2-pyrazoline derivatives in neuropharmacology: Synthesis, adme prediction, molecular docking and in vivo biological evaluation

Article abstract of DOI:10.17179/excli2017-250

A novel series of 1,3,5-trisubstituted-2-pyrazoline derivatives (PFC-1 to PFC-16) were synthesized in a three step reaction using conventional and microwave assisted green chemistry approach. The synthesized derivatives were characterized and their chemic

Full text of DOI:10.17179/excli2017-250

EXCLI Journal 2017;16:628-649 – ISSN 1611-2156
Received: March 04, 2017, accepted: April 28, 2017, published: May 08, 2017
Original article:
2-PYRAZOLINE DERIVATIVES IN NEUROPHARMACOLOGY:
SYNTHESIS, ADME PREDICTION, MOLECULAR DOCKING AND
IN VIVO BIOLOGICAL EVALUATION
Savita Upadhyay1, Avinash C. Tripathi1, Sarvesh Paliwal2, Shailendra K. Saraf1*
1
Division of Pharmaceutical Chemistry, Faculty of Pharmacy, Babu Banarasi Das Northern
India Institute of Technology, Lucknow-226028, U.P., India
Professor and Head, Department of Pharmacy, Banasthali Vidyapith, Banasthali,
2
Tonk-304022, Rajasthan, India
*
Corresponding author: Prof. (Dr.) Shailendra K. Saraf, Director (Pharmacy), Division of
Pharmaceutical Chemistry, Faculty of Pharmacy, Babu Banarasi Das Northern India Insti-
tute of Technology, BBD City, Faizabad Road, Chinhat, Lucknow-226028, U.P., India.
Voice Contact: +91-522-3911052 (office), +919839228022(mobile).
Fax: +91-522-3911152, E-mail: dirpharmniec@gmail.com
http://dx.doi.org/10.17179/excli2017-250
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0/).
ABSTRACT
A novel series of 1,3,5-trisubstituted-2-pyrazoline derivatives (PFC-1 to PFC-16) were synthesized in a three step
reaction using conventional and microwave assisted green chemistry approach. The synthesized derivatives were
characterized and their chemical structures were established by various physicochemical methods such as IR,
Mass, 1H-NMR, 13C-NMR and elemental analysis. The synthesized compounds were tested for their neurophar-
macological potential. The compounds exhibited significant antidepressant and anti-anxiety activities against var-
ious behavioral in vivo models. Compounds PFC-3 and PFC-12 were found to be the most active derivatives in
the series. The 2-pyrazoline analogs, having 2-hydroxyphenyl and anthracen-9-yl substitution at 3rd position while
4-benzyloxyphenyl and 4-methylphenyl substitution at 5th position, were decisive in eliciting good antidepressant
and anxiolytic properties, respectively. The docking experiments revealed that the synthesized derivatives were
potential inhibitors of MAO-A protein, which plays a central role in managing depression and anxiety disorders.
The most potent derivatives were found to be involved in some key interactions with Tyr407, Tyr444, Phe352 and
Ala68 amino acid residues at the binding site of MAO-A protein. All the synthesized derivatives successfully
passed the pharmacokinetic barriers of absorption, distribution, metabolism and elimination as predicted using in
silico techniques without showing any substantial indication of acute and neurotoxicity. This was further con-
firmed in the laboratory by performing acute toxicity studies as per OECD guidelines.
Keywords: 4,5-Dihydro-(1H)-pyrazoles, antidepressant, anxiolytic, MAO inhibitors, neurotoxicity, microwave
synthesis, molecular docking
INTRODUCTION
the aerobic oxidation of structurally diverse
xenobiotic arylalkylamine substrates includ-
ing neurotransmitters and exogenous amines
to the corresponding aldehyde and imines
with the generation of hydrogen peroxide
(Rose et al., 1989). In mammals MAO exists
The monoamine oxidase (EC 1.4.3.2;
amine oxygen oxidoreductase) is a FAD-de-
pendent major neurotransmitter degrading en-
zyme present in the outer mitochondria of
neuronal, glial and other cells, that catalyse
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EXCLI Journal 2017;16:628-649 – ISSN 1611-2156
Received: March 04, 2017, accepted: April 28, 2017, published: May 08, 2017
in two isoforms, MAO-A and MAO-B. Sero- Lange et al., 2005; Patil and Bari, 2014; Pra-
tonin and norepinephrine are preferentially sad et al., 2005; Rathish et al., 2009; Shahar-
deaminated by MAO-A, which is selectively yar et al., 2006; Yar et al., 2009; Zampieri et
inhibited by clorgyline. However, MAO-B al., 2008; Zhang et al., 2007; Chimenti et al,
deaminates phenylethylamine and benzyla- 2004; Gokhan-Kelekci et al., 2007).
mine and is selectively inhibited by l-
In order to understand the structural re-
deprenil. The proper functioning of synaptic quirements, for both inhibition and selectivity
neurotransmission is due to rapid degradation towards the two MAO isoforms (MAO-A/
of biogenic amines, which are critically im- MAO-B), it was decided to synthesize some
portant for the regulation of emotional behav- novel 1, 3,5 trisubstituted-2-pyrazoline deriv-
iors and other brain functions (Manna et al., atives. The influence on the biological behav-
2002). The undesirable elevation in plasma ior, due to the substitutions of different aro-
concentration of MAO substrates and delete- matic/heteroaromatic rings at 1st, 3rd and 5th
rious reactivities shown by MAO-catalyzed position of the pyrazoline nucleus, was in par-
reaction products are of major concern in ticular the focus of the study and was further
mental health problems.
corroborated by the computational studies. In
The early development of MAO inhibitors addition to the conventional heating methods,
started with heterocyclic chemotypes contain- microwave assisted organic synthesis ap-
ing two or more hydrogen atoms, mostly hy- proach was employed to prepare the proposed
drazine/hydrazido derivatives (Binda et al., derivatives. A technique which can be used to
2002). However, they have been withdrawn rapidly explore “chemical space” and in-
due to serious side effects such as hyperten- crease the vast interaction of the compounds
sion crises (cheese reaction), liver toxicity, produced is known as microwave assisted or-
hemorrhage, blurred vision, dizziness and in ganic synthesis. Finally, to predict the binding
some cases death (De Colibus et al., 2005). affinity and ascertain the interactions of the
Pyrazolines are considered as cyclic hydra- proposed derivatives with the biological tar-
zine moieties having two adjacent nitrogen at- get, molecular docking studies were per-
oms within the ring in an envelope confor- formed.
mation. Substituted pyrazolines decorated
with different functional groups can be con-
sidered as cyclic benzylhydrazine moieties,
endowed with MAO inhibitory activity (Ed-
mondson et al., 2004). The stability of pyra-
zoline ring system and possible substitutions
at 1st, 3rd and 5th positions by different aro-
matic and heteroaromatic units inspired the
researchers to carry out structural modifica-
tions with an array of pharmacological activi-
ties such as antimicrobial, anti-mycobacterial,
antifungal, anti-amoebic, anti-malarial, anti-
inflammatory, anti-depressant, antiepileptic,
anticancer, anti-HIV, antitubercular, antibac-
terial, photoluminescence, polarity probes, in-
secticidal, antinociceptive, antioxidant, hypo-
tensive, cholesterol inhibitory, cannabinoid
(CB1) receptor antagonist, nitric oxide syn-
thase (NOS) inhibitor and MAO inhibitor
(Abid and Azam 2005; Abid et al., 2009;
Camacho et al., 2004; Havrylyuk et al., 2009;
MATERIALS AND METHODS
The chemicals and reagents for synthesis
were procured from S. D. Fine Chemicals and
Sigma Aldrich, Mumbai, India and the pre-
coated TLC sheets were obtained from Merck
Chemicals, India and were used as such. Rea-
gent grade solvents were used and were puri-
fied and dried by standard methods. Raga's
Scientific Microwave System (Ragatech,
Pune, Maharashtra, India) was used for the
microwave assisted organic synthesis
(MAOS). Melting points were determined by
open capillary method and are uncorrected.
IR spectra were recorded on Bruker FT-IR,
ALPHA-T (Eco-ATR) spectrophotometers,
(Bruker Corporation, USA) and the values are
1
expressed in cm-1. H-NMR and 13C-NMR
spectra were recorded on Bruker Avance-400,
FTNMR spectrometer (Bruker, Tech. Pvt.
Ltd., USA) at 400 MHz and the chemical
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Received: March 04, 2017, accepted: April 28, 2017, published: May 08, 2017
shifts are reported in parts per million (δ General procedure for the synthesis of 3,5-
value), taking TMS (δ 0 ppm for 1H NMR) as disubstituted-2-pyrazoline derivatives
the internal standard. Mass spectra were rec- (PS1-PS16)
orded on Waters UPLC-TQD Mass Spec-
Conventional synthesis: The obtained
trometer instrument (Waters Corporation, chalcone derivatives were refluxed for 3-6 hrs
USA) using LC-ESI or APCI-MS Technique. with excess of hydrazine hydrate in dry etha-
Elemental analysis was performed on Perkin nol. The hot reaction mixture was poured in
Elmer-2400, Series-II Analyzer (Waltham, ice-cold water to obtain the crude product,
Massachusetts, USA).
which was washed and re-crystallized from
appropriate solvent (ethanol/acetone/ethyl ac-
etate) to afford the respective pyrazoline de-
Chemistry
Synthesis of the proposed 2-pyrazoline rivative (Karuppasamy et al., 2010; Tripathi
analogs was carried out in three steps using et al., 2016).
conventional methods (stirring and refluxing)
MAOS: The chalcone derivatives obtained
as well as an environmentally benign green were mixed with hydrazine hydrate (in ex-
synthetic approach under the exposure of mi- cess) in dry ethanol under the exposure of mi-
crowave irradiations. It has been observed crowave irradiation (MWI: 240-350 W; 50-
that the entire reactions performed using mi- 400 s). The reaction mixture was poured in
crowave assisted organic synthesis (MAOS) ice-cold water to get the crude product, which
produced compounds in better yields and at a was washed and re-crystallized from appro-
faster rate.
priate solvent (ethanol/acetone/ethyl acetate)
to give the respective pyrazoline derivative
(Chawla et al., 2010; Insuasty et al., 2011;
Tripathi et al., 2016).
General procedure for the synthesis of
chalcones (PC1-PC16)
Conventional synthesis: Ethanolic KOH
(60 %, 10 mL) solution was added with stir- General procedure for the synthesis of 1,3,5-
ring over a period of 15 min-2 hrs to the solu- trisubstituted-2-pyrazoline derivatives
tion of substituted ketones (0.01 M) and alde- (PFC1-PFC16)
hydes (0.01 M). The stirring was continued
Conventional method: 3,5-Disubstituted-
for 24-48 hrs at a low temperature (0-10 °C). 2-pyrazolines (0.001M) were reacted with
On completion of the reaction, the reaction 0.002 M of 4-chlorobenzenesulfonylchloride
mixture was poured into ice-cold water, and by stirring, taking tetra hydro furan (10 mL)
then neutralized to pH 2 with hydrochloric as the solvent. Stirring was continued for 1-
acid (6N). The yellow colored products ob- 4 hrs. After completion of reaction, the reac-
tained were filtered, washed, and re-crystal- tion mixture was poured on a petri plate and
lized from methanol (Jayaprakash et al., 2008; solvent was evaporated to dryness. The sticky
Tripathi et al., 2016).
crude product was re-precipitated using ace-
MAOS: Substituted ketones (0.01 M) and tonitrile or methanol. Recrystallization was
aldehydes (0.01 M) were reacted, in presence done with acetonitrile or methanol to obtain
of hydro-alcoholic solution of KOH (60 %, the pure product.
10 mL), under microwave irradiation (MWI:
MAOS: Appropriately substituted 3,5-di-
120-280 W, 60-230 s). On completion of the substituted-2-pyrazolines (0.001 M) were re-
reaction, the reaction mixture was poured into acted with 0.002 M of 4-chlorobenzenesul-
ice-cold water, and then neutralized to pH 2 fonylchloride under of microwave irradiation
with hydrochloric acid (6N). The yellow col- (MWI: 210-350 W; 80-280 s), taking tetra hy-
ored products obtained were filtered, washed, dro furan (10 mL) as the solvent. After com-
and re-crystallized from methanol (Jayashree pletion of reaction, the reaction mixture was
et al., 2008; Tripathi et al., 2016).
poured on a petri plate and solvent was evap-
orated to dryness. The sticky crude product
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EXCLI Journal 2017;16:628-649 – ISSN 1611-2156
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was re-precipitated using acetonitrile or meth- 4.32; N, 6.32. Found: C, 59.70; H, 4.31; N,
anol. Recrystallization was done with acetoni- 6.35.
trile or methanol to obtain the pure product.
2-[5-(4-Benzyloxy-phenyl)-1-(4-chloro-ben-
Characterization of the synthesized 1,3,5-tri-
substituted-2-pyrazoline derivatives
zenesulfonyl)-4,5-dihydro-1H-pyrazol-3-yl]-
phenol(PFC-3)
Characterization of the intermediate com-
IR (cm-1): N-H str (3258), C-H Ar (3115),
pounds was done with the help of TLC, melt- C=N str (1605), C-H deform (1448), S=O
1
ing point and mass spectrometry. However, (1380, 1162). H NMR (DMSO, δ ppm):
complete physicochemical (Table 1) and 2.27-2.51 (dd, Jab: 16.74 Hz, Jax: 2.99 Hz, 1H,
spectral characterization was carried out for Ha), 3.67-3.94 (dd, Jab: 3.02 Hz, Jbx: 14.23 Hz,
the final derivatives and the values were in ac- 1H, Hb), 3.15-3.63 (dd, Jax: 9.86 Hz, Jbx: 15.66
cordance with the proposed derivatives.
Hz, 1H, Hx), 7.11-7.82 (m, 12H, Ar), 8.62 (s,
1H, OH). 13C NMR (DMSO, ppm): 41.5 (CH2
pyrazoline), 49.6 (CH pyrazoline), 79.2 (CH2,
methylene), 112.7-119.8 (4CH aromatic),
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): 519.6 (M+, 80 %).
Anal. Calcd. for C28H23ClN2O4S: C, 64.80; H,
4.47; N, 5.40. Found: C, 64.83; H, 4.45; N,
5.41.
2-[1-(4-Chloro-benzenesulfonyl)-5-(4-hy-
droxy-phenyl)-4,5-dihydro-1H-pyrazol-3-yl]-
phenol (PFC-1).
IR (cm-1): N-H str (3531), C-H Ar (3036),
C=N str (1596), C-H deform (1491),
1
S=O(1349,1162). H NMR (DMSO, δ ppm):
2.00-2.06 (dd, Jab: 15.63 Hz, Jax: 3.47 Hz, 1H,
Ha), 2.54-2.58 (dd, Jab: 3.78 Hz, Jbx: 16.24 Hz,
1H, Hb), 3.70-3.73 (dd, Jax: 3.51 Hz, Jbx: 16.14
Hz, 1H, Hx), 5.23-5.27 (s, 2H, Ar-OH), 6.50-
7.96 (m, 12H, Ar). 13C NMR (DMSO, ppm): 2-[1-(4-Chloro-benzenesulfonyl)-5-p-tolyl-
39.2 (CH2 pyrazoline), 45.1 (CH pyrazoline), 4,5-dihydro-1H-pyrazol-3-yl]-phenol (PFC-
113.8-146.3 (12CH benzene), 149.6-157.2 4)
(6C benzene), 161.5 (C pyrazoline). MS
IR (cm-1): N-H str (3456), C-H Ar (3083),
(m/z): 429 (M+, 100 %). Anal.Calcd. for C=N str (1582), C-H deform (1414), S=O
1
C21H17ClN2O4S: C, 59.66; H, 4.32; N, 6.32. (1365, 1168). H NMR (DMSO, δ ppm):
Found: C, 59.64; H, 4.36; N, 6.29.
2.58-2.71 (dd, Jab: 17.55 Hz, Jax: 3.27 Hz, 1H,
Ha), 3.70-3.96 (dd, Jab: 3.12 Hz, Jbx: 16.23 Hz,
1H, Hb), 3.11-3.56 (dd, Jax: 11.47 Hz, Jbx:
16.22 Hz, 1H, Hx), 4.09 (s, 3H, CH3), 7.14-
7.91 (m, 12H, Ar), 9.02 (s, 1H, OH). 13C
NMR (DMSO, ppm): 26.5 (CH3, methyl),
41.7 (CH2 pyrazoline), 48.6 (CH pyrazoline),
112.7-128.7 (12CH aromatic), 134.2-137.6
(3C benzene), 157.3 (C pyrazoline), 160.1-
161.9 (2C benzene). MS (m/z): 428.1 (M+1,
80 %). Anal. Calcd. for C22H19ClN2O3S: C,
61.89; H, 4.49; N, 6.56. Found: C, 61.91; H,
4.53; N, 6.57.
2-[1-(4-Chloro-benzenesulfonyl)-5-(4-meth-
oxy-phenyl)-4,5-dihydro-1H-pyrazol-3-yl]-
phenol (PFC-2)
IR (cm-1): N-H str (3361), C-H Ar (3049),
C=N str (1602), C-H deform (1460), S=O
1
(1349, 1165). H NMR (DMSO, δ ppm):
2.30-2.48 (dd, Jab: 17.04 Hz, Jax: 3.06 Hz, 1H,
Ha), 3.51-3.70 (dd, Jab: 2.87 Hz, Jbx: 15.24 Hz,
1H, Hb), 3.22-3.50 (dd, Jax: 9.14 Hz, Jbx: 15.11
Hz, 1H, Hx), 7.23-7.86 (m, 12H, Ar), 8.62 (s,
1H, OH). 13C NMR (DMSO, ppm): 40.8 (CH2
pyrazoline), 47.2 (CH pyrazoline), 76.5 (CH2,
methylene), 113.4-117.1 (4CH aromatic),
119.1 (2C benzene), 126.8-131.7 (8CH
aromatic), 135.2 (C benzene), 144.7 (C
benzene), 158.5 (C pyrazoline), 160.4-160.8
(2C benzene). MS (m/z): 443.2 (M+, 75 %).
Anal.Calcd. for C22H19ClN2O4S: C, 59.66; H,
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Table 1: Comparative study of physicochemical properties of synthesized 1,3,5-trisubstituted -2-pyrazoline derivatives (PFC-1 to PFC-16) using conventional and
microwave methods
Comps.
Structure
Molecular
Formula
Color and
State
Solubility
Rf
Melting Conventional
Microwave Assisted
Value Range
Synthesis (Refluxing) Synthesis
(0C)
Reaction % Yield
Time (h)
Microwave Reaction
Power (W) Time (s)
%
Yield
OH
N
PFC-1
C21H17ClN2O4S Grey col-
DMSO,
0.84
218-220 3.0
75
240-280
185
86
OH
ored amor- Methanol
phous solid
N
O
S
O
Cl
OCH3
PFC-2
PFC-3
C22H19ClN2O4S Cream col- Acetonitrile,
ored amor- acetone,
phous solid DMSO,
0.90
0.55
196-198 3.0
50
62
210
90
93
91
OH
O
N
methanol
N
S
O
Cl
OH
N
C28H23ClN2O4S Light yel-
low colored acetone,
amorphous DMSO,
Acetonitrile,
208-210 3.0
210-280
110
O
N
O
S
solid
methanol
O
Cl
CH3
PFC-4
C22H19ClN2O3S Light yel-
Acetonitrile,
0.82
170-172 2.0
20
240-350
230
82
low colored chloroform,
OH
crystalline
solid
DMSO
O
N
N
S
O
Cl
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OH
PFC-5
PFC-9
C22H19ClN2O4S Yellowish
grey col-
Methanol,
acetone,
DMSO
0.96
0.48
102-104 2.5
50
240-280
170
78
89
ored crys-
talline solid
O
O
N
N
S
H3C
O
Cl
C36H27ClN2O3S Light yel-
Chloroform,
190-192 1.5
75
280-350
120
low colored acetone,
amorphous DMSO,
solid methanol
O
N
N
S
O
O
Cl
PFC-10
C30H23ClN2O3S Yellow col- Chloroform,
ored amor- acetone,
phous solid DMSO,
0.90
200-202 0.5
96
280-350
190
85
OCH3
N
methanol
N
S
O
O
Cl
PFC-11
PFC-12
C29H21ClN2O3S Light brown Chloroform,
0.56
0.49
208-210 2.0
83
36
280-350
200
120
85
60
colored
acetone,
OH
amorphous DMSO
solid
N
N
S
O
O
Cl
C30H23ClN2O2S Dark brown Chloroform,
176-178 1.5
280
colored
amorphous DMSO, ace-
solid tonitrile
acetone,
CH3
N
N
O
S
O
Cl
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PFC-13
PFC-14
C26H22ClN3O3S Black col-
Acetonitrile,
0.73
0.67
118-120 2.0
90
240-350
140
87
93
N
ored amor- acetone,
phous solid DMSO,
methanol
H
O
N
N
S
O
O
Cl
C20H18ClN3O3S Brownish
black col-
Acetonitrile,
acetone,
162-64
3.0
54
280-350
280
N
H
OCH3
ored amor- DMSO,
phous solid methanol
N
N
S
O
O
Cl
OH
PFC-15
PFC-16
C19H16ClN3O3S Black col-
Methanol,
ored amor- acetone, ac-
phous solid etonitrile,
DMSO
0.88
0.83
100-102 3.0
40
75
280
160
175
81
88
N
O
N
O
N
H
S
Cl
CH3
C20H18ClN3O2S Grey col-
Methanol,
ored amor- acetone, ac-
phous solid etonitrile,
DMSO
158-160 4.0
210-350
O
N
N
N
H
S
O
Cl
*Rf Values were calculated taking solvent system, Chloroform (9) : Methanol (1) .
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4-[2-(4-Chloro-benzenesulfonyl)-5-(4-meth-
oxy-phenyl)-3,4-dihydro-2H-pyrazol-3-yl]-
phenol (PFC-5)
42.6 (CH2 pyrazoline), 47.3 (CH pyrazoline),
57.2 (CH3 methyl), 125.7-134.3 (17CH
aromatic), 138.4 (7C aromatic), 157.6 (C
IR (KBr, cm-1): N-H str (3459), C-H Ar pyrazoline), 160.0 (C aromatic). MS (m/z):
(2994), C=N str (1598), C-H deform (1451), 528.5 (M+1, 85 %). Anal. Calcd. for
S=O (1340, 1213). 1H NMR (DMSO, δ ppm): C30H23ClN2O3S: C, 68.37; H, 4.40; N, 5.32.
2.20-2.48 (dd, Jab: 17.04 Hz, Jax: 3.29 Hz, 1H, Found: 68.36; H, 4.43; N, 5.28.
Ha), 3.56-3.85 (dd, Jab: 3.17 Hz, Jbx: 15.23 Hz,
4-[5-Anthracen-9-yl-2-(4-chloro-benzenesul-
1H, Hb), 3.15-3.63 (dd, Jax: 9.86 Hz, Jbx: 15.66
fonyl)-3,4-dihydro-2H-pyrazol-3-yl]-phenol
Hz, 1H, Hx), 4.12 (s, 3H, CH3), 7.21-7.79 (m,
(PFC-11)
12H, Ar), 8.70 (s, 1H, OH). 13C NMR
IR (cm-1): N-H str (3340), C-H Ar (3050),
(DMSO, ppm): 41.5 (CH2 pyrazoline), 49.6
C=N str (1575), C-H deform (1421), S=O
(CH pyrazoline), 60.3 (CH2, methylene),
1
(1380, 1160). H NMR (DMSO, δ ppm):
115.5-127.3 (12CH aromatic), 128.2-136.4
2.20-2.35 (dd, Jab: 16.45 Hz, Jax: 3.09 Hz, 1H,
(3C benzene), 158.6 (C pyrazoline), 159.4-
Ha), 3.61-3.90 (dd, Jab: 3.02 Hz, Jbx: 16.23 Hz,
160.0 (2C benzene). MS (m/z): 443.7 (M+1,
1H, Hb), 3.15-3.64 (dd, Jax: 11.06 Hz, Jbx:
80 %). Anal. Calcd. for C22H19ClN2O4S: C,
15.70 Hz, 1H, Hx), 7.23-7.91 (m, 16H, Ar),
59.66; H, 4.32; N, 6.32. Found: C, 59.70; H,
13
8.55 (s, 1H, OH). C NMR (DMSO, ppm):
4.30; N, 6.33.
39.8 (CH2 pyrazoline), 49.5 (CH pyrazoline),
3-Anthracen-9-yl-5-(4-benzyloxy-phenyl)-1-
(4-chloro-benzenesulfonyl)-4,5-dihydro-1H-
pyrazole (PFC-9)
116.7-127.3 (17CH aromatic), 135.2 (7C
aromatic), 157.3 (C pyrazoline), 162.1 (C
aromatic). MS (m/z): 528.5 (M+1, 65 %).
IR (cm-1): N-H str (3301), C-H Ar (3051), Anal. Calcd. for C29H21ClN2O3S: C, 67.90; H,
C=N str (1608), C-H deform (1452), 4.13; N, 5.46. Found: C, 67.92; H, 4.16; N,
S=O(1375, 1178). 1H NMR (DMSO, δ ppm): 5.47.
2.60-2.72 (dd, Jab: 15.98 Hz, Jax: 3.33 Hz, 1H,
3-Anthracen-9-yl-1-(4-chloro-benzenesul-
Ha), 3.69-3.87 (dd, Jab: 3.16 Hz, Jbx: 15.77 Hz,
fonyl)-5-p-tolyl-4,5-dihydro-1H-pyra-
1H, Hb), 3.24-3.58 (dd, Jax: 9.15 Hz, Jbx: 16.86
zole(PFC-12)
Hz, 1H, Hx), 5.11 (s, 2H, CH2), 7.43-7.96 (m,
IR (cm-1): N-H str (3273), C-H Ar (2925),
22H, Ar). 13C NMR (DMSO, ppm): 41.8
C=N str (1668), C-H deform (1438), S=O
(CH2 pyrazoline), 49.6 (CH pyrazoline), 78.3
(1360, 1162). 1H NMR (CDCl3, δ ppm): 2.40
(CH2, methylene), 128.7-134.4 (21CH
(s, 3H,), 3.23-3.61 (dd, Jab: 15.28 Hz, Jax: 4.77
aromatic), 138.4 (5C aromatic), 160.3 (C
Hz, 1H, Ha), 5.15-5.22 (dd, Jab: 6.33 Hz, Jbx:
pyrazoline), 161.4 (C aromatic). MS
11.27 Hz, 1H, Hb), 7.25-7.58 (m, 4H, Ar),
(m/z):603.1 (M+1, 90 %). Anal. Calcd. For
7.77-8.49 (m, 8H, Ar). 13C NMR (CDCl3,
C36H27ClN2O3S: C, 71.69; H, 4.51; N, 4.64.
ppm): 21.4 (CH2 pyrazoline), 50.7 (C
Found: 71.71; H, 4.47; N, 4.65.
pyrazoline), 76.8-77.6 (2CH aromatic),
3-Anthracen-9-yl-1-(4-chloro-benzenesul-
fonyl)-5-(4-methoxy-phenyl)-4,5-dihydro-
1H-pyrazole(PFC-10)
124.5-134.3 (14CH aromatic), 136.2-138.8
(6C aromatic), 159.4 (C pyrazoline). MS
(m/z): 512.3 (M+1, 100 %). Anal. Calcd. for
IR (cm-1): N-H str (3379), C-H Ar (3105), C30H23ClN2O2S: C, 70.51; H, 4.54; N, 5.48.
C=N str (1611), C-H deform (1446), S=O Found: C, 70.50; H, 4.51; N, 5.44.
1
(1359, 1178). H NMR (DMSO, δ ppm):
5-(4-Benzyloxy-phenyl)-1-(4-chloro-ben-
3.02-3.11 (dd, Jab: 17.58 Hz, Jax: 3.13 Hz, 1H,
zenesulfonyl)-3-(1H-pyrrol-2-yl)-4,5-dihy-
Ha), 3.67-3.91 (dd, Jab: 3.29 Hz, Jbx: 15.44 Hz,
dro-1H-pyrazole (PFC-13)
1H, Hb), 327.1-3.56 (dd, Jax: 11.45 Hz, Jbx:
IR (cm-1): N-H str (3195), C-H Ar (3024),
16.31 Hz, 1H, Hx), 3.73 (s, 2H, CH3), 7.11-
C=N str (1602), C-H deform (1444), S=O
7.82 (m, 17H, Ar). 13C NMR (DMSO, ppm):
1
(1356, 1165). H NMR (DMSO, δ ppm):
635

EXCLI Journal 2017;16:628-649 – ISSN 1611-2156
Received: March 04, 2017, accepted: April 28, 2017, published: May 08, 2017
2.26-2.52 (dd, Jab: 16.73 Hz, Jax: 2.97 Hz, 1H, 135.2-139.6 (3C aromatic), 156.2 (C
Ha), 3.66-3.90 (dd, Jab: 3.05 Hz, Jbx: 14.27 Hz, pyrazoline), 162.1 (C aromatic). MS (m/z):
1H, Hb), 3.18-3.60 (dd, Jax: 9.76 Hz, Jbx: 15.61 402.8 (M+, 75 %). Anal. Calcd. for
Hz, 1H, Hx), 5.47 (s, 1H, NH), 6.14 (s, 3H, C19H16ClN3O3S: C, 56.79; H, 4.01; N, 10.46.
13
pyrrole), 7.12-7.87 (m, 12H, Ar). C NMR Found:C, 56.82; H, 3.97; N, 10.41.
(DMSO, ppm): 39.8 (CH2 pyrazoline), 52.5
1-(4-Chloro-benzenesulfonyl)-3-(1H-pyrrol-
(CH pyrazoline), 76.2 (CH2, methylene),
2-yl)-5-p-tolyl-4,5-dihydro-1H-pyrazole
112.7-129.8 (12CH aromatic), 118.4 (3CH
(PFC-16)
pyrrole), 127.3-130.7 (8CH aromatic), 137.2-
IR (cm-1): N-H str (3378), C-H Ar (3182),
139.2 (4C aromatic), 157.8 (C pyrazoline),
C=N str (1605), C-H deform (1438), S=O
159.1 (C aromatic). MS (m/z): 492.3 (M+,
1
(1353, 1169). H NMR (DMSO, δ ppm):
80 %). Anal. Calcd. for C26H22ClN3O3S: C,
2.08-2.34 (dd, Jab: 16.02 Hz, Jax: 3.27 Hz, 1H,
63.47; H, 4.51; N, 8.54. Found: C, 63.52; H,
Ha), 3.61-3.95 (dd, Jab: 3.17 Hz, Jbx: 16.34 Hz,
4.49; N, 8.71.
1H, Hb), 3.18-3.60 (dd, Jax: 9.81 Hz, Jbx: 15.29
1-(4-Chloro-benzenesulfonyl)-5-(4-methoxy-
phenyl)-3-(1H-pyrrol-2-yl)-4,5-dihydro-1H-
pyrazole (PFC-14)
Hz, 1H, Hx), 2.51 (s, 3H, CH3), 5.33 (s, 1H,
NH), 6.06 (s, 3H, pyrrole), 7.13-7.69 (m, 8H,
Ar). 13C NMR (DMSO, ppm): 39.5 (CH2
IR (cm-1): N-H str (3382), C-H Ar (3010), pyrazoline), 54.6 (CH pyrazoline), 33.7 (CH3,
C=N str (1600), C-H deform (1440), S=O methyl), 118.7-126.8 (8CH aromatic), 118.9
1
(1350, 1121). H NMR (DMSO, δ ppm): (3CH pyrrole), 137.2-138.6 (3C aromatic),
2.18-2.44 (dd, Jab: 16.22 Hz, Jax: 3.07 Hz, 1H, 153.8 (C pyrazoline), 155.6 (C aromatic).MS
Ha), 3.81-3.93 (dd, Jab: 3.12 Hz, Jbx: 15.30 Hz, (m/z): 401.3 (M+1, 90 %). Anal. Calcd. for
1H, Hb), 3.12-3.66 (dd, Jax: 9.96 Hz, Jbx: 15.21 C20H18ClN3O2S: C, 60.07; H, 4.54; N, 10.51.
Hz, 1H, Hx), 3.51 (s, 3H, CH3), 5.28 (s, 1H, Found: C, 60.11; H, 4.57; N, 10.49.
NH), 6.39 (s, 3H, pyrrole), 7.17-7.85 (m, 8H,
Ar). 13C NMR (DMSO, ppm): 39.8 (CH2 Biological evaluation
pyrazoline), 52.5 (CH pyrazoline), 59.8 (CH3,
Study animals
methyl), 112.7-129.8 (8CH aromatic), 121.1
Animals (male albino mice of BALB/c
(3CH pyrrole), 117.3-126.5 (8CH aromatic),
strain, body weight ranging between 20-
137.2-138.6 (3C aromatic), 157.8 (C
35 mg) were obtained from the animal house
pyrazoline), 160.5 (C aromatic). MS (m/z):
of Faculty of Pharmacy, BBDNIIT, Lucknow,
416.6 (M+, 60 %). Anal. Calcd. for
U.P., India and kept in polymeric cages under
C20H18ClN3O3S: C, 57.76; H, 4.36; N, 10.10.
standard living conditions of 26±2 ºC temper-
Found: C, 57.75; H, 4.39; N, 10.08.
ature and 55±5 percent humidity, with regular
4-[2-(4-Chloro-benzenesulfonyl)-5-(1H-pyr-
rol-2-yl)-3,4-dihydro-2H-pyrazol-3-yl]-phe-
nol (PFC-15)
light and dark cycles, having free access to
standard food and water. Animals were
treated humanely as per the Institutional Ani-
IR (cm-1): N-H str (3325), C-H Ar (2997), mal Ethics Committee (IAEC) guidelines.
C=N str (1623), C-H deform (1462), S=O The pharmacological studies were approved
1
(1214, 1164). H NMR (DMSO, δ ppm): by the IAEC with protocol number
2.61-2.84 (dd, Jab: 15.22 Hz, Jax: 3.34 Hz, 1H, BBDNIIT/IAEC/008/2014.
Ha), 3.63-3.99 (dd, Jab: 2.28 Hz, Jbx: 13.91 Hz,
Study design
1H, Hb), 3.44-3.71 (dd, Jax: 9.58 Hz, Jbx: 14.46
For the study, animals were divided into
Hz, 1H, Hx), 5.28 (s, 1H, NH), 6.39 (s, 3H,
29 groups, each group consisting of 6 animals
pyrrole), 7.24-7.68 (m, 8H, Ar), 8.23 (s, 1H,
(n=6). All the mice were treated at respective
OH). 13C NMR (DMSO, ppm): 40.3 (CH2
doses according to body weight. Treatments
pyrazoline), 49.8 (CH pyrazoline), 114.1-
including standard/vehicle/test compounds
128.5 (8CH aromatic), 116.7 (3CH pyrrole),
were given at 1 mL/100 g body weight per
636

EXCLI Journal 2017;16:628-649 – ISSN 1611-2156
Received: March 04, 2017, accepted: April 28, 2017, published: May 08, 2017
oral (p.o.). Imipramine (10 mg/kg body potential of test compounds, where the mouse
weight) for antidepressant activity, and Diaz- is hanged through its tail using a lever. The
epam (2 mg/kg body weight) for anti-anxiety total 6 min test duration was segregated into
activity was respectively taken as the standard agitation and immobility periods. The rodents
drugs. Carboxy methyl cellulose (0.5 % CMC showed the state of immobility once they
solution) was taken as the vehicle (control were exposed to an inescapable stress. In
group). The groups and respective doses are TST, the potential antidepressants minimize
given in Table 2.
the state of immobility of mice after active
and unsuccessful attempts to escape, when
compared with the vehicle treated controls.
The locomotor stimulant doses of the tested
compounds can be differentiated from antide-
pressant doses, when the period of serenity is
studied in conjunction with locomotor activ-
ity(Steru et al., 1985; Vogel, 2002).
Antidepressant activity
Forced swim test (FST) and Tail suspen-
sion test (TST) in mice model were used to
screen the antidepressant potential of the pre-
pared compounds (PFC1-PFC16). These be-
havioral in vivo tests have been successfully
used to screen and predict the efficacy and po-
tency of various antidepressant treatments in- Anti-anxiety activity
cluding MAO inhibitors (Porsolt, 1981; Will-
ner and Mitchell, 2002).
The most frequently used method to study
anti-anxiety behavior of the test compounds is
the maze model. Elevated plus maze has
found a better acceptance in several laborato-
ries in comparison to the other mazes, such
as-water maze, Y-maze and radial maze. This
test selectively identifies the anxiolytic (open
arm exploration time is increased and time
spent in closed arms is decreased) and anxio-
genic drugs with opposite effects. The values
are represented in percent of control. Open
arm exploratory time is increased while motor
activity is decreased by benzodiazepines and
valproates (Vogel, 2002).
-
Porsolt’s behavioral despair or
forced swim test (FST) in mice
Antidepressant potential of the synthesiz-
ed compounds was tested using Porsolt’s be-
havioral despair test, i.e. FST. Typical immo-
bility behaviors of mice are induced when
they are forced to swim in a limited space
from where they cannot flee. During the test,
mice were plunged into a glass cylinder con-
taining water and it was observed that the an-
imals, after the first 2 min of the initial strug-
gle, became motionless. The duration of still-
ness was considered when mice started float-
ing in water in a vertical position, with modest
-
Elevated plus-maze test
The elevated plus-maze is used to deter-
activities in order to prevent sinking, and was mine the anxiety-related behavior, measured
documented specially for the last 4 min of the by the extent to which the mouse avoids to
6 min test. This behavior is explained as a visit the open arm of the maze. It is a standard
state of despair which can be minimized by test of fear and anxiety where animals are po-
different therapeutically effective antidepres- sitioned at the middle of an elevated four-arm
sants, measured by comparing animals treated maze, in which two arms were open (50 X 10
at numerous doses of test or standard drugs X 40 cm) and 2 arms are closed (50 X 10 X
with control (Porsolt et al., 1977; Vogel, 40 cm), with an open roof situated on the op-
2002).
posite side. The maze was elevated to a height
of 50 cm. After, one hour of oral administra-
tion 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 % aque-
ous CMC suspension), the mice were put in
the middle of the maze, facing the closed arm.
The observations were recorded during the 6
-
Tail suspension test (TST) in mice
This procedure is simple, uses objective
test situations and the results are congruent
with the validated "behavioral despair" test
from Porsolt showing sensitivity towards var-
ious drug doses. TST is a superficial method
and was used to evaluate the antidepressant
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EXCLI Journal 2017;16:628-649 – ISSN 1611-2156
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Table 2: Data showing antidepressant, anti-anxiety and neurotoxicity studies of the synthesized 1,3,5-trisubstituted-2-pyrazoline derivatives (PFC-1 to PFC-16).
Comps.
Group
Number g/kg b.w.)
Doses(m
Antidepressant Activitya
Anti-anxiety Activityb
Neurotoxicity Studies
Rotarod Testc
Actophotometer Testd
(Mean Counts)
Duration of
Immobility in
FST (sec)
Duration of
Immobility in Entries in
TST (sec)
Number of
Time Spent in Before
Closed Arms Dose
After Dose
(After 1 h)
Before Dose After Dose
(After 1 h)
Closed Arms (sec)
(Mean±SD)
(Mean±SD)
(Mean±SD)
(Mean±SD)
290.50
307.83
287.17
290.50
282.50
306.33
283.83
423.83
411.17
306.33
419.67
306.33
299.50
288.83
302.67
280.83
321.33
288.00
295.17
285.67
1
50
105.67 ± 11.69 145.17 ±18.70 5.50±2.26
79.83 ± 7.57 178.00±10.64
104.33 ± 14.77 196.17±18.65
245.00±22.86
227.00±15.89
254.83±14.40
223.50±19.94
270.17±24.38
195.00±50.54
216.50±29.54
229.00±37.53
192.33±25.32
215.83±22.02
142.50±25.00
135.83±14.69
264.67±26.40
252.67±34.77
203.00±22.65
153.17±40.90
169.17±18.50
133.83±12.51
256.17±20.85
211.50±25.10
0/6
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
0/6
0/6
1/6
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
1/6
0/6
0/6
0/6
0/6
0/6
283.83
306.33
283.83
288.83
284.17
306.33
283.83
418.33
413.17
306.33
413.17
306.33
283.83
288.83
306.33
280.83
330.67
286.00
296.50
284.17
PFC-1
PFC-2
PFC-3
PFC-4
PFC-5
PFC-9
PFC-10
PFC-11
PFC-12
PFC-13
2
100
50
6.67±2.16
6.33±2.07
6.50±1.38
6.33±1.97
6.67±2.16
6.17±2.14
7.00±1.41
5.00±2.00
8.50±2.59
11.00±3.90
14.67±2.34
6.00±1.41
8.83±2.48
4.50±1.87
6.33±3.27
10.83±3.66
16.67±2.58
7.83±3.19
8.67±2.16
3
4
100
50
86.50 ± 8.24
55.50 ± 9.18
27.17 ± 5.42
96.67 ± 16.82
85.50 ± 10.50
82.67 ± 6.41
74.83 ± 16.82
55.17 ± 5.71
49.83 ± 9.66
196.83±12.56
151.33±29.56
22.17±9.62
5
6
100
50
7
139.33±20.17
125.50±13.75
209.17±23.61
160.33±13.44
97.50±30.25
65.50±29.61
8
100
50
9
10
11
12
13
14
15
16
17
18
19
20
100
50
100
50
105.17 ± 13.85 150.50±24.74
87.83 ± 18.79 155.83±12.80
104.17 ± 15.56 89.83±14.94
99.50 ± 14.64 199.50±15.58
129.17 ± 11.23 127.50±27.81
100
50
100
50
100
50
97.67 ± 14.19
57.00 ± 8.67
43.33 ± 10.98
93.17±11.82
107.17±27.46
100.83±16.19
100
638

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423.83
413.17
236.83
324.33
21
22
23
50
56.33 ± 10.21
21.17 ± 8.33
54.67±14.36
36.83±11.29
6.17±2.14
9.83±2.86
8.67±2.34
206.83±16.12
187.33±12.56
216.50±16.84
0/6
0/6
0/6
0/6
0/6
0/6
418.33
413.17
226.67
PFC-14
PFC-15
100
50
110.83 ± 16.65 110.17±14.95
84.67 ± 13.34 87.67±10.56
24
100
12.67±3.27
158.67±20.77
1/6
0/6
306.33
281.33
284.17
25
26
50
155.00 ± 11.64 149.17±38.29
101.67 ± 12.26 132.17±11.32
2.67±2.73
5.17±2.79
202.00±11.35
144.00±66.83
0/6
0/6
2/6
0/6
291.67
288.83
PFC-16
100
Control
(CMC)
Standard
drug
27
0.5 %
179.67± 18.67
29.33±8.43***
219.00±17.56
60.33±9.33***
0.83±0.75
281.67±41.09
0/6
0/6
305.50
309.00
313.17
10
---
---
0/6
0/6
316.17
(Imipramine)
Standard
drug
177.33±45.18*
28
2
---
---
9.33±3.08***
0/6
0/6
286.00
284.17
**
( Diazepam)
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 (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 FST and TST are established ways to evaluate effectiveness of antidepressants.
b The number of entries is increased in anxiolytic agents and decreased in anxiogenic agents and the amount of time spent in closed arms is decreased in anxiolytic agents and increased
in anxiogenic agents.
c Neurotoxicity was evaluated in rotarod test before dosing and after 1 hour of dosing (number of animals exhibiting toxicity/number of animals tested).
d Neurotoxicity recorded in actophotometer test (Counts recorded in 10 minutes duration)
639

EXCLI Journal 2017;16:628-649 – ISSN 1611-2156
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min test duration: time spent in closed arm
and total number of the arm entries(Lister,
1987; Pellow et al., 1985).
-
Locomotor activity (actophotometer/
open field test)
Locomotor activities of animals, and hu-
mans, are affected by majority of the CNS act-
ing drugs and therefore it is utilized as an in-
dex of their mental alertness. Some CNS de-
pressant drugs like barbiturates and alcohols
have been found to reduce it. However, CNS
stimulants like amphetamines and caffeine
exhibit significant increase in the motor activ-
ity. The locomotor activity was examined for
a 10 minute duration, using Medicraft Acto-
photometer (Model No. 600-4D, INCO, Am-
bala, India), to rule out such CNS depressant
or stimulant type of effects of the tested com-
pounds. During the test, animal moves in a
square arena of dimension 30x30x25 cm with
mesh wire bottom. Photoelectric cells con-
nected in circuit with a counter are used to op-
erate the apparatus, where light beam striking
on the photo cell is cut off by the animal to
record a count(Dhingra and Goyal, 2008).
Neurotoxicity study
Neurotoxicity studies are used to screen
the effect(s) of a test substance on the CNS.
Several behavioral studies (such as rotarod
test, open-field/actophotometer test, turning
on flat surface and turning on inclined plane)
are employed to identify potential neurotoxi-
city in mice model. Rotarod and actophotom-
eter tests are the two most often used methods
to identify the effect of chemical compounds
(test compound) on neuromuscular coordina-
tion and locomotor activities, respectively
(Parasuraman, 2011).
-
Neuromuscular coordination study
(rotarod test)
The rotarod test is valuable to find out the
consequences of drug on motor coordination,
i.e. neurotoxicity. This test was introduced by
Dunham and Miya in 1956, where relaxation
of skeletal muscle produced by any test com-
pound is examined by maintaining the ability
of mice or rats on a rotating rod, at a speed of
10 revolutions per min. For the test, male
mice of average weight 25±5g underwent a
pretest, and only those mice were selected for
the test which remained on the revolving rod
for at least 1 minute. The mice were placed on
the rod after 1 hour of oral administration of
test compounds/standard drug/control. At this
speed, normal mice stay on the rod for an in-
definite period of time. However, if they fall
before 1 min, the sign of neurotoxicity is ap-
parent at the tested doses. Therapeutic end
point is considered as a dose which persuades
ability of fifty per cent of the mice to stay on
the revolving rod. Number of animals falling
down from the rod is counted in a qualitative
manner and mean fall off time is recorded to
quantify the values (Bhandari et al., 2013;
Vogel, 2002).
Acute toxicity study
Acute toxicity testing is carried out to de-
termine the effect of a single dose on a partic-
ular animal species. In the present study,
acute oral toxicity (LD50) of two most active
final derivatives (PFC-3 and PFC-12) was
performed as per guidelines laid by Organiza-
tion for Economic Co-operation and Develop-
ment (OECD) guideline No 423 “Acute Oral
Toxicity - Acute Toxic Class Method”. The
compounds were administered orally at four
fixed dose levels (5, 50, 300 and 2000 mg/kg
body weights), after 4 hours of fasting. After
dose administration, food but not water was
withheld for 2 hours. The body weight of all
the mice were recorded on study days D0 (in-
itiation), D1, D7 and D14. The animals were
observed for 4 hours post dose treatment and
thereafter for 14 days for mortality, including
various signs of toxicity such as, changes in
skin and fur, behavior patterns, convulsions,
tremors and death(OECD, 2001; Parasura-
man, 2011).
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EXCLI Journal 2017;16:628-649 – ISSN 1611-2156
Received: March 04, 2017, accepted: April 28, 2017, published: May 08, 2017
Molecular docking studies
a generic tool for predicting complex toxico-
Molecular docking simulations were used logical end points, such as carcinogenicity,
to predict binding affinity and binding orien- long-term toxicity, and reproductive toxicity.
tations of the synthesized compounds with Virtual Computational Chemistry Laboratory
MAO-A protein (PDB ID: 2Z5X), using maintains OSIRIS as a fundamental part of
GLIDE program. The X-ray crystal structure Actelion's in house substance registration sys-
of MAO protein was retrieved from the Pro- tem. This calculates various drug-related
tein Data Bank (PDB) and optimized using properties of chemical structures including
“protein preparation wizard”. Ligands were some toxicity parameters and drug likeness.
prepared by LigPrep module 3.2 version Predicted outcomes are rated and color coded.
v25111(Schrödinger, LLC, New York, NY, Properties such as mutagenicity or a poor in-
2014), using OPLS (optimized potential liq- testinal absorption having higher risks of un-
uid simulations) 2005 force field to give the desired effects are shown in red, whereas a
consequent energy minima. Default settings green color indicates drug-conform behav-
were applied to other parameters. Rigid dock- ior(Klebe, 2000).
ing was performed during these calculations.
Re-docking experiment was conducted to val- Statistical analysis
idate the docking procedure, in which the co-
All the values of the experimental results
ordinates of crystal ligand were obtained from are expressed as mean±SD and analyzed by
the PDB structure of MAO-A protein-ligand one-way ANOVA, followed by Dunnett’s test
complex and docked back into the binding for the possible significance (P<0.05) identi-
pocket. GLIDE (version 6.5, Schrödinger, fication between various groups. Statistical
LLC, New York, NY, 2014) program per- analysis was carried out using Graph Pad
fectly reproduced the tentative pose of the lig- Prism 5.0 (Graph Pad Software, San Diego,
and and accurately predicted the binding CA).
mode.
RESULTS AND DISCUSSION
In silico prediction of pharmacokinetic
properties
A series of thirteen 2-pyrazoline deriva-
Majority of the drug candidates do not tives (PFC1 to PFC-16) were synthesized us-
succeed in clinical trials due to poor pharma- ing conventional as well as a green chemistry
cokinetic properties, such as absorption, dis- approach of microwave assisted organic syn-
tribution, metabolism, and excretion thesis (MAOS) as given in Figure 1. Among
(ADME). The outlay in developing new drug the two methods, MAOS showed better syn-
significantly increases due to these later-stage thetic efficiency when compared with the
failures, which can be radically reduced if the conventional procedures. The synthesized de-
problematic candidates are detected in early rivatives were characterized by various phys-
phases. In this study, the QikProp module icochemical (Table 1), spectral and elemental
(version 4.2) of Schrödinger (LLC, New analysis methods. The spectral analysis data
York, NY, 2014) software program was suc- expound C=N stretching (1509-1612 cm-1),
cessfully employed for in silico prediction of N-H stretching (3456-3105 cm-1) and C-H de-
ADME properties of the synthesized deriva- formation (1428-1357 cm-1) along with a
tives.
characteristic peak of sulfonyl group absorp-
tion bands (1340-1180 cm-1) in the corre-
sponding regions of IR spectra.
In silico toxicity prediction
There are a large number of freely availa-
ble computer programs to envisage the in sil-
ico toxicity of the compounds. Two of these
softwares, used in this study are; LAZAR and
OSIRIS Property explorer. LAZAR provides
641

EXCLI Journal 2017;16:628-649 – ISSN 1611-2156
Received: March 04, 2017, accepted: April 28, 2017, published: May 08, 2017
Conventional Method
0-10 oC, Stir, 24-48 hrs.
O
H
CH3
C
R
C
R'
R-CHO
C2H5OH, KOH
+
R'
H
O
Microwave Method
Chalcone derivatives
(PC1-PC16)
MWI: 120-280W, 40-190s
Ketone
Aldehyde
Conventional Method
R
R'
Reflux, 3-6 hrs.
N NH
NH2NH2.H2O
3,5-Disubstituted
2-pyrazoline derivatives
(PS1-PS16)
Microwave Method
MWI: 240-350W, 50-250s
R
Conventional Method
O
Stir, 1-4 hrs.
N S
Cl
N
4-Chlorobenzenesulfonyl chloride
O
R'
1,3,5-Trisubstituted
2-pyrazoline derivatives
(PFC1-PFC16)
Microwave Method
MWI: 210-350W, 80-280s
H2
C
O
OCH3
OCH3
CH3
Where R:
and R':
OH
OH
N
H
Figure 1: Synthesis of 1,3,5-Trisubstituted-2-pyrazoline derivatives
The presence of two non-equivalent pro- good range of antidepressant and anti-anxiety
tons of a methylene group (Ha/Hb) at δ 2.92– activities, as evaluated using various in vivo
3.38 ppm and 3.70–3.93, coupled with each models. Compounds PFC-3 and PFC-12
other and in turn with the vicinal methine pro- were found to be the most active compounds
ton (Hx) at δ 6.68–7.04 were recorded in the amongst all the synthesized derivatives (Ta-
1H-NMR spectra. All the other protons of var- ble 2 and Figure 2).
ious aliphatic/aromatic/heteroaromatic groups
were anticipated at respective places.
Results of the pharmacological studies
demonstrated that the synthesized 1,3,5-tri-
substituted-2-pyrazoline derivatives possess
642

EXCLI Journal 2017;16:628-649 – ISSN 1611-2156
Received: March 04, 2017, accepted: April 28, 2017, published: May 08, 2017
Figure 2: (a) Graph showing anti-anxiety activity (Number of entries in closed arms in Elevated Plus
Maze Test); (b) Graph showing anxiety activity (Time spent in closed arms in Elevated Plus Maze Test);
(c) Graph showing antidepressant activity (Forced Swim Test); (d) Graph showing antidepressant ac-
tivity (Tail Suspension Test).For each group, number of animals tested was 6 (n=6).
Detailed SAR studies revealed that 4-
All the synthesized derivatives were also
chlorobenzenesulfonyl substitution at N1 po- evaluated for their possible neurotoxicologi-
sition was decisive in exhibiting good antide- cal effects such as neuromuscular coordina-
pressant and anti-anxiety activities. It was tion (using rotarod test) and locomotor activ-
also observed that 2-hydroxyphenyl substitu- ity (using actophotometer test). It was ob-
tion at 3rd position and 4-benzyloxyphenyl served that none of the synthesized com-
substitution at 5th position of 2-pyrazoline nu- pounds showed serious neurotoxicity (dis-
cleus was instrumental for the remarkable an- turbances in the motor co-ordinations and lo-
tidepressant potency of compound PFC-3. comotor activity) threats at the tested doses
However, a bulky hydrophobic anthracen-9- (Table 2). Therefore, any possibility of CNS
yl substitution at 3rd position and 4- stimulating or depressing effects of the tested
methylphenyl substitution at 5th position of compounds was ruled out. Additionally, two
the 2-pyrazoline scaffold were favorable in most potent derivatives (PFC-3 and PFC-12)
demonstrating the significant anxiolytic pro- were screened for their acute toxicities, at the
file of compound PFC-12. The biological ac- tested doses. During these studies; behavioral
tivities of the synthesized compounds were pattern, changes in skin and fur, convulsions,
depending upon the doses administered, with tremors and death were not observed in any of
increased effects at higher tested dose the animals, in due course of 14 days of acute
(100 mg/kg b.w.).
toxicity studies, as per the OECD guidelines.
643

EXCLI Journal 2017;16:628-649 – ISSN 1611-2156
Received: March 04, 2017, accepted: April 28, 2017, published: May 08, 2017
The results obtained by animal experi- or Tyr407 amino acid residue. Due to these
mentation were further supported by the out- Hydrogen bonding interactions, substituted
comes of molecular docking experiments. phenyl ring at C5 and benzenesulfonyl ring at
The antidepressant and anxiolytic properties N1 position of pyrazoline nucleus are well
of substituted 2-pyrazoline derivatives may placed in the aromatic cage. The most potent
be attributed to their MAO-A inhibiting capa- compound from N1 benzenesulfonyl substi-
bilities. The synthesized compounds were tuted series (PFC-12) displayed additional
found to possess tremendous affinity towards key interactions of this moiety with side chain
MAO-A enzyme, and may therefore be and backbone residues of the amino acids at
deemed as imperative targets to develop po- the binding pocket. This included a hydropho-
tential neuropharmacological agents. Dock- bic interaction of benzene ring with Tyr 407,
ing studies also confirmed that the presence of Tyr444 residues, and H-bond interaction with
sulfonyl group at N1 position of pyrazoline Ala68 and Tyr444 residues (Figure 3 and 4).
nucleus establishes a hydrogen bonding inter-
action between the sulfonyl oxygen of the lig-
ands and hydroxyl hydrogen of either Tyr444
Figure 3: Ligand receptor interaction diagram of compound PFC-3 at the binding site of MAO-A protein
(PDB ID: 2Z5X) showing best anti-anxiety activity. (a) 2D Ligand receptor interaction diagram, (b) 3D
Ligand receptor interaction diagram
Figure 4: Ligand receptor interaction diagram of compound PFC-12 at the binding site of MAO-A protein
(PDB ID: 2Z5X) showing best antidepressant activity. (a) 2D Ligand receptor interaction diagram, (b)
3D Ligand receptor interaction diagram
644

EXCLI Journal 2017;16:628-649 – ISSN 1611-2156
Received: March 04, 2017, accepted: April 28, 2017, published: May 08, 2017
It was observed that one amongst the other which the computational software facility has
aromatic substitutions of previously men- been made available at the host institute. We
tioned compounds at these positions is in- also acknowledge the technical support
volved in hydrophobic and probably aromatic team/application scientists of Schrodinger
interaction. Also, favorable in silico ADME Inc. for their help during computational stud-
performances (Table 3) were obtained for all ies.
the synthesized compounds. Furthermore, the
tested compounds were found to be safe, by Conflict of interest
not presenting any potential risks of carcino-
The authors declare that they have no con-
genicity, mutagenicity, reproductive toxicity, flict of interest.
acute toxicity and irritancy as predicted by
LAZAR and OSIRIS Property Explorer pro-
grams (Table 4).
CONCLUSION
In the present study, in vivo and in silico
screening results have clearly demonstrated
the expediency of synthesized 2-pyrazoline
derivatives in neuropharmacological disor-
ders. Although, the evidence showed promis-
ing status of the tested compounds against in
vivo models of depression and anxiety, further
experiments including in vitro MAO-A inhi-
bition studies are needed to explore their con-
crete mechanism of action at molecular level.
Since, all these derivatives were found to be
effective in a dose dependent manner, further
therapeutic dose adjustment studies and de-
signing of most appropriate dosage forms are
some imperative prospects. Some of the ex-
cellent outcomes obtained from the preclini-
cal investigations demand comprehensive
clinical trials to establish the role of synthe-
sized 1,3,5-trisubstituted-2-pyrazoline deriv-
atives in the management of depression and
anxiety disorders.
Acknowledgements
We express our sincere gratitude to Cen-
tral Drugs Research Institute (CDRI), Luck-
now, India and Department of Chemistry,
Banasthali Vidyapith University, Banasthali,
Rajasthan, 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 Re-
search Promotion Scheme (Grant No.:
8023/RID/RPS/30 (Pvt.) 2011-12), through
645

EXCLI Journal 2017;16:628-649 – ISSN 1611-2156
Received: March 04, 2017, accepted: April 28, 2017, published: May 08, 2017
Table 3: In silico prediction of binding affinity (Glide gscore) and ADME parameters of the synthesized 1,3,5-trisubstituted -2-pyrazoline derivatives (PFC-1 to
PFC-16)
Comps.
Glide
gscore
MW
Volume
PSA
Donar accpt
CNS
Qlog
Po/w
QPlog
BB
QPlog
Khsa
metab % Human
Oral ab-
Violations
of Rule of
HB
HB
sorption
87.79
100
Five
0
PFC-1
-9.36
-9.10
-11.15
-8.97
-9.76
-8.55
-6.74
-10.09
-10.20
-9.19
-7.82
-10.19
-8.69
428.89
442.92
519.01
426.92
442.92
603.13
527.04
513.01
511.04
491.99
415.89
401.87
399.89
1156.63
1191.49
1432.31
1190.23
1220.09
1698.69
1458.19
1459.47
1496.30
1366.97
1127.11
1091.93
1125.05
94.96
80.71
79.62
72.43
81.08
58.48
59.58
74.92
52.38
74.17
75.25
89.50
66.96
2
1
1
1
1
0
0
1
0
1
1
2
1
7.00
7.00
7.00
6.25
7.00
6.25
6.25
6.25
5.50
6.25
6.25
6.25
5.50
-2
0
3.04
3.63
5.29
3.95
3.76
7.84
6.10
5.98
6.89
5.31
3.66
3.05
3.96
-1.025
-0.646
-0.926
-0.573
-0.736
-0.459
-0.228
-0.695
-0.174
-0.734
-0.461
-0.943
-0.389
0.180
0.27
0.34
0.88
0.53
0.41
1.67
1.05
1.21
1.41
0.90
0.37
0.28
0.55
4
4
5
4
4
4
3
3
3
4
3
3
3
PFC-2
PFC-3
2
-1
0
83.04
100
0
0
PFC-4
PFC-5
-1
0
0
100
0
PFC-9
100
2
2
PFC-10
PFC-11
PFC-12
PFC-13
PFC-14
PFC-15
PFC-16
Crystal
Ligand(2Z5X)
Standard
values
96.27
88.28
100
100
100
0
2
0
-1
0
2
1
0
-1
0
0
90.99
100
0
0
-6.89
---
212.25
733.39
33.50
1
1.75
3.11
0.222
3
100
0
130 to
725
500 to
2000
7 to
200
-2 to
+2
-2.0 to
6.5
-3.0-
1.2
-1.5 to
1.5
<20% poor,
>80% high
0 to 6
2 to 20
1 to 8
Max. is 4
#stars: Number of property or descriptor values that fall outside the 95 % range of similar values for known drugs
Volume: Total solvent accessible volume in cubic angstrom using a probe with 1.4 Ǻ radius
PSA: Van der Waals polar surface area of nitrogen and oxygen atoms
SASA: Total solvent accessible surface area in square angstrom using a probe with 1.4 Ǻ radius
donarHB: Hydrogen bond donar
accptHB: Hydrogen bond acceptor
CNS: Predicted central nervous system activity at -2 (inactive) to +2 (active) scale
QPlogBB: Predicted brain/blood partition coefficient
QlogPo/w: Predicted octanol-water partition coefficient
QPlogKhsa: Prediction of binding to human serum albumin
QPPCaco: Predicted apparent Caco-2 cell permeability in nm/sec
metab: Number of likely metabolic reactions, Number of violations of Lipinski’s rule of five
646

EXCLI Journal 2017;16:628-649 – ISSN 1611-2156
Received: March 04, 2017, accepted: April 28, 2017, published: May 08, 2017
Table 4: In silico toxicity prediction data of the synthesized 1,3,5-trisubstituted -2-pyrazoline derivatives(PFC-1 to PFC-16)
Toxicity prediction
OSIRIS Property Explorer
LAZAR
Comp.
Maximum recom-
mended daily dose icity LC50
(mmol)
Acute tox-
Reproductive
effect
Carcinogenic
potency
Mutagenic
Tumorigenic
Irritant
Mutagenicity
(mmol)
PFC-1
PFC-2
PFC-3
PFC-4
PFC-5
PFC-9
No risk
No risk
No risk
No risk
No risk
High risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
2.74e+00
4.34e+00
3.05e+00
6.99e+00
2.07e+01
5.97e+00
Inactive
9.92e+00
Inactive
Inactive
Inactive
Inactive
Inactive
Active
Inactive
Inactive
Inactive
Inactive
Inactive
1.82e+01
3.00e+01
8.88e+00
1.29e+01
1.66e+01
Rat: active
Mouse: inactive
PFC-10
High risk
No risk
No risk
No risk
5.37e+00
9.73e+00
Active
PFC-11
PFC-12
PFC-13
PFC-14
PFC-15
PFC-16
High risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
No risk
2.78e+00
1.12e+01
1.79e+01
1.89e+01
9.03e+00
1.40e+01
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
1.20e+01
1.17e+01
1.04e+01
6.81e+00
1.84e+01
1.63e+01
Active
No risk
Inactive
Inactive
Inactive
Inactive
Inactive
Medium risk
Medium risk
Medium risk
Medium risk
647

EXCLI Journal 2017;16:628-649 – ISSN 1611-2156
Received: March 04, 2017, accepted: April 28, 2017, published: May 08, 2017
Gokhan-Kelekci N, Yabanoglu S, Kupeli E, Salgin U,
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