Novel 1-(2-pyrimidin-2-yl)piperazine derivatives as selective monoamine oxidase (MAO)-A inhibitors

Abstract In the present study, a new series of 2-[4-(pyrimidin-2-yl)piperazin-1-yl]-2-oxoethyl 4-substituted piperazine-1-carbodithioate derivatives (2a-n) were synthesized and screened for their monoamine oxidase A and B inhibitory activity. The structures of compounds were elucidated using spectroscopic methods and some physicochemical properties of new compounds were predicted using Molinspiration and MolSoft programs. Compounds 2-[4-(pyrimidin-2-yl)piperazin-1-yl]-2-oxoethyl 4-(4-nitrophenyl)piperazine-1-carbodithioate (2j) and 2-[4-(pyrimidin-2-yl)piperazin-1-yl]-2-oxoethyl 4-benzhydrylpiperazine-1-carbodithioate (2m) exhibited selective MAO-A inhibitory activity with IC50 = 23.10, 24.14 µM, respectively. Some of the biological results were found in accordance with the obtained in silico data based on Lipinski’s fule of five.


Introduction
By definition depression means a serious and common disorder including symptoms like feeling of sadness, hopelessness, weight loss or gain, tiredness, changes in sleeping routine and thinking of suicide 1 . Depression is a major public health problem, and the fourth cause of the global burden of disease 2 . In the history of development of antidepressants, tricyclic antidepressants and monoamine oxidase inhibitors (MAOIs) were the first-generation antidepressants introduced in the late 1950s 3,4 . Selective serotonin reuptake inhibitors (SSRIs) noradrenaline reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors (SNRIs) and dopaminenoradrenaline reuptake inhibitors (DNRIs) were improved as the new generation of antidepressants with fewer adverse effects than traditional antidepressants 5,6 . Despite many developments in the field of antidepressants, the clinical use of currently used drugs were restricted as a result of various adverse effects and a response in less than 50% of patients 7,8 . Thereby search for new class of antidepressant agents more effective, safe and with a more advantageous benefit-risk balance is an urgent need.
Aminergic neurotransmitters such as norepinephrine and serotonin (5-HT) became as key aspects in the therapy of depression. MAOIs are one of the most widely used groups of antidepressants agents regulating the metabolism of serotonin and norepinephrine. Monoamine oxidase enzymes (MAOs) control the concentration of neurotransmitters and intracellular amines in brain and peripheral tissues through catalyzing the oxidative deamination of them 9 . MAOs are localized in outer mitochondrial membrane's of neuronal, glial, and other cells and contain the covalently linked cofactor flavin adenine dinucleotide (FAD) 10 . MAO-A mostly select serotonin, adrenaline and noradrenaline as subtrate, whereas MAO-B metabolize phenylethylamine and benzylamine 11 . Besides, tyramine and dopamine were metabolized by both forms of the enzyme 12 . Two groups of MAO enzymes namely MAO-A and MAO-B were classified on the basis of their affinities to inhibitors, specificities to substrates and tissue/cellular distribution 13,14 . MAO-A inhibitors are clinically used in the treatment of depression and anxiety 15 while MAO-B inhibitors are mostly used in Parkinson's and Alzheimer's diseases 14 .
The azapirones (buspirone, gepirone, ipsapirone, tandospirone and zalospirone, Figure 1) have been known to exhibit anxiolytic and antidepressant activities 16 . 1-(2-Pyrimidinyl)piperazinyl (1-PP) pharmacophore widely exists in the azapirones structure and it is an active metabolite of azapirones 17 . The most notable azapirone, buspirone, is a selective 5-HT1A agonist that is used clinically as an antidepressant drug [18][19][20] . In contrast to diazepam, buspirone does not cause sedation or muscle relaxation 21 . In a study, it was reported that the connection of 1-PP pharmacophores with appropriate side chains may eliminate neuroleptic-like side effects thus produce nondopaminergic agents 22 . Furthermore, between 5-HT1A receptor ligands, long-chain arylpiperazines such as 1-PP comprise one of the most important classes 23 . Due to the fact that there are some studies support that compounds bearing 1-PP moiety possess antidepressant, anxiolytic and antipsychotic activity 16,22,[24][25][26] . In vivo produced 1-PP moiety is estimated to be partly in charge of the efficacy of the azapirones in the therapy of depression 27,28 . Piperazine derivatives have been also known to exhibit antidepressant activity 29,30 .

Materials and methods
Melting points were determined by MP90 digital melting point apparatus (Mettler Toledo, Columbus, OH) and were uncorrected. Spectroscopic data were recorded on the following instruments: a Bruker Tensor 27 IR spectrophotometer; a 1 H NMR (nuclear magnetic resonance) Bruker DPX-500 FT-NMR spectrometer, 13  A mixture of 2-chloro-N-[4-(2-pyrimidinyl)piperazine]acetamide and potassium salt of appropriate piperazine dithiocarbamate derivative (10 mmol) was stirred in acetone at room temperature with the presence of potassium carbonate (10 mmol). After the reaction finished, controlled with TLC, the reaction mixture was poured into ice-water and the precipitated portion was filtered and crystallised from ethanol to gain the final products.

MAO activity assay
Enzyme activity assay was performed according to the modified fluorimetric method reported by Matsumoto et al. 31 The entire materials used in enzymatic assay were purchased from Sigma-

Enzyme kinetics
The MAO-A enzyme kinetic of the most active compound 2j was studied. The nature of MAO-A inhibition, caused by this compound, was investigated by the graphical analysis of steady-state inhibition data. Lineweaver-Burk plots identified the compound 2j as a mix-typed inhibitor, due to the different intercepts on both the y-and x-axes ( Figure 2). The values of K m and V max were calculated by nonlinear regression according to literature 31 and found as 48.37 and 5.34, respectively.

Molecular properties and drug-likeness score
Molecular properties identify some physicochemical parameters of a molecule which help to evaluate whether a compound could be a potential therapeutic agent. Also, oral bioavailability of a molecule is known to play an important role for the development of bioactive derivatives 32 . Therefore, some physicochemical properties and drug-likeness score of the synthesized compounds were calculated using Molinspiration 33 and MolSoft 34 softwares and the obtained data were represented in Table 1. The computational study for prediction of ADME properties of the molecules was performed by determination of log P, topological polar surface area (TPSA), molecular weight (MW), number of hydrogen donors (nON) and acceptors (nOHNH), number of rotable bonds (nrotb) and volume. The absorption percentage (% ABS) of the compounds were also calculated using the formula % Absorption ¼ 109 À (0.345 Â TPSA) placing the predicted TPSA values 35 . Calculated % ABS of the compounds (2a-n) were found between the range of 73.94-89.75%. Good intestinal absorption, reduced molecular flexibility (measured by the number of rotatable bonds), low polar surface area or total hydrogen bond count (sum of donors and acceptors) are important molecular descriptors for high oral bioavailability 36 . Considering Lipinski's rule of five 37 , all synthesized compounds have hydrogen bond donors and log P values smaller than five, hydrogen bond acceptors smaller than 10 and polar surface area smaller than 140. Besides, molecular weights of the compounds are in accordance with the value smaller than 500 dalton except compound 2m. According to the stated data, all compounds are in the specified values to be a potential drug with good physicochemical properties such as solubility, lipophilicity, flexibility and membrane permeability. Drug-likeness score is also assigned for all compounds and standard drugs according to MolSoft's chemical fingerprints mode consisted of 5K of marketed drugs from World Drug Index (positives) and 10K of carefully selected non-drug compounds (negatives). The values were found between À0.30 and 1.56 for synthesized compounds (2a-n) and 1.03-1.36 for standard drugs. According to all these data, two active compounds 2j bearing 4-nitrophenyl moiety and 2m bearing diphenylmethyl moiety did not correlate these informations. Compound 2j is in accordance with Lipinski's rule of five, but the predicted drug-likeness score of it is the lowest one, À0.30 among the others. Compound 2m possesses the highest drug-likeness score (1.56) which is also higher than standard drugs, although molecular weight of the compound  (MW ¼ 532) exceeds Lipinski's limit. However, compound 2m possesses the highest log P (4.00) due to bearing two aromatic rings that provides lipophilic character which is suitable to cross BBB (blood brain barrier) 38 .

Molecular docking
Docking studies were applied to discover and designate the assumed binding modes in MAO-A enzyme active site of the most potent compound 2j in the compound series and protein-ligand interactions analysis was performed using human MAO-A X-ray crystal structure complex with 7-methoxy-1-methyl-9H-b-carboline (harmine), retrieved from Protein Data Bank server (PDB ID: 2Z5X) (www.pdb.org). Docking calculations were performed with the program AutoDock Vina 39 . AutoDock Tools (ADT, Version 1.5.6) 40 were used to add polar hydrogen atoms and partial charges for protein and ligand, which were saved in pdbqt format. For docking studies initial protein was prepared. In the PDB crystallographic structure any co-crystallized solvent and the ligand were removed. Docking procedure was carried out following the same protocol described previously 41

Monoamine oxidase inhibitory activity
The synthesized compounds (2a-n) were investigated for their MAO-A and MAO-B inhibitory activity by an in vitro fluorimetric method. The fundamental of the activity determination is based on the ability of both MAO-A and MAO-B enzymes to metabolize non-fluorescent kynuramine, which is a suitable substrate for both isozymes, to the fluorescent product 4-hydroxyquinoline. MAOinhibitory effect of the test compounds was correlated to the amount of 4-hydroxyquinoline formed. DMSO was considered as control (100% activity). In the assay as reference drug, moclobemide and selegiline were used. The inhibitory activity results are shown in Tables 2 and 3. In general, when the results were analyzed, it was observed that the synthesized compounds are more potent against MAO-A enzyme as regards MAO-B enzyme. The compounds 2i, 2j and 2m displayed over 50% inhibition against MAO-A enzyme at 10 À3 M concentration. The compounds 2f, 2j, 2l and 2m exhibited significant inhibition profile against MAO-B enzyme at 10 À3 M concentration. When the percentage inhibition rates at 10 À4 M concentration were compared, the compounds 2j and 2m showed over 50% inhibition against MAO-A enzyme. So it is understood that these derivatives are selective MAO-A enzyme inhibitors.
Due to their inhibition potency (>50%) at 10 À3 and 10 À4 M, the compounds 2j and 2m were studied in more concentrations (10 À5 M to 10 À9 M) so as to calculate IC 50 values (Figure 4). According to enzyme inhibition profiles, compound 2j is the most active derivative owing to its MAO-A inhibiton with an IC 50 value of 23.10 mM.

Enzyme kinetic studies
The same materials were used in MAO enzyme kinetic assay. The compound 2e was prepared at IC 50 concentration and then added to the wells (100 mL/well). Stock solution (25 mM) of kynuramine was diluted to final concentrations of 40, 20, 10, 5, 2.5 and 1.25 mM and then added to the wells (50 mL/well). After MAO-A enzyme was added to the plate (50 mL/well), incubation period at 37 C for 20 min was started. The plate was read at 310/340 nm excitation/emission wavelength pair 31 . Control measurement without inhibitor was also determined simultaneously. The results were analyzed in Lineweaver-Burk plots using Microsoft Office Excel 2013.

Predictions of in silico physicochemical parameters
The physicochemical properties and drug-likeness score of the synthesized compounds were calculated using Molinspiration-Calculation of Molecular Properties and Bioactivity Score toolkit 33 and MolSoft-Drug-Likeness and molecular property prediction toolkit 34 .

Molecular docking
Compound 2j was docked into the active site of MAO-A using the program AutoDock Vina 39 in order to structurally understand the interactions with this target together with the inhibitor profile. Due to inhibition, data were determined on human MAOs, docking studies were run on the human model of the MAO-A. The 3D structure for human MAO-A retrieved from the Protein Data Bank server (PDB ID: 2Z5X) (www.rcsb.org) 41 . The best docking pose, showing residues in the active site, is seen in Figure 5. The docking study suggested that the compound 2j is very compatible with the active site. The active region is consisting of these amino acids: Ile180, Asn181, Phe208, Gln215, Leu337 and Phe352. The carbonyl group in the structure settles down hydrogen bond with the amino group of Gln215. The nitrogen atom of piperazine moiety, near the pyrimidine, creates formation cation-p interaction with Phe208, whereas the other piperazine moiety does the same interaction with Leu337. The oxygen atoms of the nitro group are very significant in terms of polar interactions. The docking poses reveals that the nitro substituent interacts with Ile180 and Phe352 by formation of hydrogen bond. Also, it is thought that van der Waals interactions, between the compound 2j and the active region of the enzyme, provide more steady binding.   inhibitory activities. Compounds 2j bearing 4-nitrophenyl moiety and 2m bearing diphenylmethyl moiety has exhibited the highest MAO-A inhibitory activity. Some physicochemical properties and drug-likeness scores of the final compounds (2a-n) were also predicted using online Molinspiration and MolSoft programs. The obtained biological data was found to compatible to drug-likeness score (the highest) for compound 2m. The calculated physicochemical properties were identified in the range determined by Lipinski's rule of five.