Synthesis and evaluation of some novel methylene-bridged aryl semicarbazones as potential anticonvulsant agents

Article abstract of DOI:10.1007/s00044-011-9924-6

A series of aryl semicarbazones containing a methylene bridge in their skeleton were synthesized as anticonvulsant agents. The strategy of introducing a methylene bridge was to increase the flexibility of the structures because of sp3 hybridization. Pharmacological evaluations of the compounds were performed by determination of their effects on pentylenetetrazole-induced seizure parameters and neurotoxicity in mice. The statistical analysis indicated that most of the synthesized compounds showed significant anticonvulsant activity in comparison with the control group. No remarkable neurotoxicity was observed in rotorod test. A QSAR study was performed using multiple linear regressions. The results of the QSAR study confirmed that the compounds with Br at para position of these new derivatives are more potent than para-ethoxy series. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9924-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-011-9924-6
ORIGINAL RESEARCH
Synthesis and evaluation of some novel methylene-bridged aryl
semicarbazones as potential anticonvulsant agents
•
Saeed Mozaffari Saeed Ghasemi Hoda Baher Hamidreza Khademi
•
•
•
•
Mohsen Amini Amirhossein Sakhteman Alireza Foroumadi
•
•
•
Abdolrasoul H. Ebrahimabadi Mohammad Sharifzadeh
Received: 25 July 2011 / Accepted: 18 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract A series of aryl semicarbazones containing a
methylene bridge in their skeleton were synthesized as
anticonvulsant agents. The strategy of introducing a meth-
ylene bridge was to increase the flexibility of the structures
because of sp3 hybridization. Pharmacological evaluations
of the compounds were performed by determination of their
effects on pentylenetetrazole-induced seizure parameters
and neurotoxicity in mice. The statistical analysis indicated
that most of the synthesized compounds showed significant
anticonvulsant activity in comparison with the control
group. No remarkable neurotoxicity was observed in roto-
rod test. A QSAR study was performed using multiple
linear regressions. The results of the QSAR study confirmed
that the compounds with Br at para position of these new
derivatives are more potent than para-ethoxy series.
Keywords Semicarbazones ꢀ Methylene bridge ꢀ
Anticonvulsant ꢀ Neurotoxicity ꢀ Pentylenetetrazole
S. Mozaffari ꢀ M. Amini ꢀ A. Foroumadi
Department of Medicinal Chemistry, Faculty of Pharmacy
and Pharmaceutical Sciences Research Center, Tehran
University of Medical Sciences, 14176 Tehran, Iran
Introduction
S. Ghasemi
Epilepsy is the third most common neurologic disorder after
stroke and dementia, and as a general term includes more
than 40 different types of seizure disorders characterized by
recurrent episodes of abnormal events. About 0.5–1% of the
world population are involved in this serious neurological
disorder at any one time (Sander, 2003; Yogeeswari et al.,
2005a, b). Despite an optimal drug therapy, approximately
one quarter of epileptic patients find an insufficient control
for their convulsions. Epilepsy is controlled in about 75% of
the patients, but 10% continue to have seizures at intervals of
1 month or less. This happens because of the lack of response
toward conventionally available medical therapies. In addi-
tion, the present drug therapies are associated with some
adverse effects, such as fatigue, drowsiness, headache,
mental confusion, cognitive impairment, ataxia, hirsutism,
gingival hyperplasia, megaloblastic anemia, and gastroin-
testinal disturbances. More than 25% of the patients dis-
continue the treatment because of the adverse drug reactions
caused by initial antiepileptic drugs (AED) (Perucca, 2005;
Yogeeswari et al., 2006; Kennedy and Lhatoo, 2008).
Department of Medicinal Chemistry, Faculty of Pharmacy,
Tabriz University of Medical Sciences, 51664-14766 Tabriz,
Iran
H. Baher ꢀ M. Sharifzadeh
Department of Neuroscience, Faculty of Advanced Science
and Technology in Medicine, Tehran University of Medical
Sciences, Tehran, Iran
H. Khademi ꢀ M. Sharifzadeh (&)
Department of Pharmacology and Toxicology,
Pharmaceutical Sciences Research Center, Faculty of Pharmacy,
Tehran University of Medical Sciences, P.O. Box 14155-6451,
Tehran 14176, Iran
e-mail: msharifzadeh@sina.tums.ac.ir
A. Sakhteman
Department of Medicinal Chemistry, Faculty of Pharmacy,
Shahid Sadoughi University of Medical Sciences,
Yazd, Iran
A. H. Ebrahimabadi
Essential Oils Research Center, University of Kashan,
Kashan, Iran
123

Med Chem Res
In the last 20 years, a novel designed group of com-
pounds, aryl semicarbazones and N4-substituted phenyl
aryl semicarbazones, have shown considerable anticon-
vulsant activities. Furthermore, these new compounds
showed no remarkable sedative-hypnotic effects and were
found to be less neurotoxic than usual AEDs (Pandeya
et al., 2000; Yogeeswari et al., 2003; Yogeeswari et al.,
2004; Yogeeswari et al., 2005a, b; Yogeeswari et al., 2006;
Shafiee et al., 2009). Recognized modeling on anticon-
vulsants has identified that one aryl unit, one or two elec-
tron donor atoms, and one NH group in correct spatial
arrangement are necessary for anticonvulsant activity. A
four-point pharmacophore model for anticonvulsant activ-
ity of AEDs that has been developed previously (Yogees-
wari et al., 2005a, b) includes the following:
with nine standard AEDs. It has been verified that aryl
semicarbazones fit to the essential parts of the pharmaco-
phore (Yogeeswari et al., 2005a, b). However, the distances
relating to pharmacophore A in aryl semicarbazones are
considerably different from those in routine AEDs.
One notable fact about aryl semicarbazones is attributed
to potential resonances in –NHCO– groups. It causes
duality in H-donor/-acceptor identity of the moieties at 1, 3,
and 4 positions because of the unstable amide protons
(Fig. 1). Therefore, the spatial requirements of all phar-
macophores can be anticipated by aryl semicarbazones. In
this regard, a new series of aryl semicarbazones with an
additional methylene bridge between N1 and the carbonyl
group (C2) of the semicarbazone skeleton were synthe-
sized. The reason for this modification was to introduce a
different distance between pharmacophores and a more
flexibility and lipophilicity in aryl semicarbazone structure.
The anticonvulsant and neurotoxicity properties of the
synthesized compounds were evaluated in comparison with
the control group.
(i) The aryl ring center or the lipophilic group with a
halogen substituent at para position (A)
(ii) An electron donor atom (D)
(iii) A hydrogen bond acceptor (HA)
(iv) A hydrogen bond donor (HD).
The limited lipophilic group (L) at N4-substitution
(Fig. 1) regulates the pharmacokinetic properties of aryl
semicarbazones. It could be considered as a subordinate or
the fifth pharmacophore together with the four essential
ones mentioned above. Compounds with the alkyl groups
such as methyl, ethyl, and acetomethyl replaced in L posi-
tion have displayed better anticonvulsant effects against
standard animal protocols (Yogeeswari et al., 2004;
Thirumurugan et al., 2006). In their suggested pharmaco-
phore model, the center of the aromatic ring is determined
as the reference point for A (Yogeeswari et al., 2005a, b;
Rajak et al., 2010). Several research groups have synthe-
sized different derivatives of these compounds by changing
the L and A binding sites to obtain more active and less
toxic anticonvulsant agents. On the contrary, very few
studies have been done about the distances and spatial
conformations in the pharmacophore structure of aryl
semicarbazones (Dimmock et al., 2000; Pandeya et al.,
2000; Yogeeswari et al., 2004; Yogeeswari et al., 2005a, b;
Yogeeswari et al., 2006; Azam et al., 2009; Azam et al.,
2010; Kaushik et al., 2010). The average distance range
between these four pharmacophore points has been previ-
ously calculated (Yogeeswari et al., 2005a, b). The common
structure of aryl semicarbazones has been superimposed
Results and discussion
Synthesis
Scheme 1 shows the synthesis of arylsemicarbazone
derivatives 3a(1–6) and 3b(1–6). The 4-substituted aniline
was reacted with 2-chloroacetamide in the presence of
Na2CO3 to give 1a, b. Acetohydrazide derivatives 2a,
b were prepared by an uncommon and slow reaction of 1a,
b with hydrazine hydrate as previously reported (Pandeya
et al., 1999; Dumciute et al., 2006; Singh et al., 2011).
Finally, aryl semicarbazones 3a(1–6) and 3b(1–6) were
synthesized by condensation of 2a and 2b with appropriate
aldehydes and ketones. The physicochemical characteris-
tics of the target compounds are given in Table 1.
Pharmacology
The anticonvulsant mechanism of the semicarbazones is
not clearly defined. However, it is proposed that they act
through inhibition of the GABA transaminase enzyme
(GABA-T) and consequently increase the GABA levels in
the brain. These compounds also affect seizure activity by
blocking the voltage-gated sodium ion channels (Dimmock
et al., 2000; Yogeeswari et al., 2005a, b; Thirumurugan
et al., 2006). The above functions are similar to well-
established mechanisms of anticonvulsant agents (Treiman,
D
O
HA
R
A
1
3
N
4
R2
2
N
N
H
H
L
R1
¨
2001; Bolcskei et al., 2008). Pentylenetetrazole (PTZ) by
HD
affecting the GABAA-gated chloride channels induces
seizure activities through stimulation of epileptogenic
Fig. 1 Proposed pharmacophore in aryl semicarbazones
123

Med Chem Res
Scheme 1 Synthesis of aryl
semicarbazone derivatives (3a,
b). Reagents and conditions:
(i) Na2CO3, EtOH, refluxed for
24 h; (ii) NH2NH2, EtOH,
refluxed for 24 h; (iii)
O
NH2
NH
NH2
O
i
Cl
NH2
Appropriate aldehyde or ketone,
EtOH, refluxed for 4 h
1a,b
R
R
a: R = Br
b: R = EtO
ii
R1
O
R2
O
NH N
NH NH2
NH
NH
iii
3a,b
2a,b
R
R
-1: R1 = H, R2 2-Nitrophenyl
-2: R1 = H, R2 = 4-Nitrophenyl
-3: R1 = H, R2 2-Hydroxyphenyl
-4: R1 = H, R2 2-Chlorophenyl
-5: R1 = Me, R2 Me
-6: R1 = Me, R2 Phenyl
Table 1 Physicochemical characteristics of finally synthesized compounds
Compounds
R
R1
R2
mp (8C)
Yield (%)
Mol. Formula
C15H13N4O3Br
Mol. Wt.
3a-1
3a-2
3a-3
3a-4
3a-5
3a-6
3b-1
3b-2
3b-3
3b-4
3b-5
3b-6
Br
H
2-Nitrophenyl
4-Nitrophenyl
2-Hydroxyphenyl
2-Chlorophenyl
Me
201–203
238–240
238–241
190–192
155–157
187–189
126–128
215–216
187–189
185–188
143–145
146–148
51
75
87
53
84
75
56
64
42
43
52
47
377.2
377.2
348.2
366.6
284.2
346.2
342.3
342.3
313.4
331.8
249.3
311.4
Br
H
C
C
C
C
C
C
C
C
C
C
C
15H13N4O3Br
15H14N3O2Br
Br
H
Br
H
15H13N3OBrCl
11H14N3OBr
16H16N3OBr
17H18N4O4
17H18N4O4
17H19N3O3
17H18N3O2Cl
13H19N3O2
18H21N3O2
Br
Me
Me
H
Br
Phenyl
EtO
EtO
EtO
EtO
EtO
EtO
2-Nitrophenyl
4-Nitrophenyl
2-Hydroxyphenyl
2-Chlorophenyl
Me
H
H
H
Me
Me
Phenyl
centers in the brain. PTZ-mediated seizure was used as a
clinically relevant experimental animal model for evalua-
tion of seizure latency, seizure duration, and death time
(DT) through acute administration of PTZ at CD97 (a dose
of PTZ to produce clonic seizures lasting for a period of at
least 5 s in 97% of animals tested) (Gholizadeh et al.,
2007).
seizure was 184 32 s for the control group. Our results
showed that administration of compounds 3a-1, 3a-5, 3a-6,
3b-1, 3b-3, and 3b-5 (P \ 0.01) and 3b-4 and 3b-6
(P \ 0.05) with a dose of 300 mg/kg significantly increased
seizure latency in comparison with control animals. In
addition, intraperitoneal injection of 100 mg/kg of com-
pounds 3b-2 (P \ 0.01) and 3a-2, 3a-3, 3b-3, and 3b-5
(P \ 0.05) significantly increased seizure latency. These
results are probably caused by an elevation of GABA levels
in the brain for –NO2–, –OH–, and alkyl-substituted deriv-
atives (Yogeeswari et al., 2005a, b, 2006; Rajak et al., 2010).
No significant alteration on seizure latency was found with
The target compounds were evaluated for anticonvulsant
activity against PTZ-induced tonic-clonic generalized sei-
zure. Their neurotoxicity was also measured by rotorod test.
The results for all derivatives of 3a, b on PTZ-induced sei-
zure parameters are reported in Tables 2 and 3. The onset of
123

Med Chem Res
the dose 50 mg/kg for some synthesized compounds
(Table 2).
of 100 mg/kg in comparison with control group (Table 2).
Based on our results, it can be deduced that substitution of
4-Br at R position in combination with each group of nitro-
phenyl, methyl, or phenyl at R2 position of these new syn-
thesized compounds leads to a better anticonvulsant effect
compared with the other functional groups.
The mean of total seizure duration for the control group
was 9.9 1.3 s. Evaluation of seizure duration indicated
that injection of compounds 3a-1, 3a-5, and 3b-5 (P \ 0.01)
and 3a-2 and 3a-6 (P \ 0.05) with dose 300 mg/kg signifi-
cantly decreased total seizure duration compared with con-
trol animals. Compounds 3a-2 and 3b-5 caused significant
(P \ 0.05) decrease on the total seizure duration at the dose
The mean time for incidence of death following PTZ
injection (death time) was 416 58.2 s in the control group.
It was indicated that DT significantly increased after
administration of 300 mg/kg of 3a-1, 3a-5, and 3b-2
(P \ 0.001), 3a-6 and 3b-3 (P \ 0.01), and 3a-2 and 3a-3
(P \ 0.05). Pretreatment of animalswith 100 mg/kg of 3a-2,
3a-3, 3a-6, and 3b-2 (P \ 0.001), 3b-1 and 3b-4 (P \ 0.01),
and 3a-4 and 3a-5 (P \ 0.05) also caused significant
increase in DT (Table 3). These results were also confirmed
with findings of onset of seizure and seizure duration.
It is important to consider that the substitution of –OH
group at R2 position of the 4-bromo series (3a-3) increased
DT at lower doses (50 and 100 mg/kg, P \ 0.001) more
than higher concentration (300 mg/kg, P \ 0.05). One
tentative explanation for this event could be related to
increased hydrogen bonding interactions with the receptors
at lower doses (Table 3).
Table 2 Effects of methylene bridge containing aryl semicarbazones
on PTZ-induced seizure parameters
Compound
Dose (mg/kg) Onset (s)
Total seizure
time (s)
Control (vehicle)
2 ml/kg
100
300
50
184 32
144 29
9.9 1.3
11 2.0
3a-1
1,073 224** 2.8 1.1**
3a-2
3a-3
640 229
809 293*
596 240
724 290
884 276*
562 271
403 101
514 330
224 50
6.1 1.5
3.8 1.4*
3.7 0.81*
7.7 2.2
6.6 2.0
8.6 2.2
6.4 1.5
4.5 1.5
10 2.0
100
300
50
100
300
100
300
100
300
100
300
100
300
50
Our findings also indicated that the compounds with
4-bromo group at R moiety are more effective than their
counterparts with 4-ethoxy at the same position (Tables 2,
3). It seems that the lipophilicity of these molecules plays
an important role in their anticonvulsant activity. The
lipophilic property of the 4-bromo substituent is better than
4-ethoxy and this effect might lead to more affinity toward
hydrophobic region of the receptor at A position of the
proposed pharmacophore. One more possible reason is the
improved diffusion of 4-Br-substituted compounds across
the blood-brain barrier.
3a-4
3a-5
3a-6
3b-1
3b-2
1,098 212** 3.3 1.1**
498 229 11 2.7
915 268** 3.1 1.1*
275 84 8.7 1.7
626 217** 6.8 1.7
603 205 5.3 1.2
885 219** 5.2 1.0
100
300
50
High dose (300 mg/kg) of compounds 3a-1, 3a-2, 3a-3,
3a-5, 3a-6, 3b-2, and 3b-3 that emerged as promising
anticonvulsant agents was further tested for acute neuro-
logical toxicity by the rotorod method. Evaluation of all
tested compounds on minimal motor impairment indicated
no significant neurotoxicity except for 3b-2 (Fig. 2). No
delayed mortality was observed during 1 week after
neurotoxicity assessment in treated mice.
643 243
275 46
7.3 2.5
11 1.5
6.4 0.92
3b-3
100
300
100
300
100
300
100
300
15
616 106*
903 198** 5.2 1.8
3b-4
436 230
590 192*
373 70*
425 66**
243 67
8.5 1.9
9.3 1.6
3b-5
4.9 0.67*
4.2 0.74**
5.9 0.92
5.8 0.70
3b-6
Quantitative structure activity relation
414 101*
Phenobarbitala
Sodium valproateb 220
1,088 319** 2.2 1.0**
1,207 267** 2.3 1.1**
To investigate the role of structural features on the anti-
seizure activity of the synthesized compounds, quantitative
structure activity relation (QSAR) studies were conducted.
The QSAR method was first developed by Hansch and
Fujita as a way of identifying the relationship between
compound structures and their activities (Kumar et al.,
2007). QSAR is based on this assumption that biological
activity is a mathematical function of molecular properties
All compounds were injected intraperitoneally to groups of mice at
presented doses 30 min before PTZ injection (80 mg/kg). Control
a
group received DMSO solution as vehicle. Phenobarbithal and
b
sodium valproate were injected as standard drugs. Data were shown
as mean SEM of eight animals in each group. * P \ 0.05,
** P \ 0.01 and *** P \ 0.001 show significant differences com-
pared with control animals
123

Med Chem Res
The common approaches used in QSAR studies are nor-
mally based on linear methods such as multiple linear
regression (MLR) and principal component regression
(PCR) or non-linear methods such as support vector
machines, neural networks, and fuzzy mappings (Jalali-
Heravi and Asadollahi-Baboli, 2009; Afiuni-Zadeh and
Azimi, 2010). For this purpose, the ligands were drawn in
chemdraw and saved as cdx file type. A primary 3D gen-
eration of the structures was done through iterative runs of
OpenBabel in Linux (Mandriva) operating system. The
structures were then entered into Hyperchem for geometry
optimization using AM1 semiempirical method. The con-
jugate gradient algorithm Polak Ribiere with the RMS
Table 3 Effects of methylene bridge containing aryl semicarbazones
on protection (%), survived (%), and death time parameters in PTZ-
induced seizure
Compound
Dose
(mg/kg)
Protection Survived Death time
a
b
%
%
(s)
Control
(vehicle)
2 ml/kg
0
0
416 58.2
3a-1
3a-2
100
300
50
0
33
0
38
33
62
62
50
100
100
25
25
29
25
64
86
50
12
14
50
44
67
12
0
897 276
1,265 171***
1,400 205***
1,456 188***
1,122 305*
1,800 0.00***
1,800 0.00***
832 237*
100
300
50
38
16
25
38
25
0
3a-3
˚
value 0.1 kcal/Aꢀmol was used during minimization.
100
300
100
300
100
300
100
300
100
300
50
Automation of minimization for different compounds was
done by means of an application implemented in VB.net
through dynamic data exchange with Hyperchem. The
resulted ligands were then subjected to Dragon Software
for calculation of descriptors. During this stage, up to 436
descriptors were calculated for each ligand. The descriptors
included constitutional, functional, properties, topological,
and 2D autocorrelations indices. The matrix of descriptors
(each row vector representing one ligand) together with the
p-function of the total seizure time at 300 mg/kg (activity
vector) was entered into Matlab for further statistical
evaluations. To decrease autocorrelations in the data, a
pretreatment step was performed. For this purpose, Pear-
son’s correlation for all descriptors was calculated. Among
the two descriptors with high co-linearity (correla-
tion [ 0.85), the one with more correlation with the
activity vector was retained and the other was excluded.
The number of the resulted descriptors after this step was
reduced to 28 descriptors. Correlations of some remained
descriptors with the activity vector are shown in Table 4.
The resulted matrix of the descriptors and the activity
vector were subsequently entered into SPSS for MLR
analysis using stepwise method. The two descriptors,
namely, T (N..Br) and MATS7e were selected to be in
good relationship with the activity vector. To obtain a more
reasonable model compound, 3b-3 was considered as an
outlier and excluded from the final model. The final MLR
equation is as follows:
3a-4
3a-5
3a-6
3b-1
3b-2
885 225*
14
0
781 242
945 203*
45
14
38
0
1,428 160***
1,577 223***
1,161 254**
1,011 206**
734 194
14
16
22
16
0
1,137 313*
1,286 201***
1,528 182***
659 183
100
300
50
3b-3
100
300
100
300
100
300
100
300
15
0
896 200
22
14
0
33
43
12
0
1,055 225**
1,189 291**
651 180
3b-4
3b-5
0
623 130
0
0
606 106
3b-6
0
0
310 71.3
0
0
483 93.6
Phenobarbital
50
50
100
100
1,800 0.00***
1,800 0.00***
Sodium valproate 220
All compounds were injected intraperitoneally to groups of mice at
presented doses 30 min before PTZ injection (80 mg/kg). Control
group received DMSO solution as vehicle. Phenobarbithal and
sodium valproate were injected as standard drugs. Data were shown
as mean SEM of eight animals in each group. * P \ 0.05,
** P \ 0.01 and *** P \ 0.001 show significant differences com-
pared with control animals
ꢁlogðactivityÞ ¼ f0:06ðTN::BrÞ ꢂ 0:002g
þ fꢁ1:493ðMATS7eÞ ꢂ 0:260g
þ fꢁ1:082 ꢂ 0:56g
a
Protection % = (number of mice was not afflicted with seizure/
number of tested mice in each group) 9 100
b
Survived % = (number of mice was alive/number of tested mice in
each group) 9 100. The time of death after PTZ injection was shown
as death time
r ¼ 0:941
To evaluate the predictive activity of the model, leave
one out cross-validation (LOO-CV) internal test was used.
During this procedure, iterative cycles of ligand elimina-
tion and model building were preceded by predicting the
activity of the eliminated ligand. The two metrics Rcv and
predicted residual error sum of squares (PRESS) were 0.88
called descriptors (Kumar et al., 2007). The quality of a
QSAR model depends on many factors including biological
data, the choice of the descriptors, and statistical methods.
123

Med Chem Res
Cont; control
(2 ml/kg)
200
150
100
50
test compounds
(300 mg/kg)
Phb; phenobarbital
(15 mg/kg)
*
Val; sodium valproate
(220 mg/kg)
0
Cont. 3a-1 3a-2 3a-3 3a-5 3a-6 3b-2 3b-3 Phb. Val.
Test compounds (300 mg/kg)
Fig. 2 Evaluation of effective synthesized compounds on neurotox-
icity. Tested compounds were intraperitoneally injected to groups of
mice at presented doses. Neurologic toxicity was defined as the failure
of animals to remain on the rod for three trials. Each trial continues
for 1 min. The equilibrium time or time that each animal remains on
the rod was measured for total duration of three trials (180 s). Each
value represents the mean SEM of seven animals. Control group
received vehicle with the same volume (DMSO solution). Phenobar-
bithal (15 mg/kg) and sodium valproate (220 mg/kg) were intraper-
itoneally injected as standard drugs. *P \ 0.05 shows significant
difference compared with control group
and 0.073, respectively (Fig. 3). The result of CV internal
validation revealed the stability of the model to be in a
reasonable level.
(100 mg/kg) of compounds 3a-2 and 3a-3 showed better
anticonvulsant capacity in PTZ-induced seizure compared
with the higher concentration (300 mg/kg). This fact is
more obvious at death time. This kind of activity was
attributed to the presence of 4-nitrophenyl and
2-hydroxyphenyl groups as R2 substituent of these new
derivatives in combination with 4-Br as R substituent. The
result of the QSAR study indicates that the compounds
with bromine at para position are more potent than their
counterparts with ethoxy group. As a consequence of
QSAR study, two structural characteristics of these com-
pounds are important in their activities. The first is the
topological distances between N and Br and the second one
is atomic Sanderson’s electronegativity of the substituent.
The obtained mathematical equation can be used for
designing of other structures in the future studies. In all
experiments, none of these new derivatives showed more
protection than the standard drugs. However, 3a-1, 3a-5,
3a-6, and 3b-3 at the dose of 300 mg/kg and 3a-2, 3a-3,
and 3b-2 at the dose of 100 mg/kg were comparable with
the standard drugs. Neurotoxicity evaluation by rotorod test
showed that all compounds except 3b-2 did not cause any
neurological toxic effect in comparison with control group
and standard drugs.
The descriptor T (N..Br) corresponds to sum of topo-
logical distances between N and Br and is among topo-
logical descriptors. MATS7e corresponds to Moran
autocorrelation -lag7/weighted by atomic Sanderson’s
electronegativites and is among 2D autocorrelation
descriptors. The result of this study is in accord with the
fact that the compounds with bromine at para position are
more potent than their counterparts with ethoxy group. It
seems that the presence of different substituents on the
second phenyl ring can alter the conformation of the
resulted structures (Fig. 4). In this regard, the topological
distances between Br and N and consequently the activity
of the final compounds could be influenced.
Conclusion
In this study, a number of methylene-bridged aryl semi-
carbazones were designed, synthesized, and characterized
by spectral analysis. They were evaluated for preliminary
anticonvulsant activity using PTZ animal model and
rotorod neurotoxicity screening after intraperitoneal
administration.
In general, 3a-1, 3a-5, 3a-6, 3b-1, 3b-3, 3b-5, and 3b-6
derivatives exhibited better anticonvulsant activities
against PTZ-induced seizure at high dose (300 mg/kg) in
comparison with the lower concentrations (100 or 50 mg/
kg). The activity was related to the existence of methyl
groups as R1 substituent and 2-nitrophenyl, methyl, phe-
nyl, and 2-hydroxyphenyl groups as R2 substituent in new
derivatives structure. Administration of moderate dose
Experimental
Chemistry
All reagents and solvents were used from commercially
available sources (Sigma and Merck). Capillary tubes on an
electrothermal melting point were used to determine melting
point. Proton nuclear magnetic resonance (1H NMR), 13C
123

Med Chem Res
123

Med Chem Res
General procedure for the synthesis of 2-(arylamino)
acetamide (1a,b)
In a one-neck flask containing 100 ml absolute ethanol,
4-substituted aniline (0.05 mol), sodium carbonate (2.65 g,
0.025 mol), and 2-chloroacetamide (4.67 g, 0.05 mol)
were added and refluxed for 24 h with stirring (Pandeya
et al., 2000). The solvent was evaporated and 40 ml dis-
tilled water was added to residue. The precipitate was fil-
tered off, washed with extra water, and recrystallized from
90% ethanol.
2-(4-Bromophenylamino) acetamide (1a) Yield 60%, mp:
136–138°C. IR (KBr, mmax, cm-1): 3400 (N–H, NH2),
3217 (C–H, Ar), 1670 (C=O); 1HNMR (500 MHz, DMSO-
d6) (ppm) ?d: 7.66 (s, 1H, CONH2), 7.13 (d, 2H,
J = 8.1 Hz, Ar–H), 6.72 (s, 1H, CONH2), 6.45(d, 2H,
J = 8.1 Hz, Ar–H), 4.40(s, 2H, CH2), 3.61(s, 1H, Ar–NH);
Elem. Anal. Calcd. for C8H9N2OBr; C: 41.95, H: 3.96, N:
12.23, found C: 42.17, H: 3.59, N: 12.45
Fig. 3 Plot of predicted values versus experimental values for the
activity vector
2-(4-Ethoxyphenylamino) acetamide (1b) Yield 56%,
mp: 146–148°C. IR (KBr, mmax, cm-1): 3399 (N–H, NH2),
2982 (C–H, CH2), 2902 (C–H, CH3), 1647 (C=O), 1515
1
(C=C, Ar); HNMR (500 MHz, DMSO-d6) (ppm) d: 7.28
(s, 1H, CONH2), 7.07 (s, 1H, CONH2), 6.69 (d, 2H,
J = 8.4 Hz, Ar–H), 6.46 (d, 2H, J = 8.4 Hz, Ar–H), 5.45
(s, 1H, Ar–NH), 3.86 (q, 2H, J = 6.4 Hz, CH2O), 3.49 (s,
2H, CH2), 1.23 (tr, 3H, J = 6.4 Hz, CH3); Elem. Anal.
Calcd. for C10H14N2O2; C: 61.84, H: 7.27, N: 14.42. found
C: 61.97, H: 7.51, N: 14.12.
General procedure for the synthesis of 2-(arylamino)
acetohydrazide (2a,b)
Fig. 4 Superimposition of compounds 3a-1 (front molecule) and 3a-
3 (back molecule). The presence of different substituents on the
second ring can affect the activity of the resulted structures because of
changes in molecular features such as T(N..Br)
A solution of compound 1a-b (0.03 mol) in 50 ml of abso-
lute ethanol was added dropwise to hydrazine hydrate
(14.5 ml, 10 mol) with vigorous stirring and the mixture was
refluxed for 24–36 h until the reaction was completed
(Pandeya et al., 2000; Dumciute et al., 2006). The solvent
was evaporated and distilled water (50 ml) was added to the
mixture. The white resultant precipitate was filtered, washed
with extra water, and crystallized in 90% ethanol twice.
nuclear magnetic resonance (13C NMR), and infrared were
1
used for structure elucidation. H NMR and FT-IR spectra
were obtained by Brucker advance (500 MHz) and Mgna-
IR-550 instruments, respectively. 13C NMR spectra were
obtained by Brucker advance (125 MHz). Chemical shifts
(d) are reported in parts per million (ppm) by using tetra-
methyl silane as internal standard. Elemental analyses (C, H,
and N) were undertaken with Perkin–Elmer model 240C
analyzer. Ascending thin layer chromatography (TLC) on
silica gel G (Merck) was used to follow-up the reactions for
purity and completion. Iodine vapor and UV light were used
for visualization of TLC spots. Purification of derivatives
was performed on silica gel 60 (particle size 0.06–0.2).
2-(4-Bromophenylamino) acetohydrazide (2a) Yield
50%, mp: 143–145°C. IR (KBr, mmax, cm-1): 3322 and
3296 (N–H), 1684 (C=O); HNMR (500 MHz, DMSO-d6)
1
(ppm) d: 9.14 (s, 1H, CONH), 7.18 (d, 2H, J = 8.0 Hz,
Ar–H), 6.51 (d, 2H, J = 8.0 Hz, Ar–H), 6.08 (s, 1H,
Ar–NH), 4.26 (s, 2H, CH2), 3.63(s, 2H, NH2); Elem. Anal.
Calcd. for C8H10N3OBr; C: 39.37, H: 4.13, N: 17.22. found
C: 39.05, H: 4.20, N: 17.33.
123

Med Chem Res
2-(4-Ethoxyphenylamino) acetohydrazide (2b) Yield
44%, mp: 132–134°C. IR (KBr, mmax, cm-1): 3346 and
3301 (N–H), 2980 (C–H, CH2), 2869 (C–H, CH3), 1655
2-(4-Bromophenylamino)-N0-(2-hydroxybenzylidene) acet-
ohydrazide (3a-3) Yield 87%, mp: 238–241°C. IR (KBr,
mmax, cm-1): 3438 and 3175 (N–H), 3081 (C–H, Ar), 1676
(C=O); 1HNMR (500 MHz, DMSO-d6) (ppm) d: 11.40 (bs,
2H, Ar–OH and CONH), 8.44 and 8.29 (s,1H, imine-H),
7.70–7.47 (m, 1H, Ar–H), 7.30–7.10 (m, 3H, Ar–H),
6.90–6.80 (m, 2H, Ar–H), 6.55–6.50 (m, 2H, Ar–H), 6.25
and 5.98 (s, 1H, Ar–NH), 4.17 and 3.81 (s, 2H, CH2);
Elem. Anal. Calcd. for C15H14N3O2Br; C: 51.74, H: 4.05,
N: 12.07. found C: 51.65, H: 3.82, N: 12.45.
1
(C=O); HNMR (500 MHz, DMSO-d6) (ppm) d: 9.03 (s,
1H, CONH), 6.70 (d, 2H, J = 8.0 Hz, Ar–H), 6.47 (d, 2H,
J = 8.0 Hz, Ar–H), 5.45 (s, 1H, Ar–NH), 4.21 (bs, 2H,
NH2), 3.83 (q, 2H, J = 6.8 CH2O), 3.54 (s, 2H, CH2), 1.23
(t, 3H, J = 6.8, CH3); Elem. Anal. Calcd. for C10H15N3O2;
C: 57.40, H: 7.23, N: 20.08. found C: 57.35, H: 7.55, N:
20.40.
General procedure for the synthesis of 1–6 derivatives
of 3a and 3b
2-(4-Bromophenylamino)-N0-(2-chlorobenzylidene)acet-
ohydrazide (3a-4) Yield 53%, mp: 190–192°C. IR (KBr,
mmax, cm-1): 3404 and 3188 (N–H), 3071 (C–H,Ar), 1682
(C=O); 1HNMR (500 MHz, DMSO-d6) (ppm) d: 11.74 (bs,
1H, CONH), 8.63 and 8.39 (s, 1H, imine-H), 8.05–7.93 (m,
1H, Ar–H), 7.50–7.35 (m, 2H, Ar–H),7.25–7.18 (m, 1H,
Ar–H), 6.55–6.50 (m, 2H, Ar–H), 6.26 and 6.01 (s, 1H, Ar–
NH), 4.22 and 3.80 (s, 2H, CH2); 13CNMR (500 MHz,
CDCl3) (ppm) d: 171.27 (C=O), 145.42 (CH-imine),
144.80 (C1, bromophenyl), 141.65, 134.53, 132.79, 132.46,
132.07, 131.65, 131.51, 130.59, 130.12, 129.75, 128.01,
127.21, 127.16, 127.10 (aromatic), 115.19 (C4, bromo-
phenyl), 45.43 (CH2); Elem. Anal. Calcd. for
C15H13N3OBrCl; C: 49.14, H: 3.57, N: 11.46. found C:
49.41, H: 3.60, N: 11.55.
Appropriate aldehyde or ketone (5 mmol) was added
dropwise to solution of 2a,b (5 mmol) in 40 ml of ethanol
and the result mixture was refluxed for 3–4 h. The reaction
mixture was concentrated to half of the volume and
allowed to cool to room temperature (Pandeya et al., 2000;
Yogeeswari et al., 2004). The precipitate was filtered,
washed with water, and recrystallized from 90% ethanol
1
twice. HNMR of 1–6 derivatives 3a and 3b showed the
presence of both Z and E isomers. The efforts for separa-
tion of these isomers by column chromatography or TLC
were unsuccessful. Therefore, a mixture of two isomers
was used for biological studies.
2-(4-Bromophenylamino)-N0-(2-nitrobenzylidene)acetohyd-
razide (3a-1) Yield 51%, mp: 201–203°C. IR (KBr,
mmax, cm-1): 3365 and 3254 (N–H), 3088 (C–H, Ar), 1695
(C=O), 1513 and 1380 (N–O); 1HNMR (500 MHz,
DMSO-d6) (ppm) d: 11.78 (bs, 1H, CONH), 8.65 and 8.35
(s, 1H, imine-H), 8.09–8.01 (m, 2H, Ar–H), 7.75–7.65 (m,
2H, Ar–H), 7.18 (m, 2H, Ar–H), 7.57 (m, 2H, Ar–H), 6.30
and 6.01 (s, 1H, Ar–NH), 4.21 and 3.81 (s, 2H, CH2);
Elem. Anal. Calcd. for C15H13N4O3Br; C: 47.76, H: 3.47,
N: 14.85. found C: 48.09, H: 3.81, N: 14.43.
2-(4-Bromophenylamino)-N0-(propan-2-ylidene)acetohyd-
razide (3a-5) Yield 84%, mp: 155–157°C. IR (KBr,
mmax, cm-1): 3424 and 3216 (N–H), 3120 (C–H, Ar), 1673
(C=O); 1HNMR (500 MHz, DMSO-d6) (ppm) d: 10.30 and
10.03 (s, 1H, CONH), 7.17 (d, 2H, J = 8 Hz, Ar–H), 6.52
(d, 2H, J = 8 Hz, Ar–H), 6.13 and 5.90 (s, 1H, Ar–NH),
4.04 and 3.76 (s, 2H, CH2), 1.85 (s, 3H, CH3), 1.81 (s, 3H,
CH3); 13CNMR (500 MHz, CDCl3) (ppm) d: 171.35 and
165.82 (C=O), 150.90 (C-imine), 146.57 (C1, phenyl),
132.31 and 131.92 (C3, phenyl), 115.11 and 114.67 (C2,
phenyl), 109.30 (C4, phenyl), 48.53 and 45.16 (CH2), 25.46
and 16.06 (CH3); Elem. Anal. Calcd. for C11H14N3OBr; C:
46.50, H: 4.97, N: 14.79. found C: 46.23, H: 4.65, N: 14.90.
2-(4-Bromophenylamino)-N0-(4-nitrobenzylidene)acetohyd-
razide (3a-2) Yield 75%, mp: 238–240°C. IR (KBr,
mmax, cm-1): 3407 and 3185 (N–H), 3080 (C–H, Ar), 1679
(C=O), 1525 and 1340 (N–O); 1HNMR (500 MHz,
DMSO-d6) (ppm) d: 11.77 (bs, 1H, CONH), 8.33 and 8.09
(s, 1H, imine-H), 8.25–8.18 (m, 2H, Ar–H), 7.95–7.85 (m,
2H, Ar–H), 7.25–7.15 (m, 2H, Ar–H), 6.70–6.65 (m, 2H,
Ar–H), 6.21 and 6.02 (s, 1H, Ar–NH), 4.25 and 3.82 (s, 2H,
CH2); 13CNMR (500 MHz, CDCl3) (ppm) d: 172.06 and
167.62 (C=O), 148.22 and 148.08 (C4, nitrophenyl), 144.91
(CH-imine), 141.65, 141.04, 140.87, 131.85, 131.75,
128.43, 128.32, 124.48 (aromatic), 114.74 (C2, bromo-
phenyl), 107.63 and 107.14 (C4, bromophenyl), 46.23 and
44.26 (CH2); Elem. Anal. Calcd. for C15H13N4O3Br; C:
47.76, H: 3.47, N: 14.85. found C: 47.37, H: 3.70, N: 14.51.
2-(4-Bromophenylamino)-N0-(1-phenylethylidene)acet-
ohydrazide (3a-6) Yield 75%, mp: 187–189°C. IR (KBr,
mmax, cm-1): 3401 and 3172 (N–H), 3093 (C–H, Ar), 1662
(C=O); 1HNMR (500 MHz, DMSO-d6) (ppm) d: 10.75 and
10.40 (s, 1H, CONH), 7.80–7.70 (m, 2H, Ar–H), 7.45–7.35
(m, 3H, Ar–H), 7.25–7.10 (m, 2H,Ar–H), 6.60–6.50 (m,
2H, Ar–H), 6.20 and 6.00 (s, 1H, Ar–NH), 4.24 and 3.85 (s,
2H, CH2), 2.25 (s, 3H, CH3); 13CNMR (500 MHz, CDCl3)
(ppm) d: 171.70 and 171.22 (C=O), 148.52 and 147.89 (C-
imine), 146.71 (C1, bromophenyl), 142.88, 139.46, 137.86,
133.19, 133.00, 131.35, 130.11, 129.87, 129.18, 128.91,
128.64, 128.28, 128.20, 128.12, 127.93, 126.26, 125.81,
123

Med Chem Res
124.20 (aromatic), 114.39, 114.28 (C2, bromophenyl) 107.96,
107.86 (C4, bromophenyl), 46.65, 46.42 (CH2), 13.62, 13.35
(CH3); Elem. Anal. Calcd. for C16H16N3OBr; C: 55.51, H:
4.66, N: 12.14. found C: 55.38, H: 4.44, N: 12.37.
(OCH2), 47.73 and 45.08 (CH2), 15.32 (CH3); Elem. Anal.
Calcd. for C17H19N3O3; C: 65.16, H: 6.11, N: 13.41. found
C: 65.45, H: 5.83, N: 13.45.
2-(4-Ethoxyphenylamino)-N0-(2-chlorobenzylidene)acet-
ohydrazide (3b-4) Yield 43%, mp: 187–189°C. IR (KBr,
mmax, cm-1): 3407 (N–H), 2979 (C–H, CH2), 2903 (C–H,
2-(4-Ethoxyphenylamino)-N0-(2-nitrobenzylidene)acet-
ohydrazide (3b-1) Yield 56%, mp: 126–128°C. IR (KBr,
mmax, cm-1): 3331 (N–H), 2984 (C–H, CH2), 2909 (C–H,
1
CH3), 1707 (C=O); HNMR (500 MHz, DMSO-d6) (ppm)
1
CH3), 1703 (C=O); HNMR (500 MHz, DMSO-d6) (ppm)
d: 9.40 (bs, 1H, CONH), 8.30 (bs, 1H, imine-H), 7.78–7.72
(m, 2H, Ar–H), 7.57–7.47 (m, 2H, Ar–H), 7.40–7.35 (m,
2H, Ar–H), 6.85–6.70 (m, 2H, Ar–H), 6.55 (d, 2H, phenyl),
4.55 (bs, 1H, Ar–NH), 4.15 (m, 2H, CH2O), 4.00 and 3.90
(s, 2H, CH2), 1.23 (m, 3H, CH3); 13CNMR (500 MHz,
CDCl3) (ppm) d: 165.64 (C=O), 152.27 (C4, ethoxy-
phenyl), 148.93 (CH-imine), 138.21, 135.42, 135.15,
134.45, 132.00, 131.46, 130.60, 129.80, 129.20, 127.80,
127.12, 126.49, 115.82, 113.68 (aromatic), 64.01 (OCH2),
52.20 (CH2), 15.08 (CH3); Elem. Anal. Calcd. for
C17H18N3O2Cl; C: 61.54, H: 5.47, N: 12.66. found C:
61.85, H: 5.15, N: 12.54.
d: 11.76 (bs, 1H, CONH), 8.65 and 8.35 (s, 1H, imine-H),
8.00–7.90 (m, 2H, Ar–H), 7.75–7.60 (m, 2H, Ar–H),
6.70–6.50 (m, 4H, Ar–H), 5.55 and 5.30 (s, 1H, Ar–NH),
4.14 and 3.76 (s, 2H, CH2), 3.86 (m, 2H, CH2O), 1.33–1.23
(m, 3H, CH3); 13CNMR (500 MHz, CDCl3) (ppm) d:
172.53 and 168.30 (C=O), 150.91 and 150.63 (C4, eth-
oxyphenyl), 148.38 (C2, nitrophenyl), 142.91 (C-imine),
139.18, 134.10, 133.81, 130.97, 130.82, 129.17, 128.97,
128.46, 125.07, 124.85, 115.74, 113.84 (phenyl), 63.72
(OCH2), 47.41 and 45.00 (CH2), 15.29 (CH3–C=N); Elem.
Anal. Calcd. for C17H18N4O4; C: 59.64, H: 5.30, N: 16.37.
found C: 59.42, H: 5.56, N: 16.04.
2-(4-Ethoxyphenylamino)-N0-(propan-2-ylidene)acetohyd-
razide (3b-5) Yield 52%, mp: 143–145°C. IR (KBr,
mmax, cm-1): 3414 and 3201 (N–H), 2973 (C–H, CH2),
2901 (C–H, CH3), 1671 (C=O); 1HNMR (500 MHz,
DMSO-d6) (ppm) d: 10.27 and 9.96 (s, 1H, CONH),
6.75–6.65 (m, 2H, Ar–H), 6.53–6.43 (m, 2H,Ar–H), 5.50
and 5.19 (s, 1H, Ar–NH), 3.99 and 3.71 (s, 2H, CH2), 3.85
(m, 2H, CH2O), 1.90 (m, 3H, CH3), 1.78 (m, 3H, CH3),
1.25 (m, 3H, CH3); Elem. Anal. Calcd. for C13H19N3O2; C:
62.63, H: 7.68, N: 16.85. found C: 62.45,H: 7.83, N: 16.62.
2-(4-Ethoxyphenylamino)-N0-(4-nitrobenzylidene)acet-
ohydrazide (3b-2) Yield 64%, mp: 215–216°C. IR (KBr,
mmax, cm-1): 3366 (N–H), 2936 (CH, CH3), 1671 (C=O);
1HNMR (500 MHz, DMSO-d6) (ppm) d: 11.75 (bs, 1H,
CONH), 8.35–8.25 (m, 3H, imine-H and Ar–H), 8.10–7.91
(m, 2H, Ar–H), 6.80–6.69 (m, 2H, Ar–H), 6.65–6.50 (m,
2H, Ar–H), 5.55 and 5.35 (s, 1H, Ar–NH), 4.19 and 3.81(s,
2H, CH2), 3.88 (m, 2H, CH2O), 1.35–1.24 (m, 3H, CH3);
13CNMR (500 MHz, CDCl3) (ppm) d: 172.68 and 168.34
(C=O), 150.91 and 150.58 (C4, ethoxyphenyl), 148.16 (C4,
nitrophenyl), 144.85 (CH-imine), 143.03, 142.80, 141.50,
141.08, 140.91, 128.42, 128.30, 124.52, 115.76, 113.87,
113.82 (aromatic), 63.78 (OCH2), 47.41 and 45.02 (CH2),
15.35 (CH3); Elem. Anal. Calcd. for C17H18N4O4; C:
59.64, H: 5.30, N: 16.37. found C: 59.50,H: 5.21, N: 16.10.
2-(4-Ethoxyphenylamino)-N0-(1-phenylethylidene)acet-
ohydrazide (3b-6) Yield 47%, mp: 146–148°C. IR (KBr,
mmax, cm-1): 3414 and 3187 (N–H), 2978 (C–H, CH2),
2902 (C–H, CH3), 1669 (C=O); 1HNMR (500 MHz,
DMSO-d6) (ppm) d: 10.75 and 10.38 (s, 1H, CONH),
7.85–7.70 (m, 2H, Ar–H), 7.47–7.35 (m, 3H, Ar–H),
6.75–6.69 (m, 2H, Ar–H), 6.65–6.50 (m, 2H, Ar–H), 5.50
and 5.30 (s, 1H, Ar–NH), 4.21 and 3.63 (s, 2H, CH2), 3.85
(m, 2H, CH2O), 2.22 (bs, 3H, CH3), 1.24 (bs, 3H, CH3);
13CNMR (500 MHz, CDCl3) (ppm) d: 173.00 and 167.80
(C=O), 150.55 (C4, ethoxyphenyl), 148.36 (C-imine),
143.07 (C1, ethoxyphenyl), 138.60, 129.74, 129.54, 128.89,
128.78, 126.77, 126.55, 116.00, 113.93, 113.76 (aromatic),
63.75 (OCH2), 47.00 and 45.46 (CH2), 15.34 (CH3), 14.01
(CH3–C=N); Elem. Anal. Calcd. for C18H21N3O2; C:
69.43, H: 6.80, N: 13.49. found C: 69.75, H: 6.59, N: 13.70.
2-(4-Ethoxyphenylamino)-N0-(2-hydroxybenzylidene) acet-
ohydrazide (3b-3) Yield 42%, mp: 187–189°C. IR (KBr,
mmax, cm-1): 3322 and 3276 (N–H), 2975 (C–H, CH2),
2903 (C–H, CH3), 1679 (C=O); 1HNMR (500 MHz,
DMSO-d6) (ppm) d: 11.75–11.10 (bs, 2H, Ar–OH and
CONH), 8.46 and 8.31 (s, 1H, imine-H), 7.41–7.30 (m,
2H,Ar–H), 7.05–6.94 (m, 2H, Ar–H), 6.78–6.70 (m, 2H,
Ar–H), 6.68–6.55 (m, 2H, Ar–H), 5.65 and 5.33 (s, 1H,
Ar–NH), 4.13 and 3.76 (s, 2H, CH2), 3.83 (m, 2H, CH2O),
1.24 (m, 3H, CH3); 13CNMR (500 MHz, CDCl3) (ppm) d:
171.78 and 167.73 (C=O), 157.80 and 156.86 (C2,
hydroxyphenyl), 150.94 and 150.61 (C4, ethoxyphenyl),
147.93 (CH-imine), 143.00, 142.85, 141.71, 131.76,
131.56, 129.87, 127.07, 120.25, 119.05, 119.92, 119.77,
116.81, 116.58, 115.79, 113.88, 113.81 (aromatic), 63.75
Determination of anticonvulsant activity
Male albino mice (from Pasteur Institute of Iran) weighing
20–28 g were used for these experiments. Animals were
123

Med Chem Res
kept at controlled room temperature (25°C) with 12 h light/
12 h dark cycle. They were placed in standard cages and
allowed for free access to food and water except during the
experiment. All animal manipulations were carried out
according to the ethical committee for the use and care of
laboratory animals of Tehran University of Medical Sci-
ences. On the day of experiment, the animals were trans-
ferred into separate cages randomly and allowed to
habituate for 30 min before any administration. Anticon-
vulsant activity was evaluated according to the standard
method (Campagna et al., 1993; Shafaroodi et al., 2004;
Narayana et al., 2006; Amin et al., 2008; Abdel-Aziz et al.,
2009; Wagle et al., 2009; Hosseini-Zare et al., 2011). In
brief, each test compound was intraperitoneally injected
(ip) to groups of eight mice 30 min before PTZ (Sigma,
USA) injection. An acute intraperitoneal injection of PTZ
(80 mg/kg, CD97 for generalized tonic-clonic seizure in
the current experiment) was administered in a freshly
prepared solution for induction of convulsions in mice.
Each animal was placed into the cylinder (30 9 25 9
25 cm3) instantly after PTZ administration and its behavior
was observed carefully for 30 min to determine the latency
of onset of generalized tonic-clonic seizure (myoclonic
jerks and other pre-convulsive chewing behavior were not
noted), total seizure duration, and DT. A latency of 1,800 s
was recorded for instances in which no generalized tonic-
clonic seizure was occurred. Protection percent (number of
mice was not afflicted with seizure/number of tested mice
in each group) 9 100 and Survived percent (number
of mice was alive/number of tested mice in each
group) 9 100 were calculated in each group.
test compounds intraperitoneally at the dose of 300 mg/kg.
Neurologic toxicity was defined as the failure of animals to
remain on the rod for one block. Each block includes three
trials and each trial continues for 1 min. The equilibrium
time or time that each animal remains on the rod was
measured for total duration of three trials (180 s).
Statistical analysis
Data were presented as mean SEM of eight animals in
each group. One-way analysis of variance followed by
Dunnett’s multiple comparison post hoc test were used for
comparison between treated groups and control animals.
The differences less than 0.05 were considered statistically
significant.
Acknowledgments This study was supported by grants from
Pharmaceutical Sciences Research Center, Tehran University of
Medical Sciences and Kerman Neuroscience Research Center, Ker-
man University of Medical Sciences. We thank Ms. Marjan Jalili
Baleh and Ali Kazemi for their technical assistance.
References
Abdel-Aziz M, Abuo-Rahma A, Gamal El-Din A, Hassan AA (2009)
Synthesis of novel pyrazole derivatives and evaluation of their
antidepressant and anticonvulsant activities. Eur J Med Chem
44:3480–3487
Afiuni-Zadeh S, Azimi G (2010) A QSAR study for modeling of
8-azaadenine analogues proposed as A1 adenosine receptor
antagonists using genetic algorithm coupling adaptive Neuro-
Fuzzy inference system (ANFIS). Anal Sci 26:897–902
Amin KM, Rahman DEA, Al-Eryani YA (2008) Synthesis and
preliminary evaluation of some substituted coumarins as
anticonvulsant agents. Bioorg Med Chem 16:5377–5388
Azam F, Alkskas IA, Khokra SL, Prakash O (2009) Synthesis of some
novel N4-(naphtha[1,2-d]thiazol-2-yl)semicarbazides as poten-
tial anticonvulsants. Eur J Med Chem 44:203–211
Azam F, El-gnidi BA, Alkskas IA (2010) Combating oxidative stress
in epilepsy: design, synthesis, quantum chemical studies and
anticonvulsant evaluation of 1-(substituted benzylidene/ethyli-
dene)-4-(naphthalen-1-yl)semicarbazides. Eur J Med Chem 45:
2817–2826
PTZ was dissolved in distilled water (DW) (0.1 ml/10 g
of body weight of the mouse) and the test compounds were
dissolved or suspended in DMSO solution (0.02 ml/10 g of
body weight of the mouse). The control group received the
same concentration of vehicle. Phenobarbital (15 mg/kg
dissolved in DMSO: 0.02 ml/10 g of body weight of the
mouse) and sodium valproate (220 mg/kg dissolved in
DMSO: DW, 1:1) were administered as reference drugs.
For the compounds that showed significant changes with
dose of 100 mg/kg, the effects of lower dose (50 mg/kg)
were also evaluated.
¨
Bolcskei H, Tarnawa I, Kocsis P (2008) Voltage-gated sodium channel
blockers, 2001–2006: an overview. Med Chem Res 17:356–368
Campagna F, Carotti A, Casini G, Palluotto F, Genchi G, De Sarro
GB (1993) 2-Aryl-2,5-dihydropyridazino[4,3-b]lindol-3(3H)-
ones: novel rigid planar benzodiazepine receptor ligands. Bioorg
Med Chem 1:437–446
Dimmock JR, Vashishtha SC, Stables JP (2000) Anticonvulsant
properties of various acetylhydrazones, oxamoylhydrazones and
semicarbazones derived from aromatic and unsaturated carbonyl
compounds. Eur J Med Chem 35:241–248
Dumciute J, Martynaitis V, Holzer W, Mangelinckx S, Kimpe ND,
Sackus A (2006) Synthesis and ring transformations of 1-amino-
1,2,3,9a-tetrahydroimidazo[1,2-a]indol-2(9H)-ones. Tetrahedron
62:3309–3319
Gholizadeh S, Shafaroodi H, Ghasemi M, Bahremand A, Sharifzadeh
M, Dehpour AR (2007) Ultra-low dose cannabinoid antagonist
AM251 enhances cannabinoid anticonvulsant effects in the
Evaluation of neurotoxicity-minimal
motor impairment (MMI)
The rotorod test was used to evaluate minimal motor
impairment (neurotoxicity) in mice. The male albino mice
(20–28 g) were trained to stay on an accelerating knurled
plastic rod rotating at seven revolutions per minute
(Acceler Rota-Rod 7650, UGO Basile,Varese, Italy). Non-
toxic animals can remain on a rod rotating at this speed
indefinitely. Previously trained mice were given selected
123

Med Chem Res
pentylenetetrazole-induced seizure in mice. Neuropharmacology
53:763–770
Shafiee A, Rineh A, Kebriaeezadeh A, Foroumadi A, Sheibani V,
Afarinesh MR (2009) Synthesis and anticonvulsant activity of
4-(2-phenoxyphenyl)semicarbazones. Med Chem Res 18:758–
769
Singh HP, Pandeya SN, Chauhan CS, Sharma CS (2011) Synthesis
and pharmacological screening of some novel chalconyl deriv-
atives of substituted phenyl semicarbazide. Med Chem Res 20:
74–80
Thirumurugan R, Sriram D, Saxena A, Stables J, Yogeeswari P
(2006) 2,4-Dimethoxyphenylsemicarbazones with anticonvul-
sant activity against three animal models of seizures: synthesis
and pharmacological evaluation. Bioorg Med Chem 14:
3106–3112
Hosseini-Zare MS, Salehi F, Seyedi SY, Azami K, Ghadiri T,
Mobasseri M, Gholizadeh S, Beyer C, Sharifzadeh M (2011)
Effects of pentoxifylline and H-89 on epileptogenic activity of
bucladesine in pentylenetetrazol-treated mice. Eur J Pharmacol
670:464–670
Jalali-Heravi M, Asadollahi-Baboli M (2009) Quantitative structure-
activity relationship study of serotonin (5-HT7) receptor inhibitors
using modified ant colony algorithm and adaptive neuro-fuzzy
interference system (ANFIS). Eur J Med Chem 44:1463–1470
Kaushik D, Khan SA, Chawla G, Kumar S (2010) N0-[(5-chloro-
3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene] 2/4-substituted
hydrazides: synthesis and anticonvulsant activity. Eur J Med
Chem 45:3943–3949
Treiman DM (2001) GABAergic mechanisms in epilepsy. Epilepsia
42(3):8–12
Kennedy GM, Lhatoo SD (2008) CNS adverse events associated with
antiepileptic drugs. CNS Drugs 22:739–760
Kumar M, Thurow K, Stoll N, Stoll R (2007) Robust fuzzy mappings
for QSAR studies. Eur J Med Chem 42:675–685
Wagle S, Adhikari AV, Kumari NS (2009) Synthesis of some new
4-styryltetrazolo[1,5-a]quinoxaline and 1-substituted-4-sty-
ryl[1,2,4]triazolo[4,3-a]quinoxaline derivatives as potent anti-
convulsants. Eur J Med Chem 44:1135–1143
Narayana B, Vijaya Raj KK, Ashalatha BV, Kumari NS (2006)
Synthesis of some new substituted triazolo [4,3-a][1,4] benzo-
diazepine derivatives as potent anticonvulsants. Eur J Med Chem
41:417–422
Yogeeswari P, Sriram D, Brahmandam A, Sridharan I, Thirumurugan
R, Stables JP (2003) Synthesis of novel aryl semicarbazones as
anticonvulsants with GABA-mediated mechanism. Med Chem
Res 12:57–68
Pandeya SN, Aggarwal N, Jain JS (1999) Evaluation of semicarba-
zones for anticonvulsant and sedative hypnotic properties.
Pharmazie 54:300–302
Pandeya SN, Yogeeswari P, Stables JP (2000) Synthesis and
anticonvulsant activity of 4-bromophenyl substituted aryl semi-
carbazones. Eur J Med Chem 35:879–886
Yogeeswari P, Thirumurugan R, Kavya R, Samuel JS, Stables J,
Sriram D (2004) 3-Chloro-2-methylphenyl-substituted semicar-
bazones: synthesis and anticonvulsant activity. Eur J Med Chem
39:729–734
Yogeeswari P, Sriram D, Thirumurugan R, Raghavendran JV, Sudhan
K, Pavana RK, Stables J (2005a) Discovery of N-(2,6-dimeth-
ylphenyl)-substituted semicarbazones as anticonvulsants: hybrid
pharmacophore-based design. J Med Chem 48:6202–6211
Yogeeswari P, Sriram D, Veena V, Kavya R, Rakhra K, Ragavendran
JV, Mehta S, Thirumurugan R, Stables JP (2005b) Synthesis of
aryl semicarbazones as potential anticonvulsant agents. Biomed
Pharmacother 59:51–55
Perucca E (2005) An introduction to antiepileptic drugs. Epilepsia
46(4):31–37
Rajak H, Deshmukh R, Veerasamy R, Sharma AK, Mishra P, Kharya
MD (2010) Novel semicarbazones based 2,5-disubstituted-1,3,4-
oxadiazoles: one more step towards establishing four binding site
pharmacophoric model hypothesis for anticonvulsant activity.
Bioorg Med Chem 20:4168–4172
Sander JW (2003) The epidemiology of epilepsy revisited. Curr Opin
Neurol 16:165–170
Shafaroodi H, Samini M, Moezi L, Homayoun H, Sadeghipour H,
Tavakoli S, Hajrasouliha AR, Dehpour AR (2004) The interac-
tion of cannabinoids and opioids on pentylenetetrazole-induced
seizure threshold in mice. Neuropharmacology 47:390–400
Yogeeswari P, Sriram D, Thirumurugan R, Jit LRJS, Ragavendran
JV, Kavya R, Rakhra K, Saraswat V (2006) Synthesis of N4-
(2,4-dimethylphenyl) semicarbazones as 4-aminobutyrate ami-
notransferase inhibitors. Acta Pharm 56:259–572
123