Synthesis, in vitro enzyme activity and molecular docking studies of new benzylamine-sulfonamide derivatives as selective MAO-B inhibitors

Abstract Many studies have been conducted on the selective inhibition of human monoamine oxidase B (hMAO-B) enzyme using benzylamine-sulphonamide derivatives. Using various chemical modifications on BB-4h, which was reported previously by our team and showed a significant level of MAO-B inhibition, novel benzylamine-sulphonamide derivatives were designed, synthesised, and their MAO inhibition potentials were evaluated. Among the tested derivatives, compounds 4i and 4t achieved IC50 values of 0.041 ± 0.001 µM and 0.065 ± 0.002 µM, respectively. The mechanism of hMAO-B inhibition by compounds 4i and 4t was studied using Lineweaver–Burk plot. The nature of inhibition was also determined to be non-competitive. Cytotoxicity tests were conducted and compounds 4i and 4t were found to be non-toxic. Molecular docking studies were also carried out for compound 4i, which was found as the most potent agent, within hMAO-B catalytic site.


Introduction
Monoamine oxidase (MAO) is the enzyme responsible for catalysing the oxidative deamination of intracellular amines and monoamine neurotransmitters, which contributes to the regulation of the concentrations of these chemicals in the brain and in peripheral tissues 1,2 . MAOs, which are flavin adenine dinucleotide (FAD)containing enzymes, are localised in the outer mitochondrial membranes of glial, neuronal, and other types of cells; they are particularly abundant in the liver and the brain. MAOs have two different isoforms, MAO-A and MAO-B, with 70% homology. The genes that code for the two isoforms are linked in opposite orientation on the X chromosome, differ in the specificity of their substrates and the selectivity of their inhibitors 3 . For example, MAO-B is selectively inhibited by selegiline, and utilises phenylethylamine and benzylamine as substrates. On the contrary, MAO-A is selectively inhibited by clorgiline, and utilises adrenaline, noradrenaline and serotonin as substrates. However, both isoforms may also act on the same substrates such as dopamine and tyramine 4 .
MAOs are of extensive pharmacological importance due to their roles in the metabolism of certain neurotransmitters. Selective MAO-A inhibitors are used clinically as antidepressants and anxiolytics, while MAO-B inhibitors are used to reduce the progression of Parkinson's disease, and manage symptoms related to Alzheimer's disease 5 . Moreover, MAO-catalyzed deamination reactions produce hydrogen peroxide as a byproduct, which may typically contribute to the oxidative stress state. In this context, MAO inhibitors are thought to act as neuroprotective agents in degenerative processes 6,7 .
Parkinson's disease (PD), which affects more than 5 million people worldwide, is the second most common disease after Alzheimer's disease. Considering the loss of nigrostriatal dopaminergic cells as a pathological hallmark of PD, therapeutic strategies have been established to boost the levels of dopamine in the brain [8][9][10] . Although dopamine is metabolised by both MAO isoforms, MAO-B is the more common isoform present in the basal ganglia and is therefore responsible for dopamine metabolism in this region 11 .
Currently, the Protein Data Bank contains more than 40 crystal structures of MAO (most of them MAO-B) in complex with different reversible and irreversible inhibitors, as observed through X-ray diffraction at refinements of 3.0-1.7 Å. Additionally, MAO-A shows a markedly different monopartite cavity ($550 Å) compared to the bipartite cavity (290 Å) found in MAO-B. The "aromatic cage"-a hydrophobic binding pocket containing the FAD cofactor-is considered the active region 4,7 . The FAD is covalently attached to the cysteine residue of the protein, and the 8a-thioether linkage provides this connection. It is believed that the catalytic activity of the two tyrosine residues, Tyr398 and Tyr435, found in the hMAO-B structure is due to the polarisation of the amine N pair of the substrate 12 . Therefore, in designing a new inhibitor compound, it is desirable to have the amine group in the structure.
In light of the above-mentioned information, this study was conducted to develop new and potent MAO inhibitors. It has been thought that the proven MAO inhibition of benzylamine derivatives may provide MAO-B inhibitory activity due to strong interactions on the enzyme active side [13][14][15] . In our recent study 2 , we reported a new benzothiazole-benzylamine hybrid compound, 2-((5-chlorobenzothiazol-2-yl)thio)-N-(4-fluorophenyl)-N-(3-nitrobenzyl)acetamide (BB-4h), as shown in Figure 1, with significant IC 50 (2.95 ± 0.09 mM) against MAO-B. Moreover, sulphonamides and various heterocyclic ring systems have been identified as inhibitors of MAO in previous studies 3, [16][17][18][19] . Therefore, we considered the compound (BB-4h) as a lead compound, and we performed some modifications, such as removing nitro and fluoro groups, introducing a sulphonamide group, and changing heterocyclic rings in order to improve biological activity. Subsequently, 20 benzylamine derivatives containing a sulphonamide moiety and different heterocyclic ring moieties were synthesised, and their MAO inhibitory activities were evaluated in this study. 4-((4-methylbenzylidene)amino)benzenesulfonamide (1b) (3.507 g, 0.0128 mol) was dissolved in MeOH. Sodium borohydride was added to the reaction medium in portions of 0.5 moles. It was observed by controlling the end of the reaction with TLC that the reaction was complete when the total amount of sodium borohydride reached 1.5 moles. After completion of the reaction, the MeOH was removed by a rotavapor. The precipitated product was washed with deionised water to remove the excess of the sodium borohydride, dried, and recrystallized from EtOH.

MAO-A and MAO-B inhibition assay
Ampliflu TM Red (10-Acetyl-3,7-dihydroxyphenoxazine), hMAO-A, hMAO-B, peroxidase from horseradish, tyramine hydrochloride, H 2 O 2 , clorgiline and selegiline were acquired from Sigma-Aldrich (Steinheim, Germany) and retained under the proposed conditions by supplier. A Biotek Precision XS robotic system (USA) was used for all pipetting operations. Measurements were performed with the use of BioTek-Synergy H1 microplate reader (USA) based upon the fluorescence generated (excitation, 535 nm, emission, 587 nm) over a 30 min period, in which the fluorescence increased linearly.
Enzymatic assay was performed according to recent method pronounced by our research group 17,[20][21][22] . Control, blank and all concentrations of obtained compounds were tested in quadruplicate and inhibition percent was calculated with following equation: FCt 2 : Fluorescence of a control well measured at t 2 time, FCt 1 : Fluorescence of a control well measured at t 2 time, FIt 2 : Fluorescence of an inhibitor well measured at t 2 time, FIt 1 : Fluorescence of an inhibitor well measured at t 1 time, The IC 50 values were calculated using a dose-response curve achieved by plotting the percentage inhibition versus the log concentration using GraphPad 'PRISM' software (version 5.0). The results were showed as mean ± SD.

Enzyme kinetic studies
The same materials were used in the MAO inhibition assay. The most active compounds 4i and 4t determined according to the result of the MAO inhibition assay were experienced in three different concentrations of IC 50 /2, IC 50 and 2(IC 50 ) in accordance with the assay assigned in our final study. 17,[20][21][22] . All processes were evaluated in quadruplicate. The results were analysed as Lineweaver-Burk plots by means of Microsoft Office Excel 2013. The V max values of the Lineweaver-Burk plots were replotted versus the inhibitor concentration, and the K i values were determined from the x-axis intercept as ÀK i .

Cytotoxicity assay
The NIH/3T3 mouse embryonic fibroblast cell line (ATCC V R CRL-1658 TM , London, UK) was used for cytotoxicity assays. The incubation period of NIH/3T3 cells was based on the supplier's recommendation. NIH/3T3 cells were seeded at 1 Â 10 4 cells into each well of 96-well plates. MTT assay was carried out in accordance with the standards previously described manner 23,24 . The compounds were tested between 1 and 0.000316 mM concentrations. Inhibition % for each concentration was calculated according to the following formula and IC 50 values were reported by plotting the% inhibition dose response curve against the compound concentrations tested. [23][24][25] .

Prediction of ADME parameters and BBB permeability
Physicochemical parameters were performed with the use of QikProp 4.8 software 26 to predict pharmacokinetic profiles and BBB permeability of obtained compounds (4a-4u).

Molecular docking
A structure based in silico procedure was applied to discover the binding modes of compound 4i to hMAO-B enzyme active site. The crystal structures of hMAO-B (PDB ID: 2V5Z) 27 , which was crystallised with safinamide, were retrieved from the Protein Data Bank server (www.pdb.org). The structures of ligands were built using the Schr€ odinger Maestro 28 interface and then were submitted to the Protein Preparation Wizard protocol of the Schr€ odinger Suite 2016 Update 2 29 . The ligands were prepared by the LigPrep 3.8 30 to assign the protonation states at pH 7.4 ± 1.0 and the atom types, correctly. Bond orders were assigned, and hydrogen atoms were added to the structures. The grid generation was formed using Glide 7.1 31 . The grid box with dimensions of 20 Å Â 20 Å Â 20 Å was centred in the vicinity of the flavin (FAD) N5 atom on the catalytic site of the protein to cover all binding sites and neighbouring residues [32][33][34] . Flexible docking runs were performed with single precision docking mode (SP).
The synthesised compounds were elucidated by instrumental analyses such as infra-red spectroscopy (IR), mass spectrometry (MS), and nuclear magnetic resonance (NMR). The N-H bond of the sulphonamide group appeared as IR bands between 3238 and 3483 cm À1 , and as a singlet between 7.30 and 7.60 ppm on the 1 H-NMR spectrum. The presence of the carbonyl group was shown by IR bands between 1645 cm À1 and 1666 cm À1 , and a 13 C-NMR peak over 160 ppm. The CH 2 group bound to the nitrogen atom gave a singlet 1 H-NMR peak around 4.90 ppm, and a 13 C-NMR peak over 50 ppm. The other CH 2 group between the carbonyl and sulphur groups was recorded in 1 H-NMR peak around 4.20 ppm and a 13 C-NMR peak between 33.4 and 38.5 ppm. The carbons of aromatic groups were observed from 105.0 to 166.7 ppm in the 13 C-NMR spectrum, and the protons of the same groups were between 6.90 and 8.51 ppm in the 1 H-NMR spectrum. In mass spectroscopy, the masses were found to differ by at most 5 ppm from the expected masses.
The assay was carried out in two steps. The first step was carried out using 10 À3 and 10 À4 M concentrations of all synthesised compounds and reference agents, namely selegiline and clorgiline. The enzyme activity results of first step are presented in Table 1. Then, the selected compounds that displayed more than 50% inhibitory activity at 10 À3 and 10 À4 M concentrations were further tested, along with reference agents, at concentrations of 10 À5 to 10 À9 M. The IC 50 values of the test compounds and reference agents are presented in Table 2.
According to the enzyme inhibition results, none of the synthesised compounds showed a significant activity against hMAO-A enzyme. All of the obtained compounds displayed selective inhibition on hMAO-B. At 1 0 À3 M concentration, all of the compounds showed more than 50% inhibitory activity. Compounds 4b, 4d, 4f, 4i and 4t could pass the second step of enzyme activity assay and the IC 50 values of them were calculated by performing enzyme inhibition study at 10 À5 -10 À9 M concentration. The most active compounds, 4i and 4t, exhibited IC 50 values of 0.041 ± 0.001 mM and 0.065 ± 0.002 mM, respectively, against hMAO-B, while the reference agent, selegiline, had an IC 50 of 0.037 ± 0.001 mM.
These findings from the screening of inhibitory activities against hMAO-B revealed that the compounds containing 5-chlorobenzothiazole exhibited more potent inhibitory activity than the other obtained compounds as in the previously synthesised and reported BB-4h derivative, which has a 5-chlorobenzothiazole ring. Moreover, the increased inhibitory activity of the synthesised compounds, compared to that of BB-4h, is likely due to the contribution of the sulphonamide group, which displaced the fluorine group, and the removal of the nitro group from the structure.

Kinetic studies of enzyme inhibition
Enzyme kinetics studies were performed to determine the mechanism of hMAO-B inhibition by using a procedure similar to that of the MAO inhibition assay. Compounds 4i and 4t, which were found to be the most potent agents, were included in these studies. In order to estimate the type of inhibition of these compounds, linear Lineweaver-Burk graphs were used. Substrate velocity curves in the absence and presence of compounds 4i and

Compounds
Ar R 4t were recorded. These compounds were prepared at concentrations of IC 50 /2, IC 50 , and 2(IC 50 ) for enzyme kinetic studies. In each case, the initial velocity measurements were obtained at different substrate (tyramine) concentrations ranging from 20 lM to 0.625 lM. The secondary plots of slope (K m /V max ) versus varying concentrations (0, IC 50 /2, IC 50 , and 2(IC 50 )) were created to calculate the K i (intercept on the x-axis) value of these compounds. The graphical analyses of steady-state inhibition data for compounds 4i and 4t are shown in Figures 2 and 3. The type of inhibition can be determined as either reversible or irreversible by using the Lineweaver-Burk plots. The reversible inhibition type can be classified as mixed-type, uncompetitive, competitive, or non-competitive 17,[20][21][22] . According to Lineweaver-Burk plots, a graph that shows parallel lines without any cross-overs is observed in the uncompetitive type of inhibition. For mixed-type inhibition, a graph with lines that do not intersect at the x-axis or the y-axis is formed. Competitive inhibition is seen if the lines intersect on the y-axis, and the slopes and x-intercepts are different. On the contrary, non-competitive inhibition has the opposite result: the plotted lines have the same xintercept but there are diverse slopes and y-intercepts. Therefore, as shown in Figures 2 and 3, compounds 4i and 4t are reversible and non-competitive inhibitors with similar inhibition features as the substrates. K i values for compounds 4i and 4t were calculated as 0.036 and 0.055 lM, respectively, for the inhibition of hMAO-B.
Irreversible enzymatic inhibition involves covalent interactions between the substrate and the enzyme. In contrast, there are non-covalent interactions such as hydrophobic interactions, ionic bonds, and hydrogen bonds involved in reversible inhibition. In this type of inhibition, inhibitors bind to the enzymes without forming any chemical bonds; thus, the enzyme-inhibitor complex could be separated quickly because non-covalent interactions can form rapidly and break easily. Furthermore, reversible inhibitors have a lower risk of side effects than irreversible inhibitors owing to their non-covalent binding ability. Consequently, compounds 4i and 4t, whose inhibition types were determined to be reversible and non-competitive, have pharmaceutical importance in contrast to irreversible hydrazine-type MAO inhibitors.

Cytotoxicity
Compounds 4i and 4t displayed potent hMAO-B inhibition profiles and were further tested for toxicity using the MTT assay in the NIH3T3 cell line; the IC 50 values of compounds 4i and 4t are shown in Table 3. Both compounds showed an IC 50 value of >1000 mM against NIH3T3 cells, which was significantly higher  Consequently, compounds 4i and 4t were found to be non-cytotoxic at their effective concentrations against hMAO-B. This result further increases the biological importance of the compounds.

Prediction of ADME parameters and BBB permeability
Intrinsic pharmacological activity and low toxicological effects are not sufficient for a compound to become a drug nominee 35 . Most new drug nominees fail in clinical trials due to their reduced absorption, distribution, metabolism, and excretion (ADME) properties. These late-stage failures result in increased drug development costs 36 . The ability to identify problematic issues early can dramatically reduce the amount of wasted time and funds, and can streamline the overall development process. Therefore, the pharmacokinetic properties of new drug candidates are very important, and it is beneficial to assess them as early as possible in the drug development process 37 . ADME estimation can be used to focus on precursor compound optimisation thereby improving the preferred properties of a compound 38 . Predictions of ADME parameters of the obtained compounds (4a-4u) were performed using QikProp 4.8 software 26 . The violations of Jorgensen's "Rule of Three" 39 and Lipinski's rule of five 40 , which assess the ADME properties of new drug nominees, are crucial for the optimisation of a biologically active compound. The calculated ADME parameters, including molecular weight (MW), number of rotatable bonds (RB), dipole moment (DM), molecular volume (MV), number of hydrogen donors (DHB), number of hydrogen acceptors (AHB), polar surface area (PSA), octanol/water partition coefficient (log P), aqueous solubility (log S), apparent Caco-2 cell permeability (PCaco), number of likely primer metabolic reactions (PM), percent of human oral absorption (%HOA), and the violations of the rules of three (VRT) and five (VRF) are presented in Table 4. In keeping with Jorgensen's "Rule of Three" and Lipinski's rule of five, the obtained compounds (4a-4u) are in accordance with the set parameters as they did not cause more than one violation. Drugs that specifically target the CNS must first pass the blood-brain barrier (BBB). Although the BBB is protective in nature, the use of drug candidates with CNS effects in a clinical setting is unlikely if such drug molecules are unable to penetrate it. Therefore, this feature should be examined earlier on in the drug discovery process. Accordingly, predicting the BBB permeability of new compounds is of great significance 41 . Thereby, the BBB permeability of the obtained compounds (4a-4u) was also evaluated using QikProp 4.8 software 26 . Brain/blood partition coefficient (logBB) and apparent MDCK cell permeability (PMDCK) were calculated for this purpose. In keeping with the software estimates, the PMDCK values of <25 and >500 nm/sec are advised as poor and great for non-active transport of compounds. In order to assess for a compound's capacity to pass through the BBB, logBB is the other significant parameter to consider, with recommended values between À3 and þ1.2. The PMDCK and logBB values of the synthesised compounds are within the advised ranges as shown in Table 4. Thus, it can be postulated that the obtained compounds are capable of crossing the BBB, which is crucial for CNS-associated drugs.
Considering the results of the ADME and BBB permeability studies, the synthesised compounds have pharmacokinetic profiles that may be appropriate for clinical use.

Molecular docking
As observed in the MAO inhibition assay studies, compounds 4i and 4t were found to be the most active derivatives in the hMAO-B enzyme inhibition series. Furthermore, compound 4i was determined to be the most potent agent with an IC 50 value of 0.041 ± 0.001 mM; hence, docking studies were carried out to evaluate its inhibition capability in silico. Using the X-ray crystal structure of hMAO-B (PDB ID: 2V5Z) 27 , docking studies were performed and the binding modes of compound 4i were assigned. Also, this compound was subjected to the molecular docking procedure with the X-ray crystal structure of hMAO-A (PDB ID: 2Z5X) to compare its binding modes on hMAO-A and hMAO-B enzymes. Unfortunately, it was determined that compound 4i did not be settle down to the active site of hMAO-A enzyme (data not shown). Thus, no important and significant interactions were observed. Actually, this evidence is consistent with in vitro enzyme inhibition assay and supports the selective effect of compound 4i and the other derivatives in the series on hMAO-B enzyme.
The docking poses of this compound are presented in Figures  4-7. Compound 4i adequately binds to the amino acid residues lining the cavity of hMAO-B enzyme and is located very near the FAD cofactor. When analysing the docking poses of this compound, it is clear that there is a p-p interaction, formation of three hydrogen bonds, and formation of a halogen bond. Compound 4i has a sulphonamide group at the 4th position of the phenyl ring. This group is essential for polar interactions. The amino moiety of sulphonamide forms a hydrogen bond with the carbonyl of  Pro102. In addition, there is another hydrogen bond between the oxygen atom of sulphonamide and the amino group of Thr201. As mentioned above, one of the main structural modifications of BB-4b, as previously reported by our research group 2 , is the substitution of the fluorine atom with a sulphonamide group (Figure 1). All the detected interactions of the sulphonamide group prove that the structural modification of compound BB-4b is a suitable approach. The addition of the sulphonamide group made a positive contribution to the MAO enzyme inhibition profile. Another formation of hydrogen bond is observed between the carbonyl of the amide group in the structure and the amino group of Leu171. Compound 4i has a benzothiazole ring as a heterocyclic ring. The benzene on the benzothiazole ring interacts with the phenyl of Thr398. Interaction with the Thr398 amino acid is very important in terms of catalytic activity, and the binding of    inhibitor candidates in the substrate cavity of the MAO-B enzyme. This finding indicates that compound 4i binds very effectively to the MAO-B enzyme active site.
The main structural difference between compound 4i and the other compounds in the series is that it carries a chlorine atom at the 5th position of the benzothiazole ring. It is clearly observed in Figure 5 that this halogen atom establishes a halogen bond with the hydrogen of the hydroxyl group of Tyr188. This additional interaction ensures that it binds more strongly to the active site. Furthermore, all these interactions explain why compound 4i exhibits a stronger inhibition profile than the other compounds.
In order to analyse the contribution of van der Waals and electrostatic interactions in binding to the enzyme active site, docking studies were performed using Glide, according to the Per-Residue Interaction panel. Figures 6 and 7 present the van der Waals and electrostatic interactions of compound 4i. As shown in the figures, this compound has favourable van der Waals interactions with Leu88, Phe99, Phe103, Pro104, Tyr119, Leu167, Phe168, Leu171, Cys172, Tyr188, Ile198, Ile199, Ser200, Gln206, Ile316, Tyr326, Phe343, Tyr398 and Tyr435, which are displayed in pink and red colours as described in the user guide of Glide 31 . Similarly, promising electrostatic contributions of compound 4i have been determined with Pro102, Phe168, Leu171, Ile199, Ser200 and Ile316 amino acids.

Conclusion
In conclusion, a new series of benzylamine-sulphonamide derivatives were designed, and their inhibition profile of MAO isozymes was evaluated. None of the synthesised compounds displayed a remarkable enzyme activity on hMAO-A enzyme. All of the compounds showed selectivity against hMAO-B enzyme. Among the obtained compounds, labelled 4i and 4t derivatives were found to be most active agents. Compound 4i, which contained 5-chlorobenzothiazole ring, was determined to be the most effective inhibitor candidate with an IC 50 value of 0.041 ± 0.001 mM. It is thought that the 5-chlorobenzothiazole ring and sulphonamide groups were very essential for inhibiting hMAO-B enzyme by docking studies. Hence, these findings showed that the new benzylamine-sulphonamide derivatives inhibited hMAO-B enzyme and suggested that benzylamine-sulphonamide derivatives could be improved in future studies with modifications to design and gain more potent MAO enzyme inhibitor candidates.