Synthesis and docking studies of novel antitumor benzimidazoles

Article abstract of DOI:10.1016/j.bmc.2012.10.010

In this work, the benzimidazole-pyrrole conjugates 6a-h and benzimidazole-tetracycles conjugates 12-14 were prepared. The cytotoxicity of the compounds 3, 4a-h, 6a-h, 8, 10 and 12-14 was tested against lung cancer cell line A549. Compound 6b exhibited higher activity than the bis-benzoxazole natural product (UK-1), the standard. The tested 4g,h, 6a-h, 10 and 12-14 exhibited remarkable cytotoxicity activity against breast cancer cell line MCF-7 with higher activity than tamoxifen. Furthermore, compound 4h was found to be also more potent than doxurubicin. The antitumor promotion activity of synthesized compounds 4g,h, 6a-h, 10 and 12-14 has been estimated by studying their possible inhibitory effects on EBV-EA activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Among the studied compounds, the inhibitory activities of compounds 8, 13 and 14 demonstrated strong inhibitory effects on the Epstein-Barr virus early antigen (EBV-EA) activation without showing any cytotoxicity on the Raji cells and their effects being stronger than that of a representative control, oleanolic acid. Moreover, the molecular docking of the new compounds into plasminogen activator (uPA) receptor has been in correlation with the antitumor activity. All synthesized compounds 3, 4a-h, 6a-h, 8, 10 and 12-14 were docked into same groove of the binding site of the native co-crystalized (4-iodobenzo[b]thiophene-2-carboxamidine) ligand (PDB code:1c5x) for activity explaination. Compounds 4h, 6b and 13, giving the best docking results, were further studied to estimate their effect on the level of uPA using AssayMax human urokinase (uPA) ELISA kit. In case of A549 cell line, compound 6 exhibited similar activity to MMC, and for MCF-7 cell line, compound 4h exhibited similar activity to doxorubicin, in inhibiting the expression of uPA.

Full text of DOI:10.1016/j.bmc.2012.10.010

Bioorganic & Medicinal Chemistry 20 (2012) 6989–7001
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and docking studies of novel antitumor benzimidazoles
Mohamed A. Omar a, Yasser M. Shaker a, , Shadia A. Galal a, Mamdouh M. Ali b, Sean M. Kerwin c,
⇑
Jing Li c, Harukuni Tokuda d, Raghda A. Ramadan e, Hoda I. El Diwani a
a Chemistry of Natural and Microbial Products Department, National Research Center, Dokki, 12311 Cairo, Egypt
b Biochemistry Department, Division of Genetic Engineering and Biotechnology, National Research Centre, Cairo, Egypt
c Phar-Med Chem, The University of Texas at Austin, 1 University Station, A1935, Austin, TX 78712-0128, USA
d Department of Complementary and Alternative Medicine, R&D, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8640, Japan
e Youssef Jameel Science and Technology Research Center, The American University in Cairo, New Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work, the benzimidazole-pyrrole conjugates 6a–h and benzimidazole-tetracycles conjugates 12–
14 were prepared. The cytotoxicity of the compounds 3, 4a–h, 6a–h, 8, 10 and 12–14 was tested against
lung cancer cell line A549. Compound 6b exhibited higher activity than the bis-benzoxazole natural prod-
uct (UK-1), the standard. The tested 4g,h, 6a–h, 10 and 12–14 exhibited remarkable cytotoxicity activity
against breast cancer cell line MCF-7 with higher activity than tamoxifen. Furthermore, compound 4h
was found to be also more potent than doxurubicin. The antitumor promotion activity of synthesized
compounds 4g,h, 6a–h, 10 and 12–14 has been estimated by studying their possible inhibitory effects
on EBV-EA activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Among the studied com-
pounds, the inhibitory activities of compounds 8, 13 and 14 demonstrated strong inhibitory effects on the
Epstein–Barr virus early antigen (EBV-EA) activation without showing any cytotoxicity on the Raji cells
and their effects being stronger than that of a representative control, oleanolic acid.
Moreover, the molecular docking of the new compounds into plasminogen activator (uPA) receptor has
been in correlation with the antitumor activity. All synthesized compounds 3, 4a–h, 6a–h, 8, 10 and 12–
14 were docked into same groove of the binding site of the native co-crystalized (4-iodobenzo[b]thio-
phene-2-carboxamidine) ligand (PDB code:1c5x) for activity explaination. Compounds 4h, 6b and 13, giv-
ing the best docking results, were further studied to estimate their effect on the level of uPA using
AssayMax human urokinase (uPA) ELISA kit. In case of A549 cell line, compound 6 exhibited similar activ-
ity to MMC, and for MCF-7 cell line, compound 4h exhibited similar activity to doxorubicin, in inhibiting
the expression of uPA.
Received 24 September 2012
Accepted 11 October 2012
Available online 16 October 2012
Keywords:
Benzimidazoles
Cytotoxicity
Epstein–Barr virus early antigen (EBV-EA)
activation
Docking study
Plasminogen activator (uPA) receptor
uPA enzyme
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
membrane uPA receptor (uPAR).17,18 The uPA is involved in many
physiological functions and, along with members of the matrix
Benzimidazole derivatives are structural isosteres of naturally
occurring compounds such as purine, consequently, they can easily
interact with biomolecules of the living systems possessing diverse
biological activity as well as anticancer effects against various
types of cell lines.1–7 Different modes of action of benzimidazole
compounds have been described in the literature, such as DNA
groove binding,8 topoisomerase I,9–11,8 or II,12 checkpoint kinase
(Chk2)13–15, polo-like kinase 116 inhibition.
Urokinase plasminogen activator (uPA) is a serine protease that
functions in the conversion of the circulating plasminogen to the ac-
tive, broad-spectrum, serine protease plasmin. uPA is secreted as an
inactive single-chain proenzyme by many different cell types and
exists in a soluble or cell-associated form by binding to a specific
metalloproteinases (MMPs) family; it has been implicated in cancer
invasion and metastatization.19–21 Besides the proteolytic function,
upon binding to uPAR, uPA is involved in initiating versatile intracel-
lular signal pathways that regulate cell proliferation, adhesion, and
migration through its interaction with various integrins and vitro-
nectin.22 Urokinase is implicated in a large number of malignancies,
for example, cancers of breast, lung, bladder, cervix, kidney, stomach
and brain.23,24 The role of uPA in human cancer progression is fur-
ther supported by clinical evidences demonstrating that high tissue
levels of its components correlate with a poor prognosis in different
types of cancer as breast, gastrointestinal carcers.25,26
In the literature, benzimidazole-heterocycles conjugates linked
at the 2-position were synthesized and evaluated for their antitu-
mor activity. Coupling of benzimidazole-4-carboxamide to 1-pro-
pylpiperidin-4-yl at the 2-position of the benzimidazole, i.e.
compound I (Fig. 1), has been discovered to display a very good
⇑
Corresponding author. Tel.: +20 233371615; fax: +20 23337093.
E-mail address: yabdelrahman@yahoo.com (Y.M. Shaker).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.010

6990
M. A. Omar et al. / Bioorg. Med. Chem. 20 (2012) 6989–7001
NC
NC
NH
CONH2
Br
Abs. EtOH
NaOH
N
N
NC
CN
N
+
O
N
O
2
N
N
H
N
H
1
3
CH3
R-NH2
I
II
Abs. EtOH
Conc. HCl
Reflux
NH
NH2
NH2
HO
N
N
H2N
NH2
N
H
N
H
NC
N
5
HO
OC2H5
N
4 N HCl
N
R
H2N
N
H
III
IV
R
H2N
6a-h
4a-f
Figure 1.
R
a
2-pyridyl
4-pyridyl
b
c
d
e
f
I
3-bromophenyl
4-bromophenyl
3-chlorophenyl
4-chlorophenyl
2-benzimidazolyl
NH
NH2
S
g
h
Left hand side Right hand side
2-methylbenzimidazolyl
Figure 2.
Scheme 1.
been performed to explain activity and to find out the possible
interaction between the synthesized compounds and Urokinase
plasminogen activator (uPA) receptor (uPAR). The high molecular
docking scores of the new compounds into uPAR was in correlation
with the potent antitumor activity of the new compounds. Com-
pounds 4h, 6b and 13, giving the best docking results, were further
studied to estimate their effect on the level of uPA using AssayMax
human urokinase (uPA) ELISA kit. In case of A549 cell line, com-
pound 6 exhibited similar activity to MMC, and for MCF-7 com-
pound 4h exhibited similar activity to doxorubicin, in inhibiting
the expression of uPA.
potency against poly(ADP-ribose)polymerase (PARP), possessing
oral efficacy in several preclinical murine tumor models potentiat-
ing the efficacy of radiation and many cytotoxic agents e.g. tem-
ozolomide and cis-platin.27
Heterocyclic benzimidazole derivatives bearing amidino sub-
stituents at C-5 of benzimidazole ring have been prepared by intro-
ducing pyridine, imidazole or N-methylpyrrole at C-2 as in
compound II (Fig. 1), which was found to be the most potent anti-
tumor one.28
Benzimidazole derivatives were found to be active as urokinase
plasminogen activator inhibitors as 2-(2-hydroxy-3-ethoxy-
phenyl)-1H-benzimidazole-5-carboxamidine III,29 2-aminobenz-
imidazole and 2-amino-5-hydroxybenzimidazole IV (Fig. 1).30
Based on bioisosterism theory, we had for target in this
manuscript the synthesis of benzimidazoles acting as urokinase
inhibitors as analogues to the lead compound, 4-iodobenzo[b]thio-
phene-2-carboxamidine (B428, IC50 value of 320 nM,Fig. 2).31–35
B428 is a potent and selective inhibitor of the plasminogen activa-
tor urokinase.36 In our syntheses, the left-hand side of the lead com-
pound was changed to benzimidazole and the right-hand side to
substituted 2-aminopyrroles or the highly basic and hydrophobic
quinoxalino-1,4-benzodiazepine moiety. We aimed to increase
the number of amidine skeleton in the stucture of the new com-
pounds due to its important role in activity.36
The cytotoxicity of the new compounds against breast cancer,
MCF-7, human lung adenocarcinoma, A549 and the in vitro pro-
moting activity by estimating the inhibitory effect on Epstein–Barr
virus early antigen (EBV-EA) activation was tested as well as the ef-
fect of structure variation on activity was studied. Successfuly, the
activity of all the compounds against MCF-7, except compound 6c,
was higher than tamoxifen, especially compound 4h, which was
found to be more potent than tamoxifen and doxurubicin, and
was chosen for further optimization. Also compounds 8, 13 and
14 were potent against Burkitt’s lymphoma Epstein-Barr virus,
and most of the compounds express high activity against human
adenocarcinoma. The new class of 2-substituted benzimidazoles
by the quinoxalino-1,4-diazepine moiety was discovered to be of
interesting antitumor activity. Molecular docking studies have
2. Results and discussion
2.1. Chemistry
Compound 3 was prepared by the dehydrohalogenation reac-
tion of phenacyl bromide 1 and malononitrile 2 as previously re-
ported.37 Cycloaddition of various aromatic and heteroaromatic
amines to compound 3 was accomplished in the presence of few
drops of conc. hydrochloric acid in ethanol, to afford the 2-ami-
no-3-cyano-5-phenyl pyrrole derivatives 4a–h.38 Compounds 4a–
h were used as starting materials to prepare the desired 2-pyrrol-
ylbenzimidazole derivatives. Refluxing 4a–h with o-phenylenedi-
amine 5 in 4 N HCl, followed by neutralization with NH4OH,
yielded 3-(1H-benzimidazol-2-yl)-5-phenyl-1-(aryl)- 1H- pyrrol-
2-amine 6a–h (Scheme 1).
The formation of the novel bis-benzimidazoles 6g and 6h was
performed by using the same strategy. The reaction of 3 with 2-
aminobenzimidazole or 2-aminomethylbenzimidazole yielded
compounds 4g and 4h which were further reacted with o-phenyl-
enediamine 5 in presence of 4 N HCl to form 6g and 6h, respec-
tively (Scheme 1).
3-Amino-1H-quinoxalin-2-one 8,39 prepared previously by
refluxing o-phenylenediamine 5 with ethyl oxamate 7 in pyridine,
was cyclocondensed with anthranilic acid 9 to afford the tetracy-
clic product 10 (Scheme 2).40 Introduction of 2-benzimidazolyl-
methyl moiety into the quinoxalinyl NH of compound 10, was

M. A. Omar et al. / Bioorg. Med. Chem. 20 (2012) 6989–7001
6991
H2N
O
N
O
pyridine
Reflux
H2N
H2N
NH2
O
N
H
+
O
COOH
NH2
8
5
7
Δ
9
O
N
H
N
O
N
H
N
N
N
N
H
11
Cl
+
N
H
N
N
N
10
NH
Ethylbromoacetate
12
Allyl bromide
O
H
N
N
N
N
N
O
H
N
N
O
N
N
O
N
N
N
14
13
Scheme 2.
O
A549. For comparison, mitomycin C (MMC, Fig. 3) and the bis-
benzoxazole natural product (UK-1, Fig. 3) were also tested (Ta-
ble 1). The most active compound 6b exhibited higher activity than
CH3
O
O
NH
N
O
O
UK-1 (IC50 = 1.1 lM). Most of the synthetic intermediates and
N
O
HO
H2N
benzimidazoles were of moderately strong cytotoxicity against
A549 cell line, with the exception of the pyrrole derivative 4a,
which has moderate activity. The order of activity of the com-
pounds was 6b, 13, 4d, 6h, 6e, 4f, 6g, 4e = 6a, 14, 12, 8, 3, 6f in a
descending order.
N
O
O
O
H2N
Mitomycin C (MMC)
UK-1
2.2.2. Cytotoxicity against breast cancer cell line MCF7
Cytotoxicity of compounds 4g, 4h, 6a–h, 10 and 12–14 was
tested using by SRB assay using the method of Skehan et al43 in
breast cancer cell line MCF-7. For comparison doxorubicin (DOX)
and tamoxifen (TAM) (Fig. 4) were also tested (Table 2). All of
the tested compounds exhibited higher activity than tamoxifen,
and compound 4h was more potent than doxorubicin. The order
of reactivity was 4h, 13, 6b, 6d, 6f = 14, 6a, 6h, 4g, 10, 6g, 6e, 12,
6c in a descending order (Table 2).
Figure 3.
achieved by its reaction with 2-chloromethylbenzimidazole 1141 in
DMF and anhydrous potassium carbonate to form the desired
product 12. Subsequent alkylation of 12 with allyl bromide and
ethyl bromoacetate in DMF and anhydrous potassium carbonate
yielded 13 and 14 respectively (Scheme 2).
2.2. Antitumor activity
2.2.3. Antitumor promoting effect in vitro (Inhibition of
Epstein–Barr virus activation test)
2.2.1. Cytotoxicity against lung cancer cell line A549
A primary screening test was carried out using a short-term
in vitro synergistic assay on Epstein–Barr virus early antigen
The cytotoxicity of the compounds 3, 4a–h, 6a–h, 8, 10 and 12–
14 was tested using the AlamarBlue assay42 in lung cancer cell line

6992
M. A. Omar et al. / Bioorg. Med. Chem. 20 (2012) 6989–7001
Table 1
Table 3
The cytotoxicity of compounds 3, 4a–h, 6a–h, 8, 10, 12–14 as well as Mitomycin C
The Relative ratioa of EBV-EA activation with respect to positive control (100%) in the
(MMC) and UK-1 against A549 cell line
presence of compounds 4g,h, 6a–h, 8, 10, 12–14 and Oleanolic acid
Compound
IC50 for A549a
(
lM)
Compound
Concentration (mol ratio/TPA) % to control (% viability)
3
9.7 4.2
32.0 19.2
10.7 2.8
13.0 2.9
4.6 1.3
8.0 1.5
7.7 0.4
12.1 2.8
12.0 5.2
8.0 1.5
1.1 0.7
13.5 1.2
12.0 2.6
7.0 4.6
9.9 3.9
7.7 2.0
5.1 3.5
9.1 0.6
13.2 4.5
8.5 1.6
3.4 0.5
8.4 2.2
0.4 0.1
2.0 0.5
% to control (% viability) 500 mol
100 mol
ratio/
10 mol
ratio/
TPAb
4a
4b
4c
4d
4e
4f
4g
4h
6a
6b
6c
6d
6e
6f
6g
6h
8
10
12
13
14
MMC
UK-1
1000 mol ratio/TPAbc
ratio/
TPAb
TPAb
4g
4h
6a
6b
6c
6d
6e
6f
6g
6h
8
10
12
13
14
Oleanolic
acid
19.7(60)
18.0(60)
15.9(60)
15.3(60)
19.2(60)
18.9(60)
17.8(60)
20.1(60)
16.3(60)
18.7(60)
12.6(70)
22.6(60)
20.5(60)
11.6(60)
10.3(60)
12.7 (70)
49.6
48.3
47.3
46.9
49.9
48.0
48.3
50.3
48.2
48.3
46.3
54.6
53.1
45.8
43.7
3 0.0
83.5
82.0
79.1
78.6
84.0
83.2
80.1
85.6
80.0
82.6
78.1
86.3
85.6
77.9
74.4
80.0
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
Data are expressed as mean S.E. of three separate experiments.
a
Values represent percentages relative to the positive control value (100%).
TPA concentration was 20 ng/mL (32 pmol/mL).
Values in parentheses are the viability percentages of Raji cells.
b
c
Data are expressed as mean S.E.of three separate experiments.
Determined using the AlamarBlue assay.43
a
(EBV-EA) activation.44–46 The inhibitory effect of benzimidazole
derivatives 4g,h, 6a–h, 8, 10 and 12–14 on the EBV-EA activation
induced by TPA and the associated viability of Raji cells was listed
in Table 3. In this assay, all the tested compounds showed inhibi-
tory effects on EBV-EA activation without cytotoxicity on Raji cells.
All compounds exhibited dose dependent inhibitory activities, and
the viability percentages of Raji cells treated with the test com-
pounds were 60% or 70% at the highest concentration of
1000 mol ratio/TPA. As shown in Table 3, the inhibitory activities
of compounds 8, 13, and 14 were stronger at concentration
(1,000) than that of oleanolic acid,47 known as a representive
anti-tumor promoting agent. The relative ratio of compound 14
with respect to TPA (100%) was 10.3, 43.7, 74.4 and 100% at the
concentrations of 1000, 500, 100 and 10 mol ratio/TPA, respec-
tively, (Table 3); meaning 89.7, 56.3, 25.6% and 0.0% inhibition of
the EBV-EA activation by TPA, respectively. Compounds 8 and 13
showed 87.4%, 53.7%, 21.9% and 0% and 88.4%, 54.2%, 22.1% and
0% inhibition of the EBV-EA activation by TPA, respectively, at con-
centrations of 1000, 500, 100 and 10 mol ratio/TPA.
O
O
O
OH
OH
OCH2CH2N(CH3)2
OH
OMe
OH
O
O
C2H5
H2N
OH
Tamoxifen (TAM)
Doxorubicin (DOX)
Figure 4.
2.3. Molecular docking study
Table 2
The cytotoxicity of compounds 4g,h, 6a–h, 10, 12–14 as well as doxurubicin and
tamoxifen against MCF-7 cell line
Investigation of the binding affinity of the new benzimidazole
derivatives in to uPA receptor (PDB code:1c5x) was performed
for the purpose of lead optimization and to find out the interaction
between the new synthesized benzimidazole compounds and Uro-
kinase plasminogen activator receptor.
Molecular modeling calculations and local docking were done
using MOE48 (molecular modeling environment) to evaluate the
binding free energies of these inhibitors into the target to uPA
receptor.
Compound
IC50 for MCF7 lg/well
4g
1.28
4h
6a
6b
6c
0.515
0.934
0.705
2.12
6d
0.82
6e
1.7
6f
6g
0.934
1.62
2.3.1. Validation of the docking performance and accuracy
To validate the docking accuracy of the program used, docking
of the native co-crystallized (4 (4-iodobenzo[b]thiophene-2-car-
boxamidine) ligand was done into its binding site of uPA receptor.
The docked ligand was exactly superimposed on the native co-
crystallized one with RMSD being 0.32 Å and binding free energies
of ꢀ13.3818 kcal/mol. The hydrogen bonds between the docked li-
6h
10
12
13
14
DOX
TAMOX
0.972
1.39
1.66
0.667
0.934
0.629
1.82

M. A. Omar et al. / Bioorg. Med. Chem. 20 (2012) 6989–7001
6993
Figure 5. The interaction between the native ligand (4-iodobenzo[b]thiophene-2-carboxamidine) with uPA receptor, it exhibit 4 H-bonds with SER 190,GLY 219 and 2 with
ASP 189.
gand and the amino acids were the same as those between the na-
tive ligand and the amino acids (Fig. 5).
are deeply embedded into the binding cleft of uPA receptor domain
in S1 pocket with S-score of ꢀ16.6664 kcal/mol for compound 4h
and ꢀ17.0890 kcal/mol for compound 13. Also, Compound 4h
and 13 has the lowest binding free energy 16.6664 kcal/mol for
compound 4h and ꢀ17.0890 kcal/mol for compound 13 (highest
2.3.2. The binding affinities of the synthesized compounds into
uPA receptor
Molecular docking study was performed to find out interactions
between ligand and receptor and to compare affinities of the syn-
thesized compounds to the target uPA receptor.
For docking calculations, the protein structure (PDB code:1c5x)
was first separated from the inhibitor molecule and refined using
molecular minimization with added hydrogens.
Docking calculations were carried out using standard default
variables for the MOE program.48 The binding affinity was evalu-
ated by the binding free energies (S-score, kcal/mol) and hydrogen
bonds.
All synthesized compounds were docked into same groove of
the binding site of the native co-crystalized (4-iodobenzo[b]thio-
phene-2-carboxamidine) ligand.
The affinity of the compounds is represented in Table 4 with the
hydrogen bonds with the target receptor.
The compounds giving the best docking scores, based on the
binding free energy and the H-bonds with their distance between
the amino acids in the receptor, were compounds 4h, 6a, 6b, 6g,
13 and 14.
affinity) and has highest biological activity (IC50:0.515
l
g/well
and IC50:0.667
lg/well) for compounds 4h and 13, respectively.
For compounds 6b and 13 their docking results are highly corre-
lated to their antitumor activity against A549 cell lines and com-
pound 6b is deeply embedded into the binding cleft of uPA
receptor domain in S1 pocket with S-score of ꢀ16.3476 kcal/mol
and IC50 of 1.1 0.7 lM. The docking orientations for Compound
13 and compound 6b along with the native ligand into uPA recep-
tor are represented (Fig. 8).
The overall correlation between the binding free energy (S) and
the biological activity of the synthesized compounds against MCF-
7 is shown in Figure 9. Most of the synthesized compounds’ biolog-
ical activities are correlating with their binding free energy.
Hydrophobic interaction between the ligand and the receptor
also indicate good fitting of the compound in the binding site,
our compound show good corresponding between their hydropho-
bic moieties and the hydrophobic alpha spheres found in the bind-
ing site of uPA.
Most of the synthesized compounds gave higher score than the
native (4-iodobenzo[b]thiophene-2-carboxamidine) which binding
free energy was ꢀ13.3818 Kcal/mol, while the score of binding free
energy of most of the new synthesized compounds was around
ꢀ15 Kcal/mol. H-bonds between all synthesized compounds and
the amino acids in uPA were the same of those between the native
co-crystalized ligand with the amino acids of the receptor.
The active-site residues in uPA are Cys42, His57, Asp189, His99,
Asp194, Ser190, Ser195, and GLY 219. These residues of uPA are the
important residues in S1 site. Most of the synthesized compounds
form H-bonds with His 57, Ser 190, Asp 189 and Ser 195 (Figs. 6–8).
The docking results of compounds 4h and 13 are highly corre-
lated to their antitumor MCF7 activities. Compounds 4h and 13
2.4. The level of uPA protein expression
The compounds giving the best docking results, 4h, 6b and 13
by binding to uPA receptor were further used to estimate their ef-
fects on the level of uPA using AssayMax human urokinase (uPA)
ELISA kit (Assaypro, USA).
The level of uPA protein expressed in cell extracts of A549 and
MCF7 cell lines was represented in Tables 5 and 6.
The level of uPA protein expressed in cell extracts of A549 and
MCF7 cell lines was represented in Tables 5 and 6.
In case of A549 cell lines, the concentration of uPA protein re-
duced in a dose-dependent manner when the cells treated with
gradual doses of compounds 6b and 13 ranged from 0 to the

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M. A. Omar et al. / Bioorg. Med. Chem. 20 (2012) 6989–7001
Table 4
Docking scores of the synthesized benzimidazole compounds into uPA receptor
Compound
S-score (kcal/
mol)
Ki value
Hydrogen bonds between atoms of compounds and amino acids
Atom of compound Involved receptor
Involved receptor
residues
Type of Hydrogen bond Distance
atoms
(Å)
(Native
ligand)
ꢀ13.3818
1.51 E-10 H 4086
H 4088
OD 3165
OD 3164
OG 3175
O 3564
ASP
189 ASP
189
SER 190
GLY 219
HIS 57
SER 195
—
HIS 57
—
SER 190
—
SER 190
—
SER 195 HIS 57
SER 195
H-don
H-don
H-don
H-don
2.11
1.99
2.11
1.81
H 4087
H 4085
3
ꢀ12.9846
2.96 E-10 O 5131
O 5131
NE 840
OG 3232
—
NE 840
—
OG 3175
—
OG 3175
—
OG 3232
NE 840
OG 3232
O 3184
OG 3232
NE 840
OG 3232
OG 3232
NE 840
OG 3232
N 3208
N 3208
OG 3232
OG 3232
NE 840
OD 3164 OG 3175
O 3564
H-acc
H-acc
—
H-acc
—
H-acc
—
H-acc
–
2.73
2.80
—
3.04
—
2.65
—
2.66
—
4a
4b
4c
4d
4e
4f
ꢀ14.2778
ꢀ15.1534
ꢀ15.5169
ꢀ15.6518
ꢀ15.8661
ꢀ15.6487
ꢀ15.6977
ꢀ16.6664
3.33 E-11
7.59 E-12 N 5150
4.11 E-12
3.27 E-12 N 5122
2.27 E-12
—
—
—
3.29 E-12 N 5122
3.02 E-12 —-
5.89 E-13 N 5128
N 5128
N 5128
6.53 E-12 H 5116
H 5115
N 5111
1.00 E-12 N 5108
N 5108
4g
4h
H-don
H-acc
H-acc
H-don
H-don
H-acc
H-don
H-acc
H-acc
H-don
H-acc
H-acc
H-don
H-don
H-acc
H-don
H-don
H-don
H-don
H-acc
H-acc
H-don
H-acc
H-acc
H-acc
2.62
2.89
2.62
1.90
2.07
2.60
2.78
2.78
2.95
1.68
3.12
3.08
2.63
2.16
2.55
1.95
1.45
1.91
1.98
2.73
2.65
2.24
2.24
2.67
2.50
6a
6b
ꢀ15.2428
ꢀ16.3476
CYS 191
SER 195
HIS 57
SER 195 SER 195
6c
6d
6e
6f
6g
6h
ꢀ14.8792
ꢀ14.9337
ꢀ15.4696
ꢀ15.5963
ꢀ16.1000
ꢀ15.8928
1.2 E-11
1.1 E-11
4.4 E-12
N 5110
H 5121
N 5110
HIS 57
SER 195
GLY 193
GLY 193
SER 195
SER 195
HIS 57
ASP 189
SER 190
GLY 219
SER 195
HIS 57
4.45 E-12 N 5110
1.53 E-12 H 5122
2.17 E-12 H 5123
N 5136
8
ꢀ10.4686
ꢀ12.0956
ꢀ15.2572
2.08 E-8
H 5114
H 5113
H 5109
H 5118
O 5119
O 5119
10
12
1.33 E-9
OG 3232
NE 840
OG 3232
OG 3232
OG 3232
NE 840
SER 195
SER 195
SER 195
HIS 57
6.36 E-12 N 5131
N 5131
2.88 E-13 N 5116
13
14
ꢀ17.0890
ꢀ16.0152
1.7 E-12
N 5131
OG 3232
SER 195
S- score: binding free energy.
Native Ligand: The native co-crystallized (4-iodobenzo[b]thiophene-2-carboxamidine) bound ligand of uPA (PDB code:1c5x).
Ki value = Exp (delta G/RT).
R = 1.986 cal/mol-K, T = 298 K.
IC50. For compound 6b, the uPA concentration of untreated cells
(control) was 11.00 1.60 ng/ml, while that of cells treated were
6.20 0.75, 5.20 0.65, 4.80 0.50 and 2.70 0.30 ng/ml, respec-
tively (Table 5), this mean that the level of uPA decreased by
44%, 53%, 56% and 75%, respectively, when compared with control.
For compound 13, the uPA concentration of untreated cells (con-
trol) was 11.00 1.60 ng/ml, while that of cells treated were
7.40 0.85, 6.20 0.72, 5.30 0.50 and 3.80 0.45 ng/ml, respec-
tively, (Table 5) and they decreased by 33%, 44%, 52% and 65% as
compared with control. From the result, compound 6b exhibited
higher activity than compound 13 and similar to mitomycin in
inhibition the expression of uPA.
In case of MCF7 cell line, the concentration of uPA protein re-
duced in a dose-dependent manner when the cells treated with
gradual doses of compounds 4h and 13 ranged from 0 to the
IC50. For compound 4h, the uPA concentration of untreated cells
(control) was 11.40 1.40 ng/ml, while that of cells treated were
7.40 0.80, 5.70 0.63, 4.00 0.45 and 2.80 0.30 ng/ml, respec-
tively, with 35%, 50%, 65% and 75% decrease level as compared with
control. For compound 13, the uPA concentration of untreated cells
(control) was 11.40 1.40 ng/ml, while that of cells treated were
8.80 1.20, 7.70 0.85, 7.00 0.80 and 4.40 0.50 ng/ml, respec-
tively (Table 6) and they showed 23%, 33%, 39% and 61% decrease
level, respectively, comparing to control. From the foregoing re-
sults, compound 4h exhibited higher activity toward suppression
the expression of uPA than compound 13 and it was similar to
doxorubicin in inhibition the expression of uPA.
3. Structure-activity relationship
Regarding adenocarcinoma inhibition, the most active com-
pound was found to be 6b, having the 4-pyridyl as substituent in
position 1 of the pyrrole ring which may act as hydrogen bonding
acceptor. The role of the N atom of the pyridine ring was also pro-
ven by the decrease in activity if the position of N atom in pyridine
differs with respect to the link to pyrrole as 6a having the 2-pyridyl
ring was less potent than 6b having the 4-pyridyl ring. The pro-
nounced cytotoxicity of benzimidazole derivative 6b relative to
the pyrrolo intermediate 4b was remarkable, indicating the impor-
tance of the contribution of the benzimidazole moiety in activity.
Compound 13 having the quinoxalinobenzodiazepine moiety as
well as the allyl group on the N atom of the benzimidazole moiety,
was the 2nd most potent compound in the series. The importance
of benzimidazole substitution to the tetracyclic scaffold as well as

M. A. Omar et al. / Bioorg. Med. Chem. 20 (2012) 6989–7001
6995
Figure 6. Ligand interaction and the binding mode of compound 4h with uPA receptor, it exhibited 2 H-bonds with SER 195 and 1 H-bond with HIS 57. The hydrogen bond
formed is colored in green.
Figure 7. Ligand interaction and the binding mode of compound 6a with uPA receptor, it exhibited 3 H-bond with CYS 191, SER 195 and HIS 57. The hydrogen bond formed
colored in green.
the allyl substitution on the benzimidazole NH were emphasized
by the fact that 12 was more potent than 10 and 13 was more po-
tent than 12, respectively. The reason of this activity may be dis-
covered from the docking of compounds 6b and 13 which
showed that they were found to be deeply embedded into the
binding cleft of the uPA receptor domain. Also the allyl substitution
on the benzimidazole NH was more favored than the ethyl acetate
substitution as 13 was more potent than 14.
Substitution of the pyrrole ring of compound 6e by 3-chloro-
phenyl was more favoured than substitution by 4-chorophenyl as
for compound 6f, as 6e was more potent than 6f. Also, the chloro-
phenyl substitution was more favoured than the bromophenyl as
6f and 6e were more potent than 6d and 6c, which may be attrib-
uted to the larger volume of Br with respect to Cl.
Compound 4h was found to be highly potent against MCF-7.
This may be due to the presence of the hydrophobic phenyl group,
the polar and hydrogen bond donor NH2 group and the electron
withdrawing CN group. Also the CH2 linker between the benzimid-
azole moiety and the pyrrole ring might also have a certain influ-
ence on activity, as 4h and 6h were more potent than 4g and 6g
respectively. This may be attributed to the hydrophobic interaction
of the benzimidazole moiety with the active site of the enzyme,
due to more flexibility when linked to CH2 group. Replacement of
the pyrrole ring with the quinixalinobenzodiazepine moiety was

6996
M. A. Omar et al. / Bioorg. Med. Chem. 20 (2012) 6989–7001
Figure 8. Docking orientations for Compound 13 colored in red and compound 6b in ball and stick mode along with the native ligand colored in yellow into uPA receptor. A
Gaussian Contact surface is drown around the binding site, this is the surface that surrounds the van der Waals surface of a molecule (filling in solvent inaccessible gaps).
Table 6
The level of uPA protein measured by ELISA after exposure the MCF7 cells to gradual
concentration of compounds 4h, 6b, 13 and doxorubicin (DOX)
Compound
Concentration
l
g/well
uPA level (ng/ml)
4h
0
11.4000 1.40
7.4000 0.80a
5.7000 0.63a
4.0000 0.45a
2.8000 0.30a
0.0625
0.125
0.25
0.50
6b
0
11.4000 1.40
9.8000 1.10
8.7000 0.90
9.0000 0.86
9.4000 0.80
0.087
0.175
0.35
0.70
Figure 9. The overall correlation between the binding free energy
biological activity synthesized compounds against MCF7.
S and the
13
0
11.4000 1.40
8.8000 1.20a
7.7000 0.85a
7.0000 0.80a
4.4000 0.50a
0.084
0.167
0.335
0.67
Table 5
The level of uPA protein measured by ELISA after exposure the A549 cells to gradual
DOX
concentration of compounds 4h, 6b, 13 and mitomycin C (MMC).
0
11.4000 1.40
3.4000 0.48a
3.2000 0.30a
3.0000 0.35a
2.7500 0.30a
0.075
0.15
0.30
0.60
Compound
Concentration
l
M
uPA level (ng/ml)
4h
0
1,5
3
6
12
11.00 1.60
11.6000 1.30
9.7000 1.20
9.8000 1.50
9.2000 1.15
Data are expressed as mean S.E. of three separate experiments.
a
Is significant difference from control untreated at p <0.05.
successful, especially when the benzimidazole has an allyl group or
an ethyl acetate group in position1 for compounds 13 and 14, and
the products were more active than tamoxifen. This may also indi-
cate the importance of substitution on the N atom by these two
groups which enhance the activity as 13 and 14 were more potent
than the unsubstituted compound 12. The allyl group presence was
favored than ethyl acetate group as 13 was more potent than 14.
Similar to the case of adenocarcinoma cytotoxicity, substitution
with 4-pyridyl led to higher activity than with 3-pyridyl as 6b
was more active than 6a. The position of substitution of the halo-
gen atoms on the phenyl ring as the 4-substitution increases the
activity more the 3-substitution, as 6d and 6f were twice as potent
as 6c and 6e, respectively.
6b
0
11.0000 1.60
6.2000 0.75a
5.2000 0.65a
4.8000 0.50a
2.7000 0.30a
0.137
0.275
0.55
1.10
13
0
11.0000 1.60
7.4000 0.85a
6.2000 0.72a
5.3000 0.50a
3.8000 0.45a
0.425
0.85
1.70
3.40
MMC
0
11.0000 1.60
4.3000 0.50a
3.3000 0.40a
2.6000 0.20a
2.4000 0.30a
0.05
0.10
0.20
0.40
For the inhibitory effect against Burkitt’s lymphoma induced by
Epstein virus, the most active compounds were 13 and 14 having
the highly hydrophobic quinoxalinobenzodiazepine scaffold linked
to the benzimidazole moiety carrying an ester group in the first
and an allyl group in the second compound.
Data are expressed as mean S.E. of three separate experiments.
a
Is significant difference from control untreated at p <0.05.

M. A. Omar et al. / Bioorg. Med. Chem. 20 (2012) 6989–7001
6997
Halogen atoms substitution on the phenyl ring of the pyrrole
ring of compounds 6c–f has negative effect on activity within all
the tests performed. The role of allyl and ethyl acetate substitution
seemed to be crucial for activity as 13 and 14 were twice as potent
as their precursor 12. Also allyl substitution had more positive ef-
fect on activity than did the ethyl acetate substitution as 14 was
more potent than 13. The quinoxalino derivative 8, having NH2,
C@O, NH and C@N, was of high activity. The pyridyl substituted
compounds 6a and 6b were of moderate activity.
5.1.1. General procedure for the preparation of compounds (4a–
h)
A mixture of hetero/aromatic aniline (20 mmol) and 3–5 drops
of conc. hydrochloric acid was added to a solution of compound 3
(20 mmol) in ethanol (50 mL). This mixture was refluxed for 7 h.
The reaction mixture was then allowed to cool at room tempera-
ture. The precipitate was collected by filtration, dried and recrys-
tallized from appropriate solvent.
5.1.2. 2-Amino-5-phenyl-1-(pyridin-2-yl)-1H-pyrrole-3-
carbonitrile (4a)
4. Conclusion
Crystallized from chloroform/methanol (2:5) as a green powder.
Rf = 0.27 (petroleum ether/ethyl acetate, 1:1). Yield: (71%); mp
121–132 °C; 1H NMR (DMSO-d6) d: 6.29 (s, 1H, CH), 7.13–7.59
(m, 8H, H4+H5+H6+H2 –H6 ), d 8.2 (br, NH2, D2O exchangeable),
8.53 (d, J = 7.45 Hz, 1H, CH pyridine). IR (cmꢀ1): 3439, 3343
(NH2), 2223 (CN). MS: m/z 261.0, 2.1% (M++1); m/z 232.0, 24.5%
(M+—CN–2H); m/z 231.0, 100.0% (M+—CN–3H); m/z 182.0, 15.4%
[M++1—py]. Anal. Calcd for C16H12N4 (260.11): C, 73.83; H, 4.65;
N, 21.52. Found: C, 73.88; H, 4.62; N, 21.49.
New benzimidazole-pyrrole conjugates 6a–h have been syn-
thesized by the reaction of the synthesized pyrrole derivatives
4a–h with o-phenylene diamine. 3-amino-1H-quinoxalin-2-one
(8) reacted with anthranilic acid (9) to form the fused tetracyclic
compound 10. Reaction of 2-chloromethylbenzimidazole (11)
with compound 10 yielded benzimidazole-tetracyclic conjugate
12. N-substituted benzimidazoles 13 and 14 were obtained by
reaction of compound 12 with halo compounds. The antitumor
activity of new benzimidazole conjugates has been evaluated by
studying their cytotoxicity against lung cancer cell line A549
and breast cancer cell line MCF-7 besides their inhibitory effects
on Epstein–Barr virus early antigen (EBV-EA) activation. One of
the tested compounds against lung cancer A549, 6b, was found
to be more potent than mitomycin C. Most of the tested com-
pounds against breast cancer MCF7 cell line showed higher activ-
ity than tamoxifen and 4h was of higher potency than
doxurubicin. The cancer chemopreventive activity of the new
derivatives 4g, 4h, 6a–h, 10 and 12–14 has been estimated by
studying their inhibitory effects on Epstein–Barr virus early anti-
gen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-
13-acetate (TPA). The tested compounds showed dose dependent
inhibitory activities, most of them showed significant activity at
the highest concentration of 1000 mol ratio/TPA. Compounds 14,
13, and 8 showed stronger effect than that of oleanolic acid. The
Molecular Docking investigation of the synthesized derivatives
was carried out for lead optimization and they were docked into
plasminogen activator (uPA) receptor. There was good correlation
between the binding free energies (DGb, kcal/mol) predicted by
MOE and the antitumor activity of the new compounds against
MCF-7. The results of the ELISA study were consistent with the
docking results indicating that the anticancer effect of the pre-
pared compounds may exert its anticancer activity by inhibiting
uPA. It can be concluded from the results obtained that the tested
compounds possess significant anticancer activity comparable to
the activity of the commonly used anticancer drugs.
0
0
5.1.3. 2-Amino-5-phenyl-1-(pyridin-4-yl)-1H-pyrrole-3-
carbonitrile (4b)
=
Crystallized from chloroform as a brown powder. Rf
0.25
(petroleum ether /ethyl acetate, 1:1). Yield: 79%; mp 117–119 °C;
1H NMR (DMSO-d6) d: 6.20 (s, 1H, CH), 7.50–7.34 (m, 7H, Aromatic
protons), 8.2 (br, NH2, D2O exchangeable), 8.62 (d, J = 7.4 Hz, 1H,
CH pyridine). 13C NMR (500 MHz, DMSO-d6): 15.33, 66.74,
107.26, 109.31, 116.63, 122.59, 124.55, 128.77, 130.39, 142.34,
164.55. IR (cmꢀ1): 3414, 3325 (NH2), 2257 (CN). MS: m/z 260.0,
1.9% (M+); m/z 184.0, 5.3% (M+—Ph); m/z 168.0, 10.6% [M+—Ph—
NH2], m/z 140, 7.7% [M+ — Ph — NH2—CN—2H]. Anal. Calcd for
C16H12N4 (260.11): C, 73.83; H, 4.65; N, 21.52. Found: C, 73.79;
H, 4.66; N, 21.57.
5.1.4. 2-Amino-1-(3-bromophenyl)-5-phenyl-1H-pyrrole-3-
carbonitrile (4c)
Crystallized from ethyl acetate as a reddish brown powder.
Rf = 0.32 (petroleum ether /ethyl acetate, 1:1). Yield: 75%; m.p.
178–180 °C; 1H NMR (DMSO-d6) d: 6.60 (s, 1H, CH), 7.52-7.68 (m,
8H, Aromatic protons), 8.3 (broad band, NH2, D2O exchangeable).
IR (cmꢀ1): 3370, 3337 (NH2), 2258 (CN). MS: m/z 338.0, 100.0%
(M+); m/z 182.0, 20.5% (M+—m-Br-Ph); m/z 155.0, 45.4% (M+—m-
Br-Ph— CN—1H). Anal. Calcd for C17H12BrN3 (338.02): C, 60.37;
H, 3.58; Br, 23.63; N, 12.42 Found: C, 60.39; H, 3.59; Br, 23.71; N,
12.39.
5.1.5. 2-Amino-1-(4-bromophenyl)-5-phenyl-1H-pyrrole-3-
carbonitrile (4d)
Crystallized from ethyl acetate as a yellow powder. Rf = 0.31
(petroleum ether/ethyl acetate, 1:1). Yield 83%; mp 110–112 °C;
1H NMR (DMSO-d6) d 6.45 (s, 1H, CH), d 6.97 (d, J = 8.1 Hz, 2H,
5. Experimental
5.1. Physical measurements
H2+H6Aromatic
protons),
d
7.10–7.20
(m,
5H,
H30ꢀH40+H50+H3+H5), d 7.50 (d, J = 8.1 Hz, 2H, H20+H60), 8.14 (br,
NH2, D2O exchangeable). 13C NMR (500 MHz, DMSO-d6): 14.49,
71.36, 109.13, 116.64, 122.20, 124.34, 127.18, 128.77, 131.18,
132.36, 135.77, 144.99, 149.82. IR (cmꢀ1): 3458, 3379 (NH2),
2257 (CN). Anal. Calcd for C17H12BrN3 (337.02): C, 60.37; H, 3.58;
Br, 23.63; N, 12.42. Found: C, 60.31; H, 3.50; Br, 23.61; N, 12.47.
Microanalyses, spectral data of the compounds were performed
in the Microanalytical, National Research Centre, Cairo, Egypt. The
IR spectra (4000–400 cmꢀ1) were recorded using KBr pellets in a
Jasco FT/IR 300E Fourier transform infrared spectrophotometer
on a Perkin Elmer FT-IR 1650 (spectrophotometer). The NMR spec-
tra were recorded using Joel EX-270 MHz and 500 MHz NMR spec-
trophotometers. Chemical shifts are reported in parts per million
(ppm) from the tetramethylsilane resonance in the indicated sol-
vent. Coupling constants are reported in Hertz (Hz); spectral split-
ting partners are designed as follow: singlet (s); doublet (d); triplet
(t); multiplet (m). The mass spectra were carried out using Finni-
gan mat SSQ 7000 (Thermo. Inst. Sys. Inc., USA) spectroscopy at
70 ev.
5.1.6. 2-Amino-1-(3-chlorophenyl)-5-phenyl-1H-pyrrole-3-
carbonitrile (4e)
Crystallized from ethyl acetate as yellow powder. Rf = 0.29
(petroleum ether/ethyl acetate, 1:1). Yield: 83%; mp 185–187 °C;
1H NMR (DMSO-d6) d: 6.41 (s, 1H, CH), 6.94–7.41 (m, 9H, Aromatic
protons), 8.42 (br, NH2, D2O exchangeable). IR (cmꢀ1): 3440, 3337

6998
M. A. Omar et al. / Bioorg. Med. Chem. 20 (2012) 6989–7001
(NH2), 2258 (CN). MS: m/z 294.0, 100.0% (M++1); m/z 257.0, 24.6%
(M+—Cl), m/z 181.0, 12.6% (M+—m-ClPh). Anal. Calcd for C17H12ClN3
(293.07): C, 69.51; H, 4.12; Cl, 12.07; N, 14.30. Found: C, 59.55; H,
4.14; Cl, 12.02; N, 14.36.
5.1.12. 3-(1H-Benzo[d]imidazol-2-yl)-5-phenyl-1-(pyridin-4-yl)-
1H-pyrrol-2-amine (6b)
Crystallized from ethyl acetate as a green powder. Rf = 0.38
(petroleum ether/ethyl acetate/methanol, 1:1:1/2). Yield: 1.9 g
(70%); mp 135–137 °C; 1H NMR (DMSO-d6) d: 6.09 (s, 1H, CH),
7.80–7.09 (m, 9H, Aromatic protons), 7.98–8.16 (m, 2H, Aromatic
protons), 8.22 (br, NH2, D2O exchangeable), 8.60 (d, J = 4 Hz 2H,
CH pyridine), 12.59 (br, NH, benzimidazole, D2O exchangeable).
IR (cmꢀ1): 3393 (NH), 3323, 3260 (NH2). Anal. Calcd for C22H17N5
(351.15): C, 75.19; H, 4.88; N, 19.93. Found: C, 75.26; H, 4.85; N,
19.89.
5.1.7. 2-Amino-1-(4-chlorophenyl)-5-phenyl-1H-pyrrol-3-
carbonitrile (4f)
Crystallized from chloroform as a yellow powder. Rf = 0.34
(petroleum ether/ethyl acetate, 1:1). Yield: 79%; mp 98–100 °C;
1H NMR (DMSO-d6): d 6.41 (s, 1H, CH), 7.63–6.78 (m, 9H, Aromatic
protons), 8.22 (br, NH2, D2O exchangeable). IR (cmꢀ1): 3321, 3225
(NH2), 2208 (CN). MS: m/z 294.0, 10.6% (M++1); m/z 214.0, 1.8%
(M+—Ph—2H); m/z 104, 9.5% (M+—Ph—p-ClPh). Anal. Calcd for
5.1.13. 3-(1H-Benzo[d]imidazol-2-yl)-1-(3-bromophenyl)-5-
phenyl-1H-pyrrol-2-amine (6c)
C17H12ClN3 (293.07): C, 69.51; H, 4.12; Cl, 12.07; N, 14.30. Found:
Crystallized from ethyl acetate/methanol (2:5) as a brown pow-
der. Rf = 0.44 (petroleum ether/ethyl acetate/methanol, 1:1:1/2).
Yield: 1.8 g (72%); mp 192–194 °C; 1H NMR (DMSO-d6) d 6.45 (s,
1H, CH), d 7.48–8.05 (m, 13H, Aromatic protons), 8.32 (br, NH2,
D2O exchangeable), 12.55 (br, NH, benzimidazole, D2O exchange-
able). IR (cmꢀ1): 3374 (NH), 3318, 3249 (NH2). MS: m/z 415.0,
1.3% (M++2 –NH2); m/z 313.0, 4.3% (M++1—benzimidazole part).
Anal. Calcd for C23H17BrN4 (429.06): C, 64.35; H, 3.99; Br, 18.61;
N, 13.05. Found: C, 64.38; H, 4.01; Br, 18.59; N, 13.10.
C, 69.55; H, 4.13; Cl, 12.15; N, 14.28.
5.1.8. 2-Amino-1-(1H-benzo[d]imidazol)-5-phenyl-1H-pyrrole-
3-carbonitrile (4g)
Crystallized from chloroform as a brown powder. Rf = 0.28
(petroleum ether/ethyl acetate, 1:1). Yield: 82%; mp 122–124 °C;
1H NMR (DMSO-d6) d: 6.34 (s, 1H, CH), 7.69–7.33 (m, 9H, Aromatic
protons), 8.40 (br, NH2, D2O exchangeable) 12.55(br, NH, benz-
imidazole, D2O exchangeable). 13C NMR (500 MHz, DMSO-d6):
18.56, 66.76, 107.25, 114.93, 122.59, 128.75, 134.75, 135.35,
142.34, 164.55, 194.86. IR (cmꢀ1): 3359, 3283, 3206 (NH) and
(NH2), 2258 (CN). MS: m/z 300.0, 0.89% (M++1); m/z 183.0, 29.0%
(M++1—benzimidazole part); m/z 104, 100.0% (M++1—benzimid-
azole part—Ph—2H). Anal. Calcd for C18H13N5 (299.12): C, 72.23;
H, 4.38; N, 23.40. Found: C, 72.27; H, 4.40; N, 23.36.
5.1.14. 3-(1H-Benzo[d]imidazol-2-yl)-1-(4-bromophenyl)-5-
phenyl-1H-pyrrol-2-amine (6d)
Crystallized from chloroform/methanol (2:5) as a brown pow-
der. Rf = 0.37 (petroleum ether/ethyl acetate/methanol, 1:1:1/2).
Yield: 72%; mp 200–202 °C; 1H NMR (DMSO-d6) d 6.40(s, 1H, CH
pyrrol), d 6.93–6.91 (m, 5H, Aromatic protons), d 7.11–7.14 (m,
2H, Aromatic protons), d 7.43(d, J = 8.4 Hz, 2H, Aromatic protons),
d 7.59(d, J = 8.4 Hz, 2H, Aromatic protons), 8.34 (br, NH2, D2O
exchangeable), 12.65 (br, NH, benzimidazole, D2O exchangeable).
IR (cmꢀ1): 3423, 3299, 3212 (NH) and (NH2). MS: m/z 339.0,
0.02% (M++2—Ph—NH); m/z 273.0, 6.2% (M+—p-Br-Ph); m/z 144,
100.0% [M++2—p-Br-Ph—NH2—benzimidazole unit +2H], m/z 143,
48.9% [M++2—p-Br-Ph—NH2—benzimidazole unit +1H]. Anal. Calcd
for C23H17BrN4 (429.06): C, 64.35; H, 3.99; Br, 18.61; N, 13.05
Found: C, 64.37; H, 3.90; Br, 18.67 N, 13.10.
5.1.9. 1-((1H-benzo[d]imidazol-2-yl)methyl)-2-amino-5-
phenyl-1H-pyrrole-3-carbonitrile (4h)
Crystallized from chloroform as a green powder. Rf = 0.24
(petroleum ether/ethyl acetate, 1:1). Yield: 4.9 g (73%); mp 131–
133 °C; 1H NMR (DMSO-d6): d 5.07 (s, 2H, CH2), 6.21 (s, 1H, CH),
7.56–7.54 (m, 5H, H5+H6+H30+H4+H50), 7.68 (d, J = 6.9HZ,2H,
H20+H60), 7.68 (d, J = 6.9HZ, 2H, H4+H7), 8.35 (br, NH2, D2O
exchangeable), 12.52(br, NH, benzimidazole, D2O exchangeable).
13C NMR (500 MHz, DMSO-d6): 18.55, 39.79, 114.96, 128.77,
129.49, 134.78, 135.31,194.89. IR (cm-1): 3411, 3329, 3225 (NH),
(NH2), 2206 (CN), 1677 (C@N). Anal. Calcd for C19H15N5
(313.13): C, 72.83; H, 4.82; N, 22.35. Found: C, 72.79; H, 4.39;
N, 2.41.
5.1.15. 3-(1H-Benzo[d]imidazol-2-yl)-1-(3-chlorophenyl)-5-
phenyl-1H-pyrrol-2-amine (6e)
Crystallized from ethyl acetate/methanol (2:5) as a brown pow-
der. Rf = 0.39 (petroleum ether/ethyl acetate/methanol, 1:1:1/2).
Yield: 66%; mp 200–202 °C; 1H NMR (DMSO-d6) d 6.51 (s, 1H, CH
pyrrol), 7.52–7.49 (m, 5H, 2H benzimidazole +4H Aromatic pro-
tons), 7.63–7.60 (m, 4H, Aromatic protons), 7.65 (d, J = 7.65, 2H,
Aromatic protons), 7.98 (d, J = 7.65, 2H, Aromatic protons), 8.28
(br, NH2, D2O exchangeable), 12.25 (br, NH, benzimidazole, D2O
exchangeable). IR (cmꢀ1): 3331 (NH), 3274, 3258 (NH2). MS: m/z
382.0, 0.03% (M+—2H); m/z 272.0, 3.5% (M+—m-Cl-Ph); m/z 143,
59.1% [M++2 –m-Cl-Ph—NH2—benzimidazole unit + 1H]. Anal.
Calcd for C23H17ClN4 (384.11): C, 71.78; H, 4.45; Cl, 9.21; N,
14.56. Found: C, 71.72; H, 4.38; Cl, 9.18; N, 14.51.
5.1.10. General procedure for the preparation of compounds
(6a–h)
A mixture of o-phenylenediamine (0.8 g, 7.5 mmol) and pyrrole
derivatives (7.5 mmol) in 4 N HCl (20 mL) was refluxed for 6 h. The
reaction mixture was allowed to cool and then neutralized with
NH4OH. The resulting precipitate was filtered off and recrystallized
from appropriate solvent.
5.1.11. 3-(1H-Benzo[d]imidazol-2-yl)-5-phenyl-1-(pyridin-2-yl)-
1H-pyrrol-2-amine (6a)
Crystallized from ethyl acetate as a yellowish green powder.
Rf = 0.18 (petroleum ether/ethyl acetate/methanol, 1:1:1/2). Yield:
1.9 g (70%); mp 170–172 °C; 1H NMR (DMSO-d6) d: 6.26 (s, 1H, CH),
7.35–7.45 (m, 12H, Aromatic protons), d 8.56 (d, J = 4HZ, 1H, CH
pyridine), 8.22 (br, NH2, D2O exchangeable), 12.69 (br, NH, benz-
imidazole, D2O exchangeable). IR (cmꢀ1): 3455 (NH), 3376, 3325
(NH2). MS: m/z 350.0, 1.3% (M+—1H); m/z 273.0, 2.4% (M++1—py);
m/z 259.0, 10.3% (M++2—Ph–NH2—1H). Anal. Calcd for C22H17N5
(351.15): Anal. Calcd for C22H17N5 (351.15): C, 75.19; H, 4.88; N,
19.93. Found: C, 75.18; H, 4.79; N, 19.99.
5.1.16. 3-(1H-Benzo[d]imidazol-2-yl)-1-(4-chlorophenyl)-5-
phenyl-1H-pyrrol-2-amine (6f)
Crystallized from ethyl acetate/methanol (2:5) as a brown pow-
der. Rf = 0.36 (petroleum ether/ethyl acetate/methanol, 1:1:1/2).
Yield: 73%; mp 133–135 °C; 1H NMR (DMSO-d6) d 6.38 (s, 1H,
CH), 7.59–7.33 (m, 13H, Aromatic protons), 8.34 (br, NH2, D2O
exchangeable), 12.49 (br, NH, benzimidazole, D2O exchangeable).
IR (cmꢀ1): 3411, 3329, 3243, (NH) and (NH2). MS: m/z 383.0,
1.7% (M+—1H). Anal. Calcd for C23H17ClN4 (384.11): C, 71.78; H,
4.45; Cl, 9.21; N, 14.56. Found: C, 71.72; H, 4.48; Cl, 9.29; N, 14.61.

M. A. Omar et al. / Bioorg. Med. Chem. 20 (2012) 6989–7001
6999
5.1.17. 1,3-Di (1H-Benzo[d]imidazol-2-yl)-5-phenyl-1H-pyrrol-
2-amine (6g)
formamide (30 mL), 2-chloromethylbenzimidazole42 (2.32 g,
0.015 mol) was added and the reaction mixture was stirred for
8 h at room temperature, then poured onto ice water. The obtained
precipitate was filtered off, dried and recrystallized from ethanol as
a green powder. Rf = 0.31 (petroleum ether/ethyl acetate, 2:1).
Yield: 1.8 g (65%); mp 181–183 °C; 1H NMR (DMSO-d6) d: 4.51 (s,
2H, CH2), 7.49–7.01 (m, 10H, Aromatic protons), 8.12 (d,
J = 8.4 Hz, 2H), 9.36 (s, 1H, NH, D2O exchangeable), 12.78 (br, NH,
benzimidazole, D2O exchangeable). IR (cmꢀ1): 3368 (NH), 1673
(C@O), 1592 (C@N). MS: m/z 394, 1.4% (M++2); m/z 261, 3.1%
(M+—benzimidazole part—CH2). Anal. Calcd for C23H16N6O
(392.14): C, 70.40; H, 4.11; N, 21.42. Found: C, 70.35; H, 4.13; N,
21.38.
Crystallized from ethyl acetate/methanol (2:5) as a green pow-
der. Rf = 0.42 (petroleum ether/ethyl acetate/methanol, 1:1:1/2).
Yield: 74%; mp 160–162 °C; 1H NMR (DMSO-d6): d 6.36 (s, 1H,
CH), 7.17–7.42 (m, 13H, Aromatic protons), 8.12 (br, NH2, D2O
exchangeable), 12.78 (br, NH, benzimidazole, D2O exchangeable).
IR (cmꢀ1): 3473, 3333, 3298, 3212, 2(NH) and (NH2). MS: m/z
391, 6.12% (M++1). Anal. Calcd for C24H18N6 (390.16): C, 73.83; H,
4.65; N, 21.52. Found: C, 73.79; H, 4.67; N, 21.56.
5.1.18. 1-((1H-Benzo[d]imidazol-2-yl)methyl)-3-(1H-
benzo[d]imidazol-2-yl)-5-phenyl-1H-pyrrol-2-amine (6h)
Crystallized from ethyl acetate/methanol (2:5) as a brown pow-
der. Rf = 0.41(petroleum ether/ethyl acetate/methanol, 1:1:1/2).
Yield: 76%; mp 185–187 °C; 1H NMR (DMSO-d6) d 5.11 (s, 1H,
CH2), 6.12 (s, 1H, CH2), 7.68–7.53 (m, 13H, Aromatic protons),
8.10 (br, NH2, D2O exchangeable), 12.65 (br, NH, benzimidazole,
D2O exchangeable). 13C NMR (500 MHz, DMSO-d6): 23.00, 28.44,
33.65, 36. 01, 114.72, 122.63, 128.47, 129.24, 133.73, 136.84,
136.96, 137. 43, 154.89, 174.39, 198.85, 199.02. IR (cmꢀ1): 3224,
3274, 3086, 3136, 2(NH) and (NH2), 2208 (CN). MS: m/z 404.9,
22% (M+); m/z 286.1, 0.45% (M+—Benzimidazole moiety); m/z
295, 6.6% [M++2-(NH2+CN)]. Anal. Calcd for C25H20N6 (404.17): C,
74.24; H, 4.98; N, 20.78. Found: C, 74.20; H, 4.99; N, 20.73.
5.1.22. General procedure for the preparation of compounds
(13, 14)
To a solution of compound 12 (0.005 mol) and anhydrous potas-
sium carbonate (0.005 mol) in N,N-dimethylformamide (30 mL),
allyl bromide or ethylbromoacetate (0.005 mol) was slowly added
dropwise. The mixture was stirred at room temperature for 5 h, the
mixture was then poured onto ice water, and the resulting precip-
itate was collected by filtration and recrystallized from appropriate
solvent.
5.1.23. [2-(13-Oxo-12,13-dihydro-5,6,11,12-tetraaza-
benzo[4,5]cyclohepta[1,2-b]naphth-6-ylmethyl)-benzimidazol-
1-yl]prop-1-ene (13)
5.1.19. 3-Amino-1H-quinoxalin-2-one (8).41
A mixture of o-phenylenediamine (0.04 mmol) and ethyl oxam-
ate (0.06 mmol) in pyridine (25 mL) was stirred under reflux for
8 h. The reaction mixture was poured into water and the precipi-
tate formed was filtered off, washed and crystallized from ethanol
as a buff powder. Rf = 0.36 (petroleum ether/ethyl acetate/metha-
nol, 1: 1: 0.1).Yield: 95%, mp >300 °C. 1H NMR (500 MHz, DMSO-
d6): 7.25(m, 4H); 11.21(br., 2H, NH2, D2O exchangeable),
13.19(br, 1H, NH, D2O exchangeable). 13C NMR (500 MHz, DMSO-
d6): 115.85, 123.95, 125.8, 129.1, 131.6, 142.7, 155.3, 157.5. IR
(cmꢀ1): 3492.45(NH quinoxaline), 3288, 3208 (NH2), 3050, 2968
(CH, aromatic), 1682 (C@O), 1613 (C@N), 1529 (C@C). MS: [m/z
(rel. abundance)]: 161(M+, 100%). Anal. Calcd for C8H7N3O (FW:
161.16): C, 59.62; H, 4.38; N, 26.07. Found: C, 59.58; H, 4.41; N,
26.19.
Crystallized from ethanol as a brown powder. Rf = 0.61 (petro-
leum ether/ethyl acetate, 2:1). Yield: 72%; mp 98—100 °C; 1H
NMR (DMSO-d6) d 5.12 (d, J = 4.56, 2H, CH2), 5.42(m, 2H, @CH2),
6.05(m, 1H, @CH), 7.05–8.45 (m, 10H, Aromatic Protons), 8.16 (d,
J@8.11, 2H), 9.54 (s, 1H, NH, D2O exchangeable). IR (cmꢀ1): 3325
(NH), 1653 (CO). MS: m/z 277, 100.0% (M++2—propene group—
benzimidazole part); m/z 263, 1.2% (M+—propene group—benz-
imidazole part—CH2). Anal. Calcd for C26H20N6O (432.17): C,
72.21; H, 4.66; N, 19.43. Found: C, 72.37; H, 4.58; N, 19.49.
5.1.24. [2-(13-Oxo-12,13-dihydro-5,6,11,12-tetraaza-
benzo[4,5]cyclohepta[1,2-b]naphth- alene-6-ylmethyl)-
benzimidazol-1-yl]-acetic acid ethyl ester (14)
Crystallized from ethanol as a brown powder. Rf = 0.44 (petro-
leum ether/ethyl acetate, 2:1). Yield: 70%; mp 121—123 °C; 1H
NMR (DMSO-d6) d 1.02–1.2 (t, 3H, CH3), 4.16 (s, 2H, COCH2), 4.53
(q, 2H, OCH2), 5.18 (s, 2H, CH2), 7.03–8.34 (m, 10H, Aromatic Pro-
tons), 8.11 (d, J = 8.11, 2H), 9.43 (s, 1H, NH, D2O exchangeable). IR
(cmꢀ1): 3302 (NH), 1741 (OCO), 1682 (CO amide). MS: m/z 479,
1.6% (M++1); m/z 389, 1.3% (M+—C2H5OCOCH2—2H). Anal. Calcd
for C27H22N6O3 (478.18): C, 67.77; H, 4.63; N, 17.56. Found: C,
67.72; H, 4.55; N, 17.52.
5.1.20. 6,12-Dihydro-5,6,11,12-tetraaza-
benzo[4,5]cyclohepta[1,2-b]naphthalene-13-one (10)
A mixture of anthranilic acid (0.012 mol) and compound 8
(0.012 mol) was heated on a sand path at 120–130 °C for 4 h. Sub-
sequently the melt was allowed to cool for 30 min. at room tem-
perature during this period the melted was solidified. It was
treated with an aqueous solution of sodium bicarbonate (10%) in
order to dissolve any unreacted acid into the cyclized product.
The solid was filtered off and washed with water in order to re-
move any inorganic materials and recrystallized from ethanol as
a green powder. Rf = 0.74 (petroleum ether/ethyl acetate, 2:1).
Yield: 84%; mp 168–170 °C; 1H NMR (DMSO-d6) d 7.5–7.2 (m, 4H,
H2+H3+H9+H10), 7.7–7.6 (m, 4H, H1+H4+H8+H11), 9.41 (s, 1H,
NH, D2O exchangeable). IR (cmꢀ1): 3370 (NH), 3316 (NH), bonded
OH centered at 2911, 1675 (C@O), 1592 (C@N). MS: m/z 263, 24%
(M++1); m/z 237, 100% (M++2—CO). Anal. Calcd for C15H10N4O
(262.09): C, 68.69; H, 3.84; N, 21.36. Found: C, 68.76; H, 3.79; N,
21.41.
5.2. Antitumor activity
5.2.1. Cytotoxicity against lung cancer cell line A549
Cell culture cytotoxicity assays were performed using the ala-
marBlue method, as previously described.42 Briefly, aliquots of
100
l
L of cell suspension (1.0–2.5 ꢁ 103 cells) were placed in 96-
well microtiter plates in an atmosphere of 5% CO2 at 37 °C. After
24 h, 100 lL of culture medium and 2 lL of compound in vehicle
(culture media with 40% pyridine) or vehicle alone were added,
and the plates incubated an additional 72 h. The final pyridine con-
centration in all cases was 0.4%; at this pyridine concentration,
there was no effect on the growth of cells compared to cells in cul-
ture media without added pyridine. Compounds, along with mito-
mycin C as positive control, were evaluated in duplicate at final
5.1.21. 6-((1H-Benzo[d]imidazol-2-yl)methyl)-6,12-dihydro-
5,6,11,12-tetraaza-benzo[4,5]cyclohepta[1,2-b]naphthalene-13-
one (12)
To a stirred solution of compound 10 (4 g, 0.015 mol) and anhy-
drous potassium carbonate (2.1 g, 0.015 mol) in N,N-dimethyl-
concentrations ranging from 0.001 to 100
media had been removed from each well, 200
l
M. After the culture
l
L of fresh media

7000
M. A. Omar et al. / Bioorg. Med. Chem. 20 (2012) 6989–7001
and 20
l
L of 90% alamarBlue reagent were added, followed by
5.3.1. Preparation of ligands and target Urokinase plasminogen
activator (uPA) receptors
additional 6 h incubation. The fluorescent intensity was measured
using a SpectrafluorPlus plate reader with excitation at 530 nm and
emission at 590 nm. Results are reported as IC50 values, the aver-
age concentration required to produce a decrease of fluorescent
intensity of 50% relative to vehicle-treated controls in two separate
determinations.
The compounds involved in this study as ligands are com-
pounds which were studied for their binding affinity into Uroki-
nase plasminogen activator (uPA) receptors. The Molecule
Builder tool in MOE was used to construct a three-dimensional
model of the structures .Energy minimization was done through
Force field MMFF94x Optimization using gradient of 0.0001 for
determining low energy conformations with the most favorable
(lowest energy) geometry.
The crystal structures of uPA receptors were obtained from the
Protein Data Bank http://www.rcsb.org/pdb/Welcome.do (PDB
code: 1c5x). Hydrogens and partial charges were added to the pro-
tein with the Protonate3D application in MOE. This application is
to assign ionization states and position hydrogens in the macromo-
lecular structure.
5.2.2. Cytotoxicity against Breast cancer MCF-7 cell line
The antitumor activity against MCF-7 was performed in the Na-
tional Cancer Institute, Cancer Biology Department, Cairo, Egypt.
Potential cytotoxicity of compounds 4g,h, 6a–h, 10–14 was tested
using the method of Skehan et al.44 Cells were plated in 96-multi-
well plate (104 cells/well) for 24 h before treatment with the com-
pounds to allow attachment of cell to wall of the plate. Different
concentrations of the compound under test (0, 1, 2.5, 5 and
10
l
g/mL) were added to the cell monolayer. Triplicate wells were
As most of protein structures obtained from the Protein Data
Bank contain little or no hydrogen coordinate data due to limited
resolution. Yet, the hydrogen bond network and ionization states
can have a dramatic effect on simulations results.
prepared for each individual dose. Monolayer cells were incubated
with the compounds for 48 h at 37 °C and in atmosphere of 5% CO2.
After 48 h cells were fixed, washed and stained with Sulforhoda-
mine B stain. Excess stain was washed with acetic acid and at-
tached stain was recovered with Tris EDTA buffer. Color intensity
was measured in an ELISA reader. The relation between surviving
fraction and drug conc. is plotted to get the survival curve of each
tumor cell line after the specified compound.
5.3.2. Molecular modeling and analysis of the docked results
The binding free energy was used to rank the binding affinity of
the synthesize compounds to uPA receptor. Also, Hydrogen bonds
between the ligand and amino acids in uPA receptors were used
in the ranking of the compounds. Evaluation of the hydrogen bonds
was done by measuring the hydrogen bond length which doesn’t
exceed 3 Å. The mode of interaction of the native ligand uPA recep-
tors within their crystal structures was used as a standard docked
model as well as for RMSD calculation.
5.2.3. Antitumor promoting effect in vitro (Epstein–Barr virus
activation test)
Compounds 4g, 4h, 6a–h, 8,10,12,13 and 14 were tested for Ep-
stein–Barr virus (EBV) Inhibitory Activity. The inhibition of EBV-EA
activation was assayed using methods reported in the literature.
46–48
The cells were incubated at 37 °C for 48 h in a medium con-
5.4. Determination the level of uPA protein expression
taining butyric acid (4 nM), TPA (32 pM), and various amounts of
test compounds. Smears were made from the cell suspensions
and the EBV-EA inducing cells were stained using an indirect
immunofluorescence technique. In each assay, at least 500 cells
were counted, and the number of stained cells (positive cells)
was recorded. Triplicate assays were per-formed for each data
point. The EBV-EA inhibitory activity of the test compound was
compared with that of the control experiment with butyric acid
plus TPA.
In control experiments, the EBV-EA inhibitory activities were
generally around 40%, and these values were taken as a positive
control. The viability of the cells was assayed by the Trypan-blue
staining method. For the determination of cytotoxicity, the cell via-
bility was required to be more than 60%.
The level of uPA protein expression was determined using
AssayMax human urokinase (uPA) ELISA kit (Assaypro, USA)
according to manufacturer’s instructions, with absorption mea-
sured at 450 nm. Compounds 4h, 6b and 13 as well as standards
were incubated for 48 h with A549 and MCF7 cells at concentration
ranging from 0 to IC50 values of each compound which shown in
Tables 5 and 6.
After 48 h from compounds treatment, medium was collected
and centrifuged at 2000ꢁ g for 10 min to remove cellular debris.
Add 50
were washed with 200
ylated uPA antibody to each well and incubate for 1 h at 25 °C.
After washing, plates were incubated with 50 l of streptavidin-
peroxidase conjugate per well and incubate for 30 min then wash
the microplate as described above. Add 50 l of chromogen sub-
strate per well and incubate for about 10 min or till the optimal
blue color density develops. Add 50 l of stop solution to each well.
ll of the cell extract per well and incubate for 2 h. Wells
l
l of wash buffer then add 50 ll of biotin-
l
5.3. Molecular docking study
l
The docking studies were carried out using Molecular Operating
Environment (MOE) 2008.10. First, a Gaussian Contact surface
around the binding site was drawn. This surface surrounds the
van der Waals surface of a molecule (filling in solvent inaccessible
gaps). Then docking studies were carried out to evaluate the bind-
ing free energy of the inhibitors within the macromolecules. The
Dock scoring in MOE software was done using London dG scoring
function and has been enhanced by using two different refinement
methods, the Forcefield and GridMin pose have been updated to
ensure that refined poses satisfy the specified conformations. We
allowed rotatable bonds; the best 10 poses were retained and ana-
lyzed for the binding poses best score. The database browser was
used in MOE to compare the docking poses to the ligand in the
co-crystallized structure and to compare the docking pose of the
synthesized compounds to the co-crystal ligand position.
l
The color will change from blue to yellow. Read the absorbance on
a microplate reader at a wavelength of 450 nm immediately. The
concentrations of uPA in the samples were determined based on
the uPA standard curves (0–12 ng).
Acknowledgements
This study was supported by the ‘US–Egypt Joint Science and
Technology Board Fund’ administered through the USDA (BIO9-
002-015). The authors are grateful to the National Cancer Institute,
Cancer Biology Department, Cairo, Egypt, to perform the antitumor
activity against MCF-7. Deep thanks are expressed to the ‘Science &
Technology Development Fund’ for financially supporting the man-
uscript through the fund of the STDF project No 1517.

M. A. Omar et al. / Bioorg. Med. Chem. 20 (2012) 6989–7001
24. Meyer, T.; Hart, I. R. Eur. J. Cancer 1998, 34, 214.
7001
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