Design, synthesis and?in?vivo anticonvulsant screening in?mice of?Novel phenylacetamides

Article abstract of DOI:10.1016/j.ejmech.2006.03.013

A set of seven novel N-substituted 2-anilinophenylacetamides were designed by pharmacophore generation and using flexible alignment module of MOE software. The novel molecules were synthesized and screened for anticonvulsant activity in Swiss albino mice by MES and ScPTZ induced seizure tests. Test compounds were found to be potent in MES test. Compounds 12 and 14 were found to be more potent with ED₅₀ values 24.0 and 8.0?mg kg^-1, respectively, and their activity was comparable to standard drugs (Phenytoin, Carbamazepine). Test compounds did not show significant activity in ScPTZ test. Compounds 12 and 14 also exhibited higher protective indices (20.3 and 87.5, respectively) when assessed for neurotoxicity by rotarod test as compared to the standards.

Full text of DOI:10.1016/j.ejmech.2006.03.013

European Journal of Medicinal Chemistry 41 (2006) 786–792
http://france.elsevier.com/direct/ejmech
Laboratory note
Design, synthesis and in vivo anticonvulsant
screening in mice of Novel phenylacetamides
A.V. Shindikar *, F. Khan, C.L. Viswanathan
The Bombay College of Pharmacy, Kalina, Santacruz (East), Mumbai 400098, Maharashtra, India
Accepted 6 March 2006
Available online 02 May 2006
Abstract
A set of seven novel N-substituted 2-anilinophenylacetamides were designed by pharmacophore generation and using flexible alignment
module of MOE software. The novel molecules were synthesized and screened for anticonvulsant activity in Swiss albino mice by MES and
ScPTZ induced seizure tests. Test compounds were found to be potent in MES test. Compounds 12 and 14 were found to be more potent with
ED50 values 24.0 and 8.0 mg kg–1, respectively, and their activity was comparable to standard drugs (Phenytoin, Carbamazepine). Test com-
pounds did not show significant activity in ScPTZ test. Compounds 12 and 14 also exhibited higher protective indices (20.3 and 87.5, respec-
tively) when assessed for neurotoxicity by rotarod test as compared to the standards.
© 2006 Elsevier SAS. All rights reserved.
Keywords: Anticonvulsants; phenylacetamides; 2-anilinophenylacetamides; MES test; ScPTZ test
1. Introduction
insight into the molecular mechanisms by which these might
be controlled [6]. Inhibition of neuronal cation conductance
via block of voltage gated sodium channels is a proven me-
chanism by which anticonvulsants phenytoin, carbamazepine
and valproic acid act to control seizures [7].
The present study aims to generate pharmacophore for po-
tent anticonvulsant activity and from it design the structure of a
lead molecule. It is then proposed to synthesize a series of
analogs of the lead molecule and screen them for anticonvul-
sant activity using maximal electroshock seizure (MES) and
sub-cutaneous pentylenetetrazole (ScPTZ) induced seizure
models.
Epilepsy is the most common serious disorder of the brain
and is characterized by recurrent unprovoked seizures. It is es-
timated that there are 50 million people with epilepsy world-
wide and the majority of cases are in the developing countries
[1,2]. The disorder, if untreated, can lead to impaired intellec-
tual function or death and is typically accompanied by psycho-
pathological consequences such as loss of self-esteem [3]. The
established antiepileptic drugs like phenytoin, carbamazepine,
ethosuximide, valproic acid and barbiturates though widely
prescribed, produce adverse effects such as ataxia, hepatotoxi-
city, gingival hyperplasia and megaloblastic anaemia [4,5].
Studies indicate that a significant group of patients (20–30%)
are resistant to the currently used therapeutic agents. Thus,
there is a constant need for improved antiepileptic drugs i.e.
agents with more effective anticonvulsant activity and exhibit-
ing lower toxicity.
2. Results and discussion
2.1. Design and synthesis of test series
A group of potent anticonvulsants which act by binding to
the Na+ ion channel were selected for pharmacophoric studies.
A two-point pharmacophore was then generated wherein an
aromatic center separated from an H-bonding acceptor/donor
group was found to be essential for activity (Fig. 1). 3D data-
base searching lead to identification of phenylacetamide as the
Intensive study into the physiological and biochemical
events that take place during epileptic seizures has provided
* Corresponding author.
E-mail address: anandshindikar@yahoo.com (A.V. Shindikar).
0223-5234/$ - see front matter © 2006 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.03.013

A.V. Shindikar et al. / European Journal of Medicinal Chemistry 41 (2006) 786–792
787
Fig. 1. Pharmacophore for anticonvulsant activity.
lead molecule. To further refine the search, a flexible alignment
2.2. Anticonvulsant screening
2.2.1. MES test
study was carried out using structures of potent anticonvulsants
flunarizine and PD85639. Seven substituted phenylacetamides
which showed good alignment (low scores) were chosen for
further development (Table 1 and Fig. 2).
The results of preliminary anticonvulsant screening are
given in Table 2. Compound 12 and 14 were active at
30 mg kg–1 body weight dose. Compound 10 and 13 were
active at 100 mg kg–1 body weight dose while compound 9
was active only at a dose of 300 mg kg–1 body weight. Com-
pounds 11, 12 and 13 induced drowsiness to varying degrees.
Compound 11 was active at a dose of 100 mg kg–1 body
weight but activity could be recorded at 4 h as the compound
induced severe drowsiness in the initial stages.
The substituted phenylacetamides were synthesized using 2-
anilinophenylacetic acid (2-[2-(phenylamino) phenyl] acetic
acid) as an important intermediate. Synthesis of 2-[2-(phenyla-
mino) phenyl] acetic acid was achieved by two different routes
(Methods A and B, Scheme 1), where the percentage yield in
Method A was 43% and in Method B was 85.5%. Method B
had advantages with respect to not only yield but also in the
efficiency of the process (cost and number of steps). The con-
densation of diphenylamine with chloroacetylchloride (Method
B) was achieved by using DMF in catalytic quantity and with-
out the use of solvent. It led to improved yields of 1-(dipheny-
lamino)-2-chloro-ethan-1-one. The condensation of diphenyla-
mine with oxalyl chloride (Method A), was similarly achieved
in the absence of solvent and without any reduction in yield
when compared with the literature process [9]. Thus the use
of solvent was avoided and cost effectiveness achieved.
2.2.2. ScPTZ test
No seizure abolition could be achieved with any of the test
compounds. However, compound 14 could reduce the severity
of the seizures and prevent death in 75% mice at 100 mg kg–1
body weight dose, whereas Compounds 12, 13 could prevent
death in 50% mice at a dose of 300 mg kg–1 body weight.
2.2.3. Neurotoxicity test
The Median minimal neurotoxic dose (TD50) in mice was
determined by the rotarod procedure. No ataxia, sedation, hy-
perexcitability was observed for groups treated with compound
12 (up to 400 mg kg–1) and compound 14 (up to 600 mg kg–1).
As the dose was further increased mice were found to be
lethargic.
On the basis of their preliminary anticonvulsant potential,
compounds 12 and 14 were selected for quantitative evaluation
of their pharmacological parameters. Median effective doses
The Wolff–Kishner reduction of N-phenyl-indolin-2,3-dione
was achieved by refluxing with hydrazine hydrate in alcohol
for 6 hours, effectively reducing the reaction time and avoiding
the use of any high boiling solvents. In the final step 2-[2-(phe-
nylamino) phenyl] acetyl chloride was condensed with various
amines readily at room temperature except in the case of pyr-
rolidin-2-one which required high temperature due to its de-
creased reactivity.
Table 1
Pharmacophore generation and flexible alignment studies
Pharmacophore studies
Flexible alignement
Training set
Test set
Dist.a (°A)
Test set
Score
Flunarizine
Compound
Phenytoin
Carbamazepine
Flunarizine
Zonisamide
Lamotrigine
Nafimidone
Rufinamide
Remacemide
Dezinamide
Denzimol
Dist.a (°A)
3.46
3.43
4.42
4.46
4.46
4.70
4.75
Compound
8
9
10
11
12
13
14
Compound
8
9
10
11
12
13
14
PD85639
208.47
210.32
117.47
223.37
117.37
119.34
131.15
4.26
4.18
4.49
4.14
6.71
6.88
5.23
218.63
225.26
129.04
236.29
133.28
136.70
137.49
5.20
6.37
6.84
a
Distance between centroid of aromatic portion and centroid of H.A/D group.

788
A.V. Shindikar et al. / European Journal of Medicinal Chemistry 41 (2006) 786–792
Fig. 2. Flexible alignment of Compounds 12 and 14 with Flunarizine and PD85639.
(ED50s) and median neurotoxic doses (TD50s) were determined
at the time of peak anticonvulsant and neurotoxic effect and
this data is given in Table 3. The data includes the correspond-
ing values for phenytoin and carbamazepine taken from litera-
ture [13,16].
it included phenytoin, carbamazepine, remacemide, flunarizine,
rufinamide, zonisamide, nafimidone, dezinamide, denzimol
and lamotrigine. The software ‘Molecular Operating Environ-
ment’ was used. (MOE of Chemical Computing Group. Inc. on
a Pentium IV 1.6 GHz workstation). Upon superposition of
minimum energy conformers of the training set molecules,
the presence of at least one phenyl ring and a hydrogen bond
acceptor/donor (H.A/D) group could be identified in each mo-
lecule. Therefore, the distance between the centroid of phenyl
ring/aromatic moiety and H.A/D unit was measured and based
on this; a two point pharmacophore model was built up
(Table 1 and Fig. 1). Further, 3-D database searching to find
molecules obeying the above distance constraints resulted in
identification of substituted phenylacetic acid derivatives.
3. Conclusions
Potent anticonvulsant profile was displayed by compounds
12 (ED50 = 24.0 mg kg–1) and 14 (ED50 = 8.0 mg kg–1). They
also had higher protective index, Compound12 (P.I. = 20.3)
and 14 (P.I = 87.5) when compared to standard drugs. Good
separation between anticonvulsant activity and neurotoxicity
was thus observed.
4. Experimental protocols
4.1.2. Flexible alignment
Phenylacetamides in general were found to have sodium
channel blocking activity, one such derivative PD 85639 was
equipotent with flunarizine in neuroprotection assays [8].
Therefore, the designed molecules were subjected to flexible
alignment with PD 85639 and flunarizine using descriptors like
aromaticity, H-bond acceptor/donor groups, LogP, molar re-
fractivity and volume. Out of twenty molecules used in the
4.1. Design
4.1.1. Generation of pharmacophore for potent anticonvulsant
activity
A training set of 10 molecules known to possess potent an-
ticonvulsant activity was utilized for pharmacophoric study and

A.V. Shindikar et al. / European Journal of Medicinal Chemistry 41 (2006) 786–792
789
Table 2
Anticonvulsant screening of test compounds 8–14 in MES and ScPTZ models
Compound administered
Protection against
Maximal Electroshock
induced seizures
Protection against
subcutaneous
Pentylenetetrazole
induced seizures
0.5 h
+ +
+
4.0 h
+ +
+
0.5 h
–
–
4.0 h
–
–
8
9
10
11
12
13
+ +
N.D.
+ + +
+ +
+ + +
+ + +
+ + +
–
+ +
+ +
+ +
+ +
+ + +
+ + +
+ + +
–
–
N.D.
–
–
–
–
–
+ + +
+ + +
–
–
–
–
–
–
–
–
14
Phenytoin
Carbamazepine
Diazepam
Phenobarbitone
Vehicle treated
+ + +
+ + +
–
–
–
–
–
Statistical analysis was conducted by the method of Litchfield and Wilcoxon
[15], P < 0.025 compared to vehicle treated control groups; Key: + + +: activ-
ity at 30 mg kg–1; + +: activity at 100 mg kg–1; +: activity at 300 mg kg–1; –:
no activity even at 300 mg kg–1; N.D.: not determined.
Table 3
Quantitative anticonvulsant data of test compounds 12 and 14 in mice
a
a
Compound
12
14
MES ED50
24.0 [0.5]
8.0 [0.5]
TD50
Protective index (PI)b
20.33
87.5
6.9
500 [0.5]
700 [0.5]
66 [2.0]
Phenytoin
9.5 [2.0]
Carbamazepine
11.7 [0.5]
71.6 [0.5]
6.11
a
ED50 and TD50 values are in mg kg–1. Numbers in parentheses are 95%
confidence intervals. The dose data was obtained at the ‘time of peak effect’
(indicated in hours in square brackets).
b
PI calculated as (TD50/ED50).
tus. All intermediates were characterized by infrared spectro-
1
scopy (KBr disc method) on a JASCO FT/IR 5300. The H
NMR spectrum was recorded on Varian–VXR-300S at
300 MHz. Elemental analysis results were within 0.4% of
theoretical values.
Scheme 1. (I) Oxalyl chloride, DCM, RT, 2 h; (II) AlCl3, CS2, RT, 2 h; (III)
hydrazine hydrate, EtOH, reflux, 6 h; (IV) chloroacetyl chloride, DMF, 80 °C,
2 h; (V) AlCl3, 180 °C, 1 h; (VI) alkaline hydrolysis, EtOH; (VII) SOCl2, RT,
1 h; (VIII) aq. ammonia, RT, 2 h; (IX) 70% ethylamine, RT, 2 h; (X) 90%
hydrazine hydrate, RT, 2 h; (XI) aniline, RT, 2 h; (XII) Semicarbazide HCl,
(C2H5)3N, RT, 2 h; (XIII) thiosemicarbazide, RT, 2 h; (XIV) pyrrolidin-2-one,
pyridine, 90 °C, 2.5 h.
4.2.1. Synthesis of 2-[2-(phenylamino) phenyl] acetic acid (6)
(Scheme 1)
4.2.1.1. Method A
4.2.1.1.1. 1-(Diphenylamino)-2-chloro-ethanedione (2). Diphe-
nylamine 1 (20 g, 0.11 mol) was added in small portions to
cold oxalyl chloride 17 g (11 ml, 0.4 mol) with stirring and
the mixture was stirred at room temperature for 2 hours and
excess of oxalyl chloride was removed on a rotary evaporator.
The residue on recrystallization from absolute ethanol gave
pale white colored crystals. Yield: 30.06 g (98%), m.p.:
140 °C; IR (KBr) cm–1 3069, 2934, 1776, 1684, 1587, 1491,
670.
study, seven molecules showed good alignment with PD 85639
and flunarizine (Table 1, Fig. 2).
4.2. Synthesis
Progress of all the reactions was monitored by thin-layer
chromatography using Merck precoated silica gel GF254.
Compounds were purified by Column Chromatography using
silica gel 60 (0.062–0.200 mm) from Merck. Melting points
were recorded on an electrically heated melting point appara-
4.2.1.1.2. N-phenyl-indolin-2,3-dione (3). To a solution of 30 g
(0.11 mol) of 2 in 160 ml of carbon disulfide at 10 °C was
added 47 g (0.35 mol) of anhydrous aluminum chloride in

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A.V. Shindikar et al. / European Journal of Medicinal Chemistry 41 (2006) 786–792
small portions. The mixture was stirred at room temperature for
2 hours and was poured into a mixture of ice and 50 ml of 2 N
HCl. The solid obtained was filtered, washed well with water
and air-dried. Recrystallization using ethanol yielded pale buff
colored crystals. Yield: 24.90 g (83%), m.p.: 121 °C; IR (KBr)
cm–1 3061, 2930, 1739, 1610, 1500, 1464.
and then treated with crushed ice and 70 ml of 1 N hydrochlo-
ric acid with stirring. The crude product, which solidified, was
filtered, washed well with water and air-dried. Recrystallization
from absolute ethanol gave pale golden colored crystals. Yield:
18.75 g (75%), m.p.: 119 °C (117–119 °C) [9].
4.2.2. General procedure for the synthesis of N-substituted 2-
[2-(phenylamino) phenyl] acetamides (8–11) (Scheme 1)
4.2.1.1.3. N-phenyl-indolin-2-one (5). To a solution of 20 g
(0.07 mol) of 3 in 60 ml of ethanol in a round bottom flask
fitted with a reflux condenser was added 30 ml of 90% hydra-
zine hydrate. The mixture was heated under reflux for 6 hours,
the excess hydrazine hydrate was removed by distillation and
the residual solution concentrated. The residual mixture was
then diluted with water. The precipitated crystals were filtered,
washed with water and air-dried. Recrystallization from etha-
nol and water mixture (80:20) gave white colored leaflets.
Yield: 11.6 g (58%), m.p.: 119 °C (115–118 °C) [9]; IR (KBr)
cm–1 3005, 2945, 1682, 1589; H1 NMR (CDCl3) δ ppm 7.53–
7.36 (m, 5H, Ar), 7.21, 7.10–7.08, 6.90–6.80 (d, 4H, Ar), 3.72
(s, 2H, CH2). Anal. Calcd. for C14H11NO: C, 80.36; H, 5.30;
N, 6.69. Found: C, 80.40; H, 5.27; N, 6.67.
2-[2-(Phenylamino) phenyl] acetic acid 6 (1 g, 0.004 mol)
was reacted with redistilled thionyl chloride (0.38 ml,
0.005 mol) for an hour. The 2-[2-(phenylamino) phenyl] acetyl
chloride 7 thus obtained was cooled and reacted in turn with
various amines (0.008 mol) stirred for 2 hours. On dilution
with water, the product precipitated out. It was filtered, washed
with sodium bicarbonate solution, followed by washing with
water and then air-dried. Products were recrystallized with
ethanol.
4.2.2.1. 2-[2-(Phenylamino) phenyl] acetamide (8). Reaction
of 7 (1.15 g, 0.04 mol) and 30% aq. ammonia solution gave
8. Yield: 0.7 g (71.08%), m.p.: 272–274 °C; IR (KBr) cm–1
3405, 3060, 2950, 1695, 1500; H1 NMR (CDCl3) δ ppm 9.23
(d, 1H, CONH), 7.42–7.50 (m, 5H, Ar), 7.32, 7.27, 7.10 (m,
3H, Ar), 6.76 (d, 1H, Ar), 3.44 (s, 2H, CH2), 3.88 (s, 2H,
NH2,), 2.2 (s, 1H, Ar-NH). Anal. Calcd. for C14H14N2O: C,
74.31; H, 6.24; N, 12.38. Found: C, 74.27; H, 6.19; N, 12.42.
4.2.1.1.4. 2-[2-(Phenylamino) phenyl] acetic acid (6). A solu-
tion of 10 g of 5 in 33 ml of 2 N NaOH and 33 ml of ethanol
was refluxed for 4 hours. Ethanol was removed by distillation.
The residual mixture was then acidified using hydrochloric
acid. The precipitate obtained was then filtered, washed with
water and air-dried. Recrystallization from ethanol-water mix-
ture using decolorizing charcoal offered colorless crystals.
Yield: 9.0g (90%), m.p.: 117 °C (114–116 °C) [10]; IR (KBr)
cm–1 3398, 2926, 1711, 1367; H1 NMR (CDCl3) δ ppm 7.55–
7.42 (m, 5H, Ar), 7.32, 7.10–7.08, 6.95–6.78 (d, 4H, Ar), 3.7
(s, 2H, CH2), 2.17 (s, 1H, Ar–NH). Anal. Calcd. for
C14H13NO2: C, 73.99; H, 5.77; N, 6.16. Found: C, 74.05; H,
5.81; N, 6.13.
4.2.2.2. N-ethyl-2-[2-(phenylamino) phenyl] acetamide (9).
Reaction of 7 (1.15 g, 0.04 mol) and 70% aq. ethylamine solu-
tion gave 9. Yield: 0.81 g (73.28%), m.p.: 129–131 °C; IR
(KBr) cm–1 3398, 3058, 2944, 1697, 1500; H1 NMR (CDCl3)
δ ppm 9.23 (d, 1H, CONH), 7.46–7.55 (m, 5H, Ar), 7.36, 7.31,
7.10 (m, 3H, Ar), 6.76 (d, 1H, Ar), 3.44 (s, 2H, CH2), 3.22 (s,
2H, CH2,), 2.2 (s, 1H, Ar–NH), 1.23 (2H, CH3). Anal. Calcd.
for C16H18N2O: C, 75.56; H, 7.13; N, 11.01. Found: C, 75.49;
H, 7.02; N, 10.94.
4.2.1.2. Method B
4.2.2.3. 2-{[2-(Phenylamino) phenyl] acetyl} hydrazine (10).
Reaction of 7 (1.15 g, 0.04 mol) and 90% aq. hydrazine hy-
drate solution gave 10. Yield: 0.69 g (66%), m.p.: 95 °C; IR
(KBr) cm–1 3393, 3057, 2947, 1699, 1500; H1 NMR (CDCl3)
δ ppm 9.23 (d, 1H, CONH), 7.46–7.55 (m, 5H, Ar), 7.36, 7.31,
7.10 (m, 3H, Ar), 6.76 (d, 1H, Ar), 3.47 (s, 2H, CH2), 3.08 (s,
2H, NH2, hydrazide), 2.2 (s, 1H, Ar-NH). Anal. Calcd. for
C14H15N3O: C, 69.69; H, 6.27; N, 17.41. Found: C, 69.74;
H, 6.30; N, 17.44.
4.2.1.2.1. 1-(Diphenylamino)-2-chloro-ethan-1-one (4). To a
solution of 20 g (0.11 mol) of 1 and chloroacetyl chloride
(16.0 ml, 0.14 mol) was added 3 ml of N,N-dimethylforma-
mide (DMF) and heated at a temperature of 80 °C on water
bath for a period of 2 hours. The reaction mixture was then
diluted with water wherein the product precipitated out. It
was filtered, washed with water, dried and recrystallized using
ethanol to get pale white crystals. Yield: 28.05 g (97%). m.p.:
115 °C (113–116 °C) [9]; IR (KBr) cm–1 3005, 2945, 1680,
1589, 1491, 700.
4.2.2.4. N-phenyl-2-[2-(phenylamino) phenyl] acetamide (11).
Reaction of 7 (1.15 g, 0.04 mol) with aniline (0.401 ml,
4.2.1.2.2. N-phenyl-indolin-2-one (5). A mixture of 25 g
(0.10 mol) of 4 and 31 g (0.23 mol) of anhydrous aluminum
chloride was heated in flask fitted with CaCl2 guard tube until
an internal temperature of 180–190 °C was attained. There was
copious evolution of hydrogen chloride; mixture was heated
for an additional 10 min. The molten mass was allowed to cool
0.004 mol) gave 11. Yield: 0.74
g (55.71%), m.p.:
125–127 °C; IR (KBr) cm–1 3327, 3038, 2930, 1626, 1500;
H1 NMR (CDCl3) δ ppm 7.54–7.42 (m, 6H, Ar), 7.32(d, 1H,
Ar), 7.24, 7.05 (m, 2H, Ar), 7.18–7.08 (m, 4H, Ar), 6.81 (d,
1H, Ar), 3.72 (s, 1H, CONH), 3.5 (s, 2H, CH2), 2.2 (s, 1H, Ar-

A.V. Shindikar et al. / European Journal of Medicinal Chemistry 41 (2006) 786–792
791
NH). Anal. Calcd. for C20H18N2O: C, 79.44; H, 6.00; N, 9.26.
–CH2–), 0.88 (m, 2H, –CH2–). Anal. Calcd. for C18H18N2O2:
Found: C, 79.50; H, 6.08; N, 9.23.
C, 73.45; H, 6.16; N, 9.52. Found: C, 73.41; H, 6.13; N, 9.49.
4.2.3. 2-{[2-(Phenylamino) phenyl] acetyl} hydrazine
4.3. Anticonvulsant screening
carboxamide (12)
Semicarbazide HCl (0.9 g, 0.008 mol) was taken in 2 ml of
DMF and to it was added 1.16 ml (0.008 mol) of triethylamine
and the mixture warmed on a water-bath for 20 min. This mix-
ture was added slowly to a cold solution of 7 (1.15 g,
0.04 mol) and then stirred at room temperature for 2 hours. It
was then diluted with water when the product precipitated out.
The precipitate was filtered off, washed with NaHCO3 solution
followed by washing with water and air-dried. Recrystalliza-
tion with ethanol offered dark brown crystals. Yield: 0.67 g
(53.62%), m.p.: 100–101 °C; IR (KBr) cm–1 3398, 3204,
3059, 2930, 1697, 1500; H1 NMR (CDCl3) δ ppm 9.23 (d,
1H, CONH), 7.58–7.45 (m, 5H, Ar), 7.30, 7.26 (d, 2H, Ar),
7.08 (m, 1H, Ar), 6.76 (d, 1H, Ar), 3.7 (s, 2H, CH2), 2.2(s,
1H, Ar–NH), 2.17 (s, 2H, –CONH2), 1.4 (s, 1H, N–NH). Anal.
Calcd. for C15H16N4O2: C, 63.37; H, 5.67; N, 19.71. Found: C,
63.31; H, 5.62; N, 19.68.
The Institutional Ethics Committee, Regd. No: 242/
CPCSEA approved the use of animals and animal models.
Swiss albino (male) mice in the weight range 25–30 g were
used. Mice were housed in cages. Temperature was maintained
at 25 3 °C and humidity at 50–60%. Lighting was artificial
with alternate 12 hour light and 12 hour dark cycle. Mice were
fed on standard laboratory diet and supplied with drinking
water [11].
4.3.1. MES test [12,13]
Mice were divided into 10 groups with four mice in each
group. Suspension of test compounds 8–14/standard (pheny-
toin, carbamazepine) in 0.5% tween80 in saline were adminis-
tered intraperitoneally (i.p.) at three dose levels (30, 100,
300 mg kg–1). The untreated group was administered the same
volume of the vehicle. A drop of 0.9% saline was instilled in
each eye prior to application of electrodes (Centroniks Electro-
convulsiometer). The mice were subjected to electrical shock
delivered through the corneal electrodes for 0.2 s (40 mA,
50 Hz, ac). Failure to extend the hindlimbs to an angle with
the trunk greater than 90° is defined as protection. Seizure pat-
tern was recorded at 0.5 and 4 hours after administration of the
dose (Table 2).
4.2.4. 2-{[2-(Phenylamino) phenyl] acetyl} hydrazine
thiocarboxamide (13)
To the cold acid chloride 7 (1.15 g, 0.04 mol) was added
0.8 g (0.008 mol) of thiosemicarbazide in 2 ml of DMF and
stirred at room temperature for 2 hours. Reaction mixture was
then diluted with water when the product precipitated out,
which was filtered off, washed with NaHCO3 solution, fol-
lowed by washing with water and air-dried. The product was
purified by column chromatography (petroleum ether 70:
chloroform30), the second fraction being the pure product.
Yield: 0.83 g (62.84%), m.p.: 240–242 °C; IR (KBr) cm–1
3408, 3238, 3144, 2928, 1699, 1500, 1180,1105; H1 NMR
(CDCl3) δ ppm 9.23 (d, 1H, CONH), 7.54–7.44 (m, 5H, Ar),
7.30, 7.26 (d, 2H, Ar), 7.05 (m, 1H, Ar), 6.73 (d, 1H, Ar), 3.0
(s, 2H, CH2), 2.9 (s, 1H, Ar–NH), 2.7 (s, 2H, –CSNH2), 1.25
(s, 1H, N–NH). Anal. Calcd. for C15H16N4OS: C, 59.98; H,
5.37; N, 18.65; S, 10.68. Found: C, 60.02; H, 5.40; N, 18.63;
S, 10.61.
4.3.2. ScPTZ test [13,14]
Mice were divided into 10 groups of four mice each. Sus-
pension of test compounds 8–14/standard (diazepam, pheno-
barbitone) in 0.5% tween80 in saline were administered intra-
peritoneally at three dose levels (30, 100, 300 mg kg–1). Thirty
minutes later, pentylenetetrazole at a dose of 85 mg kg–1 was
administered subcutaneously to all groups. One group with ve-
hicle served as control. Protection was defined as a failure to
observe a single episode of clonic spasms of at least 5-s dura-
tion during a 30-min period following administration of test/
standard compound. The observations made are given in Ta-
ble 2.
4.2.5. 1-{[2-(Phenylamino) pheny] acetyl}-2-pyrrolidinone
(14)
4.3.3. Neurotoxicity test [14]
To the cold acid chloride 7 (1.15 g, 0.04 mol) was added
(0.36 ml, 0.0048 mol) of pyrrolidin-2-one in 5 ml of dry pyr-
idine and heated at 90 °C for 2.5 hours. Pyridine was removed
on rotary evaporator. The solid obtained was washed with 5%
HCl, washed with NaHCO3 solution, followed by washing
with water and air-dried. The product was purified by column
chromatography (petroleum ether 70: chloroform 30), the sec-
ond fraction being the pure product. Yield: 0.82 g (64.51%), m.
p.: 160–163 °C; IR (KBr) cm–1 3398, 3059, 2924, 1720, 1500;
H1 NMR (CDCl3) δ ppm 7.55–7.44 (m, 5H, Ar), 7.36, 7.24 (d,
2H, Ar), 7.15 (m, 1H, Ar), 6.76 (d, 1H, Ar), 4.4 (s, 2H, CH2),
2.17 (s, 1H, Ar–NH), 1.6 (m, 2H, –CH2–), 1.27 (m, 2H,
The median minimal neurotoxic dose (TD50) in mice was
determined by the rotarod procedure. Mice were divided into
eight groups of four mice each. Suspensions of test compounds
12 and 14 in 0.5% tween80 in saline were administered intra-
peritoneally with dose levels in increasing order (200–800 mg
kg–1). The mice were placed on a 1-inch diameter knurled plas-
tic rod rotating at 6 rpm. Unimpaired mice can easily remain
on a rod rotating at this speed. The inability of the mice to
maintain their balance for at least 1 min in three successive
trials was interpreted as demonstration of motor impairment
or toxicity.

792
A.V. Shindikar et al. / European Journal of Medicinal Chemistry 41 (2006) 786–792
[8] I. Roufos, S.J. Hays, D.J. Doolay, R.D. Schwarz, G.W. Campbell, A.W.
Acknowledgements
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The authors acknowledge the financial support of Council
of Scientific and Industrial Research (C.S.I.R.), Govt. of India.
[11] OECD (Organization for Economic Cooperation and Development),
Guidelines, USA, 1998.
[12] B.B. Gallagher, in: J.A. Vida (Ed.), Anticonvulsants: A series of mono-
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