Augmentation of GABAergic neurotransmission by novel N-(substituted)-2-[4- (substituted)benzylidene]hydrazinecarbothioamides - A potential anticonvulsant approach

Article abstract of DOI:10.1016/j.ejmech.2013.04.019

New N-(substituted)-2-[4-(substituted)benzylidene]hydrazinecarbothioamides were designed, 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 N-(4-methoxyphenyl)-2-[4-(4-methylphenoxy) benzylidene]hydrazinecarbothioamide PT 30 which showed 100% protection in both MES and 6 Hz test. Compound PT 30 showed protection at three different time points in 6 Hz test at a dose of 100 mg/kg. Compound 2-[4-(4-Chlorophenoxy)benzylidene]-N-cyclohexylhydrazine carbothioamide PT 4 was also found to be active in both MES and 6 Hz test. Titled compounds exhibited good binding properties with epilepsy molecular targets GABA (A) delta and GABA (A) alpha-1 receptors, in LGA based flexible docking studies. Compounds PT 30 and PT 4 were found to elevate γ-aminobutyric acid (GABA) levels in the midbrain and medulla oblongata regions of rat brain. A computational study was carried out for calculation of pharmacophore pattern and prediction of pharmacokinetic properties.

Full text of DOI:10.1016/j.ejmech.2013.04.019

European Journal of Medicinal Chemistry 64 (2013) 477e487
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Augmentation of GABAergic neurotransmission by novel
N-(substituted)-2-[4-(substituted)benzylidene]
hydrazinecarbothioamidesdA potential anticonvulsant approach
*
Laxmi Tripathi , Praveen Kumar
Department of Pharmaceutical Chemistry, S.D. College of Pharmacy and Vocational Studies, Muzaffarnagar 251001, UP, India
a r t i c l e i n f o
a b s t r a c t
Article history:
New N-(substituted)-2-[4-(substituted)benzylidene]hydrazinecarbothioamides were designed, synthe-
sized 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
N-(4-methoxyphenyl)-2-[4-(4-methylphenoxy) benzylidene]hydrazinecarbothioamide PT 30 which
showed 100% protection in both MES and 6 Hz test. Compound PT 30 showed protection at three
different time points in 6 Hz test at a dose of 100 mg/kg. Compound 2-[4-(4-Chlorophenoxy)benzyli-
dene]-N-cyclohexylhydrazine carbothioamide PT 4 was also found to be active in both MES and 6 Hz test.
Titled compounds exhibited good binding properties with epilepsy molecular targets GABA (A) delta and
GABA (A) alpha-1 receptors, in LGA based flexible docking studies. Compounds PT 30 and PT 4 were
Received 30 January 2013
Received in revised form
6 April 2013
Accepted 8 April 2013
Available online 16 April 2013
Keywords:
Hydrazinecarbothioamides
Anticonvulsant activity
Neurotoxicity
found to elevate g-aminobutyric acid (GABA) levels in the midbrain and medulla oblongata regions of rat
brain. A computational study was carried out for calculation of pharmacophore pattern and prediction of
pharmacokinetic properties.
Docking
GABA assay
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
epilepsy and competitive NMDA receptor antagonists are proposed
as potential anti-epileptic drugs [1].
Epilepsy is a disease of high prevalence, being well known since
thousands of years as “morbus sacer”. Inspite of intensive in-
vestigations, the pathophysiology of epilepsy is still poorly under-
stood. Studies with various animal models have provided ample
evidence for heterogeneity in the mechanisms of epileptogenesis.
Several biochemical hypotheses have been advanced, involving the
inhibitory GABAergic system and the system of the excitatory
amino acids glutamate and aspartate. Agents which reduce GABAA
synaptic function provoke convulsions. A convulsive state is
induced by the direct blockade of GABAA receptors or a reduction in
the GABA mediated opening of the chloride ion channel. One major
factor in epileptogenesis seems to be a decreased function of GABAA
synapses. More recently, research has focused on the therapeutic
potential of blocking excitatory amino acids, in particular gluta-
Thus, it is well documented that attenuation of GABAergic
neurotransmission is involved in the pathophysiology of epilepsy.
With regard to epilepsy, it has been shown that convulsions can
occur when the level of GABA in the brain diminishes below a
critical amount and that direct administration of GABA into the
brain terminates the seizures. However, GABA does not cross the
bloodebrain barrier, a protective membrane that prevents xeno-
biotics from entering the brain. Consequently, GABA is not an
effective anticonvulsant agent. Some of the clinically effective ap-
proaches are the direct agonism of GABA receptors, the inhibition of
enzymatic breakdown of GABA, and the inhibition of the uptake of
GABA into neuronal and glial cell bodies [2].
Several GABA analogs have been designed and synthesized in an
attempt to synthesize effective anticonvulsants. Bryans et al. re-
mate. Of the three receptors of glutamate, the NMDA (N-methyl-
D-
ported gabapentin, a g-amino acids to have anticonvulsant, anxi-
aspartate) receptor is considered the one of most interest in
olytic and analgesic actions [3]. Receveur et al. synthesized a series
of conformationally restricted analogs of gabapentin. The pyrroli-
dine analog (R)-2-aza-spiro [4,5] decane-4-carboxylic acid hydro-
chloride had an IC50 of 120 nM, similar to that of gabapentin
* Corresponding author. Tel.: þ91 9897112288.
E-mail addresses: tripathilaxmi@rediffmail.com (L. Tripathi), praveensha1977@
rediffmail.com (P. Kumar).
(IC50 ¼ 140 nM) [4]. Cundy et al. reported XP13512 [(ꢀ)-1-([(
a-
isobutanoyloxyethoxy)carbonyl]aminomethyl)-1-cyclohexane
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.04.019

478
L. Tripathi, P. Kumar / European Journal of Medicinal Chemistry 64 (2013) 477e487
lamotrignine, rufinamide, remacemide and phenobarbitone led to
the proposal of general model for anticonvulsant activity
Distal hydrophobic centre
Electron donor atom
a
comprising of two aromatic rings or their equivalent in a favored
orientation and a third region containing a number of hydrogen
bond forming functional groups. In terms of interaction at the
binding site, as proposed by Dimmock et al., the pharmacophoric
elements are 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. The
specific placement of hydrogen bonding groups in this region
appeared to be less important than the correct conformational
arrangement of the hydrophobic elements [11e13].
Two dimensional (2D) modeling and structure activity rela-
tionship of potential anticonvulsant agents propose some essential
pharmacophoric features for anticonvulsant activity also suggested
by Unverferth et al. [14] The essential pharmacophoric features
include at least one aryl group which acts as a lipophilic domain,
one or two electron donor atoms, and/or an NH group in a special
spatial arrangement which acts as a H-bonding domain, and a distal
hydrophobic center.
S
H
H
Lipophilic domain
R
N
C
N
N
C
O Ar
H
NH group acting as a H-bonding domain
Fig. 1. Essential pharmacophoric requirements of PT 1e36 for anticonvulsant activity.
acetic acid] as a novel prodrug of gabapentin designed to be
absorbed throughout the intestine by high capacity nutrient
transporters [5]. The presence of cyclohexyl moiety in gabapentin
and analogs suggests its role in anticonvulsant activity. Several
other investigations have recognized aryl thiosemicarbazones as
structurally novel class of anticonvulsants [6e10]. These cyclo-
hexyl/aryl thiosemicarbazones do not possess the dicarboxamide
group as found in conventional antiepileptic drugs e.g. barbiturates,
hydantoins, oxazolidiones etc., which may be associated with
toxicity and side effects.
All the synthesized titled compounds comprised these essential
pharmacophoric elements (Fig. 1) that are necessary for good
anticonvulsant activity. The pharmacophoric distance mapping
study was done to confirm whether the titled compounds fulfill the
essential demands of the pharmacophore when compared to the
average distance requirement. 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 viewer version 12.0 and Argus Lab 4.0
Mark A. Thompson Planaria Software LLC (Table 1). Our analysis of
the distance relationship showed that N-(substituted)-2-[4-
(substituted)benzylidene]hydrazinecarbothioamides did fulfill the
essential demands of the pharmacophore when compared to the
average distance requirement (Fig. 2). Thus, it was decided to
synthesize the titled compounds and evaluate their anticonvulsant
activity.
The present work reports the design, synthesis and anticon-
vulsant activity of N-(substituted)-2-[4-(substituted)benzylidene]
hydrazinecarbothioamides PT 1e36. Their chemical structures
were characterized using IR, 1H NMR, MS and elemental analysis
techniques. Their anticonvulsant activity was evaluated by using
three experimental epilepsy models, i.e., maximal electroshock
(MES), subcutaneous metrazole (scMET) and psychomotor seizure
(6 Hz) test in mice. The rotarod assay was performed in mice to
evaluate the neurotoxicity of the compounds. The docking study of
the active compounds was done using AutoDock 4.0 along with its
LGA algorithm six established epilepsy molecular targets. The
docking affinity and count of probable hydrogen bonds was eval-
uated. As per docking results, the active compounds were subjected
to biochemical investigation to determine the molecular mecha-
nism of action of active compounds. Computational study was also
carried out for prediction of pharmacokinetic parameters and log P.
2. Results and discussions
2.2. Chemistry
2.1. Design
The reaction sequence leading to the formation of the titled
compounds, viz. N-(Substituted)-2-[4-(substituted)benzylidene]
hydrazinecarbothioamides PT 1e36 is shown in Scheme 1. The N-
(substituted)hydrazinecarbothioamides 2aed were obtained by
The conformational analysis of the older generation clinically
active anticonvulsant drugs such as phenytoin, carbamazepine,
Table 1
Distance range between the essential structural elements A, D and HAeHD.a
Compounds
AeHA
AeHD
AeD
HAeHD
HDeD
HAeD
Albutoin
5.37
4.28
4.26
5.30
3.78
6.20
9.51
8.30
7.51
6.02
6.05
(3.78e9.51)
6.54
2.72
4.28
4.93
7.42
5.50
4.01
9.23
5.55
8.75
6.22
5.86
(2.72e9.23)
5.24
5.21
5.0
4.51
4.25
3.83
4.54
4.81
4.35
3.79
4.56
5.38
3.01
4.30
(3.01e5.38)
4.14
2.72
2.33
2.23
2.42
2.34
2.63
2.41
2.75
3.87
1.78
2.54
(1.78e3.87)
1.43
4.03
5.75
3.57
4.94
4.63
3.88
6.72
2.52
3.96
3.71
4.37
(2.52e6.72)
2.61
5.40
5.67
4.50
4.25
5.23
5.17
6.79
4.85
2.43
4.31
4.86
(2.43e6.79)
3.12
Carbamazepine
Gabapentin
Lamotrigine
Mephobarbital
Phenytoin
Progabide
Ralitoline
Remacemide
Zonisamide
Average distance
(Range)
PT 4
PT 7
PT 19
6.55
6.35
4.17
4.04
1.43
1.41
2.61
2.58
3.19
3.22
PT 28
PT 30
6.32
6.31
5.02
5.01
4.03
4.04
1.40
1.42
2.57
2.55
3.20
3.19
a
Distances calculated for 3D optimized structures using MM3 and CHARMm parameterization (Argus Lab 4.0 and ACD/3D viewer).

L. Tripathi, P. Kumar / European Journal of Medicinal Chemistry 64 (2013) 477e487
A- Hydrophobic Unit
479
reacting cyclohexyl amine 1a/4-(substituted)aniline 1bed with
carbon disulphide in the 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,N0-DMF in presence of potassium
carbonate. The N-(substituted) hydrazinecarbothioamides 2ae
d were refluxed with 4-substituted benzaldehyde 5aei in the
presence of catalytic amount of glacial acetic acid to yield the titled
compounds PT 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 2. The formation of the titled
compounds was confirmed by its IR and 1H NMR spectral studies.
D- Electron Donar Group
HA- Hydrogen Bond Acceptor Unit
HD- Hydrogen Bond Donar Unit
D
4.30
(3.01-5.38)
4.86
(2.43-6.79)
C
C
C
C
4.37
(2.52-6.72)
A
C
C
6.05
(3.78-9.51)
5.86
(2.72-9.23)
HA
2.3. Anticonvulsant activity and neurotoxicity
2.54
(1.78-3.87)
HD
The newly synthesized N-(substituted)-2-[4-(substituted)ben-
zylidene]hydrazinecarbothioamides PT 1e36 were subjected to
anticonvulsant screening according to the anticonvulsant drug
development (ADD) program protocol. The profile of anticonvul-
sant activity was established after i.p. injections into mice and
evaluated in the maximal electroshock (MES) and subcutaneous
Fig. 2. Four-point 3D pharmacophore model for anticonvulsants derived by using
MM3 and CHARMm parametrization (Argus Lab 4.0 and ACD/3D viewer).
NaOH, CS2, DMF
stirring (at 0-50C for 1a
& at 20-350Cfor 1b-d)
S
H
R
NH2
R
N
C
NHNH2
for 1 h, NH2NH2.H2O
stirring at 60-700C for
1h.
2 a-d
1a-d
O
F
K2CO3, N,N'-DMF
reflux
Ar
+
OH
Ar
CHO
CHO
5a-i
3a-i
4
O
Ar
O
CHO
S
Ar
S
H
5a-i
H
H
R
N
C
NHNH2
R
N N C N
Reflux, 4-6 h
C
H
ethanol
glacial acetic acid
2 a-d
PT 1-36
PT 1-9:
R =
R =
PT 10-18:
R =
PT 19- 27:
R =
PT 28-36:
OCH3
CH3
PT 1, 10, 19 & 28: Ar =
PT 6, 15, 24 & 33: Ar =
PT 7, 16, 25 & 34: Ar =
F
CH3
Cl
NO2
CH3
Cl
PT 2, 11, 20 & 29: Ar =
PT 3, 12, 21 & 30: Ar =
PT 4, 13, 22 & 31: Ar =
PT 8, 17, 26 & 35: Ar =
PT 9, 18, 27 & 36: Ar =
O
O
PT 5, 14, 23 & 32: Ar =
Br
Scheme 1. Synthesis of N-(substituted)-2-[4-(substituted)benzylidene]hydrazinecarbothioamides PT 1e36.

480
L. Tripathi, P. Kumar / European Journal of Medicinal Chemistry 64 (2013) 477e487
Table 2
Physical and elemental analyses data of N-(4-substituted phenyl)-2-[4-(substituted)benzylidene]-hydrazinecarbothioamides PT 19e36.
Compound
Ar
R
Molecular Formula
(M.W.)
Mp (ꢁC)
Yield (%)
Elemental analysis (%): found (cal.)
C
H
N
PT 19
PT 20
PT 21
PT 22
PT 23
PT 24
PT 25
PT 26
PT 27
PT 28
PT 29
PT 30
PT 31
PT 32
PT 33
PT 34
PT 35
PT 36
Phenyl
4-CH3 phenyl
4-CH3 phenyl
4-CH3 phenyl
4-CH3 phenyl
4-CH3 phenyl
4-CH3 phenyl
4-CH3 phenyl
4-CH3 phenyl
4-CH3 phenyl
4-OCH3 phenyl
4-OCH3 phenyl
4-OCH3 phenyl
4-OCH3 phenyl
4-OCH3 phenyl
4-OCH3 phenyl
4-OCH3 phenyl
4-OCH3 phenyl
4-OCH3 phenyl
C21H19N3OS (361.5)
C21H18N4O3S (406.5)
C22H21N3OS (375.5)
C21H18ClN3OS (395.9)
C21H18BrN3OS (440.4)
C21H18FN3OS (379.5)
C22H20ClN3OS (409.9)
C25H21N3OS (411.5)
C22H19N3O3S (405.47)
C21H19N3O2S (377.5)
C21H18N4O4S (422.5)
C22H21N3O2S (391.5)
C21H18ClN3O2S (411.9)
C21H18BrN3O2S (456.4)
C21H18FN3O2S (395.5)
C22H20ClN3O2S (425.9)
C25H21N3O2S (427.5)
C22H19N3O4S (421.5)
142
144
134
150
146
144
148
130
102
143
180
146
184
176
152
158
124
109
68
74
77
81
80
78
73
77
75
73
77
80
79
78
81
80
74
68
69.75 (69.78)
62.01 (62.05)
70.32 (70.37)
63.67 (63.71)
57.22 (57.28)
66.43 (66.47)
64.42 (64.46)
72.98 (72.97)
65.10 (65.17)
66.77 (66.82)
59.68 (59.70)
67.47 (67.50)
61.20 (61.23)
55.26 (55.27)
63.76 (63.78)
62.00 (62.04)
70.20 (70.24)
62.61 (62.69)
5.29 (5.30)
4.42 (4.46)
5.60 (5.64)
4.56 (4.58)
4.10 (4.12)
4.75 (4.78)
4.88 (4.92)
5.08 (5.14)
4.70 (4.72)
5.04 (5.07)
4.25 (4.29)
5.38 (5.41)
4.35 (4.40)
3.92 (3.98)
4.53 (4.59)
4.68 (4.73)
4.91 (4.95)
4.50 (4.54)
11.57 (11.6)
13.74 (13.8)
11.16 (11.2)
10.58 (10.6)
9.51 (9.54)
11.05 (11.1)
10.21 (10.3)
10.17 (10.2)
10.34 (10.4)
11.10 (11.1)
13.23 (13.3)
10.70 (10.7)
10.16 (10.2)
9.17 (9.21)
10.56 (10.6)
9.81 (9.87)
9.80 (9.83)
9.93 (9.97)
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
metrazole (scMET) using doses of 30, 100 and 300 mg/kg at two
different time intervals. The results are shown in Table 3. Neuro-
toxicity was observed by minimal motor impairment which was
measured by the rotarod test. Some compounds were screened in
6 Hz model to identify their activity at five different time points, i.e.,
0.25, 0.5, 1.0, 2.0 and 4.0 h after i.p. administration in mice. The
results are shown in Table 4. Potential compounds PT 4, PT 28 and
PT 30 were also subjected to quantification studies in 6 Hz test.
Some compounds were screened in pilocarpine induced status
prevention (PISP) model. Compounds PT 4 and PT 30 were evalu-
ated in the primary screen experiment of in vitro hippocampal slice
culture neuroprotection assay (NP).
Compounds PT 4 and PT 30 were found to be active in MES test,
indicative of the compounds ability to prevent seizure spread. The
compound PT 4 showed 100% protection (1/1, 4.0 h) at a dose of
100 mg/kg. The compound PT 30 showed 100% protection (1/1,
4.0 h) at a dose of 300 mg/kg. Titled compounds did not exhibit
protection in 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
6 Hz model. Compound PT 30 was the most active one in this series
with protection at three different time points, i.e. 100% (4/4, 0.5 h)
and 50% (2/4, 0.25 h; 2/4, 2.0 h) at a dose of 100 mg/kg. Compound
PT 4 displayed 75% protection at two different time points i.e. (3/4,
0.5 h) and (3/4, 1.0 h) at a dose of 100 mg/kg. Compound PT 28
showed 75% protection (3/4, 1.0 h) and 50% protection (2/4, 0.5 h) at
a dose of 100 mg/kg. Compound PT 7 showed 75% protection (3/4,
1.0 h) at a dose of 100 mg/kg. Compound PT 6 showed 50% pro-
tection (2/4, 2.0 h) at a dose of 100 mg/kg. Compound PT 19 showed
50% protection (2/4, 0.5 h) at a dose of 100 mg/kg. Other com-
pounds showed minor activity in 6 Hz test. Compounds PT 4 and PT
30 showed an ED50 of >100 mg/kg at a time of peak effect (TPE)
of 1.0 h.
Compound PT 4, PT 28 and PT 30 were subjected to acute
toxicity test of pilocarpine induced status prevention (PISP) model.
In acute toxicity test the compounds did not show any toxicity up to
600 mg/kg but they also did not exhibit any protection in
pilocarpine-induced status prevention model. No significant pro-
tection was observed against either KA- or NMDA-induced cyto-
toxicity in the primary screen experiment of in vitro hippocampal
slice culture neuroprotection assay (NP).
2.4. Docking study
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. Their affinity (kcal/mol) and count of
Table 4
Anticonvulsant activity of some N-(substituted)-2-[4-(substituted)benzyli-
dene]hydrazinecarbothioamides by 6 Hz model.
Table 3
Compound
Dose
(mg/kg)
Time (h) to peak effect (N/F)a
Anticonvulsant activity (MES and scMET test) of N-(substituted)-2-[4-(substituted)
benzylidene]hydrazinecarbothioamides.
0.25
0.5
1.0
2.0
4.0
3
3
PT 4
PT 5
PT 6
PT 7
PT 14
PT 15
PT 16
PT 19
PT 25
PT 28
PT 30
PT 33
PT 34
100
100
100
100
100
100
100
100
100
100
100
100
100
0/4
¼
¼
/
4
/
¼
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
4
Compound
Intraperitoneal injection in micea
0/4
0/4
¼
¼
0/4
¼
2/4
0/4
2/4
0/4
¼
0/4
0/4
2/4
0/4
¼
0/4
0/4
0/4
¼
MES screen (h)
scMET
Neurotoxicity
screen (h)
0/4
3
screen (h)
0/4
0/4
0/4
0/4
0/4
1/4
1/4
2/4
0/4
0/4
/
4
¼
¼
0/4
0/4
0.5
2.0
4.0
0.5
4.0
0.5
4.0
PT 4
PT 30
Phenytoin
Sodium valproate
e
e
100a
300a
30a
e
e
e
e
e
e
e
e
e
e
e
e
e
30a
e
30a
e
e
300a
100a
e
100a
e
¼
3
/
0/4
2/4
¼
4
4/4
¼
0/4
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).
¼
0/4
a
N/F ¼ number of animals active or toxic over the number tested.

L. Tripathi, P. Kumar / European Journal of Medicinal Chemistry 64 (2013) 477e487
481
Table 5
Docking study of compounds PT 4, PT 7, PT 19, PT 28 and PT 30.a
Ligand
Receptor
Affinity
H-Bonds
H-Binding ligand
H-Binding receptor
(kcal/mol)
Element
Atom No.
Type
Residue
Element
Atom No.
Type
PT 4
GABA (A) alpha-1
GABA (A) delta
Glutamate
GABA (A) alpha-1
GABA (A) delta
ꢂ5.9
ꢂ5.1
ꢂ6.3
ꢂ6.2
ꢂ6.0
01
00
01
01
02
N
e
N
N
N
O
N
N
N
2
e
Donor
e
Trp 315
e
O
e
O
O
O
N
O
O
O
329
e
Acceptor
e
PT 7
PT 19
12
07
11
21
8
Acceptor
Donor
Donor
Acceptor
Donor
Donor
Donor
Tyr 462
Try 315
Glu 327
Ala 324
Asp 311
Phe 323
Phe 320
177
329
479
448
317
334
338
Both
Acceptor
Acceptor
Donor
Acceptor
Acceptor
Acceptor
PT 28
PT 30
GABA (A) delta
GABA (A) alpha-1
GABA (A) delta
ꢂ6.0
ꢂ6.6
ꢂ7.4
01
01
01
03
03
a
Affinity and count of probable hydrogen bonds were determined by docking studies using AutoDock 4.0 software.
probable hydrogen bonds were evaluated (Table 5). Docking images
of compounds PT 4, PT 7, PT 19, PT 28 and PT 30 with the target
receptors is shown in Fig. 3.
Compound PT 4 exhibited good binding properties with GABA
(A) alpha-1 receptor (affinity value ꢂ5.9 kcal/mol and 1 H-bonds).
Compound PT 4 showed affinity for GABA (A) delta receptor (af-
finity value ꢂ5.1 kcal/mol), but did not exhibit binding properties.
Compound PT 7 exhibited good binding properties with glutamate
receptor (affinity value ꢂ6.3 kcal/mol and 1 H-bonds) but did not
show affinity and H-bonding with any other receptor. Compound
PT 19 exhibited good binding properties with GABA (A) delta re-
ceptor (affinity value ꢂ6.0 kcal/mol and 2 H-bonds) and GABA (A)
alpha-1 receptor (affinity value ꢂ6.2 kcal/mol and 1 H-bond).
Compound PT 28 exhibited good binding properties with GABA (A)
PT 4 with GABA (A) alpha-1 PT 7 with glutamate receptor
PT 19 with GABA(A) alpha-
1
PT 19 with GABA(A) delta
PT 28 with GABA (A) delta
PT 30 with GABA (A) alpha-1
PT 30 with GABA (A) delta
Fig. 3. Docking image of compounds PT 4, PT 7, PT 19, PT 28 and PT 30.

482
L. Tripathi, P. Kumar / European Journal of Medicinal Chemistry 64 (2013) 477e487
Table 6
effects on the levels of GABA in different regions of rat brain viz.,
olfactory lobe, midbrain, medulla oblongata and cerebellum. The
compounds PT 4 and PT 30 were found to significantly increase the
GABA level in the medulla oblongata and midbrain regions (Table 6).
Effect of PT 4 and PT 30 (30 mg/kg, i.p.) on GABA levels in rat brain.
Compound Concentration of GABA (mg/100 mg tissue)
Olfactory lobe Midbrain
Cerebellum
Medulla oblongata
Control
(PEG)
PT 4
13.56 ꢀ 1.22
40.76 ꢀ 1.26 21.43 ꢀ 1.32 38.92 ꢀ 1.32
2.6. Prediction of ADME properties
14.81 ꢀ 1.29
15.84 ꢀ 1.63
51.76 ꢀ 2.35 24.43 ꢀ 1.99 47.24 ꢀ 1.82
58.09 ꢀ 1.80 27.02 ꢀ 2.16 50.08 ꢀ 2.14
PT 30
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 vio-
lations of Lipinski’s rule of five by using Molinspiration online
property calculation toolkit (Table 7). Calculated miLog P for syn-
thesized compounds were then compared with the experimental
log P data of these compounds (Table 7).
From all these parameters, it can be observed that all titled
compounds exhibited a great %ABS ranging from 74.2 to 93.2%. All
compounds of this series except PT 18 violated Lipinski’s parame-
ters. 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.
delta receptor (affinity value ꢂ6.0 kcal/mol and 1 H-bond). Com-
pound PT 30 exhibited good binding properties with GABA (A)
alpha-1 receptor (affinity value ꢂ6.6 kcal/mol and 1 H-bonds)
and GABA (A) delta receptor (affinity value ꢂ7.4 kcal/mol and 1 H-
bonds).
Docking study results show that the compounds PT 4, PT 19, PT
28 and PT 30 exhibited good H-bonding properties with GABA (A)
alpha and GABA (A) delta, hence could be considered as potential
anticonvulsants with GABA mimetic/facilitatory mode of action.
2.5. Neurochemical investigations
2.7. Log P determination
In order to explore the mechanism of anticonvulsant activity
compounds (PT 4 and PT 30), which were very active in anticon-
vulsant models and showed good docking property with GABA re-
ceptor, were subjected to neurochemical investigation to study their
Titled compounds showed dependence of biological activity on
lipophilic character in a congeneric series. In particular, for drugs
acting on central nervous system to be potent, they have to cross
Table 7
Pharmacokinetic parameters important for good oral bioavailability and log P value of compounds N-(substituted)-2-[4-(substituted)benzylidene]hydrazinecarbothioamide
PT 1e36.
Compound
% ABS
TPSA (A2)
n-ROTB
MW
Molecular
volume
n-OHNH
donors
n-OH
acceptors
Lipinski’s
violation
Theoretical
miLog P
Experimental
log P
Rule
PT 1
PT 2
PT 3
PT 4
PT 5
PT 6
PT 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
8
9
8
8
8
8
8
8
8
<500
353.4
e
<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
5
8
5
5
5
5
5
5
7
ꢃ1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
ꢃ5
5.9
5.8
6.3
6.5
6.7
6.0
6.9
7.0
5.8
5.0
5.0
5.5
5.7
5.8
5.2
6.1
6.2
4.9
5.5
5.4
5.9
6.1
6.3
5.6
6.5
6.7
5.4
5.1
5.0
5.5
5.8
5.9
5.2
6.1
6.3
5.0
e
93.1
77.4
93.1
93.1
93.1
93.1
93.1
93.1
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
90.0
74.2
90.0
90.0
90.0
90.0
90.0
90.0
83.7
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
54.8
100.7
54.8
54.8
54.8
54.8
54.8
54.8
73.3
330.3
353.6
346.9
343.8
348.2
335.2
360.4
374.3
354.2
311.7
335.1
328.3
325.3
329.6
316.7
341.8
355.7
335.7
328.3
351.6
344.8
341.8
346.2
333.2
358.4
372.3
352.2
337.3
360.6
353.8
350.8
355.2
342.2
367.4
381.3
361.2
5.7
398.4
367.5
387.9
432.3
371.4
401.9
403.5
397.5
347.4
392.4
361.4
381.8
426.3
365.4
395.9
397.5
391.4
361.4
406.4
375.4
395.9
440.3
379.4
409.9
411.5
405.4
377.4
422.4
391.4
411.9
456.3
395.4
425.9
427.5
421.4
5.51
6.17
6.44
6.36
5.82
6.76
6.79
5.56
4.9
PT 8
PT 9
PT 10
PT 11
PT 12
PT 13
PT 14
PT 15
PT 16
PT 17
PT 18
PT 19
PT 20
PT 21
PT 22
PT 23
PT 24
PT 25
PT 26
PT 27
PT 28
PT 29
PT 30
PT 31
PT 32
PT 33
PT 34
PT 35
PT 36
4.95
5.39
5.52
5.62
5.09
6.03
6.07
4.80
5.28
5.23
5.84
6.71
6.23
5.48
6.30
6.56
5.34
4.99
4.90
5.44
5.68
5.84
5.09
6.03
6.11
4.90
%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, P. Kumar / European Journal of Medicinal Chemistry 64 (2013) 477e487
483
bloodebrain barrier (BBB), thus potency has been correlated with
optimum lipophilicity (log P) near 2. In this study, we attempted to
correlate the anticonvulsant activity of congeners with their
calculated log P value. The experimental log P values were deter-
mined using the octanol-phosphate buffer method. As observed,
some of the experimental values were in good agreement with the
theoretical values (Table 7). All the titled compounds showed
lipophilic character. Optimum log P value ensures good pharma-
cokinetic property of drugs. Drugs possessing optimum log P value
will be least inhibited in their movement through the aqueous and
lipophilic phases of living tissue.
The compound PT 30 displayed significant protection and
emerged as a lead in this series. Further, compounds PT 4, PT 7, PT
19 and PT 28 came out as a potential candidate for further inves-
tigation. The results show that both N-cyclohexyl and N-aryl sub-
stitution in 2-[4-(substituted) benzylidene]hydrazinecarbothioam-
ides leads to active anticonvulsant compounds. Docking study
shows that the active compounds exhibited good binding proper-
ties with GABA (A) delta and GABA (A) alpha-1 receptor. The
docking study reveals the possible role of these receptors in
observed anticonvulsant activity of N-(substituted)-2-[4-
(substituted)benzylidene]hydrazinecarbothioamides.
Finally,
these compounds were found to elevate -aminobutyric acid
g
2.8. Structure activity relationship
(GABA) levels in the midbrain and medulla oblongata regions of rat
brain.
Various N-(substituted)-2-[4-(substituted)benzylidene]hydra-
zinecarbothioamide PT 1e36 were synthesized in order to establish
the pharmacophoric substituents, responsible for the activity. The
aryl/cyclohexyl 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. 1). Thus
it may be stated that compounds PT 1e36 fulfill the essential
pharmacophoric requirements for anticonvulsant activity.
4. Experimental section
4.1. Pharmacophoric distance mapping
The present work involves the correlation of the structural
requirement of well known and structurally different anticonvulsant
compounds with the titled compounds. The two-dimensional (2D)
modelingon anticonvulsants has identified that at leastone 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 well known and structurally different
compounds with anticonvulsant activity e albutoin, carbamazepine,
gabapentin, lamotrigine, mephobarbital, phenytoin, progabide, rali-
toline, remacemide and zonisamide (Fig. 4) with different mecha-
nisms of action, were selected so as to propose a generalized
pharmacophore model. The pharmacophore group’s distance esti-
mation was done by molecular mechanics calculation with the force
fields based on both CHARMm force fields and MM3 parameteriza-
tion. In the present work, energy minimization was performed on
above mentioned ten well known anticonvulsants and N-
(substituted)-2-[4-(substituted)benzylidene]hydrazinecarbothioa-
mides using Argus Lab 4.0. Distance between the various structural
components essential foractivity was determined by ACD/3D viewer.
The crucial structural components that were included in the four-
point pharmacophore model were the aryl ring center or the lipo-
philic group (A), an electron donor atom (D), a hydrogen bond
acceptor (HA), and a hydrogen bond donor (HD) (Fig. 2). An average
distance range for every point was obtained and compared to that of
N-(substituted)-2-[4-(substituted)benzylidene]hydrazinecarbo-
thioamides. Now it may be interesting to examine whether the N-
(substituted)-2-[4-(substituted) benzylidene]hydrazinecarbothioa-
mides reflect the conditions of the derived pharmacophore model.
The presence of cyclohexyl moiety in gabapentin and analogs
suggest its role in anticonvulsant activity. Based on these facts, the N-
cyclohexyl-2-[4-(substituted)]hydrazinecarbothioamide were syn-
thesized and evaluated for anticonvulsant activity. Compounds PT
1e9 displayed moderate protection and supported the pharmaco-
phoric character of cyclohexyl moiety for anticonvulsant activity. The
most active compound PT 4 has 4-chlorophenyl at position 19 of
cyclohexylhydrazine carbothioamide backbone structure. Com-
pounds PT 5 and PT 6 having 4-bromo phenyl and 4-fluoro phenyl
substituents respectively, also displayed mild to moderate protec-
tion. The orderof protection among halogenated phenyl substituents
is Cl > F > Br. The 4-chlorophenyl substituent also contributed to
longer duration of action. This observation supports the fact that
substitution in the aryl ring by halogen led to a number of thio-
semicarbazones with low ED50 value andhigh protective index value.
This observation also indicated the role of electronic factors effecting
anticonvulsant activity. Compound PT 7 having 4-chloro-3-methyl
phenyl substitutions at position 19 displayed significant protection
with short duration of action. The non-halogenated substituents like
phenyl, 4-nitro phenyl, 4-methyl phenyl, naphthalene-2-yl, 1,3-
benzodioxol-5-yl at position 19 yielded inactive compounds. Com-
pounds PT 10e27 displayed mild protection, showing that phenyl
and 4-methyl phenyl substituents were less active than cyclohexyl
substituent. The 4-methoxy substituent at position 18 of N-phenyl-
benzylidenehydrazinecarbothioamide backbone structure yielded
the compounds PT 28e36 with significant protection and longer
duration of action. This observation supports the fact that electron
rich substituent at para position of the aryl ring showed increased
potency in anticonvulsant screen. The most active compound PT 30
has 4-methoxy substituent at position 18 and 4-methyl phenyl
substituent at position 7.
4.2. Chemistry
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 FT-IR spectra were recorded on Perkin Elmer Spectrum
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 Perkin Elmer Model 240C analyzer.
3. Conclusion
A series of N-(substituted)-2-[4-(substituted)benzylidene]hydra-
zinecarbothioamides were designed, synthesized, and their anti-
convulsant activity and neurotoxicity were evaluated after
intraperitoneal administration in three seizure models, which
include the MES, scMET and 6 Hz model. Docking study was per-
formed on the active compounds to ascertain the probable molecular
mechanism of action of drugs. Neurochemical investigations were
done to support the docking results. A computational study was also
carried out for the prediction ofpharmacokinetic properties and log P.

484
L. Tripathi, P. Kumar / European Journal of Medicinal Chemistry 64 (2013) 477e487
D
D
N
D
N
H
N
H
N
CH3
A
D
H
H
HD
OH
S
CH3
A
Cl
HD
N
N
O
HA
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
HA
Mephobarbital
Phenytoin
O
D
H3C
D
O
N
CH3
O
N
A
S
A
H
HD
A
R
N
C
N
N
C
O Ar
O
S
HA
N
H
H
H
H
S
N
Cl
H
HD
HA
O
H
HD
Ralitoline
Fig. 4. Pharmacophoric pattern of well-known anticonvulsants.
PT 1-36
Zonisamide
4.2.1. Synthesis of N-(substituted) hydrazinecarbothioamides (2ae
d) and 4-substituted benzaldehyde (5aei)
(s, 1H, NHeAr, D2O exchangeable), 9.10 (s, 1H, NHeN, D2O
exchangeable), 7.81 (s, 1H, CH]N), 7.02e7.83 (a set of signals,
The titled compounds were prepared following the reported
procedures [15e17].
13H, AreH), 2.36 (s, 3H, CH3); IR (KBr)
n
¼ 3314, 3138 (NH),
1589 (N]CH), 1243 (eOe), 1163 (C]S) cmꢂ1; MS (ES, 1.16 eV)
m/z (%): 362.17 (68.11) [Mþ þ 1].
4.2.1.1. N-(4-Methyl
phenyl)
hydrazinecarbothioamides
(1c).
Yield: 72%; mp: 170e172 ꢁC; 1H NMR (300 MHz, CDCl3)
d
¼ 9.57 (s,
4.2.2.2. N-(4-Methylphenyl)-2-[4-(4-nitrophenoxy)benzylidene]
1H, AreNH, D2O exchangeable), 9.09 (s, 1H, NHeN, D2O
exchangeable), 6.87e7.65 (a set of signals, 4H, AreH), 4.66 (s, 2H,
hydrazinecarbothioamide (PT 20). 1H NMR (300 MHz, CDCl3)
d
¼ 9.71 (s, 1H, NHeAr, D2O exchangeable), 9.13 (s, 1H, NHeN, D2O
NH2), 2.37 (s, 3H, CH3); IR (KBr)
n
¼ 3347 (NHNH2), 1166 (C]S)
exchangeable), 7.85 (s, 1H, CH]N), 6.98e7.70 (a set of signals, 12H,
cmꢂ1; MS (ES, 1.16 eV) m/z (%): 182.07 (56.87) [Mþ þ 1].
AreH), 2.35 (s, 3H, CH3); IR (KBr)
n
¼ 3328, 3136 (NH), 1592 (N]
CH), 1526 (N]O), 1240 (eOe), 1168 (C]S) cmꢂ1; MS (ES, 1.16 eV)
m/z (%): 407.17 (100.00) [Mþ þ 1].
4.2.1.2. N-(4-Methoxy phenyl) hydrazinecarbothioamides (1d).
Yield: 66%; mp: 178e180 ꢁC; 1H NMR (300 MHz, CDCl3)
1H, AreNH, D2O exchangeable), 9.05 (s, 1H, NHeN, D2O
exchangeable), 6.82e7.66 (a set of signals, 4H, AreH), 4.69 (s, 2H,
d
¼ 9.59 (s,
4.2.2.3. N-(4-Methylphenyl)-2-[4-(4-methylphenoxy)benzylidene]
hydrazinecarbothioamide (PT 21). 1H NMR (300 MHz, CDCl3)
NH2), 2.34 (s, 3H, CH3); IR (KBr)
n
¼ 3344 (NHNH2), 1163 (C]S)
d
¼ 9.79 (s, 1H, NHeAr, D2O exchangeable), 9.13 (s, 1H, NHeN, D2O
cmꢂ1; MS (ES, 1.16 eV) m/z (%): 198.04 (43.33) [Mþ þ 1].
exchangeable), 7.86 (s, 1H, CH]N), 7.03e7.73 (a set of signals, 12H,
AreH), 2.36 (s, 6H, CH3); IR (KBr)
n
¼ 3211, 3140 (NH),1596 (N]CH),
4.2.2. Synthesis of N-(substituted)-2-[(4-substituted)benzylidene]
hydrazinecarbothioamides (PT 1e36)
1242 (eOe), 1164 (C]S) cmꢂ1; MS (ES, 1.16 eV) m/z (%): 376.17
(23.89) [Mþ þ 1].
Equimolar quantities (0.01 mol) of 4-substituted benzaldehydes
5aei and N-(substituted) hydrazinecarbothioamides 2aed were
dissolved inwarm 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 recrystallized from 90% ethanol.
The physical data, elemental analysis data and spectral data of
N-cyclohexyl-(2-(4-substituted)benzylidene)hydrazinecarbothioa-
mide (PT 1e9) and N-(phenyl)-(2-(4-substituted)-benzylidene)
hydrazinecarbothioamide (PT 10e18) are reported earlier [13,14].
Physical and elemental analysis data of the titled compounds PT
19e36 are depicted in Table 1.
4.2.2.4. N-(4-Methylphenyl)-2-[4-(4-chlorophenoxy)benzylidene]
hydrazinecarbothioamide (PT 22). 1H NMR (300 MHz, CDCl3)
d
¼ 9.80 (s, 1H, NHeAr, D2O exchangeable), 9.06 (s, 1H, NHeN, D2O
exchangeable), 7.83 (s, 1H, CH]N), 7.04e7.82 (a set of signals, 12H,
AreH), 2.36 (s, 3H, CH3); IR (KBr)
n
¼ 3324, 3148 (NH), 1597 (N]
CH), 1242 (eOe), 1165 (C]S) cmꢂ1; MS (ES, 1.16 eV) m/z (%): 396.13
(100.00) [Mþ þ 1] for 35Cl, 398.14 (37.13) [Mþ þ 1] for 37Cl.
4.2.2.5. 2-[4-(4-Methylphenoxy)benzylidene]-N-(4-bromophenyl)
hydrazinecarbothioamide (PT 23). 1H NMR (300 MHz, CDCl3)
d
¼ 9.73 (s, 1H, NHeAr, D2O exchangeable), 9.10 (s, 1H, NHeN, D2O
exchangeable), 7.82 (s, 1H, CH]N), 6.97e7.69 (a set of signals,
12H, AreH), 2.36 (s, 3H, CH3); IR (KBr)
¼ 3316, 3146 (NH), 1593
4.2.2.1. N-(4-Methylphenyl)-2-(4-phenoxybenzylidene)hydrazine-
carbothioamide (PT 19). 1H NMR (300 MHz, CDCl3)
d
¼ 9.78
n

L. Tripathi, P. Kumar / European Journal of Medicinal Chemistry 64 (2013) 477e487
485
(N]CH), 1244 (eOe), 1167 (C]S) cmꢂ1; MS (ES, 1.16 eV) m/z (%):
442.08 (100.00) [Mþ þ 1] for 81Br, 440.10 (98.10) [Mþ þ 1] for 79Br.
4.2.2.14. 2-[4-(4-Bromophenoxy)benzylidene]-N-(4-methoxyphenyl)
hydrazinecarbothioamide (PT 32). 1H NMR (300 MHz, CDCl3)
d
¼ 3.76 (s, 3H, OCH3), 6.87e7.75 (a set of signals, 12H, AreH), 7.84
4.2.2.6. N-(4-Methylphenyl)-2-[4-(4-fluorophenoxy)benzylidene]
hydrazinecarbothioamide (PT 24). 1H NMR (300 MHz, CDCl3)
(s, 1H, CH]N), 8.80 (s, 1H, NHeN, D2O exchangeable), 9.95 (s, 1H,
NHeAr, D2O exchangeable); IR (KBr)
n
¼ 3278, 3028 (NH), 1607
d
¼ 9.79 (s, 1H, NHeAr, D2O exchangeable), 9.14 (s, 1H, NHeN, D2O
(N]CH), 1241 (eOe), 1165 (C]S) cmꢂ1; MS (ES, 1.16 eV) m/z (%):
exchangeable), 7.87 (s, 1H, CH]N), 7.06e7.73 (a set of signals, 12H,
458.09 (67.92) [Mþ þ 1] for 81Br, 456.10 (64.18) [Mþ þ 1] for 79Br.
AreH), 2.36 (s, 3H, CH3); IR (KBr)
n
¼ 3320, 3136 (NH), 1589 (N]
CH), 1246 (eOe), 1167 (C]S) cmꢂ1; MS (ES, 1.16 eV) m/z (%): 380.17
(100.00) [Mþ þ 1].
4.2.2.15. 2-[4-(4-Fluorophenoxy)benzylidene]-N-(4-methoxyphenyl)
hydrazinecarbothioamide (PT 33). 1H NMR (300 MHz, CDCl3)
d
¼ 9.85 (s, 1H, NHeAr, D2O exchangeable), 8.93 (s, 1H, NHeN, D2O
4.2.2.7. N-(4-Methylphenyl)-2-[4-(4-chloro-3-methylphenoxy)ben-
exchangeable), 7.89 (s, 1H, CH]N), 6.83e7.76 (a set of signals, 12H,
zylidene]hydrazinecarbothioamide (PT 25). 1H NMR (300 MHz,
AreH), 3.78 (s, 3H, OCH3); IR (KBr)
n
¼ 3302, 3036 (NH), 1604 (N]
CDCl3)
d
¼ 9.81 (s,1H, NHeAr, D2O exchangeable), 9.08 (s,1H, NHeN,
CH), 1242 (eOe), 1165 (C]S) cmꢂ1; MS (ES, 1.16 eV) m/z (%): 396.15
(100.00) [Mþ þ 1].
D2O exchangeable), 7.79 (s, 1H, CH]N), 6.98e7.79 (a set of signals,
11H, AreH), 2.36(m, 6H, CH3);IR (KBr)
n
¼ 3327, 3144 (NH),1597 (N]
CH), 1242 (eOe), 1163 (C]S) cmꢂ1; MS (ES, 1.16 eV) m/z (%): 410.17
4.2.2.16. 2-[4-(4-Chloro-3-methylphenoxy)benzylidene]-N-(4-
(100.00) [Mþ þ 1] for 35Cl, 412.13 (38.43) [Mþ þ 1] for 37Cl.
methoxyphenyl)hydrazinecarbothioamide
(300 MHz, CDCl3)
(s, 1H, NHeN, D2O exchangeable), 7.84 (s, 1H, CH]N), 6.88e7.89 (a
set of signals, 11H, AreH), 3.76 (s, 3H, OCH3), 2.34 (s, 3H, CH3); IR
(PT
34). 1H
NMR
d
¼ 9.89 (s, 1H, NHeAr, D2O exchangeable), 9.02
4.2.2.8. N-(4-Methylphenyl)-2-[4-(naphthalen-2-yloxy)benzylidene]
hydrazinecarbothioamide (PT 26). 1H NMR (300 MHz, CDCl3)
d
¼ 9.76 (s, 1H, NHeAr, D2O exchangeable), 9.08 (s, 1H, NHeN, D2O
exchangeable), 7.88 (s, 1H, CH]N), 7.06e7.85 (a set of signals, 15H,
AreH and naphthyleH), 2.36 (s, 3H, CH3); IR (KBr)
¼ 3316, 3143
-naphthyl)
(KBr)
n
¼ 3288, 3035 (NH), 1607 (N]CH), 1247 (eOe), 1166 (C]S)
cmꢂ1; MS (ES, 1.16 eV) m/z (%): 426.15 (100.00) [Mþ þ 1] for 35Cl,
n
428.13 (36.11) [Mþ þ 1] for 37Cl.
(NH), 1595 (N]CH), 1247 (eOe), 1165 (C]S), 830, 816 (
b
cmꢂ1; MS (ES, 1.16 eV) m/z (%): 412.22 (66.34) [Mþ þ 1].
4.2.2.17. N-(4-Methoxyphenyl)-2-[4-(naphthalen-2-yloxy)benzyli-
dene]hydrazinecarbothioamide (PT 35). 1H NMR (300 MHz, CDCl3)
4.2.2.9. 2-[4-(1,3-Benzodioxol-5-yloxy)benzylidene]-N-(4-
d
¼ 9.96 (s, 1H, NHeAr, D2O exchangeable), 9.04 (s, 1H, NHeN, D2O
exchangeable), 7.80 (s, 1H, CH]N), 6.93e7.85 (a set of signals, 15H,
AreH and naphthyleH), 3.79 (s, 3H, OCH3); IR (KBr)
¼ 3295, 3035
-naphthyl)
methylphenyl)hydrazinecarbothioamide (PT 27). 1H NMR (300 MHz,
CDCl3)
d
¼ 10.17 (s, 1H, NHeAr, D2O exchangeable), 9.10 (s, 1H, NHe
n
N, D2O exchangeable), 7.89 (s, 1H, CH]N), 7.03e7.87 (a set of sig-
nals, 11H, AreH and benzodioxolyleH), 5.98 (s, 2H, OCH2), 2.36 (s,
(NH),1606 (N]CH),1244 (eOe), 1165 (C]S), 833, 818 (b
cmꢂ1; MS (ES, 1.16 eV) m/z (%): 428.19 (33.12) [Mþ þ 1].
3H, CH3); IR (KBr)
n
¼ 3312, 3138 (NH), 1596 (N]CH), 1238 (eOe),
1167 (C]S) cmꢂ1; MS (ES, 1.16 eV) m/z (%): 406.15 (53.44) [Mþ þ 1].
4.2.2.18. 2-[4-(1,3-Benzodioxol-5-yloxy)benzylidene]-N-(4-methox-
yphenyl)hydrazinecarbothioamide (PT 36). 1H NMR (300 MHz,
4.2.2.10. N-(4-Methoxyphenyl)-2-(4-phenoxybenzylidene)hydrazine-
CDCl3)
d
¼ 9.95 (s, 1H, NHeAr, D2O exchangeable), 8.79 (s, 1H, NHe
carbothioamide (PT 28). 1H NMR (300 MHz, CDCl3)
d
¼ 9.94 (s, 1H,
N, D2O exchangeable), 7.83 (s, 1H, CH]N), 6.92e7.80 (a set of sig-
nals, 11H, AreH and benzodioxolyleH), 5.94 (s, 2H, OCH2), 3.77
NHeAr, D2O exchangeable), 8.68 (s, 1H, NHeN, D2O exchangeable),
7.85 (s, 1H, CH]N), 6.81e7.78 (a set of signals, 13H, AreH), 3.79 (s,
(s, 3H, OCH3); IR (KBr)
n
¼ 3302, 3042 (NH), 1596 (N]CH), 1244 (e
3H, OCH3); IR (KBr)
n
¼ 3292, 3033 (NH),1599 (N]CH),1241 (eOe),
Oe), 1164 (C]S) cmꢂ1; MS (ES, 1.16 eV) m/z (%): 422.16 (62.73)
1163 (C]S) cmꢂ1; MS (ES, 1.16 eV) m/z (%): 378.17 (33.90) [Mþ þ 1].
[Mþ þ 1].
4.2.2.11. N-(4-Methoxyphenyl)-2-[4-(4-nitrophenoxy)benzylidene]
4.3. Pharmacology
hydrazinecarbothioamide (PT 29). 1H NMR (300 MHz, CDCl3)
d
¼ 9.88 (s, 1H, NHeAr, D2O exchangeable), 8.66 (s, 1H, NHeN, D2O
The evaluation of anticonvulsant activity and neurotoxicity was
carried out by the Epilepsy Branch, National Institute of Neuro-
logical Disorder and Stroke, National Institute of Health, Bethesda,
USA following the reported procedures.
exchangeable), 7.88 (s, 1H, CH]N), 6.88e7.83 (a set of signals, 12H,
AreH), 3.79 (s, 3H, OCH3); IR (KBr)
n
¼ 3301, 3029 (NH), 1604 (N]
CH), 1523 (N]O), 1240 (eOe), 1164 (C]S) cmꢂ1; MS (ES, 1.16 eV)
m/z (%): 423.14 (Mþ þ 1, 53.65).
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. 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.
4.2.2.12. N-(4-Methoxyphenyl)-2-[4-(4-methylphenoxy)benzylidene]
hydrazinecarbothioamide (PT 30). 1H NMR (300 MHz, CDCl3)
d
¼ 9.90 (s, 1H, NHeAr, D2O exchangeable), 8.59 (s, 1H, NHeN, D2O
exchangeable), 7.83 (s, 1H, CH]N), 6.83e7.80 (a set of signals, 12H,
AreH), 3.77 (s, 3H, OCH3), 2.35 (s, 3H, CH3); IR (KBr)
n
¼ 3298, 3027
(NH), 1605 (N]CH), 1244 (eOe), 1169 (C]S) cmꢂ1; MS (ES, 1.16 eV)
m/z (%): 392.11 (42.22) [Mþ þ 1].
4.2.2.13. 2-[4-(4-Chlorophenoxy)benzylidene]-N-(4-methoxyphenyl)
hydrazinecarbothioamide (PT 31). 1H NMR (300 MHz, CDCl3)
d
¼ 3.74 (s, 3H, OCH3), 6.79e7.81 (a set of signals, 12H, AreH), 7.78
(s, 1H, CH]N), 8.67 (s, 1H, NHeN, D2O exchangeable), 9.88 (s, 1H,
NHeAr, D2O exchangeable); IR (KBr)
n
¼ 3290, 3030 (NH), 1599
(N]CH), 1244 (eOe), 1164 (C]S) cmꢂ1; MS (ES, 1.16 eV) m/z (%):
412.14 (100.00) [Mþ þ 1] for 35Cl, 414.11 (35.11) [Mþ þ 1] for 37Cl.

486
L. Tripathi, P. Kumar / European Journal of Medicinal Chemistry 64 (2013) 477e487
4.3.1. Maximal electroshock (MES) test
4.3.6. 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.
Organotypic hippocampal slice cultures are treated with N-
For 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 rat MES screen.
methyl-
D
-aspartate (NMDA) or kainic acid (KA) to induce
membrane-
neuronal cell death. Propidium iodide (PI),
a
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 indi-
cated above, with the excitotoxin and either one or two investi-
gational compounds at the concentrations indicated. If
neuroprotection occurs as a consequence of the added com-
pound, slice cultures will have a visibly reduced fluorescent in-
tensity when compared to the slice cultures that have been
treated with the excitotoxin alone.
4.3.2. Subcutaneous metrazol seizure threshold (scMET) test
For scMET test animals are pretreated with various doses of the
test compound. At a previously determined TPE of the test com-
pound the dose of metrazol which will induce convulsion 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.
4.3.3. Neurotoxicity-minimal motor impairment (MMI)
4.4. Docking study
Minimal motor impairment was measured by the rotarod
(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.
Compounds PT 4, PT 7, PT 19, PT 28 and PT 30 were selected
as ligands for docking studies with six established epilepsy re-
ceptors namely GABA (A) alpha-1, GABA (A) delta, glutamate, Na/
H exchanger, Na channel and T-type calcium channel receptor.
These receptors are among the most important targets in the
design and discovery of successful antiepileptic drugs [18]. In
present study, AutoDock 4.0 with its Lamarckian genetic algo-
rithm (LGA) was used for automated flexible ligand docking of
PT 4, PT 7, PT 19, PT 28 and PT 30 with above mentioned
receptors.
4.3.4. Minimal clonic seizure (6 Hz) test
Minimal clonic seizure (6 Hz) test was used to assess com-
pound’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 be-
haviors, 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.
The grid maps were calculated using AutoGrid. In all dockings, a
ꢀ
grid map with 60 ꢄ 60 ꢄ 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 ꢄ 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.
4.3.5. 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).
4.5. Neurochemical investigation
Adult wistar rats were divided into three groups of three ani-
mals each. After 2 h of drug administration (30 mg/kg, i.p.) the
animals were decaptivated and the brain regions, olfactory lobe,
midbrain, cerebellum and medulla oblongata were dropped into
separate vials containing 4e6 ml of ice-cold 80% ethanol. The GABA
assay was performed in brain tissue extracts enzymatically as
previously described [19].
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 pre-
vent acute pilocarpine induced status an initial qualitative ef-
ficacy screen was performed. Administration of the candidate
drug was given to male albino Sprague Dawley rats (150e
180 g) via the i.p. route of administration. Then a challenge
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.
4.6. Prediction of ADME properties
A computational study of titled compounds was performed for
prediction of ADME properties. Polar surface area (TPSA) [20],
miLog P, number of rotatable bonds, molecular volume, number of
hydrogen donor and acceptor atoms and violations of Lipinski’s rule
of five [21] were calculated using Molinspiration online property
calculation toolkit [22]. Absorption (%ABS) was calculated by: %
ABS ¼ 109 ꢂ (0.345 ꢄ TPSA) [23].

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24 h at room temperature and then transferred to a separating
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octanol phase were separated and filtered through membrane filter
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Acknowledgments
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The authors would like to express their gratitude to Dr. James P.
Stables, Preclinical Pharmacology Section, Epilepsy Branch, National
Institute of Neurological Disorder and Stroke, National Institute of
Health, Bethesda for undertaking anticonvulsnt screening under
their anticonvulsant drug development (ADD) program. The authors
convey their sincere thanks to IIT, Delhi and CDRI, Lucknow for
providing the spectral and elemental data. The authors also appre-
ciate the help and support by Mr. Om Prakash during docking study.
[22] Molinspiration Cheminformatics, Bratislava, Slovak Republic, Available from:
http://www.molinspiration.com/services/properties.html, (accessed on 16.09.
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