An expeditious one-pot microwave facilitated versus conventional syntheses: In vivo biological screening and molecular docking studies of some 3,5-disubstituted-4,5-dihydro-(1H)-pyrazole derivatives

Article abstract of DOI:10.1007/s00044-015-1489-3

A series of 3,5-disubstituted-2-pyrazoline derivatives (2a-2t) were synthesized by reacting different aromatic/heteroaromatic aldehydes and ketones, in a two-step reaction through Claisen Schmidt condensation, followed by cyclization of the resulted chalc

Full text of DOI:10.1007/s00044-015-1489-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1489-3
ORIGINAL RESEARCH
An expeditious one-pot microwave facilitated versus conventional
syntheses: in vivo biological screening and molecular docking
studies of some 3,5-disubstituted-4,5-dihydro-(1H)-pyrazole
derivatives
Avinash C. Tripathi1 Savita Upadhyay1 Sarvesh Paliwal2 Shailendra K. Saraf1
•
•
•
Received: 15 May 2015 / Accepted: 15 December 2015
Ó Springer Science+Business Media New York 2016
Abstract
A
series of 3,5-disubstituted-2-pyrazoline
b.w.), when compared to the standard drugs imipramine
and diazepam, respectively. In order to ascertain the
binding interactions of the synthesized derivatives to the
MAO-A target protein, molecular docking was employed
which demonstrated the key interactions with the amino
acid residues Asn181, Phe208, Tyr69, Tyr197, Tyr444 and
Met445 at the binding site. In addition, the most active
derivatives 2i and 2b showed some imperative conserved
interactions of the PDB co-crystal ligand 2Z5X with the
amino acid residues at the binding site of MAO-A protein.
The results of the study also demonstrated that the Glide
gscores of the synthesized derivatives were in close cor-
relation with the in vivo biological activity data, in par-
ticular with the forced swim test of the antidepressant
activity with a very good correlation coefficient of
0.754103. Furthermore, the ADME properties of the syn-
thesized derivatives were predicted and found to be within
the affirmed limits.
derivatives (2a–2t) were synthesized by reacting different
aromatic/heteroaromatic aldehydes and ketones, in a two-
step reaction through Claisen Schmidt condensation, fol-
lowed by cyclization of the resulted chalcones with
hydrazine hydrate in the presence of a base using con-
ventional and microwave approaches. The synthesized
derivatives were characterized by various physicochemical
methods, and their chemical structures were established by
IR, Mass, 1H-NMR, 13C-NMR spectroscopic data and
elemental analysis. The antidepressant with tail suspension
test and forced swim test and anti-anxiety with Elevated
Plus Maze Test activities were evaluated using suit-
able animal models. Compounds 2i, and 2j showed
noticeable antidepressant activity, by reducing the duration
of immobility in both the tests, while compounds 2a and 2b
were found to possess good anxiolytic activity, by
increasing the number of arm entries and open arm
exploratory time at the tested doses (50 and 100 mg/kg
Keywords 2-Pyrazolines Á Antidepressant Á
Anti-anxiety Á MAO inhibitors Á Neurotoxicity Á
Microwave synthesis Á Molecular docking Á
In silico ADME prediction
& Shailendra K. Saraf
dirpharmniec@gmail.com
Avinash C. Tripathi
aviniec31@gmail.com
Introduction
Savita Upadhyay
savvypharma@gmail.com
Monoamine oxidase (MAO) [E.C. 1.4.3.4] is a flavin-
containing key enzyme located on the outer membrane of
the mitochondria, bound via a C-terminal transmembrane
polypeptide segment (Mitoma and Ito, 1992) and inserted
in the membrane by means of ubiquitin, with energy pro-
vided by ATP (Zhuang et al., 1992), in neuronal, glial, and
other cells, regulating monoaminergic homeostasis and
possibly neurotransmission. Low level of certain
Sarvesh Paliwal
paliwalsarvesh@yahoo.com
1
Division of Pharmaceutical Chemistry, Faculty of Pharmacy,
Babu Banarasi Das Northern India Institute of Technology,
BBD City, Faizabad Road, Chinhat, Lucknow, UP 226028,
India
2
Department of Pharmacy, Banasthali Vidyapith,
Banasthali, Tonk, Rajasthan 304022, India
123

Med Chem Res
neurotransmitters (NTs) such as serotonin, norepinephrine, 2010). Pyrazolines are also well-established pharma-
dopamine and gamma amino butyric acid (GABA) in the cophores for activities other than nervous system such as
brain is the cause of depression and other mental disorders. anti-amebic (Bhat et al., 2009; Budakoti et al., 2009;
NTs are released by the process of neurotransmission and Husain et al., 2008; Wanare et al., 2010), anti-cancer
are broken down by the MAOs. Monoamine oxidase (Congiu et al., 2010; Havrylyuk et al., 2009; Insuasty et al.,
inhibitors (MAOIs) block this enzyme, and hence, the 2011; Parekh et al., 2011), anti-viral (Diamond 2009;
concentration of NTs is increased in the brain (Meyer et al., Ramajayam et al., 2010), anti-malarial (Acharya et al.,
2006).
2010; Wanare et al., 2010), anti-inflammatory-analgesic
Serendipitous finding of antidepressant effects in (Fioravanti et al., 2010; Girisha et al., 2010; Khode et al.,
patients treated with iproniazid, a hydrazine-based antitu- 2009), anti-microbial (Abdel-Wahab et al., 2009; Dawane
bercular agent structurally similar to isoniazid, started the et al., 2010; Ozdemir et al., 2007; Siddiqui et al., 2011;
journey of development of MAO inhibitors. This discov- Sivakumar et al., 2010) etc.
ery, together with the demonstration that iproniazid was a
Studies by different workers have demonstrated 1,3,5-
potent MAO inhibitor (Zeller and Barsky, 1952), led to the trisubstituted-2-pyrazolines to be very promising scaffolds
design and production of other MAO inhibitors such as for CNS activities such as antidepressant, anti-anxiety and
phenelzine. MAOIs were the first-generation antidepres- anticonvulsant. In view of these observations, it was
sants used for decades in the treatment for patients with decided to synthesize and characterize a series of 3,5-dis-
atypical depression (Pletscher, 1991), high level of anxiety, ubstituted-2-pyrazolines, with novel aromatic and hetero-
anergic bipolar depression and treatment resistant depres- aromatic substitutions at 3rd and 5th positions, respec-
sion (Thase, 2012), specific phobias, posttraumatic stress tively, and to evaluate them as antidepressant and anti-
disorder and migraine headaches resistant to other thera- anxiety agents using suitable models. In addition to the
pies (Gareri et al., 2000). Nowadays, the therapeutic conventional heating methods, microwave-assisted syn-
interest in MAOIs falls into two major categories. MAO-A thetic approach was employed to prepare the proposed
inhibitors have been used mostly in the treatment of mental derivatives and the results were compared with those of the
disorders, in particular depression and anxiety (Amrein former method. It has been observed that most of the
et al., 1999), while MAO-B inhibitors have shown thera- conventional heat reactions could be performed more
peutic value in a variety of diseases (Youdim et al., 2006), conveniently using this technique, at a faster rate and in
especially neurodegenerative (Foley et al., 2000) such as good yields (Lidstrom et al., 2001). Finally, the molecular
Parkinson’s (Cesura and Pletscher, 1992) and Alzheimer’s docking studies were performed to predict the binding
(Volz and Gleiter, 1998). Hydrazine-based MAO inhibitors affinity and ascertain the interactions of the proposed
were devoid of serious side effects such as liver toxicity derivatives with the biological target.
and ‘‘cheese reaction’’ (potentially lethal hypertensive
crises with cerebral hemorrhages, following the consump-
tion of cheese, wine and other fermented foods, typically Results and discussion
rich in tyramine and sympathomimetic amines) (Brown
et al., 1989). These side effects were hypothesized to be A series of fourteen 2-pyrazoline derivatives (2a–2t) were
related to nonselective and irreversible MAO inhibition. synthesized via Claisen-Schmidt condensation and char-
The quest for MAO inhibitors devoid of untoward effects acterized by physicochemical (Table 1), spectral and ele-
prompted research to characterize selective MAO-A and mental analysis methods. The IR spectra of the synthesized
MAO-B inhibitors.
The synthesis of pyrazolines and their derivatives have responding to C=N stretching (1509–1612 cm-1), N–H
engrossed substantial attention for many years. Increasing stretching (3456–3105 cm-1
and C–H deformation
compounds afforded absorption bands in the regions cor-
)
evidence suggests that pyrazoline is an important scaffold (1428–1357 cm-1). 1H-NMR spectra of the compounds
since it is known to be associated with multiple biological exhibited the presence of two non-equivalent protons of a
activities such as tranquilizer, muscle relaxant, anticon- methylene group (Ha/Hb) at d 2.92–3.38 ppm, 3.70–3.93
vulsant and antidepressant (Bilgin et al., 1993; Kaplancikli coupled with each other and in turn with the vicinal
et al., 2010; Ozdemir et al., 2008; Palaska et al., 2001; methine proton (Hx) at d 6.68–7.04. All the other aliphatic,
Rajendra Prasad et al., 2005; Ruhoglu et al., 2005), psy- aromatic and heteroaromatic protons were also observed at
choanaleptic and MAO inhibitory (Chimenti et al., 2004, their expected ppm values.
2010; Gokhan-Kelekci et al., 2009, 2007; Gokhan et al.,
The results of pharmacological studies, presented in
2003; Jagrat et al., 2011; Jayaprakash et al., 2008; Table 2 and Fig. 1, showed that compounds 2i and 2j
Karuppasamy et al., 2010; Maccioni et al., 2010; Manna exhibited very good antidepressant activity at the tested
et al., 1998, 2002; Mishra and Sasmal, 2011; Sahoo et al., doses in both, FST and TST models (Fig. 1a, b).
123

Med Chem Res
Table 1 Comparison of physicochemical properties of the synthesized 3,5-disubstituted-2-pyrazoline derivatives (2a–2t) using conventional and
microwave methods
Comps. Structure
Molecular
formula
Color and
state
Solubility
Rf
Melting Conventional synthesis Microwave assisted synthesis
valuea
range
(°C)
(refluxing)
Reaction
time (h)
% Yield
54.7
Microwave
power (W)
Reaction
time (s)
% Yield
86.3
HO
OCH3
OCH3
C
17H18N2O3
178–
180
Colorless
crystalline
solid
Hot methanol, 0.41
chloroform,
acetone,
4.5
240–280
190
2a
N
NH
NH
DMSO
HO
Cl
C15H13ClN2O
210–
212
Colorless
crystalline
solid
Hot methanol, 0.38
chloroform,
acetone,
4.5
67.2
60.5
240
50
90.1
91.4
2b
2c
N
DMSO
HO
C13H12N2O2
148–
150
Cream
Methanol,
acetone,
DMSO
0.48
0.31
5.0
240–280
110
O
colored
crystalline
solid
Colorless
crystalline
solid
Colorless
crystalline
solid
N
NH
HO
Cl
C13H12N2OS
C15H12Cl2N2
88–91
Methanol,
acetone,
DMSO
Hot methanol, 0.83
Chloroform,
acetone,
6.0
8.0
78.8
68.9
240–350
240–280
230
270
82.8
78.6
2d
2f
S
N
NH
Cl
118–
120
N
NH
DMSO
Cl
Cl
C13H11ClN2O
80–82
90–92
Brownish
black
colored
amorphous
solid
Brown
colored
amorphous
solid
Hot methanol, 0.96
Chloroform,
acetone,
5.0
57.4
280–350
220
88.2
2g
O
N
NH
DMSO
C13H11ClN2S
C21H20N2O2
Hot methanol, 0.88
chloroform,
acetone,
4.5
6.0
59.6
280–350
280–350
400
200
85.0
85.3
2h
2i
S
N
NH
DMSO
OCH3
OCH3
112–
114
Cream
Hot methanol, 0.91
Chloroform,
acetone,
63.09
yellow
colored
crystalline
solid
N
NH
DMSO
Cl
C19H15ClN2
118–
120
Cream
White
Methanol,
chloroform,
0.50
5.0
86.7
280
120
90.8
2j
colored
crystalline
acetone,
DMSO
N
NH
solid
C
17H14N2O
88–90
Light brown Methanol,
0.90
0.97
4.5
4.5
71.3
73.5
240–350
280–350
320
380
87.4
94.7
2k
2l
O
colored
crystalline
solid
chloroform,
acetone,
DMSO
N
NH
C17H14N2S
98–100
Cream
Methanol,
chloroform,
acetone,
S
yellow
colored
crystalline
solid
N
NH
DMSO
OCH3
OCH3
C
16H17N3O2
110–
113
Brown
colored
crystalline
solid
Methanol,
chloroform,
acetone,
0.65
0.72
0.54
3.0
3.0
4.0
93.8
48.2
51.6
280
160
175
300
81.5
68.2
72.5
2q
2r
2t
N
N
NH
DMSO
Cl
C14H12ClN3
C12H11N3S
170–
172
Light orange Methanol,
280–350
280–350
colored
crystalline
solid
chloroform,
acetone,
DMSO
Methanol,
chloroform,
acetone,
N
N
NH
82–84
Brown
S
colored
crystalline
solid
N
N
NH
DMSO
123

Med Chem Res
Table 2 Data showing antidepressant, anti-anxiety and neurotoxicity studies of the synthesized derivatives (2a–2t)
Comps.
Doses
(mg/kg
b.w.)
Antidepressant activitya
Anti-anxiety activityb
Neurotoxicity
studiesc
Duration of immobility Duration of immobility Number of entries in Time spent in closed At
in FST
(s) (mean SD)
At
1.0 h 4.0 h
in TST
(s) (mean SD)
closed arm
(mean SD)
arms
(s) (mean SD)
2a
2b
2c
2d
2f
50
129.17 11.23***
97.67 14.19***
96.67 16.82***
85.50 10.50***
82.67 6.41***
74.83 16.82***
124.33 9.71***
97.67 14.19***
105.17 13.85***
87.83 18.79**
104.17 15.56***
99.50 14.64***
155.00 11.64***
101.67 12.26***
56.33 10.21***
21.17 – 8.33***
62.17 14.08***
43.33 10.98***
94.33 14.18***
73.33 25.40***
112.50 19.15***
84.67 13.34***
76.50 21.82***
68.17 18.88***
155.00 11.64***
101.67 12.26***
105.50 11.78***
78.17 11.39***
179.67 18.67
196.17 18.65***
196.83 12.56***
107.17 27.46***
100.83 16.19***
139.33 20.17***
125.50 13.75***
209.17 23.61***
160.33 13.44***
97.50 30.25***
65.50 29.61***
150.50 24.75***
155.83 12.80***
89.83 14.93***
149.17 42.10***
62.33 16.69***
36.83 – 11.28***
151.33 29.56***
29.67 – 15.13***
129.50 37.25***
132.17 11.32***
133.33 23.85***
107.67 17.96***
99.17 19.67***
76.67 6.59***
127.67 19.66***
98.50 14.27***
107.83 18.12***
76.67 6.59***
219.00 17.56
11.17 4.17***
15.50 2.35***
12.33 2.58***
17.67 – 3.01***
7.00 1.55***
6.33 – 2.16***
8.83 3.06***
10.00 4.77***
4.50 1.87***
6.17 – 1.83***
5.83 2.14***
9.17 2.64***
3.17 1.94***
4.17 2.14***
5.50 1.87***
7.17 2.93***
7.67 2.16***
8.50 2.43***
6.50 1.87***
6.83 1.72***
7.67 3.20***
9.00 2.37***
11.17 4.17***
15.50 2.35***
1.00 0.63***
2.67 2.80***
8.17 2.93***
5.17 2.40***
0.83 0.75
162.00 21.54*** 0/6
126.33 11.50*** 0/6
155.33 18.69*** 0/6
117.33 – 17.42*** 0/6
216.50 29.54*** 0/6
242.33 43.58*** 0/6
151.17 18.37*** 0/6
143.83 16.04*** 1/6
200.67 18.20*** 0/6
228.67 30.90*** 0/6
289.00 20.68*** 0/6
283.50 24.81*** 0/6
203.00 22.65*** 1/6
260.50 32.12*** 0/6
256.17 20.85*** 0/6
211.50 25.10*** 0/6
224.17 20.15*** 0/6
193.67 20.22*** 1/6
206.33 15.87*** 0/6
174.17 24.24*** 0/6
169.17 18.50*** 0/6
134.17 12.73*** 0/6
222.50 24.19*** 0/6
162.33 24.08*** 0/6
259.50 22.29*** 0/6
233.33 22.65*** 0/6
270.17 24.38*** 0/6
244.17 39.50*** 0/6
0/6
1/6
0/6
1/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
2/6
0/6
0/6
0/6
1/6
0/6
0/6
1/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
100
50
100
50
100
50
100
50
100
50
2g
2h
2i
100
50
100
50
100
50
2j
100
50
2k
2l
100
50
100
50
2q
2r
2t
100
50
100
50
100
Control (CMC) 0.5 %
281.67 41.09
0/6
Standard
(Imipramine
Diazepam)
10
2
29.33 8.43***
–
60.33 9.33***
–
–9.33 3.08***
–177.33 45.18*** 0/6
0/6
Bold data represent the most active compounds in the series; n = 6 (number of animals tested at each dose level); *** P \ 0.05
Control: Carboxy methyl cellulose (CMC, 0.5 % suspension). Standard: Imipramine (10mg/kg, b.w.) for antidepressant activity and Diazepam
(2mg/kg, b.w.) for anti-anxiety activity
a
The reduction in time of immobility in FST and TST is established way to evaluate effectiveness of antidepressants
b
The number of entries is increased in anxiolytic agents and decrease in anxiogenic agents and the amount of time spent in closed arms is
decreased in anxiolytic agents and increase in anxiogenic agents
c
Neurotoxicity was evaluated in rotarod test after 1.0 and 4.0 h (number of animals exhibiting toxicity/number of animals tested)
Compounds 2a and 2b were found to be the most active
compounds for anti-anxiety activity in the elevated plus
maze test, with the number of entries in different arms
exploration time (Fig. 1c, d). Most of the synthesized
derivatives showed biological activities in a dose-depen-
dent manner, with increasing magnitude of the effects at
higher tested dose (100 mg/kg b.w.). None of the
increasing significantly with
a
lesser closed arm
123

Med Chem Res
Fig. 1 a Graph showing
antidepressant activity (Forced
Swim Test); b graph showing
antidepressant activity (Tail
Suspension Test); c graph
showing anti-anxiety activity
(number of entries in closed
arms in Elevated Plus Maze
Test); d graph showing anti-
anxiety activity (time spent in
closed arms in Elevated Plus
Maze Test)
synthesized derivatives exhibited neurotoxicity (distur-
bances in the motor co-ordinations) at the tested doses, as
evaluated by the rotarod test (Table 2), since the mean fall
off time for all the mice was found to be more than 180 s.
All the synthesized derivatives demonstrated excellent in
silico ADME properties in predictions using QikProp
module (Table 3). The 3,4-dimethoxyphenyl and 4-chlor-
ophenyl substitution at the 5th position (2i, and 2j) and
1-naphthyl substitution at 3rd position of the 2-pyrazoline
nucleus was found to be crucial in eliciting antidepressant
action, while a polar 4-hydroxyphenyl substitution at 3rd
position of the 2-pyrazoline nucleus (2a and 2b) was found
to be decisive in exhibiting good anxiolytic properties.
Previous studies have demonstrated that the 2-pyrazo-
line derivatives possess excellent affinity toward MAO
proteins and are therefore considered as important targets
for developing antidepressants and anti-anxiety agents
(Chimenti et al., 2004; Gokhan-Kelekci et al., 2007;
Jayaprakash et al., 2008). In view of these observations,
docking studies were performed to gain some insights into
the binding mode of these synthesized compounds. The
stable ligand–receptor complex conformations and the
123

Med Chem Res
Fig. 1 continued
interactions of the most active compounds 2i and 2b with
amino acid residues have been depicted in Figs. 2, 3 and 4,
in the typical binding pocket of MAO-A protein. It may be
inferred from the docking studies that Asn181, Phe208,
Phe352, Tyr69, Tyr197, Tyr444 and Met445 are the key
interacting residues at the binding site of MAO-A protein
2Z5X, making crucial contacts with the 3,5-disubstituted 2-
pyrazoline ring. In such an orientation, the bound confor-
mations of compound 2i engrossed in two hydrogen bond
interactions of 2-pyrazoline nitrogen and C5-methox-
yphenyl substituent with the side chain residues; Asn181,
Tyr197; and two pi–pi stacking interactions of C3 naphthyl
ring and 3,4-dimethoxyphenyl ring at C5 position with
Phe208 and Tyr444 backbone residues, respectively
(Fig. 2a, b). Also, one of the most active compounds 2b
(for anti-anxiety activity) is involved in three pi–pi stack-
ing interactions; first two between 4-chlorophenyl ring at
C3 position with Tyr69 and Phe352 residues; and third
between 4-hydroxyphenyl ring of 2-pyrazoline nucleus and
Tyr444 residue (Fig. 3a, b). In addition, two H-bond
interactions are also present between 4-hydroxyphenyl
substituent at C3 position Tyr69 and Met445 backbone
123

Med Chem Res
Table 3 Glide gscore and in silico ADME prediction data of the synthesized derivatives (2a–2t)
Comps.
Glide MW
gscore
donarHB accptHB QlogPo/w QPlogBB
Predicted
metab QPlog
Khsa
% Human
Oral
absorption
Volume PSA
Violations
of Rule of
Five
2a
2b
2c
2d
2f
-8.02 298.341
28.32 272.733
-7.15 228.25
-7.15 244.311
-7.40 291.179
-7.60 246.696
-7.29 262.756
29.70 332.401
-9.28 306.794
-8.45 262.31
-7.80 278.371
-8.07 283.329
-7.73 257.722
-6.63 229.299
-7.09 212.251
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
4.25
2.75
3.25
2.75
2
3.079
3.44
-0.567
-0.179
-0.43
-0.283
0.489
0.335
0.382
0.052
0.317
0.306
0.208
-0.1
5
3
4
4
2
3
3
4
2
3
3
5
3
4
3
0.282
0.319
-0.007
0.138
0.651
0.268
0.471
0.837
0.908
0.472
0.688
0.25
100
100
93.135
100
100
100
100
100
100
100
100
100
100
100
100
978.429 65.316 0
869.116 50.204 0
767.276 61.538 0
797.983 51.868 0
891.902 28.776 0
2.174
2.751
4.587
3.438
4.016
4.852
5.05
2g
2h
2i
2.5
2
783.703 36.91
0
826.674 30.083 0
1096.888 39.532 0
3.5
2
2j
984.621 29.11
1
2k
2l
2.5
2
3.871
4.352
3.279
3.446
2.964
3.11
857.101 36.256 0
904.637 28.966 0
2q
2r
2t
4.5
3
949
837.625 40.379 0
762.261 39.19
52.796 0
0.124
0.212
0.182
0.321
0.125
0.222
3
0
PDB-
2Z5X
crystal
ligand
1.75
733.386 33.499 0
Standard
values/
range
–
130–725 0–6
2–20
-2.0 to
6.5
-3.0 to 1.2 1–8
-1.5 to 1.5 [80 % is
high;
500–2000 7–200 Maximum
is 4
\20 % is
poor
donarHB: hydrogen bond donar; accptHB: hydrogen bond acceptor; metab: number of likely metabolic reactions; QPlogKhsa: prediction of
binding to human serum albumin; QPlogBB: predicted brain/blood partition coefficient; Volume: total solvent accessible volume in cubic
´
˚
angstrom using a probe with 1.4 A radius; PSA: Van der Waals polar surface area of nitrogen and oxygen atoms; Number of violations of
Lipinski’s rule of five
residues, respectively. Further, compound 2i and 2b were
found to possess the conserved interactions (Fig. 4) of the
PDB co-crystal ligand 2Z5X with the amino acid residues
at the binding site to MAO-A protein. It was also evident
from the results that the predicted binding affinity (Glide
scores) of the synthesized derivative showed a close cor-
relation with the in vivo biological activity data, in par-
ticular with the forced swim test of the antidepressant
activity with a very good correlation coefficient of
0.754103 (Table 4; Fig. 5).
series. A polar 4-hydroxyphenyl or pyridin-2-yl substitu-
tion at 3rd position (compounds 2a, 2b and 2q) was
detrimental in eliciting anxiolytic activity among the syn-
thesized derivatives. Also, favorable in silico ADME per-
formance was observed for all the synthesized derivatives
without any significant neurotoxicity. The docking studies
established a fair interaction of the synthesized derivatives
with the MAO-A protein plays a vital role in neurological
disorders such as depression and anxiety. Thus, it may be
concluded that the synthesized 2-pyrazoline derivatives
could be promising candidates for antidepressant and
anxiolytic activities.
Conclusion
In the present study, some new 2-pyrazoline derivatives
were synthesized and evaluated in vivo for their antide-
pressant and anti-anxiety potential. It was observed that the
3,4-dimethoxyphenyl and 4-chlorophenyl substitution at
5th position as well as naphthyl substitution at 3rd position
of the 2-pyrazoline nucleus were crucial for the antide-
pressant activity, as derivatives bearing these substitutions
(2i and 2j) were found to be the most active among the
Experimental
The chemicals and reagents were procured from Sigma
Aldrich and S. D. Fine Chemicals, Mumbai, India, and
were used as such. Solvents were of reagent grade and were
purified and dried by standard procedures. Microwave-as-
sisted synthesis was performed using Raga’s Scientific
Microwave Systems (Ragatech, Pune, Maharashtra, India).
123

Med Chem Res
Fig. 2 Ligand–receptor
interaction diagram of
compound 2i at the binding site
of MAO-A protein (PDB ID:
2Z5X) showing good
antidepressant activity. a 2D
Ligand–receptor interaction
diagram. b 3D Ligand–receptor
interaction diagram
Melting points were determined by open capillary method
and are uncorrected. IR spectra were recorded on Shimadzu
8400S and Perkin Elmer RX1 FTIR spectrophotometers
(Shimadzu Corporation, Japan) using KBr disks and values
performed on Vario EL III Elemental analyzer (Elementar,
Germany).
Chemistry
1
are expressed in cm-1. H NMR spectra were recorded on
Bruker DRX-300 spectrometer (Bruker Instruments Inc.,
USA) at 300 MHz and the chemical shifts are reported in
In the first step, substituted aldehydes and ketones were
reacted in a basic medium to form the substituted chal-
cones, through Claisen-Schmidt condensation, which in the
second step were cyclized into the final derivatives (2a–2t)
using hydrazine hydrate in excess as given in Scheme 1
(Derivatives 2e and 2s could not be synthesized by both,
1
parts per million (d value), taking TMS (d 0 ppm for H
NMR) as the internal standard. Mass spectra were recorded
on Agilent 6520-QTOF LCMS mass spectrometer instru-
ment using ESI technique. Elemental analysis was
123

Med Chem Res
Fig. 3 Ligand–receptor
interaction diagram of
compound 2b at the binding site
of MAO-A protein (PDB ID:
2Z5X) showing good anti-
anxiety activity. a 2D Ligand–
receptor interaction diagram.
b 3D Ligand–receptor
interaction diagram
conventional and microwave methods). Progress of the
reactions was monitored by thin layer chromatography
(TLC) on precoated silica gel G plates, using iodine vapors
and UV light as the visualizing agents.
20–36 h, with occasional shaking. After 36 h, it was
poured into ice-cold water and then neutralized to pH 2
using 6 N hydrochloric acid. The yellow colored interme-
diates (chalcones) obtained were filtered, washed, dried,
and re-crystallized from methanol (Jayaprakash et al.,
2008; Karuppasamy et al., 2010; Mishra and Sasmal, 2011;
Sahoo et al., 2010; Stirrett et al., 2008).
General procedure for the synthesis of chalcones
Conventional method To a solution of different ketones
(0.01 M) and suitably substituted aldehydes (0.01 M) in
ethanol (10 mL), aqueous solution of potassium hydroxide
(60 %) was added drop wise and with continuous stirring at
0 °C over a period of 15 min–2 h. The reaction mixture
was stirred at a low temperature (0–10 °C) for about
Microwave-assisted method Different aromatic/hetero-
aromatic ketones (0.01 M) and suitably substituted alde-
hydes (0.01 M) were reacted, in the presence of hydro-
alcoholic solution of KOH (60 %, 10 mL), under micro-
wave irradiation (MWI: 120–280 W, 60–230 s). The
reaction mixture was poured into ice-cold water and then
123

Med Chem Res
Fig. 4 Compounds 2i (spring
green color) and 2b (plum
color) showing conserved
H-bond interactions of 2Z5X
crystal ligand (orange color) at
the receptor site of MAO-A
protein 2Z5X, showing good
antidepressant and anti-anxiety
activities (Color figure online)
Table 4 Correlation of in vivo biological activity (antidepressant and anti-anxiety) data with Glide scores
Correlation coefficient in relation with
Antidepressant activity
Anti-anxiety activity
Duration of
Immobility
in FST (s)
Duration of
immobility
in TST (s)
Number of
entries in
closed
Time spent in
closed arm (s)
Glide score
0.754103
0.47171
-0.24201
0.28982
neutralized to pH 2 using 6 N hydrochloric acid. The
yellow colored intermediates (chalcones) obtained were
filtered, washed, dried, and re-crystallized from methanol
(Jayashree et al., 2008).
ethanol/acetone/ethyl acetate to afford the respective
pyrazolines (Chawla et al., 2010; Insuasty et al., 2011).
Characterization of the synthesized 3,5-disubstituted-2-
pyrazoline derivatives
General procedure for the synthesis of 3,5-disubstituted-2-
pyrazoline derivatives
Intermediates were characterized by TLC, melting point
and mass spectra, while final derivatives were subjected to
complete physicochemical (Table 1) and spectral charac-
terization and the values were found to be in accordance
with the proposed derivatives.
Conventional method Appropriate chalcone was treated
with 10 times excess of hydrazine hydrate (80 %) in dry
ethanol and refluxed for 3–6 h. The hot reaction mixture
was then poured into ice-cold water. The separated out
solid was filtered, washed, dried and re-crystallized from
ethanol/acetone/ethyl acetate to afford the respective
pyrazoline (Karuppasamy et al., 2010; Mishra and Sasmal,
2011).
4-[5-(3,4-Dimethoxy-phenyl)-4,5-dihydro-1H-pyrazol-3-
yl]-phenol (2a) IR (KBr, cm-1): N–H str (3278), C–H Ar
(3044), C=N str (1597), C–H deform (1429). 1H NMR
(DMSO, d ppm): 2.35–3.00 (dd, Jab: 13.29 Hz, Jax:
4.21 Hz, 1H, Ha), 3.60–3.68 (dd, Jab: 5.60 Hz, Jbx:
11.33 Hz, 1H, Hb), 3.72 (m, 6H), 6.59–6.66 (m, 4H, Ar),
7.22 (s, 2H, Ar), 6.88 (s, 1H, NH), 9.27 (s, 1H, OH). 13C
NMR (CDCl3, ppm): 41.7 (CH2 pyrazoline), 48.9 (CH
pyrazoline), 57.2 (2CH3 aliphatic), 114.3–116.1 (4CH
aromatic), 121.6 (CH aromatic), 125.7 (2C aromatic),
Microwave-assisted method Appropriate chalcone was
treated with 10 times excess of hydrazine hydrate (80 %) in
dry ethanol, under the exposure of microwave irradiation
(MWI: 240–350 W; 50–400 s). The reaction mixture was
then poured into ice-cold water. The separated out solid
was filtered, washed, dried and re-crystallized from
123

Med Chem Res
Fig. 5 Graphs correlating in vivo biological activity (antidepressant and anti-anxiety) data with Glide scores. a Glide score versus FST. b Glide
score versus TST. c Glide score versus number of entries in closed arms. d Glide score versus time spent in closed arms
Conventional Method
Conventional Method
C2H5OH, KOH
Reflux, 3-6 hrs.
Hb
Ha
O
0-10 oC, Stir, 24-48 hrs.
R
Hx
CH3
O
R'
H
C
R-CHO
+
R'
NH2NH2.H2O
R
C
H
R'
N
NH
MWI: 120-280W;
60-230s
3,5-Disubstituted
Aldehyde
Ketone
MWI: 240-350W;
50-400s
Chalcones
1a-1t
2-pyrazoline derivatives
(2a-2t)
Microwave Method
OCH3
Microwave Method
S
O
Cl
Where R:
and R':
OCH3
Cl
OH
N
Scheme 1 Synthesis of 3,5-disubstituted-2-pyrazoline derivatives
123

Med Chem Res
128.4–130.1 (3CH aromatic), 151.6 (3C aromatic), 156.8
(C pyrazoline). MS (m/z): 299.2 (M ? 1). Anal. Calcd. for
C17H18N2O3: C, 68.44; H, 6.08; N, 9.39. Found: C, 68.13;
H, 6.55; N, 10.30.
(s, 1H, NH), 7.26–7.65 (s, 8H, Ar). 13C NMR (DMSO,
ppm): 44.7 (CH2 pyrazoline), 53.4 (CH pyrazoline),
128.7–129.4 (8CH aromatic), 129.7 (2C aromatic), 135.1
(2C aromatic), 160.1 (C pyrazoline). MS (m/z): 288.8
(M ? 1). Anal. Calcd. for C15H12Cl2N2: C, 61.87; H, 4.15;
N, 9.62. Found: C, 61.70; H, 4.56; N, 9.41.
4-[5-(4-Chloro-phenyl)-4,5-dihydro-1H-pyrazol-3-yl]-phe-
nol (2b) IR (KBr, cm-1): N–H str (3359), C–H Ar
(3046), C=N str (1572), C–H deform (1461). 1H NMR
(DMSO, d ppm): 2.76–3.02 (dd, Jab: 17.11 Hz, Jax:
3.62 Hz, 1H, Ha), 3.82–3.89 (dd, Jab: 3.15 Hz, Jbx:
12.29 Hz, 1H, Hb), 7.05 (s, 1H, NH), 6.94–7.38 (d, 8H,
Ar), 9.22 (s, 1H, OH). 13C NMR (CDCl3, ppm): 42.9 (CH2
pyrazoline), 49.1 (CH pyrazoline), 113.9–115.1 (2CH
aromatic), 120.2 (C aromatic), 141.6 (C aromatic),
129.2–131.1 (6CH aromatic), 133.8 (C aromatic), 154.7 (C
pyrazoline), 159.5 (C aromatic). MS (m/z): 272.8 (M?).
Anal. Calcd. for C15H13ClN2O: C, 66.06; H, 4.80; N,
10.27. Found: C, 65.97; H, 5.03; N, 10.28.
3-(4-Chloro-phenyl)-5-furan-2-yl-4,5-dihydro-1H-pyrazole
(2g) IR (KBr, cm-1): N–H str (3315), C–H Ar (3100),
1
C=N str (1586), C–H deform (1397). H NMR (CDCl3, d
ppm): 3.01–3.22 (dd, Jab: 17.04 Hz, Jax: 8.11 Hz, 1H, Ha),
3.73–3.90 (dd, Jab: 6.25 Hz, Jbx: 13.38 Hz, 1H, Hb),
5.97–6.04 (dd, Jax: 11.10 Hz, Jbx: 15.60 Hz, 1H, Hx), 6.18
(s, 2H, Ar), 7.20 (s, 1H, NH), 7.26–7.42 (m, 5H, Ar). 13C
NMR (CDCl3, ppm): 42.7 (CH2 pyrazoline), 49.5 (CH
pyrazoline), 106.3–107.0 (2CH furan), 129.1–130.8 (4CH
aromatic), 135.2 (C aromatic), 141.4 (CH furan), 153.6 (C
pyrazoline), 158.2 (C furan). MS (m/z): 247.0 (M ? 1)
Anal. Calcd. for C13H11ClN2O: C, 63.29; H, 4.49; N,
11.36. Found: C, 63.13; H, 4.52; N, 11.44.
4-(5-Furan-2-yl-4,5-dihydro-1H-pyrazol-3-yl)-phenol (2c)
IR (KBr, cm-1): N–H str (3283), C–H Ar (3112), C=N str
1
(1600), C–H deform (1385). H NMR (DMSO, d ppm):
3-(4-Chloro-phenyl)-5-thiophen-2-yl-4,5-dihydro-1H-pyra-
zole (2h) IR (KBr, cm-1): N–H str (3382), C–H Ar
(3110), C=N str (1585), C–H deform (1347). 1H NMR
(CDCl3, d ppm): 3.12–3.28 (dd, Jab: 16.53 Hz, Jax: 6.35 Hz,
1H, Ha), 3.69–3.90 (dd, Jab: 4.16 Hz, Jbx: 13.76 Hz, 1H, Hb),
6.22–6.29 (dd, Jax: 10.38 Hz, Jbx: 17.36 Hz, 1H, Hx),
6.60–6.73 (m, 3H, Ar), 7.20–7.73 (s, 4H, Ar), 7.05 (s, 1H,
NH). 13C NMR (CDCl3, ppm): 40.5 (CH2 pyrazoline), 50.23
(CH pyrazoline), 125.1–125.7 (3CH thiophene), 128.16 (C
aromatic), 130.9–131.5 (4CH aromatic), 139.10 (C thio-
phene), 153.6 (C pyrazoline). MS (m/z): 363.0 (M ? 1).
Anal. Calcd. for C13H11ClN2S: C, 59.42; H, 4.22; N, 10.66.
Found: C, 60.02; H, 4.19; N, 10.88.
3.15–3.29 (dd, Jab: 16.74 Hz, Jax: 2.99 Hz, 1H, Ha),
3.67–3.94 (dd, Jab: 3.02 Hz, Jbx: 14.23 Hz, 1H, Hb),
6.41–6.75 (dd, Jax: 9.86 Hz, Jbx: 15.66 Hz, 1H, Hx), 7.03 (s,
1H, NH), 6.70–7.16 (dd, 4H, aromatic), 6.00–6.26 (s, 2H,
Ar), 8.37 (s, 1H, OH). 13C NMR (DMSO, ppm): 41.5 (CH2
pyrazoline), 49.6 (CH pyrazoline), 79.2 (CH2, methylene),
102.7–109.8 (4CH aromatic), 118.2 (2CH benzene), 130.7
(2CH aromatic), 136.2 (CH furan), 157.3 (2C pyrazoline,
furan), 154.4 (CH pyrazoline). MS (m/z): 229.13 (M?). Anal.
Calcd. for C13H12N2O2: C, 68.41; H, 5.30; N, 12.27. Found:
C, 68.55; H, 5.33; N, 12.24.
4-(5-Thiophen-2-yl-4,5-dihydro-1H-pyrazol-3-yl)-phenol
(2d) IR (KBr, cm-1): N–H str (3452), C–H Ar (3089),
5-(3,4-Dimethoxy-phenyl)-3-naphthalen-1-yl-4,5-dihydro-
1H-pyrazole (2i) IR (KBr, cm-1): N–H str (3345), C–H
1
Ar (3056), C=N str (1625), C–H deform (1419). H NMR
1
C=N str (1532), C–H deform (1411). H NMR (CDCl3, d
ppm): 3.07–3.24 (dd, Jab: 16.75 Hz, Jax: 4.06 Hz, 1H, Ha),
3.62–3.96 (dd, Jab: 3.11 Hz, Jbx: 12.33 Hz, 1H, Hb),
6.12–6.43 (dd, Jax: 11.27 Hz, Jbx: 16.93 Hz, 1H, Hx),
6.79–6.85 (s, 5H, Ar), 7.18 (s, 1H, Ar), 8.36 (s, 1H, NH),
9.55 (s, 1H, OH). 13C NMR (CDCl3, ppm): 41.5 (CH2
pyrazoline), 49.6 (CH pyrazoline), 115.3 (2CH aromatic),
123.2 (CH benzene), 124.1–126.8 (3CH thiophene), 130.7
(2CH aromatic), 138.2 (C thiophene), 159.3 (C aromatic),
154.9 (CH pyrazoline). MS (m/z): 243.0 (M - 1). Anal.
Calcd. for C13H12N2OS: C, 63.91; H, 4.95; N, 11.47.
Found: C, 63.79; H, 5.03; N, 11.52.
(CDCl3, d ppm): 3.03–3.10 (dd, Jab: 16.34 Hz, Jax:
5.02 Hz, 1H, Ha), 3.62–3.94 (dd, Jab: 6.76 Hz, Jbx:
10.47 Hz, 1H, Hb), 6.55–6.74 (dd, Jax: 11.22 Hz, Jbx:
16.43 Hz, 1H, Hx), 3.85 (s, 6H), 6.40–6.55 (s, 3H, Ar),
7.25–7.76 (m, 7H, Ar), 8.12 (s, 1H, NH). 13C NMR
(CDCl3, ppm): 40.4 (CH2 pyrazoline), 51.5 (C pyrazoline),
60.3 (2CH3 aliphatic), 114.6–117.2 (3CH aromatic),
126.4–129.1 (6CH aromatic), 130.6–131.2 (4C aromatic),
157.1 (C pyrazoline). MS (m/z): 333.3 (M ? 1). Anal.
Calcd. for C21H20N2O2: C, 75.88; H, 6.06; N, 8.43. Found:
C, 75.70; H, 5.91; N, 8.40.
3,5-Bis-(4-chloro-phenyl)-4,5-dihydro-1H-pyrazole (2f)
IR (KBr, cm-1): N–H str (32777), C–H Ar (2991), C=N str
5-(4-Chloro-phenyl)-3-naphthalen-1-yl-4,5-dihydro-1H-
pyrazole (2j) IR (KBr, cm-1): N–H str (3266), C–H Ar
(2994), C=N str (1635), C–H deform (1421). 1H NMR
(CDCl3, d ppm): 2.11 (s, 3H,), 3.00–3.17 (dd, Jab:
15.28 Hz, Jax: 4.77 Hz, 1H, Ha), 3.74–3.89 (dd, Jab:
1
(1540), C–H deform (1387). H NMR (DMSO, d ppm):
3.10–3.18 (dd, Jab: 17.55 Hz, Jax: 3.88 Hz, 1H, Ha),
3.76–3.98 (dd, Jab: 4.03 Hz, Jbx: 11.64 Hz, 1H, Hb),
6.05–6.21 (dd, Jax: 10.38 Hz, Jbx: 17.36 Hz, 1H, Hx), 7.09
123

Med Chem Res
6.33 Hz, Jbx: 11.27 Hz, 1H, Hb), 7.30–8.00 (m, 11H, Ar),
8.12 (s, 1H, NH). 13C NMR (CDCl3, ppm): 40.4 (CH2
pyrazoline), 51.5 (C pyrazoline), 127.2–128.6 (11CH aro-
matic), 136.6–138.2 (4C aromatic), 157.1 (C pyrazoline).
MS (m/z): 307.1 (M ? 1). Anal. Calcd. for C19H15ClN2: C,
74.38; H, 4.93; N, 9.13. Found: C, 74.40; H, 4.96; N, 8.99.
3.41 Hz, 1H, Ha), 3.68–3.90 (dd, Jab: 4.01 Hz, Jbx:
16.23 Hz, 1H, Hb), 6.44–6.51 (dd, Jax: 3.28 Hz, Jbx:
17.46 Hz, 1H, Hx), 7.0–7.15 (m, 4H, Ar), 7.58 (s, 1H, NH),
7.63–7.80 (m, 4H, Ar). 13C NMR (CDCl3, ppm): 46.3 (CH2
pyrazoline), 52.5 (C pyrazoline), 126.4–128.7 (5CH aro-
matic), 131.5–133.2 (4CH pyridine), 153.2 (C pyr-
idine),139.6 (C aromatic), 155.4 (C pyrazoline). MS (m/z):
258.0 (M ? 1, M - 1). Anal. Calcd. for C14H12ClN3: C,
65.25; H, 4.69; N, 16.30. Found: C, 65.02; H, 4.98; N,
16.42.
5-Furan-2-yl-3-naphthalen-1-yl-4,5-dihydro-1H-pyrazole
(2k) IR (KBr, cm-1): N–H str (3281), C–H Ar (2958),
1
C=N str (1623), C–H deform (1437). H NMR (CDCl3, d
ppm): 3.11–3.26 (dd, Jab: 15.09 Hz, Jax: 4.70 Hz, 1H, Ha),
3.77–3.91 (dd, Jab: 6.35 Hz, Jbx: 11.54 Hz, 1H, Hb),
6.32–6.44 (dd, Jax: 13.38 Hz, Jbx: 18.57 Hz, 1H, Hx),
6.12–6.23 (d, 2H, Ar), 7.25–7.76 (m, 7H, Ar), 8.12 (s, 1H,
NH). 13C NMR (CDCl3, ppm): 40.4 (CH2 pyrazoline), 51.5
(C pyrazoline), 108.6 (2CH fruran), 125.8–127.1 (7CH
aromatic), 130.6–131.2 (3C aromatic), 138.9 (CH fruran),
157.1 (C pyrazoline), 161.5 (C furan). MS (m/z): 263.1
(M ? 1). Anal. Calcd. for C17H14N2O: C, 77.84; H, 5.38;
N, 10.68. Found: C, 77.72; H, 5.14; N, 10.54.
2-(5-Thiophen-2-yl-4,5-dihydro-1H-pyrazol-3-yl)-pyridine
(2t) IR (KBr, cm-1): N–H str (3430), C–H Ar (3136),
1
C=N str (1615), C–H deform (1381). H NMR (CDCl3, d
ppm): 3.02–3.05 (dd, Jab: 17.12 Hz, Jax: 3.41 Hz, 1H, Ha),
3.68–3.90 (dd, Jab: 4.01 Hz, Jbx: 16.23 Hz, 1H, Hb),
6.44–6.51 (dd, Jax: 3.28 Hz, Jbx: 17.46 Hz, 1H, Hx),
6.5–6.83 (m, 3H, Ar), 7.58 (s, 1H, NH), 7.79–8.03 (m, 4H,
Ar). 13C NMR (CDCl3, ppm): 46.1 (CH2 pyrazoline), 52.5
(C pyrazoline), 124.4–126.7 (3CH thiophene), 125.2–128.6
(2CH pyridine), 139.6 (C thiophene), 153.2 (C pyridine),
155.4 (C pyrazoline). MS (m/z): 229.8 (M?). Anal. Calcd.
for C12H11N3S: C, 62.86; H, 4.84; N, 18.33. Found: C,
62.90; H, 4.98; N, 18.42.
3-Naphthalen-1-yl-5-thiophen-2-yl-4,5-dihydro-1H-pyra-
zole (2l) IR (KBr, cm-1): N–H str (3292), C–H Ar
(3047), C=N str (1560), C–H deform (1353). 1H NMR
(CDCl3, d ppm): 3.09–3.12 (dd, Jab: 17.41 Hz, Jax:
3.65 Hz, 1H, Ha), 3.57–3.84 (dd, Jab: 5.18 Hz, Jbx:
16.42 Hz, 1H, Hb), 6.43–6.74 (dd, Jax: 3.38 Hz, Jbx:
11.36 Hz, 1H, Hx), 6.50–6.73 (d, 3H, Ar), 7.41–7.88 (m,
7H, Ar), 8.11 (s, 1H, NH). 13C NMR (CDCl3, ppm): 40.4
(CH2 pyrazoline), 51.5 (C pyrazoline), 123.6–124.2 (3CH
thiophene), 127.2–129.6 (7CH aromatic), 132.5–134.1 (3C
aromatic), 139.2 (CH thiophene), 153.7 (C pyrazoline). MS
(m/z): 279.1 (M ? 1). Anal. Calcd. for C17H14N2S: C,
73.35; H, 5.07; N, 10.06. Found: C, 73.10; H, 5.02; N,
10.37.
Biological evaluation
Animals were procured from the Animal House, Faculty of
Pharmacy, BBDNIIT, Lucknow, U.P., India, and housed in
polypropylene cages with steel net, in a temperature con-
trolled room under standard living conditions of;
26 2 °C and relative humidity of; 55 5 % with reg-
ular 12-h light and 12-h dark cycles and allowed free
access to standard laboratory food and water. All the ani-
mals were treated humanely in accordance with the
guidelines laid down by the Institutional Animal Ethics
Committee (IAEC). The biological activities were
approved by the IAEC with protocol number BBDNIIT/
IAEC/008/2014. The mice were divided into 14 groups,
and each group consisted of 6 animals for each dose level
in the study.
2-[5-(3,4-Dimethoxy-phenyl)-4,5-dihydro-1H-pyrazol-3-
yl]-pyridine (2q) IR (KBr, cm-1): N–H str (3321), C–H
1
Ar (2955), C=N str (1647), C–H deform (1473). H NMR
(DMSO, d ppm): 2.96–3.10 (dd, Jab: 17.12 Hz, Jax:
3.60 Hz, 1H, Ha), 3.69–3.88 (dd, Jab: 3.14 Hz, Jbx:
12.03 Hz, 1H, Hb), 6.05–6.21 (dd, Jax: 11.23 Hz, Jbx:
14.36 Hz, 1H, Hx), 3.49 (s, 6H), 6.44–6.62 (m, 3H, Ar),
7.19 (s, 1H, NH), 7.69–8.10 (m, 4H, Ar). 13C NMR
(CDCl3, ppm): 42.2 (CH2 pyrazoline), 50.1 (CH pyrazo-
line), 62.5 (CH3 aliphatic), 117.2–120.8 (3CH aromatic),
124.2–126.5 (3CH pyridine), 146.4–149.7 (3C aromatic),
152.6–153.2 (2C pyridine), 159.3 (C pyrazoline). MS (m/
z): 283.8 (M ? 1). Anal. Calcd. for C16H17N3O2: C, 67.83;
H, 6.05; N, 14.83. Found: C, 68.05; H, 5.95; N, 14.11.
Antidepressant activity
The antidepressant activity of the synthesized derivatives
was evaluated by Porsolt’s behavioral despair or forced
swim test (FST) and tail suspension test (TST) in mice.
These behavioral tests are used to evaluate the efficacy of
antidepressant treatments and in predicting the activity of a
wide variety of antidepressants, such as MAO inhibitors
and atypical antidepressants (Porsolt, 1981). They have
good predictive value for the assessment of antidepressant
potency in humans (Willner and Mitchell, 2002).
2-[5-(4-Chloro-phenyl)-4,5-dihydro-1H-pyrazol-3-yl]-pyr-
idine (2r) IR (KBr, cm-1): N–H str (3400), C–H Ar
(3119), C=N str (1635), C–H deform (1374). 1H NMR
(CDCl3, d ppm): 3.02–3.05 (dd, Jab: 17.12 Hz, Jax:
123

Med Chem Res
Porsolt’s behavioral despair or forced swim test (FST)
in mice
Anti-anxiety activity
The maze model in mice (Vogel, 2002) is widely used for
the evaluation of anti-anxiety activity. Out of the many
possibilities to modify maze tests, water maze, the Y-maze,
the radial maze, and the elevated plus maze have found
acceptance in many laboratories. The test has been pro-
posed for selective identification of anxiolytic and anxio-
genic drugs. Anxiolytic compounds, by decreasing anxiety,
increase the open arm exploration time and decrease the
time spent in closed arm; anxiogenic compounds have the
opposite effect. The values of the treated groups are reg-
istered and expressed as percentage of controls. Benzodi-
azepines and valproates decrease motor activity and
increase open arm exploratory time.
The synthesized compounds were screened for their
antidepressant activity using Porsolt’s behavioral despair
test FST (Porsolt et al., 1977; Vogel, 2002). It was sug-
gested that mice or rats forced to swim in a restricted space
from which they cannot escape are induced to a charac-
teristic behavior of immobility. This behavior reflects a
state of despair which can be reduced by several agents,
which are therapeutically effective in human depression.
Duration of immobility is measured in controls and animals
treated with various doses of a test drug or standard.
Tail suspension test (TST) in mice
Another method, TST (Steru et al., 1985; Vogel, 2002) has
been described as a facile means of evaluating potential
antidepressants. TST, a novel test procedure for antide-
pressants, was designed in which a mouse is suspended by
the tail from a lever and the movements of the animal are
recorded. The total duration of the test (6 min) can be
divided into periods of agitation and immobility. Antide-
pressant drugs decrease the duration of immobility and, if
coupled with measurement of locomotor activity in different
conditions, the test can separate the locomotor stimulant
doses from antidepressant doses. The main advantages of
this procedure are the use of a simple, objective test situa-
tion, the concordance of the results with the validated ‘‘be-
havioral despair’’ test from Porsolt and the sensitivity to a
wide range of drug doses. The immobility displayed by
rodents, when subjected to an unavoidable and inescapable
stress, has been hypothesized to reflect behavioral despair
which in turn may reflect depressive disorders in humans.
Clinically effective antidepressants reduce the immobility
that mice display after active and unsuccessful attempts to
escape, when suspended by the tail. The percentage of ani-
mals showing the passive behavior is counted and compared
with the vehicle treated controls.
Elevated plus-maze test
The elevated plus-maze is used to determine the mouse’s
unconditioned response to a potentially dangerous envi-
ronment and the anxiety-related behavior is measured by
the degree to which the mouse avoids the unenclosed arms
of the maze. It is a standard test of fear and anxiety for
which the animal was placed in the center of an elevated
4-arm maze, in which 2 arms are open 50 9 10 9 40 cm
and 2 arms are enclosed 50 9 10 9 40 cm, with an open
roof. The two open arms were opposite to each other. The
maze was elevated to a height of 50 cm. One hour after
oral administration of the standard drug (Diazepam at a
dose of 2 mg/kg b.w.), test compound (at doses; 50 and
100 mg/kg b.w.) and control (0.5 % aqueous CMC sus-
pension), the mice were placed in the center of the maze,
facing one of the enclosed arms. During a 6-min test per-
iod, the following observations were recorded: time spent
in the enclosed arms and total number of the arm entries.
The test is rapid and sensitive to the effects of both anxi-
olytic and anxiogenic agents, anxiolytic agents decreasing,
and anxiogenic agents increasing the amount of time spent
in closed arms; anxiolytic agents increasing and anxiogenic
agents decreasing the number of entries (Lister, 1987;
Pellow et al., 1985).
The synthesized compounds (50 and 100 mg/kg b.w.)
and imipramine (10 mg/kg b.w.), a reference antidepres-
sant drug, were suspended separately in a 0.5 % aqueous
suspension of CMC. The drugs were administered orally in
a standard volume of 0.5 ml/20 g body weight, 1 h prior to
the test. Control animals received 0.5 % aqueous suspen-
sion of CMC. Then, the mice were dropped individually
into the Plexiglas cylinder and left in water for 6 min. After
the first 2 min of the initial vigorous struggling, the animals
were immobile. A mice were judged immobile if it floated
in water in an upright position and made only slight
movements in order to prevent sinking. The duration of
immobility was recorded during the last 4 min of the 6-min
test.
Neurotoxicity study (Rotarod test)
The rotarod test is used to evaluate the activity of drugs
interfering with motor coordination, i.e., neurotoxicity. In
1956, Dunham and Miya suggested that the skeletal muscle
relaxation induced by a test compound could be evaluated
by testing the ability of mice or rats to remain on a
revolving rod. Male mice, with a weight between 20 and
30 g, undergo a pretest on the apparatus. Only those ani-
mals which have demonstrated their ability to remain on
123

Med Chem Res
Statistical analysis
the revolving rod for at least 1 min are used for the test.
The test compounds are administered intraperitoneally or
orally. One hour after the oral administration of the test
compounds/control, the mice are placed for 1 min on the
rotating rod. Normal mice remain on a rod rotating at this
speed indefinitely. Neurologic toxicity was defined as the
failure of the animal to remain on the rod for 1 min at the
tested doses. The number of animals falling from the roller
during this time is counted. The dose which impairs the
ability of 50 % of the mice to remain on the revolving rod
is considered the endpoint (Vogel, 2002).
All the values of the experimental results are expressed as
mean SD and analyzed by one-way ANOVA followed
by Dunnett’s test for the possible significant (P \ 0.05)
identification between various groups. Statistical analysis
was carried out using Graph Pad Prism 5.0 (Graph Pad
Software, San Diego, CA).
Acknowledgments We express our sincere gratitude to Central
Drugs Research Institute (CDRI), Lucknow, India, for providing the
library and sophisticated analytical instrument facilities. Authors are
thankful to the All India Council for Technical Education (AICTE),
New Delhi, India, for providing grant under the Research Promotion
Scheme (RPS), through which the computational software facility has
been made available at the host institute. We also acknowledge the
technical support team/application scientists of Schrodinger Inc. for
their help during computational studies.
Molecular docking studies
In order to gain structural insights into the binding mode of
the ligand with biological target, all the synthesized com-
pounds were computationally docked into the MAO-A
enzyme crystal structure (PDB ID: 2Z5X) using GLIDE.
The MAO protein crystal structure used for molecular
docking was retrieved from the Protein Data Bank (PDB)
and subsequently optimized and minimized with the
‘‘protein preparation wizard’’ workflow. The ligands were
built using Maestro 9.3 build panel and prepared by Lig-
Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.
References
¨
Prep 2.5 version v25111(Schrodinger, LLC, USA) appli-
Abdel-Wahab BF, Abdel-Aziz HA, Ahmed EM (2009) Synthesis and
antimicrobial evaluation of 1-(benzofuran-2-yl)-4-nitro-3-
arylbutan-1-ones and 3-(benzofuran-2-yl)-4,5-dihydro-5-aryl-1-
cation that uses optimized potential liquid simulations
(OPLS) 2005 force field and it gave the corresponding
energy minima 3D conformers of the ligands. The default
settings were used for all the other parameters. All ligand
atoms, but no protein atoms, were allowed to move during
the calculations. To validate the docking protocol, re-
docking experiment was performed in which the ligand
conformation of co-crystal ligand was extracted from the
crystal structure of the corresponding MAO-A protein/li-
gand complex and later docked back into the binding
pocket. GLIDE was able to perfectly reproduce the
experimental position of the ligand, confirming the ability
of the method to accurately predict the binding
conformation.
[4-(aryl)-1,3-thiazol-2-yl]-1H-pyrazoles. Eur
44:2632–2635
J Med Chem
Acharya BN, Saraswat D, Tiwari M, Shrivastava AK, Ghorpade R,
Bapna S, Kaushik MP (2010) Synthesis and antimalarial evaluation
of 1,3,5-trisubstituted pyrazolines. Eur J Med Chem 45:430–438
Amrein R, Martin JR, Cameron AM (1999) Moclobemide in patients
with dementia and depression. Adv Neurol 80:509–519
Bhat AR, Athar F, Azam A (2009) Bis-pyrazolines: synthesis,
characterization and antiamoebic activity as inhibitors of growth
of Entamoeba histolytica. Eur J Med Chem 44:426–431
Bilgin AA, Palaska E, Sunal R (1993) Studies on the synthesis and
antidepressant activity of some 1-thiocarbamoyl-3,5-diphenyl-2-
pyrazolines. Arzneimittelforschung 43:1041–1044
Brown CS, Kent TA, Bryant SG, Gevedon RM, Campbell JL,
Felthous AR, Barratt ES, Rose RM (1989) Blood platelet uptake
of serotonin in episodic aggression. Psychiatry Res 27:5–12
Budakoti A, Bhat AR, Azam A (2009) Synthesis of new 2-(5-
substituted-3-phenyl-2-pyrazolinyl)-1,3-thiazolino[5,4-b]quinox-
aline derivatives and evaluation of their antiamoebic activity.
Eur J Med Chem 44:1317–1325
In silico prediction of pharmacokinetic properties
Nearly 40 % of drug candidates fail in clinical trials due to
poor ADME (absorption, distribution, metabolism, and
excretion) properties. These late-stage failures contribute
significantly to the rapidly escalating cost of new drug
development. The ability to detect the problematic candi-
dates early can dramatically reduce the amount of wasted
time and resources, and streamline the overall development
Cesura AM, Pletscher A (1992) The new generation of monoamine
oxidase inhibitors. Prog Drug Res 38:171–297
Chawla R, Sahoo U, Arora A, Sharma PC, Radhakrishnan V (2010)
Microwave assisted synthesis of some novel 2-pyrazoline deriva-
tives as possible antimicrobial agents. Acta Polo Pharm 67:55–61
Chimenti F, Bolasco A, Manna F, Secci D, Chimenti P, Befani O,
Turini P, Giovannini V, Mondovi B, Cirilli R, La Torre F (2004)
Synthesis and selective inhibitory activity of 1-acetyl-3,5-
diphenyl-4,5-dihydro-(1H)-pyrazole derivatives against mono-
amine oxidase. J Med Chem 47:2071–2074
¨
process. QikProp, version 3.5, Schrodinger, LLC, New
York, NY, 2012, program was used for in silico prediction
of pharmacokinetic properties of the synthesized
compounds.
Chimenti F, Carradori S, Secci D, Bolasco A, Bizzarri B, Chimenti P,
Granese A, Yanez M, Orallo F (2010) Synthesis and inhibitory
activity against human monoamine oxidase of N1-thiocarbamoyl-
123

Med Chem Res
3,5-di(hetero)aryl-4,5-dihydro-(1H)-pyrazole derivatives. Eur
Med Chem 45:800–804
Congiu C, Onnis V, Vesci L, Castorina M, Pisano C (2010) Synthesis
and in vitro antitumor activity of new 4,5-dihydropyrazole
derivatives. Bioorg Med Chem 18:6238–6248
Dawane BS, Konda SG, Mandawad GG, Shaikh BM (2010)
Poly(ethylene glycol) (PEG-400) as an alternative reaction
solvent for the synthesis of some new 1-(4-(40-chlorophenyl)-
2-thiazolyl)-3-aryl-5-(2-butyl-4-chloro-1H-imidazol-5yl)-2-
pyrazolines and their in vitro antimicrobial evaluation. Eur J
Med Chem 45:387–392
J
Kaplancikli ZA, Ozdemir A, Turan-Zitouni G, Altintop MD, Can OD
(2010) New pyrazoline derivatives and their antidepressant
activity. Eur J Med Chem 45:4383–4387
Karuppasamy M, Mahapatra M, Yabanoglu S, Ucar G, Sinha BN,
Basu A, Mishra N, Sharon A, Kulandaivelu U, Jayaprakash V
(2010) Development of selective and reversible pyrazoline based
MAO-A inhibitors: synthesis, biological evaluation and docking
studies. Bioorg Med Chem 18:1875–1881
Khode S, Maddi V, Aragade P, Palkar M, Ronad PK, Mamledesai S,
Thippeswamy AH, Satyanarayana D (2009) Synthesis and
pharmacological evaluation of a novel series of 5-(substi-
tuted)aryl-3-(3-coumarinyl)-1-phenyl-2-pyrazolines as novel
anti-inflammatory and analgesic agents. Eur J Med Chem
44:1682–1688
Lidstrom P, Tierney J, Wathey B, Westman J (2001) Microwave
assisted organic synthesis—a review. Tetrahedron 57:9225–9283
Lister RG (1987) The use of a plus-maze to measure anxiety in the
mouse. Psychopharmacology 92:180–185
Diamond MS (2009) Progress on the development of therapeutics
against West Nile virus. Antiviral Res 83:214–227
Fioravanti R, Bolasco A, Manna F, Rossi F, Orallo F, Ortuso F,
Alcaro S, Cirilli R (2010) Synthesis and biological evaluation of
N-substituted-3,5-diphenyl-2-pyrazoline
derivatives
as
cyclooxygenase (COX-2) inhibitors. Eur J Med Chem 45:
6135–6138
Foley P, Gerlach M, Youdim MB, Riederer P (2000) MAO-B
inhibitors: multiple roles in the therapy of neurodegenerative
disorders? Parkinsonism Relat Disord 6:25–47
Gareri P, Falconi U, De Fazio P, De Sarro G (2000) Conventional and
new antidepressant drugs in the elderly. Prog Neurobiol
61:353–396
Maccioni E, Alcaro S, Orallo F, Cardia MC, Distinto S, Costa G,
Yanez M, Sanna ML, Vigo S, Meleddu R, Secci D (2010)
Synthesis of new 3-aryl-4,5-dihydropyrazole-1-carbothioamide
derivatives. An investigation on their ability to inhibit mono-
amine oxidase. Eur J Med Chem 45:4490–4498
Manna F, Chimenti F, Bolasco A, Bizzarri B, Befani O, Pietrangeli P,
Mondovi B, Turini P (1998) Inhibitory effect of 1,3,5-triphenyl-
4,5-dihydro-(1H)-pyrazole derivatives on activity of amine
oxidases. J Enzyme Inhib 13:207–216
Manna F, Chimenti F, Bolasco A, Secci D, Bizzarri B, Befani O, Turini
P, Mondovi B, Alcaro S, Tafi A (2002) Inhibition of amine
oxidases activity by 1-acetyl-3,5-diphenyl-4,5-dihydro-(1H)-pyra-
zole derivatives. Bioorg Med Chem Lett 12:3629–3633
Meyer JH, Ginovart N, Boovariwala A, Sagrati S, Hussey D, Garcia
A, Young T, Praschak-Rieder N, Wilson AA, Houle S (2006)
Elevated monoamine oxidase a levels in the brain: an explana-
tion for the monoamine imbalance of major depression. Arch
Gen Psychiatry 63:1209–1216
Mishra N, Sasmal D (2011) Development of selective and reversible
pyrazoline based MAO-B inhibitors: virtual screening, synthesis
and biological evaluation. Bioorg Med Chem Lett 21:1969–1973
Mitoma J, Ito A (1992) Mitochondrial targeting signal of rat liver
monoamine oxidase B is located at its carboxy terminus.
J Biochem 111:20–24
Girisha KS, Kalluraya B, Narayana V (2010) Synthesis and pharma-
cological study of 1-acetyl/propyl-3-aryl-5-(5-chloro-3-methyl-
1-phenyl-1H-pyrazol-4-yl)-2-pyrazoline. Eur
45:4640–4644
J Med Chem
Gokhan N, Yesilada A, Ucar G, Erol K, Bilgin AA (2003) 1-N-
substituted thiocarbamoyl-3-phenyl-5-thienyl-2-pyrazolines:
synthesis and evaluation as MAO inhibitors. Arch Pharm
(Weinheim) 336:362–371
Gokhan-Kelekci N, Yabanoglu S, Kupeli E, Salgin U, Ozgen O, Ucar
G, Yesilada E, Kendi E, Yesilada A, Bilgin AA (2007) A new
therapeutic approach in Alzheimer disease: some novel pyrazole
derivatives as dual MAO-B inhibitors and antiinflammatory
analgesics. Bioorg Med Chem 15:5775–5786
Gokhan-Kelekci N, Koyunoglu S, Yabanoglu S, Yelekci K, Ozgen O,
Ucar G, Erol K, Kendi E, Yesilada A (2009) New pyrazoline
bearing 4(3H)-quinazolinone inhibitors of monoamine oxidase:
synthesis, biological evaluation, and structural determinants of
MAO-A and MAO-B selectivity. Bioorg Med Chem 17:675–689
Havrylyuk D, Zimenkovsky B, Vasylenko O, Zaprutko L, Gzella A,
Lesyk R (2009) Synthesis of novel thiazolone-based compounds
containing pyrazoline moiety and evaluation of their anticancer
activity. Eur J Med Chem 44:1396–1404
Ozdemir A, Turan-Zitouni G, Kaplancikli ZA, Revial G, Guven K
(2007) Synthesis and antimicrobial activity of 1-(4-aryl-2-
thiazolyl)-3-(2-thienyl)-5-aryl-2-pyrazoline derivatives. Eur J
Med Chem 42:403–409
Husain K, Abid M, Azam A (2008) Novel Pd(II) complexes of 1-N-
substituted 3-phenyl-2-pyrazoline derivatives and evaluation of
antiamoebic activity. Eur J Med Chem 43:393–403
Insuasty B, Garcia A, Quiroga J, Abonia R, Ortiz A, Nogueras M,
Cobo J (2011) Efficient microwave-assisted synthesis and
antitumor activity of novel 4,40-methylenebis[2-(3-aryl-4,5-di-
Ozdemir Z, Kandilci HB, Gumusel B, Calis U, Bilgin AA (2008)
Synthesis and studies on antidepressant and anticonvulsant
activities of some 3-(2-thienyl)pyrazoline derivatives. Arch
Pharm (Weinheim) 341:701–707
Palaska E, Aytemir M, Uzbay IT, Erol D (2001) Synthesis and
antidepressant activities of some 3,5-diphenyl-2-pyrazolines. Eur
J Med Chem 36:539–543
Parekh S, Bhavsar D, Savant M, Thakrar S, Bavishi A, Parmar M,
Vala H, Radadiya A, Pandya N, Serly J, Molnar J, Shah A (2011)
Synthesis of some novel benzofuran-2-yl(4,5-dihyro-3,5-substi-
tuted diphenylpyrazol-1-yl) methanones and studies on the
antiproliferative effects and reversal of multidrug resistance of
human MDR1-gene transfected mouse lymphoma cells in vitro.
Eur J Med Chem 46:1942–1948
Pellow S, Chopin P, File SE, Briley M (1985) Validation of
open:closed arm entries in an elevated plus-maze as a measure
of anxiety in the rat. J Neurosci Methods 14:149–167
Pletscher A (1991) The discovery of antidepressants: a winding path.
Experientia 47:4–8
hydro-1H-pyrazol-5-yl)phenols]. Eur
2436–2440
J
Med Chem 46:
Jagrat M, Behera J, Yabanoglu S, Ercan A, Ucar G, Sinha BN,
Sankaran V, Basu A, Jayaprakash V (2011) Pyrazoline based
MAO inhibitors: synthesis, biological evaluation and SAR
studies. Bioorg Med Chem Lett 21:4296–4300
Jayaprakash V, Sinha BN, Ucar G, Ercan A (2008) Pyrazoline-based
mycobactin analogues as MAO-inhibitors. Bioorg Med Chem
Lett 18:6362–6368
Jayashree BS, Arora S, Venugopala KN (2008) Microwave assisted
synthesis of substituted coumarinyl chalcones as reaction
intermediates for biologically important coumarinyl heterocy-
cles. Asian J Chem 20:1–7
123

Med Chem Res
Porsolt RD (1981) Antidepressants. In: Enna SJ, Malick JB,
Richelson E (eds) Neurochemical, behavioural and clinical
perspectives. Raven Press, New York, pp 129–139
Porsolt RD, Bertin A, Jalfre M (1977) Behavioral despair in mice: a
primary screening test for antidepressants. Arch Int Pharmaco-
dyn Ther 229:327–336
Rajendra Prasad Y, Lakshmana Rao A, Prasoona L, Murali K, Ravi
Kumar P (2005) Synthesis and antidepressant activity of some
1,3,5-triphenyl-2-pyrazolines and 3-(200-hydroxy naphthalen-100-
yl)-1,5-diphenyl-2-pyrazolines. Bioorg Med Chem Lett
15:5030–5034
Ramajayam R, Tan KP, Liu HG, Liang PH (2010) Synthesis and
evaluation of pyrazolone compounds as SARS-coronavirus 3C-
like protease inhibitors. Bioorg Med Chem 18:7849–7854
Ruhoglu O, Ozdemir Z, Calis U, Gumusel B, Bilgin AA (2005)
Synthesis of and pharmacological studies on the antidepressant
and anticonvulsant activities of some 1,3,5-trisubstituted pyra-
zolines. Arzneimittelforschung 55:431–436
Sahoo A, Yabanoglu S, Sinha BN, Ucar G, Basu A, Jayaprakash V
(2010) Towards development of selective and reversible pyra-
zoline based MAO-inhibitors: synthesis, biological evaluation
and docking studies. Bioorg Med Chem Lett 20:132–136
Siddiqui ZN, Musthafa TN, Ahmad A, Khan AU (2011) Thermal
solvent-free synthesis of novel pyrazolyl chalcones and pyrazo-
lines as potential antimicrobial agents. Bioorg Med Chem Lett
21:2860–2865
Stirrett KL, Ferreras JA, Jayaprakash V, Sinha BN, Ren T, Quadri LE
(2008) Small molecules with structural similarities to side-
rophores as novel antimicrobials against Mycobacterium tuber-
culosis and Yersinia pestis. Bioorg Med Chem Lett
18:2662–2668
Thase ME (2012) The role of monoamine oxidase inhibitors in
depression treatment guidelines. J Clin Psychiatry 73(Suppl
1):10–16
Vogel HG (ed) (2002) Drug discovery and evaluation: pharmacolog-
ical assays, 2nd edn. Springer, Berlin
Volz HP, Gleiter CH (1998) Monoamine oxidase inhibitors. A
perspective on their use in the elderly. Drugs Aging 13:341–355
Wanare G, Aher R, Kawathekar N, Ranjan R, Kaushik NK, Sahal D
(2010) Synthesis of novel alpha-pyranochalcones and pyrazoline
derivatives as Plasmodium falciparum growth inhibitors. Bioorg
Med Chem Lett 20:4675–4678
Willner P, Mitchell PJ (2002) The validity of animal models of
predisposition to depression. Behav Pharmacol 13:169–188
Youdim MB, Edmondson D, Tipton KF (2006) The therapeutic
potential of monoamine oxidase inhibitors. Nat Rev Neurosci
7:295–309
Zeller EA, Barsky J (1952) In vivo inhibition of liver and brain
monoamine oxidase by 1-isonicotinyl-2-isopropyl hydrazine.
Proc Soc Exp Biol Med 81:459–461
Zhuang ZP, Marks B, McCauley RB (1992) The insertion of
monoamine oxidase A into the outer membrane of rat liver
mitochondria. J Biol Chem 267:591–596
Sivakumar PM, Prabhu Seenivasan S, Kumar V, Doble M (2010)
Novel 1,3,5-triphenyl-2-pyrazolines as anti-infective agents.
Bioorg Med Chem Lett 20:3169–3172
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
123