Synthesis and pharmacological evaluation of new arylpiperazines N-{4-[4-(aryl) piperazine-1-yl]-phenyl}-amine derivatives: Putative role of 5-HT1A receptors

Article abstract of DOI:10.1016/j.bmc.2009.01.043

In an attempt to design novel 5-HT_1A agonists/partial agonists, based on an arylpiperazine nucleus, a series of N-{4-[4-(aryl)piperazine-1-yl]-phenyl}-amine derivatives were synthesized and biologically tested. The anxiolytic effect of the comp

Full text of DOI:10.1016/j.bmc.2009.01.043

Bioorganic & Medicinal Chemistry 17 (2009) 1890–1897
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Bioorganic & Medicinal Chemistry
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Synthesis and pharmacological evaluation of new arylpiperazines
N-{4-[4-(aryl) piperazine-1-yl]-phenyl}-amine derivatives:
Putative role of 5-HT1A receptors
Manisha Khatri a, Santosh Kumar Rai a, Sameena Alam a, Anjana Vij b, Manisha Tiwari a,
*
a Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110 007, India
b Department of Neurochemistry and Haematology, Defence Institute of Physiology and Allied Sciences, Delhi 110 054, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In an attempt to design novel 5-HT1A agonists/partial agonists, based on an arylpiperazine nucleus, a ser-
ies of N-{4-[4-(aryl)piperazine-1-yl]-phenyl}-amine derivatives were synthesized and biologically tested.
The anxiolytic effect of the compounds was investigated employing the Elevated plus Maze (EPM) task.
On the basis of in vivo functional test, compound 1c (3 mg/kg) and 4c (3 mg/kg) induced significant incre-
ments in open arm entries and time on EPM as compared to Buspirone. The anxiolytic effects of com-
pounds 1c and 4c were effectively antagonized by WAY-100635, a 5-HT1A receptor antagonist (0.5 mg/
kg). Furthermore, we have also evaluated the concentration of 5-HT in the brain tissue using HPLC with
fluorescent detection. Our result showed that serotonin levels were significantly decreased by ꢀ38%
(p < 0.001) and ꢀ32% (p < 0.001) after acute administration of compounds 1c and 4c, respectively. These
findings suggest that the anxiolytic like activity of these new arylpiperazines is mediated via 5-HT1A
receptors in the brain.
Received 17 December 2008
Revised 20 January 2009
Accepted 21 January 2009
Available online 24 January 2009
Keywords:
Serotonin receptors
5-HT1A Receptor agonist/antagonist
Elevated plus Maze
Anxiolytic like activity
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
still a topical subject of investigations and lies within the area of
our interest.
Anxiety is a common disorder affecting one eighth of the total
population worldwide and has become an important area of re-
search interest in psychopharmacology.1 Now a days with increas-
ing competition anxiety disorders have already become one of the
most wide spread psychiatric diseases. Currently many reports
suggest that the occurrence of anxiety is associated with the dys-
function of central monoamine neurotransmitter serotonin.
The serotonergic system has been consistently implicated in the
pathophysiology of a number of psychiatric disorders including
depression and anxiety.2 5-HT (serotonin) is found throughout
the CNS in high levels and participates in modulating mood.
According to the classic serotonin hypothesis, anxiety is usually
associated with increased endogenous 5-HT, and anxiolytics tend
to decrease endogenous 5-HT.3 Among the 13 different serotonin
receptors belonging to ‘G’ protein coupled receptor superfamily,4
the 5-HT1A and 5-HT2A subtypes are most frequently considered
to be the targets for anxiolytic and antidepressant drugs.5 Indeed,
5-HT1A agonists and partial agonists (e.g., Buspirone (Fig. 1) and
tandospirone)6 demonstrate clinical effectiveness in the treatment
of either disorder. However, development of agents of this type is
Several structurally different compounds are known to bind 5-
HT1A R sites. Among these arylpiperazine derivatives represent one
of the most important classes of 5-HT1A R ligands.7 Numerous stud-
ies have indicated that even minor modifications in the chemical
structure of arylpiperazines, strongly affect the affinity and selectiv-
ity for the 5-HT1A receptor sites8 and their effect on central nervous
system.9 The significance of the respective parts of arylpiperazines
for 5-HT1A affinity, intrinsic activity and selectivity has been the sub-
ject of many structure–activity relationship studies.10
While searching for new agents for a possible treatment of anxi-
ety and depressive disorder, we designed a novel class of arylpiper-
* Corresponding author. Tel.: +91 11 27666272; fax: +91 11 27666248.
E-mail addresses: manisha_07@hotmail.com, skrai7@gmail.com (M. Tiwari).
Figure 1. Structure of 5-HT1A partial agonist Buspirone and synthesized arylpip-
erazine derivatives 1c and 4c.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.043

M. Khatri et al. / Bioorg. Med. Chem. 17 (2009) 1890–1897
1891
azine derivatives containing different N-4 terminal amide and ester
fragments. Further modifications involved diversifying the nature of
the N-aryl substituent and the length and nature of an alkyl chain
connecting the amide/ester fragment to the basic nitrogen of aryl-
piperazine. In the present paper, we described the synthesis of 12
compounds, the derivatives of N-{4-[4-(aryl) piperazine-1-yl]-phe-
nyl}-amine and tested them for their anxiolytic like activity on Ele-
vated plus maze in a rat model. On the basis of obtained results the
two most promising compounds 1c (Fig. 1) and 4c (Fig. 1) were fur-
ther tested for the activity of biochemical markers for anxiety glyox-
alase1 and glutathione reductase. In addition, it was of interest to
investigate the involvement of 5-HT receptor system through co-
administration of the 5-HT1A receptor antagonist WAY-100635.
We have also investigated the effects of these synthesized com-
pounds on serotonin levels in brain homogenates, using reverse
phase high performance liquid chromatography (HPLC) with fluo-
rescence detection The aim is to determine the neurochemical
mechanisms of the antianxiety activity of these derivatives.
raised platform) that can induce anxiety in humans.11 An anxio-
lytic agent increases the frequency of entries into the open arms
and increases the time spent in open arms of EPM. According to
the main goal of our work, functional profile of all the derivatives
was determined in vivo; to find out whether slight changes in
the structure of the terminal amide/ester fragment and aryl frag-
ment influence their anxiolytic activity.
The effects of single administration of all the 12 aryl piperazine
derivatives, Diazepam, Buspirone and vehicle on spent time and the
number of entries into the open arms of EPM are shown in Table 1.
One way analysis of variance revealed a significant increase in
percentage time spent on the open arms after administration of
both positive controls Diazepam and Buspirone as compared to
control. The compounds were tested on EPM at different doses
(data not shown) and the best results were found at the dose of
3 mg/kg. The majority of tested compounds produced a significant
anxiolytic action displayed as a considerably increased number of
open arm entries. Among all the 12 compounds, 1c (3 mg/kg bwt)
prolonged the percentage of entries in open arms with ꢀ82%
(p < 0.001) and 4c (3 mg/kg bwt) with ꢀ60% (p < 0.001) as com-
pared to control. These results are found to be comparable with
Buspirone. The total number of arm entries was not significantly
different for all treatment groups.
2. Results and discussion
2.1. Synthesis
The results presented in Table 1 have conclusively proven that
the derivatives with terminal ester fragment significantly in-
creased the entry as well as time spent in open arms (1c to 4c)
than the corresponding amide fragment (1d to 4e). Within a set
of respective structural analogs, unsubstituted phenyl piperazines
displayed good anxiolytic activity. Introduction of electron with-
drawing groups such as –Cl introduced at the ortho position into
the phenyl piperazine moiety decreases the entry as well as time
spent in open arms (2c, 2d, 2e). The same effect was observed with
the introduction of linker alkyl between phenyl ring and pipera-
zine (3c, 3d, 3e). While methoxy group (–OCH3) introduced at
the ortho position of phenyl piperazines showed anxiolytic activity
(4c, 4d, 4e) comparable to unsubstitued phenyl piperazines (1c,
1d, 1e), irrespective of ester or amide terminal fragment. Com-
pound 1c containing unsubstituted phenyl ring and 4c containing
an o-OCH3 substituent turned out to be the most active in behav-
ioral test. Hence, on the basis of results obtained from elevated
plus maze, compounds 1c and 4c were analyzed further for their
effect on biomarkers of anxiety.
The synthetic methodology employed to develop target com-
pounds is summarized in Scheme 1. 1-Fluoro4-nitro benzene was
reacted with N-arylpiperazine in dry DMSO to yield 1-(4-nitro phe-
nyl)-4-arylpiperazine (1a to 4a). The nitro group was reduced in
the presence of Sn/HCl to afford the aniline, 4-[4-(aryl)-pipera-
zine-1-yl]-phenyl amine (1b to 4b). Then condensation of amine
with acetic anhydride in methylene chloride gave amide with an
approximate yield of ꢀ75% (1d to 4d). Alternatively, ethyl chloro
formate was coupled with amine to yield the desired compound
N-[4-(4-aryl-piperazine-1-yl)-phenyl]carbamic acid ethyl ester
(1c to 4c) in the presence of tri ethyl amine with an approximate
yield of ꢀ80%. Additionally, coupling of amine (1b to 4b) with 2-
(6-chloro)-nicotinic acid in acetonitrile at room temperature using
the coupling agent, EDC/HCl in the presence of 1-hydroxybenzotri-
azole hydrate (HOBt) gave 6-chloro-N-{4-[4-(2-aryl)-piperazin-1-
yl]-phenyl}-nicotinamide (1e to 4e). All the compounds were fully
characterized by IR, 1H NMR and mass spectroscopy.
2.2. Effect of arylpiperazine derivatives on the rat behavior in
elevated plus maze test
2.3. Effect of arylpiperazine derivatives on glyoxalase1 and
glutathione reductase activity
The elevated plus maze (EPM) is considered to be an etiologi-
cally valid animal model of anxiety because it uses natural stimuli,
(fear of novel space and fear of balancing on a relatively narrow
Two enzymes, glyoxalase 1 (Glo1) and glutathione reductase
(GSHRd) regulate anxiety12 and are considered as biomarkers for
anxiety. Glo1 levels are found to be increased in anxiogenic condi-
tions and low in anxiolytic conditions.12 GSHRd is functionally re-
lated to Glo1 because reduced glutathione (GSH) is the cofactor for
the enzymatic reaction that is catalyzed by Glo1 and levels of GSH
are maintained by GSHRd.13 Hovatta et al.12 and Kromer et al.14
have shown that there is an increased neuronal damage by methyl
glyoxal (di carbonyl) glycation in anxiety.
In order to confirm the anxiolytic activities of compounds 1c
and 4c, we tested the levels of these enzymes immediately after
EPM experiment. Our results showed increased glyoxalase levels,
induced by anxiety, were significantly decreased by both the com-
pounds (Fig. 2A and B). Glo1 and GSHRd activity was found to be
decreased by ꢀ20% (p < 0.05) and ꢀ15% (p < 0.05), respectively,
with compound 1c and by ꢀ17% (p < 0.05) and ꢀ12% (p < 0.05),
respectively, with compound 4c. Hence, our results further confirm
that both these compounds are anxiolytic in nature, as the in-
creased level of both the enzymes were significantly decreased
after administration of compounds 1c and 4c.
Scheme 1. Reagents and conditions: (a) DMSO,rt,1 h (b) Sn/HCl, 60–70 °C, 3–4 h (c)
ethyl chloro formate (ClCOOC2H5), DCM, Et3 N, 2 h (d) acetic an hydride, DCM, Et3 N,
2 h (e) 2-(6-chloro) nicotinic acid, EDC/HCl, HOBt, acetonitrile, 3–4 h.

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M. Khatri et al. / Bioorg. Med. Chem. 17 (2009) 1890–1897
Table 1
Effects of aryl piperazines (1c to 4e) on the relevant anxiolytic parameters in the elevated plus maze test
Compound
Spent time into open
arms
Number of entries into open
arms
Number of entries into closed
arms
Total number of
entries
% Number of entries into open
arms
1c
2c
3c
4c
1d
2d
3d
4d
1e
2e
3e
4e
Bus
Dzp
Veh
87.6 21.40*
58.98 23.97
53.94 19.26
84.36 23.68*
62.67 15.24
57.3 20.28
53.67 18.47
58.77 21.26
56.94 24.40
53.67 18.68
50.58 14.86
54.69 17.28
85.56 26.98*
105.36 22.46*
42.84 17.51
6.29 2.4
9.19 4.58
9.75 2.98
9.91 3.65
11.80 3.57
10.08 4.2
12.30 3.4
10.26 3.87
9.01 2.86
11.94 4.12
9.80 3.18
11.17 3.65
10.93 3.87
8.91 4.20
10.13 3.76
10.23 3.40
15.48 5.28
13.98 2.86
13.85 3.92
18.21 4.8
14.28 5.24
16.82 4.8
13.86 4.27
15.45 3.45
16.46 4.28
12.98 4.19
14.48 5.21
15.26 4.89
14.26 5.35
18.47 3.89
13.26 4.2
40.64 8.01*
30.27 13.45
28.49 12.68
35.23 16.27*
29.42 9.89
26.89 13.67
25.98 10.2
28.74 11.12
27.48 12.26
24.56 8.79
22.90 11.28
28.42 12.82
37.52 12.38*
45.16 14.7*
22.86 10.28
4.23 1.32
3.94 1.41
6.41 2.12
4.20 1.59
4.52 1.68
3.60 1.10
4.44 1.01
4.52 0.98
3.18 1.25
3.31 1.68
4.33 1.27
5.35 1.42
8.34 1.56
3.03 1.20
Data represent the mean SEM; n = 8. Bus, Buspirone; Dzp, Diazepam; Veh, Tween 80 (1%), DMSO (1%).
*p < 0.001 compared with vehicle.
2.4. 5-HT1A Antagonism study using EPM test
of Buspirone and compounds 1c and 4c. In the case of compounds
1c and 4c the time spent in the open arms was decreased by ꢀ35%
(p < 0.001) and ꢀ20% (p < 0.01), respectively. Similar effects were
observed for the number of entries in open arms. An intraperito-
neal injection of WAY-100635 (0.5 mg/kg) completely blocked
the effects of compound 1c (3 mg/kg).
The anxiolytic effects of compounds 1c and 4c administration
were shown to be attributable to actions on 5-HT1A receptors, be-
cause they were antagonized by the 5-HT1A receptor antagonist
WAY-100635. These results suggest the possible involvement of
the 5-HT1A receptor complex in the mechanism of action for these
arylpiperazine derivatives.
Activation of pre-synaptic receptors by a 5-HT1A agonist re-
duces the firing rate of 5-HTergic neurones, leading to suppression
of 5-HT synthesis, turnover and release in the diverse projection
areas.15 Forster et al. have reported that WAY-100635 is a highly
selective 5-HT1A receptor antagonist that reverses the effects of
5-HT1A R agonist induced behavioral 5-HT syndrome in rodents.16
The aim of this study was to determine whether any of the effects
of Buspirone and compounds 1c and 4c on the elevated plus-maze
were also reversed by WAY-100635.
After administration of Buspirone as well as both the com-
pounds 1c and 4c, a significant increase in percentage time spent
in the open arms compared to the control group has been noticed,
while WAY-100635 alone was not significantly different with re-
spect to control. (Fig. 3A and B). The percentage time spent in
the open arms was significantly decreased, when WAY-100635
was injected intraperitoneally (ip) 15 min before administration
2.5. Tissue concentration of 5-HT in the brain
In order to determine the neurochemical basis of anxiety, we
have evaluated the extracellular serotonin (5-HT) levels in brain
extracts of drug administered rats using HPLC with fluorescent
detection. The neurotransmitter 5-HT plays a crucial role in anxiety
related behavior in animals and humans.17 Drugs acting at various
serotonergic receptors and decreasing or increasing the activity of
the central serotonergic system have ‘anxiolytic’ or ‘anxiogenic’ ef-
fects in animals, respectively.18
A representative chromatogram of 5-HT (serotonin) is shown in
Figure 4A. The retention time obtained for 5-HT was 6.37 min. The
peak identical to 5-HT in the chromatogram obtained from the
brain extract of the drug administered rat was attributed on the
basis of retention time. The difference between retention time for
brain samples and that of authentic 5-HT was less than ꢀ2%.
Figure 4B shows a typical overlay of chromatograms obtained from
brain homogenate of rats administered with vehicle and com-
pounds 1c and 4c. It is evident from Figure 4B that compounds
1c and 4c decreased the extracellular serotonin levels as compared
to control. It has been shown that exposure of animals to an animal
test of anxiety, as the elevated plus maze test, causes an increase in
5-HT release.19 This rise in extracellular 5-HT could be attenuated
by administration of anxiolytic drugs,20 indicating a central inter-
action of serotonergic transmission system. Our results have
shown that the extracellular levels of 5-HT were significantly de-
creased by ꢀ38% (p < 0.001) and ꢀ32% (p < 0.001) following
administration of compounds 1c (3 mg/kg) and 4c (3 mg/kg),
respectively, as compared to control (Fig. 4C). These results also
confirmed that compounds 1c and 4c have anxiolytic properties.
This decrease in the extracellular 5-HT was also comparable with
Buspirone.
Figure 2. Effect of Buspirone, compounds 1c and 4c on glyoxalase activity (A) and
glutathione reductase activity (B). Results are expressed as mean S.E.M, n = 6 each
group *p < 0.05 compared with vehicle.

M. Khatri et al. / Bioorg. Med. Chem. 17 (2009) 1890–1897
1893
Figure 3. Influence of WAY-100635 on the anxiolytic effect of Buspirone and compounds 1c and 4c, expressed by the time spent in open arms (A), and the % number of open
arm entries (B) in the elevated plus maze test. Results are expressed as mean S.E.M. n = 8, ***p < 0.001 compared with Bus, ***p < 0.001 compared with 1c, ++p < 0.01 compared
with 4c.
Additionally, we have also analyzed the extracellular serotonin
levels after co-administration of 5-HT1A receptor antagonist viz
WAY-100635. When rats were administered alone with WAY-
100635, the serotonin levels were increased by ꢀ28% (p < 0.01)
as compared to control (Fig. 4D). The increased levels of extracellu-
lar serotonin were significantly brought down by ꢀ30% (p < 0.001)
and ꢀ24% (p < 0.01) by compounds 1c and 4c with respect to WAY-
100635 treated animals. These results suggest the involvement of
5-HT receptors, as the extracellular levels of 5-HT was significantly
decreased after administration of compounds 1c and 4c. These re-
sults were also consistent with our findings of behavioral test data
that compounds 1c and 4c are good anxiolytic.
for anxiety, glyoxalase1 and glutathione reductase and found to
decrease the levels of these two enzymes further confirming their
anxiolytic activity. The study on the elevated plus maze using the
antagonist WAY-100635 demonstrates that the anxiolytic effect
of compounds 1c and 4c is mediated via 5-HT1A receptor system.
In addition to this, the extracellular serotonin levels were also
found to be decreased after administration of these two com-
pounds indicating that these compounds exhibit anxiolytic activity
mediated by the serotonergic system. The effects observed on brain
extracellular serotonin levels after co administration of compounds
1c and 4c with 5-HT1A receptor antagonist WAY-100635, were also
found to be consistent with our behavioral antagonism test data
tentatively, confirming the involvement of 5-HT1A receptors. The
results discussed above are in line with the general view on the
serotonin receptor affinity of arylpiperazine derivatives; however,
additional information related to the activity and receptor specific-
ity of these two compounds would be helpful in the elucidation of
the mechanism of their anti anxiety activity, which will be re-
ported at a later date.
3. Conclusion
Here we report the synthesis, structure–activity studies and dis-
cussion of pharmacological results of a series of 12 new derivatives
of arylpiperazine, N-{4-[4-(aryl)-piperazine-1-yl]-phenyl}-amine.
All the compounds were tested in the behavioral test-elevated plus
maze and the differences in anxiolytic activity were explained on
the basis of the substituents used on a phenyl piperazine fragment
and the N-4 terminal fragment. Compound 1c having an unsubsti-
tuted phenyl piperazine and 4c having o-methoxy substituted phe-
nyl piperazine were found to be the most active in the EPM test.
Both compounds had ester functionality at their terminal N-4
fragment.
4. Experimental
4.1. General
All organic solvents and common reagents were procured from
the Merck India Ltd. All the arylpiperazines were procured from Al-
drich Chemical Company, Inc. USA. TLC was carried out on com-
mercially available flexible TLC silica gel (silica gel 60 F254)
Since compounds 1c and 4c were found to be good anxiolytics;
they were further examined for the activity of biochemical markers

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M. Khatri et al. / Bioorg. Med. Chem. 17 (2009) 1890–1897
Figure 4. Chromatogram of serotonin standard (A) and an overlay chromatogram of brain extract of Vehicle, compounds 1c and 4c administered rats (B). Effect of Buspirone,
compounds 1c and 4c on serotonin concentration in brain ***p < 0.001, **p < 0.01 compared with Veh control (C) and influence of WAY-100635 on the effect of Buspirone,
compounds 1c and 4c on serotonin concentration in brain (D). Results are expressed as mean S.E.M., n = 6, ***p < 0.001 compared with Bus, ***p < 0.001 compared with 1c,
++p < 0.01 compared with 4c.
plates (E Merck, Germany). The purity of all organic compounds
was confirmed by TLC, 1H NMR, IR, and mass spectroscopy. 1H
NMR spectra were recorded in Bruker Spectrospin Avance 300
instrument operating at 300 MHz in CDCl3 or DMSO using TMS
as an internal standard. The chemical shifts are reported in parts
per million (d) downfield from TMS and coupling constants are re-
ported in Hertz (Hz). IR spectra (KBr) were recorded on a Perkin–
Elmer BX FT-IR instrument. Melting points were determined on a
Buchi melting point B-450 instrument. Mass spectra were recorded
on a Qstar (Applied biosystem) ESI-MS mass spectrometer. Ele-
mental analysis was performed on Heraeus CHN rapid analyzer.
2829.85 (C–H str), 828.10 (C–N str); 798 (C–Cl str); 1H NMR (CDCl3,
300 MHz): d 3.42 (s, 4H, piperazine), 3.58–3.61 (d, 4H, piperazine),
6.99–8.2 (m, 8H, Ar–H); LC–MS: m/e 317(M+); Anal. Calcd for
C16H16ClN3O2: C, 62.15; H, 6.37; N, 12.08. Found: C, 61.98; H,
6.39; N, 11.98.
4.2.3. 1-(4-Nitro phenyl)-4-phenethyl-piperazine (3a)
Yield: 80%; mp: 196–198 °C; Rf = 0.77 (SiO2, CHCl3/CH3OH = 98/
02); IR (KBr)
m
cmÀ1: 1332 (NO2 str, sym), 1596.11 (–NO2 str,
asym), 2823.85 (C–H str), 836.30 (C–N str); 1H NMR (CDCl3,
300 MHz): d 2.52–2.59 (d, 2H, Ph–CH2, J = 8.0 Hz), 2.79–2.81 (t,
2H, N–CH2, J = 8.1 Hz), 3.23 (s, 4H, piperazine), 3.58 (s, 4H, pipera-
zine), 6.8–8.15 (m, 9H, Ar–H); LC–MS: m/e 311(M+); Anal. Calcd for
C18H21N3O2: C, 69.43; H, 7.40; N, 13.41. Found: C, 69.28; H, 7.35; N,
13.36.
4.2. General procedure for the synthesis of compounds 1a, 2a,
3a, 4a
A solution of 1-fluoro4-nitro benzene (2.120 ml, 20 mM) and N-
arylpiperazine (20 mM) in 40 ml of dimethylsulfoxide was stirred
at room temperature for 2 h. The resulting mixture was then
washed with water and extracted with chloroform. The organic
layer was dried over anhydrous sodium sulfate and concentrated
on a rotary evaporator to obtain the product.
4.2.4. 1-(2-Methoxy-phenyl)-4-(nitro phenyl)-piperazine (4a)
Yield: 85%; mp: 210–212 °C; Rf = 0.77 (SiO2, CHCl3/CH3OH = 98/
02); IR (KBr)
m
cmÀ1: 1329.20 (NO2 str, sym), 1596 (–NO2 str,
asym), 2834.85 (C–H str), 830.30 (C–N str); 1H NMR (CDCl3,
300 MHz): d 3.38–3.40 (d, 4H, piperazine), 3.59 (s, 4H, piperazine),
3.81 (s, 3H,OCH3), 6.86–8.1 (m, 8H, Ar–H); LC–MS: m/e 313(M+);
Anal. Calcd for C17H19N3O3: C, 66.45; H, 7.34; N, 12.24. Found: C,
66.35; H, 7.28; N, 12.16.
4.2.1. 1-(4-Nitro phenyl)-4-phenyl-piperazine (1a)
Yield: 80%; mp: 174–176 °C; Rf = 0.77 (SiO2, CHCl3/CH3OH = 98/
02); IR (KBr)
m
cmÀ1: 1328.20 (–NO2 str, sym), 1590.51 (–NO2 str,
asym), 2831.85 (C–H str), 830.30 (C–N str); 1H NMR (CDCl3,
300 MHz): d 3.36 (s, 4H, piperazine), 3.58–3.59 (d, 4H, piperazine),
6.86–8.1 (m, 9H, Ar–H); LC–MS: m/e 283 (M+), 284 (M++1); Anal.
Calcd for C16H17N3O2: C, 68.98; H, 7.40; N, 13.41. Found: C,
70.12; H, 7.36; N, 13.27.
4.3. General procedure for the synthesis of compounds 1b, 2b,
3b, 4b
To a solution of 1-(4-nitro phenyl)-4-arylpiperazine (10 mM) in
20 ml of ethyl acetate was added tin (1.780 g, 15 mM) with 15 ml
of concd HCl. The reaction mixture was refluxed for 2 h. After cool-
ing the reaction mixture was alkalized by 10% NaOH and extracted
with ethyl acetate. The organic layer was dried over anhydrous so-
dium sulfate and concentrated on rotary evaporator.
4.2.2. 1-(2-Chloro-phenyl)-4-(nitro phenyl)-piperazine (2a)
Yield: 78%; mp: 204–206 °C; Rf = 0.77 (SiO2, CHCl3/CH3OH = 98/
02); IR (KBr)
m
cmÀ1: 1334 (NO2 str, sym), 1579 (–NO2 str, asym),

M. Khatri et al. / Bioorg. Med. Chem. 17 (2009) 1890–1897
1895
4.3.1. 4-(4-Phenyl piperazine-1-yl)-phenylamine (1b)
300 MHz): d 1.18–1.25 (q, 1 Â 3H, CH2–CH3, J = 7.2 Hz), 3.14–3.15
(d, 4H, piperazine, J = 2.7), 3.23–3.24 (d, 4H, piperazine, J = 3.6),
4.10–4.17 (q, 2H, –COO–CH2, J = 6.9 Hz), 6.38–7.32 (m, 9H, Ar–H,
N–H); LC–MS: m/e 359(M+); Anal. Calcd for C19H22ClN3O2: C,
63.42; H, 6.16; N, 11.68. Found: C, 63.48; H, 6.29; N, 11.82.
Yield: 80%; mp: 128–130 °C; Rf = 0.77 (SiO2, CHCl3/CH3OH = 98/
02); IR (KBr)
m
cmÀ1: 3359.52 (N–H str), 1599.68 (N–H def),
1231.39 (C–N str); 1H NMR (CDCl3, 300 MHz): d; 3.17–3.20 (m,
4H, piperazine), 3.32–3.35 (m, 4H, piperazine), 3.71 (s, 2H, NH2),
6.66–7.31 (m, 9H, Ar–H); LC–MS: m/e 253 (M+); Anal. Calcd for
C16H19N3: C, 75.85; H, 7.56; N, 16.59. Found: C, 75.68; H, 7.62; N,
16.70.
4.4.3. N-[4-(4-Phenethyl-piperazin-1-yl)-phenyl]-carbamic acid
ethyl ester (3c)
Colourless solid. Yield: 80%; mp: 210–212 °C; Rf = 0.69 (SiO2,
4.3.2. 4-[4-(2-Chloro phenyl)-piperazine-1-yl]-phenylamine
(2b)
CHCl3/CH3OH = 98/02); IR (KBr) m
cmÀ1: 3313 (N–H str), 1689
(C@O str), 1520 (N–H def), 1314 (C–N str); 1H NMR (CDCl3,
300 MHz): d 1.25–1.29 (t, 1 Â 3H, CH2–CH3, J = 6.6 Hz), 2.55–2.62
(d, 2H, Ph–CH2, J = 8.1 Hz), 2.79–2.81 (t, 2H, N–CH2, J = 8.1 Hz),
3.09 (s, 4H, piperazine), 3.40 (s, 4H, piperazine), 4.10–4.17 (q, 2H,
–COO–CH2, J = 6.9 Hz), 6.89–7.33 (m, 10H, Ar–H, N–H); LC–MS:
m/e 353(M+); Anal. Calcd for C21H27N3O2: C, 71.36; H, 7.70; N,
11.89. Found: C, 71.46; H, 7.86; N, 11.82.
Yield: 78%; mp: 156–158 °C; Rf = 0.77 (SiO2, CHCl3/CH3OH = 98/
02); IR (KBr)
m
cmÀ1: 3345.52 (N–H str), 1589.86 (N–H def),
1223.39 (C–N str), 798 (C–Cl str); 1H NMR (CDCl3, 300 MHz): d
3.18–3.20 (m, 4H, piperazine), 3.32–3.35 (m, 4H, piperazine), 4.1
(s, 2H, NH2), 6.56–7.25 (m, 8H, Ar–H); LC–MS: m/e 287 (M+); Anal.
Calcd for C16H18ClN3: C, 66.78; H, 6.30; N, 14.60. Found: C, 66.92;
H, 6.25; N, 14.52.
4.4.4. N-{4-[4-(2-Methoxy-phenyl)-piperazin-1-yl]-phenyl}-
carbamic acid ethyl ester (4c)
4.3.3. 4-(4-Phenyl piperazine-1-yl)-phenethylamine (3b)
Yield: 80%; mp: 149–151 °C; Rf = 0.77 (SiO2, CHCl3/CH3OH = 98/
creamish solid. Yield: 85%; mp: 230–232 °C; Rf = 0.70 (SiO2,
02); IR (KBr)
m
cmÀ1: 3345.72 (N–H str), 2831.85 (C–H str), 1587.54
CHCl3/CH3OH = 98/02); IR (KBr) m
cmÀ1; 3253 (N–H str), 1685
(N–H def), 1230.39 (C–N str); 1H NMR (CDCl3, 300 MHz): d 2.52–
2.59 (d, 2H, Ph–CH2, J = 8.0 Hz), 2.79–2.81 (t, 2H, N–CH2,
J = 8.1 Hz), 3.17(s, 4H, piperazine), 3.32–3.35 (m, 4H, piperazine),
3.79 (s, 2H, NH2), 6.66–7.31 (m, 9H, Ar–H); LC–MS: m/e 281
(M+); Anal. Calcd for C18H23N3: C, 76.83; H, 8.24; N, 14.93. Found:
C, 76.62; H, 8.45; N, 14.93.
(C@O str), 1535 (N–H def), 1316 (C–N str); 1H NMR (CDCl3,
300 MHz); d 1.18–1.25 (t, 3H, CH2–CH3, J = 6.9 Hz), 3.15 (s, 4H,
piperazine), 3.23 (s, 4H, piperazine), 3.81 (s, 3H,OCH3), 4.10–4.17
(q, 2H, –COO–CH2, J = 6.9 Hz), 6.37–7.22 (m, 9H, Ar–H, N–H); LC–
MS: m/e 355(M+); Anal. Calcd for C20H25N3O3: C, 67.58; H, 7.09;
N, 11.82. Found: C, 67.72; H, 7.19; N, 11.85.
4.3.4. 4-[4-(2-Methoxy phenyl)-piperazine-1-yl]-phenylamine
(4b)
4.5. General procedure for the synthesis of compounds 1d, 2d,
3d, 4d
Yield: 80%; mp: 165–167 °C; Rf = 0.77 (SiO2, CHCl3/CH3OH = 98/
02); IR (KBr)
m
cmÀ1: 3350.57 (N–H str), 1599.68 (N–H def),
To a solution of 4-(4-arylpiperazine-1-yl)-phenylamine (10 mM)
and triethyl amine (15 mM) in 50 ml of dry dichloro methane was
added acetic an hydride (945 ll, 10 mM) at room temperature.
The resulting mixture was stirred at room temperature for 2 h.
The mixture was then washed with 5% aq NaHCO3 followed by
water. The organic layer was dried over anhydrous sodium sulfate
and concentrated on a rotary evaporator.
1228.39 (C–N str); 1H NMR (CDCl3, 300 MHz): d; 3.19 (s, 4H, piper-
azine), 3.32–3.35 (m, 4H, piperazine), 3.81 (s, 3H, OCH3), 3.97 (s,
2H, NH2), 6.66–7.31 (m, 8H, Ar–H); LC–MS: m/e 283 (M+); Anal.
Calcd for C17H21N3O: C, 72.06; H, 7.47; N, 14.83. Found: C, 71.97;
H, 7.34; N, 14.90.
4.4. General procedure for the synthesis of compounds 1c, 2c,
3c, 4c
4.5.1. N-[4-(4-Phenyl piperazin-1-yl)-phenyl]-acetamide (1d)
Yield: 90%; mp: 188–190 °C; Rf = 0.24 (SiO2, CHCl3/CH3OH = 98/
A solution of 4-(4-aryl piperazine-1-yl)-phenylamine (10 mM)
02); IR (KBr) m
cmÀ1: 3276.83 (N–H str), 1654.53 (C@O str),
and ethylchloro formate (953
l
l, 10 mM) in 40 ml dry dichloro-
1317.59 (C–N str), 1511.62 (N–H def); 1H NMR (CDCl3,
300 MHz): d 2.15 (s, 3H, CO-CH3), 3.30–3.32 (d, 8H, piperazine),
6.89–7.41 (m, 10H, Ar–H, N–H); LC–MS: m/e 296 (M++1); Anal.
Calcd for C18H21N3O: C, 73.19; H, 7.17; N, 14.23. Found: C, 73.30;
H, 7.23; N, 14.37.
methane was stirred at room temperature for 2 h in the presence
of triethylamine (15 mM). The resulting mixture was then washed
with 5% aq NaHCO3 followed by water. The organic layer was dried
over anhydrous sodium sulfate and concentrated on a rotary
evaporator.
4.5.2. N-{4-[4-(2-Chloro-phenyl)-piperazin-1-yl]-phenyl}-
acetamide (2d)
4.4.1. N-[4-(4-Phenyl-piperazin-1-yl)-phenyl]-carbamic acid
ethyl ester (1c)
Yield: 85%; mp: 280–283 °C; Rf = 0.29 (SiO2, CHCl3/CH3OH = 98/
Yellowish solid. Yield: 80%; mp: 170–172 °C; Rf = 0.77 (SiO2,
02); IR (KBr) m
cmÀ1: 3297 (N–H str), 1655 (C@O str), 1513 (N–H
CHCl3/CH3OH = 98/02); IR (KBr)
m
cmÀ1
:
3295.38 (N–H str),
def), 1316 (C–N str); 1H NMR (CDCl3, 300 MHz): d 2.05 (s, 3H,
COCH3), 3.17(s,4H, piperazine), 3.27 (s, 4H, piperazine), 6.97–
7.51(m, 9H, Ar–H, N–H); LC–MS: m/e 329(M+); Anal. Calcd for
C18H20ClN3O: C, 65.55; H, 6.11; N, 12.74. Found: C, 65.68; H,
6.23; N, 12.87.
1691.74 (C@O str), 1526.35 (N–H def), 2827.52 (C–H str); 1H
NMR (CDCl3, 300 MHz): d 1.007–1.055 (t, 3H, CH2–CH3, J = 7.2),
2.48–2.55 (q, 2H, –COO–CH2, J = 7.2), 3.28–3.30 (d, 8H, piperazine),
6.83–7.25 (m, 10H, Ar–H, N–H); LC–MS: m/e 325 (M+), 326 (M++1);
Anal. Calcd for C19H23N3O2: C, 70.13; H, 7.12; N, 12.91. Found: C,
70.21; H, 7.24; N, 13.02.
4.5.3. N-[4-(4-Phenethyl-piperazin-1-yl)-phenyl]-acetamide
(3d)
4.4.2. N-{4-[4-(2-Chloro-phenyl)-piperazin-1-yl]-phenyl}-
carbamic acid ethyl ester (2c)
Yield: 78%; mp: 267–270 °C; Rf = 0.23 (SiO2, CHCl3/CH3OH = 98/
02); IR (KBr)
m
cmÀ1: 3297 (N–H str), 1654 (C@O str), 1524 (N–H
Colourless solid. Yield: 80%; mp: 225–227 °C; Rf = 0.73 (SiO2,
def), 1370 (C–N str); 1H NMR (CDCl3, 300 MHz): d 2.07 (s, 3H,
COCH3), 2.76–2.81 (t, 2H, Ar-CH2, J = 6.6), 3.12 (s, 2H, N–CH2),
2.62 (s, 8H, piperazine), 6.81–7.31 (m, 10H, Ar–H, N–H); LC–MS:
CHCl3/CH3OH = 98/02); IR (KBr)
m
cmÀ1: 3253 (N–H str), 1684
(C@O str), 1501 (N–H def), 1316 (C–N str); 1H NMR (CDCl3,

1896
M. Khatri et al. / Bioorg. Med. Chem. 17 (2009) 1890–1897
m/e 323(M+); Anal. Calcd for C20H25N3O: C, 74.27; H, 7.79; N, 12.99.
Found: C, 74.36; H, 7.56; N, 13.16.
piperazine), 3.33 (s, 4H, piperazine), 3.79 (s, 3H, OCH3), 6.8–8.2
(m, 9H, Ar–H, N–H), 8.39–8.42 [d, 1H(X), J = 9.1 Hz], 8.65 [s,
1H(Y)], 8.81 [s, 1H(Z)]; LC–MS: m/e 423 (M+); Anal. Calcd for
C23H23ClN4O2: C, 65.32; H, 5.48; N, 13.25. Found: C, 65.19; H,
5.32; N, 13.22.
4.5.4. N-{4-[4-(2-Methoxy-phenyl)-piperazin-1-yl]-phenyl}-
acetamide (4d)
Yield: 85%; mp: 290–292 °C; Rf = 0.32 (SiO2, CHCl3/CH3OH = 98/
02); IR (KBr)
m
cmÀ1: 3262.55 (N–H str), 1642.22 (C@O str), 1517
4.7. Animals and drug treatment
(N–H def), 1315 (C–N str); 1H NMR (CDCl3, 300 MHz): d 2.08 (s,
3H, COCH3), 3.15(s,4H, piperazine), 3.18 (s, 3H, OCH3), 3.25–3.26
(s, 4H, piperazine), 6.80–7.33(m, 9H, Ar–H, N–H); LC–MS: m/e
325(M+); Anal. Calcd for C19H23N3O2: C, 70.13; H, 7.12; N, 12.91.
Found: C, 70.25; H, 7.23; N, 12.74.
Six to eight weeks old male Wistar rats, weighing 120–150 g
each, were selected from the stock colony maintained in our ani-
mal facility with free access to food and water. Animals were main-
tained in an air-conditioned room. The room was maintained at
25 2 °C with natural daytime and no light after 1900 h, until
morning. All the experiments were performed during the light
phase according to the guidelines of the Institutional Animal Ethics
committee.
4.6. General procedure for the synthesis of compounds 1e, 2e,
3e, 4e
2-(6-Chloro)-nicotinic acid (1.575 g, 10 mM) and 1-Hydroxy
benzotriazole hydrate (HOBt) (1.621 g, 12 mM) were slurried in
dry acetonitrile (50 ml). Addition of EDC/HCl (2.108 g, 11 mM) to
this mixture at room temperature generates the HOBt ester. Then
4-(4-arylpiperazine-1-yl)-phenylamine (2.53 g, 10 mM) was added
and the mixture was stirred at room temperature for 3–4 h. The
resulting mixture was then filtered and filtrate was concentrated
on rotary evaporator. Then the compound was partitioned with
ethyl acetate and aqueous bicarbonate solution. The organic layer
was dried over anhydrous sodium sulfate and concentrated on a
rotary evaporator. Finally after evaporation of the solvent the res-
idue was purified by column chromatography and elution with
hexane/ethyl acetate (70:30).
Diazepam ampoules (10 mg/2 ml; Ranbaxy Labs, India) and
Buspirone hydrochloride (Sigma–Aldrich Co., St. Louis, MO, USA)
were used as reference drugs. Diazepam and Buspirone were di-
luted in deionized water (Millipore quality). 5-HT1A receptor
antagonist WAY-100635, (N-[2-[4-(2-methoxyphenyl)-1-piperazi-
nyl]-ethyl]-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochlo-
ride) (Sigma–Aldrich Co., St. Louis, MO, USA) was dissolved in 0.9%
NaCl solution (saline). Six different concentrations (0.25, 0.5, 1, 2, 3,
4 mg/kg) of each compound were used in the form of freshly pre-
pared suspensions in 1% tween 80 and 1% DMSO. All solutions were
prepared freshly on test days and given intraperitoneally (ip) in a
volume of 2 ml/kg body weight of rats.
The experimental animals were treated with Diazepam (1 mg/
kg, n = 8), Buspirone (5 mg/kg, n = 8) or the compounds (0.25, 0.5,
1, 2, 3, 4 mg/kg) 60 min before evaluation in the maze. The control
group was given saline with 1% tween 80 and 1% DMSO. The antag-
onist WAY-100635 (0.5 mg/kg, n = 8) was administered 15 min be-
fore the treatments.
4.6.1. 6-Chloro-N-[4-(4-phenyl-piperazin-1-yl)-phenyl]-
nicotinamide (1e)
Yield: 65%; mp: 430–432 °C; Rf = 0.56 (SiO2, CHCl3/CH3OH = 98/
02); IR (KBr)
m
cmÀ1: 3313 (N–H str), 1637 (C@O str), 1522 (N–H
def), 1324 (C–N str), 756 (C–Cl str); 1H NMR (CDCl3, 300 MHz): d
3.37 (m, 8H, piperazine), 6.92–7.55 (m, 10H, Ar–H, N–H), 8.16–
8.19 [d, 2H(X + Y)], 8.85 [s, 1H(Z)]; LC–MS: m/e 392 (M+); Anal.
Calcd for C22H21ClN4O: C, 67.26; H, 5.39; N, 14.26. Found: C,
67.37; H, 5.21; N, 14.32.
4.8. Elevated plus maze test
The elevated plus maze was comprised of two open arms (50
cm  10 cm) and two closed arms (50 cm  10 cm  30 cm).
The arms extend from a central platform (5 cm  5 cm) that was
elevated to a height of 50 cm above floor level. The experiments
were performed between 9:00 h and 15:00 h. Each rat was placed
in the central area of the maze with its head facing an open arm.
The following behavioral parameters were recorded during 5 min
exposure using FW: Camera O: Fire - iBBW 1.3 interface (Columbus
Instruments): (1) time in open arms; (2) time in closed arms; (3)
time in central area; (4) number of open arm entries; (5) number
of closed arm entries; (6) number of central area entries. Any rat,
which dropped of the plus maze, was excluded in the result.21
4.6.2. 6-Chloro-N-{4-[4-(2-chloro-phenyl)-piperazin-1-yl]-
phenyl}-nicotinamide (2e)
Yield: 59%; mp: 412–415 °C; Rf = 0.55 (SiO2, CHCl3/CH3OH = 98/
02); IR (KBr)
m
cmÀ1: 3334 (N–H str), 1623 (C@O str), 1513 (N–H
def), 1319 (C–N str); 1H NMR (CDCl3, 300 MHz): d 3.06 (s, 4H,
piperazine), 3.29 (s, 4H, piperazine), 6.9–7.8 (m, 9H, Ar–H, N–H),
8.09–8.12 [d, 1H(X), J = 8.4 Hz], 8.65 [s, 1H(Y)], 8.96 [s, 1H(Z)];
LC–MS: m/e 426 (M+); Anal. Calcd for C22H20Cl2N4O: C, 61.83; H,
4.72; N, 13.11. Found: C, 61.67; H, 4.86; N, 13.02.
4.6.3. 6-Chloro-N-[4-(4-phenethyl-piperazin-1-yl)-phenyl]-
nicotinamide (3e)
4.9. Assay of glyoxalase1 and glutathione reductase activity
Yield:57%; mp: 395–398 °C; Rf = 0.51 (SiO2, CHCl3/CH3OH = 98/
02); IR (KBr)
After the elevated plus maze experiment the Wistar rats were
dissected to remove their brain tissue, which was washed in ice
cold saline (0.85% NaCl). The extraneous material was removed.
The 20% homogenate of brain tissue in 0.1 M potassium phosphate
buffer (pH 7.4) with 0.25 M sucrose was centrifuged at 800g for
5 min at 4 °C in an IEC-20 refrigerated centrifuge (Rotar no. 894)
to separate the nuclear debris. The supernatant obtained was cen-
trifuged at 10,500g for 20 min at 4 °C to obtain the post mitochon-
drial supernatant (PMS) which was used as a source for estimation
of glutathione reductase. The PMS was centrifuged further at
36,000g for 1 h at 4 °C to obtain cytosolic fraction which was used
as a source for estimation of glyoxalase 1. The activity of glyoxalase
1 was assayed by a slight modification of the technique described
by Racker.22 Glutathione reductase activity was assayed by the
m
cmÀ1: 3280 (N–H str), 1642 (C@O str), 1534 (N–H
def), 1373 (C–N str); 1H NMR (CDCl3, 300 MHz): d 2.4–2.5 (d, 2H,
Ph–CH2), 2.71 (t, 2H, N–CH2), 3.05 (s, 4H, piperazine), 3.82 (s, 4H,
piperazine), 6.8–7.6 (m, 10H, Ar–H, N–H), 8.26 [s,
1 Â H(X) + 1 Â 1H(Y)], 8.85 [s, 1 Â 1H(Z)]; LC–MS: m/e 420 (M+);
Anal. Calcd for C24H25ClN4O: C, 68.48; H, 5.99; N, 13.31. Found:
C, 68.29; H, 6.05; N, 13.22.
4.6.4. 6-Chloro-N-{4-[4-(2-methoxy-phenyl)-piperazin-1-yl]-
phenyl}-nicotinamide (4e)
Yield: 62%; mp: 380–382 °C; Rf = 0.54 (SiO2, CHCl3/CH3OH = 98/
02); IR (KBr)
m
cmÀ1: 3305 (N–H str), 1639 (C@O str), 1525 (N–H
def), 1327 (C–N str); 1H NMR (CDCl3, 300 MHz): d 3.21 (s, 4H,

M. Khatri et al. / Bioorg. Med. Chem. 17 (2009) 1890–1897
1897
method of Carlberg and Mannervik (1975).23 The protein content
was measured by the method of Lowry et al. (1951) using bovine
serum albumin as standard.24
difference between means of two groups was obtained with one-
way analysis of variance (ANOVA), Tukey–Kramer multiple com-
parisons test, using Graph pad Prism 4.00 computer software,
p < 0.05 was considered to be statistically significant.
4.10. Determination of tissue concentration of 5-HT in brain
Acknowledgments
One hour after last administration, the experimental animals
were decapitated by cervical dislocation, and the brains were re-
moved immediately and placed on an ice-cold plate. The tissue
samples were weighed and stored at À80 °C until homogenization.
Each frozen tissue sample was homogenized by ultrasonication
The author M. Khatri thanks the Indian Council of Medical Re-
search (ICMR), India, for the award of JRF and SRF. The author
M.T. thanks the Department of Science and Technology, Govern-
ment of India, for financial support.
in 500
were kept on ice bath for 1 h and then centrifuged at 12,000g for
20 min at 4 °C. The pellets were discarded. An aliquot of 300
of supernatant was added to 150 l of solution B (containing 0.2
ll of 0.4 M perchloric acid (solution A). The homogenates
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Identification (by retention time) and measurement of the com-
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Calculation of the percentage time and number of entries on the
open arms with 95% confidence limits and comparisons of the re-
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San Diego, CA, USA). All the measurements were made in triplicate
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