College of Science, Guangdong
Ocean University, Zhanjiang 524088, China
e-mail: huzhangqyx@126.com
L. Ou
College of Pharmaceutical Science, Zhejiang University,
Hangzhou 310058, China
123

Med Chem Res
substituted aryl guanidines could be successfully carried
out to form 1-cyanobenzimidazoles by employing a CuI/
2,20-biimidazole catalyst system (Hu et al., 2011). In con-
tinuation with our efforts in search of potential anticancer
agents, we have synthesized the novel series of cyano-
substituted benzimidazole derivatives by introducing dif-
ferent substituted amino groups at the second position of
benzimidazole nucleus. Full details about the synthesis,
evaluation of antitumor activity in vitro of benzimidazole
derivatives are reported herein.
cytotoxic activities of these compounds was carried out
using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide (MTT) assay. Doxorubicin hydrochlo-
ride was employed as the reference drug. The cytotoxic
potency of these compounds was indicated by IC50 values.
The results are summarized in Table 1.
As shown in Table 1, most of the 1-cyano-2-amino-
benzimidazole derivatives exhibited inhibitions on the
growth of selected tumor cell lines, especially on A549 and
K562 cell lines. Several compounds showed moderate to
potent cytotoxic activities against all the tested cell lines
(IC50 \ 50 lmol L-1). Compounds 4c and 4d were the
most promising compounds among the tested derivatives
Results and discussions
with IC50 values of 3.33–33.52 and 2.69–18.51 lmol L-1
,
Chemistry
respectively. Preliminary structure–activity relationship of
1-cyano-2-amino-benzimidazole derivatives was investi-
gated. In general, cyclic secondary amines with R1R2NH
substitutions were beneficial for increasing the cytotoxicity
of 1-cyano-2-amino-benzimidazoles compared with open
chain secondary amines. It was found that the introduction
of morpholine (4c) or methylpiperazine (4d) at the R1R2NH
position led to compounds with good cytotoxic activity
against A549 and K562 cell lines. Particularly noteworthy is
that the methylpiperazine substitution yielded compound 4d
which was discovered as the most potent inhibitor against
A549, K562 and PC-3 cell lines with IC50 values of 6.48,
2.69 and 18.51 lmol L-1, respectively. Additionally, a
heterocyclic derived piperazine analog, 4j showed a similar
potency against K562 with an IC50 of 3.39 lmol L-1 when
compared with 4d. Among R1R2NH substitutions of the
open chain secondary amines, little steric hindrance sub-
stitutions were beneficial for 1-cyano-2-amino-benzimida-
zole’s cytotoxicity on A549 and K562 cell lines, such as
compounds 4k and 4l. And bulky groups were detrimental to
the compounds’ cytotoxicity, such as compounds 4m and
4n. When the acyclic aliphatic amines were replaced by
diphenylamine, 4o showed a decreased cytotoxic activity.
To shed light on the mechanisms responsible for the
cytotoxic effect of compound 4d, we examined its influ-
ence on the growth and division of K562 cells by mea-
suring the DNA content of eukaryotic cells. Cells were
cultured for 24 h in the presence of 4d with 10, 30, and
The synthesis of a series of 1-cyano-2-amino-benzimida-
zole derivatives were accomplished via intramolecular
cyclization with a CuI/2,20-biimidazole catalyst system. As
shown in Scheme 1, condensation of the commercially
available dimethyl N-cyanodithioiminocarbonate 1 with
2-iodoaniline in the presence of Cs2CO3 in DMF at 100 °C
for 8 h gave adduct 2. Compounds 2 reacted with the
secondary amines in fusion to afford the corresponding aryl
guanidines 3. Finally, 1-cyano-2-amino-benzimidazole
derivatives 4 were obtained by intramolecular cyclization
of aryl guanidines 3 under the conditions of 5 mol% of CuI
with 10 mol% of 2,20-biimidazole in the presence of
Cs2CO3. The final compounds 4 were purified by silica gel
column chromatography using ethyl acetate/hexane as
eluent and obtained in good yields. Structures of all the
final compounds, 4, were established through FTIR, NMR
spectra, ESI–MS and elemental analysis.
Biological activities
All the obtained 1-cyano-2-amino-benzimidazole deriva-
tives were evaluated for their cytotoxic activities against
three human cancer cell lines derived from the various
human cancer types, including A549 (human lung carci-
noma cells), K562 (human leukemia cells), and PC-3
(human prostate cancer cells). In vitro evaluation of the
R1
Scheme 1 Synthesis of
1-cyano-2-amino-
benzimidazole derivatives
SCH3
R2
N
R1
R2
N
N
H3CS
N
I
C
N
a
c
b
NH
CN
N
C
N
CN
NH
CN
H3CS
I
CN
1
3
4
2
Reagents and conditions: (a) 2-iodoaniline, Cs2CO3, DMF, 100°C; (b) R1R2NH, ethanol, reflux; (c) CuI,
2,2'-biimidazole, Cs2CO3, DMF, 80°C.
123

Med Chem Res
Table 1 Cytotoxicity of 1-cyano-2-amino-benzimidazole derivatives against human cancer lines in vitro
Compounds
R1R2NH
Cytotoxicity (IC50, lmol L-1 a
)
A549
K562
PC-3
Doxorubicin
–
3.36 0.57
1.15 0.18
2.37 0.16
4a
19.11 4.01
12.35 3.16
38.76 2.57
NH
4b
4c
12.38 6.24
8.55 3.37
18.47 5.17
3.33 2.15
41.39 0.12
33.52 4.21
NH
O
NH
N
4d
4e
4f
6.48 0.77
13.21 1.24
17.16 2.33
15.69 0.35
16.22 5.01
11.16 2.52
28.26 5.21
2.69 0.65
23.66 3.03
22.03 5.12
18.32 4.11
28.83 2.35
37.64 1.20
3.39 0.26
18.51 0.33
[50
HN
N
NH
39.65 3.18
[50
N
NH
4g
4h
4i
O
N
NH
42.15 2.26
[50
Cl
N
NH
O2N
N
NH
4j
31.25 3.09
N
NH
N
N
F3C
4k
4l
17.03 3.62
18.31 2.29
40.35 3.72
21.25 3.15
20.49 2.15
[50
[50
[50
[50
NH
O2N
N
NH
4m
H
N
4n
4o
37.13 5.26
36.82 3.47
[50
[50
N
H
[50
[50
H
N
a
Each experiment was independently performed three times and expressed as mean SD
123

Med Chem Res
N-(2-Iodophenyl)-N0-cyano-S-methylisothiourea (2)
Table 2 Cell cycle distribution by flow cytometry in K562 cells
treated with compound 4d for 24 h
Dimethyl cyanodithioimido-carbonate (1.46 g, 10 mmol)
was dissolved in DMF (20 mL). To the stirred solution
were added 2-iodoaniline (10 mmol) and Cs2CO3 (4.89 g,
15 mmol). The solution was kept at 100 °C for 8 h and
then poured into ice water. Precipitates formed immedi-
ately and were collected by filtration. The crude products
were chromatographed on silica gel with petroleum ether/
ethyl acetate to afford the desired compound. It was
obtained as a yellow solid, mp 178–180 °C, yield 41 %. IR
(KBr), m (cm-1): 3332 (NH), 3046 (Ar–H), 2241 (C:N),
Concentration
(lmol L-1
G0/G1 (%)
S (%)
G2/M (%)
)
Control
10
44.7 0.63
38.3 3.26
27.0 1.37
10.3 1.72
49.0 0.31
46.5 0.28
45.7 0.86
44.5 0.37
6.3 0.54
15.2 2.59
27.3 2.17
45.2 0.53
30
50
50 lmol L-1, and then evaluated. Flow cytometric ana-
lysis of cell cycle was carried out and reported in Table 2.
The results showed that compound 4d arrested the cells in
the G2/M phase of the cell cycle, which was accompanied
by a dose-dependent manner.
1
1587 (C=N). H NMR (DMSO-d6) d: 2.63 (s, 3H, SCH3),
7.12–7.14 (m, 1H, ArH), 7.36–7.48 (m, 2H, ArH),
7.92–7.94 (m, 1H, ArH), 10.35 (s, 1H, NH). 13C NMR
(DMSO-d6) d: 13.3 (S–C), 83.6 (C–I), 116.1 (C:N),
124.1, 128.5, 130.3, 139.2 (aromatic carbons), 158.1 (C–N,
iodophenyl), 161.3 (C=N). MS: m/z [M?1]? 318.
Conclusion
General procedure for the synthesis of aryl guanidines
A series of 1-cyano-2-amino-benzimidazole derivatives
were synthesized via intramolecular cyclization. Their
cytotoxicity against a variety of cancer cell lines was
evaluated. Most of them displayed potent cytotoxic activ-
ities in the micromolar range, especially against A549 and
K562 cell lines. The preliminary structure–activity rela-
tionship of these compounds was investigated. The cell
cycle analysis in K562 cells showed that compound 4d
caused the significant arrest of the cell cycle at the G2/M
phase in a dose-dependent manner.
(3a–o)
A mixture of 2 (10 mmol) and secondary amines
(12 mmol) in ethanol (30 mL) was refluxed for 24 h, then
the solvent was removed under the reduced pressure. The
resulting residue was purified by column chromatography
on silica gel with petroleum ether/ethyl acetate to provide
the desired aryl guanidines 3a–o.
N0-(2-Iodophenyl)-N-cyanopyrrolidine-1-carboximidamide
(3a)
This compound was prepared by the reaction of 2 and
pyrrolidine. It was obtained as a pale yellow solid, mp
82–84 °C, yield 63 %. IR (KBr), m (cm-1): 3386 (NH),
3075 (Ar–H), 2846 (CH2, aliphatic), 2232 (C:N), 1594
Experimental
Materials and methods
1
(C=N). H NMR (CDCl3, 400 MHz) d: 1.93 (t, 4H, CH2
Unless otherwise indicated, all reactions were carried out
under a dry nitrogen atmosphere. DMF was freshly dis-
tilled from calcium hydride. The other reagents were used
directly without further purification. The melting points
(mp) were obtained on a B-540 Bu¨chi melting-point
apparatus and are uncorrected. FTIR spectra were recorded
on a PerkinElmer Spectrum 100 infrared spectrophotome-
ter (KBr pellet). 1H NMR (400 MHz) or 13C NMR
(100 MHz) data were recorded on a DPX-400 instrument
with CDCl3 or DMSO-d6 as solvents and tetramethylsilane
(TMS) as the internal standard. Chemical shifts are given
in ppm and spin–spin coupling constants, J, are given in
Hz. Abbreviations used were s (singlet), d (doublet), t
(triplet), q (quartet), and m (multiplet). Mass spectra (MS)
were recorded on a HP5989A mass spectrometer. Ele-
mental analyses were carried out on a PE EA2400 CHN
analyzer.
pyrrolidin), 3.44 (t, 4H, NCH2, pyrrolidin), 6.91–6.94 (m,
1H, ArH), 7.34–7.36 (m, 2H, ArH), 7.82–7.84 (m, 1H,
ArH), 9.12 (s, 1H, NH). 13C NMR (CDCl3, 100 MHz) d:
25.2 (2C, pyrrolidin), 49.3 (2C, N–C, pyrrolidin), 83.5 (C–
I), 116.4 (C:N), 123.6, 128.7, 131.4, 139.5 (aromatic
carbons), 159.3 (C–N, iodophenyl), 162.6 (C=N). MS: m/z
[M?1]? 341.
N0-(2-Iodophenyl)-N-cyanopiperidine-1-carboximidamide
(3b)
This compound was prepared by the reaction of 2 and
piperidine. It was obtained as a pale yellow solid, mp
88–89 °C, yield 67 %. IR (KBr), m (cm-1): 3351 (NH),
3066 (Ar–H), 2911 (CH2, aliphatic), 2238 (C:N), 1610
1
(C=N). H NMR (DMSO-d6) d: 1.57–1.59 (m, 6H, CH2
123

Med Chem Res
piperidin), 3.51–3.53 (m, 4H, NCH2, piperidin), 6.96–7.00
(m, 1H, ArH), 7.20–7.22 (m, 1H, ArH), 7.35–7.39 (m, 1H,
ArH), 7.84–7.86 (m, 1H, ArH), 9.07 (s, 1H, NH). 13C NMR
(DMSO-d6) d: 24.4 (piperidin), 25.6 (2C, piperidin), 49.5
(2C, N–C, pyrrolidin), 83.8 (C–I), 115.7 (C:N), 123.9,
128.8, 130.2, 138.8 (aromatic carbons), 158.2 (C–N,
iodophenyl), 159.7 (C=N). MS: m/z [M?1]? 355.
(2C, N–C, piperazin), 54.6 (2C, N–C, piperazin), 83.6 (C–
I), 116.2 (C:N), 116.6 (2C), 120.8, 123.5, 128.4, 130.3,
130.7 (2C), 138.9 (aromatic carbons), 155.0 (C–N, phenyl),
158.2 (C–N, iodophenyl), 160.5 (C=N). MS: m/z [M?1]?
432.
N0-(2-Iodophenyl)-N-cyano-4-p-tolylpiperazine-1-
carboximidamide (3f)
N0-(2-Iodophenyl)-N-cyanomorpholine-4-carboximidamide
(3c)
This compound was prepared by the reaction of 2 and 1-p-
tolylpiperazine. It was obtained as a pale yellow solid, mp
106–108 °C, yield 58 %. IR (KBr), m (cm-1): 3358 (NH),
3079 (Ar–H), 2942 (CH3), 2879 (CH2, aliphatic), 2235
(C:N), 1600 (C=N). 1H NMR (CDCl3, 400 MHz) d: 2.18
(s, 3H, CH3), 2.72 (t, 4H, NCH2, piperazin), 3.20 (t, 4H,
NCH2, piperazin), 6.76–6.78 (d, J = 7.2 Hz, 2H, ArH),
6.93–6.95 (d, J = 7.2 Hz, 2H, ArH), 7.01–7.03 (m, 1H,
ArH), 7.23–7.26 (m, 1H, ArH), 7.33–7.35 (m, 1H, ArH),
7.77–7.81 (m, 1H, ArH), 9.10 (s, 1H, NH). 13C NMR
(CDCl3, 100 MHz) d: 24.1 (CH3), 47.3 (2C, N–C, pip-
erazin), 53.6 (2C, N–C, piperazin), 83.5 (C–I), 116.3
(C:N), 115.4 (2C), 123.1, 128.0, 128.4, 130.0 (2C), 130.3,
139.1 (aromatic carbons), 154.1 (C–N, 1-p-tolyl), 158.3
(C–N, iodophenyl), 160.3 (C=N). MS: m/z [M?1]? 446.
This compound was prepared by the reaction of 2 and
morpholine. It was obtained as a pale yellow solid, mp
163–165 °C, yield 61 %. IR (KBr), m (cm-1): 3375 (NH),
3053 (Ar–H), 2889 (CH2, aliphatic), 2232 (C:N), 1620
(C=N). 1H NMR (CDCl3, 400 MHz) d: 3.54 (t, 4H, NCH2,
morpholino), 3.66 (t, 4H, OCH2, morpholino), 6.98–7.01
(m, 1H, ArH), 7.24–7.26 (m, 1H, ArH), 7.36–7.39 (m, 1H,
ArH), 7.84–7.87 (m, 1H, ArH), 9.26 (s, 1H, NH). 13C NMR
(CDCl3, 100 MHz) d: 43.7 (2C, N–C, morpholino), 63.9
(2C, O–C, morpholino), 83.6 (C–I), 116.0 (C:N), 123.3,
128.3, 130.7, 139.0 (aromatic carbons), 158.6 (C–N,
iodophenyl), 160.1 (C=N). MS: m/z [M?1]? 357.
N0-(2-Iodophenyl)-N-cyano-4-methylpiperazine-1-
carboximidamide (3d)
N0-(2-Iodophenyl)-N-cyano-4-(4-
methoxyphenyl)piperazine-1-carboximidamide (3g)
This compound was prepared by the reaction of 2 and
1-methylpiperazine. It was obtained as a pale yellow solid,
mp 113–116 °C. yield 73 %; IR (KBr), m (cm-1): 3362
(NH), 3088 (Ar–H), 2867 (CH2, aliphatic), 2236 (C:N),
This compound was prepared by the reaction of 2 and 1-(4-
methoxyphenyl)piperazine. It was obtained as a pale yel-
low solid, mp 138–140 °C, yield 62 %. IR (KBr), m (cm-1):
3350 (NH), 3086 (Ar–H), 2956 (CH3), 2892 (CH2, ali-
phatic), 2243 (C:N), 1603 (C=N). 1H NMR (CDCl3,
400 MHz) d: 2.70 (t, 4H, NCH2, piperazin), 3.16 (t, 4H,
NCH2, piperazin), 3.76 (s, 3H, OCH3), 6.82–6.84 (d,
J = 7.2 Hz, 2H, ArH), 6.88–6.90 (d, J = 7.2 Hz, 2H,
ArH), 6.99–7.02 (m, 1H, ArH), 7.24–7.26 (m, 1H, ArH),
7.34–7.36 (m, 1H, ArH), 7.79–7.82 (m, 1H, ArH), 9.09 (s,
1H, NH). 13C NMR (CDCl3, 100 MHz) d: 48.5 (2C, N–C,
piperazin), 54.7 (2C, N–C, piperazin), 57.2 (OCH3), 83.7
(C–I), 116.1 (C:N), 116.5 (2C), 118.6(2C), 123.3, 128.5,
130.6, 139.0 (aromatic carbons), 154.9 (C–N, methoxy-
phenyl), 156.5 (C–O, methoxyphenyl), 158.4 (C–N, iodo-
phenyl), 160.8 (C=N). MS: m/z [M?1]? 462.
1
1608 (C=N). H NMR (CDCl3, 400 MHz) d: 2.20 (s, 3H,
NCH3), 2.35 (t, 4H, NCH2, piperazin), 3.57 (t, 4H, NCH2,
piperazin), 6.96–6.99 (m, 1H, ArH), 7.21–7.23 (m, 1H,
ArH), 7.35–7.37 (m, 1H, ArH), 7.83–7.85 (m, 1H, ArH),
9.10 (s, 1H, NH). 13C NMR (CDCl3, 100 MHz) d: 43.6 (N–
CH3), 45.7 (2C, N–C, piperazin), 54.3 (2C, N–C, pipera-
zin), 83.7 (C–I), 116.4 (C:N), 123.1, 128.7, 131.2, 139.3
(aromatic carbons), 158.5 (C–N, iodophenyl), 161.6
(C=N). MS: m/z [M?1]? 370.
N0-(2-Iodophenyl)-N-cyano-4-phenylpiperazine-1-
carboximidamide (3e)
This compound was prepared by the reaction of 2 and
1-phenylpiperazine. It was obtained as a pale yellow solid,
mp 121–123 °C, yield 56 %. IR (KBr), m (cm-1): 3379
(NH), 3051 (Ar–H), 2883 (CH2, aliphatic), 2238 (C:N),
N0-(2-Iodophenyl)-N-cyano-4-(4-chlorophenyl)piperazine-
1-carboximidamide (3h)
1
1607 (C=N). H NMR (CDCl3, 400 MHz) d: 2.68 (t, 4H,
This compound was prepared by the reaction of 2 and 1-(4-
chlorophenyl)piperazine. It was obtained as a pale yellow
solid, mp 131–133 °C, yield 42 %. IR (KBr), m (cm-1):
3346 (NH), 3072 (Ar–H), 2881 (CH2, aliphatic), 2237
(C:N), 1602 (C=N). 1H NMR (CDCl3, 400 MHz) d: 2.71
NCH2, piperazin), 3.20 (t, 4H, NCH2, piperazin), 6.92–6.94
(m, 2H, ArH), 6.98–7.02 (m, 2H, ArH), 7.23–7.26 (m, 3H,
ArH), 7.34–7.36 (m, 1H, ArH), 7.79–7.82 (m, 1H, ArH),
9.12 (s, 1H, NH). 13C NMR (CDCl3, 100 MHz) d: 48.9
123

Med Chem Res
(t, 4H, NCH2, piperazin), 3.42 (t, 4H, NCH2, piperazin),
6.83–6.85 (d, J = 7.2 Hz, 2H, ArH), 7.00–7.03 (m, 1H,
ArH), 7.21–7.24 (m, 1H, ArH), 7.32–7.34 (m, 1H, ArH),
7.36–7.39 (d, J = 7.2 Hz, 2H, ArH), 7.82–7.84 (m, 1H,
ArH), 9.12 (s, 1H, NH). 13C NMR (CDCl3, 100 MHz) d:
48.3 (2C, N–C, piperazin), 54.1 (2C, N–C, piperazin), 83.5
(C–I), 116.3 (C:N), 116.5 (2C), 123.5, 124.2, 127.6 (2C),
128.3, 130.1, 138.9 (aromatic carbons), 157.8 (C–N,
4-chlorophenyl), 158.1 (C–N, iodophenyl), 160.5 (C=N).
MS: m/z [M?1]? 466.
89–92 °C, yield 55 %. IR (KBr), m (cm-1): 3386 (NH),
3083 (Ar–H), 2972 (CH3), 2864 (CH2), 2240 (C:N), 1613
1
(C=N). H NMR (CDCl3, 400 MHz) d: 2.52 (s, 6H, CH3),
6.98–7.01 (m, 1H, ArH), 7.20–7.23 (m, 1H, ArH),
7.33–7.35 (m, 1H, ArH), 7.81–7.84 (m, 1H, ArH), 9.14 (s,
1H, NH). 13C NMR (CDCl3, 100 MHz) d: 35.2 (2C, N–
CH3), 83.3 (C–I), 116.5 (C:N), 123.1, 128.6, 130.7, 139.0
(aromatic carbons), 159.2 (C–N, iodophenyl), 162.5
(C=N). MS: m/z [M?1]? 315.
1,1-Diethyl-2-(2-iodophenyl)-3-cyanoguanidine (3l)
N0-(2-Iodophenyl)-N-cyano-4-(4-nitrophenyl)piperazine-1-
carboximidamide (3i)
This compound was prepared by the reaction of 2 and
diethylamine. It was obtained as a pale yellow solid, mp
76–79 °C, yield 64 %. IR (KBr), m (cm-1): 3393 (NH),
3071 (Ar–H), 2987 (CH3), 2848 (CH2), 2242 (C:N), 1622
This compound was prepared by the reaction of 2 and 1-(4-
nitrophenyl)piperazine. It was obtained as a pale yellow
solid, mp 126–129 °C, yield 48 %. IR (KBr), m (cm-1): 3411
(NH), 3079 (Ar–H), 2886 (CH2, aliphatic), 2228 (C:N),
1610 (C=N), 1521, 1372 (NO2). 1H NMR (CDCl3,
400 MHz) d: 2.68 (t, 4H, NCH2, piperazin), 3.44 (t, 4H,
NCH2, piperazin), 6.81–6.83 (d, J = 7.2 Hz, 2H, ArH),
7.01–7.03 (m, 1H, ArH), 7.25–7.27 (m, 1H, ArH), 7.33–7.35
(m, 1H, ArH), 7.82–7.85 (m, 1H, ArH), 8.09–8.11 (d,
J = 7.2 Hz, 2H, ArH), 9.13 (s, 1H, NH). 13C NMR (CDCl3,
100 MHz) d: 48.8 (2C, N–C, piperazin), 54.6 (2C, N–C,
piperazin), 83.7 (C–I), 116.1 (C:N), 116.9 (2C), 123.4,
127.2 (2C), 128.4, 130.4, 138.8 (aromatic carbons), 140.5
(C–NO2, nitrophenyl), 158.2 (C–N, nitrophenyl), 158.5 (C–
N, iodophenyl), 160.9 (C=N). MS: m/z [M?1]? 477.
1
(C=N). H NMR (CDCl3, 400 MHz) d: 1.34 (t, 6H, CH3),
3.32 (q, 4H, NCH2), 6.97–7.00 (m, 1H, ArH), 7.22–7.25
(m, 1H, ArH), 7.33–7.36 (m, 1H, ArH), 7.84–7.86 (m, 1H,
ArH), 9.16 (s, 1H, NH). 13C NMR (CDCl3, 100 MHz) d:
13.4 (2C, CH3), 43.0 (2C, N–C), 83.2 (C–I), 116.3 (C:N),
123.4, 128.8, 131.0, 139.2 (aromatic carbons), 159.1 (C–N,
iodophenyl), 162.3 (C=N). MS: m/z [M?1]? 343.
1,1-Diisopropyl-2-(2-iodophenyl)-3-cyanoguanidine (3m)
This compound was prepared by the reaction of 2 and
diisopropylamine. It was obtained as a pale yellow solid,
mp 71–73 °C, yield 58 %. IR (KBr), m (cm-1): 3426 (NH),
3063 (Ar–H), 2953 (CH3), 2924 (CH), 2239 (C:N), 1617
(C=N), 1386 (CH3). 1H NMR (CDCl3, 400 MHz) d:
1.40–1.41 (12H, d, J = 6.8 Hz, CH3), 3.11 (m, 2H, CH),
6.98–7.00 (m, 1H, ArH), 7.23–7.25 (m, 1H, ArH),
7.30–7.34 (m, 1H, ArH), 7.82–7.85 (m, 1H, ArH), 9.07 (s,
1H, NH). 13C NMR (CDCl3, 100 MHz) d: 22.1 (4C, CH3),
43.3 (2C, N–C), 83.3 (C–I), 116.2 (C:N), 123.5, 128.6,
131.0, 139.0 (aromatic carbons), 158.9 (C–N, iodophenyl),
160.5 (C=N). MS: m/z [M?1]? 371.
N0-(2-Iodophenyl)-N-cyano-3-(trifluoromethyl)-5,6-
dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-
carboximidamide (3j)
This compound was prepared by the reaction of 2 and 3-(tri-
fluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyra-
zine. It was obtained as a pale yellow solid, mp 173–175 °C,
yield 43 %. IR (KBr), m (cm-1): 3369 (NH), 3060 (Ar–H),
2869 (CH2, aliphatic), 2235 (C:N), 1601 (C=N). 1H NMR
(CDCl3, 400 MHz) d: 4.32 (s, 2H, NCH2, piperazin), 4.44 (t,
2H, NCH2, piperazin), 5.36 (t, 2H, NCH2, piperazin),
7.00–7.03 (m, 1H, ArH), 7.24–7.26 (m, 1H, ArH), 7.33–7.35
1,1-Dibenzyl-2-(2-iodophenyl)-3-cyanoguanidine (3n)
(m, 1H, ArH), 7.80–7.83 (m, 1H, ArH), 9.08 (s, 1H, NH). 13
C
This compound was prepared by the reaction of 2 and
dibenzylamine. It was obtained as a pale yellow solid, mp
103–105 °C, yield 46 %. IR (KBr), m (cm-1): 3395 (NH),
NMR (CDCl3, 100 MHz) d: 29.0 (N–C, piperazin), 42.7 (N–
C, piperazin), 43.0 (N–C, piperazin), 83.6 (C–I), 116.0
(C:N), 119.9 (C–F), 123.5, 128.6, 130.5, 139.0 (aromatic
carbons), 143.3 (C=N, triazolo), 153.0 (C=N, triazolo), 158.6
(C–N, iodophenyl), 160.8 (C=N). MS: m/z [M?1]? 462.
1
3078 (Ar–H), 2936 (CH2), 2223 (C:N), 1583 (C=N). H
NMR (CDCl3, 400 MHz) d: 4.18 (s, 4H, CH2), 6.97–7.00
(m, 1H, ArH), 7.22–7.27 (m, 5H, ArH), 7.31–7.38 (m, 7H,
ArH), 7.84–7.86 (m, 1H, ArH), 9.19 (s, 1H, NH). 13C NMR
(CDCl3, 100 MHz) d: 50.5 (2C, CH2), 83.4 (C–I), 116.2
(C:N), 123.3, 126.9 (2C), 128.3 (4C), 128.6, 128.8 (4C),
131.1, 138.4 (2C), 139.3 (aromatic carbons), 158.9 (C–N,
iodophenyl), 161.7 (C=N). MS: m/z [M?1]? 467.
1,1-Dimethyl-2-(2-iodophenyl)-3-cyanoguanidine (3k)
This compound was prepared by the reaction of 2 and
dimethylamine. It was obtained as a pale yellow solid, mp
123

Med Chem Res
1,1-Diphenyl-2-(2-iodophenyl)-3-cyanoguanidine (3o)
piperidin), 3.53–3.59 (m, 4H, NCH2, piperidin), 7.16–7.20
(m, 1H, ArH), 7.25–7.28 (m, 1H, ArH), 7.40–7.44 (m, 2H,
ArH). 13C NMR (DMSO-d6, 100 MHz) d: 23.9 (piperidin),
25.1 (2C, piperidin), 49.8 (2C, N–C, pyrrolidin), 105.6
(C:N), 109.8, 117.9, 122.4, 125.9 (aromatic carbons),
132.6 (C-5, imidazol), 141.4 (C-4, imidazol), 154.8 (C-2,
imidazol). MS: m/z [M?1]? 227. Anal. Calcd. for
C13H14N4 (%): C, 69.00; H, 6.24; N, 24.76. Found: C,
68.79; H, 6.21; N, 24.70.
This compound was prepared by the reaction of 2 and
diphenylamine. It was obtained as a pale yellow solid, mp
121–123 °C, yield 48 %. IR (KBr), m (cm-1): 3376 (NH),
3073 (Ar–H), 2246 (C:N), 1620 (C=N). 1H NMR
(CDCl3, 400 MHz) d: 6.97–7.00 (m, 1H, ArH), 7.03–7.07
(m, 2H, ArH), 7.15–7.26 (m, 5H, ArH), 7.31–7.39 (m, 5H,
ArH), 7.82–7.85 (m, 1H, ArH), 9.22 (s, 1H, NH). 13C NMR
(CDCl3, 100 MHz) d: 83.2 (C–I), 116.5 (C:N), 119.2
(2C), 120.2 (4C), 123.7, 128.3, 130.0 (4C), 131.0, 139.6
(aromatic carbons), 144.6 (2C, C–N, phenyl), 159.2 (C–N,
iodophenyl), 161.4 (C=N). MS: m/z [M?1]? 439.
2-Morpholino-1H-benzo[d]imidazole-1-carbonitrile (4c)
This compound was prepared by the intramolecular C–N
coupling reaction of 3c. It was obtained as a white solid;
mp 83–85 °C. IR (KBr), m (cm-1): 3088 (Ar–H), 2883
(CH2, aliphatic), 2239 (C:N), 1621 (C=N). 1H NMR
(CDCl3, 400 MHz) d: 3.75 (t, 4H, NCH2, morpholino),
3.91 (t, 4H, OCH2, morpholino), 7.01–7.05 (m, 1H, ArH),
7.13–7.18, (m, 1H, ArH), 7.27–7.31 (m, 2H, ArH). 13C
NMR (CDCl3, 100 MHz) d: 44.3 (2C, N–C, morpholino),
65.6 (2C, O–C, morpholino), 105.2 (C:N), 108.6, 117.1,
121.4, 125.8 (aromatic carbons), 132.9 (C-5, imidazol),
143.1 (C-4, imidazol), 152.3 (C-2, imidazol). MS: m/z
[M?1]? 229. Anal. Calcd. for C12H12N4O (%): C, 63.15;
H, 5.30; N, 24.55. Found: C, 63.32; H, 5.41; N, 24.47.
General procedure for the synthesis of 1-cyano-2-amino-
benzimidazole derivatives (4a–o)
A
mixture of aryl guanidine 3 (0.5 mmol), CuI
(0.025 mmol), 2,20-biimidazole (0.05 mmol), Cs2CO3
(1.0 mmol) and DMF (5 mL) was heated at 80 °C for 10 h
under nitrogen atmosphere. The resulting suspension was
cooled to room temperature, diluted with ethyl acetate,
filtered through a pad of silica gel, and washed with ethyl
acetate. The combined filtrate was concentrated in vacuo,
and the resulting residue was purified by column chroma-
tography on silica gel with cyclohexane/ethyl acetate to
provide the desired product.
2-(4-Methylpiperazin-1-yl)-1H-benzo[d]imidazole-1-
carbonitrile (4d)
2-(Pyrrolidin-1-yl)-1H-benzo[d]imidazole-1-carbonitrile
(4a)
This compound was prepared by the intramolecular C–N
coupling reaction of 3d. It was obtained as a white solid;
mp 78–81 °C. IR (KBr), m (cm-1): 3091 (Ar–H), 2932
(CH3), 2856 (CH2, aliphatic), 2234 (C:N), 1611 (C=N).
1H NMR (CDCl3, 400 MHz) d: 2.23 (s, 3H, NCH3), 2.47 (t,
4H, NCH2, piperazin), 3.92 (t, 4H, NCH2, piperazin),
7.00–7.06 (m, 1H, ArH), 7.15–7.20 (m, 1H, ArH),
7.28–7.34 (m, 2H, ArH). 13C NMR (CDCl3, 100 MHz) d:
42.3 (N–CH3), 45.9 (2C, N–C, piperazin), 54.1 (2C, N–C,
piperazin), 105.4 (C:N), 108.9, 117.2, 121.5, 126.0
(aromatic carbons), 133.2 (C-5, imidazol), 143.3 (C-4,
imidazol), 152.6 (C-2, imidazol). MS: m/z [M?1]? 242.
Anal. Calcd. for C13H15N5 (%): C, 64.71; H, 6.27; N,
29.02. Found: C, 64.57; H, 6.20; N, 28.91.
This compound was prepared by the intramolecular C–N
coupling reaction of 3a. It was obtained as a white solid;
mp 59–60 °C. IR (KBr), m (cm-1): 3081 (Ar–H), 2886
(CH2, aliphatic), 2235 (C:N), 1612 (C=N). 1H NMR
(CDCl3, 400 MHz) d: 2.00 (t, 4H, CH2, pyrrolidin), 3.75 (t,
4H, NCH2, pyrrolidin), 7.03–7.07 (m, 1H, ArH), 7.16–7.22
(m, 1H, ArH), 7.26–7.32 (m, 2H, ArH). 13C NMR (CDCl3,
100 MHz) d: 25.6 (2C, pyrrolidin), 49.0 (2C, N–C, pyrr-
olidin), 105.7 (C:N), 109.4, 117.0, 121.2, 125.4 (aromatic
carbons), 132.4 (C-5, imidazol), 142.2 (C-4, imidazol),
151.5 (C-2, imidazol). MS: m/z [M?1]? 213. Anal. Calcd.
for C12H12N4 (%): C, 67.90; H, 5.70; N, 26.40. Found: C,
67.79; H, 5.81; N, 26.26.
2-(Piperidin-1-yl)-1H-benzo[d]imidazole-1-carbonitrile
2-(4-Phenylpiperazin-1-yl)-1H-benzo[d]imidazole-1-
(4b)
carbonitrile (4e)
This compound was prepared by the intramolecular C–N
coupling reaction of 3b. It was obtained as a white solid;
mp 68–69 °C. IR (KBr), m (cm-1): 3093 (Ar–H), 2891
(CH2, aliphatic), 2241 (C:N), 1618 (C=N). 1H NMR
(DMSO-d6, 400 MHz) d: 1.61–1.67 (m, 6H, CH2
This compound was prepared by the intramolecular C–N
coupling reaction of 3e. It was obtained as a white solid;
mp 128–130 °C. IR (KBr), m (cm-1): 3057 (Ar–H), 2879
(CH2, aliphatic), 2236 (C:N), 1603 (C=N). 1H NMR
(CDCl3, 400 MHz) d: 3.31 (t, 4H, NCH2, piperazin), 3.97
123

Med Chem Res
2-(4-(4-Chlorophenyl)piperazin-1-yl)-1H-
(t, 4H, NCH2, piperazin), 6.90–6.93 (m, 2H, ArH),
6.99–7.05 (m, 2H, ArH), 7.14–7.18 (m, 1H, ArH),
7.23–7.25 (m, 2H, ArH), 7.37–7.39 (m, 2H, ArH). 13C
NMR (CDCl3, 100 MHz) d: 49.1 (2C, N–C, piperazin),
54.9 (2C, N–C, piperazin), 105.6 (C:N), 109.2, 116.6
(2C), 117.3, 120.8, 122.1, 126.5 (aromatic carbons), 130.6
(2C), 134.4 (C-5, imidazol), 142.8 (C-4, imidazol), 153.3
(C-2, imidazol), 155.1 (N–C, phenyl). MS: m/z [M?1]?
304. Anal. Calcd. for C18H17N5 (%): C, 71.27; H, 5.65; N,
23.09. Found: C, 71.31; H, 5.57; N, 23.01.
benzo[d]imidazole-1-carbonitrile (4h)
This compound was prepared by the intramolecular C–N
coupling reaction of 3h. It was obtained as a white solid;
mp 141–143 °C. IR (KBr), m (cm-1): 3069 (Ar–H), 2870
(CH2, aliphatic), 2238 (C:N), 1605 (C=N). 1H NMR
(CDCl3, 400 MHz) d: 2.73 (t, 4H, NCH2, piperazin), 3.44
(t, 4H, NCH2, piperazin), 6.84–6.86 (d, J = 7.2 Hz, 2H,
ArH), 6.97–7.03 (m, 1H, ArH), 7.15–7.20 (m, 1H, ArH),
7.23–7.30 (m, 2H, ArH), 7.35–7.39 (d, J = 7.2 Hz, 2H,
ArH). 13C NMR (CDCl3, 100 MHz) d: 48.5 (2C, N–C,
piperazin), 54.2 (2C, N–C, piperazin), 105.3 (C:N),
109.0, 116.7 (2C), 117.5, 122.0, 123.6, 126.4, 127.5 (2C,
aromatic carbons), 134.4 (C-5, imidazol), 142.1 (C-4,
imidazol), 153.3 (C-2, imidazol), 157.6 (C–N, 4-chloro-
phenyl). MS: m/z [M?1]? 338. Anal. Calcd. for
C18H16ClN5 (%): C, 64.00; H, 4.77; N, 20.73. Found: C,
64.23; H, 4.81; N, 20.65.
2-(4-p-tolylpiperazin-1-yl)-1H-benzo[d]imidazole-1-
carbonitrile (4f)
This compound was prepared by the intramolecular C–N
coupling reaction of 3f. It was obtained as a white solid;
mp 115–117 °C. IR (KBr), m (cm-1): 3083 (Ar–H), 2962
(CH3), 2885 (CH2, aliphatic), 2240 (C:N), 1608 (C=N).
1H NMR (CDCl3, 400 MHz) d: 2.18 (s, 3H, CH3), 3.30 (t,
4H, NCH2, piperazin), 3.89 (t, 4H, NCH2, piperazin),
6.83–6.85 (d, J = 7.2 Hz, 2H, ArH), 6.85–6.89 (d,
J = 7.2 Hz, 2H, ArH), 7.01–7.05 (m, 1H, ArH), 7.18–7.21
(m, 1H, ArH), 7.28–7.36 (m, 2H, ArH). 13C NMR (CDCl3,
100 MHz) d: 24.2 (CH3), 47.5 (2C, N–C, piperazin), 53.8
(2C, N–C, piperazin), 105.5 (C:N), 109.2, 115.4 (2C),
117.3, 120.8, 122.1, 128.1, 130.2 (2C, aromatic carbons),
134.2 (C-5, imidazol), 142.4 (C-4, imidazol), 153.6 (C-2,
imidazol), 154.1 (N–C, 1-p-tolyl). MS: m/z [M?1]? 318.
Anal. Calcd. for C19H19N5 (%): C, 71.90; H, 6.03; N,
22.07. Found: C, 71.78; H, 6.09; N, 21.98.
2-(4-(4-Nitrophenyl)piperazin-1-yl)-1H-
benzo[d]imidazole-1-carbonitrile (4i)
This compound was prepared by the intramolecular C–N
coupling reaction of 3i. It was obtained as a white solid; mp
147–149 °C. IR (KBr), m (cm-1): 3084 (Ar–H), 2877 (CH2,
aliphatic), 2239 (C:N), 1616 (C=N), 1530, 1366 (NO2).
1H NMR (CDCl3, 400 MHz) d: 3.35 (t, 4H, NCH2, pip-
erazin), 3.98 (t, 4H, NCH2, piperazin), 6.83–6.87 (d,
J = 7.2 Hz, 2H, ArH), 6.98–7.05 (m, 1H, ArH), 7.16–7.20
(m, 1H, ArH), 7.25–7.32 (m, 2H, ArH), 8.08–8.11 (d,
J = 7.2 Hz, 2H, ArH). 13C NMR (CDCl3, 100 MHz) d:
49.0 (2C, N–C, piperazin), 54.7 (2C, N–C, piperazin),
105.7 (C:N), 109.1, 117.0 (2C), 117.3, 122.3, 126.2,
127.4 (2C, aromatic carbons), 134.2 (C-5, imidazol), 140.8
(C-NO2, nitrophenyl), 142.6 (C-4, imidazol), 153.5 (C-2,
imidazol), 158.4 (C–N, nitrophenyl). MS: m/z [M?1]?
349. Anal. Calcd. for C18H16N6O2 (%): C, 62.06; H, 4.63;
N, 24.12. Found: C, 62.31; H, 4.58; N, 24.01.
2-(4-(4-Methoxyphenyl)piperazin-1-yl)-1H-
benzo[d]imidazole-1-carbonitrile (4g)
This compound was prepared by the intramolecular C–N
coupling reaction of 3g. It was obtained as a white solid;
mp 152–154 °C. IR (KBr), m (cm-1): 3103 (Ar–H), 2954
(CH3), 2885 (CH2, aliphatic), 2241 (C:N), 1594 (C=N).
1H NMR (CDCl3, 400 MHz) d: 3.32 (t, 4H, NCH2, pip-
erazin), 3.76 (s, 3H, OCH3), 3.95 (t, 4H, NCH2, piperazin),
6.81–6.84 (d, J = 7.2 Hz, 2H, ArH), 6.87–6.91 (d,
J = 7.2 Hz, 2H, ArH), 6.99–7.06 (m, 1H, ArH), 7.15–7.19
(m, 1H, ArH), 7.26–7.33 (m, 2H, ArH). 13C NMR (CDCl3,
100 MHz) d: 48.3 (2C, N–C, piperazin), 54.6 (2C, N–C,
piperazin), 57.2 (OCH3), 105.6 (C:N), 108.8, 116.4 (2C),
117.2, 118.5 (2C), 121.3, 126.2 (aromatic carbons), 134.2
(C-5, imidazol), 142.5 (C-4, imidazol), 153.1 (C-2, imi-
dazol), 154.8 (C–N, methoxyphenyl), 156.3 (C–O,
methoxyphenyl). MS: m/z [M?1]? 334. Anal. Calcd. for
C19H19N5O (%): C, 68.45; H, 5.74; N, 21.01. Found: C,
68.27; H, 5.83; N, 21.11.
2-(3-(Trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-
a]pyrazin-7(8H)-yl)-1H-benzo[d]imidazole-1-carbonitrile
(4j)
This compound was prepared by the intramolecular C–N
coupling reaction of 3j. It was obtained as a white solid;
mp 216–219 °C. IR (KBr), m (cm-1): 3062 (Ar–H), 2865
(CH2, aliphatic), 2225 (C:N), 1610 (C=N). 1H NMR
(CDCl3, 400 MHz) d: 4.34 (s, 2H, NCH2, piperazin), 4.45
(t, 2H, NCH2, piperazin), 5.31 (t, 2H, NCH2, piperazin),
7.00–7.08 (m, 1H, ArH), 7.17–7.21 (m, 1H, ArH),
7.30–7.36 (m, 2H, ArH). 13C NMR (CDCl3, 100 MHz) d:
29.1 (N–C, piperazin), 42.9 (N–C, piperazin), 43.2 (N–C,
123

Med Chem Res
piperazin), 105.7 (C:N), 109.3, 117.1, 119.9 (C–F),
121.3, 125.8 (aromatic carbons), 133.0 (C-5, imidazol),
142.7 (C-4, imidazol), 143.4 (C=N, triazolo), 151.6 (C-2,
imidazol), 153.1 (C=N, triazolo). MS: m/z [M?1]? 334.
Anal. Calcd. for C14H10F3N7 (%): C, 50.45; H, 3.02; N,
29.42. Found: C, 50.62; H, 3.06; N, 29.33.
for C14H18N4 (%): C, 69.39; H, 7.49; N, 23.12. Found: C,
69.23; H, 7.53; N, 23.06.
2-(Dibenzylamino)-1H-benzo[d]imidazole-1-carbonitrile
(4n)
This compound was prepared by the intramolecular C–N
coupling reaction of 3n. It was obtained as a white solid;
mp 113–115 °C. IR (KBr), m (cm-1): 3062 (Ar–H), 2924
(CH2), 2241 (C:N), 1596 (C=N). 1H NMR (CDCl3,
400 MHz) d: 4.64 (s, 4H, CH2), 7.05–7.08 (m, 1H),
7.14–7.19 (m, 1H, ArH), 7.23–7.32 (m, 4H, ArH),
7.34–7.37 (m, 8H, ArH). 13C NMR (CDCl3, 100 MHz) d:
50.5 (2C, CH2), 105.2 (C:N), 108.7, 117.0, 121.1, 125.6,
126.6 (2C), 128.1 (4C), 128.5 (4C, aromatic carbons),
132.7 (C-5, imidazol), 138.1 (2C), 142.5 (C-4, imidazol),
151.7 (C-2, imidazol). MS: m/z [M?1]? 339. Anal. Calcd.
for C22H18N4 (%): C, 78.08; H, 5.36; N, 16.56. Found: C,
78.31; H, 5.29; N, 16.35.
2-(Dimethylamino)-1H-benzo[d]imidazole-1-carbonitrile
(4k)
This compound was prepared by the intramolecular C–N
coupling reaction of 3k. It was obtained as a white solid;
mp 86–88 °C. IR (KBr), m (cm-1): 3089 (Ar–H), 2978
1
(CH3), 2837 (CH2), 2236 (C:N), 1605 (C=N). H NMR
(CDCl3, 400 MHz) d: 2.53 (s, 6H, NCH3), 7.05–7.08 (m,
1H, ArH), 7.21–7.25 (m, 1H, ArH), 7.29–7.35 (m, 2H,
ArH). 13C NMR (CDCl3, 100 MHz) d: 35.1 (2C, N–CH3),
106.0 (C:N), 109.1, 117.4, 121.5, 125.7 (aromatic car-
bons), 132.6 (C-5, imidazol), 143.1 (C-4, imidazol), 152.1
(C-2, imidazol). MS: m/z [M?1]? 187. Anal. Calcd. for
C10H10N4 (%): C, 64.50; H, 5.41; N, 30.09. Found: C,
64.43; H, 5.45; N, 29.95.
2-(Diphenylamino)-1H-benzo[d]imidazole-1-carbonitrile
(4o)
2-(Diethylamino)-1H-benzo[d]imidazole-1-carbonitrile
This compound was prepared by the intramolecular C–N
coupling reaction of 3o. It was obtained as a white solid;
mp 156–159 °C. IR (KBr), m (cm-1): 3061 (Ar–H), 2240
(C:N), 1611 (C=N). 1H NMR (CDCl3, 400 MHz) d:
7.00–7.06 (m, 3H, ArH), 7.13–7.23 (m, 5H, ArH),
7.29–7.38 (m, 6H, ArH). 13C NMR (CDCl3, 100 MHz) d:
105.7 (C:N), 109.4, 117.0, 117.6 (2C), 119.3 (4C), 121.2,
125.4, 130.5 (4C, aromatic carbons), 132.4 (C-5, imidazol),
142.2 (C-4, imidazol), 146.1 (2C, C–N, phenyl), 151.5 (C-
2, imidazol). MS: m/z [M?1]? 311. Anal. Calcd. for
C20H14N4 (%): C, 77.40; H, 4.55; N, 18.05. Found: C,
77.52; H, 4.48; N, 17.89.
(4l)
This compound was prepared by the intramolecular C–N
coupling reaction of 3l. It was obtained as a white solid; mp
72–74 °C. IR (KBr), m (cm-1): 3075 (Ar–H), 2981 (CH3),
2845 (CH2), 2232 (C:N), 1602 (C=N). 1H NMR (CDCl3,
400 MHz) d: 1.32 (t, 6H, CH3), 3.76 (q, 4H, NCH2),
7.03–7.06 (m, 1H, ArH), 7.19–7.25 (m, 1H, ArH),
7.27–7.34 (m, 2H, ArH). 13C NMR (CDCl3, 100 MHz) d:
13.7 (2C, CH3), 43.3 (2C, N–C), 105.6 (C:N), 109.2,
117.2, 121.3, 125.8 (aromatic carbons), 132.8 (C-5, imi-
dazol), 143.0 (C-4, imidazol), 151.9 (C-2, imidazol). MS:
m/z [M?1]? 215. Anal. Calcd. for C12H14N4 (%): C, 67.27;
H, 6.59; N, 26.15. Found: C, 67.34; H, 6.51; N, 26.02.
Cytotoxic assay
The tumor cell lines panel consisted of A549, K562, and
PC-3. The tumor cells were cultured in proper medium in a
5 % CO2 atmosphere at 37 °C. The cells were seeded to
96-well plates at a density of 5 9 103 cells/well. After
24 h, the cells were treated with 0.01, 0.1, 1, 10, and
100 lmol L-1 chemicals dissolved in DMSO (final con-
centration 0.1 %) and the positive control cells were trea-
ted with doxorubicin hydrochloride. 72 h later, 20 lL of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bro-
mide (MTT, 5 mg mL-1) solution was added per well and
the cells were cultured for another 4 h. Following this, the
medium was discarded, and DMSO was added. The optical
density (OD) of the resultant solution was measured at a
wavelength of 570 nm with a microplate reader. Doxoru-
bicin hydrochloride was used as a control antitumor agent.
2-(Diisopropylamino)-1H-benzo[d]imidazole-1-
carbonitrile (4m)
This compound was prepared by the intramolecular C–N
coupling reaction of 3m. It was obtained as a white solid;
mp 69–71 °C. IR (KBr), m (cm-1): 3059 (Ar–H), 2968
(CH3), 2921 (CH), 2235 (C:N), 1606 (C=N), 1382 (CH3).
1H NMR (CDCl3, 400 MHz) d: 1.42–1.44 (12H, d,
J = 6.8 Hz, CH3), 3.12 (m, 2H, CH), 7.00–7.05 (m, 1H,
ArH), 7.20–7.26 (m, 1H, ArH), 7.28–7.36 (m, 2H, ArH).
13C NMR (CDCl3, 100 MHz) d: 22.4 (4C, CH3), 48.7 (2C,
N–C), 105.3 (C:N), 109.7, 117.6, 122.3, 125.6 (aromatic
carbons), 132.7 (C-5, imidazol), 142.2 (C-4, imidazol),
152.4 (C-2, imidazol). MS: m/z [M?1]? 243. Anal. Calcd.
123

Med Chem Res
benzimidazo[1,2-a] quinolines and fluorenes: Synthesis, antitu-
mor evaluation in vitro and crystal structure determination. Eur J
Med Chem 45:2405–2417
The IC50 values were calculated with LOGIT method.
Assays were performed in triplicate on three independent
experiments (Hu et al., 2009).
Hu Z, Ma T, Chen Z, Ye Z, Zhang G, Lou Y, Yu Y (2009) Solid-
phase synthesis and antitumor evaluation of 2,4-diamino-6-aryl-
1,3,5-triazines. J Comb Chem 11:267–273
Cell cycle analysis
Hu Z, Li S, Hong P, Wu Z (2011) Copper (I)-catalyzed intramolecular
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voc 2011:147–155
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Design, synthesis, and evaluation of 5-sulfamoyl benzimidazole
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Kohara Y, Kubo K, Imamiya E, Wada T, Inada Y, Naka T (1996)
Synthesis and angiotensin II receptor antagonistic activities of
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K562 cells were seeded into 6-well plates and treated with
different concentrations of 4d or without any addition for
24 h. The cells were harvested, washed with PBS, and
fixed overnight in 70 % ethanol at -20 °C. The fixed cells
were treated with 100 lg mL-1 Rnase A in PBS for 1 h at
room temperature and followed by staining with
50 lg mL-1 propidium iodide for 30 min in the dark.
DNA content was analyzed by flow cytometry.
Ku¨hler TC, Swanson M, Shcherbuchin V, Larsson H, Mellgard B,
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Acknowledgments This work was supported by the Program for
Excellent Talents in Guangdong Ocean University (Grant No.
0912118) and the Natural Science Foundation of Guangdong Ocean
University (Grant No. 1012043).
Kus C, Ayhan-Kilcigil G, Tuncbilek M, Altanlar N, Coban T, Can-
Eke B, Iscan M (2009) Synthesis, antimicrobial and antioxidant
activities of some benzimidazole derivatives. Lett Drug Des
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