Design and synthesis of some new 1-phenyl-3/4-[4-(aryl/heteroaryl/alkyl-piperazine1-yl)-phenyl-ureas as potent anticonvulsant and antidepressant agents

Article abstract of DOI:10.1007/s12272-016-0720-1

A series of 1-phenyl-3/4-[4-(aryl/heteroaryl/alkyl-piperazine1-yl)-phenyl-urea derivatives (29–42) were designed, synthesized and evaluated for their anticonvulsant activity by using maximal electroshock (MES), subcutaneous pentylenetetrazole (scPTZ) seizure tests. The acute neurotoxicity was checked by rotarod assay. Most of the test compounds were found effective in both seizure tests. Compound 30 (1-{4-[4-(4-chloro-phenyl)-piperazin-1-yl]-phenyl}-3-phenyl-urea) exhibited marked anticonvulsant activity in MES as well as scPTZ tests. The phase II anticonvulsant quantification study of compound 30 indicates the ED₅₀value of 28.5?mg/kg against MES induced seizures. In addition, this compound also showed considerable protection against pilocarpine induced status epilepticus in rats. Seizures induced by 3-mercaptopropionic acid model and thiosemicarbazide were significantly attenuated by compound 30, which suggested its broad spectrum of anticonvulsant activity. Interestingly, compound 30 displayed better antidepressant activity than standard drug fluoxetine. Moreover, compound 30 appeared as a non-toxic chemical entity in sub-acute toxicity studies.

Full text of DOI:10.1007/s12272-016-0720-1

Arch. Pharm. Res.
DOI 10.1007/s12272-016-0720-1
RESEARCH ARTICLE
Design and synthesis of some new 1-phenyl-3/4-[4-(aryl/
heteroaryl/alkyl-piperazine1-yl)-phenyl-ureas as potent
anticonvulsant and antidepressant agents
Chandra Bhushan Mishra1 Shikha Kumari1 Manisha Tiwari1
•
•
Received: 11 September 2015 / Accepted: 5 February 2016
Ó The Pharmaceutical Society of Korea 2016
Abstract A series of 1-phenyl-3/4-[4-(aryl/heteroaryl/
alkyl-piperazine1-yl)-phenyl-urea derivatives (29–42) were
designed, synthesized and evaluated for their anticonvul-
sant activity by using maximal electroshock (MES), sub-
cutaneous pentylenetetrazole (scPTZ) seizure tests. The
acute neurotoxicity was checked by rotarod assay. Most of
the test compounds were found effective in both seizure
tests. Compound 30 (1-{4-[4-(4-chloro-phenyl)-piperazin-
1-yl]-phenyl}-3-phenyl-urea) exhibited marked anticon-
vulsant activity in MES as well as scPTZ tests. The phase
II anticonvulsant quantification study of compound 30
indicates the ED50 value of 28.5 mg/kg against MES
induced seizures. In addition, this compound also showed
considerable protection against pilocarpine induced status
epilepticus in rats. Seizures induced by 3-mercaptopropi-
onic acid model and thiosemicarbazide were significantly
attenuated by compound 30, which suggested its broad
spectrum of anticonvulsant activity. Interestingly, com-
pound 30 displayed better antidepressant activity than
standard drug fluoxetine. Moreover, compound 30
appeared as a non-toxic chemical entity in sub-acute tox-
icity studies.
Keywords Epilepsy Á Maximal electroshock Á Seizure Á
Neurotoxicity Á Anticonvulsant Á Thiosemicarbazide
Introduction
Epilepsy is a chronic neurological disorder symbolized by
spontaneous, recurrent unpredictable occurrence of sei-
zures arises from brain’s neurons due to hyper-syn-
chronous neuronal firing and neuronal hyper-excitability
(Scharfman 2007). Epilepsy affects approximately 50
million people worldwide and almost 80 % of the
epileptic patients live in developing countries (WHO
2015). The past decade has seen a substantial progress in
the development of many antiepileptic drugs (AEDs)
However, none of the AEDs which has been introduced
since 1993 can be considered as a ‘magic bullet’ which
can consistently prevent patient’s seizures (Bialer and
White 2010). There are still nearly 30–40 % of therapy
resistant epileptic patients remain live with uncontrolled
seizures (Laxer et al. 2014). Moreover, the recent
antiepileptic therapy is occupied with severe adverse
effects and other dose related toxicities (Greenwood
2000). Today, a large number of studies have confirmed a
relationship between epilepsy and depression. Epileptic
patients are generally at amplified risk for developing
depression and earlier reports showed that the symptoms
of depression may be present in 40–60 % of patients with
epilepsy (Grzyb et al. 2006). Some research reports have
also been shown that many AEDs such as oxcarbazepine,
lamotrigine, phenytoin, topiramate displayed intrinsic
antidepressant properties (Bourin et al. 2009). Therefore,
current research is envisaged for development of potent
and least toxic anticonvulsant agent which endowed with
depression modifying capability.
Chandra Bhushan Mishra and Shikha Kumari have contributed
equally to this article.
Electronic supplementary material The online version of this
article (doi:10.1007/s12272-016-0720-1) contains supplementary
material, which is available to authorized users.
& Manisha Tiwari
mtiwari07@gmail.com
1
Bio-Organic Chemistry Laboratory, Dr. B.R. Ambedkar
Center for Biomedical Research, University of Delhi,
New Delhi, India
123

C. Mishra et al.
The literature survey reveals that piperazine is one of the
most encountered heterocycles in designing central ner-
vous system (CNS) active agents (Asif 2015). Piperazine
and its derivatives are well known for their anticonvulsant
(Patel et al. 1990) and antidepressant activity (Waszkiele-
wicz et al. 2015). Previously, we have also reported
numerous substituted piperazine derivatives containing
ester fragment as promising anticonvulsant agents (Kumari
et al. 2015). Here, we have replaced ester fragment of
piperazine derivatives with phenyl urea fragment (Fig. 1a).
It is anticipated that this replacement may increase the
anticonvulsant activity of desired compounds because most
of the clinically used AEDs contains either urea or amide
moieties (for example carbamazepine, phenobarbital, rufi-
namide and phenytoin). In due course 1-[4-(aryl/heteroaryl/
group (D) and (iii) hydrogen donor/acceptor unit (HAD)
that are required for exerting anticonvulsant effect (Un-
verferth et al. 1998) (Fig. 1b), were designed and
synthesized.
All the synthesized compounds (29–42) were screened
for their anticonvulsant activity in two most commonly
used seizure models named maximal electroshock (MES)
and subcutaneous pentylenetetrazole (scPTZ) tests using
mice. The degree of motor impairment was determined
by using standard rotarod assay. The most potent com-
pound 30 was subjected in pilocarpine induced status
prevention (PISP), 3-mercaptopropionic acid (3-MPA),
thiosemicarbazide (TSC) and 4-aminopyridine (4-AP)
induced seizures. The antidepressant effect of compound
30 was determined in forced swim test (FST) and tail
suspension test (TST) using mice. Additionally, sub-acute
toxicity study of compound 30 was assessed in adult
Wistar rats.
alkyl-piperazine1-yl)phenyl]-3-phenyl-urea
derivatives
(29–42) endowed with pharmacophoric structural necessi-
ties such as (i) hydrophobic unit (A), (ii) an electron donor
Fig. 1 a Lead modification of
previously developed
anticonvulsant agents with
newly synthesized aryl/
heteroaryl/alkyl-substituted
(a)
O
O
piperazine urea derivatives.
b Essential pharmacophoric
necessities of well-known AEDs
and synthesized compounds
(29–42): (a) A represents
NH
O
N
N
NH
R
N
N
NH
R
Previously developed lead
Modified lead
hydrophobic unit (b) dotted
circle (D) represents electron
donor group and (c) dotted
rectangle represents a hydrogen
donor/acceptor unit (HAD)
(b)
D
D
H
N
CH
A
O
O
HAD
A
H
N
A
HN
N
O
HAD
O
O
H
A
N
H
D
O
N
H
HAD
Phenytoin
Carbamazepine
Phenobarbital
D
O
H
N
H
D
N
N
N
D
A
A
A
O
O
S
Cl
N
N
H
O
HAD
N
O
Cl
H
HAD
Cl
N
H
N
H
H
S
H
HAD
Zonisamide
Lamotrignine
Ralitoline
F
D
N
R
N
N
HAD
N
H
A
N
O
F
A
A
O
HAD
N
H
N
H
N
H
Model compound structure (29-42)
Ruf
inamide
123

Design and synthesis of some new 1-phenyl-3/4-[4-(aryl/heteroaryl/alkyl-piperazine1-yl)-phenyl-ureas…
1-{4-[4-(4-Chloro-phenyl)-piperazin-1-yl]-phenyl}-3-
phenyl-urea (30)
Materials and methods
Chemistry
White solid (yield 76 %); mp: 238–240 °C; IR (KBr):
1639.8 (C=O), 2813.5 (=CH str, Ar), 3309.3 (NH) cm-1;1H
NMR (DMSO-d6, 400 MHz): 3.16–3.18 (m, 4H, piper-
azine), 3.24–3.27 (m, 4H, piperazine), 6.91–6.95(m, 3H,
Ar–H), 7.00 (d, 2H, Ar–H, J = 9.1 Hz), 7.23–7.25 (m, 4H,
Ar–H), 7.31 (d, 2H, Ar–H, J = 9.1 Hz), 7.42 (d, 2H, Ar–H,
J = 8.4 Hz), 8.40 (s, 1H, NH), 8.54 (s, 1H, NH); 13C NMR
(DMSO-d6, 100 MHz)d: 152.6, 149.7, 146.2, 139.8, 132.0,
128.6, 122.5, 121.5, 119.5, 117.9, 117.0, 116.5, 48.1, 40.1;
LC–MS: m/e 407 (M?1); Anal. Calcd for C23H23Cl, N4 O:
C, 67.89; H, 5.70; N, 13.77; Found: C, 67.80; H, 5.78; N,
13.87.
All the chemicals and reagents were obtained from Sigma
Aldrich (USA), Alfa Aesar (Massachusetts), S.D Fine
Chemicals (India) and Merck (Germany). Melting points
were determined in open capillaries using model KSPII,
KRUSS, (Germany). The infrared (IR) spectra (KBr, thin
film) were recorded on a Nicolet iS5 IR spectrophotometer
(Thermo scientific, United States). The nuclear magnetic
resonance (NMR) spectra were obtained on high resolution
Jeol-400 MHz NMR spectrophotometer (USA). Mass
spectra were recorded on an Agilent 6310 Ion trap LC/MS
and elemental analysis (C, H and N) was carried on Ele-
mentar analysensysteme.
1-{4-[4-(4-Fluoro-phenyl)-piperazin-1-yl]-phenyl}-3-
phenyl-urea (31)
General procedure for synthesis of 1-phenyl-3/4-[4-
(aryl/heteroaryl/alkyl-piperazine1-yl)-phenyl-ureas
(29–42)
White lustrous solid (yield 84 %); mp: 232–234 °C; IR
(KBr): 1648.6 (C=O), 2815.0 (=CH str, Ar), 3316.8 (NH)
cm-1;1H NMR (DMSO-d6, 400 MHz): 3.18 (s, 8H,
piperazine), 6.91–7.08 (m, 7H, Ar–H), 7.23–7.32 (m, 4H,
Ar–H), 7.42 (d, 2H, Ar–H, J = 8.0 Hz), 8.40 (s, 1H, NH),
8.54 (s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz)d:152.6,
147.8, 146.2, 139.9, 132.0, 128.7, 121.5119.5, 117.9,
117.4, 116.5, 115.4, 115.1, 49.1; LC–MS: m/e 391 (M?1);
Anal. Calcd for C23H23F,N4O: C, 70.75; H, 5.94; N, 14.35;
Found: C, 70.84; H, 5.88; N, 14.42.
The intermediates (1–28) were synthesized and character-
ized according to our previously reported procedure (Ku-
mari et al. 2015). Briefly, an equimolar ratio of 4-(4-aryl/
heteroaryl/alkylpiperazine-1-yl) phenylamines (15–28) and
phenylisocyanate were either refluxed in dried acetonitrile
(5–8 h) or stirred at 0–10 °C in dried dimethylformamide
(2–4 h). After that, the reaction mixture was diluted with
water and product was extracted with ethyl acetate. The
organic layer was dried over anhydrous sodium sulphate
and evaporated under reduced pressure. The crude solid
products (29–42) were washed thoroughly with petroleum
ether. All the final compounds were purified by column
chromatography using chloroform/methanol (95/05 as
eluent) to yield pure products.
1-Phenyl-3-{4-[4-(4-trifluoromethyl-phenyl)-
piperazin-1-yl]-phenyl}-urea (32)
White solid (yield 58 %); mp: 218–220 °C; IR (KBr):
1653.9 (C=O), 2923.9 (=CH str, Ar), 3301.3(NH) cm-1; 1H
NMR (DMSO-d6, 400 MHz): 3.18 (t, 4H, piperazine,
J = 5.1 Hz), 3.41 (t, 4H, piperazine, J = 5.0 Hz), 6.93 (d,
3H, Ar–H, J = 8.8 Hz), 7.11 (d, 2H, Ar–H, J = 8.8 Hz),
7.24 (t, 2H, Ar–H, J = 7.6 Hz), 7.31 (d, 2H, Ar–H,
J = 9.5 Hz), 7.43 (d, 2H, Ar–H, J = 7.3 Hz),7.51(d, 2H,
Ar–H, J = 8.8 Hz), 8.39 (s, 1H, NH), 8.53 (s, 1H, NH);
13C NMR (DMSO-d6, 100 MHz)d: 152.7, 146.1, 139.9,
132.1, 128.7, 126.1, 121.4, 119.5, 117.8, 116.5, 114.3,
48.9, 47.0; LC-MS: m/e 441 (M?1); Anal. Calcd for
C24H23F3,N4O: C, 65.44; H, 5.26; N, 12.72; Found: C,
65.51; H, 5.18; N, 12.82.
1-Phenyl-3-[4-(4-phenyl-piperazin-1-yl)-phenyl]-
urea (29)
White solid (yield 89 %); mp: 241–243 °C; IR (KBr):
1640.8 (C=O), 2920.8 (=CH str, Ar), 3315.0 (NH) cm-1;1H
NMR(DMSO-d6, 400 MHz): 3.16–3.18 (m, 4H, piper-
azine), 3.24–3.26 (m, 4H, piperazine), 6.79 (t, 1H, Ar–H,
J = 7.3 Hz), 6.91–6.98 (m, 5H, Ar–H), 7.20–7.26 (m, 4H,
Ar–H), 7.31 (d, 2H, Ar–H, J = 8.7 Hz), 7.42 (d, 2H, Ar–H,
J = 7.3 Hz), 8.40 (s, 1H, NH), 8.54 (s, 1H, NH); 13C NMR
(DMSO-d6, 100 MHz)d: 152.6, 150.9, 146.3, 139.8, 131.9,
128.9, 128.7, 121.5, 119.5, 119.0, 117.9, 116.4, 115.6,
49.1, 48.3; LC–MS: m/e 373 (M?1); Anal. Calcd for
C23H24, N4, O: C, 74.17; H, 6.49; N, 15.04; Found: C,
74.26; H, 6.55; N; 14.98.
1-Phenyl-3-[4-(4-p-tolyl-piperazin-1-yl)-phenyl]-
urea (33)
White-Creamish solid (yield 77 %); mp: 220–222 °C; IR
(KBr): 1647.3 (C=O), 2966.1 (=CH str, Ar), 3312.8 (NH)
cm-1;1H NMR (DMSO-d6, 400 MHz): 2.19 (s, 3H, CH3),
123

C. Mishra et al.
3.18 (s, 8H, piperazine), 6.87–6.93 (m, 5H, Ar–H), 7.03 (d,
2H, Ar–H, J = 7.3 Hz), 7.23–7.32 (m, 4H, Ar–H), 7.43 (d,
2H, Ar–H, J = 7.3 Hz), 8.41 (s, 1H, NH), 8.56 (s, 1H,
NH); 13C NMR (DMSO-d6, 100 MHz)d: 152.6148.9,
146.3, 139.7, 132.1, 129.3, 128.7, 127.6, 121.4, 119.5,
117.9, 116.4, 115.9, 49.1, 48.8, 19.9; LC-MS: m/e
387(M?1); Anal. Calcd for C24H26,N4O: C, 74.58; H,
6.78; N, 14.50; Found: C, 74.67; H, 6.84; N; 14.42.
MS: m/e 463 (M?1); Anal. Calcd for C30H30,N4O: C,
77.89; H, 6.54; N, 12.11; Found: C, 77.81, H, 6.59; N,
12.21.
1-(4-{4-[Bis-(4-fluoro-phenyl)-methyl]-piperazin-1-
yl}-phenyl)-3-phenyl-urea (37)
White-Pinkish (yield 78 %); mp: 243–245 °C; IR (KBr):
1651.2 (C=O), 2817.2 (=CH str, Ar), 3317.2 (NH) cm-1
;
1-{4-[4-(4-Methoxy-phenyl)-piperazin-1-yl]-phenyl}-
3-phenyl-urea (34)
1H NMR (DMSO-d6, 400 MHz): 2.41 (s, 4H, piperazine),
3.06 (s, 4H, piperazine), 4.39 (s,1H, CH), 6.83 (d, 2H, Ar–
H, J = 9.1 Hz), 6.91 (t, 1H, Ar–H, J = 8.4 Hz), 7.11–7.27
(m, 8H, Ar–H), 7.40–7.46 (m, 6H, Ar–H), 8.37 (s, 1H,
NH), 8.53 (s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz)d:
162.2, 159.8, 152.6, 146.3, 139.8, 138.6, 131.7, 129.3,
128.6, 121.4, 119.6, 117.9, 116.0, 115.0, 73.0, 51.3, 48.9;
LC-MS: m/e 499 (M?1); Anal. Calcd for C30H28,F2,N4O:
C, 72.27; H, 5.66; N, 11.24; Found: C, 72.38; H, 5.74; N,
11.16.
White solid (yield 69 %); mp: 228–230 °C; IR (KBr):
1653.3 (C=O), 2920.6 (=CH str, Ar), 3300.0 (NH) cm-1;1H
NMR (DMSO-d6, 400 MHz): 3.13–3.18 (m, 8H, piper-
azine), 3.68 (s, 3H, OCH3), 6.83 (d, 2H, Ar–H,
J = 8.8 Hz), 6.92–6.95 (m, 5H, Ar–H), 7.25 (t, 2H, Ar–H,
J = 7.6 Hz), 7.31 (d, 2H, Ar–H, J = 9.5 Hz), 7.41 (d, 2H,
Ar–H, J = 7.3 Hz), 8.39 (s, 1H, NH), 8.54 (s, 1H, NH);
13C NMR (DMSO-d6, 100 MHz)d: 153.1, 152.6, 146.3,
145.3, 139.9, 131.9, 128.7, 121.5, 119.6, 118.0, 117.6,
116.4, 114.2, 55.1, 49.7, 49.2; LC-MS: m/e 403 (M?1);
Anal. Calcd for C24H26,N4O2: C, 71.62; H, 6.51; N, 13.92;
Found: C, 71.57; H, 6.55; N, 14.01.
1-Phenyl-3-[4-(4-pyridin-4-yl-piperazin-1-yl)-
phenyl]-urea (38)
White solid (yield 66 %); mp: 140–142 °C; IR (KBr):
1-{4-[4-(4-Acetyl-phenyl)-piperazin-1-yl]-phenyl}-3-
phenyl-urea (35)
1639.8 (C=O), 2973.9 (=CH str, Ar), 3306.9 (NH) cm-1
;
1H NMR (DMSO-d6, 400 MHz): 3.15 (t, 4H, piperazine,
J = 5.8 Hz), 3.45 (t, 4H, piperazine, J = 5.4 Hz), 6.87 (d,
2H, Ar–H, J = 8.0 Hz), 6.89–6.94 (m, 3H, Ar–H), 7.31 (d,
2H, Ar–H, J = 9.2 Hz), 7.34–7.40 (m, 4H, Ar–H), 8.15 (d,
2H, Ar–H, J = 6.0 Hz), 8.40 (s, 1H, NH), 8.54 (s, 1H,
NH); 13C NMR (DMSO-d6, 100 MHz)d: 155.4, 152.6,
150.3, 146.3, 139.9, 137.5, 132.0, 128.7, 121.5, 119.5,
118.0, 116.6, 108.9, 49.0, 44.6. LC–MS: m/e 374 (M ? 1);
Anal. Calcd for C22H23,N5O: C, 70.76; H, 6.21; N, 18.75
Found: C, 70.88; H, 6.28; N, 18.65.
Yellowish-Whitish solid (yield 88 %); mp: 239–241 °C; IR
(KBr): 1675.8 (C=O), 2829.4 (=CH str, Ar), 3307.0 (NH)
cm-1; 1H NMR (DMSO-d6, 400 MHz): 2.44 (s, 3H, CH3),
3.17 (s, 4H, piperazine), 3.46 (s, 4H, piperazine), 6.93 (d,
3H, Ar–H, J = 8.0 Hz), 7.02 (d, 2H, Ar–H, J = 8.0 Hz),
7.22–7.33 (m, 4H, Ar–H), 7.42 (d, 2H, Ar–H, J = 7.3 Hz),
7.81 (d, 2H, Ar–H, J = 8.0 Hz), 8.42 (s, 1H, NH), 8.56 (s,
1H, NH); 13C NMR (DMSO-d6, 100 MHz)d: 195.6, 153.8,
152.6, 146.0, 139.8, 132.2, 130.1, 128.7, 126.5, 121.4,
119.5, 118.0, 116.6, 113.3, 48.9, 46.6, 26.1; LC-MS: m/e
415 (M ? 1); Anal. Calcd for C25H26,N4O2: C, 72.44; H,
6.32; N, 13.52 Found: C, 72.52; H, 6.38; N, 13.44.
1-[4-(4-Benzyl-piperazin-1-yl)-phenyl]-3-phenyl-
urea (39)
White-Creamish solid (yield 83 %); mp: 166–168 °C; IR
(KBr): 1663.5 (C=O), 2934.3 (=CH str, Ar), 3368.5(NH)
cm-1;1H NMR (DMSO-d6, 400 MHz): 2.47 (t, 4H, piper-
azine, J = 4.7 Hz), 3.02 (t, 4H, piperazine, J = 4.7 Hz),
3.49 (s, 2H, CH2), 6.84 (d, 2H, Ar–H, J = 8.8 Hz), 6.92 (t,
1H, Ar–H, J = 7.3 Hz), 7.21–7.31 (m, 9H, Ar–H), 7.42 (d,
2H, Ar–H, J = 7.3 Hz), 8.37 (s, 1H, NH), 8.53 (s, 1H,
NH); 13C NMR (DMSO-d6, 100 MHz)d: 152.7, 146.4,
139.8, 138.0, 131.6, 128.8, 128.7, 128.1, 126.9, 121.4,
119.6, 117.9, 116.1, 62.0, 52.6, 49.0; LC-MS: m/e
387(M?1); Anal. Calcd for C24H26,N4O: C, 74.58; H,
6.78; N, 14.50 Found: C, 74.49; H, 6.86; N, 14.39.
1-[4-(4-Benzhydryl-piperazin-1-yl)-phenyl]-3-
phenyl-urea (36)
White solid (yield 82 %); mp: 258–260 °C; IR (KBr):
1647.0 (C=O), 2923.0 (=CH str, Ar), 3329.4 (NH) cm-1
;
1H NMR (DMSO-d6, 400 MHz): 2.43 (s, 4H, piperazine),
3.06 (s, 4H, piperazine), 4.30 (s,1H, CH), 6.83 (d, 2H, Ar–
H, J = 8.3 Hz), 6.92 (t, 1H, Ar–H, J = 6.4 Hz), 7.16–7.31
(m, 10H, Ar–H), 7.40–7.46 (m, 6H, Ar–H), 8.37 (s, 1H,
NH), 8.53 (s, 1H, NH); 13C NMR (DMSO-d6, 100 MHz)d:
152.6, 146.3, 142.9, 139.7, 131.6, 128.6, 128.5, 127.5,
126.9, 121.5, 119.6, 117.9, 116.0, 75.0, 51.5, 48.9; LC–
123

Design and synthesis of some new 1-phenyl-3/4-[4-(aryl/heteroaryl/alkyl-piperazine1-yl)-phenyl-ureas…
1-{4-[4-(Furan-2-carbonyl)-piperazin-1-yl]-phenyl}-
3-phenyl-urea (40)
percentage absorption was calculated using the formula:
Absorption (%ABS) = 109 - (0.345 9 TPSA) (Zhao
et al. 2002).
Pinkish-Whitish solid (yield 79 %); mp: 198–200 °C; IR
(KBr):1647.8 (C=O), 2856.0 (=CH str, Ar), 3312.1(NH)
cm-1 1H NMR (DMSO-d6, 400 MHz): 3.06 (t, 4H,
;
Pharmacokinetic studies
piperazine, J = 4.5 Hz), 3.58 (t, 4H, piperazine,
J = 4.9 Hz), 6.92–6.94 (m, 3H, Ar–H), 7.20–7.27 (m, 3H,
Ar–H), 7.31 (d, 2H, Ar–H, J = 8.4 Hz), 7.41–7.47 (m, 3H,
Ar–H), 8.40 (s, 1H, Ar–H), 8.54 (s, 1H, NH), 8.59 (s, 1H,
NH); 13C NMR (DMSO-d6, 100 MHz)d: 155.0, 152.6,
146.3, 140.4, 139.9, 132.1, 128.7, 128.2, 121.7, 121.4,
119.6, 117.9, 116.7, 49.2, 43.7.LC-MS: m/e 391(M?1);
Anal. Calcd for C22H22,N4O3: C, 67.68; H, 5.68; N, 14.35
Found: C, 67.77; H, 5.75; N, 14.26.
All the synthesized compounds (29–42) were studied for in
silico ADME-toxicity profile by using Discovery Studio
3.5 software (Accelrys, San Diego, CA, USA). The
chemical structures of all compounds (29–42) were drawn
on Chemdraw version 10 to generate the possible confor-
mations and obtained optimized structures were saved in
Mol 2 format which were further utilized for pharma-
cokinetic evaluations.
1-[4-(4-Methyl-piperazin-1-yl)-phenyl]-3-phenyl-
urea (41)
Pharmacology
Adult Swiss Albino mice (25–30 g) and Wistar rats
(100–150 g) of either sex were used as experimental ani-
mals and were kept under standard animal laboratory
conditions at Dr. B.R Ambedkar Center For Biomedical
Research, University of Delhi, India. The MES, scPTZ and
rotarod tests were performed according to the standard
protocols described by Antiepileptic Drug Development
(ADD) program of National Institute of Health (NIH, USA)
(Hester et al. 1979). All the test compounds were used as
fresh suspension (0.5–1 % gum acacia) and fluoxetine was
suspended in aqueous tween 80 (0.5 %). Vehicle represents
1 % gum acacia in normal saline. The standard AEDs
(carbamazepine, phenobarbital and diazepam) and chemo-
convulsants [pentylenetetrazole (PTZ), 3-MPA, TSC, 4-AP
and pilocarpine] were dissolved in normal sterile saline
solution. Biochemical estimations were done quickly and
all the experimental protocols were prior approved by
Institutional Animal Ethics Committee (IAEC) for animal
care.
White solid (yield 81 %); mp: 163–165 °C; IR (KBr):
1646.2 (C=O), 2930.3 (=CH str, Ar), 3293.6 (NH)
cm-1;1HNMR(DMSO-d6,400 MHz):2.19 (s, 3H, CH3),
2.42 (t, 4H, piperazine, J = 4.5 Hz), 3.03 (t, 4H, piper-
azine, J = 4.9 Hz), 6.85 (d, 2H, Ar–H, J = 9.1 Hz), 6.92
(t, 1H, Ar–H, J = 7.6 Hz), 7.22–7.28 (m, 4H, Ar–H), 7.41
(d, 2H, Ar–H, J = 8.4 Hz), 8.36 (s, 1H, NH), 8.53 (s, 1H,
NH); 13C NMR (DMSO-d6, 100 MHz)d: 152.6, 146.4,
139.9, 131.6, 128.7, 121.4, 119.5, 117.9, 116.1, 54.7, 48.8,
45.8. LC-MS: m/e 311(M?1); Anal. Calcd for
C18H22,N4O: C, 69.65; H, 7.14; N, 18.05 Found: C, 69.77;
H, 21; N, 18.16.
1-[4-(4-Ethyl-piperazin-1-yl)-phenyl]-3-phenyl-urea
(42)
White solid (yield 79 %); mp: 160–162 °C; IR (KBr):
1675.9 (C=O), 2970.1 (=CH str, Ar), 3300.8 (NH) cm-1;1H
NMR (DMSO-d6, 400 MHz): 1.01 (t, 3H, CH3,
J = 6.4 Hz), 2.34 (q, 2H, CH2, J = 8.0 Hz), 2.46 (s, 4H,
piperazine), 3.03 (t, 4H, piperazine, J = 4.9 Hz), 6.86 (d,
2H, Ar–H, J = 9.1 Hz), 6.92 (t, 1H, Ar–H, J = 7.2 Hz),
7.22–7.28 (m, 4H, Ar–H), 7.41 (d, 2H, Ar–H, J = 8.4 Hz),
8.36 (s, 1H, NH), 8.52 (s, 1H, NH); 13C NMR (DMSO-d6,
100 MHz)d: 152.6, 146.2, 139.9, 131.6, 128.7, 121.5,
119.6, 117.9, 116.0, 52.4, 51.6, 48.9, 12.0; LC–MS: m/e
325(M?1); Anal. Calcd for C19H24,N4O: C, 70.34; H,
7.46; N, 17.27 Found: C, 70.46; H, 7,39; N, 17.38.
MES test (Loscher and Nolting 1991)
In the MES seizure test, an electric stimulus of 50 mA
(mice) and 150 mA (rats) for 0.2 s was delivered through
ear clip electrodes. In preliminary evaluation, the test
compounds (29–42) were administered intraperitoneally
(ip) in mice at the doses of 30, 100 and 300 mg/kg and the
anti-MES activity was evaluated after 0.5 and 4 h time
intervals. To study oral bioavailability, compound 30 at the
doses of 30 and 100 mg/kg was administered orally to rats
and the anti-MES activity assessed up to 4 h within small
time intervals of 0.25, 0.5, 1.0, 2.0 and 4.0 h. Complete
abolition of hind limb extensor phase was taken considered
as protection.
Physicochemical parameters
The physicochemical properties of the synthesized com-
pounds (29–42) were calculated using Molinspiration
online property explorer (Cheminformatics 2015).The
123

C. Mishra et al.
PTZ test (Krall et al. 1978)
3-MPA. The mice were kept in individual cages and
observed for 0.5 h. The number of tonic–clonic seizures as
well as number of deaths was recorded.
The test compounds (29–42) were administered (ip) at the
doses of 30, 100 and 300 mg/kg in mice and after 0.5 h and
4 h; PTZ was injected (sc) at the dose of 85 mg/kg (CD97).
The number of clonic seizures, tonic seizures and deaths
were noted carefully. For oral bioavailability in rats,
compound 30 was given orally (30 and 100 mg/kg) and
then PTZ at the dose of 70 mg/kg was administered sc. The
anti-PTZ effect was ascessed up to 2 h within time inter-
vals of 0.25, 0.5, 1.0 and 2.0 h.
TSC induced convulsions (Chaubey and Pandeya
2012)
After 0.5 h of the administration of compound 30 at the
doses of 30, 100 and 300 mg/kg (ip) and standard drug
150
Rotarod test (Dunham and Miya 1957)
***
***
##
a
100
50
0
The motor coordination test was conducted according to
the procedure described by Dunham and Miya with some
minor modifications. The animals (mice) were trained to
ride over rotating rod of diameter 3.2 cm (Techno Rotarod
system, Techno Electronics, Lucknow, India) at 10 rpm.
On the next day, trained animals injected with the test
compounds (29–42) at the doses of 30, 100 and 300 mg/kg
(ip) and after 30 min, motor impairment was measured as
the inability of the animal to stay on the rotating rod for at
least 1 min in each of the trial.
***
***
Groups
Anticonvulsant quantification studies (Litchfield
and Wilcoxon 1949)
Fig. 2 Effect of administration of compound 30 (CPP) at the doses of
30, 100 and 300 mg/kg and fluoxetine (FLX) at dose of 50 mg/kg in
mice on the immobility time during forced swim test. Results are
expressed as mean SEM with n = 8 in each group. ***p \ 0.0001
compared to control group, ##p \ 0.001 when CPP 30 mg/kg
compared to FLX 50 mg/kg, a = p \ 0.05 when CPP 300 mg/kg
compared FLX 50 mg/kg
The median effective dose (ED50) of compound 30 was
evaluated in MES test using mice and was administered ip to
each group of animals at the varied doses until three-five
points was established between the dose level of 0 % pro-
tection and 100 % protection. The ED50 and the 95 % con-
fidence interval were calculated by the Graphpad prism 5.
250
200
***
Pilocarpine induced status prevention (PISP) model
(Racine 1972)
##
a
150
100
50
***
The test compound 30 was administered at the dose of 30 and
100 mg/kg via ip route. The anticonvulsant activity was
assessed after providing challenge dose of pilocarpine
(400 mg/kg, ip) at the time zero, namely the time from first III
seizures and at 30 min after a post-first stage III seizure. The
seizure severity score was assigned according to Racine scale.
0
3-MPA induced convulsions (Arnoldi et al. 1990)
Groups
The animals of control group received vehicle only while,
group II was administered with standard drug carba-
mazepine (50 mg/kg, ip). Rest groups of animals were
administered with compound 30 at the dose of 30, 100 and
300 mg/kg, ip (10 mice per group). After 30 min, all
groups of animals received an sc dose of 60 mg/kg of
Fig. 3 Effect of administration of compound 30 (CPP) at the doses of
30, 100 and 300 mg/kg and fluoxetine (FLX) at dose of 50 mg/kg in
mice on the immobility time during tail suspension test. Results are
expressed as mean SEM with n = 8 in each group. ***p \ 0.0001
compared to control group, ##p \ 0.001 when CPP 30 mg/kg
compared to FLX 50 mg/kg, a = p \ 0.05 when CPP 300 mg/kg
compared FLX 50 mg/kg
123

Design and synthesis of some new 1-phenyl-3/4-[4-(aryl/heteroaryl/alkyl-piperazine1-yl)-phenyl-ureas…
diazepam (10 mg/kg, ip), a sc dose of TSC (20 mg/kg) was
given to all groups of mice (10 mice per group). The
occurrence of clonic seizures, tonic seizures and death or
recovery was recorded after 2.5 h.
used for biomarker studies by using automated hemato-
logical analyzers by machine (Nihon Kohden Japan) and
biochemical auto-analyzer (Agappe, India) respectively.
Statistical analysis
4-AP induced convulsions (Chaubey and Pandeya
2012)
Statistical analysis was performed by one-way analysis of
variance ANOVA followed by Tukey’s test. The p value
less than 0.05 was considered to be statistically significant.
Statistical analysis was done using the GraphPad Prism 51
software (La Jolla, USA). Data were represented using
standard error of the mean ( SEM).
In this method, the test compound 30 (30, 100 and 300 mg/
kg, ip) was given to mice and after 0.5 h a sc injection of
4-AP (13.3 mg/kg) was given to all groups of mice (10 mice
per group) to induce convulsions. All mice were carefully
observed for 30 min for characteristic behavioral signs. The
ability of test compound to protect the mice from threshold
convulsion and lethality was designated as protection.
Result and discussion
Porsolt’s FST (Porsolt et al. 1977)
The synthetic methodology to prepare intermediates (1–14;
15–28) and target compounds (29–42) is depicted in
Scheme 1. All the intermediates (1–28) were synthesized
The mice were randomly divided into five groups (n = 8 for
each group) and the test compound 30 (30, 100 and 300 mg/
kg) and the standard antidepressant drug fluoxetine (50 mg/
kg) were given through ip route. After 0.5 h, the mice were
dropped one at a time in a test chamber. The test apparatus
consists of a circular tank (25 cm height 10 cm in diameter)
filled with tap water (23 2 °C). The animal was consid-
ered immobile when remained floating without struggling
and making only slight movements necessary to maintain
the head above the water. The total period of immobility
was recorded in the last 4 min of a total duration of 6 min.
a
O2N
F
HN
N
R
O2N
N
N R
+
1-14
b
O
c
HN
N
N R
H2N
N
N R
HN
29-42
15-28
TST (Steru et al. 1985)
Compound
Number
R-Group
Compound
Number
R-Group
1, 15, 29
8, 22, 36
The capability of the test compound to reduce the immo-
bility behavior was undertaken as a measure of antide-
pressant like effect. The mice were arbitrarily divided into
five groups (n = 8 for each group) similar to FST experi-
ment and after 0.5 h, the mice were individually hanged by
the tail above the floor and affixed with adhesive tape
placed approximately 1–2 cm from the tip of the tail. When
the mouse was hung passively without any motions, con-
sidered as immobile time duration. The immobility period
was recorded in the last 6 min of a total period of 7 min.
N
N
F
F
2, 16, 30
9, 23, 37
Cl
F
N
N
3, 17, 31
4, 18, 32
5, 19, 33
6, 20, 34
10, 24, 38
11, 25, 39
12, 26, 40
13, 27, 41
N
F3C
O
H3C
O
O
H3C
H3C
Toxicological studies (hematology and biochemistry)
CH3
O
7, 21, 35
14, 28, 42
A total of 16 rats were divided into two groups and the
control group received only vehicle (1 % gum acacia in
normal saline). The other group was administered with the
test compound 30 at the dose of 100 mg/kg (per oral, po)
daily for two weeks. After 14 days, blood was collected by
cardiac puncture under mild ether anesthesia and blood
samples were collected. The heparinized blood was used
for hematological study and non-heparinized blood was
CH3
Scheme 1 Synthesis of 1-Phenyl-3-[4-(4-aryl/heteroaryl/alkyl-piper-
azin-1-yl)-phenyl]-urea derivatives (1-42). Reagents and conditions
a dimethylsulfoxide (DMSO)/dimethylformamide (DMF), rt, 4–8 h;
b Ethyl acetate and Sn/Hcl reflux, 3–4 h; c phenyl isocyanate,
acetonitrile (ACN, reflux, 5–8 h) or phenyl isocyanate, DMF
(0–10 °C, 2–4 h)
123

C. Mishra et al.
Table 1 Physicochemical parameters of synthesized compounds (29–42)
Compound
Rule
%ABSa
TPSA
(A2)b
–
n-
Molecular
weightd
\500
n–OH NH
donorse
\5
n-ON
acceptorsf
\10
Lipinski’s
violation
B1
Theoretical
log P
B5
ROTBc
–
–
29
30
31
32
33
34
35
36
37
38
39
40
41
42
92.5
92.5
92.5
92.5
92.5
89.4
86.7
92.5
92.5
88.1
92.5
82.1
92.5
92.5
47.6
47.6
47.6
47.6
47.6
56.8
64.6
47.6
47.6
60.4
47.6
77.8
47.6
47.6
4
4
4
5
4
5
5
6
6
4
5
4
3
4
372
406
390
440
386
402
414
462
498
373
386
390
310
324
2
2
2
2
2
2
2
2
2
2
2
2
2
2
5
5
5
5
5
6
5
5
5
6
5
7
5
5
0
0
0
0
0
0
0
1
1
0
0
0
0
0
4.0
4.6
4.1
4.9
4.4
4.0
3.9
5.7
5.9
3.0
4.0
3.2
2.5
2.9
All values were calculated using molinspiration software (www.molinspiration.com)
a
Absorption percentage
b
Topological surface area
c
Number of rotatable bonds
d
Molecular weight
e
Number of H-bond donors
f
Numberof H-bond acceptors
according to our previously reported procedure (Kumari
et al. 2015) and structures of all key intermediates were
reconfirmed by both spectral data and melting point
The pharmacokinetic properties are also very crucial for
drug development process (Lin and Lu 1997). Therefore,
several important ADMET parameters were studied which
includes BBB level, absorption level, aqueous solubility,
CYP2D6 and hepatotoxicity (Table 2). According to
ADMET predictor software, the BBB penetration level ran-
ges from very high to low (0 = very high, 1 = high,
2 = medium, 3 = low, 4 = undefined). Compounds have
shown very high (36, 37), high (29–35, 39) and medium (38,
40, 41, 42) BBB penetration levels. All thecompounds 29–42
showed a human intestinal absorption (HIA) value ‘0’
excluding 36 and 37 which showed an HIA value of ‘1’ and
results suggested that majority of all compounds are endowed
with good as well as moderate HIA properties (Poonan et al.
2013). The aqueoussolubility logarithmic levelof mostof the
compounds was found to be 3, 2 and 1 which indicates low to
good aqueous solubility levels. Additionally, titled com-
pounds (29–42) displayed hepatotoxicity level ‘0’ which is
indicative of their non-toxicity. Moreover, all compounds
except36, 41and42 belongtonon-inhibitorclassofCYP2D6
enzyme system and this indicates that these compounds could
be efficiently metabolized and excreted well. Overall results,
showed that all compounds (29–42) have good pharmacoki-
netic properties and could be further developed as promising
anticonvulsant agents.
1
determination. The H NMR spectra of final compounds
displayed a pair of singlet in the region d 8.83 and
8.85 ppm that corresponded to two –NH protons and dis-
appearance of broad singlet of NH2, confirmed the for-
mation of urea linkage. These peaks of two –NH protons
were also D2O exchangeable.
The in silico physiochemical properties of the target
compounds (29–42) are listed in Table 1. All synthesized
derivatives 29–42 showed log P value ranges from 2.5 to
4.9 except compounds 36 and 37. This result clearly indi-
cates that most of the compounds showed excellent
lipophilicity to cross blood brain barrier (BBB) easily.
TPSA (topological polar surface area) is an important
descriptor that was shown to correlate well with molecular
transport through membranes (Ertl et al. 2000). All the
tested compounds (29–42) displayed TPSA values within
˚
satisfactory range (47.6–77.8 A). Moreover, all derivatives
exhibited promising %ABS ranging from 82.1 to 92.5 %
which is indicative of good bioavailability upon oral
administration. In addition, the number of rotatable bonds
(n-ROTB), molecular weight, hydrogen bond donor/ac-
ceptor (Lipinski et al. 2001) were also in acceptable range.
123

Design and synthesis of some new 1-phenyl-3/4-[4-(aryl/heteroaryl/alkyl-piperazine1-yl)-phenyl-ureas…
123

C. Mishra et al.
Table 3. In the MES seizure test, majority of compounds
showed promising anticonvulsant activity and thus, prevent
seizure spread effectively. Compounds 29, 30, 31, 32 and
35 showed significant protections at the lowest dose of
30 mg/kg at both time intervals and the protection offered
by these compounds was indicative of their fast as well as
longer duration of action similar to standard AED pheny-
toin. A similar kind of rapid onset and extended duration of
action (up to 4 h) was observed by compound 33, but at the
higher dose of 100 mg/kg. The compounds 38 and 39
showed 100 % anti-MES protection at the dose of 30 mg/
kg (0.5 h) and 100 mg/kg (4 h), respectively. Compounds
34 and 41 displayed anti-MES activity only after 4 h at the
higher dose of 300 mg/kg which validates their slow but
longer period of action. An interesting pattern of seizure
shield was seen with the compounds 40 and 42 which
showed protections at the lowest dose of 30 mg/kg at 0.5 h
interval and at the higher dose of 300 mg/kg at 4 h interval.
Results suggested that all the compounds exhibited excel-
lent protection against MES test, which indicated the
potential of these compounds against generalized tonic–
clonic seizures, similar to human grand mal epileptic
disorder.
Table 3 Anticonvulsant and neurotoxicity (NT) evaluation of the
synthesized compounds (29–42)
Intraperitoneal injection in mice (h)a
Compounds
MES screenb
scPTZ screenc
NT-screend
0.5 h 4.0 h 0.5 h
4.0 h
05 h 4.0 h
29
30
30
30
301
100
–
30
–
–
–
–
30
30
30
–
100
100
–
3002
300
300
–
–
–
31
30
–
–
32
30
100
–
NT
–
NT
–
33
100
300
30
34
–
100
–
–
35
30
100
300
30
301
30
–
–
–
36
300
300
100
1001
300
300
300
30
–
–
–
37
–
–
–
–
38
100
–
–
3003
–
300
NT
300
–
39
NT
–
40
300
–
–
41
–
3002
–
42
Phenytoine
Ethosuximidee
Carbamazepinee 30
Valproic acide
30
30
–
–
–
–
–
–
100
–
100
–
–
300
100
300
–
100
–
300
–
100
–
300
–
The scPTZ seizure test utilizes chemically induced sei-
zures and is capable of identifying the clinical anticon-
vulsant candidate that acts by raising the seizure threshold
(Swinyard et al. 1989). PTZ is a well-known chemo-con-
vulsant and binds to the c-amino butyric acid ‘A’ subtype
(GABAA) receptor in the brain. In scPTZ screening,
compound 30 had shown significant protection by raising
seizure threshold at both time intervals at the lower doses
(30 and 100 mg/kg) and it was better than standard AED
ethosuximide. Compound 31 at the dose of 100 mg/kg
(4 h) had shown total abolition of myoclonic jerks against
scPTZ induced seizures. Two compounds 32 and 38 have
shown protection only after 0.5 h at the doses of 100 mg/
kg whereas compound 40 was active at the dose of 300 mg/
kg. Other active compounds 34 and 35 delayed seizure
onset (4 h) at the higher dose of 300 mg/kg better than the
reference AED sodium valproate. As shown in Table 3,
compounds 29–31, 33, 35–37, 41–42 did not show any
neurological motor impairments at the maximum dose
administered (300 mg/kg) at 0.5 and 4 h time intervals.
Compound 34 showed minimal motor impairment at the
dose of 100 mg/kg after 0.5 h interval whereas the other
compounds 38 and 40 exhibited neurotoxic effects at the
higher dose of 300 mg/kg only after 4 h.
–
Response comments: 1 sedated, 2 myoclonic jerks, 3 deaths following
tonic extension
a
30, 100, and 300 mg/kg of doses were administered intraperi-
toneally (i.p.). The figures in the table indicate the minimal dose
where by bioactivity was demonstrated 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) (n = 4 mice for each tested dose
in MES test)
c
Subcutaneous pentylenetetrazole test (scPTZ) (n = 4 mice for each
tested dose in scPTZ test)
d
Neurotoxicity (NT) screening using rotarod test (n = 8 mice for
each tested dose)
e
Data taken from reference (Dimmock et al. 1996, Porter et al. 1984
and Flaherty et al. 1996)
The early identification of novel anticonvulsant agents
heavily relies on the use of predictable animal models that
must afford same seizure generations similar to human
epileptic disorders. MES and the scPTZ seizure tests are
widely used by antiepileptic drug discovery (ADD) pro-
gram and both are considered as ‘gold standards’ for
screening new chemical entities for anticonvulsant activity
(Krall et al. 1978). Keeping this in mind, all the target
compounds 29–42 were evaluated for their anticonvulsant
activity in MES and scPTZ tests. The results of anticon-
vulsant and motor coordination tests are summarized in
The structure activity relationship (SAR) was deter-
mined to understand the effect of aryl/heteroaryl/alkyl
substitutions at the piperazine ring that could be responsi-
ble for influencing anticonvulsant activity in MES and
scPTZ seizure models. It has been seen that phenyl
piperazine containing derivative 29 provided noteworthy
123

Design and synthesis of some new 1-phenyl-3/4-[4-(aryl/heteroaryl/alkyl-piperazine1-yl)-phenyl-ureas…
protection in MES test but was completely inactive in
scPTZ test. It was observed that the presence of less
electronegative chlorine atom (30) was more favorable for
eliciting anticonvulsant activity in both MES as well as
scPTZ tests at both time intervals. However, highly elec-
tronegative fluorine atom substitution (31) retained similar
anticonvulsant activity as compound 30 but failed to pro-
tect animals in scPTZ seizure test at 0.5 h. Introduction of
strong electron-withdrawing and lipophilic group –CF3
(32) did not alter the activity very much. The presence of
electron donating groups such as methyl (33) and methoxy
(34) at the para position of phenyl piperazine moiety
showed anticonvulsant activity at the doses of 100 and
300 mg/kg in both tests. Introduction of bulkier group (36,
37) did not exert satisfactory effects in MES test and
completely inactive in scPTZ test. Replacement of phenyl
ring attached to piperazine moiety (29) with pyridyl (38)
and furoyl ring (40) does not significantly change the
anticonvulsant activity in MES test but showed rapid onset
of action in scPTZ test at 0.5 h. Insertion of one carbon
linker in phenyl piperazine derivative (29) leads to benzyl
piperazine derivative (39) which showed improvement in
scPTZ test at 4 h (300 mg/kg). Next, we studied the impact
of aliphatic substitution at the piperazine ring and it was
viewed that methyl piperazine derivative (41) failed to
produce remarkable anticonvulsant activity in both seizure
models. However, one carbon homologation gave ethyl
piperazine derivative (42) which displayed anti-MES
activity at the lower dose (30 mg/kg, 0.5 h) and anti-PTZ
activity at the higher dose (300 mg/kg, 4 h). SAR studies
suggested that aromatic substituent at the piperazine ring
were more capable of exerting good anticonvulsant activity
as compared to aliphatic substituents. By looking at the
preliminary anticonvulsant screening results, it was
observed that compound 30 exerted remarkable protections
against seizures induced by MES and scPTZ in mice.
Therefore, extensive anticonvulsant activity of compound
30 was further evaluated in various seizure models.
Table 4 Evaluation of compound 30 in MES test after oral admin-
istration to rats
Compound 30 and dose
Oral administration to rats (h)a
0.25
0.5
1.0
2.0
4.0
30 mg/kg
100 mg/kg
Phenytoinb
a
1
2
1
2
3
4
2
3
3
2
3
3
0
3
3
Figure indicates the total number of rats which are protected out of
four
b
Data taken from reference (Yogeeswari et al. 2005)
Table 5 Evaluation of compound 30 in scPTZ test after oral
administration to rats
Compound 30 and dose
Oral administration to rats (h)a
0.25
0.5
1.0
2.0
30 mg/kg
100 mg/kg
Ethosuximideb
a
0
0
0
1
1
2
1
1
1
0
1
1
Figure indicates the total number of rats which are protected out of
four
b
Data taken from reference (Azam et al. 2009)
scPTZ activity from 0.5 to 2 h (1/4, 0.5 h; 1/4, 1 h; 1/4,
2 h) as comparable to ethosuximide. As result, the in vivo
efficacy of compound 30 upon oral administration con-
firmed its absorption from gastrointestinal tract and also its
penetration to CNS that leads to protect seizures.
Status epilepticus (SE) is life threatening condition
comes from either failure of seizure termination mecha-
nism or the initiation of mechanisms, which lead to
abnormally, prolonged seizures (Trinka et al. 2015). The
PISP model is one of the most acknowledged animal
models of SE. Pilocarpine, is a muscarinic cholinergic
agonist, induces robust limbic seizures when systemically
administered to rats and mice. In the PISP test, compound
30 at the dose of 30 mg/kg, resulted in fair protection (4/8)
at time-zero but failed to protect at the 0.5 h (Table 6).
However, at the dose of 100 mg/kg, protected more than
50 % of animals at 0.5 h after post-first stage III seizure.
3-MPA is a powerful chemo-convulsant rapidly inhibits the
synthesis of GABA, resulting into decreased GABA levels
in the brain and thus induces spontaneous tonic–clonic
seizures in mice or rats (Horton and Meldrum 1973).The
standard AED, carbamazepine at the dose of 50 mg/kg
completely inhibited tonic extensions and mortality
(Table 7). In a similar way, compound 30 at the dose of
300 mg/kg successfully abolished tonic seizures and pro-
tected all animals from mortality. Thiosemicarbazide
The anticonvulsant activity of most potent compound 30
was also expressed in terms of (ED50). The estimated time
of peak effect (TPE) was evaluated for after ip adminis-
tration of compound 30 in mice at 0.5 h. Results of anti-
convulsant quantification studies showed that compound 30
has an ED50 value of 28.5 mg/kg against MES test. A
valued property of an anticonvulsant agent is its ability to
inhibit convulsions when given by the oral route. Most
active compound 30 (30 and 100 mg/kg) was evaluated for
its oral efficacy in rat MES and scPTZ seizure tests. In
MES screen, compound 30 at the dose of 100 mg/kg,
compound 30 displayed an increase in anti-MES activity
from 50 % (2/4) to 75 % (3/4) and the results were almost
equivalent to phenytoin (Table 4). In scPTZ oral evaluation
(Table 5), compound 30 (100 mg/kg) gave 25 % anti-
123

C. Mishra et al.
Table 6 Anticonvulsant activity of compound 30 in pilocarpine-induced status prevention (PISP) model in rats
Compound 30 dose (mg/kg)
Time (h)a
Number of rat protected/number of rat tested
Average weight change (g) SEMb
Protected rats
Non-protected rats
30
0
4/8
1/8c
3/8
5/8
10.5 2.3
25.2 1.9
9.7 2.1
18.5 3.3
11.4 0.5
23.8 2.5
11.1 1.6
20.5 3.0
30
0.5
0
100
100
0.5
a
Post first III seizure
b
Weight change 24 h Post first III seizure
Two rats were died
c
Table 7 Anticonvulsant activity of compound 30 in 3-mercaptopropionic acid (3-MPA) induced convulsions in mice
Test compound with dose (mg/kg)
Test time (h)
Tonic-seizure (%)
Clonic-seizure (%)
Mortality (%)
Control (1 % gum acacia in saline)
Carbamazepine (50 mg/kg)
Compound 30 (30 mg/kg)
Compound 30 (100 mg/kg)
Compound 30 (300 mg/kg)
0.5
0.5
0.5
0.5
0.5
100
0
100
90
50
0
80
50
0
100
100
100
20
10
0
Table 8 Anticonvulsant activity of compound 30 in Thiosemicarbazide (TSC) induced convulsions in mice
Test compound with dose (mg/kg)
Test time (h)
Tonic-seizure (%)
Clonic-seizure (%)
Mortality (%)
Control (1 % gum acacia in saline)
Diazepam (10 mg/kg)
2.5
2.5
2.5
2.5
2.5
100
0
100
40
100
0
Compound 30 (30 mg/kg)
Compound 30 (100 mg/kg)
Compound 30 (300 mg/kg)
50
60
10
100
80
50
10
0
60
(TSC) is a pyridoxal phosphate antagonist and interferes
with either the synthesis or the coenzymic function of
pyridoxal phosphate. Systemic administration of
thiosemicarbazide produces severe tonic–clonic seizures in
mice or rats (Horton and Meldrum 1973). In TSC induced
convulsion test, compound 30 (300 mg/kg) in comparison
with control group successfully reduces the percentage of
tonic extensions from 100 to 10 % and also saved all
animals from death similar to standard drug diazepam
(Table 8).
4-AP is a K? channel antagonist and behaves as potent
convulsant in animals as well as in humans (Szente and
Baranyi 1987). The seizure like activity is chiefly facili-
tated by non-NMDA (N-methyl-D-aspartate) type excita-
tory amino acid receptors. In this test, compound 30 failed
to significantly protects animals from tonic seizures as
compared to phenobarbital (Table 9). The inability of
compound 30 even at higher doses to produce significant
activity against 4-AP induced seizure suggests that it may
likely not be interacting with K? channel. The results
clearly demonstrated that compound 30 successfully
inhibited seizures induced by MES and various chemo-
convulsants such as PTZ, 3-MPA and TSC. Thus, com-
pound 30 appeared as a broad spectrum anticonvulsant
agent.
A number of literature reports revealed that epileptic
patients are at greater risks of having depression and often
require treatment with antidepressants (Kanner et al.
2012).Therefore, antidepressant potential of compound 30
was studied. In FST test animals treated with compound 30
and fluoxetine displayed a noteworthy (p \ 0.0001) atten-
uation in immobility time as compared to control group
(Fig. 2). Moreover, compound 30 at the dose of 30 mg/kg
significantly (p \ 0.001) reduced the immobility time in
123

Design and synthesis of some new 1-phenyl-3/4-[4-(aryl/heteroaryl/alkyl-piperazine1-yl)-phenyl-ureas…
Table 9 Anticonvulsant activity of compound 30 in 4-Aminopyridine (4-AP) induced convulsions in mice
Test compound with dose (mg/kg)
Test time (h)
Tonic-seizure (%)
Clonic-seizure (%)
Mortality (%)
Control (1 % gum acacia in saline)
Phenobarbital (30 mg/kg)
0.5
0.5
0.5
0.5
0.5
100
0
100
10
100
0
Compound 30 (30 mg/kg)
Compound 30 (100 mg/kg)
Compound 30 (300 mg/kg)
100
60
80
100
100
100
50
60
40
Table 10 Hematological parameters after oral administration of compound 30 for 14 days in rats
Hematological parameters with units
Control (1 % Gum acacia in normal saline, p.o.), SD
Compound 30 (100 mg/kg, p.o), SD
Hb (g/dl)
TLC (103/ll)
14.7 1.66
11.7 0.98
15.7 3.53
79.3 7.01
3.3 1.52
2.6 0.57
0
13.3 1.93
11.5 0.75
15.3 3.05
79.0 2.64
3.6 1.10
2.0 1.00
0
Neutrophils (%)
Lymphocytes (%)
Eosinophils (%)
Monocytes (%)
Basophils (%)
RBC (106/ll)
7.40 0.24
960 5.85
7.68 0.43
984 13.5
Platelet count (103/ll)
Table 11 Biochemical estimations after oral administration of compound 30 for 14 days in rats
Biochemical parameter (liver and
kidney) with units
Control (1 % Gum acacia in normal
saline, p.o.), SD
Compound 30 (30 mg/kg, p.o.),
SD
SGOT (U/l)
61.7 3.29
65.1 4.25
158.1 6.85
0.24 0.04
7.3 0.40
59.8 4.15
67.0 2.11
158.9 3.18
0.26 0.04
7.0 0.42
SGPT (U/l)
Alkaline phosphatase (U/l)
Total bilirubin (mg/dl)
Total protein (g/dl)
Blood urea (mg/dl)
Creatinine (mg/dl)
Uric acid (mg/dl)
Calcium (mg/dl)
Phosphorus (mg/dl)
Na? (mEq/l)
34.9 2.90
0.83 0.08
6.0 1.37
34.4 3.13
0.8 0.09
6.05 1.14
11.8 0.66
6.3 0.53
11.9 1.27
6.5 0.54
146.2 3.90
4.7 0.54
144.8 2.0
4.4 0.34
K? (mEq/l)
Cl- (mEq/l)
104.2 4.89
105.8 1.83
mice as compared to fluoxetine. Thus, the results suggest
that compound 30 has modified the depressive symptoms in
mice better than fluoxetine in FST. Similarly, in TST test
compound 30 at the dose of 30 mg/kg significantly
(p \ 0.001) reduced the time duration of immobility
behavior as compared to fluoxetine at the dose of 50 mg/kg
(Fig. 3). This study also affirmed that compound 30 has an
antidepressant-like effect on mice.
The notion that chemicals can cause toxicity is not new.
More than a 1000 drugs of the modern pharmacopoeia can
cause liver damage with different clinical presentations
(Larrey 2000). Therefore, it is mandatory to evaluate tox-
icity as well as safety profile of newer chemical agent
before its further development for therapeutic use. Toxi-
cological studies results have shown that the hematological
values of the animals treated with compound 30 were not
123

C. Mishra et al.
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(Table 10). Moreover, treatment with compound 30 did not
showed any alteration in liver function and renal function
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pound 30 does not induced any pathological changes in
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Conclusion
We have successfully accomplished the design and syn-
thesis of new 1-phenyl-3/4-[4-(aryl/heteroaryl/alkyl-piper-
azine1-yl)-phenyl-urea derivatives and evaluated for their
anticonvulsant activity in vivo model of epilepsy. Studies
have shown that compound 30 was found to be the most
active compound in this series and exhibited broad spec-
trum anticonvulsant activity in various seizure models.
Moreover, compound 30 displayed good antidepressant
activity in FST and TST. Toxicological studies revealed
that compound 30 did not show significant toxicity after
administration for 14 days (sub-acute toxicity study) in
rats. From these studies it may be concluded that com-
pound 30 has potential to overcome seizures episodes as
well as depressive symptoms.
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Acknowledgments The author C.B Mishra is thankful to the
University Grants Commission (UGC), Delhi, India for award of Dr.
D.S. Kothari post-doctoral fellowship, Shikha Kumari is thankful to
UGC for financial support and Manisha Tiwari thanks the University
of Delhi, Delhi, India for providing financial assistance for this
research work. University Science Instrumentation Center (USIC),
University of Delhi is acknowledged for giving NMR spectral data of
the synthesized compounds.
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