Synthesis and Evaluation of N-[ 1-( ( ( 3 , 4-Diphenylthiazol-2 ( 3 H )-ylidene ) amino ) methyl ) cyclopentyl ] acetamide Derivatives for the Treatment of Diseases Belonging to MAOs

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskişehir, Turkey Department of Chemistry, Faculty of Sciences, Mentouri University, 325 Constantine, Algeria Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Aden University, 6075 Aden, Yemen Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Anadolu University, 26470 Eskişehir, Turkey Doping and Narcotic Compounds Analysis Laboratory, Faculty of Pharmacy, Anadolu University, 26470 Eskişehir, Turkey


Introduction
e frequency of Parkinson's disease (PD) is the most minimal in China, Japan, and Africa.In any case, it is emerging all through the world, in spite of stamped contrasts in demography, atmosphere, diet, sociocultural foundation, and industrialization [1].In early stages of PD, there seems to be a compensatory increment in the quantity of dopamine receptors to suit the underlying loss of dopamine neurons.As the disease raise, number of dopamine receptors diminishes clearly because of the attending degeneration of dopamine in striatal neurons.
e loss of dopaminergic neurons in PD [2] results in enhanced metabolism of dopamine, enlarging the arrangement of H 2 O 2 and thus leading to generation of highly neurotoxic hydroxyl radicals (OH) [3].
e free radicals can also be produced by 6hydroxydopamine or MPTP, which breaks down striatal dopaminergic neurons and causes Parkinsonism in experimental animals as well as human beings [4].
Monoamine oxidase (MAO) enzymes have two isoforms such as MAO-A and MAO-B, which share around 70% amino acids arrangement homology, and however vary in their cell localization, substrate preference, and inhibitor affectability [5,6].Enzyme classes have critical parts in mental health.ey manage the neurotransmitter capacity by metabolism in the brain, and by this way, they keep up proper concentration of intracellular amines [7].In any case, overactivation of MAOs leads to production of neurotoxic by-products coming from neuronal variations.
is biochemical event may result in disorders such as stroke, PD, and Alzheimer's disease (AD) [8,9].In the same way, modifications in other neurotransmitter frameworks are thought to be responsible for the behavioral unsettling influences [10].
iazole derivatives have potential against neurodiseases in the new drug development studies [11].Furthermore, thiazole derivatives additionally can cross the blood-brain barrier [12].In addition to thiazoles, acetamide derivatives are already known for their stimulatory effect on the central nervous system [13].In the light of previously stated information and as a supplement of our researches on the same topic, we report herein the synthesis of some N- [1-(((3,4-diphenylthiazol-2(3H)-ylidene)amino)methyl)cyclopentyl]acetamide derivatives in order to investigate their ability to inhibit MAO enzymes.

Experimental
2.1.Chemistry.All chemicals were purchased from commercial supplier and used without purification.Melting points (mp) were determined on an Electrothermal 9100 melting point apparatus (Weiss-Gallenkamp, Loughborough, UK) and are uncorrected.IR spectra were recorded on Shimadzu 8400S spectrophotometer (Shimadzu, Tokyo, Japan), 1 H-NMR and 13 C-NMR spectra of the synthesized compounds were recorded on a Bruker 500 MHz spectrometer in DMSO d 6 using TMS as internal standard, and mass spectra were recorded on a LC/MS/MS Mass Spectrometer (3200 QTRAP, AB Sciex Instruments).Purity of synthesized compounds (4a-4i) was checked by Shimadzu LC-20A prominence HPLC system (Shimadzu, Tokyo, Japan), using acetonitrile (95%) and water (5%) mixture as a mobile phase at a flow rate of 0.8 ml/min.
e product was crystallized from ethanol.

MAO-A and MAO-B Inhibition
Assay.MAO enzyme inhibition assay, based on fluorometric measurements, was used to determine the inhibition profiles of all synthesized compounds as previously defined by us [15,16].e reagents used in the enzymatic assay including Ampliflu ™ Red (10-  acetyl-3,7-dihydroxyphenoxazine), peroxidase from horseradish, hMAO-A, hMAO-B, H 2 O 2 , tyramine hydrochloride, selegiline, and moclobemide were purchased from Sigma-Aldrich (Steinheim, Germany).All dilution and pipetting processes were carried out by the robotic system, BioTek Precision XS (BioTek Instruments, Winooski, VT, USA).BioTek-Synergy H1 microplate reader was used to record the fluorescence measurements (excitation, 535 nm; emission, 587 nm) over a 30 min period, in which the fluorescence increased linearly.

Enzyme Kinetic Studies.
In order to investigate the inhibition type of compound 4b on hMAO-B, enzyme kinetic Journal of Chemistry study was applied according to the previous studies reported by us [15,16].

Cytotoxicity Test.
MTT test was performed to determine the cytotoxic profile of the most active compounds 4a, 4b, and 4c using the NIH/3T3 mouse embryonic fibroblast cell line (ATCC ® CRL-1658 ™ , London, UK). e method was carried out in the same way as we have reported in our previous studies [15][16][17][18].

Molecular Docking Studies.
A structure-based in silico procedure was applied to discover the binding modes of compound 4b hMAO-B enzyme active site.
e crystal structures of hMAO-B (PDB ID: 2V5Z) [19], which was crystallized with the reversible inhibitor safinamide, was retrieved from the Protein Data Bank server (http://www.pdb.org).e docking procedure was applied as previously described by our research group [16].

Chemistry.
e synthesis of thiazoline derivatives (4a-4i) was carried out according to the steps shown in Scheme 1.
In the IR spectra of compounds 4a-4i, C�N and C�C stretching vibrations were observed in the region 1645-1470 cm −1 .
e aromatic C-H stretching vibrations gave rise to bands at 3120-3010 cm −1 .
In the 1 H-NMR spectra, the protons corresponding to cyclopentyl, resonated as multiplets at d 0.78-0.85ppm, 1.34-1.40ppm, 1.56-1.65 ppm, and 2.25-2.35ppm, and CH 2 -N protons appeared as singlet at d 4.09 ppm.e protons of acetamide appeared as a singlet at d 5.80 ppm.All the other aromatic and aliphatic protons were observed at expected regions. 13 e mass spectra of compounds (4a-4i) are also in agreement with their molecular formula.According to HPLC analysis, purity ratio was found between 98.4 and 99.7%.Peak purity index of all compounds was also checked, and no impurity was determined in observed peaks.

MAO-A and MAO-B Inhibition Assay.
In order to investigate hMAO-A and hMAO-B inhibitory activity of all end compounds (4a-4i), an in vitro fluorometric method, which is on the strength of the detection of H 2 O 2 in a horseradish peroxidase-coupled reaction using Amplex Red reagent, was used.
e activity results against both isoenzymes by using all compounds at the concentration of 10 −3 and 10 −4 M were obtained as a result of second stage of the activity tests.In this second step, compounds showing more than 50% inhibition in the first step were selected and their further concentrations (10 −5 -10 −9 M) were studied.Also, IC 50 graphs, obtained from GraphPad "Prism" software (version 5.0), of these selected compounds can be observed in Figures 1 and 2.
ese compounds, 4a, 4b, and 4c, displayed hMAO-B inhibition profile with IC 50 values of 0.42 ± 0.012 µM, 0.36 ± 0.014 µM, and 0.69 ± 0.020 µM, respectively.According to all these results, it can be understood that compound 4b is the most potent derivative against MAO-A and MAO-B isoenzymes.It can also be suggested that compound 4b could be a selective MAO-B inhibitor when its IC 50 values on these enzymes are considered.

Kinetic Studies of Enzyme Inhibition.
e mechanism of hMAO-B inhibition was investigated by enzyme kinetics, following a similar procedure to the MAO inhibition assay.
e linear Lineweaver-Burk graphics were used to estimate the type of inhibition.Enzyme kinetics were analyzed by recording substrate velocity curves in the absence and presence of the most potent compound 4b, which was prepared at concentrations of IC 50 /2, IC 50 , and 2xIC 50 .In each case, the initial velocity measurements were gained at different substrate (tyramine) concentrations ranging from 20 µM to 0.625 µM. e K i (intercept on the x-axis) values of compound 4b were determined from the secondary plot of the K m /V max (slope) versus varying concentrations.e graphical analysis of steady-state inhibition data for compound 4b is shown in Figure 3.
Based on the type of interaction with the enzyme, inhibitor binding can be classified as either reversible or irreversible.e type of inhibition can be determined by the Lineweaver-Burk plot as mixed type, uncompetitive, competitive, or noncompetitive, which are the indicators of a reversible inhibitor [22].It is known that, in the uncompetitive type inhibition, a graphic, including the parallel lines without any cross, is observed.If the lines cross neither the x-nor the y-axis at the same point, the inhibition type is called mixed type.Competitive inhibitors possess the same intercept on the y-axis, but there are diverse slopes and intercepts on the x-axis between the two datasets.On the other hand, noncompetitive inhibition has plots with the same intercept on the x-axis, but there are different slopes and intercepts on the y-axis, which is observed in Figure 3.
erefore, this pattern indicates that the compound 4b is reversible and noncompetitive inhibitor and can bind to either the free enzyme or the enzyme-substrate complex.K i value for compound 4b was calculated as 0.346 μM for the inhibition of hMAO-B.
Reversible inhibitors bind to enzymes by noncovalent interactions as hydrophobic interactions, ionic bonds, and hydrogen bonds without forming any chemical bonds or reactions with the enzyme.ese interactions are formed rapidly and can be easily removed; hence, the enzyme and inhibitor complex are quickly dissociated opposite to irreversible inhibition.Due to reversible binding ability to biomolecules, such inhibitors carry a lower risk of side effects compared to irreversible inhibitors.As a result, reversible-noncompetitive inhibition potency of compound 4b has enhanced their biological importance.
Scheme 1: Synthesis pathway of targeted compounds.

Molecular Docking Studies.
As stated in the MAO inhibition studies, compound 4b was found to be the most active and selective derivative against hMAO-B isoform.In order to assess this in vitro activity and find out the binding modes of compound 4b, docking studies were performed by using Maestro interface.X-ray crystal structure of hMAO-B, obtained from Protein Data Bank server (http://www.pdb.org), Journal of Chemistry was used (PDB ID: 2V5Z) [19].e docking pose of compound 4b is presented in Figure 4.
It can be seen that compound 4b is located in close proximity to the FAD cofactor and binds tightly to the amino acid residues in the cavity.According to the docking pose, compound 4b interacts with the active site by establishing three hydrogen bonds and a p-π interaction.
is p-π interaction is observed between the phenyl ring attached to the nitrogen atom of thiazole and phenyl of Phe343.Two of the other hydrogen bonds are associated with the amide carbonyl in the structure.is moiety creates these hydrogen bonds with amino groups of Ser59 and Tyr60.e last hydrogen bond is formed between the methoxy group, stated at the C-4 position of the phenyl ring, and hydroxyl of Tyr326.It is thought that this additional bond is very important in terms of enzyme activity.
When the chemical structures of the synthesized compounds are compared with each other, it is seen that varying substituents at the C-2, C-3, C-4, and C-5 positions of the phenyl ring is the main reason of structural difference.It is considered by analyzing especially the activity results on hMAO-B that the substituents at the C-4 position of the phenyl increase the activity and selectivity.Furthermore, according to the results obtained from docking studies, as in compound 4b, functional groups such as methoxy moiety with the capacity to establish hydrogen bonds at this position bind more strongly to active region of enzyme and thus are important for activity.On the contrary, incorporation of both methoxy and hydroxyl groups in the C-2 and C-4 positions of phenyl ring as in the compound 4h showed no activity.is might be referred to the steric hindrance in general which may increase as the distance between two substituents decreases [24].

Conclusion
Another class of N- thiazol-2(3H)ylidene)amino)methyl)cyclopentyl]acetamide derivatives (4a-4i), bearing active thiazole moiety which contributes to MAO inhibition, was designed and synthesized.According to activity results, compounds 4a, 4b, and 4c showed good activity on both isoenzymes.Among the series, compound 4b was found as most active derivative and selective MAO-B inhibitor.Furthermore, enzyme kinetic, toxicological, and docking evaluations of compound 4b were undertaken in the current study.
e type of inhibition of this compound was found as reversible and noncompetitive.In addition, this active compound was not cytotoxic at MAO inhibitors IC 50 value given.Docking studies clearly indicated interactions between compound 4b and hMAO-B.Consequently, the study gave significant data to further improvement and change of medications for the treatment of diseases connected with MAOs.e information of this study recommends these compounds as promising leads for the development of novel MAO inhibitors with a decent inhibitory power, which in turn may fill the gap in the curative weapons store for Parkinson's disease (PD) in particular.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Figure 4 :
Figure 4: Docking studies including interacting modes of compound 4b in the active region of hMAO-B.e inhibitor and the important residues in the active site of the enzyme are presented by tube model.e FAD molecule is colored orange with ball and stick model.