Design and development of oxobenzimidazoles as novel androgen receptor antagonists

Article abstract of DOI:10.1007/s00044-016-1504-3

Antiandrogens are a novel class of anticancer agents that inhibit cancer cell proliferation and induce apoptosis in various cell lines. To find the lead compound from the oxobenzimidazole derivatives, receptor-ligand docking studies were initially performed using Schr?dinger software. The best fit molecules were synthesized and characterized through IR, ¹H-NMR, ¹³C-NMR and HRMS analyses. The structure of compound (9b) was further confirmed by single-crystal XRD analysis. The cell viability of the compounds was determined by MTT assay to find IC₅₀ values against prostate cancer and breast cancer cell lines (PC-3, LNCaP, MCF-7 and MDA-MB-231). The ADME/T property studies were performed to rationalize the inhibitory properties of these compounds. It can be concluded from the study that 9b is the most active compound from the series against PC-3 and LNCaP cell lines.

Full text of DOI:10.1007/s00044-016-1504-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2016) 25:539–552
DOI 10.1007/s00044-016-1504-3
ORIGINAL RESEARCH
Design and development of oxobenzimidazoles as novel androgen
receptor antagonists
1
2
1
•
R. Elancheran
3
•
K. Saravanan
2
•
Bhaswati Choudhury
3
4
•
S. Divakar
1
•
S. Kabilan
Jibon Kotoky
•
M. Ramanathan
•
Babulal Das
1
R. Devi
•
Received: 5 February 2015 / Accepted: 6 January 2016 / Published online: 21 January 2016
Ó Springer Science+Business Media New York 2016
Abstract Antiandrogens are a novel class of anticancer
agents that inhibit cancer cell proliferation and induce
apoptosis in various cell lines. To find the lead compound
from the oxobenzimidazole derivatives, receptor-ligand
docking studies were initially performed using Schr o¨ dinger
software. The best fit molecules were synthesized and
Introduction
Androgen receptor (AR), belongs to the nuclear receptor
subfamily, plays an important role in the development,
growth, function and homeostasis of the prostate. The
National Cancer Institute (NCI) has estimated that 220,800
men will be diagnosed with and 27,540 men will die of
prostate cancer in the USA in 2015 (Ferlay et al., 2010;
Howlader et al., 2015). Recently, we reported that AR is
one of the attractive targets to treat prostate cancer (PCa)
(Elancheran et al., 2015b). Although androgens are often
considered to be ‘‘male’’ hormones, they are also found at
lower levels in women. Recent studies have found that AR
is frequently expressed in primary breast tumors, is esti-
mated to be 50–90 %, depending on the subtypes of breast
cancer, and could respond to antiandrogen treatment
1
13
characterized through IR, H-NMR, C-NMR and HRMS
analyses. The structure of compound (9b) was further
confirmed by single-crystal XRD analysis. The cell via-
bility of the compounds was determined by MTT assay to
find IC50 values against prostate cancer and breast cancer
cell lines (PC-3, LNCaP, MCF-7 and MDA-MB-231). The
ADME/T property studies were performed to rationalize
the inhibitory properties of these compounds. It can be
concluded from the study that 9b is the most active com-
pound from the series against PC-3 and LNCaP cell lines.
(Fioretti et al., 2014). Antiandrogens, such as bicalutamide
Keywords Androgen receptor Á Prostate cancer Á
Antiandrogen Á Breast cancer Á Oxobenzimidazoles
(Chen et al., 2005; Gao et al., 2006), cyproterone acetate
(Figg et al., 2010; Neumann and T o¨ pert, 1986), flutamide
(
Cleve et al., 2011; Brogden and Clissold, 1989) and
nilutamide (Hsieh and Ryan, 2008; Moguilewsky et al.,
987; Kassouf et al., 2003) have been used to block the
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-016-1504-3) contains supplementary
material, which is available to authorized users.
1
androgen signal, but they have several side effects.
Recently, novel AR antagonist, enzalutamide (Scher et al.,
2010; Tran et al., 2009), has demonstrated efficacy against
castration-resistant prostate cancer (CRPC) and estrogen
receptor-negative tumors. Among the subtypes, triple-
negative breast cancers (ER-, PR-, HER2-) were positive
for AR expression and may respond to treatment with
antiandrogen drugs (Ni et al., 2011). ARN-509
(IC50 = 16 nmol/L) binds AR with seven to tenfold
greater affinity than the clinically approved antiandrogen,
bicalutamide (median IC50 = 160 nmol/L), and competes
for the same binding site in the ligand-binding pocket of
the receptor (Clegg et al., 2012). As a result, there is an
&
Jibon Kotoky
jkotoky@gmail.com
1
Drug Discovery Laboratory, Life Sciences Division, Institute
of Advanced Study in Science and Technology, Guwahati,
Assam 781035, India
2
3
4
Department of Chemistry, Annamalai University,
Annamalai Nagar, Tamilnadu 608002, India
Department of Pharmacology, PSG College of Pharmacy,
Coimbatore, Tamilnadu 641 004, India
Department of Chemistry, Indian Institute of Technology,
Guwahati, Assam 781035, India
123

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Med Chem Res (2016) 25:539–552
increasing need for the development of new therapy for
prostate cancer and breast cancer with better activity profile
and less toxicity. Therefore, the concept of non-steroidal,
AR antagonists emerged as an attractive target to overcome
these problems.
yield, reaction time, reaction temperature and sensitivity
toward moisture. Here, we report the design, synthesis and
biological evaluation of few 4-(1-substituted-1,2-dihydro-
2-oxobenzo[d]imidazol-3-yl)-2-(trifluoromethyl) benzoni-
trile as novel AR antagonists.
Carbamide (CO(NH ) ), thiocarbamide (SC(NH ) ) and
2
2
2 2
sulfonamide (SO NH ) derivatives are the basis of several
2
2
groups of drugs that have very good biological activities
Results and discussion
against cancer (Balasubramaninan et al., 2006; Balaji et al.,
2
014). Oxobenzimidazoles which are highly useful syn-
In an attempt to understand the binding mode of our
oxobenzimidazole analogues, the ligands (100 compounds)
were docked with the crystal structure of the human
androgen receptor ligand-binding domain (hARLBD) in
complex with dihydrotestosterone agonist (PDB:2AMA)
from the Protein Data Bank (J e´ sus-Tran et al., 2006). To
identify molecular determinants responsible for this selec-
tivity, we have taken the crystal structure which contains
the hydrophobic, flexible pockets with two deep residues
(ARG 752, GLN 711). The metabolic product, DHT, has
greater affinity (GS = -10.423 kcal/mol) toward AR
which forms hydrogen bonds through the 17b hydroxyl
group with two residues (ASN 705, THR 877) in the Helix
12 shown in Fig. 2a, b. The protein was first prepared using
the Protein Preparation wizard, and the docking studies
were performed using the Schr o¨ dinger Glide software
(Maestro 9.5) with extra-precision (XP) mode (Schr o¨ -
dinger, 2013; Friesner et al., 2004; Halgren et al., 2004;
Friesner et al., 2006). Out of 100 compounds, we got the
appreciable results from the compounds (9a–n). The nitrile
thetic intermediates have found a myriad of applications in
the area of therapeutics (Guo et al., 2012). Oxobenzimi-
dazole analogues were designed by the structural modifi-
cations of the three potent structures of the AR antagonist
(
Fig. 1). Taking into account the interesting properties of
these benzimidazole derivatives and in continuation of our
research, we designed a series of novel 4-(1-substituted-
1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)-2-(trifluoromethyl)
benzonitrile using the Schr o¨ dinger (Maestro 9.5) software.
Since there is no reported full antagonist-bound AR crystal
structure, the docking studies were carried out with the
available crystal structure of AR bound to steroid agonists
such as dihydrotestosterone (DHT) (J e´ sus-Tran et al.,
2
006). The compound, 4-(1-allyl-1,2-dihydro-2-oxobenzo
[
d]imidazol-3-yl)-2-(trifluoromethyl) benzonitrile has
shown similar binding characters like bicalutamide. In
recent years, cesium carbonate has found extensive appli-
cations as an excellent base for a variety of synthetic
transformations and is superior to other bases in terms of
Fig. 1 Three potent structures of AR antagonist discovered to date (1a, 1b, 1c) together and their structural modifications (Oxobenzimidazole
analogues)
1
23

Med Chem Res (2016) 25:539–552
541
protein interaction as detected by glide molecular docking
is shown in Tables 1 and 2.
Initially, the compounds were designed with the differ-
ent hydrogen acceptors (CN, Methyl amine, Morpholine) at
para position and hydrophobic groups (CF3, Cl, F) at the
meta position (7a–d). From the results, we found that CF3
and Cl at meta position, CN at para positions are essential
for getting good glide scores. Further, the compounds
3
extended with the substitution at R showed a better G
score compared to the compounds (7a–d). The 2D diagram
interactions of active AR antagonists (9a, 9b, 9d, 9f,
9
we found that the compounds (9a and 9b) showed the
m and 9n) are shown in Fig. 3. From the docking studies,
-
highest G scores (-9.19 and -9.12 kcal mol , respec-
1
tively) comparatively similar to the standard drug, bicalu-
-
1
tamide (-9.94 kcal mol ). Figure 4 represents the three
dimensional model of hARLBD in complex with
oxobenzimidazoles.
Based on the molecular docking results, the synthesis of
the ligands was considered to be important. The general
synthesis of oxobenzimidazoles (7a, 7b and 9a–c) is
depicted in schemes 1 and 2. These compounds were
synthesized by prepared bromo substituted benzene, com-
mercially available oxobenzimidazole and allyl or aryl
3
bromide (R ). The 4-Bromo-2-(substituted) benzonitrile
were obtained from 2-(substituted) aniline through the
successive bromination (Bartoli et al., 2009) cyanation and
diazotization followed by halogenations (Dalton et al.,
2008). Oxobenzimidazole coupling with substituted phenyl
bromide was affected in the presence of cesium carbonate
at N1 position to afford the requisite N-substituted
Oxobenzimidazoles, in low to moderate yield (60–90 %),
8–12 h. The similar reaction was reported by using potas-
sium carbonate, 6 days (Guo et al., 2012). Subsequently,
0
N-alkylation with allyl bromide and 2-Bromo-4 -fluo-
roacetophenone in the presence of cesium carbonate at N3
position yielded compounds (9a–c).
Fig. 2 a 3D model of dihydrotestosterone in docking with hARLBD
residues in the active site (colored by atom types: C tan, N blue, O
red, H light gray, F pink), b 2D diagram interactions of hARLBD
with Dihydrotestosterone (Color figure online)
New compounds, 7a, 7b and 9a–c were well charac-
terized by IR, NMR and HRMS, and all the data are in
accordance with the proposed structures. The generation of
1
the heterocyclic structure was confirmed by H NMR since
group of the ligand makes the hydrogen bond interactions
with ARG752 and GLN711 residues directly and also by
way of water-mediated interaction. This pattern of hydro-
gen bond interaction is similar to that seen with the nitrile
group of (R)-bicalutamide (Piatnitski Chekler et al., 2014).
The Oxobenzimidazole and its N-substituted rings are
oriented toward the GLY 708, THR 877, ASN 705 residues
to pick up additional hydrophobic interactions. It is inter-
esting to note that the TRP741 residue completely encloses
the binding cavity in AR X-ray structures complexes with
steroidal agonists such as DHT. Lowest binding energy for
the standard and designed ligand-AR (PDB, 2AMA)
the spectra exhibited the characteristic signals of oxoben-
zimidazole. Indeed, in addition to the signals at d 4.59
(doublet), d 5.34 (doublet of doublets) and d 5.90–5.98
(multiplet) correspond to the N-allyl group. ESI–MS of all
compounds showed M?H, M–H or M?K adduct as the
molecular ion.
The structure of the compound 9b was reconfirmed by
single-crystal X-ray diffraction method (Sheldrick, 1990,
1997). The Compound 9b was crystallized under triclinic
system with space group P-1. The unit cell parameters of
˚
the compound 9b were as follows: a = 7.6795(5) A,
˚
˚
b = 9.0374(6) A, c = 12.9452(9) A, a = 108.996(4)°,
123

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Med Chem Res (2016) 25:539–552
Table 1 Lowest binding energy for the Standard ligand-AR (PDB:2AMA) protein interaction as detected by GLIDE molecular docking
ligand
G Score
Lipophilic EvdW
PhobEn
HBond
Electro
Sitemap
LowMW
Penalties
RotPenal
Bicalutamide
Andarine
-9.94
-8.82
-6.47
-6.32
-5.86
-5.82
-5.22
-6.85
-5.71
-5.04
-4.78
-4.09
-4.79
-3.79
0
-1.11
-2.02
-0.04
-0.28
-0.7
-0.55
-0.76
-0.1
-1.6
-0.07
-0.41
-0.5
0
0.23
0.45
0
0
-0.45
-0.32
-0.4
0.08
0
Bexlosterid
Dutasteride
Nilutamide
Epristeride
Flutamide
-0.46
-0.41
0
-0.42
-0.15
0.13
-0.2
0
0.16
0.17
0.16
0.25
-0.59
-0.2
-0.5
0
-0.21
0
-0.73
-0.7
-0.17
-0.5
0
-0.19
-0.28
0
b = 100.334(4)°, g = 98.951(4)°. Figure 5 represented the
ORTEP diagrams of 9b (Elancheran et al., 2015a).
drugs: -3.0 to 1.0) the predicted apparent Madin-Darby
canine kidney (MDCK) cell permeability in nm s-1 (\25
poor, [500 great); calculated from the predicted Polariz-
ability, QPpolrz, IC50 value for blockage of HERG
K ? channels, log HERG apparent Caco-2 cell perme-
ability, QPP Caco in nm/s, metabolic reactions, #metab,
Polarizability, QP polrz.
The human prostate cancer cell lines (PC-3 and LNCaP)
and breast cancer cell lines (MCF-7 and MDA-MB-231)
were used for the cytotoxicity studies. Cellular viability in
the presence and absence of experimental agents was
determined using the standard MTT assay as described in
the Experimental section (Mosmann, 1983; Rubeinstein
et al., 1990; Ishioka et al., 2002; Schayowitz et al., 2008;
Khanfar and Sayed, 2010; Khatik et al., 2011; Nunez-
Nateras and Castle, 2011). The results of the cytotoxic
potential of compounds (9a–c, 7a and 7b) are listed in
Conclusion
Thanks to the recent disclosure of the hARLBD X-ray
structure, we were able to perform a structure-based design
of a novel class of AR antagonists. We designed a series of
oxobenzimidazoles using molecular docking, and the glide
scores were in good agreement with the observed IC50
values. It is evident from the docking calculations that the
3
Table 3. It is evident that the N-substitution at position, R
of the oxobenzimidazole core was influencing the activity
in the cell-based assay: N-allyl (9b), N-(4-Fluorphenyl)
ethanonyl (9a and 9c) displayed similar more active com-
pared to the compounds (7a and 7b). One of the most
active compounds of the series, 9b, contained 4-cyano-3-
3
substitution at R has better binding affinities than the free
(
trifluoromethyl) phenyl, which is present in many AR
amide. The compounds (7a, 7b and 9a–c) were prepared
from 2-(substituted) anilines for the cost reduction. The
compounds 9a–c exhibited the significant in vitro cyto-
toxicity. In particular, compound 9b was identified as the
most promising of the series, especially selective against
PC-3 and LNCaP cell lines. The efficacy of the compound
(9b) against MCF-7 and MDA-MB-231 is also significant,
which indicates that AR antagonists may be also used
against breast cancer. Further work for this will be con-
tinued. We, further, intend to investigate the target site and
to study the in vivo anticancer activity of the active
compounds.
ligands. In MTT assays, 9b was specifically for AR with
the IC50 values (LNCap: 10.02 ± 0.23 lM and PC-3:
1
0.91 ± 0.34 lM). In addition, it has evidence that 9b
showed good activity in breast cancer cell lines also.
Recent reports depict that antiandrogens are used as a
targeted therapy for breast cancer (Niemeier et al., 2010;
Garay and Park, 2012).
We have predicted the ADME/T properties of the test
compounds (7a–d and 9a–n) and the reference compounds
(
Nilutamide, Flutamide, Testosterone) for the pharmaceu-
tical relevant properties to assess the drug likeness and
pharmacokinetic properties (Jorgensen and Duffy, 2002).
The qikpro within Maestro 9.5 (Schr o¨ dinger, 2013; Fries-
ner et al., 2004, 2006; Halgren et al., 2004) was used for
the evaluation of some important absorption, distribution,
metabolism and elimination (ADME) parameters and its
permissible rangers are listed in Table 4. In the present
study, the test compounds showed good drug-like proper-
ties based on the logarithm of predicted binding constant to
human serum albumin, log KHSA (range for 95 % of
drugs: -1.5 to 1.2); the logarithm of predicted blood/brain
barrier partition coefficient, log B/B (range for 95 % of
Experimental section
In silico molecular docking
The protein was first prepared using the Protein Preparation
wizard and the docking studies were performed using the
Schr o¨ dinger Glide software within Maestro 9.5, which
executes the correction of raw PDB structure, where
amendments such as the addition of hydrogen atoms,
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Med Chem Res (2016) 25:539–552
543
Table 2 Lowest binding energy for the ligand-AR (PDB:2AMA) protein interaction as detected by GLIDE molecular docking
R
1
R
2
O
R
3
N
N
Compd
R1
R2
R3
G score Lipophilic
HBond
Electro
7
7
a
CF
3
CN
CN
H
H
−
−
6.26
6.15
−5.32
−6.88
0
0
−0.04
−0.06
b
Cl
7
7
c
F
F
NH–
H
H
CH
3
−5.22
−3.79
−0.7
−0.19
O
d
N
H
−5.05
−5.18
−6.91
−0.07
−0.7
−0.08
−0.31
9
a
CF
3
CN
F
F
−
9.19
9.12
O
O
9
9
b
c
CF
Cl
3
CN
CN
−
−6.35
−1.39
−0.47
−8.79
−6.33
−4.85
−1.51
−0.7
−0.38
−0.2
9
9
9
9
9
9
d
e
f
Cl
CN
−8.5
CF3
Cl
F
CN
CN
−6.46
−6.35
−5.27
−5.09
−4.91
−4.89
−4.33
−4.16
0
0.02
0
0.02
g
h
i
NH–
−0.35
−0.35
−0.27
−0.27
CH
3
F
NH–
CH
3
F
NH–
CH
3
F
O
−5.49
−7.99
−4.56
−6.82
0
0
−0.03
−0.09
9
j
F
O
N
H
123

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Med Chem Res (2016) 25:539–552
Table 2 continued
O
9
9
k
l
F
F
N
H
−7.82
−7.64
−7.23
−0.47
−0.11
O
F
N
H
O
−4.83
−6.35
−4.96
−0.72
0
−0.37
−0.23
−0.41
9
9
m
n
H
H
CN
−6.4
CN
−6.36
−0.7
Fig. 3 2D diagram interactions of active AR antagonists (9a, 9b, 9d, 9f, 9m, 9n)
1
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Med Chem Res (2016) 25:539–552
545
Fig. 5 ORTEP representation of the compound 9b
virtual screening. Glide molecular docking needs an X-ray
crystal structure of protein binding with ligands for deter-
mining active site receptor grid. Receptor grid-based
molecular docking assists the ligands to bind in more than
one possible conformation. Glide searches for favorable
interactions between one or more ligand molecules and a
protein. The van der Waals radii of the receptor atoms with
partial atomic charges are to be less than the specified
Fig. 4 Three-dimensional model of oxobenzimidazoles in the AR-
binding site (colored by atom types: C green, N blue, O red, S yellow,
H light gray) (Color figure online)
assigning bond orders, creating disulfide bonds, fixing of
the charges and orientation of groups were incorporated.
Ligand preparation was performed using LigPrep for the
corrections on the ligands such as the addition of hydro-
gens, 2D to 3D conversion, bond lengths and bond angles,
low energy structure, and ring conformation followed by
minimization and optimization in optimized potential for
liquid simulations (OPLS, 2005) force field. Glide is a
ligand docking program for predicting protein–ligand
binding modes and ranking ligands via high-throughput
cutoff. The scaling factor and partial charge cutoff are 1
and 0.25 A˚ , respectively. The G score is calculated in
kcal/mol, and it includes ligand–protein interaction ener-
gies, hydrophobic interactions, hydrogen bonds, internal
energy, p–p stacking interactions, and root mean square
deviation (RMSD) and desolvation. Glide utilizes two
different scoring functions, SP and XP Glide Score, to
rank-order compounds. Glide module of the XP visualizer
Scheme 1 Synthesis of
compounds 5a and 5b. Reagents
and conditions: a N-
R
R
R
R
2
NC
Br
NC
(
a)
(b)
(c)
Bromosuccinimide, DMF, RT,
b CuCN, DMF, reflux,
c NaNO , CuBr, Conc.HCl,
Br
2
NH2
NH2
NH2
0
°C to RT
4
5a (R= CF )
3
3
5
b (R= Cl)
1
1
R
R
2
1
2
R
R
R
O
O
2
3
R
R
(
d)
N
NH
(
e)
N
N
Br
5
a-b
7
a-b
9
a-c
Scheme 2 Synthesis of compound 7a, b, 9a–c. Reagents and conditions: (d) 2-hydroxy benzimidazole (6), Cs
Cs CO
, DMF, 110 °C
2
CO
3
, DMF, 110 °C (e) R-X (8),
2
3
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Table 3 Cytotoxicity studies expressed by IC50 in lM for compounds 9a–c, 7a and 7b and for the positive controls, bicalutamide and
doxorubicin
a
a
a
a
Compounds
IC50 (lM) on LNCaP
IC50 (lM) on PC-3
IC50 (lM) on MCF-7
IC50 (lM) on MDA-MB-231
9
9
9
7
7
a
b
c
11.2 ± 0.13
10.02 ± 0.23
20.56 ± 0.19
50.91 ± 0.85
65.54 ± 2.1
1.71 ± 0.12
12.19 ± 0.25
10.91 ± 0.34
15.71 ± 0.52
45.21 ± 0.92
55.23 ± 0.56
77
20.2 ± 0.73
15.6 ± 0.82
25.61 ± 0.95
[100
26.2 ± 0.13
22.25 ± 0.25
52.3 ± 0.62
[100
a
b
[100
[100
b
c
c
nd
Bicalutamide
b
Doxorubicin
nd
c
c
nd
nd
0.36 ± 0.03
0.20 ± 0.01
a
The values are the mean ± standard deviation (SD) of three independent experiments performed in triplicate
b
c
Positive control
nd not determined
analyzes the specific ligand–protein interactions. Grids
in DMF 100 ml dropwise. The reaction mixture was stirred
at room temperature for 3 h, then diluted with ethyl acetate
500 ml and washed with brine 2 9 150 ml. The organic
phase was dried Na SO , filtered and concentrated to give
were generated using Glide version 9.5 following the
standard procedure recommended by Schrodinger.
2
4
Chemistry
the title compound 3a or 3b as brownish solid, 90–92 %
yield. To a solution of 2a or 2b (0.8 mmol) in the selected
solvent (1 ml), a solution of NBS (0.8 mmol) in the same
solvent (1 ml) was added batch wise at room temperature,
and the reaction was monitored by LC–MS/MS. Mixtures
were obtained with traces of dibrominated products.
General
All reagents and solvents were purchased from Sigma-
Aldrich, Spectrochem and Merck Scientific, and used
without further purification unless otherwise stated. Melt-
ing points were recorded on a Buchi Melting Point M-565
system. All the reaction mixtures and column eluents were
monitored by thin-layer chromatography (TLC) on Merck
silica gel 60 F254 pre-coated aluminum plates with a
thickness of 0.2 mm and were developed with the mixtures
of hexane/ethyl acetate or CH Cl /Methanol and were
4-Bromo-3-(trifluoromethyl) aniline (3a) Yield: 92 %;
mp 48–50 °C; brownish powder; IR (KBr) m max:
3485–3362 (N–Hstretch), 2219 (C:N), 1627 (N–Hbend),
1615 1566, 1509, 1467 (C=Carom), 1361, 1185, 1121, 1044
-
1
1
(C–Hbend), 1276, 550 (CF3) cm
; H NMR (CDCl3,
400 MHz, ppm): d 7.38 (d, 1H, J = 8.8 Hz, C5-H), 6.95
(d, 1H, J = 2.8 Hz, C2–H), 6.63 (dd, 1H, J = 2 Hz, J =
2
2
visualized with UV light at 254 and 365 nm. Column
chromatography was performed under ‘‘flash’’ conditions
on Merck silica gel (230–400 mesh). Infrared spectra (IR)
1
3
8.4 Hz, C6-H), 3.84 (br s, 2H, –NH2);
C NMR
(100 MHz, CDCl , ppm): d 145.7 (C1),135.5 (C5), 130.6
3
-
1
were recorded using KBr discs, c (cm ), on a Thermo
Scientific IR spectrophotometer. Accurate masses (HRMS)
were determined using a Thermo Scientific Exactive plus
(C3), 124.3 (C6), 121.6 (CF3), 119.07 (C2), 114.1 (C4);
7
9
HRMS (ESI): m/z calculated for C H BrF N [M–H] :
-
7
5
3
8
5
1
237.94847; Found: 237.94478; Calculated for C H BrF N
7
3
1
13
-
[M–H] : 239.94620; Found: 239. 94250.
LC–MS/MS mass spectrometer. H and C NMR spectra
were recorded in CDCl3 or DMSO-d6 using a Bruker
4
00 MHz NMR instrument. Chemical shifts are given in d
4-Bromo-3-chloro aniline (3b) Yield: 90 %; mp
63–64 °C; brownish Powder; IR (KBr) m max: 3486, 3371
(N–Hstretch), 2224 (C:N), 1627 (N–Hbend), 1605, 1547,
1506 (C=Carom), 1333, 1253, 1152, 1040 (C–Hbend), 823,
values (ppm) using tetramethylsilane as the internal stan-
dard. Peak multiplicities are expressed as follows: s, sin-
glet; d, doublet; t, triplet; q, quartet; dd, doublet of doublet;
br, broad; br s, broad singlet; m, multiplet.
-
1 1
713 (C–Clbend) cm ; H NMR (CDCl3, 400 MHz, ppm):
d 7.33 (d, J = 8.67 Hz, 1H, C5-H), 6.79 (d, J = 2.64 Hz,
1H, C2-H), 6.45 (dd, J = 8.67, 2.64 Hz, 1H, C6-H), 3.74
General procedure for the synthesis of 4-Bromo-3-
1
3
(
substituted) aniline (3a–b)
(broad s, 2H, -NH2); C NMR (100 MHz, CDCl , ppm):
3
d 146.7 (C1),134.6 (C3), 133.9 (C5), 116.4 (C6), 115.1
7
9
To a solution of substituted aniline 2a or 2b in DMF
00 ml was added to a solution of NBS (92 mmol, 1.0 eq)
(C2), 109.8 (C4); HRMS (ESI): m/z Calcd for C H
6 5
3
Br ClN [M?H] : 205.93667; Found: 205.93427; Calcd
5
?
1
1
23

Med Chem Res (2016) 25:539–552
547
123

5
48
Med Chem Res (2016) 25:539–552
7
5
9
for C H Br ClN [M?H] : 207.93396; Found:
35
?
to the stirred solution with the temperature being kept
within (0 and 5 °C). This diazonium salt solution was
then poured into a flask containing CuBr (53 mmol,
1.4 eq) and 22 ml of conc. HCl. The solution was stirred
for 2 h, and then, the reaction mixture was poured into
ice water (50 ml) extracted with Ethyl acetate (100 ml).
The organic layer was separated, and the aqueous layer
was extracted with ethyl acetate (2 9 50 ml). The com-
bined extracts were washed with saturated aqueous
6
2
07.93156.
General procedure for the synthesis of 4-cyano-3-
substituted) aniline (4a, b)
(
A mixture of compound 3a or 3b (19 mmol) and CuCN
29 mmol, 1.5 eq) in DMF (20 ml) was heated at 145 °C
under nitrogen overnight. The reaction mixture was cooled
to room temperature and then poured into ice water
(
NaHCO solution (1 9 150 ml), brine (3 9 100 ml) and
3
(
50 ml). Ethyl acetate (100 ml) was added and the insol-
uble solid was filtered off (washed with ethyl acetate
2 9 30 ml]). The organic layer was separated, and the
aqueous layer was extracted with ethyl acetate
2 9 50 ml). The combined extracts were washed with
saturated aqueous NaHCO solution (1 9 150 ml), brine
dried over Na SO . The resulting material was concen-
2
4
trated, and the residue was purified by silica gel chro-
matography (petroleum ether:ethyl acetate, 10:1) to
afford the title compound (5a or 5b) as white solid
(72–75 % yield).
[
(
3
(
3 9 100 ml) and dried over Na SO . The resulting
2
4-Bromo-2-(trifluoromethyl) benzonitrile (5a) Yield
75 %; White Solid; IR (KBr) m max: 3101, 3040 (C–
4
material was concentrated and the residue was purified by
silica gel chromatography (petroleum ether:ethyl acetate,
Harom), 2924, 2853 (C–H ), 2235 (C:N), 1594, 1558,
alk
1
0:1) to afford the title compound (4a or 4b) as a gray solid
60–65 % yield).
1542, 1508, 1484, 1405 (C=Carom), 1182, 1142, 1057 (C–
1
-
1
(
Hbend), 1304, 1270, 551 (C–CF3) cm
;
H NMR
(
400 MHz, CDCl , ppm): d 7.96 (s, 1H, H-2), d 7.87 (d,
3
1
3
4
6
3
1
-Amino-2-(trifluoromethyl) benzonitrile (4a) Yield:
5 %; mp 143–145 °C, Gray Powder; IR (KBr) m max:
485–3362 (N–Hstretch), 2219 (C:N), 1627 (N–Hbend),
615 1566, 1509, 1467 (C=Caroma), 1361, 1185, 1121, 1044
J = 8.0 Hz, 1H, H-6), 7.74 (d, J = 8.0 Hz, 1H, H-5);
C
NMR (100 MHz, CDCl , ppm): d 135.8 (C6), 134.4, 133.8
(q, J = 65.5 Hz, C2), 130.3 (q, CF3), 128.2 (d, J = 26 Hz,
3
C5), 122.9 (C3), 120.2 (C4), 114.8 (CN), 108.9 (C1);
?
-
1
1
79
HRMS (ESI): m/z Calcd for C H BrF N [M?H] : 249.
8 3 3
(
C–Hbend), 1276, 550 (CF3) cm
;
H NMR (CDCl3,
8
1
?
4
00 MHz, ppm): d 7.46 (d, J = 3.6 Hz, 1H, C6-H), 7.01
d, J = 2 Hz, 1H, C3-H), 6.82 (dd, J = 2.4 Hz, J =
94737 Found: 249.94696; Calcd for C H BrF N [M?H] :
8 3 3
(
251.94520; Found: 251.94479.
1
3
.2 Hz, 1H, C5-H), 6.12 (br s, 2H, –NH2); C NMR
5
(
(
100 MHz, CDCl , ppm): d 152.6 (C4), 135.1 (C6), 132.7
C2), 123.3 (C5), 120.5 (CF3), 116.7 (C3), 114.9 (CN),
4-Bromo-2-chlorobenzonitrile (5b) Yield 72 %; White
Solid; IR (KBr) m max: 3083 (C–Harom), 2937, 2835 (C–
3
1
10.7 (C1); HRMS (ESI): m/z calculated for C H F N
8
Halk), 2233 (C:N), 1579, 1548, 1542, 1467 (C=C
),
5
3
2
arom
-1
1182, 1151, 1117 (C–Hbend), 830, 692 (C–Clbend) cm ; H
-
M–H] : 185.03321; Found: 185.03133.
1
[
NMR (400 MHz, CDCl , ppm): d 7.71 (s, 1H, H-2), 7.55
3
1
(d, J = 1.2 Hz, 2H, H-5, H-6); C NMR (100 MHz,
3
4
-Amino-2-chlorobenzonitrile (4b) Yield: 60 %; mp
16–118 °C, Gray powder; IR (KBr) m max: 3486, 3371
1
CDCl , ppm): d 134.8 (C6), 127.8 (C2), 126.5 (C5), 123.7
3
(
N–Hstretch), 2224 (C:N), 1627 (N–Hbend), 1605, 1547,
(C3), 123.3 (C4), 110.3 (CN), 109.2 (C1); HRMS (ESI): m/
?
7
9
35
1
506 (C=Carom), 1333, 1253, 1152, 1040 (C–Hbend), 823,
z Calcd for C H Br ClN [M?K] : 255.4624 Found: 255.
7 3
-
13 (C–Clbend) cm ; H NMR (CDCl3, 400 MHz, ppm):
1
1
79 37
?
7
96956; Calcd for C H Br ClN [M?K] : 257.4624;
7 3
d 7.32 (d, J = 8.4 Hz, 1H, C6-H), 6.64 (d, 1H, C3-H),
6
Found: 257.96722.
1
.47(d, J = 8.4 Hz, 1H, C5-H), 4.23 (br s, 2H); C NMR
3
(
(
100 MHz, CDCl , ppm): d 150.3 (C4), 137.1 (C2), 134.0
C6), 116.3 (C5), 113.7 (C3), 111.8 (CN), 99.9 (C1);
?
General procedure for the synthesis of compounds (7a, b)
3
HRMS (ESI): m/z calculated for C H ClN [M?H] :
7
4-Bromo-2-(substituted) benzonitrile 5a or 5b (5 mmol)
was added to a mixture of 1H-benzo[d]imidazol-2(3H)-one
5
2
1
53.02140; Found: 153.02168.
(
6) (10 mmol) and Cs CO (10 mmol) in DMF (25.0 ml).
2 3
General procedure for the synthesis of 4-Bromo-2-
substituted) benzonitrile (5a, b)
After 12 h, 50 ml of ice water was added dropwise. The
resulting suspension was filtered, and the filter cake was
dried in a vacuum oven. The solid was purified by flash
chromatography (50 % ethyl acetate in hexane) to afford
4-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)-2-(sub-
stituted) benzonitrile (7a or 7b).
(
4
-Cyano-3-(substituted) aniline 4a or 4b (38 mmol) was
dissolved in conc. HCl (27 ml). After cooling to 0 °C,
sodium nitrite (39 mmol, 1.5 eq) was added very slowly
1
23

Med Chem Res (2016) 25:539–552
549
00 00
134.2, 133.6 (q, J = 60 Hz, C2), 133.2 (C 1), 130.6 (C 2,
2-(Trifluoromethyl)-4-(1,2-dihydro-2-oxobenzo[d]imidazol-3-
0
0
0
0
yl) benzonitrile (7a) Yield 62 %; White Solid; mp
C 6), 129.1 (C 6), 127.6 (d, J = 34 Hz, C5), 125.6 (C 3),
0
0
0
232–233 °C; IR (KBr) m max: 3189, 3149 (N–Hstretch),
3072 (C–Harom), 2957, 2895, 2820 (C–Halk), 2230 (C:N),
1720 (C=O), 1611 (N–Hbend), 1575, 1507, 1481, 1439 (C=
124.0 (C 5), 122.8, 122.5 (q, CF3), 120.6 (C 2), 118.8 (C
0
0
00
4), 115.6 (C 3, C 5), 115.8 (C3), 114.2 (CN), 102.3 (C1),
44.9 (CH2); HRMS (ESI): m/z calculated C H F N O
13 4 3 2
-
[M–H] : 438.08711; Found: 438.087.
23
Carom), 1385, 1319, 1240, 1185, 1127, 1053 (C–Hbend),
-
1
1
1
272, 558 (C–CF3) cm ; H NMR (400 MHz, CDCl ,
3
ppm): d 9.372 (s, 1H, NH), 8.14 (s, 1H, H-2), 8.03 (d, 2H,
H-5,H-6), 7.2 (m, 4H, phenyl); C NMR (100 MHz,
4-(1-Allyl-1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)-2-(tri-
fluoromethyl) benzonitrile (9b) Yield 85 %; Colorless
needles; mp 107–108 °C; IR (KBr) m max: 3070 (C–Harom),
1
3
CDCl , ppm): d 152.9 (C=O), 139.2 (C4), 135.6 (C6),
3
0
0
1
33.8, 133.1 (q, J = 65 Hz, C2), 128.5 (C 1), 127.6
0
2925, 2853 (C–H ), 2232 (C:N), 1709 (C=O), 1607,
alk
(
d, J = 34 Hz, C5), 123.3 (C 6), 122.8 (C 3), 122.5
0
1572, 1506, 1489, 1435, 1400 (C=Carom), 1347, 1308,
0
(
q, CF3), 121.6 (C), 120.4 (C 5), 114.8 (CN), 110.0 (C 2),
0
1280, 1225, 1177, 1146, 1132, 1052 (C–Hbend), 1280, 557
-1
1
1
08.7 (C 4), 106.6 (C1); HRMS (ESI): m/z calculated for
(C–CF3) cm
8.15(s, 1H, H-2), 8.01 (d, J = 1.2 Hz, 2H, H-5, H-6),
.06–7.27 (m, 4H, phenyl), 5.90–5.98 (m, 1H,CH), 5.34
(dd, J = 5.6 Hz, 2 H, = CH2), 4.59 (d, J = 5.2 Hz, 2 H,
; H NMR (400 MHz, CDCl , ppm): d
3
?
C H F N O [M?H] : 304.06922; Found: 304.069.
1
5 8 3 3
7
2-(Chloro)-4-(1,2-dihydro-2-oxobenzo[d]imidazol-3-yl) ben-
1
3
zonitrile (7b) Yield 62 %; White Solid; mp 225–226 °C;
IR (KBr) m max: 3188, 3150(N–Hstretch), 3076 (C–Harom),
–CH2–); C NMR (100 MHz, CDCl , ppm): d 152.3 (C=
3
O), 139.4 (C4), 136.0 (C6), 134.7, 134.0 (q, J = 66 Hz,
0 0 0 00
2
924, 2853 (C–Halkane), 2230 (C:N), 1719 (C=O), 1611
N–Hbend), 1575, 1510, 1481, 1438 (C=Carom), 1387, 1319,
240, 1195, 1127, 1053 (C–Hbend), 830, 701 (C–Clbend
C2), 132.1(C 1), 131.2(C 6), 129.5(C 3), 127.3(C 2),
0 0
123.5, 123.0 (q, CF3), 123.0(d, C5), 122.2(C 5), 121.3(C
(
0
00
1
)
2),120.6 (C3), 118.5 (C 4), 117.6 (C 3), 115.1(CN), 108.0
00
-
1
1
cm ; H NMR (400 MHz, CDCl , ppm): d 8.17 (s, 1H),
(C1), 43.7 (C 1); HRMS (ESI): m/z calculated for
?
C H F N O [M?H] : 344.10052; Found: 344.0986.
3
1
.01 (d, 2H), 7.39 (m, 4H); C NMR (100 MHz, CDCl3,
3
8
18
12 3 3
0
ppm): d 152.3 (C=O), 139.4 (C4), 136.4 (C2), 134.5 (C 1),
0
0
0
1
33.9 (C6), 123.3 (C 6), 122.9 (C 3), 122.2 (C 5), 119.5
0
2-Chloro-4-{3-[2-(4-fluorophenyl)-2-oxoethyl]-2-oxo-2,3-
dihydro-1H-benzimidazol-1-yl}benzonitrile (9c) Yield
0
(
C 2), 117.6 (C5), 116.2 (C 4), 115.1 (C3), 114.2 (CN),
09.1 (C), 105.2 (C1); HRMS (ESI): m/z calculated for
-
C H ClN O [M–H] : 268.02831; Found: 268.02609.
1
82 %; White Solid; mp 175–176 °C; IR (KBr) m max: 3072
(C–Harom), 2925, 2852 (C–Halk), 2229 (C:N), 1720, 1693
1
4
8
3
(2 C=O), 1602, 1510, 1484, 1437 (C=Carom), 1382, 1330,
General procedure for the synthesis of compounds (9a–c)
-(Substituted)-4-(1,2-dihydro-2-oxobenzo[d]imidazol-3-
1240, 1195, 1130, 1054 (C–Hbend), 829, 695 (C–Clbend
-
)
1 1
cm ; H NMR (400 MHz, CDCl , ppm): d 3.7 (s, 2 H,
3
2
–CH2) 7.09–7.13 (m, 4H,), d 7.13–7.16 (m, 4H, phenyl),
7.37–7.41 (m, 2H, H-5, H-6), 8.01 (s, 4H, phenyl) 8.28 (s,
yl) benzonitrile 7a or 7b (5 mmol) was added to a mixture
of compound (R–X) and Cs CO (10 mmol) in DMF
1
3
2
3
1H, H-2); C NMR (100 MHz, CDCl , ppm): d 192.05
3
00
(
25.0 ml). After 12 h, 50 ml of ice water was added
(C=O), 157.2 (C 4), 152.3 (N–C=O), 139.4 (C4), 136.4
0
00
00
00
dropwise. The resulting suspension was filtered, and the
filter cake was dried in a vacuum oven. The solid was
purified by flash chromatography (50 % ethyl acetate in
hexane) to afford the title compound (9a–c).
(C2), 134.5 (C 1), 133.9 (C6), 131.7(C 1), 130.5 (C 2, C
0
0
0
0
6), 127.3 (C 6), 123.5 (C 3), 122.7 (C 5), 119.5 (C 2),
00
0
00
118.2 (C5), 116.5 (C 4), 115.6 (C 3, C 5), 115.1 (C3),
114.0 (CN), 109.1 (C), 104.2 (C1), 44.1 (CH2); HRMS
?
ESI): m/z calculated for C H ClFN O [M?H] :
(
2
2
13
3 2
4
1
-{3-[2-(4-Fluorophenyl)-2-oxoethyl]-2-oxo-2,3-dihydro-
H-benzimidazol-1-yl}-2-(trifluoromethyl) benzonitrile
406.8089; Found: 406.9141.
(
9a) Yield 80 %; Colorless needles; mp 178–180 °C; IR
X-ray diffraction analysis
(
KBr) m max: 3063 (C–Harom), 2954, 2916, 2851 (C–
Halkane), 2236 (C:N), 1712, 1696 (2 C=O), 1602, 1508,
The single crystal of the compound was developed by slow
evaporation of chloroform and alcohol (1:1 ratio). The
1
489, 1437 (C=Caromatic), 1365, 1347, 1312, 1240, 1180,
-1 1
161, 1135, 1055 (C–Hbend), 1276, 550 (C–CF3) cm ; H
1
XRD data for
a
good-quality single crystal
3
NMR (400 MHz, CDCl , ppm): d 8.23(s, 1H, H-2), 8.03 (s,
(0.40 9 0.34 9 0.22 mm ) was collected at 296 K tem-
perature with a Bruker 3-circle diffractometer (Bruker
Nonius SMART APEX 2) equipped with CCD area
detector. Bruker SMART software was used for data col-
lection and also for indexing reflections and unit cell
3
4
H, phenyl), 7.19–7.23 (m, 2H, H-5, H-6), 7.09–7.13 (m,
3
1
4
H, phenyl), 3.72 (s, 2 H, –CH2–CO);
C NMR
0
0
(
100 MHz, CDCl , ppm): d 193.5 (C=O), 157.2 (C 4),
3
0
1
53.3 (N–C=O), 138.3 (C4), 136.5 (C 1), 135.6 (C6),
123

5
50
Med Chem Res (2016) 25:539–552
parameters. The data were integrated using SAINT soft-
ware. The structure was solved by direct methods and
refined by full-matrix least squares calculation using
SHELXL software. Lattice parameters were determined
from h values in the range 1.72 \ h \ 24.50.
physically relevant descriptors and uses them to perform
ADMET predictions. The methods implemented were
developed by Jorgensen and Duffy, the logarithm of
aqueous solubility, log Swat (range for 95 % of drugs:
-6.0 to 0.5); the logarithm of predicted binding constant to
human serum albumin, log KHSA (range for 95 % of
drugs: -1.5 to 1.2); the logarithm of predicted blood/brain
barrier partition coefficient, log B/B (range for 95 % of
drugs: -3.0 to 1.0) the predicted apparent Madin-Darby
In vitro cytotoxicity evaluation
Cell lines and cell culture
-
1
canine kidney (MDCK) cell permeability in nm s (\25
poor, [500 great); the index of cohesion interaction in
solids, Indcoh, calculated from the predicted Polarizability,
QPpolrz, IC50 value for blockage of HERG K? channels,
log HERG, apparent Caco-2 cell permeability, QPP Caco
in nm/s, metabolic reactions, #metab, Polarizability, QP
polrz, Absorption, Distribution, Metabolism, Excretion,
and Toxicity (ADME/T) properties of the selected best
docked ligands molecules were predicted using QikProp
tool of Schrodinger 2012. It predicted the properties such
as the number of hydrogen bond acceptors (HBA), donors
Human prostate cancer cell lines, PC-3 and LNCaP and
breast cancer cell lines, MCF-7 and MDA-MB-231 were
obtained from National Centre for Cell Science (NCCS),
Pune, India, and were cultured in RPMI1640 and DMEM,
respectively, with 10 % fetal bovine serum (FBS), MTT,
EDTA, trypsin and 1 % penicillin–streptomycin. DMEM,
RPMI1640, FBS, 1 % penicillin–streptomycin, bicalu-
tamide and dextrorubicin were purchased from Sigma-
Aldrich. Cell cultures were maintained in flasks under
standard conditions: incubation at 37 °C and 5 % CO . All
2
(HBD), QP log Po/w, skin permeability, log Kp, percentage
the subcultures were used prior to passage 15. Cells were
routinely passaged using 0.25 % trypsin/0.1 % EDTA. For
treatment, cells were cultured in the presence of increasing
concentrations (1–100 lM) of compounds for 24 h.
of human oral absorption, etc.
Supplementary data
In vitro cytotoxicity measurements
Supplementary data associated HRMS and NMR spectra of
compounds (3a, 3b, 4a, 4b, 5a, 5b, 7a, 7b and 9a–c). The
Crystallographic data for compound 9b has been deposited
with the Cambridge Crystallographic Data Centre, CCDC
No. 1042893. Copies of the data can be obtained free of
charge on application to CCDC, 12 Union Road, Cam-
bridge CB2 1EZ, UK (fax: ?44 (0) 1223 336033 or e-mail:
deposit@ccdc.cam.ac.uk).
All in vitro experiments for cell proliferation/inhibition
were performed in triplicates. For the PC-3 cell growth
inhibition assay, cells were cultured in RPMI1640 sup-
4
plement and trypsinized, further diluted to 2.0 9 10 cell/
ml with RPMI1640 supplemented with 10 % fetal bovine
serum. This cell suspension was transferred to 96-well
microtiter plates, and incubated in the presence or absence
of increasing concentration of positive control (Bicalu-
tamide) or the test compounds (1–100 lM) at 37 °C and
Acknowledgments Authors are thankful to the Director, Institute of
Advanced Study in Science and Technology, Assam, India, for giving
us the facility to carry out this work. Thanks are attributable to DBT,
Govt. of India for financial support as a Project.
5
% CO . After 24-h incubation, cells were treated with an
2
MTT solution for 4 h in a cell culture incubator at 37 °C
and 5 % CO . Cell proliferation was determined by the
2
MTT method. MTT which is a tetrazolium salt is converted
into insoluble formazan by mitochondrial dehydrogenases
in live cells. Formazan is dissolved in DMSO (Merck), and
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