Design, synthesis, and neuroprotective effects of a series of pyrazolines against 6-hydroxydopamine-induced oxidative stress

Article abstract of DOI:10.3390/molecules23092151

Parkinson’s disease (PD) is a chronic, progressive, and age-related neurodegenerative disorder characterized by the loss of midbrain dopaminergic neurons caused by the accumulation of free radicals and oxidative stress. Based on the neuroprotective properties of 2-pyrazoline derivatives, in the current work, 1-(phenyl/4-substituted phenyl)-3-(2-furanyl/thienyl)-5-aryl-2-pyrazolines (3a–i, 4a–i) were synthesized via the cyclization of the chalcones (1, 2) with suitable phenylhydrazine hydrochloride derivatives. All these compounds were investigated for their neuroprotective effects using an in vitro 6-hydroxydopamine (6-OHDA)-induced neurotoxicity model of PD in the rat pheochromocytoma (PC-12) Adh cell line. In addition, some different pharmacokinetic parameters of all compounds were in silico predicted by the QikProp module of Schr?dinger’s Maestro molecular modeling package. 4-Methylsulfonylphenyl substituted compounds 3h (20%) and 4h (23%) were determined as the most promising neuroprotective agents related to their inductive roles in cell viability when compared with the 6-OHDA-positive control group (43% and 42%, respectively). Moreover, in silico pharmacokinetic results indicated that all compounds were within the acceptable range intended for human use. According to both in vitro and in silico studies, compounds 3h and 4h draw attention as potential orally bioavailable therapeutic drug candidates against neurodegeneration in PD.

Full text of DOI:10.3390/molecules23092151

molecules
Article
Design, Synthesis, and Neuroprotective Effects of a
Series of Pyrazolines against
6-Hydroxydopamine-Induced Oxidative Stress
Ahmet Özdemir 1, ID , Belgin Sever 1 ID , Mehlika Dilek Altıntop 1 ID , Elif Kaya Tilki 2 and
*
Miris¸ Dikmen 2
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, Eskis¸ehir 26470,
Turkey; belginsever@anadolu.edu.tr (B.S.); mdaltintop@anadolu.edu.tr (M.D.A.)
Department of Pharmacology, Faculty of Pharmacy, Anadolu University, Eskis¸ehir 26470, Turkey;
elif_kaya@anadolu.edu.tr (E.K.T.); mirisd@anadolu.edu.tr (M.D.)
2
*
Correspondence: ahmeto@anadolu.edu.tr; Tel.: +90-222-335-0580 (ext. 3780)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 26 July 2018; Accepted: 22 August 2018; Published: 27 August 2018
Abstract: Parkinson’s disease (PD) is a chronic, progressive, and age-related neurodegenerative disorder
characterized by the loss of midbrain dopaminergic neurons caused by the accumulation of free radicals
and oxidative stress. Based on the neuroprotective properties of 2-pyrazoline derivatives, in the
current work, 1-(phenyl/4-substituted phenyl)-3-(2-furanyl/thienyl)-5-aryl-2-pyrazolines (3a
–i, 4a–i)
were synthesized via the cyclization of the chalcones ( ) with suitable phenylhydrazine hydrochloride
1, 2
derivatives. All these compounds were investigated for their neuroprotective effects using an in vitro
6-hydroxydopamine (6-OHDA)-induced neurotoxicity model of PD in the rat pheochromocytoma
(PC-12) Adh cell line. In addition, some different pharmacokinetic parameters of all compounds were
in silico predicted by the QikProp module of Schrödinger’s Maestro molecular modeling package.
4-Methylsulfonylphenyl substituted compounds 3h (20%) and 4h (23%) were determined as the most
promising neuroprotective agents related to their inductive roles in cell viability when compared with the
6-OHDA-positive control group (43% and 42%, respectively). Moreover, in silico pharmacokinetic results
indicated that all compounds were within the acceptable range intended for human use. According to
both in vitro and in silico studies, compounds 3h and 4h draw attention as potential orally bioavailable
therapeutic drug candidates against neurodegeneration in PD.
Keywords: Parkinson’s disease; neurodegeneration; 2-pyrazoline; chalcone; 6-hydroxydopamine;
pharmacokinetic parameters
1. Introduction
Neurodegenerative diseases (NDDs) are rapidly rising in prevalence in many countries and are
highly linked to the expected aging of the population, because these disorders mainly occur in the
elderly. NDDs can be classified according to extrapyramidal and pyramidal movement problems
or cognitive and behavioral problems. The most common forms of NDDs are Alzheimer’s disease
(AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), and
dementia with Lewy bodies (DLB) [1–4]. These diseases reveal common pathological properties such
as the formation of insoluble protein-based aggregates in deteriorated neurons and glial cells, whereas
there is a big difference in clinical features of patients suffering from them [5].
PD, firstly described as “paralysis agitans” by James Parkinson in 1817, is the second most
common NDD affecting 2–3% of those >65 years of age [
6,7]. The cause of PD is not clear, but the
defects in mitochondrial functions and brain iron regulation, inflammation, and energy metabolism
Molecules 2018, 23, 2151; doi:10.3390/molecules23092151
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2151
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problems have been proven to be substantial in underlying mechanisms [8]. It is neuropathologically
characterized by the loss of progressive dopaminergic neurons in the substantia nigra pars compacta
and the accumulation of aggregated -synuclein protein forming intracellular Lewy bodies in affected
regions [ ]. Moreover, the loss of dopaminergic neurons is associated with the decrease in the activity
α
8,9
of catalase enzymes and the increase of monoamine oxidase-B (MAO-B) in glial cells. These enzymatic
changes lead to emerging oxidative stress through the formation of higher levels of quinones, peroxides,
and other reactive oxygen species (ROS), which then contribute to lipid and protein peroxidation and
finally to neuronal death [8–11].
The cardinal clinical features of PD patients are motor impairment, including resting tremors,
muscular rigidity, bradykinesia, and postural instability. Furthermore, these patients also mainly
have speech and swallowing difficulties, a masklike facial expression, and micrographia [12,13].
Approved treatment procedures for PD to date, such as pharmacotherapy and neurosurgery, have
mainly focused on the recovery of these clinical symptoms. However, recently a major approach
for the treatment is to discover new beneficial, potential, and disease-modifying drugs targeting the
underlying mechanisms related to the neurodegenerative process of PD [14–16].
The main aspect of oxidative stress in PD has been investigated with toxin-based models such as
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA). 6-OHDA
(Figure 1) is a neurotoxin, which is capable of generating ROS in neurons, and is detected in the
urine of PD patients treated with long-term L-3,4-dihydroxyphenylalanine (L-DOPA) (Figure 1),
which is the natural precursor of dopamine (Figure 1) and the typically prescribed drug for the
symptomatic treatment of PD. Neurodegeneration with a 6-OHDA-based model can mimic idiopathic
PD pathogenesis in vitro, as well as in vivo. The primary advantages of this model are measurable
motor deficit (rotation) and proven usage in the pharmacological screening of compounds effective on
dopamine and its receptors [17–20].
Figure 1.
Chemical structures of 6-hydroxydopamine (6-OHDA), dopamine, and
L-3,4-dihydroxyphenylalanine (L-DOPA).
Rat pheochromocytoma (PC-12) cells have been widely used for in vitro studies exploring the
mechanisms of NDDs. PC-12 Adh is a rat pheochromocytoma-derived cell-line, which responds
to nerve growth factor (NGF) by switching from an immature chromaffin-cell-like phenotype to a
sympathetic-neuron-like one. Therefore, this cell line has been widely used as an in vitro assay system
for screening the neuroprotective effects of compounds [21–24].
Pyrazoline is a five-membered heterocyclic ring bearing two adjacent nitrogen atoms and one
endocyclic double bond. It has three tautomeric forms called as 1-pyrazoline, 2-pyrazoline, and
3-pyrazoline, but among them, 2-pyrazoline is the most common one. 2-Pyrazoline-based compounds
possess a wide range of pharmacological applications including antiepileptic, antidepressant, and
anti-neurodegenerative activities. Moreover, these compounds can cross the blood–brain barrier (BBB)
easily by means of their lipophilic characters [25–28].
Having the above aspects in mind, a series of 2-pyrazoline-based compounds 3a
–i and 4a–i
were designed and synthesized via the reaction of chalcones ( ). All the compounds were in vitro
1, 2
evaluted for their neuroprotective potentials against 6-OHDA-induced neurotoxicity in PC-12 Adh
cells. In addition, in silico absorption, distribution, metabolism, and excretion (ADME) studies were
performed to predict their physicochemical properties particularly their abilities to cross the BBB.

Molecules 2018, 23, 2151
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2. Results and Discussion
The synthesis of new pyrazoline derivatives (3a
in Scheme 1. Initially, 1,3-diaryl-substituted chalcones (1, 2
–
i
,
4a
–
i
) followed the general pathway depicted
) were synthesized via the base-catalyzed
Claisen–Schmidt condensation of 2-acetylfuran/2-acetylthiophene with the appropriate aromatic
aldehydes [29 32]. Then, the final compounds, 1-(phenyl/4-substituted phenyl)-3-(2-furanyl/thienyl)-
5-aryl-2-pyrazolines (3a 4a ), were obtained via the cyclization of the chalcones ( ) with suitable
phenylhydrazine hydrochloride derivatives in the presence of hot acetic acid [33].
–
–i,
–i
1, 2
Scheme 1. The synthetic route for the preparation of the compounds (1, 2, 3a–i, 4a–i). Reagents and
conditions: (i) 40% (w/v) sodium hydroxide, ethanol, rt, 24 h; (ii) appropriate phenylhydrazine
hydrochloride derivative, CH3COOH, reflux, 8 h.
1
The structures of all the compounds were elucidated by FTIR, H NMR, 13C NMR, mass
1
spectral data, and elemental analyses. In the H NMR spectra of the compounds, 3a
–
i
and 4a
–
i,
the CH2 protons of the 2-pyrazoline ring resonated as a pair of doublets of doublets at 2.95–3.20 ppm
(HA) (JAM = 17.13–17.64 Hz, JAX = 4.65–7.41 Hz) and 3.69–3.96 ppm (HM) (JMA = 17.18–17.64 Hz,
JMX = 11.85–12.09 Hz). The CH proton appeared as a doublet of doublets at 5.29–5.59 ppm (HX)
(JMX = 11.76–12.09 Hz, JAX = 4.68–7.38 Hz) owing to the vicinal coupling with two magnetically
non-equivalent protons of the methylene group at the 4th position of the pyrazoline ring. All the
other aromatic and aliphatic protons were observed in the expected regions. Mass spectral data and
elemental analysis were also in agreement with the proposed structures of the compounds [33,34].
According to the IC50 values given in Table 1, apart from compound
compounds at 100 g/mL did not reduce cell viability significantly. The compounds demonstrated
their protective effects against cell death in differentiated PC-12 Adh cells treated with 6-OHDA
1, treatment with the
µ

Molecules 2018, 23, 2151
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(150
36, 29, 35, 37, 30, 31, 29, 20, 30% with compounds 3a
However, compound was found to be highly cytotoxic, and it reduced cell viability with the values
of 97% (100 g/mL) and 40% (10 g/mL). In addition, the reduction in cell viability was evaluated
as 34.1% with compound and 59, 54, 76, 56, 41, 62, 69, 23, 87% with compounds 4a (100 g/mL),
respectively, when compared with 6-OHDA (150 M) (42.13%) (Figure 2; Table 2). According to
these results, all the compounds reduced cell viability compared with the control group after 24 h
6-OHDA exposure (* p < 0.05, *** p < 0.001, **** p < 0.0001 for compounds and 3a **** p < 0.0001
for compounds and 4a ). Compounds 3a , and 4h might have neuroprotective potential against
µM) for 24 h. The reduction in cell viability was determined as 43% with 6-OHDA (150
µM) and
–
i
(100 µg/mL), respectively (Figure 2; Table 2).
1
µ
µ
2
–
i
µ
µ
1
–i;
2
–
i
–i, 2
6-OHDA induced neurotoxicity in PC-12 Adh cells but only the induction in cell viability percentage
with compounds 3h and 4h was found significant compared with the 6-OHDA-positive control
group related to the statistical analysis (** p < 0.01). This outcome pointed out that the presence of
4-methylsulfonylphenyl moiety enhanced the neuroprotective potency of the 2-pyrazoline ring as
observed in both compounds 3h and 4h.
Table 1. IC50 values of compounds 1, 2, 3a–i and 4a–i and 6-OHDA according to the cell viability assay.
Compound
IC50 (µg/mL)
Compound
IC50 (µg/mL)
1
3a
3b
3c
3d
3e
3f
3g
3h
12
>400
204
353
>400
398
>400
>400
>400
205
2
4a
4b
4c
4d
4e
4f
4g
4h
345
>400
>400
>400
>400
>400
239
252
>400
>400
150 µM
3i
4i
6-OHDA
150 µM
6-OHDA
Table 2. Cell viability values of 6-OHDA and compounds
1,
2,
3a–
i
,
4a–
i
according to the cell viability
assay results. The results of the cell viability were presented as a % of the control (the O.D. value).
Compound
Cell Viability %
Compound
Cell Viability %
Control
6-OHDA
1 (100 µg/mL)
1 (10 µg/mL)
100
57
3
Control
6-OHDA
100
58
66
41
46
24
44
59
38
31
77
13
2
60
64
71
65
63
70
69
71
80
70
4a
4b
4c
4d
4e
4f
4g
4h
4i
3a
3b
3c
3d
3e
3f
3g
3h
3i

Molecules 2018, 23, 2151
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(A)
(B)
Figure 2. Neuroprotective effects of compounds
neurotoxicity. PC-12 Adh cells were treated with 100
before exposure to 150 M 6-OHDA for 24 h. The cell viability was detected at the 24th hour. The results
1
and 3a–i (A); 2 and 4a–i (B) against 6-OHDA induced
µg/mL concentration of the compounds for 6 h
µ
of the cell viability were presented as a % of the control (the O.D. value). Data are shown as mean
three experiments. ((n = 8), p > 0.05 n.s., * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).
±
SD of
ADME properties of compounds 1, 2, 3a–i, and 4a–i were in silico assessed to enlighten the
biological, pharmaceutical, and drug similarities of these compounds. The results given in Table 3
were found to be within the acceptable range intended for human use, making these derivatives
promising drug candidates [35]. All these compounds were detected to exhibit excellent absorption %
(92–100%) in human oral absorption on a 0–100% scale, and based on the parameters of the brain/blood
partition coefficient (QPlogBB) (
they were also found to pass through the BBB, which restrains drug entry from blood into brain by
multiple mechanisms [36 37]. Solvent accessible surface area (SASA) is used to define the accessibility
−
1.08 to 0.78) and central nervous system (CNS) activity ( 1 to 0),
−
,
of the residue to the solvent; either it is between lipid or water accessibility, and it is essential to BBB
permeability [38]. The SASA values of all the compounds were found within the range (467–680).
The results also indicated that all the compounds obey Lipinski’s rule of five and Jorgensen’s rule of

Molecules 2018, 23, 2151
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three; due to the fact that a potential orally active drug candidate should reveal no more than one
violation of these rules [39,40].
Table 3. Predicted ADME properties of compounds 1, 2, 3a–i, and 4a–i.
Human Oral
SASA *
(300.0 to
1000.0)
Rule
of Five
**
QplogBB *
(−3 to 1.2)
CNS *
(−2 to 2)
Absorption% *
(>80% Is High,
<25% Is poor)
Rule of
Three ***
Compound
1
2
−0.12
0.02
0.49
0
1
2
0
2
2
2
2
1
482
467
594
632
603
618
623
626
631
680
666
582
620
591
606
611
614
619
669
654
100
100
100
100
100
100
100
100
100
100
93
100
100
100
100
100
100
100
100
92
0
0
1
0
1
1
1
1
1
0
0
1
0
1
1
1
1
1
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
3a
3b
3c
3d
3e
3f
3g
3h
3i
4a
4b
4c
4d
4e
4f
4g
4h
4i
−0.31
0.60
0.66
0.67
0.48
0.42
−0.40
−1.08
0.59
0
−2
2
−0.16
0
2
2
2
2
2
0
−1
0.71
0.77
0.78
0.59
0.53
−0.26
−0.91
* QPlogBB: brain/blood partition coefficient, CNS: predicted central nervous system activity, SASA: total solvent
accessible surface area, in square angstroms using a probe with a 1.4 Å Radius, and Percent Human Oral Absorption:
human oral absorption on a 0–100% scale. ** Rule of Five: Number of violations of Lipinski’s rule of five. The rules
are as follows: mol_MW (molecular weight of the molecule) < 500, QPlogPo/w (predicted octanol/water partition
coefficient) < 5, donorHB (hydrogen-bond donor atoms)
Compounds that provide these rules are considered as drug-like molecules. *** Rule of Three: Number of violations
of Jorgensen’s rule of three. The three rules are as follows: QPlogS (predicted aqueous solubility) > 5.7, QPPCaco
≤
5, and accptHB (hydrogen-bond acceptor atoms)
≤
10.
−
(predicted apparent Caco-2 cell permeability in nm/s) > 22 nm/s, Primary Metabolites < 7. Compounds with fewer
(and preferably no) violations of these rules are more likely to be orally available (Schrödinger Release 2016-2:
QikProp, Schrödinger, LLC, New York, NY, 2016).
3. Materials and Methods
3.1. Chemistry
All reagents were purchased from commercial suppliers and were used without further
purification. Melting points (M.p.) were determined on an Electrothermal 9100 melting point apparatus
(Weiss-Gallenkamp, Loughborough, UK) and are uncorrected. IR spectra were recorded on an
1
IRPrestige-21 Fourier Transform Infrared spectrophotometer (Shimadzu, Tokyo, Japan). H NMR and
13C NMR spectra were recorded by a Bruker digital FT-NMR spectrometer (Bruker Bioscience, Billerica,
MA, USA) in DMSO-d6. 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) and the results were within
the theoretical values. Thin Layer Chromatography (TLC) was performed on TLC Silica gel 60 F254
aluminium sheets (Merck, Darmstadt, Germany) to check the purity of the compounds.
±
0.4% of

Molecules 2018, 23, 2151
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3.1.1. General Procedure for the Synthesis of the Compounds
1-(2-Furanyl/thienyl)-3-aryl-2-propen-1-one (1, 2)
2-Acetylfuran/2-acetylthiophene (0.02 mol), proper aromatic aldehyde (0.02 mol) and 40% (w/v)
sodium hydroxide (5 mL) in ethanol (30 mL) were stirred at room temperature for 24 h. Then, the
reaction mixture was poured into ice and the precipitated solid was filtered, washed with water, and
dried. The product was crystallized from ethanol [29–34].
1-(2-Furanyl)-3-aryl-2-propen-1-one (1 ν
). Yield: 55%. M.p.: 111–113 ◦C. Lit. M.p.: 96–98 ◦C [41]. IR
max
(cm−1): 3126.61 (aromatic C-H stretching), 2927.94 (aliphatic C-H stretching), 1651.07 (C=O stretching),
1597.06, 1558.48, 1512.19, 1462.04 (C=C stretching), 1411.89, 1392.61, 1332.81, 1300.02, 1288.45, 1247.94,
1207.44, 1182.36, 1161.15, 1083.99, 1053.13, 1010.70 (C-O stretching and aromatic C-H in plane bending),
981.77, 927.76, 883.40, 864.11, 842.89, 813.96, 802.39, 765.74, 727.16 (aromatic C-H out of plane bending).
1H-NMR (300 MHz, DMSO-d6)
δ (ppm): 2.35 (3H, s, CH3), 6.79 (1H, dd, J = 3.57 Hz, 1.71 Hz aromatic
proton), 7.28 (2H, d, J = 7.92 Hz, aromatic protons), 7.65 (1H, d, J = 15.72 Hz, C3-H), 7.70 (1H, d,
J = 8.76 Hz, aromatic proton), 7.74–7.76 (2H, m, C2-H and aromatic proton), 7.81 (1H, dd, J = 3.60 Hz,
0.63 Hz, aromatic proton), 8.07 (1H, dd, J = 1.65 Hz, 0.66 Hz, aromatic proton). 13C-NMR (75 MHz,
DMSO-d6)
δ (ppm): 21.54 (CH3), 113.16 (CH), 119.79 (CH), 121.37 (C2-H), 129.52 (2CH), 130.03 (2CH),
132.19 (C), 141.20 (C), 143.30 (C3-H), 148.74 (CH), 153.46 (C), 177.15 (C, C=O). Anal. Calcd. for
C14H12O3: C, 79.22; H, 5.70; Found: C, 79.12; H, 5.64. MS (ESI) (m/z): [M + H]+ 212.08.
◦ ◦
2). Yield: 60%. M.p.: 126–128 C. Lit. M.p.: 117–119 C [35,42].
1-(2-Thienyl)-3-aryl-2-propen-1-one (
IR
(cm−1): 3105.39 (aromatic C-H stretching), 2989.66, 2906.73 (aliphatic C-H stretching), 1643.55
ν
max
(C=O stretching), 1604.77, 1583.56, 1498.69, 1487.12, 1444.68 (C=C stretching), 1411.89, 1369.46, 1346.31,
1305.81, 1246.02, 1228.66, 1215.15, 1192.01, 1105.21, 1058.92, 1028.06 (C-O stretching and aromatic C-H
in plane bending), 972.12, 925.83, 916.19, 860.25, 831.32, 800.46, 727.16, 700.16 (aromatic C-H out of
1
plane bending and C-S stretching). H-NMR (300 MHz, DMSO-d6)
δ
(ppm): 6.12 (2H, s, O-CH2-O),
7.00 (1H, d, J = 8.01 Hz, aromatic proton), 7.30–7.36 (2H, m, aromatic protons), 7.64-7.69 (2H, m, C2-H
and aromatic protons), 7.76 (1H, d, J = 15.51 Hz, C3-H), 8.04 (1H, dd, J = 4.92 Hz, 0.99 Hz, aromatic
proton), 8.32 (1H, dd, J = 3.78 Hz, 0.99 Hz, aromatic proton). 13C-NMR (75 MHz, DMSO-d6)
δ (ppm):
102.15 (CH2), 107.42 (CH), 109.01 (CH), 120.35 (CH), 126.46 (C2-H), 129.29 (CH), 129.50 (C), 133.83
(CH), 135.70 (CH), 143.61 (C3-H), 146.26 (C), 148.58 (C), 150.10 (C), 182.00 (C, C=O). Anal. Calcd. for
C14H10O3S: C, 65.10; H, 3.90; Found: C, 64.98; H, 3.84. MS (ESI) (m/z): [M + H]+ 258.04.
1-(Phenyl/4-Substituted phenyl)-3-(2-furanyl/thienyl)-5-aryl-2-pyrazolines (3a–i, 4a–i)
A mixture of appropriate chalcone (1, 2) (10.0 mmol) and phenylhydrazine hydrochloride
derivative (20.0 mmol) was refluxed for 8 h in absolute ethanol (30 mL) in the presence of acetic acid
(10 mL) to get 2-pyrazolines. Then the reaction mixture was poured into crushed ice. The precipitate
was separated by filtration, washed with water and crystallized from methanol [33].
1-Phenyl-3-(2-furanyl)-5-(4-methylphenyl)-2-pyrazoline (3a) [43]. Yield: 67%. M.p.: 162–163 ◦C. IR
ν
max
(cm−1): 3113.11, 3034.03 (aromatic C-H stretching), 2918.30, 2848.86 (aliphatic C-H stretching), 1595.13,
1498.69, 1487.12 (C=N and C=C stretching), 1413.82, 1365.60, 1334.74, 1319.31, 1247.94, 1176.58, 1128.36,
1072.42, 1051.20, 1033.85, 1022.27 (C-N, C-O stretching and aromatic C-H in plane bending), 997.20,
960.55, 923.90, 885.33, 869.90, 842.89, 813.96, 744.52, 731.02, 690.52, 673.16 (aromatic C-H out of plane
1
bending). H-NMR (300 MHz, DMSO-d6)
δ (ppm): 2.25 (3H, s, CH3), 2.98 (1H, dd, JAM = 17.28 Hz,
J
AX = 6.15 Hz, C4-HA pyrazoline), 3.82 (1H, dd, JMA = 17.28 Hz, JMX = 12.12 Hz, C4-HM pyrazoline),
5.40 (1H, dd, JMX = 12.09 Hz, JAX = 6.12 Hz, C5-HX pyrazoline), 6.61 (1H, dd, J = 3.42 Hz, 1.80 Hz,
aromatic proton), 6.68–6.77 (2H, m, aromatic protons), 6.95 (2H, d, J = 8.72 Hz, aromatic protons),
7.10–7.16 (6H, m, aromatic protons), 7.80 (1H, dd, J = 1.71 Hz, 0.63 Hz). 13C-NMR (75 MHz, DMSO-d6)
δ
(ppm): 22.11 (CH3), 43.39 (CH2), 62.85 (CH), 111.12 (CH), 112.42 (CH), 113.45 (2CH), 119.05 (CH),
126.22 (2CH), 129.30 (2CH), 129.66 (C), 130.01 (2CH), 137.08 (CH), 139.66 (C, d, J = 3.75 Hz), 144.62 (2C,

Molecules 2018, 23, 2151
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d, J = 6.00 Hz), 147.97 (C). Anal. Calcd. for C20H18N2O: C, 79.44; H, 6.00; N, 9.26; Found: C, 79.39; H,
6.04; N, 9.25. MS (ESI) (m/z): [M]+ 302.00, [M + H]+ 303.00.
◦
1-(4-Cyanophenyl)-3-(2-furanyl)-5-(4-methylphenyl)-2-pyrazoline (3b). Yield: 55%. M.p.: 77–78 C. IR
ν
max
(cm−1): 3118.90 (aromatic C-H stretching), 2918.30 (aliphatic C-H stretching), 2212.35 (C
N stretching),
1598.99, 1510.26 (C=N and C=C stretching), 1415.75, 1373.32, 1323.17, 1174.65, 1122.57, 1004.91 (C-N,
C-O stretching and aromatic C-H in plane bending), 812.03, 740.67 (aromatic C-H out of plane bending).
≡
1H-NMR (300 MHz, DMSO-d6)
δ (ppm): 2.25 (3H, s, CH3), 3.06 (1H, dd, JAM = 17.58 Hz, JAX = 4.65 Hz,
C4-HA pyrazoline), 3.90 (1H, dd, JMA = 17.49 Hz, JMX = 11.91 Hz, C4-HM pyrazoline), 5.58 (1H, dd,
JMX = 11.76 Hz, JAX = 4.68 Hz, C5-HX pyrazoline), 6.64–6.65 (1H, m, aromatic proton), 6.90 (1H, d,
J = 3.30 Hz, aromatic proton), 7.01 (2H, d, J = 8.73 Hz, aromatic protons), 7.07–7.16 (4H, m, aromatic
protons), 7.54 (2H, d, J = 8.73 Hz, aromatic protons), 7.86 (1H, s, aromatic proton). 13C-NMR (75 MHz,
DMSO-d6) δ (ppm): 21.10 (CH3), 43.44 (CH2), 61.91 (CH), 99.40 (C), 112.66 (CH), 112.98 (CH), 113.25
(2CH), 120.39 (C), 126.04 (2CH), 130.19 (2CH), 133.76 (2CH), 137.50 (C), 138.56 (C), 142.89 (2C), 145.53
(CH), 146.89 (C). Anal. Calcd. for C21H17N3O: C, 77.04; H, 5.23; N, 12.84; Found: C, 77.06; H, 5.24; N,
12.82. MS (ESI) (m/z): [M + H]+ 327.90.
◦
1-(4-Fluorophenyl)-3-(2-furanyl)-5-(4-methylphenyl)-2-pyrazoline (3c). Yield: 53%. M.p.: 87–89 C. IR
ν
max
(cm−1): 2987.74, 2900.94 (aliphatic C-H stretching), 1597.06, 1508.33 (C=N and C=C stretching), 1413.82,
1373.32, 1321.24, 1172.72, 1002.98 (C-N, C-O stretching and aromatic C-H in plane bending), 812.03,
742.59 (aromatic C-H out of plane bending). 1H-NMR (300 MHz, DMSO-d6)
δ
(ppm): 2.25 (3H, s,
CH3), 2.98 (1H, dd, JAM = 17.25 Hz, JAX = 6.57 Hz, C4-HA pyrazoline), 3.82 (1H, dd, JMA = 17.25 Hz,
MX = 12.03 Hz, C4-HM pyrazoline), 5.37 (1H, dd, JMX = 11.97 Hz, JAX = 6.54 Hz, C5-HX pyrazoline),
J
6.61 (1H, dd, J = 3.36 Hz, 1.77 Hz, aromatic proton), 6.76 (1H, d, J = 3.33 Hz, aromatic proton), 6.93–6.99
(4H, m, aromatic protons), 7.15 (4H, s, aromatic protons), 7.80 (1H, d, J = 1.44 Hz, aromatic proton).
13C-NMR (75 MHz, DMSO-d6)
δ (ppm): 21.09 (CH3), 43.57 (CH2), 63.42 (CH), 111.20 (CH), 112.43 (CH),
114.65 (CH, d, J = 7.5 Hz), 115.84 (CH, d, J = 22.5 Hz), 126.32 (2CH), 130.04 (2CH), 137.18 (CH), 139.36
(CH), 139.93 (C), 141.56 (C), 144.70 (CH), 147.56 (C), 147.88 (C), 154.79 (C), 157.90 (C). Anal. Calcd. for
C20H17FN2O: C, 74.98; H, 5.35; N, 8.74; Found: C, 74.94; H, 5.33; N, 8.76. MS (ESI) (m/z): [M + H]−
318.90, [M]+ 320.90, [M + H]+ 321.90.
◦
1-(4-Chlorophenyl)-3-(2-furanyl)-5-(4-methylphenyl)-2-pyrazoline (3d). Yield: 83%. M.p.: 115–117 C.
IR ν
max (cm−1): 2987.74, 2900.94 (aliphatic C-H stretching), 1598.99, 1492.90 (C=N and C=C stretching),
1375.25, 1076.28, 1051.20 (C-N, C-O stretching and aromatic C-H in plane bending), 817.82, 734.88
1
(aromatic C-H out of plane bending). H-NMR (300 MHz, DMSO-d6) δ (ppm): 2.25 (3H, s, CH3), 3.00
(1H, dd, JAM = 17.37 Hz, JAX = 5.85 Hz, C4-HA pyrazoline), 3.84 (1H, dd, JMA = 17.37 Hz,
C4-HM pyrazoline), 5.43 (1H, dd, JMX = 12.06 Hz, JAX = 5.82 Hz, C5-HX pyrazoline), 6.61 (1H, dd,
J = 3.45 Hz, 1.80 Hz, aromatic proton), 6.79 (1H, d, J = 3.93 Hz, aromatic proton), 6.93 (2H, d, J = 7.94 Hz
aromatic protons), 7.13 (4H, s, aromatic protons), 7.17 (2H, d, J = 9.00 Hz, aromatic protons), 7.81 (1H,
dd, J = 1.68 Hz, 0.63 Hz, aromatic proton). 13C-NMR (75 MHz, DMSO-d6)
(ppm): 21.08 (CH3), 43.49
JMX = 12.06 Hz,
,
δ
(CH2), 62.75 (CH), 111.60 (CH), 112.48 (CH), 114.85 (2CH), 122.60 (C), 126.20 (2CH), 129.11 (2CH),
130.07 (2CH), 137.23 (C), 139.11 (C), 140.51 (C), 143.36 (C), 144.89 (CH), 147.73 (C). Anal. Calcd. for
C20H17ClN2O: C, 71.32; H, 5.09; N, 8.32; Found: C, 71.28; H, 5.03; N, 8.35. MS (ESI) (m/z): [M + H]−−
334.90, [M]+ 336.90, [M + H]+ 337.90.
◦
1-(4-Bromophenyl)-3-(2-furanyl)-5-(4-methylphenyl)-2-pyrazoline (3e). Yield: 79%. M.p.: 133–135 C.
IR
ν
(cm−1): 2999.31, 2920.23 (aliphatic C-H stretching), 1593.20, 1512.19, 1492.90, 1483.26 (C=N
max
and C=C stretching), 1365.60, 1319.31, 1122.57, 1087.85, 1074.35, 1051.20, 1004.91 (C-N, C-O stretching
and aromatic C-H in plane bending), 883.40, 862.18, 815.89, 792.74, 732.95 (aromatic C-H out of plane
1
bending). H-NMR (300 MHz, DMSO-d6)
δ (ppm): 2.25 (3H, s, CH3), 3.00 (1H, dd, JAM = 17.37 Hz,
J
AX = 5.76 Hz, C4-HA pyrazoline), 3.83 (1H, dd, JMA = 17.34 Hz, JMX = 12.06 Hz, C4-HM pyrazoline),
5.43 (1H, dd, JMX = 12.03 Hz, JAX = 5.70 Hz, C5-HX pyrazoline), 6.61 (1H, dd, J = 3.42 Hz, 1.80 Hz,

Molecules 2018, 23, 2151
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aromatic proton), 6.79 (1H, d, J = 3.39 Hz, aromatic proton), 6.88 (2H, d, J = 9.00 Hz, aromatic protons),
7.12 (4H, s, aromatic protons), 7.29 (2H, d, J = 8.97 Hz, aromatic protons), 7.81 (1H, t, J = 1.65 Hz, 1.08 Hz,
0.57 Hz, aromatic proton). 13C-NMR (75 MHz, DMSO-d6)
δ (ppm): 21.09 (CH3), 43.48 (CH2), 62.64
(CH), 110.21 (C), 111.65 (CH), 112.49 (CH), 115.35 (2CH), 126.19 (2CH), 130.07 (2CH), 131.94 (2CH),
137.24 (C), 139.05 (C), 140.57 (C), 143.67 (C), 144.91 (CH), 147.72 (C). Anal. Calcd. for C20H17BrN2O: C,
−−
63.00; H, 4.49; N, 7.35; Found: C, 63.04; H, 4.53; N, 7.32. MS (ESI) (m/z): [M + H]
378.80, [M]+ 380.80,
[M + H]+ 381.90, [M + H]+++ 383.00.
◦
1-(4-Methylphenyl)-3-(2-furanyl)-5-(4-methylphenyl)-2-pyrazoline (3f). Yield: 53%; M.p. 67–68 C. IR
ν
max
(cm−1): 2972.31, 2900.94 (aliphatic C-H stretching), 1614.42, 1512.19 (C=N and C=C stretching), 1408.04,
1365.60, 1249.87, 1074.35, 1049.28 (C-N, C-O stretching and aromatic C-H in plane bending), 883.40,
1
804.32, 738.74 (aromatic C-H out of plane bending). H-NMR (300 MHz, DMSO-d6)
δ (ppm): 2.15 (3H,
s, CH3), 2.24 (3H, s, CH3), 2.95 (1H, dd, JAM = 17.19 Hz, JAX = 6.36 Hz, C4-HA pyrazoline), 3.79 (1H, dd,
MA = 17.22 Hz, JMX = 12.09 Hz, C4-HM pyrazoline), 5.36 (1H, dd, JMX = 12.09 Hz, JAX = 6.33 Hz, C5-HX
J
pyrazoline), 6.60 (1H, dd, J = 3.42 Hz, 1.80 Hz, aromatic proton), 6.72–6.74 (1H, m, aromatic proton),
6.83–6.86 (2H, m, aromatic protons), 6.94 (2H, d, J = 8.34 Hz, aromatic protons), 7.09–7.13 (4H, m,
aromatic protons), 7.78 (1H, dd, J = 1.71 Hz, 0.63 Hz, aromatic proton). 13C-NMR (75 MHz, DMSO-d6)
δ
(ppm): 20.54 (CH3), 21.08 (CH3), 43.34 (CH2), 63.13 (CH), 110.79 (CH), 112.39 (CH), 113.63 (2CH),
126.28 (2CH), 127.76 (CH), 128.80 (C), 129.73 (2CH, d, J = 16.50 Hz), 137.00 (C), 139.16 (C), 139.67 (CH),
142.51 (2C), 144.51 (CH), 148.08 (C). Anal. Calcd. for C21H20N2O: C, 79.72; H, 6.37; N, 8.85; Found: C,
79.66; H, 6.28; N, 8.89. MS (ESI) (m/z): [M]+ 316.90, [M + H]+ 318.00.
◦
1-(4-Methoxyphenyl)-3-(2-furanyl)-5-(4-methylphenyl)-2-pyrazoline (3g). Yield: 27%; M.p. 70–72 C.
IR
ν
(cm−1): 3118.90 (aromatic C-H stretching), 2918.30, 2831.50 (aliphatic C-H stretching), 1606.70,
max
1568.13, 1506.41, 1463.97 (C=N and C=C stretching), 1359.82, 1292.31, 1238.30, 1178.51, 1120.64, 1097.50,
1035.77 (C-N, C-O stretching and aromatic C-H in plane bending), 883.40, 873.75, 815.89, 746.45
(aromatic C-H out of plane bending). 1H-NMR (300 MHz, DMSO-d6)
δ
(ppm): 2.25 (3H, s, CH3),
2.95 (1H, dd, JAM = 17.13 Hz, JAX = 7.14 Hz, C4-HA pyrazoline), 3.63 (3H, s, OCH3), 3.69 (1H, dd,
MA = 17.18 Hz, JMX = 12.00 Hz, C4-HM pyrazoline), 5.30 (1H, dd, JMX = 11.97 Hz, JAX = 7.17 Hz, C5-HX
J
pyrazoline), 6.59 (1H, dd, J = 3.42 Hz, 1.80 Hz, aromatic proton), 6.71–6.77 (3H, m, aromatic protons),
6.86–6.90 (2H, m, aromatic protons), 7.12–7.18 (4H, m, aromatic protons), 7.78 (1H, dd, J = 1.71 Hz,
0.66 Hz, aromatic proton). 13C-NMR (75 MHz, DMSO-d6)
δ (ppm): 21.08 (CH3), 43.47 (CH2), 55.63
(CH3), 63.94 (CH), 106.80 (CH), 110.58 (CH), 112.36 (CH), 114.88 (2CH, d, J = 12.75 Hz), 126.42 (2CH),
127.34 (C), 130.01 (2CH, d, J= 8.25 Hz), 137.03 (C), 138.97 (C), 139.16 (C), 139.69 (CH), 144.41 (CH),
148.14 (C), 153.15 (C). Anal. Calcd. for C21H20N2O2: C, 75.88; H, 6.06; N, 8.43; Found: C, 75.90; H, 6.04;
N, 8.50. MS (ESI) (m/z): [M]+ 332.00, [M + H]+ 333.00.
◦
1-(4-Methylsulfonylphenyl)-3-(2-furanyl)-5-(4-methylphenyl)-2-pyrazoline (3h). Yield: 68%. M.p.: 162–163 C.
IR ν
max (cm−1): 2985.81, 2900.94 (aliphatic C-H stretching), 1589.34, 1506.41 (C=N and C=C stretching),
1406.11, 1379.10, 1292.31, 1242.16, 1132.21, 1066.64 (C-N, C-O stretching and aromatic C-H in plane
1
bending), 960.55, 883.40, 815.89, 769.60 (aromatic C-H out of plane bending and C-S stretching). H-NMR
(300 MHz, DMSO-d6)
δ (ppm): 2.24 (3H, s, CH3), 3.00 (1H, dd, JAM = 17.37 Hz, JAX = 5.76 Hz, C4-HA
pyrazoline), 3.06 (3H, s, SO2CH3), 3.91 (1H, dd, JMA = 17.55 Hz, JMX = 11.97 Hz, C4-HM pyrazoline), 5.59
(1H, dd, JMX = 11.88 Hz, JAX = 4.74 Hz, C5-HX pyrazoline), 6.64 (1H, dd, J= 3.39 Hz, 1.77 Hz, aromatic
proton), 6.89 (1H, d, J = 3.39 Hz, aromatic proton), 7.07 (2H, d, J = 8.88 Hz, aromatic protons), 7.14 (4H, s,
aromatic protons), 7.65 (2H, d, J = 8.97 Hz, aromatic protons), 7.86 (1H, t, J = 1.59 Hz, 1.20 Hz, 0.39 Hz,
aromatic proton). 13C-NMR (75 MHz, DMSO-d6)
δ (ppm): 21.10 (CH3), 43.45 (CH2), 44.62 (CH3), 61.98
(CH), 112.76 (2CH, d, J = 18.75 Hz), 113.67 (CH), 126.06 (2CH), 129.02 (2CH), 129.55 (CH), 129.85 (C),
130.20 (2CH), 137.46 (C), 139.63 (C), 142.64 (C), 145.45 (CH), 147.35 (2C, d, J = 8.25 Hz). Anal. Calcd. for
C21H20N2O3S: C, 66.29; H, 5.30; N, 7.36; Found: C, 66.26; H, 5.34; N, 7.39. MS (ESI) (m/z): [M]+ 380.90,
[M + H]+ 382.00.

Molecules 2018, 23, 2151
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1-(4-Sulfonamidophenyl)-3-(2-furanyl)-5-(4-methylphenyl)-2-pyrazoline (3i). Yield: 88%. M.p.: 139–140 ◦C.
IR
(cm−1): 3369.64, 3257.77 (N-H stretching), 3120.82, (aromatic C-H stretching), 2920.23, 2852.72
ν
max
(aliphatic C-H stretching), 1589.34, 1504.48 (C=N and C=C stretching), 1413.82, 1373.32, 1325.10, 1305.81,
1149.57, 1095.57, 1002.98 (C-N, C-O stretching and aromatic C-H in plane bending), 883.40, 866.04,
1
815.89, 738.74 (aromatic C-H out of plane bending and C-S stretching). H-NMR (300 MHz, DMSO-d6)
δ
(ppm): 2.24 (3H, s, CH3), 3.05 (1H, dd, JAM = 17.46 Hz, JAX = 4.92 Hz, C4-HA pyrazoline), 3.89 (1H, dd,
JMA = 17.49 Hz, JMX = 12.00 Hz, C4-HM pyrazoline), 5.57 (1H, dd, JMX = 11.94 Hz, JAX = 4.83 Hz, C5-HX
pyrazoline), 6.63 (1H, dd, J = 3.45 Hz, 1.80 Hz, aromatic proton), 6.85 (1H, d, J = 3.41 Hz, aromatic
proton), 6.99–7.03 (4H, m, aromatic and NH2 protons), 7.12–7.13 (4H, m, aromatic protons), 7.57 (2H, d,
J = 8.97 Hz, aromatic protons), 7.84 (1H, dd, J = 1.71 Hz, 0.66 Hz). 13C-NMR (75 MHz, DMSO-d6)
δ
(ppm): 21.08 (CH3), 43.33 (CH2), 62.06 (CH), 112.48 (2CH, d, J = 12.00 Hz), 126.12 (2CH), 127.59 (2CH),
130.11 (2CH), 133.51 (C), 137.35 (CH), 138.73 (2CH), 141.85 (C), 145.24 (2C), 146.22 (C), 147.47 (C). Anal.
Calcd. for C20H19N3O3S: C, 62.97; H, 5.02; N, 11.02; Found: C, 62.94; H, 5.03; N, 11.05. MS (ESI) (m/z):
[M]+ 381.90, [M + H]+ 382.90.
◦
1-Phenyl-3-(2-thienyl)-5-(1,3-benzodioxol-5-yl)-2-pyrazoline (4a) [44]. Yield: 83%. M.p.: 179–180 C.
IR
ν
(cm−1): 3105.39, 3070.68 (aromatic C-H stretching), 2916.37 (aliphatic C-H stretching), 1593.20,
max
1498.69, 1481.33, 1442.75 (C=N and C=C stretching), 1379.10, 1317.38, 1238.30, 1109.07, 1037.70 (C-N,
C-O stretching and aromatic C-H in plane bending), 937.40, 823.60, 748.38, 719.45 (aromatic C-H
out of plane bending and C-S stretching). 1H-NMR (300 MHz, DMSO-d6)
δ
(ppm): 3.10 (1H, dd,
J
AM = 17.37 Hz, JAX = 6.33 Hz, C4-HA pyrazoline), 3.87 (1H, dd, JMA = 17.37 Hz, JMX = 12.09 Hz, C4-HM
pyrazoline), 5.40 (1H, dd, JMX = 12.00 Hz, JAX = 6.30 Hz, C5-HX pyrazoline), 5.97 (2H, d, J = 0.57 Hz,
O-CH2-O), 6.70–6.79 (3H, m, aromatic protons), 6.87 (1H, d, J = 8.13 Hz, aromatic proton), 6.95 (2H, d,
J = 7.89 Hz, aromatic protons), 7.08–7.18 (3H, m, aromatic protons), 7.25 (1H, d, J = 3.51 Hz, aromatic
proton), 7.59 (1H, d, J = 5.04 Hz, aromatic proton). 13C-NMR (75 MHz, DMSO-d6)
δ (ppm): 44.18 (CH2),
63.51 (CH), 101.52 (CH2), 106.54 (CH), 109.07 (CH), 113.46 (2CH), 119.16 (CH), 119.57 (CH), 127.90
(2CH), 128.28 (CH), 129.35 (2CH), 136.15 (C), 136.59 (C), 144.21 (C), 144.50 (C), 146.99 (C), 148.19 (C).
Anal. Calcd. for C20H16N2O2S: C, 68.95; H, 4.63; N, 8.04; Found: C, 68.93; H, 4.64; N, 8.05. MS (ESI)
(m/z): [M + H]− 346.90, [M]+ 347.90, [M + H]+ 348.90.
1-(4-Cyanophenyl)-3-(2-thienyl)-5-(1,3-benzodioxol-5-yl)-2-pyrazoline (4b). Yield: 93%. M.p.: 156–157 ◦C.
IR
(C
ν
(cm−1): 3105.39 (aromatic C-H stretching), 2916.37, 2848.86 (aliphatic C-H stretching), 2208.49
N stretching), 1600.92, 1510.26, 1481.33, 1442.75 (C=N and C=C stretching), 1396.46, 1325.10,
max
≡
1242.16, 1174.65, 1095.57, 1035.77 (C-N, C-O stretching and aromatic C-H in plane bending), 935.48,
840.96, 821.68, 802.39, 727.16 (aromatic C-H out of plane bending and C-S stretching). 1H-NMR
(300 MHz, DMSO-d6)
3.95 (1H, dd, JMA = 17.64 Hz, JMX = 11.94 Hz, C4-HM pyrazoline), 5.57 (1H, dd, JMX = 11.82 Hz,
AX = 4.92 Hz, C5-HX pyrazoline), 5.99 (2H, d, J = 1.71 Hz, O-CH2-O), 6.73 (1H, dd, J = 7.98 Hz, 1.71 Hz,
δ (ppm): 3.20 (1H, dd, JAM = 17.64 Hz, JAX = 4.98 Hz, C4-HA pyrazoline),
J
aromatic proton), 6.77 (1H, d, J = 1.56 Hz, aromatic proton), 6.87 (1H, d, J = 7.95 Hz, aromatic proton),
7.01 (2H, d, J = 8.91 Hz, aromatic protons), 7.14 (1H, dd, J = 5.04 Hz, 3.66 Hz, aromatic proton), 7.36
(1H, dd J = 3.60 Hz, 1.05 Hz, aromatic proton), 7.57 (2H, d, J = 8.94 Hz, aromatic protons), 7.69 (1H,
dd, J = 5.04 Hz, 1.02 Hz, aromatic proton). 13C-NMR (75 MHz, DMSO-d6)
δ (ppm): 44.26 (CH2), 62.52
(CH), 99.45 (C), 101.65 (CH2), 106.41 (CH), 109.20 (CH), 113.21 (2CH), 119.42 (CH), 120.42 (C), 128.48
(CH), 129.18 (CH), 129.47 (CH), 133.80 (2CH), 135.24 (C), 135.46 (C), 146.75 (C), 147.24 (C), 147.51 (C),
148.33 (C). Anal. Calcd. for C21H15N3O2S: C, 67.54; H, 4.05; N, 11.25; Found: C, 67.56; H, 4.04; N, 11.24.
MS (ESI) (m/z): [M]+ 373.90.
1-(4-Fluorophenyl)-3-(2-thienyl)-5-(1,3-benzodioxol-5-yl)-2-pyrazoline (4c). Yield: 76%. M.p.: 108–110 ◦C.
IR
ν
(cm−1): 3086.11 (aromatic C-H stretching), 2970.38, 2916.37 (aliphatic C-H stretching), 1504.48,
max
1483.26, 1442.75 (C=N and C=C stretching), 1240.23, 1220.94, 1035.77 (C-N, C-O stretching and aromatic
C-H in plane bending), 931.62, 802.39, 705.95 (aromatic C-H out of plane bending and C-S stretching).
1H-NMR (300 MHz, DMSO-d6)
δ (ppm): 3.11 (1H, dd, JAM = 17.31 Hz, JAX = 6.81 Hz, C4-HA pyrazoline),

Molecules 2018, 23, 2151
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3.87 (1H, dd, JMA = 17.31 Hz, JMX = 11.97 Hz, C4-HM pyrazoline), 5.36 (1H, dd, JMX = 11.91 Hz,
AX = 6.75 Hz, C5-HX pyrazoline), 5.98 (2H, d, J = 0.78 Hz, O-CH2-O), 6.77–6.81 (2H, m, aromatic
J
protons), 6.87 (1H, d, J = 7.83 Hz, aromatic proton), 6.91–6.94 (2H, m, aromatic protons), 6.95–7.02 (2H,
m, aromatic protons), 7.04–7.11 (1H, m, aromatic proton), 7.24–7.26 (1H, m, aromatic proton), 7.60 (1H,
dd, J = 5.07 Hz, 1.05 Hz, aromatic proton). 13C-NMR (75 MHz, DMSO-d6)
δ (ppm): 44.36 (CH2), 64.11
(CH), 101.55 (CH2), 106.62 (CH), 109.08 (CH), 114.65 and 114.75 (2CH), 115.75 and 116.05 (2CH), 119.70
(2CH), 127.98 and 128.28 (2CH), 136.04 and 136.31 (2C), 141.49 (C), 144.46 (C), 147.06 (C), 148.22 (C),
154.85 and 157.96 (C). Anal. Calcd. for C20H15FN2O2S: C, 65.56; H, 4.13; N, 7.65; Found: C, 65.54; H,
4.14; N, 7.66. MS (ESI) (m/z): [M + H]− 364.90, [M]+ 365.90, [M + H]+ 366.90.
1-(4-Chlorophenyl)-3-(2-thienyl)-5-(1,3-benzodioxol-5-yl)-2-pyrazoline (4d). Yield: 92%. M.p.: 130–132 ◦C.
IR
ν
(cm−1): 3072.60 (aromatic C-H stretching), 2916.37, 2848.86 (aliphatic C-H stretching), 1595.13,
max
1492.90, 1481.33, 1442.75 (C=N and C=C stretching), 1382.96, 1319.31, 1244.09, 1136.07, 1093.64, 1037.70
(C-N, C-O stretching and aromatic C-H in plane bending), 933.55, 813.96, 796.60, 725.23, 707.88
1
(aromatic C-H out of plane bending and C-S stretching). H-NMR (300 MHz, DMSO-d6)
δ (ppm): 3.13
(1H, dd, JAM = 17.40 Hz, JAX = 6.09 Hz, C4-HA pyrazoline), 3.89 (1H, dd, JMA = 17.43 Hz, JMX = 12.03
Hz, C4-HM pyrazoline), 5.42 (1H, dd, JMX = 11.94 Hz, JAX = 6.03 Hz, C5-HX pyrazoline), 5.98 (2H, d,
J = 1.59 Hz, O-CH2-O), 6.74–6.78 (2H, m, aromatic protons), 6.87 (1H, d, J = 7.89 Hz, aromatic proton),
6.94 (2H, d, J = 9.03 Hz, aromatic protons), 7.11 (1H, dd, J = 5.07 Hz, 3.63 Hz, aromatic proton), 7.20
(2H, d, J = 9.00 Hz, aromatic protons), 7.27 (1H, dd, J = 3.57 Hz, 1.08 Hz, aromatic proton), 7.62 (1H, dd,
J = 5.07 Hz, 1.05 Hz, aromatic proton). 13C-NMR (75 MHz, DMSO-d6)
δ (ppm): 44.30 (CH2), 63.41 (CH),
101.57 (CH2), 106.52 (CH), 109.10 (CH), 114.85 (2CH), 116.52 (C), 119.58 (CH), 122.70 (CH), 128.27 (2CH,
d, J = 6.75 Hz), 129.21 (2CH, d, J = 6.75 Hz), 135.85 (C), 136.06 (C), 143.26 (C), 145.04 (C), 147.09 (C),
148.24 (C). Anal. Calcd. for C20H15ClN2O2S: C, 62.74; H, 3.95; N, 7.32; Found: C, 62.76; H, 3.94; N, 7.31.
MS (ESI) (m/z): [M + H]−− 378.80, [M]+ 380.80, [M + H]+ 381.90, [M + H]++ 382.80, [M + H]+++ 383.90.
1-(4-Bromophenyl)-3-(2-thienyl)-5-(1,3-benzodioxol-5-yl)-2-pyrazoline (4e). Yield: 90%. M.p.: 136–138 ◦C.
IR
ν
(cm−1): 3070.68 (aromatic C-H stretching), 2916.37 (aliphatic C-H stretching), 1589.34, 1481.33,
max
1442.75 (C=N and C=C stretching), 1382.96, 1319.31, 1244.09, 1128.36, 1091.71, 1035.77 (C-N, C-O
stretching and aromatic C-H in plane bending), 935.48, 812.03, 707.88 (aromatic C-H out of plane
1
bending and C-S stretching). H-NMR (300 MHz, DMSO-d6)
δ
(ppm): 3.13 (1H, dd, JAM = 17.40 Hz,
J
AX = 5.91 Hz, C4-HA pyrazoline), 3.88 (1H, dd, JMA = 17.43 Hz, JMX = 12.09 Hz, C4-HM pyrazoline),
5.42 (1H, dd, JMX = 11.88 Hz, JAX = 5.85 Hz, C5-HX pyrazoline), 5.98 (2H, d, J = 1.56 Hz, O-CH2-O),
6.73–6.77 (2H, m, aromatic protons), 6.83–6.94 (3H, m, aromatic protons), 7.10 (1H, dd, J = 4.89 Hz,
3.75 Hz, aromatic proton), 7.27–7.33 (3H, m, aromatic protons), 7.62 (1H, d, J = 4.95 Hz, aromatic
proton). 13C-NMR (75 MHz, DMSO-d6)
δ (ppm): 44.29 (CH2), 63.29 (CH), 101.57 (CH2), 106.51 (CH),
109.11 (CH), 110.32 (CH), 115.34 (2CH), 119.56 (CH), 128.31 (2CH, d, J = 5.25 Hz), 132.00 (2CH), 135.82
(C), 136.01 (2C), 143.57 (C), 145.13 (C), 147.10 (C), 148.24 (C). Anal. Calcd. for C20H15BrN2O2S: C, 56.22;
H, 3.54; N, 6.56; Found: C, 56.24; H, 3.53; N, 6.55. MS (ESI) (m/z): [M + H]— 426.80, [M]+ 427.80.
1-(4-Methylphenyl)-3-(2-thienyl)-5-(1,3-benzodioxol-5-yl)-2-pyrazoline (4f). Yield: 47%. M.p.: 153–155 ◦C.
IR
ν
(cm−1): 3070.68 (aromatic C-H stretching), 2916.37, 2848.86 (aliphatic C-H stretching), 1593.20,
max
1481.33, 1442.75 (C=N and C=C stretching), 1382.96, 1319.31, 1244.09, 1130.29, 1091.71, 1035.77 (C-N,
C-O stretching and aromatic C-H in plane bending), 933.55, 812.03, 802.39, 723.31, 707.88 (aromatic
1
C-H out of plane bending and C-S stretching). H-NMR (300 MHz, DMSO-d6)
δ (ppm): 2.16 (3H, s,
CH3), 3.07 (1H, dd, JAM = 17.28 Hz, JAX = 6.57 Hz, C4-HA pyrazoline), 3.84 (1H, dd, JMA = 17.25 Hz,
MX = 12.03 Hz, C4-HM pyrazoline), 5.35 (1H, dd, JMX = 11.97 Hz, JAX = 6.54 Hz, C5-HX pyrazoline),
J
5.97 (2H, d, J = 1.26 Hz, O-CH2-O), 6.76 (2H, d, J = 6.84 Hz, aromatic protons), 6.86 (3H, d, J = 8.64 Hz,
aromatic protons), 6.97 (2H, d, J = 8.43 Hz, aromatic protons), 7.09 (1H, dd, J = 5.04 Hz, 3.63 Hz,
aromatic proton), 7.22 (1H, dd, J = 3.54 Hz, 0.99 Hz, aromatic proton), 7.57 (1H, dd, J = 5.04 Hz, 0.99 Hz,
aromatic proton). 13C-NMR (75 MHz, DMSO-d6)
δ
(ppm): 20.54 and 20.57 (CH3), 44.14 (CH2), 63.80
(CH), 101.50 (CH2), 106.57 (CH), 109.02 (CH), 113.65 (2CH), 119.63 (CH), 127.61 (CH), 127.88 (CH),

Molecules 2018, 23, 2151
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128.24 (CH), 129.78 (2CH), 136.29 (C), 136.63 (2C), 142.43 (C), 143.67 (C), 146.95 (C), 148.14 (C). Anal.
Calcd. for C21H18N2O2S: C, 69.59; H, 5.01; N, 7.73; Found: C, 69.61; H, 5.00; N, 7.72. MS (ESI) (m/z):
[M + H]−− 360.90, [M]+ 361.90, [M + H]+ 362.90.
◦
1-(4-Methoxyphenyl)-3-(2-thienyl)-5-(1,3-benzodioxol-5-yl)-2-pyrazoline (4g). Yield: 39%. M.p.: 140–142 C.
IR
ν
(cm−1): 3107.32 (aromatic C-H stretching), 2916.37, 2848.86 (aliphatic C-H stretching), 1587.42,
max
1504.48, 1487.12, 1444.68 (C=N and C=C stretching), 1375.25, 1230.58, 1180.44, 1116.78, 1083.99, 1033.85
(C-N, C-O stretching and aromatic C-H in plane bending), 935.48, 829.39, 804.32, 707.88 (aromatic
1
C-H out of plane bending and C-S stretching). H-NMR (300 MHz, DMSO-d6)
δ
(ppm): 3.07 (1H, dd,
J
J
AM = 17.16 Hz, JAX = 7.41 Hz, C4-HA pyrazoline), 3.65 (3H, s, OCH3), 3.83 (1H, dd, JMA = 17.19 Hz,
MX = 11.94 Hz, C4-HM pyrazoline), 5.29 (1H, dd, JMX = 11.85 Hz, JAX = 7.38 Hz, C5-HX pyrazoline),
5.98 (2H, d, J = 0.81 Hz, O-CH2-O), 6.76–6.81 (4H, m, aromatic protons), 6.85–6.91 (3H, m, aromatic
protons), 7.09 (1H, dd, J = 5.04 Hz, 3.60 Hz, aromatic proton), 7.21 (1H, dd, J = 3.54 Hz, 1.02 Hz,
aromatic proton), 7.56 (1H, dd, J = 5.04 Hz, 1.02 Hz, aromatic proton). 13C-NMR (75 MHz, DMSO-d6)
δ
(ppm): 44.24 (CH2), 55.64 and 55.67 (CH3), 64.66 (CH), 101.50 (CH2), 106.71 (CH), 109.01 (CH), 114.83
(2CH), 115.05 (2CH), 119.82 (CH), 127.46 (2CH, d, J = 6.00 Hz), 128.22 (CH), 136.37 (C), 136.63 (C),
139.11 (C), 143.49 (C), 146.97 (C), 148.14 (C), 153.25 (C). Anal. Calcd. for C21H18N2O3S: C, 66.65; H,
4.79; N, 7.40; Found: C, 66.67; H, 4.78; N, 7.39. MS (ESI) (m/z): [M]+ 377.90, [M + H]+ 378.90.
1-(4-Methylsulfonylphenyl)-3-(2-thienyl)-5-(1,3-benzodioxol-5-yl)-2-pyrazoline (4h). Yield: 63%. M.p.:
◦
172–173 C. IR
ν
(cm−1): 3097.68 (aromatic C-H stretching), 2985.81, 2900.84 (aliphatic C-H
max
stretching), 1589.34, 1502.55, 1483.26, 1442.75 (C=N and C=C stretching), 1392.61, 1381.03, 1296.16,
1247.94, 1078.21, 1051.20 (C-N, C-O stretching and aromatic C-H in plane bending), 821.68, 769.60,
705.95 (aromatic C-H out of plane bending and C-S stretching). 1H-NMR (300 MHz, DMSO-d6)
δ
(ppm): 3.07 (3H, s, SO2CH3), 3.20 (1H, dd, JAM = 17.64 Hz, JAX = 5.07 Hz, C4-HA pyrazoline), 3.96 (1H,
dd, JMA = 17.61 Hz, JMX = 11.94 Hz, C4-HM pyrazoline), 5.57 (1H, dd, JMX = 11.85 Hz, JAX = 4.98 Hz,
C5-HX pyrazoline), 5.98 (2H, d, J = 1.62 Hz, O-CH2-O), 6.75 (1H, dd, J = 7.95 Hz, 1.74 Hz, aromatic
proton), 6.79 (1H, d, J = 1.56 Hz, aromatic proton), 6.88 (1H, d, J = 7.92 Hz, aromatic proton), 7.07
(2H, d, J = 8.94 Hz, aromatic protons), 7.13 (1H, dd, J = 5.07 Hz, 3.66 Hz, aromatic proton), 7.36 (1H,
dd, J = 3.57 Hz, 1.05 Hz, aromatic proton), 7.65–7.69 (3H, m, aromatic protons). 13C-NMR (75 MHz,
DMSO-d6)
δ (ppm): 44.29 (CH2), 44.63 (CH3), 62.61 (CH), 101.64 (CH2), 106.43 (CH), 109.21 (CH),
112.61 (2CH), 119.45 (CH), 128.47 (CH), 129.05 (2CH), 129.38 (2CH), 129.61 (C), 135.30 (C), 135.56 (2C),
147.27 (2C), 148.32 (C). Anal. Calcd. for C21H18N2O4S2: C, 59.14; H, 4.25; N, 6.57; Found: C, 59.16; H,
4.24; N, 6.56. MS (ESI) (m/z): [M]+ 426.80, [M + H]+ 427.80.
1-(4-Sulfonamidophenyl)-3-(2-thienyl)-5-(1,3-benzodioxol-5-yl)-2-pyrazoline (4i) [45]. Yield: 83%. M.p.:
◦
129–130 C. IR
ν
(cm−1): 3321.42, 3250.05 (N-H stretching), 3103.46, 3074.53 (aromatic C-H
max
stretching), 2916.37, 2848.86 (aliphatic C-H stretching), 1591.27, 1502.55, 1485.19, 1442.75 (C=N and C=C
stretching), 1394.53, 1323.17, 1307.74, 1238.30, 1151.10, 1095.57, 1035.77, 1001.06 (C-N, C-O stretching
and aromatic C-H in plane bending), 933.55, 904.61, 860.25, 812.03, 713.66 (aromatic C-H out of plane
1
bending and C-S stretching). H-NMR (300 MHz, DMSO-d6)
δ
(ppm): 3.18 (1H, dd, JAM = 17.52 Hz,
JAX= 5.10 Hz, C4-HA pyrazoline), 3.93 (1H, dd, JMA = 17.52 Hz, JMX = 11.94 Hz, C4-HM pyrazoline), 5.56
(1H, dd, JMX = 11.85 Hz, JAX = 5.01 Hz, C5-HX pyrazoline), 5.98 (2H, d, J = 2.04 Hz, O-CH2-O), 6.72–6.77
(2H, m, aromatic protons), 6.87 (1H, d, J = 7.86 Hz, aromatic proton), 7.01–7.04 (4H, m, aromatic and
NH2 protons), 7.12 (1H, dd, J = 5.04 Hz, 3.63 Hz, aromatic proton), 7.33 (1H, dd, J = 3.57 Hz, 1.05 Hz,
aromatic proton), 7.60 (2H, d, J = 8.94 Hz, aromatic protons), 7.66 (1H, dd, J = 5.04 Hz, 1.02 Hz, aromatic
proton). 13C-NMR (75 MHz, DMSO-d6)
δ (ppm): 44.83 (CH2), 64.86 (CH), 101.66 (CH2), 106.85 (CH),
109.87 (CH), 115.65 (2CH), 119.87 (CH), 127.47 (CH), 127.96 (CH), 128.05 (CH), 131.88 (2CH), 135.77
(C), 136.45 (C), 146.03 (C), 147.49 (C), 148.90 (C), 159.36 (2C). Anal. Calcd. for C20H17N3O4S2: C, 56.19;
H, 4.01; N, 9.83; Found: C, 56.24; H, 4.08; N, 9.80. MS (ESI) (m/z): [M]+ 427.80, [M + H]+ 428.80.

Molecules 2018, 23, 2151
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3.2. Pharmacology
3.2.1. Cell Culture
PC-12 Adh cells were cultured in 10% horse serum, 5% fetal bovine serum, and 1%
◦
penicillin-streptomycin containing DMEM growth medium, at 37 C in a humidified incubator
with 5% CO2. The proliferating cells were passaged 1:2 to new 25 and/or 75 cm2 flasks and cell
stocks were prepared in order to use in future experiments. In order to induce PC-12 Adh cell
differentiation into neuronal phenotype, growth medium was changed with 1% fetal bovine serum,
1% penicillin-streptomycin and 100 nM NGF containing DMEM differentiation medium.
Differentiated PC-12 Adh cells were stained with Trypan blue solution and counted by a cell
counter device (Cedex XS, Innovatis, Malvern, PA, USA) in order to determine the appropriate cell
numbers before the experiments.
3.2.2. Determination of Non-Cytotoxic Concentrations
In order to obtain non-cytotoxic concentrations of the compounds, the viability of neuronal
cells was measured by using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium
sodium salt (WST-1) assay (Roche, Mannheim, Germany). The test is based on the cleavage of the
tetrazolium salt WST-1 in formazan by mitochondrial dehydrogenases in viable cells. The formazan
dye was quantified in a scanning multiwell spectrophotometer by measuring the absorbance of the
dye at 420 nm. The differentiated PC-12 Adh cells were scratched and plated onto 96-well culture
plates at 5
25 g/mL concentrations of compounds and 400, 200, 100, 50 and 25
incubation period, the cell proliferation reagent WST-1 (10 L per well) was added to the wells; and
×
103 density per well. After 24 h, the cells were treated with 400, 200, 100, 50, and
M 6-OHDA for 24 h. After the
µ
µ
µ
absorbances were measured after 3 h using a Cytation 3 cell imaging multi-mode reader at 420 nm
(BioTek, Winooski, VT, USA). The measured absorbances directly correlated to the number of viable
cells. The cell viability rates were expressed as a percentage of the controls, and IC50 values of the
compounds and 6-OHDA were calculated according to the control group [46].
3.2.3. Determination of Neuroprotective Activity against 6-OHDA-Induced Neurodegeneration
In order to determine the in vitro neuroprotective potentials of the compounds, 6-OHDA-induced
neurotoxicity model of PD was conducted [47
and plated onto 96-well culture plates at 5
103 density per well. After 24 h, the cells were treated with
100 g/mL concentration (only 10 g/mL and 100 g/mL were tested for compound , which was
found to be highly cytotoxic) of the compounds for 6 h. 100 g/mL concentration of the compounds
,48]. The differentiated PC-12 Adh cells were scratched
×
µ
µ
µ
1
µ
was determined as the non-cytotoxic concentration, and the IC50 value of the 6-OHDA (150 µM) was
used as a positive control according to the WST-1 cell viability assay results. After a 6-h incubation
period with the compounds, the medium was removed, and the cells were treated with 150
6-OHDA in order to induce neurodegeneration via oxidative stress for 24 h. The control cells were
cultured in differentiation medium containing 0.1% DMSO, and the cells cultured in 150 M 6-OHDA
µM
µ
were used as a positive control. After 24 h, the neuroprotective effects of the compounds were
determined by WST-1 cell viability assay as explained above. The graphics were drawn according to
the cell viability, which was expressed as percentage of the surviving control cells in the study.
3.2.4. Statistical Analysis
The graphics were drawn with Graphpad Prism 6.0 software and statistically analyzed using
one-way ANOVA and Tukey’s post hoc test. The results are expressed as mean
±
standard deviation
and the means of three independent experiments (n = 8), n.s; p > 0.05, * p < 0.05, ** p < 0.01,
*** p < 0.001, and **** p < 0.0001 were considered significant compared with the control group and the
6-OHDA-positive control group.

Molecules 2018, 23, 2151
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3.3. Prediction of Pharmacokinetic Parameters
ADME properties of compounds
1
,
2
,
3a–
i
, and 4a
–
i
were in silico predicted using QikProp
program (Schrödinger Release 2016-2: QikProp, Schrödinger, LLC, New York, NY, USA, 2016).
This program computes physically significant descriptors and pharmaceutically relevant properties,
such as QPlogBB, CNS activity, SASA (in square angstroms using a probe with a 1.4 Å Radius) and a
percentage of human oral absorption. The acceptability of compounds 1, 2, 3a–i and 4a–i, based on the
Lipinski’s rule of five [39] and Jorgensen’s rule of three [40], was also determined.
4. Conclusions
In the recent work, 2-pyrazoline based compounds 3a
–i and 4a–i were synthesized based
on the reaction of chalcones ( ) and phenylhydrazine hydrochloride derivatives. All these
1, 2
compounds were evaluated for their neuroprotective effects against toxicity induced by 6-OHDA
in rat pheochromocytoma (PC-12) cells. According to in vitro studies, 4-methylsulfonylphenyl
containing compounds 3h and 4h induced cell viability percentage notably when compared with
the 6-OHDA-positive control group. In silico ADME prediction also pointed out that all compounds
were within the acceptable range for some pharmacokinetic parameters especially important for CNS
activity. Consequently, compounds 3h and 4h stand out as potential orally bioavailable CNS acting
drug candidates for further neuroprotective studies associated with PD.
Author Contributions: A.Ö., B.S., and M.D.A. designed the research, A.Ö., B.S., and M.D.A. performed the
synthetic work. B.S. carried out the ADME studies and mainly wrote the manuscript. M.D. and E.K.T. performed
the whole pharmacology part of this study. A.Ö. was also responsible for the correspondence of the manuscript.
All the authors discussed, edited, and approved the final version of the manuscript.
Funding: This study was supported by Anadolu University Scientific Research Projects Commission under the
grant no: 1805S208.
Conflicts of Interest: The authors declare no conflicts of interest.
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Sample Availability: Samples of the compounds 1, 2, 3a-i, 4a-i are available from the authors.
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