Design, Synthesis, and Neuroprotective Effects of a Series of Pyrazolines against 6-Hydroxydopamine-Induced Oxidative Stress

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 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.


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][2][3][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 characterized by the loss of progressive dopaminergic neurons in the substantia nigra pars compacta and the accumulation of aggregated α-synuclein protein forming intracellular Lewy bodies in affected regions [8,9]. Moreover, the loss of dopaminergic neurons is associated with the decrease in the activity 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][9][10][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][15][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][18][19][20]. 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][22][23][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 3pyrazoline, 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][26][27][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 (1,2). All the compounds were in vitro 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. 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][22][23][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][26][27][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 (1,2). All the compounds were in vitro 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.
(150 µM) for 24 h. The reduction in cell viability was determined as 43% with 6-OHDA (150 µM) and 36,29,35,37,30,31,29,20, 30% with compounds 3a-i (100 µg/mL), respectively ( Figure 2; Table 2). However, compound 1 was found to be highly cytotoxic, and it reduced cell viability with the values of 97% (100 µg/mL) and 40% (10 µg/mL). In addition, the reduction in cell viability was evaluated as 34.1% with compound 2 and 59, 54, 76, 56, 41, 62, 69, 23, 87% with compounds 4a-i (100 µg/mL), respectively, when compared with 6-OHDA (150 µM) (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 1 and 3a-i; **** p < 0.0001 for compounds 2 and 4a-i). Compounds 3a-i, 2, and 4h might have neuroprotective potential against 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.   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) (−1.08 to 0.78) and central nervous system (CNS) activity (−1 to 0), 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 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 three; due to the fact that a potential orally active drug candidate should reveal no more than one violation of these rules [39,40]. 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) (−1.08 to 0.78) and central nervous system (CNS) activity (−1 to 0), 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 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 three; due to the fact that a potential orally active drug candidate should reveal no more than one violation of these rules [39,40].

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% CO 2 . The proliferating cells were passaged 1:2 to new 25 and/or 75 cm 2 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.

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 × 10 3 density per well. After 24 h, the cells were treated with 400, 200, 100, 50, and 25 µg/mL concentrations of compounds and 400, 200, 100, 50 and 25 µM 6-OHDA for 24 h. After the incubation period, the cell proliferation reagent WST-1 (10 µL per well) was added to the wells; and 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 IC 50 values of the compounds and 6-OHDA were calculated according to the control group [46].

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 [47,48]. The differentiated PC-12 Adh cells were scratched and plated onto 96-well culture plates at 5 × 10 3 density per well. After 24 h, the cells were treated with 100 µg/mL concentration (only 10 µg/mL and 100 µg/mL were tested for compound 1, which was found to be highly cytotoxic) of the compounds for 6 h. 100 µg/mL concentration of the compounds was determined as the non-cytotoxic concentration, and the IC 50 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 µM 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 µM 6-OHDA 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.

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.

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.

Conclusions
In the recent work, 2-pyrazoline based compounds 3a-i and 4a-i were synthesized based on the reaction of chalcones (1, 2) and phenylhydrazine hydrochloride derivatives. All these 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.