Design, synthesis and evaluation of N-(substituted benzothiazol-2-yl)amides as anticonvulsant and neuroprotective

Article abstract of DOI:10.1016/j.ejmech.2012.10.002

A series of N-(substituted benzothiazol-2-yl)amide derivatives 2a-h and 4a-h were synthesized by the EDC coupling reactions of substituted-benzothiazol- 2-amine with 4-oxo-4-phenylbutanoic acid/2-benzoyl benzoic acid and evaluated for their anticonvulsant and neuroprotective effect. N-(6-methoxybenzothiazol-2- yl)-4-oxo-4-phenylbutanamide (2f) emerged as the most effective anticonvulsant with median doses of 40.96 mg/kg (MES ED₅₀), 85.16 mg/kg (scPTZ ED₅₀) and 347.6 mg/kg (TD₅₀). Furthermore, compound 2f displayed promising neuroprotective effect by lowering the levels of MDA and LDH; therefore, it represents a potential lead in search for safer and effective anticonvulsants having neuroprotective effects.

Full text of DOI:10.1016/j.ejmech.2012.10.002

European Journal of Medicinal Chemistry 58 (2012) 206e213
Contents lists available at SciVerse ScienceDirect
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Short communication
Design, synthesis and evaluation of N-(substituted benzothiazol-2-yl)amides as
anticonvulsant and neuroprotectiveq
*
Mohd. Zaheen Hassan, Suroor A. Khan, Mohd. Amir
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, New Delhi 110062, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of N-(substituted benzothiazol-2-yl)amide derivatives 2aeh and 4aeh were synthesized by
the EDC coupling reactions of substituted-benzothiazol-2-amine with 4-oxo-4-phenylbutanoic acid/2-
benzoyl benzoic acid and evaluated for their anticonvulsant and neuroprotective effect. N-(6-
methoxybenzothiazol-2-yl)-4-oxo-4-phenylbutanamide (2f) emerged as the most effective anticonvul-
sant with median doses of 40.96 mg/kg (MES ED50), 85.16 mg/kg (scPTZ ED50) and 347.6 mg/kg (TD50).
Furthermore, compound 2f displayed promising neuroprotective effect by lowering the levels of MDA
and LDH; therefore, it represents a potential lead in search for safer and effective anticonvulsants having
neuroprotective effects.
Received 5 June 2012
Received in revised form
11 September 2012
Accepted 2 October 2012
Available online 10 October 2012
Keywords:
Benzothiazole
Amides
Ó 2012 Elsevier Masson SAS. All rights reserved.
Anticonvulsants
Neuroprotective agents
1. Introduction
of safer agents and novel mechanistic approaches in the treatment
of epilepsy.
Epilepsy is a chronic neurological disorder characterized by an
enduring predisposition to generate seizures due to hyper-
synchronous neuronal activity in the brain. According to epidemi-
ological studies, epilepsy is the third most devastating neurological
disorder, affecting more than 50 million people globally with
almost 90% of these patients being in developing countries [1].
Antiepileptic drugs (AEDs) comprise a diverse range of molecules
Anticonvulsants having neuroprotective actions have received
considerable attention in the treatment of epilepsy. Several studies
reported that neurodegeneration is the major neurobiological
abnormality in epileptic brain [5]. The pathomechanisms of neu-
rodegeneration are complex involving glutamate overload,
decreased GABA level due to inhibition of enzyme glutamic acid
decarboxylase (GAD) by oxygen free radicals and multiple lipid and
protein damages via lipid peroxidation (LPO) [6]. Since epileptic
seizure and neurodegeneration share aspects of their underlying
pathophysiology therefore, some AEDs are equally effective neu-
roprotectives [7]. Riluzole (2-amino-6-trifluoromethoxy benzo-
thiazole) is an anticonvulsant drug with phenytoin-like spectrum
of activity. It was also found to be effective for treating some forms
of neurodegeneration. Although riluzole is known to modulate
excitatory neurotransmission, the precise neuroprotective mecha-
nisms remain largely speculative [8]. As riluzole is a simple mole-
cule having benzothiazole moiety in its structure and a wide range
of CNS activity, it is a good target for structure modification and
structureeactivity relationship (SAR) studies. Many studies have
been conducted in an attempt to find derivatives superior to rilu-
zole [9,10]. The synthesis of various riluzole derivatives showed
that strong neuroprotective and anti-seizure activity required the
presence of six substituents containing either an unchanged or
oxidized sulfur atom or a nitrogen atom [9]. On the other hand,
a variety of benzothiazole derivatives have been reported to possess
acting mostly through: enhancement of
g-aminobutyric acid
(GABA) mediated inhibitory neurotransmission, modulation of
voltage-gated ion channels (Naþ, Caþþ), and reduction of excitatory,
particularly glutamate-mediated neurotransmitter [2]. Despite
these therapeutic arsenals, there is yet no complete cure for
epileptic conditions and current AEDs suppress the seizure symp-
toms of epilepsy (w70% epileptic patients) but do not affect the
natural course of the epileptogenic process [3]. Finally, the efficacy
of many AEDs are limited by the dose related toxicities and diverse
array of adverse drug reactions from minimal brain impairment,
megaloblastic anemia to death from aplastic anemia or hepatic
failure [4]. Thus, there is a substantial need for further development
q
Part of the work was presented at 8th AFMC International Medicinal Chemistry
Symposium "Frontier of Medicinal Science" held in Nov. 2011 at Tokyo, Japan.
* Corresponding author. Tel.: þ91 11 2605 9688x5307; fax: þ91 11 26059663.
E-mail address: mamir_s2003@yahoo.co.in (Mohd. Amir).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.002

Mohd.Z. Hassan et al. / European Journal of Medicinal Chemistry 58 (2012) 206e213
207
anticonvulsant activity in a number of animal models. Therefore,
these are promising candidates for the design of novel antiepileptic
drugs [11,12].
2.98 and 3.44e3.51 ppm due ethylene protons and disappearance of
signal of amino protons of substituted-2-aminobenzothiazoles. The
13C NMR spectra of a prototype compound 2f revealed three upfield
Encouraged by these observations and in continuation of our
ongoing research program [13e15], some amide derivatives of
benzothiazole were designed and synthesized as anticonvulsant
agents. We propose that the GABA like pharmacophore on the
benzothiazole nucleus might sufficiently bind with the receptors
and the resultant molecules will have a synergistic anticonvulsant
effect due to increased lipophilicity. The presence of an amide
group as hydrogen bonding domain (HBD) is an optimal anticon-
vulsant pharmacophoric feature in the title compounds. The addi-
tional features include benzothiazolyl hydrophobic-domain (A), S
atom as electron donor system (D) with distal phenyl residue (R),
which influences the blood brain barrier (BBB) diffusion and
pharmacokinetic properties of the anticonvulsants (Fig. 1) [16]. In
our present work, we aimed to synthesize a series of N-(substituted
benzothiazol-2-yl)amide derivatives in order to investigate their
behavior not only as anticonvulsants but as neuroprotective agents
too. Since, none of the currently available AEDs is perfect and most
of them have chronic and acute adverse effects therefore, the
present study also focuses on the toxicity studies of the synthesized
compounds.
peaks at d 28.42, 33.41 and 55.86 due to a pair of ethylene carbons
and one methoxy carbon, respectively while carbonyl carbons of
amide (CONH) and benzoyl (COPh) groups appeared downfield at
d
178.32 and 198.21, respectively. The other peaks of carbon were
observed at 105.53, 113.87, 118.14, 128.08, 128.63, 130.00, 133.27,
d
136.51, 143.04, 155.74 and 167.03 confirming the presence of eigh-
teen carbons in the compound.
2.2. Pharmacological evaluation
The results of the in vivo pharmacological screening are shown
in Tables 1e3. The titled compounds were initially evaluated
against maximal electroshock seizure (MES) and subcutaneous
pentylenetetrazol (scPTZ) (Table 1), the two most widely used
seizure models in the search for new AEDs. These tests aim to
detect compounds possessing activity against generalized tonic-
clonic (grand mal) and generalized absence (petit mal) seizures
respectively. The most potent compound was also subjected to
Phase II quantitative determination of median effective dose
(ED50), median toxic dose (TD50
) and protective index (PI)
(Table 3). In the preliminary screening, all the compounds showed
protection against MES test, which indicates the potential of these
compounds against generalized tonic-clonic seizure. Compound
2f showed protection against seizures in the MES model at the
30 mg/kg dose after both 0.5 and 4.0 h of the administration. This
indicates that compound 2f has a rapid onset and long duration
action at a lower dose. Compound 2e was active at 100 mg/kg
after 0.5 and 4.0 h in MES tests, which is indicative of its quick
onset and long duration of action at relatively higher dose while
remaining compounds were active at 300 mg/kg. In the scPTZ
screening, only compound 2f raised the seizure threshold at
a dose of 100 mg/kg after both 0.5 and 4.0 h time intervals,
showing the promising nature of compound against the absence
seizure. In the neurotoxicity screen, compounds 2a, 2b, 2eeh, 4a
and 4eeg did not exhibit any neurological deficit at the highest
dose. Rest of the compounds showed neurotoxicity but at a higher
dose.
We further explored the CNS depressant effects of all the
compounds by Porsolt’s swimming pool test. Their immobility time
was compared with respect to the reference drug carbamazepine
(Table 1). Compounds 2a, 2e and 2f had no significant CNS
depression, whereas rest of the compounds possessed CNS
depressant effects as they increased the immobility time signifi-
cantly compared to control at p < 0.05. Hepatotoxicity is the key
limiting factor for the clinical use of AEDs. Therefore, selected
compounds 2a, 2e and 2f were further analyzed for their hepato-
toxic profiles. Any significant alterations in the level of enzymes
and proteins are indicative of liver toxicity. As shown in Table 2,
activities of liver enzymes SGOT, SGPT, alkaline phosphatase,
albumin and total protein remained almost the same with respect
to control values (p > 0.05) except for the compound 2e, which
slightly increased the total protein level (p < 0.05). The liver
histological examination of compound 2f-treated animals showed
no symptoms of necrosis, inflammation or hepatocytes degenera-
tion as compared to control animals. The hepatic parenchyma
appeared normal, which indicates the nontoxic effect of compound
on liver (Figs. 2 and 3).
2. Results and discussion
2.1. Chemistry
The synthetic routes of compounds are outlined in Scheme 1.
Synthesis of 4-oxo-4-phenylbutanoic acid (1) and 2-benzoyl benzoic
acid (3) involves the Lewis acid-catalyzed Friedel Crafts acylation of
benzene with succinic anhydride and phthalic anhydride, respec-
tively. Reactions of the above mentioned acids 1 or 3 with 1-(3-
dimethylaminopropyl)-3-ethyl-carbodiimide (EDC) and hydrox-
ybenzotriazole (HOBt) in presence of DCM resulted in the in situ
formation of activated acyl compounds which on further addition of
substituted-2-aminobenzothiazole and triethylamine, produced the
desired amides 2aeh and 4aeh, respectively in good yields. The
structures of the amides 2aeh and 4aeh were confirmed by
elemental analyses and spectral data. The IR spectra of compounds
2aeh and 4aeh showed the appearance of C¼O and NeH absorption
bands in the range of 1632e1680 and 3265e3345 cmꢀ1, respec-
tively. The 1H NMR spectra revealed a broad singlet signal at
10.76 ppm due to CONH proton. The 1H NMR spectra of compounds
2aeh revealed the appearance of a pair of triplets signals at 2.80e
d 8.77e
d
Compound 2f appeared as the most effective anticonvulsant in
phase I screening without any significant toxicities like minimal
motor impairment, CNS depression and hepatotoxic effect. This led
us to further investigate and quantify its pharmacological proper-
ties in phase II anticonvulsant screening (Table 3). In the MES
Fig. 1. Design of the titled compounds as anticonvulsant-neuroprotective agents
possessing the essential pharmacophoric elements: aryl unit/Hydrophobic domain (A),
electron donor atom (D), and coupled hydrogen donor (HD)ehydrogen acceptor (HA)
group.

208
Mohd.Z. Hassan et al. / European Journal of Medicinal Chemistry 58 (2012) 206e213
O
O
S
N
OH
(iii)
(iii)
(i)
N
H
R
O
O
1
O
2(a-h)
AlCl3
C6H6
OH
O
O
O
(ii)
S
N
H
R
N
3
4(a-h)
Scheme 1. Reagent and conditions: (i) succinic anhydride, reflux; (ii) phthalic anhydride, reflux, (iii) substituted 2-aminobenzothiazole, EDC, HOBt, DCM, stirring.
screen, compound 2f exhibited moderate efficacy with an ED50 of
40.96 mg/kg, which is higher than that of phenytoin (9.5 mg/kg)
and carbamazepine (8.8 mg/kg) and lower than sodium valproate
(272 mg/kg). In the scPTZ screen, it offered protection with an ED50
of 85.16 mg/kg, which is considerably lower than that of phenytoin,
carbamazepine and sodium valproate. The median toxic dose
(TD50) in rotarod test was also higher (347.6 mg/kg) compared to all
the standard drugs. Calculation of protective indices (PI) resulted in
higher PI values of 8.4 in MES and 4.0 in scPTZ screen, indicating
that compound 2f is indeed a safer and effective anticonvulsant
agent.
(p < 0.01). This GABA-mediatory action might be due the molecular
hybridization of lipophilic benzothaizole with GABA like moiety.
The neuroprotective effects of the compounds were investigated
against the acrylamide (ACR)-induced neurotoxity. ACR is a potent
neurotoxin in both humans and experimental animals [18]. The
release of tissue malonaldehyde (MDA) and cytosolic enzyme
lactate dehydrogenase (LDH) are the peripheral markers of cells
intoxication [19]. Neuroprotective agents ameliorate seizure
induced neuronal cells intoxications by lowering the LDH and MDA
levels which may eventually alters the clinical course of the
seizures [20,21]. The data depicted in Fig. 5 demonstrated that,
ACR-administration in mice induced degenerative effect on the
brain tissue as manifested by the significant increase in the levels of
MDA and LDH (224% and 67%, respectively) compared to the control
group (p < 0.01). Compounds 2a, 2e and 2f administered 10 min
before ACR intoxication revealed the decrease in brain MDA and
LDH activity, however significant decrease in the activity was
shown by only compound 2f. These results confirmed the powerful
neuroprotective actions of compounds and suggested possibly an
influence of neuroprotective benzothiazole on intoxicated neuronal
cells.
2.3. GABA assay and neuroprotective studies
PTZ produces seizures by inhibiting GABA neurotransmission
[17]. The promising anti-PTZ effects of compounds 2a, 2e and 2f
prompted us to estimate whole brain GABA levels, to gain insight
into the mechanism of anticonvulsant behavior of benzothiazole
derivatives. Results of GABA estimation (Fig. 4) showed that oral
treatment of compound 2f for 7 days increased the GABA level
significantly (by 1.8-fold) compared to the control animals
Table 1
Preliminary anticonvulsant, minimal motor impairment and CNS depression studies of compounds (2aeh and 4aeh).
b
Compound
C log P
i.p. injection in micea
Porsolt’s swimming pool test
Mean average immobility
Control (24 h before)
MES
scPTZ
NT
0.5 h
4 h
0.5 h
4 h
0.5 h
4 h
Post-treatment (60 min after)
2a
2b
2c
2d
2e
2f
2g
2h
4a
4b
4c
4d
4e
4f
4.12
3.57
4.29
3.25
3.98
3.44
2.97
4.28
4.60
4.06
4.78
3.74
4.47
3.92
3.45
4.76
e
300
e
300
300
300
e
300
e
300
e
e
e
120 ꢁ 6.38
127.5 ꢁ 5.22
117 ꢁ 5.13
109 ꢁ 5.33
113.3 ꢁ 5.42
118.5 ꢁ 5.18
115 ꢁ 4.81
125.6 ꢁ 6.20
112 ꢁ 4.87
124 ꢁ 5.03
109.1 ꢁ 5.04
121 ꢁ 5.26
131.5 ꢁ 5.96
104.1 ꢁ 4.66
110 ꢁ 5.16
111.8 ꢁ 5.03
X
134.5 ꢁ 5.30NS
160 ꢁ 5.91
162 ꢁ 5.77
166.1 ꢁ 6.27
130 ꢁ 5.46NS
135.6 ꢁ 5.73NS
168.3 ꢁ 6.33
146.6 ꢁ 5.62
157 ꢁ 6.50
147.3 ꢁ 5.57
179.1 ꢁ 6.02
154.3 ꢁ 5.78
180 ꢁ 6.53
177 ꢁ 5.62
172.3 ꢁ 5.78
158.1 ꢁ 6.10
X
e
e
e
e
e
e
300
300
e
300
100
30
e
e
e
e
100
30
100
300
100
100
e
300
100
e
300
100
300
e
e
e
e
e
e
300
e
e
e
e
e
300
300
e
e
e
e
e
300
300
300
e
300
300
300
e
300
300
300
e
e
e
e
e
e
300
300
e
e
100
300
300
30
300
300
e
e
e
4g
4h
PHTc
CBZc
e
e
e
300
30
30
e
300
100
100
300
100
300
e
e
e
100
100
300
112.1 ꢁ 5.85
179.5 ꢁ 5.53
a
Doses of 30, 100 and 300 mg/kg were administered (n ¼ 6). The data indicates the minimum dose whereby bioactivity/neurotoxicity was demonstrated in half or more of
the mice while dash indicates the absence of anticonvulsant activity/neurotoxicity at the maximum dose administered (300 mg/kg).
b
Each value represents the mean average immobility time in seconds ꢁ SEM of six mice, significantly different from the control at p < 0.05, and NS denotes not significant at
p > 0.05 (Student’s t test). X indicates that the compound was not screened.
c
PHT, phenytoin; CBZ, carbamazepine.

Mohd.Z. Hassan et al. / European Journal of Medicinal Chemistry 58 (2012) 206e213
209
Table 2
Effect of selected benzothiazole derivatives on serum enzymatic activity in mice.
Compounda
SGOT
SGPT
Alkaline phosphatase
Total albumin
Total protein
2a
2e
38.20 ꢁ 1.62
35.86 ꢁ 1.40
34.63 ꢁ 1.70
34.08 ꢁ 1.51
28.65 ꢁ 1.60
30.51 ꢁ 1.21
29.36 ꢁ 1.75
27.83 ꢁ 1.37
58.73 ꢁ 1.60
60.41 ꢁ 2.41
57.80 ꢁ 2.09
56.11 ꢁ 2.00
1.63 ꢁ 0.022
1.61 ꢁ 0.024
1.58 ꢁ 0.017
1.56 ꢁ 0.021
5.20 ꢁ 0.26
6.12 ꢁ 0.17*
5.28 ꢁ 0.24
5.21 ꢁ 0.21
2f
Controlb
a
Relative to control and data were analyzed by ANOVA followed by Student’s t test for n ¼ 6 at *p < 0.05.
b
Control group were treated with 0.5% methyl cellulose for 15 days.
2.4. Structureeactivity relationship
inhibition. It binds in a mode very similar to that of GABA, with
the -proton pointing toward the Lys 329 so that it can be
g
Benzothiazole scaffold was substituted with different func-
tional groups having varied sizes and electronic attributes to
assess their stereo-electronic effect on anticonvulsant activity.
These substituents were also considered on the basis of lipophilic
successfully abstracted. Furthermore, it forms three hydrogen
bonds (one with PLP cofactor and two with Arg 192 protein
residue) in the active site. In contrast, compound 2f lacks zwitter
ion but hydrogen bonds to the enzyme through its carbonyl
oxygen and benzothiazolyl nitrogen atom with the guanidine
group of Arg 192 residue i.e. at the same residue where the
carboxylate moiety of the natural substrate GABA binds [25]. The
amide group of 2f seems to have an important role in strong
hydrogen bonding because the lone pair electrons on nitrogen of
the amide are delocalized into the carbonyl group. Lipophilicity
also appears to play crucial role in 2f inhibitory activity, as ben-
zothiazole group is properly oriented to the more lipophilic area
(
p
) and electronic (
lipophilic electron-withdrawing groups (þ
hydrophilic electron-donating groups (ꢀ /ꢀ
electron-withdrawing groups (þ /ꢀ COCH3; and lipophilic
electron-releasing groups (ꢀ /þ CH3 [22]. The general
s
) attributes as defined by Craig’s plot, like
/þ ) Cl, F, Br and NO2;
) OCH3; hydrophilic
s
p
s
p
s
p)
s
p)
structureeactivity relationship of these compounds showed that
the derivatives having electron-releasing groups were more
active than derivatives having electron-withdrawing groups.
Among the electron-donating groups, hydrophilic methoxy
derivatives were more active than the lipophilic methyl deriva-
tives. On the other hand among the different isoelectronic halo-
gens, the lipophilic chloro substituent at the 6th position of
benzothiazole ring was more potent and less neurotoxic than the
bromo, fluoro, and nitro derivatives. Also, it is interesting to note
that the introduction of hydrophilic electron-withdrawing group
such as acetyl group at the benzothiazole ring caused a significant
improvement in anticonvulsant activity with abolition of
neurotoxicity.
of GABA-AT binding site and formed CHep interaction with Phe
189. All these are corroborated well by the results of the docking
calculations. The glide score value of vigabatrin was found to be
3.16, while that of 2f was 4.17, suggesting that molecule 2f
interacts better. Therefore, the increased concentration of GABA
in brain tissue may be attributed to the inhibition of enzyme
GABA-AT.
2.6. Pharmacophore distance mapping
A pharmacophore distance mapping provides a useful tool for
designing anticonvulsant molecules and explanation of plausible
interactions. Therefore, the present work involves the correlation of
pharmacophoric pattern of different class of anticonvulsants with
the prototype compound 2f. Comparison of pharmacophore
distance showed that the compound 2f was in good conformity
with the clinically available AEDs (Table 4).
2.5. Molecular docking studies
To gain further evidence regarding the GABAergic effect of
compound 2f, a molecular docking study was carried out on
g-
aminobutyric acid aminotransferase (GABA-AT) enzyme using
Maestro 9.0 program (Schrodinger Inc. USA). GABA-AT is a vali-
dated target for AEDs and being a catabolic in nature, its selective
inhibition raises GABA concentrations in brain [23]. GABA-AT
used in the current study was prepared from the raw PDB
structure 1OHV, which has 96% similarity with the human brain
enzyme and all active site residues in the vicinity of cofactor have
exact counterparts [24]. The best binding modes of vigabatrin, an
irreversible GABA-AT inhibitor and compound 2f in the active site
of GABA-AT are presented in Figs. 6 and 7. The selectivity of
vigabatrin has been shown to result from the carboxylate and
amino groups, which are essential for the mechanism-based
3. Conclusion
A series of N-(substitutedbenzothiazol-2-yl)amide derivatives
were synthesized and tested for anticonvulsant activity using MES
and scPTZ screens. N-(6-methoxybenzothiazol-2-yl)-4-oxo-4-
phenylbutanamide (2f) represents a valuable lead in the explora-
tion of agents controlling both development of seizure and intox-
ication during epilepsy. In various toxicity studies, no overt effects
were noted that would be considered to confound any findings of
Table 3
Phase II quantitative anticonvulsant evaluation in mice.
a
b
Compound
ED50
MES
TD50
PIc
scPTZ
MES
scPTZ
2f
40.96 (36.69e45.23)d
9.5 (8.1e10.4)
8.8 (5.5e14.1)
85.16 (83.32e87)
>300
>100
347.60 (343.74e351.46)
65.5 (52.5e72.9)
71.6 (45.9e135)
8.4
6.9
8.1
1.6
4.0
<0.22
<0.22
2.9
Phenytoin
Carbamazepine
Sod. valproate
272 (247e338)
149 (123e177)
426 (369e450)
a
Dose in milligrams per kilogram body mass (n ¼ 10).
b
c
Minimal toxicity was determined by rotarod test 30 min after the test drug was administered.
PI ¼ protective index (TD50/ED50).
d
Data in parentheses are the 95% confidence limits. Probit analyses were done using Statplus 2007 professional 4.0.3 program by Finney method.

210
Mohd.Z. Hassan et al. / European Journal of Medicinal Chemistry 58 (2012) 206e213
Fig. 2. Control: Section of liver showing normal Portal Triad structures. PV ¼ portal
vein, HA ¼ hepatic artery (400ꢂ).
Fig. 4. Effects of some selected synthesized derivatives on GABA levels in mice brain
tissues (
significantly different from the control at *p < 0.05; **p < 0.01 (Student’s t test).
m
g/100 mg wet tissue). Each value represents the mean ꢁ SEM of six rats,
its efficacy. Compound 2f administration significantly increased the
GABA level and also reduced the progression of ACR induced
neurotoxicity. Their decreased MDA and LDH enzyme activity
suggested that neuroprotective mechanisms may also be involved
in observed anticonvulsant activity of the benzothiazole deriva-
tives. However, further molecular and clinical studies are still
required to ascertain its effectiveness and mechanism of action
during epilepsy.
measured on Bruker-300 instrument (75 MHz) with complete
proton decoupling. Mass spectra were recorded at Waters Synapt
Mass spectrometer. The homogeneity of the compounds was
checked by thin layer chromatography (TLC) on silica gel 60 F254
-
coated plates (Merck, Germany) by using ethyl acetate/hexane (1:2)
as solvent system. Iodine chamber and UV lamp were used for the
visualization of TLC spots.
4.1.1. General procedure for the preparation of N-(substituted-
benzothiazol-2-yl)amide derivatives (2aeh and 4aeh)
4. Experimental
HOBt (0.01 mol) dissolved in 25 mL of dry DCM was added
dropwise to a stirred mixture of EDCl (0.01 mol) and 4-oxo-4-
phenylbutanoic acid (1) (for the synthesis of compound 2aeh) or
2-benzoyl benzoic acid (3) (for the synthesis of compound 4aeh)
(0.01 mol) in 100 mL of dry DCM. The resulting solution was stir-
red for 15 min and then a mixture of substituted 1,3-benzothiazol-
2-amine (0.01 mole) and triethylamine (0.01 mole) in dry DCM
(25 mL) was added to this. The reaction mixture was further stirred
for 5 h at room temperature. Water (15 mL) was added, and the
organic layer was separated. The aqueous phase washed three
times with 20 mL of DCM. The organic extracts were combined and
4.1. Chemistry
The melting points were determined in open capillary tubes in
a Hicon melting point apparatus (Hicon, India) and are uncorrected.
The elemental analyses (C, H, N) of all compounds were performed
on the CHNS Elimentar (Analysen systime, GmbH) Germany Vario
EL III and results were within ꢁ0.4% of the theoretical values.
Fourier transform infrared (FT-IR) spectra were recorded in KBr
pellets on a Shimadzu FTIR spectrometer. 1H NMR spectra were
recorded on a Brucker Model-300 NMR Spectrometer in CDCl3
and DMSO-d6 using Tetramethysilane (TMS) as the internal refer-
ence (chemical shifts in
d
ppm). 13C NMR spectra were also
Fig. 5. Levels of LPO and LDH enzyme activity in ACR-intoxicated and treated mice
brain. Each value represents the mean ꢁ SEM of six rats, significantly different from the
control at **p < 0.01; NS, p > 0.05 (Student’s t test).
Fig. 3. Compound 2f: Section of liver showing normal Portal Triad structures.
PV ¼ portal vein, BD ¼ bile duct (400ꢂ).

Mohd.Z. Hassan et al. / European Journal of Medicinal Chemistry 58 (2012) 206e213
211
4.1.3. N-(6-Fluorobenzothiazol-2-yl)-4-oxo-4-phenylbutanamide
(2b)
Yield 64%; m.p. 112e114 ꢃC; IR (KBr) ymax in cmꢀ1: 3336 (NeH
str.), 1676 (C¼O), 1612 (C¼N). 1H NMR (CDCl3, 300 MHz)
d ppm:
2.94 (t, 2H, J ¼ 6.4 Hz, CH2), 3.48 (t, 2H, J ¼ 6.4 Hz, CH2), 7.10e7.14
(m, 2H, 30 and 50 AreH), 7.22e7.25 (d, 1H, J ¼ 8.4 Hz, 5th AreH
benzoth.), 7.54e7.59 (t, 1H, J ¼ 7.2, 6.2 Hz, 40 AreH), 7.58e7.61 (d,
1H, J ¼ 8.4 Hz, 4th AreH benzoth.), 7.86e7.88 (d, 2H, J ¼ 7.4 Hz, 20
and 60 AreH), 8.12 (s, 1H, 7th AreH benzoth.), 9.82 (s, 1H, CONH,
D2O exchangeable); MS m/z (%): Mþ þ 1, 329 (16), Mþ, 328 (100),
312 (8), 168 (15), 152 (12), 142 (6), 108 (10), 105 (8). Anal. Calcd. for
C17H13FN2O2S: C, 62.18; H, 3.99; N, 8.53%. Found: C, 62.22; H, 4.02;
N, 8.51%.
4.1.4. N-(6-Bromobenzothiazol-2-yl)-4-oxo-4-phenylbutanamide
(2c)
Yield 65%; m.p. 124e126 ꢃC; IR (KBr) ymax in cmꢀ1: 3316 (NeH
str.), 1644 (C¼O), 1608 (C¼N). 1H NMR (CDCl3, 300 MHz)
d ppm:
2.83 (t, 2H, J ¼ 6.3 Hz, CH2), 3.48 (t, 2H, J ¼ 6.3 Hz, CH2), 7.26e7.52
(m, 6H, 4, 5, 7th, 30, 40 and 50 AreH and benzoth.), 8.00e8.03 (d,
2H, J ¼ 7.5 Hz, 20 and 60 AreH), 8.82 (s, 1H, CONH, D2O exchange-
able); MS m/z (%): Mþ þ 2, 391 (33), Mþ, 389 (100), 373 (12), 229
(20), 213 (8), 203 (10), 169 (7). Anal. Calcd. for C17H13BrN2O2S: C,
52.45; H, 3.37; N, 7.20%. Found: C, 52.47; H, 3.40; N, 7.18%.
Fig. 6. Binding mode of vigabatrin into GABA-AT pocket (PDB code: 1OHV) showing
ꢀ
ꢀ
three hydrogen bonds (yellow dotted lines) with PLP (1.46 A) and Arg 192 (1.79 A and
2.00 A).
ꢀ
washed three times with brine, dried over MgSO4, filtered, and
evaporated under reduced pressure. The obtained products were
purified by crystallization using ethyl acetate/hexane mixture (1:3)
to give (62ꢀ73% yield) of white crystals.
4.1.5. N-(6-Nitrobenzothiazol-2-yl)-4-oxo-4-phenylbutanamide
(2d)
Yield 70%; m.p. 166e168 ꢃC; IR (KBr) ymax in cmꢀ1: 3320 (NeH
str.), 1632 (C¼O), 1602 (C¼N). 1H NMR (DMSO-d6, 300 MHz)
4.1.2. N-(6-Chlorobenzothiazol-2-yl)-4-oxo-4-phenylbutanamide
(2a)
d
ppm: 2.92 (t, 2H, J ¼ 6.3 Hz, CH2), 3.46 (t, 2H, J ¼ 6.3 Hz, CH2),
7.12e7.16 (d, 1H, J ¼ 8.6 Hz, 5th AreH benzoth.), 7.20e7.48 (m, 3H,
30, 40 and 50 AreH), 7.60e7.64 (d, 1H, J ¼ 8.1 Hz, 4th AreH benzoth.),
7.82e7.85 (d, 2H, J ¼ 7.6 Hz, 20 and 60 AreH), 7.98 (s, 1H, 7th AreH
benzoth.), 9.84 (s, 1H, CONH, D2O exchangeable); MS m/z (%): Mþ,
355 (100), Mþ þ 1, 356 (18), 339 (10) 310 (12), 150 (17), 134 (8), 124
(14), 90 (6), 105 (5). Anal. Calcd. for C17H13N3O4S: C, 57.46; H, 3.69;
N, 11.82%. Found: C, 57.50; H, 3.71; N, 11.86%.
Yield 68%; m.p. 166e168 ꢃC; IR (KBr) ymax in cmꢀ1: 3340 (NeH
str.), 1672 (C¼O), 1604 (C¼N). 1H NMR (CDCl3, 300 MHz)
d ppm:
2.98 (t, 2H, J ¼ 6.3 Hz, CH2), 3.45 (t, 2H, J ¼ 6.3 Hz, CH2), 7.02e7.06
(m, 2H, 30 and 50 AreH), 7.20e7.23 (d, 1H, J ¼ 8.4 Hz, 5th AreH
benzoth.), 7.57e7.62 (t, 1H, J ¼ 7.2, 6.3 Hz, 40 AreH), 7.65e7.68 (d,
1H, J ¼ 8.7 Hz, 4th AreH benzoth.), 7.75 (s, 1H, 7th AreH benzoth.),
7.92e8.01 (d, 2H, J ¼ 7.2 Hz, 20 and 60 AreH), 9.66 (s, 1H, CONH, D2O
exchangeable); MS m/z (%): Mþ þ 2, 346 (32), Mþ, 344 (100), 328
(10), 183 (22), 168 (10), 158 (8), 124 (16), 105 (7). Anal. Calcd. for
4.1.6. N-(6-Methylbenzothiazol-2-yl)-4-oxo-4-phenylbutanamide
(2e)
C
17H13ClN2O2S: C, 59.21; H, 3.80; N, 8.12%. Found: C, 59.24; H, 3.82;
Yield 63%; m.p. 178e180 ꢃC; IR (KBr) ymax in cmꢀ1: 3286 (NeH
N, 8.16%.
str.), 1680 (C¼O), 1607 (C¼N). 1H NMR (CDCl3, 300 MHz)
d ppm:
2.28 (s, 3H, CH3), 2.80 (t, 2H, J ¼ 6.2 Hz, CH2), 3.44 (t, 2H, J ¼ 6.2 Hz,
Table 4
Calculated distance ranges between the pharmacophoric elements.
O
S
.
N
.
.
.
D
A
O
HBD
.
.
N
H
Compound
Gabapentin
Carbamazepine
Phenytoin
Remacemide
Progabide
2f
A-HBDa
A-Da
D-HBDa
4.02
5.11
4.97
5.06
6.65
4.30
4.14
5.22
4.61
6.74
4.49
4.20
4.45
4.63
3.44
4.37
5.12
2.95
a
Fig. 7. Binding mode of compound (2f) into GABA-AT pocket (PDB code: 1OHV)
showing two hydrogen bonds (yellow dotted lines) with Arg 192 (1.88 A and 2.21 A).
Distance calculated for 3D optimized structures using ACD/Chemsketch/3-D
viewer 12.01 version program.
ꢀ
ꢀ

212
Mohd.Z. Hassan et al. / European Journal of Medicinal Chemistry 58 (2012) 206e213
CH2), 7.07e7.10 (d, 1H, J ¼ 7.8 Hz, 5th AreH benzoth.), 7.38e7.40 (m,
2H, 4 and 7th AreH benzoth.), 7.43e7.48 (t, 2H, J ¼ 7.2, 7.8 Hz, 30, 50
AreH), 7.54e7.59 (t, 1H, J ¼ 7.5, 7.2 Hz, 40 AreH), 7.97e8.0 (d, 2H,
J ¼ 7.5 Hz, 20, 60 AreH), 8.77 (s, 1H, CONH, D2O exchangeable); MS
m/z (%): Mþ þ 1, 325 (19), Mþ, 324 (100), Mþ ꢀ 1, 323 (10), 308 (12),
163 (16), 147 (7), 104 (12), 105 (6). Anal. Calcd. for C18H16N2O2S: C,
66.64; H, 4.97; N, 8.64%. Found: C, 66.68; H, 5.02; N, 8.60%.
d
ppm:7.22e7.26 (d, 1H, J ¼ 8.0 Hz, 5th AreH benzoth.), 7.42e7.78
(m, 10H, 4th benzoth. and AreH), 7.94 (s, 1H, 7th AreH benzoth.),
9.42 (s,1H, CONH, D2O exchangeable); MS m/z (%): Mþ þ 1, 377 (24),
Mþ, 376 (100), 299 (17), 168 (18), 152 (10), 108 (8), 142 (7), 105 (7).
Anal. Calcd. for C21H13FN2O2S: C, 67.01; H, 3.48; N, 7.44%. Found: C,
67.04; H, 3.50; N, 7.47%.
4.1.12. 2-Benzoyl-N-(6-bromobenzothiazol-2-yl)benzamide (4c)
Yield 65%; m.p. 202e204 ꢃC; IR (KBr) ymax in cmꢀ1: 3316 (NeH
4.1.7. N-(6-Methoxybenzothiazol-2-yl)-4-oxo-4-phenylbutanamide
(2f)
str.), 1640 (C¼O), 1608 (C¼N). 1H NMR (CDCl3, 300 MHz)
d ppm:
Yield 72%; m.p. 190e192 ꢃC; IR (KBr) ymax in cmꢀ1: 3265 (NeH),
7.12e7.16 (d, 1H, J ¼ 8.1 Hz, 5th AreH benzoth.), 7.36e7.80 (m, 10H,
4th benzoth. and AreH), 7.98 (s, 1H, 7th AreH benzoth.), 9.58 (s, 1H,
CONH, D2O exchangeable); MS m/z (%): Mþ þ 2, 439 (30), Mþ, 437
(100), 360 (18), 229 (15), 213 (7), 203 (10), 169 (10), 105 (8). Anal.
Calcd. for C21H13BrN2O2S: C, 57.68; H, 3.00; N, 6.41%. Found: C,
57.65; H, 3.06; N, 6.43%.
1669 (C¼O), 1607 (C¼N); 1H NMR (CDCl3, 300 MHz)
d ppm: 2.96 (t,
2H, J ¼ 6.3 Hz, CH2), 3.51(t, 2H, J ¼ 6.3 Hz, CH2), 3.85 (s, 3H, OCH3),
7.02e7.06 (dd, 1H, J ¼ 6.6, 2.4 Hz, 5th AreH benzoth.), 7.27e7.28 (d,
1H, J ¼ 2.7 Hz, 7th AreH benzoth.), 7.47e7.52 (t, 2H, J ¼ 7.5, 7.8 Hz,
30, 50 AreH), 7.58e7.63 (t, 1H, J ¼ 7.2, 7.5 Hz, 40 AreH), 7.71e7.74 (d,
1H, J ¼ 9.0 Hz, 4th AreH benzoth.), 8.01e8.03 (d, 2H, J ¼ 7.5 Hz, 20, 60
AreH), 10.39 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3,
4.1.13. 2-Benzoyl-N-(6-nitrobenzothiazol-2-yl)benzamide (4d)
Yield 65%; m.p. 176e178 ꢃC; IR (KBr) ymax in cmꢀ1: 3320 (NeH
75 MHz)
d ppm: 28.42 (CH2), 33.41 (CH2), 55.86 (OCH3), 105.53,
113.87, 118.14, 128.08, 128.63, 130.00, 133.27, 136.51, 143.04, 155.74,
str.), 1656 (C¼O), 1602 (C¼N); 1H NMR (CDCl3, 300 MHz)
d ppm:
167.03, 178.32 (CONH), 198.21 (COPh); MS m/z (%): Mþ þ 2, 342 (5),
7.28e7.32 (d, 1H, J ¼ 8.2 Hz, 5th AreH benzoth.), 7.68e7.92 (m, 10H,
4th benzoth. and AreH), 8.10 (s, 1H, 7th AreH benzoth.), 10.02 (s,
1H, CONH, D2O exchangeable); MS m/z (%): Mþ þ 1, 404 (23), Mþ,
403 (100), 358 (12), 281 (17), 150 (20), 134 (16), 124 (10), 90 (8).
Anal. Calcd. for C21H13N3O4S: C, 62.52; H, 3.25; N, 10.42%. Found: C,
62.56; H, 3.26; N, 10.46%.
M
þ þ 1, 341 (18), Mþ, 340 (48), Mþ ꢀ 1, 339 (100), 324 (8), 268 (6),
180 (6). Anal. Calcd. for C18H16N2O3S: C, 63.51; H, 4.74; N, 8.23%.
Found: C, 63.53; H, 4.72; N, 8.28%.
4.1.8. N-(6-Acetylbenzothiazol-2-yl)-4-oxo-4-phenylbutanamide
(2g)
Yield 73%; m.p. 212e214 ꢃC; IR (KBr) ymax in cmꢀ1: 3314 (NeH
str.), 1660 (C¼O), 1600 (C¼N); 1H NMR (DMSO-d6, 300 MHz)
4.1.14. 2-Benzoyl-N-(6-methylbenzothiazol-2-yl)benzamide (4e)
Yield 63%; m.p. 216e218 ꢃC; IR (KBr) ymax in cmꢀ1: 3316 (NeH
d
ppm: 2.50 (s, 3H, COCH3), 2.88 (t, 2H, J ¼ 6.2 Hz, CH2), 3.50(t, 2H,
str.), 1640 (C¼O), 1608 (C¼N). 1H NMR (DMSO-d6, 300 MHz)
d ppm:
J ¼ 6.2 Hz, CH2), 7.12e7.15 (d, 1H, J ¼ 8.4 Hz, 5th AreH benzoth.),
7.42e7.58 (m, 3H, 30, 40 and 50 AreH), 7.72e7.75 (d, 1H, J ¼ 8.4 Hz,
4th AreH benzoth.), 7.96e8.0 (d, 2H, J ¼ 7.6 Hz, 20, 60 AreH), 8.02
(s, 1H, 7th AreH benzoth.), 9.64 (s, 1H, CONH, D2O exchangeable);
MS m/z (%): Mþ þ 1, 353 (20), Mþ, 352 (100), 337 (18), 336 (12),
310 (7), 150 (10), 134 (8), 90 (8), 105 (6). Anal. Calcd. for
2.12 (s, 3H, CH3), 6.65e7.89 (m, 12H, AreH and benzoth.), 9.15 (s,
1H, CONH, D2O exchangeable); MS m/z (%): Mþ þ 1, 373 (25), Mþ,
372 (100), Mþ ꢀ 1, 371 (20), 294 (22), 163 (12), 147 (10), 105 (5), 104
(7). Anal. Calcd. for C22H16N2O2S: C, 70.95; H, 4.33; N, 7.52%. Found:
C, 70.98; H, 4.36; N, 7.56%.
C
19H16N2O3S: C, 64.76; H, 4.58; N, 7.95%. Found: C, 64.77; H, 4.59;
4.1.15. 2-Benzoyl-N-(6-methoxybenzothiazol-2-yl)benzamide (4f)
Yield 65%; m.p. 172e174 ꢃC; IR (KBr) ymax in cmꢀ1: 3306 (NeH),
N, 7.92%.
1662 (C¼O), 1600 (C¼N). 1H NMR (CDCl3, 300 MHz)
d ppm: 3.83 (s,
4.1.9. N-(5-Chloro-6-fluorobenzothiazol-2-yl)-4-oxo-4-
phenylbutanamide (2h)
3H, OCH3), 7.47e8.14 (m, 12H, AreH and benzoth.), 10.76 (s, 1H,
CONH, D2O exchangeable); 13C NMR (CDCl3, 100 MHz)
d ppm: 55.86
Yield 67%; m.p. 192e194 ꢃC; IR (KBr) ymax in cmꢀ1: 3312 (NeH
(OCH3), 105.51, 114.03, 117.66, 127.32, 128.37, 129.38, 129.44, 130.35,
130.81, 132.12, 132.82, 137.49, 142.17, 155.89, 167.11 (CONH), 170.40
(C¼N), 196.78 (COPh); MS m/z (%): Mþ þ 1, 389 (25), Mþ, 388 (100),
str.), 1646 (C¼O), 1606 (C¼N). 1H NMR (CDCl3, 300 MHz)
d ppm:
2.90 (t, 2H, J ¼ 6.1 Hz, CH2), 3.44 (t, 2H, J ¼ 6.1 Hz, CH2), 7.12e7.15
(m, 2H, 30 and 50 AreH), 7.46e7.50 (t, 1H, J ¼ 7.2, 7.2 Hz, 40 AreH),
7.54 (s, 1H, 4th AreH benzo.), 7.76e7.78 (d, 2H, J ¼ 7.5 Hz, 20 and
60 AreH), 8.02 (s, 1H, 7th AreH benzoth.), 9.64 (s, 1H, CONH, D2O
exchangeable); MS m/z (%): Mþ þ 2, 364 (37), Mþ þ 1, 363 (22), Mþ,
362 (100), 346 (10), 202 (14), 186 (8), 142 (6), 175 (10), 105 (5). Anal.
Calcd. for C17H12ClFN2O2S: C, 56.28; H, 3.33; N, 7.72%. Found: C,
56.30; H, 3.30; N, 7.76%.
M
þ ꢀ 1, 387 (8), 373 (12), 311 (15), 180 (10). Anal. Calcd. for
C22H16N2O3S: C, 68.02; H, 4.15; N, 7.21%. Found: C, 68.07; H, 4.18; N,
7.26%.
4.1.16. N-(6-Acetylbenzothiazol-2-yl)-2-benzoylbenzamide (4g)
Yield 68%; m.p. 226e228 ꢃC; IR (KBr) ymax in cmꢀ1: 3312 (NeH
str.), 1660 (C¼O), 1605 (C¼N); 1H NMR (DMSO-d6, 300 MHz)
d
ppm: 3.0 (s, 3H, COCH3), 7.02e8.17 (m, 12H, AreH and benzoth.),
4.1.10. 2-Benzoyl-N-(6-chlorobenzothiazol-2-yl)benzamide (4a)
Yield 62%; m.p. 220e212 ꢃC; IR (KBr) ymax in cmꢀ1: 3345 (NeH
9.83 (s,1H, CONH, D2O exchangeable); MS m/z (%): Mþ þ 1, 401 (26),
Mþ, 400 (100), 386 (22), 358 (10), 281 (18), 150 (17), 134 (7), 124 (8),
105 (6). Anal. Calcd. for C23H16N2O3S: C, 68.98; H, 4.03; N, 7.00%.
Found: C, 69.00; H, 4.08; N, 7.02%.
str.), 1674 (C¼O), 1603 (C¼N). 1H NMR (CDCl3, 300 MHz)
d ppm:
7.41e7.43 (d, 1H, J ¼ 7.2 Hz, 5th AreH benzoth.), 7.47e7.76 (m, 10H,
7th benzoth. and AreH), 7.98e8.01 (d, 1H, J ¼ 7.8 Hz, 4th AreH
benzoth.), 9.10 (s, 1H, CONH, D2O exchangeable); MS m/z (%):
4.1.17. 2-Benzoyl-N-(5-chloro-6-fluorobenzothiazol-2-yl)
benzamide (4h)
M
þ þ 2, 394 (33), Mþ þ 1, 393 (24), Mþ, 392 (100), 315 (18),183 (14),
168 (8), 158 (7), 124 (8), 105 (4). Anal. Calcd. for C21H13ClN2O2S: C,
64.20; H, 3.34; N, 7.13 %. Found: C, 64.22; H, 3.38; N, 7.10%.
Yield 62%; m.p. 162e164 ꢃC; IR (KBr) ymax in cmꢀ1: 3316 (NeH),
1670 (C¼O), 1612 (C¼N); 1H NMR (CDCl3, 300 MHz)
d ppm: 7.42
(s, 1H, 4th benzoth.), 7.54e7.80 (m, 9H, AreH and benzoth. ), 7.98 (s,
1H, 7th AreH benzoth.), 9.64 (s, 1H, CONH, D2O exchangeable); MS
m/z (%): Mþ þ 2, 412 (39), Mþ þ 1, 411 (24), Mþ, 410 (100), 333 (16),
202 (20), 186 (8), 174 (8), 142 (12), 105 (8). Anal. Calcd. for
4.1.11. 2-Benzoyl-N-(6-fluorobenzothiazol-2-yl)benzamide (4b)
Yield 70%; m.p. 178e180 ꢃC; IR (KBr) ymax in cmꢀ1: 3329 (NeH
str.), 1671 (C¼O), 1603 (C¼N). 1H NMR (CDCl3, 300 MHz)

Mohd.Z. Hassan et al. / European Journal of Medicinal Chemistry 58 (2012) 206e213
213
[4] M. Bialer, S.I. Johannessen, H.J. Kupferberg, R.H. Levy, E. Perucca, T. Tomson,
Epilepsy Res. 61 (2004) 1e48.
[5] J.R. Naegele, Epilepsia 48 (2007) 107e117.
C21H12ClFN2O2S: C, 61.39; H, 2.94; N, 6.82%. Found: C, 61.42; H,
2.98; N, 6.80%.
[6] K. Sudha, A.V. Rao, A. Rao, Clin. Chim. Acta 303 (2001) 19e24.
[7] J.C. Stanis1aw, F. Renata, B. Barbara, K.B. Kinga, J. Pre-Clin. Clin. Res. 1 (2007)
1e5.
4.2. Pharmacology
[8] B.C. Cheah, S. Vucic, A.V. Krishnan, M.C. Kiernan, Curr. Med. Chem. 17 (2010)
1942e1959.
[9] P. Jimonet, M. Barreau, J.C. Blanchard, A. Boireau, A. Doble, P. Laduron,
J. Lavayre, C. Malgouris, O. Piot, J. Pratt, J. Rataud, M. Reibaud, S. Mignani,
J.M. Stutzmann, Bioorg. Med. Chem. 2 (1994) 793e798.
[10] P. Jimonet, F. Audiau, M. Barreau, J.C. Blanchard, A. Boireau, Y. Bour,
M.A. Coleno, A. Doble, G. Doerflinger, C. Do Huu, M.H. Donat, J.M. Duchesne,
P. Ganil, C. Gueremy, E. Honore, B. Just, R. Kerphirique, S. Gontier, P. Hubert,
P.M. Laduron, J. Le Blevec, M. Meunier, J.M. Miquet, C. Nemecek, M. Pasquet,
O. Piot, J. Pratt, J. Rataud, M. Reibaud, J.M. Stutzmann, S. Mignani, J. Med.
Chem. 42 (1999) 2828e2843.
[11] N.D. Amnerkar, K.P. Bhusari, Eur. J. Med. Chem. 45 (2010) 149e159.
[12] N. Siddiqui, A. Rana, S.A. Khan, M.A. Bhat, S.E. Haque, Bioorg. Med. Chem. Lett.
17 (2007) 4178e4182.
[13] M. Amir, S. Asif, I. Ali, M.Z. Hassan, Med. Chem. Res. 21 (2012) 2661e2670.
[14] M. Shaquiquzzaman, S.A. Khan, M. Amir, M.M. Alam, J. Enzyme Inhib. Med.
Chem. (2011). http://dx.doi.org/10.3109/14756366.2011.618129.
[15] M. Amir, M.J. Ahsan, I. Ali, Ind. J. Chem. 49B (2010) 1509e1514.
[16] K. Unverferth, J. Engel, N. Hofgen, A. Rostock, R. Gunther, H.J. Lankau,
M. Menzer, A. Rolfs, J. Liebscher, B. Muller, H.J. Hofmann, J. Med. Chem. 41
(1998) 63.
[17] R. Okada, N. Negishi, H. Nagaya, Brain Res. 480 (1989) 383e387.
[18] H. Igisu, I. Goto, Y. Kawamura, M. Kato, K. Izumi, Y. Kuroiwa, J. Neurol. Neu-
rosurg. Psychiatry 38 (1975) 581e584.
[19] R.M. Mohareb, H.H. Ahmed, G.A. Elmegeed, M.M. Abd-Elhalim, R.W. Shafic,
Bioorg. Med. Chem. 19 (2011) 2966e2974.
[20] R.M. Freitas, S.M.M. Vasconcelos, F.C.F. Souza, G.S.B. Viana, M.M.F. Fonteles,
FEBS J. 272 (2005) 1307e1312.
[21] H. Ehrenreich, A. Siren, Eur. Arch. Psych. Clin. Neurosci. 251 (2001) 149e151.
[22] H.V. Waterbeemed, Quantitative approaches to structureeactivity relation-
ships, in: C.G. Wermuth (Ed.), The Practice of Medicinal Chemistry, Academic
Press, London, 1996, p. 367.
[23] S.M. Nanavati, R.B. Silverman, J. Med. Chem. 32 (1989) 2413e2421.
[24] P. Storici, G. Capitani, D. De Biase, M. Moser, R.A. John, J.N. Jansonius,
T. Schirmer, Biochemistry 38 (1999) 8628e8634.
All the pharmacological experiments were conducted in
compliance with ethical principles after Institutional Animal Ethics
Committee (IAEC) approval. The investigations were conducted on
albino mice of either sex (25e30 g; animals were obtained from
central animal house facility, Hamdard University, New Delhi-62.
Registration no. 173/CPCSEA and date of registration is 28 January
2000). The animals were housed under standard conditions and
allowed free access to standard pellet diet and water (ad libitum).
The preliminary anticonvulsant (phase I) screening of the test
compounds was assessed by following the standard protocols of
Antiepileptic Drug Development (ADD) program by NINDS, US [26]
using two widely used models namely, maximal electroshock
seizure (MES) [27], subcutaneous pentylenetetrazol (scPTZ) test [28]
and minimal neurological toxicity was evaluated by rotarod test
[29]. The most potent compound was also subjected to phase II
quantitative determination of ED50, TD50, PI and probit calculations
were done by means of computer program BioStat 2009 using Fin-
ney’s method [30]. Anticonvulsant activity of the title compounds
were compared using data of standard AEDs reported in the litera-
ture [31,32]. Behavioral depression was measured by evaluating the
immobility time of the mice using Porsolt’s swim pool test [33].
Hepatotoxicity studies of the compounds werealso carried out using
procedures described elsewhere [34e37]. Most active anticonvul-
sant compounds in phase I screening were also subjected to
neurochemical estimation of GABAs levels in the mouse whole brain
[38]. Neuroprotective effects of some selected compounds were also
evaluated against the ACR induced neurotoxicity [39e41].
[25] P. Storici, D. De Biase, F. Bossa, S. Bruno, A. Mozzarelli, C. Peneff,
R.B. Silverman, T. Schirmer, J. Biol. Chem. 279 (2004) 363.
[26] Anticonvulsant Screening Project, Antiepileptic Drug Development Program,
National Institute of Health, DHEW Publ (NIH), USA, 1978, pp. 78e1093.
[27] R.L. Krall, J.K. Penry, B.G. White, H.J. Kupferbeng, E.A. Swinyard, Antiepileptic
drug development: II. Anticonvulsant drug screening, Epilepsia 19 (1978)
409e428.
[28] E.A. Swinyard, J.H. Woodhead, H.S. White, M.R. Franklin, General principles:
experimental selection, quantification, and evaluation of anticonvulsants, in:
R.H. Levy, R.H. Mattson, B. Melrum, J.K. Penry, F.E. Dreifuss (Eds.), Antiepileptic
Drugs, third ed., Raven-Press, New York, 1989, pp. 85e102.
[29] N.W. Dunham, T.S. Miya, J. Am, Pharm, Assoc 46 (1957) 208e209.
[30] D.J. Finney, Probit Analysis, third ed., Cambridge University Press, London,
1971.
Acknowledgment
The authors are thankful to the Head of Pharmaceutical Chem-
istry Department, Hamdard University, New Delhi for providing
necessary research facilities. Also we thank Dr. A. K. Tiwary, In
charge animal house, Hamdard University for providing experi-
mental animals, and Dr. A. Mukherjee, MD, Department of
Pathology, All India Institute of Medical Sciences (AIIMS), New
Delhi, for carrying out histopathological studies.
[31] J.R. Dimmock, S.C. Vashishtha, J.P. Stables, Eur. J. Med. Chem. 35 (2000) 241e248.
[32] N. Siddiqui, W. Ahsan, Eur. J. Med. Chem. 45 (2010) 1536e1543.
[33] R.D. Porsolt, G. Anton, N. Blanet, M. Jalbre, Eur. J. Pharmacol. 47 (1978) 379e386.
[34] S. Reitman, S. Frankel, Am. J. Clin. Pathol. 28 (1957) 56e63.
[35] P.R.N. Kind, E.J. King, J. Clin. Pathol. 7 (1954) 322e326.
[36] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 (1951)
265e275.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.10.002
[37] G.L. Luna, Manual of Histological Staining Methods of the Armed Forces
Institute of Pathology, third ed., McGraw-Hill, New York, 1968, p. 567.
[38] V.N. Gokani, M.U. Thakker, J.G. Patel, S.K. Ghosh, S.K. Chatterjee, Ind. J. Physiol.
Pharmacol. 23 (1979) 101e104.
[39] J. Fournier, R. Steinberg, T. Gauthier, P.E. Keane, U. Guzzi, F.X. Coude, I. Bougault,
J.P. Maffrand, P. Soubrie, G. Le Fur, Neuroscience 55 (1993) 629e641.
[40] H. Ohkawa, N. Ohishi, K. Yagi, Anal. Biochem. 95 (1979) 351e358.
[41] J.A. Lott, E. Nemensanszky, Lactate dehydrogenase, in: J.A. Lott, P.L. Wolf
(Eds.), Clinical Enzymology, Case-oriented Approach, Year Book Medical, New
York, 1987, pp. 213e244.
References
[1] The Global Campaign Against Epilepsy, Information Pack for the launch of the
Global Campaign’s Second Phase, 12e13 February 2001, World Health Orga-
nization, Geneva, 2000.
[2] G. Saravanan, V. Alagarsamy, C.R. Prakash, Bioorg. Med. Chem. Lett. 22 (2012)
3072e3078.
[3] S. Shorvon, E. Perucca, J. EngelJr (Eds.), The Treatment of Epilepsy, third ed.,
Wiley-Blackwell, Oxford, 2009, p. 123.