Facile One-Pot Synthesis Methodology for Nitrogen-Containing Heterocyclic Derivatives of 3,5-Disubstituted 4,5-Dihydro-1H-Pyrazole, Their Biological Evaluation and Molecular Docking Studies

Article abstract of DOI:10.1007/s11094-017-1655-7

A series of 2-pyrazoline derivatives (PS-1 to PS-16) were synthesized by reacting different aromatic/heteroaromatic aldehydes and ketones, in a two-step reaction through Claisen – Schmidt condensation, followed by cyclization of the resulting chalcones wi

Full text of DOI:10.1007/s11094-017-1655-7

DOI 10.1007/s11094-017-1655-7
Pharmaceutical Chemistry Journal, Vol. 51, No. 7, October, 2017 (Russian Original Vol. 51, No. 7, July, 2017)
FACILE ONE-POT SYNTHESIS METHODOLOGY FOR
NITROGEN-CONTAINING HETEROCYCLIC DERIVATIVES
OF 3,5-DISUBSTITUTED 4,5-DIHYDRO-1H-PYRAZOLE, THEIR
BIOLOGICAL EVALUATION AND MOLECULAR DOCKING STUDIES
Savita Upadhyay,1 Avinash C. Tripathi,1 Sarvesh Paliwal,2
and Shailendra K. Saraf1,*
Original article submitted January 27, 2016.
A series of 2-pyrazoline derivatives (PS-1 to PS-16) 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 resulting chalcones with hydrazine hydrate in the presence of a base using conven-
tional and microwave approaches. The synthesized derivatives were characterized by various physicoche-
1
mical methods including IR, H-NMR, 13C-NMR, and mass spectroscopic data and elemental analysis. The
antidepressant and anti-anxiety activities were evaluated using suitable animal models. Compounds PS-3, and
PS-14 showed noticeable antidepressant activity, by reducing the duration of immobility in both tests, while
compounds PS-9 and PS-12 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 b.w.) in comparison to standard
drugs imipramine and diazepam, respectively. In order to elucidate binding interactions of the synthesized de-
rivatives to the MAO-A target protein, molecular docking was employed which demonstrated the key interac-
tions with amino acid residues Phe208, Asn181, and Tyr407 at the binding site. Further, the ADME properties
of the synthesized derivatives were predicted and found to fall within the stated limits.
Keywords: 3, 5-Disubstituted-4,5-dihydro-(1H)-pyrazoles; antidepressant; anti-anxiety: MAO inhibitors;
microwave synthesis; molecular docking.
1. INTRODUCTION
and sensitivity to acetylenic inhibitors clorgyline and l-de-
prenyl (selegiline) [36]. Due to their key role, both MAO
isoforms provide a better understanding of the pharmaco-
phoric requirements needed for the rational design of potent
and selective enzyme inhibitors [1, 8]. MAO inhibitors are
best known for their powerful antidepressant effects and are
efficient in treating certain anxiety disorders including panic
disorder and social phobia [31, 37]. In particular, reversible
and selective MAO-A inhibitors are used as anti-depressant
and anti-anxiety drugs [21], while MAO-B inhibitors have
proved to be useful in the treatment of Parkinson’s disease
(PD) and Alzheimer’s disease (AD). Under some other con-
ditions including affective disorders, neurodegenerative dis-
eases, stroke, and ageing, MAO inhibitors have proved to be
of great therapeutic value [24, 36].
Amine oxidases (AOs) are a heterogeneous superfamily
of enzymes that catalyze the oxidative deamination of
mono-, di- and polyamines [4, 20]. Monoamine oxidases
(MAOs) are present in the outer mitochondrial membranes
of neuronal glial and other mammalian tissues, anchored by
C-terminal domain. The oxidative deamination of biogenic
amines in the brain and the peripheral tissues are catalyzed
by MAOs. The proper functioning of synaptic neurotrans-
mission is due to rapid degradation of these molecules and is
critically important for the regulation of emotional behaviors
and other brain functions [2]. MAOs exist in two isoforms,
MAO-A and MAO-B, which differ in substrate specificity
Pyrazolines are well known nitrogen-containing 5-mem-
bered heterocyclic scaffolds with diverse chemical reactivity.
2-Pyrazolines seem to be the most frequently studied
pyrazoline type compounds arising from structural modifica-
tion of the prototype drug molecule. They have several pro-
1
Faculty of Pharmacy, Babu Banarasi Das Northern India Institute of Tech-
nology, Lucknow, U. P., 227105 India.
2
Department of Pharmacy, Banasthali Vidyapith, Banasthali, Tonk,
Rajasthan, 304022 India.
*
e-mail: dirpharmniec@gmail.com
564
0091-150X/17/5107-0564 © 2017 Springer Science+Business Media New York

Facile One-Pot Synthesis Methodology
565
TABLE 1. Physicochemical Characterization of Synthesized 3,5-Disubstituted 2-Pyrazoline Derivatives (PS-1 to PS-16) with Comparison be-
tween Conventional and Microwave-Assisted Methods
Melting
Conventional
Microwave-assisted method
*
range (°C) method (reflux)
Molecular
formula
Rf
Compd.
Structure
Color and state
Solubility
value
Reaction
%
Microwave Reaction
%
time (h) Yield power (W) time (s) Yield
OH
PS-1
C15H14N2O2 Cream colored Hot methanol,
amorphous solid chloroform, ace-
tone, DMSO
0.68 110 – 112
4.0
4.5
6.0
65.0 240 – 280 145
88.1
75.9
72.3
OH
N
N
H
OCH3
PS-2
PS-3
C16H16N2O2 Light yellow
Hot methanol,
0.93 72 – 74
68.8 240 – 350 110
OH
colored crystal- chloroform, ace-
line solid
tone, DMSO
NH
N
C22H20N2O2 White amor-
phous solid
Hot acetone,
DMSO
0.66 78 – 80
54.6
240
70
50
OH
O
N
N
H
CH3
PS-4
C16H16N2O Cream colored Hot methanol,
crystalline solid chloroform, ace-
tone, DMSO
0.75 90 – 92
4.0
77.3
77.9
240
240
90.0
OH
NH
N
OH
PS-5
PS-9
C16H16N2O2 Cream colored Hot methanol, ac- 0.52 186 – 188
crystalline solid etone, DMSO
4.5
5.5
50
70
87.5
74.0
H3C
O
NH
N
C30H24N2O Light yellow
Chloroform, ace- 0.86 120 – 122
57.4 240 – 280
colored amor- tone, DMSO
phous solid
O
N
N
H
PS-10
PS-11
C24H20N2O Light brown
Hot methanol,
0.81 108 – 110
4.0
5.0
55.6
240
95
81.4
70.6
amorphous solid chloroform, ace-
tone, DMSO
OCH3
N
NH
C23H18N2O Brown colored Chloroform, ace- 0.82 114 – 118
amorphous solid tone, DMSO
60.9 240 – 280 110
OH
N
NH
PS-12
C24H20N2 Yellow colored Hot methanol
amorphous solid chloroform, ace-
tone, DMSO
0.70 84 – 86
5.0
62.3 240 – 280 250
62.5
CH3
N
NH
PS-13
PS-14
PS-15
C20H19N3O Brown colored Hot methanol,
amorphous solid chloroform, ace-
tone, DMSO
0.79 124 – 126
6.0
5.5
7.0
75.0
71.3
53.7
240
240
240
75
80
70
94.2
91.7
60.3
N
H
O
N
N
H
C14H15N3O White colored Methanol, chloro- 0.78 130 – 132
N
H
crystalline solid form, acetone,
DMSO
OCH3
N
N
H
OH
C13H13N3O Light brown
Chloroform, ace- 0.64 138 – 140
colored amor- tone, DMSO
phous solid
N
H
NH
N
CH3
PS-16
C14H15N3 Light brown
line solid
Methanol, chloro- 0.86 136 – 138
5.5
93.8
240
60
96.4
colored crystal- form, acetone,
DMSO
N
H
NH
N
* R Values were calculated for chloroform – methanol (9 : 1) solvent system.
f

566
Savita Upadhyay et al.
TABLE 2. Data on Antidepressant, Anti-anxiety and Neurotoxicity Properties of Synthesized Pyrazoline Derivatives (PS-1 to PS-16)
Neurotoxicity
testingc
Anti-depressant activitya,
Anti-anxiety activityb
Dose
(mg/kg
b.w.)
Duration
of immobility
in FST (s)
Duration
of Immobility
in TST (s)
Number
Compd.
Time spent
in closed arm (s)
(Mean ± SD)
of entries
1.0 h
4.0 h
in closed arm
(Mean ± SD)
(Mean ± SD)
(Mean ± SD)
105.67 ± 11.69**
79.83 ± 7.57**
104.33 ± 14.77**
86.50 ± 8.24**
55.5 ± 9.18**
145.17 ± 18.70*
178 ± 10.63955*
199.43 ± 11.05*
196.17 ± 18.65*
151.33 ± 29.56*
22.17 ± 9.62*
5.50 ± 2.26*
6.67 ± 2.16*
5.83 ± 2.48*
6.50 ± 1.26*
8.16 ± 3.25*
4.83 ± 2.31*
6.17 ± 2.14*
6.00 ± 2.19*
10.33 ± 3.61*
13.67 ± 2.66*
3.67 ± 1.37*
6.17 ± 1.60*
5.83 ± 1.72*
8.33 ± 2.73*
3.67 ± 2.16*
4.17 ± 1.22*
11.17 ± 4.17*
15.50 ± 2.35*
7.33 ± 3.56*
8.83 ± 1.94*
5.17 ± 1.17*
6.83 ± 3.06*
11.33 ± 2.94*
16.50 ± 2.74*
0.83 ± 0.72*
2.50 ± 1.87*
1.83 ± 1.94
–
253.17 ± 12.58*
232.00 ± 8.85*
259.50 ± 22.29*
233.33 ± 22.65*
270.16 ± 24.38*
244.17 ± 39.50*
216.50 ± 29.54*
242.33 ± 43.58*
151.17 ± 18.37*
142.67 ± 15.51*
200.67 ± 18.20*
228.67 ± 30.89*
288.50 ± 41.46*
283.50 ± 24.81*
203.00 ± 22.65*
260.50 ± 32.12*
169.17 ± 18.50*
133.83 ± 12.51*
256.16 ± 20.85*
211.50 ± 25.10*
224.16 ± 20.15*
193.67 ± 20.22*
231.33 ± 24.53*
166.50 ± 28.12*
206.33 ± 15.87*
174.17 ± 24.24*
280.33 ± 34.52
–
PS-1
PS-2
PS-3
PS-4
PS-5
PS-9
50
0/6
0/6
0/6
0/6
0/6
0/6
0/6
1/6
0/6
0/6
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
1/6
0/6
0/6
0/6
0/6
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
0/6
0/6
0/6
1/6
0/6
0/6
1/6
0/6
0/6
0/6
0/6
0/6
100
50
100
50
27.16 ± 5.42**
96.67 ± 16.42**
85.50 ± 10.50**
81.00 ± 8.51**
74.83 ± 16.82**
55.17 ± 5.71**
49.83 ± 9.66**
105.16 ± 13.85**
87.83 ± 18.79**
104.17 ± 15.56**
99.50 ± 14.64**
129.17 ± 11.23**
97.67 ± 14.19**
57.00 ± 8.61**
43.33 ± 10.98**
56.33 ± 10.21**
21.16 ± 8.32**
112.50 ± 19.15**
84.67 ± 13.34**
155.00 ± 11.64**
101.67 ± 12.26**
179.67 ± 18.67
36.16 ± 10.61**
–
100
50
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 ± 24.74*
155.83 ± 12.79*
89.83 ± 14.93*
199.50 ± 15.58*
127.50 ± 27.81*
93.17 ± 11.82*
107.17 ± 27.46*
96.33 ± 9.63*
100
50
100
50
100
50
PS-10
100
50
PS-11
100
50
PS-12
100
50
PS-13
100
50
54.67 ± 14.36*
36.83 ± 11.29*
110.17 ± 14.95*
87.67 ± 10.56*
149.17 ± 38.29*
132.17 ± 11.32*
219.00 ± 16.03
60.33 ± 8.52*
PS-14
100
50
PS-15
100
50
PS-16
100
0.5%
10
Control (CMC)
Standard
(Imipramine
Diazepam)
9.33 ± 3.08*
192.33 ± 27.75*
2
–
n = 6 (Number of animals tested at each dose level); control: carboxymethylcellulose (CMC, 0.5% solution); standard: imipramine (10 mg/kg,
b.w.) for antidepressant activity and diazepam (2 mg/kg, b.w.) for anti-anxiety activity.
a
The reduction in time of immobility in forced swim test (FST) and tail suspension test (TST) are established to evaluate effectiveness of anti-
depressants.
b
The number of entries increases for anxiolytic agents and decreases in anxiogenic agents; 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 h and 4.0 h (number of animals exhibiting toxicity/number of animals tested).
* p < 0.05, ** p < 0.01.

Facile One-Pot Synthesis Methodology
567
minent effects such as antimicrobial, anti-mycobacterial,
antifungal, anti-amoebic, anti-malarial, anti-inflammatory,
analgesic, anti-depressant, anticancer, anti-HIV, cannabinoid
(CB1) receptor antagonist, nitric oxide synthase (NOS) in-
hibitor, and MAO inhibitor activities [5 – 7, 9, 10, 14, 17, 22,
27, 32]. One of the most common structural features of sub-
strates and substituted hydrazine based MAO inhibitors is
the presence of an amino or imino group involved in crucial
interactions [33] at the active site of the enzyme. 2-Pyra-
zolines are considered as cyclic hydrazine moieties, and have
demonstrated impressive MAO inhibition activity in previ-
ous investigations [18].
produced. It has been found that all the previous convention-
ally heated reactions can be performed more conveniently
using this technique, at a faster rate and in good yields [15].
Finally, the molecular docking studies were performed to
predict the binding affinity and ascertain the interactions of
proposed derivatives with biological targets.
2. RESULTS AND DISCUSSION
A series of thirteen 2-pyrazoline derivatives (PS-1 to
PS-13) were synthesized via Claisen-Schmidt condensation
and characterized by physicochemical methods (Table 1),
spectral techniques, and elemental analysis. The IR spectra
of the synthesized compounds displayed absorption bands in
the regions corresponding to C=N stretching (1509 –
1612 cm-1), N – H stretching (3456 – 3105 cm-1) and C – H
In order to better understand the structural requirements
for both inhibition and selectivity toward the two MAO
isoforms (MAO-A/MAO-B), it was decided to synthesize
some novel 3,5-disubstituted 2-pyrazoline derivatives. The
influence on their biological behavior, related to the intro-
duction of different aromatic/heteroaromatic rings at 3rd and
5th positions of the pyrazoline nucleus, was a particular aim
of the study that was further corroborated by numerical eval-
uations. In addition to the conventional heating methods, mi-
crowave-assisted synthetic approach was employed to pre-
pare the proposed derivatives and the results were compared
to those of the former method. Microwave-assisted organic
synthesis is a technique that can be used to rapidly explore
“chemical space” and increase the diversity of compounds
1
deformation (1428 – 1357 cm-1). The H-NMR spectra con-
firmed the presence of two non-equivalent protons of methy-
lene group (H /H ) at d = 2.92 – 3.38 ppm and 3.70 –
a
b
3.93 ppm coupled with each other and in turn with the vici-
nal methine proton (H ) at d = 6.68 – 7.04. All the other
x
aliphatic, aromatic, and heteroaromatic protons were also ob-
served with their expected chemical shifts.
The results of pharmacological studies are presented in
Table 2 and Fig. 1 and show that compounds PS-3 and PS-14
exhibit very good antidepressant activity at tested doses in
TABLE 3. Glide Score and in silico ADME Prediction for Synthesized Derivatives (PS-1 to PS-16)
Glide
score
Donor
HB
Acceptor
HB
QlogPo/w
predicted
% Human (oral
absorption)
RO5
Compd.
MW
Metab QPlog Khsa
Volume
PSA
violation
PS-1
PS-2
PS-3
PS-4
PS-5
PS-9
–7.83
–7.67
–8.80
–7.68
–8.48
–10.10
–7.99
–8.59
–8.78
–7.60
–8.35
–7.52
–7.07
254.29
268.32
344.41
252.32
268.32
430.55
352.44
338.41
336.44
317.39
241.29
227.27
225.29
212.25
3
2
2
2
2
1
1
2
1
2
2
3
2
1
3.5
3.5
2.16
3.29
4.64
3.22
3.26
7.43
5.37
4.59
6.02
4.52
3.12
2.06
3.23
3.11
4
4
5
5
4
6
4
0.03
0.21
0.75
0.36
0.25
1.83
1.09
0.90
1.40
0.73
0.21
–0.03
0.33
0.22
86.95
100
100
100
100
100
100
100
100
100
89.78
100
100
100
850.84
885.23
1128.65
882.60
901.86
1412.08
1116.94
1086.90
1162.22
1058.68
827.37
773.63
814.57
732.80
72.43
56.82
56.71
50.52
60.45
34.43
35.72
51.21
26.19
51.65
52.25
63.96
41.60
33.71
0
0
0
0
0
1
1
0
1
0
0
0
0
0
3.5
2.75
3.5
2.75
2.75
2.75
2
PS-10
PS-11
PS-12
PS-13
PS-14
PS-15
PS-16
4
4
4
3
3
3
3
2.75
2.75
2.75
2.0
PDB-2Z5X crys- –6.91
tal ligand
1.75
Standard value/
range
–
130 to 725 0 to 6
2 to 20 –2.0 to 6.5 1 to 8 –1.5 to 1.5 >80% is high; 500 to 2000 7 to 200
<20% is poor
Maxi-
mum is 4
Donor HB: hydrogen bond donor; acceptor HB: hydrogen bond acceptor; Metab: number of likely metabolic reactions; QPlogKhsa: prediction
of binding to human serum albumin; Volume: total solvent accessible volume in cubic angstrom for a probe with 1.4 Å radius; PSA: Van der
Waals polar surface area of nitrogen and oxygen atoms; RO5 violation: number of violations of Lipinski’s rule of five.

568
Savita Upadhyay et al.
Fig. 1. Histograms showing (a, b) antidepressant activity in forced swim test and tail suspension test, respectively, and (c, d) anti-anxiety activ-
ity in elevated plus maze test as characterized by the number of entries in closed arms and closed arm exploration time, respectively.
both FST and TST models (Figs. 1a and 1b, respectively).
These behavioral tests are used to evaluate the efficacy of an-
tidepressant treatments and ensure effective prediction of ac-
tivity of a wide variety of antidepressants such as MAO in-
hibitors and atypical antidepressants [25]. They have also
good predictive ability for the assessment of antidepressant
potency in humans [35]. Compounds PS-9 and PS-12 were
found to be the most active compounds for anti-anxiety ef-
fect in the elevated plus maze test, with the number of entries
in different arms increasing significantly at a shorter closed
arm exploration time (Figs. 1c and 1d). Most of the synthe-
sized derivatives showed biological activity in a dose de-
pendent manner, with increasing magnitude of effects at
higher dose (100 mg/kg b.w.). No one of the synthesized de-
rivatives exhibited neurotoxicity (disturbances in the motor
co-ordinations) at tested doses, as evaluated by the rotarod
test, since the mean fall off time for all mice was found to be
more than 180 sec. All the synthesized derivatives demon-
strated excellent in silico ADME properties in predictions
using QikProp module (Table 3). The p-benzyloxy (PS-3 and
PS-13) and p-methoxy (PS-14) substitution at the 5th posi-
tion and pyrol-2-yl substitution at 3rd position of the
2-pyrazoline nucleus was found to be crucial in eliciting anti-
depressant action, while a hydrophobic bulky substitution of
anthracen-9-yl at 3rd position of the 2-pyrazoline nucleus
(PS-9 and PS-12) was found to be decisive for exhibiting
good anxiolytic properties.
Previous studies by different researchers have clearly
demonstrated that 2-pyrazoline derivatives possess excellent
affinity toward MAO proteins [4, 9, 11] and can therefore be
considered as important targets for developing antidepres-
sants and anti-anxiety agents. In view of these observations,
docking studies were performed to gain some insights into
binding mode of the synthesized compounds. Stable ligand-
receptor complex conformations and the interactions of the
most active compounds PS-3 and PS-14 (against antidepres-
sant screening) and PS-9 and PS-12 (against anti-anxiety
screening) with amino acid residues are depicted in Figs. 2
and 3 for the typical binding pocket of MAO-A protein. It
may be inferred from these docking studies that Phe208,
Asn181, and Tyr407 are the key interacting residues at the
binding site of MAO-A protein 2Z5X, making crucial con-

Facile One-Pot Synthesis Methodology
569
Fig. 2. 2D ligand – receptor interaction diagrams at the binding site of MAO-A protein 2Z5X, showing good antidepressant activity for com-
pounds (a) PS-3 and (b) PS-14 and good anti-anxiety activity for compounds (c) PS-9 and (d) PS-12.
tacts with the 3,5-disubstituted 2- pyrazoline ring. In such an
orientation, the bound conformations of compound PS-3 in-
clude two hydrogen bond interactions of 2-pyrazoline nitro-
gen and C3-hydroxy phenyl substituent with the side chain
residues Asn181 and Phe208, and p-p stacking interaction of
benzyloxy group of substituted 2-pyrazoline with Tyr444
backbone residues (Figs. 2a and 3a). Another active com-
pound of the series, PS-14, was engaged in two hydrogen
bonding interactions of 2-pyrazoline and C3-pyrrole nitro-
gens with the backbone residue Asn181 and Phe208 residue
of MAO-A protein (Figs. 2b and 3b). Also, one of the most
active compounds for anti-anxiety activity, PS-12, was found
to make two p-p stacking interactions: the first between
anthracen-9-yl ring substituent and Tyr407 residue and the
second between C5-substituted phenyl ring of 2-pyrazane
nucleus and backbone Phe208 residue (Figs. 2d and 3d).
sant and anti-anxiety potential along with their neurotoxicity
studies. The results obtained were further correlated by mo-
lecular docking and prediction of ADME profiles. It was
found that the benzoyloxy substitution at 5th position and
pyrrol-2-yl substitution at 3rd position of the 2-pyrazoline
nucleus was important for the antidepressant activity, as most
of the derivatives bearing this substitution (PS-3 and PS-14)
were found to be active. A hydrophobic bulky substitution of
anthracen-9-yl at 3rd position (compounds PS-9 and PS-12)
was detrimental in eliciting anxiolytic activity among the
synthesized derivatives. Also, favorable in silico ADME per-
formance was observed for all synthesized derivatives with-
out any significant neurotoxicity. Moreover, docking studies
established a fair interaction of the synthesized derivatives
with the MAO-A protein, which plays a crucial role in neuro-
logical disorders such as depression and anxiety. Thus, it
may be concluded that the synthesized 2-pyrazoline deriva-
tives can be promising candidates for antidepressant and
anxiolytic drugs.
3. CONCLUSION
In the present study, some new 2-pyrazoline derivatives
were synthesized and evaluated in vivo for their antidepres-

570
Savita Upadhyay et al.
Fig. 3. 3D ligand – receptor interaction diagrams at the binding site of MAO-A protein 2Z5X, showing good antidepressant activity of com-
pounds (a) PS-3 and (b) PS-14 and good anti-anxiety activity of compounds (c) PS-9 and (d) PS-12.
4. EXPERIMENTAL PART
many). The data of elemental analyses corresponded to the
calculated values.
4.1. Materials and Methods
4.2. Chemical Synthesis
All chemicals and reagents were procured from Sigma
Aldrich and S. D. Fine Chemicals, Mumbai, India and were
used as received. Solvents were of reagent grade and were
purified and dried by standard procedures. Microwave as-
sisted synthesis was performed using Raga’s Scientific Mi-
crowave Systems (Ragatech, Pune, Maharashtra, India).
Melting points were determined by open capillary method
and are uncorrected. The IR spectra were recorded using KBr
discs on Shimadzu 8400S (Shimadzu Corporation, Japan)
and Perkin Elmer RX1 FTIR spectrophotometers, and values
are expressed in cm-1. The 1H NMR spectra were recorded on
Bruker DRX-300 (300 MHz) spectrometer (Bruker Instru-
ments Inc., United States) and chemical shifts (ä values) are
In the first step, substituted aldehydes and ketones react
in a basic medium to form substituted chalcones through
Claisen – Schmidt condensation, which in second step were
cyclized into the target derivatives (PS-1 to PS-16) using
hydrazine hydrate in excess as shown in Scheme 1. Progress
of the reactions was monitored by thin layer chromatography
(TLC) on precoated silica gel G plates, using iodine vapors
and UV chamber as visualizing agents.
4.2.1. General procedure for the synthesis of chalco-
nes:
Method 1 (conventional method). To solutions of various
ketones (0.01 M) and substituted aldehydes (0.01 M) in etha-
nol (10 mL), aqueous solution of potassium hydroxide (60%)
was added dropwise with continuous stirring at 0°C over a
time period of 15 min – 2 h. The reaction mixture was stirred
at a low temperature (0 – 10°C) for about 24 – 48 h, with oc-
casional shaking. After 48 h, the mixture was poured into
ice-cold water and then neutralized to pH 2 using 6 N hydro-
1
reported in parts per million, taking TMS (d = 0 ppm for H
NMR) as an internal standard. The mass spectra were re-
corded on Agilent 6520-QTOF LCMS mass spectrometer in-
strument using ESI source. Elemental analysis was per-
formed on Vario ELIII elemental analyzer (Elementar, Ger-

Facile One-Pot Synthesis Methodology
571
Scheme 1. Synthesis of 3,5-disubstituted-2-pyrazoline derivatives.
chloric acid. Yellow colored intermediates (chalcones) ob-
tained were filtered, washed, dried, and recrystallized from
methanol [11, 13, 19, 28, 30].
2-[5-(4-Hydroxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl]ph
enol (PS-1). IR (KBr, cm-1): N-H str (3287), C-H Ar (3036),
1
C=N str (1594), C-H deform (1439). H NMR (DMSO, d
Method 2 (microwave assisted method). Solutions of var-
ious aromatic/hetero-aromatic ketones (0.01 M) and substi-
tuted aldehydes (0.01 M) were reacted in the presence of hy-
dro-alcoholic solution of KOH (60%, 10 mL), under micro-
wave irradiation (MWI: 120 – 280 W, 40 – 190 sec). The
reaction mixture was poured into ice-cold water and then
neutralized to pH 2 using 6 N hydrochloric acid. The yellow
colored intermediates (chalcones) obtained were filtered,
washed, dried, and recrystallized from methanol [12].
4.2.2. General procedure for the synthesis of 3,5-disub-
stituted-2-pyrazoline derivatives:
ppm): 2.23 – 3.01 (dd, J : 13.25 Hz, J : 4.14 Hz, 1H, H ),
ab
ax
a
3.78 – 3.99 (dd, J : 5.60 Hz, J : 11.33 Hz, 1H, H ),
ab
bx
b
6.70 – 7.66 (m, 8H, Ar), 9.22 (s, 2H, OH). 13C NMR (CDCl ,
3
ppm): 41.8 (CH pyrazoline), 48.5 (CH pyrazoline), 114.2
2
(2CH aromatic), 119.1 – 121.7 (C aromatic), 121.6 (CH aro-
matic), 128.4 – 132.1 (4CH aromatic), 156.2 (C pyrazoline),
156.8 – 158.3 (2C aromatic). MS (m/z): 254.8 (M+);
C H N O .
15 14
2
2
2-[5-(4-Methoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl]p
henol (PS-2). IR (KBr, cm-1): N-H str (3361), C-H Ar
(3049), C=N str (1568), C-H deform (1455). 1H NMR
(DMSO, d ppm): 2.99 – 3.14 (dd, J : 17.04 Hz, J : 3.65 Hz,
Method 1 (conventional method). Appropriate chalcone
was treated with tenfold excess of hydrazine hydrate (80%)
in dry ethanol and refluxed for 3 – 6 h. The hot reaction mix-
ture was poured into ice-cold water. The precipitated solid
was filtered, washed, dried and recrystallized from etha-
nol/acetone/ethyl acetate to afford the corresponding
pyrazoline [13, 19].
ab
ax
1H, H ), 3.73 – 3.90 (dd, J : 3.11 Hz, J : 12.33 Hz, 1H,
a
ab
bx
H ), 6.05 – 6.21 (dd, J : 11.23 Hz, J : 14.36 Hz, 1H, H ),
b
ax
bx
x
7.05 (s, 1H, NH), 6.72 – 7.85 (m, 8H, Ar), 9.31 (s, 1H, OH).
13C NMR (CDCl , ppm): 42.9 (CH pyrazoline), 49.1 (CH
3
2
pyrazoline), 56.0 (CH aliphatic), 113.9 – 115.1 (3CH aro-
3
matic), 119.7 (C aromatic), 120.2 (CH aromatic),
129.5 – 130.1 (3CH aromatic), 133.6 (C aromatic), 155.6 (C
pyrazoline), 157.8 – 159.1 (2C aromatic). MS (m/z): 269.2
(M+1); C H N O .
Method 2 (microwave assisted method). Appropriate
chalcone was treated with tenfold excess of hydrazine hy-
drate (80%) in dry ethanol, under the exposure to microwave
irradiation (MWI: 240 – 350 Wm, 50 – 250 sec). The reac-
tion mixture was poured into ice-cold water. The precipitated
solid was filtered, washed, dried, and recrystallized from eth-
anol/acetone/ethyl acetate to afford the corresponding
pyrazolines [3].
16 16
2
2
2-[5-(4-Benzyloxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl]
phenol (PS-3). IR (KBr, cm-1): N-H str (3258), C-H Ar
(3115), C=N str (1601), C-H deform (1387). 1H NMR
(DMSO, d ppm): 3.15 – 3.29 (dd, J : 16.74 Hz, J : 2.99 Hz,
ab
ax
1H, H ), 3.67 – 3.94 (dd, J : 3.02 Hz, J : 14.23 Hz, 1H,
a
ab
bx
H ), 6.41 – 6.75 (dd, J : 9.86 Hz, J : 15.66 Hz, 1H, H ),
b
ax
bx
x
4.2.3. Characterization of the synthesized 3,5-disub-
stituted-2-pyrazoline derivatives.
7.43 (s, 1H, NH), 6.61 – 7.93 (m, 12H, Ar), 9.37 (s, 1H, OH).
13C NMR (DMSO, ppm): 41.5 (CH pyrazoline), 49.6 (CH
2
Intermediates were characterized by TLC, melting points
and mass spectra, while final derivatives were subjected to
complete physicochemical (Table 1) and spectral character-
ization and the values were found to be in accordance with
the proposed derivatives.
pyrazoline), 79.2 (CH , methylene), 112.7 – 119.8 (4CH aro-
2
matic), 118.4 (C benzene), 127.3 – 130.7 (8CH aromatic),
135.2 (C benzene), 141.6 (C benzene), 157.3 (C pyrazoline),
159.4 – 160.0 (2C benzene). MS (m/z): 345.30 (M+1);
C H N O .
22 20
2
2

572
Savita Upadhyay et al.
2-(5-p-Tolyl-4,5-dihydro-1H-pyrazol-3-yl)-phenol
(PS-4). IR (KBr, cm-1): N-H str (3456), C-H Ar (3087), C=N
6.55 – 6.74 (dd, J : 11.22 Hz, J : 16.43 Hz, 1H, H ), 8.12
(s, 1H, NH), 6.44 – 7.96 (m, 8H, Ar), 9.84 (s, 1H, OH). 13C
ax
bx
x
1
str (1527), C-H deform (1405). H NMR (CDCl , d ppm):
NMR (CDCl , ppm): 40.4 (CH pyrazoline), 51.5 (C
3
2
3
pyrazoline), 114.3 (2CH aromatic), 126.1 – 127.9 (10CH ar-
omatic), 133.6 – 135.5 (5C aromatic), 157.1 (C pyrazoline).
MS (m/z): 339.2 (M+1); C H N O.
3.07 – 3.24 (dd, J : 16.75 Hz, J : 4.06 Hz, 1H, H ),
ab
ax
a
3.62 – 3.96 (dd, J : 3.11 Hz, J : 12.33 Hz, 1H, H ),
ab
bx
b
6.12 – 6.43 (dd, J : 11.27 Hz, J : 16.93 Hz, 1H, H ), 8.85
ax
bx
x
23 18
2
(s, 1H, NH), 6.69 – 7.88 (m, 8H, Ar), 9.74 (s, 1H, OH). 13C
3-Anthracen-9-yl-5-p-tolyl-4,5-dihydro-1H-pyrazole
(PS-12). IR (KBr, cm-1): N-H str (3266), C-H Ar (2994),
NMR (CDCl , ppm): 22.6 (CH aliphatic), 40.9
3
3
1
C=N str (1635), C-H deform (1421). H NMR (CDCl , d
(CH pyrazoline), 50.3 (CH pyrazoline), 115.8 – 118.3 (2CH
2
3
aromatic), 118.4 (C aromatic), 127.8 – 128.5 (4CH aro-
matic), 131.2 – 1.33.7 (2CH aromatic), 134.8 (C aromatic),
138.2 (C aromatic), 156.1 (C pyrazoline), 158.4 (C aro-
matic). MS (m/z): 253.2 (M+1); C H N O.
ppm): 2.11 (s, 3H,), 3.00 – 3.17 (dd, J : 15.28 Hz, J :
ab
ax
4.77 Hz, 1H, H ), 3.74 – 3.89 (dd, J : 6.33 Hz, J :
a
ab
bx
11.27 Hz, 1H, H ), 7.60 (s, 1H, NH), 6.92 – 7.06 (m, 4H,
b
Ar), 7.45 – 7.88 (m, 8H, Ar). 13C NMR (CDCl , ppm): 42.3
16 16
2
3
4-[5-(2-Methoxy-phenyl)-3,4-dihydro-2H-pyrazol-3-yl]-
phenol (PS-5). IR (KBr, cm-1): N-H str (3271), C-H Ar
(2994), C=N str (1560), C-H deform (1378). 1H NMR
(DMSO, d ppm): 3.10 – 3.18 (dd, J : 17.55 Hz, J : 3.88 Hz,
(CH pyrazoline), 49.5 (C pyrazoline), 114.3 (2CH aro-
2
matic), 126.2 – 128.8 (14CH aromatic), 131.5 – 132.7 (4C
aromatic), 136.2 – 138.8 (2C aromatic), 155.6 (C
pyrazoline). MS (m/z): 337.7 (M+1); C H N .
ab
ax
24 20
2
1H, H ), 3.76 – 3.98 (dd, J : 4.03 Hz, J : 11.64 Hz, 1H,
5-(4-Benzyloxy-phenyl)-3-(1H-pyrrol-2-yl)-4,5-dihydro-
1H-pyrazole(PS-13). IR (KBr, cm-1): N-H str (3279), C-H
a
ab
bx
H ), 6.05 – 6.21 (dd, J : 10.38 Hz, J : 17.36 Hz, 1H, H ),
b
ax
bx
x
1
Ar (2966), C=N str (1612), C-H deform (1415). H NMR
7.56 (s, 1H, NH), 6.46 – 7.77 (m, 8H, Ar), 10.06 (s, 1H, OH).
13C NMR (DMSO, ppm): 44.5 (CH pyrazoline), 52.3 (CH
2
(CDCl , d ppm): 3.13 – 3.26 (dd, J : 15.04 Hz, J : 4.79 Hz,
3
ab
ax
pyrazoline), 59.4 (CH aliphatic), 116.2 – 119.7 (4CH aro-
3
1H, H ), 3.77 – 3.93 (dd, J : 6.35 Hz, J : 11.54 Hz, 1H,
a
ab
bx
matic), 117.6 (C aromatic), 128.7 – 131.4 (4CH aromatic),
135.0 (C aromatic), 155.1 – 156.8 (2C aromatic), 162.0 (C
aromatic). MS (m/z): 267.4 (M-1); C H N O .
H ), 6.32 – 6.44 (dd, J : 13.38 Hz, J : 18.57 Hz, 1H, H ),
b
ax
bx
x
7.34 (s, 1H, NH), 6.71 – 7.88 (m, 12H, pyrrole, Ar), 10.06 (s,
1H, OH). 13C NMR (CDCl , ppm): 46.3 (CH pyrazoline),
16 16
2
2
3
2
3-Anthracen-9-yl-5-(4-benzyloxy-phenyl)-4,5-dihydro-1
H-pyrazole (PS-9). IR (KBr, cm-1): N-H str (3301), C-H Ar
(3109), C=N str (1589), C-H deform (1393). 1H NMR
(CDCl , d ppm): 3.01 – 3.22 (dd, J : 17.04 Hz, J : 8.11 Hz,
53.28 (C pyrazoline), 78.5 (CH aliphatic), 109.14 (2CH
2
pyrrole), 117.4 (2CH aromatic), 128.3 – 129.6 (5CH aro-
matic), 137.5 – 139.8 (2C aromatic), 154.8 (C pyrazoline),
161.4 (C aromatic). MS (m/z): 316.30 (M+1); C H N O.
3
ab
ax
20 19
3
1H, H ), 3.73 – 3.90 (dd, J : 6.25 Hz, J : 13.38 Hz, 1H,
a
ab
bx
5-(4-Methoxy-phenyl)-3-(1H-pyrrol-2-yl)-4,5-dihydro-1
H-pyrazole (PS-14). IR (KBr, cm-1): N-H str (3380), C-H Ar
(3010), C=N str (1582), C-H deform (1383). 1H NMR
(CDCl , d ppm): 3.09 – 3.12 (dd, J : 17.41 Hz, J : 3.65 Hz,
H ), 5.97 – 6.04 (dd, J : 11.10 Hz, J : 15.60 Hz, 1H, H ),
b
ax
bx
x
7.58 (s, 1H, NH), 6.68 – 7.97 (m, 16H, Ar), 9.48 (s, 1H, OH).
13C NMR (CDCl , ppm): 43.4 (CH pyrazoline), 49.1 (CH
3
2
3
ab
ax
pyrazoline), 77.8 (CH aliphatic), 116.3 (2CH aromatic),
2
1H, H ), 3.57 – 3.84 (dd, J : 5.18 Hz, J : 16.42 Hz, 1H,
a
ab
bx
127.1 – 128.8 (10CH aromatic), 129.4 – 131.7 (9CH aro-
matic), 135.2 (C aromatic), 141.4 (C aromatic), 153.6 (C
pyrazoline), 159.0 (C aromatic). MS (m/z): 429.2 (M+1);
C H N O.
H ), 6.43 – 6.74 (dd, J : 3.38 Hz, J : 11.36 Hz, 1H, H ),
b
ax
bx
x
7.51 (s, 1H, NH), 6.72 – 7.85 (m, 7H, pyrrole, Ar), 9.26 (s,
1H, OH). 13C NMR (CDCl , ppm): 43.6 (CH pyrazoline),
3
2
30 24
2
49.8 (CH pyrazoline), 56.7 (CH aliphatic), 111.4 (2CH
3-Anthracen-9-yl-5-(4-methoxy-phenyl)-4,5-dihydro-1H-
pyrazole (PS-10). IR (KBr, cm-1): N-H str (3380), C-H Ar
(3105), C=N str (1593), C-H deform (1357). 1H NMR
(CDCl , d ppm): 3.14 – 3.28 (dd, J : 16.53 Hz, J : 6.35 Hz,
3
pyrrole), 112.9 (2CH aromatic), 118.3 (2CH pyrrole), 127.5
(2CH aromatic), 135.8 (C aromatic), 156.1 (C pyrazoline),
160.2 (C aromatic). MS (m/z): 242.1 (M+1), 240.3 (M-1);
C H N O.
3
ab
ax
1H, H ), 3.69 – 3.90 (dd, J : 4.16 Hz, J : 13.76 Hz, 1H,
14 15
3
a
ab
bx
4-[5-(1H-Pyrrol-2-yl)-3,4-dihydro-2H-pyrazol-3-yl]-phe
nol (PS-15). IR (KBr, cm-1): N-H str (3325), C-H Ar (2997),
1
C=N str (1623), C-H deform (1462). H NMR (DMSO, d
H ), 6.22 – 6.29 (dd, J : 10.38 Hz, J : 17.36 Hz, 1H, H ),
b
ax
bx
x
7.75 (s, 1H, NH), 6.60 – 8.13 (m, 3H, Ar), 10.04 (s, 1H, OH).
13C NMR (CDCl , ppm): 40.5 (CH pyrazoline), 50.23 (CH
3
2
ppm): 2.96 – 3.10 (dd, J : 17.12 Hz, J : 3.60 Hz, 1H, H ),
pyrazoline), 57.5 (CH aliphatic), 113.9 (2CH aromatic),
ab
ax
a
3
3.69 – 3.88 (dd, J : 3.14 Hz, J : 12.03 Hz, 1H, H ),
125.3 – 128.7 (10CH aromatic), 132.9 – 133.1 (C aromatic),
155.6 (C pyrazoline), 161.0 (C benzene). MS (m/z): 353.2
(M+1); C H N O.
ab
bx
b
6.05 – 6.21 (dd, J : 11.23 Hz, J : 14.36 Hz, 1H, H ), 7.11
ax
bx
x
(s, 1H, NH), 6.70 – 7.81 (m, 5H, Ar), 9.05 (s, 1H, OH). 13C
24 20
2
4-(5-Anthracen-9-yl-3,4-dihydro-2H-pyrazol-3-yl)-phe-
nol (PS-11). IR (KBr, cm-1): N-H str (3340), C-H Ar (3050),
NMR (CDCl , ppm): 42.2 (CH pyrazoline), 50.1 (CH
3
2
pyrazoline), 109.2 (2CH aromatic), 113.2 – 115.8 (3CH aro-
matic), 119.4 (C aromatic), 134.6 (C aromatic), 155.8 (C
pyrazoline), 157.9 (C aromatic). MS (m/z): 213.8 (M-15);
C H N O.
1
C=N str (1616), C-H deform (1421). H NMR (CDCl , d
3
ppm): 3.03 – 3.10 (dd, J : 16.34 Hz, J : 5.02 Hz, 1H, H ),
ab
ax
a
3.62 – 3.94 (dd, J : 6.76 Hz, J : 10.47 Hz, 1H, H ),
ab
bx
b
13 13
3

Facile One-Pot Synthesis Methodology
573
3-(1H-Pyrrol-2-yl)-5-p-tolyl-4,5-dihydro-1H-pyrazole
(PS-16). IR (KBr, cm-1): N-H str (3438), C-H Ar (3146),
stress, has been hypothesized to reflect behavioral despair
which in turn may reflect depressive disorder 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 passive behavior is counted and compared to
the vehicle treated control.
1
C=N str (1605), C-H deform (1386). H NMR (CDCl , d
3
ppm): 3.02 – 3.05 (dd, J : 17.12 Hz, J : 3.41 Hz, 1H, H ),
ab
ax
a
3.68 – 3.90 (dd, J : 4.01 Hz, J : 16.23 Hz, 1H, H ),
ab
bx
b
6.44 – 6.51 (dd, J : 3.28 Hz, J : 17.46 Hz, 1H, H ), 7.58 (s,
ax
bx
x
1H, NH), 6.70 – 7.81 (m, 7H, Ar), 9.31 (s, 1H, OH). 1.9 (s,
1H), 2.35 (d, 2H), 3.9 (s, 1H), 5. 01(d, 5H). 13C NMR
(CDCl , ppm): 23.7 (CH aliphatic), 46.3 (CH pyrazoline),
The synthesized compounds (50 and 100 mg/kg b.w.)
and imipramine (10 mg/ kg b.w.), a reference antidepressant
drug, were suspended separately in a 0.5% aqueous solution
of carboxymethylcellulose (CMC). The drugs were adminis-
tered orally in a standard volume of 0.5 mL/20 g b.w., 1 h
prior to test. Control animals received 0.5% aqueous solution
of CMC. Then, the mice were dropped individually into a
Plexiglas cylinder and left in water for 6 min. After the first
2 min of initial vigorous struggling, the animals were immo-
bile. A mice was 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.
3
3
2
52.5 (C pyrazoline), 111.7 – 115.2 (3CH pyrrole),
127.4 – 128.2 (4CH aromatic), 138.3 – 139.6 (2C aromatic),
155.4 (C pyrazoline). MS (m/z): 226.0 (M+1); C H N .
14 15
3
4.3. 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 temperature
(26 ± 2°C), relative humidity (55 ± 5%) with regular 12 h
light/12 h dark cycles and free access to standard laboratory
food and water. All test animals were treated in accordance
with guidelines of 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.
4.3.2. Anti-anxiety activity:
The maze model in mice [34] is widely used for evalua-
tion of the anti-anxiety activity. Among many possibilities to
modify maze tests (water maze, Y-maze, radial maze), the el-
evated plus maze have been accepted in many laboratories.
The test has been proposed for selective identification of
anxiolytic and anxiogenic drugs: anxiolytic compounds – by
decreasing anxiety, increasing the open arm exploration time,
and decreasing the time spent in closed arm; anxiogenic
compounds produce the opposite effects. Data obtained in
the treated groups are registered and expressed as percentage
relative to control. Benzodiazepines and valproates decrease
the motor activity and increase the open arm exploratory
time.
4.3.1. Antidepressant activity:
Porsolt’s behavioral despair or forced swim test (FST) in
mice. The synthesized compounds were screened for their
antidepressant activity using Porsolt’s behavioral despair
FST [26, 34]. It was suggested that mice or rats forced to
swim in a restricted space from which they cannot escape are
induced to a characteristic behavior of immobility. This be-
havior 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 control
group and animals treated with various doses of test drug or
standard.
Elevated plus maze test. The elevated plus maze is used
to determine the mouse unconditioned response to a poten-
tially dangerous environment and the anxiety-related behav-
ior 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 in which the animal is placed in the center of an ele-
vated 4-arm maze, in which two arms (50 ´ 10 ´ 40 cm) are
open and two arms (50 ´ 10 ´ 40 cm) are enclosed with open
roof. The two open arms are 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 solution), the mice
were placed in the center of the maze, facing one of the en-
closed arms. During a 6 min test period, the following mea-
surements are taken: the time spent in enclosed arms and the
total number of arm entries. The test is rapid and sensitive to
the effects of both anxiolytic and anxiogenic agents, whereby
anxiolytic agents decrease and anxiogenic agents increase
Tail suspension test (TST) in mice. TST [29, 34] has been
described as a facile means of evaluating potential antide-
pressants. According to this novel test procedure for antide-
pressants, a mouse is suspended by the tail from a lever and
movements of the animal are recorded. The total duration of
the test (6 min) can be divided into periods of agitation and
immobility. Antidepressants decrease the duration of immo-
bility and, if coupled with measurement of locomotor activ-
ity under various conditions, the test can separate the loco-
motor stimulant doses from antidepressant doses. The main
advantages of this procedure are the use of a simple, objec-
tive test situation, the concordance of results with the vali-
dated Porsolt’s “behavioral despair” test, and the sensitivity
to a wide range of drug doses. The immobility displayed by
rodents, when subjected to an unavoidable and inescapable

574
Savita Upadhyay et al.
the amount of time spent in closed arms and the anxiolytic
agents increase and anxiogenic agents decrease the number
of entries 16, 23].
4.4. 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 devel-
opment. The ability to detect the problematic candidates
early can dramatically reduce the amount of wasted time and
resources, and streamline the overall development process.
QikProp, version 3.5 (Schrödinger LLC, New York)
2012 program was used for in silico prediction of
pharmacokinetic properties of the synthesized compounds.
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. The apparatus con-
sists of a horizontal wooden rod or rubber coated metal rod
of 3 cm diameter attached to a motor, with the speed adjusted
to 2 rpm. The rod is 75 cm in length and is divided into
6 sections by plastic discs, thereby allowing the simulta-
neous testing of 6 mice. The rod is in a height of about 50 cm
above the table top in order to discourage the animals from
jumping off the roller. Cages below the sections serve to re-
strict the movements of the animals when they fall from the
roller. Male mice with a weight between 20 and 30 g undergo
a pretest on the apparatus. Only those animals which have
demonstrated their ability to remain on the revolving rod for
at least 1 minute 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. Neuro-
logic toxicity was defined as the failure of animal to remain
on the rod for 1 min at the tested doses. The number of ani-
mals falling from the roller during this time is counted. The
dose which impairs the ability of 50% of test mice to remain
on the revolving rod is considered the endpoint [34].
4.5. Statistical Analysis
All values of the experimental data are expressed as
mean ± SD and analyzed by one-way ANOVA followed by
Dunnett’s test for the possible significant (P < 0.01) identifi-
cation between various groups. Statistical analysis was car-
ried out using Graph Pad Prism 5.0 (Graph Pad Software,
San Diego, CA).
ACKNOWLEDGEMENTS
We express our sincere gratitude to Central Drugs Re-
search Institute (CDRI), Lucknow, India for providing the li-
brary and sophisticated analytical instrument facilities. Au-
thors are thankful to the All India Council for Technical Edu-
cation (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 want to acknowledge the technical
support team/application scientists of Schrodinger Inc. for
their help during computational studies.
4.3. Molecular Docking Studies
In order to gain some structural insights into the binding
mode of the ligand with biological target, all the synthesized
compounds were computationally docked into the MAO-A
enzyme crystal structure (PDB ID: 2Z5X) using GLIDE.
The, MAO protein crystal structure used for molecular dock-
ing was retrieved from the Protein Data Bank (PDB), and
subsequently optimized and minimized with the “protein
preparation wizard” worklow. The ligands were built using
Maestro 9.3 build panel and prepared by LigPrep 2.5 version
v25111 (Schrödinger LLC, United States) application 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 at-
oms, were allowed to move during the calculations. To vali-
date the docking protocol, re-docking experiment was per-
formed in which the ligand conformation of co-crystal ligand
was extracted from the crystal structure of the corresponding
MAO-A protein/ ligand complex and later docked back into
the binding pocket. GLIDE was able to perfectly reproduce
the experimental position of the ligand, confirming the abil-
ity of the method to accurately predict the binding conforma-
tion.
Conflict of Interest
The authors declare no conflict of interest.
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