Newer GABA derivatives for the treatment of epilepsy including febrile seizures: A bioisosteric approach

Article abstract of DOI:10.1016/j.ejmech.2008.01.036

The present study aims at design and synthesis of newer γ-aminobutyric acid (GABA) derivatives with the combination of thiosemicarbazone and GABA pharmacophores in order to develop newer anticonvulsants. The reported compounds were designed as bioisosteric analogues of GABA semicarbazones. The structures of the synthesized compounds were confirmed by the use of their spectral data besides elemental analysis. Initial anticonvulsant screening was performed using intraperitoneal (i.p.) maximal electroshock-induced seizure (MES), subcutaneous pentylenetetrazole (scPTZ), subcutaneous strychnine (scSTY), and subcutaneous picrotoxin (scPIC)-induced seizure threshold tests. A model involving 22-day old rat pups was also employed to further screen the effects of the test compounds against hyperthermia-induced febrile seizures. Only compounds 1 and 11 were found to be active in the MES test. Most of the compounds were found to be effective in the scPIC and febrile seizure models and very few compounds showed protection in scPTZ and scSTY models. This is the first report on these new GABA derivatives effective in the treatment of febrile seizures.

Full text of DOI:10.1016/j.ejmech.2008.01.036

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 2650e2655
http://www.elsevier.com/locate/ejmech
Original article
Newer GABA derivatives for the treatment of epilepsy
including febrile seizures: A bioisosteric approach
Jegadeesan Vaigunda Ragavendran a, Dharmarajan Sriram a, Srikanth Kotapati a,
James Stables b, Perumal Yogeeswari a,
*
a Medicinal Chemistry Research Laboratory, Pharmacy Group, Birla Institute of Technology and Science, Vidya Vihar, Pilani 333031, Rajasthan, India
b Epilepsy Branch, National Institutes of Health, Bethesda, MD, USA
Received 15 September 2007; received in revised form 10 January 2008; accepted 24 January 2008
Available online 6 February 2008
Abstract
The present study aims at design and synthesis of newer g-aminobutyric acid (GABA) derivatives with the combination of thiosemicarbazone
and GABA pharmacophores in order to develop newer anticonvulsants. The reported compounds were designed as bioisosteric analogues of
GABA semicarbazones. The structures of the synthesized compounds were confirmed by the use of their spectral data besides elemental anal-
ysis. Initial anticonvulsant screening was performed using intraperitoneal (i.p.) maximal electroshock-induced seizure (MES), subcutaneous pen-
tylenetetrazole (scPTZ), subcutaneous strychnine (scSTY), and subcutaneous picrotoxin (scPIC)-induced seizure threshold tests. A model
involving 22-day old rat pups was also employed to further screen the effects of the test compounds against hyperthermia-induced febrile sei-
zures. Only compounds 1 and 11 were found to be active in the MES test. Most of the compounds were found to be effective in the scPIC and
febrile seizure models and very few compounds showed protection in scPTZ and scSTY models. This is the first report on these new GABA
derivatives effective in the treatment of febrile seizures.
Ó 2008 Published by Elsevier Masson SAS.
Keywords: Thiosemicarbazones; Anticonvulsants; GABA; Pentylenetetrazole; Picrotoxin; Strychnine; Febrile seizures
1. Introduction
performed since this neurotransmitter is able to cross the
bloodebrain diffusion barrier (BBB) only when extremely
high doses are applied, which produce severe adverse side ef-
fects [9]. Hence, over the past few decades, research aimed at
achieving successful delivery of GABA into the CNS has re-
sulted in the discovery of various GABA analogues with im-
proved pharmacological activities [10]. Recently, we reported
the anticonvulsant properties of variously substituted N,N-
phthaloyl GABA amides and acid hydrazones [11]. One of
the approaches to analog-based drug discovery is the concept
of bioisosteric replacement (Fig. 1), which continues to play
an important role in bioorganic and medicinal chemistry in
the design of novel pharmacological tools as well as new ther-
apeutic agents with optimal pharmacological profile and im-
proved pharmacokinetic properties [12]. In the past decade,
aryl semicarbazones had been designed that were structurally
dissimilar from many common anticonvulsants containing the
dicarboximide function (CONRCO), which may contribute to
Epilepsy is the most common primary neurological disorder
known, affecting 0.4e0.8% of the population and up to 50 mil-
lion people worldwide [1,2]. Epilepsy is the tendency to expe-
rience seizures-intermittent, usually unprovoked and
stereotyped episodes that result from abnormal, paroxysmal
electrical discharge of neurons of the cerebral cortex [3]. 4-
Aminobutyric acid (GABA) is the principal inhibitory neuro-
transmitter in the mammalian brain [4,5]. It is well documented
that attenuation of GABAergic neurotransmission is involved in
the pathophysiology of several central nervous system (CNS)
disorders in humans, namely anxiety, pain, and epilepsy [6e
8]. The peripheral administration of GABA cannot be usefully
* Corresponding author. Tel.: þ91 1596 244684; fax: þ91 1596 244183.
E-mail address: pyogee@bits-pilani.ac.in (P. Yogeeswari).
0223-5234/$ - see front matter Ó 2008 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2008.01.036

J.V. Ragavendran et al. / European Journal of Medicinal Chemistry 43 (2008) 2650e2655
2651
R2
HO
O
R2
N
HO
O
R1
Bioisosteric Replacement
R1
NH N
O
NH
NH
NH
S
2-16
Fig. 1. Anticonvulsant compounds designed by bioisosteric replacement.
toxic side effects [13]. Consistent advances in the design of
novel anticonvulsant agents have been obtained through the
works of Dimmock and his colleagues [14], which included var-
ious aryl semicarbazones and (aryloxy) aryl semicarbazones.
Recently, we reported the anticonvulsant and antinociceptive
activities of GABA semicarbazones designed as pharmaco-
phoric hybrids [15]. Moreover, various aryl thiosemicarbazides
and thiosemicarbazones have also been reported by our group to
exhibit anticonvulsant activity in maximal electroshock seizure
(MES) and subcutaneous pentylenetetrazole (scPTZ) tests.
These agents were also found to block the expression of fully
kindled seizures [16]. Hence, given the promising biological
profile of GABA derivatives, aryl semicarbazones/thiosemicar-
bazones, we initiated a drug discovery program focusing on the
design and synthesis of newer GABA thiosemicarbazones as bi-
oisosteric analogues of GABA semicarbazones. These GABA
thiosemicarbazones were found to exhibit anticonvulsant activ-
ity in various animal models of seizure including the febrile sei-
zure model with lesser neurotoxicity.
yielded 85% of 1. Finally the required thiosemicarbazones
(2e16) were prepared by the reaction between appropriate
aryl/alkyl aldehydes or ketones and 4-(hydrazine carbothioa-
mido)butanoic acid in the presence of glacial acetic acid in
ethanol. The yields ranged from 45% to 78%. The purity
was assessed by TLC; and the assignments of the structures
were based on elemental and spectroscopic methods. The
physical properties of the synthesized compounds are pre-
sented in Table 1. The chemical shifts obtained from 1H
NMR spectra supported the proposed structures. The 1H
NMR spectrum revealed that the hydrazino proton (]NeNH)
showed a singlet at d ¼ 11.27e11.68 ppm and the alkyl NH
at 1.99e2.03 ppm both of which were D2O exchangeable.
All of the compounds (2e16) showed a characteristic D2O
exchangeable singlet due to OH proton of the acid function
at d ¼ 12.3e12.36 ppm. The aryl ring protons resonated at
d ¼ 6.9e8.41 ppm. The singlet due to 1H of carbimino proton
was observed at d ¼ 8.0e8.2 ppm and the singlet due to 3H of
carbimino CH3 was observed at d ¼ 0.91 ppm.
2. Synthesis
3. Results and discussion
The synthesis of GABA thiosemicarbazones was accom-
plished as per earlier reported procedure [17,18] and presented
in Scheme 1. The 4-(hydrazine carbothioamido)butanoic acid
(1) was synthesized from 4-aminobutanoic acid via a one-pot
procedure. 4-Aminobutanoic acid on treatment with carbon di-
sulphide in the presence of potassium hydroxide in ethanol
gave the potassium salt of the corresponding 4-dithiocarba-
mate derivative, which on reaction with hydrazine hydrate
The synthesized compounds (1e16) were evaluated at dose
levels of 30, 100 and 300 mg/kg intraperitoneally in mice for
anticonvulsant activity. In our anticonvulsant drug develop-
ment program, our approach was to test the new compounds
preliminarily in two standard models MES and scPTZ (at
NIH) for their ability to reduce seizure spread and to elevate
seizure threshold, respectively. Further to understand the
mechanistic aspects, we undertook testing in the other two
models scSTY and scPIC in our laboratory. From our initial
screening results we found that some compounds showed
broad spectrum anticonvulsant activity, which prompted us
to evaluate their efficacy in a stress-induced febrile seizure
model. Table 2 lists the results obtained from the initial anti-
convulsant evaluation of the synthesized compounds com-
pared to the clinically proven antiepileptics such as
phenytoin and ethosuximide. The acute neurological toxicity
was determined by the rotorod test.
Two compounds (1 and 11) showed activity in the MES
screen at 300 mg/kg with a shorter duration of action (0.5 h)
indicative of their ability to prevent seizure spread. In the
scPTZ screen, a test used to identify compounds that elevate
seizure threshold, six compounds (2, 4, 5, 7, 15, and 16)
showed protection. Compound 16 was the most effective in
this model exhibiting protection at 30 mg/kg for a shorter
S
(a)
CS2, KOH, Ethanol
0-5°C, 1h
COOH
HN
H2N
COOH
S
(b) NH2NH2.H2O,
50°C, 2h
NH2
1
Aldehyde or Ketone
Glacial CH3COOH
Ethanol
COOH
R1
S
HN
R1
COOH
N
HN
N
R2
2-12
13-16
R2
Scheme 1. Synthetic route to GABA thiosemicarbazones.

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J.V. Ragavendran et al. / European Journal of Medicinal Chemistry 43 (2008) 2650e2655
Table 1
Physical data of 4-(alkylidene/arylidene hydrazine carbothioamido)butanoic acids
Compound
R1
R2
Yield (%)
M.P. (ꢀC)a
C log Pb
Rfc
1
e
e
85
54
65
68
55
70
54
45
62
54
62
75
56
54
52
78
202e204
214e216
213e215
206e208
184e187
256e258
121e123
116e119
258e260
134e137
52e55
ꢁ0.58
1.18
0.73
2.76
1.33
1.74
1.91
2.18
0.96
0.40
3.17
4.05
2.87
1.84
1.35
1.78
0.42
0.63
0.61
0.65
0.72
0.59
0.62
0.67
0.71
0.65
0.62
0.61
0.66
0.59
0.62
0.69
2
H
2-OH
4-NO2
4-Cl
3
H
4
H
5
H
3-NO2
4-N(CH3)2
H
6
H
CH3
7
8
CH3
CH3
4-CH3
3-NH2
4-NO2
H
9
10
11
12
13
14
15
16
a
CH3
C6H5
C6H5
4-Br
CH2eC6H5
74e76
84e87
CH2eC6H5
Cyclohexylene
Cyclopentylene
Isatinyl
127e129
124e127
117e120
Elemental analyses for C, H, N were within ꢂ0.4% of the theoretical values.
C log P was calculated using www.logp.com.
Mobile phase CHCl3eCH3OH (9:1).
b
c
duration. Compounds 2, 4, and 15 exhibited activities at
100 mg/kg and compounds 5 and 7 showed activity at
300 mg/kg at 4 h and 0.5 h, respectively. Five compounds (2,
4e6, and 12) showed protection in the subcutaneous strych-
nine-induced seizure model. Compounds 2 and 12 showed ac-
tivity at 100 mg/kg and other compounds showed activity at
300 mg/kg, in which only compounds 4 and 6 showed activity
till 4 h period of observation. Therefore, these compounds
may be useful in treating not only generalized toniceclonic
and complex partial seizures, but also absence seizures. In
the scPIC-induced seizure threshold test, most of the com-
pounds exhibited activity indicative of the possible involve-
ment of GABA-mediation in the anticonvulsant action. All
of the compounds except 1, 8, 10 and 15 showed protection
Table 2
Anticonvulsant activity of the synthesized compounds
Compound
MESa
scPTZa
scSTYa
0.5 h
scPICa
0.5 h
Hyperthermia-induced seizures b
Neurotoxicitya
0.5 h
0.5 h
4 h
4 h
4 h
Duration of
seizures (s)
% Protection
0.5 h
4 h
1
300
e
e
100
e
e
e
e
e
300
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
100
e
e
e
e
300
e
300
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
215 ꢂ 3.2NS
25 ꢂ 1.2
45 ꢂ 1.5
150 ꢂ 1.9
145 ꢂ 2.2
35 ꢂ 1.7
30 ꢂ 1.2
60 ꢂ 2.2
215 ꢂ 2.8NS
160 ꢂ 4.5NS
50 ꢂ 2.6
240 ꢂ 1.2NS
80 ꢂ 2.5
65 ꢂ 1.5
85 ꢂ 2.8
25 ꢂ 1.5
e
27
92
85
49
51
88
90
80
27
46
83
19
73
78
71
92
e
300
e
300
e
2
100
300
300
100
300
100
e
100
300
300
100
300
100
e
3
e
e
e
4
e
100
e
e
300
300
300
e
100
e
e
e
5
e
e
e
e
6
7
e
300
e
e
e
8
e
e
e
e
9
10
e
e
e
e
e
e
300
e
300
e
e
100
e
e
100
e
11
12
300
e
e
e
e
300
100
300
300
e
300
100
e
e
e
100
e
300
e
e
300
e
13
14
e
e
e
300
e
e
15
16
e
100
30
e
e
e
e
e
30
e
e
e
300
e
300
e
e
100
e
e
100
e
Phenytoin
Ethosuximide
Lamotrigine
Hyperthermic control
Vehicle-control
a
300
30
e
e
e
e
e
e
30
e
e
e
e
e
145 ꢂ 2.2
330 ꢂ 10.0
295 ꢂ 7.0
51
e
e
100
e
e
100
e
e
e
e
e
e
e
e
e
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 animals were examined at 0.5 h and 4 h. (e) Indicates an absence of effect at the maximum dose tested.
b
Compounds were administered intraperitoneally at a single dose of 100 mg/kg; each value represents the mean ꢂ SEM of four rat pups significantly different
from the vehicle-control at P < 0.05 and NS denotes not significant at P < 0.05 (Student’s t-test).

J.V. Ragavendran et al. / European Journal of Medicinal Chemistry 43 (2008) 2650e2655
2653
in the scPIC test. Compounds that showed activity at 100 mg/
kg till the 4 h period included 2, 5, 7, and 12. All other active
compounds except 13 showed activity till 4 h period at
300 mg/kg. Overall it appears that three compounds (2, 4,
and 5) were effective in three animal models of seizure, five
(6, 7, 11, 12, and 16) were effective in at least two models
and six compounds (1, 3, 9, and 13e15) showed anticonvul-
sant activity in one model. Compounds 8 and 10 were com-
pletely devoid of anticonvulsant activity.
with theoretical values (within ꢂ0.4%) unless indicated. The
homogeneity of the compounds was monitored by ascending
thin layer chromatography (TLC) on silicagel-G (Merck)
coated aluminium plates, visualized by iodine vapor and UV
light. Developing solvents were chloroformemethanol (9:1).
5.1.1. Synthesis of 4-(hydrazine carbothioamido)butanoic
acid (1)
4-Aminobutanoic acid (0.1 mol, 10.32 g) was dissolved in
30 mL of ethanol and was stirred at 0e5 ꢀC vigorously. To
the stirred solution, an equimolar amount of potassium hy-
droxide (0.1 mol, 5.60 g) and double the molar amount of car-
bon disulphide (0.2 mol, 15.20 mL) were added and stirred for
the next 1 h. The reaction mixture was brought back to room
temperature, after which hydrazine hydrate (99%) (0.1 mol,
4.85 mL) was added. Stirring was continued for 2 h, while si-
multaneously heating the reaction mixture to 50 ꢀC. The re-
sulting precipitate was filtered, dried and recrystallized using
ethanol, M.P. 202e204 ꢀC, IR (KBr) nmax 3300, 1740, 1640,
Due to the promising anticonvulsant activity profile of these
derivatives, the effect of newer GABA analogues against febrile
seizures was studied in a rat immature model (22-day old rat
pups) of hyperthermia (Table 2). Lamotrigine is one of the
first-line drugs prescribed for febrile seizures and hence em-
ployed as the standard drug. Since the compounds were
screened for the first time in an excitotoxic model of febrile sei-
zure, to avoid death of the animals, we started with the single
dose (100 mg/kg). Of a total of 16 compounds screened, 11
compounds (2, 3, 5e8, 11, 13e16) demonstrated activity with
50% or more percentage protection and also shortened the
duration of seizures significantly (P < 0.05) at a single dose
of 100 mg/kg. Compounds 2, 7, and 16 showed percentage pro-
tections more than or equal to 90%. This is the first report on
such derivatives with significant anticonvulsant activity against
febrile seizures. Promising compounds could be carried on for
lower doses evaluation in the quantitative studies in the future.
In the acute neurological toxicity screen, the compounds 2,
3, 5e7, 9, 11, 12, and 14e16 emerged as promising anticon-
vulsants with less or no neurotoxicity. There was no separation
between the anticonvulsant dose and the neurotoxic dose for
compounds 1, 4, and 13. Compound 10, which was ineffective
in the seizure model showed neurotoxicity at 100 mg/kg.
1550, 1270, 835 cmꢁ1
;
1H NMR (DMSO-d6, 300 MHz,
d ppm) 1.82 (m, 2H, CH2 b to COOH), 2.03 (s, 2H, NHCSNH,
D2O exchangeable), 2.23 (t, 2H, CH2 a to COOH), 3.36 (t, 2H,
CH2 g to COOH), 4.34 (s, 2H, NH2, D2O exchangeable),
12.24 (s, 1H, OH of COOH, D2O exchangeable).
5.1.2. General procedure for the synthesis of 4-(alkylidene/
arylidene hydrazine carbothioamido)butanoic acids (2e16)
To a solution of 4-(hydrazinecarbothioamido)butanoic acid
(0.005 mol, 0.89 g) in ethanol (20 mL), an equimolar amount
of appropriate alkyl/aryl aldehydes or ketones (including isatin)
was added and refluxed in the presence of glacial acetic acid for
1e3 h until the completion of the reaction. The solvent was then
removed in vacuo and the residue obtained was dried and re-
crystallized from ethanol. The physical data of the compounds
are presented in Table 1. The IR spectra of the compounds were
identical in the following aspects: 3300, 1640, 1590, 1170,
1015 cmꢁ1; 1H NMR (DMSO) d (ppm) spectra of some repre-
sentative compounds are as follows.
4. Conclusions
Thepresent studyreportsthe synthesis of GABA thiosemicar-
bazones designed as bioisostericanalogues of GABA semicarba-
zones. These thiosemicarbazono derivatives of GABA were
found to exhibit anticonvulsant activity invarious animal models
of seizure including the febrile seizure model. Overall, the syn-
thesized compounds emerged as more active and less neurotoxic
when compared to their semicarbazono counter parts.
5.1.2.1. 4-(2-(2-Hydroxybenzylidene)hydrazine carbothioami-
do)butanoic acid (2). 1.80 (m, 2H, CH2 b to COOH), 2.01
(s, 1H, NHCS, D2O exchangeable), 2.20 (t, 2H, CH2 a to
COOH), 3.40 (t, 2H, CH2 g to COOH), 6.9e7.4 (m, 4H,
AreH), 8.0 (s, 1H, carbimino H), 9.9 (s, 1H, AreOH, D2O
exchangeable), 11.56 (s, 1H, NHN], D2O exchangeable),
12.3 (s, 1H, OH of COOH, D2O exchangeable).
5. Experimental protocols
5.1. Chemistry
Melting points were measured in open capillary tubes on
a Buchi 530 melting point apparatus and are uncorrected. Infra-
red (IR) and proton nuclear magnetic resonance (1H NMR)
spectra were recorded for the compounds on Jasco IR Report
100 (KBr) and Brucker Avance (300 MHz) instruments, respec-
tively. Chemical shifts are reported in parts per million (ppm)
using tetramethyl silane (TMS) as an internal standard. All
exchangeable protons were confirmed by addition of D2O. Ele-
mental analyses (C, H and N) were undertaken with a Perkine
Elmer model 240C analyzer and all analyses were consistent
5.1.2.2. 4-(2-(4-Nitrobenzylidene)hydrazine carbothioamido)-
butanoic acid (3). 1.81 (m, 2H, CH2 b to COOH), 2.03 (s,
1H, NHCS, D2O exchangeable), 2.21 (t, 2H, CH2 a to
COOH), 3.43 (t, 2H, CH2 g to COOH), 7.93e8.41 (m, 4H,
AreH), 8.2 (s, 1H, carbimino H), 11.62 (s, 1H, NHN],
D2O exchangeable), 12.32 (s, 1H, OH of COOH, D2O
exchangeable).
5.1.2.3. 4-(2-(4-Chlorobenzylidene)hydrazine carbothioami-
do)butanoic acid (4). 1.79 (m, 2H, CH2 b to COOH), 2.02

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J.V. Ragavendran et al. / European Journal of Medicinal Chemistry 43 (2008) 2650e2655
(s, 1H, NHCS, D2O exchangeable), 2.24 (t, 2H, CH2 a to
COOH), 3.42 (t, 2H, CH2 g to COOH), 7.47e7.74 (m, 4H,
AreH), 8.13 (s, 1H, carbimino H), 11.64 (s, 1H, NHN],
D2O exchangeable), 12.35 (s, 1H, OH of COOH, D2O
exchangeable).
exchangeable), 2.24 (t, 2H, CH2 a to COOH), 3.44 (t, 2H,
CH2 g to COOH), 11.27 (s, 1H, NHN], D2O exchangeable),
12.35 (s, 1H, OH of COOH, D2O exchangeable).
5.1.2.11. 4-(2-(2-Oxoindolin-3-ylidene)hydrazine carbothioa-
mido)butanoic acid (16). 1.84 (m, 2H, CH2 b to COOH),
1.99 (s, 1H, NHCS, D2O exchangeable), 2.21 (t, 2H, CH2
a to COOH), 3.47 (t, 2H, CH2 g to COOH), 7.1e7.8 (m,
4H, AreH), 11.20 (s, 1H, NH of isatinyl, D2O exchangeable),
11.31 (s, 1H, NHN], D2O exchangeable), 12.32 (s, 1H, OH
of COOH, D2O exchangeable).
5.1.2.4. 4-(2-(4-(Dimethylamino)benzylidene)hydrazine carbo-
thioamido)butanoic acid (6). 1.83 (m, 2H, CH2 b to
COOH), 2.01 (s, 1H, NHCS, D2O exchangeable), 2.22 (t,
2H, CH2 a to COOH), 3.04 (s, 6H, N(CH3)2), 3.46 (t, 2H,
CH2 g to COOH), 6.81e7.48 (m, 4H, AreH), 8.11 (s, 1H, car-
bimino H), 11.68 (s, 1H, NHN], D2O exchangeable), 12.33
(s, 1H, OH of COOH, D2O exchangeable).
5.2. Pharmacology
5.1.2.5. 4-(2-(1-Phenylethylidene)hydrazine carbothioamido)-
butanoic acid (7). 0.88 (s, 3H, carbimino CH3), 1.81 (m,
2H, CH2 b to COOH), 2.02 (s, 1H, NHCS, D2O exchange-
able), 2.24 (t, 2H, CH2 a to COOH), 3.43 (t, 2H, CH2 g to
COOH), 7.51e7.62 (m, 5H, AreH), 11.27 (s, 1H, NHN],
D2O exchangeable), 12.35 (s, 1H, OH of COOH, D2O
exchangeable).
The preliminary anticonvulsant and neurotoxicity evalua-
tions were done using reported procedures [19,20]. Male al-
bino mice (Swiss strain, 18e25 g) were used as
experimental animals. All of the test compounds were sus-
pended in 0.5% w/v methyl cellulose in the case of MES
and scPTZ screens and 30% v/v PEG 400 for screening in
the picrotoxin and strychnine-induced seizure models. The an-
imals were maintained at an ambient temperature of
22 ꢂ 1 ꢀC, in groups of 5 per cage under standard laboratory
conditions, receiving standard laboratory chow and water ad
libitum. A 12 h:12 h light/dark cycle was maintained through-
out the experimental studies. All the tests have been performed
in accordance with the guidelines laid out by the Institutional
Animal Ethics Committee.
5.1.2.6. 4-(2-(1-p-Tolylethylidene)hydrazine carbothioamido)-
butanoic acid (8). 0.91 (s, 3H, carbimino CH3), 1.83 (m,
2H, CH2 b to COOH), 2.03 (s, 1H, NHCS, D2O exchange-
able), 2.22 (t, 2H, CH2 a to COOH), 2.41 (s, 3H, AreCH3),
3.44 (t, 2H, CH2 g to COOH), 7.20e7.65 (m, 4H, AreH),
11.28 (s, 1H, NHN], D2O exchangeable), 12.36 (s, 1H, OH
of COOH, D2O exchangeable).
5.2.1. Anticonvulsant screening
5.1.2.7. 4-(2-(1-(3-Aminophenyl)ethylidene)hydrazine carbo-
thioamido)butanoic acid (9). 0.89 (s, 3H, carbimino CH3),
1.81 (m, 2H, CH2 b to COOH), 2.02 (s, 1H, NHCS, D2O ex-
changeable), 2.24 (t, 2H, CH2 a to COOH), 3.43 (t, 2H, CH2 g
to COOH), 5.82 (s, 2H, AreNH2, D2O exchangeable), 6.52e
7.10 (m, 4H, AreH), 11.30 (s, 1H, NHN], D2O exchange-
able), 12.33 (s, 1H, OH of COOH, D2O exchangeable).
All of the test compounds were administered intraperitone-
ally in a volume of 0.01 mL/g for mice at doses of 30, 100 and
300 mg/kg. Anticonvulsant activity was assessed after 30 min
and 4 h of drug administration. Activity in the scSTY and
scPIC tests was established according to the earlier reported
procedures [21,22] and the data are presented in Table 2.
5.2.1.1. Hyperthermia-induced seizures. Wistar rat pups ob-
tained at age 15 days were housed with their mothers under
standard laboratory conditions until they were weaned at 21
days. At age 22 days, the pups had a mean weight of 100 g.
They were then housed in groups of four with ad libitum
access to food and water. A daylight cycle of 12 h:12 h was
maintained. The majority of our studies began with animals
aged 22 days. Exposure to hyperthermia was carried out as re-
ported earlier [23]. Briefly, the rat pups were placed in a glass
chromatography tank (30 ꢃ 30 ꢃ 60 cm), which contained wa-
ter to a depth that the animal could stand upright supported by
the side of the tank with only its head above water level. Ex-
posure to hyperthermia was achieved by maintaining the water
in the tank at a temperature of 45 ꢀC by placing it in a temper-
ature-controlled water bath. This temperature (45 ꢀC) does not
produce skin damage at exposures <1 h. In human subjects,
only a mild ‘‘pricking pain’’ is reported at 45 ꢀC, and this sen-
sation disappears in a few seconds. The pups were placed in
the water unrestrained for 4 min or until a seizure occurred
(whichever was shorter). They were immediately removed
5.1.2.8. 4-(2-(Diphenylmethylene)hydrazine carbothioamido)-
butanoic acid (11). 1.83 (m, 2H, CH2 b to COOH), 2.03 (s,
1H, NHCS, D2O exchangeable), 2.22 (t, 2H, CH2 a to
COOH), 3.46 (t, 2H, CH2 g to COOH), 7.32e7.82 (m, 10H,
AreH), 11.27 (s, 1H, NHN], D2O exchangeable), 12.32 (s,
1H, OH of COOH, D2O exchangeable).
5.1.2.9. 4-(2-(1,3-Diphenylpropan-2-ylidene)hydrazine carbo-
thioamido)butanoic acid (13). 1.84 (m, 2H, CH2 b to
COOH), 2.02 (s, 1H, NHCS, D2O exchangeable), 2.25 (t,
2H, CH2 a to COOH), 2.63 (s, 4H, CH2ePhenyl) 3.43 (t,
2H, CH2 g to COOH), 7.05e7.30 (m, 10H, AreH), 11.31
(s, 1H, NHN], D2O exchangeable), 12.33 (s, 1H, OH of
COOH, D2O exchangeable).
5.1.2.10. 4-(2-Cyclohexylidene hydrazine carbothioamido)bu-
tanoic acid (14). 1.29 (m, 4H, o-position of cyclohexane
ring), 1.65 (m, 6H, m- and p-positions of cyclohexane ring),
1.81 (m, 2H, CH2 b to COOH), 2.03 (s, 1H, NHCS, D2O

J.V. Ragavendran et al. / European Journal of Medicinal Chemistry 43 (2008) 2650e2655
2655
from the water and placed in an observation chamber at the
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