Journal of Chemistry
Volume 2018 (2018), Article ID 3547942, 10 pages
https://doi.org/10.1155/2018/3547942

Research Article

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

Gülhan Turan-Zitouni,¹ Aouatef Tabbi,1,2 Weiam Hussein,1,3 Abdullah Burak Karaduman,⁴ Begüm Nurpelin Sağlık,1,5 and Yusuf Özkay1,5

¹Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskişehir, Turkey
²Department of Chemistry, Faculty of Sciences, Mentouri University, 325 Constantine, Algeria
³Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Aden University, 6075 Aden, Yemen
⁴Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Anadolu University, 26470 Eskişehir, Turkey
⁵Doping and Narcotic Compounds Analysis Laboratory, Faculty of Pharmacy, Anadolu University, 26470 Eskişehir, Turkey

Correspondence should be addressed to Gülhan Turan-Zitouni; gturan@anadolu.edu.tr

Received 30 January 2018; Revised 18 March 2018; Accepted 13 August 2018; Published 13 September 2018

Academic Editor: Joaquin Campos

Copyright © 2018 Gülhan Turan-Zitouni et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

A series of N-[1-(((3,4-diphenylthiazol-2(3H)-ylidene)amino)methyl)cyclopentyl]acetamide derivatives (4a-4i) were synthesized in good yield and assayed for their inhibitory potency against monoamine oxidase (MAO) isoforms. Structures of newly synthesized compounds were characterized by IR, ¹H-NMR, ¹³C-NMR, and mass spectroscopic methods. The inhibitory activity of compounds (4a-4i) against hMAO-A and hMAO-B enzymes was elucidated by using in vitro fluorometric method using Amplex Red® reagent. In the hMAO-A inhibition assay, compounds 4a, 4b, 4c, and 4i exhibited similar activity with standard drug moclobemide (IC₅₀ = 6.061 ± 0.262 µM) with IC₅₀ values of 7.06 ± 0.18 µM, 6.56 ± 0.20 µM, 6.78 ± 0.15 µM, and 7.09 ± 0.17 µM, respectively. According to hMAO-B inhibition results, compounds 4a, 4b, and 4c displayed significant activity with IC₅₀ values of 0.42 ± 0.012 µM, 0.36 ± 0.014 µM, and 0.69 ± 0.020 µM, respectively. In the wake of all these results, it was understood that compound 4b was found to be the most potent derivative in the series against both isoforms and selective as MAO-B inhibitor. The cytotoxicity test was performed for compounds 4a, 4b, and 4c, and it was found that these compounds were noncytotoxic at the concentration of their IC₅₀ values. Also, enzyme kinetic and docking studies of compound 4b were performed against MAO-B. It was observed that 4b showed a reversible and noncompetitive inhibition type. The important binding modes of this compound with active site of hMAO-B were shown owing to in silico studies.

1. Introduction

The 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. The loss of dopaminergic neurons in PD [2] results in enhanced metabolism of dopamine, enlarging the arrangement of H₂O₂ and thus leading to generation of highly neurotoxic hydroxyl radicals (OH) [3]. The free radicals can also be produced by 6-hydroxydopamine 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. They 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. This 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].

Thiazole 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.

2. 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), ¹H-NMR and ¹³C-NMR spectra of the synthesized compounds were recorded on a Bruker 500 MHz spectrometer in DMSO d₆ 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.

2.1.1. General Procedure for the Synthesis of the Intermediate Compounds (1, 2, 3)

N-(1-cyanocyclopentyl)acetamide (1) was synthesized via the method of Reihlen, Hessling, Hühn, and Weinbrenner [14]. The reduction of nitriles (1) gives N-[(1-aminomethyl)cyclopentyl]acetamide (2) by nickel Raney. A mixture of amine (2) (0.1 mol) and phenylisothiocyanate (0.1 mol) was refluxed in ethanol for 2 h. N-[(1-((3-phenylthioureido)methyl)cyclopentyl]acetamide (3) was filtered, washed with water, and dried. The product was crystallized from ethanol.

2.1.2. N-[(1-((3-Phenylthioureido) methyl)cyclopentyl]acetamide (3)

Yield: 87%, Mp 230°C. IR νmax (cm⁻¹): 3470.70, 3375.20 (N–H stretching), 3260 (N–H stretching of acetamide), 1680 (C=O stretching of acetamide), 1560.95, 1510.32, 1467.46 (C=N and C=C stretching), 1210.17, 1168.00, 1095.45, 1010.01 (C–N stretching and aromatic C–H bending).

2.1.3. General Procedure for Compounds (4a-4i)

N-[(1-((3-phenylthioureido)methyl)cyclopentyl]acetamide (3) (0.001 mol) and appropriate 2-bromo acetophenone derivative (0.001 mol) were refluxed in ethanol (20 mL) for 4 h. The reaction mixture was cooled and filtered.

2.1.4. N-[1-(((3-Phenyl-4-(p-tolyl)thiazol-2(3H)-ylidene)amino)methyl)cyclopentyl]acetamide (4a)

¹H-NMR (500 MHz, DMSO-d₆) d (ppm): 0.78–0.81 (2H, m, CH₂), 1.35–1.38 (2H, m, CH₂), 1.60–1.62 (2H, m, CH₂), 1.68 (3H, s, CH₃), 1.78 (3H, s, CH₃), 2.24–2.28 (2H, m, CH₂), 4.06 (2H, s, CH₂–N), 5.83 (1H, s, NH), 7.10–7.41 (9H, m, Ar–H), 8.71 (1H, s, thiazol-H). ¹³C-NMR (125 MHz, DMSO-d₆, ppm): 22.83 (CH₂–CH₂), 21.25 (CH₃), 23.90 (COCH₃), 34.52 (2 CH₂), 47.63 (CH₂–NH), 65.42 (C), 98.27 (CH-thiazol), 120.40 (Ar-CH) 121.42 (Ar-2CH), 126.18 (Ar–C), 128.07 (Ar-2CH), 129.12 (Ar-2CH), 130.07 (Ar-2CH), 133.05 (Ar–C), 136.62 (Ar–C), 138.55 (Ar–C), 140.30 (Ar–C), 149.34 (thiazol-C), 159.15 (N=C-thiazol), 170.12 (CO). MS [M + 1]+: m/z 406 (100%). HPLC: 99.4% purity.

2.1.5. N-[1-(((4-(4-Methoxyphenyl)-3-phenylthiazol-2(3H)-ylidene)amino)methyl)cyclopentyl]acetamide (4b)

¹H-NMR (500 MHz, DMSO-d₆) d (ppm): 0.78–0.80 (2H, m, CH₂), 1.34–1.37 (2H, m, CH₂), 1.56–1.62 (2H, m, CH₂), 1.79 (3H, s, CH₃), 2.26–2.32 (2H, m, CH₂), 3.88 (3H, s, OCH₃), 4.07 (2H, s, CH₂–N), 5.79 (1H, s, NH), 7.00 (2H, d J = 8.60 Hz, Ar–H), 7.11–7.13 (3H, m, Ar–H), 7.30 (2H, d J = 8.70, Ar–H), 7.38–7.41 (2H, m, Ar–H), 8.90 (1H, s, thiazol-H). ¹³C-NMR (125 MHz, DMSO-d₆, ppm): 22.82 (CH₂–CH₂), 23.95 (COCH₃), 34.61 (2 CH₂), 48.63 (CH₂–NH), 55.69 (O–CH₃), 65.67 (C), 96.17 (CH-thiazol), 114.48 (Ar-2CH), 121.39 (Ar-2CH), 123.11 (Ar-2CH), 129.10 (Ar-CH), 130.02 (Ar-2CH), 130.07 (Ar-2CH), 133.05 (Ar–C), 133.69 (Ar–C), 139.57 (Ar–C), 151.61 (thiazol-C), 161.16 (N=C-thiazol), 169.18 (CO). MS [M + 1]+: m/z 422 (100%). HPLC: 98.6% purity.

2.1.6. N-[1-(((4-(4-Nitrophenyl)-3-phenylthiazol-2(3H)-ylidene)amino)methyl)cyclopentyl]acetamide (4c)

¹H-NMR (500 MHz, DMSO-d₆) d (ppm): 0.96 (2H, br, CH₂), 1.48 (2H, br, CH₂), 1.61 (2H, br, CH₂), 1.76 (3H, s, CH₃), 2.21 (2H, br, CH₂), 4.22 (2H, br, CH₂–N), 5.98 (1H, br, NH), 7.15 (3H, m, Ar–H), 7.41 (2H, d J = 8.2 Hz, Ar–H), 7.60 (2H, d J = 8.1 Hz, Ar–H), 7.78 (1H, br, thiazol-H), 8.37 (2H, d J = 7.35 Hz, Ar–H). ¹³C-NMR (125 MHz, DMSO-d₆, ppm): 22.66 (CH₂–CH₂), 23.63 (COCH₃), 35.09 (2 CH₂), 48.27 (CH₂–NH), 65.67 (C), 100.40 (CH-thiazol), 121.19 (Ar-2CH), 123.46 (Ar–C), 124.28 (Ar-2CH), 129.83 (Ar-2CH), 130.12 (Ar-2CH), 138.59 (Ar–C), 138.90 (Ar–C), 147.33 (thiazol-C), 151.46 (Ar–C), 161.17 (N=C-thiazol), 169.67 (CO). MS [M + 1]+: m/z 437 (100 %). HPLC: 99.2% purity.

2.1.7. N-[1-(((4-(4-Fluorophenyl)-3-phenylthiazol-2(3H)-ylidene)amino)methyl)cyclopentyl]acetamide (4d)

¹H-NMR (500 MHz, DMSO-d₆) d (ppm): 0.79–0.80 (2H, m, CH₂), 1.34–1.39 (2H, m, CH₂), 1.59–1.62 (2H, m, CH₂), 1.78 (3H, s, CH₃), 2.24–2.29 (2H, m, CH₂), 4.07 (2H, s, CH₂–N), 5.83 (1H, s, NH), 7.11–7.13 (3H, m, Ar–H), 7.18 (2H, t, Ar–H), 7.36–7.42 (4H, m, Ar–H), 8.72 (1H, s, thiazol-H). ¹³C-NMR (125 MHz, DMSO-d₆, ppm): 22.76 (CH₂–CH₂), 23.88 (COCH₃), 34.80 (2 CH₂), 48.40 (CH₂–NH), 65.61 (C), 97.21 (CH-thiazol), 115.88 (Ar-CH), 116.17 (Ar-CH), 121.31 (Ar-2CH), 123.18 (Ar–C), 128.73 (Ar–C), 128.77 (Ar-2CH), 129.10 (Ar-CH), 131.45 (Ar-2CH), 139.70 (Ar–C), 151.61 (thiazol-C), 161.20 (Ar–C), 164.17 (N=C-thiazol), 169.18 (CO). MS [M + 1]+: m/z 410 (100 %). HPLC: 98.7% purity.

2.1.8. N-[1-(((4-(4-Chlorophenyl)-3-phenylthiazol-2(3H)-ylidene)amino)methyl)cyclopentyl]acetamide (4e)

¹H-NMR (500 MHz, DMSO-d₆) d (ppm): 0.83–0.85(2H, m, CH₂), 1.35–1.39 (2H, m, CH₂), 1.60–1.64 (2H, m, CH₂), 1.78 (3H, s, CH₃), 2.24–2.28 (2H, m, CH₂), 4.09 (2H, s, CH₂–N), 5.84 (1H, s, NH), 7.11–7.14 (3H, m, Ar–H), 7.28 (2H, d J = 8.40, Ar–H), 7.38 (2H, t, Ar–H), 7.46 (2H, d J = 8.35 Hz, Ar–H), 8.54 (1H, s, thiazol-H). ¹³C-NMR (125 MHz, DMSO-d₆, ppm): 22.75 (CH₂–CH₂), 23.77 (COCH₃), 34.89 (2 CH₂), 48.33 (CH₂–NH), 65.68 (C), 97.77 (CH-thiazol), 121.28 (Ar-2CH), 123.21 (Ar-2CH), 129.10 (Ar-CH), 130.02 (Ar-2CH), 130.07 (Ar-Ar-(Ar-2CH), 124.57 (Ar–C), 129.98 (Ar-2CH), 130.75 (Ar-CH), 140.59 (Ar–C), 151.66 (thiazol-C), 159.86 (Ar–C), 161.41 (N=C-thiazol), 169.07 (CO). MS [M + 1]+: m/z 426 (100 %). HPLC: 99.5% purity.

2.1.9. N-[1-(((4-(4-Bromophenyl)-3-phenylthiazol-2(3H)-ylidene)amino)methyl)cyclopentyl]acetamide (4f)

¹H-NMR (500 MHz, DMSO-d₆) d (ppm): 0.85–0.90 (2H, m, CH₂), 1.37–1.39 (2H, m, CH₂, 1.59–1.63 (2H, m, CH₂), 1.78 (3H, s, CH₃), 2.26–2.28 (2H, m, CH₂), 4.09 (2H, s, CH₂–N), 5.84 (1H, s, NH), 7.10–7.15 (3H, m, Ar–H), 7.25–7.28 (2H, d J = 8.40 Hz, Ar–H), 7.38–7.41 (2H, t, Ar–H), 7.62–7.64 (2H, d J = 8.40 Hz, Ar–H), 8.50 (1H, s, thiazol-H). ¹³C-NMR (125 MHz, DMSO-d₆, ppm): 22.75 (CH₂–CH₂), 23.76 (COCH₃), 34.91 (2 CH₂), 48.32 (CH₂–NH), 65.57 (C), 97.75 (CH-thiazol), 121.28 (Ar-2CH), 122.34 (Ar–C), 123.21 (Ar–C), 130.03 (Ar-2CH), 131.09 (Ar-2CH), 131.51 (Ar–C), 132.02 (Ar-2CH), 139.62 (Ar–C), 151.61 (thiazol-C), 161.16 (N=C-thiazol), 169.18 (CO). MS [M + 1]+: m/z 470 (100 %). HPLC: 98.9% purity.

2.1.10. N-[1-(((4-(3,4-Dichlorophenyl)-3-phenylthiazol-2(3H)-ylidene)amino)methyl)cyclopentyl]acetamide (4g)

¹H-NMR (500 MHz, DMSO-d₆) d (ppm): 0.96–0.97 (2H, m, CH₂), 1.42–1.44 (2H, m, CH₂), 1.61–1.66 (2H, m, CH₂), 1.76 (3H, s, CH₃), 2.24–2.27 (2H, m, CH₂), 4.11 (2H, s, CH₂–N), 5.86 (1H, s, NH), 7.09–7.14 (3H, m, Ar–H), 7.22 (1H, d, Ar–H), 7.38–7.41 (2H, t, Ar–H), 7.51–7.515 (1H, d J = 2.0 Hz, Ar–H), 7.57–7.58 (1H, d J = 8.25 Hz, Ar–H), 8.19 (1H, d, thiazol-H). ¹³C-NMR (125 MHz, DMSO-d₆, ppm): 22.75 (CH₂–CH₂), 23.62 (COCH₃), 35.09 (2 CH₂), 48.27 (CH₂–NH), 65.63 (C), 98.75 (CH-thiazol), 121.20 (Ar-2CH), 123.27 (Ar-CH), 129.09 (Ar-CH), 130.07 (Ar–C), 130.87 (Ar-2CH), 131.11 (Ar–C), 131.56 (Ar–C), 131.82 (Ar-CH), 132.80 (Ar–C), 138.35 (Ar–C), 57 (Ar–C), 151.55 (thiazol-C), 160.96 (N=C-thiazol), 169.14 (CO). MS [M + 1]+: m/z 460 (100 %). HPLC: 98.4% purity.

2.1.11. N-[1-(((4-(2-Hydroxy-5-methoxyphenyl)-3-phenylthiazol-2(3H)-ylidene)amino)methyl) cyclopentyl]acetamide (4h)

¹H-NMR (500 MHz, DMSO-d₆) d (ppm): 1.06 (2H, br, CH₂), 1.28 (2H, br, CH₂), 1.62 (2H, br, CH₂), 1.86 (3H, s, CH₃), 2.30 (2H, br, CH₂), 3.79 (2H, s, CH₂–N), 3.85 and 3.88 (2H, d s, OCH₃), 5.77 and 5.88 (1H, d s, NH), 6.53–6.59 (2H, m, Ar–H), 7.07–7.15 (4H, t, Ar–H), 7.37–7.40 (2H, t, Ar–H), 7.98 (1H, s, thiazol-H). ¹³C-NMR (125 MHz, DMSO-d₆, ppm): 22.70 (CH₂–CH₂), 23.74 (COCH₃), 36.08 (2 CH₂), 48.07 (CH₂–N), 56.03 (OCH₃), 64.07 (C), 100.02 (CH-thiazol) 111.70 (Ar-CH), 114.02 (Ar-CH), 119.03 (Ar-CH), 121.95 (Ar-CH), 122.80 (Ar-2CH), 130.02 (Ar-2CH), 140.87 (Ar–C), 146.11 (thiazol-C), 149.92 (Ar-C-OH), 152.50 (Ar-C-OCH₃), 158.55 (N=C-thiazol), 170.14 (CO). MS [M + 1]+: m/z 437 (100 %). HPLC: 99.6% purity.

2.1.12. N-[1-(((4-(3-Nitrophenyl)-3-phenylthiazol-2(3H)-ylidene)amino)methyl)cyclopentyl]acetamide (4i)

¹H-NMR (500 MHz, DMSO-d₆) d (ppm): 1.02 (2H, br, CH₂), 1.27 (2H, br, CH₂), 1.59 (2H, br, CH₂), 1.81 (3H, s, CH₃), 2.25 (2H, br, CH₂), 4.29 (2H, s, CH–N), 6.1 (1H, s, NH), 7.28 (1H, s, Ar–H), 7.43–7.45 (3H, t, Ar–H), 7.74–7.78 (4H, m, Ar–H), 8.32 (1H, s, thiazol-H), 8.37 (1H, d, Ar–H). ¹³C-NMR (125 MHz, DMSO-d₆, ppm): 22.72 (CH₂–CH₂), 23.42 (COCH₃), 35.39 (2 CH₂), 49.21 (CH₂–NH), 65.61 (C), 99.85 (CH-thiazol), 120.02 (Ar-CH), 121.90 (Ar-CH), 123.00 (Ar-2CH), 123.01 (Ar-CH), 129.54 (Ar-2CH), 130.07 (Ar–C), 130.87 (Ar-CH), 134.11 (Ar-CH), 135.53 (Ar–C), 140.02 (Ar–C), 147.00 (Ar–C)), 151.55 (thiazol-C), 157.08 (N=C-thiazol), 160.96 (N=C-thiazol), 169.14 (CO). MS [M + 1]+: m/z 437 (100 %). HPLC: 99.7% purity.

2.2. 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]. The reagents used in the enzymatic assay including Ampliflu™ Red (10-acetyl-3,7-dihydroxyphenoxazine), peroxidase from horseradish, hMAO-A, hMAO-B, H₂O₂, 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.

2.3. Enzyme Kinetic Studies

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

2.4. 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). The method was carried out in the same way as we have reported in our previous studies [15–18].

2.5. Molecular Docking Studies

A structure-based in silico procedure was applied to discover the binding modes of compound 4b hMAO-B enzyme active site. The 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). The docking procedure was applied as previously described by our research group [16].

3. Result and Discussion

3.1. Chemistry

The synthesis of thiazoline derivatives (4a-4i) was carried out according to the steps shown in Scheme 1.

Scheme 1: Synthesis pathway of targeted compounds.

N-(1-cyanocyclopentyl)acetamide (1) was synthesized via the method of Reihlen, Hessling, Hühn, and Weinbrenner [14]. The reduction of nitriles (1) gives N-[(1-aminomethyl)cyclopentyl]acetamide (2) by nickel Raney [20]. Secondly, N-[(1-((3-phenylthioureido)methyl)cyclopentyl]acetamide (3) was synthesized via the reaction of compound 2 with phenyl isothiocyanates [21] (Scheme 1). The ring closure reaction of the intermediate compound (3) with phenacyl bromides afforded the title compounds (4a-4i) (Scheme 1 and Table 1). The structures of the compounds were confirmed by ¹H-NMR, ¹³C-NMR, mass spectroscopy, and elemental analysis.

Table 1: Newly synthesized compounds (4a-4i).

Compounds
R₁
R₂
R₃
R₄
MW

4a
H
H
CH₃
H
405.19

4b
H
H
OCH₃
H
421.18

4c
H
H
NO₂
H
436.16

4d
H
H
F
H
409.16

4e
H
H
Cl
H
425.13

4f
H
H
Br
H
469.08

4g
H
Cl
Cl
H
459.09

4h
OH
H
H
OCH₃
437.18

4i
H
NO₂
H
H
436.16

In the IR spectra of compounds 4a-4i, C=N and C=C stretching vibrations were observed in the region 1645–1470 cm⁻¹. The aromatic C–H stretching vibrations gave rise to bands at 3120–3010 cm⁻¹.

In the ¹H-NMR spectra, the protons corresponding to cyclopentyl, resonated as multiplets at d 0.78–0.85 ppm, 1.34–1.40 ppm, 1.56–1.65 ppm, and 2.25–2.35 ppm, and CH₂–N protons appeared as singlet at d 4.09 ppm. The protons of acetamide appeared as a singlet at d 5.80 ppm. All the other aromatic and aliphatic protons were observed at expected regions.

¹³C-NMR chemical shift values of the carbon atoms at 22.60–22.85 and 34.50–36.10 ppm (cyclopentyl CH₂), 23.60–23.90 ppm (COCH₃), 47.60–48.65 ppm (CH₂–NH), 159.10–161.45 ppm (N=C-thiazol), and 169.05–170.15 ppm (CO) corroborate the compounds deduced from the ¹H-NMR data. All the other aromatic carbon atoms were observed at expected areas.

The 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.

3.2. 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 H₂O₂ in a horseradish peroxidase-coupled reaction using Amplex Red reagent, was used. The 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, IC₅₀ graphs, obtained from GraphPad “Prism” software (version 5.0), of these selected compounds can be observed in Figures 1 and 2.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

Figure 1: IC₅₀ (μM) of the selected compounds (4a, 4b, 4c, 4d, 4e, 4f, 4g, and 4i) and control drugs against MAO-A enzyme. (a) Moclobemide (6.06 μM); (b) 4a (7.06 μM); (c) 4b (6.56 μM); (d) 4c (6.78 μM); (e) 4d (10.98 μM); (f) 4e (13.08 μM); (g) 4f (12.41 μM); (h) 4g (13.45 μM); (i) 4i (7.09 μM).

(a)

(b)

(c)

(d)

Figure 2: IC₅₀ (μM) of the selected compounds (4a, 4b, and 4c) and control drugs against MAO-B enzyme. (a) Selegiline (0.038 μM); (b) 4a (0.42 μM); (c) 4b (0.36 μM); (d) 4c (0.69 μM).

All compounds except for compound 4h showed high percent inhibition against hMAO-A in the first step of activity studies. Among these compounds, 4a, 4b, 4c, and 4i exhibited similar activity with standard drug moclobemide (IC₅₀ = 6.061 ± 0.262 µM) with IC₅₀ values of 7.06 ± 0.18 µM, 6.56 ± 0.20 µM, 6.78 ± 0.15 µM, and 7.09 ± 0.17 µM, respectively. When analyzing hMAO-B activity results, none of the compounds except for compounds 4a, 4b, and 4c displayed remarkable activity. These compounds, 4a, 4b, and 4c, displayed hMAO-B inhibition profile with IC₅₀ 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 IC₅₀ values on these enzymes are considered.

3.3. Kinetic Studies of Enzyme Inhibition

The mechanism of hMAO-B inhibition was investigated by enzyme kinetics, following a similar procedure to the MAO inhibition assay. The 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 IC₅₀/2, IC₅₀, and 2xIC₅₀. In each case, the initial velocity measurements were gained at different substrate (tyramine) concentrations ranging from 20 µM to 0.625 µM. The Ki (intercept on the x-axis) values of compound 4b were determined from the secondary plot of the Km/Vmax (slope) versus varying concentrations. The graphical analysis of steady-state inhibition data for compound 4b is shown in Figure 3.

(a)

(b)

Figure 3: (a) Lineweaver–Burk plots for the inhibition of hMAO-B by compound 4b. [S], substrate concentration (μM); V, reaction velocity (nmol/min/mg protein). Inhibitor concentrations are shown at the left. Vmax values from 2xIC₅₀ to control: 172.565, 243.902, 277.778, and 525.120 (nmol/min/mg protein). Km value of the noncompetitive inhibition: 0.443 ± 0.018 (µM). (b) Secondary plot for calculation of steady-state inhibition constant (Ki) of compound 4b. Ki was calculated as 0.346 μM.

Based on the type of interaction with the enzyme, inhibitor binding can be classified as either reversible or irreversible. The 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. Therefore, 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. Ki 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. These 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.

3.4. Cytotoxicity Test

The cytotoxic activity of compounds 4a, 4b, and 4c was evaluated against healthy NIH/3T3 mouse embryonic fibroblast cell line (ATCC CRL1658), which is suggested for preliminary cytotoxicity screening by ISO (10993-5, 2009) [23]. The IC₅₀ values of the compounds are represented in Table 2. Compounds 4a, 4b, and 4c displayed higher IC₅₀ values against NIH/3T3 cells than their IC₅₀ values (0.42, 0.36, and 0.69 µM, resp.) against hMAO-B. This result reveals that compounds 4a, 4b, and 4c are not cytotoxic at their effective concentration against hMAO-B.

Table 2: Cytotoxic activity of compounds 4a, 4b, and 4c against NIH/3T3 cell line.

Compound
IC50 (µM)

4a
>1000

4b
>1000

4c
>1000

3.5. 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), was used (PDB ID: 2V5Z) [19]. The docking pose of compound 4b is presented in Figure 4.

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

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. This 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. This moiety creates these hydrogen bonds with amino groups of Ser59 and Tyr60. The 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. This might be referred to the steric hindrance in general which may increase as the distance between two substituents decreases [24].

4. Conclusion

Another class of N-[1-(((3-Phenyl-4-(p-tolyl)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. The type of inhibition of this compound was found as reversible and noncompetitive. In addition, this active compound was not cytotoxic at MAO inhibitors IC₅₀ 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. The 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

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

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Acknowledgments

This study was financially supported by Anadolu University Scientific Projects Fund, Project no. 1805S189.

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