Synthesis of new benzimidazole and phenylhydrazinecarbothiomide hybrids and their anticonvulsant activity

Article abstract of DOI:10.1007/s00044-016-1570-6

A series of new benzimidazole derivatives (4a–p) were synthesized and evaluated for anticonvulsant activity in albino mice against two most adopted models, i.e. maximal electroshock seizure (MES)- and subcutaneous pentylenetetrazole (scPTZ)-induced seizures. Synthesized compounds were also screened for possible neurotoxicity using rotarod test. Among the synthesized compounds, 4p showed the most promising activity in MES and scPTZ screens, which was further subjected for oral activity in rats. At a dose of 30?mg/kg, it showed tremendous activity in the scPTZ screen. The acute toxicity study (LD₅₀) of compounds showed that only two tested compounds 4f and 4m did not produce any mortality at any of the dose level. Molecular properties and pharmacokinetic parameters of the titled compounds were also determined using Lipinski’s rule of five. The promising results encourage future investigation on the rational modification of this nucleus for development of better compounds.

Full text of DOI:10.1007/s00044-016-1570-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1570-6
ORIGINAL RESEARCH
Synthesis of new benzimidazole
and phenylhydrazinecarbothiomide hybrids and their
anticonvulsant activity
Nadeem Siddiqui1 Md. Shamsher Alam1 Ruhi Ali1 M. Shahar Yar1 Ozair Alam1
•
•
•
•
Received: 18 June 2015 / Accepted: 7 April 2016
Ó Springer Science+Business Media New York 2016
Abstract A series of new benzimidazole derivatives (4a–
p) were synthesized and evaluated for anticonvulsant
activity in albino mice against two most adopted models,
i.e. maximal electroshock seizure (MES)- and subcuta-
neous pentylenetetrazole (scPTZ)-induced seizures. Syn-
thesized compounds were also screened for possible
neurotoxicity using rotarod test. Among the synthesized
compounds, 4p showed the most promising activity in
MES and scPTZ screens, which was further subjected for
oral activity in rats. At a dose of 30 mg/kg, it showed
tremendous activity in the scPTZ screen. The acute toxicity
study (LD50) of compounds showed that only two tested
compounds 4f and 4m did not produce any mortality at any
of the dose level. Molecular properties and pharmacoki-
netic parameters of the titled compounds were also deter-
mined using Lipinski’s rule of five. The promising results
encourage future investigation on the rational modification
of this nucleus for development of better compounds.
Keywords Anticonvulsant Á Glutamate Á Benzimidazole Á
In silico Á In vivo Á Molecular docking
Introduction
Epilepsy is simply defined as the tendency to developed
seizures. Epilepsy is characterized clinically by periodic
development of seizures which are often distressing, and
educe fear and misunderstanding. The distribution of epi-
lepsy is uniform globally with no racial, geographical or
social limits (Sander, 2003). This has led to social conse-
quences and has added effect to the disease burden. With
the advancement in neurological science, a number of
antiepileptic drugs (AEDs), pharmacotherapies and neu-
rosurgical techniques have been developed and new ones
are under development.
GABAA receptors, voltage gated K? and Na? channels,
T-type Ca2? channels and glutamate receptors are primary
targets for which antiepileptic drugs have been developed
(Brauner-Osborne et al., 2000). The identification of
gamma-amino butyric acid (GABA) as an important inhi-
bitory neurotransmitter and glutamate (Glu) as most
important excitatory neurotransmitter in mammalian brain
led to the identification of agents which can be targeted to
inhibit seizures through enhancing the activity of GABA.
Glutamate performs through the activation of either iono-
tropic or metabotropic receptors (Dingledine et al., 1999).
It was proposed that pharmacological intervention with
iGluRs antagonists may have a potential therapeutic benefit
in case of epilepsy (Lees, 2000; Auberson, 2001). Espe-
cially, the non-competitive NMDA antagonists acting at
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-016-1570-6) contains supplementary
material, which is available to authorized users.
& Nadeem Siddiqui
nadeems_03@yahoo.co.in
Md. Shamsher Alam
w_shams123@yahoo.co.in
Ruhi Ali
ruhi.pharma@gmail.com
M. Shahar Yar
msyar@jamiahamdard.ac.in
Ozair Alam
dr.ozairalam@gmail.com
1
Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Jamia Hamdard, Hamdard Nagar,
New Delhi 110062, India
123

Med Chem Res
the glutamate coagonist glycine binding site on the NMDA
receptor complex have been identified as possible thera-
peutic agents for the epilepsy (Danysz and Parsons, 1998;
Kulagowski, 1996; Kulagowski and Leeson, 1995; Donati
and Micheli, 2000; Leeson and Iversen, 1994). Antagonists
of these glutamate receptors help against epilepsy. Con-
vulsion can be prevented by antagonism of ionotropic
glutamate receptors, which are the main grounds of seizing
(Sarro et al., 2005).The BCATc is expressed in neuronal
tissue of CNSand catalyses the transfer of an amino group
from branched chain amino acids to a-ketoglutarate led to
synthesis of glutamate in CNS (Lain-Yen et al., 2006). The
inhibition of BCATc can reduce the release of glutamate
synthesis during excitation in neuronal tissues which is
likely to be useful for the treatment variety of neurode-
generative disorder such as epilepsy.
Materials and methods
Chemistry
All chemicals used in the synthesis were procured from E.
Merck and S D Fine-Chem Limited. Thin-layer chro-
matography (TLC) was performed with silica gel 60 F254
TLC aluminium sheet (Merck) using toluene:ethyl
acetate:formic acid (5:4:1) and benzene:acetone (9:1) as
eluents. Spots were visualized under UV light (254 nm)
and in iodine chamber. Ashless Whatmann No. 1 filter
paper was used for vacuum filtration. Melting points were
determined by using open capillary tubes in a Hicon
melting point apparatus (Hicon, India) and are uncor-
rected. The purity of the compounds was confirmed
through elemental analysis. The elemental analyses (C, H,
N and S) of all compounds were performed on the CHNS
Elimentar (Analysen systime, GmbH) Germany Vario EL
III, and results were within 0.4 % of the theoretical
values. Fourier transform infrared (FT-IR) spectra were
recorded in KBr pellets on a Shimadzu FT-IR spectrom-
Within the vast range of heterocycles, benzimidazoles are
found to be trendy scaffold used for finding of drugs in the
pharmaceutical and medicinal chemistry field. The exclusive
structural features of benzimidazole and a vast range of
biological activities of its derivatives made it privileged
structure in drug discovery. Benzimidazole is a heterocyclic
aromatic system, found in some natural and many synthetic
compounds. Benzimidazoles and its derivatives are found to
be biologically active against many diseases which have
made it a crucial anchor for development of promising
therapeutic agents (Kruse et al., 1989; Jun et al., 2005; Castro
et al., 2011; Anelia et al., 2006; Kaur et al., 2014; Iemura
et al., 1986; Cole et al., 1974; Kubo et al., 1993; Blaszczak-
Swiqtkiewicz and Mikiciuk-Olasik, 2015; Porcari et al.,
1998). Recently, benzimidazole scaffold has emerged as a
pharmacophore of choice for designing anticonvulsant
agents active on different clinically approved targets. The
structural modification of existing leads (arylsemicar-
bazone) to aryl thiosemicarbazide by biosteric replacement
of oxygen with sulphur and saturation of imine, led to
development of newer aryl hydrazinecarbothioamide as
promising anticonvulsant agents (Dimmock et al., 1996). In
designing a drug molecule, computational biology and
bioinformatics display a most important role and have the
potential of speeding up the drug discovery process.
Molecular docking of the drug molecule with the receptor
gives important information about drug–receptor interac-
tions and is commonly used to find out the binding orienta-
tion of drug candidates to their protein targets in order to
predict the affinity and activity. On the basis of above facts
and in continuation of our research on antiepileptics, we
report here the synthesis, molecular docking and anticon-
vulsant activity of a new series of benzimidazole derivatives.
1
eter. HNMR and 13CNMR spectra in DMSO-d6/CDCl3
solutions were, respectively, recorded at 400 and
100 MHz with Bruker 400 ultrashield TM NMR spec-
trometer using TMS [(CH3)4Si] as internal standard.
Splitting patterns are nominated as follows: s, singlet; bs,
broad singlet; d, doublet; t, triplet; m, multiplet. The NH
protons were D2O exchanged for their spectral character-
ization. The mass spectra were recorded using Waters
micromass ZQ 2000 spectrophotometer (Jamia Hamdard,
New Delhi, India).
2-[(1-(2-Substitutedbenzyl)-1H-benzo[d]imidazol-
2-yl)methyl]-N-substitutedphenylhydrazinecarbothio
amides (4a–p) were synthesized and presented in
Scheme 1.
(Chloromethyl)-1H-benzo[d]imidazole (1) was prepared
from heating a mixture of o-phenylene diamine and 2-chlor-
oacetic acid in a dry conical flask. Compounds (2a, b) were
synthesized by stirring a mixture of compound (1), anhydrous
potassium carbonate in acetone and substitutedbenzyl chlo-
ride. On refluxing, compounds (2a, b) with hydrazine hydrate
in the presence of ethanol, 1-[(1-substitutedbenzyl-1H-
benzo[d]imidazol-2-yl) methyl]hydrazines (3a, b) were pre-
pared. Finally titled compounds 2-[(1-(2-substitutedbenzyl)-
1H-benzo[d]imidazol-2-yl)methyl]-N-substitutedphenylhy-
drazinecarbothioamides (4a–p) were prepared on refluxing
different substitutedphenylisothiocyanates with compounds
3a, b. The physicochemical parameters of all the final com-
pounds (4a–p) are presented in Table 1.
123

Med Chem Res
Scheme 1 Reagents and conditions: (i) Heated for 1.5 h, cooled and basified with 10 % NaOH soln, (ii) Anhy. K2CO3, acetone,
substitutedbenzyl chloride, stirred for 8 h, (iii) NH2NH2ÁH2O, C2H5OH, refluxed for 20–22 h, (iv) ArNCS, absolute ethanol, refluxed for 5–6 h
General procedure for the synthesis of titled
compounds 4a–p
the flask was plugged with cotton. The mixture was
heated on a boiling water bath for 1.5 h. Flask was cooled
down under tap water, and the product was basified with
10 % NaOH solution, filtered and washed with cold
water, and compound 1 obtained was recrystallized with
hot water.
2-(Chloromethyl)-1H-benzo[d]imidazole (1): A mixture
of o-phenylenediamine (0.01 mol) and 2-chloroacetic acid
(0.01 mol) was taken in a dry conical flask, and mouth of
123

Med Chem Res
Table 1 Physicochemical parameters of compounds (4a–p)
Compd.
R
R’
Mol. Formulaa
LogPb found (calculated.)
cRf (Rm)d
eElemental analysis (%)
C
H
N
4a
4b
4c
4d
4e
4f
H
H
C22H21N5S
4.21 (3.99)
4.57 (4.70)
4.20 (4.48)
4.38 (4.48)
4.21 (4.48)
4.19 (3.90)
4.14 (3.90)
4.10 (3.90)
4.67 (4.70)
4.33 (4.41)
5.16 (5.20)
5.41 (5.20)
5.52 (5.20)
4.25 (4.62)
4.44 (4.62)
4.51 (4.62)
0.79 (-0.57)
0.82 (-0.65)
0.80 (-0.60)
0.90 (-0.95)
0.91 (-1.00)
0.94 (-1.19)
0.83 (-0.68)
0.94 (-1.19)
0.83 (-0.68)
0.94 (-1.19)
0.77 (-0.52)
0.76 (-0.50)
0.94 (-1.19)
0.77 (-0.52)
0.94 (-1.19)
0.78 (-0.54)
68.19
62.62
68.80
68.62
68.95
66.16
66.56
66.34
62.60
57.90
63.36
63.72
63.52
61.12
61.36
61.02
5.46
4.78
5.77
5.98
5.57
5.55
5.15
5.37
4.74
4.20
5.09
5.34
5.28
4.91
4.62
5.09
18.07
16.60
17.44
17.04
17.20
16.77
16.34
16.67
16.57
15.35
16.06
16.37
16.46
15.50
15.79
15.85
H
2-Cl
C22H20ClN5S
H
2-CH3
3-CH3
4-CH3
2-OCH3
3-OCH3
4-OCH3
H
C23H23N5S
C23H23N5S
C23H23N5S
C23H23N5OS
C23H23N5OS
C23H23N5OS
H
H
H
4g
4h
4i
H
H
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
C
22H20ClN5S
22H19Cl2N5S
4j
2-Cl
C
4k
4l
2-CH3
3-CH3
4-CH3
2-OCH3
3-OCH3
4-OCH3
C23H22ClN5S
C23H22ClN5S
C23H22ClN5S
C23H22ClN5OS
C23H22ClN5OS
C23H22ClN5OS
4m
4n
4o
4p
a
Solvent of crystallization-ethanol
b
c
d
e
LogP was determined by octanol:phosphate buffer method; CLogP was calculated using software ChemDraw Ultra 8.0
Solvent system—toluene:ethyl acetate:formic acid (5:4:1)
A logarithmic function of Rf value was also calculated; Rm = log (1 - 1/Rf)
Elemental analysis for C, H, N were within 0.4 % of the theoretical value
2-(Chloromethyl)-1-(2-substitutedbenzyl)-1H-benzo[d]im-
idazoles (2a, b): To a solution of compound 1 (0.01 mol)
and anhydrous potassium carbonate (0.01 mol) in acetone
(30 mL), substitutedbenzyl chloride (0.01 mol) was added
dropwise. The mixture was stirred at room temp for about
8 h. The mixture was then poured into water and extracted
with ethyl acetate, dried over sodium sulphate anhydrous
and concentrated under vacuum to give the pure com-
pound. Desired compound 2a, b was finally recrystallized
with ethanol.
overnight at room temperature. The needle-shaped crystals
thus obtained were purified by repeated washing with petro
leum ether (see Supplementary material).
2-[(1-Benzyl-1H-benzo[d]imidazol-2-yl)methyl]-N-
phenylhydrazinecarbothioamide (4a)
Yield: 64 %; m.p. 180 °C; IR (KBr) t cm-1: 3576 (NHstr.),
3373 (NHstr.), 3200 (NHstr.-thioamide), 3089 (Ar–CHstr.),
2957 (CHstr.), 1602 (C=N, cyclic), 1528 (C=Sstr.); 1H NMR
(CDCl3) d (ppm): 3.66 (s, 2H, CH2), 4.05 (s, 2H, CH2–Ar),
6.59–7.37 (m, 10H, Ar–H), 7.01–7.67 (m, 4H, Bz–H), 8.46
(s, 1H, NH), 8.67 (s, 1H, NH), 10.50 (s, 1H, NH–Ar, D2O
exchangeable); 13C NMR (DMSO-d6) d (ppm): 42.24, 45.63,
109.41, 117.32, 120.24, 122.62, 123.53, 124.64, 126.42,
127.12, 127.35, 127.63, 128.23, 129.52, 130.54, 130.87,
134.23, 135.12, 138.54, 148.46, 153.26, 161.24; MS: m/z 386
(M-1).
1-[(1-Substitutedbenzyl-1H-benzo[d]imidazol-2-yl)methyl]
hydrazines (3a, b): Hydrazine hydrate (10 mL) was placed
in a round bottom flask, and compound 2a, b (0.01 mol)
was added. Contents were diluted with a sufficient quantity
of dry ethanol till clear solution was obtained, and the
reaction mixture was refluxed for 20–22 h. After comple-
tion of the reaction, ethanol was distilled off till a small
volume was left. On cooling, crystals of compounds (3a,
b) were formed and were filtered and recrystallized with
ethanol.
2-[(1-Benzyl-1H-benzo[d]imidazol-2-yl)methyl]-N-
2-[(1-(2-Substitutedbenzyl)-1H-benzo[d]imidazol-2-yl)
methyl]-N-substitutedphenylhydrazinecarbothioamides (4a–
p): A mixture of compound (3a, b, 0.01 mol) and substi-
tutedphenylisothiocyanates (0.01 mol) in 20 mL of abso-
lute ethanol was refluxed for 5–6 h. After completion of the
reaction the reaction, mixture was concentrated and kept
(2-chlorophenyl)hydrazinecarbothioamide (4b)
Yield: 52 %, m.p. 190 °C. IR (KBr) t cm-1: 3525 (NHstr.),
3345 (NHstr.), 3265 (NHstr.-thioamide), 3008 (Ar–CHstr.),
2985 (CHstr.), 1608 (C=N, cyclic), 1537 (C=Sstr.), 805 (C–
Cl); 1H NMR d (ppm) (CDCl3): 3.58 (s, 2H, CH2), 4.25 (s,
123

Med Chem Res
2H, CH2–Ar), 6.64–7.86 (m,9H, Ar–H), 7.04–7.36 (m, 4H,
Bz–H), 8.24 (s, 1H, NH, D2O exchangeable), 8.95 (s, 1H,
NH, D2O exchangeable), 10.24 (s, 1H, NH–Ar, D2O
exchangeable); 13C NMR (DMSO-d6) d (ppm): 43.44,
45.53, 110.28, 118.22, 119.82, 122.82, 123.63, 125.25,
126.19, 127.28, 127.39, 127.84, 128.81, 129.28, 130.10,
130.92, 133.43, 135.27, 137.34, 148.28, 152.16, 163.82.
126.92, 127.17, 127.82, 127.44, 128.21, 129.34, 130.28,
131.83, 132.23, 134.35, 138.32, 147.45, 152.39, 160.91; MS:
m/z 402 (M?1).
2-[(1-Benzyl-1H-benzo[d]imidazol-2-yl)methyl]-N-(2-
methoxyphenyl)hydrazinecarbothioamide (4f)
Yield: 63 %; m.p. 170 °C; IR (KBr) t cm-1: 3424 (NHstr.),
3299 (NHstr.), 3117 (NHstr.-thioamide), 2961 (Ar–CHstr.),
2901 (CHstr.), 1601 (C=N, cyclic), 1536 (C=Sstr.), 1169
2-[(1-Benzyl-1H-benzo[d]imidazol-2-yl) methyl]-N-o-
tolylhydrazinecarbothioamide (4c)
1
(OCH3); H NMR (DMSO-d6) d(ppm): 3.17 (s, 2H, CH2),
Yield: 51 %; m.p. 175 °C; IR (KBr) t cm-1: 3371 (NHstr.),
3194 (NHstr.), 3091 (NHstr.-thioamide), 2990 (Ar–CHstr.),
2943 (CHstr.), 1685 (C=N, cyclic), 1540 (C=Sstr.); 1H NMR
(CDCl3) d (ppm): 2.48 (s, 3H, CH3) 3.89 (s, 2H, CH2), 4.09
(s, 2H, CH2–Ar), 7.07–7.97 (m, 9H, Ar–H), 7.22–7.91 (m,
4H, Bz–H), 8.00 (s, 1H, NH, D2O exchangeable), 9.25 (s,
1H, NH, D2O exchangeable), 10.01 (s, 1H, NH–Ar, D2O
exchangeable); 13C NMR (DMSO-d6) d (ppm): 18.24,
41.27, 43.48, 110.17, 118.29, 119.83, 121.29, 124.10,
125.53, 126.82, 127.16, 127.23, 127.72, 128.73, 129.18,
130.28, 130.96, 133.14, 134.47, 136.17, 149.27, 151.92,
164.92; MS: m/z 403 (M?2).
3.40 (s, 3H, OCH3) 4.15 (s, 2H, CH2–Ar), 6.58–8.15 (m,
9H, Ar–H), 7.22–8.76 (m, 4H, Bz–H), 8.82 (s, 1H, NH,
D2O exchangeable), 9.19 (s, 1H, NH, D2O exchangeable),
11.11 (s, 1H, NH–Ar, D2O exchangeable); 13C NMR
(DMSO-d6) d (ppm): 39.24, 42.84, 55.25, 114.26, 119.28,
120.27, 121.38, 124.84, 125.28, 126.94, 127.21, 127.23,
127.49, 128.48, 129.28, 130.47, 131.23, 133.40, 134.35,
138.24, 147.36, 150.58, 163.41; MS: m/z 419 (M?2).
2-[(1-Benzyl-1H-benzo[d]imidazol-2-yl)methyl]-N-(3-
methoxyphenyl)hydrazinecarbothioamide (4g)
Yield: 61 %; m.p. 151 °C; IR (KBr) t cm-1: 3458 (NHstr.),
3273 (NHstr.), 3123 (NHstr.-thioamide), 2984 (Ar–CHstr.),
2928 (CHstr.), 1648 (C=N, cyclic), 1521 (C=Sstr.), 1114
2-[(1-Benzyl-1H-benzo[d]imidazol-2-yl)methyl]-N-m-
tolylhydrazinecarbothioamide (4d)
1
Yield: 44 %; m.p. 210 °C; IR (KBr) t cm-1: 3345 (NHstr.),
3173 (NHstr.), 3065 (NHstr.-thioamide), 2985 (Ar–CHstr.),
2932 (CHstr.), 1647 (C=N, cyclic), 1564 (C=Sstr.); 1H NMR
(CDCl3) d (ppm): 2.45(s, 3H, CH3) 3.58 (s, 2H, CH2), 4.05
(s, 2H, CH2–Ar), 7.04–7.85 (m, 9H, Ar–H), 7.12–7.99 (m,
4H, Bz–H), 8.05 (s, 1H, NH, D2O exchangeable), 9.29 (s,
1H, NH, D2O exchangeable), 10.17 (s, 1H, NH–Ar, D2O
exchangeable); 13C NMR (DMSO-d6) d (ppm): 17.26,
42.47, 44.29, 112.21, 117.13, 120.15, 121.72, 124.27,
125.17, 126.73, 127.33, 127.74, 127.85, 128.83, 129.92,
130.12, 130.83, 133.26, 134.18, 136.92, 147.22, 153.12,
162.61; MS: m/z 401 (M?1).
(OCH3); H NMR (CDCl3) d (ppm): 3.25 (s, 2H, CH2),
3.63 (s, 3H, OCH3), 4.27 (s, 2H, CH2–Ar), 6.24–8.56
(m,9H, Ar–H), 7.02–8.21 (m, 4H, Bz–H), 8.15 (s, 1H, NH,
D2O exchangeable), 9.01 (s, 1H, NH, D2O exchangeable),
11.23 (s, 1H, NH–Ar, D2O exchangeable); 13C NMR
(DMSO-d6) d (ppm): 41.25, 43.95, 57.28, 111.37, 119.83,
120.73, 121.49, 122.39, 125.25, 126.29, 127.35, 127.57,
127.21, 128.24, 129.35, 130.64, 131.24, 133.87, 137.25,
141.23, 148.26, 150.35, 165.45.
2-[(1-Benzyl-1H-benzo[d]imidazol-2-yl)methyl]-N-(4-
methoxyphenyl)hydrazinecarbothioamide (4h)
Yield: 59 %; m.p. 200 °C; IR (KBr) t cm-1: 3445 (NHstr.),
3273 (NHstr.), 3154 (NHstr.-thioamide), 2921 (Ar–CHstr.),
2958 (CHstr.), 1625 (C=N, cyclic), 1556 (C=Sstr.), 1147
2-[(1-Benzyl-1H-benzo[d]imidazol-2-yl)methyl]-N-p-
tolylhydrazinecarbothioamide (4e)
Yield: 65 %; m.p. 215 °C; IR (KBr) t cm-1: 3321 (NHstr.),
3173 (NHstr.), 3054 (NHstr.-thioamide), 2974 (Ar–CHstr.),
2965 (CHstr.), 1627 (C=N, cyclic), 1538 (C=Sstr.); 1H NMR
(CDCl3) d (ppm): 2.44(s, 3H, CH3), 3.48 (s, 2H, CH2), 4.04
(s, 2H, CH2–Ar), 7.06–7.84 (m, 9H, Ar–H), 7.14–7.47 (m,
4H, Bz–H), 8.45 (s, 1H, NH, D2O exchangeable), 9.04 (s, 1H,
NH, D2O exchangeable), 10.14 (s, 1H, NH–Ar, D2O
exchangeable); 13C NMR (DMSO-d6) d (ppm): 18.25, 40.23,
43.18, 113.41, 118.92, 120.62, 121.16, 124.68, 125.15,
1
(OCH3); H NMR (CDCl3) d (ppm): 3.26 (s, 2H, CH2),
3.58 (s, 3H, OCH3), 4.15 (s, 2H, CH2–Ar), 6.14–8.56
(m,9H, Ar–H), 7.05–8.12 (m, 4H, Bz–H), 8.17 (s, 1H, NH,
D2O exchangeable), 9.06 (s, 1H, NH, D2O exchangeable),
11.31 (s, 1H, NH–Ar, D2O exchangeable); 13C NMR
(DMSO-d6) d (ppm): 40.34, 45.24, 53.26, 114.26, 118.26,
120.23, 121.24, 123.34, 125.36, 126.45, 127.56, 127.68,
127.48, 128.23, 129.42, 129.62, 132.54, 133.35, 139.24,
140.24, 146.36, 149.85, 164.43.
123

Med Chem Res
Cl); 1H NMR (CDCl3) d (ppm): 2.55 (s, 3H, CH3), 3.42 (s,
2H, CH2), 5.66 (s, 2H, CH2–Ar), 6.63–7.95 (m,8H, Ar–H),
7.69–7.14 (m, 4H, Bz–H), 9.12 (s, 1H, NH, D2O
exchangeable), 10.74 (s, 1H, NH, D2O exchangeable),
10.34 (s, 1H, NH–Ar, D2O exchangeable); 13C NMR
(DMSO-d6) d (ppm): 24.35, 40.95, 44.57, 117.96, 119.85,
120.74, 121.73, 124.42, 125.58, 126.37, 127.15, 127.27,
127.34, 128.43, 129.79, 130.23, 131.42, 132.47, 135.35,
138.32, 143.35, 155.27, 158.23.
2-[(1-(2-Chlorobenzyl)-1H-benzo[d]imidazol-2-yl)methyl]-
N-phenylhydrazinecarbothioamide (4i)
Yield: 67 %; mp 173 °C. IR (KBr) t cm-1: 3576 (NHstr.),
3471 (NHstr.), 3223 (NHstr.-thioamide), 2960 (Ar–CHstr.),
2905 (CHstr.), 1625 (C=N, cyclic), 1595 (C=Sstr.), 745 (C–
Cl); 1H NMR (CDCl3) d (ppm): 2.52 (s, 2H, CH2), 3.31 (s,
2H, CH2–Ar), 7.14–8.20 (m, 9H, Ar–H), 7.20–8.23 (m, 4H,
Bz–H), 8.76 (s, 1H, NH, D2O exchangeable), 10.42 (s, 1H,
NH, D2O exchangeable), 11.52 (s, 1H, NH–Ar, D2O
exchangeable); 13C NMR (DMSO-d6) d (ppm): 38.23,
42.65, 106.43, 119.39, 120.47, 122.68, 123.35, 125.24,
126.24, 127.35, 127.68, 127.58, 128.37, 129.28, 130.68,
130.76, 133.12, 137.46, 138.35, 147.17, 156.58, 161.97;
MS: m/z 423 (M?2).
2-[(1-(2-Chlorobenzyl)-1H-benzo[d]imidazol-2-yl)methyl]-
N-p-tolylhydrazinecarbothioamide (4m)
Yield: 53 %; m.p. 180 °C; IR (KBr) t cm-1: 3565 (NHstr.),
3484 (NHstr.), 3442 (NHstr.-thioamide), 3432 (Ar–CHstr.),
2985 (CHstr.), 1612 (C=N, cyclic), 1442 (C=Sstr.), 812 (C–
Cl); 1H NMR (CDCl3) d (ppm): 2.54 (s, 3H, CH3), 3.45 (s,
2H, CH2), 5.32 (s, 2H, CH2–Ar), 6.52–7.85 (m, 8H, Ar–H),
7.56–7.46 (m, 4H, Bz–H), 9.85 (s, 1H, NH, D2O
exchangeable), 10.32 (s, 1H, NH, D2O exchangeable),
10.47 (s, 1H, NH–Ar, D2O exchangeable); 13C NMR
(DMSO-d6) d (ppm): 26.67, 41.24, 46.43, 118.57, 119.34,
120.34, 121.48, 124.97, 125.97, 126.45, 127.24, 127.74,
127.64, 128.23, 129.96, 130.35, 131.75, 132.95, 135.86,
136.98, 144.78, 154.26, 157.22.
2-[(1-(2-Chlorobenzyl)-1H-benzo[d]imidazol-2-yl)methyl]-
N-(2-chlorophenyl)hydrazinecarbothioamide (4j)
Yield: 57 %; m.p. 144 °C; IR (KBr) t cm-1: 3545 (NHstr.),
3473 (NHstr.), 3365 (NHstr.-thioamide), 3023 (Ar–CHstr.),
2941 (CHstr.), 1632 (C=N, cyclic), 1575 (C=Sstr.), 712 (C–
Cl); 1H NMR (CDCl3) d (ppm): 2.12 (s, 2H, CH2), 3.62 (s,
2H, CH2–Ar), 7.11–8.25 (m,8H, Ar–H), 7.24–8.56 (m, 4H,
Bz–H), 8.25 (s, 1H, NH, D2O exchangeable), 10.23 (s, 1H,
NH, D2O exchangeable), 11.74 (s, 1H, NH–Ar, D2O
exchangeable); 13C NMR (DMSO-d6) d (ppm): 39.43,
40.65, 108.44, 119.35, 121.42, 122.46, 124.89, 125.84,
126.45, 127.74, 127.84, 127.57, 128.36, 129.32, 130.14,
132.46, 135.58, 137.34, 139.43, 144.58, 154.23, 165.27.
2-[(1-(2-Chlorobenzyl)-1H-benzo[d]imidazol-2-yl)methyl]-
N-(2-methoxyphenyl)hydrazinecarbothioamide (4n)
Yield: 57 %; m.p. 185 °C; IR (KBr) t cm-1: 3497 (NHstr.),
3484 (NHstr.), 3474 (NHstr.-thioamide), 3074 (Ar–CHstr.),
2826 (CHstr.), 1670 (C=N, cyclic), 1488 (C=Sstr.), 1105
2-[(1-(2-Chlorobenzyl)-1H-benzo[d]imidazol-2-yl)methyl]-
1
N-o-tolylhydrazinecarbothioamide (4k)
(OCH3), 733 (C–Cl); H NMR (CDCl3) d (ppm): 3.31 (s,
2H, CH2), 3.80 (s, 3H, OCH3), 3.85 (s, 2H, CH2–Ar),
6.97–7.89 (m, 8H, Ar–H), 7.00–7.96 (m, 4H, Bz–H), 7.98
(s, 1H, NH, D2O exchangeable), 9.08 (s, 1H, NH, D2O
exchangeable), 10.72 (s, 1H, NH–Ar, D2O exchangeable);
13C NMR (DMSO-d6) d (ppm): 37.24, 44.45, 59.37,
109.43, 118.32, 118.35, 120.42, 123.54, 125.53, 126.56,
127.17, 127.32, 127.35, 128.47, 129.09, 130.85, 132.56,
133.75, 137.64, 140.43, 147.32, 149.32, 165.35.
Yield: 64 %; m.p. 300 °C; IR (KBr) t cm-1: 3506 (NHstr.),
3484 (NHstr.), 3463 (NHstr.-thioamide), 3063 (Ar–CHstr.),
2950 (CHstr.), 1610 (C=N, cyclic), 1409 (C=Sstr.), 801 (C–
Cl); 1H NMR (CDCl3) d (ppm): 2.50 (s, 3H, CH3), 3.37 (s,
2H, CH2), 5.24 (s, 2H, CH2–Ar), 6.47–7.96 (m,8H, Ar–H),
7.02–7.97 (m, 4H, Bz–H), 9.85 (s, 1H, NH, D2O
exchangeable), 10.39 (s, 1H, NH, D2O exchangeable),
10.67 (s, 1H, NH–Ar, D2O exchangeable); 13C NMR
(DMSO-d6) d (ppm): 22.36, 39.35, 47.74, 118.87, 119.37,
120.34, 121.72, 124.87, 125.53, 126.57, 127.45, 127.93,
127.95, 128.35, 129.49, 130.86, 131.09, 132.46, 133.67,
136.42, 144.67, 153.37, 158.94.
2-[(1-(2-Chlorobenzyl)-1H-benzo[d]imidazol-2-yl)methyl]-
N-(3-methoxyphenyl)hydrazinecarbothioamide (4o)
Yield: 54 %; m.p. 300 °C; IR (KBr) t cm-1: 3466 (NHstr.),
3454 (NHstr.), 3412 (NHstr.-thioamide), 3021 (Ar–CHstr.),
2854 (CHstr.), 1654 (C=N, cyclic), 1484 (C=Sstr.), 1131
2-[(1-(2-Chlorobenzyl)-1H-benzo[d]imidazol-2-yl)methyl]-
1
N-m-tolylhydrazinecarbothioamide (4l)
(OCH3), 745 (C–Cl); H NMR (CDCl3) d (ppm): 3.32 (s,
2H, CH2), 3.75 (s, 3H, OCH3), 3.56 (s, 2H, CH2–Ar),
6.54–7.86 (m,8H, Ar–H), 7.05–7.32 (m, 4H, Bz–H), 7.85
(s, 1H, NH, D2O exchangeable), 9.12 (s, 1H, NH, D2O
exchangeable), 10.41 (s, 1H, NH–Ar, D2O exchangeable).
Yield: 56 %; m.p. 300 °C; IR (KBr) t cm-1: 3545 (NHstr.),
3444 (NHstr.), 3412 (NHstr.-thioamide), 3085 (Ar–CHstr.),
2953 (CHstr.), 1641 (C=N, cyclic), 1485 (C=Sstr.), 805 (C–
123

Med Chem Res
13C NMR (DMSO-d6) d (ppm): 39.34, 46.75, 55.76,
105.35, 117.54, 118.38, 121.27, 124.25, 125.43, 126.54,
127.35, 127.65, 127.76, 128.23, 129.67, 131.23, 132.55,
133.46, 136.24, 139.44, 145.46, 147.57, 161.38.
Subcutaneous pentylenetetrazole (scPTZ)-induced seizure
test
This model primarily identifies those compounds that rai-
ses seizure threshold. The subcutaneous dose of PTZ
(85 mg/kg) at which 97 % of the animals showed clonic
seizure induction was calculated using dose response
method. Animals were divided into different groups, and
test drugs were administered. The test compounds were
administered intraperitoneally at three different doses, i.e.
30, 100, 300 mg/kg. At the anticipated time of testing, PTZ
was administered subcutaneously as 0.5 % solution in the
posterior midline of mice. Animals were observed after
30 min and 4.0 h. The absence of clonic spasm in half or
more animals was regarded as endpoint protection against
scPTZ-induced clonic seizures (Swinyard et al., 1989).
2-[(1-(2-Chlorobenzyl)-1H-benzo[d]imidazol-2-yl)methyl]-
N-(4-methoxyphenyl)hydrazinecarbothioamide (4p)
Yield: 57 %; m.p. 190 °C; IR (KBr) t cm-1: 3423 (NHstr.),
3412 (NHstr.), 3358 (NHstr.-thioamide), 3054 (Ar–CHstr.),
2896 (CHstr.), 1612 (C=N, cyclic), 1431 (C=Sstr.), 1154
1
(OCH3), 736 (C–Cl); H NMR (CDCl3) d (ppm): 3.12 (s,
2H, CH2), 3.14 (s, 2H, CH2–Ar), d 3.50 (s, 3H, OCH3),
6.65–7.84 (m,8H, Ar–H), 7.01–7.32 (m, 4H, Bz–H), 7.45
(s, 1H, NH, D2O exchangeable), 9.22 (s, 1H, NH, D2O
exchangeable), 10.51 (s, 1H, NH–Ar, D2O exchangeable);
13C NMR (DMSO-d6) d (ppm): 37.36, 44.56, 56.47,
103.38, 117.56, 118.43, 120.37, 121.23, 124.45, 125.46,
127.23, 127.37, 127.87, 128.16, 129.48, 131.48, 132.47,
133.65, 136.67, 138.36, 144.26, 146.32, 167.84.
Rotarod test
Mice were divided into different experimental groups and
trained to stay on a knurled rotarod of diameter 3.2 cm
rotating at 10 rpm. Trained animals were injected with the
test compounds. Motor impairment was indicated by the
inability of the animal to maintain equilibrium on the rod
for at least 1 min in each of the three trials (Dunham and
Miya, 1957).
Pharmacology
The pharmacological testing of all the final compounds has
been performed by National Institute of Neurological
Disorders and Stroke (NINDS), USA, under Anticonvul-
sant Screening Program (ASP), following the protocol
adopted by the Antiepileptic Drug Development (ADD)
programme. The screening for anticonvulsant activity was
carried out by maximal electroshock seizure (MES) test
and subcutaneous pentylenetetrazole (scPTZ)-induced sei-
zure test, whereas neurotoxicity evaluation was done by the
rotarod method. The tested compounds (4a–p) were
injected intraperitoneally (i.p) at a dose of 30, 100 and
300 mg/kg for both methods. Phenytoin and carba-
mazepine were used as standard drugs for comparison.
Quantification studies
For the determination of effective dose ED50, that is, the
dose of the drug required to elicit the biological response in
50 % of animals and TD50 values, that is, median toxic
dose, groups of six mice were given a range of i.p. doses of
the test drug until at least three points were established in
the range of 10–90 % seizure protection or minimal
observed neurotoxicity. From the plot of these data, the
respective ED50 and TD50 values, 95 % confidence inter-
vals, slope of the regression line and the standard error of
the slope were calculated by means of the computer pro-
gram (Litchfield and Wilcoxon, 1949).
Maximal electroshock seizure (MES) test
MES test is a model for generalized tonic–clonic seizures
and identifies those compounds which prevent seizure
spread. Animals were randomly divided into different
treatment groups. For induction of seizures in mice, a
60 Hz alternating current of 50 mA intensity was used. The
current was applied for 0.2 s using ear clip electrodes,
coated with an electrolyte solution containing an anaes-
thetic agent. Abolition of the hind limb and tonic maximal
extension was regarded as end point protection against
MES-induced seizures (Krall et al., 1978). The observa-
tions were taken at two different time intervals (0.5 h in
one group and 4.0 h in other group, respectively).
Computational studies
MM-GBSA binding free energy estimation
To calculate ligand binding energies and ligand strain
energies, binding energy estimation can be used for a set of
ligand and a single receptor human cytosolic branched chain
amino transferase (hBCATc) using the Prime molecular
mechanics-generalized Born surface area (MM-GBSA)
method, Maestro 10.1. LigPrep and protein preparation
wizard was used to prepare the ligands and the receptors.
123

Med Chem Res
1HNMR, 13C NMR and mass spectrometry, and the data
confirmed the structures of synthesized compounds. The
structures and purity of the final compounds were con-
firmed on the basis of spectral and elemental analyses, and
the data were within 0.4 % of the theoretical values. The
FT-IR bands at the 3576–3173, 3474–3054, 3432–2921,
1685–1602 and 1576–1409 cm-1 confirmed the presence
of NHstr., NHstr.-thioamide, Ar–CHstr., C=N, cyclic and
C=Sstr. functionalities, respectively. The 1H NMR spectrum
showed multiplet at d 7.01–8.76 confirmed benzimidazole
aromatic protons and singlet at d 3.12–3.89, d 8.00–10.74
and d 10.01–11.74 confirms methylene, thiourea NH and
NH-aromatic protons, respectively. 13C NMR of different
carbon atoms of the compounds were found to be in the
range of d 17.26–167.84. The mass spectrometry of the
prototype compound 4a was performed, and it showed a
base peak at the m/z value 386 that represents the M-1 peak
corresponding to the molecular weight of the compound.
Molecular docking
To predict binding modes of ligands to receptor and good
anticonvulsant activity on the basis of structures, molecular
docking studies of all the compounds were carried out by
taking X-ray crystal structure data to establish the inter-
action of the synthetic compounds with hBCATc enzyme
(PDB code: 2COI) using Glide extra precision (XP) Mae-
stro 10.1 Schrodinger, running on Linux 64 operating
¨
system (Schrodinger, 2015a, b). It involves few steps, i.e.
selection and preparation of appropriate protein, grid gen-
eration, ligand preparation followed by docking and its
analysis. The docking score, binding free energy and
hydrogen bonds and pi–pi interaction formed with the
surrounding amino acids are used to envisage their binding
affinities and proper alignment of these compounds at the
active site of the hBCATc enzyme.
Pharmacology
Results and discussion
Chemistry
Synthesized titled compounds 4a–p of Scheme 1 were
screened for protection against MES and scPTZ seizures.
Some of these compounds showed promising results in Phase
1 screening in mice (Table 2). The compounds 4e, 4f, 4j, 4l,
All the final compounds were characterized by different
spectral analytical techniques such as IR spectroscopy,
Table 2 Anticonvulsant screening project (ASP)
Compd. MES screening
30 mg/kg
scPTZ screening
30 mg/kg 100 mg/kg
Minimal motor impairment screening
100 mg/kg
300 mg/kg
300 mg/kg
30 mg/kg
100 mg/kg
300 mg/kg
0.5 h 4.0 h 0.5 h 4.0 h 0.5 h 4.0 h 0.5 h 4.0 h 0.5 h 4.0 h 0.5 h 4.0 h 0.5 h 4.0 h 0.5 h 4.0 h 0.5 h 4.0 h
N/F
N/F
N/F
N/F
N/F
N/F
N/F
N/F
N/F
N/F
N/F
N/F
N/F
N/F
N/F
N/F
N/F
N/F
4a
4b
4c
4d
4e
4f
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
0/1
9
0/1
9
0/3
9
0/3
9
0/1
9
0/1
9
0/1
9
0/1
9
0/1
9
0/1
9
0/1
9
0/1
9
0/4
9
0/2
9
0/8
9
0/4
9
0/4
9
0/2
9
0/1
0/1
0/1
0/1
0/1
9
0/1
0/1
0/1
0/1
0/1
9
0/3
0/3
0/3
0/3
0/3
9
0/3
1/3
0/3
0/3
0/3
9
0/1
0/1
0/1
0/1
0/1
9
0/1
1/1
0/1
0/1
0/1
9
0/1
0/1
0/1
0/1
0/1
9
0/1
0/1
0/1
0/1
0/1
9
0/1
0/1
0/1
0/1
0/1
9
0/1
0/1
1/5
0/1
0/1
9
0/1
0/1
0/1
0/4
0/1
9
0/1
0/1
0/1
0/2
0/1
9
0/4
0/4
0/4
0/4
0/4
9
0/2
0/2
0/2
0/2
0/2
9
0/8
0/8
0/8
0/8
0/8
9
0/4
0/4
0/4
0/4
0/4
9
0/4
0/4
0/4
0/4
0/4
9
0/2
0/2
0/2
0/2
0/2
9
4g
4h
4i
4j
0/1
0/1
0/1
0/1
9
0/1
0/1
0/1
0/1
9
0/3
0/3
0/3
0/3
9
0/3
0/3
0/3
0/3
9
0/1
0/1
0/1
0/1
9
1/1
0/1
1/1
0/1
9
0/1
0/1
0/1
0/1
9
0/1
0/1
0/1
0/1
9
0/1
0/1
0/1
0/1
9
0/1
0/1
0/1
0/1
9
0/1
0/1
0/1
0/1
9
0/0
0/1
0/1
0/1
9
0/4
0/4
0/4
0/4
9
0/2
0/2
0/2
0/2
9
0/8
0/8
0/8
0/8
9
0/4
0/4
0/4
0/4
9
0/4
0/4
0/4
0/4
9
1/2
0/2
0/2
0/2
9
4k
4l
4m
4n
4o
4p
0/1
0/1
0/1
0/1
0/3
0/3
0/3
2/3
0/1
0/1
0/1
0/0
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1/1
0/1
4/5
0/1
0/0
0/4
0/4
0/2
0/2
0/8
0/8
0/4
3/4
0/4
1/4
0/2
2/2
Bold values indicate that molecules were active at at least one of the tested dose and did not show any toxicity
Phase I anticonvulsant screening of synthesized compounds (4a–p)
‘9’ indicated not tested
MES maximal electroshock seizure, scPTZ subcutaneous pentylenetetrazole, route of administration: ip
123

Med Chem Res
4m and 4p were most potent anticonvulsants. In MES test,
compounds 4e and 4p were active at dose of 100 mg/kg (1/3
and 2/3 animals protected) 4.0 h after administration; how-
ever, compound 4p showed toxicity at 100 and 300 mg/kg
dose levels. Interestingly compound 4e was also active at
dose of 300 mg/kg after 4.0 h of administration. Further,
compounds 4e, 4j and 4l were active 4.0 h after adminis-
tration of 300 mg/kg dose. In this case also, toxicity was
observed with 4j, 4.0 h after 300 mg/kg dose administration.
In scPTZ screening, compounds 4f and 4p were active at
doses of 100 mg/kg (1/5 and 1/1 animals protected) 4.0 h
after administration; interestingly, compound 4f did not
show any toxicity at any dose. Dramatically compound 4p
was found to be most potent at the dose of 100 mg/kg (1/1
animal protected) after 4.0 h and at the dose of 300 mg/kg (4/
5 animals protected) after 0.5 h administration; however, it
showed some toxicity at higher doses of 100 and 300 mg/kg.
The compound 4p that displayed a marked MES activity in
mice at the dose of 100 mg/kg i.p (2/3 animals protected) was
evaluated for oral activity in rats, Test 2 results (Table 3).
Compound 4p did not show any activity in the MES screen at
30 mg/kg (p.o. dose) but tremendously showed far-fetched
activity in the scPTZ screen at 30 mg/kg (p.o. dose) (1/4, 1/4
and 2/4 animals protected) 1.0, 2.0 and 4.0 h after adminis-
tration. It is interesting that compound 4p showed lack of any
toxicity at 30 mg/kg p.o. dose) in any time interval.
Compound 4m was evaluated for 6 Hz assay in mice, Test
7 results (Table 4). Compound 4m showed protection in the
minimal clonic seizure (6 Hz) test at 100 mg/kg (i.pdose) (1/
4 animal protected) 0.5 h after administration. Interestingly
it lacks any toxicity at given dose in any time intervals.
The acute toxicity study (LD50) was performed on the
albino mice in order to assess any mortality or toxic effects
shown by the synthesized compounds at increased doses,
and the data are presented in Table 5. It was found that the
tested compounds 4f and 4m did not produce any mortality
or gross effect on the central nervous system at any of the
dose levels, i.e. 5, 25, 125, 250 and 400 mg/kg. However,
the animals have exhibited some of the toxic signs such as
chewing, licking, salivation followed by brief period of
sedation. But at the doses of 125, 250 and 400 mg/kg,
compound 4p showed mortality (1/6, 3/6 and 3/6, respec-
tively). There was not any toxicity seen in the vehicle alone
as evident in the control group of animals.
These results suggest that thioamide as hydrogen
bonding domain (HBD), nitrogen of benzimidazole as
electron donor (D), substitution of 2/4-methoxy or methyl
as electron donating agent in hydrophobic domain (A) and
unsubstitution or 2-chloro substitution in distal hydropho-
bic domain (R) made the compound potent in the MES
screen at 100 and 300 mg/kg doses, scPTZ screen at 100
and 300 mg/kg (i.p dose) in mice and 30 mg/kg (p.o. dose)
Table 3 Anticonvulsant screening project (ASP)
Compd.
Test
Time (h)
0.25
0.5
1.0
2.0
4.0
4p
MESa
scPTZb
TOXc
0/4
0/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
0/4
1/4
0/4
0/4
2/4
0/4
Bold values indicate active compounds in one row and compounds without toxicity in other row
Test 2 results in rats
Dose = 30 mg/kg
a
Maximal electroshock test (number of animal protected/number of animals tested)
b
Subcutaneous pentylenetetrazole test (number of animal protected/number of animals tested)
c
Rotarod toxicity (number of animals exhibiting toxicity/number of animals tested) endpoint: unable to maintain balance on rotarod
Table 4 Anticonvulsant screening project (ASP)
Compd.
Test
Time (h)
0.25
0.5
1.0
2.0
4.0
4m
6 Hza
TOXb
0/4
1/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
Bold values indicate active compounds in one row and compounds without toxicity in other row
Test 7 results in mice; 6 Hz assay
Dose = 100 mg/kg
a
Qualitative 6 Hz assay (number of animal protected/number of animals tested)
b
Rotarod toxicity (number of animals exhibiting toxicity/number of animals tested) endpoint: unable to maintain balance on rotarod
123

Med Chem Res
Table 5 Acute toxicity study data (LD50) of active compounds
Compd.
Number of animals dead/Total number of animals tested
Dose (mg/kg), i.p.
5
25
125
250
400
Control
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
1/6
0/6
0/6
0/6
0/6
0/6
0/6
3/6
3/6
3/6
3/6
0/6
0/6
0/6
3/6
4/6
3/6
3/6
4f
4m
4p
PHY
Carbamazepine
Phenobarbital
Vehicle used: polyethylene glycol (0.4 mL, i.p.)
in rats as well as minimal clonic seizure (6 Hz) test at
100 mg/kg (i.p dose) in mice.
Table 6 Docking score and binding free energy of all the compounds
at the active sites of hBCATc (PDB ID: 2COI) GBN-A-420
Comp.
Docking score
Binding free energy
(kcal/mol)
Computational studies
4a
-5.679
-4.652
-6.286
-6.172
-4.102
-4.468
-5.082
-4.732
-5.094
-5.732
-4.631
-4.219
-4.180
-4.685
-5.038
-5.879
-4.367
-41.313
-64.205
-60.438
-76.141
-66.226
-69.816
-72.537
-63.909
-70.725
-57.073
-49.009
-69.571
-63.286
-56.504
-71.011
-64.231
-38.147
MM-GBSA binding free energy estimation
4o
4p
All the waters were deleted from the designed complexes
prior to dG binding free energy calculation using solvation
model VSGB by Prime MM-GBSA, Maestro 10.1. All the
compounds are well fitted in the active sites of human
cytosolic branched chain amino transferase (hBCATc). The
binding poses for each compound were analysed by exam-
ining their free energy scores. The more energetically
favourable conformation was selected as the best pose. The
dG binding energy of compounds was found in the range of
-41.313 to -76.141 kcal/mol for enzyme hBCATc active
sites (Table 6). Allthe compoundsdisplayeda higherbinding
free energy (dG bind) for hBCATc which indicates that
compounds may have higher selectivity towards hBCATc
enzyme except 4a. However, there are considerable differ-
ences in free energy binding for the compounds towards
4n
4m
4k
4l
4j
4i
4h
4g
4f
4e
4d
4c
4b
Co-crystal ligand
Fig. 1 2D ligand interaction
representation of compound 4p
showing hydrogen bond
interaction with THR 260
(purple colour arrow line) and
ARG 119 (purple colour dash
arrow line) and pi–pi stacking
(green line) with TYR 227,
ARG 163 and ARG 119; pi-
cation with LYS 222 (red line)
(Color figure online)
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Med Chem Res
Fig. 2 Docked pose of
compound 4p represented as
stick in the binding site of
hBCATc showing hydrogen
bond interaction with THR 260
and ARG 119; pi–pi stacking
with TYR 227, ARG 163 and
ARG 119; pi-cation with LYS
222. The red dash lines, sky
blue dash lines and green dash
lines denote the hydrogen
bonds, pi–pi interaction and pi-
cation, respectively. The amino
acids are represented as green
colour thin line (Color
figure online)
hBCATc. The binding energy of co-crystal ligand was found
to be -38.147 kcal/mol and less than the synthesized com-
pounds (4a–p). This result justify that the synthesized com-
pounds have high affinity for hBCATc than co-crystal.
with the active site of human cytosolic branched chain
amino transferase (hBCATc) are summarized in Table 6.
The crystal structure of hBCATc was prepared for docking
with the protein preparation wizard workflow of Maestro
that allows addition of hydrogen atoms which were sub-
sequently minimized with OPLS-2005 force field and
optimize the protonation state. The receptor grid was
generated by applying a van der Waals radii of non-polar
atoms, which decreases penalties for close contacts (scaling
Molecular docking
The glide scores, binding energy and interaction with
amino acids of the titled compounds and co-crystal ligand
Fig. 3 Receptor surface model
of compound 4p represented as
ball and stick in the binding site
of hBCATc showing hydrogen
bond interactions
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Med Chem Res
Compliance with ethical standards
factor = 1.00 and partial charge cut off = 0.25). Before
docking calculation, all the compounds (4a–p) were sub-
jected to ligand preparation with the LigPrep tool. Finally
the ligand docking were run using receptor grid file and
LigPrep out file in the Glide tool of application view.
Among all the titled compounds for hBCATc, com-
pound 4p was found to be most potent and have high
docking score. The compound 4p also assumes favourable
orientation within the hBCATc binding site. The 2D ligand
interaction diagram of 4p is represented in Fig. 1. The
nitrogen atom of benzimidazole ring and hydrogen atom of
NH of hydrazinecarbothioamide linker of compound 4p
formed strong hydrogen bonds with THR 260 approxi-
Conflict of interest Authors declare no conflict of interest.
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