Synthesis and evaluation of 2-[2-(phenylthiomethyl)-1H-benzo[d] imidazol-1-yl)acetohydrazide derivatives as antitumor agents

Article abstract of DOI:10.1016/j.bmcl.2012.03.061

A novel class of acetylhydrazone derivatives (5a-x) containing 2-(phenylthiomethyl)-1H-benzo-[d]-imidazole moieties are synthesizer, and their antitumor activities against A549, HCT116, HepG2, PC-9, and A375 were determined by the MTT assay. Among them ar

Full text of DOI:10.1016/j.bmcl.2012.03.061

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3122–3125
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of 2-[2-(phenylthiomethyl)-1H-benzo[d]
imidazol-1-yl)acetohydrazide derivatives as antitumor agents
Ting Liu a, , Changyan Sun b, , Xiumei Xing a, Lingling Jing a, Rui Tan a, Youfu Luo b, Wencai Huang a,
b,
Hang Song a, Zicheng Li a, , Yinglan Zhao
⇑
⇑
a Department of Pharmaceutical and Bioengineering, School of Chemical Engineering, Sichuan University, Chengdu, Sichuan 610065, PR China
b State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan 610041, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of acetylhydrazone derivatives (5a–x) containing 2-(phenylthiomethyl)-1H-benzo-[d]-
imidazole moieties are synthesizer, and their antitumor activities against A549, HCT116, HepG2,
PC-9, and A375 were determined by the MTT assay. Among them are N-(2,4-dihydroxybenzylidene)-
2-(2-(phenylthiomethyl)-1H-benzo[d]-imidazol-1-yl)acetohydrazide (5a) and N-(5-bromo-2-hydroxy-
benzylidene)-2-(2-(phenylthiomethyl)-1H-benzo[d]-imidazol-1-yl)acetohydrazide (5d) which displayed
Received 21 November 2011
Revised 5 March 2012
Accepted 15 March 2012
Available online 21 March 2012
excellent cancer inhibitory activity against the tested cancer cells (IC50 4–17 lM), compared with 5-FU
and SU11248. The others have moderate to weak inhibitory activity against the tested cancer cell lines.
Keywords:
Acetohydrazide derivatives
2-(Phenylthiomethyl)-1H-benzo[d]-imidazole
Antitumor activity assay
Ó 2012 Elsevier Ltd. All rights reserved.
The benzimidazole unit is the key building block for a variety of
compounds, which have crucial roles in the functions of biologically
important molecules, its derivatives have a wide range of well
known biologic activities, such as anticancer,1–3 antimicrobial,4,5
proton pump inhibitor,6 antiviral,7,8 etc. Several bibenzimidazole
and terbenzimidazole derivativies have recently been identified
as topoisomerase II poisons (Hoechst-33258 and Hoechst-
33342)9,10 (Fig. 1). It was found that the topoisomerase II was
The hydrazone compounds have unique biologic activity and
strong coordination ability, and they were widely used in the field
of sterilization, antibacterial, anti-tuberculosis and anti-tumor.
They are also hot topics in the field of pharmaceutical research.
Galal et al.14,15 synthesizer novel benzimidazole-5-carboxylic acid
and hydrazide derivatives and complexes with transition metal
as topo II inhibitors, among them, two compounds inhibited topo
II activity at 10 times lower concentration than etoposide in relax-
ation assay.
significantly inhibited by compound 1 with IC50 of 17.0
l
M,11
M).12 Abdel-Mohsen
which is higher than etoposide (IC50 is 21.8
l
In this Letter, taking into account of the wide bioactivities of
benzimidazole derivatives and hydrazones, and basing on analyz-
ing of the structure of compound 1, the hydrazone group was
et al.13 reported novel benzimidazole–pyrimidine conjugates with
marked potency against twelve cancer cell lines.
OR
N+
Cl-
HOOC
N
N
H
N
N
H
NH
N
N
N
Hoechst 33342 R=Et
Hoechst 33258 R=H
Compound 1
Figure 1. Compounds with good antitumor activity.
⇑
Corresponding authors. Tel./fax: +86 28 85405221.
E-mail addresses: sculzc@scu.edu.cn (Z. Li), alancenxb@sina.com (Y. Zhao).
These authors contributed equally to this Letter.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.061

T. Liu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3122–3125
3123
H
N
Cl
(a)
(b)
S
N
N
S
N
N
N
H
2
CH2COOC(CH3)3
1
3
(c)
S
S
N
N
N
N
(d)
CH2CONHN CH Ar
5a-x
CH2CONHNH2
4
Cl
OH
Br
Ar
OH
OH
Br
Cl
OMe
OH
F
Br
5g
Cl
5h
OMe
OH
5d
5f
5e
5b
5c
5a
N
OMe
OMe
OH
N
H
NO2
F
CH3
OMe
5p
5x
5j
5o
5m
5n
5k
5l
OMe
5i
CN
N
OCH3
NO2
5r
CH2CO2Bu-t
F
5s
CF3
5u
OMe
5t
5q
5v
5w
Scheme 1. Synthetic pathway of benzimidazole derivatives (5a–x). Reagents and conditions: (a) K2CO3 and benzenethiol, stirred for 0.5 h at rt; then compound 1 in DMF was
added, and stirred for 2 h at rt; (b) K2CO3, tert-butyl 2-bromopropanoate, stirred for 6 h at rt; (c) EtOH, NH2NH2 H2O; (d) Ar-CHO, reflux for 2 h in ethanol.
Table 1
Physical data and proliferation inhibitory effect of compounds 5a–x against tumor cells in vitro
a,b
Compound
mp,(°C)
Yield (%)
Z/E
IC50
A549
HCT116
HepG2
PC-9
A375
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
5x
272–274
157–160
222–224
194–196
267–270
185–189
182
86
77
84
88
86
76
74
78
79
77
75
72
76
75
73
77
78
75
76
73
78
77
75
76
42/58
25/75
29/71
29/71
27/73
19/81
23/77
20/80
20/80
23/77
22/78
22/78
23/77
20/40
28/72
25/75
26/74
20/80
20/80
20/80
34/66
20/80
19/81
22/78
12.36
45.25
58.32
10.75
53.1
41.23
34.98
36.07
64
49.06
51.51
31.89
38.51
46.94
42.42
50.03
51.35
62.16
38.11
54.41
31.22
41.92
46.21
36.89
35.05
12.35
4.86
>20c
50.24
71.7
17.03
78.21
42.47
32.04
33.79
>80
14.63
30.52
39.19
12.2
42.03
38.9
5.75
43.46
>80
4.09
>80
37.15
26.81
21.41
5.44
48.03
76.22
8.33
63.9
40.92
31.12
31.17
>80
29.84
30.15
38.58
10.92
39.31
28.11
30.58
34.13
34.07
34.17
42.61
42.21
31.03
44
18.09
44.56
57.31
32.81
23.47
10.97
201–203
198
215–218
178–180
105–107
125–127
171–175
271–273
198–199
245–248
201–202
120–122
202
69.3
>80
>80
55.06
30.55
43.99
49.63
40.35
44.37
77.64
44.05
31.93
>80
10.05
40.78
62.02
35.62
31.72
18.35
59.93
31.55
38.2
50.85
47.31
52.36
>80
61.85
44.76
>80
29.55
56.54
70.01
48.7
53.51
21.24
42.12
40.96
45.37
58.04
76.14
77.56
>80
>80
190–192
136–138
215–218
160–162
18.05
52.11
>80
42.2
46.55
11.58
5-FU
SU11248
46.31
13.24
a
b
c
The IC50 values represent the concentration that causes 50% growth inhibition.
The IC50 values were the mean values of three repeated experiments.
The concentartion of compounds 5a and 5d ranges from 0.625 to 20 lM.
incorporated into the 2-mercapto-1H-benzimidazole, novel hydra-
zone derivatives with benzimidazole moieties have been designed
and synthesizer, and their biologic activity against five cancer cells
were studied by the MTT assay.

3124
T. Liu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3122–3125
Figure 2. Relative distribution of A375 cells after 24 h treatment with compounds 5a and 5d at various concentrations.
Figure 3. Statistic results of the effect of 5a/5d on G0/G1, S and G2/M phase in A375 cells. Data were obtained from three independent experiments, with triplicates in each
experiment.
In this Letter, taking into account of the wide bioactivities of
benzimidazole derivatives and hydrazones, and basing on analyzing
of the structure of compound 1, the hydrazone group was
incorporated into the 2-mercapto-1H-benzimidazole, novel hydra-
zone derivatives with benzimidazole moieties have been designed
and synthesizer, and their biologic activity against five cancer cells
were studied by the MTT assay.
The target compounds 5a–x were synthesizer via the general
synthetic route shown in Scheme 1. Compound 2 was prepared from
potassium benzenethiolate reacted with 2-(chloromethyl)-1H-ben-
zo[d]-imidazole (1), which was produced from o-phenylenediamine
and 2-chloroacetic acid by the procedures reported,17,18 Compound
3 was obtained by combining of 2 and tert-butyl 2-bromoacetate in
dry acetone in the presence of anhydrous K2CO313,14, which was
then treated with 85% hydrazine hydrate to afford 4; the target com-
pounds 5a–x were obtained by the condensation of compound 4
with corresponding phenylcarbaldehyde in the presence of glacial
acetic acid19,20, and then recrystallized from methanol. The struc-
tures of 5a–x were characterized by 1H NMR, IR, and ESI-HRMS anal-
ysis and all the analytical data were documented in Supplementary
data. The purity of all compounds was above 97.0%, which is deter-
mined by HPLC normalization method.
The characteristic data of the target compounds are presented
in Table 1. The ratio of Z/E isomers caused by imine (CH@NH) bond
can be calculated from splitting of hydrogen. According to the 1H
NMR spectra of compounds 5a–x, each group of NH, N@CH,
CH2C@O, SACH2, OH, OCH3, CH3, displaying two single peaks,
showed the cis–trans isomerism of the prepared compounds. The
ratio of Z/E of 5a–x ranged were from 1/4 to 1/3, and the E isomer
is main isomer, the ratio of Z/E for 5a reached 42:58. It seems that
the existence of hydroxy group may cause a higher Z/E value, and
the more the hydroxy groups has, the higher the value of Z/E is,
which could be due to the intramolecular hydrogen bond.9,16
The proliferation inhibitory effect of all target compounds
against cancer cells was determined by the MTT assay.21 The anti-
tumor screening results, presented in Table 1, revealed that most
target compounds were exhibited potent antitumor activity
against A549, HCT116, HepG2, PC-9 and A375 cancer cell lines.
Among them were 5a and 5d exhibiting excellent antitumor activ-
ity against HCT116 (IC50 = 4.86
lM and 8.33
lM, respectively) and
A375 (IC50 = 5.75 M and 4.09
l
lM, respectively), the result of
which is compared with 5-FU and SU11248. Compound 5i exhib-
ited enchanced antiproliferation activity against A375 cell with
IC50 of 5.44 lM, 5j showed moderate cancer inhibitory activity

T. Liu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3122–3125
3125
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against PC-9 with IC50 of 10.92
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l
M, and 5u showed moderate
M.
l
These results showed that compounds containing electron with-
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To examine the mechanism of compounds 5a and 5d responsible
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using flow cytometry22,23 .The data are shown in Figures 2 and 3.
As can be seen in Figure 2, exposure of A375 cells to growth
suppressive concentration of compounds 5a and 5d resulted in a
significant accumulation of cells in G0/G1 phases, which was
accompanied by a decrease in cells with S and G2/M DNA content.
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treatment with 5d at 40 lM for 24 h.
In summary, a novel class of acetylhydrazone derivatives with
benzimidazole moieties were synthesizer and tested for their
in vitro antitumor activity against five strains of cancer cell lines.
compounds 5a and 5d showed excellent cancer inhibitory activity
in vitro against the tested cancer cell lines (IC50 4–17 lM). Flow
cytometric analysis revealed that compound 5a and 5d induce sig-
nificant levels of apoptosis in A375 cells in vitro at low micromolar
concentrations. The hydroxyl at 2 (and 4) position on the phenyl
ring of hydrazone play a significant role in the antitumor activity.
This kind of benzimidazole derivatives may constitute a novel class
of antiproliferative agents, which deserve further study.
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Acknowledgments
We thank the Analytical and Testing Center of Sichuan University
for 1H NMR, IR and MS measurements.
20. Desai, K. G.; Desai, K. R. Bioorg. Med. Chem. 2006, 14, 8271.
21. Briefly, Cells (4–5 Â 103/well) were seeded in 96-well plates and cultured for
24 h, followed by various concentration of compounds treatment for 48 h.
Supplementary data
10
l
l of 10 mg/ml MTT was added per well and incubated for another 2.5 h at
Supplementary data (the list of experimental details and spec-
troscopic characterization of 5a–x, and their intermediates) associ-
ated with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmcl.2012.03.061.
37 °C, then the supernatant fluid was removed and DMSO was added 150
ll/
well for 15–20 min. The light absorptions (OD) were measured at 570 nm with
SpectraMAX M5 microplate spectrophotometer (Molecular Devices). The effect
of compounds on tumor cells viability was expressed by IC50 of each cell lines.
22. Jiang, P.-D.; Zhao, Y.-L.; Shi, W.; Deng, X.-Q.; Xie, G.; Mao, Y.-Q.; Li, Zh.-G.;
Zheng, Y.-Z. h.; Yang, Sh.-Y.; Wei, Y.-Q. Cell Physiol. Biochem. 2008, 22, 431.
23. A375 (1 Â 105cells/well) were seeded in 6-well plates and cultured for 24 h,
followed by treatment with 5a or 5d at various concentrations for 24 h. Then,
cells were harvested and fixed with 70% ethanol for 1 h. The fixed cells were
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