Synthesis, characterization and pharmacological evaluation of (E)-N′-(substituted-benzylidene)isonicotinohydrazide derivatives as potent anticonvulsant agents

Article abstract of DOI:10.1007/s00044-011-9739-5

A series of (E)-N′-(substituted-benzylidene)isonicotinohydrazide derivatives were synthesized by coupling it with different substituted aldehydes, acetophenone, and benzophenones in presence of absolute ethanol along with catalytic amount of glacial aceti

Full text of DOI:10.1007/s00044-011-9739-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:2145–2152
DOI 10.1007/s00044-011-9739-5
ORIGINAL RESEARCH
Synthesis, characterization and pharmacological evaluation
0
of (E)-N -(substituted-benzylidene)isonicotinohydrazide
derivatives as potent anticonvulsant agents
Manav Malhotra
•
•
Vikramdeep Monga
•
•
Sagun Sharma
James Stables
Jainendra Jain
Aakash Deep
Abdul Samad
•
•
Received: 5 March 2011 / Accepted: 1 July 2011 / Published online: 15 July 2011
Ó Springer Science+Business Media, LLC 2011
0
Abstract A series of (E)-N -(substituted-benzylidene)ison-
icotinohydrazide derivatives were synthesized by cou-
pling it with different substituted aldehydes, acetophenone,
and benzophenones in presence of absolute ethanol along
with catalytic amount of glacial acetic acid. All the synthe-
sized compound were confirmed and characterized by using
30, 100, and 300 mg/kg body weight and anticonvulsant
activity was noted at 0.5 h and 4 h time intervals after the
0
drug administration. Compound 1a (E)-N -2-benzylidene
0
isonicotinohydrazide, 1g (E)-N -2-ethoxybenzylidene
0
isonicotinohydrazide, 1k (E)-N -3-flourobenzylidene ison-
0
icotinohydrazide and 3a (E)-N -diphenylmethylene ison-
1
13
various spectral technique like IR, H NMR, C NMR, and
mass spectroscopy studies. Anticonvulsant evaluations of all
the synthesized compounds were done using various seizures
models like maximal electroshock-induced seizure (MES)
and subcutaneous pentylenetetrazole (scPTZ) at a dose of
icotinohydrazide showed protection in MES model, which
indicates that these compounds have the ability to prevent
the spread of seizure at 300 mg/kg dose and showed pro-
tection at 0.5 h duration. Compound 3a was also found to
be active in scPTZ screen at a dose of 300 mg/kg. In
neurotoxicity screen, all the synthesized compounds were
found non-toxic except compounds 1n, 2a, and 3b. Further
compounds 1a, 1g, 1k, and 3a were also evaluated in the
minimal clonic seizure model and exhibited potent anti-
convulsant activity with lower neurotoxicity. Among all
synthesized derivatives, analogue 3a was found to exhibit
protection in MES and scPTZ seizure models. This study
proved that isonicotinoyl hydrazides synthesized by con-
densing isoniazid with various aldehydes and ketones dis-
played moderate to potent anticonvulsant activity.
M. Malhotra ꢀ V. Monga ꢀ S. Sharma
Department of Pharmaceutical Chemistry,
ISF College of Pharmacy, Ferozepur Road,
Moga 142 001, India
V. Monga
Adesh Institute of Pharmacy and Biomedical Sciences,
Adesh Institute of Medical Sciences and Research,
Bathinda 151001, India
J. Jain
Department of Pharmaceutical Chemistry,
Ram-Eash Institute of Technical and Vocational Studies,
Greater Noida 201310, India
Keywords Isoniazid ꢀ
0
(
E)-N -(substituted-benzylidene)isonicotinohydrazide ꢀ
Anticonvulsant ꢀ Pentylenetetrazole ꢀ Neurotoxicity
A. Samad
Department of Pharmaceutical Chemistry, College of Pharmacy
in Al-Kharj, King Saud University, Riyadh, Saudi Arabia
J. Stables
Preclinical Pharmacology, National Institute of Neurological
Disorders and Stroke (NINDS), National Institute of Health
Introduction
(
NIH), Bethesda, MD 20892-9020, USA
In the modern world, the drugs affecting central nervous
system plays significant role. Centrally acting drugs are of
special interest to mankind. So, over the last century, large
numbers of compounds with specific effects on brain and
behavior have been discovered (Aggarwal et al., 2009).
A. Deep (&)
Department of Pharmaceutical Sciences,
Maharshi Dayanand University, Rohtak 124001, India
e-mail: aakashdeep82@gmail.com
123

2
146
Med Chem Res (2012) 21:2145–2152
Epilepsy is a central nervous system disorder characterize
by paroxysmal and excessive discharges of large number of
neurons (Kaushik et al., 2010). There is urgent need for
improved drugs for the treatment because the presence of
large number of patients who are resistant to the available
antiepileptic drugs. The long established AEDs control
seizures in 50% of patients developing partial seizures and
in 60–70% of those developing generalized seizures
attenuation of GABAergic neurotransmission is involved in
the pathophysiology of several CNS disorders in humans,
namely anxiety, pain, and epilepsy. It is one of the
important targets in the design and discovery of successful
antiepileptic drugs (Gajcy et al.; 2010, Loscher et al., 1985;
Curtis et al., 1978). Until now, aryl and heteroaryl semi-
carbazones and thiosemicarbazones are very well-estab-
lished anticonvulsants. By considering the anticonvulsant
potential displayed by these class of compounds, we tried
to develop aryl acid hydrazide–hydrazones as new cong-
eners to join this, as this group mimics the cyclic ureide
group of semicarbazone along with the other fundamental
requirement. In terms of interaction at the binding site, as
suggested by Dimmock et al. (1996; Puthucode et al.,
1998) the pharmacophoric elements were thought to be
lipophilic aryl ring and hydrogen bonding acid hydrazide
moiety. The conformational analysis of previously well
established drugs showed that for anticonvulsant activity,
the model must be comprised of two aromatic rings
(Yogeeswari et al., 2005) as proximal aryl ring and distal
ring to increase the van der Waal’s bonding at the binding
site and to increase the potency as reported in a favoured
orientation and a third region, usually the number of
hydrogen bonding forming functional groups. Also the
specific placement of these hydrogen bonding groups in
this region appeared to be less important than the proper
orientation and correct conformation (Wong et al., 1986).
The presence of electron rich group at ortho and para
position of the aryl ring has shown increased potency in the
MES screen. The presence of halogens particularly,
showed low ED50 values in the rat oral MES screen. It was
further associated with higher protection index values
(Yogeeswari et al., 2005). Halogen substituted semicarba-
zone was reported as the prototype molecule from the class
of semicarbazones (Dimmock and Baker 1994). Keeping
all this in background, we tried to develop and explore this
particular class of compounds as potential anticonvulsant
agents with minimal number of side effects. In this work,
we highlight the synthesis, characterization, anticonvul-
sant, and neurotoxicity profile of aryl acid hydrazones.
(
Kulandasamy et al., 2009a, b; Duncan, 2002). During past
decade numerous new drugs were approved but despite the
advances in the new drug treatment of epilepsy, approved
antiepileptic drugs have dose-related toxicity and idiosyn-
cratic side effects (Ghogare et al., 2010). Mechanistic
approaches are increasingly being facilitated by the wave
of research in the epileptics (Eadie et al., 1989). Some of
the currently available active drugs have not been directly
linked with any specific receptor within the brain. It is too
difficult to identify common pharmacophore responsible
for prevention or arrest of seizure activity mainly because
of the chemical diversity of organic compounds and their
multiple mechanism of action in controlling the seizures.
Hence, the search for antiepileptic compounds with more
selective activity and minimum toxicity continues to be an
area of investigation in medicinal chemistry (Kulig et al.,
2
011). So, the anticonvulsant drug discovery has given a
high priority. Further anticonvulsant properties displayed
by various established drugs containing hydrazones group
open a new path for the treatment of epilepsy. The Semi-
carbazones have been considered as a new class of anti-
convulsants with oral activity (Angelusiu et al., 2010).
Semicarbazone have documented consistent advances in
the design of novel anticonvulsant agents, through the work
of Dimmock and his collogues (Dimmock et al., 1999). A
number of (aryl-oxy) aryl semicarbazones possessed
greater protection in the maximal electroshock seizure
MES screen (Dimmock et al., 1996). Semicarbazones
displaying activity in the MES screen interact at a specific
binding site referred as the hydrogen bonding area and aryl
binding site, respectively (Dimmock et al., 1996). In the
initial studies on aryl semicarbazones, it was found to
possess potent anticonvulsant activity and the importance
of the terminal amino group of semicarbazone was impli-
cated in hydrogen bonding (Pandeya et al., 1998, Pandeya
et al., 2002). The aryl acid hydrazones contains large
hydrophobic groups similar to semicarbazones perform the
structural requirements of maximal electroshock (MES)
screen (Dimmock et al., 1995). Semicarbazones act at a
specific binding site called as hydrogen bonding area and
aryl binding site (Yogeeswari et al., 2004, 2005). The
gamma amino butyric acid an inhibitory neurotransmitter
gets degradated by pyridoxal phosphate dependent enzyme
known as gamma amino butyrate amino transferase (Jones
and Woodbury 1982). Also, it is well documented that
Chemistry
The synthesis of target compounds were carried outline in
Scheme 1. Compounds 1a–1n, 2a, and 3a–3b is readily
prepared in good yields and purity. Equimolar quantity of
substituted benzaldehydes, acetophenone, and benzophe-
nones with equimolar quantity of isoniazid in ethanolic
medium with catalytic amount of glacial acetic acid was
refluxed for 3–9 h to form acid hydrazones. The comple-
tion of reaction was confirmed by thin layer chromatog-
raphy (TLC). The reaction mixture was then poured in ice
1
23

Med Chem Res (2012) 21:2145–2152
2147
Scheme 1 Synthetic pathway
for the formation of the title
compounds
R1
R1
O
C
O
C
R
C
O
C
C H OH
2 5
N
NH NH2
R
+
N
NH
N
CH COOH
3
Isoniazid
Substituted benzaldehydes,
Substituted aryl acid hydrazones
acetophenone and benzophenones
(1a-1n, 2a, 3a-3b)
1
3
cold water and the precipitate obtained was filtered and
dried in oven at low temperature. The products were re-
crystallised from absolute ethanol. The percentage yield
was calculated. Physical Data of Substituted aryl acid
hydrazones is shown in Table 1. The purity of the com-
pounds was checked by TLC, elemental analyses, and
characterized by spectral data. In general, IR spectra of
most compounds 1a–1n, 2a, and 3a–3b showed absorption
band at around 3365–3199, 3071–3029, 1691–1659, 1662–
C-NMR spectra of most compounds have characteristic
C=O signals appeared at around d 164.52–162.92 ppm,
pyridine d 149.89–121.11 ppm, –N=C–H d 143.72–
142.69 ppm, benzylidene d 168.17–113.64 ppm.
Experimental
Melting points of the synthesized compounds were deter-
mined in open-glass capillaries on Stuart SMP10 melting
point apparatus and were uncorrected. The purity of the
compounds was checked by thin layer chromatography
-
1
1
625, 1569–1542 cm regions, conforming the presence
1
of NH, CH, C=N, C=O, C=C, respectively. The H NMR
spectra, the signals of the respective prepared most deriv-
atives were verified on the basis of their chemical shifts,
multiplicities, and coupling constants. The spectra of most
compounds showed the characteristic NH proton d 12.63–
(TLC). Silica gel plates kiesel gel 0.25 mm, 60 GF254
,
precoated sheets obtained from Merck, Darmstadt
(Germany) were used for TLC and the spots were visual-
ized by iodine vapors/ultraviolet light as visualizing agent.
1
1.52 ppm, 1H proton of –N=C–H at d 8.92–8.30 ppm, 4H
-
1
proton of pyridine were at around d 8.92–7.50 ppm, and
characteristic protons of benzylidene at d 8.79–6.75 ppm.
The IR spectra (t, cm ) were obtained with a Perkin-
Elmer 1600 FTIR spectrometer in KBr pellets. H-NMR
1
spectra (d, ppm) were recorded in DMSO-d solutions on a
6
Table 1 Physical Data of Substituted aryl acid hydrazones
Varian-Mercury 300 MHz spectrometer using tetrameth-
1
ylsilane as the internal reference. C NMR spectra were
3
O
C
R
C
R1
recorded on in DMSO-d solutions on a Bruker Avance II
6
N
NH
N
400 spectrometer at 400 MHz using tetramethylsilane as
the internal reference. Mass spectra were recorded on a
shimadzu GCMS-QP 1000 EX. Elemental analyses were
performed on an ECS 4010 Elemental Combustion System.
The necessary chemicals were purchased from Sigma
Aldrich companies.
a
Compounds
R
R
1
Yield (%) MP (°C)
R
f
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
3
3
a
a
b
c
d
e
f
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH
H
82
75
72
68
72
58
64
58
72
65
62
68
71
69
74
68
71
205–207 0.45
218–220 0.27
215–217 0.38
222–224 0.35
220–222 0.42
147–149 0.55
205–207 0.47
157–159 0.45
212–214 0.39
188–190 0.48
210–212 0.53
203–205 0.55
187–189 0.52
207–209 0.43
169–171 0.47
113–115 0.38
158–160 0.44
2-Cl
4-Br
3-NO
General procedure for the synthesis of aryl acid
hydrazones
2
2,4-NO
2
4- OCH
3
The synthesis of the 17 isonicotinohydrazide derivatives
g
2-OC
4-OC
2
H
5
1
a–1n, 2a and 3a–3b were synthesized, among them 1e,
g, 1h, 1i, 1l, 1m, 1n, 3a, and 3b being unpublished, was
h
3
H
7
1
i
4-OH, 3-OCH
3
performed with good yields from commercially available
materials. The compounds 1a–1d, 1f, 1j, 1k, and 2a have
been reported with their anti-tubercular activity elsewhere
j
3,4-(OCH
3-F
3 2
)
k
l
2-OH
4-OH
(
Lourenco et al., 2007, 2008). The corresponding substi-
tuted benzaldehydes otherwise acetophenone or else
benzophenones (0.01 mol) was reacted with Isoniazid
m
n
a
a
b
3 2
4-N(CH )
3
H
(1.37 g, 0.01 mol) in ethanolic medium with catalytic
C
6
H
H
5
5
–
amount of glacial acetic acid was refluxed for 3–9 h to
form acid hydrazones. The completion of reaction was
confirmed by thin layer chromatography. The reaction
C
6
4-Br
f
R calculated in chloroform:ethyl acetate (1:1)
123

2
148
Med Chem Res (2012) 21:2145–2152
mixture was then poured in ice cold water and the pre-
cipitate obtained was filtered and dried the solid residue in
a desiccator, was recrystallised from absolute ethanol to
yield compounds. The percentage yield was calculated.
benzylidene), 7.09 (d, 1H, benzylidene, J = 8.2), 5.08 (s,
1
1H, OH, D O exchangeable), 3.82 (s, 3H, CH ); C-NMR
3
2
3
(400 MHz, DMSO-d , d ppm): 163.42, 151.94, 149.53,
6
147.65, 142.83, 140.38, 128.12, 123.74, 122.91, 116.85,
1
13.64, 55.89. MS (ESI) m/z = 272 (M ? 1): Anal. Calcd.
for C H N O (271.27): C 61.99, H 4.83, N 15.49.
0
(
E)-N -(2,4-dinitrobenzylidene)isonicotinohydrazide (1e)
1
4 13 3 3
Found: C 61.97, H 4.76, N 15.58.
-
1
IR (t, cm ): 3199, 3052, 1685, 1629, 1556, 1529, 1342,
1
0
1
163. H NMR (300 MHz, DMSO-d , d ppm): 12.63 (s,
H, –NH–N=), 8.92 (s, 1H, –N=C–H), 8.79 (s, 1H, ben-
(E)-N -(3,4-dimethoxybenzylidene)isonicotinohydrazide
(1j)
6
1
zylidene), 8.79 (d, 2H, pyridine, J = 4.2 Hz), 8.61 (d, 2H,
pyridine, J = 3.7 Hz), 7.85 (d, 2H, benzylidene,
-
1
IR (t, cm ): 3232, 3047, 1675, 1645, 1560, 1249, 1026.
1
3
1
J = 8.2 Hz); C-NMR (400 MHz, DMSO-d , d ppm):
H NMR (300 MHz, DMSO-d , d ppm): 11.82 (s, 1H,
6
6
1
62.92, 153.17, 149.79, 142.84, 139.66, 131.76, 129.72,
–NH–N=), 8.56 (d, 2H, pyridine, J = 4.8 Hz), 8.43 (s, 1H,
–N=C–H), 7.80 (d, 2H, pyridine, J = 4.3), 7.35 (d, 2H,
benzylidene, J = 8.4), 7.11 (s, 1H, benzylidene), 3.82 (s,
127.34, 121.18, 117.66. MS (ESI) m/z = 316 (M ? 1):
Anal. Calcd. for C H N O (315.24): C 49.53, H 2.88, N
13 9 5 5
1
3
2
2.22. Found: C 49.47, H 2.91, N 22.25.
6H, 2CH3); C-NMR (400 MHz, DMSO-d , d ppm):
6
1
63.59, 152.98, 149.71, 149.12, 143.72, 139.82, 126.89,
122.82, 122.11, 114.28, 56.72. MS (ESI) m/z = 286
M ? 1): Anal. Calcd. for C H N O (285.32): C 63.15,
0
(
E)-N -(2-ethoxybenzylidene)isonicotinohydrazide (1g)
(
1
5 15 3 3
-
1
IR (t, cm ): 3249, 3045, 1659, 1635, 1550, 1467, 1372,
253, 1023. H NMR (300 MHz, DMSO-d , d ppm): 12.07
6
s, 1H, –NH–N=), 8.82 (s, 1H, –N=C–H), 8.75 (d, 2H,
pyridine, J = 4.4 Hz), 7.89 (d, 2H, pyridine, J = 3.7 Hz),
.84 (d, 2H, benzylidene, J = 7.5 Hz), 7.38 (d, 2H, ben-
zylidene, J = 7.2 Hz), 4.08 (m, 2H, CH ) 1.33 (t, 3H,
H 5.30, N 14.73. Found: C 63.14, H 5.16, N 14.88.
1
1
0
(
(E)-N -(2-hydroxybenzylidene)isonicotinohydrazide (1l)
-
1
7
IR (t, cm ): 3345, 3284, 3071, 1677, 1648, 1553, 1283.
1
H NMR (DMSO-d , 300 MHz) d: 12.47 (s, 1H, –NH–
2
6
1
3
CH3); C-NMR (400 MHz, DMSO-d , d ppm): 163.54,
58.74, 149.59, 143.52, 139.79, 132.12, 128.79, 122.72,
N=), 8.92 (d, 2H, pyridine, J = 4.8), 8.79 (s, 1H, –N=C–
H), 7.85 (d, 2H, pyridine, J = 4.2), 7.54 (d, 2H, benzyli-
dene, J = 8.3), 7.38 (d, 2H, benzylidene, J = 7.7), 4.84 (s,
6
1
1
20.18, 117.94, 114.72, 64.92, 13.89. MS (ESI) m/z = 270
M ? 1): Anal. Calcd. for C H N O (269.32): C 66.90,
1
3
(
1H, OH, D O exchangeable);
2
C-NMR (400 MHz,
1
5
15
3
2
H 5.61, N 15.60. Found: C 66.86, H 5.59, N 15.66.
DMSO-d , d ppm): 163.75, 149.77, 143.18, 139.78,
6
1
30.57, 124.89, 121.85, 116.45. MS (ESI) m/z = 242
(M ? 1): Anal. Calcd. for C H N O (283.33): C 64.72,
0
(
E)-N -(4-propoxybenzylidene)isonicotinohydrazide (1h)
1
3 11 3 2
H 4.60, N 17.42 Found: C 64.65, H 4.55, N 17.54.
-
1
IR (t, cm ): 3259, 3029, 1665, 1642, 1546, 1464, 1371,
1
0
1
251, 1023. H NMR (300 MHz, DMSO-d , d ppm): 11.92
(E)-N -(4-hydroxybenzylidene)isonicotinohydrazide (1m)
6
(s, 1H, –NH–N=), 8.77 (d, 2H, pyridine, J = 4.5 Hz), 8.39
(s, 1H, –N=C–H), 7.81 (d, 2H, pyridine, J = 4.1 Hz), 7.68
(d, 2H, benzylidene, J = 8.2 Hz), 7.01 (d, 2H, benzyli-
-
1
IR (t, cm ): 3368, 3238, 3059, 1671, 1638, 1556, 1289.
1
H NMR (DMSO-d , 300 MHz) d: 12.42 (s, 1H, –NH–
6
dene, J = 7.5 Hz), 3.97 (t, 2H,CH ), 1.75 (m, 2H, CH )
2
N=), 8.89 (d, 2H, pyridine, J = 4.7), 8.74 (s, 1H, –N = C–
H), 7.89 (d, 2H, pyridine, J = 4.2), 7.64 (d, 2H, benzyli-
dene, J = 7.8), 7.35 (d, 2H, benzylidene, J = 7.2), 5.08 (s,
2
1
3
0
.98 (t, 3H, CH3); C-NMR (400 MHz, DMSO-d , d
6
ppm): 163.49, 159.79, 149.82, 143.23, 139.79, 129.57,
25.55, 121.87, 114.18, 72.29, 22.36, 11.88. MS (ESI)
m/z = 298 (M ? 1): Anal. calcd. for C H N O (283.33):
1
3
1
1H, OH, D O exchangeable);
2
C-NMR (400 MHz,
DMSO-d , d ppm): 163.72, 159.89, 143.52, 139.76,
1
6
17
3
2
6
C 67.83, H 6.05, N 14.83. Found: C 67.85, H 6.07, N 14.79.
130.44, 125.57, 121.94, 116.37. MS (ESI) m/z = 242
(
M ? 1): Anal. Calcd. for C H N O (241.25): C 64.72,
13 11 3 2
0
(E)-N -(4-hydroxy-3-methoxybenzylidene)
isonicotinohydrazide (1i)
H 4.60, N 17.42 Found: C 64.69, H 4.62, N 17.43.
0
(E)-N -(4-dimethylaminobenzylidene)isonicotinohydrazide
(1n)
-
1
IR (t, cm ): 3336, 3256, 3029, 1661, 1639, 1542, 1292,
251, 1029. H NMR (300 MHz, DMSO-d , d ppm): 11.86
6
s, 1H, –NH–N=), 8.76 (d, 2H, pyridine, J = 4.3), 8.34 (s,
H, –N=C–H), 7.80 (d, 2H, pyridine, J = 3.9), 7.32 (s, 1H,
1
1
-
1
1
(
IR (t, cm ): 3245, 3064, 1675, 1637, 1555, 1168. H
NMR (300 MHz, DMSO-d , d ppm): 11.76 (s, 1H,
1
6
1
23

Med Chem Res (2012) 21:2145–2152
2149
–
NH–N=), 8.75 (d, 2H, pyridine, J = 4.7), 8.30 (s, 1H,
N=C–H), 7.79 (d, 2H, pyridine, J = 4.2), 7.55 (d, 2H,
benzylidene, J = 8.7), 6.75 (d, 2H, benzylidene, J = 8.2),
Maximal electroshock seizure
–
Seizures were elicited in mice with (50 mA) or in rats
(150 mA) 60 Hz alternating current. The current was
applied via corneal electrodes for 0.2 s. Protection against
the spread of MES-induced seizures was defined as the
abolition of the hind leg tonic maximal extension compo-
nent of the seizure. At 0.5 h and 4.0 h after administration
of the test compounds, activities were evaluated in the
MES test.
1
3
2
.98 (s, 6H, CH3); C-NMR (400 MHz, DMSO-d , d
6
ppm): 163.18, 152.16, 149.77, 143.27, 139.86, 130.55,
21.79, 114.88, 41.56. MS (ESI) m/z = 269 (M ? 1):
Anal. Calcd. for C H N O (268.31): C 67.15, H 6.01, N
1
15 16 4
2
0.83 Found: C 67.18, H 5.95, N 20.31.
0
(
E)-N -(diphenylmethylene)isonicotinohydrazide (3a)
-
1
1
IR (t, cm ): 3244, 3062, 1691, 1662, 1569. H NMR
300 MHz, DMSO-d , d ppm): 11.52 (s, 1H, –NH–N=),
(
Subcutaneous pentylene tetrazole-induced seizures
6
8
.67 (d, 2H, pyridine, J = 4.6), 7.84 (d, 2H, pyridine,
3
1
J = 4.1) 7.58 (m, 10 ArH, benzylidene);
(
C-NMR
Seizures were produced in mice and rats with subcutaneous
administration of pentylene tetrazole (85 mg/kg for mice
and 70 mg/kg for rats). The test compounds were admin-
istered intraperitoneally in mice and orally in rats followed
by subcutaneous injection of pentylene tetrazole. Protec-
tion against the spread of scPTZ-induced seizures was
defined as the absence of clonic spasm. At 0.5 h and 4.0
after administration of the compounds, activities were
evaluated in the scPTZ test.
400 MHz, DMSO-d6) d = 163.37, 156.18, 149.64,
1
39.79, 133.12, 131.28, 127.15, 122.84. MS (ESI) m/
z = 302 (M ? 1): Anal. Calcd. for C H N O (301.34): C
1
9 15 3
7
5.73, H 5.02, N 13.94 Found: C 75.82, H 5.05, N 13.82.
0
(E)-N -4-(bromophenylphenylmethylene)
isonicotinohydrazide (3b)
-
1
1
IR (t, cm ): 3256, 3054, 1689, 1662, 1557, 649. H NMR
400 MHz, DMSO-d , d ppm): 11.65 (s, 1H, –NH–N=),
(
6
8
.68 (d, 2H, pyridine, J = 4.8), 7.72 (d, 2H, pyridine,
J = 4.2), 7.50 (m, 4H, benzylidene), 7.37 (m, 5H, phenyl);
Rotarod test
1
3
C-NMR (400 MHz, DMSO-d , d ppm): 163.33, 154.46,
At 30 min after the administration of test compounds, the
animals were tested on a 1-in. diameter knurled plastic rod
rotating at 6 rpm for 1 min. Neurotoxicity was indicated by
the inability of an animal to maintain equilibrium in each
of three trials.
6
1
1
49.77, 138.74, 131.81, 131.45, 131.15, 127.91, 125.43,
21.68. MS (ESI) m/z = 381 (M ? 1): Anal. Calcd. for
C H BrN O (268.31): C 60.02, H 3.71, N 11.05 Found: C
3
1
9
14
6
0.05, H 3.69, N 11.04.
Pharmacology
Minimal clonic seizure test
Maximal electroshock seizures (MES), subcutaneous pent-
ylene tetrazole-induced seizures (scPTZ), minimal clonic
seizure (6 Hz), and rotarod test were carried out by the
Antiepileptic Drug Development Program, Epilepsy
Branch, National Institute of Neurological Disorders and
Stroke (NINDS) program following previously described
testing procedures (Krall et al., 1978; Porter et al., 1984;
Barton et al., 2001). Compounds were dissolved in poly-
ethylene glycol 400 and evaluated for anticonvulsant
activity with both sexes of C57B/6 mice in the 18–22 g
weight range and Sprague-Dawley rats in the 120–125 g
weight range in both qualitative and quantitative screening
methods. In quantitative screening, groups of eight mice
were given a range of intraperitoneal doses (per oral for
rats) of the test drug until at least three points were
established in the range of 10–90% seizure protection or
minimal-observed neurotoxicity.
The minimal clonic seizure test is used to assess a com-
pound’s efficacy against electrically induced seizures but
uses low frequency (6 Hz) and longer duration of stimu-
lation (3 s). Test compounds are pre-administered to mice
via i.p. injection. At varying times, individual mice are
challenged with sufficient current delivered through cor-
neal electrodes to elicit a psychomotor seizure in 97% of
animals (32 mA for 3 s). Protection was defined as the
absence of minimal clonic phase with stereotyped, au-
tomatistic behaviors. This behavior is described as being
similar to the aura of human patients with partial seizures.
Result and discussions
All the synthesized hydrazones were subjected to anti-
convulsant screening by maximal electroshock seizures test
123

2
150
Med Chem Res (2012) 21:2145–2152
(
MES) and subcutaneous pentylenetetrazole test (scPTZ)
attached to it. In addition to these tests, compound dis-
playing activity in MES screen, were selected for evalua-
tion of their anticonvulsant activity in minimal clonic
seizure model (6 Hz) where smaller amount of electric
current was given for longer duration of time as shown in
Table 3. In this screen, compounds 1a, 1g, 1k, and 3a
displayed strong anticonvulsant activity with lower
neurotoxicity.
using doses of 30, 100, and 300 mg/kg and observations
were carried out at two different time intervals (0.5 and
4
.0 h). Among all the synthesized derivatives the com-
pounds 1a, 1g, 1k, 1n, 2a, 3a–3b was found to be active
and the rest of the compounds were inactive as shown in
Table 2. The result of the MES results revealed that
compound 1a, 1g, 1k and 3a exhibited anti MES activity at
a dose of 300 mg/kg at 0.5 h duration. The compound
From the results it has been noticed that the introduction
of phenyl group to hydrazone (1a) has caused moderate
activity while the substitution by 2-hydroxyphenyl (1l)
group has resulted in loss of activity on the other hand
replacement of hydroxy group with ethoxy group (1g) in
position 2 of phenyl ring imparts activity to the compounds
as reported by (Ghogare et al., 2010) even the presence of
electron donating group impart a very good anticonvulsant
activity. Thus the presence of electron donating group at
o-position of phenyl ring (1g) contributes toward anticon-
vulsant activity as the analogue if having electronegative
group (1b) at 2-position of phenyl ring resulted in the loss
of activity. Further, the compounds having substituent at
the 4-position (1c, 1f, 1h, 1m, and 3b) were found to be
inactive. Even the disubstituted compounds 1e and 1i does
not exhibit any protection. It is intresting to note that the
incorporation of 4-fluoro phenyl group to hydrazone (1k)
impart anticonvulsant activity to the compound activity
probably due to the bioisosteric resemblance of flourine
with hydrogen atom. As earlier reported by (Sinha et al.,
2010) six nicotinic acid hydrazone derivatives D , D , D ,
1
a and 3a have phenyl group to hydrazone has shown
very good anticonvulsant activity. As it was reported by
Kulandasamy et al., 2009a, b) the presence of phenyl
(
group is necessary for a compound to exhibit potent anti-
convulsant activity. However, none of the compound was
found to be active at 4 h thus, indicate that these com-
pounds had the ability to prevent the spread of seizure and
having a rapid onset and short duration of action.
All the test compounds were found to be inactive except
3
a in the scPTZ test, a test used to identify compounds that
elevate seizure threshold. The compound (3a) displayed
anticonvulsant activity at a dose of 300 mg/kg at 0.5 h of
time period indicating that the compound is short acting.
As seen from the results of neurotoxicity screening,
compounds 1n and 2a exhibited toxicity at a dose of
3
1
00 mg/kg where as compound (3b) showed at a dose of
00 mg/kg at 4 h but did not exhibit any toxicity at 5 h
interval. It is interesting to note that the remaining com-
pounds did not exhibit any neurotoxicity at a maximum
administered dose level of 300 mg/kg. These results clearly
indicated that toxicity is not attributed due to the basic
moiety but it is due to the presence of other substituents
2
4
8
D , D and D17 were found to be exhibit significant
9 10
anticonvulsant activity, all the derivatives contain electron
withdrawing group. Thus from the results it has been
noticed the nature and position of the substituent has
marked effect on anticonvulsant activity. Analogue (3a),
diphenyl hydrazone derivative in which the hydrogen atom
of NH group was replaced by a phenyl group exhibit
protection in both MES, scPTZ induced seizures model at a
dose of 300 mg/kg and devoid of any neurotoxicity. Thus,
from the results, it has been noticed the nature and position
Table 2 Anticonvulsant evaluation of compounds 1a, 1g, 1k, 1n, 2a,
3
a–3b
a
Comp. no.
Intraperitoneal injection in mice
MES screen scPTZ screen
Neurotoxicity
0
.5
4.0
0.5
4.0
0.5
4.0
1
1
1
1
2
3
3
a
g
k
n
a
a
b
300
300
300
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Table 3 Anticonvulsant activity of compounds 1a, 1g, 1k, and 3a at
6
–
–
–
300
300
–
Hz
–
–
–
300
–
Comp. no.
Dose
mg/kg)
MES screen
300
–
–
300
–
(
0
.25 h
0.5 h
1.0 h
2.0 h
4.0 h
–
–
100
100
Phenytoin
30
30
–
100
1
a
100
100
100
100
3/4
3/4
2/4
3/4
2/4
3/4
1/4
3/4
1/4
0/4
0/4
2/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
The figures in the table indicate the minimum dose whereby bioac-
tivity was demonstrated in half or more of the mice. – indicates
absence of activity at maximum dose administered (300 mg/kg) a
doses of 30, 100, and 300 mg/kg were administered
1g
1
3
k
a
Compounds 1a, 1g, 1k, 1n, 2a, 3a–3b was found to be active and the
rest of the compounds were inactive as shown in Table 2
Dose of 100 mg/kg was administered. The data indicate the number
of mice of four that were protected
1
23

Med Chem Res (2012) 21:2145–2152
2151
of the substituent has marked effect on anticonvulsant
activity of the synthesized compounds.
semicarbazones, thiosemicarbazones and bis-carbohydrazones of
some aryl alicyclic ketones for anticonvulsant and other
biological properties. Eur J Med Chem 30:303–314. doi:
1
0.1016/0223-5234(96)88238-9
Dimmock JR, Puthucode RN, Smith JM, Hetherington M, Quail JW,
Pugazhenthi U, Lechler T, Stables JP (1996) Aryloxy aryl
semicarbazones and related compounds: a novel class of
anticonvulsant agents possessing high activity in the maximal
electroshock dcreen. J Med Chem 39:3984–3997. doi:10.1021/
jm9603025
Conclusion
This study reports the synthesis of (E)-N’-(mono-
substituted-benzylidene)isonicotinohydrazide derivative and
all were characterized by spectral and elemental analysis.
All the compounds were subjected to antiepileptic screen-
ing by standard methods. The compounds 1a, 1g, 1k, and
Dimmock JR, Sample HA, John S, Beazely MA, Abrams GD (1999)
Isolation and identification of the major urinary metabolite of
4-(4-fluorophenoxy) benzaldehyde semicarbazone after oral
dosing to rats. Die Pharmazie 54:260–262
3
a were found to exhibit protection in MES model, were
Duncan JS (2002) The promise of new antiepileptic drugs. Br
J Clin Pharmacol 53:123–131. doi:10.1046/j.0306-5251.2001.
also found to exhibit potent anticonvulsant activity in 6 Hz
model at a dose of 100 mg/kg and displayed lesser
neurotoxicity. Among the tested model the 6 Hz results
were promising. Thus, hydrazone derivatives of isoniazid
displayed moderate to good anticonvulsant activity. Here
the activity is attributed to the presence of favorable
structural environment such as aryl binding site with a
hydrophobic group, hydrogen bonding domain –NHCO–
group, electron donar, electron withdrawing group and
another hydrophobic aryl ring in these hydrazone. Rather
increase in the hydrophobicity in the synthesized molecules
brings about same degree of activity in the series. So,
hydrazone derivative 3a found to be the most promising
analogue displaying protection in all tested seizures models
with least contribution toward the neurotoxicity and
emerged as lead in these series. Further, the 1a, 1g, and 1k
come out as potential candidates for further investigation.
Finally, it can be readily conclude that the substitution
pattern in the phenyl ring influences the activity as well as
toxicity of the different substituted hydrazones.
0
1540
Eadie MJ, Tyrer JH, Kutt H (1989) Anticonvulsant therapy:
pharmacological basis and practice. Churchill Livingstone,
Edinburgh
Gajcy K, Kala JP, Fra BZ, Wojtasek C, Librowski T, Lochyn S (2010)
Stereochemistry of terpene derivatives. Part 7: Novel rigidified
amino acids from (?)-3-carene designed as chiral GABA
analogues. Tetrahedron Asymmetr 21:2015–2020. doi:10.1016/
j.tetasy.2010.06.026
Ghogare JG, Bhandari SV, Bothara KG, Madgulkar AR, Parashar
GA, Sonawane BG, Inamdar PR (2010) Design, synthesis and
pharmacological screening of potential anticonvulsant agents
using hybrid approach. Eur J Med Chem 45:857–863. doi:
10.1016/j.ejmech.2009.09.014
Jones GL, Woodbury DM (1982) Anticonvulsant structure–activity
relationships: historical development and probable causes of
failure. Drug Dev Res 2:333–355
Kaushik D, Khan SA, Chawla G, Kumar S (2010) N’-[(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. doi:10.1016/j.ejmech.2010.05.049
Krall RJ, Penry JK, White BG, Kupferberg HJ, Swinyard EA (1978)
Antiepileptic drug development: II. Anticonvulsant drug screen-
ing. Epilepsia 19(4):409–428
Kulandasamy R, Adhikari AV, Stables JP (2009a) Synthesis and
anticonvulsant activity of some bishydrazone derived from 3,4-
dipropyloxythiophene. Eur J Med Chem 44:3672–3679. doi:
1
0.1016/j.ejmech.2009.02.009
References
Kulandasamy R, Adhikari AV, Stables JP (2009b) A New class of
anticonvulsant possessing 6 Hz activity: 3,4-Dialkyloxythioph-
ene bishydrazones. Eur J Med Chem 44:4376–4384. doi:
10.1016/j.ejmech.2009.05.026
Kulig K, Wieckowski K, Wieckowski A, Gajda J, Pochwat B, Hofner
GC, Wanner KT, Malawska B (2011) Synthesis and biological
evaluation of new derivatives of 2-substituted 4-hydroxybutana-
mides as GABA uptake inhibitors. Eur J Med Chem 46:183–190.
doi:10.1016/j.ejmech.2010.11.001
Loscher W, Bartholini G, Bossi L, Lloyd KG, Morselli PL (1985)
Epilepsy and GABA receptor agonists: basic and therapeutic
research. L.E.R.S. monograph series. Raven Press, New York,
pp 109–119
Lourenco MCS, Souza MVND, Pinheiro AC, Ferreira MDL,
Goncalves RSB, Nogueira TCM, Peralta MA (2007) Evaluation
of anti-tubercular activity of nicotinic and isoniazid analogue.
Arkivoc 15:181–191
Aggarwal N, Mishra P, Nagori BP, Aggarwal R, Jain J (2009)
Anticonvulsant and neurotoxicity evaluation of Some N4 phenyl
substituted pyridyl semicarbazones. Cent Nerv Syst Agents Med
Chem 9:295–299
Angelusiu MA, Barbuceanu SF, Draghici C, Almajan GL (2010) New
Cu (II), Co (II), Ni (II) complexes with aroyl-hydrazone based
ligand. Synthesis, spectroscopic characterization and in vitro
antibacterial evaluation. Eur J Med Chem 45:2055–2062. doi:
1
0.1016/j.ejmech.2010.01.033
Barton ME, Klein BD, Wolf HH, White HS (2001) Pharmacological
characterization of the 6 Hz psychomotor seizure model of
partial epilepsy. Epilepsy Res 47:217–227
Curtis DR, Larsen KP, Kruger SJ, Kofoed H (1978) GABA
Neurotransmitters: pharmacochemical biochemical and pharma-
cological aspects. Munksgaard, Copenhagen, pp 17–27
Dimmock JR, Baker GB (1994) Anticonvulsant activities of
4-bromobenzaldehyde semicarbazone. Epilepsia 35:648–655.
doi:10.1111/j.1528-1157.1994.tb02486
Dimmock JR, Pandeya SN, Quail JW, Pugazhenthi U, Allen TM,
Kao GY, Balzarini J, Declercq E (1995) Evaluation of the
Lourenco MCDS, Ferreira MDL, Souza MVND, Peralta MA,
Vasconcelos TRA, Henriques MDGM (2008) Synthesis and
anti-mycobacterial activity of (E)-N’-(substituted-benzylidene)-
isonicotinohydrazide derivatives. Eur J Med Chem 43:1344–
1347. doi:10.1016/j.ejmech.2007.08.003
123

2
152
Med Chem Res (2012) 21:2145–2152
Pandeya SN, Mishra V, Singh PN, Rupainwar DC (1998) Anticon-
vulsant activity of thioureido derivatives of acetophenone
semicarbazone. Pharmacol Res 37:17–22
Sinha R, Sara UVS, Khosa RL, Stables J, Jainendra J (2010) Nicotinic
acid hydrazone: a novel anticonvulsant pharmacophore. Med
Chem Res. doi:10.1007/s0004–010-9396–0
Pandeya SN, Raja AS, Stables JP (2002) Synthesis of isatin
semicarbazones as potential anticonvulsants. Role of hydrogen
bonding. J Pharm Pharm Sci 5:266–271
Porter RJ, Cereghino JJ, Gladding GD, Hessie BJ, Kupferburg HJ,
Scoville B (1984) Antiepileptic drug development program.
Cleve Clin Q 51:293–305
Wong MG, Dejina JA, Andrews PR (1986) Conformational analysis of
clinically active anticonvulsant drugs. J Med Chem 29:562–572
Yogeeswari P, Sriram D, Pandeya SN, Stables JP (2004) 4-
Sulphamoyl phenyl semicarbazones with anticonvulsant activity.
IL Farmaco 59:609–613. doi:10.1016/j.farmac.2004.01.009
Yogeeswari P, Sriram D, Veena V, Kavya R, Rakhra K, Mehta S,
Raghuvendran JV, Thirumurugan R, Stables JP (2005) Synthesis
of aryl semicarbazones as potential anticonvulsant agents.
Biomed Pharmacother 59:51–55. doi:10.1016/j.biopha.2004.
04.013
Puthucode RN, Pugazhenthi U, Quail JW, Stables JP, Dimmock JR
(
1998) Anticonvulsant activity of various aryl, arylidene and
aryloxyaryl semicarbazones. Eur J Med Chem 33:595–607. doi:
0.1016/S0223-5234(98)80018-4
1
1
23