Design, synthesis and pharmacological evaluation of N-[4-(4-(alkyl/aryl/heteroaryl)-piperazin-1-yl)-phenyl]-carbamic acid ethyl ester derivatives as novel anticonvulsant agents

Article abstract of DOI:10.1016/j.bmcl.2015.01.004

A series of alkyl/aryl/heteroaryl piperazine derivatives (37-54) were designed and synthesized as potential anticonvulsant agents. The target compounds are endowed with satisfactory physicochemical as well as pharmacokinetic properties. The synthesized compounds were screened for their in vivo anticonvulsant activity in maximal electroshock (MES) and subcutaneous pentylenetetrazole (sc-PTZ) seizure tests. Further, neurotoxicity evaluation was carried out using rotarod method. Structure activity relationship studies showed that compounds possessing aromatic group at the piperazine ring displayed potent anticonvulsant activity. Majority of the compounds showed anti-MES activity whereas compounds 39, 41, 42, 43, 44, 50, 52, and 53 exhibited anticonvulsant activity in both seizure tests. All the compounds except 42, 46, 47, and 50 did not show neurotoxicity. The most active derivative, 45 demonstrated potent anticonvulsant activity in MES test at the dose of 30 mg/kg (0.5 h) and 100 mg/kg (4 h) and also delivered excellent protection in sc-PTZ test (100 mg/kg) at both time intervals. Therefore, compound 45 was further assessed in PTZ-kindling model of epilepsy which is widely used model for studying epileptogenesis. This compound was effective in delaying onset of PTZ-evoked seizures at the dose of 5 mg/kg in kindled animals and significantly reduced oxidative stress better than standard drug phenobarbital (PB). In result, compound 45 emerged as a most potent and safer anticonvulsant lead molecule.

Full text of DOI:10.1016/j.bmcl.2015.01.004

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and pharmacological evaluation of N-[4-(4-(alkyl/
aryl/heteroaryl)-piperazin-1-yl)-phenyl]-carbamic acid ethyl ester
derivatives as novel anticonvulsant agents
⇑
Shikha Kumari, Chandra Bhushan Mishra, Manisha Tiwari
Bio-Organic Chemistry Laboratory, Dr. B.R. Ambedkar Centre for Biomedical Research, North Campus, University of Delhi, Delhi 110007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of alkyl/aryl/heteroaryl piperazine derivatives (37–54) were designed and synthesized as poten-
tial anticonvulsant agents. The target compounds are endowed with satisfactory physicochemical as well
as pharmacokinetic properties. The synthesized compounds were screened for their in vivo anticonvul-
sant activity in maximal electroshock (MES) and subcutaneous pentylenetetrazole (sc-PTZ) seizure tests.
Further, neurotoxicity evaluation was carried out using rotarod method. Structure activity relationship
studies showed that compounds possessing aromatic group at the piperazine ring displayed potent anti-
convulsant activity. Majority of the compounds showed anti-MES activity whereas compounds 39, 41, 42,
43, 44, 50, 52, and 53 exhibited anticonvulsant activity in both seizure tests. All the compounds except
42, 46, 47, and 50 did not show neurotoxicity. The most active derivative, 45 demonstrated potent anti-
convulsant activity in MES test at the dose of 30 mg/kg (0.5 h) and 100 mg/kg (4 h) and also delivered
excellent protection in sc-PTZ test (100 mg/kg) at both time intervals. Therefore, compound 45 was fur-
ther assessed in PTZ-kindling model of epilepsy which is widely used model for studying epileptogenesis.
This compound was effective in delaying onset of PTZ-evoked seizures at the dose of 5 mg/kg in kindled
animals and significantly reduced oxidative stress better than standard drug phenobarbital (PB). In result,
compound 45 emerged as a most potent and safer anticonvulsant lead molecule.
Received 27 September 2014
Revised 14 December 2014
Accepted 5 January 2015
Available online xxxx
Keywords:
Anticonvulsant
Epilepsy
Piperazine
Kindling
Oxidative stress
Ó 2015 Elsevier Ltd. All rights reserved.
Epilepsy is a most prevalent chronic neurological disorder and
is characterized by recurrent unprovoked seizures of cerebral ori-
gin presenting with episodes of sensory, motor, autonomic and
psychic origin.1 According to the World Health Organization,
Epilepsy affects almost 50 million people worldwide.2 Although a
large number of antiepileptic drugs (AEDs) that suppress or pre-
vents seizures are now available but 25–40% of patients remain
live with uncontrolled seizures and experiences number of drug
induced side effects.3 The treatment of epilepsy involves chronic
consumption of AEDs and in addition concomitant administration
of other drugs increases the risk of adverse drug interactions.4
Among all present AEDs, phenobarbital (PB) is used as first line
therapy in management of status epilepticus and generalized epi-
lepsy in many areas of world due to its high efficacy and low cost.5
Nevertheless the number of adverse effects such as sedation, hypo-
tonia, ataxia, cognitive decline are associated with the use of PB.
Therefore, it is mandatory to search new chemical entities which
possess better efficacy and safer therapeutic index as compared
to existing AEDs.
Piperazine and its derivatives constitute an important class of
heterocyclic compounds and comprising broad range of biological
activities such as antioxidant,6 anti-cancer,7 antipsychotic,8 anxio-
lytic,9,10 antidepressant,11 anti-anginal,12 antimicrobial,13 anti-
fungal,14 antihistamine,15 anti-HIV16 protease activity. Literature
survey also exposed that piperazine derivatives are widely used
to design central nervous system (CNS) active agents and have
been extensively investigated by many research groups as potent
anticonvulsant agents.17,18 For example, the compound
(3-phosphonopropyl)piperazine-2-carboxylic acid ( -CPP) and its
unsaturated analogue (
(À)(E)-4-(3-phosphonoprop-2-enyl) piper-
azine-2-carboxylic acid ( -CPPene) have been reported as potent
D-(À)4-
D
D
D
anticonvulsants.19 Moreover, befuraline (psychoactive drug) and
flunarizine (calcium channel blocker) also possessed piperazine
ring and were reported with significant anticonvulsant activities
in mice and rats.20–22 With this background in mind, piperazine
scaffold was selected for the development of effective anticonvul-
sant agents. Pharmacophoric structural necessities23 such as a (i)
hydrophobic domain, (ii) one electron donor atom, and (iii) a
⇑
Corresponding author. Tel.: +91 011 27666272; fax: +91 011 27666248.
E-mail address: mtiwari07@gmail.com (M. Tiwari).
http://dx.doi.org/10.1016/j.bmcl.2015.01.004
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kumari, S.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.01.004

2
S. Kumari et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
hydrogen donor/acceptor unit (HAD), required for anticonvulsant
activity were also incorporated in these newly designed molecules
(Fig. 1). In the present study, we synthesized a series of N-[4-(4-
verified the conjugation of ester fragment. This conjugation was
also validated by the appearance of broad singlet of NH at 6.1–
6.5 ppm in 1H NMR spectra. All the compounds were purified by
column chromatography using either DCM/methanol or chloro-
form/methanol as an eluent and all the compounds were fully
characterized by IR, NMR and Mass spectroscopy and the purity
of final compounds was assessed by elemental analysis (see refer-
ence 25 and Supplementary data).
The physicochemical properties plays crucial role in drug devel-
opment and potential CNS active agents must requires favorable
physicochemical properties such as noticeable range of molecular
weight, molecular necessities and topological indices. In silico
physicochemical parameters of compounds (37–54) were calcu-
lated by using online molinspiration cheminformatics26 (Table 1).
TPSA (topological polar surface area) is an important descriptor
in characterizing drug absorption and blood brain barrier (BBB)
penetration capability. TPSA values 6140 Å2 for prevailing drug
absorption through intestine and 690 Å2 for a molecule to pene-
trate BBB are considered as standard values.27,28 The results
showed that all the test compounds (37–54) demonstrated TPSA
values within acceptable range (44.8–75.0). Additionally, TPSA
was used to calculate the percentage of absorption (%ABS) and
compounds 37–54 displayed the %ABS range of 83–93%, which
indicates their good bioavailability by oral administration. Reason-
able physicochemical properties such as molecular weight, number
(alkyl/aryl/heteroaryl)-piperazin-1-yl)-phenyl]-carbamic
acid
ethyl ester derivatives (37–54) to evaluate their anticonvulsant
potential. Initial anticonvulsant screening was performed in mice
using maximal electroshock seizure (MES) and subcutaneous
pentylenetetrazole (sc-PTZ) seizure tests and the neurotoxicity
was determined by the rotarod test. After correlating the structures
of the tested compounds with their anticonvulsant activity, struc-
ture activity relationship (SAR) was established. Compound 45 was
found most active in preliminary MES and sc-PTZ seizure models.
These findings encouraged us to further evaluate the anticonvul-
sant property of compound 45 in pentylenetetrazole induced kind-
ling (PTZ-induced kindling) model of epilepsy. In addition, the
antioxidant potential of 45 was assessed by measuring oxidative
stress biomarkers such as malondialdehyde (MDA), reduced gluta-
thione (GSH), glutathione peroxidase (GSH-Px) and nitric oxide
(NO) levels in kindled mice.
Synthesis of the titled compounds was carried out according to
synthetic protocols earlier standardized in our laboratory with sev-
eral modifications.10 Synthesized derivatives 37–39 were previ-
ously reported by our research group and were studied for their
anxiolytic10 and anti-Alzheimer activity.24 Rest of all derivatives
are new and herein reported for the first time. The preparation of
target compounds was accomplished by using the general method
outlined in Scheme 1. Concisely, an equimolar mixture of 1-fluoro-
4-nitrobenzene and N-alkyl/aryl/heteroaryl piperazine in dried
dimethylsulfoxide (DMSO)/dimethylformamide (DMF) was stirred
at room temperature for 4–8 h to give 1-(4-nitrophenyl)-4-alkyl/
aryl/heteroaryl piperazine (1–18). Next, the nitro group was
reduced with Sn/HCl to afford the key intermediates 4-(4-alkyl/
aryl/heteroaryl piperazine-1-yl)-phenylamine (19–36) in good
yields. The appearance of an intense absorption band at 3170–
3450 cmÀ1 (free primary amino group) in infrared (IR) spectra,
and the broad singlet at 3.1–3.9 ppm in 1H NMR spectra confirmed
the complete reduction of nitro to amine group. In third step, the
mixture of 4-(4-alkyl/aryl/heteroaryl piperazine-1-yl)-phenyla-
mine (19–36), ethylchloroformate (equal molar) and dried triethyl-
amine (TEA, in catalytic amount) in dried dichloromethane (DCM)/
DMF was stirred at 0–20 °C for 6–8 h to give the target compounds
(37–54). The appearance of NH (3300–3400 cmÀ1) and C@O
(1650–1700 cmÀ1) absorption bands of amide group in IR spectra
of rotatable bonds, hydrogen bond donor/acceptors along with
29
non-violation of Lipniski ‘Rule of Five’
advocated the suitability
of these compounds as drug like candidates.
Pharmacokinetic properties also impart a vital role in the devel-
opment of successful therapeutic agents. Pharmacokinetic proper-
ties such as absorption, distribution, metabolism, excretion and
toxicity (ADMET) of compounds 37–54 were calculated using in
silico Discovery Studio software (Table 2). The ADMET absorption
and BBB levels generally predicts human intestinal absorption
(HIA) and penetration through BBB, respectively. According to
ADMET predictor software, the range for HIA lies from 0 to 3 which
briefly indicates good (0), moderate (1), low (2) and very low (3)
absorption levels.30 All the compounds demonstrated ‘0’ which
showed their good HIA properties. The BBB penetration level
ranges from very high to low (0 = very high, 1 = high, 2 = medium,
3 = low and 4 = undefined) and all compounds disclosed acceptable
BBB penetration capability. ADMET aqueous solubility plays pre-
carious role in defining bioavailability of drug candidate and in
F
CH
N
N
N
H
N
N
F
O
O
O
H
N
H
H
N
H
N
O
H
Rufinamide
Carbamazepine
Phenytoin
O
O
O
O
NH
O
N
NH
NH2
N
N
H
HN
N
O
F
Representative
Compound 45
O
Retigabine
Mephenytoin
Figure 1. Essential pharmacophoric pattern of well-known AEDs and representative compound 45: (a) red circle represents hydrophobic domain; (b) green circle represents
hydrogen donor/acceptor unit; (c) blue circle represents electron donor group.
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S. Kumari et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
A
R
N
N
NO2
R
N
NH
+
O2N
F
(1-18)
B
O
O
C
R
N
N
NH
R
N
N
NH2
(19-36)
(37-54)
CH3
O
CH3
1,19, 37:R=
2, 20, 38:R=
7, 25, 43:R=
3, 31, 49:R=
C2H5
O
8, 26, 44:R=
9, 27, 45:R=
F3C
14, 32, 50:R=
3, 21, 39:R=
4, 22, 40:R=
O
O
F
H3C
15, 33, 51:R=
16, 34, 52:R=
O
10, 28, 46:R=
11, 29, 47:R=
12, 30, 48:R=
Cl
5, 23, 41:R=
N
N
6, 24, 42:R=
H3C
O
F
F
N
N
17, 35, 53:R=
18, 36, 54:R=
O
Scheme 1. Synthesis of piperazine carbamic acid ethyl ester derivatives (1–54). Reagents and conditions: (A) dimethylsulfoxide (DMSO)/dimethylformamide (DMF), rt; (B)
ethyl acetate (EA) and Sn/HCl, reflux; (C) ethylchloroformate, dichloromethane (DCM), triethyl amine (TEA), 0–20 °C.
Table 1
Physicochemical parameters of synthesized compounds (37–54)
Compound
Rule
%ABSa
—
TPSA (Å2)b
—
n-ROTBc
—
Molecular Weightd
<500
n-OH NH donorse
<5
n-ON acceptorsf
<10
Lipinski’s Violation
61
Theoretical logP
65
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
93.5
93.5
93.5
93.5
93.5
93.5
90.2
93.5
87.6
89.0
89.0
84.6
93.5
87.6
83.1
93.5
93.5
87.1
44.8
44.8
44.8
44.8
44.8
44.8
54.4
44.8
61.8
57.7
57.7
70.5
44.8
61.8
75.0
44.8
44.8
63.2
4
5
5
5
5
5
6
6
6
5
5
5
6
5
5
7
7
6
263.3
277.3
325.4
343.4
359.0
339.4
355.4
393.4
367.4
326.4
326.3
327.3
339.4
353.5
343.3
415.5
451.5
383.4
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
5
5
5
5
5
5
6
5
6
6
6
7
5
6
7
5
5
7
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
2.2
2.6
3.9
4.1
4.6
4.3
3.9
4.8
3.8
3.0
2.6
2.6
3.6
2.8
2.5
5.4
5.7
3.5
All values were calculated using online software www.molinspiration.com.
a
Percentage of absorption.
Topological polar surface area.
Number of rotatable bonds.
Molecular weight.
Number of H-bond donors.
Number of H-bond acceptors.
b
c
d
e
f
due course aqueous solubility logarithmic level for all compounds
was found 2 or 3 which specifies low to good solubility levels
except compound 53. The ADMET hepatotoxicity probabilities for
all compounds (37–54) lies within range 0.1–0.2 and thus are not
likely to possess any kind of hepatotoxic reactions. Another impor-
tant parameter is cytochrome P450 2D6 (CYP2D6), an important
enzyme belonging to family of cytochrome P450 mixed-function
oxidase system and responsible for metabolism as well as excre-
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S. Kumari et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 2
ADMET study of synthesized compounds (37–54)
Compound ADMET
absorption
ADMET
ADMET
solubility
ADMET
solubility
level
ADMET
hepatotoxicity
probability
ADMET
hepatotoxicity
level
ADMET
ADMET CYP2D6
probability
ADMET
BBB levela
CYP2D6b
PPB levelc
level
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
2
1
1
1
1
1
1
2
2
2
2
1
2
3
0
0
2
À2.92
À3.15
À4.44
À4.76
À5.14
À4.90
À4.43
À5.61
À4.24
À4.0
3
3
2
2
2
2
2
2
2
3
3
3
2
3
3
2
1
2
0.152
0.218
0.238
0.231
0.192
0.231
0.218
0.198
0.198
0.211
0.231
0.251
0.125
0.178
0.251
0.132
0.218
0.251
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0.435
0.461
0.405
0.405
0.415
0.475
0.435
0.306
0.376
0.336
0.336
0.336
0.465
0.485
0.386
0.653
0.623
0.425
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
À3.52
À3.52
À4.33
À3.81
À3.12
À5.81
À6.33
À4.45
a
b
c
Blood brain barrier level.
Cytochrome P450 2D6.
Plasma protein binding.
tion of any xenobiotics. According to ADMET predictor software,
CYP2D6 is designated to have two classes; class ‘0’ and class ‘1’
which signifies compounds are likely to be non-inhibitor and
inhibitor of CYP2D6, respectively. Compounds 37–54 were found
non-inhibitor of CYP2D6 enzyme and may be metabolized and
excreted successfully. The result suggests that compounds 37–54
have good physicochemical and pharmacokinetic profile.
The preclinical discovery and development of new chemical
agents for the treatment of epilepsy heavily relies on the use of
predictable animal models that must provide same seizure gener-
ations as in case of human epileptic syndromes. Therefore at pres-
ent time there are two in vivo screens named MES, the sc-PTZ are
used regularly by most of antiepileptic drug discovery (ADD) pro-
grams and are considered as the ‘gold standards’ in early stages of
anticonvulsant testing.31 Almost all clinically important AEDs are
protective in at least one of these primarily seizure tests. The
MES and sc-PTZ models mimics human generalized tonic–clonic
seizures (grand mal) and generalized absence seizures (petit
mal), respectively.32 The anticonvulsant activity of target com-
pounds (37–54) were primarily evaluated in MES and sc-PTZ sei-
zure tests at 0.5 and 4 h time interval with three different doses
of 30, 100 and 300 mg/kg doses in adult swiss albino male mice
(Table 3). The experimental procedures used for in vivo studies
are described in Supplementary data.
It is generally proposed that compounds active in MES test pos-
sess ability to prevent seizure spread. We found that majority of
compounds have moderate to good anticonvulsant activity in
MES test. The compounds 42 and 45 revealed significant protec-
tions at the lowest dose of 30 mg/kg after 0.5 h as compare to stan-
dard drug phenytoin. Similarly compounds 46 and 49 provide
protection at the dose of 100 mg/kg in both time intervals specify-
ing their fast onset as well as longer duration of action. Other
active compounds 40, 50, 51, 52 and 53 showed anti-MES activity
only after 4 h which demonstrates their slow but longer duration
of action. Among these compounds, 50 and 51 were active at
100 mg/kg and the remaining compounds were active at relatively
higher doses of 300 mg/kg. Three compounds 37, 38 and 54 were
completely inactive in MES test whereas compounds 41, 47 and
48 exhibited unique protections at dose 100 mg/kg (0.5 h) and
300 mg/kg (4 h). Compounds 43 and 44 delivered protection at
dose of 100 mg/kg only after 0.5 h that implies their rapid onset
and short duration of action.
In sc-PTZ model myoclonic seizures are induced chemically
through administration of pentylenetetrazole at the convulsive
dose (CD97) of 85 mg/kg subcutaneously and this test is largely
used to identify agents that could provide protection by elevating
the seizure threshold. It was perceived that few numbers of deriv-
atives were active in sc-PTZ test. Compounds 39, 41 and 45 have
shown total abolition of myoclonic jerks against sc-PTZ induce sei-
zures and thus providing 100% protection at both time intervals.
Two compounds 42 and 44 have shown protection only after
0.5 h at the doses of 100 mg/kg and 300 mg/kg, respectively. Other
compounds 50 and 52 delayed seizure onset at higher dose
300 mg/kg however compounds 43 and 53 were active at dose of
100 mg/kg after 4 h. The rotarod test has been widely used for
screening of newer anticonvulsant agents to determine their
neurotoxicity which comprises of minimal neurological deficits
such as sedation, ataxia, hyperexcitability and impaired motor
functions.36 Compounds 42, 46, 47, and 50 exhibited neurotoxicity
at higher doses which was found similar to standard drugs phenyt-
oin and carbamazepine. It is noteworthy that most tested com-
pounds did not show any sign of neurotoxicity. Nevertheless
animals administered with compounds 41 and 44 were slightly
sedated and became unable to grasp rotating rods in three succes-
sive trials therefore the neurotoxicity was not determined.
The SAR was established to understand the effect of various
substitutions at the piperazine ring that could be responsible for
exerting anticonvulsant activity in MES and sc-PTZ seizure tests.
It was observed that all aromatic ester derivatives (39–54) were
active either in MES or sc-PTZ seizure tests yet compounds 37
and 38 contains aliphatic side chain on piperazine ring were devoid
of anticonvulsant activity in any of seizure tests. The 1-phenyl
piperazine derivative (39) demonstrated good anticonvulsant
activity in both seizure tests; whereas replacement of phenyl ring
with benzyl ring (49) leads to complete abolition of activity in sc-
PTZ test. Compound 40 substituted with fluorine at para position
on phenyl piperazine ring was only active in MES test while
replacement of fluorine atom with less electronegative chlorine
atom (41) led to improvement of activity in both tests. Trifluoro-
methyl-phenyl piperazine derivative (44) was also active in both
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S. Kumari et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
Table 3
activity. The above mentioned findings distinctly revealed that
compound 45 is most promising candidate with remarkable pro-
tections against MES as well as sc-PTZ seizure tests and therefore
was selected for assessment in chronic model of epilepsy using
PTZ-induced kindling.
In vivo anticonvulsant and NT screening of N-[4-(4-(aryl/heteroaryl/alkyl)-piperazin-
1-yl)-phenyl]-carbamic acid ethyl ester derivatives
Compound
Intraperitoneal injection in micea (h)
MESb screen sc-PTZc screen NTd-screen
Animal kindling models are most widely used models for study-
ing epileptogenesis through which epilepsy can be modified or
prevented. PTZ (pentylenetetrazole)-induced kindling is character-
ized by repeated administration of sub-convulsive dose of PTZ (a
blocker of the chloride channel of gamma-aminobuytric acid
(GABAA) receptor), resulting in progressive multiplication of sei-
zure activity, which culminates in generalized seizures resembling
human partial complex epilepsy.37 PB is one of the highly effective
antiepileptic drug and has been used since the early twentieth cen-
tury for the successful treatment of various types of epileptic dis-
orders.38 Hence, PB is taken as positive control in kindling model
to compare with compound 45. Experiments were carried out on
adult swiss albino male mice, randomly divided in to the seven
experimental groups (n = 8 in each group except group 2 where
n = 9), (refer Supplementary data for complete kindling procedure).
In order to investigate the role of compound 45 at varying doses in
PTZ-induced kindling, the treatment was given for 35 days along
with PTZ injection on every alternate day. Repeated administration
of PTZ in mice at sub-convulsive dose (30 mg/kg, intraperitoneal
(i.p.), alternate days) for a period of 5 weeks induce kindling and
results in to progressive increase of convulsive activity leading to
generalized tonic–clonic seizures (Fig. 2). In the present experi-
ment, animals of control group received only saline therefore did
not shown any seizure like activity and control group was taken
to produce reliable biological data. In PTZ group all animals ini-
tially developed behavioral patterns of seizure and later recognized
with secondary generalized seizures that was very similar to
chronic epileptic disorder. In some animals belonging to PTZ group,
concomitant high pitch vocalization with flipping activity was seen
and three mice were died due to severe tonic–clonic seizures.
Additionally seizure severity score in this group was increased
with subsequent number of PTZ injection and significant difference
(p <0.001) was also seen as compare to control group. PB is
reported to exert its effect by potentiating GABAnergic function
by altering the conductance of chloride channel at the GABAA
receptor.39 Pretreatment with the PB (30 mg/kg, i.p.) significantly
(p <0.05) reduced both the incidence and seizure severity score
as compared to PTZ group. Animals treated with compound 45
(5 mg/kg, i.p.) also displayed a noteworthy (p <0.05) attenuation
in kindling score as compared to PTZ group. Moreover at the sim-
ilar dose compound 45 showed no significant difference in kindling
score compare to PB, thus study indicate that compound 45 have
modified course of kindling similar to PB. However, the administra-
tion of compound 45 at other doses of 10, 20 & 30 mg/kg sup-
pressed kindling score to reach stage 5 or 6. Mortality was not
observed among animals treated with compound 45. Finding
clearly suggested that compound 45 provided significant protec-
tion against PTZ-induced kindling. At the end of kindling phase,
all animals were killed under ether anesthesia and the brains were
decapitated immediately and stored in a deep freeze (À20 °C) until
processing (maximum 10 h). The pathophysiology of epilepsy has
been extensively studied and many reports indicate that oxidative
stress imparts important role in the initiation and progression of
epilepsy after prolong seizures.40 It has been reported that
increased production of reactive oxygen species (ROS) alters the
neuronal excitability and synaptic neurotransmission.41 At the cel-
lular level seizure like activity significantly initiates influx of cal-
0.5 h
4.0 h
0.5 h
4.0 h
0.5 h
4.0 h
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
—
—
30
—
—
—
—
—
300
—
—
—
300
—
100
—
100
—
100
—
—
—
—
300
—
300
1002
—
—
—
—
—
NT
100
—
NT
—
100
300
—
—
300
—
—
—
—
100
—
100
—
—
—
—
—
NT
100
—
NT
—
100
100
—
—
300
—
—
—
—
100
—
300
—
300
300
300
100
—
1001
30
100
1001
30
100
100
1003
100
—
100
1002
—
—
3001
100
—
100
100
3003
300
100
100
100
300
300
—
—
—
—
—
—
—
—
—
—
—
—
30
—
30
—
53
54
3002
—
100
100
—
PHYe
ETXe
CBZe
VALe
30
—
100
—
—
300
300
300
Response comments:
1
Sedated.
2
Myoclonic jerks.
Deaths following tonic extension.
30, 100, and 300 mg/kg of doses were administered intraperitoneally (ip). The
3
a
figures in the table indicate the minimal dose where by bioactivity was demon-
strated in half or more of the mice. The animals were examined at 0.5 h and 4.0 h
after injections were administered. A dash indicates an absence of activity at the
maximum dose administered (300 mg/kg).
b
Maximal electroshock test (MES).
Subcutaneous pentylenetetrazole test (sc-PTZ).
Neurotoxicity screening using rotarod test.
Data taken from references 33–35.
c
d
e
seizure tests; it seemed that increasing electronegative cloud by
one carbon linker at N-phenyl ring leads to restoration of the anti-
convulsant activity. The substitution at N-phenyl piperazine ring
with electron donating groups does not significantly affect activity.
Substitution with para-tolyl-ethanone at N-phenyl piperazine ring
(45) exhibit excellent anticonvulsant activity in both seizure tests
as compare to standard drugs carbamazepine and ethosuximide.
It may be hypothesized that H-bond acceptors at the both ends
of this pharmacophore imparts suitable interactions at the biolog-
ical active site to produce excellent anticonvulsant activity. We
next examined the impact of substitution with pyridyl ring (com-
pounds 46 and 47) resulted improvement of anti-MES activity and
it may be due to enhancement in polarity. Replacement of pyridyl
ring with pyrimidine ring (48) also showed almost similar potency.
Insertion of ketonic group between nitrogen of piperazine and phe-
nyl ring leads to benzoyl derivative (50) which produced moderate
activity in both seizure tests but when benzoyl ring was replaced
with furoyl ring (51), the activity remained only in MES test. Study
indicates that benzoyl ring is more favorable to display anticonvul-
sant activity in both MES and sc-PTZ test. The benzhydryl pipera-
zine (52) derivative as well as fluoro-substituted benzhydryl
piperazine (53) derivative could not display good activity. It may
be due to presence of bulkier diphenyl ring in the structure. The
piperonyl piperazine derivative (54) was not able to produce good
anticonvulsant activity. SAR studies clearly evoke that compounds
bearing aromatic substituents (39–54) at the piperazine ring were
active in either of two tests and we conclude that the presence of
aromatic substituents is essential for exerting anticonvulsant
cium via voltage gated and N-methylated-D aspartate (NMDA)
dependent ion channels which further induce high levels of ROS
production.42 Recently, novel therapies targeting protection
against seizures along with potential to overcome the oxidative
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6
S. Kumari et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
GSH Levels
6
5
4
3
2
1
0
a
Control
200
180
160
140
120
100
80
60
40
20
0
a
b
e
a
PTZ group
###
a
PTZ+PB(30mg/kg)
PTZ+45(5mg/kg)
PTZ+45(10mg/kg)
PTZ+45(20mg/kg)
PTZ+45(30mg/kg)
b
b
###
b
***
***
b
c
d
a
c
e
e
d
b
1
2
3
4
5
Weeks of Treatment
Figure 2. The visual assessment of seizure score in PTZ-induced kindling in all
groups for 5 weeks. The mean seizure scores expressed ( SEM) for each group of
mice (n = 8) and for PTZ group (n = 9), a = p <0.001, b = p <0.01, c = p <0.05 compared
to control group. And d = p <0.05, e = p <0.05 compared to PTZ group. (ANOVA
followed by Tukey’s test.)
Groups
Figure 4. Effect of administration of compound 45 (5, 10, 20 and 30 mg/kg, i.p.) on
brain GSH levels which were expressed in micrograms per gram of wet tissue in
PTZ-treated mice (n = 8 for each group while for PTZ group, n = 6).Values are given
as mean SEM. ⁄⁄⁄p <0.0001 compared to control group. ###p <0.0001 compared to
PTZ group. a = p <0.0001, b = p <0.001 compared to PTZ + PB (30 mg/kg). (ANOVA
followed by Tukey’s test.)
MDA Levels
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
***
***
###
GSH-Px
a
###
2.0
###
a
###
###
###
a
###
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
b
##
***
***
***
Groups
Figure 3. Effect of administration of compound 45 (5, 10, 20 and 30 mg/kg, i.p.) on
brain lipid peroxidation (LPO) levels which were expressed in nanomoles of MDA
per microgram of protein in PTZ-treated mice (n = 8 for each group while for PTZ
group, n = 6). Values are given as mean SEM. ⁄⁄⁄p <0.0001 compared to control
group. ###p <0.0001 compared to PTZ group. a = p <0.0001 compared to PTZ + PB
(30 mg/kg). (ANOVA followed by Tukey’s test.)
Groups
Figure 5. Effect of administration of compound 45 (5, 10, 20 and 30 mg/kg, i.p.) on
brain GSH-Px levels which were expressed in unit per gram protein in PTZ-treated
mice (n = 8 for each group while for PTZ group, n = 6). Values are given as
mean SEM. ⁄⁄⁄p <0.0001 compared to control group. ###p <0.0001, ##p <0.001
compared to PTZ group. a = p <0.0001, b = p <0.001 compared to PTZ + PB (30 mg/
kg). (ANOVA followed by Tukey’s test.)
stress has drawn considerable attention for the treatment of epi-
lepsy. MDA is an end product of lipid peroxidation and is used as
an indicator of oxidative stress. PTZ-induced kindling produce sig-
nificant rise (p <0.0001) in whole brain MDA content as compare to
control group (Fig. 3). Pretreatment with PB (30 mg/kg, ip) consid-
erably (p <0.0001) lessens brain MDA levels as compare to PTZ
group however, compound 45 at the lower dose of 5 mg/kg, signif-
icantly (p <0.0001) reduces MDA levels as compare to both PTZ and
PTZ + PB group. It was observed that compound 45 at other doses
of 10, 20 & 30 mg/kg also decreased brain high MDA levels better
than standard drug PB. Superior restoration of MDA levels by com-
pound 45 as compared to standard drug PB proves its potential as
better antioxidant than PB.
GSH is well recognized marker to quantify the oxidative stress
and plays a key role in protecting cellular components against
the oxidative damage by scavenging free radicals. The whole brain
GSH levels were measured in all groups and PTZ kindled group
showed a significant (p <0.0001) depletion in cellular GSH levels
as compare to control group (Fig. 4). Compound 45 at the doses
of 5 mg/kg and 10 mg/kg, significantly (p <0.0001) augmented
reduced brain GSH levels as compared to PTZ group. In addition
compound 45 at the dose of 30 mg/kg notably elevated GSH levels
as compared to PTZ+PB group whereas pretreatment with PB could
not reinforce the GSH levels effectively. The increment of GSH lev-
els in PTZ-kindled mice further confirms the antioxidant potential
of compound 45.
GSH-Px is one of the major endogenous antioxidant enzymes
and extent of eventual oxidative injury could be monitored via
determining GSH-Px activity in the biological system. In the pres-
ent study, the marked decline in brain’s GSH-Px activity was seen
in PTZ group that indicates their high consumption (Fig. 5). It may
be thought that PTZ-induced kindling decreases GSH-Px activity
which simultaneously causes failure of H2O2 detoxification pro-
cesses. Moreover these H2O2 radical accumulates with iron ions
present in brain regions and catalyzes Fenton’s reaction and conse-
quently hydroxyl free radical are produced. These hydroxyl radi-
cals are highly reactive and further initiate’s lipid peroxidation in
brain. Therefore it could be concluded that GSH-Px activity is
inversely proportional to lipid peroxidation levels. In PTZ group,
GSH-Px activity was significantly (p <0.0001) lower than control
Please cite this article in press as: Kumari, S.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.01.004

S. Kumari et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
7
NO Levels
dose of 5 mg/kg in PTZ kindled animals without causing mortality.
Moreover, the antioxidant potential of compound 45 was found
significantly better than PB in PTZ-induced kindling model. Our
studies evidently indicates that compound 45 is endowed with
good anticonvulsant activity, satisfactory pharmacokinetic proper-
ties and excellent antioxidant property and may provide a useful
lead for further development of potent antiepileptic agents.
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
*
*
**
**
Acknowledgments
The author Shikha Kumari acknowledges the University Grant
Commission (UGC), New Delhi, India for financial support. C. B.
Mishra is thankful to the UGC for providing Dr. D. S. Kothari
post-doctoral fellowship and the author Manisha Tiwari is thankful
to University of Delhi, Delhi, India for providing the funds to carry
out this research work. University Science Instrumentation Center
(USIC), University of Delhi is acknowledged for giving NMR spec-
tral data of the synthesized compounds.
Groups
Figure 6. Effect of administration of compound 45 (5, 10, 20 and 30 mg/kg, ip) on
brain NO levels which were expressed in micromole per gram protein in PTZ-
treated mice (n = 8 for each group while for PTZ group, n = 6). Values are given as
mean SEM. ⁄⁄P <0.01, ⁄P <0.05 compared to PTZ group (ANOVA followed by
Tukey’s test).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.01.
004.
group. Substantial dropped GSH-Px activity was seen animals trea-
ted with PB as compare to control group. Pretreatment with com-
pound 45 markedly ameliorated GSH-Px activity at both doses of 5
and 10 mg/kg (p <0.0001) as compare to PTZ and PTZ + PB group.
PTZ-induced kindling produces a non-significant increase in the
NO levels in brain when compared to control group (Fig. 6). NO
plays a messenger role in nervous system and peripheral tissues
therefore considered as an important neurotransmitter in the
brain. It is well studied that NO deprivation by nitric oxidase syn-
thase (NOS) inhibitors and NO scavengers prevents initial seizure
like events. Consequently endogenous NO is regarded as a key pro-
moting factor for initiation of seizures.43 Earlier studies have
shown that seizures induced by PTZ activates calcium release via
NMDA glutamate receptors in brain that consequently activates
calcium–calmodulin pathway to increase nNOS protein expression
and NO levels elevates the induction of generalized seizures.44 The
NO measurement is very difficult in biological samples because the
half-life of NO is very short therefore we estimated total tissue
nitrite (NOÀ2 ) and nitrate (NOÀ3 ) levels as an index of NO production.
Pretreatment with compound 45 considerably suppressed brain
NO level at the three different doses (p <0.01, 10 and 20 mg/kg),
(p <0.05, 30 mg/kg) as compared to PTZ group. However, there
was no statistical significance in the NO activities between
PTZ + PB (30 mg/kg) and PTZ + compound 45 (5 mg/kg). Our results
showed that compound 45 efficiently reduced NO levels in kindled
animals resulting suppression of epileptic seizures and oxidative
stress.
In summary, N-[4-(4-(alkyl/aryl/heteroaryl)-piperazin-1-yl)-
phenyl]-carbamic acid ethyl ester derivatives were synthesized
and evaluated for anticonvulsant activity. Most of the synthesized
compounds showed anticonvulsant activity in both or either of two
seizure models. The most promising compound 45 disclosed good
anticonvulsant activities in MES and sc-PTZ seizure tests that des-
ignate 45 as newer broad spectrum anticonvulsant agent. SAR
analysis showed that significant anticonvulsant activity available
mostly in compounds consisting aromatic substituents and it is
plausibly that aromatic residues are more favorable to bind with
allied receptor in brain. The SAR studies also revealed the impor-
tance of H-bond acceptors present at both ends of compound 45
in exerting excellent anticonvulsant action. This compound was
also effective in delaying the onset of PTZ-evoked seizure at the
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