Synthesis and anticonvulsant evaluation of 2-(substituted benzylidene/ethylidene)-N-(substituted phenyl)hydrazinecarboxamide analogues

Article abstract of DOI:10.1007/s00044-012-0271-z

In the present investigation, we described herein the molecular properties prediction by Molinspiration (2008) and synthesized a series of 17 2-(substituted benzylidene/ethylidene)-N-(substituted phenyl) hydrazinecarboxamide analogues. All the title compounds (4a-q) followed the Lipinski "Rule of Five." The synthesized compounds were characterised by elemental analyses and spectral data followed by anticonvulsant activity according to the Antiepileptic Drug Development Programme Protocol. 2-(4-Hydroxybenzylidene)-N-(2-chlorophenyl)hydrazinecarboxamide (4j) was found to be the most active compound of the series showing protection at 4.0 h at a dose of 100 mg/kg against maximal electroshock seizure test and 50 % (2/4, 0.25, 1-2 h) and 100 % (4/4, 0.5 h) protection in 6 Hz psychomotor seizure test without showing any neurotoxicity. N-(2-chlorophenyl)hydrazine carboxamide (3b) showed 100 % (4/4, 0.25-2 h) and 66.6 % (2/3, 4 h) protection in 6 Hz psychomotor seizure test.

Full text of DOI:10.1007/s00044-012-0271-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2746–2754
DOI 10.1007/s00044-012-0271-z
ORIGINAL RESEARCH
Synthesis and anticonvulsant evaluation of 2-(substituted
benzylidene/ethylidene)-N-(substituted phenyl)hydrazine
carboxamide analogues
•
Mohamed Jawed Ahsan Habibullah Khalilullah
•
•
Sabina Yasmin Surender Singh Jadav
•
•
James P. Stables Jeyabalan Govindasamy
Received: 10 March 2012 / Accepted: 3 October 2012 / Published online: 18 October 2012
Ó Springer Science+Business Media New York 2012
Abstract In the present investigation, we described
herein the molecular properties prediction by Molinspira-
tion (2008) and synthesized a series of 17 2-(substituted
benzylidene/ethylidene)-N-(substituted phenyl)hydrazine-
carboxamide analogues. All the title compounds (4a–
q) followed the Lipinski ‘‘Rule of Five.’’ The synthesized
compounds were characterised by elemental analyses and
spectral data followed by anticonvulsant activity according
to the Antiepileptic Drug Development Programme Pro-
tocol. 2-(4-Hydroxybenzylidene)-N-(2-chlorophenyl)hy-
drazinecarboxamide (4j) was found to be the most active
compound of the series showing protection at 4.0 h at a
dose of 100 mg/kg against maximal electroshock seizure
test and 50 % (2/4, 0.25, 1–2 h) and 100 % (4/4, 0.5 h)
protection in 6 Hz psychomotor seizure test without
showing any neurotoxicity. N-(2-chlorophenyl)hydrazine
carboxamide (3b) showed 100 % (4/4, 0.25–2 h) and
66.6 % (2/3, 4 h) protection in 6 Hz psychomotor seizure
test.
Keywords Anticonvulsant agents Á
6 Hz psychomotor seizure test Á
Maximal electroshock seizure Á
Molecular properties prediction Á Neurotoxicity Á
Semicarbazones
Introduction
Epilepsy is a common neurological disorder, affecting 1–2 %
of the world’s population (White, 2003). In spite of new
developments in the antiepileptic drug (AED) research,
roughly 28–30 % patients still remain poorly treated with the
available AEDs (Meador, 2003). Also most of the AEDs have
dose-related toxicity and idiosyncratic side effects (Dimmock
et al., 1995a; Duncan, 2002). Thus, the search for the new and
effective AEDs continues to be an active area of investigation
in medicinal chemistry. CPP 115 and vigabatrin were found to
be effective in 6 Hz psychomotor seizure test (Antiepileptic
Drug and Device Trials XI Conference, 2011).
M. J. Ahsan (&) Á H. Khalilullah Á J. Govindasamy
New Drug Discovery Research, Department
of Pharmaceutical Chemistry,
Alwar Pharmacy College, Alwar 301 030, Rajasthan, India
e-mail: jawedpharma@gmail.com
Semicarbazones have been reported as potent anticonvul-
sant agents possibly act by inhibiting voltage sensitive Na?
channel (Dimmock et al., 1995b). The pharmacophore model
for anticonvulsant activity includes hydrophobic domain, a
hydrogen binding site and an electron donor moiety (Dimmock
et al., 2000; Pandeya and Raja, 2002). Aryl semicarbazones, a
structurally dissimilar to existing AEDs, can be assumed as
such novel compounds that would not have the side effects
commonly seen with many of the currently available medica-
tions (Azam et al., 2010). Their activity is mainly attributed to
the presence of aryl binding site with aryl/alkyl hydrophobic
group, hydrogen bonding domain and electron donor group as
M. J. Ahsan
Department of Pharmaceutical Sciences,
Nims University, Jaipur 303 121, Rajasthan, India
S. Yasmin Á S. S. Jadav
Department of Pharmaceutical Chemistry,
Birla Institute of Technology, Mesra, Ranchi 835 215,
Jharkhand, India
J. P. Stables
Preclinical Pharmacology Section,
Epilepsy Branch, National Institute of Health, Bethesda,
MD 20892-9020, USA
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Med Chem Res (2013) 22:2746–2754
2747
Synthesis of substituted phenyl urea (2a–b)
H-bonding site
Electron donor
Aromatic aniline (0.1 mol) was dissolved in 20 ml of
glacial acetic acid and 10 ml of hot water and sodium
cyanate (6.5 g, 0.1 mol) in 80 ml of hot water was added
with stirring. It was allowed to stand for 30 min, then
cooled in ice bath and filtered with suction, dried, and
recrystallized from boiling water (Pandeya and Raja, 2002;
Singhal and Paul, 2011).
O
H
N
Ar
NH
N
R1
R
Distal hydrophobic domain
Hyrdrophobic domain
Synthesis of substituted phenyl semicarbazide (3a–b)
Equimolar quantities (0.05 mol) of substituted phenyl urea
(2a–b) and hydrazine hydrate (AR 99–100 %) (2.5 ml,
0.05 mol) in ethanol were refluxed for 48 h with stirring. The
two-third volume of alcohol was distilled by vacuum distil-
lation and then poured into the crushed ice. The resultant
precipitate was filtered, washed with water, and dried. The
solid mass was recrystallized from 50 ml of absolute ethanol
(Pandeya and Raja, 2002; Singhal and Paul, 2011).
H-bonding site
Electron donor
Fig. 1 Pharmacophore model for anticonvulsant activity
Synthesis of substituted phenyl semicarbazone (4a–q)
reported earlier. Furthermore, it was proposed that introduc-
tion of the one more aryl ring in the structure brings about the
enhanced van der Waals bonding at the receptor site that
leads to increased potency (Pandeya and Raja, 2002). The
proposed element for anticonvulsant activity is shown in
Fig. 1 for in vivo interaction with macromolecules. In the
current work, we studied the molecular properties prediction
of 2-(substituted benzylidene/ethylidene)-N-(substituted
phenyl)hydrazinecarboxamide analogues by Molinspira-
tion and synthesized 17 compounds (4a–q) followed by
anticonvulsant evaluation. Earlier we have reported the
anticonvulsant and antimicrobial activities of semicarba-
zone analogues (Amir et al., 2010; Ahsan et al., 2011).
To a solution of substituted phenyl semicarbazide (3a–b)
(0.005 mol) in 1 ml conc. HCl and 25 ml water, a solution
of sodium acetate (0.41 g, 0.005 mol) in 2 ml water was
added. About 25 ml ethanol was added to clear turbidity.
This solution mixture was added to an equimolar quantity
of the appropriate aldehyde or ketone in alcohol. The
solution was stirred for 15 min immediately precipitation
occurred (if immediate precipitation was not occurred,
reaction mixture was allowed to stand for 1–2 h) and the
solids were filtered, dried, and recrystallized from hot
ethanol (Amir et al., 2010).
2-(2-Chlorobenzylidene)-N-(4-methylphenyl)hydrazinecarb-
oxamide (4a) IR (KBr) cm-1: 3375 (NH), 1675 (C=O), 1532
(C=N). 1H NMR (DMSO-d6) ppm: 2.16 (3H, s, CH3),
7.16–7.91 (8H, m, aromatic), 7.93 (1H, s, CH=N), 9.17 (1H, s,
ArNH, D2O exchangeable), 11.02 (1H, s, CONH, D2O
exchangeable). EI-MS (m/z): 287 (M?1), 288 (M?H)?; Cal/
Ana: [C (62.61) 62.59 H (4.90) 4.92 N (14.60) 14.63].
Materials and methods
Chemistry
The entire chemicals were supplied by E. Merck (Germany),
and S.D. Fine Chemicals (India). Melting points were
determined by open tube capillary method and are uncor-
rected. The completion of reactions was monitored by TLC
(silica gel G) using mobile phase chloroform–methanol
(9:1), and the spots were located under iodine vapours or UV
light. IR spectra were obtained on a Schimadzu 8201 PC,
2-(3-Methoxy-4-hydroxybenzylidene)-N-(4-methylphenyl)
hydrazinecarboxamide (4b) IR (KBr) cm-1: 3378 (NH),
1685 (C=O), 1542 (C=N). 1H NMR (DMSO-d6) ppm: 2.36
(3H, s, CH3), 3.79 (3H, s, OCH3), 7.19–7.89 (7H, m, aro-
matic), 8.09 (1H, s, CH=N), 9.12 (1H, s, ArNH, D2O
exchangeable), 10.09 (1H, s, CONH, D2O exchange-
able), 11.57 (1H, s, OH). EI-MS (m/z): 299 (M?1), 300
(M?H)?; Cal/Ana: [C (64.24) 64.20 H (5.70) 5.72 N
(14.06) 14.04].
1
FT-IR spectrometer, Japan (KBr pellets). H NMR spectra
were recorded on a Bruker AC 300 MHz spectrometer,
Germany using TMS as internal standard in DMSO-d6. Mass
spectra were recorded on a Bruker Esquire LCMS, Germany
using ESI and elemental analyses were performed on Perkin-
Elmer 2400 Elemental Analyzer, USA.
123

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Med Chem Res (2013) 22:2746–2754
2-(4-Hydroxybenzylidene)-N-(4-methylphenyl)hydrazine-
carboxamide (4c) IR (KBr) cm-1: 3372 (NH), 1682
(C=O), 1540 (C=N). 1H NMR (DMSO-d6) ppm: 2.24 (3H, s,
CH3), 7.17–7.92 (8H, m, aromatic), 8.19 (1H, s, CH=N),
9.16 (1H, s, ArNH, D2O exchangeable), 10.29 (1H, s,
CONH, D2O exchangeable), 11.02 (1H, s, OH). EI-MS (m/z):
269 (M?1), 270 (M?H)?; Cal/Ana: [C (66.92) 66.90 H
(5.63) 5.61 N (15.59) 15.60].
2-(2,4-Dichlorobenzylidene)-N-(2-chlorophenyl)hydrazine-
carboxamide (4i) IR (KBr) cm-1: 3377 (NH), 1670
(C=O), 1531 (C=N). 1H NMR (DMSO-d6) ppm: 7.22–7.98
(7H, m, aromatic), 8.83 (1H, s, CH=N), 9.02 (1H, s, ArNH,
D2O exchangeable), 10.72 (1H, s, CONH, D2O exchange-
able). EI-MS (m/z): 342 (M?1), 343 (M?H)?; Cal/Ana:
[C (49.08) 49.09 H (2.94) 2.93 N (12.26) 12.27].
2-(4-Hydroxybenzylidene)-N-(2-chlorophenyl)hydrazine-
carboxamide (4j) IR (KBr) cm-1: 3375 (NH), 1675
(C=O), 1532 (C=N). 1H NMR (DMSO-d6) ppm: 7.02–7.64
(8H, m, aromatic), 8.10 (1H, s, CH=N), 8.27 (1H, s, ArNH,
D2O exchangeable), 10.02 (1H, s, CONH, D2O exchange-
able). 13C NMR (DMSO-d6) ppm: 116.21, 122.23, 126.12,
130.42, 129.13, 130.11, 134.02, 143.01, 159.09 (C=O),
160.12. EI-MS (m/z): 389 (M?1), 390 (M?H)?; Cal/Ana:
[C (58.04) 58.05 H (4.17) 4.16 N (14.50) 14.51].
2-[(2-Hydroxyphenyl)ethylidene)]-N-(4-methylphenyl)
hydrazinecarboxamide (4d) IR (KBr) cm-1: 3378
(NH), 1672 (C=O), 1534 (C=N). 1H NMR (DMSO-d6)
ppm: 2.28 (3H, s, CH3), 7.19–7.96 (8H, m, aromatic), 9.24
(1H, s, ArNH, D2O exchangeable), 10.24 (1H, s, CONH,
D2O exchangeable), 10.92 (1H, s, OH). EI-MS (m/z): 283
(M?1), 284 (M?H)?; Cal/Ana: [C (67.86) 67.83 H (6.04)
6.05 N (14.81) 14.83].
2-[(2,4-Dihydroxyphenyl)ethylidene)]-N-(4-methylphenyl)-
hydrazinecarboxamide (4e) IR (KBr) cm-1: 3376 (NH),
1682 (C=O), 1540 (C=N). 1H NMR (DMSO-d6) ppm: 2.18
(3H, s, CH3), 7.22–7.98 (7H, m, aromatic), 9.02 (1H, s,
ArNH, D2O exchangeable), 10.27 (1H, s, CONH, D2O
exchangeable), 11.02 (1H, s, OH). EI-MS (m/z): 299
(M?1), 300 (M?H)?; Cal/Ana: [C (64.18) 64.20 H (5.70)
5.72 N (14.05) 14.04].
2-(3,4-Dimethoxybenzylidene)-N-(2-chlorophenyl)hydra-
zinecarboxamide (4k) IR (KBr) cm-1: 3372 (NH), 1678
1
(C=O), 1538 (C=N). H NMR (DMSO-d6) ppm: 3.81 (3H,
s, OCH3), 3.83 (3H, s, OCH3), 7.39–7.98 (7H, m, aro-
matic), 8.98 (1H, s, CH=N), 9.17 (1H, s, ArNH, D2O
exchangeable), 11.67 (1H, s, CONH, D2O exchangeable).
EI-MS (m/z): 333 (M?1), 334 (M?H)?; Cal/Ana:
[C (57.58) 59.33 H (4.83) 4.66 N (12.59) 13.85].
2-(3-Hydroxybenzylidene)-N-(2-chlorophenyl)hydrazine-
carboxamide (4l) IR (KBr) cm-1: 3377 (NH), 1674
(C=O), 1542 (C=N). 1H NMR (DMSO-d6) ppm: 7.21–7.98
(8H, m, aromatic), 8.62 (1H, s, CH=N), 9.18 (1H, s, ArNH,
D2O exchangeable), 10.97 (1H, s, CONH, D2O exchange-
able), 11.04 (1H, s, OH). EI-MS (m/z): 289 (M?1), 290
(M?H)?; Cal/Ana: [C (58.05) 58.04 H (4.18) 4.17 N
(14.49) 14.50].
2-Cyclohexylidene-N-(4-methylphenyl)hydrazinecarboxa-
mide (4f) IR (KBr) cm-1: 3373 (NH), 1672 (C=O), 1533
(C=N). 1H NMR (DMSO-d6) ppm: 2.16 (3H, s, CH3),
2.22–2.54 (10H, m, cyclohexyl), 7.16–7.74 (4H, m, aro-
matic), 9.17 (1H, s, ArNH, D2O exchangeable), 10.67 (1H, s,
CONH, D2O exchangeable). EI-MS (m/z): 245 (M?1); Cal/
Ana: [C (68.54) 68.55 H (7.81) 7.83 N (17.13) 17.14].
2-N-(4-methylphenyl)-2-(4-oxopentan-2-ylidene)hydrazin-
ecarboxamide (4g) IR (KBr) cm-1: 3376 (NH), 1678
(C=O), 1537 (C=N). 1H NMR (DMSO-d6) ppm: 1.12 (3H, s,
CH3), 2.36 (3H, s, CH3), 2.52 (2H, s, CH2), 7.06–7.51 (4H,
m, aromatic), 8.64 (1H, s, ArNH, D2O exchangeable), 9.67
(1H, s, CONH, D2O exchangeable). EI-MS (m/z): 247
(M?1); Cal/Ana: [C (63.13) 63.14 H (6.91) 6.93 N (16.97)
16.99].
2-[1-(4-Methoxyphenyl)ethylidene]-N-(2-chlorophenyl)
hydrazinecarboxamide (4m) IR (KBr) cm-1: 3377
(NH), 1680 (C=O), 1531 (C=N). 1H NMR (DMSO-d6)
ppm: 3.82 (3H, s, OCH3), 7.29–7.88 (8H, m, aromatic),
9.17 (1H, s, ArNH, D2O exchangeable), 10.37 (1H, s,
CONH, D2O exchangeable). EI-MS (m/z): 317 (M?1), 318
(M?H)?; Cal/Ana: [C (60.47) 60.48 H (5.08) 5.09 N
(13.22) 13.23].
2-(4-Chlorobenzylidene)-N-(2-chlorophenyl)hydrazine-
carboxamide (4h) IR (KBr) cm-1: 3379 (NH), 1685
2-[1-(4-Methylphenyl)ethylidene]-N-(2-chlorophenyl)
hydrazinecarboxamide (4n) IR (KBr) cm-1: 3370 (NH),
1678 (C=O), 1539 (C=N). 1H NMR (DMSO-d6) ppm: 2.18
(3H, s, OCH3), 7.19–7.79 (8H, m, aromatic), 9.26 (1H, s,
ArNH, D2O exchangeable), 10.57 (1H, s, CONH, D2O
exchangeable). EI-MS (m/z): 301 (M?1), 302 (M?H)?;
Cal/Ana: [C (63.67) 63.68 H (5.33) 5.34 N (13.93) 13.92].
1
(C=O), 1543 (C=N). H NMR (DMSO-d6) ppm: 7.19–7.74
(8H, m, aromatic), 8.96 (1H, s, CH=N), 9.07 (1H, s, ArNH,
D2Oexchangeable),10.87(1H,s,CONH,D2Oexchangeable).
EI-MS (m/z): 308 (M?1), 309 (M?H)?; Cal/Ana: [C (54.57)
54.55 H (3.60) 3.63 N (13.64) 13.65].
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Med Chem Res (2013) 22:2746–2754
2749
2-[(4-Chlorophenyl)ethylidene)]-N-(2-chlorophenyl)hydra-
zinecarboxamide (4o) IR: 3373 (NH), 1671 (C=O), 1531
(C=N). 1H NMR (DMSO-d6) ppm: 7.42–8.09 (8H, m,
aromatic), 9.02 (1H, s, ArNH, D2O exchangeable), 10.52
(1H, s, CONH, D2O exchangeable). EI-MS (m/z): 322
(M?1), 323 (M?H)?; Cal/Ana: [C (55.92) 55.93 H (4.07)
4.06 N (13.04) 13.05].
from scMET-induced seizures upon the absence of episode
of clonic spasms, approximately 3–5 s, of the fore and/or
hindlimbs, jaws, or vibrissae.
Hz psychomotor seizure test
Some clinically useful AEDs are ineffective in the standard
MES and scMET tests but still have anticonvulsant activ-
ities in vivo. In order to identify potential AEDs with this
profile, compounds were tested in the minimal clonic sei-
zure (6 Hz or ‘‘psychomotor’’) test. The title compounds
were tested in the minimal clonic seizure test at dose of
100 mg/kg to four mice. Like the MES test, the minimal
clonic seizure (6 Hz) test is used to assess a compound’s
efficacy against electrically induced seizures but uses a
lower frequency (6 Hz) and longer duration of stimulation
(3 s).
2-(Furan-2-ylmethylidene)-N-(2-chlorophenyl)hydrazine-
carboxamide (4p) IR (KBr) cm-1: 3375 (NH), 1674
(C=O), 1531 (C=N). 1H NMR (DMSO-d6) ppm: 7.02–7.58
(7H, m, aromatic), 8.38 (1H, s, CH=N), 9.17 (1H, s, ArNH,
D2O exchangeable), 10.12 (1H, s, CONH, D2O exchange-
able). EI-MS (m/z): 263 (M?1), 264 (M?H)?; Cal/Ana:
[C (54.66) 54.65 H (3.38) 3.39 N (15.94) 15.93].
2-Cyclopentylidene-N-(2-chlorophenyl)hydrazinecarbox-
amide (4q) IR (KBr) cm-1: 3378 (NH), 1682 (C=O), 1543
(C=N). 1H NMR (DMSO-d6) ppm: 2.31–2.63 (8H, m,
cyclopentyl), 7.16–7.54 (4H, m, aromatic), 9.02 (1H, s,
ArNH, D2O exchangeable), 10.17 (1H, s, CONH, D2O
exchangeable). EI-MS (m/z): 251 (M?1), 252 (M?H)?; Cal/
Ana: [C (57.22) 57.26 H (5.62) 5.61 N (16.67) 16.69].
Neurotoxicity or minimal motor impairment
Minimal motor impairment was measured in mice by the
rotorod test. The mice were trained to stay on an acceler-
ating rotorod that rotate at 10 rpm. The rod diameter was
3.2 cm trained animals were given i.p. injection of the test
compound in doses of 30, 100, and 300 mg/kg, neurotox-
icity was indicated by the inability of the mice to maintain
equilibrium on the rod for at least 1 min in each of the
three trails.
Pharmacology
Maximal electroshock (MES)
The anticonvulsant evaluations were undertaken by the
National Institute of Health, using their reported proce-
dures. For all tests based on MES convulsions, 60 Hz of
alternating current (50 mA in mice and 150 mA in rat) was
delivered for 0.2 s by corneal electrodes which have been
primed with an electrolyte solution containing anaesthetic
agent (0.5 % tetracaine HCl). The mice were tested at
various (0.5 and 4 h) intervals following doses of 30, 100,
and 300 mg/kg of test compound given by i.p. injection of
a volume of 0.01 ml/g. An animal is considered ‘‘pro-
tected’’ from MES-induced seizures upon abolition of the
hindlimb tonic extensor component of the seizure.
In vitro hippocampal slice culture neuroprotection
assay (NP): primary screen experiment
The ‘‘Primary Screen Experiment’’ is a qualitative assess-
ment of the ability of a compound to prevent excitotoxic
cell death. Organotypic hippocampal slice cultures are
treated with N-methyl-D-aspartate (NMDA) or kainic acid
(KA) to induce neuronal cell death. Propidium iodide, a
membrane-impermeant compound, is included in all wells
of the culture plate. Dying cells have compromised cell
membranes, thus, propidium iodide may diffuse into the
cell, intercalate with DNA and fluoresce. Thus, the inten-
sity of the propidium iodide fluorescence is proportional to
the amount of cell death in the individual slices. Hippo-
campal slice cultures are treated with the excitotoxin alone,
or where indicated above, with the excitotoxin and either
one or two investigational compounds at the concentrations
indicated. If NP occurs as a consequence of the added
compound, slice cultures will have a visibly reduced
fluorescent intensity when compared to the slice cultures
that have been treated with the excitotoxin alone.
Subcutaneous metrazole-induced seizure (scMET)
Subcutaneous injection of the convulsant metrazole pro-
duces clonic seizures in laboratory animals. The scMET
test detects the ability of a test compound to raise the
seizure threshold of an animal, and thus, protect it from
exhibiting a clonic seizure. Animals were pre-treated with
various doses of the test compound (in a similar manner to
the MES test, although a dose of 50 mg/kg (p.o.) is the
standard for scMET). An animal is considered ‘‘protected’’
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Med Chem Res (2013) 22:2746–2754
Results and discussion
Molecular properties prediction
according to the equation: %ABS = 109 0.345 9 TPSA
(Wang et al., 1997). From all these parameters, it can be
observed that all the title compounds (4a–q) exhibited a great
%ABS ranging from 76.59 to 90.55 % and followed the
Lipinski ‘‘Rule of Five’’ which states that the molecules with
good membrane permeability have log P B5, molecular
weight B500, number of hydrogen bond acceptors B10, and
number of hydrogen bond donors’ B5 (Lipinski et al., 2001).
This rule is widely used as a filter for drug-like properties.
The pharmacokinetic parameters were calculated online
from Molinspiration Chemoinformatics (http://www.molin
spiration.com) and are given in Table 1.
Molecular properties, mainly hydrophobicity, molecular
size, flexibility and the presence of various pharmacophoric
features influence the behaviour of molecules in the living
organism, including bioavailability. Thus, in order to
achieve good oral drugs, we have subjected a series of
2-(substituted benzylidene/ethylidene)-N-(substituted phenyl)-
hydrazinecarboxamide analogues (4a–q) for prediction of
lipophilicity and Lipinski ‘‘Rule of Five’’ and other prop-
erties for filtering compounds for synthesis and anticon-
vulsant evaluation. For good membrane permeability, log P
value should be B5. All the title compounds (4a–q) have
miLog P value 1.997–5.327. It is revealed that for passing
oral bioavailability criteria, number of rotatable bond
should be B10 (Veber et al., 2002). The compounds in this
series (4a–q) in general possess sufficient number of
rotatable bonds (1–5) and therefore, exhibit good confor-
mational flexibility. Molecular polar surface area (TPSA) is
a very useful parameter for the prediction of drug transport
properties. TPSA is a sum of surfaces of polar atoms (usually
oxygen, nitrogen and attached hydrogen) in a molecule.
TPSA and volume is inversely proportional to percentage of
absorption (%ABS). TPSA was used to calculate the %ABS
Chemistry
The aryl semicarbazone analogues (4a–q) described in this
study are shown in Table 2 and the reaction sequence for
the synthesis is summarized in Scheme 1. In the initial
step, substituted aromatic amines were treated with sodium
cyanate in glacial acetic acid at room temperature giving
substituted phenyl urea (2a–b). In the subsequent step,
substituted phenyl urea was treated with hydrazine hydrate
in ethanol giving phenyl semicarbazide (3a–b). In the final
step, treatment of phenyl semicarbazide with appropriate
aldehyde/ketone in the presence in sodium acetate in eth-
anol furnished the title compounds (4a–q). The yields of
Table 1 Pharmacokinetic parameters important for good oral bioavailability of 2-(substituted benzylidene/ethylidene)-N-(substituted phenyl)-
hydrazinecarboxamide analogues (4a–q)
Compound
% ABS
Volume
(A3)
TPSA
(A2)
NROTB
n-ON
acceptor
n-OHNH
donors
miLog P
MW
Lipinski’s
violations
Rule
4a
4b
4c
4d
4e
4f
–
–
–
–
3
4
3
3
3
2
4
3
3
3
5
3
4
3
3
3
2
\10
4
\5
2
3
3
3
4
2
2
2
2
3
2
3
2
2
2
2
2
B5
\500
287
299
269
283
299
245
247
308
342
289
333
289
317
301
322
263
251
B1
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
90.55
80.38
83.57
83.57
76.59
90.55
84.66
90.55
90.55
83.57
84.17
83.57
87.36
90.55
90.55
86.01
90.55
252.598
272.625
247.08
53.489
82.951
73.717
73.717
93.945
53.489
70.56
4.492
3.201
3.359
3.716
3.213
3.723
1.997
4.721
5.327
3.564
3.69
6
5
263.641
271.658
240.82
5
6
4
4g
4h
4i
236.561
249.572
263.108
244.054
287.128
244.054
278.143
269.159
266.133
217.605
220.993
5
53.489
53.489
73.717
71.957
73.717
62.723
53.489
53.489
66.629
53.489
4
4
4j
5
4k
4l
6
5
3.54
4m
4n
4o
4p
4q
5
4.013
4.405
4.635
3.3
4
4
5
4
3.398
%ABS percentage of absorption, TPSA topological polar surface area, NROTB number of rotatable bonds, MW, molecular weight, miLog
P logarithm of compound partition coefficient between n-octanol and water, n-OHNH number of hydrogen bond donors, n-ON number of
hydrogen bond acceptors
123

Med Chem Res (2013) 22:2746–2754
2751
Table 2 Physical constant of N-(substituted phenyl)hydrazinecarboxamide and 2-(substituted benzylidene/ethylidene)-N-(substituted phenyl)
hydrazinecarboxamide analogues
H
N
H
N
H
N
H
N
H
N
H
N
R
H
N
H
N
NH
N
N
Ar
1
1
N
2
1
1
R
R
R
R
O
O
O
O
4a-e, 4g-p
4f
4q
3a-b
Compound
ADD No.
R1
R
Ar
Yield (%)
Rfa
Mp (°C)
3a
3b
4a
4b
4c
4d
4e
4f
433050
439081
433052
433054
439077
433055
433058
433059
433060
433061
433062
433063
433064
433065
433067
433068
439085
433070
433069
Methyl-
–
–
–
86
82
78
62
58
84
72
66
76
72
69
67
92
82
84
88
62
64
62
0.64
0.58
0.65
0.72
0.48
0.70
0.81
0.59
0.79
0.60
0.73
0.68
0.78
0.77
0.75
0.61
0.66
0.63
0.56
125
105
172
188
122
168
220
178
70
2-Chloro
4-Methyl-
4-Methyl-
4-Methyl-
4-Methyl-
4-Methyl-
4-Methyl-
4-Methyl-
2-Chloro-
2-Chloro-
2-Chloro-
2-Chloro-
2-Chloro-
2-Chloro-
2-Chloro-
2-Chloro-
2-Chloro-
2-Chloro-
–
H
2-Chlorophenyl-
H
3-Methoxy-4-hydroxyphenyl-
3-Hydroxyphenyl-
2-Hydroxyphenyl-
2,4-Dihydroxyphenyl-
–
H
Methyl-
Methyl-
–
4g
4h
4i
Methyl
2-Propanonyl-
H
4-Chlorophenyl-
2,4-Dichlorophenyl-
4-Hydroxyphenyl-
3,4-Dimethoxyphenyl-
3-Hydroxyphenyl-
4-Methoxyphenyl-
4-Methylphenyl-
4-Chlorophenyl-
2-Furfuryl-
182
184
202
172
186
72
H
4j
H
4k
4l
H
H
4m
4n
4o
4p
4q
Methyl-
Methyl-
Methyl-
H
169
92
78
–
–
128
ADD No. Antiepileptic Drug Development Number (National Institute of Neurodegenerative Disorders and Stroke, National Institute of Health,
USA)
a
Mobile phase: chloroform: methanol (9:1)
the titled compounds were ranging from 62 to 92 % after
recrystallization with absolute ethanol. The completion of
reactions was monitored TLC using mobile phase chloro-
form: methanol (9:1) and purity was checked by elemental
analyses. Both the analytical and spectral data (IR, 1H
NMR) of all the synthesized compounds were in full
accordance with the proposed structures. In general, the IR
spectra of the compounds afforded imine C=N stretching at
1,528–1,545 cm-1, and carbamoyl group N–H stretching at
3,366–3,390 cm-1 and C=O stretching at 1,670–1,686 cm-1
bands. In the Nuclear Magnetic Resonance spectra
(1H NMR), the signals of the respective protons of the
synthesized titled compounds were verified on the basis of
their chemical shift and multiplicities in DMSO-d6. The
spectra showed singlet at d 2.02–2.96 ppm corresponding
to CH3; a singlet at d 3.80–3.85 ppm corresponding to
OCH3 group; multiplet at d 7.06–7.98 ppm corresponding
to aromatic protons; a singlet at d 7.93–9.08 ppm
corresponding to imine H; a singlet at d 9.02–9.27 ppm
corresponding to aromatic NH (D2O exchangeable); broad
singlet at d 9.97–11.87 ppm corresponding to CONH (D2O
exchangeable) and a singlet at 10.92–11.67 ppm corre-
sponding to aromatic OH. The elemental analysis results
were within 0.4 % of the theoretical values.
Anticonvulsant activity
All the compounds were evaluated for their anticonvulsant
activity according to the Antiepileptic Drug Development
(ADD) Programme Protocol reported elsewhere (Swing-
yard et al., 1989; White et al., 1995a, b; Dunham and Miya,
1957; Barton et al., 2001; Toman et al., 1952). Initially, the
compounds were administered i.p. at doses of 30, 100 and
300 mg/kg in mice and activity was established using the
MES-induced seizure and scMET model to identify their
anticonvulsant activity at two intervals (0.5 and 4.0 h).
123

2752
Med Chem Res (2013) 22:2746–2754
Scheme 1 Synthetic protocol:
(a) reagents: NaCNO/AcOH,
(b) NH2NH2ÁH2O/EtOH and
(c) aldehyde/ketone/EtOH
H
N
H
N
H
N
NH
NH
2
2
NH
2
1
1
a
b
1
R
R
R
O
O
3a-b
1a-b
2a-b
c
H
N
H
N
R
H
N
H
N
H
N
H
N
N
Ar
1
1
N
1
N
R
R
R
O
O
O
4q
4f
4a-e, 4g-p
Table 3 Anticonvulsant evaluation of N-(substituted phenyl)hydrazinecarboxamide and 2-(substituted benzylidene/ethylidene)-N-(substituted
phenyl)hydrazinecarboxamide analogues
Compound
Intraperitoneal injection in micea
MES screen Toxicity screen
Intraperitoneal injection in mice (100 mg/kg)
Time to peak effect (N/F)b
Toxicity
0.5 h
4.0 h
0.5 h
4.0 h
0.25 h 0.5 h 1.0 h 2.0 h 4.0 h 0.25 h 0.5 h 1.0 h 2.0 h 4.0 h
3a
300
–
–
–
300d
–
4/4
4/4
9
2/4
4/4
9
2/4
4/4
9
1/4c
4/4
9
0/3d
2/3
9
0/4
1/4
9
0/4
1/4
9
0/4
1/4
9
1/4
0/4
9
2/4
1/4e
9
3b
–
–
4a
–
–
–
–
4b
–
–
–
–
9
9
9
9
9
9
9
9
9
9
4c
–
–
–
–
2/4
9
1/4
9
0/4
9
1/4
9
0/4
9
0/4
9
0/4
9
0/4
9
0/4
9
0/4
9
4d
–
–
–
–
4e
–
–
–
–
9
9
9
9
9
9
9
9
9
9
4f
–
300
–
–
–
1/4
9
4/4
9
2/4
9
0/4
9
1/4
9
0/4
9
0/4
9
0/4
9
0/4
9
0/4
9
4g
–
–
–
4h
–
–
–
–
9
9
9
9
9
9
9
9
9
9
4i
–
–
–
–
9
9
9
9
9
9
9
9
9
9
4j
–
100
100
–
–
–
2/4
1/4
9
4/4
0/4
9
2/4
0/4
9
2/4
1/4
9
0/4
1/4
9
0/4
0/4
9
0/4
0/4
9
0/4
0/4
9
0/4
0/4
9
0/4
0/4
9
4k
–
–
–
4l
–
–
–
4m
4n
300
–
300
–
–
300
–
9
9
9
9
9
9
9
9
9
9
–
9
9
9
9
9
9
9
9
9
9
4o
–
100
–
–
–
0/4
9
0/4
9
2/4
9
0/4
9
0/4
9
0/4
9
0/4
9
0/4
9
0/4
9
0/4
9
4p
–
–
–
4q
Phenytoinf
–
–
–
–
9
9
9
9
9
9
9
9
9
9
30
Carbamazepinef 30
30
100
100
100
100
300
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
A dash (–) indicate lack of activity
A cross (9) indicates activity not determined
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 mice. The animals were examined 0.5 and 4 h after administration
b
N/F = number of animals active or toxic over the number tested at a dose of 100 mg/kg
c
Clonic seizure in 1/4 animal
d
Death in 2/3 animal
e
Death of one mice followed by clonic seizure
f
Activity reported (Pandeya and Raja, 2002)
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Med Chem Res (2013) 22:2746–2754
2753
Neurotoxicity or toxicity was observed by minimal motor
impairment which was measured in mice by rotorod test.
Seven compounds were screened in 6 Hz psychomotor
seizure test to identify their anticonvulsant activity at five
different time points viz. 0.25, 0.5, 1.0, 2.0 and 4.0 h after
i.p. administration in mice. Two compounds were also
evaluated in the MES test and toxicity after oral adminis-
tration to rat at 30 mg/kg dose and in vitro hippocampal
slice culture NP assay (Noraberg et al., 2005).
4-hydroxy, 4-methoxy and 3,4-dimethoxy group showed
increased activity in MES screening. 4-Hydroxy group
present on distal hydrophobic aryl domain showed maxi-
mum activity in 6 Hz psychomotor seizure test without any
neurotoxicity or toxicity. Also semicarbazone analogue
with cyclohexyl group (4f) showed significantly more
activity than cyclopentyl group (4q).
All the compounds were evaluated for their anticon-
vulsant activity against MES, scMET model and toxicity at
two intervals (0.5 and 4.0 h), and the results are given in
Table 3. None of the compounds showed any protection in
scMET screen while 6 compounds showed significant
protection in MES screening. In 6 Hz psychomotor seizure
test four among the seven compounds showed significant
protection against MES-induced seizure. N-(4-methyl-
phenyl)hydrazinecarboxamide 3a, showed protection in
MES screening at 0.5 h at 300 mg/kg dose but associated
with toxicity followed by death of the mice. 2-Cyclo-
hexylidene-N-(4-methylphenyl)hydrazinecarboxamide 4f,
2-(4-hydroxybenzylidene)-N-(2-chlorophenyl)hydrazine-
carboxamide 4j and 2-(3,4-dimethoxybenzylidene)-N-(2-
chlorophenyl)hydrazinecarboxamide 4k showed protec-
tion against MES-induced seizure at 4.0 h at 300, 100
and 300 mg/kg dose, respectively, devoid of any toxicity.
2-[1-(4-Methoxyphenyl)ethylidene]-N-(2-chlorophenyl)
hydrazinecarboxamide 4m showed protection against
MES-induced seizure at 0.5 and 4.0 h at 300 mg/kg dose
and associated with neurotoxicity at 4.0 h (300 mg/kg).
The compounds, 4j and 4k did not show any protection
against MES-induced seizure test in rat at 30 mg/kg dose
after oral administration. Also the compounds 4j and 4k in
pairs did not show NP against KA or NMDA-induced
cytotoxicity in vitro hippocampal slice culture NP assay.
The compounds 3a, 3b, 4f, and 4j showed significant
protection in 6 Hz psychomotor seizure test. The com-
pound 3a showed 100 % (4/4, 0.25 h), 50 % (2/4,
0.5–1.0 h) and 25 % (1/4, 2.0 h) protection; compound 3b
showed 100 % (4/4, 0.25–2.0 h) and 66.6 % (2/3, 4.0 h)
protection; compound 4f showed 25 % (1/4, 0.25 and
4.0 h), 100 % (4/4, 0.5 h) and 50 % (2/4, 1.0 h) protection;
compound 4j showed 50 % (2/4, 0.25 and 1–2 h) and
100 % (4/4, 0.5 h) protection. The compound 4k showed
25 % (1/4, 0.25, 2.0 and 4.0 h), while compound 4o
showed 50 % (2/4, 1.0 h) in 6 Hz psychomotor seizure test.
We have observed that 4j was the most active compounds
of the series. In the title compounds (4a–p), aryl semicar-
bazone with electronegative group substitution such as
2-chloro, 4-chloro, 2,4-dichloro and electron releasing
group such as 2-hydroxy and 3-hydroxy in the distal
hydrophobic aryl domain showed decreased activity in
MES screening while electron releasing group such as
Conclusion
All the compounds were synthesized in good yields. Aryl
semicarbazone with electronegative group substitution
such as 2-chloro, 4-chloro, 2,4-dichloro and electron
releasing group such as 2-hydroxy and 3-hydroxy in the
distal hydrophobic aryl domain showed decreased activity
in MES screening while electron releasing group such as
4-hydroxy, 4-methoxy and 3,4-dimethoxy group showed
increased activity in MES screening. 4-Hydroxy group
present on distal hydrophobic aryl domain showed maxi-
mum activity in 6 Hz psychomotor seizure test without any
neurotoxicity or toxicity. Also semicarbazone analogue
with cyclohexyl group showed significantly more activity
than cyclopentyl group.
Acknowledgments The authors would like to express their grati-
tude to the management people of Alwar Pharmacy College, Alwar,
Rajasthan, India for providing research facilities, and all the staffs of
Antiepileptic Drug Development (ADD) programme, National Insti-
tute of Health (NIH), USA for anticonvulsant screening.
Conflict of interest The authors reported no conflict of interest.
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