Synthesis and anticancer activity of novel benzimidazole and benzothiazole derivatives against HepG2 liver cancer cells

Article abstract of DOI:10.2174/157340612800493719

Most of cancer chemotherapeutics and chemopreventives exert their effects by triggering apoptotic cell death. In this study, novel benzimidazole and benzothiazole derivatives have been synthesized to investigate their effects on HepG2 liver cancer cell lines after initial screening study. A dose response curve was constructed and the most active derivatives were further studied for apoptotic analysis. Six active benzimidazole derivatives (8, 9, 10, 12, 13 and 14) significantly induced apoptosis compared to control group. Two compounds 10 and 12 induced apoptosis by arresting cells in G1 phase of cell cycle which is confirmed by increased expression level of p21. The activity of caspase-3 which is well known as one of the key executioners of apoptosis was determined in the presence and absence of the tested derivatives. Our results indicated that compounds 10 and 12 significantly increased caspase-3 activity compared to control group. Moreover, a docked pose of compounds 10 and 12 was obtained bound to caspase-3 active site using Molecular Operating Environment module. This study demonstrated that benzimidazole derivatives 10 and 12 provoke cytotoxicity and induced apoptosis in liver cancer cells HepG2.

Full text of DOI:10.2174/157340612800493719

Medicinal Chemistry, 2012, 8, 151-162
151
Synthesis and Anticancer Activity of Novel Benzimidazole and Benzo-
thiazole Derivatives against HepG2 Liver Cancer Cells
1
2
1
1,
ꢀ
2,3,
ꢀ
Amal M. Youssef , Ahmed Malki , Mona H. Badr , Rasha Y. Elbayaa and Ahmed S. Sultan
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt
2Department of Biochemistry, Faculty of Science, Alexandria University, Alexandria, Egypt
3Department of Oncology, Georgetown University Medical Center, Washington DC, USA
Abstract: Most of cancer chemotherapeutics and chemopreventives exert their effects by triggering apoptotic cell death.
In this study, novel benzimidazole and benzothiazole derivatives have been synthesized to investigate their effects on
HepG2 liver cancer cell lines after initial screening study. A dose response curve was constructed and the most active de-
rivatives were further studied for apoptotic analysis. Six active benzimidazole derivatives (8, 9, 10, 12, 13 and 14) signifi-
cantly induced apoptosis compared to control group. Two compounds 10 and 12 induced apoptosis by arresting cells in
G1 phase of cell cycle which is confirmed by increased expression level of p21. The activity of caspase-3 which is well
known as one of the key executioners of apoptosis was determined in the presence and absence of the tested derivatives.
Our results indicated that compounds 10 and 12 significantly increased caspase-3 activity compared to control group.
Moreover, a docked pose of compounds 10 and 12 was obtained bound to caspase-3 active site using Molecular Operating
Environment module. This study demonstrated that benzimidazole derivatives 10 and 12 provoke cytotoxicity and
induced apoptosis in liver cancer cells HepG2.
Keywords: Benzimidazole and benzothiazole derivatives/p21/Apoptosis/Caspase-3 activity/liver cancer.
INTRODUCTION
[Hoechst-33342], 2'-(4-ethoxyphenyl)-5-(4-methyl-1-pipera-
zinyl)-2,5'-bis-1H-benzimidazole, has been demonstrated
being an inhibitor of topoisomerase I [16,17]. Albendazole, a
benzimidazole carbamate (methyl 5-propylthio-1H- benzimi-
dazol-2-yl carbamate) with extensive clinical use as an an-
thelmintic drug, can also inhibit hepatocellular carcinoma
cell proliferation under both in vitro and in vivo experimen-
tal conditions [18].
Cancer is a disease of striking significance in the world
today. It represents the second leading cause of death, after
cardiovascular diseases [1]. Hepatocellular carcinoma, a
form of cancer originating in liver cells, is a challenging ma-
lignancy with high patient mortality rates worldwide [2].
Although therapies are available, drawbacks such as cytotox-
icity have prompted researchers to seek more effective
means of treatment. Chemotherapeutics are cytotoxic agents
used to treat cancer. Ideally, these are developed to selec-
tively cause toxicity to cancer cells with minimal cytotoxic
effects to normal cells [3]. Given the toxicity, development
of resistance, and lack of broad spectrum treatments, there is
a continuing need for the development of new chemothera-
peutic agents for the treatment of cancer. Accordingly, many
diverse strategies have been employed to develop new thera-
pies or to improve existing treatments. Literature survey re-
vealed that, benzimidazole derivatives are endowed with
different types of biological activities espically antitumor
activity [4-7]. Pyrrolo[1,2-a]benzimidazoles represent a new
class of antitumor agents exhibiting cytotoxic activity
against a variety of cancer cell lines. The mechanism of cyto-
toxicity involves reductive alkylation of DNA accompanied
by cleavage of G and A bases [8-15]. An anticancer agent,
On the other hand, benzothiazole ring comprises a class
of therapeutic compounds proved to exert a wide range of
anticancer activity [19-23]. Literature survey revealed that,
2-(4-aminophenyl) benzothiazoles represent a new class of
antitumor agents exhibiting inhibitory activity against human
breast cancer cell lines [24-26].
Apoptosis is a selective process of physiological cell de-
letion that plays an important role in the balance between
cellular replication and death. Apoptotic signaling can pro-
ceed via two pathways, i.e., via death receptors expressed on
the plasma membranes of cells or alternatively via mito-
chondria, which contain several proteins that regulate apop-
tosis. The death receptor pathway is initiated by the ligation
of membrane bound tumor necrosis factor (TNF) or Fas re-
ceptors, which result in a caspase-8-dependent cascade and
subsequent cell death. During this cascade, caspase-8 cleaves
BH3-interacting domain (Bid) and induces cytochrome c
release and/or directly activates caspase-3 [27-29]. One of
the key events in apoptosis is the activation of a cascade of
intracellular cysteine proteases known as caspases. On pro-
teolytic activation by upstream caspases, caspase-3 is able to
cleave a variety of substrates, including poly (ADP-ribose)
*Address correspondence to these authors at the Department of Pharmaceu-
tical Chemistry, Faculty of Pharmacy, Alexandria University, 1 Elkhartoum
square-Azarita, 21521, Alexandria, Egypt; Tel: +2 (03) 4871317;
Fax: +2 (03) 4873273; E-mail: rasha_bayaa@yahoo.com; Department of
Biochemistry, Faculty of Science, Alexandria University, Alexandria,
Egypt; E-mail: dr_asultan@univ.alex.edu.eg
1875-6638/12 $58.00+.00
© 2012 Bentham Science Publishers

152 Medicinal Chemistry, 2012, Vol. 8, No. 2
Youssef et al.
polymerase (PARP). The cleavage of various substrates con-
tributes to the typical morphological and biochemical fea-
tures observed in apoptosis [30-32].
3H, CH2-CH3); 3.84 (s, 2H, CH2); 4.25 (q, J = 6.95 Hz, 2H,
CH2-CH3); 6.40 (s, 1H, pyridobenzothiazole-C2-H); 7.50 (t,
1H, J = 7.65 Hz, pyridobenzothiazole-C8-H); 7.57 (t, 1H, J =
7.65 Hz, pyridobenzothiazole-C7-H); 7.85 (d, J = 8.4 Hz, 1H,
pyridobenzothiazole-C9-H); 8.28 (d, J = 7.65 Hz, 1H, pyri-
dobenzothiazole-C6-H). Anal. Calcd for C16H12N2O3S
(312.34): C, 61.53; H, 3.87; N, 8.97; S, 10.27. Found: C,
61.19; H, 3.65; N, 8.68. S, 10.34.
Encouraged by the above findings a number of benzimi-
dazole and benzothiazole derivatives were synthesized and
evaluated for their antitumor activity against liver cancer
HepG2 cells. In vivo studies are in progress to investigate the
mechanism of action of the interesting derivatives.
7,8,9,10-Tetrahydro-11-oxo-11H-benzothiazolo[3,2-
b]isoquinoline-6-carbonitrile (5)
MATERIALS AND METHODS
Chemistry
A mixture of 2-(benzothiazol-2-yl)acetonitrile 2 (0.70 g,
0.004 mole), ethyl cyclohexanone-2-carboxylate ( 0.68 g,
0.004 mole) and ammonium acetate (0,62 g, 0.008 mole)
was heated in an oil bath at 140-150°C for 45 min; during
this time ethanol and ammonia were liberated and the reac-
tion mixture gradually solidified. After cooling, the solid was
treated with ethanol and the product was filtered, washed
with ethanol, dried and crystallized from dioxane. Yield:
Melting points were determined in open glass capillaries
on a Gallenkamp melting point apparatus and were uncor-
rected. The infrared (IR) spectra were recorded on Perkin-
Elmer 1430 infrared spectrophotometer using the KBr plate
1
technique. H-NMR spectra were determined on Jeol spec-
trometer (500 MHz) at the Microanalytical unit, Faculty of
Science, Alexandria University using tetramethylsilane
(TMS) as the internal standard and DMSO-d6 as the solvent
(Chemical shifts in ꢀ, ppm). Splitting patterns were desig-
nated as follows: s: singlet; br s: broad singlet; d: doublet; t:
triplet; q: quartet; m: multiplet. Microanalyses were per-
formed at the Microanalytical Unit, Faculty of Science,
Cairo University, Egypt and the found values were within
±0.4% of the theoretical values. Follow up of the reactions
and checking the homogeneity of the compounds were made
by TLC on silica gel-protected glass plates and the spots
were detected by exposure to UV-lamp at 254 nm.
o
76%, mp: 220-222 C. IR (cm-1): 2212 (CꢁN); 1660 (C=O);
1
1543 (C=C); 1275, 1095 (C-S-C). H-NMR (ꢀ ppm): 1.68-
1.70 (m, 4H, benzothiazoloisoquinoline-C8,9-H2); 2.62-2.64
(m, 4H, benzothiazoloisoquinoline-C7,10-H2); 7.52-7.55 (m,
2H, benzothiazoloisoquinoline-C2,3-H); 8.04 (d, J = 7.65 Hz,
1H, benzothiazoloisoquinoline-C1-H); 8.97 (d, J = 6.9 Hz,
1H, benzothiazoloisoquinoline-C4-H). Anal. Calcd for
C16H12N2OS (280.34): C, 68.55; H, 4.31; N, 9.99; S, 11.44.
Found: C, 68.23; H, 4.65; N, 10.11; S, 11.62.
2-[4,6-Bis(4-chlorophenyl)-2-imino-1H-pyridin-3-
yl]benzothiazole (6)
Ethyl 4-cyano-1-oxo-1H,5H-pyrido[2,1-b]benzimidazole-
3-acetate (3) and ethyl 4-cyano-1-oxo-1H-pyrido[2,1-
b]benzothiazole-3-acetate (4)
A mixture of 2-(benzothiazol-2-yl)acetonitrile 2 (0.35 g,
0.002 mole), 4-chlorobenzaldehyde (0.28 g, 0.002 mole), 4-
chloroacetophenone (0.31 g, 0.002 mole) and ammonium
acetate (1.23 g, 0.016 mole) in dry dioxane (5 ml) was hea-
ted under reflux for 12 hours. The solvent was evaporated
under reduced pressure and the remaining solid was tritu-
rated with water, filtered, dried and crystallized from etha-
A mixture of 2-(1H-benzimidazol-2-yl)acetonitrile 1 or
2-(benzothiazol-2-yl)acetonitrile 2 (0.004 mole), diethyl ace-
tone-1,3-dicarboxylate (0.81 g, 0.004 mole) and ammonium
acetate (0,62 g, 0.008 mole) was heated in an oil bath at 140-
150°C for 30-45 min; during this time ethanol and ammonia
were liberated and the reaction mixture gradually solidified.
After cooling, the solid was treated with ethanol and the
product was filtered, washed with ethanol, dried and crystal-
lized from the proper solvent.
o
nol. Yield: 32%, mp: 118-120 C. IR (cm-1): 3421, 3282
1
(NH); 1610, 1527 (C=N, C=C); 1254, 1081 (C-S-C). H-
NMR (ꢀ ppm): 7.49-7.51 (m, 3H, benzothiazole-C4,5,6-H);
7.53 (s, 1H, pyridine-C5-H); 7.64, 7.77 (2d, J = 8.4 Hz, each 2H,
p-chlorophenyl-C2,6-H); 7.91, 8.14 (2d, J = 8.4 Hz, each 2H, p-
chlorophenyl-C3,5-H); 8.24, 10.17 (2s, each 1H, NH and =NH,
D2O exchangeable); 8.27 (d, J = 7.65 Hz, 1H, benzothiazole-
C7-H). Anal. Calcd for C24H15Cl2N3S (448.37): C, 64.29; H,
3.37; N, 9.37; S, 7.15. Found: C, 64.15; H, 3.63; N, 9.28; S,
6.96.
Ethyl 4-cyano-1-oxo-1H,5H-pyrido[2,1-b]benzimidazole-
3-acetate (3)
o
Yield: 67%, mp: 231-233 C (Dioxane). IR (cm-1): 3125
(NH); 2218 (CꢁN); 1726, 1684 (C=O ester, C=O amide);
1529 (C=C). 1H-NMR (ꢀ ppm): 1.16 (t, J = 6.9 Hz, 3H, CH2-
CH3); 3.11 (s, 2H, CH2); 4.04 (q, J = 6.9 Hz, 2H, CH2-CH3);
6.36 (s, 1H, pyridobenzimidazole-C2-H); 7.06-7.11 (m, 3H,
pyridobenzimidazole-C7,8,9-H); 7.21-7.22 (m, 1H, pyridoben-
zimidazole-C6-H); 10.27 (s, 1H, NH, D2O exchangeable).
Anal. Calcd for C16H13N3O3 (295.29): C, 65.08; H, 4.44; N,
14.23. Found: C, 65.13; H, 4.65; N, 14.38.
2-(1H-Benzimidazol-2-yl)acetimidohydrazide (7)
A mixture of compound 1 (7.2 g, 0.046 mole) and hydra-
zine hydrate (99%) (8.3 ml, 0.138 mole) was heated under
reflux for 30 min. After cooling, the solid formed was tritu-
rated with ether, filtered, dried and crystallized from ethanol.
Ethyl 4-cyano-1-oxo-1H-pyrido[2,1-b]benzothiazole-3-
acetate (4)
o
Yield: 73%, mp: 231-233 C. IR (cm-1): 3482, 3316 (NH2);
3142 (NH); 1626, 1541 (C=N, C=C). 1H-NMR (ꢀ ppm): 3.45
(br s, 2H, NH2, D2O exchangeable); 4.27 (s, 2H, CH2); 5.10
(s, 1H, benzimidazole-NH, D2O exchangeable), 6.35-6.37 (m,
2H, benzimidazole-C5,6-H), 6.47-6.49 (m, 2H, benzimidazo-
o
Yield: 59%, mp: 182-184 C (EtOH). IR (cm-1): 2220
(CꢁN); 1726, 1674 (C=O ester, C=O amide); 1510 (C=C);
1246, 1072 (C-S-C). 1H-NMR (ꢀ ppm): 1.23 (t, J = 6.95 Hz,

Synthesis and Anticancer Activity of Novel Benzimidazole
Medicinal Chemistry, 2012, Vol. 8, No. 2 153
le-C4,7-H). Anal. Calcd for C9H11N5 (189.22): C, 57.13; H,
5.86; N, 37.01. Found: C, 57.23; H, 5.74; N, 37.12.
General Procedure for the Preparation of Compounds
13, 14 and 15
A solution of 12 (0.45 g, 0.002 mole) and the appropriate
amine (0.002 mole) in DMF (5 ml) was stirred at 100 °C for
2 h. The white solid obtained after cooling was filtered,
washed with ethanol, dried and crystallized from the proper
solvent.
General Procedure for the Preparation of Compounds 8,
9 and 10
A solution of compound 7 (1.5 g, 0.008 mole) in ethanol
(30 ml) was treated with sodium ethoxide (1.84 g, 0.08 mole
sodium metal in 10 ml ethanol). Thionyl chloride, ethyl chlo-
roformate or ethyl bromoacetate (0.008 mole) was gradually
added and the reaction mixture was stirred at room tempera-
ture for 1-2 hours during which a yellowish white crystalline
product was separated. The product was filtered, washed
with ethanol, dried and crystallized from methanol.
1-(4-Methoxyphenethyl)-3-(1H-benzimidazol-2-yl)urea
(13)
o
Yield: 71%, mp: 285-287 C (DMF). IR (cm-1): 3237,
3124 (NH); 1642 (C=O); 1608, 1553 (C=N, C=C); 1258,
1
1153, 1074 (C-O-C). H-NMR (ꢀ ppm): 2.70 (t, J = 6.9 Hz,
2H, CH2); 3.32 (t, J = 6.9 Hz, 2H, CH2); 3.68 (s, 1H, OCH3);
6.84 (d, J = 8.4 Hz, 2H, p-methoxyphenyl-C3,5-H); 6.96-6.98
(m, 2H, benzimidazole-C5,6-H); 7.14 (d, J = 8.4 Hz, 2H, p-
methoxyphenyl-C2,6-H); 7.28-7.30 (m, 2H, benzimidazole-
C4,7-H); 9.93, 11.49 (2s, each 1H, 2 NH, D2O exchangeable).
Anal. Calcd for C17H18N4O2 (310.35): C, 65.79; H, 5.85; N,
18.05. Found: C, 66.13; H, 5.65; N, 18.38.
4-((1H-Benzimidazol-2-yl)methyl)-2,3-dihydro-1,2,3,5-
thiatriazol-1-oxide (8)
Yield: 71%, mp: 267-269 oC. IR (cm-1): 3413, 3165
1
(NH); 1619, 1539 (C=N, C=C); 1383 (SO). H-NMR (ꢀ
ppm): 3.40 (s, 2H, CH2), 5.03 (s, 1H, benzimidazole-NH,
D2O exchangeable), 6.30-6.31 (m, 2H, benzimidazole-C5,6-
H), 6.38-6.44 (m, 2H, benzimidazole-C4,7-H). Anal. Calcd
for C9H9N5OS (235.27): C, 45.95; H, 3.86; N, 29.77; S,
13.63. Found: C, 45.87; H, 3.82; N, 29.53; S, 13.42.
1-(1H-Benzimidazol-2-yl)-3-cyclohexylurea (14)
o
Yield: 65%, mp: > 300 C (DMF). IR (cm-1): 3329, 3146
1
(NH); 1640 (C=O); 1620, 1571 (C=N, C=C). H-NMR (ꢀ
5-((1H-Benzimidazol-2-yl)methyl)-1H-1,2,4-triazol-
3(2H)-one (9)
ppm): 1.18-1.31 (m, 4H, cyclohexyl-C3,5-H2); 1.48-1.50 (m,
2H, cyclohexyl-C4-H2); 1.62-1.80 (m, 4H, cyclohexyl-C2,6-
H2); 3.51-3.53 (m, 1H, cyclohexyl-C1-H); 6.95-6.98 (m, 2H,
benzimidazole-C5,6-H); 7.30-7.32 (m, 2H, benzimidazole-
C4,7-H); 9.69, 11.42 (2s, each 1H, 2 NH, D2O exchangeable).
Anal. Calcd for C14H18N4O (258.32): C, 65.09; H, 7.02; N,
21.69. Found: C, 65.13; H, 7.05; N, 21.45.
o
Yield: 56% , mp: > 300 C. IR (cm-1): 3440, 3180 (NH);
1
1681 (C=O); 1617, 1520 (C=N, C=C). H-NMR (ꢀ ppm):
3.42 (s, 2H, CH2), 5.13 (s, 1H, benzimidazole-NH, D2O ex-
changeable), 6.33-6.35 (m, 2H, benzimidazole-C5,6-H), 6.48-
6.50 (m, 2H, benzimidazole-C4,7-H). Anal. Calcd for
C10H9N5O (215.21) C, 55.81; H, 4.22; N, 32.54. Found: C,
55.72; H, 4.11; N, 32.66.
1-(1H-Benzimidazol-2-yl)-3-(4-methoxyphenyl)urea (15)
Yield: 54%, mp: > 300 oC (DMF/EtOH). IR (cm-1): 3336,
3136 (NH); 1645 (C=O); 1611, 1573 (C=N, C=C); 1264,
1149, 1067 (C-O-C). 1H-NMR (ꢀ ppm): 3.72 (s, 1H, OCH3);
6.73 (d, J = 8.4 Hz, 2H, p-methoxyphenyl-C3,5-H); 6.96-6.99
(m, 2H, benzimidazole-C5,6-H); 7.39-7.35 (m, 2H, benzimi-
dazole-C4,7-H); 7.64 (d, J = 8.4 Hz, 2H, p-methoxyphenyl-
C2,6-H); 9.72, 11.69 (2s, each 1H, 2 NH, D2O exchangeable).
Anal. Calcd for C15H14N4O2 (282.30): C, 63.82; H, 5.00; N,
19.85. Found: C, 64.10; H, 4.69; N, 19.68.
3-((1H-Benzimidazol-2-yl)methyl)-1,2-dihydro-1,2,4-
triazin-5(6H)-one (10)
o
Yield: 69% , mp: > 300 C. IR (cm-1): 3428, 3180 (NH);
1
1685 (C=O); 1621, 1528 (C=N, C=C). H-NMR (ꢀ ppm):
3.63 (s, 2H, CH2), 4.52 (s, 2H, triazine-C6-H2), 6.21 (s, 1H,
benzimidazole-NH), 6.59-6.61 (m, 2H, benzimidazole-C5,6-
H), 6.81-6.84 (m, 2H, benzimidazole-C4,7-H), 12.00 (s, 1H,
NH, D2O exchangeable). Anal. Calcd for C11H11N5O
(229.24): C, 57.63; H, 4.84; N, 30.55. Found: C, 57.60; H,
4.74; N, 30.67.
Anticancer Studies
Cell Culture and Drug Treatment
N-(1H-Benzimidazol-2-yl)-1H-imidazole-1-carboxamide
HepG2 cells were maintained in Dulbecco’s modified es-
sential media (DMEM, Gibco) supplemented with 10 % Fe-
tal Bovine Serum (FBS), 100 Units/ml penicillin and 100
μg/ml streptomycin at 37oC in a 5% CO2 atmosphere.
(12)
A solution of 1,1'-carbonyldiimidazole (1.33 g, 0.01
mole) and 2-amino-1H-benzimidazole 11 (1.62 g, 0.01 mole)
in acetonitrile (50 ml) was stirred at room temperature for 20
h. The resulting precipitate was filtered, washed with etha-
nol, dried and crystallized from N,N-dimethylformamide
Cell Proliferation (MTT Assay)
4000- 5000 cells/well in 100 ꢀL of medium were seeded
in a 96-well plate for 24 h prior to drug treatment. The media
was then changed to media with tested compounds. At the
end of incubation (24 h), 10 ꢀL of 5 mg/ml 3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (AT
CC) was added to each well for 4 h. After incubation,
100 ꢀL of 0.01 M HCl to one tube containing 1 gm of SDS
was added to each well to dissolve the formazan crystals.
(DMF). Yield: 68%, mp: > 300 C. IR (cm-1): 3335 (NH);
o
1
1630 (C=O); 1608, 1593 (C=N, C=C). H-NMR (ꢀ ppm):
6.94-6.97 (m, 2H, benzimidazole-C5.6-H); 7.03-7.05 (m, 2H,
benzimidazole-C4,7-H); 7.37-7.51 (m, 2H, imidazole-C4,5-H);
8.10 (s, 1H, imidazole-C2-H); 11.71 (s, 1H, NH, D2O ex-
changeable). Anal. Calcd for C11H9N5O (227.22): C, 58.14;
H, 3.99; N, 30.82. Found: C, 58.13; H, 3.65; N, 31.27.

154 Medicinal Chemistry, 2012, Vol. 8, No. 2
Youssef et al.
The absorbance was determined at 570 nm. Assays were
performed in triplicate and standard deviation determined.
HCl pH 7.5, 1 mM EDTA, 1% triton X-100, 150 mM NaCl,
1 mM dithiothretol, 10% glycerol, 0.2 mM phenylmethysul-
phonyl fluoride and protease inhibitors). Protein concentra-
tions were determined using BCA protein assay kit (Pierce;
Rockford, IL). 20 ꢀg of total protein was loaded to 8% so-
dium dodecyl sulphate – polycrylamide gel electrophoresis
(SDS-PAGE) gels. The protein of interest was probed using
anti-p21 (B-9) mouse IgG (Santa Cruz; Santa Cruz, CA) and
the secondary antibody goat anti-mouse IgG – HRP (Santa
Cruz; Santa Cruz, CA). ꢁ-actin is the loading control
(Sigma-Aldrich; St. Louis, MO). The proteins were visual-
ized using ECL western blot chemiluminescence kit (GE
health care; Piscataway, NJ).
Enzyme Linked Immunosorbent Apoptosis Assay
Cells were seeded at a density of 2 X 104/ well in a 96-
well plate and incubated for 24 hours. Media was changed to
media containing the tested drugs (IC50 = 0.2~ 0.25 mM)
dose. Cells then incubated for extra 24 hours. An ELISA
assay was performed, using Cell Death Detection ELISAPLUS
kit (Roche-Applied Science, Indianapolis, USA) that meas-
ures histone release from fragmented DNA in apoptosing
cells. Cells were treated with 200-μL lysis buffer for 30 min
at room temperature. Cells lysate was subjected to centrifu-
gation for 10 min and 200 μL of supernatant was collected,
of which 20-μL was incubated with anti-histone biotin and
anti-DNA peroxidase at room temperature for 2 h. After
washing with incubation buffer three times, 100 μL of sub-
strate solution (2, 2`azino-di (3-ethylbenzthiazolin-sulphuric
acid) was added to each well and incubated for 15-20 min at
room temperature. The absorbance was measured using an
ELISA reader (Spectra Max Plus) at 405 nm. Each assay was
done in triplicate and standard deviation was determined.
Modeling Studies
Computer-assisted simulated docking experiments were
carried out under an MMFF94X force field in caspase-3
structure (PDB ID: 1GFW) using Chemical Computing
Group's Molecular Operating Environment (MOE-dock
2009) software, Montréal, Canada.
RESULTS AND DISCUSSION
Chemistry
Caspase-3 Activity
Caspase-3 activity was assayed according to manufac-
turer’s protocol (Assay designs, USA). 5X106 cells were
lysed in 100 μL lysis buffer containing 10 mM HEPES (4-
(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.4, 2
mM EDTA, 0.1% CHAPS (3-[(3-cholamidopropyl)di-
methylammonio]-1-propanesulfonicacid, 5 mM, 350 μg/ml
PMSF (phenylmethanesulfonylfluoride or phenylmethylsul-
fonyl fluoride) and 5 mM DTT (Dithiothreitol). Cell were
homogenized by three cycles of freezing and thawing and
then centrifuged to remove the cellular debris. Each sample
was then incubated in buffer containing 10 mM HEPES, pH
7.4, 2 mM EDTA, 0.1% CHAPS, 5 mM EDTA supple-
mented with its substrate (Ac-Asp-Glu-Val-Asp-AFC) Ac-
DEVD-AFC for 1 hour at room temperature and then reac-
tion was stopped with 1N HCl. OD405 was measured using a
spectrophotometer (Spectra Max Plus). Each assay was done
in triplicate and standard deviation was determined.
The synthetic strategies to obtain the target compounds
are depicted in Schemes 1, 2 and 3. Ethyl 4-cyano-1-oxo-
1H,5H-pyrido[2,1-b]benzimidazol-3-acetate (3) and ethyl 4-
cyano-1-oxo-1H-pyrido[2,1-b]benzothiazole-3-acetate (4)
were prepared by fusion of 2-(1H-benzimidazol-2-
yl)acetonitrile (1) [33] and 2-(benzothiazol-2-yl)acetonitrile
(2) [34] with diethyl acetone-1,3-dicarboxylate in the pres-
ence of ammonium acetate at 140-150 °C, respectively. 2-
(Benzothiazol -2-yl)acetonitrile (2) was fused with ethyl cyc-
lohexanone-2-carboxylate in the presence of ammonium
acetate to afford 7,8,9,10-tetrahydro-11-oxo-11H-benzothia-
zolo[3,2-b]isoquinoline-6-carbonitrile (5). Reacting 2 with 4-
chlorobenzaldehyde and 4-chloroacetophenone in dry dio-
xane in the presence of ammonium acetate following re-
ported reaction method [35] yielded the corresponding 2-
[4,6-bis(4-chlorophenyl)-2-imino-1H-pyridin-3-yl]benzo-
thiazole (6) (Scheme 1).
Flow Cytometric Analysis
Cells were seeded at a density of 3-5 X 105/ 10-cm2 plate
and incubated for 24 h before radiation. Media was changed
to media containing 10 and 12 (200 μM), 30 min before irra-
diation. After 24 hours, cells were harvested by trypsiniza-
tion. The cells were washed with PBS and fixed with ice-
cold 70 % ethanol while vortexing. Finally, the cells were
washed and resuspended in phosphate buffer saline (PBS)
containing 5 μg/mL RNase A (Sigma, St. Louis, MO USA)
and 50 μg/mL propidium iodide (Sigma, St. Louis, MO
USA) for analysis. Cell cycle analysis was performed using
FACScan Flow Cytometer (Becton Dickson) according to
the manufacturer’s protocol.
Scheme 2 starts with the key intermediate, 2-(1H-
benzimidazol-2-yl)acetimidohydrazide (7) which was pre-
pared by refluxing 1 in excess hydrazine hydrate. Cycliza-
tion of the amidrazone derivative 7 was performed by reflux-
ing with thionyl chloride, ethyl chloroformate or ethyl bro-
moacetate in ethanol and sodium ethoxide to obtain 1,2,3,5-
thiatriazole derivative 8, 1,2,4-triazol-3-one derivative 9 and
1,2,4-triazin-5-one derivative 10, respectively.
The precursor N-(1H-benzimidazol-2-yl)-1H-imidazole-
1-carboxamide (12) was prepared in analogy to a previously
reported method [33,34] by reaction of 2-amino-1H-
benzimidazole (11) with 1,1'-carbonyldiimidazole in acetoni-
trile. 1-(4-Methoxyphenethyl)-3-(1H-benzimidazol-2-yl)urea
(13), 1-(1H-benzimidazol-2-yl)-3-cyclohexylurea (14) and 1-
(1H-benzimidazol-2-yl)-3-(4-methoxyphenyl)urea (15) were
prepared by reacting 12 with the appropriate amines namely;
2-(4-methoxyphenyl)ethanamine, cyclohexylamine or 4-
methoxy benzenamine in DMF, respectively (Scheme 3).
Western Blot Analysis
For p21 protein analysis, HepG2 cells were grown to
60% confluency and then the culture media was switched to
media supplemented with 10 mM Metformin and/or 10 μM
of MEK inhibitor I (Calbiochem; San Diego, CA) (26,27).
After 24 h, cells were lysed with lysis buffer (10 mM Tris

Synthesis and Anticancer Activity of Novel Benzimidazole
Medicinal Chemistry, 2012, Vol. 8, No. 2 155
X
N
O
O
O
4-ClC6H5CHO
CN
OC2H5
C2H5O
Cl
+
4-ClC6H5COCH3
1, X = NH
2; X = S
+
CH3COONH4
X
N
CN
O
O
S
CH2COOC2H5
OC2H5
N
HN
O
N
H
3; X = NH
4; X = S
Cl
6
S
CN
N
O
5
Scheme 1.
O
H
H
N
H
N
N
Br
NH2NH2
OC2H5
N
NH
N
CN
N
N
HN
HN
HN
O
NH2
1
7
10
O
SOCl2
Cl
OC2H5
H
H
N
N
N
N
N
8
N
HN
HN
SO
O
N
N
H
H
9
Scheme 2.
Anticancer Studies
[38,39]. Despite the combined efforts of governments and
scientists worldwide, there is constant increase in the inci-
dence of Hepatocelluar carcinoma during the last two dec-
ades [40]. Successful drug treatment of a human disease re-
quires an adequate therapeutic index reflecting the treat-
ment’s specific effects on target cells and its lack of clini-
cally significant effects on the host. In the present study, a
series of benzimidazole and benzothiazole derivatives were
designed and synthesized to examine their effects on viabil-
ity and proliferation in HepG2, liver cancer cell lines. The
concentrations at which 50% inhibition of HepG2 cell viabil-
ity (IC50) were calculated using a semilogarithmic plotting of
the % of cell viability versus concentration of the tested
compounds. The (IC50 = 0.2~ 0.25 mM) dose of synthesized
compounds (3, 4, 5, 7, 8, 9, and 10) showed significant de-
crease in cell survival of HepG2 cells compared to control
group (Fig. 1).
Chemoprevention is now regarded as a promising strat-
egy to control cancer [36]. Cancer is usually associated with
aberrant cell cycle progression and defective apoptosis in-
duction due to the activation of proto-oncogenes and/or inac-
tivation of tumor suppressor genes [37]. We screened our
novel derivatives against various cancer cell lines including
breast cancer cells (MCF-7), colon cancer cells (RKO), lung
cancer cells (H1299), Melanoma (B16F10) and liver cancer
cells (HepG2), our initial screening study showed benzimi-
dazole derivatives decreased proliferation of liver cancer
cells (HepG2) (data not shown). In this study, we report
novel benzimidazole and benzothiazole derivatives that
showed cytotoxicity against liver cancer HepG2 cells.
Hepatocellular carcinoma (HCC) is one of the most
common malignant tumor worldwide and in Egypt as well

156 Medicinal Chemistry, 2012, Vol. 8, No. 2
Youssef et al.
O
H
N
N
N
H
N
N
N
NH
O
NH2
N
N
N
11
N
12
OMe
H2N
OMe
H2N
H2N
H
N
H
N
NH
NH
N
NH
H
N
N
NH
O
O
NH
N
NH
13
15
O
OMe
OMe
14
Scheme 3.
100
90
80
70
60
50
40
30
20
10
0
control
3
4
5
7
8
9
10
Fig. (1). Effect of benzimidazole and benzothiazole derivatives on cell viability of HepG2 cells. MTT assay was performed to detect living
cells. Each data point is an average of results from three independent experiments performed in triplicate and presented as M±SD.
The cell viability of the most potent derivatives (3, 5, 8,
9, and 10) was tested via the 3-(4,5-Dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide assay (MTT) and the ef-
fects of compounds were confirmed. Our data showed a sig-
nificant decrease in cell viability of HepG2 cells in dose de-
pendent manner, using (IC50 range = 0.2~ 0.25 mM) dose
that was determined based on dose response curve as shown
in Fig. (2).
cal factors and molecular mechanism associated with hepa-
tocelluar carcinoma improves screening and treatment of the
disease [41]. To investigate if the decrease in cells viability
could be due to induced-apoptosis, Enzyme linked immu-
nosorbent apoptosis assay (ELISA) was performed, which
detected histone release from apoptotic cells. We next exam-
ined whether the tested compounds at toxic concentrations
induced cell death or not. HepG 2 cells were treated with
(IC50 = 0.2~ 0.25 mM) dose of compounds (8, 9, 10, 12, 13,
and 14) and induction of apoptosis was determined as indi-
cated by histone release according to manufacturer's proto-
col. Our data indicated that previously mentioned derivatives
significantly induced apoptosis in HepG2 cells compared to
control untreated group (Fig. 4).
In addition to the previous tested compounds, we exam-
ined the effect of different series of compounds (6, 12, 13,
14, and 15) on cell viability of HepG 2 cells. Our data indi-
cated that compounds 12, 13, and 14 significantly decreased
cell viability compared to control group (Fig. 3).
It is possible that the decrease in cell viability as deter-
mined by the tested compounds could be due to either cell
growth arrest or cell death. Understanding the epdimiologi-
Cell death was assayed by trypan blue staining, which
determines membrane integrity. Compounds (8, 9, 10, 12,

Synthesis and Anticancer Activity of Novel Benzimidazole
Medicinal Chemistry, 2012, Vol. 8, No. 2 157
100
90
80
70
60
50
40
30
20
10
0
3
5
8
9
10
0
0.5
1
1.5
2
2.5
3
3.5
Log conc.(micrM)
Fig. (2). Dose response curve of the tested compounds for HepG2 cells. Each data point is an average of three independent experiments and
expressed as M±SD.
100
90
80
70
*
*
60
50
40
*
30
20
10
0
Control
6
12
13
14
15
Fig. (3). Effect of benzimidazole and benzothiazole derivatives on cell viability of HepG2 cells. Each data point is an average of three inde-
pendent experiments and expressed as M±SD.
13, and 14) induced cell death was a relatively late event,
occurring only after 24 h of treatment with 200 μM. The
number of both the trypan blue-positive cells and the con-
densed and fragmented nuclei increased with compounds (8,
9, 10, 12, 13, and 14) treatment (data not shown), confirming
that the tested compounds-induced cell death was due to
apoptosis in HepG 2 cells.
As shown in Fig. (5) the tested compounds (8, 9, 10, 12,
13, and 14) at the apoptosis-inducing concentration (IC50
range = 0.2~ 0.25 mM) dose strongly stimulated caspase-3-
like activity in HepG2 cells. These results demonstrate that
the active tested compounds induce apoptosis of HepG2 cells
in a caspase-dependent manner.
Cell cycle checkpoints in G1 and G2 are activated in re-
sponse to UV or gamma radiation allowing time for DNA
repair to take place. After complete repair, the cells resume
cycling. When the damage in cells is severe and cannot be
repaired, the cells undergo apoptosis and die. Mammalian
cell cycle is regulated by complex machinery, in which cy-
clin depenedent kinases (CDKs), cyclin depenedent kinase
inhibitors (CDKIs) and cyclins play essential roles [44]. The
increased expression of G1 cyclins in cancer cells provide
them an uncontrolled growth advantage because most of
Because of the diversity of its substrates, caspase-3 is
thought to be a general mediator of physiological as well as
stress-induced apoptosis [42,43]. To investigate if the tested
compounds would activate caspase-3 in treated HepG2 cells,
Caspase-3 activity was assayed according to manufacturer’s
protocol (more details under the materials and methods). Our
data indicated that the compounds (8, 9, 10, 12, 13, and 14)
showed a significant increase in caspase-3 activity in treated
HepG2 cells when compared with untreated group.

158 Medicinal Chemistry, 2012, Vol. 8, No. 2
Youssef et al.
4
*
3.5
3
*
*
*
*
*
2.5
2
1.5
1
0.5
0
Control
8
9
10
12
13
14
Fig. (4). ELISA assay was applied for apoptotic cell detection. Cells were treated with (IC50 range = 0.2~ 0.25 mM) dose of 8, 9, 10, 12, 13 d
14 for 24 h. Cells without drug treatment were used as controls. * p < 0.05 as compared to the mock-treated controls using the unpaired Stu-
dent t-test. Each condition was performed in triplicate. Data are presented as M±SD.
2500
*
2000
*
*
*
*
1500
1000
500
0
*
control
8
9
10
12
13
14
Fig. (5). Effect of benzimidazole derivatives on caspase-3 activity (As indicated by (DEVD-pNA) cleavage) in HepG2 cells in the presence
of 200 μM. Each data point is an average of three independent experiments and expressed as M±SD.
these cells either lack CDKI or possess non-functional CDKI
or have low expression of CDKI [45]. Consistent with the
results from ELISA, sub-G1 apoptotic cells were shown by
flow cytometry assay to have a significant increase in
HepG2 cells (~ 30 and 25 % compared to the non-treated
control of ~ 19%) by arresting cells in G1 phase (45 and
40% compared by untreated group) (Fig. 6A). p21, a key
regulator of G1 phase, was determined by Western blot
analysis, our data indicated that both derivatives 10 and 12
increased its expression by 2 and 2.7 fold; respectively com-
pared to control group (Fig. 6B, C). Our data indicated that
two benzimidazole derivatives 10 and 12 induced apoptosis
compared to control group via arresting cells in G1 phase of
cell cycle, increasing p21 expression level and increasing
caspase-3 activity.
Docking Study
The docking study was carried out for the two active
benzimidazole derivatives 10 and 12 using the enzyme pa-
rameters obtained from the crystallographic structure of the
complex between caspase-3 with the co-crystallized isatin
sulfonamide derivative MSI (PDB ID: 1GFW) [46]. The
docking simulation for the ligands was carried out using mo-
lecular operating environment (MOE) software supplied by
the Chemical Computing Group, Inc., Montréal Canada [47].
The x-ray co-crystal structure of the complex between
recombinant human caspase-3 and isatin sulfonamide reveals
that a tetrahedral intermediate is formed between the cata-
lytic cysteine thiolate and the isatin ketone carbonyl group.
The three hydrophobic residues (Tyr204, Trp206, and

Synthesis and Anticancer Activity of Novel Benzimidazole
Medicinal Chemistry, 2012, Vol. 8, No. 2 159
(A)
100
90
80
70
60
50
40
30
20
10
0
Control
10
12
*
G1
S
G2
Apoptosis
(B)
(C)
3.5
3
*
2.5
2
*
1.5
1
0.5
0
C
10
12
Fig. (6). Effect of benzimidazole derivatives on cell cycle progression. Cells were treated as described under the materials and methods. The
% of cell cycle phases was determined in HepG2 cells after treatment with 200 ꢀM of 10 and 12 for 24 h (A). Expression level of p21 was
determined for p21 after treating cells with the same concentration of derivatives 10 and 12 and ꢁ-actin was used as control (B). Relative
increase in p21 expression was illustrated as bar graph and data are presented as M±SD (C).
Phe256) in the S2 pocket are involved in extensive hydro-
phobic contacts with the pyrrolidine ring of the inhibitor. In
the caspase-3/inhibitor MSI structure, the oxygen of the tet-
rahedral intermediate and the amide carbonyl oxygen are
within hydrogen bonding distance of Cys163NH and
Gly122NH, respectively. There is a hydrophobic and/or
aromatic interaction between the edge of Tyr204 with one
face of the bicyclic isatin core, which likely contributes to
the binding of the inhibitor. Whereas in compound 10, hy-
drogen bonding is observed between nitrogen of triazine ring
and Cys163NH as well as Gly122NH. Hydrogen bonding is
also seen between nitrogen of triazine ring and carbonyl
oxygen of Gly122. The bezimidazole ring of compound 10 is
bound in a hydrophobic cavity formed by Tyr204 and
Phe256 (Fig. 7).
Fig. (8). shows caspase-3 superimposition of MSI and the
docked compound 12. In this case, hydrogen bonding is ob-
served between the amide carbonyl oxygen of compound 12
and both the NH of Cys163 and Gly122. Hydrogen bonding
is also seen between the nitrogen of imidazole ring of com-
pound 12 and Arg207NH as well as Arg64NH. The ben-
zimidazole ring is surrounded by hydrophobic residues
Gly165, Thr166 and Ser205.

160 Medicinal Chemistry, 2012, Vol. 8, No. 2
Youssef et al.
GLU_123
1
50%
84%
HIS_121
GLY_122
ARG_164
16%
TYR_204
PHE_256
CYS_163
23%
SER_120
ALA_162
MSI_1
SER_205
ARG_207
GLN_161
TRP_206
Fig. (7). Caspase-3 superimposition of the co-crystallized isatin derivative MSI (colored green) and the docked compound 10 (colored red).
Pink dashed lines depict hydrogen bond interactions. Viewed using MOE module. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this paper).
THR_166
GLY_165
GLU_123
ARG_164
GLY_122
TYR_204
CYS_163
PHE_256
10%
92%
23%
68%
ME_ 61
23%
ALA_162
HIS_12
SER_205
MSI_1
TRP_206
SER_120
46%
ARG_64
93%
GLN_161
ARG_207
Fig. (8). Caspase-3 superimposition of the co-crystallized isatin derivative MSI (colored green) and the docked compound 12 (colored red).
Pink dashed lines depict hydrogen bond interactions. Viewed using MOE module. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this paper).
The docking study shows that compounds 10 and 12
form hydrogen bonds with the same residues (Cys163 and
Gly122) as that observed in the crystal structure of the MSI
complex. Moreover, compound 10 have a binding pattern in
the caspase-3 site which is close to the pattern observed in
the crystallographic structure of the complex between
caspase-3 with the co-crystallized isatin sulfonamide deriva-
tive MSI. This may account for increased caspase-3 activity
recorded for compound 10.
sponsible for the traced activity as it is responsible for the
interaction with hydrophobic residues of the enzyme, on
condition that the spacer between this ring and the SP2 nitro-
gen atom or its isoster does not exceed two carbon atoms.
Elongation of the spacer might decrease or abolish the activ-
ity.ꢀ
CONCLUSION
In conclusion, our novel benzimidazole series of com-
pounds exhibit cytotoxicity to HepG2 and induced apoptosis
via activation of caspase-3 activity which is confirmed by
docking experiments. Additionally, they increased expres-
sion of p21 as key play in cell cycle by arresting cells in G1
Structure-Activity Relationship
We can contrive from the anticancer studies together
with docking studies that the benzimidazole nucleus is re-

Synthesis and Anticancer Activity of Novel Benzimidazole
Medicinal Chemistry, 2012, Vol. 8, No. 2 161
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Received: June 02, 2011
Revised: November 05, 2011
Accepted: November14, 2011