Synthesis and evaluation of antidepressant-like activity of some 4-substituted 1-(2-methoxyphenyl) piperazine derivatives

Article abstract of DOI:10.1111/cbdd.12394

A series of new derivatives of N-(2-methoxyphenyl) piperazine have been synthesized for their affinity toward serotonergic receptors and for their potential antidepressant-like activity. They have been evaluated toward receptors 5-HT_1A, 5-HT₆, and 5-HT₇, as well as in vivo in the tail suspension, locomotor activity, and motor co-ordination tests. All the tested compounds proved very good affinities toward 5-HT_1A and 5-HT₇ receptors. The most promising compound was 1-[(2-chloro-6-methylphenoxy)ethoxyethyl]-4-(2-methoxyphenyl) piperazine hydrochloride, exhibiting affinity toward receptors K_i <1 nM (5-HT_1A) and K_i = 34 nM (5-HT₇). Antidepressant-like activity (tail suspension test) was observed at 2.5 mg/kg b.w. (mice, i.p.), and the effect was stronger than that observed for imipramine (5 mg/kg b.w.). Sedative activity was observed at ED₅₀ (locomotor test, mice, i.p.) = 17.5 mg/kg b.w. and neurotoxicity was observed at TD₅₀ (rotarod, mice, i.p.) = 53.2 mg/kg b.w.

Full text of DOI:10.1111/cbdd.12394

Chem Biol Drug Des 2015; 85: 326–335
Research Article
Synthesis and Evaluation of Antidepressant-like
Activity of Some 4-Substituted 1-(2-methoxyphenyl)
Piperazine Derivatives
Anna M. Waszkielewicz1,*, Karolina Pytka2, Anna
Received 7 May 2014, revised 17 June 2014 and accepted
for publication 25 June 2014
2
2
2
_
Rapacz , Elzbieta Wełna , Monika Jarzyna ,
Grzegorz Satała3, Andrzej Bojarski3, Jacek
4
5
2
_
Sapa , Paweł Zmudzki , Barbara Filipek and
Henryk Marona1
Depression is a prevalent psychiatric disorder, concerning
about 21% of human world population. It is characterized
by anhedonia or loss of interest or pleasure in normal
activity. It affects quality of life and productivity and is a
significant factor limiting length of life (1). Despite many
antidepressant drugs, still their effectiveness is unsatisfac-
tory. Among many types of depression, major depressive
disorder or unipolar depression is predominant and is a
leading indication in the interest of drug development
efforts.
1Department of Bioorganic Chemistry, Chair of Organic
Chemistry, Faculty of Pharmacy, Jagiellonian University
Medical College, Medyczna 9, 30-688 Krakow, Poland
2Department of Pharmacodynamics, Faculty of Pharmacy,
Jagiellonian University Medical College, Medyczna 9,
30-688 Krakow, Poland
3Department of Medicinal Chemistry, Institute of
Pharmacology, Polish Academy of Sciences, Smetna 12,
z
31-343 Krakow, Poland
4Department of Pharmacological Screening, Faculty of
Pharmacy, Jagiellonian University Medical College,
Medyczna 9, 30-688 Krakow, Poland
Serotonin metabotropic receptors are known to influence
mood and cognitive performance of the central nervous
system, especially 5-HT1A, 5-HT6, and 5-HT7 receptors
have been well described for their role in depression, anxi-
ety, memory deficits, as well as other disorders character-
ized by change of cholinergic neurotransmission (2).
Moreover, it has been reported that 5-HT1A antagonists
may serve as adjunctive therapies for selective serotonin
reuptake inhibitors (SSRIs), due to their potential of reduc-
ing the delay of onset of SSRIs activity. Moreover, 5-HT1A
ligands may present antidepressant activity themselves (3).
As anxiety is often concomitant to depression, the phar-
macological effect of anxiolytics may be observed also in
antidepressant-like activity in animal models.
5Department of Medicinal Chemistry, Faculty of Pharmacy,
Jagiellonian University Medical College, Medyczna 9,
30-688 Krakow, Poland
*Corresponding author: Anna M. Waszkielewicz,
awaszkie@cm-uj.krakow.pl
A series of new derivatives of N-(2-methoxyphenyl)
piperazine have been synthesized for their affinity
toward serotonergic receptors and for their potential
antidepressant-like activity. They have been evaluated
toward receptors 5-HT1A, 5-HT6, and 5-HT7, as well as
in vivo in the tail suspension, locomotor activity, and
motor co-ordination tests. All the tested compounds
proved very good affinities toward 5-HT1A and 5-HT7
receptors. The most promising compound was 1-[(2-
chloro-6-methylphenoxy)ethoxyethyl]-4-(2-methoxyphe-
nyl)piperazine hydrochloride, exhibiting affinity toward
receptors Ki <1 nM (5-HT1A) and Ki = 34 nM (5-HT7).
Antidepressant-like activity (tail suspension test)
was observed at 2.5 mg/kg b.w. (mice, i.p.), and the
effect was stronger than that observed for imipramine
(5 mg/kg b.w.). Sedative activity was observed at ED50
(locomotor test, mice, i.p.) = 17.5 mg/kg b.w. and
neurotoxicity was observed at TD50 (rotarod, mice,
i.p.) = 53.2 mg/kg b.w.
Among selective 5-HT1A ligands, one can find derivatives
of 1-alkyl-4-aryl-piperazine. Typical examples are lecozotan
(antagonist), BMY 7378, or buspirone (partial agonists)
(Figure 1) (4). Moreover, aroxyalkyl derivatives of N-(2-
methoxyphenyl)piperazine have been in the field of our
interest in the recent years (5,6). Some of them, for exam-
ple, 1-[(2,6-dimethylphenoxy)propyl]-4-(2-methoxyphenyl)
piperazine hydrochloride (Figure 1), exhibited satisfactory
affinity toward a1 versus a2-adrenoreceptors, Ki = 2.4 and
341.1 nM, respectively (6). As a continuation of our studies
on phenylpiperazine derivatives, four new substituted
phenoxypropyl derivatives of N-(2-methoxyphenyl)pipera-
zine have been synthesized for their affinity toward seroto-
nergic receptors as well as antidepressant-like activity.
Key words: (2-methoxyphenyl)piperazine, 5-HT, antidepres-
sant-like activity, chimney test, locomotor test, neurotoxicity,
rotarod, tail suspension test
326
ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12394

Activity of Piperazine Derivatives
Figure 1: Chemical structures of some
derivatives of piperazine - reference
compounds (4–6).
The choice of substituents and their positions in the phenyl
ring was based on our former experience—the position 2
and additional position 3, 5, or 6 have specifically proved to
be beneficial. Therefore, our aim was to continue research
among these derivatives, comparing them together with
other parameters such as length of the linker between phen-
oxyl and N-(2-methoxyphenyl)piperazine groups.
36 057 Hz spectral width. Standard pulse sequences from
Bruker library were used for 2D spectra. Gradient-enhanced
sequences were used for the homo- and heteronuclear 2D
experiments. All processing and analysis were performed
using BRUKER’S TOPSPIN 3.0 software suite. Results are pre-
sented in the following format: chemical shift d in ppm, mul-
tiplicity, coupling constant J in Hertz (Hz), number of
protons, and proton’s position. Multiplicities are showed as
the abbreviations: s (singlet), bs (broad singlet), d (doublet),
dd (doublet of doublets), ddd (doublet of doublets of dou-
blets), t (triplet), and m (multiplet). Elemental analyses were
performed on an Elementar Vario EL III (Elementar Analysan-
systeme, Hanau, Germany). Analyses of percentage content
of carbon, hydrogen, and nitrogen were within 0.4% of the
theoretical values. Purity was checked with use of LC-MS
system, which consisted of a Waters Acquity UPLC, cou-
pled to a Waters TQD mass spectrometer (electrospray ioni-
zation mode ESI-tandem quadrupole). All the analyses were
carried out using an Acquity UPLC BEH C18, 1.7 lm, 2.1 _
100 mm column. A flow rate of 0.3 mL/min and a gradient
of (5–95)% B over 10 min and then 100% B over 2 min
were used. Eluent A: water/0.1% HCOOH; eluent B: aceto-
nitrile/0.1% HCOOH. LC/MS data were obtained by scan-
ning the first quadrupole in 0.5 second in a mass range
from 50 to 1000 Da; eight scans were summed up to pro-
duce the final spectrum.
The linker, as in our former studies, usually had the length
of two or three methylene groups, and the bonds were
single. Use of either propyl (compounds 1-4) or ethoxyeth-
yl (compounds 5-6) linker was a modification that could
substantially change pharmacological properties of our
compounds, especially that the alkyl linker in reference
compounds has 2-4 methylene groups (Figure 1).
Methods and Materials
Chemistry
All reagents were purchased from Alfa Aesar (Karlsruhe,
Germany). Solvents were commercially available materials
of reagent grade. Melting points (mp) are uncorrected and
€
€
were determined using a Buchi SMP-20 apparatus (Buchi
Labortechnik, Flawil, Switzerland). The infrared spectra were
recorded on potassium bromide pellets using a Jasco FT/IR
1
410 spectrometer (Jasco Inc., Easton, MD, USA). H NMR
and 13C NMR spectra for compounds were recorded on a
Bruker AVANCE III 600 (Bruker, Karlsruhe, Germany) (reso-
General procedure for preparation of the tested
compounds
1
nance frequencies 600.20 MHz for H and 150.94 MHz for
13C) equipped a 5-mm probehead: PABBO with z-gradient
or TBI with XYZ gradients. The 1H spectra were recorded
with 16 scans, 1 second relaxation delay, 4 seconds acqui-
sition time, 128 kW FID size, and 16234 Hz spectral width.
0.02 mole of N-(2-methoxyphenyl)piperazine was added to a
solution of 0.02 mole of appropriately (2-chloro-5-methyl-
phenoxy)-, (2,5-dimethylphenoxy)-, (2,3,5-trimethylphenoxy)-,
or (2,4,6-trimethylphenoxy)propyl, or (2,6-dimethyl- or
(2-chloro-6-methylphenoxy)ethoxyethyl bromide in 50 mL
of toluene, and the reaction mixture was refluxed in
the presence of 0.02 mole (excess) anh. K2CO3 for 12 h.
1
The 13C spectra were recorded with WALTZ-16 H broad-
band decoupling, a few thousand scans, 2 seconds relaxa-
tion delay, 0.9 second acquisition time, 64 kW FID size, and
Chem Biol Drug Des 2015; 85: 326–335
327

Waszkielewicz et al.
Inorganic salts were filtered off from the hot mixture and
washed with hot toluene (5 mL). The solvent was distilled
from the filtrate under reduced pressure. After addition of
acetone saturated with gaseous HCl to the residue, the
mixture was refluxed and cooled. The crystals formed
were collected by filtration and dried. Recrystallization was
performed from mixture acetone/ethanol 1:1 (v/v).
1-[(2,4,6-trimethylphenoxy)propyl]-4-(2-
methoxyphenyl)piperazine hydrochloride (4)
C23H33N2O2Cl; white solid, mp 234–236 °C (base:
calcd
95–97 °C (acetone)); el. anal.
/
C62.57
/
;
62.32
found
H7.96 8.08; N6.34 6.49; IR (KBr, cmꢀ1) v: 3436, 3061, 2988,
/
/
2919, 2873, 2838, 2666, 2591, 2520, 2450, 1608, 1501,
1463, 1246, 749; 1H NMR (DMSO-d6, d ppm): 11.49 (bs,
1H, CH2-NH+); 7.05 (ddd, J = 8.1, J = 7.1, J = 1.7, 1H,
H-50); 7.00 (dd, J = 8.1, J = 1.6, 1H, H-60); 6.99 (dd,
J = 7.9, J = 1.7, 1H, H-30); 6.92 (ddd, J = 7.9, J = 7.1,
J = 1.6, 1H, H-40); 6.82 (s, 2H, H-3, H-5); 3.81 (s, 3H, Ar-
O-CH3); 3.77 (t, J = 5.9, 2H, Ar-O-CH2); 3.65–3.47 (m,
4H, N-CHH-pip(a)); 3.40–3.33 (m, 2H, -CH2-NH+); 3.28–
3.14 (m, 4H, N-CHH-pip(e)); 2.32–2.22 (m, 2H, CH2-CH2-
In case of compounds 1-4, the base product was recrys-
tallized from acetone.
1-[(2-chloro-5-methylphenoxy)propyl]-4-(2-
methoxyphenyl)piperazine hydrochloride (1)
C21H28N2O2Cl2; white solid, mp 200–202 °C (base: 83–
CH2);
2.19
(s,
6H,
2*
calcd
85 °C (acetone)); el. anal.
N6.81
/
C61.31
/
60.90; H6.86
/
;
7.16
Ar-CH3 (2,6)); 2.18 (s, 3H, Ar-CH3(4)); [M+H]+ 369.44,
found
/
6.65; IR (KBr, cmꢀ1) v: 3480, 3059, 3010, 2979,
100%.
2963, 2928, 2879, 2837, 2621, 2497, 2305, 2205, 1487,
1260, 1065, 760; 1H NMR (DMSO-d6, d ppm): 11.30 (bs,
1H, CH2-NH+); 7.29 (d, J = 8.0, 1H, Ar); 7.07–6.88 (m,
5H, Ar); 6.79 (ddd, J = 8.0, J = 1.9, J = 0.7, 1H, Ar); 4.16
(t, J = 6.2, 2H, Ar-O-CH2); 3.80 (s, 3H, Ar-O-CH3); 3.65–
3.44 (m, 4H, N-CHH-pip(a)); 3.33–3.27 (m, 2H, -CH2-
NH+); 3.27–3.09 (m, 4H, N-CHH-pip(e)); 2.30 (s, 3H,
Ar-CH3); 2.33–2.25 (m, 2H, -CH2-CH2-CH2); [M+H]+
375.36, 98.97%.
1-[(2,6-dimethylphenoxy)ethoxyethyl]-4-(2-
methoxyphenyl)piperazine hydrochloride (5)
C23H33N2O3Cl; white solid, mp 146–148 °C; IR (KBr,
cmꢀ1) v: 3501, 3444, 3015, 2978, 2952, 2925, 2625,
2491, 2296, 2200, 1609, 1453, 1262, 1205, 766; 1H
NMR (DMSO-d6, d ppm): 11.25 (bs, 1H, NH+); 7.06–6.90
(m, 7H, Ar); 3.98 (ddd, J = 6.0, J = 4.0, J = 2.0, 2H,
Ar-O-CH2); 3.91 (ddd, J = 4.6, J = 3.3, J = 2.8, 2H, -O-
CH2-CH2-NH); 3.78 (ddd, J = 6.0, J = 4.0, J = 2.0, 2H,
Ar-O-CH2-CH2-O); 3.78 (s, 3H, -O-CH3); 3.62–3.56 (m,
2H, Ar-N-CHH-(pip)a); 3.50–3.45 (m, 2H, CH2-N-CHH-
(pip)a); 3.39 (ddd, J = 4.6, J = 3.3, J = 2.8, 2H, -O-CH2-
CH2-NH); 3.32–3.24 (m, 2H, Ar-N-CHH-(pip)e); 3.17–3.09
1-[(2,5-dimethylphenoxy)propyl]-4-(2-
methoxyphenyl)piperazine hydrochloride (2)
C22H31N2O2Cl; white solid, mp 208–210 °C (base: 70–
72 °C (acetone)); IR (KBr, cmꢀ1) v: 3439, 3056, 2980,
2953, 2922, 2842, 2680, 2584, 2522, 2474, 1502, 1247,
750; 1H NMR (DMSO-d6, d ppm): 11.07 (bs, 1H, CH2-
NH+); 7.08–6.88 (m, 5H, Ar); 6.77 (d, J = 1.7, 1H, Ar);
6.66 (ddd, J = 7.5, J = 1.7, J = 0.9, 1H, Ar); 4.05 (t,
J = 6.1, 2H, Ar-O-CH2); 3.80 (s, 3H, Ar-O-CH3); 3.63–
3.47 (m, 4H, N-CHH-pip(a)); 3.34–3.27 (m, 2H, -CH2-
NH+); 3.27–3.07 (m, 4H, N-CHH-pip(e)); 2.26 (s, 3H, Ar-
CH3(5)); 2.33–2.25 (m, 2H, -CH2-CH2-CH2); 2.13 (s, 3H,
Ar-(CH3)2); [M+H]+ 355.42, 99.69%.
(m, 2H, -CH2-N-CHH-(pip)e); 2.24 (s, 6H, Ar-(CH3)2).13
C
NMR: 155.26 (C-1); 151.70 (C-20); 139.17 (C-10); 130.26
(C-2); 128.60 (C-3); 123.60 (C-50); 123.43 (C-40); 120.74
(C-4); 118.15 (C-30); 111.86 (C-4); 70.77 (Ar-O-CH2-CH2-
O); 69.74 (Ar-O-CH2-CH2-O); 64.89 (-O-CH2-CH2-NH+);
55.26 (O-CH3); 54.79 (-O-CH2-CH2-NH+); 51.64 (Ar-N-
CH2-(pip)); 46.74 (-CH2-N-CH2-(pip)); 15.84 (Ar-(CH3)2);
[M+H]+ 385.40; 99.22%.
1-[(2-chloro-6-methylphenoxy)ethoxyethyl]-4-(2-
methoxyphenyl)piperazine hydrochloride (6)
1-[(2,3,5-trimethylphenoxy)propyl]-4-(2-
methoxyphenyl)piperazine hydrochloride (3)
C22H30N2O3Cl2, white solid, mp 155–157 °C; IR (KBr,
cmꢀ1) v: 3387, 3007, 2966, 2614, 2401, 1707, 1610,
1457, 1263, 1018, 765; 1H NMR (DMSO-d6, d ppm):
11.65 (bs, 1H, NH+); 7.31 (ddd, J = 8.0, J = 1.7, J = 0.7,
1H, H-4); 7.20 (ddd, J = 7.6, J = 1.7, J = 0.8, 1H, H-6);
7.08–6.99 (m, 4H, H-5, H-30, H-50, H-60); 6.92 (ddd,
J = 8.7, J = 7.2, J = 1.6, 1H, H-40); 4.06 (t, J = 4.6, 2H,
Ar-O-CH2-); 4.00 (t, J = 5.0, 2H, -O-CH2-CH2-NH-);
3.82–3.80 (m, 2H, Ar-O-CH2-CH2-O-); 3.80 (s, 3H, -O-
CH3); 3.65–3.58 (m, 2H, Ar-N-CHH- (pip)a); 3.52–3.46 (m,
2H, CH2-N-CHH- (pip)a); 3.43–3.37 (m, 2H, -O-CH2-CH2-
NH-); 3.37–3.29 (m, 2H, Ar-N-CHH- (pip)e); 3.29–3.21
(m, 2H, -CH2-N-CHH- (pip)e); 2.30 (s, 3H, CH3-Ar
C23H33N2O2Cl; white solid, mp 224–226 °C; IR (KBr, cmꢀ1
)
v: 3427, 2976, 2359, 1610, 1485, 1263, 1213, 1022, 1455,
1307; 1H NMR (DMSO-d6, d ppm): 11.43 (bs, 1H, CH2-
NH+); 7.04 (ddd, J = 8.1, J = 7.1, J = 1.7, 1H, H-50); 7.00
(dd, J = 8.1, J = 1.6, 1H, H-60); 6.98 (dd, J = 7.8, J = 1.7,
1H, H-30); 6.92 (ddd, J = 7.8, J = 7.1, J = 1.6, 1H, H-40);
6.63 (d, J = 2.3, 1H, H-6); 6.59 (d, J = 2.3, 1H, H-4); 4.01
(t, J = 6.1, 2H, Ar-O-CH2); 3.80 (s, 3H, Ar-O-CH3);
3.64–3.46 (m, 4H, N-CHH-(pip)a); 3.42–3.26 (m, 2H,
CH2-NH+); 3.26–3.14 (m, 4H, N-CHH-(pip)e); 2.31–2.23
(m, 2H, CH2-CH2-CH2); 2.22 (s, 3H, Ar-CH3(5)); 2.17 (s,
3H, Ar-CH3(3)); 2.05 (s, 3H, Ar-CH3(2)); [M+H]+ 369.44,
99.29%.
13
(1)); C NMR: 152.60 (C-2); 151.71(C-20); 138.46 (C-10);
328
Chem Biol Drug Des 2015; 85: 326–335

Activity of Piperazine Derivatives
133.23 (C-3); 129.89 (C-4); 127.69 (C-6); 126.62
(C-1); 124.92 (C-5); 123.94 (C-50); 120.75 (C-40); 118.43
(C-30); 111.99 (C-60); 71.46 (Ar-O-CH2-CH2-O-); 69.48
(Ar-O-CH2-CH2-O-); 64.91 (-O-CH2-CH2-NH+-); 55.31
(-O-CH3); 54.76 (-O-CH2-CH2-NH+-); 51.43 (Ar-N-CH2-
(pip)); 46.82 (-CH2-N-CH2- (pip)); 15.99 (CH3-Ar (1)).
[M+H]+ 405.34; 99.89%.
Membrane preparation and general assay procedures for
cloned receptors were adjusted to 96-microwell format
based on protocols described by us previously (8,9).
Tail suspension test in mice
The experiments were conducted on Swiss albino (Krf:
CD-1) mice according to the method described by Steru
et al. (10). Each animal was fastened with medical adhe-
sive tape 50 cm below the surface. The total time of
immobility was measured during the entire 6 min of the
testing session. Immobility was scored manually and was
defined when the animals hung passively without limb
movements. The compounds were injected at the doses
1.25–10 mg/kg b.w. If the compound given at the lowest
dose was activated in the tail suspension test (TST), the
dose was reduced by half, until the disappearance of the
antidepressant-like activity, or statistically insignificant
results.
Pharmacology
Cells preparation
HEK293 cells with stable expression of human serotonin
5-HT1AR, 5-HT6, or 5-HT7bR (prepared with the use of
Lipofectamine 2000) were maintained at 37 °C in a humid-
ified atmosphere with 5% CO2 and were grown in Dulb-
eco’s modified Eagle’s medium containing 10% dialyzed
fetal bovine serum and 500 lg/mL G418 sulfate. For
membranes preparation, cells were subcultured in 10-cm-
diameter dishes, grown to 90% confluence, washed twice
with a phosphate-buffered saline (PBS) prewarmed to
37 °C, and were pelleted by centrifugation (200 9 g) in
PBS containing 0.1 mM EDTA and 1 mM dithiothreitol.
Prior to membrane preparation, the pellets were stored at
ꢀ80 °C.
Spontaneous locomotor activity
The locomotor activity was measured with photoresistor
actometers (Ugo Basile, Italy) connected to a counter for
the recording of light-beam interruptions, and the num-
ber of light-beam crossings was counted during the ses-
sion (either 6-min or 30-min). The number of crossings
of the light beams was then recorded as the locomotor
activity. The studied compounds were administered at
the doses active in TST to determine whether the
observed effect is specific, and for the statistical analysis,
the data obtained in the 6th min of the observation were
used. To evaluate whether compounds possess the abil-
ity to induce CNS depression, the data obtained in the
30th min of the observation were used and median
Radioligand binding assays
Cell pellets were thawed and homogenized in 20 volumes
of assay buffer using an Ultra Turrax tissue homogenizer
and centrifuged twice at 35 000 9 g (20 000 r.p.m.) for
20 min at 4 °C, with incubation for 15 min at 37 °C in
between. The composition of the assay buffers was as fol-
lows: for 5-HT1AR: 50 mM Tris–HCl, 0.1 mM EDTA, 4 mM
MgCl2, 10 lM pargyline, and 0.1% ascorbate; for 5-HT6R:
50 mM Tris–HCl, 0.5 mM EDTA, and 4 mM MgCl2; and for
5-HT7bR: 50 mM Tris–HCl, 4 mM MgCl2, 10 lM pargyline,
and 0.1% ascorbate.
effective doses (ED50
) were calculated. To determine
ED50 values, the compounds were administered at the
lowest doses active in the TST, and then, the dose was
gradually increased until the appearance of sedative
effects.
All assays were incubated in a total volume of 200 lL
in 96-well microtiter plates for 1 h at 37 °C, except for
5-HT1A
for 1 h. The process of equilibration is terminated by rapid
filtration through Unifilter plates with 96-well cell
harvester, and radioactivity retained on the filters was
quantified on a Microbeta plate reader. For displacement
studies, the assay samples contained as radioligands:
1.5 nM [3H]8-OH-DPAT (187 Ci/mM) for 5-HT1AR; 2 nM
[3H]LSD (85.2 Ci/mM for 5-HT6R or 0.6 nM [3H]5-CT
(39.2 Ci/mM) for 5-HT7R.
R which were incubated at room temperature
Rotarod test
a
The test was performed according to the method
described by Sałat et al. (11) with some minor modifica-
tions. Mice were trained for three consecutive days on
the rotarod apparatus (rotarod apparatus, May Commat
RR0711, Turkey; rod diameter: 2 cm), rotating at a con-
stant speed of 24 r.p.m. During each training session,
the animals were placed on a rotating rod for 3 min with
an unlimited number of trials. The proper experiment was
conducted at least 24 h after the final training trial. On
the test day, mice were given the test compound or vehi-
cle and tested on the rotarod, revolving at 24 r.p.m.
Motor impairment was defined as the inability to remain
on the rotating rod for 1 min. It was measured and
expressed as the number of animals that fell off the rotat-
ing rod. ED50 values were then calculated. To determine
Non-specific binding was defined with 10 lM of 5-HT in 5-
HT1AR and 5-HT7R binding experiments, whereas 10 lM
methiothepin was used in 5-HT6R assays. Each com-
pound was tested in triplicate at 7–8 concentrations
(10ꢀ11–10ꢀ4 M). The inhibition constants (Ki) were calcu-
lated from the Cheng-Prusoff equation (7). Results were
expressed as means of at least two separate experiments.
Chem Biol Drug Des 2015; 85: 326–335
329

Waszkielewicz et al.
ED50 values, the compounds were administered at the
lowest doses active in the TST, and then, the dose was
gradually increased until the appearance of neurotoxic
effects.
The log-probit method described by Litchfield and Wilco-
xon (13) was used to determine ED50 for compounds in
locomotor activity, rotarod, and chimney tests. In locomo-
tor activity test, ED50 is defined as the dose of the tested
compound that decreases spontaneous locomotor activity
in 50% of mice compared with vehicle-treated group. In
rotarod and chimney tests, TD50 is defined as the dose of
investigated compound that impairs motor co-ordination in
50% of mice compared with vehicle-treated group.
Chimney test
The chimney test was performed according to a method
described by Boissier et al. (12). Previously trained and
selected animals were placed in a 25 cm long and
2.5 cm in diameter, horizontally located tube, which was
reversed in such a way that the mice were able to leave
it only climbing backward up until they reached another
end. The inability of mice to perform the test within
60 seconds indicated motor impairment. The number of
animals that were unable to climb backwards was
recorded, and ED50 values were calculated. To deter-
mine ED50 values, the compounds were administered at
the dose active in the TST, and then, the dose was
gradually increased until the appearance of neurotoxic
effects.
Results
Chemistry
Compounds 1-6 were obtained by N-alkylation of
N-(2-methoxyphenyl)piperazine using, respectively, phen-
oxypropyl or phenoxyethoxyethyl bromide. The reaction
was performed in the presence of K2CO3 as a proton
acceptor in toluene solution. The yield of alkylation was
in the range 40–55%. Appropriate phenoxypropyl or phen-
oxyethoxyethyl bromides were achieved according to ear-
lier-published methodology (14), and crude products were
used for further alkylation. All amines received as oily prod-
ucts were converted into hydrochlorides upon treatment
with excess of ethanolic solution of gaseous HCl. The raw
hydrochlorides were recrystallized from the mixture of ace-
tone/ethanol 1:1 (v/v). The synthesis and chemical struc-
tures of the tested compounds are shown in Scheme 1.
Data analysis
The data obtained were presented as means ꢁ SEM and
evaluated using Student’s t-test or one-way analysis of
variance (ANOVA), followed by Dunnett’s test. Differences
between groups were considered significant if p < 0.05.
Scheme 1: Synthesis and structures of
compounds 1–6.
330
Chem Biol Drug Des 2015; 85: 326–335

Activity of Piperazine Derivatives
Table 2: Effect of the studied compounds and imipramine on the
Pharmacology
duration of immobility time in tail suspension test in mice
Compounds 2, 4, and 6 showed very high affinity toward
5-HT1A receptor (Ki <1 nM). The results are superior to
both serotonin and buspirone. Compounds 2, 3, and 6
showed high affinity toward 5-HT7 receptor (Ki <35 nM),
and the results are comparable to clozapine. None of
the tested compounds proved nanomolar affinity toward
5-HT6 receptor (Table 1).
Dose
[mg/kg
Treatment
b.w.]
Immobility time [s]
Vehicle
(0.5% MC)
1
–
131.0 ꢁ 11.6
1.25
2.5
5
95.7 ꢁ 12.4
69.4 ꢁ 12.1**
Compounds 1, 2, and 4 decreased immobility time in mice
at the dose 2.5 mg/kg b.w. by 47.0%, 60.3%, and 53.4%,
respectively, but no effect was detectable using either
lower or higher concentrations of the compounds. The
reason for this fact may be a possible characteristic of
antidepressants—U-shape of effect–dose curve in such
tests. Compound 3 did not influence immobility time at
the doses 2.5–10 mg/kg b.w. Compound 5 at the doses
2.5–10 mg/kg b.w. statistically significantly decreased
immobility time in mice by 56.8% to 59.2%. Compound 6
decreased immobility time in mice by 41.6% and 54.5% at
the doses 2.5 and 5 mg/kg b.w., respectively. Imipramine
at the doses 5 and 10 mg/kg b.w. statistically significantly
decreased immobility time in mice by 52.4% and 83.5%,
respectively. The results of TST in mice are summarized in
Table 2.
99.3 ꢁ 12.1
10
125.2 ꢁ 15.4
F(4,41) = 3.741, p < 0.05
128.7 ꢁ 14.5
Vehicle
(0.5% MC)
2
–
1.25
2.5
5
121.6 ꢁ 15.5
51.1 ꢁ 14.7**
131.5 ꢁ 20.1
10
122.6 ꢁ 15.2
F(4,41) = 4.011, p < 0.01
126.9 ꢁ 14.4
Vehicle
(0.5% MC)
3
–
2.5
5
106.7 ꢁ 14.3
132.5 ꢁ 12.4
127.2 ꢁ 18.1
F(3,36) = 0.561, NS
129.7 ꢁ 14.8
10
Vehicle
(0.5% MC)
4
–
1.25
2.5
5
84.9 ꢁ 12.1
None of the compounds affected locomotor activity in mice
at the doses active in TST in mice (Table 3). Compounds 1,
2, 4, and 6 statistically significantly decreased locomotor
activity in mice at the doses 30 and 60 mg/kg b.w. by
94.8% and 99.7% (F(4,41) = 11.170, p < 0.0001), 72.3%
and 98.3% (F(3,29) = 8.413, p < 0.001), 64.9% and 97.5%
(F(3,30) = 8.750, p < 0.001), and 51.4% and 87.8% (F
(4,45) = 26.750, p < 0.0001), respectively. Compound 5
statistically significantly decreased locomotor activity in mice
at the doses 20 and 40 mg/kg b.w. by 37.5% and 94.8%,
respectively (F(3,36) = 12.420, p < 0.0001). ED50 values for
all compounds are shown in Table 3.
59.1 ꢁ 13.7**
91.8 ꢁ 14.9
10
111.8 ꢁ 11.5
F(4,45) = 3.980, p < 0.01
138.1 ꢁ 12.4
Vehicle
(0.5% MC)
5
–
1.25
2.5
5
96.0 ꢁ 14.8
59.6 ꢁ 10.1***
59.4 ꢁ 9.4***
10
56.3 ꢁ 11.6****
F(4,43) = 8.650, p < 0.0001
147.3 ꢁ 7.7
Vehicle
(0.5% MC)
6
–
1.25
2.5
5
125.6 ꢁ 14.3
86.0 ꢁ 18.4**
Table 1: Results of binding to serotoninergic receptors of tested
and reference compounds
67.0 ꢁ 11.4***
10
110.9 ꢁ 12.5
F(4,42) = 4.292, p < 0.01
140.2 ꢁ 7.4
Ki [nM]
Vehicle (DW)
Imipramine
–
2.5
5
116.0 ꢁ 11.2
Compd.
5-HT1A
15
5-HT6
5-HT7
78.2 ꢁ 10.9***
1
–
–
10
34.4 ꢁ 10.3****
F(3,34) = 20.830, p < 0.0001
2
<1
6
<1
41
<1
1809
4465
8058
9967
9617
–
23
25
157
156
34
–
3
4
All compounds were administered i.p. 30 min before the test. The
values are expressed as mean ꢁ SEM, n = 9–10 mice per group.
Statistical analysis: one-way ANOVA (Dunnett’s post hoc)
**p < 0.01, ***p < 0.001, ****p < 0.0001 versus respective
vehicle-treated group. NS, non significant; MC, methylcellulose;
DW, distilled water.
5
6
Serotonin
Buspirone
Imipramine
Clozapine
3
12
–
–
>10 000
143
190
4
1000
18
Inhibition constants (Ki) were calculated according to the equation
of Cheng and Prusoff (7). Radioligand binding assays to rats brain
tissues using [3H]8-OH-DPAT for 5-HT1A, [3H]-LSD for 5-HT6,
[3H]-5-CT for 5-HT7; n = 3.
None of the compounds affected motor co-ordination in
rotarod and chimney tests at the doses active in TST in
mice (Table 4).
Chem Biol Drug Des 2015; 85: 326–335
331

Waszkielewicz et al.
Table 3: The influence of the studied
compounds on locomotor activity in mice
Dose active
in the TST
Treatment
[mg/kg b.w.]
Number of crossings
ED50 [mg/kg b.w.] (CI)
Vehicle (0.5% MC)
1
–
2.5
349 ꢁ 58
–
377 ꢁ 48
12.6 (5.4–29.5)
t(18) = 0.376, NS
383 ꢁ 63
2
2.5
15.0 (6.8–33.0)
t(18) = 0.405, NS
345 ꢁ 46
Vehicle (0.5% MC)
4
–
2.5
–
337 ꢁ 47
34.6 (21.4–56.1)
t(18) = 0.123, NS
347 ꢁ 27
Vehicle (0.5% MC)
5
–
2.5
5
–
356 ꢁ 28
21.8 (16.3–29.3)
342 ꢁ 24
10
355 ꢁ 51
F(3,33) = 0.034, NS
387 ꢁ 30
Vehicle (0.5% MC)
6
–
2.5
5
–
322 ꢁ 32
17.5 (11.9–25.8)
342 ꢁ 39
F(2,27) = 0.972, NS
All compounds were administered i.p. 30 min before the test. The values are expressed
as mean ꢁ SEM, n = 9–10 mice per group. Statistical analysis: Student’s t-test or one-
way ANOVA (Dunnett post hoc). ED50 values were calculated using the log-probit method
described by Litchfield and Wilcoxon 18; NS, non significant; MC, methylcellulose. Com-
pounds 1 and 2 were evaluated versus the same control (vehicle); CI, confidence interval;
TST, Tail suspension test.
Table 4: The influence of the studied compounds on motor coordination in rotarod and chimney tests
Rotarod test
Chimney test
Dose active
in the TST
[mg/kg b.w.]
% of animals
that fell from
rotating rod
% of animals
that did not climb
out the chimney
Treatment
TD50 [mg/kg b.w.] (CI)
TD50 [mg/kg b.w.] (CI)
1
2
4
5
2.5
2.5
2.5
2.5
5
10
2.5
5
0
0
0
0
0
0
0
0
46.0 (34.5–61.3)
106.0 (72.0–156.1)
19.6 (10.6–36.1)
46.6 (41.2–52.7)
0
0
0
0
0
0
0
0
18.7 (13.1–26.8)
30.5 (23.3–39.8)
14.8 (8.9–24.8)
22.4 (11.5–43.6)
6
53.2 (44.1–62.2)
20.1 (13.2–30.5)
All compounds were administered i.p. 30 min before the test. n = 10 mice per group. TD50 values were calculated using the log-probit
method described by Litchfield and Wilcoxon 17; CI, confidence interval; TST, Tail suspension test.
Compound 1 at the doses 20, 30, and 40 mg/kg statisti-
cally significantly increased time spent in the chimney
compared with the vehicle-treated animals by 84%, 111%,
and 136%, respectively (F(4,41) = 6.642, p < 0.001).
Compound 2 at the doses 30, 40, and 50 mg/kg statisti-
cally significantly increased time spent in the chimney
compared with control group by 122%, 134%, and 161%,
spent in the chimney by 218% and 304%, respectively
(F(3,36) = 4.434, p < 0.01). Compound 6 at the doses 25
and 40 mg/kg b.w. statistically significantly increased time
spent in the chimney compared with control group by
210%
and
260%,
respectively
(F(3,36) = 7.789,
p < 0.001). ED50 values in rotarod and chimney tests for
both compounds are presented in Table 4.
respectively (F(4,40) = 5.520, p < 0.01). Compound
4
at the doses 20 and 40 mg/kg statistically significantly
increased time spent in the chimney compared with
control group by 223% and 296%, respectively
(F(3,36) = 14.230, p < 0.0001). In comparison with
vehicle-treated group, compound 5 at the doses 40 and
50 mg/kg b.w. statistically significantly increased time
Discussion
It is commonly known that 5-HT1A receptors have been
implicated in many CNS disorders, including anxiety and
depression. Numerous researches show that 5-HT1A
332
Chem Biol Drug Des 2015; 85: 326–335

Activity of Piperazine Derivatives
receptor ligands possess antidepressant-like properties
(15,16). Given the fact that all studied compounds
showed high affinity for serotonergic 5-HT1A receptors,
and compounds 2, 3, and 6 exhibit favorable 5-HT7 bind-
ing properties, their antidepressant-like activity was investi-
gated in tail suspension test in mice, which is widely used
to evaluate antidepressant properties. In this test, all com-
pounds except for 3 showed significant activity, stronger
than that of imipramine (Table 2). From in vivo observa-
tions, compounds 5 and 6 seem the most promising, as
they were active also at other doses apart from 2.5 mg/
kg (Table 2). This is very interesting taking into account
receptor-binding properties, as activity of compound 5
does not correlate with its receptor binding to 5-HT1A or
5-HT7. This can be explained by other possible mecha-
nism(s) of action.
In terms of various substitution of the phenyl ring, com-
pounds 2, 4, and 5-6 exhibit the most favorable pharma-
cological properties, which suggest that substitution with
methyl in position 2, as a common feature of all com-
pounds may be significant, as well as use of at least 2
lipophilic substituents (methyl, chloro).
As the reference compound constituted 4-[(2,6-dimethyl-
phenoxy)propyl]-1-(2-methoxyphenyl)piperazine hydrochlo-
ride (Figure 1), the modifications were made in terms of
changing position of methyl in the phenyl ring from 6 to 5
(compound 2) as well as addition of an extra methyl group
in the phenyl ring in various positions—2,3,5 as well as
2,4,6 (as in compounds 3-4). These changes resulted in
most favorable results for 2,5-dimethyl isomer (2) of the
reference compound—its affinities toward 5-HT1A and
5-HT7 remain below 1 nM (Table 1), its ED50 in locomotor
activity is equal to 15 mg/kg b.w. (mice, i.p., Table 3), and
the compound is the least neurotoxic of all the synthesized
compounds, revealing TD50 (mice, i.p.) = 106 mg/kg b.w.
(rotarod) and 30.5 mg/kg b.w. (chimney test).
As not only antidepressants but also psychostimulants
show activity in TST, the influence on locomotor activity in
mice was also evaluated. None of the compounds affected
locomotor activity in mice at the doses active in TST, and
this fact suggests that the observed antidepressant-like
effect was specific. It has been shown that arylpiperazines
produce antidepressant-like activity in many behavioral
tests and models of depression (17–21). The research
results are convergent with our findings.
Then, substitution in compound 2 of one methyl into chloro
in the phenyl ring resulted in compound 1–4-[(2-chloro-6-
methylphenoxy)propyl]-1-(2-methoxyphenyl)piperazine hydro-
chloride with locomotor activity ED50 = 12.6 mg/kg b.w.
The results of our study clearly show that all active
compounds possess sedative properties. In patients suf-
fering from depression with agitation or insomnia, seda-
tion can be desirable. On the other hand, excessive
sleepiness during antidepressant therapy is regarded as
side-effect and is the most common reason for drug
discontinuation. Taking into account that studied com-
pounds showed sedative properties at the doses around
five to fourteen times higher than the lowest dose active
in TST (Table 3), the risk of excessive sedative effect
seems to be low. Nevertheless, this requires further
investigation.
Compound 5 is ethoxyethyl analog of the reference com-
pound (Figure 1) (6). Its antidepressant-like activity was
comparable to imipramine (Table 2). Therefore, similar to
modifications in compounds 1 and 2, compound 6 is
chloro analog of compound 5. The affinities of 5 and 6
toward 5-HT1A receptor can be compared—compound 6
exhibits Ki <1 nM and 5 Ki = 41 nM. Compound 6 also
exhibits more favorable antidepressant-like activity than
both 5 and imipramine—the observed doses at a signifi-
cant effect are 2.5 mg/kg b.w. for 6 and 5 mg/kg b.w. for
imipramine (Table 2). Its ED50 in locomotor activity is
17.5 mg/kg b.w. (Table 3) and TD50 = 53.2 and 20.1 mg/
kg b.w. in rotarod and chimney test, respectively (Table 4).
It is well known that drugs acting within CNS may have
negative influence on brain function. None of the com-
pounds impaired motor co-ordination in mice at antide-
pressant-like doses, and this observation suggests that
none of the compounds possesses neurotoxic properties
at active doses (Table 4). The neurotoxic effect was
observed at much higher doses (rotarod—around 19–42
times as high, chimney test—around 8–12 times as high)
(Table 4).
Conclusions
As a continuation of our former studies, it has been
revealed that variously substituted aroxypropyl derivatives
of 1-(2-methoxyphenyl)piperazine can present very good
affinities toward 5-HT1A and 5-HT7 receptors. The in vivo
results reflect the in vitro activity, which may suggest some
favorable ADME properties. However, this should be fur-
ther explored. As an unobvious result of the performed
studies, exchange of the propyl linker into ethoxyethyl
resulted in achievement of both favorable in vitro and in
vivo characteristics. As a conclusion, the title group of
compounds is promising in terms of antidepressant-like
activity, and some further research in terms of in vivo
properties should be performed, especially for ethoxyethyl
derivatives of 1-(2-methoxyphenyl)piperazine. Moreover,
Structure–activity relationship
Among the synthesized group of compounds, 1-4 and 5-6
represent variously substituted (phenoxy)propyl or (phen-
oxy)ethoxyethyl derivatives of 1-(2-methoxyphenyl)pipera-
zine, respectively. It can be observed that compounds 5-6
are at least as active as 1-4 (Tables 1–3), but less neuro-
toxic (rotarod and chimney test, Table 4).
Chem Biol Drug Des 2015; 85: 326–335
333

Waszkielewicz et al.
the observed affinities toward 5-HT7 receptors suggest
that it is probable to find cognitive activity, and this possi-
bility also should be explored.
(2007) Structure-activity relationship studies of CNS
agents. Part 9: 5-HT1A and 5-HT2 receptor affinity of
some 2- and 3-substituted 1,2,3,4-tetrahydro-beta-
carbolines. Pharmazie;48:289–294.
ꢀ
9. Paluchowska M.H., Bugno R., Duszynska B., Tata-
ꢀ
ꢀ
Acknowledgments
rczynska E., Nikiforuk A., Lenda T., Chojnacka-Wojcik
E. (2007) The influence of modifications in imide frag-
ment structure on 5-HT(1A) and 5-HT(7) receptor affin-
ity and in vivo pharmacological properties of some new
1-(m-trifluoromethylphenyl)piperazines. Bioorg Med
Chem;15:7116–7125.
The research was performed with approval of Local Ethics
Committee for Research on Animals. The research was
co-funded by Jagiellonian University Students’ Grant No.
28/2012. Radioligand binding studies for 5-HT receptors
were co-funded by statutory funds of Department of
Medicinal Chemistry IP PAS. Synthesis was supported by
Jagiellonian University Medical College in Krakow (grant no
10. Steru L., Chermat R., Thierry B., Simon P. (1985) The
tail suspension test: a new method for screening
antidepressants in mice. Psychopharmacology;85:
367–370.
1
K/ZDS/003328). H and 13C NMR spectra were performed
at Faculty of Chemistry, Jagiellonian University, and were
supported by the European Regional Development Fund
through the Innovative Economy Program.
11. Sałat K., Gawlik K., Witalis J., Pawlica-Gosiewska D.,
Filipek B., Solnica B., Wieckowski K., Malawska B.
z
(2013) Evaluation of antinociceptive and antioxidant
properties of 3-[4-(3-trifluoromethyl-phenyl)-piperazin-
1-yl]-dihydrofuran-2-one in mice. Naunyn Schmiede-
bergs Arch Pharmacol;386:493–505.
Conflict of Interest
12. Boissier J.R., Tardy J., Diverres J.C. (1960) A simple
novel method to explore tranquilizer activity: the chim-
ney test. Med Exp (Basel);3:81–84.
There is no conflict of interests among the authors.
13. Litchfield J., Wilcoxon F. (1949) A simplified method of
evaluating dose-effect experiments. J Pharmacol Exp
Ther;96:99–113.
References
1. Schechter L.E., Ring R.H., Beyer C.E., Hughes Z.A.,
Khawaja X., Malberg J.E. (2005) Innovative approaches
for the development of antidepressant drugs: current
and future strategies. NeuroRx;2:590–611.
2. Wesołowska A. (2002) In the search for selective
ligands of 5-HT5, 5-HT6 and 5-HT7 serotonin recep-
tors. Pol J Pharmacol;54:327–341.
3. Blier P., Bergeron R. (1998) The use of pindolol to
potentiate antidepressant medication. J Clin Psychia-
try;59(Suppl 5):16–23.
4. Goetz A.S., King H.K., Ward S.D.C., True T.A., Rimele
T.J., Saussy D.L. (1995) BMY7378 is a selective antag-
onist of the D subtype of a1-adrenoceptors. Eur J
Pharmacol;272:R5–R6.
5. Marona H., Szkaradek N., Kubacka M., Bednarski M.,
Filipek B., Cegla M., Szneler E. (2008) Synthesis and
evaluation of some xanthone derivatives for anti-
arrhythmic, hypotensive properties and their affinity for
adrenergic receptors. Arch Pharm (Weinheim);341:90–
98.
6. Marona H., Kubacka M., Filipek B., Siwek A., Dyba M.,
Szneler E., Pociecha T., Gunia A. (2011) Synthesis,
alpha-adrenoceptors affinity and alpha 1-adrenoceptor
antagonistic properties of some 1,4-substituted pipera-
zine derivatives. Pharmazie;66:733–739.
14. Waszkielewicz A.M., Szneler E., Cegła M., Marona H.
(2013) Synthesis and evaluation of anticonvulsant
activity of some N-(4-Chlor-2-methylphenoxy)ethyl-
and N-(4-Chlor-2-methylphenoxy)acetylaminoalkanols.
Lett Drug Des Discov;10:34–42.
15. Savitz J., Lucki I., Drevets W.C. (2009) 5-HT(1A)
receptor function in major depressive disorder. Prog
Neurobiol;88:17–31.
16. Artigas F. (2013) Developments in the field of antidepres-
sants, where do we go now? Eur Neuropsychopharma-
col doi:10.1016/j.euroneuro.2013.04.013. [e-pub ahead
of print].
17. Kang S.Y., Park E.-J., Park W.-K., Kim H.J., Jeong D.,
Jung M.E., Song K.-S. et al. (2010) Arylpiperazine-con-
taining pyrrole 3-carboxamide derivatives targeting
serotonin 5-HT(2A), 5-HT(2C), and the serotonin trans-
porter as a potential antidepressant. Bioorg Med Chem
Lett;20:1705–1711.
18. Kim J.Y., Kim D., Kang S.Y., Park W.-K., Kim H.J.,
Jung M.E., Son E.-J., Pae A.N., Kim J., Lee J. (2010)
Arylpiperazine-containing pyrimidine 4-carboxamide
derivatives targeting serotonin 5-HT(2A), 5-HT(2C), and
the serotonin transporter as a potential antidepressant.
Bioorg Med Chem Lett;20:6439–6442.
€
€
€
€
19. Demir Ozkay U., Yurttas L., Ozkay Y., Ucel U.I., Can
ꢀ
ꢀ
€
€
€
7. Cheng Y., Prusoff W.H. (1973) Relationship between the
inhibition constant (K1) and the concentration of inhibitor
which causes 50 per cent inhibition (I50) of an enzymatic
reaction. Biochem Pharmacol;22:3099–3108.
O.D., Ozturk Y. (2013) Synthesis of new 1-phenyl-2-(4-
substituted-piperazin-1-yl)-propanol derivatives and
evaluation of their antidepressant-like effects. Arch
Pharm Res;36:802–811.
8. Bojarski A.J., Cegła M.T., Charakchieva-Minol S., Mok-
20. Prashanth M.K., Revanasiddappa H.D., Lokanatha Rai
K.M., Veeresh B. (2012) Synthesis, characterization,
ꢀ
rosz M.J., Mackowiak M., Misztal S., Mokrosz J.L.
334
Chem Biol Drug Des 2015; 85: 326–335

Activity of Piperazine Derivatives
Figure S1. Compound 1.
antidepressant and antioxidant activity of novel pipera-
mides bearing piperidine and piperazine analogues.
Bioorg Med Chem Lett;22:7065–7070.
Figure S2. Compound 2.
Figure S3. Compound 3.
Figure S4. Compound 4.
Figure S5. Compound 5.
Figure S6. Compound 6.
21. Pandey D.K., Mahesh R., Kumar A.A., Rao V.S., Arjun
M., Rajkumar R. (2010) A novel 5-HT(2A) receptor
antagonist exhibits antidepressant-like effects in a bat-
tery of rodent behavioural assays: approaching early-
onset
antidepressants.
Pharmacol
Biochem
Behav;94:363–373.
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Chem Biol Drug Des 2015; 85: 326–335
335