Synthesis, Molecular Docking Studies, and Anticonvulsant Evaluation of Novel bis-Phenylhydrazones against Chemically induced Seizures in Mice

Article abstract of DOI:10.1002/jccs.201700038

A series of novel bis-phenylhydrazones were synthesized by the condensation of dialdehydes with phenylhydrazine to evaluate them for their anticonvulsant activity. Efficacy of newly synthesized compounds against pentylenetetrazole (PTZ)-induced, strychnin

Full text of DOI:10.1002/jccs.201700038

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Article
Synthesis, Molecular Docking Studies, and Anticonvulsant Evaluation of Novel
bis-Phenylhydrazones against Chemically induced Seizures in Mice
a
a
b
b
*
Humaira Masood Siddiqi, Aneela Tabasum, Sumera Qasim, Muhammad Shoaib Akhtar,
Saima Kalsooma¶ and Farzana Latif Ansaric
aDepartment of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
bFaculty of Pharmacy, University of Sargodha, Sargodha, Pakistan
cPakistan Council for Science and Technology, Islamabad, Pakistan
(Received: February 3, 2017; Accepted: May 18, 2017; DOI: 10.1002/jccs.201700038)
A series of novel bis-phenylhydrazones were synthesized by the condensation of dialdehydes with
phenylhydrazine to evaluate them for their anticonvulsant activity. Efficacy of newly synthesized
compounds against pentylenetetrazole (PTZ)-induced, strychnine-induced, and picrotoxin-induced
convulsions was tested after administration of these compounds to albino mice via the oral route.
All the five tested compounds showed anticonvulsant activity against strychnine-induced and
picrotoxin-induced convulsions in a dose-dependent manner. However, the effect was more signifi-
cant against PTZ-induced convulsions. Behavioral pattern studies on mice suggested that these
compounds are less neurotoxic compared to phenytoin. Molecular docking studies were carried out
to correlate the experimental and theoretical results. It was concluded that these compounds exerted
their anticonvulsant effect through the modification of the function of GABA receptor-mediated
chloride channels. Molecular docking studies revealed a good correlation, which indicated that
in silico studies could provide an alternate tool for the identification and design of more potent anti-
convulsant agents.
Keywords: bis-Phenylhydrazones; Anticonvulsant; Molecular docking; Molecular operating
environment (MOE).
INTRODUCTION
Epilepsy, a neurodegenerative disorder, is mani-
simultaneous administration of other drugs predisposes
the patient to the risk of drug interaction. Nevertheless,
newer antiepileptics such as gabapentin, vigabatrin,
lamotrigine, etc. are used as supplements to the conven-
tional agents.1 Moreover, the current drug therapy is
associated with adverse side effects such as drowsiness,
ataxia, gastrointestinal disturbance, gingival hyperpla-
sia, hirsutism, and megaloblastic anemia.6
Hence, the search for curative antiepileptic agents
that are effective as well as safe in terms of drug-related
toxicity has been going on. Earlier, two-dimensional
(2D) modeling on anticonvulsants had identified that
one aryl unit, one or two electron-donor atoms, and/or
an NH group in a spatial arrangement are to be recom-
mended for anticonvulsant activity.7,8 A pharmaco-
phore model based on some well-known voltage-gated
sodium channel blockers such as phenytoin and
fested by seizures induced mainly by the imbalance
between inhibitory and excitatory neurotransmitters.1,2
Many epileptic patients not only encounter stigmatiza-
tion but also face other problems like depression, mus-
cle spasm, strange feelings, abnormal behavioural
alterations, convulsions, and unconsciousness, besides
being highly susceptible to suicide and sudden unex-
pected death.3 Furthermore, repeated and increased
excitation of neurons or increased duration of convul-
sions can lead to neuronal death and loss of memory.4
The antiepileptic agents conventionally used in clinical
settings, such as phenytoin, carbamazepine, and sodium
valporate, produce many serious side effects and
remarkable neurotoxicity.5 As they provide only symp-
tomatic relief and have to be consumed life long,
*Corresponding author. humairas@qau.edu.pk, humaira_siddiqi@yahoo.com
¶Present address: Pakistan Institute of Engineering and Applied Sciences (PIEAS), 45650 Pakistan.
J. Chin. Chem. Soc. 2017
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1

Article
Siddiqi et al.
lamotrigine was also proposed.9 Since hydrazone deri-
vatives had been reported to exhibit a variety of biolog-
ical activities such as antimicrobial10 antitubercular,11
anticonvulsant,12 and anti-inflammatory,13 in the pres-
ent work five bis-phenylhydrazone derivatives were
synthesized and selected especially for the evaluation of
their anticonvulsant activity by standard pharmacologi-
cal screening methods and also to see their possible
mechanism of the antiepileptic effect.
Here
n=2, D-1
n=3, D-2
n=5, D-3
n=6, D-4
n=8, D-5
H2
C
OHC
O
O
n
CHO
D
Here
n=2, PH-1
n=3, PH-2
n=5, PH-3
n=6, PH-4
n=8, PH-5
H2
C
H
H
N
N
C
O
O
n
C N N
H
H
PH
Scheme 1. Synthesis of bis-phenylhydrazones.
RESULTS AND DISCUSSION
Chemistry
Eager Xperience CHNS 20120104 analyzer using dried
samples. The reactions were monitored by thin-layer
chromatography (TLC) using precoated silica gel alumi-
num plates (0.2 mm thickness, HF-254, E. Merck) and
visualized using a UV lamp.
The following dialdehydes (D-1 to D-5) used as
starting material were prepared by a method in the
literature.15
Dialdehydes used as reactants were prepared by
the reported procedure.1 Symmetrical derivatives of bis-
phenylhydrazone were also synthesized by a reported
procedure.14 Reaction of dialdehydes with phenylhydra-
zine in the presence of dry and distilled ethanol and gla-
cial acetic acid produced bis-phenylhydrazone under
reflux in excellent yield. A representative structure and
codes of the synthesized compounds are shown in
Table 1 and Scheme 1.
Reagents and conditions were (1) K2CO3 anhy-
drous/dry distilled dimethyl formamide (DMF)/reflux/
36–48 h, and (2) dry distilled EtOH/glacial acetic acid/
PhNHNH2/reflux/2 h.
The melting points were determined on a Mel-
Temp. apparatus (Mitamura Riken Kogyo, Inc. Tokyo,
Japan) using open capillary tubes. The solid-state Four-
ier transform infrared (FTIR) spectra (4000–400 cm−1
were recorded on a Thermo Scientific Nicolet 6700
instrument. Nuclear magnetic resonance (1H NMR and
13C NMR) experiments were carried out on a Bruker
300 MHz digital NMR instrument using the solvent as
indirect reference. CHNS analysis was carried out on an
General procedure for the synthesis of dialdehydes.
4-
Hydroxybenzaldehyde (4.88 g, 0.04 mol) and cesium
carbonate anhydrous (5.52 g, 0.04 mol) were taken in a
250-mL, two-necked, round-bottom flask equipped with
a magnetic stirrer. Fifty milliliters of DMF was added
as solvent. The reaction mixture was stirred under N2
for 1 h. Then, dibromoalkane (0.02 mol) dissolved in
10 mL of DMF was added dropwise into the reaction
mixture through a dropping funnel. After the addition
of dibromoalkane, the reaction mixture was refluxed
for 5–10 h, and the reaction was monitored by TLC.
When reaction was complete, the mixture was poured
into 300 mL of ice-cooled distilled water. Precipitates
formed, which were filtered under suction and washed
with water.
)
Synthesis of dimethylene 1,2-bis(4-oxybenzaldehyde) (D-
Table 1. Codes and IUPAC names of bis-phenylhydrazones
1).
D-1 was prepared according to the literature
method using 4-hydroxybenzaldehyde (4.88 g, 0.04 mol),
potassium carbonate anhydrous (5.52 g, 0.04 mol), and
dibromoethane (3.76 g, 1.72 mL, 0.02 mol). The product
was recrystallized from ethanol. Yield: 56%, m.p.:
101–102 ꢀC (lit. m.p.: 100–102 ꢀC), Rf: 0.39 in n-hexane/
ethyl acetate (1:1) solvent system. FTIR: V (cm−1): 2757
(Cald H), 1677 (C O), 1598, 1505 (C C).
No.
1
Codes
IUPAC name
PH-1
1,2-bis[4-(Phenylhydrazonomethyl)
phenoxy]ethane
1,3-bis[4-(Phenylhydrazonomethyl)
phenoxy]propane
1,5-bis[4-(Phenylhydrazonomethyl)
phenoxy]pentane
1,6-bis[4-(Phenylhydrazonomethyl)
phenoxy]hexane
2
3
4
5
PH-2
PH-3
PH-4
PH-5
Synthesis of trimethylene 1,3-bis(4-oxybenzaldehyde) (D-
1,8-bis[4-(Phenylhydrazonomethyl)
phenoxy]octane
2).
D-2 was prepared according to the literature
method
using
4-hydroxybenzaldehyde
(4.88 g,
2
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bis-Phenylhydrazones, Anticonvulsant, Docking
0.04 mol), potassium carbonate anhydrous (5.52 g,
0.04 mol), and dibromopropane (4.32 g, 2.36 mL,
0.02 mol). The product was recrystallized from ethanol.
Yield: 76%, m.p.: 131–133 ꢀC (lit. m.p.: 133 ꢀC), Rf:
0.42 in n-hexane/ethyl acetate (1:1) solvent system.
FTIR: V (cm−1): 2759 (Cald H), 1682 (C O), 1596,
1509 (C C).
under vacuum. The dried product was recrystallized
from chloroform. Yield: 96%, m.p.: 173–174 ꢀC.
Synthesis of 1,2-bis[4-(phenylhydrazonomethyl)phenoxy]ethane
(PH-1).
D-1 (1.5 g, 5.6 mmol), anhydrous phenyl-
hydrazine (2.42 g, 23 mmol), absolute ethanol (30 mL),
and 2–3 drops of glacial acetic acid were heated
together, and PH-1 was synthesized by the same proce-
ꢀ
Synthesis of pentamethylene 1,5-bis(4-oxybenzaldehyde) (D-
dure as cited above. Yield: 96%, m.p.: 173–174 C, Rf:
0.79 in n-hexane/ethyl acetate (1:1) solvent system.
3).
D-3 was prepared according to the literature
using 4-hydroxybenzaldehyde (4.88 g,
FTIR: V (cm−1): 3316 (N H), 2926, 2874 (C
H),
method
sp3
0.04 mol), potassium carbonate anhydrous (5.52 g,
0.04 mol), and dibromopentane (4.88 g, 3.04 mL,
0.02 mol). The product was recrystallized from metha-
nol. Yield: 94%, m.p.: 77–78 C (lit. m.p.: 77 C), Rf:
0.54 in n-hexane/ ethyl acetate (1:1) solvent system.
FTIR: V (cm−1): 2756 (Cald H), 1687 (C O), 1601,
1509 (C C).
1595 (C N), 1505 (C C), 1258 (C O), 826 (para sub-
stitution). 1H NMR: (300 MHz, DMSO-d6) δ (ppm):
10.21 (d, 2H, J = 15 Hz, H5), 7.83 (s, 2H, H4), 7.60 (m,
ꢀ
ꢀ
0
0
4H, H6,6 ), 7.20 (t, 4H, J = 7.9 Hz, H2,2 ), 7.12 (m, 4H,
0
0
H7,7 ), 4.36 (s, 4H, H8), 7.03 (m, 4H, H3,3 ), 6.72 (t, 2H,
J = 7.3 Hz, H1), 13C NMR: (75 MHz, DMSO-d6) δ
(ppm): (C1–C10) 158.79, 149.95, 129.30, 136.75, 127.49,
130.62, 115.21, 112.30, 66.83.
Synthesis of hexamethylene 1,6-bis(4-oxybenzaldehyde) (D-
4).
D-4 was prepared according to the literature
using 4-hydroxybenzaldehyde (4.88 g,
Synthesis of 1,3-bis[4-(phenylhydrazonomethyl)phenoxy]pro-
method
pane (PH-2).
D-2 (1.5 g, 5.3 mmol), anhydrous phe-
0.04 mol), potassium carbonate anhydrous (5.52 g,
0.04 mol), and dibromohexane (5.44 g, 3.70 mL,
0.02 mol). The product was recrystallized from ethanol.
Yield: 91%, m.p.: 102–104 ꢀC (lit. m.p.: 104 ꢀC), Rf:
0.65 in n-hexane/ethyl acetate (1:1) solvent system.
FTIR: V (cm−1): 2757 (Cald H), 1683 (C O), 1596,
1506 (C C).
nylhydrazine (2.29 g, 21.2 mmol), absolute ethanol
(30 mL), and 2–3 drops of glacial acetic acid were
heated together, and PH-2 was synthesized by the same
procedure as cited above. Yield: 95%, m.p.: 175–177
ꢀC, Rf: 0.79 in n-hexane/ethyl acetate (1:1) solvent sys-
tem. FTIR: V (cm−1): 3307 (N H), 2910, 2868
(C
H), 1597 (C N), 1504 (C C), 1238 (C O),
sp3
Synthesis of octamethylene 1,8-bis(4-oxybenzaldehyde) (D-
1
824 (para substitution). H NMR: (300 MHz, DMSO-
5).
D-5 was prepared according to the literature
d6) δ (ppm): 10.16 (s, 2H, H5), 7.81 (s, 2H, H4), 7.58 (d,
method using 4-hydroxybenzaldehyde (4.88 g, 0.04 mol),
potassium carbonate anhydrous (5.52 g, 0.04 mol), and
dibromooctane (5.44 g, 3.70 mL, 0.02 mol). The product
was recrystallized from ethanol. Yield: 84%, m.p.: 75–78
0
0
4H, J = 9.0 Hz, H6,6 ), 7.19 (t, 4H, J = 7.95 Hz, H2,2 ),
0
7.02 (m, 4H, H7,7 ), 6.70 (m, 2H, H1), 7.02 (m, 4H,
0
H3,3 ), 4.17 (t, 4H, J = 6.3 Hz, H8), 2.20 (t, 2H,
J = 6.3 Hz, H9). 13C NMR: (75 MHz, DMSO-d6) δ
(ppm): (C1–C11) 158.98, 146.00, 136.97, 129.54, 129.03,
127.51, 118.78, 115.16, 112.24, 64.73, 29.10.
ꢀ
ꢀC (lit. m.p.: 78 C), Rf: 0.64 in n-hexane/ethyl acetate
(1:1) solvent system. FTIR: V (cm−1): 2738 (Cald H),
1684 (C O), 1595, 1509 (C C).
Synthesis of 1,5-bis[4-(phenylhydrazonomethyl)phenoxy]pen-
General
procedure
for
the
synthesis
of
bis-
tane (PH-3).
D-3 (1.5 g, 4.8 mmol), anhydrous phe-
phenylhydrazones16.
A
mixture
of
dialdehyde
nylhydrazine (2.0 g, 19 mmol), absolute ethanol
(30 mL), and 2–3 drops of glacial acetic acid were
heated together, and PH-4 was synthesized by the same
procedure as cited above. Yield: 92%, m.p.: 160–161
ꢀC, Rf: 0.80 in n-hexane/ethyl acetate (1:1) solvent sys-
tem. FTIR: V (cm−1): 3312 (N H), 2940, 2868
(5.6 mmol), anhydrous phenylhydrazine (2.42 g,
23 mmol), absolute ethanol (30 mL), and glacial acetic
acid (2–3 drops) was heated at reflux for 2 h. During
this period, the yellow solution became off-white with
the formation of the product. The progress of reaction
was monitored by TLC in n-hexane/ethyl acetate (1:1)
solvent system. After the completion of reaction, the
mixture was cooled and the crude product was filtered
(C
H), 1597 (C N), 1506 (C C), 1241 (C O),
sp3
1
828 (para substitution). H NMR: (300 MHz, DMSO-
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Article
Siddiqi et al.
d6) δ (ppm): 10.16 (s, 2H, H5), 7.82 (s, 2H, H4), 7.57 (d,
4H, J = 8.7Hz, H6,6 ), 7.19 (t, 4H, J = 7.8 Hz, H2,2 ),
USA), picrotoxin (Sigma Aldrich), phenol (Riedel-de-
Haen), absolute alcohol (Riedel-de-Haen), ninhydrin
(AnalaR), phenytoin (received as a gift sample), and
phenobarbitone (Amros, Pharmaceuticals, Karachi,
Pakistan).
Adult Swiss albino mice of both sexes, which
weighed between 22 and 30 g, were used for experimen-
tal studies. The animals were housed in metabolic ani-
mal cages and maintained at controlled room
conditions of 25 ꢁ 10 ꢀC and relative humidity
60–70%. All the animals had free access to food and
water ad libitum. Treatment of experimental animals
was carried out according to protocols approved by the
local ethical committee. The mice used for experimental
work were divided into 17 groups of six mice each.
0
0
0
7.03 (d, 4H, J = 7.8 Hz, H7,7 ), 6.95 (d, 4H,
0
J = 8.7 Hz, H3,3 ), 6.71 (t, 2H, J = 7.4 Hz, H1), 4.01 (t,
4H, J = 6.3 Hz, H8), 1.79 (m, 4H, H9), 1.59 (m, 2H,
H10). 13C NMR: (75 MHz, DMSO-d6) δ (ppm): (C1–
C12) 159.18, 146.02, 137.03, 129.54, 128.83, 127.49,
118.76, 115.11, 112.22, 67.89, 28.91, 22.72.
Synthesis of 1,6-bis[4(phenylhydrazonomethyl)phenoxy]hexane
(PH-4).
D-4 (1.5 g, 4.6 mmol), anhydrous phenyl-
hydrazine (1.98 g, 16.8 mmol), absolute ethanol
(30 mL), and 2–3 drops of glacial acetic acid were
heated together, and PH-5 was synthesized by the same
procedure as cited above. Yield: 90%, m.p.: 161–163
ꢀC, Rf: 0.81 in n-hexane/ethyl acetate (1:1) solvent sys-
tem. FTIR: V (cm−1): 3291 (N H), 2932, 2865
Evaluation
of
anticonvulsant
activity
against
(C
H), 1595 (C N), 1506 (C C), 1244 (C O),
sp3
pentylenetetrazole-induced convulsions.
To study the
1
anticonvulsant effect of the synthesized compounds
against pentylenetetrazole (PTZ)-induced convulsions,
the following method was used.16 Swiss albino mice of
either sex were divided into 17 groups of six mice each.
All the animals were fasted for 24 h before the start of
the experiments; however, they had free access to water.
Group I served as control and received 2% DMSO
solution 1 mL/kg, p.o. only. Group II served as the
standard drug-treated group and was treated with the
standard drug phenytoin (25 mg/kg). Likewise all the
other groups of animals picked up three different doses
of the newly synthesized agents, which were dissolved
in DMSO. Synthetic test compounds were administered
to mice via the oral route. After 1 h of treatment, con-
vulsions were induced in the mice by injecting i.
p. 0.1 mL of freshly prepared PTZ in distilled water.
After administration of PTZ, the following para-
meters were observed: (1) latency (onset of clonus),
(2) onset of tonic convulsions, (3) status of animal after
30 min, (4) status of animal after 24 h, and (5) percent-
age protection.
823 (para substitution). H NMR: (300 MHz, DMSO-
d6) δ (ppm): 7.81 (s, 2H, H4), 10.51 (s, 2H, H5), 7.57 (d,
4H, J = 9.0 Hz, H6,6 ), 7.19 (t, 4H, J = 7.8 Hz, H2,2 ),
0
0
0
7.03 (d, 4H, J = 7.5 Hz, H7,7 ), 6.94 (d,4H, J = 8.7 Hz,
0
H3,3 ), 6.71 (t, 2H, J = 7.4 Hz, H1), 3.99 (t, 4H,
J = 6.3 Hz, H8), 1.75 (t, 4H, J = 6.0 Hz, H9), 1.48 (s,
4H, H10). 13C NMR: (75 MHz, DMSO-d6) δ (ppm):
(C1–C12) 159.19, 146.02, 137.04, 129.53,128.81, 127.48,
118.75, 115.11, 112.23, 67.89, 29.12, 25.80.
Synthesis of 1,8-bis[4-(phenylhydrazonomethyl)phenoxy]octane
(PH-5).
D-5 (1.5 g, 4.2 mmol), anhydrous phenyl-
hydrazine (1.81 g, 16.8 mmol), absolute ethanol
(30 mL), and 2–3 drops of glacial acetic acid were
heated together, and PH-5 was synthesized by the same
procedure as cited above. Yield: 90%, m.p.: 154–155
ꢀC, Rf: 0.83 in n-hexane/ethyl acetate (1:1) solvent sys-
tem. FTIR: V (cm−1): 3292 (N H), 2914, 2850
(C
H), 1596 (C N), 1506 (C C), 1246 (C O),
sp3
1
831 (para substitution). H NMR: (300 MHz, CDCl3)
δ (ppm): 7.85 (s, 2H, H4), 9.89 (s, 2H, H5), 7.56 (m, 4H,
0
0
0
H6,6 ), 7.19 (m, 4H, H2,2 ), 7.03 (m, 4H, H7,7 ), 6.94 (m,
Strychnine-induced convulsions.
To study the anticon-
0
4H, H3,3 ), 6.71 (m, 2H, H1), 4.03 (m, 4H, H8), 1.82 (m,
vulsant effect of the synthesized test compounds against
strychnine-induced convulsions, the method suggested
by Vogel was followed.17 Swiss albino mice of either
sex were divided into 17 groups of six mice each. All
animals were fasted for a period of 24 h before the
commencement of the experiments; however, they had
free access to water. Group I was the untreated control
4H, H9), 1.42 (m, 4H, H10), 0.91 (m, 4H, H11).
Biological methods
The following chemicals and drugs were
employed, with their manufacturer’s name given in
parenthesis: DMSO (Riedel-de-Haen,USA), strychnine
(AnalaR, UK), pentylenetetrazole (Sigma Aldrich,
4
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bis-Phenylhydrazones, Anticonvulsant, Docking
group and received 2% DMSO solution 1 mL/kg
p.o. only. Group II served as standard group and was
treated with the standard drug phenytoin (25 mg/kg).
Similarly, all the other groups of animals received three
different doses of the newly synthesized test agents,
which were dissolved in DMSO. The synthesized test
compounds were administered to mice via the oral
route. After 1 h of treatment with the test compound,
convulsions were induced in mice by injecting i.
p. 0.1 mL of freshly prepared strychnine solution in dis-
tilled water, and the parameters mentioned above were
recorded.
and subsequently dried in air. Thereafter, a ninhydrin
salt solution was sprayed on chromatographic paper
and heated at 65 C for 10 min. The spot developed as
ꢀ
a result of the chromatographic mobility of GABA was
cut and put in a 3-mL solution of absolute alcohol for
elution. The optical density was read by a spectropho-
tometer at the wavelength of 509 nm and compared
with that of a standard GABA solution. GABA levels
were determined by the following formula: GABA level
(pmol/mL) = optical density of the sample/optical den-
sity of the standard GABA solution.
Phenobarbitone-induced hypnotic activity.
Phenobarbital-
induced hypnotic activity test was performed according
to the method described by Williamson19 in albino
mice. Group I was untreated control and had 2%
DMSO solution (1 mL/kg p.o.). Group II was treated
with the standard drug phenytoin, 25 mg/kg. Similarly,
three different doses of the test agents were adminis-
tered after dissolving in DMSO via the oral route. After
30 min, phenobarbitone sodium (45 mg/kg) was admi-
nistered to mice via the i.p. route. The time required for
the onset of sleep and the total duration of sleep were
recorded in all the groups.
Picrotoxin-induced convulsions.
Anticonvulsant effect
of the synthesized test compounds against picrotoxin-
induced convulsions was studied following the method
of Vogel. Group I (control) received 2% DMSO solu-
tion 1 mL/kg p.o. Group II was treated with the stand-
ard drug phenytoin 25 mg/kg. Other groups were
treated with three different doses of the test agents dis-
solved in DMSO via the oral route. After 1 h of treat-
ment, convulsions were induced in the animals by
injecting i.p. 0.1 mL freshly prepared picrotoxin in dis-
tilled water, and the parameters mentioned were
recorded.
Acute toxicity and behavioural pattern studies.
For the
preliminary screening of the toxic effects of the synthe-
sized test compounds, the mice were treated with differ-
ent doses of the test compounds and monitored closely
for 12 h for 1 week. The animals were observed for any
symptoms such as restlessness, muscular spasm, convul-
sions, difference in body weights, motor incoordination,
and respiratory distress. Any mortality occurring during
the succeeding 2 weeks was also recorded.19,20
Estimation
treatment.
of
brain
GABA
level
after
drug
Effect of the synthesized test compounds
on brain GABA level was ascertained by a standard
method.18 Group I (control) received 1 mL/kg p.o. 2%
DMSO once daily for 7 days. Group II received gaba-
pentin (20 mg/kg i.p.) only on first day, and after
30 min of gabapentin administration, blood sample was
collected via cardiac puncture. Other groups of animals
were administered three doses of the test compounds
once daily for a period of 7 days after dissolving them
in DMSO. On the seventh day, 60 min after the admin-
istration of the vehicle and test compounds, blood sam-
ples were collected from all the animals via cardiac
puncture. Blood was transferred into glass tubes for
separation of the serum. All the glass and propylene
tubes used for trace element analysis were soaked in
10% (v/v) nitric acid for 12 h and then washed thor-
oughly with distilled water. The serum sample (0.1 mL)
was added to 1.5 mL of absolute alcohol and centri-
fuged at 3000 g for 15 min. The upper layer was aspi-
rated, and 0.3 mL was placed on a Whatman filter
paper that had been dipped in phenol solution for 24 h
Statistical analysis.
The data obtained were shown as
means ꢁ SEM (standard error of means), and the
observed results were compared with control group by
one-way ANOVA followed by Tukey’s post-trial test.
p < 0.05 values were considered as statistically
significant.21
Molecular docking
In vitro studies of the synthesized compounds were
also correlated with in silico studies technique using the
MOE (molecular operating environment) software,
which showed good correlation between experimental
and theoretical results. The structure of the target 4-
aminobutyrate-aminotransferase was obtained from
http://www.rcsb.org/pdb server with PDB ID 1OHV22
J. Chin. Chem. Soc. 2017
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5

Article
Siddiqi et al.
(Figure 1), resolution 2.30 Å, and possessing four iden-
tical chains. It consists of 472 residues of which 461 resi-
dues were identified in the domain region.22
selected based on the lowest energy. The resulting
compound–enzyme complex model was then used for
the interpretation of compound–enzyme interactions at
the active site. The quality level of the docking complex
was similar to that of the original crystal structure. The
important contact residues were Tyr59, His44, Arg53,
Ser52, Gly437, Tyr69, His444, Asp67, Asn55, Ser52,
Arg100, Arg192, and Ser137.22
The test compounds were built using the builder
interface of the MOE program and subjected to energy
minimization. Water molecules of protein complex were
removed. The edited crystal structure was imported into
MOE, and all hydrogen atoms were added to the com-
plex with their standard geometry followed by their
energy minimization using MOPAC 7.0. The resulting
model was further subjected to systematic conforma-
tional search at default parameters with the root mean
squared (RMS) gradient of 0.001 kcal/mol. The enzyme
was searched for its active pocket, and dummy atoms
were created from the resulting alpha spheres.23 The
residues and backbone were kept fixed, and the energy
minimization was performed. Initially, the MOE dock-
ing performance was assessed by redocking the co-
crystallized structure from PDB ID 1OHV. The co-
crystallized ligand, namely PLP (pyridoxal-50-phos-
phate), was docked into the active site of the protein to
ensure that this program regenerates the bound confor-
mation correctly. The RMS deviation (RMSD) of the
docked conformation to the experimental conformation
was ~1.3. All compounds were docked following the
above procedure. Ten different conformations were
selected for each compound. The remaining parameters
were maintained at their default settings. The best con-
formation of each compound–enzyme complex was
EXPERIMENTAL
Efficacy of test agents against PTZ-induced convulsions
The onset of seizures was significantly delayed in
the mice treated with three different doses of the test
agents. As shown in Table 2, the onset was significantly
delayed by PH-1 at 400 mg/kg when compared with the
vehicle-treated group. Also, PH-2 at 400 mg/kg was
found to produce the most significant reduction in the
duration of clonic and tonic seizures. The mortality was
also prevented in the test groups in a dose-dependent
manner, and the most marked anticonvulsant effect was
demonstrated by PH-2 at 400 mg/kg dose.
Efficacy against strychnine-induced convulsions
The onset of seizure was less significantly delayed
in the animals treated with different doses of the test
synthetic agents; however, PH-2 at the dose of 400 mg/
kg showed better results, as summarized in Table 3. It
was also observed that PH-1 at 400 mg/kg significantly
reduced the duration of seizures, but the results were
not as impressive as that of PTZ-induced seizures,
including percentage protection.
Efficacy against picrotoxin-induced convulsions
The onset time was not significantly delayed by
the test compounds except for PH-2 in the 300 mg/kg
dose. However, the duration of convulsion was signifi-
cantly reduced by all the test compounds in a dose-
dependant manner, with the most significant reduction
produced by PH-1 at 400 mg/kg dose, as summarized
in Table 4. The compounds also prevented mortality in
a dose-dependant manner, providing better protection
as compared to protection provided against strychnine-
induced convulsions.
Effect on brain GABA level in various treatment groups
All the tested synthetic agents increased the brain
GABA levels in a dose-dependent manner, as shown
in Table 5. However, PH-2 at the dosage level of
Fig. 1. Structure of target GABA (PDB ID: 1OHV).
6
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J. Chin. Chem. Soc. 2017

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
bis-Phenylhydrazones, Anticonvulsant, Docking
Table 2. Effect of the tested synthetic compounds against PTZ-induced convulsions
Treatment
given
Mean onset
Dose (mg/kg) of HLTE (s)
Mean
difference
Tukey’s Mean duration
alphabets of HLTE (s)
Mean
difference
Tukey’s
Alphabets protection
%age
Control
(DMSO)
PH-1
PH-1
PH-1
PH-2
PH-2
PH-2
PH-3
PH-3
PH-3
PH-4
PH-4
PH-4
PH-5
PH-5
PH-5
1 mL/kg
172.50 ꢁ 3.48
–
a
436 ꢁ 5.99
14.00000 gh
0
300
350
400
300
350
400
300
350
400
300
350
400
300
350
400
25
388.66 ꢁ 8.21
465 ꢁ 9.66
216.16667*
d
450 ꢁ 8.26
353.33 ꢁ 8.13
305.50 ꢁ 6.94
319.50 ꢁ 4.92
289.66 ꢁ 3.51
179.66 ꢁ 3.01
410.83 ꢁ 3.27
395 ꢁ 8.16
−82.66*
−130.50*
−116.50*
−146.33*
−256.33*
−25.16*
−41.0*
hi
e
cd
d
c
a
fg
f
c
i
fg
b
Fg
Cd
B
0
0
292.50000* ef
501 ꢁ 5.09
328.50000*
156.66667*
215.66667*
g
b
d
33.33
50
50
83.33
0
0
329.16 ꢁ 10.52
388.16 ꢁ 6.08
482.50 ꢁ 3.81
310.00000* fg
155.83 ꢁ 10.98 −16.66667
a
b
b
320.83 ꢁ 5.83
335.83 ꢁ 7.79
352.50 ꢁ 3.81
368.33 ꢁ 3.33
435.50 ꢁ 4.95
325.83 ꢁ 3
148.33333*
163.33333*
−145.16*
41.50*
290.83 ꢁ 3.27
477.50 ꢁ 4.42
410 ꢁ 2.88
50
180.00000* bc
195.83333* cd
−26.0*
33.33
33.33
66.66
0
50
66.66
−196.0*
−15.50
263.00000*
153.33333*
e
b
240 ꢁ 2.88
420.50 ꢁ 2.92
299.50 ꢁ 7.86
233.16 ꢁ 7.09
--------
−136.50*
−202.83*
371 ꢁ 2.8*
198.50000* cd
290.83333* ef
463.33 ꢁ 5.25
--------
Phenytoin
--------
--------
----------
---------
100
Data is expressed as mean ꢁ SEM. Mean difference with “*” shows significant result with control, while Tukey’s results shows
means with the same letter are not significantly different at the level of significance 0.05.(n = 6).
Table 3. Effect of the tested synthetic compounds against strychnine-induced convulsions
Treatment
given
Dose
(mg/kg)
Mean onset
of HLTE (s)
Mean
difference
Tukey’s
alphabets
Mean duration
of HLTE (s)
Mean
Tukey’s
%age
protection
difference Alphabets
Control
(DMSO)
PH-1
PH-1
PH-1
PH-2
PH-2
PH-2
PH-3
PH-3
PH-3
PH-4
PH-4
PH-4
PH-5
PH-5
PH-5
1 mL/kg 136.66 ꢁ 5.28 ----------------
a
585 ꢁ 4.02
-------------
H
0
300
350
400
300
350
400
300
350
400
300
350
400
300
350
400
25
140.66 ꢁ 5.74
244.83 ꢁ 3.63
286.16 ꢁ 5.06
182.66 ꢁ 2.43
253.33 ꢁ 3.11
304.16 ꢁ 1.57
172.16 ꢁ 1.74
247.00 ꢁ 1.80
288.66 ꢁ 2.76
186.83 ꢁ 1.42
243.16 ꢁ 2.08
292.50 ꢁ 2.21
131.83 ꢁ 2.48
192.83 ꢁ 1.51
277.00 ꢁ 1.03
---------
4.00
108.16*
149.50*
46.00*
a
d
ef
bc
d
g
b
d
ef
bc
d
fg
a
537.83 ꢁ 2.38
462.16 ꢁ 3.85
357.83 ꢁ 2.13
598.50 ꢁ 2.02
523.16 ꢁ 3.46
459.83 ꢁ 3.17
589.33 ꢁ 1.52
484.66 ꢁ 1.68
466.66 ꢁ 1.76
588.66 ꢁ 2.37
534.33 ꢁ 2.36
473.83 ꢁ 1.60
603.66 ꢁ 1.45
545.83 ꢁ 1.95
425.16 ꢁ 1.62
---------
−47.16*
−122.83*
−227.16*
13.50*
G
Cd
A
ij
f
c
0
0
50
33.33
0
33.33
0
0
0
0
116.66*
167.50*
35.50*
−61.83*
−125.16*
4.33
hi
e
110.33*
152.00*
50.16*
−100.33*
−118.33*
3.66
cd
hi
fg
de
j
106.50*
155.83*
−4.83
−50.66*
−111.16*
18.66*
0
33.33
0
0
33.33
100
56.16*
d
e
−39.16*
−159.83*
g
b
140.33*
Phenytoin
Data is expressed as mean ꢁ SEM. Mean differences with “*” shows significant result with control, while Tukey’s results shows
means with the same letter are not significantly different at the level of significance 0.05 (n = 6).
J. Chin. Chem. Soc. 2017
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7

Article
Siddiqi et al.
%age
Table 4. Effect of synthesized compounds against picrotoxin-induced convulsions
Dose
(mg/kg)
Mean onset
of HLTE (s)
Mean
Tukey’s
Mean duration Mean
Tukey’s
Treatment given
difference alphabets of HLTE (s)
difference Alphabets protection
Control (DMSO) 1 mL/kg 172.33 ꢁ 2.21
–
a
587.16 ꢁ 2.12
335.50 ꢁ 1.11
306.16 ꢁ 1.66
237.50 ꢁ 1.33
330.50 ꢁ 2.09
327.00 ꢁ 1.52
243.66 ꢁ 1.92
423.16 ꢁ 1.40
401.66 ꢁ 1.28
295.16 ꢁ 1.66
416.50 ꢁ 1.56
375.33 ꢁ 1.52
245.66 ꢁ 1.45
468.66 ꢁ 0.91
408.50 ꢁ 1.23
292.50 ꢁ 4.63
–
–
j
0
0
0
PH-1
PH-1
PH-1
PH-2
PH-2
PH-2
PH-3
PH-3
PH-3
PH-4
PH-4
PH-4
PH-5
PH-5
PH-5
Phenytoin
300
350
400
300
350
400
300
350
400
300
350
400
300
350
400
25
366.16 ꢁ 1.60
462.16 ꢁ 1.47
486.33 ꢁ 1.14
863.5 ꢁ 110.3
475.66 ꢁ 1.40
561.50 ꢁ 1.23
368.66 ꢁ 1.20
484.00 ꢁ 1.67
534.16 ꢁ 2.24
355.33 ꢁ 1.58
444.66 ꢁ 1.54
539.33 ꢁ 1.80
368.83 ꢁ 1.42
495.00 ꢁ 1.87
535.50 ꢁ 1.52
193.83
289.83
314.00
691.16*
303.33
389.16
196.33
311.66
361.83
183.00
272.33
367.00
196.50
322.66
363.16
ab
ab
ab
b
−251.66*
−281.00*
−349.66*
−256.66*
−260.16*
−343.50*
−164.00*
−185.50*
−292.00*
d
c
a
d
d
a
h
f
33.33
33.33
33.33
66.66
0
ab
ab
ab
ab
ab
ab
ab
ab
ab
ab
ab
0
b
33.33
0
33.33
50
−170.66* gh
−211.83*
−341.50*
−118.50*
e
a
i
0
−178.66* fg
33.33
66.66
100
−294.66*
b
Data is expressed as mean ꢁ SEM. Mean differences with “*” shows significant result with control, while Tukey’s results shows
means with the same letter are not significantly different at level of significance 0.05 (n = 6).
Table 5. Brain GABA level in various treatment groups by spectrophotometric method
Treatment given
Dose (mL/kg) Mean absorbance GABA level (pmol/mL) Mean difference Tukey’s alphabets
Control
Standard GABA solution
1 mL/kg
0.1 μg
25
300
350
400
300
350
400
300
350
400
300
350
400
300
350
0.023
0.138
0.084
0.041
0.062
0.065
0.034
0.044
0.069
0.044
0.065
0.077
0.035
0.058
0.057
0.038
0.045
0.052
166.20 ꢁ 7.66
1000 ꢁ 0.00
–
A
M
L
D
H
Hi
B
E
J
De
I
K
B
833.79*
443.34*
133.80*
285.62*
304.44*
83.21*
Gabapentin
PH-1
PH-1
PH-1
PH-2
PH-2
PH-2
PH-3
PH-3
PH-3
PH-4
PH-4
PH-4
PH-5
PH-5
PH-5
609.55 ꢁ 6.61
300.01 ꢁ 1.50
451.83 ꢁ 2.70
470.64 ꢁ 3.81
249.42 ꢁ 2.72
320.99 ꢁ 3.01
502.46 ꢁ 4.83
314.01 ꢁ 3.43
472.54 ꢁ 1.49
314.01 ꢁ 3.43
256.17 ꢁ 2.49
420.01 ꢁ 1.58
415.44 ꢁ 2.83
277.13 ꢁ 2.33
325.57 ꢁ 3.08
384.86 ꢁ 3.46
154.78*
336.26*
147.80*
306.34*
392.40*
89.96*
253.80*
249.24*
110.92*
159.37*
218.65*
G
G
C
E
400
F
Data is expressed as mean ꢁ SEM. Mean difference with “*” shows significant result with control, while Tukey’s results shows
means with the same letter are not significantly different at level of significance 0.05 (n = 3).
8
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J. Chin. Chem. Soc. 2017

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
bis-Phenylhydrazones, Anticonvulsant, Docking
Table 6. Effect of synthesized compounds on phenobarbitone-induced sleeping time
Treatment
given
Dose
(mL/kg)
Mean onset of
hypnosis (min)
Mean
difference
Tukey’s
alphabets
Mean duration of
hypnosis (min)
Control
Phenytoin
PH-1
PH-1
PH-1
PH-2
PH-2
PH-2
PH-3
PH-3
PH-3
PH-4
PH-4
PH-4
PH-5
PH-5
PH-5
1 mL/kg
25
23 ꢁ 1.064
34.16 ꢁ 1.57
24.83 ꢁ 1.35
24.33 ꢁ 0.88
23.83 ꢁ 1.30
24.66 ꢁ 1.22
26.33 ꢁ 1.14
27 ꢁ 0.85
–
bcd
f
93.66 ꢁ 1.28
62.33 ꢁ 1.05
101.33 ꢁ 1.02
108.66 ꢁ 1.02
114 ꢁ 1.31
–
bcd
a
11.16*
1.83
1.33
0.833
1.66
3.33
4
−7.66*
3.83
4.83
−6
−0.33
6.33*
−4.50
2.33
8.83*
−31.33*
7.66
15
20.33*
6.83
16.50
23.50*
−14.66
−10.33
45.66*
−10
300
350
400
300
350
400
300
350
400
300
350
400
300
350
400
cde
bcd
bcd
cde
de
def
a
def
def
ab
abcd
def
abc
cde
ef
bcde
def
def
bcde
def
efg
ab
abc
gh
abc
cdef
gh
100.50 ꢁ 1.11
110.16 ꢁ 1.13
117.16 ꢁ 1.49
79 ꢁ 2.52
15.33 ꢁ 2.07
26.83 ꢁ 1.55
27.83 ꢁ 1.51
17 ꢁ 1.48
83.33 ꢁ 4.04
139.33 ꢁ 10.49
83.66 ꢁ 3.56
106.66 ꢁ 8.71
139.33 ꢁ 10.49
92.50 ꢁ 2.29
128.33 ꢁ 2.12
143.50 ꢁ 3.46
22.66 ꢁ 1.47
29.33 ꢁ 1.74
18.50 ꢁ 2.01
25.33 ꢁ 1.68
31.83 ꢁ 1.57
13
45.66*
−1.16
34.66*
49.83*
bcd
fgh
h
Data is expressed as mean ꢁ SEM. Mean difference with “*” shows significant result with control, while Tukey’s results shows
means with the same letter are not significantly different at level of significance 0.05 (n = 6).
400 mg/kg produced the most marked increase in the
brain GABA levels.
visible signs of acute toxicity such as restlessness, respira-
tory distress, convulsions, coma, or death. Even the visual
behavioral pattern studies in mice revealed no prominent
change in the awareness, motor activity, mood, posture,
motor coordination, CNS (central nervous system) excita-
tion, food consumption, and body weight.
Effect of tested compounds on phenobarbitone-induced
sleeping time
All the tested compounds did not significantly
decrease the time of onset of sleep except PH-3
(300 mg/kg), PH-4 (400 mg/kg), and PH-5 (400 mg/
kg). However, they significantly increased the duration
of sleep in a dose-dependant manner, with the most sig-
nificant prolongation of duration of sleep produced by
the PH-5 at 400 mg/kg dose, as shown in Table 6.
Molecular docking
All the compounds were docked into the active
site of 1OHV (4-aminobutyrate-aminotransferase) using
the MOE docking method to determine their most suit-
able docking conformations. The Active Site Finder
tool of MOE was used to dock phenytoin and test com-
pounds into the binding site of 1OHV. All the synthe-
sized compounds that adopted an almost identical
Acute toxicity and behavioural pattern studies
Mice treated with different doses of the synthesized
bis-hydrazones remained alive without showing any
Table 7. Binding interaction of the synthesized compounds in active site of 1OHV
H-bonding
Compds
%age protection
Distance (Å)
Score (%)
Amino acids
π–π Interactions
π–cation
PH-1
PH-2
PH-3
PH-4
PH-5
Phenytoin
33.33
83.33
50
66.66
66.66
100
–
–
41, 10
51
63
70
64, 72
–
–
Arg100, Arg192
His44
Arg192
–
2.82, 1.79
2.91
Arg53, Ser52
Ser52
Arg53
Arg53
Arg53, His44
Tyr59
–
1.87
2.34
Tyr59
–
Arg100
Arg100
1.92, 1.63
Arg192
J. Chin. Chem. Soc. 2017
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9

Article
Siddiqi et al.
Table 8. Binding energies of docked complexes with
protein 1OHV
position at the surface of the 1OHV protein comprised
a hydrophobic and a hydrophilic cage. The docked pat-
tern of all active compounds showed that they were
involved in strong binding interactions with amino
acids in the active site22 (Tyr59, His44, Arg53, Ser52,
Gly437, Tyr69, His444, Asp67, Asn55, Ser52, Arg100,
Arg192, and Ser137), as shown in Table 7.
However, no correlation was observed between
binding energies and experimental percent protection
(PTZ-induced convulsions) values, as shown in Table 8.
The oxygen and the amine group of the most
active compound, i.e., PH-2 (% protection = 83), were
involved in strong hydrogen bonding with Arg53 at a
distance of 2.82 (94%) and with Ser52 at a distance of
1.79 (10%). In addition to hydrogen bonding, it also
had π–cation interactions with HisA44 and π–π interac-
tion with TyrA59, as shown in Figure 2. The putative
% protection
(PTZ-induced
Binding
energy
Compounds
convulsion)
(kcal/mol)
PH-1
PH-2
PH-3
33.33
83.33
50
−1.58
−1.26
0.40
4.59
4.58
PH-4
PH-5
Phenytoin
66.66
66.66
100
−0.34
Fig. 2. Binding mode of PH-2 in 1OHV active site.
Fig. 3. Docked pose of PH-1 in 1OHV active site.
(a) 2D. (b) 3D.
(a) 2D. (b) 3D.
10
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CHEMICAL SOCIETY
bis-Phenylhydrazones, Anticonvulsant, Docking
binding mode of compound PH-1 (% protection = 30)
is shown in Figure 3. The low percent protection of this
compound may be due to its binding pattern in the
lower site of the pocket, as shown in Figure 3. It had
only π–π and π–cation interactions.
In addition, strychnine, another very potent convul-
sant, has been shown to act on the spinal cord and pro-
duce inhibitory effect on the inhibitory neurotransmitter
glycine, which produces hyper excitability in the CNS
and ultimately convulsions.29 Strychnine counteracts the
action of excitatory neurotransmitters like AMPA and
NMDA and also produces inhibitory effects on calcium-
induced excitotoxicity in neurons. These factors are pro-
epileptogenic and precipitate seizures. So, suppression of
the inhibitory neurotransmitter glycine leads to tonic-
clonic seizures in experimental animals.30 In our study, all
test compounds showed anticonvulsant activity against
strychnine-induced convulsions in a dose-dependent man-
ner. However, the effect was more potent against PTZ-
induced convulsions (Table 3). The anticonvulsion effect
of the tested compounds against strychnine-induced con-
vulsions indicates their ability to interfere with glycine
transmission in the spinal cord. Furthermore, picrotoxin,
a noncompetitive antagonist at GABA receptors, has
been reported to block the GABA-activated chloride
ionophor.17 All the tested compounds have also demon-
strated anticonvulsant activity against picrotoxin-induced
convulsions (Table 4). This also indicates that these com-
pounds exert their anticonvulsant effect as a result of the
modification of function of GABA receptor-mediated
chloride channels.
Our data on the brain GABA level clearly show that
there is significantly increased concentration of GABA in
the serum of the treated animals, particularly with the
high dose of the tested compounds (Table 5). This
increase in concentration of GABA with high doses of
tested compounds and also with gabapentin treatment
indicates that the tested compounds might have increased
the synthesis of GABA in GABAergic neurons.29
This study also shows that all the tested com-
pounds increase phenobarbitone-induced sleeping time
in mice (Table 6). Nevertheless, the tested compounds
did not significantly change the sleep latency. Potentia-
tion of phenobarbitone-induced hypnotic activity could
be due to effects on fundamental mechanisms that regu-
late sleep or inhibit the metabolism of phenobarbi-
tone.31 Several neurotransmitters and endogenous
molecules are involved in the regulation of sleep and
wakefulness. The neurons involved in promoting sleep,
located in the anterior hypothalamus, release GABA to
inhibit the activity of wakefulness-producing regions of
the brain.32 Phenobarbitone is known to act at GABA
DISCUSSION
Among many experimental models used for the
induction of convulsions, PTZ has been most frequently
employed for preliminary screening of the anticonvul-
sant effect. PTZ produces petit mal-type of epilepsy and
convulsions in experimental animals by counteracting
the action of the inhibitory neurotransmitter GABA
and thus producing hyperexcitation and excessive neu-
ronal firing, which leads to the development of epi-
lepsy.24 PTZ also produces convulsions by destabilizing
neuronal membranes.25,26 GABA is the major inhibi-
tory neurotransmitter in the CNS, and even a slight
deficiency in GABAergic transmission may lead to
hyper excitability and pathological neuronal discharges
leading to epilepsy. GABA is an endogenous agonist at
the GABAA receptor (ionotropic receptor) that opens
the channels to Cl−ions in the neuronal membrane. An
enhancement of GABAergic inhibitory transmission is
responsible for the antiepileptic effect of drugs that
directly bind and activate GABAA receptors or influ-
ence GABA release, transport, and metabolism.26 Since
PTZ has been established to be a noncompetitive antag-
onist, it blocks GABA-mediated Cl¯ influx. Hence, this
accentuates the excitatory neurons to release neuro-
transmitters such as α-amino-3-hydroxy-5-methyl-4-
isoxazolepropionic acid receptor (AMPA) and N-
methyl-D-aspartic acid (NMDA). This contributes to
neuronal excitotoxicity, leading to seizures and convul-
sions characterized by clonic and tonic phases. PTZ-
induced convulsions decrease the GABA content in the
brain when compared to the vehicle group.27
The results of the present study have clearly
shown potent anticonvulsant activity of all the five
tested derivatives of bis-phenylhydrazones. The tested
compounds were found to exert anticonvulsant effect
due to their potent ability to increase the time of onset
of convulsions and to decrease the time of duration of
convulsions caused by the administration of PTZ
(Table 2).The anticonvulsant action of the tested com-
pounds against PTZ-induced convulsions might, there-
fore, be due to their action as GABA agonists.28
J. Chin. Chem. Soc. 2017
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Article
Siddiqi et al.
receptors’ ionophore complex and favor the binding of
GABA. All the tested compounds significantly poten-
tiated the sleep duration. Hence, it can be suggested
that the tested compounds might be acting through
potentiating the GABA-mediated inhibition of neuro-
transmission. Regarding the results on phenobarbitone-
induced sleep in mice, there is the possibility of pharma-
cokinetic interaction between phenobarbitone and the
test compounds, which might have affected the metabo-
lism of phenobarbitone leading to the prolongation of
sleeping time.
ACKNOWLEDGMENTS
The authors are thankful to the Quaid-i-Azam
University Research Fund, Islamabad, Pakistan, for
providing funds for synthesis and biological assays.
REFERENCES
1. E. M. Williamson, D. T. Okpako, F. J. Evans, Pharmaco-
logical Methods in Phytotherapy Research, Vol. 1, John
Wiley & Sons, New York, 1996, p. 238.
2. H. P. Rang, M. M. Dale, J. M. Ritter, P. K. Moore,
Pharmacology, 4 ed., Churchill Livingstone, Edinburgh,
New York, 2000, p. 470.
During molecular docking studies, it was observed
that all the compounds adopted almost identical positions
on the surface of the 1OHV protein comprised of a
hydrophobic and a hydrophilic cage. The binding pattern
of all active compounds showed that they were involved
in hydrogen bonding, π–π, and arene–cation interactions
with the amino acids in the active site, as shown and tabu-
lated in the results section. Anticonvulsant activity of all
these synthesized compounds may be due to their strong
binding interaction behavior as well as their binding pat-
tern, as shown in the above figures 2,3.
3. T. Sunmonu, M. Komolafe, A. Ogunrin, B. Oladimeji,
A. Ogunniyi, Acta Neurol. Scand. 2008, 118, 395.
4. M. Levite, Y. Ganor, Neurology 2008, 56, 1111.
5. Y. Gupta, J. Malhotra, B. George, S. Kulkarni, Indian
J. Physiol. Pharmacol. 1999, 43, 25.
6. P. Yogeeswari, D. Sriram, R. Thirumurugan, R. J. Jit,
J. V. Ragavendran, R. Kavya, V. Saraswat, Acta Pharm.
2006, 56, 259.
7. A. Camerman, N. Camerman, Adv. Neurol. 1979,
27, 223.
8. M. Wong, J. Defina, P. Andrews, J. Med. Chem. 1986,
29, 562.
9. K. Unverferth, J. Engel, N. Hofgen, A. Rostock,
R. Gunther, H. Lankau, M. Menzer, A. Rolfs, J.
Liebscher, B. Muller, H. J. Hofmann, J. Med. Chem.
1998, 41, 63.
10. P. Vicini, F. Zani, P. Cozzini, I. Doytchinova, Eur.
J. Med. Chem. 2002, 37, 553.
CONCLUSIONS
In the light of the above discussion, it is conceiva-
ble that all five bis-hydrazones tested in the present
investigation possess significant anticonvulsant activity
against different types of convulsant drugs in the ani-
mal model used. It can also be concluded from the
results obtained that this anticonvulsant effect of the
tested compounds is probably due to their ability to
increase the release of GABA from GABAergic neu-
rons. These findings, therefore, suggest conducting fur-
ther pharmacological and toxicological studies and
clinical trials on these newly synthesized compounds for
treating different types of epilepsy. Further studies
would be essentially required to find their actual worth
and safety in treating epilepsy and other CNS disorders.
Hence, it can be safely concluded that the current study
has resulted in a successful understanding of the anti-
convulsant behavior of these synthesized compounds by
correlating experimental and in silico results. On the
basis of in silico studies, it is anticipated that these com-
pounds may be potential candidates for developing new
anticonvulsant agents that can be used for analog
designing and proving their safety for human
consumption.
ꢀ
11. B. K. Kaymakçıoglu, S. Rollas, II Farmaco 2002,
57, 595.
12. J. V. Ragavendran, D. Sriram, S. K. Patel, I. V. Reddy,
N. Bharathwajan, J. Stables, J. Yogeeswari, Eur. J. Med.
Chem. 2007, 42, 146.
13. S. Rollas, N. Gulerman, H. Erdeniz, II Farmaco 2002,
57, 171.
14. G. Newkome, D. Fishel, Org. Synth. 1988, 50, 12.
15. P. Kannan, S. Raja, P. Sakthivel, Polymer 2004,
45, 7895.
16. H. S. White, J. H. Woodhead, M. R. Franklin, In Antiepi-
leptic Drugs, R. H. Levy, R. H. Mattson, B. S. Meldrum
Eds., Raven Press, New York, 1995, p. 99.
17. H. G. Vogel, Drug Discovery and Evaluation: Pharmaco-
logical Assays, Vol. 40, Springer Science & Business
Media, Springer International Publishing, 2002, p. 260,
Including a CD-ROM.
18. S.
Chintawar,
R.
Somani,
V.
S.
Kasture,
J. Ethanopharmacol. 2002, 81, 299.
19. M. Ramanathan, S. Sivakumar, P. Anandvijayakumar,
C. Saravanababu, P. R. Pandian, Indian J. Exp. Biol.
2007, 45, 425.
12
www.jccs.wiley-vch.de
© 2017 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Chin. Chem. Soc. 2017

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
bis-Phenylhydrazones, Anticonvulsant, Docking
20. D. R. Laurence, A. L. Bacharach, Evaluation of Drug
Activities, Pharmacometrics, Vol. 1, Academic Press,
London, New York, 1964, p. 33.
21. F. Anwar, R. Singh, H. Maurya, I. Kazmil, M. Afzal,
G. Kandpal, G. Gupta, P. Kumar, Am. J. Phytomed.
Clin. Therap. 2013, 1, 478.
25. D. Curtis, A. Duggan, D. Felix, G. Johnston,
H. McLennan, Brain Res. 1971, 33, 57.
26. K. Tripathi, Essentials of Medical Pharmacology, Jaypee
Medical Publisher, New Delhi, 2013, p. 1020.
27. \J. W. Choi, H. J. Park, Nat. Prod. Sci. 2007,
13, 384.
22. P. Storici, D. Biase, F. Bossa, S. Bruno, A. Mozzarelli,
C. Peneff, R. B. Silverman, T. Schirmer, J. Biol. Chem.
2004, 279, 363.
23. S. Kalsoom, U. Rashid, A. Shaukat, O. M. Abdalla,
K. Hussain, W. Khan, S. Nazir, M. A. Mesaik, Zaheer-
ul-Haq, F. L. Ansari, Med. Chem. Res. 2013, 22, 5739.
24. J. H. Ha, D. U. Lee, J. T. Lee, J. S. Kim, C. S. Yong,
J. A. Kim, J. S. Ha, K. Huh, J. Ethnopharmacol. 2000,
73, 329.
28. E. N. Bum, M. Schmutz, C. Meyer, A. Rakotonirina,
M. Bopelet, C. Portet, A. Jeker, S. V. Rakonirina, H. R.
Olpe, P. Herrling, J. Ethnopharmacol. 2001, 76, 145.
29. O. Adeyemi, A. Akindele, O. Yemitan, F. Aigbe,
F. Fagbo, J. Ethnopharmacol. 2010, 130, 191.
30. M. A. Rogawski, Epilepsy Curr. 2011, 11, 56.
31. P. N. Kaul, S. K. Kulkarni, J. Pharm. Sci. 1978, 67, 1293.
32. O. K. Hassani, P. Henny, M. G. Lee, B. E. Jones, Eur.
J. Neurosci. 2010, 32, 448.
J. Chin. Chem. Soc. 2017
© 2017 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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