Design and synthesis of novel N-(4-(Pyridin-2-yloxy)benzylidene)-4-[4-(substituted)phenyl]semicarbazides as potential anticonvulsant agents

Article abstract of DOI:10.14233/ajchem.2018.21368

A new series of N-(4-(pyridin-2-yloxy)benzylidene)-4-[4-(substituted)phenyl]semicarbazides (PSSD1-8) were designed and synthesized keeping in view the structural requirement of pharmacophore and evaluated for their possible anticonvulsant activity. All the derivatives were synthesized by the given scheme and reaction process was monitored by thin layer chromatography. The structure of synthesized derivatives was confirmed by FT-IR, ¹H NMR, mass spectroscopy and elemental analysis. The anticonvulsant activity was established after intraperitoneal administration in MES and scMET seizure models. The most active compound of the series was 1-(4-(pyridin-2-yloxy)-benzylidene)-4-p-tolylsemicarbazide (PSSD5). A molecular docking study was carried out in order to assess the interaction and binding modes with target receptor/enzyme. Titled compounds were found to strongly bind to human gamma-aminobutyric acid receptor (GABAAR-β3). A computational study was also carried to predict the pharmacokinetic properties of the synthesized compounds.

Full text of DOI:10.14233/ajchem.2018.21368

Asian Journal of Chemistry; Vol. 30, No. 10 (2018), 2193-2200
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2018.21368
Design and Synthesis of Novel N-(4-(Pyridin-2-yloxy)benzylidene)-4-
[4-(substituted)phenyl]semicarbazides as Potential Anticonvulsant Agents
1
2,*
PREM SINGH and LAXMI TRIPATHI
1
2
IFTM University, Moradabad-244 001, India
Moradabad Educational Trust, Group of Institutions, Faculty of Pharmacy, Moradabad-244 001, India
Corresponding author: E-mail: tripathilaxmi1979@gmail.com
Received: 6 April 2018; Accepted: 23 July 2018;
*
Published online: 31 August 2018;
AJC-19042
A new series of N-(4-(pyridin-2-yloxy)benzylidene)-4-[4-(substituted)phenyl]semicarbazides (PSSD1-8) were designed and synthesized
keeping in view the structural requirement of pharmacophore and evaluated for their possible anticonvulsant activity. All the derivatives
were synthesized by the given scheme and reaction process was monitored by thin layer chromatography. The structure of synthesized
1
derivatives was confirmed by FT-IR, H NMR, mass spectroscopy and elemental analysis. The anticonvulsant activity was established
after intraperitoneal administration in MES and scMET seizure models. The most active compound of the series was 1-(4-(pyridin-2-
yloxy)-benzylidene)-4-p-tolylsemicarbazide (PSSD5). A molecular docking study was carried out in order to assess the interaction and
binding modes with target receptor/enzyme. Titled compounds were found to strongly bind to human gamma-aminobutyric acid receptor
(
GABA
A
R-β ). A computational study was also carried to predict the pharmacokinetic properties of the synthesized compounds.
3
Keywords: Semicarbazone, Anticonvulsant Activity, Neurotoxicity, Molecular docking, Computational study.
based pharmacophore model consisting of four binding sites
INTRODUCTION
is used. The model consists of an aryl hydrophobic binding
site, hydrogen-bonding domain, an electron donating group
and another hydrophobic-hydrophilic site regulating the
pharmacokinetic properties of the anticonvulsant [3].
Anticonvulsants constitute a varied group of drugs used
for treating epileptic seizures. The objective of administering
anticonvulsants is to suppress the rapid and uncontrolled activa-
tion of neurons that start a seizure. The non-selectivity and
adverse side effects caused by the use of currently available
antiepileptic drugs have propelled the search for antiepileptic
compounds with selective activity and relatively low toxicity
In this study, we reported the synthesis and anticonvulsant
activitiesof N-(4-(pyridin-2-yloxy)benzylidene)-4-[4-(substituted)-
1
phenyl]semicarbazides. FT-IR, MS, H NMR and elemental
analysis were used for the characterization of the synthesized
semicarbazides. All the synthesized compounds consisted of
the pharmacophoric elements (Fig. 1) that are essential for acce-
ptable anticonvulsant activity, namely a hydrophobic binding
site (A), a hydrogen-bonding domain (HBD), a hydrophobic-
hydrophilic site (C) and an electron-donating group (D) [4].
The anticonvulsant activities of semicarbazides were experi-
mentally evaluated using the maximal electroshock and sub-
cutaneous metrazole tests in epilepsy mouse models. The neuro-
toxicity of the semicarbazides in mice was evaluated through
the rotarod assay. The molecular docking studies of designed
molecules were performed to assess the interaction and modes
of binding of the synthesized compounds with target receptor/
[
1]. A survey of literature revealed that the semicarbazones
have emerged as compounds with a myriad of biological
activities including anticonvulsant, anticancer, antitubercular
and antimicrobial activities [2]. Semicarbazones are currently
being used to develop versatile anticonvulsant pharmaco-
phores. These compounds were reported to exhibit potent anti-
convulsant effects in various preclinical anticonvulsant models.
Thus far, various semicarbazone derivatives containing 1,3,4-
thiadiazole,1,3,4-oxadiazole, pyrimidine, benzothiazole and
substituted phenyl or aryl rings have been synthesized. The
anticonvulsant activities of these derivatives have been evaluated.
Anticonvulsant activity is only observed when a semicarbazone
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(

2
194 Singh et al.
Asian J. Chem.
D
of 4-(pyridin-2-yloxy)benzaldehyde (3)(0.002mol), 4-(substituted)
-phenylsemicarbazides (6) (0.002 mol) and 10 mL of ethanol
were taken in a round bottom flask. Few drops of glacial acetic
acid was added and refluxed for 2-3 h. The reaction was monitored
by TLC and the reaction mixture was poured into ice. The preci-
pitate was filtered and washed with sodium acetate (0.005 mol).
The crude solid was dried and recrystallized with hot ethanol
to obtain compounds (PSSD1-PSSD8) (Scheme-I) [7].
N-(4-(pyridin-2-yloxy)benzylidene)-4-phenylsemi-
carbazide (PSSD1): Yield: 62.15 %, m.p. 168-170 ºC, m.f.
A
O
O
HBD
C
N
N
C
H
N
N
H
R
H
N-(4-(pyridin-2-yloxy)benzylidene)-4-
4-(substituted) phenyl]semicarbazides
[
Fig. 1. Essential pharmacophoric elements for anticonvulsant activity
enzyme using the Glide extra precision (XP) Maestro 10.1
Schrodinger software.A computational study was also performed
to predict the pharmacokinetic parameters and Log P values.
-1
C
3
19
H
16
N
4
O
2
; m.w. 332.36, R
429, 3315 (N-H str.), 3030 (C-H str.), 2875 (=C-H str.), 2000-
1670 (over-tone for mono-substitution on aromatic ring), 1659
C=O str.), 1637 (azomethine C=N str.), 1597 (phenyl ring str.),
465 (phenyl C-H in plane bending), 1282 (C-N str. coupled
with N-H bending), 1252 (C-O str.), 1042 (N-N str.), 785 & 708
f
0.53. FT-IR (KBr, νmax, cm ):
EXPERIMENTAL
(
1
All the chemicals were purchased from Sigma and Himedia
Chemicals, supplied by S.K. Traders Indore, India. Melting
points were determined on a Tempo capillary melting point
apparatus, Mumbai, India and are uncorrected. The FT-IR
spectrum was recorded (λmax in cm ) on Bruker Tensor 27 FT-
IR spectrometer. H NMR spectrum were recorded at 300 MHz,
1
(
(
7
C-H out of plane bending for mono-substitution). H NMR
CDCl , 300 MHz) δ in ppm: 8.35 (s, 1H, Ph-CH=N at N ), 7.78-
.76 (d, 2H, o-benzyl ring protons at N ), 7.54-7.42 (d, 1H, pyri-
dine ring proton at C ), 7.32-7.30 (d, 2H, o-phenyl ring protons
at N ), 7.22-7.17 (t, 3H, m- & p-phenyl ring protons at N ),
.94-6.80 (m, 3H, pyridine ring proton at C3 & C5), 6.66-
.58 (d, 2H, m-benzyl ring protons at N ), 6.23 (s, 1H, -C=N-
-
1
3
1
1
1
6
after dissolving in a suitable solvent (DMSO or D O) on Bruker
2
4
4
Avance II 400 MHz, USA FT-NMR spectrometer using tetra-
methylsilane as internal standard and chemical shifts (δ) are
reported in ppm. The purity of the compounds was ascertained
by thin layer chromatography (TLC) and elemental analysis.
Plates for TLC were prepared with silica gel G and activated
at 110 ºC for 30 min. Iodine vapours was used to develop the TLC
plates. The mass spectra were recorded on a Waters Micromass
ZQ 2000 mass spectrometer. Elemental analyses were performed
on a Vario EL-III analyzer.
6
6
1
NH semicarbazide proton), 6.21 (s, 1H, O=C-NH semicarbazide
+
+
+
proton). MS (m/z, %): M : 332.13, (M +1: 333.18), M -C
6
H :
6
+
+
2
52.50, M -C
found) (%): C, 68.66 (68.69); H, 4.85 (4.83); N, 16.86 (16.83);
O, 9.63 (9.67).
N-(4-(Pyridin-2-yloxy)benzylidene)-4-(4-chlorophenyl)-
semicarbazide (PSSD2): Yield: 68.94 %, m.p. 163-165 ºC,
Cl; m.w. 366.80, R 0.58. FT-IR (KBr, νmax
cm ): 3428, 3317 ( N-H str.), 3032 (C-H str.), 2877 (=C-H str.),
000-1670 (overtone for disubstitution on aromatic ring), 1659
C=O str.), 1639 (azomethine C=N str.), 1590 (phenyl ring str.),
467 (phenyl C-H in plane bending), 1282 (C-N str. coupled with
N-H bending), 1252 (C-O str.), 738 (C-Cl str.), 1042 (N-N str.),
H N
7 8 2
O: 204.20, M -C
5
H
5
N: 257.11.Anal. Calcd.
(
Synthesis of 4-(pyridin-2-yloxy)benzaldehyde (3): A
mixture of o-chloropyridine (1) (0.002 mol), p-hyroxybenzal-
dehyde (2) (0.002 mol), potassium carbonate (0.002 mol) and
m.f. C19
H
15
N O
4 2
f
,
-1
2
(
1
1
0 mL of dry dimethyl formamide was taken in a round bottom
flask and heated at 70 ºC for 3-4 h under reduced pressure.
Reaction mixture was poured into water and extracted with
ethyl acetate. The combined extracts were washed with water
and dried over anhydrous sodium sulfate.Yield: 66.8 %; m.w.
99.21; m.p.: 79-81 ºC. The purity of compound was checked
by TLC using silica gel G; solvent system ethyl acetate:ethanol
3:2) and detecting agent: iodine vapours. Only one sport was
obtained (R 0.65) [5].
Synthesis of 4-(substituted)phenyl urea (5):Aniline and/
or p-substituted aniline (4) (0.1 mol) was dissolved in glacial
acetic acid (10 mL) and diluted with water (upto 100 mL). To
this solution an equimolar (0.1 mol) quantity of sodium cyanate
in warm water (50 mL) was added with stirring. The reaction
mixture was allowed to stand for 30 min, and then compounds
were filtered, washed with water and dried after recrystalliz-
ation from boiling water [6].
1
8
46 (C-H out of plane bending for disubstitution). H NMR
CDCl , 300 MHz) δ in ppm: 8.59 (s, 1H, Ph-CH=N), 7.94-7.92
d, 2H, o-benzyl ring protons at N ), 7.90-7.88 (d, 2H, o-phenyl
ring protons at N ), 7.70-7.62 (t, 1H, pyridine ring proton at
), 7.46-7.44 (d, 2H, m-phenyl ring protons at N ), 7.24-7.20
d, 1H, pyridine ring proton at C ), 7.04-7.02 (d, 2H, m-benzyl
ring protons at N ), 6.60-6.58 (d, 1H, pyridine ring proton at
), 6.39-6.33 (t, 1H, pyridine ring proton at C ), 6.04 (s, 2H,
(
3
1
(
1
4
(
C
4
4
f
(
6
1
C
3
5
+
+
semicarbazide NH protons). MS (m/z, %): M : 366.09, (M +1
3
5
+
37
+
for Cl : 367.18, M +2 for Cl: 368.09 ), M -C
H
6 5
Cl: 257.16,
N: 291.66.Anal. Calcd. (found)
%): C, 62.21 (62.24); H, 4.12 (4.09); Cl, 9.67 (9.65); N, 15.27
15.25); O, 8.72 (8.76).
N-(4-(Pyridin-2-yloxy)benzylidene)-4-(4-bromophenyl)-
semicarbazide (PSSD3): Yield: 67.51 %, m.p. 190-192 ºC,
+
+
M -C
7
H
7
ClN
2
O: 198.08, M -C
5 5
H
(
(
Synthesis of 4-(substituted)phenyl semicarbazides (6):
To an aqueous solution of 4-(substituted)phenyl urea (5) (0.1
mol), an equimolar quantity of hydrazine hydrate and ethanol
-1
m.f. C19
H
15
N O
4 2
Br; m.w. 411.25, R
442, 3326 (N-H str.), 3018 (C-H str.), 2917 ( =C-H str.), 2000-
670 (overtone for disubstitution on aromatic ring), 1665 (C=O
f
0.52. FT-IR (KBr, νmax, cm ):
3
1
(
2 mL) was added. The reaction mixture was refluxed for 30 min
and cooled in ice. Sodium hydroxide (4 g) was added to make
the reaction mixture alkaline, before refluxing. The product
was filtered under suction and recrystallized from ethanol [6].
Synthesis of N- (4-(pyridin-2-yloxy)benzylidene)-4-[4-
str.), 1645 (azomethine C=N str.), 1586 (phenyl ring str.), 1462
phenyl C-H in plane bending), 1276 (C-N str. coupled with N-H
bending), 1248 (C-O str.), 588 (C-Br str.), 1046 (N-N str.), 841
(
1
(
C-H out of plane bending for disubstitution). H NMR (CDCl ,
3
(
substituted)phenyl]semicarbazide (PSSD1-8) : A mixture

Vol. 30, No. 10 (2018) Design and Synthesis of Novel N-(4-(Pyridin-2-yloxy)benzylidene)-4-[4-(substituted)phenyl]semicarbazides 2195
Cl
HO
H N
R
2
+
N
CHO
(4)
(1)
(2)
NaCNO, Glacial Acetic acid
O
H N
CHN
R
2
O
(5)
NH NH .H O
N
2
2
2
CHO
O
(3)
H NHN
CHN
R
2
Glacial acetic acid
(6)
O
O
N
N
C
N
H
N
R
H
H
N-(4-(pyridin-2-yloxy)benzylidene)-4-[4-(substituted)phenyl]semicarbazide
Scheme-I: Synthetic scheme for synthesis of N-(4-(pyridin-2-yloxy)benzylidene)-4-[4-(substituted) phenyl]semicarbazide (PSSD1-PSSD8)
3
2
00 MHz) δ in ppm: 8.62 (s, 1H, Ph-CH=N), 7.92-7.90 (d,
H, o-benzyl ring protons at N ), 7.86-7.82 (d, 2H, o-phenyl
), 7.72-7.64 (t, 1H, pyridine ring proton at
), 7.48-7.41 (d, 2H, m-phenyl ring protons at N ), 7.26-7.22
d, 1H, pyridine ring proton at C ), 7.08-7.06 (d, 2H, m-benzyl
ring protons at N ), 6.64-6.62 (d, 1H, pyridine ring proton at
), 6.39-6.35 (t, 1H, pyridine ring proton at C ), 6.08 (s, 2H,
C
4
), 7.54-7.52 (d, 2H, m-phenyl ring protons at N
(d, 1H, pyridine ring proton at C ), 7.11-7.08 (d, 2H, m-benzyl
ring protons at N ), 6.60-6.58 (d, 1H, pyridine ring proton at
), 6.52-6.48 (t, 1H, pyridine ring proton at C ), 6.05 (s, 2H,
4
), 7.34-7.32
1
6
ring protons at N
C
(
4
1
4
4
C
3
5
+
+
6
semicarbazide NH protons). MS (m/z, %): M : 350.12, (M +1
for F: 351.11), M -C
M -C
H, 4.32 (4.28); N, 15.99 (15.88); O, 9.13 (9.11); F, 5.42 (5.46).
N-(4-(Pyridin-2-yloxy)benzylidene)-4-p-tolylsemi-
carbazide (PSSD5): Yield: 78.86 %, m.p. 134-136 ºC, m.f.
19
+
+
1
6
H
5
F: 258.10, M -C
7 7 2
H N OF: 199.08,
+
C
3
5
5 5
H N: 274.09.Anal. Calcd. (found) (%): C, 65.14 (65.18);
+
+
semicarbazide NH protons). MS (m/z, %): M : 410.04, (M +1
79
+
81
+
for Br: 412.11, M +2 for Br: 411.06 ), M -C
6
H
5
Br: 257.10,
N: 334.01.Anal. Calcd. (found)
%): C, 55.49 (55.56); H, 3.68 (3.72); Br, 19.43 (19.39); N, 13.62
+
+
M -C
7
H
7
BrN
2
O: 199.08, M -C
5 5
H
-1
(
(
C
20
H
18
N
4
O
2
; m.w. 346.38, R 0.56. FT-IR (KBr, νmax, cm ):
f
13.65); O, 7.78 (7.81).
N-(4-(Pyridin-2-yloxy)benzylidene)-4-(4-fluorophenyl)-
semicarbazide (PSSD4): Yield: 62.14 %, m.p. 160-162 ºC,
F; m.w. 350.35m, R 0.62. FT-IR (KBr, νmax
cm ): 3434, 3322 (N-H str.), 3026 (C-H str.), 2897 (=C-H str.),
000-1675 (overtone for disubstitution on aromatic ring), 1675
C=O str.), 1632 (azomethine C=N str.), 1594 (phenyl ring str.),
459 (phenyl C-H in plane bending), 1284 (C-N str. coupled
3422, 3312 (N-H str.), 3028, 2967, 2876 (C-H str.), 2000-1670
(overtone for disubstitution on aromatic ring), 1657 (C=O str.),
1642 (azomethine C=N str.), 1598 (phenyl ring str.), 1464 (phenyl
C-H in plane bending), 1450 (asym. methyl C-H bending), 1377
(sym. methyl C-H bending), 1280 (C-N str. coupled with N-H
m.f. C19
H
15
N O
4 2
f
,
-1
2
(
1
bending), 1250 (C-O str.), 1045 (N-N str.), 844 (C-H out of plane
bending for disubstitution). H NMR (CDCl
1
3
, 300 MHz) δ in
ppm: 8.38 (s, 1H, Ph-CH=N), 7.86-7.82 (t, 1H, pyridine ring
proton at C ), 7.72-7.70 (d, 1H, pyridine ring proton at C ),
7.64-7.62 (d, 2H, m-phenyl ring protons at N ), 7.52-7.50 (d,
2H, o-benzyl ring protons at N ), 7.14-7.12 (d, 2H, m-phenyl
ring protons at N ), 7.98-7.96 (d, 2H, m-benzyl ring protons
at N ), 6.74-6.72 (d, 1H, pyridine ring proton at C ), 6.48-6.44
with N-H bending), 1258 (C-O str.), 1138 (C-F str.), 1039 (N-N
str.), 852 (C-H out of plane bending for disubstitution). H NMR
4
6
1
4
(
CDCl
d, 2H, o-benzyl ring protons at N
ring protons at N ), 7.74-7.64 (t, 1H, pyridine ring proton at
3
, 300 MHz) δ in ppm: 8.43 (s, 1H, Ph-CH=N), 7.86-7.84
1
(
1
), 7.80-7.78 (d, 2H, o-phenyl
4
4
1
3

2
196 Singh et al.
Asian J. Chem.
(
t, 1H, pyridine ring proton at C
5
), 6.11 (s, 2H, semicarbazide
protons at phenyl ring). MS
1375 (sym. methyl C-H bending), 1287 (C-N str. coupled with
NH protons), 2.27 (s, 3H, CH
(
C
3
N-H bending), 1247 (C-O str.), 1042 (N-N str.), 849 (C-H out
+
+
+
+
1
m/z, %): M : 346.14, (M +1: 347.18), M -C
7 8
H : 246.47, M -
of plane bending for disubstitution). H NMR (CDCl
3
, 300
+
8
H
10
N
2
O: 201.22, M -C
5
H
5
N: 271.12. Anal. Calcd. (found)
MHz) δ in ppm: 8.36-8.34 (d, 2H, m-phenyl ring protons at
), 8.22 (s, 1H, Ph-CH=N), 7.98-7.96 (d, 2H, m-phenyl ring
protons at N ), 7.88-7.86 (d, 1H, pyridine ring proton at C ),
7.72-7.70 (d, 1H, pyridine ring proton at C ), 7.52-7.50 (d,
2H, o-benzyl ring protons at N ), 7.02-7.00 (d, 2H, o-benzyl
ring protons at N ), 6.92-6.90 (d, 1H, pyridine ring proton at
), 6.42-6.40 (t, 1H, pyridine ring proton at C ), 6.08 (s, 2H,
(
(
%): C, 69.35 (69.37); H, 5.24 (5.25); N, 16.17 (16.12); O, 9.24
9.26).
N
4
4
4
N-(4-(Pyridin-2-yloxy)benzylidene)-4-(4-ethylphenyl)-
6
semicarbazide (PSSD6): Yield: 67.56 %, m.p. 163-165 ºC,
1
-1
m.f. C21
H N O
20 4 2
; m.w. 360.41, R
390, 3325 (N-H str.), 3041, 2935, 2868 (C-H str.), 2000-1670
over-tone for disubstitution on aromatic ring), 1672 (C=O str.),
648 (azomethine C=N str.), 1592 (phenyl ring str.), 1471 (phenyl
C-H in plane bending), 1453 (asym. methyl C-H bending), 1372
sym. methyl C-H bending), 1278 (C-N str. coupled with N-H
bending), 1254 (C-O str.), 1048 (N-N str.), 849 (C-H out of plane
f
0.49. FT-IR (KBr, νmax, cm ):
1
3
(
1
C
3
5
+
+
semicarbazide NH protons). MS (m/z, %): M : 377.11, (M +1:
378.12), M -HNO
199.08, M -C
+
+
+
2
: 333.14, M -C
6
H
5
NO
2
: 257.10, M - C
N: 302.09. Anal. Calcd. (found) (%): C, 60.47
7
H
7
N
3
3
O :
+
H
5 5
(
(60.52); H, 4.01 (4.04); N, 18.56 (18.61); O, 16.96 (16.92).
Pharmacological evaluation: The newly synthesized N-
(4-(pyridin-2-yloxy)benzylidene)-4-[4-(substituted)phenyl]-
semicarbazides (PSSD1-8) were subjected to anticonvulsant
screening according to the protocols of anticonvulsant drug
development (ADD) program of National Institute of Health,
Bethesda, USA. Albino mice of either sex weighing between
25-30 g were used in this study. All albino mice employed in
this study were approved by the Institutional Animal Ethics
Committee of Sapience Bioanalytical Research Laboratory,
Bhopal, India (1413/PO/E/S/11/cpcsea) and experiment was
carried out as per CPCSEA guidelines. The animals were kept
in large spacious hygienic cages during the course of the
experimental period. The animals had free access to standard
commercial diet and water ad libitum and were kept in rooms
maintained at 22 ± 1 ºC with 12 h light-dark cycle. The animals
were divided into three groups of six animals each: Group I:
Control group (distilled water treated). Group II: Test group
(test compounds were dissolved in polyethylene glycol-400
and administered at a dose of 30, 100, 300 mg/kg i.p. doses),
Group III: Standard group on reference drug (phenytoin 30
mg/kg i.p.).Anticonvulsant activity was established by maximal
electroshock (MES) and subcutaneous metrazole (scMET) models
at drug doses of 30, 100 and 300 mg/kg at three different time
intervals. Neurotoxicity of the compound was determined by
rotorod method.
1
bending for disubstitution). H NMR (CDCl
ppm: 8.26 (s, 1H, Ph-CH=N), 7.82-7.80 (t, 1H, pyridine ring
proton at C ), 7.78-7.76 (d, 1H, pyridine ring proton at C ),
.72-7.70 (d, 2H, m-phenyl ring protons at N ), 7.56-7.54 (d,
H, o-benzyl ring protons at N ), 7.24-7.22 (d, 2H, m-benzyl
), 6.92-6.90 (d, 2H, o-benzyl ring protons at
), 6.62-6.60 (d, 1H, pyridine ring proton at C ), 6.54-6.48
t, 1H, pyridine ring proton at C ), 6.14 (s, 2H, semicarbazide
NH protons), 2.68 (s, 3H, CH protons at phenyl ring), 1.45
3
, 300 MHz) δ in
4
6
7
2
4
1
ring protons at N
4
N
(
1
3
5
3
+
(s, 2H, CH
2
protons at phenyl ring). MS (m/z, %): M : 360.16,
+
+
+
+
(
M +1: 361.17), M -C
3
H
5
: 333.13, M -C
8
H
10: 258.10, M -
+
C
(
8
9
H
12
N
2
O: 198.08, M -C
%): C, 69.98 (69.96); H, 5.59 (5.60); N, 15.55 (15.57); O,
.88 (8.92).
N-(4-(Pyridin-2-yloxy)benzylidene)-4-(4-methoxy-
phenyl)semicarbazide (PSSD7): Yield: 62.86 %, m.p. 130-
5 5
H N: 284.14. Anal. Calcd. (found)
1
ν
32 ºC, m.f. C20
max, cm ): 3438, 3318 (N-H str.), 3025, 2976, 2881 (C-H str.),
H
18
N
4
O
3
; m.w. 362.38, R
f
0.49. FT-IR (KBr,
-1
2
(
1
000-1670 (overtone for disubstitution on aromatic ring), 1666
C=O str.), 1647 (azomethine C=N str.), 1587 (phenyl ring str.),
461 (phenyl C-H in plane bending), 1452 (asym. methyl C-H
bending), 1381 (sym. methyl C-H bending), 1272 (C-N str.
coupled with N-H bending), 1254 (C-O str.), 1055 (N-N str.),
8
1
43 (C-H out of plane bending for disubstitution). H NMR
(
(
CDCl
3
, 300 MHz) δ in ppm: 8.31 (s, 1H, Ph-CH=N), 7.72-7.70
d, 2H, o-benzyl ring protons at N ), 7.68-7.64 (t, 1H, pyridine
), 7.59-7.56 (d, 2H, m-phenyl ring protons at
), 7.40-7.38 (d, 1H, pyridine ring proton at C ), 7.06-7.02 (d,
H, m-benzyl ring protons at N ), 6.82-6.80 (d, 2H, m-phenyl
ring protons at N ), 6.64-6.62 (d, 1H, pyridine ring proton at
), 6.52-6.48 (t, 1H, pyridine ring proton at C ), 6.08 (s, 2H,
semicarbazide NH protons), 3.81 (s, 3H, CH protons at phenyl
Maximum electroshock (MES) seizure test: Each treat-
ment group and vehicle control group consisted of six animals;
60 Hz of alternating current (50 mA in mice) was applied via
ear-clip electrodes for 0.2 s. Mice were tested at 30 min and 4 h
following doses of 30, 100 and 300 mg/kg of test compound.
The test compounds were dissolved polyethylene glycol (PEG)
and injected intraperitoneally (i.p.) at dose of 30, 100 and 300
mg/kg for 30 min before seizures induction. Phenytoin was
used as reference drugs. Abolition of hind limb tonic extensor
component indicated the test compound's ability to inhibit MES
induced seizure spread and was defined as protection [8].
Subcutaneous pentylenetetrazole (scPTZ) seizure test:
Animals were divided into three groups each comprising of
six animals. One group was used for studying the effect of
pentylenetetrazole, the second group for control and the third
group to study effect with reference to the standard. The subcut-
aneous dose of pentylenetetrazole (85 mg/kg) at which 95 %
of the animals showed convulsive reaction was determined by
a dose-percent effect curve. The synthesized compounds were
1
ring proton at C
N
2
4
4
6
1
4
C
3
5
3
+
+
+
ring). MS (m/z, %): M : 362.14, (M +1: 363.13), M -CH
3
2
(
4
O:
N:
86.11. Anal. Calcd. (found) (%): C, 66.29 (66.31); H, 5.01
+
+
+
33.18, M -C
7 8
H
O: 257.10, M -C
H
8 10
N
2
O: 198.15, M -C
H
5 5
5.04); N, 15.46 (15.42); O, 13.25 (13.22).
N-(4-Pyridin-2-yloxy)benzylidene)-4-(4-nitrophenyl)-
semicarbazide (PSSD8): Yield: 60.25 %, m.p. 218-220 ºC,
-1
m.f. C19
H N O
15 5 4
; m.w. 377.35, R
432, 3321 (N-H str.), 3025, 2972, 2886 (C-H str.), 2000-1670
overtone for disubstitution on aromatic ring), 1661 (C=O str.),
641 (azomethine C=N str.), 1592 (phenyl ring str.), 1462 (phenyl
C-H in plane bending), 1445 (asym. methyl C-H bending),
f
0.67. FT-IR (KBr, νmax, cm ):
3
(
1

Vol. 30, No. 10 (2018) Design and Synthesis of Novel N-(4-(Pyridin-2-yloxy)benzylidene)-4-[4-(substituted)phenyl]semicarbazides 2197
administered at the three graded doses, namely 30, 100 and
00 mg/Kg, intraperitoneally. At the anticipated time, pentyl-
Determination of Log P value:Within a congeneric series,
the biological activity of the synthesized compounds depended
on the lipophillic character. Drugs acting on central nervous
system are potent only if they cross blood-brain barrier; there-
fore, potency has been correlated with optimum the lipophili-
city (Log P), which is near 2. In this study, we attempted to
identify the correlation between the anticonvulsant activities
of congeners and their calculated Log P values. The values of
Log P were experimentally determined using octanol-phosphate
buffer method [15].
3
enetetrazole was then administered subcutaneously in the post-
erior midline of mice. The absence of clonic spasm in half or
more of the animals in the observed time periods indicates the
compounds capacity to terminate the effect of pentylenete-
trazole on seizure threshold. The results of test compound were
compared with control and standard compounds [9].
Neurotoxicity screening: The activity of the drugs inter-
fering with motor coordination was checked by the rotarod
test. The mice were trained to stay on an accelerating rotarod
that rotates at 6 revolutions per minute. Trained animals were
given i.p. injection of the test compounds in doses of 30, 100
and 300 mg/kg. The rod diameter was 3.2 cm. Neurotoxicity
indicated by the inability of animal to maintain equilibrium
on the rotarod for at least 1 min in each of three trials. The dose,
at which the animals were unable to grasp the rotarod was
determined. The number of animals in each group (N) = 6;
solvent used polyethylene glycol; dosed with 30, 100 and 300
mg/kg were administered i.p. The figures indicate the minimum
dose whereby bioactivity was demonstrated in half or more
mice. The (-) indicates an absence of activity at maximum
dose administered (300 mg/kg). In neurotoxicity screen, the
figure indicates the dose at which no neurotoxicity was observed
and the dash (-) indicates compounds are not tested [10].
Molecular docking: The molecular docking study of
designed molecules was carried out in order to assess the inter-
action and binding modes with target receptor/enzyme using
Glide extra precision (XP) Maestro 10.1 Schrodinger, running
onWindowsi5(intelcoreprocessor)operatingsystem[Schrodinger,
Version 10.1, 2016]. The 2D structure for synthesized compounds
was generated and then converted to their respective 3D
structures with use of Ligplot. The PDB file of X-ray crystal
RESULTS AND DISCUSSION
A new series of N-(4-(pyridin-2-yloxy)benzylidene)-4-
[4-(substituted)phenyl]semicarbazides (PSSD1-8) was synth-
esized in an effort to obtain the drugs with improved anticon-
vulsant activity. The target compounds PSSD1-8 have been
synthesized by reaction of 4-(pyridinyl-2-oxy)benzaldehyde
and 4-(substituted)phenylsemicarbazides. All the synthesised
compounds were characterized by analytical and spectroscopic
methods. The compounds were evaluated for their anticonvul-
sant activity in MES and scMET models. Compounds PSSD5
and PSSD2 were found to be active in MES model. The maximal
electroshock test is the most widely used animal model in
evaluation of antiepileptic drugs. In the MES screen, compound
PSSD2 showed protection (1/1, 4 h) at a dose of 300 mg/kg.
Compound PSSD5 showed protection (2/3, 2 h) at a dose of
100 mg/kg and protection (1/1, 4 h) at a dose of 300 mg/kg.
None of the compounds showed protection in scMET test, a
test used to identify compounds that elevate seizure threshold.
Minor toxicity (1/2, 4 h) as motor impairment was observed
at a dose of 300 mg/kg with compound PSSD4. None of the
other compounds showed neurotoxicity in the highest admini-
stered dose (300 mg/kg). The present study revealed that comp-
ounds PSSD5 and PSSD2 attenuated MES induced tonic seizures
indicating anticonvulsant activity. The MES test activity of
compounds showed their potential to prevent seizure spread
and identified agents with profound activity against generalized
tonic clonic seizures (Table-1).
structure of human γ-aminobutyric acid receptor, GABA R-
A
β3 homopentamer (PDB ID: 4COF) was downloaded from
RCSB protein data bank. The protein was prepared using the
protein preparation wizard and grid was generated for co-crystal
ligand, benzamidine using receptor grid generation. The water
residues beyond 5 Å were eliminated. The protein was optimized
by assigning H-bonds and minimization at OPLS 2005 force
field. The docked pose of ligands and their interactions were
analyzed after the end of molecular docking. The valproic acid
is used as reference for docking studies.
The molecular docking study of the synthesized molecules
was carried out in order to assess the interaction and binding
modes with target receptor/enzyme. All the compounds were
found to strongly bind by completely occupying the active sites
(at the site of benzamine, co-crystal ligand of the protein as
Determination of ADME properties: The absorption,
distribution, metabolism and excretion (ADME) properties of
the synthesized compounds were determined using the results
of a computational study. The descriptor topological polar
surface area (TPSA), surface belonging to polar atoms, was
shown to be strongly correlated with passive molecular transport
through membranes; therefore, it facilitates the prediction of
transport properties of drugs from the intestine and blood-brain
barrier (BBB) crossingbydrugs [11]. The TPSA, mi Log P,
number of rotatable bonds, number of hydrogen donor and
acceptor atoms, molecular volume and violations of Lipinski's
rule of five [12] of each synthesized compound were calculated
using the Mol inspiration online property calculation tool kit
predicted by SiteMap) in the target protein (GABA
A
R-β ). Only
3
one compound found to have less docking score compared to
valproic acid, the standard anti-convulsant drug and others
outscored it. Almost all the compounds showed high docking
scores than valproic acid, the standard anti-convulsant drug
(Table-2).
Among all the titled compounds, PSSD7 was found to be
most potent and have high docking score of (-5.43) (Table-2).
The binding mode of compound PSSD7 assumes favourable
orientation due to two hydrogen bonds compared with reference
valproic acid (-4.03 as Dock Score). The docked pose (Fig. 2)
of compound PSSD7 showed the hydrogen bond interaction
by ALAB:201 of protein with N of C=N and GLUB:155 with
NH of pyrimidine ring in the compound along with two π-π
staking of pyrimidine ring with TYRA: 62 and TYRB: 157, a
[
13]. The percentage of absorption (% ABS) was calculated
using TPSA [14].

2
198 Singh et al.
Asian J. Chem.
TABLE-1
ANTICONVULSANT ACTIVITY AND NEUROTOXICITY OF
N-(4-(PYRIDIN-2-YLOXY)BENZYLIDENE)-4-[4-(SUBSTITUTED)PHENYL]SEMICARBAZIDES (PSSD 1-8)
a
Intraperitoneal injection in rat
Compounds
MES screen (h)
scMET screen (h)
Neurotoxicity screen (h)
0
–
–
–
.5
2.0
–
–
4.0
–
300
–
0.5
–
–
2.0
–
–
4.0
–
–
0.5
–
–
4.0
–
–
PSSD1
PSSD2
b
PSSD3
PSSD4
PSSD5
PSSD6
–
–
–
–
–
–
c
–
–
–
–
–
100
–
–
–
300
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
300
–
–
b
b
PSSD7
–
–
PSSD8
–
–
–
–
–
–
–
–
Phenytoin
Normal saline
30
–
30
–
30
–
–
–
–
–
–
–
100
–
100
–
a
Doses of 30, 100 and 300 mg/kg were administered. The figures in the table indicate the minimum dose whereby bioactivity was demonstrated in
half or more of the rat. The dash (–) indicates an absence of activity at maximum dose administered (300 mg/kg).
TABLE-2
DOCKING SCORES OF PSSD 1-8 AND VALPROIC ACID
Dock
score
Lipophilic
EvdW
Phob
En
Phob
EnHB
Expos
penal
Ligand
GScore
H-Bond
Electro
PiCat
Rot penal
PSSD7
PSSD8
PSSD4
PSSD3
PSSD2
PSSD1
PSSD5
PSSD6
-5.43
-5.25
-4.86
-4.83
-4.80
-4.43
-4.13
-2.60
-4.03
-5.43
-5.25
-4.86
-4.83
-4.80
-4.43
-4.13
-2.60
-4.03
-2.87
-2.48
-3.03
-3.12
-2.89
-2.86
-3.05
-3.08
-1.29
-1.52
-1.55
-1.65
-1.01
-1.67
-1.48
-0.97
-0.60
-1.40
-0.75
-0.75
-0.75
0.00
-0.75
0.00
0.00
0.00
-1.00
-0.01
-0.42
0.00
-0.60
-0.48
0.00
-0.54
-0.08
0.00
-0.40
-0.61
-0.30
-0.31
-0.49
0.00
-0.48
-0.24
-0.35
0
0
0
0
0
0
0
0
0
0.48
0.54
1.22
0.44
0.90
0.41
0.18
1.25
0.00
0.44
0.41
0.40
0.30
0.37
0.44
0.48
0.44
0.60
Valproic acid
*Note: Results are obtained in XP visualizer (application which shows results obtained by Extra precision docking) and the short forms used stands
for G-score: Glide Sore; PhobEnHB: Reward for hydrophobically packed H-bond; PhobEn: Hydrophobic enclosure reward; Penalties: Polar atom
burial and desolvation penalties, and penalty for intra-ligand contacts; ExposPenal: Penalty for solvent-exposed ligand groups; cancels van der
Waals terms; RotPenal: Rotatable bond penalty.
Fig. 2. 3D Docked pose of PSSD7 (left) and valproic acid (right)
salt bridge of NH with GLUB: 155 and negative charges formed
with TYRB: 205,157 of the same ring and that of valproic acid
in which one carbonyl group forms H-bonds with GLYA 64.
Our lead enjoys good hydrophobic enthalpy reward (-2.87)

Vol. 30, No. 10 (2018) Design and Synthesis of Novel N-(4-(Pyridin-2-yloxy)benzylidene)-4-[4-(substituted)phenyl]semicarbazides 2199
TABLE-3
PHARMACOKINETIC PARAMETERS IMPORTANT FOR GOOD ORAL BIOAVAILABILITY OF
N-(4-(PYRIDIN-2-YLOXY) BENZYLIDENE)-4-[4-(SUBSTITUTED)PHENYL]SEMICARBAZIDES (PSSD1-8)
2
n-OHNH
donors
n-ON
acceptors
Lipinski’s
violations
Compounds
R
% ABS
TPSA (A )
n-ROTB
MW
MV
Rule
–
H
Cl
Br
F
–
–
–
5
5
5
5
5
6
6
5
< 500
232.36
366.81
411.26
350.35
346.39
360.42
362.39
377.36
–
< 5
2
2
2
2
2
2
2
2
< 10
6
6
6
6
6
6
7
9
≤ 1
0
0
1
0
0
1
0
0
PSSD1
PSSD2
PSSD3
PSSD4
PSSD5
PSSD6
PSSD7
PSSD8
82.9
82.9
82.9
82.9
82.9
82.9
79.7
67.1
75.61
75.61
75.61
75.61
75.61
75.61
84.85
121.44
298.75
312.27
316.62
303.67
315.30
298.75
324.28
322.07
CH3
C H
2
5
OCH3
NO2
compared to standard (-1.29) over other compounds and is having
very less rotatable bond penalty of 0.44 whereas the standard
compound valproic acid suffers with high rotatable bond penalty
TABLE-4
LOG P VALUE FOR N-(4-(PYRIDIN-2-YLOXY)BENZYLIDENE)-
-[4-(SUBSTITUTED)PHENYL]SEMICARBAZIDES (PSSD1-8)
4
Experimental Log P
0
.60. Whereas in-case of surface exposed penalty, it suffers a
Compounds
Experimental Log P
(miLog Pa)
little bit (0.48) which is of less importance in front of rotatable
bond penalty as shown in (Fig. 3). High rewards and less penalties
of ligand PSSD7 is the rationale for high docking score and
potency towards the receptor. The docked pose of second most
dock ranked PSSD8 with d-score -5.25 has no surface penalty
but rotatable bond penalty (0.41) which is comparable to the
best one and slightly less hydrophobic enthalpy reward -2.48
possessing good hydrogen bond interactions with receptor
Rule
–
≤ 5
PSSD1
PSSD2
PSSD3
PSSD4
PSSD5
PSSD6
PSSD7
PSSD8
4.02
4.67
4.88
4.26
4.56
4.92
3.98
3.87
4.27
4.95
4.95
4.43
4.72
4.97
4.32
4.23
[
15].
A computational study for determination ofADME prop-
erties of the synthesized compounds was performed.All titled
compounds exhibited a good % ABS ranging from 67.1 to
Conclusion
8
2.9 % (Table-3). Except for compounds PSSD3 and PSSD6,
The above study concluded that the compounds N-(4-(pyridi-
2-yloxy)benzylidene)-4-(4-chlorophenyl)semicarbazide
(PSSD2) and N-(4-(pyridin-2-yloxy)benzylidene)-4-p-tolyl-
semicarbazide (PSSD5) showed anticonvulsant activity in MES
model and thus have the potential to prevent seizure spread.
N-(4-(pyridin-2-yloxy)benzylidene)-4-[4-(substituted)phenyl]-
semicarbazides (PSSD1-8) showed good docking with human
none other compound violated Lipinski's parameters, making
them potentially promising agents for treatment of epilepsy.
It was attempted to correlate the anticonvulsant activity of
congeners with their calculated Log P value (Table-4).All com-
pounds showed lipophilic character. As observed some of the
experimental values were in good agreement with the theore-
tical values suggesting that these have an excellent potential
for CNS activity.
A
γ-aminobutyric acid receptor (GABA R-β3) and the total docking
score is found to be a comparable to reference drug valproic
Fig. 3. 3D pose (in left) of PSSD7 (represented in green) enjoying enthalpy reward (portion of the ligand denoted in ball & socket form). At
right, less but completely, the standard VPA is rewarded

2
200 Singh et al.
Asian J. Chem.
4
5
6
7
.
.
.
.
S.N. Pandeya, A.A. Khan and A. Srivastava, J. Chem. Pharm. Res., 3,
acid. This shows that the probable mechanism of action of these
compounds may be augmentation of GABAergic activity in
the brain.
456 (2011).
L. Tripathi, R. Singh and J.P. Stables, Eur. J. Med. Chem., 46, 509 (2011);
https://doi.org/10.1016/j.ejmech.2010.11.030.
A. Jain, S. Patil and A. Gajbhiye, Int. J. Pharmacol. Pharm. Technol., 1,
ACKNOWLEDGEMENTS
2
277 (2015).
L. Tripathi, P. Kumar, R. Singh and J.P. Stables, Eur. J. Med. Chem., 47,
53 (2012);
The authors gratefully acknowledge Prof.Ashish Singhai,
Director & HOD, Department of Natural Chemistry, Sapience
Bioanalytical Research Lab, Bhopal, India; Prof. M. Shaharyar,
Department of Pharmaceutical Chemistry, Jamia Hamdard,
New Delhi, India and Prof. Dr. A.K. Ghosh, Vice-Chancellor,
IFTM University, Moradabad, India for providing help and
support during the research work.
1
https://doi.org/10.1016/j.ejmech.2011.10.038.
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41 (2003);
https://doi.org/10.1016/j.ejps.2003.08.002.
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3. Molinspiration Cheminformatics, Bratislava, Slovak Republic.
http://www.molinspiration.com/servies/properties.html
The authors declare that there is no conflict of interests
regarding the publication of this article.
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