Synthesis and preliminary evaluation of selected 2-aryl-5(6)-nitro- 1H-benzimidazole derivatives as potential anticancer agents

Article abstract of DOI:10.1007/s12272-011-0201-5

In this study we report the synthesis and preliminary evaluation of a series of six 2-aryl-5(6)-nitro-1H-benzimidazole derivatives (1-6) as potential anticancer agents. Cytotoxicity was evaluated against seven human neoplastic cell lines using the MTT assay. Compound 6 [2-(4-chloro-3-nitrophenyl)-5(6)- nitro-1H-benzimidazole] was the most active of the series, showing an IC ₅₀ of 28 nM against the A549 cell line. This compound displayed a selective in vitro cytotoxic activity index (>700) in non neoplastic HACAT cells (IC₅₀ = 22.2 μM). Compounds 3 and 6 induce arrest in the S phase of the cell cycle, and compounds 1-6 induce apoptosis in the K562 cell line. Compound 6 induces poly (ADP-ribose) polymerase (PARP) inhibition activity as a potential mechanism of action. These results suggest that compound 6 could be a potent anticancer agent. Compound 3 displayed the best inhibitory activity against PARP with an IC₅₀ value of 0.05 μM, compared to the activity shown by the positive control 3-aminobenzamide (IC₅₀ = 28.5 μM).

Full text of DOI:10.1007/s12272-011-0201-5

Arch Pharm Res Vol 34, No 2, 181-189, 2011
DOI 10.1007/s12272-011-0201-5
Synthesis and Preliminary Evaluation of Selected 2-Aryl-5(6)-nitro-
1H-benzimidazole Derivatives as Potential Anticancer Agents
Aurelio Romero-Castro1, Ismael León-Rivera2, Laura Citlalli Ávila-Rojas3, Gabriel Navarrete-Vázquez1, and
Alejandro Nieto-Rodríguez1
2
1Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, 62209 México, Centro de
Investigaciones Químicas, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, 62209 México, and
3Facultad de Biología, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, 62209 México
(Received January 13, 2010/Revised July 23, 2010/Accepted July 27, 2010)
In this study we report the synthesis and preliminary evaluation of a series of six 2-aryl-5(6)-
nitro-1
H
-benzimidazole derivatives (
1
-
6
) as potential anticancer agents. Cytotoxicity was eval-
[2-(4-
-benzimidazole] was the most active of the series, showing
an IC50 of 28 nM against the A549 cell line. This compound displayed a selective in vitro cyto-
toxic activity index (>700) in non neoplastic HACAT cells (IC50 = 22.2 M). Compounds and
induce arrest in the S phase of the cell cycle, and compounds induce apoptosis in the
K562 cell line. Compound induces poly (ADP-ribose) polymerase (PARP) inhibition activity
as a potential mechanism of action. These results suggest that compound could be a potent
anticancer agent. Compound displayed the best inhibitory activity against PARP with an
M, compared to the activity shown by the positive control 3-aminobenza-
M).
uated against seven human neoplastic cell lines using the MTT assay. Compound
chloro-3-nitrophenyl)-5(6)-nitro-1
6
H
µ
3
6
1-6
6
6
3
IC50 value of 0.05
mide (IC50 = 28.5
µ
µ
Key words: Anticancer, Benzimidazole, Nitrocompounds, Cytotoxicity, PARP
ety of biologically important molecules with different
therapeutic properties, such as anti-parasitic, anti-
INTRODUCTION
Cancer is a leading cause of death worldwide and microbial, antiviral, anti-helminthic, anti-hyperten-
had accounted for 7.9 million deaths (approximately sive, analgesic, anti-inflammatory, anti-ulcer, anti-
13% of all deaths) in 2007. Lung, stomach, liver, colon allergic (Özden et al., 2005) and antitumor activity
and breast cancer cause the most cancer deaths each (Bielawski et al., 2002; Ghodousi et al., 2004). Studies
year (WHO, 2009). Current treatments have many showed that, 5-phenylterbenzimidazole is a cytotoxic
limitations such as side effect of the drugs as well as agent against the RPMI 8402 cell line (Kim et al.,
drug resistance. Hence, the identification and syn- 1997). A series of 2-aryl-5(6)-substituted-1H-benzimi-
thesis of novel, efficient and less toxic anticancer dazole derivatives were synthesized which showed
agents remains an important and challenging task for cytotoxicity against human cancer cell lines and 5-
cancer treatment. In medicinal chemistry the benzi- nitro-2-(4-methoxyphenyl)-1
H-benzimidazole was chosen
midazole nucleus is the key building block for a vari- as the lead compound (Min et al., 2005). Bendamus-
tine (TREANDA®) contains benzimidazole, which has
been approved by FDA for the treatment of leukemia.
Correspondence to: A. Romero-Castro, Facultad de Farmacia,
Universidad Autónoma del Estado de Morelos, Cuernavaca,
Morelos, 62209 México
Tel, Fax: 52 777 3297089
E-mail: cdr.romero@live.com.mx
A. Nieto-Rodríguez, Facultad de Farmacia, Universidad Autónoma
del Estado de Morelos, Cuernavaca, Morelos, 62209 México
Tel, Fax: 52 777 3297089
In addition, 1-substituted-2-methyl-5-nitrobenzimida-
zoles have been reported as antitumoral compounds
(Ramla et al., 2006).
It is reported that the cytotoxicity of nitro com-
pounds against hypoxic cells is attributed to the re-
duction of the nitro group, yielding several reactive
metabolites including the nitro anion (R-NO2−), hydro-
E-mail: directorfarmacia@yahoo.com.mx
181

182
A. Romero-Castro et al.
nitroxide (R-HNO•), and amino cation free radical (R- MPA120 automated melting point device from Stanford
NH2+) (Helsby et al., 2003). In addition, novel targets Research Systems and were used uncorrected. Reac-
against cancer have been discovered. The poly (ADP- tions were monitored by TLC on 0.2 mm precoated
1
ribose) polymerases (PARP) are a family of nuclear silica gel 60 F254 plates (Merck). H-NMR spectra
enzymes. PARP1 is involved in the regulation of cellu- were recorded on a Varian INOVA 400 (400 MHz).
lar processes such as DNA repair, gene transcription, Chemical shifts are given in ppm relative to tetra-
cell cycle progression, cell death, chromatin function methylsilane (TMS,
δ = 0) in DMSO-d6 and J values
and genomic stability (Jagtap and Szabó, 2005; Tentori are given in Hz. The following abbreviations are used:
and Graziani, 2005; Moree et al., 2008). Because of the s, singlet; d, doublet; q, quartet; dd, doublet of doublet;
role of PARP in DNA repair, many PARP-1 inhibitors t, triplet; m, multiplet; bs, broad signal. EIMS were
have been developed in order to enhance the efficacy of recorded on a JEOL JMS-700 spectrometer (JEOL
chemotherapy and radiotherapy. These inhibitors inter- Ltd.). Predictive values for antineoplastic activities
act with the nicotinamide binding domain of the enzyme were investigated using the chemistry software server
(Curtin et al., 2004; White et al., 2004; Ashworth, 2008). PASS (http://195.178.207.233/PASS/). All starting
In this work, we designed a series of novel 2-aryl- materials were commercially available from Sigma-
5(6)-nitro-1
H-benzimidazole derivatives taking into Aldrich and used without purification.
consideration the physicochemical properties of com-
pounds that were predicted using Lipinski's Rule
(Rule of five, Ro5) that is widely accepted for pre-
dicting the permeability behavior of compounds in bio-
Chemistry
Synthesis of compounds 1-4
Four benzimidazole derivatives (1-4) were synthe-
logical membranes. We also predicted biological activity sized by the reaction of 4-nitro-1,2-phenylenediamine
of the compounds in silico using the computational with appropriate aromatic substituted aldehydes and
program PASS (Institute of Biomedical Chemistry of sodium metabisulfite under microwave irradiation
Russian Academy of Medical Sciences). The novelty of (Scheme 1), using a method previously reported by our
this study was the addition of an extra nitro group in laboratory (Navarrete-Vázquez et al., 2006).
the phenyl ring of the 2-arylbenzimidazole core. Since
previous reports showed that 2-(4-methoxyphenyl)-5-
nitrobenzimidazole possessed a strong cytotoxic action,
Synthesis of compounds 5 and 6
Compounds and were obtained using a classical
5
6
we decided to exchange the substituent of the phenyl method. A mixture of 4-nitro-1,2-phenylenediamine
ring (methoxy) and replace it with another nitro group. (0.0313 mol), 1.01 equivalents of 3,5-dinitrobenzoic
It is well known that the nitro group can be used in acid or 4-chloro-3-nitrobenzaldehyde and 2.0 equiva-
the chemotherapy of hypoxic cells. In these series, we lents of phosphorous oxychloride was heated to reflux
changed the position of the extra-nitro group in the for 30 h (Scheme 2), the reaction was stopped by
phenyl ring, with the aim to improve anticancer activ- neutralizing with ammonium hydroxide. The precipi-
ity, and determine the correct pattern of the dinitro- tate was collected by filtration. Purification was done
substitution.
by chromatography on a silica gel using chloroform:
We report the synthesis and screening of antiproli- methanol (95:5) to elute and then dried. Some physi-
ferative activities such cytotoxicity, PARP inhibition cochemical properties are listed in Table I. The struc-
activity, cell cycle effect and apoptosis induction of a ture of the pure compounds (1-6) was established by
series of novel 2-aryl-5(6)-nitro-1
H-benzimidazole deriva- spectroscopic and spectrometric data.
tives as potential chemotherapeutic agents (Table I).
5(6)-Nitro-2-phenyl-1H-benzimidazole (1)
1
Yield 0.97 g (63%) of brown solid, mp 147-149oC. H-
MATERIALS AND METHODS
NMR (400 MHz, DMSO-d6) 7.60 (m, 3H, H-3', H-4', H-
5), 7.75 (bs, 1H, H-7, J = 7.8 Hz), 8.12 (dd, 1H, H-6,
J
Chemicals
Melting points were determined on an EZ-Melt
=
2.0, 2.0, 8.8 Hz), 8.21 (dd, 2H, H-2', H-6', J = 1.6, 8.0
Scheme 1. Chemical synthesis of 5(6)-nitro-1H-benzimidazole derivatives 1-4

Evaluation of Benzimidazole Derivatives as Anticancer Agents
183
2-(4-Chloro-3-nitrophenyl)-5(6)-nitro-1H-benzimi-
dazole (6)
1
Yield 1.95 g (95%) of brown solid, mp 285-286oC. H-
NMR (400 MHz, DMSO-d6) 7.73 (d, 1H, H-5', J = 8.8
Hz), 7.94 (d, 1H, H-7, J = 8.4 Hz), 8.08 (dd, 1H, H-6',
J
=
2.0, 8.8 Hz), 8.37 (dd, 1H, H-6, J = 2.0, 8.4 Hz), 8.42
(d, 1H, H-2', J = 2.4 Hz), 8.75 (d, 1H, H-4, J = 2.4 Hz).
EIMS: m/z (% rel. int.) 318 (M+, 10), 283 (100), 267
(50).
Biological assays
The biological assays were performed using the
MTT assay (Monks et al., 1991) against the following
human cell lines: A549 (lung, carcinoma), K562 (bone
marrow, chronic myelogenous leukemia), KB (HeLa
Scheme 2. Preparation of compounds
5 and 6
Hz), 8.50 (bs, 1H, H-4, J = 8.5 Hz). EIMS:
int.) 239 (M+, 100), 223 (2), 193 (30), 166 (20).
m
/
z
(% rel. contaminant), HL60 (peripheral blood, acute promy-
elocytic leukemia), MDA231 (breast, adenocarcinoma),
MCF7 (breast, adenocarcinoma) and HT29 (colon,
5(6)-Nitro-2-(2-Nitrophenyl)-1
Yield 1.43 g (78%) of yellow crystals, mp 130-132 neoplastic control. Cell cycle arrest and apoptosis
oC. 1H-NMR (400 MHz, DMSO-d6) 7.81 (d, 1H, H-7,
induction were evaluated in K6562 cells. The PARP
H-benzimidazole (2)
adenocarcinoma). HACAT cells were used as a non
J
= 8.8 Hz), 7.85 (dd, 1H, H-5', J = 1.6, 1.2, 8.0 Hz), 7.92 inhibition activity was evaluated using the PARP Uni-
(ddd, 1H, H-4', J = 1.6, 1.6, 7.6 Hz), 8.01 (dd, 1H, H-3', versal Colorimetric Assay Kit (R&D Systems) (Zhu et
J = 1.6, 7.6 Hz), 8.13 (dd, 1H, H-6', J = 1.2, 8.0 Hz), al., 2008).
8.17 (dd, 1H, H-6, J = 2.4, 8.8 Hz), 8.53 (d, 1H, H-4,
J
=
2.4 Hz). EIMS: m/z (% rel. int.) 284 (M+, 100), 268
Cell culture
(80), 238 (5), 192 (20).
A549, K562, HL60, KB, MDA-231, MCF-7 and
HACAT cell lines were obtained from the National
Cancer Institute of México (INCAN). All cells were
5(6)-Nitro-2-(3-Nitrophenyl)-1H-benzimidazole (3)
Yield 1.16 g (63%) of Brown solid, mp 281-283oC. H- cultured in Dulbecco’s modified Eagle’s medium
1
NMR (400 MHz, DMSO-d6) 7.79 (d, 1H, H-7, J = 8.8 (DMEM) supplemented with heat-inactivated 10%
Hz), 7.87 (d, 1H, H-5', J = 8.0 Hz), 8.13 (dd, 1H, H-6,
J
fetal bovine serum (FBS), 1% l-glutamine, at 37oC in a
=
2.0, 8.8 Hz), 8.37 (dd, 1H, H-6', J = 2.8, 8.0 Hz), 8.49 water-jacketed CO2 incubator.
(bs, 1H, H-2'), 8.60 (dd, 1H, H-4', J = 2.8, 8.0 Hz), 8.99
(d, 1H, H-4, J = 2.0 Hz). EIMS: m/z (% rel. int.) 284
(M+, 100), 238 (30), 192 (17).
In vitro cytotoxicity assay
For cytotoxicity assays, the activity of synthesized
compounds on each cell line was determined by the
5(6)-Nitro-2-(4-Nitrophenyl)-1H-benzimidazole (4) MTT assay. Cells were seeded in DMEM containing
1
Yield 0.93 g (51%) of brown crystals, mp 267oC. H- 10% FBS into wells of 96-well ELISA-type plates and
NMR (400 MHz, DMSO-d6) 7.40 (m, 4H, H-2', H-3', H- exposed to a range of drug concentrations (0.01, 0.1,
4 , H-5'), 7.85 (d, 1H, H-7, J = 8.4 Hz), 8.01 (dd, 1H, H- 1.0, 10.0 and 100.0
3', J = 1.6, 7.6 Hz), 8.06 (d, 1H, H-6, J = 8.5 Hz), 8.46 seeding densities ranged from 2
(s, 1H, H-4). EIMS: m/z (% rel. int.) 284 (M+, 100), viability was assessed by Trypan blue dye exclusion at
µ
M) for 48 h at 37oC. The initial
×
104 cells/well. Cell
268 (4), 238 (25), 192 (25).
the beginning of each experiment and was always
higher than 95%. At the end of the drug exposure
2-(3,5-Dinitrophenyl)-5(6)-nitro-1H-benzimidazole period, the cells were incubated in the medium and
(5)
MTT (0.5 mg/mL, 100 µL) was added. Then, the plates
Yield 1.39 g (60%) of brown crystals, mp 189-190oC. with MTT were incubated in the dark for 4 h. After
1H-NMR (400 MHz, DMSO-d6) 7.84 (d, 1H, H-7, J = that, the medium was removed and the water-insol-
8.0 Hz), 8.16 (dd, 1H, H-6, J = 2.0, 8.0 Hz), 8.55 (s, 1H, uble MTT-formazan crystals were dissolved in DMSO
N-H), 8.90 (d, 1H, H-4, J = 2.0 Hz), 9.17 (d, 1H, H-4', and the absorbance was determined in an ELISA
J = 2.4 Hz), 9.32 (d, 2H, H-2', H-6', J = 2.4 Hz). EIMS: microtiter plate reader at 570 nm with a reference
m/z (% rel. int.) 329 (M+, 4), 283 (4), 237 (3), 192 (11). filter of 630 nm. Viability was defined as the ratio (ex-

184
A. Romero-Castro et al.
pressed as a percentage) of absorbance of treated cells of each experiment and was always higher than 95%.
to untreated cells. Drug concentration dependence At the end of the drug exposure period, the cells were
and time-dependence of cell death were calculated as collected in 12 × 75 Falcon tubes and centrifuged at
a toxic effect (TE): TE = [DC]/[Cell] control, where 1500 rpm, 4oC for 5 min, after 1 mL of cold PBS was
[Cell] control is cell concentration, in control wells added to the cell pellet. Then cells were fixed by
(cells incubated for the same time length with no adding 4 mL of
compound) and [DC] is the dead cell concentration. stored for 72 h at
−
20oC absolute ethanol. The cells were
−
20oC in this fixation buffer until
[DC] is calculated as [Cell] control [Cell] final, were ready for analysis. For staining, at the end of 72 h, the
[Cell] final, is the live cell concentration in wells pellet was washed in 1 mL of PBS containing: 0.1% (v/
exposed to the drug. Cell concentration was measured v) of Triton X-100, 10
µg/mL of propidium iodide and
using a calibration curve made for all cell lines tested 5 units (k unitz) of DNase-free RNase and incubated
using MTT staining. The cytotoxic effect (IC50) of the at 37oC for 30 min. Finally, samples were analyzed on
tested compounds was estimated as 100 × (T
− T0)/T0 a Becton Dickinson FACSCalibur flow cytometer and
=
−50, where T0 and T are optical densities of the test the ModFit LT V.3.0 program was used for analysis.
well at time zero (when the compound is added) and
after exposure to test compound. In this study, we use
Carboplatin as the positive control, and the cell line
HACAT (immortalized fibroblasts) as the non cancer
cell line.
RESULTS AND DISCUSSION
Synthesis and in silico activity
We designed a series of six compounds which were
2-arylbenzimidazole derivatives and predicted their
biological activity in silico. In this series, we changed
In vitro PARP inhibition assay
PARP Universal Colorimetric Assay Kit (Catalog the position of the nitro group in the phenyl ring, with
Number 4672-096-K, R&D Systems) measures the the goal to improve the activities described above. For
incorporation of biotinylated poly (ADP-ribose) onto this purpose, the chemistry software server PASS
histone proteins in a 96-well plate. Briefly, we added (prediction of activity spectra for substances) was
25 µL of 1 × PARP cocktail to each well. and prepared used; the values obtained for antineoplastic activities
an activity control (PARP-HSA enzyme without in- are shown in Table I. Results of prediction are pre-
hibitors, 100% of activity) and a negative control with- sented as the probability Pa for the compounds
out PARP to determine background absorbance. Then, activity (Stepanchikova et al., 2003). For the designed
we added 10
as well as 1 unit/well of PARP-HSA enzyme (the final pounds are very likely to reveal antineoplastic activity.
reaction volume were 50 L). The plate was incubated
at room temperature for 90 min. For detection, the
plate was washed 4 times with 1 PBS (200 L/well),
and 50
dase) was added to each well and incubated for 20 min HL60 cell line (IC50 = 4.9
at room temperature. The plate was then washed with toxicity against the HACAT cell line (IC50 = 15.2
PBS, and 50 L of TACS-Sapphire (a HRP sub- Compound also displayed strong potency against
strate that generate a soluble blue color) was added to HT29 cells (IC50 = 6.4 M). However, this compound
each well and incubated for 30 min, after which absor- was cytotoxic against the non cancer cell line HACAT.
µL of the inhibitor of interest to the wells compounds 1-6, Pa values were > 0.8; then, these com-
µ
MTT assay
×
µ
Biological results reported in Table II showed that
µ
L of diluted strep-HRP (horseradish-peroxi- compound
2
was the most potent agent against the
M), and showed low cyto-
M).
µ
µ
1
×
µ
3
µ
bance was determined at 630 nm.
The features of these compounds are the presence of a
nitro group in the phenyl ring at positions 2 and 3,
respectively. Compound
1
was active against cell lines
M),
Cell cycle arrest and apoptosis induction
K6562 cells were cultured in Dulbecco’s modified MDA231 (IC50 = 1.3
µM) and A549 (IC50 = 2.2 µ
Eagle’s medium (DMEM) supplemented with heat- but this compound was less active against the HT29
inactivated 10% fetal bovine serum (FBS), 1% l-gluta- and KB cell lines than HACAT (non cancer cell line).
mine, at 37oC in a water-jacketed CO2 incubator. Cells Interestingly, compound
were seeded in ELISA-type 6-well plates and exposed group in the phenyl ring.
1 did not bear the nitro
to the following concentrations of compounds
1-6: 0.1
Compound 4 (with nitro group at para-position of
µ
M for cell cycle and 100 M for apoptosis, over a the phenyl ring), was the most active agent against
µ
period of 12 h at 37oC. The initial seeding densities the HL60 cell line, showing an IC50 of 0.9
µM, and
ranged from 1 × 106 cells/well. Cell viability was as- good cytotoxic activity against MCF7, MDA231, A549
sessed by Trypan blue dye exclusion at the beginning and HT29 cell lines. This compound was the most

Evaluation of Benzimidazole Derivatives as Anticancer Agents
185
Table I. Physicochemical properties, antineoplastic activity predicted by PASS, and PARP inhibition activity of com-
pounds 1-6
Antineoplastic
Pa value*
Unoptimized Inhibition of PARP
Compound
R1
R2
R3
R4
Mp (oC)
yield (%)
activity IC50 (µM)
1
2
3
4
5
-H
-NO2
-H
-H
-H
-H
-H
-NO2
-H
NO2
NO2
-H
-H
-H
-NO2
-H
Cl
-H
-H
-H
-H
-NO2
-H
0.855
0.799
0.843
0.857
0.834
0.793
147 - 149
130 - 132
281 - 283
267 (dec)
189 - 190
285 - 286
63
78
63
51
60
95
2.18
0.08
0.05
1.27
40.97
38.59
28.54
6
3-ab
-H
*Pa: Prediction activity using PASS program.
Table II. In vitro cytotoxic activity of synthesized 2-aryl-5(6)-nitro-1
H-benzimidazole derivatives 1-6 against the eight
cell lines used in this study
IC50
MDA231
(µ
M)a
A549
Compound
K562
HL60
> 100
MCF7
> 100
HT29
KB
HACAT
1
2
10.1 ± 2.3
> 100
11.3 ± 0.2
11.0 ± 0.2
12.0 ± 1.0
11.2 ± 0.5
10.4 ± 0.1
14.0 ± 0.1
12.6 ± 0.4
2.211 ± 0.1 8.9 ± 0.2 17.11 ± 0.4 19.91 ± 2.4
8.611 ± 0.5 4.8 ± 1.2 18.11 ± 1.1 15.21 ± 1.2
9.311 ± 0.2 7.0 ± 1.2 29.41 ± 1.4 16.41 ± 2.6
2.811 ± 0.3 9.7 ± 3.1 11.21 ± 1.2 10.39 ± 0.2
0.811 ± 0.1 0.4 ± 0.3 12.67 ± 0.2 11.21 ± 0.8
0.028 ± 0.1 1.8 ± 0.3 13.01 ± 0.7 22.21 ± 2.1
0.051 ± 0.0 0.9 ± 0.1 14.11 ± 0.7 10.61 ± 0.1
14.9 ± 0.4
22.2 ± 1.2
10.9 ± 0.4
11.1 ± 0.5
12.1 ± 0.7
10.3 ± 0.1
74.5 ± 3.8
50.4 ± 0.6
12.4 ± 0.4
10.6 ± 0.2
18.0 ± 0.2
18.9 ± 0.1
3
4
19.2 ± 0.2
> 100
5
6
12.4 ± 0.2
14.9 ± 0.9
11.5 ± 0.2
Carboplatin
aData are presented as the mean ± S.D. of six independent series of experiments with five to six repetitions in each.
Statistic analysis was performed on the Student’s t-test. The Dunnet test with 95% confidence was applied to compare
the means. Statistic analyses were done using the Prisma V3.0 software program.
cytotoxic agent against HACAT non cancer cell line.
Compound displayed strong cytotoxic activity as Carboplatin.
against all cancer cell lines tested: K562 (IC50 = 2.4 Compound
M), HL60 (IC50 = 1.1 M), MCF7 (IC50 = 0.6 M), IC50 value of 0.028
MDA231 (IC50 = 0.4 M), A549 (IC50 = 0.8 M), HT29 activity was similar to that of Carboplatin in cancer
(IC50 = 0.4 M) and KB (IC50 = 2.7 M). Unfortunately, cell lines. Interestingly, this compound was the least
cancer cell line HACAT. This compound was as active
5
6
was the most active derivative, with
M against A549 cell line and the
µ
µ
µ
µ
µ
µ
µ
µ
this compound was also cytotoxic against the non cytotoxic agent against the non-cancer cell line HACAT,
Table III. Selectivity Index (SI) of synthesized derivatives 1-6
SIb
Compound
K562
HL60
MCF7
MDA231
A549
HT29
KB
1
2
3
4
5
6
1.0
ND
0.7
ND
0.5
4.5
0.4
ND
13.1
10.3
10.4
11.1
10.6
12.0
ND
0.2
0.1
0.2
2.0
2.8
0.1
7.6
1.4
0.5
0.3
3.0
5.6
0.2
114.5
111.8
110.7
110.1
111.5
740.0
112.0
11.11
13.21
10.91
10.04
13.01
12.31
10.71
1.41
0.81
0.21
0.03
0.41
7.41
0.11
Carboplatin
b SI = IC50 HACAT / IC50 Cell line.

186
A. Romero-Castro et al.
with a selective index > 700 (Table III). This com- the activity of the positive control 3-ab (IC50 = 28.54
pound has a chloro atom at position 4 and a nitro
group at position 3 of the phenyl ring. The nanomolar
activity of this compound makes it a suitable lead for
further in vivo research.
µM).
Cell cycle and apoptosis
We were interested in understanding the mecha-
nism by which these compounds induce cell death. In
order to study whether compounds could inhibit cell
PARP inhibition
Furthermore, in order to identify a potential mech- proliferation by cell cycle arrest or cell cycle-mediated
anism of action, we assayed the activity of this novel apoptosis, we performed DNA quantification in the
series of benzimidazoles as PARP inhibitors. To ini- K562 cell line, searching for an alteration in cell cycle
tiate this study, we considered a series of compounds and apoptosis using the Sub-G1 method, in which
developed by other research groups which explore the after DNA fragmentation, there are small fragments
interaction of many structures with the nicotinamide of DNA that are eluted following a washing step in
binding domain of PARP-1. There are similarities either PBS or a specific phosphate-citrate buffer. This
between the structures of the series developed in our means that after staining with a quantitative DNA-
study and other inhibitors that have been reported binding dye, cells that have lost DNA will take up less
and designed as nicotinamide mimics (Zhu et al., stain and appear to the left of the G1 peak. We used a
2008). For this reason, we proposed that our com- Becton Dickinson FACSCalibur flow cytometer. Inter-
pounds could act as nicotinamide mimics. Our results estingly, we found that only compounds
3 and 6 induce
obtained were consistent with recent studies which arrest in S phase of the cell cycle, and these com-
reported a new class of potent benzimidazole PARP pounds have a nitro group in position 3 of the phenyl
inhibitors (Zhu et al., 2008). In this assay, 3-amino- ring. Compounds
1, 2, 4, and 5 did not affect the cell
benzamide (3-ab) was used as a control inhibitor.
cycle of K562 cells (Fig. 2). The advantage of this method
From this screening effort, we identified that com- was its rapid ability to detect cumulative apoptosis as
pounds 1-6 inhibited the PARP activity in a concen- well as being applicable to all cell types. Results
tration-dependent manner (Fig. 1).
Under our experimental conditions, the data obtained K562 cells at a concentration of 100
indicated that synthesized compounds have similar or exposure (Fig. 3).
showed that compounds
1
-
6
induced apoptosis in
M before 12 h of
µ
even better activity than 3-ab (Table I). Compounds
1-4 showed excellent inhibitory PARP activity. Com- compounds
pound displayed the best inhibitory activity against inhibition could be mediated through a DNA replica-
PARP with an IC50 value of 0.05
Analyzing the cell cycle and apoptosis results for
3
and , our studies suggested that growth
6
3
µ
M, compared with tion defect followed by the cell cycle arrest and finally
leading to apoptosis. It is reported that the arrest in
the S phase of the cell cycle is associated to topoi-
somerase I poisons mediated by ATR (mediated ataxia
telangiectasia-Rad-3-related kinase) which modulates
the response of cells to S phase-associated DNA double-
stranded breaks induced by topoisomerase poisons
(Cliby et al., 2002). However, the assay for correlate
topo I poisoning activity with cytotoxicity was not
realized in our study, it is a possibility that compounds
3
and 6 work with a similar mechanism of action.
In conclusion, a series of six novel 2-aryl-5(6)-nitro-
H-benzimidazoles derivatives were synthesized, char-
1
acterized and subjected to preliminary evaluation as
anticancer agents against seven human cancer cell
lines of various histological origins. Cytotoxicity, in-
duction of cell cycle arrest, induction of apoptosis and
PARP inhibition activity were assayed in these lines.
Fig. 1. In vitro PARP inhibition activity of the compounds
1-
6. The results represent the mean of two independent ex-
Compound
5 had two nitro groups at position 3 and 5
periments by triplicate. Measurement was made using the
PARP Universal Colorimetric Assay Kit, from R&D Sys-
tems. In this assay the compounds interact directly with the
enzyme.
of the phenyl ring and displayed strong cytotoxic
activity. Toxicity of these types of compounds incre-
ased when the compound had a chlorine atom in posi-

Evaluation of Benzimidazole Derivatives as Anticancer Agents
187
Fig. 2. Compounds
M for 12 h. Cells without treatment were the control and cells treated with vincristine (induce G2/M phase arrest) were
the positive control.
3 and 6 induce S phase arrest in K562 cells. Leukemic cells were exposed with compounds 1 - 6 at 0.1
µ
tion 3 of the phenyl ring. Compound
6 was the most cancer agents. These results will direct the focus of our
cytotoxic agent against all cancer cell lines tested, but future research program to evaluate the dual activity
interestingly displayed less cytotoxicity against the of these compounds alone or in addition with alkylating
cell line HACAT which is non-neoplastic. Compounds agents or chemotherapy in an in vivo tumor model.
5
and
was used a positive control. Compounds which bear a
nitro group at position 3 of the phenyl ring (
induce arrest in the S phase of the cell cycle in K562
6 displayed similar activity to carboplatin which
ACKNOWLEDGEMENTS
3, 6)
This work was supported in part by grants from
cells. These compounds are good candidates as anti- PROMEP-SEP, UAEMOR-PTC (A. Nieto-Rodriguez).

188
A. Romero-Castro et al.
Fig. 3. Compounds 1 - 6 induce apoptosis in K562 cells. Leukemic cells were exposed with compounds (100 µM) for 12 h.
Aurelio Romero-Castro acknowledges the fellowship
(181811) awarded by CONACyT to carry out graduate
studies.
H. A., Hostomsky, Z., and Newell, D. R., Novel poly(ADP-
ribose) polymerase-1 inhibitor, AG14361, restores sensi-
tivity to temozolomide in mismatch repair-deficient cells.
J. Clin. Cancer Res., 10, 881-889 (2004).
Ghodousi, A., Huang, X., Cheng, Z., and Skibo, E., Pyrrolo-
benzimidazoles linked to heterocycles and peptides. Design
of DNA base pair specific phosphate hydrolyzing agents
and novel cytotoxic agents. J. Med. Chem., 47, 90-100
(2004).
Helsby, N. A., Wheeler, S. J., Pruijn, F. B., Palmer, B. D.,
Yang, S., Denny, W. A., and Wilson, W. R., Effect of nitro-
reduction on the alkylating reactivity and cytotoxicity of
the 2,4-dinitrobenzamide-5-aziridine CB 1954 and the
corresponding nitrogen mustard SN 23862: distinct mech-
anisms of bioreductive activation. Chem. Res. Toxicol., 16,
469-478 (2003).
REFERENCES
Ashworth, A., A synthetic lethal therapeutic approach: poly
(ADP) ribose polymerase inhibitors for the treatment of
cancers deficient in DNA double-strand break repair. J.
Clin. Oncol., 26, 3785-3790 (2008).
Bielawski, K., Bielawska, A., and Wolczynski, S., Synthesis,
DNA-binding activity and cytotoxicity of carbamate de-
rivatives of Hoechst 33258 in breast cancer MCF-7 cells.
Biol. Pharm. Bull., 25, 916-919 (2002).
Cliby, W. A., Lewis, K. A., Lilly, K. K., and Kaufmann, S. H.,
S phase and G2 arrests induced by topoisomerase I
poisons are dependent on ATR kinase function. J. Biol.
Chem., 277, 1599-1606 (2002).
Curtin, N. J., Wang, L. Z., Yiakouvaki, A., Kyle, S., Arris, C.
A., Canan-Koch, S., Webber, S. E., Durkacz, B. W., Calvert,
Jagtap, P. and Szabó, C., Poly (ADP-ribose) polymerase and
the therapeutic effects of its inhibitors. Nat. Rev. Drug
Discov., 4, 421-440 (2005).
Karali. N., Synthesis and primary cytotoxicity evaluation of

Evaluation of Benzimidazole Derivatives as Anticancer Agents
189
new 5-nitroindole-2,3-dione derivatives. Eur. J. Med.
Chem., 37, 909-918 (2002).
Kim, J. S., Yu. C., Liu, A., Liu, L. F., and LaVoie, E. J., Ter-
benzimidazoles: influence of 2"-, 4-, and 5-substituents on
cytotoxicity and relative potency as topoisomerase I
poisons. J. Med. Chem., 40, 2818-2824 (1997).
Min, J. K., Kim, H. M., Kim, D.-D., and Kim, J. S., The effect
of 5-substitution on the cytotoxicity of 2-(p-methoxyphenyl)-
1H-benzimidazoles in human cancer cell lines. Key Eng.
Mater. Vols., 23, 277-279 (2005).
Diwani, H. I., Synthesis and antitumor activity of 1-
substituted-2-methyl-5-nitrobenzimidazoles. Bioorg. Med.
Chem., 14, 7324-7332 (2006).
Stepanchikova, A. V., Lagunini, A. A., Filimonov, D. A., and
Poroikov, V. V., Prediction of biological activity spectra for
substances: evaluation on the diverse sets of drug-like
structures. Curr. Med. Chem., 10, 225-233 (2003).
Tentori, L. and Graziani, G., Chemopotentiation by PARP
inhibitors in cancer therapy. Pharmacol. Res., 52, 25-33
(2005).
Monks, A., Scudiero, D., Skehan, P., Shoemaker, R., Paull,
K., and Vistica, D., Feasibility of a high-flux anticancer
drug screen using a diverse panel of cultured human
tumor cell lines. J. Natl. Cancer Inst., 83, 757 (1991).
Moree, W. J., Goldman, P., Demaggio, A. J., Christenson, E.,
Herendeen, D., Eksterowicz, J., Kesicki, E. A., McElligott,
D. L., and Beaton, G., Identification of ring-fused pyrazolo
pyridin-2-ones as novel poly(ADP-ribose)polymerase-1 in-
hibitors. Bioorg. Med. Chem. Lett., 18, 5126-5129 (2008).
Navarrete-Vázquez, G., Moreno-Diaz, H., Aguirre-Crespo,
F., León-Rivera, I., Villalobos-Molina, R., Muñoz-Muñiz,
O., and Estrada-Soto, S., Design, microwave-assisted syn-
thesis, and spasmolytic activity of 2-(alkyloxyaryl)-1H-
benzimidazole derivatives as constrained stilbene bioiso-
steres. Bioorg. Med. Chem. Lett., 16, 4169-4173 (2006).
Özden, S., Atabey, D., Yildiz, S., and Göker, H., Synthesis
and potent antimicrobial activity of some novel methyl or
ethyl 1H-benzimidazole-5-carboxylates derivatives carry-
ing amide or amidine groups. Bioorg. Med. Chem., 13,
1587-1597 (2005).
White, A. W., Curtin, N. J., Eastman, B. W., Golding, B. T.,
Hostomsky, Z., Kyle, S., Li, J., Maegley, K. A., Skalitzky,
D. J., Webber, S. E., Yu, X. H., and Griffin, R. J., Poten-
tiation of cytotoxic drug activity in human tumour cell
lines, by amine-substituted 2-arylbenzimidazole-4-car-
boxamide PARP-1 inhibitors. Bioorg. Med. Chem. Lett.,
14, 2433-2437 (2004).
Yun-Fei, L., Gui-Feng, W., Pei-Lan, H., Wei-Gang, H., Feng-
Hua, Z., He-Yong, G., Wei, T., Yu, L., Chun-Lan, F., Li-
Ping, S., Yu-Dan, R., Wei, L., and Jian-Ping, Z., Synthesis
and anti-hepatitis B virus activity of novel benzimidazole
derivatives. J. Med. Chem., 49, 4790-4794 (2006).
Zhu, G. D., Gandhi, V. B., Gong, J., Thomas, S., Luo, Y., Liu, X.,
Shi, Y., Klinghofer, V., Johnson, E. F., Frost, D., Donawho,
C., Jarvis, K., Bouska, J., Marsh, K. C., Rosenberg, S. H.,
Giranda, V. L., and Penning, T. D., Synthesis and SAR of
novel, potent and orally bioavailable benzimidazole in-
hibitors of poly(ADP-ribose) polymerase (PARP) with a
quaternary methylene-amino substituent. Bioorg. Med.
Chem. Lett., 18, 14, 3955-3958 (2008).
Ramla, M. M., Omar, M. A., El-Khamry, A. M., and El-