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Medicinal Chemistry Research Laboratory, SLT Institute of Pharmaceutical Sciences, Guru Ghasidas University, Bilaspur 495 009, CG, India
Faculty of Pharmacy, AIMST University, Semeling, 08100 Bedong, Kedah Darul Aman, Malaysia
c
Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 136 119, Haryana, India
d
VNS Institute of Pharmacy, Vidhya Vihar, Neelbad, Bhopal 462 044, MP, India
e
Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar 470 003, MP, India
b
a r t i c l e
i n f o
Article history:
Received 28 September 2011
Revised 2 November 2012
Accepted 13 November 2012
Available online 27 November 2012
Keywords:
1,3,4-Oxadiazoles
Semicarbazones
Limonene
Citral
Anticonvulsant activity
a b s t r a c t
Two novel series of N4-(5-(2/3/4-substituted-phenyl)-1,3,4-oxadiazol-2-yl)-N1-(2-methyl-5-(prop-1-en2-yl)cyclohex-2-enylidene)semicarbazide and N4-(5-(2/3/4-substituted-phenyl)-1,3,4-oxadiazol-2-yl)N1-(3,7-dimethylocta-3,6-dienylidene)-semicarbazide were synthesized to meet structural prerequisite
indispensable for anticonvulsant activity. The anticonvulsant activities of the compounds were investigated using maximal electroshock seizure (MES), subcutaneous pentylenetrtrazole (scPTZ) and subcutaneous strychnine (scSTY) models. The rotorod test was conducted to evaluate neurotoxicity. Some of the
selected active compounds were subjected to GABA assay to conrm their mode of action. The outcome of
the present investigations proved that the four binding sites pharmacophore model is vital for anticonvulsant activity. The efforts were also made to establish structureactivity relationships among test
compounds.
2012 Elsevier Ltd. All rights reserved.
865
Cl
F
A
N
Cl
N
H
O
N N
N
H3C
NH2
O
NH
HBD
Phenobarbitone
N N
R
HBD
Rufinamide
HBD
Lamotrigine
NH
N
H
N
H
HBD
N N
R
H
N
H
CH
HBD
C
N
N
H D
10-18
01-09
Figure 1. Pharmacophoric structural features in anticonvulsant drugs and title compounds: hydrophobic aryl ring system (A), hydrogen binding domain (HBD), electron
donor moiety (D), hydrocarbon moiety (C).
Table 1
Four binding site hypothesis for anticonvulsant activity
Report
Hydrophobic site
S
Hydrogen-bonding
site
H2N
N
N
C
O
NH2
Electron-donor
site
N
Distal hydrocarbon
moiety
Reference
CH
13
HO
OH
H2N
O
N
H
N
H2N
S
H2N
O
C
O
C
O
C
O
NH2
Cl
14
NH2
N
15
NH2
N
16
N
H
OH
H2N
N
C
O
NH2
N
17
N
H2N
6
N
7
H2N
H2N
O
C
O
C
O
C
O
NH2
NH2
18
19
Br
NH2
N
OCH3
N
H
N N
O
9
H2N
C
O
20
N
21
OH
866
a
R
H2N
b
R
CHO
I (a-i)
CH
NNHCONH2
O
R
N N
II (a-i)
III (a-i)
N N
O
R
4-H
4-Cl
4-OCH3
4-NO2
4-F
NH CO NH N CH
10-18
IV (a-i)
N N
NH CO NH N
01-09
Compound
Code
1
2
3
4
5
NH CO NH2
N N
O
V (a-i)
N N
d
NH CO NHNH2
Compound
Code
6
7
8
9
10
R
4-CH3
2-OH
3-OH
4-OH
4-H
Compound
Code
11
12
13
14
15
R
4-Cl
4-OCH3
4-NO2
4-F
4-CH3
Compound
Code
16
17
18
R
2-OH
3-OH
4-OH
Scheme 1. Synthesis of limonene and citral based semicarbazone analogues 118. Reagents and conditions: (a) H2NNHCONH2HCl, CH3COONa, 5880%; (b) Br2, CH3COOH,
30 min stirring, 6275%; (c) NaCNO, CH3COOH, 5066%; (d) NH2NH2H2O, EtOH, NaOH, 28 h reux 5565%; (e) limonene, glacial acetic acid, ethanol, 45 min to 1.5 reux,
4860%; (f) citral, glacial acetic acid, ethanol, 45 min to 1.5 h reux, 5464%.
Table 2
Anticonvulsant and neurotoxicity screening of limonene and citral based 2,5-disubstituted 1,3,4-oxadiazoles
Compounda
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Phenytoin
Carbamazepine
Na valproate
Ethosuximide
MES screeningb
scPTZ screeningb
scSTY screeningc
NT screeningb
0.5 h
4.0 h
0.5 h
4.0 h
0.5 h
4.0 h
0.5 h
4.0 h
100
100
100
100
100
100
30
30
300
300
300
300
300
300
300
300
300
300
300
300
300
300
30
100
100
100
300
100
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
100
300
300
300
300
100
100
300
300
300
300
100
300
Abbreviations: MES = maximal electroshock seizure; scPTZ = subcutaneous pentylenetetrazole; scSTY = subcutaneous strychnine; NT = neurotoxicity.
a
30, 100 and 300 mg/kg of doses were administered ip in the animals. The data in the table indicates the minimal dose whereby biological activity was demonstrated in
half or more of the animals. The activity was measured after 0.5 and 4.0 h of dose administration of test compounds. The sign(dash) represents an absence of activity at
maximum dose administered (300 mg/kg).
b
Mice were employed in this biological evaluation.
c
Rats were employed in this biological evaluation.
867
Compound
MES Screening
4
5
9
13
Phenytoin
NT Screening
0.25 h
0.5 h
1h
2h
4h
0.25 h
0.5 h
1h
2h
4h
++++
++
++
+++
++++
++
++
+
+
+++
++
+
++
++
+++
+++
++
+
++
++++
+++
+++
+++
+
a
The compounds were administered in a dose of 30 mg/kg. The elaborations of symbols is as follows: ++++: activity in 75100% of rats; +++: activity in 5075% of rats; ++:
activity in 2550% of rats; +: activity in 025% of rats;(dash): no activity or neurotoxicity.
Table 4
GABA assay for compounds found active in anticonvulsant screening
S. no.
Compound
1
2
3
4
5
5
6
4b
5b
9b
13b
18b
Clobazamc
Control
Medulla oblangata
Cerebellum
66.60 1.93
43.56 3.84
47.42 3.52
62.56 2.58
52.82 2.62
69.51 3.76
35.42 2.14
48.59 3.12
42.40 2.47
43.48 2.45
36.72 2.78
34.42 1.74
47.53 1.96
31.86 4.65
28.64 1.76
28.42 1.43
30.64 1.88
32.04 2.32
28.25 2.66
35.44 2.39
23.48 2.48
a
Each value represents the mean SEM of six rats signicantly different from the
control at P <0.001.
b
The compounds were tested at a dose of 100 mg/kg (ip).
c
The standard drug was tested at dose of 30 mg/kg (ip).
by reaction of limonene or citral with [V(ai)].23 Most of the research groups1421 has employed different aldehydes and/or ketones in the last step for incorporation of another hydrophobic
hydrophilic site controlling with different substitutions to produce
diversity of compounds. In the present studies, limonene and citral
were employed in the last step for preparation of hitherto reported
semicarbazones based 2,5-disubstituted-1,3,4-oxadiazoles containing limonene and citral as distal hydrocarbon moiety.
The antiepileptic potential2426 of test compounds was determined using male albino mice (swiss, 1825 g) and rat (Wistar
100150 g). The anticonvulsant activity was established by three
models namely, maximal electroshock seizure (MES), subcutaneous pentylenetrtrazole (scPTZ), and subcutaneous strychnine
(scSTY) models. Acute neurological toxicity in mice was screened
by rotorod test.27 All the synthesized compounds were evaluated
for their anticonvulsant potential in doses of 30, 100, 300 mg/kg
by intraperitoneal (ip) injection. The data indicates that 72% of
the compounds that is, 17, 9, 11, 1315 and 18 were active in
the MES evaluation, 55% of the compounds that is, 25, 8, 9, 13,
14, 16 and 18 were active in the scPTZ test and 44% of the compounds that is, 25, 9, 12, 13 and 18 were active in scSTY test.
These results show that compounds possess some MES selectivity
(Table 2 and 3). All the compounds except 10 exhibited biological
activity in either of the MES, scPTZ or scSTY models after 4 h, indicating that the test compounds are slow acting anticonvulsants.
The activity of compounds that is, 25, 9, 12, 13 and 18 in scSTY
test indicates that their anticonvulsant activity may be through
inhibitory glycine receptors. It is pertinent to mention here that
only 44% of test compounds that is, 1, 6, 7, 8, 10, 15, 16 and 17 were
found to be neurotoxic in rotarod test.
Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system responsible for the
maintenance of inhibitory tone that counterbalances neuronal
excitation.28,29 When this balance is disturbed, seizures may result.
Various experimental studies indicate that GABA plays a vital role
868
13. Rajak, H.; Deshmukh, R.; Aggarwal, N.; Kashaw, S.; Kharya, M. D.; Mishra, P.
Arch. Pharm. 2009, 342, 453.
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23. Compound 04: Mp (C) 284286; yield 55%; IR (cm 1) (KBr) 3031.64 (aromatic
CH str), 1603.28 and 1503.63 (aromatic CC str), 1682.04 (C@O str of amide),
3452.61 (NH str of amide), 1095.74 (CO of oxadiazole nucleus), 1659.42
(C@N of oxadiazole), 844.38 (CH def para disubstituted aromatic ring);
3089.01 (@CH str of limonene nucleus), 1631.47 (C@C str of limonene),
893.52 (CH bend, terminal of limonene), 784.31 (CH bend, out of plane of
limonene), 1562.74 and 1335.80 (N@O str of Ar-NO2); 1H NMR (300 MHz,
DMSO-d6, TMS, d ppm): 7.448.32 (m, 4H, ArH), 6.12 (s, 1H, NHCONHN), 7.22
(s, 1H, NHCONHN), 4.77 (s, 2H, CH2), 1.72 (s, 3H, CH3 of limonene nucleus and
C(CH2)CH3), 5.59 (s, 1H, CH of limonene nucleus), 1.98 (t, 2H, C@CHCH2CH of
limonene nucleus), 1.42 (d, 2H, CCH2CH of limonene nucleus); ESI-MS
(Methanol) m/z 397.16 ([M+H]+); elemental analysis: C19H20N6O4% found
(calculated): C, 57.84 (57.57); H, 5.15 (5.09); N, 21.05 (21.20).
Compound 09: MP(C) 262264; yield 59%; IR (cm 1) (KBr) 3046.51 (aromatic
CH str), 1605.73 and 1505.38 (aromatic CC str), 1682.98 (C@O str of amide),
3442.74 (NH str of amide), 1090.93 (CO of oxadiazole nucleus), 1661.34
(C@N of oxadiazole), 841.83 (CH def para disubstituted aromatic ring);
3082.38 (@CH str of limonene nucleus), 2918.82 (CH str, both sp2 and sp3),
1632.85 (C@C str of limonene), 896.02 (CH bend, terminal), 789.48 (CH
bend, out of plane), 3549.27 (OH str); 1H NMR (300 MHz, DMSO-d6, TMS, d
ppm): 6.727.18 (m, 4H, ArH), 6.11 (s, 1H, NHCONHN), 7.25 (s, 1H, NHCONHN),
4.84 (s, 2H, CH2), 1.79 (s, 3H, CH3 of limonene nucleus and C(CH2)CH3), 5.55 (s,
1H, CH of limonene nucleus), 2.08 (t, 2H, C@CHCH2CH of limonene nucleus),
1.46 (d, 2H, CCH2CH of limonene nucleus), 5.21 (s, 1H, OH); ESI-MS (Methanol)
m/z 368.16 ([M+H]+); elemental analysis: C17H21N5O3% found (calculated): C,
62.35 (62.11); H, 5.51 (5.76); N, 19.28 (19.06).
Compound 13: Mp (C) 274276; yield 60%; IR (cm 1) (KBr) 3037.72 (aromatic
CH str), 1603.20 and 1506.63 (aromatic CC str), 1688.15 (C@O str of amide),
3450.48 (NH str of amide), 1090.61 (CO of oxadiazole nucleus), 1665.80
(C@N of oxadiazole), 842.93 (CH def para disubstituted aromatic ring),
1565.78 and 1337.22 (N@O str of Ar-NO2), 2957.14 (CH str, citral), 1638.28
(C@C str, citral); 1H NMR (300 MHz, DMSO-d6, TMS, d ppm): 7.698.32 (m, 4H,
ArH), 6.08 (s, 1H, NHCONHN), 7.18 (s, 1H, NHCONHN), 5.28 (t, 1H, CH of citral
moiety), 1.78 (s, 3H, CH3 of citral moiety), 2.11 (d, 2H, CH2C(CH3)@CHCH2CH of
citral moiety), 2.71 (s, 2H, CH2C(CH3)@CHCH2CH of citral moiety); ESI-MS
(methanol) m/z 399.17 ([M+H]+); elemental analysis: C19H22N6O4% found
(calculated): C, 57.39 (57.28); H, 5.65 (5.57); N, 21.24 (21.09).
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