Design, synthesis and anticonvulsant evaluation of novel N-(4-substituted phenyl)-2-[4-(substituted) benzylidene]-hydrazinecarbothio amides

Article abstract of DOI:10.1016/j.ejmech.2011.10.038

Thirty six new N-(4-substituted phenyl)-2-[4-(substituted) benzylidene]-hydrazinecarbothioamides were synthesized and evaluated for anticonvulsant activity and neurotoxicity. The anticonvulsant activity was established in three seizure models i.e. MES, scMET and 6 Hz model. The most active compound was 2-[4-(4-chlorophenoxy)benzylidene]-N-(4-fluorophenyl) hydrazinecarbothioamide PC 31 which showed 100% protection at 0.5 h in the 6 Hz test. Compound 2-[4-(4-bromophenoxy) benzylidene]-N-(4-bromophenyl) hydrazinecarbothioamide PC 23 was found to be active in both the MES and 6 Hz test. A computational study was carried out from calculation of a pharmacophore pattern and the prediction of pharmacokinetic properties. Titled compounds have also exhibited good binding properties with epilepsy molecular targets such as glutamate, GABA (A) delta and GABA (A) alpha-1 receptors, in the Lamarckian genetic algorithm based on flexible docking studies.

Full text of DOI:10.1016/j.ejmech.2011.10.038

European Journal of Medicinal Chemistry 47 (2012) 153e166
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and anticonvulsant evaluation of novel
N-(4-substituted phenyl)-2-[4-(substituted) benzylidene]-hydrazinecarbothio
amides
,
a
b
c
Laxmi Tripathi a , Praveen Kumar , Ranjit Singh , James P. Stables
*
a Department of Pharmaceutical Chemistry, S.D. College of Pharmacy and Vocational Studies, Muzaffarnagar 251001, India
b School of Pharmaceutical Sciences, Shobhit University, Meerut 250110, India
c Preclinical Pharmacology Section, Epilepsy Branch, National Institute of Health, Bethesda, MD 20892-9020, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Thirty six new N-(4-substituted phenyl)-2-[4-(substituted) benzylidene]-hydrazinecarbothioamides
were synthesized and evaluated for anticonvulsant activity and neurotoxicity. The anticonvulsant activity
was established in three seizure models i.e. MES, scMET and 6 Hz model. The most active compound was
2-[4-(4-chlorophenoxy)benzylidene]-N-(4-fluorophenyl)hydrazinecarbothioamide PC 31 which showed
100% protection at 0.5 h in the 6 Hz test. Compound 2-[4-(4-bromophenoxy) benzylidene]-N-(4-
bromophenyl) hydrazinecarbothioamide PC 23 was found to be active in both the MES and 6 Hz test.
A computational study was carried out from calculation of a pharmacophore pattern and the prediction
of pharmacokinetic properties. Titled compounds have also exhibited good binding properties with
epilepsy molecular targets such as glutamate, GABA (A) delta and GABA (A) alpha-1 receptors, in the
Lamarckian genetic algorithm based on flexible docking studies.
Received 23 September 2011
Received in revised form
14 October 2011
Accepted 19 October 2011
Available online 28 October 2011
Keywords:
N-(4-Substituted phenyl)-2-[4-(substituted)
benzylidene]-hydrazinecarbothioamides
Anticonvulsant activity
Neurotoxicity
Ó 2011 Elsevier Masson SAS. All rights reserved.
Computational study
1. Introduction
antiepileptic drugs demand the need for the development of more
effective and safer antiepileptic drugs.
Epilepsy is one of the most common disorders of the brain,
affecting more than 50 million individuals worldwide [1,2].
Epilepsy is a chronic and often progressive disorder characterized
by the periodic and unpredictable occurrence of epileptic seizures
that are caused by abnormal discharge of cerebral neurons [2].
Epilepsy is not a disease, but a syndrome of different cerebral
disorders of the CNS. This syndrome is characterized by parox-
ysmal, excessive, and hyper synchronous discharges of large
numbers of neurons [3]. About 50 million people worldwide have
epilepsy with almost 90% of these people being in developing
countries [4]. Epilepsy also affects about 4% of individuals over their
life time. Despite the development of several new anticonvulsants,
over 30% of people with epilepsy do not have seizure control and
others do so only at the expense of significant dose related toxicity
and peculiar adverse effects that range in harshness from minimal
brain impairment and megaloblastic anemia to death from aplastic
anemia or hepatic failure [5,6]. These limitations with conventional
Several investigations have recognized aryl thiosemicarbazones
as structurally novel class of anticonvulsants [7e10]. In terms of
interaction at the binding site, as proposed by Dimmock et al., the
pharmacophoric elements were thought to be in the lipophilic aryl
ring and hydrogen bonding domain. The attachment of second aryl
ring, designated as the distal ring to the proximal aryl ring to
increase the van der waal’s bonding at the binding site and to
increase potency, has also been reported [11,12]. Substitution in the
aryl ring by halogens has been found to increase potency in the MES
screen [13,14]. To test this hypothesis, the design and synthesis of
the titled compounds were carried out by molecular hybridization
of the aryl thiosemicarbazide (1) and the 4-(aryloxy)phenyl semi-
carbazones (2) (Fig. 1). These aryl thiosemicarbazones do not
possess the dicarboxamide group as found in conventional anti-
epileptic drugs e.g. barbiturates, hydantoins, oxazolidiones etc.,
which may be associated with toxicity and side effects.
The present work reports the synthesis and anticonvulsant
activity
of
N-(4-substituted
phenyl)-2-[4-(substituted)
benzylidene] hydrazinecarbothioamides PC 1e36. Their chemical
* Corresponding author. Tel.: þ91 9897112288.
structures were characterized using IR, 1H NMR, MS and elemental
E-mail address: tripathilaxmi@rediffmail.com (L. Tripathi).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.038

154
L. Tripathi et al. / European Journal of Medicinal Chemistry 47 (2012) 153e166
O
parameters. In this study, we have used AutoDock 4.0 along with its
S
H
H
H
N
H2N
C
N
N
C
O Ar
LGA algorithm for automated flexible ligand docking of compounds
with six established epilepsy molecular targets and evaluated
docking affinity and count of probable hydrogen bonds.
X
N
C
NH2
H
(2)
(1)
Molecular Hybridization
2. Chemistry
The reaction sequence leading to the formation of the titled
compounds, viz. N-(4-substituted phenyl)-2-[4-(substituted)
benzylidene]-hydrazine carbothioamides PC 1e36 is shown in
Scheme 1. The N-(4-substituted phenyl) hydrazinecarbothioamides
2aed were obtained by reacting 4-substituted aniline 1aed with
carbon disulphide in presence of sodium hydroxide and then with
hydrazine hydrate. The 4-substituted benzaldehydes 5aei were
prepared by refluxing various substituted phenol 3aei with 4-
fluoro benzaldehyde 4 in N,N’-DMF in presence of potassium
carbonate The N-(4-substituted phenyl) hydrazinecarbothioamides
2aed were refluxed with 4-substituted benzaldehyde 5aei in the
presence of catalytic amount of glacial acetic acid to yield the titled
compounds PC 1e36. Thin layer chromatography (TLC) was run
throughout the reactions to optimize the reactions for purity and
completion. The physical data for the newly synthesized
compounds are presented in Table 1.
S
H
H
X
N
C
N
N
C
O Ar
H
PC 1- 36
Fig. 1. Concept to design of new N-(4-substituted phenyl)-2-[4-(substituted) benzyli-
dene] hydrazinecarbothioamides PC 1e36.
analysis techniques. All the synthesized titled compounds
comprised of the essential pharmacophoric elements (Fig. 2) that
are necessary for good anticonvulsant activity as suggested by
Unverferth et al. [15]. Their anticonvulsant activity was evaluated
by using experimental epilepsy models, i.e., maximal electroshock
(MES), subcutaneous metrazole (scMET) and psychomotor seizure
(6 Hz) test in mice. The rotorod assay was performed in mice to
evaluate the neurotoxicity of the compounds. Computational study
was also carried out to highlight the pharmacophore distance
mapping, Log P calculation and prediction of pharmacokinetic
The formation of N-(4-substituted phenyl) hydrazinecarbo-
thioamides 2aed from 4-substituted aniline 1aed was confirmed
by its IR and 1H NMR spectral studies. The IR spectra of compound
2b showed a broad band of NHNH2 group at 3338 cmꢀ1 and C]S
stretching at 1164 cmꢀ1. Its 1H NMR spectrum showed a singlet
D
D
N
D
N
H
N
H
N
CH3
A
D
H
H
HD
HA
OH
S
CH3
A
Cl
HD
N
N
O
HD
Cl
H
N
H
A
N
HA
N
HA
O
HD H2N
S HA
H
Albutoin
Carbamazepine
Gabapentin
Lamotrignine
HA
D
HA
O
O
O
N
A
OH
H
H3C
H
N
H
D
NH
O
N
HA
HD
N
D
HD
C2H5
O
N
N
A
F
N
O
O
HA
H
H
HD
H
H
D
CH3
HD
A
Cl
Progabide
Remacemide
Mephobarbital
Phenytoin
O
D
S
H3C
CH3
R'
O
N
O
N
HA
D
A
A
H
X
A
N
C
N
H
N
C
O
R
HD
O
S
HA
N
H
H
H
S
Cl
H
N
HD
HA
O
H
HD
PC 31: X = F
R = Cl
PC 25: X = Br
PC 16: X = Cl
R = CH3
R' = Cl
PC 23: X = Br
R = Br
Ralitoline
Zonisamide
R = CH3
R' = Cl
R' = H
R' = H
Fig. 2. Pharmacophoric pattern of well-known anticonvulsants.

L. Tripathi et al. / European Journal of Medicinal Chemistry 47 (2012) 153e166
155
H
NaOH, CS2, DMF
stirring at 20-350C for
NH2
N
C
S
NHNH2
R
1 h, NH2NH2.H2O
stirring at 60-700C for
1h.
R
2 a-d
1a-d
O
F
K2CO3, N,N'-DMF
reflux
+
Ar OH
Ar
CHO
CHO
5a-i
3a-i
4
O
Ar
H
N
CHO
O
Ar
C
NHNH2
S
5a-i
H
H
N N C N
R
S
C
Reflux, 4-6 h
H
R
ethanol
2 a-d
glacial acetic acid
HPT 1-36
(HPT 1-9, R = H, HPT 10-18, R= Cl; HPT 19-27, R= Br, HPT 28-36, R= F)
HPT 1, 10, 19 & 28: Ar =
HPT 6, 15, 24 & 33: Ar =
HPT 7, 16, 25 & 34: Ar =
F
CH3
NO2
CH3
Cl
HPT 2, 11, 20 & 29: Ar =
HPT 3, 12, 21 & 30: Ar =
HPT 4, 13, 22 & 31: Ar =
Cl
HPT 8, 17, 26 & 35: Ar =
HPT 9, 18, 27 & 36: Ar =
O
O
HPT 5, 14, 23 & 32: Ar =
Br
Scheme 1. Synthesis of N-(4-substituted phenyl)-2-[4-(substituted)benzylidene]- hydrazine carbothioamides PC 1e36.
of eNH2 protons at
d
4.66 ppm. Four aromatic protons (AreH)
confirmed the formation of PC 4. Further mass spectrum confirmed
its purity and molecular weight.
The IR spectrum of the titled compound PC 13 showed NeHstr at
3265 and 3142 cmꢀ1, CH]Nstr at 1589 cmꢀ1, diaryl ether linkage
(eOe) at 1243 cmꢀ1 and C]Sstr at 1165 cmꢀ1. Its 1H NMR spectrum
showed a singlet of imine (CH]N) proton that resonated at
appeared as a set of signals in the region
proton of NHeN and NHeAr appeared at
respectively were D2O exchangeable.
d
6.98e7.64 ppm. One
9.10 and 9.48 ppm
d
d
The formation of 4-substituted benzaldehydes 5aei from 4-
substituted phenols was confirmed by its IR and 1H NMR spectral
studies. The IR spectrum of 4-(4-chloro-phenoxy) benzaldehydes
5d showed bands at 1662 and 1241 cmꢀ1 indicating the presence of
C]O str and diaryl ether linkage (eOe). The absence of broad band
of phenolic eOH at 2900e3000 cmꢀ1 confirms the conversion of 4-
chloro phenol 3d to 4-(4-chlorophenoxy) benzaldehydes 5d. In its
d
7.83 ppm. Twelve aromatic protons (AreH) appeared as a set of
signals in the region 6.90e7.64 ppm. One proton of NHeN and
NHeAr appeared at 9.08 and 9.43 ppm respectively were D2O
d
d
d
exchangeable. The mass spectrum showed Mþ þ 1 peak (m/z) at
416.16 (100.00%), 418.09 (76.62%) and 420.22 (24.30%) for 35,35Cl,
35,37Cl and 37,37Cl respectively. The presence of CH]Nstr at
1589 cmꢀ1 in IR spectrum and a singlet for imine (CH]N) proton at
1H NMR spectrum a singlet at
d 9.81 ppm indicating the presence of
eCHO group, whereas the absence of a singlet around
for phenolic eOH confirm the conversion of 3d to 5d.
d 9.288 ppm
d
7.83 ppm in 1H NMR spectrum, confirmed the formation of PC 13.
The structural assignments to new compounds were based on
their elemental analysis and spectral (FTeIR, 1H NMR and mass)
data. The IR spectrum of the titled compound PC 4 showed N-Hstr
at 3230 and 3117 cmꢀ1, CH ¼ Nstr at 1587 cmꢀ1, diaryl ether linkage
(eOe) at 1241 cmꢀ1 and C]Sstr at 1165 cmꢀ1. Its 1H NMR spectrum
showed a singlet of imine (CH]N) proton that resonated at
Further mass spectrum confirmed its purity and molecular weight.
3. Pharmacology
The newly synthesized N-(4-substituted phenyl)-2-[4-
(substituted)benzylidene] hydrazine carbothioamides PC 1e36
were subjected to anticonvulsant screening according to the anti-
convulsant drug development (ADD) program protocol. The profile
of anticonvulsant activity was established after i.p. injections into
mice and evaluated in the maximal electroshock (MES) and
subcutaneous metrazole (scMET) using doses of 30, 100 and
300 mg/kg at two different time intervals. Neurotoxicity was
observed by minimal motor impairment which was measured by
d
7.89 ppm. Thirteen aromatic protons (AreH) appeared as a set of
signals in the region 6.97e7.67 ppm. One proton of NHeN and
NHeAr appeared at 9.16 and d 9.85 ppm respectively, were D2O
d
d
exchangeable. The mass spectrum showed Mþ þ 1 peak (m/z) at
382.13 (100.00%) and 384.14 (39.52%) for 35Cl and 37Cl respectively.
The presence of CH]Nstr at 1587 cmꢀ1 in IR spectrum and a singlet
for imine (CH]N) proton at
d
7.89 ppm in 1H NMR spectrum,

156
L. Tripathi et al. / European Journal of Medicinal Chemistry 47 (2012) 153e166
Table 1
Physical and elemental analyses data of N-(4-substituted phenyl)-2-[4-(substituted)benzylidene]-hydrazinecarbothioamides PC 1e36.
Compd.
Ar
R
Molecular formula (M.W.)
Mp (ꢁC)
Yield (%)
Elemental analysis (%): found (cal.)
C
H
N
PC 1
PC 2
PC 3
PC 4
PC 5
PC 6
PC 7
PC 8
Phenyl
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
Br
Br
Br
Br
F
C20H17N3OS (347.43)
C20H16N4O3S (392.43)
C21H19N3OS (361.46)
C20H16ClN3OS (381.88)
C20H16BrN3OS (426.33)
C20H16FN3OS (365.42)
C21H18ClN3OS (395.91)
C24H19N3OS (397.49)
146
175
202
205
212
168
222
158
115
106
108
140
150
172
156
143
152
143
138
170
174
164
174
160
158
120
175
113
165
202
162
140
196
192
115
65
71
78
72
77
75
79
81
71
62
68
71
77
74
72
76
78
67
71
74
72
77
70
72
76
62
70
74
71
70
76
69
77
78
65
69.11 (69.14)
61.20 (61.21)
69.75 (69.78)
62.86 (62.90)
56.30 (56.34)
65.71 (65.74)
6.67 (63.71)
72.48 (72.52)
64.39 (64.43)
62.6 (62.90)
56.23 (56.27)
63.4 (63.71)
57.67 (57.70)
52.0 (52.13)
60.01 (60.07)
58.56 (58.61)
66.67 (66.74)
56.2 (56.34)
50.4 (50.97)
57.23 (57.28)
51.10 (52.13)
47.47 (47.55)
54.01 (54.06)
53.11 (53.12)
60.48 (60.51)
53.61 (53.63)
65.72 (65.74)
58.51 (58.53)
66.46 (66.47)
60.04 (60.07)
54.05 (54.06)
62.63 (62.65)
60.92 (60.94)
69.37 (69.38)
61.58 (61.60)
4.95 (4.93)
4.07 (4.11)
5.25 (5.30)
4.21 (4.22)
3.76 (3.78)
4.39 (4.41)
4.55 (4.58)
4.80 (4.82)
4.34 (4.38)
4.0 (4.22)
12.06 (12.09)
14.22 (14.28)
11.60 (11.63)
10.93 (11.00)
9.87 (9.86)
11.45 (11.50)
10.57 (10.61)
10.52 (10.57)
10.68 (10.73)
10.9 (11.00)
13.12 (13.12)
10.4 (10.61)
10.8 (10.09)
8.99 (9.12)
10.49 (10.51)
9.68 (9.76)
9.66 (9.73)
9.82 (9.86)
11.81 (11.89)
9.54 (9.54)
9.10 (9.12)
8.30 (8.32)
9.43 (9.46)
8.84 (8.85)
8.81 (8.82)
8.91 (8.93)
11.48 (11.50)
13.63 (13.65)
11.05 (11.07)
10.50 (10.51)
9.44 (9.46)
10.94 (10.96)
10.14 (10.15)
10.10 (10.11)
10.25 (10.26)
4-NO2 phenyl
4-CH3 phenyl
4-Cl phenyl
4-Br phenyl
4-F phenyl
3-CH3, 4-Cl phenyl
Naphthalene-2-yl
1,3-benzodioxol-5-yl
Phenyl
4-NO2 phenyl
4eCH3 phenyl
4-Cl phenyl
PC 9
C21H17N3O3S (391.44)
PC 10
PC 11
PC 12
PC 13
PC 14
PC 15
PC 16
PC 17
PC 19
PC 20
PC 21
PC 22
PC 23
PC 24
PC 25
PC 26
PC 27
PC 28
PC 29
PC 30
PC 31
PC 32
PC 33
PC 34
PC 35
PC 36
C20H16ClN3OS (381.88)
C20H15ClN4O3 (426.88)
C21H18ClN3OS (395.91)
C20H15Cl2N3OS (416.32)
C20H15BrClN3OS (460.77)
C20H15ClFN3OS (399.87)
C21H17Cl2N3OS (430.35)
C24H18ClN3OS (431.94)
C20H16BrN3OS (426.33)
C20H15BrN4O3S (471.33)
C21H18BrN3OS (440.36)
C20H15BrClN3OS (460.77)
C20H15Br2N3OS (505.23)
C20H15BrFN3OS (444.32)
C21H17BrClN3OS (474.80)
C24H18BrN3OS (476.39)
C21H16BrN3O3S (470.34)
C20H16FN3OS (365.42)
C20H15FN4O3S (410.42)
C21H18FN3OS (379.45)
C20H15ClFN3OS (399.87)
C20H15BrFN3OS (444.32)
3.3 (3.54)
4.3 (4.58)
3.55 (3.63)
3.18 (3.28)
3.68 (3.78)
3.88 (3.98)
4.18 (4.20)
3.68 (3.78)
3.17 (3.21)
4.09 (4.12)
3.18 (3.28)
2.86 (2.99)
3.37 (3.40)
3.59 (3.61)
3.79 (3.81)
3.41 (3.43)
4.40 (4.41)
3.66 (3.68)
4.77 (4.78)
3.77 (3.78)
3.39 (3.40)
3.93 (3.94)
4.12 (4.14)
4.35 (4.37)
3.93 (3.94)
4-Br phenyl
4-F phenyl
3-CH3, 4-Cl phenyl
Naphthalene-2-yl
Phenyl
4-NO2 phenyl
4-CH3 phenyl
4-Cl phenyl
4-Br phenyl
4- F phenyl
3-CH3, 4-Cl phenyl
Naphthalene-2-yl
1,3-benzodioxol-5-yl
Phenyl
4-NO2 phenyl
4-CH3 phenyl
4-Cl phenyl
4-Br phenyl
4- F phenyl
3-CH3, 4-Cl phenyl
Naphthalene-2-yl
1,3-benzodioxol-5-yl
F
F
F
F
F
F
F
F
C20H15F2N3OS (383.41)
C21H17ClFN3OS (413.90)
C24H18FN3OS (415.48)
C21H16FN3O3S (409.43)
the rotorod test. The results are shown in Table 2. Some compounds
were screened in the 6 Hz model to identify their activity at five
different time points, ie, 0.25, 0.5, 1.0, 2.0 and 4.0 h after i.p.
administration in mice. The results are shown in Table 3. Potential
compounds were also subjected to quantification studies in 6 Hz
test and the corresponding ED50 and TD50 reported in Table 4. Some
compounds were screened in the acute toxicity test of pilocarpine
induced status prevention (PISP) model (Table 5) and in vitro
hippocampal slice culture neuroprotection assay (NP).
using ACD/3D viewer version 12.0 and Argus Lab 4.0 Mark A.
Thompson Planaria Software LLC (Table 6). A computational study
of all compounds was performed for prediction of ADME properties
such as absorption (%ABS), polar surface area (TPSA), miLog P,
number of rotatable bonds, and violations of Lipinski’s rule of five
by using Molinspiration online property calculation toolkit
Table 3
Anticonvulsant activity of some N-(4-substituted phenyl)-2-[4-(substituted) ben-
zylidene] hydrazinecarbothioamides by 6 Hz model.
4. Computational study
Compound
Dose (mg/kg)
Time (h) to peak effect (N/F)a
The pharmacophore pattern studies in which the distance
between the various groups postulated as essential for anticon-
vulsant activity (Fig. 3) were done on the 3D optimized structures
0.25
0.5
1.0
2.0
4.0
PC 5
PC 6
PC 7
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
0/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
1/4
1/4
0/4
1/4
3/4
0/4
2/4
0/4
0/4
1/4
0/4
1/4
1/4
1/4
1/4
1/4
3/4
1/4
3/4
1/4
2/4
2/4
4/4
1/4
1/4
1/4
0/4
1/4
1/4
0/4
0/4
2/4
0/4
1/4
0/4
1/4
2/4
0/4
1/4
0/4
1/4
0/4
0/4
1/4
0/4
1/4
0/4
0/4
1/4
2/4
0/4
0/4
0/4
1/4
2/4
0/4
0/4
0/4
1/4
0/4
1/4
0/4
1/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
1/4
Table 2
PC 15
PC 16
PC 19
PC 21
PC 23
PC 24
PC 25
PC 28
PC 29
PC 30
PC 31
PC 32
PC 33
PC 34
PC 35
Anticonvulsant activity (MES and scMET test) and neurotoxicity of N-(4-substituted
phenyl)-2-[4-(substituted)benzylidene] hydrazinecarbothioamides.
Compound
Intraperitoneal injection in micea
MES screen (h)
scMET
screen (h)
Neurotoxicity
screen (h)
0.5
2.0
4.0
0.5
4.0
0.5
4.0
PC 23
Phenytoin
Sodium valproate
e
e
300a
30a
e
e
e
e
e
e
e
30a
e
30a
e
e
300a
100a
e
100a
e
a
Doses of 30, 100 and 300 mg/kg were administered. The figures in the table
indicate the minimum dose whereby bioactivity was demonstrated in half or more
of the mice. The dash (e) indicates absence of activity at maximum dose adminis-
tered (300 mg/kg).
a
N/F ¼ number of animals active or toxic over the number tested.

L. Tripathi et al. / European Journal of Medicinal Chemistry 47 (2012) 153e166
157
Table 4
Quantification studies of PC 31: ED50 and TD50 values.
A- Hydrophobic Unit
D- Electron Donar Group
HA- Hydrogen Bond Acceptor Unit
HD- Hydrogen Bond Donar Unit
Compound Test Time ED50
95% Confidence Slope STD PIa
D
(h) (mg/kg) interval
Err
value
4.30
(3.01-5.38)
PC 31
6 Hz 0.5
TOX 0.5
>200
>500
e
e
e
e
e
e
>2.5
a
PI value was determined by TD50/ED50.
4.86
(2.43-6.79)
C
C
C
C
4.37
(2.52-6.72)
A
C
C
(Table 7). Calculated miLog P for synthesized compounds were then
compared with the experimental Log P data of these compounds
(Table 8). Docking study of titled compounds was performed with
six established epilepsy molecular targets namely GABA (A) alpha-1
receptor, GABA (A) delta receptor, glutamate receptor, Na/H
exchanger, Na channel receptor, T-type calcium channel receptor by
using Autodoc 4.0 along with its LGA algorithm for automated
flexible ligand docking. The crystallographic structure of target
receptors is given in Fig. 4. Their affinity (Kcal/mol) and count of
probable hydrogen bonds were evaluated (Table 9). Docking images
of compounds PC 16, PC 23, PC 25 and PC 31 with the target
receptors is shown in Fig. 5.
6.05
(3.78-9.51)
5.86
(2.72-9.23)
HA
2.54
(1.78-3.87)
HD
Fig. 3. Four-point 3D pharmacophore model for anticonvulsants derived by using
MM3 and CHARMm parametrization (Argus Lab 4.0 and ACD/3D viewer).
5.2. Computational study
5.2.1. Distance mapping
5. Results and discussion
The present work involves the correlation of the structural
requirement of well known and structurally different anticonvul-
sant compounds with the titled compounds. The two-dimensional
(2D) modeling on anticonvulsants has identified that at least one
aryl unit, one or two electron donor atoms, and/or an NH group in
a special spatial arrangement is recommended for anticonvulsant
activity. In the present study, the 10 wellknown and structurally
different compounds with anticonvulsant activity- albutoin, car-
bamazepine, gabapentin, lamotrigine, mephobarbital, phenytoin,
progabide, ralitoline, remacemide and zonisamide (Fig. 2) with
different mechanisms of action, were selected so as to propose
a generalized pharmacophore model. The pharmacophore group’s
distance estimation was done by molecular mechanics calculation
with the force fields based on both CHARMm force fields and MM3
parametrization. In the present work, energy minimization was
performed on the above mentioned ten well known anticonvul-
5.1. Anticonvulsant activity and neurotoxicity
Compound PC 23 was found to be active in the MES test,
indicative of the compounds ability to prevent seizure spread. The
compound PC 23 showed 100% protection (1/1, 4.0 h) at a dose of
300 mg/kg. Titled compounds did not exhibit protection in the
scMET test, a test used to identify compounds ability to raise
seizure threshold. None of the compounds showed neurotoxicity in
the highest administered dose (300 mg/kg).
Compounds showed more promising results when tested in the
6 Hz model. Compound PC 31 was the most active one in this series
with protection at two different time points, i.e. 100% (4/4, 0.5 h)
and 75% (3/4, 0.25 h) at a dose of 100 mg/kg. Compound PC 23
showed 75% protection (3/4, 0.5 h) at a dose of 100 mg/kg.
Compound PC 25 showed 75% protection (3/4, 0.5 h) and 50%
protection (2/4, 1 h; 2/4, 2.0 h) at a dose of 100 mg/kg. Some
compounds showed 50% protection at different time points at
a dose of 100 mg/kg viz. PC 16 (2/4, 1.0 h; 2/4, 2.0 h), PC 29 (2/4,
0.5 h), PC 30 (2/4, 0.5 h) and PC 33 (2/4, 0.25 h). Other compounds
showed mild activity in the 6 Hz test. Compound PC 31 was eval-
uated in the quantitative 6 Hz test and showed an ED50 of >200 mg/
kg and TD50 > 500 mg/kg at a time of peak effect (TPE) of 0.5 h. The
PI value was >2.5.
sants
and
N-(4-substituted
phenyl)-2-[4-(substituted)
benzylidene]-hydrazine carbothioamides using Argus Lab 4.0.
Distance between the various structural components essential for
activity was determined by the ACD/3D viewer. The crucial struc-
tural components that were included in the four-point pharmaco-
phore model were the aryl ring center or the lipophilic group (A),
an electron donor atom (D), a hydrogen bond acceptor (HA), and
a hydrogen bond donor (HD). An average distance range for every
point was obtained and compared to the N-(4-substituted phenyl)-
2-[4-(substituted)benzylidene] hydrazine carbothioamides. Now it
may be interesting to examine whether the N-(4-substituted
phenyl)-2-[4-(substituted)benzylidene] hydrazine carbothioa-
mides reflect the conditions of the derived pharmacophore model.
Our analysis of the distance relationship showed that N-(4-
substituted phenyl)-2-[4-(substituted)benzylidene] hydrazine car-
bothioamides did fulfill the essential demands of the pharmaco-
phore when compared to the average distance requirement.
Compound PC 25 was subjected to the acute toxicity test of
pilocarpine induced status prevention (PISP) model. In the acute
toxicity test the compound PC 25 did not show any toxicity up to
600 mg/kg. Compounds PC 19 and PC 31 were evaluated in the
primary screen experiment of in vitro hippocampal slice culture
neuroprotection assay (NP). No significant protection was observed
against either KA- or NMDA-induced cytotoxicity.
Table 5
5.2.2. Prediction of ADME properties
Pilocarpine induced status prevention (PISP) model in acute toxicity test of PC 25.
A computational study for prediction of ADME properties of
titled compounds was performed. Topological polar surface area
(TPSA), i.e., surface belonging to polar atoms, is a descriptor that
was shown to correlate well with passive molecular transport
through membranes and, therefore, allows prediction of transport
properties of drugs in the intestines and bloodebrain barrier
crossing [16]. The percentage of absorption (%ABS) was calculated
Compound
Test
Dose (mg/kg)
Time (h) (N/F)a
0.25
0.5
1.0
2.0
4.0
PC 25
TOX
TOX
TOX
100
300
600
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
a
N/F ¼ number of animals toxic over the number tested.

158
L. Tripathi et al. / European Journal of Medicinal Chemistry 47 (2012) 153e166
Table 6
Distance range between the essential structural elements A, D and HAeHD.a
Compounds
AeHA
AeHD
AeD
HAeHD
HDeD
HAeD
Albutoin
5.37
2.72
4.51
2.72
4.03
5.40
Carbamazepine
Gabapentin
Lamotrigine
Mephobarbital
Phenytoin
Progabide
Ralitoline
Remacemide
Zonisamide
Average distance
(Range)
4.28
4.26
5.30
3.78
6.20
9.51
8.30
7.51
4.28
4.93
7.42
5.50
4.01
9.23
5.55
8.75
4.25
3.83
4.54
4.81
4.35
3.79
4.56
5.38
2.33
2.23
2.42
2.34
2.63
2.41
2.75
3.87
5.75
3.57
4.94
4.63
3.88
6.72
2.52
3.96
5.67
4.50
4.25
5.23
5.17
6.79
4.85
2.43
6.02
6.05
6.22
5.86
3.01
4.30
1.78
2.54
3.71
4.37
4.31
4.86
(3.78ꢀ9.51)
(2.72ꢀ9.23)
(3.01ꢀ5.38)
(1.78ꢀ3.87)
(2.52ꢀ6.72)
(2.43ꢀ6.79)
PC 16
6.39
5.07
4.05
1.39
2.61
3.24
PC 23
6.41
5.08
4.02
1.40
2.60
3.26
PC 25
6.40
5.07
4.05
1.41
2.61
3.25
PC 31
6.41
5.08
4.06
1.42
2.62
3.26
a
Distances calculated for 3D optimized structures using MM3 and CHARMm parameterization (Argus Lab 4.0 and ACD/3D viewer).
using TPSA. From all these parameters, it can be observed that all
titled compounds exhibited a great %ABS ranging from 77.4 to
93.2%. All compounds of this series except PC 9 violated Lipinski’s
parameters. It may be observed from the results that the violation
observed in Lipinski’s parameters were due to higher Log P value
than that reported in the rule.
5.2.3. Log P determination
Titled compounds showed dependence of biological activity on
lipophillic character in a congeneric series. In particular, for drugs
acting on central nervous system to be potent, they have to cross
bloodebrain barrier (BBB), thus potency has been correlated with
optimum lipophilicity (Log P) near 2. In this study, we attempted to
Table 7
Pharmacokinetic parameters important for good oral bioavailability of compounds N-(4-substituted phenyl)-2-[4-(substituted)benzylidene]hydrazine carbothioamide PC
1e36.
Compound
% ABS
TPSA (A2)
n-ROTB
MW
Molecular volume
miLogP
n-OHNH donors
n-OH acceptors
Lipinski’s violation
Rule
PC 1
PC 2
PC 3
PC 4
PC 5
PC 6
PC 7
e
e
e
7
8
7
7
7
7
7
7
7
7
8
7
7
7
7
7
7
7
7
8
7
7
7
7
7
7
7
7
8
7
7
7
7
7
7
7
<500
347.4
e
ꢂ5
5.0
5.0
5.5
5.7
5.8
5.2
6.1
6.2
4.9
5.7
5.7
6.1
6.4
6.5
5.9
6.8
6.9
5.6
5.8
5.8
6.3
6.5
6.6
6.0
6.9
7.0
5.7
5.2
5.1
5.6
5.9
6.0
5.3
6.2
6.4
5.1
<5
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
<10
4
7
4
4
4
4
4
4
6
4
7
4
4
4
4
4
4
6
4
7
4
4
4
4
4
4
6
4
7
4
4
4
4
4
4
6
ꢂ1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
93.2
77.4
93.2
93.2
93.2
93.2
93.2
93.2
86.8
93.2
77.4
93.2
93.2
93.2
93.2
93.2
93.2
86.8
93.2
77.4
93.2
93.2
93.2
93.2
93.2
93.2
86.8
93.2
77.4
93.2
93.2
93.2
93.2
93.2
93.2
86.8
45.6
91.4
45.6
45.6
45.6
45.6
45.6
45.6
64.1
45.6
91.4
45.6
45.6
45.6
45.6
45.6
45.6
64.1
45.6
91.4
45.6
45.6
45.6
45.6
45.6
45.6
64.1
45.6
91.4
45.6
45.6
45.6
45.6
45.6
45.6
64.1
311.7
335.1
328.3
325.3
329.6
316.7
341.8
355.7
335.7
325.3
348.6
341.8
338.8
343.1
330.2
355.4
369.3
349.2
329.6
352.9
346.2
343.1
347.5
334.5
359.7
373.6
353.5
316.7
340.0
333.2
330.2
334.5
321.6
346.8
360.6
340.6
392.4
361.4
381.8
426.3
365.4
395.9
397.5
391.4
381.8
426.8
395.9
416.3
460.7
399.8
430.3
431.9
425.8
426.3
471.3
440.3
460.7
505.2
444.3
474.8
476.3
470.3
365.4
410.4
379.4
399.8
444.3
383.4
413.9
415.4
409.4
PC 8
PC 9
PC 10
PC 11
PC 12
PC 13
PC 14
PC 15
PC 16
PC 17
PC 18
PC 19
PC 20
PC 21
PC 22
PC 23
PC 24
PC 25
PC 26
PC 27
PC 28
PC 29
PC 30
PC 31
PC 32
PC 33
PC 34
PC 35
PC 36
%ABS, percentage of absorption; TPSA, topological polar surface area; n-ROTB, number of rotatable bonds; MW, molecular weight; MV, molecular volume; n-OHNH, number of
hydrogen bond donors; n-ON, number of hydrogen bond acceptors.

L. Tripathi et al. / European Journal of Medicinal Chemistry 47 (2012) 153e166
159
Table 8
Log
correlate the anticonvulsant activity of congeners with their
calculated Log P value. The experimental Log P values were deter-
mined using the octanolephosphate buffer method. As observed
some of the experimental values were in good agreement with the
theoretical values. All the titled compounds showed lipophillic
character. The Log P determined is close to the optimum Log P
value. Optimum Log P value ensures good pharmacokinetic prop-
erty of drugs. Drugs possessing optimum Log P value will be least
inhibited in their movement through the aqueous and lipophilic
phases of living tissue.
P
value of N-(4-substituted phenyl)-2-[4-(substituted)benzylidene]
hydrazinecarbothioamide PC 1e36.
Compound
Experimental
Theoretical
Log P
miLog P
Standard
PC 1
PC 2
PC 3
PC 4
PC 5
PC 6
PC 7
PC 8
e
ꢂ5
5.0
5.0
5.5
5.7
5.8
5.2
6.1
6.2
4.9
5.7
5.7
6.1
6.4
6.5
5.9
6.8
6.9
5.6
5.8
5.8
6.3
6.5
6.6
6.0
6.9
7.0
5.7
5.2
5.1
5.6
5.9
6.0
5.3
6.2
6.4
5.1
4.9
4.95
5.39
5.52
5.62
5.09
6.03
6.07
4.80
5.64
5.52
5.97
6.20
6.37
5.84
6.59
6.96
5.54
6.38
5.68
6.17
6.24
6.53
5.82
7.89
6.93
5.52
5.09
4.89
5.48
5.78
5.88
5.19
6.07
6.20
4.99
5.2.4. Docking study
In this study, we have used AutoDock 4.0 along with its LGA
algorithm for automated flexible ligand docking of compounds PC
1e36 with six established epilepsy molecular targets namely GABA
(A) alpha-1 receptor, GABA (A) delta receptor, glutamate receptor,
Na/H exchanger, Na channel receptor, T-type calcium channel
receptor and evaluated docking affinity (Kcal/mol) and count of
probable hydrogen bonds. In this study, compound PC 16, PC 23, PC
25 and PC 31 were selected for docking with six established
epilepsy molecular targets. Compound PC 16 exhibited good
binding properties with GABA (A) alpha-1 receptor (affinity
value ꢀ4.7 kcal/mol and 3 H-bonds), GABA (A) delta receptor
(affinity value ꢀ6.7 kcal/mol and 2 H-bonds) and glutamate
receptor (affinity value ꢀ6.0 kcal/mol and 1 H-bonds). Compound
PC 16 showed affinity for Na/H exchanger (affinity value ꢀ5.5 kcal/
mol), but did not exhibit H-binding properties. Compound PC 16
did not show affinity and H-bonding with Na channel receptor and
T-type calcium channel receptor. Compound PC 23 exhibited good
binding properties with GABA (A) delta receptor (affinity
value ꢀ6.3 kcal/mol and 2 H-bonds), GABA (A) alpha-1 receptor
(affinity value ꢀ6.0 kcal/mol and 1 H-bonds), glutamate receptor
(affinity value ꢀ6.0 kcal/mol and 1 H-bonds) and Na/H exchanger
(affinity value ꢀ5.4 kcal/mol and 1 H-bonds). Compound PC 23 did
not show affinity and H-bonding with Na channel receptor and T-
type calcium channel receptor. Compound PC 25 exhibited
good binding properties with glutamate receptor (affinity
PC 9
PC 10
PC 11
PC 12
PC 13
PC 14
PC 15
PC 16
PC 17
PC 18
PC 19
PC 20
PC 21
PC 22
PC 23
PC 24
PC 25
PC 26
PC 27
PC 28
PC 29
PC 30
PC 31
PC 32
PC 33
PC 34
PC 35
PC 36
Fig. 4. Crystallographic structure of different receptors used in docking study. (a) GABA (A) alpha-1, (b) GABA(A) delta, (c) glutamate (d) Na/H exchanger (e) Na channel (f) T-type
calcium channel receptor.

160
L. Tripathi et al. / European Journal of Medicinal Chemistry 47 (2012) 153e166
Table 9
Docking study of compounds PC 16, PC 23, PC 25 and PC 31.a
Ligand
Receptor
Affinity (Kcal/mol)
H-bonds
H-binding ligand
H-binding receptor
Element
Atom No.
Type
Residue
Element
Atom No.
Type
PC 16
GABA(A) Alpha-1
ꢀ4.7
03
N
N
N
N
N
N
e
e
e
N
N
N
N
N
e
e
N
N
N
e
N
N
N
N
e
e
N
N
N
N
N
e
e
e
11
11
07
11
07
07
e
Donor
Donor
Donor
Donor
Donor
Donor
e
Ile 329
Thr 333
Thr 333
Asp 311
Asp 311
Leu 469
e
O
O
O
O
O
O
e
e
e
O
O
O
O
O
e
e
N
O
O
e
O
O
N
O
e
e
O
O
O
O
N
e
e
e
462
501
501
317
317
250
e
Acceptor
Both
Both
Acceptor
Acceptor
Acceptor
e
GABA (A) delta
ꢀ6.7
02
Glutamate
Na/H exchanger
Na channel
T-type calcium
GABA(A) alpha-1
GABA (A) delta
ꢀ6.0
ꢀ5.5
0.0
01
00
0.0
0.0
01
02
e
e
e
e
e
0.0
e
e
e
e
e
PC 23
PC 25
ꢀ6.0
07
7
7
11
11
e
Donor
Donor
Donor
Donor
Donor
e
Ala 318
Asp 311
Asp 311
Leu 455
Phe 253
e
366
317
320
102
100
e
Acceptor
Acceptor
Acceptor
Acceptor
Acceptor
e
ꢀ6.3
Glutamate
Na/H exchanger
Na channel
T-type calcium
GABA(A) alpha-1
ꢀ6.0
ꢀ5.4
0.0
0.0
ꢀ5.1
01
01
0.0
0.0
03
e
e
e
e
e
13
11
07
e
Acceptor
Donor
Donor
e
Val 287
Thr 283
Arg 282
e
74
44
27
e
Donor
Acceptor
Acceptor
e
GABA (A) delta
Glutamate
ꢀ6.4
ꢀ6.9
00
03
07
11
13
07
e
Donor
Donor
Acceptor
Donor
e
Lys 467
Ser 465
Ser 465
Ala 250
e
230
213
209
76
Acceptor
Acceptor
Donor
Acceptor
e
Na/H exchanger
Na channel
T-type calcium
GABA(A) alpha-1
GABA (A) delta
Glutamate
ꢀ5.7
0.0
0.0
ꢀ6.0
ꢀ6.2
ꢀ6.8
01
0.0
0.0
01
01
03
e
e
e
e
e
e
PC 31
13
11
07
11
13
e
Acceptor
Donor
Donor
Donor
Acceptor
e
Ser 297
Phe 320
Lys 467
Ser 465
Ser 465
e
168
414
230
213
209
e
Both
Acceptor
Acceptor
Acceptor
Donor
e
Na/H exchanger
Na channel
T-type calcium
ꢀ5.5
0.0
0.0
00
0.0
0.0
e
e
e
e
e
e
e
e
e
e
a
Affinity and count of probable hydrogen bonds were determined by docking studies using Autodock 4.0 software.
value ꢀ6.9 kcal/mol and 3 H-bonds), GABA (A) alpha-1 receptor
(affinity value ꢀ5.1 kcal/mol and 3 H-bonds) and Na/H exchanger
(affinity value ꢀ5.7 kcal/mol and 1 H-bonds). Compound PC 25
showed affinity for GABA (A) delta receptor (affinity
value ꢀ6.4 kcal/mol), but did not exhibit H-binding properties.
Compound PC 25 did not show affinity and H-bonding with Na
channel receptor and T-type calcium channel receptor. Compound
PC 31 exhibited good binding properties with glutamate receptor
(affinity value ꢀ6.8 kcal/mol and 3 H-bonds), GABA (A) delta
receptor (affinity value ꢀ6.2 kcal/mol and 1 H-bonds) and GABA (A)
alpha-1 receptor (affinity value ꢀ6.0 kcal/mol and 1 H-bonds).
Compound PC 31 showed affinity for Na/H exchanger (affinity
value ꢀ5.5 kcal/mol), but did not exhibit H-binding properties.
Compound PC 31 did not show affinity and H-bonding with Na
channel receptor and T-type calcium channel receptor.
In the present study various N-(4-substituted phenyl)-2-[4-
substituted benzylidene] hydrazinecarbothioamides were synthe-
sized in order to investigate the pharmacophoric substituent,
responsible for the activity. The 4-substituted phenyl ring acts as
a lipophilic domain, C]S group acts as electron donor, NH groups
acts as hydrogen bonding domain and the aryl substituents acts as
distal hydrophobic center (Fig. 6). Thus it may be stated that
compounds PC 1e36 fulfill the essential pharmacophoric require-
ments for anticonvulsant activity.
In the synthetic design of compounds, the various substituents
were placed at positions 7 and 18 of N-phenyl-benzylidenehy-
drazine carbothioamide backbone structure. At position 7, phenyl,
4-nitro phenyl, 4-methyl phenyl, 4-chloro phenyl, 4-bromo phenyl,
4-fluoro phenyl, 4-chloro-3-methyl phenyl, naphthalene-2-yl, 1,3-
benzodioxol-5-yl substituents were introduced. While at position
18, the substituent viz. eH, 4-chloro, 4-bromo, 4-fluoro, 4-methyl
and 4-methoxy substituents were introduced.
The 4-fluoro substituent at position 18 of N-phenyl-benzylide-
nehydrazine carbothioamide backbone structure yields the most
active compounds PC 28e36. These compounds displayed signifi-
cant protection with longer duration of action. This observation
supports the fact thatelectron rich substituent at paraposition of the
aryl ring showed increased potency in anticonvulsant screen [11].
The most active compounds of this series are PC 31. It has 4-
fluoro substituent at position 18 and 4-chloro phenyl substituent
at position 7. It was observed that 4-halogenated substituents at
position 18 and 4-halogenated phenyl or 4-methyl phenyl substit-
uents at position 7 yield compounds with moderate to significant
protection. The order of percentage protection among substituents
at position 7 is 4-chloro phenyl ꢄ 4-methyl phenyl > 4-bromo
Docking study results show that the titled compounds exhibited
good H-bonding properties with glutamate, GABA (A) alpha, GABA
(A) delta and Na/H exchanger, hence emerge as potential anticon-
vulsants with multiple modes of action.
5.2.5. StructureeActivity relationship
Two-dimensional (2D) modeling and structure activity rela-
tionship of potential anticonvulsant agents propose the following
essential pharmacophoric features for anticonvulsant activity
ꢃ at least one aryl group which acts as a lipophilic domain.
ꢃ one or two electron donor atoms.
ꢃ and/or an NH group in a special spatial arrangement which acts
as a H-bonding domain.
ꢃ and a distal hydrophobic center [11].

L. Tripathi et al. / European Journal of Medicinal Chemistry 47 (2012) 153e166
161
Fig. 5. Docking images of PC 16, PC 23, PC 25 and PC 31. (a) PC 16 with GABA(A) alpha-1, (b) PC 16 with GABA(A) delta, (c) PC 16 with glutamate receptor (d) PC 23 with GABA(A)
alpha-1 (e) PC 23 with GABA(A) delta (f) PC 23 with glutamate receptor, (g) PC 23 with Na/H exchanger, (h) PC 25 with GABA(A) alpha-1, (i) PC 25 with glutamate receptor, (j) PC 25
with Na/H exchanger, (k) PC 31 with GABA(A) alpha-1, (l) PC 31 with GABA(A) delta, (m) PC 31 with glutamate receptor.
phenyl > 3-methyl-4-chloro phenyl > 4-fluoro phenyl. The 4-H
substituent at position 18 and naphthalene-2-yl or
1,3-benzodioxol-5-yl substituent at position 7, yielded inactive
compounds.
6. Conclusion
A
series of N-(4-substituted phenyl)-2-[4-(substituted)
benzylidene]-hydrazine
carbothioamides
were
designed,

162
L. Tripathi et al. / European Journal of Medicinal Chemistry 47 (2012) 153e166
(CDCl3, 300 MHz)
d in ppm: 4.68 (s, 2H, NH2), 6.92e7.66 (a set of
Lipophilic
domain
Electron
donor
atom
Distal
hydrophobic
centre
signals, 5H, AreH), 9.07 (s, 1H, NHeN, D2O exchangeable), 9.53 (s,
1H, AreNH, D2O exchangeable); MS (m/z, %): 168.04 (Mþ þ 1,
100.00).
S
H
H
7.1.1.2. N-(4-chloro phenyl) hydrazꢀin1ecarbothioamide (2b). Yield:
S); 1H NMR (CDCl3, 300 MHz)
d in ppm: 4.66 (s, 2H, NH2), 6.98e7.64
R
N
C
N
N
C
O Ar
62%; m.p.: 162e164 ꢁC; IR (KBr, cm
) y: 3338 (NHNH2), 1164 (C]
H
(a set of signals, 4H, AreH), 9.10 (s, 1H, NHeN, D2O exchangeable),
9.48 (s, 1H, AreNH, D2O exchangeable); MS (m/z, %): 203.08
(Mþ þ 1 for 35Cl, 78.23), 205.02 (Mþ þ 1 for 37Cl, 26.52).
NH group acting as a H-bonding domain
Fig. 6. Essential pharmacophoric requirements of PC 1e36 for anticonvulsant activity.
7.1.1.3. N-(4-bromo phenyl) hydrazꢀin1ecarbothioamides (2c). Yield:
) y: 3332 (NHNH2), 1167 (C]
synthesized, and their anticonvulsant activity and neurotoxicity
were evaluated after intraperitoneal administration in three
seizure models, which include the MES, scMET and 6 Hz model. A
computational study was also carried out, including calculation of
pharmacophore pattern, prediction of pharmacokinetic properties
and docking studies. The compound PC 31 displayed significant
protection and emerged as a lead in this series. Further, compound
PC 16, PC 23 and PC 25 came out as a potential candidate for further
investigation. The results support the fact that substitution in the
aryl ring by halogens lead to more active anticonvulsant
compounds in comparision to unsubstituted derivatives. Docking
study results shows that the compounds exhibited good binding
properties with glutamate, GABA (A) delta and GABA (A) alpha-1
receptor. The docking study data strongly support the assumption
that these receptors may be involved in observed anticonvulsant
68%; m.p.: 170e172 ꢁC; IR (KBr, cm
S); 1H NMR (CDCl3, 300 MHz)
d in ppm: 4.62 (s, 2H, NH2), 6.92e7.66
(a set of signals, 4H, AreH), 9.06 (s, 1H, NHeN, D2O exchangeable),
9.42 (s, 1H, AreNH, D2O exchangeable); MS (m/z, %): 248.95
(Mþ þ 1 for 81Br, 55.23), 246.98 (Mþ þ 1 for 79Br, 54.12).
7.1.1.4. N-(4-fluoro phenyl) hydraziꢀn1ecarbothioamides (2d). Yield:
) y: 3336 (NHNH2), 1163 (C]
73%; m.p.: 181e182 ꢁC; IR (KBr, cm
S); 1H NMR (CDCl3, 300 MHz)
d in ppm: 4.64 (s, 2H, NH2), 6.96e7.60
(a set of signals, 4H, AreH), 9.03 (s, 1H, NHeN, D2O exchangeable),
9.47 (s, 1H, AreNH, D2O exchangeable); MS (m/z, %): 187.03 (Mþþ 1,
82.55).
7.1.2. Synthesis of 4-substituted benzaldehyde (5aei)
A mixture of substituted phenol 3aei (37.4 mmol), 4-fluoro
activity
of
N-(4-substituted
phenyl)-2-[4-(substituted)
benzaldehyde
4
(37.4 mmol) and potassium carbonate
benzylidene] hydrazine carbothioamides. However, further
studies need to be carried out to ascertain the precise mechanism of
action of anticonvulsant activity of these molecules.
(38.8 mmol) in N,N-dimethylformamide (30 ml) was refluxed for
16e18 h under nitrogen. After cooling, the product was extracted
from the reaction mixture and purified by chromatography.
7. Experimental protocols
7.1.2.1. 4-(4-Chloroꢀ-p1henoxy)-benzaldehyde (5d). Yield: 76%; m.p.:
75 ꢁC; IR (KBr, cm
) y
: 1662 (C]O), 1241 (eO-); 1H NMR CDCl3,
7.1. Chemistry
300 (MHz) d in ppm: 9.81 (s, 1H, eCHO), 6.22e7.80 (a set of signals,
8H, AreH); MS (m/z, %): 233.03 (Mþþ1 for 35Cl, 100.00), 235.0
All the chemicals and solvents, purchased from Merck (India),
Spectrochem (India), Himedia (India) and S. d. Fine were used
without further purification. The progress of reaction was moni-
tored by thin layer chromatography, performed on a silica gel 60
F254 coated aluminum sheet. The melting points were determined
by using Thomas- Hoover melting point apparatus and are uncor-
rected. The FTeIR spectra were recorded on PerkineElmer Spec-
trum BX-II Spectrophotometer. The 1H NMR spectra were recorded
on Bruker 300 MHz High Resolution NMR spectrometer using TMS
as an internal standard. Chemical shifts were reported in ppm (d)
and signals were described as singlet (s), doublet (d), triplet (t) and
multiplet (m). All exchangeable protons were confirmed by addi-
tion of D2O. The mass spectra were recorded on a Waters Micro-
mass ZQ 2000 mass spectrometer. Elemental analysis (C, H, N) was
undertaken with PerkineElmer Model 240C analyzer.
(Mþþ1 for 37Cl, 34.3).
7.1.3. Synthesis of N-(4-substituted phenyl)-2-[(4-substituted)
benzylidene] hydrazinecarbo- thioamides (PC 1e36)
Equimolar quantities (0.01 mol) of 4-substituted benzaldehydes
5 aei and N-(4-substituted phenyl) hydrazinecarbothioamides
2aed were dissolved in warm ethanol containing 0.5 ml of glacial
acetic acid. The reaction mixture was refluxed for 4e6 h and set
aside. The resultant solid was washed with ethanol and recrystal-
lized from 90% ethanol. Physical and elemental analysis data of the
titled compounds PC 1e36 are depicted in Table 1.
7.1.3.1. N-(phenyl)-2-(4-phenoxybenzylidene)hydrazinecarbothioa-
mide (PC 1). IR (KBr, cmꢀ1
)
y
: 3228, 3120 (NH), 1585 (N]CH), 1240
(eOe), 1167 (C]S); 1H NMR (CDCl3, 300 MHz)
d
in ppm: 6.88e7.76
(a set of signals, 14 H, AreH), 7.85 (s, 1H, CH]N), 9.17 (s, 1H, NHeN,
D2O exchangeable), 9.88 (s, 1H, NHeAr, D2O exchangeable); MS (m/
z, %): 348.10 (Mþ þ 1, 42.11).
7.1.1. Synthesis of N-(4-halogenated phenyl)
hydrazinecarbothioamides (2aed)
To a solution of 4-halogenated aniline 1aed (0.01 mol) in DMF
(15 ml), sodium hydroxide (0.012 mol) and carbon disulphide
(0.01 mol) were added. The mixture was stirred at 20e25 ꢁC for 1 h.
To the stirred mixture, hydrazine hydrate (0.03 mol) was added and
stirring continued at 60e70 ꢁC for 1 h. On addition of water, a solid
separated out which was recrystallized from 90% ethanol. The
physical and spectral data of compounds are given below.
7.1.3.2. N-(phenyl)-2-[4-(4-nitrophenoxy)benzylidene]hydrazine-
carbothioamide (PC 2). IR (KBr, cmꢀ1
)
y
: 3228, 3116 (NH), 1590 (N]
CH), 1520 (N]O), 1243 (eOe), 1166 (C]S); 1H NMR (CDCl3,
300 MHz) d in ppm: 6.93e7.72 (a set of signals, 13H, AreH), 7.87 (s,
1H, CH]N), 9.10 (s, 1H, NHeN, D2O exchangeable), 9.78 (s, 1H,
NHeAr, D2O exchangeable); MS (m/z, %): 393.1 (Mþ þ 1, 76.11).
7.1.1.1. N-Phenyl hydraziꢀn1ecarbothioamides (2a). Yield: 68%; m.p.:
7.1.3.3. N-(phenyl)-2-[4-(4-methylphenoxy)benzylidene]hydrazine-
181e183 ꢁC; IR (KBr, cm
)
y
: 3342 (NHNH2), 1163 (C]S); 1H NMR
carbothioamide (PC 3). IR (KBr, cmꢀ1
) y: 3232, 3121 (NH),1590 (N]

L. Tripathi et al. / European Journal of Medicinal Chemistry 47 (2012) 153e166
163
CH), 1247 (eO-), 1168 (C]S); 1H NMR (CDCl3, 300 MHz)
d
in ppm:
NHeAr, D2O exchangeable); MS (m/z, %): 427.10 (Mþ þ 1 for 35Cl,
100.00), 429.05 (Mþ þ 1 for 37Cl, 35.16).
2.36 (s, 3H, CH3), 6.92e7.63 (a set of signals, 13H, AreH), 7.83 (s, 1H,
CH]N), 9.18 (s, 1H, NHeN, D2O exchangeable), 9.88 (s, 1H, NHeAr,
D2O exchangeable); MS (m/z, %): 362.15 (Mþ þ 1, 100.00).
7.1.3.12. N-(4-chlorophenyl)-2-[4-(4-methylphenoxy)benzylidene]
hydrazinecarbothioamide (PC 12). IR (KBr, cmꢀ1
)
y
: 3262, 3153
(NH), 1587 (N]CH), 1247 (eOe), 1168 (C]S); 1H NMR (CDCl3,
300 MHz) in ppm: 2.35 (s, 3H, CH3), 6.96e7.68 (a set of signals,
7.1.3.4. 2-[4-(4-Chlorophenoxy)benzylidene]-N-(phenyl)hydrazine-
carbothioamide (PC 4). IR (KBr, cmꢀ1
)
y
: 3230, 3117 (NH), 1587 (N]
d
CH), 1241 (eOe), 1165 (C]S); 1H NMR (CDCl3, 300 MHz)
d
in ppm:
12H, AreH), 7.79 (s, 1H, CH]N), 9.08 (s, 1H, NHeN, D2O
exchangeable), 9.41 (s, 1H, NHeAr, D2O exchangeable); MS (m/z, %):
396.12 (Mþ þ 1 for 35Cl, 100.00), 398.07 (Mþ þ 1 for 37Cl, 34.5).
6.97e7.67 (a set of signals, 13H, AreH), 7.89 (s, 1H, CH]N), 9.16 (s,
1H, NHeN, D2O exchangeable), 9.85 (s, 1H, NHeAr, D2O
exchangeable); MS (m/z, %): 382.13 (Mþ þ 1 for 35Cl, 100.00), 384.14
(Mþ þ 1 for 37Cl, 39.52).
7.1.3.13. 2-[4-(4-Chlorophenoxy)benzylidene]-N-(4-chlorophenyl)
hydrazinecarbothioamide (PC 13). IR (KBr, cmꢀ1
)
y
: 3265, 3142
(NH), 1589 (N]CH), 1243 (eOe), 1165 (C]S); 1H NMR (CDCl3,
300 MHz) in ppm: 6.90e7.64 (a set of signals, 12H, AreH), 7.83 (s,
7.1.3.5. 2-[4-(4-Bromophenoxy)benzylidene]-N-(phenyl)hydrazine-
carbothioamide (PC 5). IR (KBr, cmꢀ1
)
y
: 3230, 3124 (NH),1593 (N]
d
CH), 1244 (eOe), 1163 (C]S); 1H NMR (CDCl3, 300 MHz)
d
in ppm:
1H, CH]N), 9.08 (s, 1H, NHeN, D2O exchangeable), 9.43 (s, 1H,
NHeAr, D2O exchangeable); MS (m/z, %): 416.16 (Mþ þ 1 for 35,35Cl,
100.00), 418.09 (Mþ þ 1 for 35,37Cl, 76.62), 420.22 (Mþ þ 1 for 37,37Cl,
24.30).
6.93e7.69 (a set of signals, 13H, AreH), 7.88 (s, 1H, CH]N), 9.12 (s,
1H, NHeN, D2O exchangeable), 9.77 (s, 1H, NHeAr, D2O
exchangeable); MS (m/z, %): 428.08 (Mþ þ 1 for 81Br, 100.00),
426.07 (Mþ þ 1 for 79Br, 96.60).
7.1.3.14. 2-[4-(4-Bromophenoxy)benzylidene]-N-(4-chlorophenyl)
7.1.3.6. N-(phenyl)-2-[4-(4-fluorophenoxy)benzylidene]hydrazine-
hydrazinecarbothioamide (PC 14). IR (KBr, cmꢀ1
)
y
: 3266, 3147
(NH), 1591 (N]CH), 1244 (eOe), 1163 (C]S); 1H NMR (CDCl3,
300 MHz) in ppm: 6.93e7.69 (a set of signals, 12H, AreH), 7.80 (s,
carbothioamide (PC 6). IR (KBr, cmꢀ1
)
y
: 3227, 3129 (NH), 1588 (N]
CH), 1244 (eOe), 1167 (C]S); 1H NMR (CDCl3, 300 MHz)
d
in ppm:
d
6.92e7.69 (a set of signals, 13H, AreH), 7.82 (s, 1H, CH]N), 9.08 (s,
1H, NHeN, D2O exchangeable), 9.76 (s, 1H, NHeAr, D2O
exchangeable); MS (m/z, %): 366.16 (Mþ þ 1, 42.89).
1H, CH]N), 9.08 (s, 1H, NHeN, D2O exchangeable), 9.42 (s, 1H,
NHeAr, D2O exchangeable); MS (m/z, %): 461.96 (Mþ þ 1 for 35,37Cl
and 79,81Br, 100.00), 459.99 (Mþ þ 1 for 35Cl and 79Br, 74.6), 463.97
(Mþ þ 1 for 37Cl and 81Br, 24.5).
7.1.3.7. 2-[4-(4-chloro-3-methylphenoxy)benzylidene]-N-(phenyl)
hydrazinecarbothioamid (PC 7). IR (KBr, cmꢀ1
)
y
: 3223, 3120 (NH),
1591 (N]CH), 1246 (eOe), 1168 (C]S); 1H NMR (CDCl3, 300 MHz)
in ppm: 2.35 (s, 3H, CH3), 6.93e7.72 (a set of signals, 12H, AreH),
7.1.3.15. N-(4-chlorophenyl)-2-[4-(4-fluorophenoxy)benzylidene]
hydrazinecarbothioamide (PC 15). IR (KBr, cmꢀ1
)
y
: 3261, 3149 (NH),
1586 (N]CH), 1241 (eOe), 1167 (C]S); 1H NMR (CDCl3, 300 MHz)
in ppm: 6.92e7.71 (a set of signals, 12H, AreH), 7.82 (s, 1H, CH]
d
7.81 (s, 1H, CH]N), 9.12 (s, 1H, NHeN, D2O exchangeable), 9.80 (s,
1H, NHeAr, D2O exchangeable); MS (m/z, %): 396.13 (Mþ þ 1 for
35Cl, 100.00), 398.10 (Mþ þ 1 for 37Cl, 37.12).
d
N), 9.08 (s, 1H, NHeN, D2O exchangeable), 9.41 (s, 1H, NHeAr, D2O
exchangeable); MS (m/z, %): 400.04 (Mþ þ 1 for 35Cl, 100.00),
402.06 (Mþ þ 1 for 37Cl, 35.8).
7.1.3.8. N-(phenyl)-2-[4-(naphthalen-2-yloxy)benzylidene]hydrazi-
necarbothioamide(PC 8). IR (KBr, cmꢀ1
)
y
: 3238, 3127 (NH), 1595
(N]CH), 1246 (eOe), 1167 (C]S), 832, 820 (
-naphthyl); 1H NMR
(CDCl3, 300 MHz) in ppm: 6.90e7.88 (a set of signals, 16H, AreH
7.1.3.16. 2-[4-(4-Chloro-3-methylphenoxy)benzylidene]-N-(4-chloro-
b
phenyl)hydrazinecarbothio amide (PC 16). IR (KBr, cmꢀ1
)
y
: 3268,
3158 (NH), 1591 (N]CH), 1246 (eOe), 1168 (C]S); 1H NMR (CDCl3,
300 MHz) in ppm: 2.34 (s, 3H, CH3), 6.93e7.70 (a set of signals,
d
and naphthyleH), 7.84 (s, 1H, CH]N), 9.13 (s, 1H, NHeN, D2O
exchangeable), 10.03 (s, 1H, NHeAr, D2O exchangeable); MS (m/z,
%): 398.18 (Mþ þ 1, 100.00).
d
11H, AreH), 7.81 (s, 1H, CH]N), 9.08 (s, 1H, NHeN, D2O
exchangeable), 9.42 (s, 1H, NHeAr, D2O exchangeable); MS (m/z, %):
430.07 (Mþ þ 1 for 35,35Cl, 100.00), 432.06 (Mþ þ 1 for 35,37Cl, 66.7),
434.11 (Mþ þ 1 for 37,37Cl, 32.4).
7.1.3.9. 2-[4-(1,
hydrazinecarbothioamide (PC 9). IR (KBr, cmꢀ1
1594 (N]CH), 1238 (eOe), 1163 (C]S); 1H NMR (CDCl3, 300 MHz)
in ppm: 5.95 (s, 2H, OCH2), 6.87e7.79 (a set of signals, 12H, AreH
3-benzodioxol-5-yloxy)benzylidene]-N-(phenyl)
)
y: 3242, 3129 (NH),
7.1.3.17. N-(4-chlorophenyl)-2-[4-(naphthalen-2-yloxy)benzylidene]
d
hydrazinecarbothioamide (PC 17). IR (KBr, cmꢀ1
)
y
: 3260, 3143 (NH),
1590 (N]CH), 1246 (eOe), 1164 (C]S), 836, 821 (
-naphthyl); 1H
NMR (CDCl3, 300 MHz) in ppm: 7.05e7.88 (a set of signals, 15H,
and benzodioxolyl-H), 7.86 (s, 1H, CH]N), 9.14 (s, 1H, NHeN, D2O
exchangeable), 10.13 (s, 1H, NHeAr, D2O exchangeable); MS (m/z,
%): 392.17 (Mþ þ 1, 34.14).
b
d
AreH and naphthyleH), 7.92 (s, 1H, CH]N), 9.13 (s, 1H, NHeN, D2O
exchangeable),10.12 (s,1H, NHeAr, D2O exchangeable); MS (m/z, %):
432.18 (Mþ þ 1 for 35Cl, 100.00), 434.31 (Mþ þ 1 for 37Cl, 47.20).
7.1.3.10. N-(4-chlorophenyl)-2-(4-phenoxybenzylidene)hydrazine-
carbothioamide (PC 10). IR (KBr, cmꢀ1
)
y
: 3268, 3148 (NH), 1587
(N]CH), 1241 (eOe), 1167 (C]S); 1H NMR (CDCl3, 300 MHz)
d
in
7.1.3.18. 2-[4-(1,3-benzodioxol-5-yloxy)benzylidene]-N-(4-chloro-
ppm: 6.78e7.77 (a set of signals, 13H, AreH), 7.80 (s, 1H, CH]N),
9.07 (s, 1H, NHeN, D2O exchangeable), 9.40 (s, 1H, NHeAr, D2O
exchangeable); MS (m/z, %): 382.12 (Mþ þ 1 for 35Cl,100.00), 384.07
(Mþ þ 1 for 37Cl, 34.5).
phenyl)hydrazinecarbothio amide (PC 18). IR (KBr, cmꢀ1
)
y
: 3258,
3149 (NH), 1597 (N]CH), 1242 (eOe), 1165 (C]S); 1H NMR (CDCl3,
300 MHz) in ppm: 5.98 (s, 2H, OCH2), 7.01e7.77 (a set of signals,
d
11H, AreH and benzodioxolyl-H), 7.90 (s, 1H, CH]N), 9.10 (s, 1H,
NHeN, D2O exchangeable),10.13 (s,1H, NHeAr, D2O exchangeable);
MS (m/z, %): 426.03 (Mþ þ 1 for 35Cl, 42.34), 428.06 (Mþ þ 1 for 37Cl,
15.27).
7.1.3.11. N-(4-chlorophenyl)-2-[4-(4-nitrophenoxy)benzylidene]hydra
zinecarbothioamide (PC 11). IR (KBr, cmꢀ1
)
y
: 3263, 3149 (NH), 1590
(N]CH), 1522 (N]O), 1245 (eO-), 1163 (C]S); 1H NMR (CDCl3,
300 MHz) in ppm: 6.91e7.77 (a set of signals, 12H, AreH), 7.81 (s,
1H, CH]N), 9.07 (s, 1H, NHeN, D2O exchangeable), 9.41 (s, 1H,
d
7.1.3.19. N-(4-bromophenyl)-2-(4-phenoxybenzylidene)hydrazine-
carbothioamide (PC 19). IR (KBr, cmꢀ1
) y: 3237, 3138 (NH), 1603

164
L. Tripathi et al. / European Journal of Medicinal Chemistry 47 (2012) 153e166
(N]CH), 1243 (eOe), 1168 (C]S); 1H NMR (CDCl3, 300 MHz)
d
in
7.1.3.27. 2-[4-(1,3-Benzodioxol-5-yloxy)benzylidene]-N-(4-bromo-
phenyl)hydrazinecarbothio amide (PC 27). IR (KBr, cmꢀ1
: 3245,
3133 (NH), 1593 (N]CH), 1240 (eOe), 1168 (C]S); 1H NMR (CDCl3,
300 MHz) in ppm: 5.96 (s, 2H, OCH2), 6.88e7.72 (a set of signals,
11H, AreH and benzodioxolyl-H), 7.89 (s, 1H, CH]N), 9.13 (s, 1H,
NHeN, D2O exchangeable),10.17 (s,1H, NHeAr, D2O exchangeable);
MS (m/z, %): 472.02 (Mþ þ 1 for 81Br, 100.00), 470.05 (Mþ þ 1 for
79Br, 96.22).
ppm: 6.76e7.67 (a set of signals, 13H, AreH), 7.82 (s, 1H, CH]N),
9.09 (s, 1H, NHeN, D2O exchangeable), 9.50 (s, 1H, NHeAr, D2O
exchangeable); MS (m/z, %): 428.04 (Mþ þ 1 for 81Br, 100.00),
426.02 (Mþ þ 1 for 79Br, 97.8).
) y
d
7.1.3.20. N-(4-bromophenyl)-2-[4-(4-nitrophenoxy)benzylidene]hydra-
zinecarbothioamide (PC 20). IR (KBr, cmꢀ1
)
y
: 3236, 3136 (NH), 1606
(N]CH), 1525 (N]O), 1244 (eO-), 1166 (C]S); 1H NMR (CDCl3,
300 MHz) in ppm: 6.73e7.61 (a set of signals,12H, AreH), 7.83 (s,1H,
d
7.1.3.28. N-(4-fluorophenyl)-2-(4-phenoxybenzylidene)hydrazine-
CH]N), 9.09 (s, 1H, NHeN, D2O exchangeable), 9.51 (s, 1H, NHeAr,
D2O exchangeable); MS (m/z, %): 473.05 (Mþ þ 1 for 81Br, 100.00),
471.02 (Mþ þ 1 for 79Br, 98.1).
carbothioamide (PC 28). IR (KBr, cmꢀ1
)
y
: 3138 (NH), 1584 (N]CH),
1240 (eOe), 1165 (C]S); 1H NMR (CDCl3, 300 MHz)
d
in ppm:
6.92e7.88 (a set of signals, 13H, AreH), 7.82 (s, 1H, CH]N), 9.05 (s,
1H, NHeN, D2O exchangeable), 9.57 (s, 1H, NHeAr, D2O
exchangeable); MS (m/z, %): 366.13 (Mþ þ 1, 35.87).
7.1.3.21. N-(4-bromophenyl)-2-[4-(4-methylphenoxy)benzylidene]
hydrazinecarbothioamide (PC 21). IR (KBr, cmꢀ1
)
y
: 3257, 3142 (NH),
1586 (N]CH), 1240 (eOe), 1242 (C]S); 1H NMR (CDCl3, 300 MHz)
in ppm: 2.34 (s, 3H, CH3), 6.83e7.65 (a set of signals, 12H, AreH),
7.1.3.29. N-(4-fluorophenyl)-2-[4-(4-nitrophenoxy)benzylidene]
d
hydrazinecarbothioamide (PC 29). IR (KBr, cmꢀ1
)
y
: 3147 (NH), 1592
(N]CH), 1527 (N]O), 1241 (eOe), 1166 (C]S); 1H NMR (CDCl3,
300 MHz) in ppm: 6.93e7.68 (a set of signals, 12H, AreH), 7.85 (s,
7.85 (s, 1H, CH]N), 9.09 (s, 1H, NHeN, D2O exchangeable), 9.56 (s,
1H, NHeAr, D2O exchangeable); MS (m/z, %): 442.03 (Mþ þ 1 for
81Br, 100.00), 440.05 (Mþ þ 1 for 79Br, 98.3).
d
1H, CH]N), 9.05 (s, 1H, NHeN, D2O exchangeable), 9.61 (s, 1H,
NHeAr, D2O exchangeable); MS (m/z, %): 411.10 (Mþ þ 1, 65.78).
7.1.3.22. N-(4-bromophenyl)-2-[4-(4-chlorophenoxy)benzylidene]
hydrazinecarbothioamide (PC 22). IR (KBr, cmꢀ1
)
y
: 3264, 3138
(NH), 1588 (N]CH), 1244 (eOe), 1168 (C]S); 1H NMR (CDCl3,
300 MHz) in ppm: 6.86e7.65 (a set of signals, 12H, AreH), 7.84 (s,
7.1.3.30. N-(4-fluorophenyl)-2-[4-(4-methylphenoxy)benzylidene]
hydrazinecarbothioamide (PC 30). IR (KBr, cmꢀ1
)
y
: 3132 (NH), 1587
d
(N]CH), 1240 (eOe), 1163 (C]S); 1H NMR (CDCl3, 300 MHz)
d
in
1H, CH]N), 9.09 (s, 1H, NHeN, D2O exchangeable), 9.55 (s, 1H,
NHeAr, D2O exchangeable); MS (m/z, %): 461.96 (Mþ þ 1 for 35,37Cl
and 79,81Br, 100.00), 459.99 (Mþ þ 1 for 35Cl and 79Br, 74.6), 463.97
(Mþ þ 1 for 37Cl and 81Br, 24.5).
ppm: 2.34 (s, 3H, CH3), 6.92e7.66 (a set of signals, 12H, AreH), 7.82
(s, 1H, CH]N), 9.06 (s, 1H, NHeN, D2O exchangeable), 9.61 (s, 1H,
NHeAr, D2O exchangeable); MS (m/z, %): 380.10 (Mþ þ 1, 100.00).
7.1.3.31. 2-[4-(4-Chlorophenoxy)benzylidene]-N-(4-fluorophenyl)
7.1.3.23. 2-[4-(4-bromophenoxy)benzylidene]-N-(4-bromophenyl)
hydrazinecarbothioamide (PC 31). IR (KBr, cmꢀ1
)
y
: 3128 (NH), 1586
hydrazinecarbothioamide (PC 23). IR (KBr, cmꢀ1
)
y
: 3259, 3145
(NH), 1585 (N]CH), 1241 (eOe), 1163 (C]S); 1H NMR (CDCl3,
300 MHz) in ppm: 6.85e7.65 (a set of signals, 12H, AreH), 7.85 (s,
(N]CH), 1238 (eOe), 1164 (C]S); 1H NMR (CDCl3, 300 Hz)
d
in
ppm: 6.96e7.65 (a set of signals, 12H, AreH), 7.86 (s, 1H, CH]N),
9.04 (s, 1H, NHeN, D2O exchangeable), 9.61 (s, 1H, NHeAr, D2O
exchangeable); MS (m/z, %): 400.12 (Mþ þ 1 for 35Cl, 100.00), 402.12
(Mþ þ 1 for 37Cl, 36.80).
d
1H, CH]N), 9.09 (s, 1H, NHeN, D2O exchangeable), 9.56 (s, 1H,
NHeAr, D2O exchangeable); MS (m/z, %): 505.84 (Mþ þ 1 for 79,81Br,
61.20), 507.92 (Mþ þ 1 for 81,81Br, 24.8), 503.86 (Mþ þ 1 for 79,79Br,
25.7).
7.1.3.32. 2-[4-(4-Bromophenoxy)benzylidene]-N-(4-fluorophenyl)
hydrazinecarbothioamide (PC 32). IR (KBr, cmꢀ1
)
y
: 3135 (NH), 1589
7.1.3.24. N-(4-bromophenyl)-2-[4-(4-fluorophenoxy)benzylidene]
(N]CH), 1240 (eOe), 1167 (C]S); 1H NMR (CDCl3, 300 Hz)
d
in
hydrazinecarbothioamide (PC 24). IR (KBr, cmꢀ1
)
y
: 3258, 3142
(NH), 1583 (N]CH), 1240 (eOe), 1168 (C]S); 1H NMR (CDCl3,
300 MHz) in ppm: 6.85e7.64 (a set of signals, 12H, AreH), 7.84 (s,
ppm: 6.89e7.68 (a set of signals, 12H, AreH), 7.83 (s, 1H, CH]N),
9.07 (s, 1H, NHeN, D2O exchangeable), 9.56 (s, 1H, NHeAr, D2O
exchangeable); MS (m/z, %): 446.02 (Mþ þ 1 for 81Br, 89.90), 444.03
(Mþ þ 1 for 79Br, 87.23).
d
1H, CH]N), 9.09 (s, 1H, NHeN, D2O exchangeable), 9.55 (s, 1H,
NHeAr, D2O exchangeable); MS (m/z, %): 446.02 (Mþ þ 1 for 81Br,
100.00), 444.03 (Mþ þ 1 for 79Br, 97.8).
7.1.3.33. 2-[4-(4-Fluorophenoxy)benzylidene]-N-(4-fluorophenyl)
hydrazinecarbothioamide (PC 33). IR (KBr, cmꢀ1
)
y
: 3135 (NH), 1584
7.1.3.25. N-(4-bromophenyl)-2-[4-(4-chloro-3-methylphenoxy)ben-
(N]CH), 1239 (eOe), 1165 (C]S); 1H NMR (CDCl3,300 Hz)
d
in
zylidene]hydrazinecarbo thioamide (PC 25). IR (KBr, cmꢀ1
)
y
: 3234,
3135 (NH), 1584 (N]CH), 1241 (eOe), 1165 (C]S); 1H NMR (CDCl3,
300 MHz) in ppm: 2.36 (s, 3H, CH3), 6.75e7.64 (a set of signals,
ppm: 6.90e7.81 (a set of signals, 12H, AreH), 7.80 (s, 1H, CH]N),
9.08 (s, 1H, NHeN, D2O exchangeable), 9.47 (s, 1H, NHeAr, D2O
exchangeable); MS (m/z, %): 384.10 (Mþ þ 1, 36.73).
d
11H, AreH), 7.84 (s, 1H, CH]N), 9.09 (s, 1H, NHeN, D2O
exchangeable), 9.51 (s, 1H, NHeAr, D2O exchangeable); MS (m/z, %):
476.04 (Mþ þ 1 for 35,37Cl and 79,81Br, 100.00), 474.06 (Mþ þ 1 for
35Cl and 79Br, 74.8), 478.03 (Mþ þ 1 for 37Cl and 81Br, 31.50).
7.1.3.34. 2-[4-(4-Chloro-3-methylphenoxy)benzylidene]-N-(4-fluoro-
phenyl)hydrazinecarbothio amide (PC 34). IR (KBr, cmꢀ1
)
y
: 3135
(NH), 1584 (N]CH), 1241 (eOe), 1163 (C]S); 1H NMR
(CDCl3,300 Hz) in ppm: 2.35 (s, 3H, CH3), 6.81e7.62 (a set of
d
7.1.3.26. N-(4-bromophenyl)-2-[4-(naphthalen-2-yloxy)benzylidene]
signals, 11H, AreH), 7.84 (s, 1H, CH]N), 9.07 (s, 1H, NHeN, D2O
exchangeable), 9.54 (s, 1H, NHeAr, D2O exchangeable); MS (m/z, %):
414.06 (Mþ þ 1 for 35Cl, 100.00), 416.08 (Mþ þ 1 for 37Cl, 37.80).
hydrazinecarbothioamide (PC 26). IR (KBr, cmꢀ1
)
y
: 3265, 3146
(NH), 1589 (N]CH), 1244 (eOe), 1166 (C]S), 836, 821 (
naphthyl); 1H NMR (CDCl3, 300 MHz)
in ppm: 6.85e7.89 (a set of
b
-
d
signals, 15H, AreH), 7.84 (s, 1H, CH]N), 9.09 (s, 1H, NHeN, D2O
exchangeable), 9.56 (s, 1H, NHeAr, D2O exchangeable); MS (m/z,
%): 478.01 (Mþ þ 1 for 81Br, 100.00), 476.04 (Mþ þ 1 for 79Br,
96.50).
7.1.3.35. N-(4-fluorophenyl)-2-[4-(naphthalen-2-yloxy)benzylidene]
hydrazinecarbothioamide (PC 35). IR (KBr, cmꢀ1
)
y
: 3141 (NH), 1589
-naphthyl); 1H NMR
in ppm: 6.80e7.86 (a set of signals, 15H, AreH
(N]CH), 1244 (eOe), 1164 (C]S), 835, 823 (
(CDCl3, 300 MHz)
b
d

L. Tripathi et al. / European Journal of Medicinal Chemistry 47 (2012) 153e166
165
and naphthyleH), 7.88 (s, 1H, CH]N), 9.11 (s, 1H, NHeN, D2O
exchangeable), 10.10 (s, 1H, NHeAr, D2O exchangeable); MS (m/z,
%): 416.13 (Mþ þ 1, 55.21).
7.2.4. Minimal clonic seizure (6 Hz) test
Minimal clonic seizure (6 Hz) test was used to assess
compound’s efficacy against electrically induced seizures but used
a lower frequency (6 Hz) and longer duration of stimulation (3 s).
Test compounds were pre-administered to mice via i.p. injection. At
varying times, individual mice (four mice per time point) were
challenged with sufficient current delivered through corneal elec-
trodes to elicit a psychomotor seizure in 97% animals (32 mA for
3 s). The untreated mice would display seizure characterized by
a minimal clonic phase followed by stereotyped, automatistic
behaviors, described originally as being similar to the aura of
human patients with partial seizure. Animals not displaying this
behavior are considered to be protected. Most potent derivatives
were tested quantitatively in the 6 Hz study and ED50 reported.
7.1.3.36. 2-[4-(1,3-Benzodioxol-5-yloxy)benzylidene]-N-(4-fluoro-
phenyl)hydrazinecarbothio amide (PC 36). IR (KBr, cmꢀ1
)
y
: 3142
(NH), 1595 (N]CH), 1240 (eOe), 1164 (C]S); 1H NMR (CDCl3,
300 Hz) in ppm: 5.96 (s, 2H, OCH2), 6.93e7.67 (a set of signals,
d
11H, AreH and benzodioxolyleH), 7.88 (s, 1H, CH]N), 9.08 (s, 1H,
NHeN, D2O exchangeable),10.11 (s,1H, NHeAr, D2O exchangeable);
MS (m/z, %): 410.13 (Mþ þ 1, 89.70).
7.2. Pharmacology
The evaluation of anticonvulsant activity and neurotoxicity was
carried out by the Epilepsy Branch, National Institute of Neuro-
logical Disorder and Stroke, National Institutes of Health, Bethesda,
USA following the reported procedures.
7.2.5. Pilocarpine induced status prevention (PISP) model
Compounds were assessed for pharmacological evaluation of
potential activity against nerve agents using the pilocarpine model
of epilepsy as an introductory screen. The pilocarpine models are
one of the most recognized animal models of status epilepticus
(SE).
Male albino mice (CF-1 strain, 18e25 g) and male albino rats
(Sprague Dawley, 100e150 g) were used as experimental animals.
The synthesized derivatives were suspended in 0.5% methyl cellu-
lose and the test compound is usually manipulated with a mortar
pestle to help preparation of suspension. In the preliminary
screening by MES and scMET tests, each compound was adminis-
tered as an i.p. injection at three dose levels (30, 100 and 300 mg/
kg) and anticonvulsant and neurotoxic effects were assessed at
30 min and 4 h intervals after administration in mice. Selected
derivatives were examined for the oral activity in rat MES screen.
Further compounds were also tested for their activity in 6 Hz model
at five different time points i.e. 0.25, 0.5, 1.0, 2.0 and 4.0 h at a dose
of 100 mg/kg administered i.p. Compounds showing significant
protection were evaluated for quantification studies in 6 Hz test and
ED50 reported. Selected derivatives were also evaluated in pilocar-
pine induced status prevention (PISP) model and in vitro hippo-
campal slice culture neuroprotection assay.
A. Acute toxicity: To determine acute motor impairment usual
starting doses of 100 and 300 mg/kg were administered via the
i.p. route to groups of Sprague Dawley rats over several time
points. The behavior of the animals was closely observed and
recorded over a 4 h period. A minimum number of four (4) rats,
two per dose were employed in the acute screen.
B. Status prevention: To determine if the test substance can
prevent acute pilocarpine induced status an initial qualitative
efficacy screen was performed. Administration of the candidate
drug was given to male albino Sprague Dawley rats
(150e180 g) via the i.p. route of administration. Then a chal-
lenge dose of pilocarpine was administered observing for
treatment-effects of the candidate drug. The outcome
measures were determined as “protection” or “no protection”.
The seizure severity was determined using the well established
Racine scale.
7.2.1. Maximal electroshock (MES) test
For the MES test, 60 Hz of alternating current (50 mA in mice,
150 mA in rats) is delivered for 0.2 s by corneal electrodes which
have been primed with an electrolyte solution containing anes-
thetic agent (0.5% tetracaine HCl). An animal is considered pro-
tected from MES-induced seizures upon abolition of hind limb
tonic extensor component of the seizure. One compound
described in this study was examined for the oral activity in the
rat MES screen.
7.2.6. In vitro hippocampal slice culture neuroprotection assay
(NP): primary screen experiment
The “Primary Screen Experiment” is a qualitative assessment of
the ability of a compound to prevent excitotoxic cell death. Orga-
notypic hippocampal slice cultures are treated with N-methyl-D
aspartate (NMDA) or kainic acid (KA) to induce neuronal cell death.
Propidium iodide (PI), a membrane-impermeant compound, is
included in all wells of the culture plate. Dying cells have
compromised cell membranes, thus PI may diffuse into the cell,
intercalate with DNA and fluoresce. Thus, the intensity of the PI
fluorescence is proportional to the amount of cell death in the
individual slices. Hippocampal slice cultures are treated with the
excitotoxin alone, or where indicated above, with the excitotoxin
and either one or two investigational compounds at the concen-
trations indicated. If neuroprotection occurs as a consequence of
the added compound, slice cultures will have a visibly reduced
fluorescent intensity when compared to the slice cultures that have
been treated with the excitotoxin alone.
7.2.2. Subcutaneous metrazol seizure threshold (scMET) test
For the scMET test animals are pretreated with various doses of
the test compound. At a previously determined TPE of the test
compound the dose of metrazol which will induce convulsions in
97% of animals is injected into a loose fold of skin in the midline of
the neck. The animals are placed in isolation cage to minimize
stress and observed for the next 30 min to see the absence of
seizure. An episode of clonic spasms, approximately 3e5 s of the
fore and/or hind limbs, jaws or vibrissae was taken as the end point.
Animals which do not meet this criterion were considered
protected.
7.2.3. Neurotoxicity-minimal motor impairment (MMI)
Minimal motor impairment was measured by the rotorod
(neurotoxicity) test. When a mouse is placed on a rod that rotates
at a speed of 6 rpm, the animal can maintain its equilibrium for
a long period of time. The compound was considered toxic if the
treated animal falls off this rotating rod 3 times during 1 min
period.
7.3. Computational study
7.3.1. Distance mapping
The pharmacophore pattern studies in which distance between
the various groups postulated as essential for anticonvulsant
activity were done on the 3D optimized structures using ACD/3D

166
L. Tripathi et al. / European Journal of Medicinal Chemistry 47 (2012) 153e166
viewer version 12.01 and Argus Lab 4.0 Mark A. Thompson Planaria
Software LLC. In conformational analysis of the ten clinically
effective, well known and structurally different anticonvulsant
drugs such as albutoin, carbamazepine, gabapentin, lamotrigine,
mephobarbital, phenytoin, progabide, ralitoline, remacemide,
zonisamide; a molecular model was suggested on the basis of
molecular dynamics distance estimations [17].
The authors also appreciate the help and support by Mr. Om Pra-
kash during docking study.
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Pharm. Res. 19 (2002) 1446e1457.
The grid maps were calculated using AutoGrid. In all dockings,
ꢁ
a grid map with 60 ꢅ 60 ꢅ 60 points, a grid spacing of 0.375 A
(roughly a quarter of the length of a carbonecarbon single bond)
were used, and the mps were centered on the ligand binding site.
For all dockings, 100 independent run with, an initial population of
random individuals with a population size of 150 individuals,
a maximum number of 2.5 ꢅ 106 energy evaluations, maximum
number of generations of 27,000, an elitism value of 1 and a number
of active torsion of 9 were used. AutoDock 4.0 was used to generate
both grid and docking parameter files (i.e., gpf and dpf files) and
docking affinity (Kcal/mol) and count of probable H-bonds were
determined.
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anticonvulsants, J. Med. Chem. 36 (1993) 3517e3525.
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13 (2007) 1e7 RA.
Acknowledgements
The authors would like to express their gratitude to IIT, Delhi
and CDRI, Lucknow for providing the spectral and elemental data.