Synthesis of some novel N4-(naphtha[1,2-d]thiazol-2-yl)semicarbazides as potential anticonvulsants

Article abstract of DOI:10.1016/j.ejmech.2008.02.007

A series of N⁴-(naphtha[1,2-d]thiazol-2-yl)semicarbazides were designed and synthesized to meet the structural requirements essential for anticonvulsant activity. Anticonvulsant activity was determined after intraperitoneal (i.p.) administration to mice by maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ)-induced seizure tests and minimal motor impairment was determined by rotorod test. A majority of the compounds exhibited significant anticonvulsant activity after intraperitoneal administration. Some of the selected compounds were evaluated orally in rats for activity in scPTZ test at several time points (50 mg/kg). The most active compounds carry bromo, fluoro and nitro substituents at 4-position in the phenyl ring. The biochemical estimations of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) from brain homogenate not only clearly implicated the role of free radicals in PTZ-induced convulsion but also explained the possible mechanism of protective effect of semicarbazides, through the reduced formation of MDA and increased formation of SOD and GSH-Px.

Full text of DOI:10.1016/j.ejmech.2008.02.007

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 203e211
http://www.elsevier.com/locate/ejmech
Original article
Synthesis of some novel N4-(naphtha[1,2-d]thiazol-2-yl)semicarbazides
as potential anticonvulsants
b
a
a
Faizul Azam a,b, , Ismail A. Alkskas , Sukhbir Lal Khokra , Om Prakash
*
a Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra, Haryana 136119, India
b Department of Chemistry, Faculty of Pharmacy, Seventh of October University, P.O. Box 2873 Misurata, Libyan Arab Jamahiriya
Received 1 July 2007; received in revised form 2 February 2008; accepted 8 February 2008
Available online 29 February 2008
Abstract
A series of N4-(naphtha[1,2-d]thiazol-2-yl)semicarbazides were designed and synthesized to meet the structural requirements essential for
anticonvulsant activity. Anticonvulsant activity was determined after intraperitoneal (i.p.) administration to mice by maximal electroshock
(MES) and subcutaneous pentylenetetrazole (scPTZ)-induced seizure tests and minimal motor impairment was determined by rotorod test. A
majority of the compounds exhibited significant anticonvulsant activity after intraperitoneal administration. Some of the selected compounds
were evaluated orally in rats for activity in scPTZ test at several time points (50 mg/kg). The most active compounds carry bromo, fluoro
and nitro substituents at 4-position in the phenyl ring. The biochemical estimations of malondialdehyde (MDA), superoxide dismutase
(SOD) and glutathione peroxidase (GSH-Px) from brain homogenate not only clearly implicated the role of free radicals in PTZ-induced con-
vulsion but also explained the possible mechanism of protective effect of semicarbazides, through the reduced formation of MDA and increased
formation of SOD and GSH-Px.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Anticonvulsant activity; Naphtha[1,2-d]thiazol-2-amine; Semicarbazides; Neuroprotection
1. Introduction
concerning this problem show the efficacy of some substances
with antioxidant properties in the therapy of convulsive disor-
ders and support the hypothesis that experimental epilepsy is
mediated by oxidative stress, leading to abnormal structural
alterations of cellular proteins, membrane lipids, DNA and
RNA [5e9].
The oxidative stress, defined by the excessive production of
free radicals, can alter dramatically the cell function and an
overproduction of these compounds has been related to sei-
zure-induced neuronal death [10]. Superoxide (Oꢀ2 ), a free rad-
ical can be generated in the brain by several mechanisms such
as inefficiency of the electron-carrying components of the
mitochondrial transport chain, monoamines degradation, xan-
thine oxidase reaction and by the metabolism of arachidonic
acid. Nevertheless, the produced Oꢀ2 could be metabolized
by superoxide dismutases (SOD), which are present in both
cytosol (copperezinc-associated isoform) and mitochondria
(manganese-associated isoform) [11]. The hydrogen peroxide
(H2O2) is not a free radical per se but in high concentration
Epilepsy is a neurological disorder of varied etiology,
affecting about 1% of the world’s population and characterized
by recurrent seizure attacks [1]. In fact, epilepsy is the third
most frequent neurological disorder encountered in the elderly
after cerebrovascular disease and dementia [2]. There is con-
tinuing demand for new anticonvulsant agents as several of
the currently available antiepileptic drugs (AEDs) have been
associated with severe side effects and fail to control seizures
in about 30% of epileptic patients [1,3,4].
The prolonged excitation of neurons during seizures can
lead to injury and death resulting from underlying biochemical
mechanisms that are not well understood. Recent data
* Corresponding author. Department of Medicinal Chemistry, Faculty of
Pharmacy, Seventh of October University, P.O. Box 2873 Misurata, Libyan
Arab Jamahiriya. Tel.: þ218 92 7677641; fax: þ218 51 628149.
E-mail address: azamfaizul@yahoo.co.in (F. Azam).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.02.007

204
F. Azam et al. / European Journal of Medicinal Chemistry 44 (2009) 203e211
can react with superoxide (HabereWeiss reaction) or iron
(Fenton reaction) producing highly reactive hydroxyl radical
(OHꢁ). The detoxification of H2O2 to H2O is made by glutathi-
one peroxidase (GSH-Px) [12,13] and involves the glutathione
(a thiol-containing tripeptide) which is a cofactor of this
enzyme [14,15]. The oxidanteantioxidant system is in equilib-
rium in normal conditions. However, an imbalance in this sys-
tem causes tissue injury by oxidant injury. Reactive oxygen
species attack membrane lipids and result in LPO. Malondial-
dehyde (MDA) is the end-product of lipid peroxidation (LPO)
and thus serves as an index of LPO. It was also shown that pre-
treatment with melatonin, an endogenous hormone with anti-
oxidant properties, suppressed PTZ-induced seizures and
decreased the concentration of LPO products in the brain [9].
Therefore, one of the main trends of current investigations
is the search for novel antiepileptic drugs with neuroprotective
properties [16]. Several anticonvulsant drugs such as zonisa-
mide [17], allopurinol [18] and drugs such as MK-801, which
is an N-methyl-D-aspartate receptor antagonist, protect the
brain against iron-induced oxidative stress in rats [19]. A vari-
ety of Naþ channel blockers, such as Phenytoin [20] and lamo-
trigine [21,22] have been demonstrated to protect neurons
against focal as well as global cerebral ischemia in rats.
Thiazoles are important nitrogen and sulfur-containing five-
membered heterocyclic compounds. Several thiazole deriva-
tives possess important pharmacological activities and therefore
they are useful materials in drug research. They are of interest as
potential neuroprotective agents [23e25]. Furthermore, various
2-benzothiazolamine derivatives including riluzole [6-(trifluor-
omethoxy) 2-benzothiazolamine] exhibited potent anticonvul-
sant activity [26e28].
In recent years, aryl and heteroaryl semicarbazones have
documented increasing advances in AED drug design and
were found to act by blocking the voltage-gated Naþ channels
[29e34]. Their role for the treatment of neuronal damage fol-
lowing global and focal ischemia has been documented [35].
Others [31,36] have suggested the identifiable features for
anticonvulsant activity like (i) hydrophobic aryl ring, (ii) a hy-
drogen bonding domain, (iii) an electron-donor group, and (iv)
another distal hydrophobic site.
The aryl group can be replaced by other hydrophobic moi-
eties with retention of anticonvulsant activity [37]. In addition,
the potency of 2-benzothiazolamine derivatives as sodium flux
inhibitors increases with increasing lipophilicity [26]. More-
over, naphthalene ring serves as the lipophilic aryl portion in
nafimidone derivatives which have been recognized as poten-
tial anticonvulsants [38e41]. Furthermore, tricyclic molecules
(e.g. dibenzazepines, dibenzocycloheptadienes, phenothia-
zines) exhibit widespread pharmaceutical application in the
chemical neurosciences, being used as drugs for such varied
indications as epilepsy, migraine, chronic pain, depression and
psychosis.
hydrophobic naphthalene ring in conjugation with thiazole
ring as tricyclic molecule, naphtha[1,2-d]thiazol-2-amine for
enhanced lipophilicity and membrane permeability. The com-
pounds were evaluated for their antiepileptic and neurotoxic
properties. The extent of oxidative stress has been evaluated
by measuring LPO, SOD and GSH-Px activities in selected
compounds.
2. Synthesis
In the present study N4-(naphtha[1,2-d]thiazol-2-yl)semi-
carbazides were synthesized as presented in Scheme 1. Naph-
tha[1,2-d]thiazol-2-amine (1) was prepared using a literature
protocol [45] with minor modifications. Naphtha[1,2-d]thia-
zol-2-amine (1) was treated with phenylchloroformate in
dichloromethane at room temperature to yield phenyl-N-
(naphtha[1,2-d]thiazol-2-yl)carbamate (2). The carbamate on
condensation with hydrazine hydrate in methylene chloride
gave N4-(naphtha[1,2-d]thiazol-2-yl)semicarbazide (3). The
semicarbazone derivatives (4e21) were prepared by reaction
of the appropriate aryl aldehyde or ketone or isatin with N4-
(naphtha[1,2-d]thiazol-2-yl)semicarbazide (3). All compounds
1
showed a single spot on TLC and had IR, H 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
Initial anticonvulsant activity and neurotoxicity (NT) data for
the semicarbazones are given in Table 2 along with the literature
data on phenytoin, carbamazepine, sodium valproate, phenobar-
bital and ethosuximide [31,46,47]. The maximal electroshock
(MES) model has served to identify AEDs that are functionally
similar to phenytoin, and most of these compounds display in
common the ability to inactivate voltage-dependent Naþ chan-
nels in a use-dependent fashion. Activity in this model seems
highly predictive of the ability of those AEDs to protect against
partial and secondarily generalized toniceclonic seizures. Car-
bamazepine, as well as several newer agents such as felbamate,
gabapentin, lamotrigine, and topiramate, fit this model of AED
activity [48,49]. The subcutaneous pentylenetetrazole (scPTZ)
model has proven to be a good predictor of clinical efficacy
against generalized spike-wave epilepsies of the absence type.
Thus, the MES and scPTZ screens have become the most widely
employed seizure models for early identification of candidate
anticonvulsants.
Preliminary anticonvulsant evaluation of all the synthesized
compounds was obtained by testing procedures described in
National Institute of Neurological Disorders and Stroke, NIH,
Bethesada, MD, USA, for Anticonvulsant Screening Project
(ASP). Each compound was administered as an i.p. injection
at three dose levels (30, 100 and 300 mg/kg) and the anti-
convulsant activity was assessed after 30 min and 4 h intervals
of administration. The anticonvulsant efficacy was evaluated by
the MES-induced seizure and scPTZ-induced seizure threshold
tests. NT in mice was measured by the rotorod test.
In view of these general requirements of activity (Fig. 1)
and in continuation of our work on biologically active nitrogen
and sulfur-containing heterocycles [42e44], we have designed
N4-(naphtha[1,2-d]thiazol-2-yl)semicarbazides in which distal
hydrophobic aryl moiety has been varied to include

F. Azam et al. / European Journal of Medicinal Chemistry 44 (2009) 203e211
205
S
NH2
NH2
HN
NH2
N
S
KSCN / HCl
1. Br2 / AcOH
2. NH4OH
1
O
Cl
CH2Cl2
HN
NH2
(C2H5)3N
O
HN
S
C
O
N
H
N
O
N
C
O
NH2NH2.H2O
CH2Cl2
S
2
3
Aldehyde
or Ketone
AcOH
or Isatin
H
N
H
N
Y
X
N
C
O
N
Z
S
4-21
Scheme 1. Synthetic route to 4e21.
All the compounds, except 5, 7 and 14 exhibited anticon-
vulsant activity. Majority of the compounds, except 6, 16, 19
and 20, were active in both MES and scPTZ tests making
them useful for broad spectrum of seizure types. Compounds
9, 11 and 13 with chloro, bromo and fluoro substituents, re-
spectively, showed activity at 100 mg/kg after 0.5 h in MES
test. Compounds 9 and 11 were found to be active at the
same dose after 4 h. The activity of compounds 9 and 11 having
CH3
H
N
H
N
R
C
O
N
R1
N
C
CH3
O
NH2
Aryl semicarbazones
Carbamazepine
F
F
O
S
N
C
F
NH2
N
O
Riluzole
Nafimidone
N
Fig. 1. Structures of some well-known anticonvulsants.

206
F. Azam et al. / European Journal of Medicinal Chemistry 44 (2009) 203e211
Table 1
Physical characterization of compounds 4e21
H
N
H
N
Y
X
N
C
O
N
Z
S
4-21
b
Compound
X
Y
Z
Yield
(%)
M.p.
(ꢂC)
Mol. formulaa
Mol.
weight
Rf
4
C
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
H
e
H
4-OMeC6H4
58
76
72
68
76
81
79
72
83
61
68
73
65
73
78
73
71
57
190
238
250
268
216
252
260
246
242
262
238
228
242
246
264
218
226
292
C19H14N4OS
C20H16N4O2S
C21H18N4O3S
C21H18N4O3S
C22H20N4O4S
C19H13BrN4OS
C19H13ClN4OS
C19H13ClN4OS
C19H13FN4OS
C19H14N4O2S
C19H14N4O2S
C20H16N4O3S
C19H13N5O3S
C19H13N5O3S
C21H18N4OS
C20H16N4OS
C21H19N5OS
C20H13N5O2S
346.41
376.43
406.46
406.46
436.48
425.3
0.67
0.58
0.72
0.57
0.69
0.78
0.71
0.73
0.76
0.89
0.92
0.71
0.61
0.65
0.59
0.57
0.64
0.61
5
C
6
C
2,5-OMeC6H3
3,5-OMeC6H3
3,4,5-OMeC6H2
4-BrC6H4
7
C
8
C
9
C
10
11
12
13
14
15
16
17
18
19
20
21
a
C
2-ClC6H4
4-ClC6H4
4-FC6H4
380.85
380.85
364.4
C
C
C
2-OHC6H4
4eOHC6H4
4-OH, 3-OMeC6H3
2-NO2C6H4
4-NO2C6H4
4-MeC6H4
C6H5
362.41
362.41
392.43
391.4
C
C
C
C
391.4
C
360.43
374.46
389.47
387.41
C
C
2-Oxoindolin-3-ylidene
4-NMe2C6H4
e
Elemental analyses for C, H, and N were within ꢃ0.4% of the theoretical values.
b
Solvent system: chloroform/methanol (95:5).
4-bromo and 4-chloro substituents, respectively, at the phenyl
ring in MES test is comparable to standard drug phenobarbital
(100 mg/kg) after 0.5 h, indicating that they have rapid onset
and shorter duration of action. Compounds 4, 6, 8, 12, 15,
17 and 18 were less active and inhibited the seizures at
a dose of 300 mg/kg in the MES screen after 0.5 h. Some com-
pounds like 8, 12, 15 and 17 were also active after 4.0 h
extended period of activity. Compound 10 with chloro substit-
uent at 2-position of the phenyl ring was active at 300 mg/kg
after 4 h in the MES screen. Majority of the compounds also
showed activity in scPTZ screen. Compounds 9 and 12 showed
activity both at 0.5 h and 4 h periods (100 mg/kg). However,
compound 17 showed activity at 100 mg/kg after 0.5 h and
at 300 mg/kg after 4 h time period which is comparable to
the standard drug carbamazepine. Introduction of the fluoro
or trifluoromethyl substituents into the aryl moiety enhanced
the anticonvulsant efficacy in comparison to respective chloro,
methoxy or methyl analogues [50]. Furthermore, introduction
of p-nitrophenyl substituent exhibited best activity in scPTZ
test [51]. The importance of the 4-bromophenyl substituent
for the anticonvulsant activity is documented in the literature
[34]. Our results are in accordance with these findings.
and 4 h include 4, 13, 15, 16, 18, 19 and 20. In a neurological
toxicity screening test, compounds 4, 9, 11, 12, 17, 18, 20 and
21 were found to be toxic at the maximum administrated dose
(300 mg/kg). Compounds 8, 13, 15, and 16 were toxic at
100 mg/kg dose.
Some of the selected compounds 9, 12 and 17 were evalu-
ated orally in rats for activity in scPTZ test at several time
points (Table 3). The compounds were tested at 50 mg/kg
and compared with standard drug ethosuximide. Compounds
9 and 12 exhibited bioactivity throughout the time period up
to 2 h similar to ethosuximide. These compounds did not
exhibit NT at the tested dose of 50 mg/kg.
Some active compounds (9, 12 and 17), were selected for
the measurement of antioxidant enzyme activities from the
brain homogenate to investigate their role in the pathophysiol-
ogy of epilepsy. The antioxidant effects of the compounds
were compared with valproate, which is one of the major
antiepileptic drugs, biochemically and clinically. Oxidative
stress in the central nervous system has been shown in several
rodent models of experimental epilepsy such as the PTZ kin-
dling model [6]. PTZ trigger a variety of biochemical pro-
cesses including the activation of membrane phospholipases,
proteases, and nucleases [52]. Marked alterations in membrane
phospholipid metabolism result in the liberation of free fatty
acids, diacylglycerols, eicosanoids, lipid peroxides and free
radicals [52]. PTZ-induced seizure activity coincides with an
On the other hand, compounds 8 and 10 showed activity at
300 mg/kg after 4 h. Compounds showing activity at 300 mg/
kg after 0.5 h included 11 and 21. Compounds that showed
moderate protection at a dose of 300 mg/kg both at 0.5 h

F. Azam et al. / European Journal of Medicinal Chemistry 44 (2009) 203e211
207
Table 2
Anticonvulsant and NT screening of synthesized compounds 4e21
PTZ treated mice. GSH-Px is an endogenous antioxidant
enzyme and it reacts with the free radicals and prevents the
generation of hydroxyl radicals, the most toxic form of free
radicals. During this defensive process, GSH-Px converts
reduced glutathione to its oxidized form. The decreased
GSH-Px activity in PTZ treated mice seen in our study indi-
cates that there was an increased generation of free radicals
and the decreased GSH-Px was depleted during the process
of combating oxidative stress. There was a marked increase
in the SOD and GSH-Px activities in the group treated with
compounds 9, 12 and 17. The results are summarized in Table
4. These findings made us consider the possibility that the
decrease in MDA levels and increase in the SOD and GSH-
Px activities in compounds 9, 12, 17 and valproate treated
groups may be due to the antioxidant property associated
with the semicarbazides and valproate.
Compound
Intraperitoneal injection in micea (h)
MES screen scPTZ screen
NT screen
0.5
4
0.5
4
0.5
4
4
300
e
e
300
300
e
100
e
300
e
5
e
eb
e
6
300
e
e
e
eb
e
300
e
e
e
7
eb
e
8
300
100
e
100
300
100
e
300
100
300
100
300
e
300
100
300
eb
100
300
e
100
100
300
100
300
100
e
100
300
e
300
300
100
e
100
100
300
300
e
9
10
100
e
300
11
12
100
300
eb
13
14
e
15
16
300
e
300
e
300
300
100
300c
300
300
300
300
30
300
300
300
300
300
300
e
300
e
17
18
300
300
e
300
e
300
e
4. Conclusions
19
20
e
100
300
100
e
In conclusion, we have identified a new series of N4-(naph-
tha[1,2-d]thiazol-2-yl)semicarbazides that are active against
tonic convulsions induced by electrical and chemical stimuli
in mice and rats. Biochemical investigations showed that
preventing oxidative injury by semicarbazides produces im-
portant neuroprotective and anticonvulsive effects against
PTZ-induced NT. Some of the most potent compounds appear
worthy of a further investigation aimed at assessing their anti-
convulsant activity in other models and at elucidating the un-
derlying mechanism of action.
e
e
300
300
e
21
e
e
300
e
Ethosuximided
Phenobarbitald
Phenytoind
Carbamazepined
Valproic acidd
a
e
100
30
30
30
100
e
30
100
100
100
e
300
100
300
e
e
e
30
100
300
300
e
e
Doses of 30, 100 and 300 mg/kg were administered. The figures in the ta-
ble 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 administra-
tion. The dash (e) indicates an absence of activity at maximum dose admin-
istered (300 mg/kg).
b
Death following tonic extension.
Myoclonic jerks.
Data from Refs. [31,46,47].
c
5. Experimental protocols
d
5.1. Chemistry
enhanced oxidative stress in brain tissue. When PTZ was
administered, the content of dihydroxy butyric acid reflected
the OHꢁ generation during the development of the convulsions,
or after fully developed seizures [6], this demonstration of
early seizure-induced OHꢁ formation fulfils the critical require-
ment in establishing the role of reactive oxygen species in the
pathophysiology of epilepsy.
The elevated level of MDA, a marker of lipid peroxidation,
indicates increased free radical generation in the PTZ treated
mice. PTZ-induced increment in MDA content of the tissue
was significantly prevented by the treatment of valproate as
well as compounds 9, 12 and 17. There was a simultaneous
significant decrease in the SOD and GSH-Px activities in
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 chroma-
tography with F254 silica-gel precoated sheets (Merck) using
chloroform/methanol 95/5 as eluent; UV light and iodine
Table 4
Biochemical estimation from brain homogenate
Compound
Lipid peroxidation
(pmol MDA/mg
protein)
GSH-Px
SOD
(Units/mg
protein)
(nM NADPH
oxidized/mg
protein)
Table 3
Evaluation of some compounds in the scPTZ test after oral administration
(50 mg/kg) to rats
9a
292 ꢃ 15*
278 ꢃ 13*
309 ꢃ 12
216 ꢃ 11*
418 ꢃ 17*
209 ꢃ 14
13.23 ꢃ 2.14*
12.38 ꢃ 1.93*
14.37 ꢃ 1.14*
17.81 ꢃ 2.31*
8.21 ꢃ 1.09*
18.41 ꢃ 2.40
60 ꢃ 15*
57 ꢃ 17*
58 ꢃ 13*
65 ꢃ 16
12a
17a
Compound
Oral administration to ratsa (h)
Valproic acid
PTZ
Control
0.25
0.5
1.0
2.0
42 ꢃ 11*
68 ꢃ 12
9
12
3
3
2
0
3
2
2
2
2
2
1
1
1
1
0
1
Each value represents the mean ꢃ SEM from 6 animals in each group;
*P < 0.05 significant as compared to control and the remaining values are
not significant at P < 0.05 (Student’s t-test).
17
Ethosuximide
a
a
The figures indicate the number of rats out of four which were protected.
Tested at 100 mg/kg i.p.

208
F. Azam et al. / European Journal of Medicinal Chemistry 44 (2009) 203e211
vapours were used for detection. IR spectra were recorded, as
KBr pellets, on a Schimadzu 8201 PC FT-IR spectrophotome-
ter and wave numbers are given in cmꢀ1. The mass spectra
reaction and the resultant precipitate was filtered and dried.
The product was recrystallized from 95% ethanol. The physi-
cal data of the compounds are presented in Table 1. The spec-
tral data of the compounds are the following.
1
were recorded on Jeol SX-102 (FAB). H NMR spectra, in
DMSO-d6 and 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
(ꢂC) were determined with an open glass capillary tube and
are uncorrected. Elemental analyses were performed on Ele-
mentar Vario EL III instrument.
5.1.4.1. 1-Benzylidene-4-(naphtha[1,2-d]thiazol-2-yl)semicar-
bazide (4). IR (KBr) nmax 3399, 3182, 3039, 2928, 1703,
1
1608, 1548, 1363, 1188 cmꢀ1; H NMR (CDCl3) d 7.29e8.27
(m, 10H, ArH), 8.38 (d, 1H, J ¼ 7.9 Hz), 9.66 (s, 1H, imine
H), 10.01 (s, 1H, CONH, D2O exchangeable), 10.89 (s, 1H,
NHNH2, D2O exchangeable); MS (FAB) m/z 347 (M þ 1)þ.
5.1.1. Synthesis of naphtha[1,2-d]thiazol-2-amine (1)
To N-naphthylthiourea (0.05 mol) in 100 ml glacial acetic
acid was added bromine (0.05 mol) in 10 ml glacial acetic
acid at 10 ꢂC during 1 h. After being stirred at room tempera-
ture for 4 h, the reaction mixture was diluted with hot water, the
solid impurities were filtered off and the filtrate was basified
with NH4OH when a soft base crystallizeable from acetonitrile
(5.8 g, 58%), m.p. 191 ꢂC, was obtained. Lit. [46] m.p. 190 ꢂC.
5.1.4.2. 1-(4-Methoxybenzylidene)-4-(naphtha[1,2-d]thiazol-2-
yl)semicarbazide (5). IR (KBr) nmax 3379, 3205, 3093, 2956,
1705, 1608, 1539, 1502, 1369, 1253, 1107, 790 cmꢀ1 1H
;
NMR (DMSO-d6) d 3.89 (s, 3H, OCH3), 6.96e7.99 (m, 9H,
ArH), 8.63 (d, 1H, J ¼ 7.5 Hz), 8.92 (s, 1H, imine H), 9.87
(s, 1H, CONH, D2O exchangeable), 10.96 (s, 1H,]NNH,
D2O exchangeable); MS (FAB) m/z 377 (M þ 1)þ.
5.1.2. Synthesis of phenyl-N-(naphtha[1,2-d]
thiazol-2-yl)carbamate (2)
5.1.4.3. 1-(2,5-Dimethoxybenzylidene)-4-(naphtha[1,2-d]thia-
zol-2-yl)semicarbazide (6). IR (KBr) nmax 3379, 3205, 3093,
2960, 1695, 1583, 1541, 1498, 1365, 1263, 1222, 1109,
To a solution of phenylchloroformate (0.1 mol) in dichloro-
methane (40 ml) was added 0.1 mol of naphtha[1,2-d]thiazol-
2-amine (1) and triethylamine (0.1 mol) dropwise and stirred
at the room temperature for 6 h. The reaction mixture was con-
centrated to one-third volume and, after cooling, 100 ml of
petroleum ether (40e60) was added to the reaction mixture.
The precipitate appeared immediately, which was filtered,
washed with a large quantity of water and dried. Yield 89%;
m.p. 286 ꢂC; IR (KBr) nmax 3148, 3043, 2867, 1739, 1536,
1018, 804, 746 cmꢀ1 1H NMR (CDCl3) d 3.85 (s, 3H,
;
ArOCH3), 3.91 (s, 3H, ArOCH3), 6.87e7.95 (m, 8H, ArH),
8.26 (s, 1H, H6-phenyl), 8.65 (s, 1H, imine H), 9.32 (s, 1H,
CONH, D2O exchangeable), 10.57 (s, 1H, ]NNH, D2O
exchangeable); MS (FAB) m/z 407 (M þ 1)þ.
5.1.4.4. 1-(3,5-Dimethoxybenzylidene)-4-(naphtha[1,2-d]thia-
zol-2-yl)semicarbazide (7). IR (KBr) nmax 3379, 3205, 3093,
3001, 2937, 1701, 1541, 1514, 1377, 1267, 1137, 1024, 800,
1
1483, 1218 cmꢀ1; H NMR (CDCl3) d 7.04e7.95 (m, 10 H,
1
ArH), 8.66e8.68 (d, 1H, J ¼ 7.3 Hz), 10.68 (s, 1H, CONH,
729 cmꢀ1; H NMR (CDCl3) d 3.51 (s, 3H, ArOCH3), 3.61
D2O exchangeable); MS (FAB) m/z 321 (M þ 1)þ.
(s, 3H, ArOCH3), 6.99 (d, 1H, J ¼ 8.2 Hz), 6.78 (d, 1H,
J ¼ 8.1 Hz), 7.01e7.54 (m, 6H, ArH), 8.19 (d, 1H,
J ¼ 7.9 Hz), 8.51 (s, 1H, imine H), 9.52 (br s, 1H, CONH,
D2O exchangeable), 10.61 (s, 1H, ]NNH, D2O exchange-
able); MS (FAB) m/z 407 (M þ 1)þ.
5.1.3. Synthesis of N4-(naphtha[1,2-d]
thiazol-2-yl)semicarbazide (3)
To a solution of 0.1 mol of phenyl-N-(naphtha[1,2-d]thia-
zol-2-yl)carbamate (2) in dichloromethane (100 ml), 9.7 ml
of hydrazine hydrate (0.2 mol) was added dropwise and the
mixture was stirred at room temperature for 24 h. The precip-
itate of 4-(naphtha[1,2-d]thiazol-2-yl)semicarbazide was sepa-
rated out, filtered, washed with dichloromethane, and dried.
Yield 89%; m.p. 198 ꢂC; IR (KBr) nmax 3340, 3267, 3222,
5.1.4.5. 1-(3,4,5-Trimethoxybenzylidene)-4-(naphtha[1,2-d]thia-
zol-2-yl)semicarbazide (8). IR (KBr) nmax 3388, 3215, 3093,
1
3001, 1701, 1577, 1541, 1375, 1332, 1134, 999, 796 cmꢀ1; H
NMR (CDCl3) d 3.89 (s, 3H, 4-OCH3), 3.97 (s, 6H, 3,5-
OCH3), 6.94e6.99 (m, 7H, ArH), 8.61 (d, 1H, J ¼ 7.7 Hz),
8.96 (s, 1H, imine H), 9.91 (br s, 1H, CONH, D2O exchange-
able), 11.10 (s, 1H, ]NNH, D2O exchangeable); MS (FAB)
m/z 437 (M þ 1)þ.
3103, 2923, 1701, 1600, 1537, 1367, 1153 cmꢀ1; H NMR
1
(CDCl3) d 6.76 (s, 2H, NH2, D2O exchangeable), 7.49e8.23
(m, 5H, ArH), 8.50e8.52 (d, 1H, J ¼ 7.4 Hz), 10.78 (s, 1H,
CONH, D2O exchangeable), 11.21 (s, 1H, NHNH2, D2O
exchangeable); MS (FAB) m/z 259 (M þ 1)þ.
5.1.4. General method for the synthesis of N4-(naphtha[1,2-
d]thiazol-2-yl)semicarbazides (4e21)
To a solution of 0.002 mol of N4-(naphtha[1,2-d]thiazol-2-
yl)semicarbazide (3) in ethanol (25 ml), an equimolar quantity
of appropriate aldehyde or ketone or isatin in ethanol (5 ml)
and glacial acetic acid (1 ml) were added. The mixture was re-
fluxed with stirring for 1e6 h until the completion of the
5.1.4.6. 1-(4-Bromobenzylidene)-4-(naphtha[1,2-d]thiazol-2-
yl)semicarbazide (9). IR (KBr) nmax 3390, 3205, 3093, 2960,
1
1701, 1542, 1375, 1323, 1070, 798 cmꢀ1; H NMR (CDCl3)
d 7.52e7.95 (m, 9H, ArH), 8.01 (s, 1H, imine H), 8.62 (d, 1H,
J ¼ 8.0 Hz), 10.18 (s, 1H, CONH, D2O exchangeable), 11.32 (s,
1H, ]NNH, D2O exchangeable); MS (FAB) m/z 426 (M þ 1)þ.
5.1.4.7. 1-(2-Chlorobenzylidene)-4-(naphtha[1,2-d]thiazol-2-
yl)semicarbazide (10). IR (KBr) nmax 3388, 3195, 3082,

F. Azam et al. / European Journal of Medicinal Chemistry 44 (2009) 203e211
209
2962, 1705, 1583, 1542, 1375, 1323, 792, 748, 729 cmꢀ1; H
NMR (CDCl3) d 7.38e7.96 (m, 7H, ArH), 8.33 (s, 1H, H3-
phenyl), 8.49 (s, 1H, H6-phenyl), 8.62 (d, 1H, J ¼ 7.6 Hz),
9.61 (s, 1H, imine H), 10.38 (br s, 1H, CONH, D2O exchange-
able), 11.47 (s, 1H, ]NNH, D2O exchangeable); MS (FAB)
m/z 382 (M þ 1)þ.
exchangeable), 11.58 (s, 1H, ]NNH, D2O exchangeable);
MS (FAB) m/z 392 (M þ 1)þ.
1
5.1.4.14. 1-(4-Nitrobenzylidene)-4-(naphtha[1,2-d]thiazol-2-
yl)semicarbazide (17). IR (KBr) nmax 3338, 3201, 2960,
1701, 1587, 1550, 1517, 1344, 1161, 1107, 800, 746 cmꢀ1; 1H
NMR (CDCl3) d 7.42e7.90 (m, 10H, ArH), 9.80 (s, 1H, imine
H), 10.10 (br s, 1H, CONH, D2O exchangeable), 11.53 (s, 1H,
]NNH, D2O exchangeable); MS (FAB) m/z 392 (M þ 1)þ.
5.1.4.8. 1-(4-Chlorobenzylidene)-4-(naphtha[1,2-d]thiazol-2-
yl)semicarbazide (11). IR (KBr) nmax 3384, 3205, 3093,
2964, 1701, 1585, 1541, 1373, 1323, 1149, 1085, 798 cmꢀ1
;
1H NMR (CDCl3) d 7.39e8.01 (m, 9H, ArH), 8.62 (d, 1H,
J ¼ 7.9 Hz), 9.38 (s, 1H, imine H), 10.14 (s, 1H, CONH,
D2O exchangeable), 11.30 (s, 1H, ]NNH, D2O exchange-
able); MS (FAB) m/z 382 (M þ 1)þ.
5.1.4.15. 4-(Naphtha[1,2-d]thiazol-2-yl)-1-(1-p-tolylethylide-
ne)semicarbazide (18). IR (KBr) nmax 3390, 3192, 3082,
2920, 1697, 1533, 1485, 1369, 1272, 1137, 794 cmꢀ1 1H
;
NMR (CDCl3) d 2.34 (s, 3H, N]CHeCH3), 2.40 (s, 3H,
AreCH3), 7.24 (d, 2H, J ¼ 7.6 Hz), 7.51e7.95 (m, 7H,
ArH), 8.59 (d, 1H, J ¼ 7.9 Hz), 10.32 (s, 1H, CONH, D2O
exchangeable), 10.99 (s, 1H, ]NNH, D2O exchangeable);
MS (FAB) m/z 375 (M þ 1)þ.
5.1.4.9. 1-(4-Fluorobenzylidene)-4-(naphtha[1,2-d]thiazol-2-
yl)semicarbazide (12). IR (KBr) nmax 3379, 3195, 3093,
2960, 1701, 1602, 1541, 1375, 1234, 1016 cmꢀ1
;
1H
NMR (CDCl3) d 6.92e8.44 (m, 10H, ArH), 9.16 (s, 1H,
imine H), 9.85 (s, 1H, CONH, D2O exchangeable), 11.05
(s, 1H, ]NNH, D2O exchangeable); MS (FAB) m/z 365
(M þ 1)þ.
5.1.4.16. 4-(Naphtha[1,2-d]thiazol-2-yl)-1-(1-phenylethylide-
ne)semicarbazide (19). IR (KBr) nmax 3390, 3192, 3082,
2920, 1697, 1533, 1485, 1369, 1272, 1137, 794 cmꢀ1 1H
;
NMR (CDCl3) d 2.41 (s, 3H, N]CHeCH3), 7.27e7.93 (m,
10H, ArH), 8.58 (d, 1H, J ¼ 7.9 Hz), 9.34 (s, 1H, CONH,
D2O exchangeable), 10.17 (s, 1H, ]NNH, D2O exchange-
able); MS (FAB) m/z 361 (M þ 1)þ.
5.1.4.10. 1-(2-Hydroxybenzylidene)-4-(naphtha[1,2-d]thiazol-
2-yl)semicarbazide (13). IR (KBr) nmax 3413, 3226, 3082,
1691, 1580, 1488, 1365, 1274, 1211, 952, 819 cmꢀ1 1H
;
NMR (CDCl3) d 6.88e7.96 (m, 9H, ArH), 8.32 (s, 1H, imine
H), 8.58 (d, 1H, J ¼ 7.8 Hz), 9.93 (s, 1H, CONH, D2O ex-
changeable), 11.01 (s, 1H, ]NNH, D2O exchangeable),
12.32 (s, 1H, OH); MS (FAB) m/z 363 (M þ 1)þ.
5.1.4.17. 1-(4-(Dimethylamino)benzylidene)-4-(naphtha[1,2-
d]thiazol-2-yl)semicarbazide (20). IR (KBr) nmax 3390,
3196, 3093, 2939, 1705, 1610, 1537, 1504, 1363,
;
1182 cmꢀ1 1H NMR (CDCl3) d 3.05 [s, 6H, N(CH3)2],
6.72e7.95 (m, 9H, ArH), 8.64 (d, 1H, J 7.9 Hz), 8.70 (s,
1H, imine H), 9.37 (s, 1H, CONH, D2O exchangeable),
10.56 (s, 1H, ]NNH, D2O exchangeable); MS (FAB) m/z
390 (M þ 1)þ.
5.1.4.11. 1-(4-Hydroxybenzylidene)-4-(naphtha[1,2-d]thiazol-
2-yl)semicarbazide (14). IR (KBr) nmax 3467, 3328, 3195,
2945, 1708, 1608, 1541, 1506, 1377, 1267, 1166, 833,
796 cmꢀ1
;
1H NMR (CDCl3) d 6.7 (d, 2H, J ¼ 8.5 Hz),
7.32e7.76 (m, 6H, ArH), 8.43 (d, 1H, J ¼ 7.8 Hz), 9.35 (s,
1H, imine H), 9.78 (s, 1H, CONH, D2O exchangeable),
10.84 (s, 1H, ]NNH, D2O exchangeable), 12.21 (s, 1H,
OH); MS (FAB) m/z 363 (M þ 1)þ.
5.1.4.18. 4-(Naphtha[1,2-d]thiazol-2-yl)-1-(2-oxoindolin-3-yli-
dene)semicarbazide (21). IR (KBr) nmax 3159, 3047, 2898,
1
1726, 1691, 1606, 1548, 1467, 1296, 1190, 1053 cmꢀ1; H
NMR (DMSO-d6) d 6.75e7.76 (m, 9H, ArH), 7.95 (d, 1H,
J ¼ 6.9 Hz), 8.26 (s, 1H, CONH-indolin, D2O exchangeable),
9.67 (s, 1H, CONH, D2O exchangeable), 10.44 (s, 1H,
]NNH, D2O exchangeable); MS (FAB) m/z 388 (M þ 1)þ.
5.1.4.12. 1-(4-Hydroxy-3-methoxybenzylidene)-4-(naphtha[1,
2-d]thiazol-2-yl)semicarbazide (15). IR (KBr) nmax 3350,
3205, 3093, 2941, 1695, 1585, 1541, 1371, 1265, 1012,
1
734 cmꢀ1; H NMR (CDCl3) d 3.92 (s, 3H, ArOCH3), 6.82
5.2. Pharmacological evaluation
(d, 1H, J ¼ 8.0 Hz), 7.03 (d, 1H, J ¼ 7.9 Hz), 7.42e7.55 (m,
2H, ArH), 7.62 (d, 1H, J ¼ 8.6 Hz), 7.75 (d, 1H, J ¼ 8.6 Hz),
7.84 (d, 2H, J ¼ 6.4 Hz), 8.52 (d, 1H, J ¼ 7.9 Hz), 8.69 (s,
1H, imine H), 8.74 (s, 1H, CONH, D2O exchangeable), 9.93
(s, 1H, ]NNH, D2O exchangeable), 10.95 (s, 1H, OH); MS
(FAB) m/z 393 (M þ 1)þ.
All the compounds were screened for anticonvulsant activ-
ity 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
5.1.4.13. 1-(2-Nitrobenzylidene)-4-(naphtha[1,2-d]thiazol-2-
yl)semicarbazide (16). IR (KBr) nmax 3467, 3388, 3195,
3072, 2960, 1685, 1587e1477, 1375, 1334, 1157, 1018,
Anticonvulsant evaluation of semicarbazones was under-
taken by following the National Institute of Health (NIH)
Anticonvulsant Drug Development (ADD) Program protocol
[53,54]. Male Swiss albino mice (18e25 g) and male albino
rats (Wistar, 100e125 g) were used as experimental animals.
800, 740 cmꢀ1 1H NMR (CDCl3) d 7.25e8.62 (m, 10H,
;
ArH), 9.78 (s, 1H, imine H), 10.22 (br s, 1H, CONH, D2O

210
F. Azam et al. / European Journal of Medicinal Chemistry 44 (2009) 203e211
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 1e4 animals. Some selected com-
pounds 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.
injection. The brains were quickly removed and were washed
twice with cold saline solution, placed into glass bottles, la-
beled, and stored in a deep freeze (ꢀ30 ꢂC) until processing
(maximum 10 h). Tissues were homogenized in four volumes
of ice-cold TriseHCl buffer (50 mM, pH 7.4) using a glass
Teflon homogenizer (Ultra Turrax IKA T18 Basic, USA) after
cutting up the brains into small pieces with scissors (for 2 min
at 5000 rpm). MDA and protein levels activities were carried
out at this stage. The homogenate was then centrifuged at
5000g for 60 min to remove debris. Clear upper supernatant
fluid was taken and GSH-Px activity was carried out at this
stage. The supernatant solution was extracted with an equal
volume of an ethanol/chloroform mixture (5:3, v/v). After
centrifugation at 5000g 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 ꢂC.
5.2.1.1. MES test. MES seizures were elicited (Techno
Convulsiometer Model No. C-2, Techno Electronics, Luck-
now, 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 animals protected/number of animals tested.
5.2.2.2.1. Lipid peroxidation assay. The extent of lipid per-
oxidation in brain homogenates 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 ꢄ 105/min/cm as described by Ohkawa et al. [56].
Briefly, the homogenate was centrifuged at 3000g for 15 min
and supernatant was used for assay. Samples of 0.1 ml homo-
genate were 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. Following
these additions, tubes were mixed and heated at 95 ꢂC 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 2200g 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 pmol MDA/mg protein.
5.2.2.2.2. Superoxide dismutase (SOD) activity determina-
tion. Total (CueZn and Mn) SOD activity was determined
according to the method of Sun et al. [57]. The principle of
the method is based on the inhibition of nitroblue tetrazolium
(NBT) reduction by the xanthineexanthine oxidase system as
a superoxide generator. Activity was assessed in the ethanol
phase of the brain homogenate after 1.0 ml ethanol/chloroform
mixture (5/3, v/v) was added to the same volume of sample
and centrifuged. One unit of SOD was defined as the enzyme
amount causing 50% inhibition in the NBT reduction rate.
SOD activity was expressed as units per mg protein.
5.2.2.2.3. Glutathione peroxidase (GSH-Px) activity deter-
mination. GSH-Px activity was measured spectrophotometri-
cally by a coupled enzyme procedure at 27 ꢂC monitoring
loss of NADPH at 340 nm as described by Lawrence and
Burk [58]. The homogenate was centrifuged at 4 ꢂC at 500g
for 15 min and the resulting supernatant at 10 000g for
20 min. The enzymatic reaction was conducted in 3 ml quartz
cuvettes of 1 cm path length in a PerkineElmer spectropho-
tometer. The reaction mixture contained 50 mM phosphate
buffer (pH 7.4), 4 mM NADPH, 1 unit glutathione reductase,
0.1 ml of supernatant sample and 0.7 mM H2O2 as substrate.
Reactions were initiated by the addition of H2O2. Direct pro-
portionality was seen with GSH-Px enzyme activity over time
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). A dose of 70 mg/kg scPTZ was used for rats. The
animals were observed during the following 4 h for the occur-
rence of seizures. A threshold convulsion is defined as one ep-
isode of clonic spasms which persists for at least 5 s. Absence
of even a threshold convulsion during the period of observa-
tion is taken as the endpoint in this test.
5.2.1.3. NT screening. The rotorod test was used to evaluate
NT. The mice were trained to stand on an accelerating rotarod
rotating at 10 revs./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 [55]. The dose at which
animals fell off the rotarod was determined. Before the begin-
ning of all experiments, the riding ability of 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.
5.2.2. Biochemical estimations
5.2.2.1. Animals. A total of 36 male Swiss albino mice (18e
25 g)wereusedinthisstudy. Theanimalswerehousedinatemper-
ature controlled room (22e25 ꢂC) with a 12:12 lightedark cycle;
water and food were given ad libitum. The experimental animals
were grouped as follows: Group 1: 0.9% NaCl, (n ¼ 6); Group
2: 0.9% NaCl 30 min prior to PTZ (n ¼ 6); Group 3: valproic
acid, 100 mg/kg i.p., 30 min prior to PTZ (n ¼ 6); Group 4: com-
pound 9, 100 mg/kg i.p., 30 min prior to PTZ (n ¼ 6); Group 5:
compound 12, 100 mg/kg i.p., 30 min prior to PTZ (n ¼ 6); Group
6: compound 17, 100 mg/kg i.p., 30 min prior to PTZ (n ¼ 6). PTZ
dissolved in 0.9% NaCl was injected at a dose of 85 mg/kg.
5.2.2.2. Sample preparation and biochemical evaluation.
Mice were sacrificed by decapitation 4 h after the last

F. Azam et al. / European Journal of Medicinal Chemistry 44 (2009) 203e211
[23] M.J. Wilby, P.J. Hutchinson, CNS Drug Rev. 10 (2004) 281e294.
211
and with sample concentration. GSH-Px activity was esti-
mated from the decrease of optical density at 340 nm due to
NADPH oxidation between 2 and 4 min after the start of the
reaction. The results are expressed as nmol NADPH oxi-
dized/mg protein. One unit of the GSH-Px activity is defined
as the amount of enzyme necessary to catalyze 1 nmol
NADPH/min g wet weight at 27 ꢂC.
5.2.2.2.4. Total protein determinations. Total protein con-
centration of brain homogenates was determined by foline
phenol reaction as described by Lowry et al. [59]. The bovine
serum albumin was used as a standard.
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