Combating oxidative stress in epilepsy: Design, synthesis, quantum chemical studies and anticonvulsant evaluation of 1-(substituted benzylidene/ethylidene) -4-(naphthalen-1-yl)semicarbazides

Article abstract of DOI:10.1016/j.ejmech.2010.02.063

A series of 1-(substituted benzylidene/ethylidene)-4-(naphthalen-1-yl) semicarbazides (3a-19a; 3b-6b) were synthesized in good yield and evaluated for their anticonvulsant and antioxidant activities. The structures of the synthesized compounds were confirmed on the basis of their spectral data and elemental analysis. All of the compounds were found to be active in MES and scPTZ tests. Quantum chemical studies were carried out on these compounds to understand the structural features essential for activity. Electronic properties were found to be the principal modulator of both anticonvulsant and antioxidant activities. A computational study was carried out for prediction of pharmacokinetic properties and none of the compounds violated Lipinski's rule of 5, making them potentially promising agents for the treatment of epilepsy.

Full text of DOI:10.1016/j.ejmech.2010.02.063

European Journal of Medicinal Chemistry 45 (2010) 2817e2826
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Combating oxidative stress in epilepsy: Design, synthesis, quantum chemical
studies and anticonvulsant evaluation of 1-(substituted benzylidene/
ethylidene)-4-(naphthalen-1-yl)semicarbazides
*
Faizul Azam , Bashir A. El-gnidi, Ismail A. Alkskas
Laboratory of Medicinal Chemistry, Faculty of Pharmacy, University of Seventh October, PO Box 2873, Misurata, Libyan Arab Jamahiriya
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1-(substituted benzylidene/ethylidene)-4-(naphthalen-1-yl)semicarbazides (3ae19a; 3be6b)
were synthesized in good yield and evaluated for their anticonvulsant and antioxidant activities. The
structures of the synthesized compounds were confirmed on the basis of their spectral data and
elemental analysis. All of the compounds were found to be active in MES and scPTZ tests. Quantum
chemical studies were carried out on these compounds to understand the structural features essential for
activity. Electronic properties were found to be the principal modulator of both anticonvulsant and
antioxidant activities. A computational study was carried out for prediction of pharmacokinetic prop-
erties and none of the compounds violated Lipinski’s rule of 5, making them potentially promising agents
for the treatment of epilepsy.
Received 29 September 2009
Received in revised form
24 January 2010
Accepted 27 February 2010
Available online 7 March 2010
Keywords:
Oxidative stress
Epilepsy
Semicarbazides
Neuroprotection
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
neuroprotection such as reduced neurodegeneration after acute
seizures or the status epilepticus, their competence for pre-
Epilepsy is recognized as one of the most common serious
neurological disorders affecting 1e2% of the world’s population [1].
The actual pathogenesis of epilepsy remains uncertain. However,
oxidative injury may play a role in the initiation and progression of
epilepsy, and therapies aimed at reducing oxidative stress may
ameliorate tissue damage and favourably alter the clinical course of
the disease [2,3]. Oxidative stress occurs when the normal balance
between oxidative events and antioxidant defences is disrupted
either by loss of reducing agents/antioxidant enzymes or by
increased production of oxidizing species [4,5]. There is abundant
in vivo evidence of oxidative injury in animal models of epilepsy
and for efficacy of antioxidant therapy in reducing this injury in
animal models of epileptogenesis [6e8]. Therefore, antioxidant
therapies aimed at reducing oxidative stress have received
considerable attention in the treatment of epilepsy [2,7,9].
venting the development of chronic epilepsy after an initial
precipitating injury is either unknown or not promising [7].
Moreover, the AED therapy is associated with significant side
effects [10]. Therefore, AEDs with neuroprotective and anti-
epileptogenic strategies that are effective for fighting the disease
progression are urgently required.
Aryl semicarbazones have documented consistent advances in
the design of novel anticonvulsant agents, due to their excellent
activity in the maximal electroshock seizure (MES) in both mice
and rats and also against clonic seizures induced by pentylenete-
trazole (PTZ) in mice, being more active than some conventional
AEDs, beside their low neurotoxicity [11e14]. Semicarbazones have
been developed due to their structural dissimilarity to existing
AEDs, so it can be assumed that such novel compounds would not
have the side effects seen with many of the currently available
medications [15]. Their activity is mainly attributed to the presence
of aryl binding site with aryl/alkyl hydrophobic group, hydrogen
bonding domain and electron donor group, which are the essential
requirements for the molecules to show potential activity as
proposed by Dimmock et al. [16,17]. Furthermore, Pandeya et al [18]
proposed that introduction of the one more aryl ring in the struc-
ture brings about the enhanced van der Waals bonding at the
receptor site that leads to increased potency. Recently, we have
investigated various aryl-substituted semicarbazones as potential
From the beginning of medical history, continuous efforts
have been made to treat seizures, and in spite of new develop-
ments in the antiepileptic drug (AED) research and availability of
almost 20 AEDs, seizures remain unmanageable in many types of
epileptic manifestations. Although the administration of certain
conventional and new generation AEDs is effective for primary
* Corresponding author. Tel.: þ218 91 3300265/51 5223831; fax: þ218 51 628149.
E-mail addresses: azamfaizul@yahoo.co.in, faizulazam@gmail.com (F. Azam).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.063

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F. Azam et al. / European Journal of Medicinal Chemistry 45 (2010) 2817e2826
anticonvulsant agents where naphthyl moiety served as aryl
binding domain (Fig 1) and most active compounds carried bromo,
fluoro and nitro substituents at 4-position in the phenyl ring [19]. In
addition, naphthalene ring serves as the lipophilic aryl portion in
nafimidone (Fig. 1) and its derivatives which have been recognized
as potential anticonvulsants with a therapeutic profile in experi-
mental animal seizure models similar to that of phenytoin [20e23].
Motivated by these findings, and in continuation of our ongoing
efforts endowed with the discovery of nitrogenated molecules with
potential biological activities [19,24e27], we report the synthesis of
new 1-(substituted benzylidene/ethylidene)-4-(naphthalen-1-yl)
semicarbazides (Scheme 1) that maintain the chief structural
features of the known anticonvulsants and accomplish the
hypothesis of structural requirements for anticonvulsant activity
suggested by Dimmock et al. [16,17]. The ADME parameters and
eigenvalues of lowest unoccupied molecular orbital (LUMO) and
highest occupied molecular orbital (HOMO) of the synthesized
compounds were calculated and correlated with the observed
biological activities because it is well known that the electronic/
steric effects and physicochemical properties of the drug molecules
influence the pharmacological activities [28].
NMR and mass spectra in accord with their anticipated structures.
The physical characterization data of the compounds are given in
Table 1.
3. Results and discussion
The preclinical discovery and development of new chemical
agents for the treatment of epilepsy are based mainly on the use of
predictable animal models. At the present time, there are many in
vivo screens used routinely that include the MES and scPTZ tests
which are recognized as the ‘gold standards’ in the early stages of
testing [1,29]. The profile of anticonvulsant activity of all the
compounds was established in the MES and scPTZ tests, after
intraperitoneal injection into mice at doses of 30, 100, and
300 mg/kg. An observation was carried out at two different time
intervals, namely 0.5 and 4 h. The acute neurological toxicity (NT)
was determined in the minimal motor impairment-rotorod screen.
The results are given in Table 2 along with the literature data on
phenytoin, carbamazepine, sodium valproate and ethosuximide
[30e32].
The initial anticonvulsant evaluation showed that all of the
synthesized derivatives were effective in ip MES and/or scPTZ
screens. Compounds that revealed protection in the MES test,
indicative of the ability of substance to prevent seizure spread, at
a dose of 100 mg/kg include 6a, 11a, 14a and 19a, whereas mole-
cules 3ae5a, 7a, 9a, 12a, 15ae18a and 3be5b provided anti-MES
protection at a dose of 300 mg/kg. Anti-MES activity exhibited by
the compound 8a was comparable to reference drug carbamaze-
pine, providing protection at 0.5 h and 4 h at the dose of 100 mg/kg.
Compounds showing activity only at 0.5 h indicate that they have
rapid onset and short duration of anticonvulsant action [33].
From this series 6a, 8a, 14a, 15a, 18a and 19a were active in the
MES model both at 0.5 and at 4 h, whereas 5a, 7a, 12a, 16a, 17a, 4b
and 5b showed activity only at 4 h. Compounds 5ae10a, 12ae19a
2. Synthesis
In the present study, 1-(substituted benzylidene/ethylidene)-4-
(naphthalen-1-yl)semicarbazides were synthesized as presented
in Scheme 1. 1-(Naphthalen-1-yl)urea (1) was prepared when
1-naphthylamine was treated with sodium cyanate in a mixture of
glacial acetic acid and water. 1 was refluxed with excess of hydra-
zine hydrate in 99% ethanol to afford N-(naphthalen-1-yl)hydrazi-
necarboxamide (2). The semicarbazone derivatives (3ae19a;
3be6b) were prepared by reaction of the appropriate aryl aldehyde
or ketone with 2 in glacial acetic acid and recrystallized with
ethanol. All compounds showed a single spot on TLC and had IR, 1H
R1
H
N
H
N
N
N
N
N
R2
S
O
O
Nafimidone [20-23]
Aryl-substituted semicarbazones [19]
O
HN
HN
N
R1
R2
1-(substituted benzylidene/ethylidene)-4-(naphthalen-1-yl)semicarbazides (3a-19a; 3b-6b)
A
B
D
C
Pharmacophore model for anticonvulsant activity [16-18]. A: hydrophobic domain, B: H-bonding site,
C: distal hydrophobic domain, D: electron donor moiety.
Fig. 1. Design of the title compounds based on some known anticonvulsants and pharmacophore model for anticonvulsant activity.

F. Azam et al. / European Journal of Medicinal Chemistry 45 (2010) 2817e2826
2819
O
NH2
HN
NH2
NaOCN / CH3COOH
1
NH2NH2.H2O
O
NaOH / EtOH
O
R2
N
NH2
HN
N
H
HN
N
H
O
C
R1
R1
R2
CH3COOH
2
3a-19a; 3b-6b
Scheme 1. Synthetic protocol of the title compounds.
and 4be6b were found to be active in the scPTZ test, which identify
substances elevating seizure threshold. Among these molecules, 6a,
18a and 19a showed anti-scPTZ activity at the dose of 100 and
300 mg/kg at time periods 0.5 and 4 h that was equivalent to
carbamazepine used as reference anticonvulsant drug. Comparable
results were obtained for 5a providing protection at a dose of
100 mg/kg at 0.5 h. Molecule 8a was active at the dose of 100 mg/kg
against scPTZ at 0.5 h and 4 h time period whereas 14a and 5b were
effective at 300 mg/kg. The other derivatives 7a, 9a, 10a, 12a, 13a,
15ae17a, 4b and 6b showed activity against scPTZ screen at a dose
of 300 mg/kg at 0.5 h. As shown in Table 2, the majority of
compounds were effective both in MES and in scPTZ tests; therefore
these molecules may be useful in treating not only generalized
toniceclonic epilepsy but also absence seizures.
In the NT screen, 6a did not show neurotoxicity in the maximum
dose administered (300 mg/kg). Compounds 4a, 7a, 8a, 10a,
12ae15a, 17a, 18a and 4be6b were less neurotoxic than phenytoin
and exhibited motor impairment at the dose of 300 mg/kg at 0.5 h
Table 1
Characterization data of the 1-(substituted benzylidene/ethylidene)-4-(naphthalen-1-yl)semicarbazides (3ae19a; 3be6b).
O
HN
HN
N
R2
R1
Compounds
R1
R2
Mol. Formulaa
Mol wt.
% yield
mp (ꢀC)
Rf
3a
4a
5a
6a
7a
8a
9a
10a
11a
12a
13a
14a
15a
16a
17a
18a
19a
3b
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
C
18H15N3O
289.33
319.36
319.36
349.38
349.38
379.41
335.36
305.33
305.33
323.78
323.78
358.22
368.23
307.32
334.33
334.33
332.40
303.36
337.80
372.25
317.38
79
84
76
82
82
77
78
79
70
75
77
81
72
80
81
95
71
75
90
72
78
188e190
208e210
198e200
210e212
234e236
218e220
224e226
234e236
216e218
217e219
206e208
252e254
236e238
214e216
226e228
261e263
188e190
212e214
244e246
230e232
232e234
0.32
0.35
0.24
0.26
0.40
0.34
0.38
0.12
0.17
0.47
0.45
0.57
0.34
0.23
0.39
0.36
0.42
0.58
0.63
0.65
0.43
4-OCH3
3-OCH3
3,4-OCH3
2,5-OCH3
3,4,5-OCH3
3-OCH3, 4-OH
2-OH
4-OH
4-Cl
2-Cl
2,4-Cl
4-Br
C19H17N3O2
C19H17N3O2
C20H19N3O3
C20H19N3O3
C21H21N3O4
C19H17N3O3
C18H15N3O2
C18H15N3O2
C18H14ClN3O
C18H14ClN3O
C18H13Cl2N3O
C18H14BrN3O
4-F
C18H14FN3O
2-NO2
4-NO2
4-N(CH3)2
H
4-Cl
2,4-Cl
4-CH3
C18H14N4O3
C18H14N4O3
C20H20N4O
C19H17N3O
C19H16ClN3O
C19H15Cl2N3O
C20H19N3O
H
CH3
CH3
CH3
CH3
4b
5b
6b
a
Elemental analyses for C, H and N were within ꢁ0.4% of the theoretical values.

2820
F. Azam et al. / European Journal of Medicinal Chemistry 45 (2010) 2817e2826
Table 2
also that the number of the chlorine atoms in the aryl ring influ-
ences the anticonvulsant activity. The molecules with two chlorine
atoms (14a and 5b) gave better results than the molecules having
one chlorine atom (12a,13a and 4b) in MES and scPTZ tests. We also
observed the same effect for the molecules that have a methoxy
group as the substituent, with better activity being observed when
it is trisubstituted (8a), followed by the disubstituted (6a and 7a)
and then monosubstituted (5a and 4a). The decreases in the activity
follow the decrease in the resonance effect of the methoxy group. In
agreement of our results, substitution in the aryl ring by halogens
[34] and methoxy group [35] led to a number of semicarbazones
with low ED50 values in the rat/mice oral MES screen accompanied
by high protective index values. In addition, our results also support
the observation of Agarwal et al. [36] where compound substituted
with two electron repelling groups (m-methoxy-p-hydroxyphenyl-
substituted) showed better response towards PTZ model than
substituted with one electron repelling group (p-methoxy-
substituted phenyl). Using the putative binding site theory
proposed by Dimmock et al. [16,17] and utilized by others [18,37] in
postulating the interaction of anticonvulsant compounds at
a specific binding site, the molecule would appear to interact with
the protein receptor as shown in Fig. 2.
Oxidative stress in the central nervous system has been shown
in several rodent models of experimental epilepsy, such as the
amygdala kindling model [38], the pilocarpine model [39], the PTZ
kindling model [40] and in acute PTZ-induced seizures [41]. Our
findings show that PTZ induces oxidative stress, which can produce
reductions in SOD, GSH-Px activities and GSH content whereas
increases malondialdehyde (MDA) level in brain homogenate. Lipid
peroxidation (LPO) in the brain can be induced by many chemical
compounds and brain injury such as epilepsy [42]. MDA is one of
the important aldehydes resulting from membrane LPO products
and can be regarded as a marker of LPO. The brain is more
vulnerable to injury by LPO products than other tissues [43].
Moreover, LPO is an index of irreversible neuronal damage of cell
membrane phospholipid and has been suggested as a possible
mechanism of epileptic activity [44,45]. Normally, reactive oxygen
species (ROS) are neutralized by an elaborate antioxidant defense
system consisting of enzymes such as SOD and glutathione
peroxidase (GPx) and non-enzymatic antioxidants such as gluta-
thione (GSH). However, when ROS production is excessive, the
intrinsic antioxidant scavenging capacity is overwhelmed resulting
Anticonvulsant and NT screening of 1-(substituted benzylidene/ethylidene)-4-
(naphthalen-1-yl)semicarbazides.
Compound
Intraperitoneal injection in micea (h)
MES screen scPTZ screen
NT screen
0.5
0.5
4
0.5
4
4
3a
4a
5a
6a
7a
8a
9a
10a
11a
12a
13a
14a
15a
16a
17a
18a
300
300
e
e
eb
e
100
300
100
e
100
e
e
eb
e
300
100
300
100
e
100
100
300
100
300
300
e
e
e
100
e
300
e
e
300
300
100
300
100
300
e
e
100
300
e
100
e
eb
300
100
300
300
e
e
100
e
e
e
300
e
300
300
300
300
300
300
100c
100c
e
e
e
e
300
300
300
100
e
100
300
e
100
300
300
300
300
100
e
300
e
eb
e
300
e
e
e
300
e
300
100
300
e
300
300
e
300
100
e
19a
3b
4b
5b
100
100
300
e
300
300
e
300
300c
300
300
e
e
e
e
300
e
300
300
e
6b
e
300
e
Ethosuximided
Phenytoind
Carbamazepined
Valproic acidd
e
e
e
30
30
e
30
100
e
e
100
100
e
100
300
e
100
300
300
e
The dash (e) indicates an absence of activity at maximum dose administered
(300 mg/kg).
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 animals were examined 0.5 and 4 h after administration.
b
Death following tonic extension.
Myoclonic jerks.
Data from Refs. [30e32].
c
d
and/or 4 h. The other semicarbazone derivatives, 3a, 5a, 9a, 16a, 19a
and 3b revealed NT at a dose of 100 mg/kg. Compound 6a emerged
as anticonvulsant without neurotoxic properties.
Four molecules 6a, 8a, 14a and 18a, which were active in both
MES and scPTZ screens in mice, were evaluated in the scPTZ test
after oral administration into rats. A dose of 50 mg/kg was
employed and the anticonvulsant activity was assessed in several
time points (Table 3). The results obtained were compared with
standard drug ethosuximide, which is the model substance effec-
tive in this screen. As can be seen, 8a protected 25%, 100%, 75% and
50% of tested animals at 0.25 h, 0.5 h, 1 h and 2 h respectively.
Compounds 8a, 14a and 18a were exhibited better activity than
ethosuximide in the scPTZ test after oral administration into rats.
The structureeactivity analysis demonstrated that semi-
carbazones with electron-rich atom/group attached to the aryl ring
is beneficial for anticonvulsant activity. It was possible to observe
Hydrogen bonding area
Hydrophobic binding site
NH
O
CHR
H
HN
N
O
O
Electron donor
Table 3
RHC
moiety
N
N
Evaluation of some selected compounds in the scPTZ test after oral administration
(50 mg/kg) to rats.
NH
R1
H
Compound
Oral administration to ratsa (h)
O
0.25
0.5
1.0
2.0
6a
8a
14a
18a
0
1
0
0
0
2
4
3
3
2
2
3
2
1
1
0
2
1
1
1
Distal hydrophobic
domain
R2
Ethosuximide
Fig. 2. Proposed binding interactions of the title compounds using putative binding
site theory [16e18,37].
a
The figures indicate the number of rats out of four which were protected.

F. Azam et al. / European Journal of Medicinal Chemistry 45 (2010) 2817e2826
2821
Table 5
in the development of oxidative stress which can induce tissue
Quantum chemical parameters of the synthesized compounds.
injury and may activate apoptosis processes [5].
Compounds 6a, 8a, 14a and 18a administered before PTZ
treatment countered the effects of PTZ and protected brain from
oxidative stress by decreasing the MDA formation and restoring the
SOD and GSH-Px activities as well as GSH content. The antioxidant
effects of the compounds were compared with valproate, which is
one of the major antiepileptic drugs, biochemically and clinically.
The results are summarized in Table 4. These results are in agree-
ment with our recent report [19] showing altered antioxidant
enzyme activities and increased LPO after PTZ treatment and
beneficial effects of semicabazone derivatives in restoring antioxi-
dant enzymes activities and decreasing MDA formation.
In recent years, there has been a rapid growth in the application
of quantum chemical methodology to QSAR. Extensive studies
using electronic parameters reveal that electronic attributes of
molecules are intimately related to their chemical reactivities and
biological activities [46]. Quantum chemical descriptors such as net
atomic charges, HOMO-LUMO energies, frontier orbital electron
densities, and superdelocalizabilities have been shown to correlate
well with various biological activities [28,47,48].
The quantum chemical parameters of the synthesized
compounds (3ae19a; 3be6b) were determined using semi-
empirical AM1 calculations. Results are summarized in Table 5. All
molecules have a higher HOMO and lower LUMO energies. The
eigenvalues of LUMO and HOMO and its energies gap reflect the
molecular reactivity and stability [49]. LUMO as an electron
acceptor represents the ability to obtain electrons, and HOMO as an
electron donor represents the ability to donate electrons [50].
Compounds that present larger values of EHOMO are more electron
donor and the compounds that present smaller values of ELUMO are
more electron acceptor. As EHOMO increases (relative to other
Compound
EHOMO (eV)
ELUMO (eV)
D
Ea
3a
4a
5a
6a
7a
8a
9a
10a
11a
12a
13a
14a
15a
16a
17a
18a
19a
3b
ꢃ8.456
ꢃ8.454
ꢃ8.467
ꢃ8.216
ꢃ8.419
ꢃ8.598
ꢃ8.472
ꢃ8.384
ꢃ8.466
ꢃ8.508
ꢃ8.459
ꢃ8.507
ꢃ8.524
ꢃ8.499
ꢃ8.456
ꢃ8.678
ꢃ8.472
ꢃ8.435
ꢃ8.499
ꢃ8.524
ꢃ8.439
ꢃ0.491
ꢃ0.494
ꢃ0.508
ꢃ0.422
ꢃ0.472
ꢃ3.069
ꢃ0.512
ꢃ0.429
ꢃ0.504
ꢃ0.632
ꢃ0.599
ꢃ0.864
ꢃ0.686
ꢃ0.584
ꢃ0.5
ꢃ7.965
ꢃ7.96
ꢃ7.959
ꢃ7.794
ꢃ7.947
ꢃ5.529
ꢃ7.96
ꢃ7.955
ꢃ7.962
ꢃ7.876
ꢃ7.86
ꢃ7.643
ꢃ7.838
ꢃ7.915
ꢃ7.956
ꢃ6.799
ꢃ7.962
ꢃ7.965
ꢃ7.947
ꢃ7.828
ꢃ7.96
ꢃ1.879
ꢃ0.51
ꢃ0.47
4b
5b
6b
ꢃ0.552
ꢃ0.696
ꢃ0.479
a
DE ¼ (EHOMO ꢃ ELUMO).
molecular transport through membranes and, therefore, allows
prediction of transport properties of drugs in the intestines and
blood-brain barrier crossing [54]. TPSA was used to calculate
the percentage of absorption (%ABS) according to the equation:
%ABS ¼ 109 ꢁ 0.345 ꢂ TPSA, as reported by Zhao et al. [55]. In
addition, the number of rotatable bonds (n-ROTB) and Lipinski’s
rule of five [56] were also calculated. From all these parameters, it
can be observed that all semicarbazone derivatives exhibited
a great %ABS ranging from 74 to 90%. Furthermore, none of the
compounds violated Lipinski’s parameters, making them poten-
tially promising agents for epilepsy therapy.
molecules), the molecule is less stable and more reactive. For ELUMO
,
the situation is the opposite [51]. The higher the difference in
HOMO and LUMO energy levels the greater is the anticonvulsant
activity profile of semicarbazones [52,53]. This coincides with our
findings that compound 8a possessed the highest protection profile
4. Conclusion
against MES and PTZ tests as well as having the highest
versa for compound 3a and 3b which exhibited the lowest
anticonvulsant activity while having the lowest E. In addition, the
DE and vice
A series of 1-(substituted benzylidene/ethylidene)-4-(naph-
thalen-1-yl)semicarbazides were designed, synthesized, and their
anticonvulsant activities were evaluated by both the MES and PTZ
tests. Antioxidant enzyme activities, GSH and MDA contents were
estimated from brain homogenate for some of the active
D
compound 8a was found most potent in reducing LPO as well as
restoring levels of GSH and antioxidant enzymes in brain homog-
enate. It was also noted by Kadam et al. [48] that potent molecules
exhibiting better pharmacological activity possessed higher energy
gap of HOMO-LUMO. Further molecular modelling studies of aryl
semicarbazones are in progress and will be reported in due course.
A computational study for prediction of ADME properties of all
molecules was performed and is presented in Table 6. Topological
polar surface area (TPSA), i.e., surface belonging to polar atoms, is
a descriptor that was shown to correlate well with passive
compounds. Highest
D
E
(HOMO-LUMO) was observed in
compound carrying trimethoxy-substituted phenyl group (8a) with
potent anticonvulsant and antioxidant activities. The data strongly
support the assumption that anti-oxidative mechanisms may be
involved in observed anticonvulsant activity of the semicarbazones.
However, further studies need to be carried out to ascertain the
precise mechanism of action of anticonvulsant and antioxidant
effects of these molecules.
Table 4
Biochemical estimations from brain homogenate.
Compound
LPO (nmol MDA/mg protein)
GSH (
mg/gm tissue)
GSH-Px (U/gm protein)
SOD (U/mg protein)
6aa
2.53 ꢁ 0.76d
1.87 ꢁ 0.53b
2.78 ꢁ 0.64b
3.39 ꢁ 0.44d
1.96 ꢁ 0.23b
4.56 ꢁ 0.15c
1.14 ꢁ 0.05
18.51 ꢁ 2.76b
24.56 ꢁ 3.19b
21.35 ꢁ 2.88b
16.76 ꢁ 1.85b
22.14 ꢁ 2.54b
5.76 ꢁ 0.98c
27.79 ꢁ 3.79
1.62 ꢁ 0.16b
1.97 ꢁ 0.23d
1.65 ꢁ 0.08b
1.47 ꢁ 0.06b
2.19 ꢁ 0.26d
0.93 ꢁ 0.12c
2.32 ꢁ 0.32
1.45 ꢁ 0.07b
1.77 ꢁ 0.14b
1.69 ꢁ 0.19b
1.73 ꢁ 0.13d
1.82 ꢁ 0.32b
1.12 ꢁ 0.39c
1.98 ꢁ 0.21
8aa
14aa
18aa
Valproic acida
PTZ
Control
The data are expressed as mean ꢁ S.E.M (n ¼ 6).
a
Tested at 100 mg/kg i.p.
P < 0.001 compared with corresponding value for PTZ-treated mice.
P < 0.001 compared with corresponding value for control mice.
P < 0.01 compared with corresponding value for PTZ-treated mice.
b
c
d

2822
F. Azam et al. / European Journal of Medicinal Chemistry 45 (2010) 2817e2826
Table 6
Pharmacokinetic parameters important for good oral bioavailability of semicarbazones (3ae19a; 3be6b).a
Comp
%ABS
TPSA
(A )
n-ROTB
MW
miLogP
n-OHNH
donors
n-ON
acceptors
Lipinski’s
violations
2
ꢀ
Rule
3a
4a
5a
6a
7a
8a
9a
10a
11a
12a
13a
14a
15a
16a
17a
18a
19a
3b
e
e
e
3
4
4
5
5
6
4
3
3
3
3
3
3
3
4
4
4
3
3
3
3
<500
ꢄ5
<5
2
2
2
2
2
2
3
3
3
2
2
2
2
2
2
2
2
2
2
2
2
<10
4
5
5
6
6
7
6
5
5
4
4
4
4
4
7
7
ꢄ1
0
0
0
0
0
0
0
0
0
1
1
1
1
0
0
0
0
0
1
1
0
90.55
87.36
87.36
84.17
84.17
80.99
80.38
83.57
83.57
90.55
90.55
90.55
90.55
90.55
74.74
74.74
89.43
90.55
90.55
90.55
90.55
53.49
62.72
62.72
71.96
71.96
81.19
82.95
73.72
73.72
53.49
53.49
53.49
53.49
53.49
99.31
99.31
56.73
53.49
53.49
53.49
53.49
289.34
319.36
319.36
349.39
349.39
379.42
335.36
305.34
305.34
323.78
323.78
358.23
368.23
307.33
334.34
334.34
332.41
303.37
337.81
372.26
317.39
4.57
4.63
4.61
4.22
4.61
4.20
3.91
4.51
4.09
5.25
5.20
5.86
5.38
4.74
4.48
4.53
4.68
4.49
5.16
5.77
4.94
5
4
4
4
4b
5b
6b
4
a
%ABS, percentage of absorption; TPSA, topological polar surface area; n-ROTB, number of rotatable bonds; MW, molecular weight; miLogP, logarithm of compound
partition coefficient between n-octanol and water; n-OHNH, number of hydrogen bond donors; n-ON, number of hydrogen bond acceptors.
5. Experimental protocols
and dried. The solid was recrystallized from 90% alcohol to afford 2
with mp 158 ꢀC. IR (KBr) ymax 3347, 3280, 3056, 1622, 1563, 1505,
5.1. Chemistry
1399, 1343, 1268, 1093, 783 cmꢃ1 1H NMR (CDCl3)
; d 8.77 (s, 1H,
NHNH2), 7.42e8.03 (m, 7H, ArH), 7.15 (s, 1H, CONH), 5.63 (s, 2H,
Synthetic starting material, reagents and solvents were of
analytical reagent grade or of the highest quality commercially
available and were purchased from Aldrich Chemical Co., Merck
Chemical Co. and were dried when necessary. The progress of the
reactions was monitored by thin layer chromatography with F254
silica-gel precoated sheets (Merck) using chloroform/ethyl acetate
90/10 as eluent; UV light and iodine vapours were used for detec-
tion. IR spectra were recorded, as KBr pellets, on a Schimadzu 8201
NH2); MS (FAB) m/z 202 (M þ 1)þ.
5.1.3. General method for the synthesis of semicarbazides
(3ae19a; 3be6b)
To a solution of 2 mmol of 2 in 99% ethanol (25 ml), an equi-
molar quantity of appropriate aldehyde or ketone in 99% ethanol
(5 ml) and glacial acetic acid (1 ml) were added. The mixture was
refluxed with stirring for 1e6 h until the completion of the reaction
and the resultant precipitate was filtered and dried. The product
was recrystallized from 90% ethanol. The physical data of the
compounds are presented in Table 1. The spectral data of the
compounds are as follows.
PC FT-IR spectrophotometer and wave numbers are given in cmꢃ1
.
The mass spectra were recorded on Jeol SX-102 (FAB). 1H NMR
spectra, in CDCl3 solution, were recorded on a Bruker DRX-300
instrument at 298 K. Chemical shifts are reported as ppm relative to
TMS as internal standard. Melting points (ꢀC) were determined
with an open glass capillary tube and are uncorrected. Elemental
analyses were performed on Elementar Vario EL III instrument.
5.1.3.1. 2-Benzylidene-N-(naphthalen-1-yl)hydrazinecarboxamide (3a).
IR (KBr) ymax 3104, 2961, 2858, 1708, 1542, 1503, 1408, 1344, 1247,
1149, 1097, 939, 786, 767, 753 cmꢃ1 1H NMR (CDCl3)
; d 11.17
5.1.1. Synthesis of 1-(naphthalen-1-yl)urea (1)
(s, 1H, ¼ NNH, D2O exchangeable), 10.79 (s, 1H, CONH, D2O
exchangeable), 9.11 (s, 1H, imine H), 7.21e8.42 (m, 12H, ArH); MS
(FAB) m/z 290 (M þ 1)þ.
1-Naphthylamine (0.1 mol, 14.3 g) was dissolved in 20 ml of
glacial acetic acid and 10 ml of water. To this, 0.1 mol of sodium
cyanate (6.5 g) in 80 ml of warm water was added with stirring. The
mixture was allowed to stand for 30 min, then cooled in ice, filtered
with suction, dried and recrystallized from boiling water to yield 1
with m.p. 198 ꢀC. IR (KBr) ymax 3440, 3296, 3199, 3048, 1652, 1607,
5.1.3.2. 2-(40-Methoxybenzylidene)-N-(naphthalen-1-yl)hydrazinecar-
boxamide (4a). IR (KBr) ymax 3411, 3205, 3104, 2956, 1702, 1609,
1543, 1317, 1248, 1168, 1096, 1027, 882, 769, 733 cmꢃ1 1H NMR
;
1528, 1503, 1400, 1358, 1334, 1276, 1100, 1018, 784, 760 cmꢃ1
;
1H
(CDCl3)
d
10.41 (s, 1H, ¼ NNH, D2O exchangeable), 9.62 (s, 1H, CONH,
NMR (CDCl3) 7.62 (s, 1H, CONH, D2O exchangeable), 6.55e7.16 (m,
d
D2O exchangeable), 8.74 (s, 1H, imine H), 6.91e7.99 (m, 11H, ArH),
3.84 (s, 3H, OCH3); MS (FAB) m/z 320 (M þ 1)þ.
7H, ArH), 5.45 (s, 2H, NH2, D2O exchangeable); MS (FAB) m/z 187
(M þ 1)þ.
5.1.3.3. 2-(30-Methoxybenzylidene)-N-(naphthalen-1-yl)hydrazinecar-
5.1.2. Synthesis of N-(naphthalen-1-yl)hydrazinecarboxamide (2)
18.6 g (0.1 mol) of 1 in the presence of NaOH (0.1 mol) and
excess of hydrazine hydrate (15 ml) in absolute ethanol (100 ml)
were refluxed with stirring for 24 h. The two-third volume of
alcohol was distilled by vacuum distillation unit and then poured
into ice. The resultant precipitate was filtered, washed with water
boxamide (5a). IR (KBr) ymax 3184, 3088, 2953, 1684, 1545, 1506,
1345, 1298, 1265, 1244, 1134, 1100, 941, 789, 770 cmꢃ1 1H NMR
;
(CDCl3)
d
11.21 (s,1H, ¼ NNH, D2O exchangeable),10.64 (s, 1H, CONH,
D2O exchangeable), 8.82 (s, 1H, imine H), 7.26e8.04 (m, 10H ArH),
6.94 (d, 1H, J ¼ 7.29 Hz), 3.87 (s, 3H, OCH3); MS (FAB) m/z 320
(M þ 1)þ.

F. Azam et al. / European Journal of Medicinal Chemistry 45 (2010) 2817e2826
2823
5.1.3.4. 2-(30,40-Dimethoxybenzylidene)-N-(naphthalen-1-yl)hydrazine-
1H, J ¼ 8.49 Hz), 8.00 (d, 1H, J ¼ 7.86 Hz), 7.33e7.9 (m, 8H, ArH); MS
(FAB) m/z 359 (M þ 1)þ.
carboxamide (6a). IR (KBr) ymax 3383, 3192, 3096, 2961, 2834, 1705,
1546, 1504, 1264, 1163, 1133, 1023, 852, 771 cmꢃ1 1H NMR (CDCl3)
;
d
10.69 (s, 1H, ¼ NNH, D2O exchangeable), 8.72 (s, 1H, CONH, D2O
5.1.3.13. 2-(40-Bromobenzylidene)-N-(naphthalen-1-yl)hydrazinecarbo-
xamide (15a). IR (KBr) ymax 3388, 3192, 3080, 2953, 2850, 1678, 1607,
1526, 1399, 1357, 1305, 1260, 1149, 1008, 869, 814, 779 cmꢃ1; 1H NMR
exchangeable), 7.97 (s, 1H, imine H), 7.49e7.68 (m, 10H, ArH), 2.95 (s,
6H, 3,4-OCH3); MS (FAB) m/z 350 (M þ 1)þ.
(CDCl3)
d
10.18 (s, 1H, ¼ NNH, D2O exchangeable), 9.54 (s, 1H, CONH,
5.1.3.5. 2-(20,50-Dimethoxybenzylidene)-N-(naphthalen-1-yl)hydrazine-
carboxamide (7a). IR (KBr) ymax 3397, 3200, 3088, 2945, 1691, 1542,
1500, 1347, 1306, 1261, 1221, 1168, 1097, 1042, 813, 793 cmꢃ1; 1H NMR
D2O exchangeable), 8.23 (s, 1H, imine H), 7.01e7.96 (m, 11H, ArH); MS
(FAB) m/z 369 (M þ 1)þ.
(CDCl3)
d
10.36 (s, 1H, ¼ NNH, D2O exchangeable), 8.65 (s, 1H, CONH,
5.1.3.14. 2-(40-Fluorobenzylidene)-N-(naphthalen-1-yl)hydrazine-
D2O exchangeable), 8.07 (s, 1H, imine H), 6.63e7.74 (m, 10H, ArH),
carboxamide (16a). IR (KBr) ymax 3207, 3104, 2973, 2866, 1708,
3.56 (s, 6H, 2,5-OCH3); MS (FAB) m/z 350 (M þ 1)þ.
1547, 1503, 1409, 1234, 1150, 1092, 832, 768 cmꢃ1 1H NMR
;
(CDCl3)
d
10.13 (s, 1H, ¼ NNH, D2O exchangeable), 9.72 (s, 1H,
5.1.3.6. N-(naphthalen-1-yl)-2-(30,40,50-trimethoxybenzylidene)hydra-
zinecarboxamide (8a). IR (KBr) ymax 3399, 3196, 3087, 2959, 1686,
1612, 1580, 1546, 1503, 1410, 1354, 1325, 1231, 1130, 1004, 818, 766,
CONH, D2O exchangeable), 8.22 (s, 1H, imine H), 6.57e7.48 (m,
11H, ArH); MS (FAB) m/z 308 (M þ 1)þ.
724 cmꢃ1
;
1H NMR (CDCl3)
d
10.59 (s, 1H, ¼ NNH, D2O exchange-
5.1.3.15. N-(naphthalen-1-yl)-2-(20-nitrobenzylidene)hydrazinecarbox-
able), 8.88 (s, 1H, CONH, D2O exchangeable), 8.82 (s, 1H, imine H),
6.99e8.13 (m, 9H, ArH), 3.96 (s, 6H, 3,5-OCH3), 3.87 (s, 3H, 4-OCH3);
MS (FAB) m/z 380 (M þ 1)þ.
amide (17a). IR (KBr) ymax 3265, 3173, 3013, 1696, 1659, 1597, 1570,
1522, 1340, 1251, 1132, 784, 700 cmꢃ1 1H NMR (CDCl3)
; d 10.64 (s,
1H, ¼ NNH, D2O exchangeable), 8.72 (s, 1H, CONH, D2O exchange-
able), 8.42 (s, 1H, imine H), 7.98e8.06 (m, 3H, ArH), 7.89 (d, 1H,
J ¼ 7.68 Hz), 7.67 (d, 1H, J ¼ 8.1 Hz), 7.33e7.57 (m, 6H, ArH); MS
(FAB) m/z 335 (M þ 1)þ.
5.1.3.7. 2-(4-Hydroxy-3-methoxybenzylidene)-N-(naphthalen-1-yl)-
hydrazinecarboxamide (9a). IR (KBr) ymax 3509, 3374, 3184, 3088,
2951, 1685, 1545,1503,1430,1344,1262,1242,1160, 1098,1031, 849,
791, 770 cmꢃ1
;
1H NMR (CDCl3)
d
11.51 (s, 1H, OH), 10.42 (s, 1H, ¼
5.1.3.16. N-(naphthalen-1-yl)-2-(40-nitrobenzylidene)hydrazinecarbox-
NNH, D2O exchangeable), 8.95 (s, 1H, CONH, D2O exchangeable),
8.34 (s, 1H, imine H), 7.2e8.31 (m, 10H, ArH), 3.32 (s, 3H, OCH3); MS
(FAB) m/z 336 (M þ 1)þ.
amide (18a). IR (KBr) ymax 3396, 3192, 3083, 2945, 1677, 1598, 1575,
1507, 1335, 1282, 1261, 1160, 1108, 922, 841, 774 cmꢃ1 1H NMR
;
(CDCl3)
d
10.24 (s, 1H, ¼ NNH, D2O exchangeable), 8.48 (s, 1H, CONH,
D2O exchangeable), 7.94 (s,1H, imine H), 6.59e7.74 (m, 11H, ArH); MS
(FAB) m/z 335 (M þ 1)þ.
5.1.3.8. 2-(20-Hydroxybenzylidene)-N-(naphthalen-1-yl)hydrazine-
carboxamide (10a). IR (KBr) ymax 3295, 3196, 3046, 2977, 1655,
1597, 1575, 1527, 1506, 1436, 1305, 1269, 1155, 949, 792, 752 cmꢃ1
;
5.1.3.17. 2-(4-(Dimethylamino)benzylidene)-N-(naphthalen-1-yl)hydra-
1H NMR (CDCl3)
d
12.53 (br s, 1H, OH), 10.62 (s, 1H, ¼ NNH, D2O
zinecarboxamide (19a). IR (KBr) ymax 3366, 3176, 3092, 2956, 1680,
exchangeable), 8.25 (s, 1H, CONH, D2O exchangeable), 8.01 (s, 1H,
imine H), 7.75 (d, 1H, J ¼ 7.8 Hz), 7.45 (d, 1H, J ¼ 7.65 Hz),
7.03e7.31 (m, 9H, ArH); MS (FAB) m/z 306 (M þ 1)þ.
1610, 1492, 1358, 1286, 1259, 947, 811, 780 cmꢃ1 1H NMR (CDCl3)
;
d
8.80 (s, 1H, ¼ NNH, D2O exchangeable), 8.71 (s, 1H, CONH, D2O
exchangeable), 7.49e8.09 (m, 10H, ArH), 7.26 (s, 1H, imine H), 6.73 (d,
1H, J ¼ 8.76 Hz), 3.03 [s, 6H, N(CH3)2]; MS (FAB) m/z 333 (M þ 1)þ.
5.1.3.9. 2-(40-Hydroxybenzylidene)-N-(naphthalen-1-yl)hydrazine-
carboxamide (11a). IR (KBr) ymax 3374, 3175, 1658, 1607, 1577,
5.1.3.18. N-(naphthalen-1-yl)-2-(1-phenylethylidene)hydrazinecarbox-
1510, 1360, 1279, 1257, 1162, 1127, 1094, 832, 788 cmꢃ1
;
1H NMR
amide (3b). IR (KBr) ymax 3394, 3208, 3104, 1684, 1544, 1505, 1409,
(CDCl3)
d
12.13 (s, 1H, OH), 10.07 (s, 1H, ¼ NNH, D2O exchange-
1333, 1282, 1258, 1141, 1099, 789 cmꢃ1 1H NMR (CDCl3)
; d 10.67 (s,
able), 9.31 (s, 1H, CONH, D2O exchangeable), 8.74 (s, 1H, imine H),
1H, ¼ NNH, D2O exchangeable), 9.73 (s, 1H, CONH, D2O exchange-
able), 8.89 (s,1H, imine H), 8.40 (d,1H, J ¼ 8.1 Hz), 7.26e8.08 (m,10H,
ArH), 2.37 (s, 3H, CH3); MS (FAB) m/z 304 (M þ 1)þ.
6.89e8.05 (m, 11H, ArH); MS (FAB) m/z 306 (M þ 1)þ.
5.1.3.10. 2-(40-Chlorobenzylidene)-N-(naphthalen-1-yl)hydrazinecarbo-
xamide (12a). IR (KBr) ymax 3200, 3104, 2966, 2866, 1708, 1602, 1542,
5.1.3.19. 2-(10-(400-Chlorophenyl)ethylidene)-N-(naphthalen-1-yl)hydra-
zinecarboxamide (4b). IR (KBr) ymax 3379, 3201, 3097, 1681, 1543,
1503, 1407, 1349, 1327, 1285, 1260, 1241, 1131, 1092, 1008, 819,
1503, 1409, 1346, 1307, 1153, 1011, 823, 770, 733 cmꢃ1 1H NMR
;
(CDCl3)
d
10.66 (s, 1H, ¼ NNH, D2O exchangeable), 9.34 (s, 1H, CONH,
D2O exchangeable), 8.72 (s, 1H, imine H), 7.39e8.01 (m, 11H, ArH); MS
(FAB) m/z 325 (M þ 1)þ.
790, 773 cmꢃ1
;
1H NMR (CDCl3)
d
10.64 (s, 1H, ¼ NNH, D2O
exchangeable), 9.33 (s, 1H, CONH, D2O exchangeable), 8.89 (s,
1H, imine H), 7.39e8.03 (m, 10H, ArH), 2.51 (s, 3H, CH3); MS
(FAB) m/z 339 (M þ 1)þ.
5.1.3.11. 2-(20-Chlorobenzylidene)-N-(naphthalen-1-yl)hydrazine-
carboxamide (13a). IR (KBr) ymax 3215, 3139, 3056, 2985, 1698,
1660, 1549, 1506, 1443, 1345, 1307, 1245, 1145, 1097, 950, 787,
5.1.3.20. 2-(10-(200,400-Dichlorophenyl)ethylidene)-N-(naphthalen-1-yl)-
768, 753 cmꢃ1
;
1H NMR (CDCl3)
d
10.23 (s, 1H, ¼ NNH, D2O
hydrazinecarboxamide (5b). IR (KBr) ymax 3372, 3195, 3082, 1682,
exchangeable), 9.76 (s, 1H, CONH, D2O exchangeable), 7.98
(s, 1H, imine H), 6.78e7.89 (m, 11H, ArH); MS (FAB) m/z 325
(M þ 1)þ.
1529, 1503, 1328, 1283, 1256, 1137, 873, 788, 767 cmꢃ1 1H NMR
;
(CDCl3)
d
10.35 (s, 1H, ¼ NNH, D2O exchangeable), 9.47 (s, 1H, CONH,
D2O exchangeable), 8.81 (s, 1H, imine H), 7.32e8.03 (m, 9H, ArH), 2.36
(s, 3H, CH3); MS (FAB) m/z 373 (M þ 1)þ.
5.1.3.12. 2-(20,40-Dichlorobenzylidene)-N-(naphthalen-1-yl)hydrazine-
carboxamide (14a). IR (KBr) ymax 3396, 3192, 3061, 2933, 1688, 1598,
1543, 1505, 1464, 1346, 1243, 1147, 1096, 1048, 930, 866, 791, 766,
5.1.3.21. N-(naphthalen-1-yl)-2-(1-p-tolylethylidene)hydrazinecarbox-
amide (6b). IR (KBr) ymax 3371, 3198, 3088, 1681, 1542, 1503, 1409,
729 cmꢃ1; 1H NMR (CDCl3)
8.89 (s, 1H, CONH, D2O exchangeable), 8.38 (s, 1H, imine H), 8.18 (d,
d
11.03 (s,1H, ¼ NNH, D2O exchangeable),
1327, 1283, 1260, 1129, 1099, 962, 811, 787, 770 cmꢃ1 1H NMR
;
(CDCl3)
d
10.54 (s, 1H, ¼ NNH, D2O exchangeable), 9.02 (s, 1H, CONH,

2824
F. Azam et al. / European Journal of Medicinal Chemistry 45 (2010) 2817e2826
D2O exchangeable), 8.62 (s, 1H, imine H), 6.84e7.69 (m, 10H, ArH),
2.02 (s, 3H, CH3), 1.97 (s, 3H, CH3); MS (FAB) m/z 318 (M þ 1)þ.
(MDA) and protein levels were carried out at this stage. The
homogenate was then centrifuged at 10500 ꢂ g for 20 min to
remove nuclear debris. Clear supernatant fluid was taken and
GSH-Px activity was carried out in this stage. The supernatant
solution was then extracted with an equal volume of an ethanol/
chloroform mixture (5:3, v/v). After centrifugation at 5000 ꢂ g for
30 min, the clear upper layer (the ethanol phase) was taken and
used in the SOD activity. All preparation procedures were
performed at þ4 ꢀC.
5.2. Pharmacological evaluation
All the compounds were screened for anticonvulsant activity in
MES-induced seizure test and scPTZ-induced seizure threshold test.
The NT was measured by the rotorod test. The results are
summarized in Table 2.
5.2.1. Anticonvulsant screening
5.3.1. LPO assay
Anticonvulsant evaluation of semicarbazones was undertaken
by following the National Institute of Health (NIH) Anticonvulsant
Drug Development (ADD) Program protocol [57,58]. Male Swiss
albino mice (18e25 g) and male albino rats (Wistar, 100e125 g)
were used as experimental animals. The semicarbazones were
suspended in 5% gum acacia/water mixture. All the compounds
were administered i.p. in doses of 30, 100 and 300 mg/kg to one to
four animals. Some selected compounds were examined for oral
activity in rats. All the tests have been performed in accordance
with the guidelines laid out by the Institutional Animal Ethics
Committee.
The extent of LPO in brain homogenate was determined by
measuring the release of thiobarbituric acid reactive substance
(TBARS) in terms of MDA equivalent using a molar extinction
coefficient of 1.56 ꢂ 105/min/cm as described by Ohkawa et al [60].
Briefly, the homogenate was centrifuged at 3000 ꢂ g for 15 min and
supernatant was used for assay. Samples of 0.1 ml homogenate was
mixed with 0.2 ml of 8.1% SDS, 1.5 ml 20% glacial acetic acid and
1.5 ml of 0.8% thiobarbituric acid (TBA). Following these additions,
tubes were mixed and heated at 95 ꢀC for 1 h on a water bath and
cooled under tap water before mixing 1 ml of distilled water and
5 ml mixture of n-butanol and pyridine (15:1). The mixture was
centrifuged at 2200 g for 10 min. The amount of MDA formed was
measured by the absorbance of upper organic layer at a wavelength
of 532 nm in Perkin Elmer spectrophotometer using appropriate
controls. The results are expressed as nmol MDA/mg protein.
5.2.1.1. MES test. MES seizures were elicited (Techno Con-
vulsiometer Model No. C-2, Techno Electronics, Lucknow, India)
with a 60 cycle altering current of 50 mA intensity (5e7 times that
necessary to elicit minimal electroshock seizures) delivered for
0.25 s via corneal electrodes. A drop of 0.9% saline was instilled in
the eye prior to application of the electrodes in order to prevent the
death of the animal. Abolition of the hind limb tonic extensor
component of the seizure is defined as protection, and results are
expressed as number of animal protected/number of animals
tested.
5.3.2. GSH determination
The amount of GSH in brain was measured according to the
method of Sedlak and Lindsay [61]. Briefly, brain tissue was
deproteinized with an equal volume of 10% TCA and was allowed to
stand at 4 ꢀC for 2 h. The contents were centrifuged at 2000 g for
15 min. The supernatant was added to 2 ml of 0.4 M Tris buffer
(pH 8.9) containing 0.02 M EDTA (pH 8.9) followed by the addition
of 0.01 M DTNB {5,50-dithiobis(2-nitrobenzoic acid)}. Finally, the
mixture was diluted with 0.5 ml of distilled water, to make the total
mixture to 3 ml and absorbance was read in a spectrophotometer at
5.2.1.2. scPTZ test. Thirty minutes after the administration of the
test compounds, mice were injected with PTZ dose of 85 mg/kg
intraperitoneally to cause seizures in more than 97% of the animals.
This is called the convulsive dose (CD97). 70 mg/kg scPTZ dose was
used for rats. The animals were observed during the following 4 h
for the occurance of seizures. A threshold convulsion is defined as
one episode of clonic spasms which persists for at least 5 s. Absence
of even a threshold convulsion during the period of observation is
taken as the endpoint in this test.
412 nm and results are expressed as mg GSH/gm Tissue.
5.3.3. SOD activity determination
Cu,Zn-SOD activity was determined according to the method of
Sun et al [62]. In this method, a xanthineexanthine oxidase complex
produces superoxide radicals, which react with nitroblue-
tetrazolium (NBT) to form the farmazan compound. In brief, a reac-
tive was prepared with 0.1 mM of xanthine, 0.1 mM of EDTA, 50 mg
of bovine serum albumin, 25 mM of NBT and 40 mM of Na2CO3 (pH
10.2). To 2.45 ml of reactive was added 0.5 ml of a ethanol/chloro-
form (5:3, v/v) extract, previously prepared from brain homogenate.
5.2.1.3. NT screening. The rotorod test was used to evaluate neuro-
toxicity. The mice were trained to stand on an accelerating rotarod
rotating at 10 rev/min. The rod diameter was 3.2 cm (Techno
Rotarod system, Techno Electronics, Lucknow, India). The mice were
placed on the rotarod to measure the effect of the drug on their
motor performance [59]. The dose at which animals fell off the
rotarod was determined. Before the beginning of all experiments,
the riding abilityof the animals in the rotarod was checked. Thus, the
mice were initially put on a rotating rod, and mice that immediately
dropped off (within 30 s) were removed from the experiment.
Subsequently, 50 ml of 9.9 nM of xanthine oxidase solution was
added, the mixture was kept in a water bath of 25 ꢀC for 20 min, and
the reaction was terminated using 1 ml of CuCl2. The absorbance of
the samples was read at 560 nm. In the control sample the amount of
the ethanol supernatant was replaced by equivalent volume of PBS
buffer. One unit SOD activity was defined as the amount of enzyme
causing 50% inhibition of NBT reduction to formazan. SOD activity
was expressed as U/mg protein.
5.3. Biochemical evaluation
Mice were sacrificed by decapitation 4 h after the last injection.
The brains were quickly removed and were washed twice with
ice-cold saline solution, placed into glass bottles, labeled, and
stored in a deep freeze (ꢃ30 ꢀC) until processing (maximum 10 h).
Tissues were homogenized in four volumes of ice-cold Tris-HCl
buffer (50 mM, pH 7.4) using a glass Teflon homogenizer (Ultra
Turrax IKA T18 Basic, USA) after cutting up the brain into small
pieces with scissors (for 2 min at 5000 rpm). Malondialdehyde
5.3.4. GSH-Px activity determination
The GSH-Px activity was measured by the method of Paglia and
Valentine [63]. The enzymatic reaction was conducted in 3 ml
quartz cuvettes of 1 cm path length in a Perkin-Elmer spectro-
photometer. Each 3 ml assay volume contained 50 mM potassium
phosphate buffer (pH 7.0), 1 mM EDTA,1 mM sodium azide, 0.2 mM
NADPH, 1 U glutathione reductase and 1 mM reduced glutathione.
The sample (0.2 ml of the tissue homogenate), after its addition,

F. Azam et al. / European Journal of Medicinal Chemistry 45 (2010) 2817e2826
2825
was allowed to equilibrate for 5 min at 25 ꢀC. The reaction was
initiated by adding 0.1 ml of 2.5 mM H2O2. Changes in absorbance
were recorded at 340 nm for 5 min. Values were expressed as units
of NADPH oxidized to NADP by using the extinction coefficient of
6.22 mMꢃ1 cmꢃ1 at 340 nm. All samples were assayed in duplicate.
GSH-Px activity was expressed as units per gram protein.
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Acknowledgements
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The authors are grateful to Head, Sophisticated Analytical
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for providing the spectral data of the synthesized compounds.
Mr Nitin is acknowledged for his kind help and support.
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