Design, Synthesis and Biological Evaluation of Novel N-Pyridyl-Hydrazone Derivatives as Potential Monoamine Oxidase (MAO) Inhibitors

A new series of N-pyridyl-hydrazone derivatives was synthesized by using a simple and efficient method. The final compounds obtained were screened for their inhibitory potency against monoamine oxidase (MAO) A and B. The newly synthesized compounds 2a–2n specifically inhibited monoamine oxidases, displaying notably low IC50 values. Compounds 2i and 2j, with a CF3 and OH group on the 4-position of the phenyl ring, respectively, showed considerable MAO-A and MAO-B inhibitory activities. Compounds 2k, 2l and 2n, with N-methylpyrrole, furan and pyridine moieties instead of the phenyl ring, were the most powerful and specific inhibitors of MAO-A, with IC50 values of 6.12 μM, 10.64 μM and 9.52 μM, respectively. Moreover, these active compounds were found to be non-cytotoxic to NIH/3T3 cells. This study supports future studies aimed at designing MAO inhibitors to obtain more viable medications for neurodegenerative disorders, such as Parkinson’s disease.


Introduction
MAO exists in two isoforms, MAO-A and MAO-B, which have contrasting substrate specificity; MAO-A specifically deaminates serotonin and norepinephrine, and MAO-B acts specifically on phenylethylamine. Given the essential role of MAOs in the inactivation of neurotransmitters, the mechanism of action of MAO-A and MAO-B have been an imperative focus for the treatment of the pathologies of many neurodegenerative diseases. In particular, MAO-B inhibitors are used as a part of the treatment for Alzheimer's and Parkinson's diseases, whereas MAO-A inhibitors are used as antidepressants and antianxiety agents [1,2]. Therefore, investigations on MAO inhibitors have increased in the recent years owing to their restorative effect on mental health. For example, specific MAO-A inhibitors such as iproniazid, clorgyline and moclobemide exert invigoration and antianxiety action [3,4]. Specific MAO-B inhibitors, for example selegiline, rasagiline and lazabemide, complement treatments for Parkinson's and Alzheimer's disease [5]. Unfortunately, the vast majority of the currently used MAO inhibitors, such as iproniazid and tranylcypromine, appear to initiate

Inhibition Potency of the Compounds
Our results indicated that a large number of the synthesized compounds have powerful inhibitory effects against both MAO-A and MAO-B, with IC 50 concentrations in the micromolar range; thus, the effects following introduction of substituents of various sizes to the phenyl ring were evaluated. For compounds 2i and 2j, the incorporation of CF 3 and OH groups on the 4-position of the phenyl ring, respectively, showed massive MAO-A and MAO-B inhibitory activities. It is well known that most of the monoamine oxidase inhibitors (MAO) belong to a group of nitrogen heterocycles, such as pyrazoline, indole, xanthine, oxadiazole, benzimidazole, pyrrole, quinoxaline, thiazole, with other related heterocycles also being reported [14]. As expected, our newly synthesized compounds with heterocyclic moieties showed strong inhibitory activity. Thus, compounds 2k, 2l and 2n with N-methylpyrrole, furan and pyridine moieties, instead of the phenyl ring, were the most powerful and specific inhibitors against MAO-A, with IC 50 values 6.12 µM, 10.64 µM and 9.52 µM, respectively. The screening results indicated that some of the tested compounds inhibited MAO-B. Compound 2j was found to be more potent and specific activity toward MAO-B, with an IC 50 of 9.30 µM. Therefore, we determined that compound 2j was a successful inhibitor of both MAO-A and MAO-B. Considering the closeness to the standard, compound 2l demonstrated an extraordinary level of MAO-A inhibitory activity. The MAO-A enzyme kinetics of the most active compound, 2j, were also studied. The nature of MAO-A inhibition caused by this compound was investigated by using the graphical analysis of steady-state inhibition data ( Figure 1). The reciprocal plots (Lineweaver-Burk plots) identified compound 2j as a mixed type inhibitor owing to the different intercepts on both the yand x-axes. The values of K m and V max were calculated by nonlinear regression based on a previous study [15], and found to be 0.157 µM and 1.903 nmol/min/mg protein, respectively. According to our results, these newly synthesized compounds may be considered as promising therapeutic agents for neurodegenerative and stress-related disorders; however, further trials on the selectivity and effects on other related enzymes should be performed.

Cytotoxicity Test
Toxicity is the main reason for drug candidate failure at all stages of the new drug development process. The major part of safety-related attrition occurs at preclinical phases while predicting preclinical safety liabilities earlier in the drug development process. This strategy enables the design and/or selection of improved drug candidates that have more possibilities to become commercialized drugs. Therefore, the MTT test was conducted in NIH/3T3 mouse embryonic fibroblast cell lines (ATCC CRL1658), which is the ISO-prescribed cell line for cytotoxicity screening (10993-5, 2009) [16]. The IC50 values of compounds 2i, 2j, 2k, 2l and 2n (for all; >1000 μM) against NIH/3T3 cells were higher than their IC50 values against MAO. Thus, the cytotoxicity test results confirmed the importance of compounds 2i, 2j, 2k, 2l and 2n as MAO inhibitors.

Molecular Docking Studies
To determine the binding modes of compound 2j, docking studies were performed by using the Maestro interface. The X-ray crystal structure of hMAO-A, obtained from the Protein Data Bank server (www.pdb.org), was used (PDB ID: 2Z5X) [17]. The docking pose of compound 2j is presented in Figure 2.
It can be observed that compound 2j is located in close proximity to the FAD cofactor and binds tightly to the amino acid residues in the cavity. As known by the docking pose, compound 2j interacts with the active site by establishing two hydrogen bonds and two π-π interactions. The first π-π interaction was observed between three substituted benzene rings and the phenyl of Phe208. The other phenyl ring in the structure interacted with the phenyl of Tyr407 by π-π interaction. The imine nitrogen of the hydrazone moiety established a hydrogen bond with the hydroxyl of Tyr407, whereas the other hydrogen bond was established between the hydroxyl para-substituted phenyl and the carbonyl of Gly443. Collectively, these interactions provide an explanation the inhibitory activity of compound 2j.

Cytotoxicity Test
Toxicity is the main reason for drug candidate failure at all stages of the new drug development process. The major part of safety-related attrition occurs at preclinical phases while predicting preclinical safety liabilities earlier in the drug development process. This strategy enables the design and/or selection of improved drug candidates that have more possibilities to become commercialized drugs. Therefore, the MTT test was conducted in NIH/3T3 mouse embryonic fibroblast cell lines (ATCC CRL1658), which is the ISO-prescribed cell line for cytotoxicity screening (10993-5, 2009) [16]. The IC 50 values of compounds 2i, 2j, 2k, 2l and 2n (for all; >1000 µM) against NIH/3T3 cells were higher than their IC 50 values against MAO. Thus, the cytotoxicity test results confirmed the importance of compounds 2i, 2j, 2k, 2l and 2n as MAO inhibitors.

Molecular Docking Studies
To determine the binding modes of compound 2j, docking studies were performed by using the Maestro interface. The X-ray crystal structure of hMAO-A, obtained from the Protein Data Bank server (www.pdb.org), was used (PDB ID: 2Z5X) [17]. The docking pose of compound 2j is presented in Figure 2.
It can be observed that compound 2j is located in close proximity to the FAD cofactor and binds tightly to the amino acid residues in the cavity. As known by the docking pose, compound 2j interacts with the active site by establishing two hydrogen bonds and two π-π interactions. The first π-π interaction was observed between three substituted benzene rings and the phenyl of Phe208. The other phenyl ring in the structure interacted with the phenyl of Tyr407 by π-π interaction. The imine nitrogen of the hydrazone moiety established a hydrogen bond with the hydroxyl of Tyr407, whereas the other hydrogen bond was established between the hydroxyl para-substituted phenyl and the carbonyl of Gly443. Collectively, these interactions provide an explanation the inhibitory activity of compound 2j.

Chemistry
All compounds and chemicals were purchased from Sigma-Aldrich Chemicals (Sigma-Aldrich Corp., St. Louis, MO, USA) or Merck KGaA (Darmstadt, Germany). The melting points of the synthesized compounds were assessed by using a MP90 digital melting point apparatus (Mettler Toledo, Ohio, OH, USA) and were uncorrected. The 1 H-NMR spectra and 13 C-NMR spectra were recorded by using a Bruker 300 MHz digital FT-NMR spectrometer (Bruker Bioscience, Billerica, MA, USA) and collected in DMSO-d6 (Supplementary Materials). The IR spectra were obtained by using a Shimadzu IR Prestige-21 instrument (Shimadzu, Kyoto, Japan). The LC-MS/MS studies were conducted by using a Shimadzu 8040 LC-MS/MS system (Shimadzu, Kyoto, Japan). The purities of the compounds were checked by TLC on silica gel 60 F254 (Merck KGaA, Darmstadt, Germany). The general synthesis method is presented in Scheme 1.

Chemistry
All compounds and chemicals were purchased from Sigma-Aldrich Chemicals (Sigma-Aldrich Corp., St. Louis, MO, USA) or Merck KGaA (Darmstadt, Germany). The melting points of the synthesized compounds were assessed by using a MP90 digital melting point apparatus (Mettler Toledo, Ohio, OH, USA) and were uncorrected. The 1 H-NMR spectra and 13 C-NMR spectra were recorded by using a Bruker 300 MHz digital FT-NMR spectrometer (Bruker Bioscience, Billerica, MA, USA) and collected in DMSO-d 6 (Supplementary Materials). The IR spectra were obtained by using a Shimadzu IR Prestige-21 instrument (Shimadzu, Kyoto, Japan). The LC-MS/MS studies were conducted by using a Shimadzu 8040 LC-MS/MS system (Shimadzu, Kyoto, Japan). The purities of the compounds were checked by TLC on silica gel 60 F254 (Merck KGaA, Darmstadt, Germany). The general synthesis method is presented in Scheme 1.

Chemistry
All compounds and chemicals were purchased from Sigma-Aldrich Chemicals (Sigma-Aldrich Corp., St. Louis, MO, USA) or Merck KGaA (Darmstadt, Germany). The melting points of the synthesized compounds were assessed by using a MP90 digital melting point apparatus (Mettler Toledo, Ohio, OH, USA) and were uncorrected. The 1 H-NMR spectra and 13 C-NMR spectra were recorded by using a Bruker 300 MHz digital FT-NMR spectrometer (Bruker Bioscience, Billerica, MA, USA) and collected in DMSO-d6 (Supplementary Materials). The IR spectra were obtained by using a Shimadzu IR Prestige-21 instrument (Shimadzu, Kyoto, Japan). The LC-MS/MS studies were conducted by using a Shimadzu 8040 LC-MS/MS system (Shimadzu, Kyoto, Japan). The purities of the compounds were checked by TLC on silica gel 60 F254 (Merck KGaA, Darmstadt, Germany). The general synthesis method is presented in Scheme 1.

MAO Activity Assay
The enzyme activity assay was performed in accordance with the procedure reported by Matsumoto et al. [15] with slight modifications. All materials used in the enzymatic assays were purchased from Sigma-Aldrich Chemicals. All pipetting protocols in the assay were performed by the Biotek After incubation for 10 min at 37 • C, kynuramine (50 µL/well) was added to initiate the enzyme-substrate reaction. The plate was incubated for 20 min at 37 • C and the reaction was subsequently terminated by the addition of 2 N NaOH (75 µL/well). Fluorescence was read from top by using BioTek-Synergy H1 multimode microplate reader at the excitation/emission wavelengths of 310 and 380 nm. The same procedure was followed for the dilutions (10 −5 -10 −9 M, 100 µL/well) of the inhibitors and selected compounds that indicated ≥50% inhibition at the initial concentrations (10 −3 and 10 −4 M, 100 µL/well) ( Table 1). The IC 50 values ( Table 2) were calculated from the plots of enzyme activity against concentrations, and the regression analyses were computed in GraphPad Prism Version 6 (GraphPad Software, Inc., La Jolla, CA, USA).

Enzyme Kinetic Studies
The same materials were used for the MAO-inhibition assay. Compound 2j was prepared at the IC 50 concentration calculated in the enzyme assay and then added to the wells (100 µL/well). The stock solution (25 mM) of kynuramine in 0.1 M phosphate buffer was diluted to final concentrations of 40, 20, 10, 5, 2.5 and 1.25 µM and then added to the wells (50 µL/well) that contained the test compound. The MAO-A enzyme was added to the plate (50 µL/well), incubated for 20 min at 37 • C, and the plate was read at excitation/emission wavelengths of 310 and 340 nm [20]. The control measurement without the inhibitor was determined simultaneously. The results were analyzed by using Lineweaver-Burk plots in Microsoft Office Excel 2013 (Microsoft Inc., Redmond, WA, USA).

Molecular Docking Studies
A structure based in silico procedure was applied to determine the binding modes of the compound 2j hMAO-A enzyme active site. The crystal structures of hMAO-B (PDB ID: 2Z5X) [17], which was crystallized with the reversible inhibitor safinamide, was retrieved from the Protein Data Bank server (www.pdb.org (access date: 14 November 2017)).
The structures of ligands were built using the Schrödinger Maestro [27] interface and were then submitted to the Protein Preparation Wizard protocol of the Schrödinger Suite 2016 Update 2 [28]. The ligands were prepared by the LigPrep 3.8 [29] to correctly assign the protonation states at pH 7.4 ± 1.0 and the atom types. The bond orders were assigned, and the hydrogen atoms were added to the structures. The grid generation was formed by using Glide 7.1 [30]. The grid box with dimensions of 20 Å × 20 Å × 20 Å, was centered in the vicinity of the flavin (FAD) N5 atom on the catalytic site of the protein to cover all binding sites and neighboring residues [31][32][33]. The flexible docking runs were performed in standard precision docking mode (SP).

Conclusions
Interest in MAO inhibitors has increased in recent years owing their restorative influence on mental health [3], therefore, a novel series of N-pyridyl-hydrazone derivatives was synthesized, starting from 2-chloro-6-ethoxy-3-nitropyridine, and their structures were confirmed by FT-IR, 1 H-NMR, 13 C-NMR and ESI mass spectroscopy. These compounds show strong and specific inhibitory activity against MAO-A and MAO-B enzymes; compounds 2i, 2j, 2k, 2l and 2n exhibited the highest activity with IC 50 values of 13.06 µM, 6.25 µM, 6.12 µM, 10.64 µM and 9.52 µM, respectively, against MAO A, whereas compound 2j exhibited the strongest MAO-B inhibitory activity, with a IC 50 value of 9.30 µM. Furthermore, these active compounds did not indicate any cytotoxic effects at the dose required for MAO inhibition. This work is in progress and future developments are expected. We have presented all the results on the figureMAO-A and MAO-B inhibition studies, as they may have potential effects on the future remedial treatment of neurodegenerative diseases. In addition, these derivatives may provide possible therapeutic options for the treatment of the motor and non-motor symptoms of Parkinson's disease, for which no treatment has been previously demonstrated.
Supplementary Materials: The following are available online.