Synthesis and evaluation of new 1,5-diaryl-3-[4-(methylsulfonyl)phenyl]-4,5-dihydro-1 H-pyrazole derivatives as potential antidepressant agents

Article abstract of DOI:10.3390/molecules20022668

In an effort to develop potent antidepressant agents, new pyrazoline derivatives 2a-s were synthesized and evaluated for their antidepressant-like activity by tail suspension test (TST) and modified forced swimming test (MFST). The effects of the compound

Full text of DOI:10.3390/molecules20022668

Molecules 2015, 20, 2668-2684; doi:10.3390/molecules20022668
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Evaluation of New 1,5-Diaryl-3-[4-(methyl-
sulfonyl)phenyl]-4,5-dihydro-1H-pyrazole Derivatives as
Potential Antidepressant Agents
Ahmet Özdemir 1,*, Mehlika Dilek Altıntop 1,2, Zafer Asım Kaplancıklı 1, Özgür Devrim Can 3,
Ümide Demir Özkay 3 and Gülhan Turan-Zitouni 1
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University,
26470 Eskişehir, Turkey; E-Mails: mdaltintop@anadolu.edu.tr (M.D.A.);
zakaplan@anadolu.edu.tr (Z.A.K.); gturan@anadolu.edu.tr (G.T.-Z.)
2
Graduate School of Health Sciences, Anadolu University, 26470 Eskişehir, Turkey
3
Department of Pharmacology, Faculty of Pharmacy, Anadolu University, 26470 Eskişehir, Turkey;
E-Mails: ozgurdt@anadolu.edu.tr (Ö.D.C.); udemir@anadolu.edu.tr (Ü.D.Ö.)
* Author to whom correspondence should be addressed; E-Mail: ahmeto@anadolu.edu.tr;
Tel.: +90-222-3350580 (ext. 3774); Fax: +90-222-3350750.
Academic Editor: Derek J. McPhee
Received: 5 January 2015 / Accepted: 30 January 2015 / Published: 4 February 2015
Abstract: In an effort to develop potent antidepressant agents, new pyrazoline derivatives
2a–s were synthesized and evaluated for their antidepressant-like activity by tail suspension
test (TST) and modified forced swimming test (MFST). The effects of the compounds on
spontaneous locomotor activity were also investigated using an activity cage apparatus.
Among these derivatives, compounds 2b, 2d, 2f, 2o, and 2r decreased both horizontal and
vertical activity number of the mice. On the other hand, compounds 2a, 2h, 2j, 2k, 2l, 2m,
and 2n, which did not induce any significant change in the locomotor activity, significantly
shortened the immobility time of mice in TST and MFST, representing the presence of the
antidepressant-like effect. Additionally, the same compounds increased the swimming time
of mice in MFST without any change in climbing duration, similar to the reference drug
fluoxetine (10 mg/kg). In the light of previous papers examining the effects of pyrazolines
on central nervous system, this study, once more, pointed out remarkable antidepressant
activity potential of pyrazoline derivatives.

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Keywords: pyrazoline; antidepressant activity; tail suspension test; modified forced
swimming test; activity cage
1. Introduction
Major depressive disorder (MDD) is a multifactorial mood disorder affecting millions of people
around the world. MDD typically arises in the third decade of life, with a high recurrence rate. Lifetime
prevalence of MDD is around 13% and an incidence rate of 4%. Approximately 15% of patients with
depression die as a consequence of the illness and MDD accounts for at least 90% of all suicides. The
total cost of depression in Europe has been estimated at €118 billion [1].
According to the World Health Organization (WHO), depression is the second leading cause of
disability (years of healthy life lost) in patients aged 15–44 years. For 2020, the WHO estimates that
depression will become the second leading cause of disability for all age groups. By 2030, depression is
expected to become the leading cause of disability in industrialized countries [1,2].
Antidepressant drugs, which increase the levels of one or more monoamines in the synaptic clefts,
can be classified as tricyclic antidepressants (amitriptyline, nortriptyline, imipramine, etc.), monoamine
oxidase inhibitors (phenelzine, moclobemide, etc.), selective serotonin reuptake inhibitors (fluoxetine,
paroxetine, citalopram, etc.), selective noradrenaline reuptake inhibitors (reboxetine), and
serotonin-noradrenaline reuptake inhibitors (venlafaxine, desvenlafaxine) [1–6].
The limited mechanistic understanding of depression pathogenesis and decreased antidepressant
treatment response have resulted in the high rate of treatment failures. As a result, pharmaceutical
industry has focused on delineating the mechanisms underlying depression as well as on the
antidepressant drug discovery [3–6].
Hydrazine-based drugs still remain in clinical use for the treatment of depression. Due to their side
effects, medicinal chemists have focused on the discovery of new antidepressant agents with enhanced
pharmacological activity and limited toxicity via the structural modification of the hydrazine group [7].
In medicinal chemistry, pyrazoline scaffold has attracted a great deal of interest owing to its high
synthetic accessibility and diverse therapeutic applications. In particular, pyrazolines are considered as
the cyclic congeners of hydrazine group and therefore considerable research on them in relation to depression
has been carried out [7–17]. Encouraged by the large number of papers regarding the antidepressant
potential of pyrazoline scaffold [7–17], herein we describe the synthesis and in vivo evaluation of some
new methylsulfonyl-substituted pyrazoline derivatives as potential antidepressant agents.
2. Results and Discussion
The synthesis of compounds 2a–s followed the general pathway outlined in Scheme 1.
Methylsulfonyl-substituted chalcones 1a–c were synthesized via the base-catalyzed Claisen-Schmidt
condensation of 4'-(methylsulfonyl)acetophenone with appropriate aromatic aldehydes [18]. The ring
closure reaction of chalcones 1a–c with phenylhydrazine hydrochloride derivatives in hot acetic acid
afforded 1,5-diaryl-3-[4-(methylsulfonyl)phenyl]-4,5-dihydro-1H-pyrazoles 2a–s.

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O
O
i
S
S
R
O
O
O
O
1a-c
ii
O
O
S
HA
HM
HX
N
N
R
R'
2a-s
Reagents and conditions: (i) aromatic aldehyde, 10% aqueous sodium hydroxide solution, ethanol,
rt, 10 h; (ii) appropriate phenylhydrazine hydrochloride derivative, CH3COOH, reflux, 8 h.
Scheme 1. The synthetic route for the preparation of compounds 2a–s.
The structures of the newly synthesized compounds were elucidated by FT-IR, 1H-NMR, 13C-NMR,
mass spectral data, and elemental analyses. In the IR spectra of compounds 2a–s, C=N and C=C
1
stretching bands were observed in the region 1596–1406 cm−1. In the H-NMR spectra of
compounds 2a–s, the CH2 protons of the pyrazoline ring resonated as a pair of doublets of doublets at δ
3.13–3.20 ppm (JAM = 17.5–18.0 Hz, JAX = 6.0–7.5 Hz) and 3.89–3.96 ppm (JMA =16.5–18.0 Hz,
JMX = 12.0–13.0 Hz). The CH proton appeared as doublet of doublets at δ 5.54–5.65 ppm
(JMX =11.0–12.5 Hz, JAX = 5.5–7.0 Hz) due to the vicinal coupling with two magnetically non-equivalent
protons of the methylene group at position 4 of the pyrazoline ring. All the other aromatic and aliphatic
protons were observed at expected regions. The 13C-NMR chemical shift values of the carbon atoms at
43–44 ppm (C-4), 62–64 ppm (C-5) and 144–155 ppm (C-3) corroborate the 2-pyrazoline character
deduced from the 1H-NMR data. In the mass spectra of compounds 2a–s, the M+1 peak is observed. All
compounds gave satisfactory elemental analysis.
Tail suspension test (TST) and modified forced swimming test (MFST) were carried out to evaluate
the antidepressant-like effects of the test compounds. Further, the effects of the test compounds on
spontaneous locomotor activity of mice were assessed by activity cage measurements.

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TST and MFST are the most common experimental models for antidepressant activity screening.
Both of these two methods based on the observation that mice, after initial escape-oriented movements,
develop an immobile posture when placed in a short-term inescapable stressful situation. This
immobility, referred to as behavioral despair in animals, is believed to reproduce a condition similar to
human depression. Thus, a reduction in the total duration of immobility indicates an antidepressant
effect [19–21]. In this study, when assessed in TST and MFST, compounds 2a, 2h, 2j, 2k, 2l, 2m,
and 2n decreased the immobility time of mice compared to the control group, indicating the
antidepressant-like effects of these pyrazoline derivatives (100 mg/kg) (Figures 1 and 2). In MFST, the
same compounds increased the swimming time of the animals without any significant change in the
climbing duration (Figures 3 and 4). Shortened immobility and prolonged swimming duration, without
any change in the climbing time, indicated that the antidepressant-like effects of the compounds may be
related to serotonergic, rather than noradrenergic mechanisms in the central nervous system [22].
Nevertheless, involvement of serotonergic system in the observed antidepressant activity must be
confirmed with further studies such as depleting neuronal serotonin by p-chlorophenylalanine
pretreatment or measuring serotonin levels in limbic areas of brain etc. Fluoxetine (10 mg/kg), a selective
serotonin reuptake inhibitor, also showed an antidepressant-like action in both of these tests, as expected.
Figure 1. Effects of test compounds (100 mg/kg) and fluoxetine (10 mg/kg) on immobility
time of mice in TST. Significance against control values * p < 0.05, ** p < 0.01, *** p < 0.001.
Values are given as mean ± SEM. One-way ANOVA, post-hoc Tukey’s test, n = 7.

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Figure 2. Effects of test compounds (100 mg/kg) and fluoxetine (10 mg/kg) on immobility
time of mice in MFST. Significance against control values * p < 0.05, ** p < 0.01, *** p < 0.001.
Values are given as mean ± SEM. One-way ANOVA, post-hoc Tukey’s test, n = 7.
Figure 3. Effects of test compounds (100 mg/kg) and fluoxetine (10 mg/kg) on swimming
time of mice in MFST. Significance against control values * p < 0.05, ** p < 0.01, *** p < 0.001.
Values are given as mean ± SEM. One-way ANOVA, post-hoc Tukey’s test, n = 7.

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Figure 4. Effects of test compounds (100 mg/kg) and fluoxetine (10 mg/kg) on climbing
time of mice in MFST. Significance against control values * p < 0.05. Values are given as
mean ± SEM. One-way ANOVA, post-hoc Tukey’s test, n = 7.
In the activity cage test, compounds 2a, 2h, 2j, 2k, 2l, 2m, and 2n possessing antidepressant-like
activity did not induce any significant alteration in the total number of spontaneous locomotor activities
(Figures 5 and 6). This means that the anti-immobility effect cannot be attributable to a stimulant activity.
In other words, the observed antidepressant-like effect is specific. On the other hand, compounds 2b,
2d, 2f, and 2o, which did not induce any alteration in the immobility or the swimming time of the
animals, significantly reduced the number of both horizontal and vertical locomotor activity (Figures 5
and 6). This decrease in the spontaneous locomotor activity may be produced by neurosedative effect of
these aforementioned compounds. Instead, the effects of these compounds on neuromuscular junction
may also cause this situation. Further detailed studies will help to clarify this issue.
Among the tested pyrazoline derivatives, compound 2r was the only compound increasing the
immobility time of the mice in TST (Figure 1). On the other hand, the same compound did not change
the immobility time in MFST (Figure 2). Furthermore, in MFST, it decreased both swimming and
climbing time of the mice (Figures 3 and 4). Therefore, the prolongation of the immobility time in TST,
may not be caused by a possible depressant-like activity of compound 2r; instead this compound
probably affected motor activity/motor coordination of the mice. As a matter of fact, in the activity cage
test, decrease in the number of spontaneous locomotor activity of 2r-treated animals (Figures 4 and 5)
confirmed this idea. However, examining motor coordination of the animals by a further experiment
such as a Rota-rod test, may provide additional information about the unexpected immobility-inducing
effect of compound 2r in the TST.
As well as their remarkable antidepressant-like activity, compounds 2a, 2h, 2j, 2k, 2l, 2m, and 2n
exhibited negligible toxicity; incurred neither deaths nor undesirable side effects such as ataxia,
paralysis, convulsions, and diarrhea, giving an idea about the safety of the compounds. However, the

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exact mechanism of action and probable side effects of these compounds should be clarify with further
detailed studies.
Figure 5. Effects of test compounds (100 mg/kg) on the number of horizontal movement of
mice in the activity cage test. Significance against control values * p < 0.05, ** p < 0.01,
*** p < 0.001. Values are given as mean ± SEM. One-way ANOVA, post-hoc Tukey’s
test, n = 7.
Figure 6. Effects of test compounds (100 mg/kg) on the number of vertical movement of
mice in the activity cage test. Significance against control values ** p < 0.01, *** p < 0.001.
Values are given as mean ± SEM. One-way ANOVA, post-hoc Tukey’s test, n = 7.

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3. Experimental Section
3.1. General Information
All reagents were purchased from commercial suppliers and were used without further purification.
Melting points were determined on an Electrothermal 9100 melting point apparatus (Weiss-Gallenkamp,
Loughborough, UK) and were uncorrected. IR spectra were recorded on a Shimadzu 8400 FT-IR
1
13
spectrophotometer (Shimadzu, Tokyo, Japan). H-NMR and C-NMR spectra were recorded on a
Bruker spectrometer (Bruker, Billerica, MA, USA). Mass spectra were recorded on an Agilent
LC-MSD-Trap-SL Mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). Elemental analyses
were performed on a Perkin Elmer EAL 240 elemental analyzer (Perkin-Elmer, Norwalk, CT, USA).
Thin Layer Chromatography (TLC) was performed on TLC Silica gel 60 F254 aluminium sheets (Merck,
Darmstadt, Germany) using petroleum ether-ethyl acetate (3:1 v/v) as eluent.
3.2. Chemistry: General Procedures for the Synthesis of Compounds
3.2.1. 3-(4-Substituted phenyl)-1-[4-(methylsulfonyl)phenyl]-2-propen-1-ones 1a–c
A mixture of 4'-(methylsulfonyl)acetophenone (0.05 mol), aromatic aldehyde (0.05 mol) and 10%
aqueous sodium hydroxide (10 mL) in ethanol (30 mL) was stirred at room temperature for 10 h. The
progress of the reaction was checked by TLC. Upon completion, the reaction mixture was poured into
crushed ice. The precipitated solid was filtered, washed with water, and dried. The product was
crystallized from ethanol [18].
3.2.2. 1,5-Diaryl-3-[4-(methylsulfonyl)phenyl]-4,5-dihydro-1H-pyrazoles 2a–s
A mixture of the appropriate chalcone 1a–c (10.0 mmol) and phenylhydrazine hydrochloride
derivative (20.0 mmol) in the presence of acetic acid (50 mL) was refluxed for 8 h, then poured into
crushed ice. The precipitate was separated by filtration, washed with water and crystallized from methanol.
1-(4-Chlorophenyl)-5-(4-fluorophenyl)-3-(4-(methylsulfonyl)phenyl)-4,5-dihydro-1H-pyrazole
(2a):
Yield: 93%; m.p. 193 °C. IR (KBr) νmax (cm−1): 3012.60 (Aromatic C-H), 2918.10 (Aliphatic C-H),
1585.38, 1490.87 (C=N and C=C), 1307.65, 1149.50, 1087.78 (SO2 and C-N), 835.12 (C-H out of plane
1
deformation). H-NMR (500 MHz, DMSO-d6) δ (ppm): 3.19 (1H, dd, JAM = 17.5 Hz, JAX = 6.0 Hz,
C4-HA pyrazoline), 3.23 (3H, s, SO2CH3), 3.96 (1H, dd, JMA = 17.5 Hz, JMX = 12.0 Hz, C4-HM
pyrazoline), 5.65 (1H, dd, JMX = 12.5 Hz, JAX = 6.0 Hz, C5-HX pyrazoline), 7.04 (2H, d, J = 9.0 Hz,
aromatic protons), 7.15–7.19 (2H, m, aromatic protons), 7.23 (2H, d, J = 9.0 Hz, aromatic protons),
13
7.29–7.32 (2H, m, aromatic protons), 7.94–8.02 (4H, m, 4-methylsulfonylphenyl protons). C-NMR
(125 MHz, DMSO-d6) δ (ppm): 42.52 (CH3), 43.51 (CH2), 62.59 (CH), 114.74 (2CH), 115.78 (CH),
115.95 (CH), 126.28 (C), 127.33 (2CH), 127.98 (CH), 128.77 (2CH), 131.53 (CH), 136.72 (CH), 137.77
(CH), 140.12 (C), 142.19 (C), 144.05 (C), 146.46 (C), 152.05 (C), 160.46 (C). Anal. Calcd. for
C22H18ClFN2O2S: C, 61.61; H, 4.23; N, 6.53; Found: C, 61.60; H, 4.25; N, 6.52. MS (ESI) (m/z):
[M+1]+ 429.

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1,5-Bis(4-fluorophenyl)-3-(4-(methylsulfonyl)phenyl)-4,5-dihydro-1H-pyrazole (2b): Yield: 85%; m.p.
221 °C. IR (KBr) νmax (cm−1): 1508.23 (C=N), 1305.72, 1228.57, 1151.42 (SO2 and C-N), 835.12 (C-H
1
out of plane deformation). H-NMR (500 MHz, DMSO-d6) δ (ppm): 3.18 (1H, dd, JAM = 18.0 Hz,
JAX = 7.0 Hz, C4-HA pyrazoline), 3.23 (3H, s, SO2CH3), 3.94 (1H, dd, JMA = 17.5 Hz, JMX = 12.5 Hz, C4-HM
pyrazoline), 5.61 (1H, dd, JMX = 12.5 Hz, JAX = 6.5 Hz, C5-HX pyrazoline), 7.04 (4H, d, J = 6.5 Hz,
aromatic protons), 7.15–7.19 (2H, m, aromatic protons), 7.31–7.34 (2H, m, aromatic protons), 7.93–7.97
(4H, m, 4-methylsulfonylphenyl protons). 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 42.54 (CH3), 43.53
(CH2), 63.13 (CH), 114.54 (CH), 114.60 (CH), 115.46 (CH), 115.76 (CH), 115.93 (CH), 126.14 (2CH),
127.33 (2CH), 128.00 (CH), 128.07 (CH), 136.92 (CH), 138.01 (C), 139.92 (C), 140.25 (C), 145.75 (C),
155.34 (C), 160.45 (C), 162.39 (C). Anal. Calcd. for C22H18F2N2O2S: C, 64.06; H, 4.40; N, 6.79; Found:
C, 64.05; H, 4.39; N, 6.80. MS (ESI) (m/z): [M+1]+ 413.
1-(4-Bromophenyl)-5-(4-fluorophenyl)-3-(4-(methylsulfonyl)phenyl)-4,5-dihydro-1H-pyrazole
(2c):
Yield: 77%; m.p. 169 °C. IR (KBr) νmax (cm−1): 3014.53 (Aromatic C-H), 1583.45, 1488.94 (C=N and
1
C=C), 1307.65, 1151.42, 1085.85 (SO2 and C-N), 835.12 (C-H out of plane deformation). H-NMR
(500 MHz, DMSO-d6) δ (ppm): 3.19 (1H, dd, JAM = 17.5 Hz, JAX = 6.0 Hz, C4-HA pyrazoline), 3.23 (3H,
s, SO2CH3), 3.95 (1H, dd, JMA = 17.5 Hz, JMX = 12.5 Hz, C4-HM pyrazoline), 5.65 (1H, dd,
JMX = 12.5 Hz, JAX = 6.0 Hz, C5-HX pyrazoline), 6.99 (2H, d, J = 9.0 Hz, aromatic protons), 7.15–7.19
(2H, m, aromatic protons), 7.29–7.32 (2H, m, aromatic protons), 7.33–7.36 (2H, m, aromatic protons),
13
7.94–7.98 (4H, m, 4-methylsulfonylphenyl protons). C-NMR (125 MHz, DMSO-d6) δ (ppm): 42.52
(CH3), 43.51 (CH2), 62.49 (CH), 115.22 (2CH), 115.80 (CH), 115.97 (CH), 125.98 (2CH), 127.35 (2CH),
127.97 (CH), 129.36 (C), 131.62 (2CH), 136.71 (CH), 137.73 (C), 140.14 (C), 142.52 (C), 146.56 (C),
160.47 (C), 162.41 (C). Anal. Calcd. for C22H18BrFN2O2S: C, 55.82; H, 3.83; N, 5.92. Found: C, 55.80;
H, 3.81; N, 5.92. MS (ESI) (m/z): [M+1]+ 474.
1-(4-Methoxyphenyl)-5-(4-fluorophenyl)-3-(4-(methylsulfonyl)phenyl)-4,5-dihydro-1H-pyrazole (2d):
Yield: 60%; m.p. 170 °C. IR (KBr) νmax (cm−1): 1510.16 (C=N), 1321.15, 1245.93, 1153.35, 1099.35
(SO2, C-N and C-O), 811.98 (C-H out of plane deformation). 1H-NMR (500 MHz, DMSO-d6) δ (ppm):
3.13 (1H, dd, JAM = 17.5 Hz, JAX = 7.0 Hz, C4-HA pyrazoline), 3.22 (3H, s, SO2CH3), 3.65 (3H, s, OCH3),
3.89 (1H, dd, JMA = 17.5 Hz, JMX = 12.5 Hz, C4-HM pyrazoline), 5.54 (1H, dd, JMX = 12.5 Hz,
JAX = 7.0 Hz, C5-HX pyrazoline), 6.80 (2H, d, J = 7.0 Hz, aromatic protons), 6.99 (2H, d, J = 7.0 Hz,
aromatic protons), 7.14–7.18 (2H, m, aromatic protons), 7.31–7.34 (2H, m, aromatic protons), 7.90–7.94
(4H, m, 4-methylsulfonylphenyl protons). 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 42.35 (CH3), 43.56
(CH2), 55.16 (CH3), 63.54 (CH), 114.41 (2CH), 114.78 (2CH), 115.67 (CH), 115.84 (CH), 125.85 (2CH),
127.32 (2CH), 128.05 (CH), 128.11 (CH), 137.19 (C), 138.31 (C), 139.51 (C), 144.53 (C), 153.18 (C),
160.41 (C), 162.34 (C). Anal. Calcd. for C23H21FN2O3S: C, 65.08; H, 4.99; N, 6.60. Found: C, 65.10; H,
4.97; N, 6.59. MS (ESI) (m/z): [M+1]+ 425.
1-(4-Methylphenyl)-5-(4-fluorophenyl)-3-(4-(methylsulfonyl)phenyl)-4,5-dihydro-1H-pyrazole
(2e):
Yield: 76%; m.p. 186 °C. IR (KBr) νmax (cm−1): 3028.03 (Aromatic C-H), 2920.03 (Aliphatic C-H),
1585.38, 1510.16 (C=N and C=C), 1305.72, 1224.71, 1151.42, 1087.78 (SO2 and C-N), 835.12, 781.12
(C-H out of plane deformation). 1H-NMR (500 MHz, DMSO-d6) δ (ppm): 2.17 (3H, s, CH3), 3.14 (1H,

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dd, JAM = 17.5 Hz, JAX = 6.0 Hz, C4-HA pyrazoline), 3.22 (3H, s, SO2CH3), 3.91 (1H, dd, JMA = 17.5 Hz,
JMX = 12.5 Hz, C4-HM pyrazoline), 5.60 (1H, dd, JMX = 12.0 Hz, JAX = 6.0 Hz, C5-HX pyrazoline),
6.95–7.00 (4H, m, aromatic protons), 7.13–7.18 (2H, m, aromatic protons), 7.28–7.35 (2H, m, aromatic
protons), 7.91–7.94 (4H, m, 4-methylsulfonylphenyl protons). 13C-NMR (125 MHz, DMSO-d6) δ (ppm):
20.08 (CH3), 43.20 (CH3), 43.56 (CH2), 62.88 (CH), 113.45 (2CH), 115.68 (CH), 115.85 (CH), 115.91
(CH), 125.97 (2CH), 127.38 (2CH), 128.00 (CH), 128.22 (CH), 129.41 (CH), 131.55 (C), 137.11 (C),
139.66 (C), 144.07 (C), 144.89 (C), 160.39 (C), 162.33 (C). Anal. Calcd. for C23H21FN2O2S: C, 67.63;
H, 5.18; N, 6.86. Found: C, 67.65; H, 5.17; N, 6.85. MS (ESI) (m/z): [M+1]+ 409.
1-(4-Methoxyphenyl)-5-(4-chlorophenyl)-3-(4-(methylsulfonyl)phenyl)-4,5-dihydro-1H-pyrazole (2f):
Yield: 76%; m.p. 152 °C. IR (KBr) νmax (cm−1): 2925.81 (Aliphatic C-H asymmetric), 2833.24 (Aliphatic
C-H symmetric), 1508.23 (C=N), 1307.65, 1242.07, 1151.42, 1087.78 (SO2, C-N and C-O), 825.48
(C-H out of plane deformation). 1H-NMR (500 MHz, DMSO-d6) δ (ppm): 3.14 (1H, dd, JAM = 18.0 Hz,
JAX = 7.5 Hz, C4-HA pyrazoline), 3.22 (3H, s, SO2CH3), 3.65 (3H, s, OCH3), 3.90 (1H, dd,
JMA = 17.0 Hz, JMX = 12.5 Hz, C4-HM pyrazoline), 5.55 (1H, dd, JMX = 12.5 Hz, JAX = 7.0 Hz, C5-HX
pyrazoline), 6.80 (2H, d, J = 9.0 Hz, aromatic protons), 6.98 (2H, d, J = 9.0 Hz, aromatic protons), 7.30
(2H, d, J = 8.5 Hz, aromatic protons), 7.39 (2H, d, J = 8.5 Hz, aromatic protons), 7.90–7.94 (4H, m,
4-methylsulfonylphenyl protons). 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 42.73 (CH3), 44.06 (CH2),
55.66 (CH3), 64.03 (CH), 114.95 (2CH), 115.25 (2CH), 126.37 (2CH), 127.82 (2CH), 128.49 (2CH),
129.46 (2CH), 131.76 (C), 138.10 (C), 140.05 (C), 141.59 (C), 145.09 (C), 153.70 (C), 160.40 (C). Anal.
Calcd. for C23H21ClN2O3S: C, 62.65; H, 4.80; N, 6.35. Found: C, 62.63; H, 4.78; N, 6.34. MS (ESI)
(m/z): [M+1]+ 441.
1-(4-Fluorophenyl)-5-(4-chlorophenyl)-3-(4-(methylsulfonyl)phenyl)-4,5-dihydro-1H-pyrazole (2g):
Yield: 87%; m.p. 203 °C. IR (KBr) νmax (cm−1): 2974.03 (Aliphatic C-H asymmetric), 2885.31 (Aliphatic
C-H symmetric), 1577.66, 1504.37 (C=N and C=C), 1305.72, 1230.50, 1151.42, 1087.78 (SO2 and
C-N), 825.48 (C-H out of plane deformation). 1H-NMR (500 MHz, DMSO-d6) δ (ppm): 3.18 (1H, dd,
JAM = 17.5 Hz, JAX = 6.5 Hz, C4-HA pyrazoline), 3.23 (3H, s, SO2CH3), 3.95 (1H, dd, JMA = 17.5 Hz,
JMX = 12.5 Hz, C4-HM pyrazoline), 5.61 (1H, dd, JMX = 12.5 Hz, JAX = 6.5 Hz, C5-HX pyrazoline),
7.02–7.07 (4H, m, aromatic protons), 7.30 (2H, d, J = 7.0 Hz, aromatic protons), 7.40 (2H, d,
J = 6.5 Hz, aromatic protons), 7.93–7.96 (4H, m, 4-methylsulfonylphenyl protons). 13C-NMR (125 MHz,
DMSO-d6) δ (ppm): 42.42 (CH3), 43.52 (CH2), 63.12 (CH), 114.51 (CH), 114.57 (CH), 115.50 (CH),
115.68 (CH), 126.16 (2CH), 127.34 (2CH), 127.93 (2CH), 129.04 (2CH), 132.13 (C), 136.85 (C), 139.95
(C), 140.78 (C), 145.81 (C), 155.36 (C), 160.41 (C). Anal. Calcd. for C22H18ClFN2O2S: C, 61.61; H,
4.23; N, 6.53. Found: C, 61.60; H, 4.22; N, 6.52. MS (ESI) (m/z): [M+1]+ 429
1-(4-Methylphenyl)-5-(4-chlorophenyl)-3-(4-(methylsulfonyl)phenyl)-4,5-dihydro-1H-pyrazole (2h):
Yield: 83%; m.p. 230 °C. IR (KBr) νmax (cm−1): 2970.17 (Aliphatic C-H asymmetric), 2883.38 (Aliphatic
C-H symmetric), 1510.16 (C=N), 1305.72, 1151.42, 1087.78 (SO2 and C-N), 825.48 (C-H out of plane
deformation). 1H-NMR (500 MHz, DMSO-d6) δ (ppm): 2.17 (3H, s, CH3), 3.15 (1H, dd, JAM = 17.5 Hz,
JAX = 6.0 Hz, C4-HA pyrazoline), 3.22 (3H, s, SO2CH3), 3.92 (1H, dd, JMA = 18.0 Hz, JMX = 12.0 Hz, C4-HM
pyrazoline), 5.60 (1H, dd, JMX = 12.5 Hz, JAX = 6.0 Hz, C5-HX pyrazoline), 6.95 (2H, d, J = 7.0 Hz,

Molecules 2015, 20
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aromatic protons), 6.99 (2H, d, J = 8.0 Hz, aromatic protons), 7.29 (2H, d, J = 7.0 Hz, aromatic protons),
7.39 (2H, d, J = 8.5 Hz, aromatic protons), 7.93 (4H, s, 4-methylsulfonylphenyl protons). 13C-NMR (125
MHz, DMSO-d6) δ (ppm): 20.07 (CH3), 42.15 (CH3), 43.55 (CH2), 62.87 (CH), 113.43 (2CH), 125.99
(2CH), 127.33 (2CH), 127.87 (2CH), 128.27 (CH), 128.96 (CH), 129.43 (CH), 132.00 (CH), 137.05 (C),
139.70 (C), 141.07 (C), 141.13 (2C), 144.96 (C), 160.43 (C). Anal. Calcd. for C23H21ClN2O2S: C, 65.01;
H, 4.98; N, 6.59. Found: C, 65.00; H, 4.99; N, 6.58. MS (ESI) (m/z): [M+1]+ 425.
1-(4-Bromophenyl)-5-(4-chlorophenyl)-3-(4-(methylsulfonyl)phenyl)-4,5-dihydro-1H-pyrazole
(2i):
Yield: 83%; m.p. 204 °C. IR (KBr) νmax (cm−1): 2918.10 (Aliphatic C-H), 1583.45, 1487.01 (C=N and
C=C), 1307.65, 1151.42, 1087.78 (SO2 and C-N), 819.69 (C-H out of plane deformation). 1H-NMR (500
MHz, DMSO-d6) δ (ppm): 3.20 (1H, dd, JAM = 18.0 Hz, JAX = 6.0 Hz, C4-HA pyrazoline), 3.23 (3H, s,
SO2CH3), 3.96 (1H, dd, JMA = 17.5 Hz, JMX = 12.5 Hz, C4-HM pyrazoline), 5.65 (1H, dd, JMX = 12.5 Hz,
JAX = 6.0 Hz, C5-HX pyrazoline), 6.99 (2H, d, J = 9.0 Hz, aromatic protons), 7.28 (2H, d, J = 8.5 Hz,
aromatic protons), 7.35 (2H, d, J = 9.5 Hz, aromatic protons), 7.41 (2H, d, J = 8.5 Hz, aromatic protons),
13
7.94–7.98 (4H, m, 4-methylsulfonylphenyl protons). C-NMR (125 MHz, DMSO-d6) δ (ppm): 42.90
(CH3), 44.01 (CH2), 63.00 (CH), 115.70 (2CH), 126.84 (2CH), 127.86 (2CH), 128.33 (2CH), 129.59
(2CH), 132.16 (2CH), 132.69 (C), 137.16 (C), 140.68 (C), 141.00 (C), 142.97 (C), 147.13 (C), 160.45
(C). Anal. Calcd. For C22H18BrClN2O2S: C, 53.95; H, 3.70; N, 5.72. Found: C, 53.93; H, 3.69; N, 5.70.
MS (ESI) (m/z): [M+1]+ 490.
1,5-Bis(4-chlorophenyl)-3-(4-(methylsulfonyl)phenyl)-4,5-dihydro-1H-pyrazole (2j): Yield: 73%; m.p.
209 °C. IR (KBr) νmax (cm−1): 2918.10 (Aliphatic C-H), 1583.45, 1488.94 (C=N and C=C), 1307.65,
1151.42, 1087.78 (SO2 and C-N), 821.62 (C-H out of plane deformation). 1H-NMR (500 MHz, DMSO-d6)
δ (ppm): 3.20 (1H, dd, JAM = 17.5 Hz, JAX = 6.0 Hz, C4-HA pyrazoline), 3.23 (3H, s, SO2CH3), 3.96 (1H,
dd, JMA = 18.0 Hz, JMX = 12.5 Hz, C4-HM pyrazoline), 5.65 (1H, dd, JMX = 12.5 Hz, JAX = 6.0 Hz, C5-HX
pyrazoline), 7.03 (2H, d, J = 9.0 Hz, aromatic protons), 7.23 (2H, d, J = 9.0 Hz, aromatic protons), 7.29
(2H, d, J = 8.5 Hz, aromatic protons), 7.41 (2H, d, J = 8.5 Hz, aromatic protons), 7.94–7.98 (4H, m,
4-methylsulfonylphenyl protons). 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 42.91 (CH3), 44.01 (CH2),
63.09 (CH), 115.23 (2CH), 126.82 (2CH), 127.86 (2CH), 128.34 (2CH), 129.32 (2CH), 129.90 (2CH),
132.69 (C), 137.18 (C), 140.66 (C), 141.05 (C), 142.64 (C), 147.04 (C), 160.48 (C). Anal. Calcd. for
C22H18Cl2N2O2S: C, 59.33; H, 4.07; N, 6.29. Found: C, 59.32; H, 4.05; N, 6.30. MS (ESI) (m/z):
[M+1]+ 446.
1-(4-Methoxyphenyl)-5-(4-bromophenyl)-3-(4-(methylsulfonyl)phenyl)-4,5-dihydro-1H-pyrazole (2k):
Yield: 68%; m.p. 145 °C. IR (KBr) νmax (cm−1): 2927.74 (Aliphatic C-H asymmetric), 2827.45 (Aliphatic
C-H symmetric), 1510.16, 1417.58 (C=N and C=C), 1313.43, 1242.07, 1151.42, 1087.78 (SO2, C-N and
1
C-O), 813.90 (C-H out of plane deformation). H-NMR (500 MHz, DMSO-d6) δ (ppm): 6.97 (d,
J = 3.6 Hz, 1H), 7.11 (d, J = 3.2 Hz, 1H), 7.43–7.49 (m, 3H), 7.83–7.89 (m, 3H), 7.93–7.95 (m, 2H),
8.13–8.14 (m, 1H), 12.37 (brs, 1H). 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 42.17 (CH3), 43.56 (CH2),
55.16 (CH3), 63.56 (CH), 114.45 (2CH), 114.73 (2CH), 120.56 (C), 125.87 (2CH), 127.33 (2CH),
128.33 (2CH), 131.88 (2CH), 137.12 (C), 137.57 (C), 139.54 (C), 141.52 (C), 144.59 (C), 153.19 (C).

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Anal. Calcd. for C23H21BrN2O3S: C, 56.91; H, 4.36; N, 5.77. Found: C, 56.90; H, 4.35; N, 5.76. MS
(ESI) (m/z): [M+1]+ 486.
1-(4-Fluorophenyl)-5-(4-bromophenyl)-3-(4-(methylsulfonyl)phenyl)-4,5-dihydro-1H-pyrazole
(2l):
Yield: 75%; m.p. 196 °C. IR (KBr) νmax (cm−1): 3031.89 (Aromatic C-H), 2918.10 (Aliphatic C-H),
1577.66, 1506.30, 1406.01 (C=N and C=C), 1307.65, 1218.93, 1151.42, 1010.63 (SO2 and C-N), 825.48
(C-H out of plane deformation). 1H-NMR (500 MHz, DMSO-d6) δ (ppm): 3.17 (1H, dd, JAM = 18.0 Hz,
JAX = 7.0 Hz, C4-HA pyrazoline), 3.23 (3H, s, SO2CH3), 3.95 (1H, dd, JMA = 16.5 Hz, JMX = 12.5 Hz,
C4-HM pyrazoline), 5.59 (1H, dd, JMX = 11.0 Hz, JAX = 6.0 Hz, C5-HX pyrazoline), 7.04 (4H, s, aromatic
protons), 7.24–7.26 (2H, m, aromatic protons), 7.53–7.55 (2H, m, aromatic protons), 7.95 (4H, s,
4-methylsulfonylphenyl protons). 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 42.36 (CH3), 43.52 (CH2),
63.17 (CH), 114.57 (CH), 115.50 (CH), 115.68 (2CH), 120.66 (CH), 126.16 (2CH), 127.34 (2CH),
128.26 (2CH), 131.96 (2C), 136.85 (CH), 139.96 (C), 140.17 (C), 141.20 (C), 145.82 (C), 155.37 (C).
Anal. Calcd. for C22H18BrFN2O2S: C, 55.82; H, 3.83; N, 5.92. Found: C, 55.82; H, 3.81; N, 5.93. MS
(ESI) (m/z): [M+1]+ 474.
1-(4-Methylphenyl)-5-(4-bromophenyl)-3-(4-(methylsulfonyl)phenyl)-4,5-dihydro-1H-pyrazole (2m):
Yield: 87%; m.p. 227 °C. IR (KBr) νmax (cm−1): 3016.46 (Aromatic C-H), 2918.10 (Aliphatic C-H),
1585.38, 1508.23, 1407.94 (C=N and C=C), 1294.15, 1242.07, 1151.42, 1085.85, 1010.63 (SO2 and
1
C-N), 821.62 (C-H out of plane deformation). H-NMR (500 MHz, DMSO-d6) δ (ppm): 2.17 (3H, s,
CH3), 3.15 (1H, dd, JAM = 17.5 Hz, JAX = 6.0 Hz, C4-HA pyrazoline), 3.22 (3H, s, SO2CH3), 3.92 (1H,
dd, JMA = 17.5 Hz, JMX = 12.5 Hz, C4-HM pyrazoline), 5.59 (1H, dd, JMX = 12.5 Hz, JAX = 6.0 Hz, C5-HX
pyrazoline), 6.94 (2H, d, J = 8.5 Hz), 6.99 (2H, d, J = 8.0 Hz), 7.22 (2H, d, J = 8.5 Hz, aromatic protons),
7.53 (2H, d, J = 8.5 Hz, aromatic protons), 7.93 (4H, s, 4-methylsulfonylphenyl protons). 13C-NMR (125
MHz, DMSO-d6) δ (ppm): 20.58 (CH3), 42.59 (CH3), 44.05 (CH2), 63.42 (CH), 113.92 (2CH), 121.02
(C), 126.50 (2CH), 127.83 (2CH), 128.73 (2CH), 128.77 (2CH), 129.94 (2CH), 132.38 (C), 137.54 (C),
140.20 (C), 141.62 (C), 142.00 (C), 145.47 (C). Anal. Calcd. for C23H21BrN2O2S: C, 58.85; H, 4.51; N,
5.97. Found: C, 58.85; H, 4.50; N, 5.95. MS (ESI) (m/z): [M+1]+ 470.
1,5-Bis(4-bromophenyl)-3-(4-(methylsulfonyl)phenyl)-4,5-dihydro-1H-pyrazole (2n): Yield: 94%; m.p.
225 °C. IR (KBr) νmax (cm−1): 3012.60 (Aromatic C-H), 2918.10 (Aliphatic C-H), 1583.45, 1487.01,
1409.87 (C=N and C=C), 1307.65, 1151.42, 1085.85, 1010.63 (SO2 and C-N), 819.69 (C-H out of plane
1
deformation). H-NMR (500 MHz, DMSO-d6) δ (ppm): 3.20 (1H, dd, JAM = 18.0 Hz, JAX = 6.0 Hz,
C4-HA pyrazoline), 3.23 (3H, s, SO2CH3), 3.96 (1H, dd, JMA = 17.5 Hz, JMX = 12.5 Hz, C4-HM
pyrazoline), 5.63 (1H, dd, JMX = 12.0 Hz, JAX = 5.5 Hz, C5-HX pyrazoline), 6.98 (2H, d, J = 8.5 Hz), 7.21
(2H, d, J = 8.0 Hz, aromatic protons), 7.35 (2H, d, J = 9.0 Hz, aromatic protons), 7.54 (2H, d,
J = 8.5 Hz, aromatic protons), 7.93–7.97 (4H, m, 4-methylsulfonylphenyl protons). 13C-NMR (125 MHz,
DMSO-d6) δ (ppm): 42.34 (CH3), 43.51 (CH2), 62.54 (CH), 110.74 (2CH), 115.19 (C), 120.72 (C),
126.33 (2CH), 127.35 (2CH), 128.15 (2CH), 131.99 (2CH), 136.64 (2CH), 140.17 (C), 140.91 (C),
142.45 (C), 146.62 (C), 151.70 (C). Anal. Calcd. for C22H18Br2N2O2S: C, 49.46; H, 3.40; N, 5.24. Found:
C, 49.44; H, 3.41; N, 5.25. MS (ESI) (m/z): [M+1]+ 535.

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1-(4-Chlorophenyl)-5-(4-bromophenyl)-3-(4-(methylsulfonyl)phenyl)-4,5-dihydro-1H-pyrazole (2o):
Yield: 95%; m.p. 222 °C. IR (KBr) νmax (cm−1): 3045.39 (Aromatic C-H), 2918.10 (Aliphatic C-H),
1583.45, 1488.94 (C=N and C=C), 1307.65, 1151.42, 1085.85 (SO2 and C-N), 819.69 (C-H out of plane
deformation). 1H-NMR (500 MHz, DMSO-d6) δ (ppm): 3.20 (1H, dd, JAM = 18.0 Hz, JAX = 6.0 Hz, C4-HA
pyrazoline), 3.23 (3H, s, SO2CH3), 3.96 (1H, dd, JMA = 18.0 Hz, JMX = 12.5 Hz, C4-HM pyrazoline), 5.63
(1H, dd, JMX = 12.5 Hz, JAX = 6.0 Hz, C5-HX pyrazoline), 7.03 (2H, d, J = 9.0 Hz, aromatic protons),
7.22–7.24 (4H, m, aromatic protons), 7.54 (2H, d, J = 8.5 Hz, aromatic protons), 7.94–7.98 (4H, m,
4-methylsulfonylphenyl protons). 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 42.35 (CH3), 43.51 (CH2),
62.64 (CH), 114.71 (2CH), 120.71 (C), 123.06 (CH), 126.31 (CH), 127.35 (2CH), 128.17 (2CH), 128.82
(2CH), 131.99 (2CH), 136.66 (C), 140.15 (2C), 140.96 (C), 142.12 (C), 146.53 (C). Anal. Calcd. for
C22H18BrClN2O2S: C, 53.95; H, 3.70; N, 5.72. Found: C, 53.93; H, 3.69; N, 5.74. MS (ESI) (m/z):
[M+1]+ 490.
1-Phenyl-5-(4-bromophenyl)-3-(4-(methylsulfonyl)phenyl)-4,5-dihydro-1H-pyrazole (2p): Yield: 86%;
m.p. 205 °C. IR (KBr) νmax (cm−1): 3020.32 (Aromatic C-H), 2921.96 (Aliphatic C-H), 1595.02,
1485.09, 1407.94 (C=N and C=C), 1303.79, 1151.42, 1010.63 (SO2 and C-N), 821.62, 750.26 (C-H out
1
of plane deformation). H-NMR (500 MHz, DMSO-d6) δ (ppm): 3.17 (1H, dd, JAM = 17.5 Hz,
JAX = 6.0 Hz, C4-HA pyrazoline), 3.23 (3H, s, SO2CH3), 3.94 (1H, dd, JMA = 17.5 Hz, JMX = 12.5 Hz,
C4-HM pyrazoline), 5.62 (1H, dd, JMX = 12.0 Hz, JAX = 6.0 Hz, C5-HX pyrazoline), 6.77 (1H, m, aromatic
protons), 7.04 (2H, d, J = 8.0 Hz, aromatic protons), 7.17–7.20 (2H, m, aromatic protons), 7.24 (2H, d,
J = 8.5 Hz, aromatic protons), 7.53 (2H, d, J = 8.5 Hz, aromatic protons), 7.93–7.97 (4H, m,
4-methylsulfonylphenyl protons). 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 42.19 (CH3), 43.54 (CH2),
62.71 (CH), 113.28 (2CH), 119.47 (C), 120.57 (CH), 126.14 (2CH), 127.34 (2CH), 128.18 (2CH),
129.00 (2CH), 131.93 (2CH), 136.92 (C), 139.90 (C), 141.45 (C), 143.28 (C), 145.60 (C). Anal. Calcd.
for C22H19BrN2O2S: C, 58.03; H, 4.21; N, 6.15. Found: C, 58.01; H, 4.20; N, 6.17. MS (ESI) (m/z):
[M+1]+ 456.
1-Phenyl-5-(4-chlorophenyl)-3-(4-(methylsulfonyl)phenyl)-4,5-dihydro-1H-pyrazole (2r): Yield: 81%;
m.p. 212 °C. IR (KBr) νmax (cm−1): 3020.32 (Aromatic C-H), 2921.96 (Aliphatic C-H), 1596.95,
1492.80, 1411.80 (C=N and C=C), 1303.79, 1151.42, 1087.78 (SO2 and C-N), 823.55, 750.26 (C-H out
1
of plane deformation). H-NMR (500 MHz, DMSO-d6) δ (ppm): 3.17 (1H, dd, JAM = 17.5 Hz,
JAX = 6.0 Hz, C4-HA pyrazoline), 3.23 (3H, s, SO2CH3), 3.94 (1H, dd, JMA = 18.0 Hz, JMX = 13.0 Hz,
C4-HM pyrazoline), 5.63 (1H, dd, JMX = 12.0 Hz, JAX = 6.0 Hz, C5-HX pyrazoline), 6.76–6.79 (1H, m,
aromatic protons), 7.04 (2H, d, J = 8.0 Hz, aromatic protons), 7.17–7.20 (2H, m, aromatic protons), 7.30
(2H, d, J = 8.5 Hz, aromatic protons), 7.40 (2H, d, J = 8.5 Hz, aromatic protons), 7.95–7.97 (4H, m,
4-methylsulfonylphenyl protons). 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 42.24 (CH3), 43.53 (CH2),
62.67 (CH), 113.29 (2CH), 119.46 (CH), 126.13 (2CH), 127.33 (2CH), 127.82 (2CH), 129.00 (3CH),
132.05 (CH), 136.93 (C), 139.89 (C), 141.01 (2C), 143.29 (C), 145.57 (C). Anal. Calcd. for C22H19ClN2O2S:
C, 64.30; H, 4.66; N, 6.82. Found: C, 64.31; H, 4.65; N, 6.80. MS (ESI) (m/z): [M+1]+ 411.
1-Phenyl-5-(4-fluorophenyl)-3-(4-(methylsulfonyl)phenyl)-4,5-dihydro-1H-pyrazole (2s): Yield: 78%;
m.p. 219 °C. IR (KBr) νmax (cm−1): 3020.32 (Aromatic C-H), 2921.96 (Aliphatic C-H), 1595.02, 1492.80

Molecules 2015, 20
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(C=N and C=C), 1380.94, 1299.93, 1224.71, 1149.50, 1087.78 (SO2 and C-N), 837.05, 748.33 (C-H out
of plane deformation). 1H-NMR (500 MHz, DMSO-d6) δ (ppm): 3.16 (1H, dd, JAM = 17.5 Hz, JAX = 6.0 Hz,
C4-HA pyrazoline), 3.23 (3H, s, SO2CH3), 3.93 (1H, dd, JMA = 17.5 Hz, JMX = 12.5 Hz, C4-HM
pyrazoline), 5.62 (1H, dd, JMX = 12.5 Hz, JAX = 6.5 Hz, C5-HX pyrazoline), 6.75–6.78 (1H, m, aromatic
protons), 7.06 (2H, d, J = 8.0 Hz, aromatic protons), 7.15–7.20 (4H, m, aromatic protons), 7.31–7.34
13
(2H, m, aromatic protons), 7.94–7.96 (4H, m, 4-methylsulfonylphenyl protons). C-NMR (125 MHz,
DMSO-d6) δ (ppm): 42.37 (CH3), 43.55 (CH2), 62.68 (CH), 113.32 (2CH), 115.72 (CH), 115.89 (CH),
119.42 (CH), 126.11 (2CH), 127.34 (2CH), 127.96 (CH), 128.96 (2CH), 137.00 (CH), 138.22 (C),
139.85 (C), 143.36 (C), 145.51 (C), 160.41 (C), 162.35 (C). Anal. Calcd. for C22H19FN2O2S: C, 66.99;
H, 4.85; N, 7.10. Found: C, 66.98; H, 4.82; N, 7.12. MS (ESI) (m/z): [M+1]+ 395.
3.3. Pharmacology
3.3.1. Animals
Adult Balb/c male mice (30–35 g), obtained from Anadolu University Research Center for Animal
Experiments, were used for the experiments. The animals were housed at room temperature of 24 ± 1 °C
with 12/12 h light/dark cycle (lights on at 08:00 h). Temperature, sound, and light conditions were not
altered during the course of the experiments. 12 h before each experiment, animals received only water,
in order to avoid food interference with substances absorption. The experimental protocols were approved
by the Local Ethical Committee on Animal Experimentation of Anadolu University, Eskişehir, Turkey.
3.3.2. Assessment of Antidepressant Activity
Tail Suspension Test
TST was carried out by a method described earlier by Steru and co-workers [21]. The method was
performed using an automatic TST apparatus (BioSeb, Vitrolles, France), as described previously [19].
The mice were taped by their tails on a metal hook in 3 test chambers (15 cm width × 19 cm height)
constructed of white plastic walls and black plastic floors. Each hook was connected to a computerized
strain gauge that was adjusted to detect all movements of the animals (Tail suspension software, Bioseb).
Immobility time of mice was measured during the last 4 min of 6 min test duration [23].
Modified Forced Swimming Test
MFST was performed as described previously [15,24]. The mice were forced to swim individually in
a glass cylinder (12 cm diameter × 30 cm height) containing 20 cm of water at 25 ± 1 °C. A 15-min
pre-test was conducted 24 h before the 5-min swim test. During the test, time for swimming (horizontal
movement on the surface of the water), climbing (upward-directed movements of the forepaws along
the side of the cylinder), and immobility (movement required just to keep the head above the water) were
recorded using a stopwatch.

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3.3.3. Assessment of Locomotor Activity
Activity Cage Test
The horizontal and vertical locomotor activity of the mice were monitored using an activity cage
apparatus (Ugo Basile, No. 7420, Varese, Italy), which contains two pairs of 16 photocells 3 cm and
12 cm above the floor. Interruptions of light beams to the photocells during horizontal and vertical
movements of the animals were automatically recorded for 4 min [15].
3.3.4. Statistical Analyses
Statistical analyses were performed on data for seven animals (n = 7) from each group by using
GraphPad Prism 3.0 software (GraphPad Software, San Diego, CA, USA). Comparisons between the
experimental groups were performed by one-way ANOVA followed by Tukey’s test. The results were
expressed as mean ± standard error of mean (SEM). Differences between the datasets were considered
significant at p < 0.05.
4. Conclusions
In conclusion, this study supports the previous papers reporting the antidepressant-like activities of
pyrazoline derivatives [7–17]. The antidepressant-like effects of compounds 2a, 2h, 2j, 2k, 2l, 2m, and
2n seem to be related with the serotonergic system rather than the noradrenergic system. However,
involvement of the serotonergic system in the observed antidepressant activity needs to be confirmed
with further detailed studies.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/02/2668/s1.
Acknowledgments
This study was supported by Anadolu University Scientific Research Projects Commission under the
grant no: 1406S315 and 1206S105.
Author Contributions
A.Ö., M.D.A., Z.A.K. and G.T.-Z. designed the research; A.Ö. and M.D.A. performed the synthetic
work, Ö.D.C. and Ü.D.Ö. were responsible for the whole pharmacological part of the manuscript (animal
experiments, statistical evaluation, writing and discussion of the pharmacological results). A.Ö. was also
responsible for the correspondence of the manuscript, whereas M.D.A. mainly wrote the manuscript. All
authors discussed, edited and approved the final version.
Conflicts of Interest
The authors declare no conflict of interest.

Molecules 2015, 20
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Sample Availability: Samples of the compounds 2a–s are available from the authors.
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