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Bioorganic & Medicinal Chemistry Letters 23 (2013) 864868

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Bioorganic & Medicinal Chemistry Letters


journal homepage: www.elsevier.com/locate/bmcl

Novel limonene and citral based 2,5-disubstituted-1,3,4-oxadiazoles:


A natural product coupled approach to semicarbazones for antiepileptic activity
Harish Rajak a,, Bhupendra Singh Thakur a, Avineesh Singh a, Kamlesh Raghuvanshi a, Anil Kumar Sah a,
Ravichandran Veerasamy b, Prabodh Chander Sharma c, Rajesh Singh Pawar d, Murli Dhar Kharya e
a

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.

Epilepsy is a common neurological disorder characterized by


excessive temporal neuronal discharges resulting in uncontrolled
convulsions. It is estimated that there are about 50 million patients
of epilepsy around the globe, with almost 90% of these patients
residing in developing countries.1 Epilepsy also affects about 4%
of individuals over their lifetime. The incidence of epilepsy is highest among children below 7 years-of-age and in individuals of
above 55 years. The commercially available anticonvulsant drugs
are associated with numerous side effects including ataxia, gingival
hyperplasia, vertigo, rash, hepatotoxicity and megaloblastic anaemia. Approximately one third of the epileptic patients being treated with existing anticonvulsant drugs do not have complete
control of their seizures and require medication for the duration
of their whole life. Thus these shortcomings demand for the development of more effectual and reliable anticonvulsant drug.2,3
Various research groups has established anticonvulsant potential of semicarbazones46 and 1,3,4-oxadiazoles.79 The conformational studies of the existing anticonvulsant drugs such as
Phenytoin, Carbamazepine, Lamotrigine, Runamide and Phenobarbitone has lead to the proposal of a general model for anticonvulsant activity10,11 This semicarbazones based pharmacophore

Corresponding author. Tel.: +91 9827911824.


E-mail address: harishdops@yahoo.co.in (H. Rajak).
0960-894X/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.051

model consists of following four vital binding sites: (i) an aryl


hydrophobic binding site (A) with halo substituent preferably at
para position; (ii) a hydrogen bonding domain (HBD); (iii) an electron donor group (D) and (iv) another hydrophobichydrophilic
site controlling the pharmacokinetic properties of the anticonvulsant (C) (Fig. 1). In earlier studies on this pharmacophoric model,
our research group has established that the presence of halogen
substituted aryl group near the semicarbazone moiety is one of
the crucial parameters for anticonvulsant activity.12,13 Various
semicarbazone based compounds fullling four binding site
hypothesis for the presence of anticonvulsant has been synthesized
and prominent one with structural similarity to test compounds
are being summarized here.1421 (Table 1)
The title compounds were prepared using the synthetic strategy
described in Scheme 1. The rst step of the synthetic strategy comprises of preparation of 4-substituted benzaldehyde semicarbazones [II(ai)] by reaction of semicarbazide hydrochloride with
different aromatic aldehydes [I(ai)]. [II(ai)] were oxidatively cyclized to 2-amino-5-aryl-1,3,4-oxadiazoles [III(ai)] using bromine
in the presence of glacial acetic acid.22 [III(ai)] were reacted with
sodium cyanate in the presence of glacial acetic acid to yield 1-[5(4-substitutedphenyl)-1,3,4-oxadiazol-2-yl]-urea [IV(ai)]. Hydrazine carboxamides [V(ai)] were prepared by reaction of
[IV(ai)] with hydrazine hydrate in the presence of sodium
hydroxide. In the last step, title compounds 118 were synthesized

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H. Rajak et al. / Bioorg. Med. Chem. Lett. 23 (2013) 864868

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

H. Rajak et al. / Bioorg. Med. Chem. Lett. 23 (2013) 864868

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.

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H. Rajak et al. / Bioorg. Med. Chem. Lett. 23 (2013) 864868


Table 3
Anticonvulsant evaluation of compounds after oral administration in rats
Oral administration in ratsa

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

GABA concentration in lg/100 mga


Midbrain

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

in the mechanism and treatment of epilepsy. It has been observed


that GABA agonists suppress seizures while GABA antagonists
cause seizures. Moreover the drugs that inhibit GABA synthesis
cause seizures and the drugs that increase synaptic GABA are effective antiepileptic agents.30,31 The aryl semicarbazones have been
found to possess anticonvulsant activity through GABA mediation.32,33 Selected compounds with promising biological activity
in initial anticonvulsant screening were subjected to neurochemical estimation of GABA level in adult Wistar rat brain. In GABA assay, the animals were sacriced after 2 h of drug administration by
decapitation and the brain regions that is, midbrain, medulla
oblongata and cerebellum were dropped into separate vessel containing 68 ml of ice-cold 80% ethanol and processed further as per
reported procedure.34,35 Some of these compounds were found to
elevate the GABA level many folds as compared to the control conrming that the presently studied aryl semicarbazones demonstrated anticonvulsant activity via GABA-mediation. (Table 4)
The limonene and citral were incorporated as moiety in the test
compounds because they are low molecular weight compounds
with high lipid solubility. Thus they are able to penetrate the
blood-brain barrier and act in the central nervous system by modulating of the function of GABA receptors.36,37 Compounds with
limonene ring 0109 exhibited considerable anticonvulsant activity in comparison to compounds with citral moiety 1018. The increased anticonvulsant activity of test compounds 0109 in
comparison to compounds 1018 may be attributed to the presence of closed ring present in the limonene with preferential conformation and limited mobility facilitating the tting of molecules
in the receptorial pocket. The results of present studies showed
that compounds bearing the groups like nitro, hydroxy on distant
phenyl ring possess high potency in MES, scPTZ, and scSTY tests.
On the other hand, replacement of these groups with methoxy or
methyl groups on the distant phenyl ring has resulted in compounds with reduced anticonvulsant activity. On comparison of
structure with their biological activity, it has been found that compound with nitro substitution possess maximum activity while
compound with methyl substitution was least active. Among all
test compounds, in terms of their substitutents, structure activity
relationship may be summarized as follows: NO2 > OH > F > Cl > OCH3 > CH3 > H. The Ortho- and meta-hydroxy analogues that
is, 7, 8, 16 and 17 was found to be lesser active than para-hydroxy
analogues that is, 9 and 18. This might be attributed to unfavorable
steric effect of ortho- and meta-substitutions. SAR of test compounds was found quite convincing with earlier reports.1421
The title compounds were designed and synthesized while
keeping in mind that a number of clinically active anticonvulsants
possess a nitrogen hetero atomic system with one phenyl rings and
at least one carbonyl group in their structure. The structure of the
title compounds 118 satised all the pharmacophoric structural
requirements that is, presence of 5-{4-substituted-phenyl}-1,3,4oxadiazol-2-yl moiety as hydrophobic portion, N as electron donor

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H. Rajak et al. / Bioorg. Med. Chem. Lett. 23 (2013) 864868

system, and another hydrophobic distal hydrocarbon moiety in the


form of limonene or citral might be responsible for controlling the
pharmacokinetic properties of the anticonvulsant. Thus, these ndings conrmed the long-established four binding site hypothesis
for semicarbazones. In the present study N4-(5-{4-nitro-phenyl}1,3,4-oxadiazol-2-yl)-N1-(2-methyl-5-{prop-1-en-2-yl}cyclohex2-enylidene)-semicarbazone 4 emerged out as the most active
compound showing considerable activity in maximal electroshock
seizure (at 100 mg/kg after 0.5 h and at 300 mg/kg after 4.0 h), subcutaneous pentylenetrtrazole model (at 100 mg/kg after 0.5 h and
at 300 mg/kg after 4.0 h) and subcutaneous strychnine model (at
300 mg/kg after 4.0 h) without any neurotoxicity (up to 300 mg/
kg after 4.0 h).
In conclusion, a series of novel N4-(5-(2/3/4-substitutedphenyl)-1,3,4-oxadiazol-2-yl)-N 1-(2-methyl-5-(prop-1-en-2-yl)
cyclohex-2-enylidene)semicarbazide 0109 and N4-(5-(2/3/4substituted phenyl)-1,3,4-oxadiazol-2-yl)-N1-(3,7-dimethylocta3,6-dienylidene)-semicarbazide 1018 were synthesized to meet
structural requirements necessary for anticonvulsant activity. The
limonene based semicarbazones were found to possess more anticonvulsant activity than citral based semicarbazones. The results
of the GABA assay indicate that the test compounds have inhibited
or attenuated seizures by facilitating GABAergic neurotransmission.
Our results validated that the pharmacophoric model with four
binding sites is essential for anticonvulsant activity.
Acknowledgments
Four of the authors, Bhupendra Singh Thakur, Avineesh Singh,
Kamlesh Raghuvanshi and Anil Kumar Sah are thankful to AICTE
New Delhi, India for awarding Research Fellowship and nancial
assistance. The help rendered by SAIF, CDRI Lucknow for elemental
and spectral analysis is gratefully acknowledged.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.11.051.
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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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