2,4-Dimethoxyphenylsemicarbazones with anticonvulsant activity against three animal models of seizures: Synthesis and pharmacological evaluation

Article abstract of DOI:10.1016/j.bmc.2005.12.041

Various 2,4-dimethoxyphenylsemicarbazones were synthesized starting from 2,4-dimethoxyaniline via a phenylcarbamate intermediate. The structures were confirmed by spectral and elemental analyses. The anticonvulsant activity of the synthesized compounds wa

Full text of DOI:10.1016/j.bmc.2005.12.041

Bioorganic & Medicinal Chemistry 14 (2006) 3106–3112
2,4-Dimethoxyphenylsemicarbazones with anticonvulsant
activity against three animal models of seizures: Synthesis
and pharmacological evaluation
Rathinasabapathy Thirumurugan,a Dharmarajan Sriram,a Amrita Saxena,a
James Stablesb and Perumal Yogeeswaria,*
aMedicinal Chemistry Research Laboratory, Pharmacy group, Birla Institute of Technology and Science, Pilani 333031, India
bPreclinical Pharmacology Section, Epilepsy Branch, National Institute of Health, Bethesda, MD 20892-9020, USA
Received 29 November 2005; revised 15 December 2005; accepted 15 December 2005
Available online 18 January 2006
Abstract—Various 2,4-dimethoxyphenylsemicarbazones were synthesized starting from 2,4-dimethoxyaniline via a phenylcarbamate
intermediate. The structures were confirmed by spectral and elemental analyses. The anticonvulsant activity of the synthesized com-
pounds was established after intraperitoneal administration in three seizure models in mice which include maximal electroshock sei-
zure, subcutaneous pentylenetetrazole, and subcutaneous strychnine-induced seizure screens. Nine compounds exhibited protection
in all the three seizure models, and N1-(2,4-dimethoxyphenyl)-N4-(propan-2-one)semicarbazone (17) emerged as the most active
compound with no neurotoxicity. These compounds were found to elevate c-aminobutyric acid (GABA) levels in the midbrain
and medulla oblongata regions equipotent to clobazam.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
We have recently been investigating various aryl-substi-
tuted semicarbazones as potential anticonvulsant
agents.9–15 While attempting to establish some correla-
tion between structure and the activity of these aryl
semicarbazones, we observed that the structural require-
ment for activity in the maximal electroshock (MES)
test was the presence of at least one phenyl ring in close
proximity to two-electron donor atoms (semicarbazono
group). And compounds displaying anti-pentylenetet-
razole (PTZ) activity, often possess an alkyl substituent
close to two-electron donor atoms.16
Epilepsy is not a disease, but a syndrome of different
cerebral disorders of the central nervous system
(CNS), which is characterized by paroxysmal, excessive,
and hypersynchronous discharges of large numbers of
neurons.1 In India, studies have reported the prevalence
rate of epilepsy varying from 1710 to 9780 cases per mil-
lion population.2 Despite the optimal use of available
antiepileptic drugs (AEDs), many patients with epilepsy
fail to experience seizure control and others do so only
at the expense of significant toxic side effects.3 The lim-
itations with the conventional AEDs highlighted the
need for developing newer agents for epilepsies and
the AED search has come a long way, particularly over
the last two decades.
In view of these general requirements for activity, we
have designed aryl-substituted semicarbazones with
various aryl and alkyl aldehydes and ketones to be active
in a variety of convulsant stimuli. Surprisingly, these aryl
semicarbazones were devoid of sedative-hypnotic
activity and exhibited anticonvulsant activity with lesser
neurotoxicity.9 These aryl semicarbazones do not possess
the dicarboximide group as found in conventional drugs
like barbiturates, hydantoins, oxazolidindiones, etc.,
which may be associated with toxicity and side effects.17
In this paper, we report the synthesis and pharmacolog-
ical evaluation of 2,4-dimethoxyphenylsemicarbazones
and compared its potential with those of established
drugs. These aryl-substituted semicarbazones have
Aryl semicarbazones have documented increasing
advances in AED drug design and were found to act
by blocking the voltage-gated sodium ion channels.4–8
Keywords: Aryl semicarbazones; Anticonvulsant; 2,4-Dimethoxy;
GABA.
*
Corresponding author. Tel.: +91 1596 244684; fax: +91 1596
244183; e-mail: pyogee@bits-pilani.ac.in
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.12.041

R. Thirumurugan et al. / Bioorg. Med. Chem. 14 (2006) 3106–3112
3107
elevated the c-aminobutyric acid levels in different re-
gions of brain.
Compounds 1, 4, 8, 9, 14–18, and 20–21 were found to
be active in the scPTZ test, a test used to identify com-
pounds that elevate seizure threshold. Compounds 1, 8,
and 21 showed activity at a dose of 100 mg/kg compara-
ble with Ethosuximide. Compounds that showed moder-
ate protection at a dose of 100 mg/kg include 9 (0.25 h,
1/5), 15 (0.25 h, 2/5), 16 (0.25 h, 1 h, 1/5), and 18 (0.25 h,
1/5). All these compounds showed 100% protection at a
dose of 300 mg/kg at 0.5 h. So these compounds have
quick onset of action but for shorter duration.
2. Results and discussion
2.1. Synthesis
The synthesis of 2,4-dimethoxyphenylsemicarbazones
was accomplished as presented in Scheme 1. The 2,4-
dimethoxyaniline was treated with phenyl chlorofor-
mate in the presence of chloroform to yield phenyl-N-
(2,4-dimethoxyphenyl)carbamate. The carbamate on
condensation with hydrazine hydrate in methylene
dichloride gave the 2,4-dimethoxyphenylsemicarbazide
(1). Finally the required 2,4-dimethoxyphenylsemicar-
bazones were prepared by the reaction between appro-
priate aryl/alkyl aldehydes or ketones and 1 in the
presence of glacial acetic acid in ethanol. The physical
properties of the synthesized compounds are presented
in Table 1. The structures were characterized by both
spectral and elemental analyses and the data were within
0.4% of the theoretical values.
The compounds were also screened in the scSTY pattern
test. All the compounds except 8, 11–12, and 21 showed
protection against scSTY-induced seizure threshold test,
indicative of their ability to prevent seizure. Compounds
1, 4, 9, 14, 17, and 18 showed activity at 100 mg/kg at
0.5 h in which compound 17 showed activity up to
4.0 h. Compounds that showed activity at 300 mg/kg in-
clude 1, 4, 9, 10, 14, 16, and 18 (4 h), 2, 3, 5, 13, and 19
(0.5 h), and 6, 7, 15, and 20 at both the time points.
In the neurotoxicity screen, compounds 1, 5, 7, 13, 17,
and 21 did not show neurotoxicity in the maximum
administered dose (300 mg/kg) and the compounds 4
and 18 were less neurotoxic compared to Phenytoin.
On the other hand, compounds 2, 8, 10, 15, 16, and
19–20 were neurotoxic at the anticonvulsant dose and
compounds 3, 6, 9, and 14 were more neurotoxic. Com-
pounds 11 and 12 exhibited neither anticonvulsant activ-
ity nor neurotoxicity.
2.2. Pharmacological activity
The new derivatives (1–21) obtained from the reactional
sequence were injected intraperitoneally into mice and
evaluated in the maximal electroshock (MES), subcuta-
neous pentylenetetrazole (scPTZ), subcutaneous strych-
nine threshold test (scSTY), and neurotoxicity screens,
using doses of 30, 100, and 300 mg/kg, and observation
carried out at two different time intervals (0.5 and 4 h).
These data are presented in Table 2. All the compounds
except 6, 9, and 16, showed anti-MES activity indicative
of their ability to prevent seizure spread. Compounds
that showed protection against MES model at 100 mg/
kg include 4, 7, 8, and 10 (0.25 h), 17–19, and 20
(0.25 h). The compounds 4, 5, 8, 15, and 17 showed
activity both at 0.5 h and 4.0h periods. Most of the com-
pounds showed activity only at 0.5 h, indicating that
they have rapid onset and shorter duration of action.
Among the compounds, the unsubstituted aryl deriva-
tive (2 and 13) exhibited activity against MES and
scSTY models. When the C-4 position of the aryl ring
was substituted with donating groups (3–5 and 14–15),
the compounds exhibited activity in more than one
model with the compounds 4, 14, and 15 being active
in all the three models. The compound with an elec-
tron-withdrawing group at C-4 position (6) exhibited
activity only in the scSTY model, while the similar com-
pound with carbimino methyl group (16) exhibited
activity against scPTZ and scSTY. The 3-chloro (7)
Scheme 1. Synthesis of the title compounds.

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R. Thirumurugan et al. / Bioorg. Med. Chem. 14 (2006) 3106–3112
Table 1. Physical data of the 2,4-dimethoxyphenylsemicarbazones
Compound
Substituents
R1
Yield (%)
Mp (°C)
Molecular formulaa
Molecular weight
Rf
logPb
R
2
3
H
H
62
55
52
50
66
64
69
60
61
57
65
61
58
53
67
54
52
52
50
51
188
159
155
137
173
139
200
179
127
156
145
161
166
151
118
159
138
141
156
147
C16H17N3O3
C17H19N3O3
C17H19N3O4
C18H22N4O3
C16H16N4O5
299.33
313.36
329.35
342.40
344.33
333.77
344.33
333.77
313.35
315.33
344.32
313.35
347.80
327.38
358.35
251.28
265.31
293.36
293.32
291.35
0.71
0.62
0.64
0.64
0.72
0.70
0.74
0.68
0.69
0.65
0.74
0.71
0.74
0.63
0.77
0.69
0.62
0.61
0.66
0.67
1.88
2.07
1.48
1.27
2.21
2.21
2.19
2.19
1.97
1.52
2.13
1.85
1.95
2.06
2.05
1.51
1.79
2.13
1.62
1.76
H
4-CH3
4-OCH3
4-N–(CH3)2
4
H
5
H
6
H
4-NO2
3-Cl
7
H
C
16H16N3O3Cl
C16H16N4O5
16H16N3O3Cl
C17H19N4O3
C16H17N3O4
8
H
3-NO2
2-Cl
9
H
C
10
11
12
13
14
15
16
17
18
19
20
21
H
2-CH3
2-OH
H
H
2-NO2
H
4-Cl
C16H16N4O5
C17H19N3O3
C17H18N3O3Cl
C18H21N3O3
C17H18N4O5
C12H17N3O3
C13H19N3O3
C15H23N3O3
C14H19N3O4
C15H21N3O3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
4-CH3
4-NO2
CH3
C2H5
CH2CH(CH3)2
CH2COCH3
CRR1 = cyclohexyl
a Elemental analyses for C, H, and N were within 0.4% of the theoretical values.
b logP was generated using Alchemy 2000 and SciLogP software.
and 3-nitro (8) substituted derivatives exhibited activity
in more than one models, viz., MES and scSTY, and
MES and scPTZ, respectively. The 2-hydroxy (11) and
2-nitro (12) substituted derivatives were inactive, while
a 2-chloro (9) and 2-methyl (10) were active in scPTZ
and scSTY, and Mes and scSTY models, respectively.
the standard drug Ethosuximide. These compounds
did not exhibit neurotoxicity at the tested dose of
50 mg/kg.
Some selected compounds (4, 5, 13, 15, and 17–20) were
studied for the CNS behavioral activity in mice using
actophotometer and Porsolt’s swim pool test in mice
and the results are presented in Table 4. In the behavior-
al study, using actophotometer, the compounds 4 and 19
showed no behavioral despair effect after 1.0 h when
compared to Phenytoin. All other compounds were
found to decrease the activity of the animals. In a similar
study using Porsolt’s swim test, the immobility time
after the administration of the test compounds was com-
pared with that of Carbamazepine. The compound 20
was found to show no significant CNS depression and
all other compounds tested were found to emerge as
CNS depressants as they increased the immobility time.
When the carbimino terminal aryl ring was replaced
with alkyl groups like methyl (17), ethyl (18), and ace-
tomethyl (20), the compounds were found to exhibit
activity against all the three animal models, whereas iso-
butyl derivative (19) was active in MES and scSTY mod-
els. The cyclohexyl derivative (21) showed activity in
both MES and scPTZ models. Hence, the compounds
1, 4, 14, 15, 17, 18, and 20 exhibited a broad spectrum
of anticonvulsant activity, viz., MES, scPTZ and scSTY.
With regard to neurotoxicity, the compounds 1, 15, 17,
and 18 showed no or lesser neurotoxicity.
In comparison to the 2,6-dimethylphenylsemicarbazones
reported earlier,15 the 2,4-dimethoxyphenylsemicarbaz-
ones were less effective except compound 5 and 17 which
were not neurotoxic compared to the 2,6-dimethylphe-
nyl analogues.
In order to explore the mechanism of anticonvulsant
activity, some selected compounds (4, 15, and 17), which
were more active, were subjected to neurochemical
investigation to study their effects on the levels of
GABA in different regions of rat brain viz., olfactory
lobe, mid brain, medulla oblongata, and cerebellum, as
there are regional differences in GABA concentration
within the CNS18 (Table 5). The compounds were found
to increase the GABA level in the midbrain region sig-
nificantly. In other brain regions, there was no signifi-
cant increase in the level of GABA, except for
Four compounds (1, 15, 17, and 18) were evaluated oral-
ly in rats for activity in scPTZ test at several time points
(Table 3). The compounds were tested at 50 mg/kg and
compared with standard drug Ethosuximide. Com-
pounds 15, 17, and 18 showed better protection than

R. Thirumurugan et al. / Bioorg. Med. Chem. 14 (2006) 3106–3112
3109
Table 2. Anticonvulsant activity and minimal motor impairment of 2,4-dimethoxyphenylsemicarbazones
Compound
Intraperitoneal injection in micea
MES screen
scPTZ screen scSTY screen
Neurotoxicity screen
0.5 h
4.0 h
0.5 h
4.0 h
0.5 h
2.0 h
0.5 h
4.0 h
b
1
300
300
300
100
300
—
—
—
—
100
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
100
300
300
100
300
300
300
—
300
—
—
—
—
2
300
100
300
—
3
—
—
100
—
4
300
300b
—
300
—
300
—
5
—
6
—
—
300
300
—
30
100
—
7
100
100
—
300b
—
—
—
8
300
—
100
300c
—
100
100
300
—
300
100
—
9
10
100
—
300
300
—
—
—
11
12
—
—
—
—
—
—
—
—
—
—
—
—
—
13
14
300
300
300
—
—
300
100
300
—
—
—
—
300
300c
300c
300
300c
—
300
300
300
100
300
—
100
300
300
—
15
16
300
—
—
—
c
—
17
18
100
100
100
300b
300
30
100
—
—
—
—
—
—
—
300
100
100
300
300
—
—
—
300
300
300
—
19
20
—
—
—
—
300
100
—
300
—
21
Phenytoin
Ethosuximide
—
30
—
—
300
—
—
100
—
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 mice. The animals were examined 0.5 and 4.0 h (scSTY test, 0.5 and 2.0 h period study) after injections were administered. The
dash (—) indicates the absence of activity at maximum dose administered (300 mg/kg) and (x) indicates not tested.
b In the MES screen at a dose of 100 mg/kg, compounds that showed protection were 1 (1 h, 1/3), 10, and 20 (0.25 h, 1/3).
c In the scPTZ screen, at a dose of 100 mg/kg, compounds that showed protection were 9 (0.25 h, 1/5), 15 (0.25 h, 2/5), 16 (0.25, 1 h, 1/5), and 18
(0.25 h, 1/5).
Table 3. Evaluation of some compounds in the scPTZ test after oral
administration (50 mg/kg) to rats
4. Experimental
Compound
Oral administration to ratsa
4.1. Chemistry
0.25 h
0.5 h
1.0 h
2.0 h
Melting points were determined with a Buchi 530 melt-
ing point apparatus and are uncorrected. Infrared (IR)
spectra were recorded for the compounds on Jasco IR
1
15
1
2
3
2
0
2
1
3
3
2
0
1
2
1
1
0
1
1
1
1
17
18
1
Report 100 (KBr). H NMR spectra were recorded on
a Bruker Avance 300 MHz spectrometer. Chemical
shifts were expressed in parts per million (d) with tetra-
methylsilane as an internal standard. The elemental
analyses (C, H, N) were performed using Perkin-Elmer
model 240 C analyzer, and all values were within
0.4% of the theoretical compositions. The purity of
the compounds was monitored by ascending thin-layer
chromatography (TLC) on silica gel-G (Merck) coated
aluminum plates, visualized by iodine vapour. Develop-
ing solvents used in TLC were chloroform/methanol
(9:1). The logP for all the synthesized compounds were
calculated using SciLogP software in Alchemy 2000
(Tripos Co.).
Ethosuximide
a The figures indicate the number of rats out of four which were
protected.
compounds 15 and 17, which increased the GABA level
in the medulla oblongata region similar to Clobazam.
3. Conclusion
The present study revealed that some of the 2,4-dimeth-
oxyphenylsemicarbazones possessed a broad spectrum
of anticonvulsant activity with less or no neurotoxicity.
Nine compounds exhibited protection in all the three
seizure models, viz., MES, scPTZ, and scSTY models
and N1-(2,4-dimethoxyphenyl)-N4-(propan-2-one)semic-
arbazone (17) emerged as the most active compound in
these three models with no neurotoxicity. These com-
pounds were found to elevate c-aminobutyric acid
(GABA) levels in the midbrain and medulla oblongata
regions equipotent to clobazam.
4.1.1. Synthesis of phenyl-N-(2,4-dimethoxyphenyl)car-
bamate. Phenylchloroformate (0.1 mol, 12.6 ml) was dis-
solved in 40 ml of chloroform and equimolar quantities
of 2,4-dimethoxyaniline (0.1 mol, 12.3 ml) and triethyl-
amine (0.1 mol, 13.9 ml) were added dropwise and stir-
red in the room temperature for 5 h. The reaction
mixture was concentrated to one-third volume and

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Table 4. CNS studies on selected compounds
Compounda
Actophotometer activity scoreb
Immobility timec (s)
Control (24 h prior) Post treatment (60 min after)
Control (24 h before)
Post treatment
0.5 h after
1.0 h after
4
350.67 17.43
406.67 19.71
395.33 17.48
417.50 15.68
488.00 12.57
414.17 17.59
322.00 10.58
338.00 11.43
247.32 21.12
—
207.00 09.56
282.00 11.08
231.17 12.68
268.67 07.53
276.50 08.09
323.33 07.91
240.67 07.85
154.17 06.44
104.11 14.56
—
305.67 11.64 NS
350.33 15.75
264.50 10.90
326.00 11.94
308.33 13.12
340.33 09.20
303.33 09.28 NS
225.50 09.63
106.23 12.44
—
154.33 6.06
157.33 7.97
170.67 7.57
162.33 3.88
158.50 4.90
165.50 6.53
176.00 6.94
171.67 8.44
—
209.50 6.23
197.17 8.03
204.33 7.87
218.67 6.08
219.00 5.01
187.33 7.32
202.33 7.80
193.33 6.09 NS
—
5
13
15
17
18
19
20
Phenytoind
Carbamazepined
131.50 9.32
207.33 8.49
a The compounds were tested at a dose of 100 mg/kg (ip).
b Each score represents the means SEM of six mice, significantly different from the control score at p < 0.05 and NS denotes not significant at
p < 0.05 (Student’s t-test).
c Each value represents the means SEM of six mice significantly different from the control at p < 0.05 and NS denotes not significant at p < 0.05
(Student’s t-test).
d Tested at 30 mg/kg (ip).
Table 5. Effect of selected compounds on GABA system
Compounda
Concentration of GABA (mg/100 mg tissue)b
Olfactory lobe
Midbrain
Medulla oblongata
Cerebellum
Control
12.01 1.74
13.00 2.56
12.71 1.80
14.58 1.78
17.67 2.62
44.31 1.78
58.87 2.61*
63.77 2.82*
64.72 3.48*
63.57 2.83*
33.28 2.04
39.51 2.74
42.18 2.54*
44.32 2.92*
45.91 2.16*
21.93 2.52
29.25 3.40
29.79 3.08
27.99 2.83
28.73 3.12
4
15
17
Clobazamc
a The compounds were tested at a dose of 100 mg/kg (ip).
b Each value represents the means SEM of six rats significantly different from the control at *P < 0.05 and the remaining values are not significant
at P < 0.05 (Student’s t-test).
c Tested at 30 mg/kg (ip).
100 ml of petroleum ether was added. The precipitate,
which appeared, was washed with water, filtered, and
dried, mp 126 °C, IR (KBr) mmax 3400, 3100–3060,
aldehyde or ketone in 5 ml ethanol and glacial acetic
acid (1–2 drops) was added. The mixture was stirred
with heating for 1–4 h until the completion of the
reaction and the resultant precipitate was filtered and
dried. The product was recrystallized from 95% ethanol.
The physical data of the compounds are presented in
Table 1. The IR spectra of the compounds were identical
in the following aspects: 3400–3320, 2900–2850, 1690–
1670, 1620–1540, 1320, 840 cmÀ1. The spectral and
elemental analyses of some representative compounds
are as follows:
1
2940, 1700, 1320, 845 cmÀ1; H NMR (CDCl3) d: 3.74
(s, 3H, ArOCH3), 3.78 (s, 3H, ArOCH3), 6.86–7.67
(m, 8H, ArH), 8.32 (s, 1H, ArNH, D2O exchangeable).
4.1.2. Synthesis of 2,4-dimethoxyphenylsemicarbazide (1).
Phenyl-N-(2,4-dimethoxyphenyl)carbamate (0.05 mol,
12.05 gm) was dissolved in 100 ml of dichloromethane.
To this solution, 4.85 ml of hydrazine hydrate
(0.1 mol) was added and refluxed with stirring for
24 h. The precipitate of 2,4-dimethoxyphenylsemicar-
bazide (1) was separated by vacuum filtration and
washed with dichloromethane, and dried, mp 190 °C,
4.1.4. N1-(2,4-Dimethoxyphenyl)-N4-(4-methylbenzalde-
hyde)semicarbazone (3). Yield: 55%; mp: 159 °C; IR
(KBr): 3340, 2890, 1670, 1610, 1590–1530, 1320 cmÀ1
;
IR (KBr) mmax 3390, 3090, 2860, 1680, 1320, 840 cmÀ1
;
1H NMR (DMSO-d6) d (ppm): 2.24 (s, 3H, ArCH3),
3.64 (s, 3H, ArOCH3), 3.68 (s, 3H, ArOCH3), 6.78–
7.86 (m, 7H, ArH), 7.92 (s, 1H, imine H), 8.46 (s, 1H,
ArNH, D2O exchangeable), 9.70 (s, 1H, CONH, D2O
exchangeable); Calculated for C17H19N3O3: C, 65.16;
H, 6.11; N, 13.41; found: C, 64.91; H, 6.09; N, 13.36.
1H NMR (DMSO-d6, 300 MHz, d ppm) 3.68 (s, 3H, Ar-
OCH3), 3.72 (s, 3H, ArOCH3), 5.42 (s, 2H, NH2, D2O
exchangeable), 7.12–7.37 (m, 3H, ArH), 8.36 (s, 1H,
ArNH, D2O exchangeable), 9.48 (br s, 1H, NHNH2,
D2O exchangeable).
4.1.3. General procedure for the synthesis of 2,4-dimeth-
oxyphenylsemicarbazones (2–21). To a solution of 2,4-
dimethoxyphenylsemicarbazide (0.003 mol, 0.84 g) in
25 ml of ethanol, an equimolar quantity of appropriate
4.1.5.
N1-(2,4-Dimethoxyphenyl)-N4-(4-nitrobenzalde-
hyde)semicarbazone (6). Yield: 66%; mp: 173 °C; IR
(KBr): 3350, 2900, 1690, 1605, 1590–1540, 1435, 1330,
1310, 1210 cmÀ1; H NMR (DMSO-d6) d (ppm): 3.70
1

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3111
(s, 3H, ArOCH3), 3.74 (s, 3H, ArOCH3), 6.84–7.96 (m,
7H, ArH), 8.02 (s, 1H, imine H), 8.62 (s, 1H, ArNH,
D2O exchangeable), 10.94 (s, 1H, CONH, D2O
exchangeable); Calculated for C16H16N4O5: C, 55.81;
H, 4.68; N, 16.27; found: C, 55.60; H, 4.66; N, 16.21.
4H, o-methyl protons of cyclohexyl ring ), 1.66 (m,
6H, m- and p-methyl protons of cyclohexyl ring), 3.72
(s, 3H, ArOCH3), 3.77 (s, 3H, ArOCH3), 6.36–6.74
(m, 3H, ArH), 8.93 (s, 1H, ArNH, D2O exchangeable),
10.55 (s, 1H, CONH, D2O exchangeable); Calculated
for C15H21N3O3: C, 61.84; H, 7.27; N, 14.42; found:
C, 61.86; H, 7.46; N, 14.50.
4.1.6.
N1-(2,4-Dimethoxyphenyl)-N4-(2-chlorobenz-
aldehyde)semicarbazone (9). Yield: 60%; mp: 179 °C;
IR (KBr): 3350, 2890, 1690, 1590–1540, 1320,
4.2. Pharmacology
1
1200 cmÀ1; H NMR (DMSO-d6) d (ppm): 3.70 (s, 3H,
ArOCH3), 3.73 (s, 3H, ArOCH3), 6.78–7.58 (m, 7H,
ArH), 8.02 (s, 1H, imine H), 8.89 (s, 1H, ArNH, D2O
exchangeable), 9.54 (s, 1H, CONH, D2O exchangeable);
Calculated for C16H16N3O3Cl: C, 57.58; H, 4.83; N,
12.59; found: C, 57.54; H, 4.86; N, 12.66.
Male albino mice (CF-1 strain, 18–25 g) and male albino
rats (Sprague–Dawley/Wistar, 100–150 g) were used as
experimental animals. The animals were housed in met-
abolic cages, and allowed free access to food and water.
The synthesized compounds (1–21) were suspended in
0.5% methylcellulose/water mixture or in polyethylene
glycol (PEG 200).
4.1.7.
N1-(2,4-Dimethoxyphenyl)-N4-(2-hydroxybenz-
aldehyde)semicarbazone (11). Yield: 57%; mp: 156 °C;
IR (KBr): 3350, 2900, 1690, 1590–1540, 1320,
4.2.1. Anticonvulsant screening. The anticonvulsant eval-
uations were undertaken by the National Institute of
Health, using their reported procedures.19–21 Initially,
all compounds were administered ip at doses of 30,
100, and 300 mg/kg to one to four mice. Activity was
established using the MES, scPTZ, and scSTY tests.
Some selected derivatives described in this study were
examined for oral activity in the rat scPTZ screen.
1
1200 cmÀ1; H NMR (DMSO-d6) d (ppm): 3.70 (s, 3H,
ArOCH3), 3.73 (s, 3H, ArOCH3), 6.55–7.28 (m, 7H,
ArH), 8.10 (s, 1H, imine H), 8.90 (s, 1H, ArNH, D2O
exchangeable), 9.40 (s, 1H, CONH, D2O exchangeable),
9.88 (s, 1H, OH, D2O exchangeable); Calculated for
C16H17N3O4: C, 60.94; H, 5.43; N, 13.33; found: C,
61.14; H, 5.60; N, 13.66.
4.1.8.
N1-(2,4-Dimethoxyphenyl)-N4-(4-methylaceto-
4.2.2. Neurotoxicity screening. Minimal motor impair-
ment was measured in mice by the rotarod test.22 The mice
were trained to stay on an accelerating rotarod that ro-
tates at 10 rpm. The rod diameter was 3.2 cm. Trained
animals were given ip injection of the test compounds in
doses of 30, 100, and 300 mg/kg. Neurotoxicity was indi-
cated by the inability of the animal to maintain equilibri-
um on the rod for at least 1 min in each of the three trials.
phenone)semicarbazone (15). Yield: 53%; mp: 151 °C;
IR (KBr): 3352, 2910, 1690, 1590–1540, 1312, 1320,
1200 cmÀ1; H NMR (DMSO-d6) d (ppm): 1.3 (s, 3H,
1
Carbimino-CH3), 2.34 (s, 3H, CH3), 3.72 (s, 3H, Ar-
OCH3), 3.74 (s, 3H, ArOCH3), 6.35–7.66 (m, 7H,
ArH), 8.90 (s, 1H, ArNH, D2O exchangeable), 10.05
(s, 1H, CONH, D2O exchangeable); Calculated for
C18H21N3O3: C, 66.04; H, 6.47; N, 12.84; found: C,
66.10; H, 6.64; N, 12.96.
4.2.3. Behavioral testing. Some selected compounds
(100 mg/kg) were screened for their behavioral effects
using actophotometer23 at 30 min and 1 h after injection.
The behavior of animals inside the photocell was recorded
as a digital score. Increased scores suggest good behavior-
al activity. The control animal was administered PEG.
The observations are tabulated as Table 3.
4.1.9.
semicarbazone (17). Yield: 54%; mp: 159 °C; IR (KBr):
3370, 2890, 1670, 1590–1545, 1320, 1200 cmÀ1 1H
NMR (DMSO-d6) d (ppm): 1.82 (s, 3H, CH3), 1.93 (s,
3H, CH3), 3.66 (s, 3H, ArOCH3), 3.70 (s, 3H, ArOCH3),
6.72–6.84 (m, 3H, ArH), 8.22 (s, 1H, ArNH, D2O
exchangeable), 9.54 (s, 1H, CONH, D2O exchangeable);
Calculated for C12H17N3O3: C, 57.36; H, 6.82; N, 16.72;
found: C, 57.14; H, 6.80; N, 16.66.
N1-(2,4-Dimethoxyphenyl)-N4-(propan-2-one)-
;
4.2.4. CNS depressant study. The forced swim pool
method described earlier was followed,24 mice were
placed in a chamber (diameter: 45 cm, height: 20 cm)
containing water up to a height of 15 cm at 25 2 °C.
Two swim sessions were conducted, an initial 15 min
pre-test, followed by a 5 min test session 24 h later.
The animals were administered an ip injection
(100 mg/kg) of the test compounds 30 min before the
test session. The period of immobility (passive floating
without struggling, making only those movements which
are necessary to keep its head above the surface of
water) during the 5 min test period was measured. The
results are presented in Table 4.
4.1.10.
semicarbazone (20). Yield: 50%; mp: 156 °C; IR (KBr):
3350, 2900, 1670, 1590–1545, 1320, 1200 cmÀ1 1H
NMR (DMSO-d6) d (ppm): 1.52 (s, 3H, CH3), 2.09 (s,
3H, CH3), 2.54 (s, 2H, CH2), 3.70 (s, 3H, ArOCH3),
3.76 (s, 3H, ArOCH3), 6.66–6.84 (m, 3H, ArH), 8.88
(s, 1H, ArNH, D2O exchangeable), 10.24 (s, 1H,
N1-(2,4-Dimethoxyphenyl)-N4-(acetylacetone-
;
CONH,
D2O
exchangeable);
Calculated
for
C14H19N3O4: C, 57.33; H, 6.53; N, 14.33; found: C,
57.56; H, 6.60; N, 14.48.
4.2.5. Isolation of rat brain regions and GABA assay. The
GABA assay for compounds 4, 15, and 17 was per-
formed in brain tissue extracts enzymatically as previ-
ously described.25 Adult Wistar rats were used for this
purpose. After 2 h of drug administration (30 mg/kg,
4.1.11. N1-(2,4-Dimethoxyphenyl)-N4-(cyclohexanone)-
semicarbazone (21). Yield: 51%; mp: 147 °C; IR (KBr):
3350, 3000, 2900, 1670, 1590–1545, 1500, 1320,
1200 cmÀ1 1H NMR (DMSO-d6) d (ppm): 1.32 (m,
;

3112
R. Thirumurugan et al. / Bioorg. Med. Chem. 14 (2006) 3106–3112
7. Dimmock, J. R.; Vashishtha, S. C.; Stables, J. P. Eur. J.
Med. Chem. 2000, 35, 241.
8. Dimmock, J. R.; Vashishtha, S. C.; Stables, J. P. Phar-
mazie 2000, 55, 490.
9. Pandeya, S. N.; Yogeeswari, P.; Stables, J. P. Eur. J. Med.
Chem. 2000, 35, 879.
ip), the animal was sacrificed by decapitation and the
brain regions, midbrain, olfactory lobe, cerebellum,
and medulla oblongata, were dropped into separate
vials containing 4–6 ml of ice-cold 80% ethanol and pro-
cessed further as described previously.26
10. Yogeeswari, P.; Sriram, D.; Suniljit, L. R. J.; Kumar, S. S.;
Stables, J. P. Eur. J. Med. Chem. 2002, 37, 231.
11. Yogeeswari, P.; Sriram, D.; Brahmandam, A.; Sridharan,
I.; Thirumurugan, R.; Stables, J. P. Med. Chem. Res. 2003,
12, 57.
12. Yogeeswari, P.; Thirumurugan, R.; Kavya, R.; Samuel, S.
J.; Stables, J. P.; Sriram, D. Eur. J. Med. Chem. 2004, 39,
729.
13. Yogeeswari, P.; Sriram, D.; Pandeya, S. N.; Stables, J. P.
Farmaco 2004, 59, 609.
14. Yogeeswari, P.; Sriram, D.; Veena, V.; Kavya, R.;
Rakhra, K.; Mehta, S.; Ragavendran, J. V.; Thirumur-
ugan, R.; Stables, J. P. Biomed. Pharmacother. 2005, 59,
51.
Acknowledgments
This work was supported by the Department of Science
and Technology (DST), India, under the SERC Fast
track scheme for young scientist (No. SR./FT/L-84/
2003). One of the authors Mr. R. Thirumurugan deeply
acknowledges the Council of Scientific and Industrial
Research for providing Senior Research Fellowship.
The authors are grateful to Mr. A. R. Subramanian,
1
CDRI, Lucknow, for the generation of H NMR and
elemental analyses data.
15. Yogeeswari, P.; Sriram, D.; Thirumurugan, R.; Ragaven-
dran, J. V.; Sudhan, K.; Kuamr, R.; Stables, J. J. Med.
Chem. 2005, 48, 6202.
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