Identification of New N-methyl-piperazine Chalcones as Dual MAO-B/AChE Inhibitors

Monoamine oxidase-B (MAO-B), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) have been considered target enzymes of depression and neurodegenerative diseases, including Alzheimer’s disease (AD). In this study, seventeen N-methyl-piperazine chalcones were synthesized, and their inhibitory activities were evaluated against the target enzymes. Compound 2k (3-trifluoromethyl-4-fluorinated derivative) showed the highest selective inhibition against MAO-B with an IC50 of 0.71 μM and selectivity index (SI) of 56.34, followed by 2n (2-fluoro-5-bromophenyl derivative) (IC50 = 1.11 μM, SI = 16.04). Compounds 2k and 2n were reversible competitive MAO-B inhibitors with Ki values of 0.21 and 0.28 μM, respectively. Moreover, 2k and 2n effectively inhibited AChE with IC50 of 8.10 and 4.32 μM, which underscored their multi-target inhibitory modes. Interestingly, compound 2o elicited remarkable inhibitions over MAO-B, AChE, and BChE with IC50 of 1.19–3.87 μM. A cell-based assay of compounds 2k and 2n against Vero normal cells pointed out their low cytotoxicity. In a docking simulation, 2k showed the lowest energy for MAO-B (−11.6 kcal/mol) with four hydrogen bonds and two π-π interactions. Furthermore, in silico studies were conducted, and disclosed that 2k and 2n are expected to possess favorable pharmacokinetic properties, such as the ability to penetrate the blood–brain barrier (BBB). In view of these findings, compounds 2k and 2n could serve as promising potential candidates for the treatment of neurodegenerative diseases.


Introduction
Alzheimer's disease (AD) and Parkinson's disease (PD) are the most prevalent neurodegenerative diseases (NDDs) that mainly affect the elderly [1]. AD and PD are characterized by heterogeneous and complex multifaceted pathologies that stem from a combination of diverse genomic, epigenomic, environmental, and metabolic factors [2]. A growing body of evidence disclosed that recovery of brain functional steadiness after a neurologic impairment is unlikely to be accomplished by focusing on a single molecular target [3]. Therefore, the research efforts for the treatment of NDDs are being oriented towards the design of multi-target directed ligands (MTDLs) [4]. However, to strike a balance between the effects of MTDLs acting at their molecular targets represents a major challenge [5].
The oxidative deamination of the biogenic amines in peripheral and central tissues is catalyzed by the flavin adenine dinucleotide (FAD) dependent monoamine oxidases (MAOs)-A and -B [6]. During the catabolism of different monoamine neurotransmitters by MAO-A/B, hydrogen peroxide and reactive oxygen species (ROS) are excessively produced as major by-products, triggering oxidative stress and neuronal cell damage associated with several NDDs [7]. Overexpression of MAO-B levels in the substantia nigra of PD patients, along with the relevance of elevated activity of MAO-B and impairment of cognitive functions in AD patients has been reported [8,9]. In this regard, MAO inhibitors are considered as up-regulating agents for neurotransmitter amines, such as dopamine and noradrenaline with neuroprotective properties [10]. Numerous investigations have reported the beneficial effects of reversible/irreversible MAO-B inhibitors in AD related therapeutic approaches [11,12]. Moreover, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors have shown remarkable roles in the preservation of cholinergic functions and symptomatic improvement in AD [13,14]. In view of MTDLs merits, the contemporary medicinal chemists have focused their efforts, over the past few decades, to develop new chemical entities as multiple-acting MAO-B and AChE/BChE inhibitors [15][16][17][18].
N-Methylpiperazine is a privileged derivative of piperazine moiety, with better lipophilic and steric characters, which renders this motif a proper structural element in maintaining the right balance of pharmacokinetic and pharmacodynamic attributes [19]. The existence of two nitrogens along with the small bulky N-methyl allows N-methylpiperazine to be engaged in various hydrophobic and charge transfer interactions with target enzymes. The favorable drug-like properties of N-methylpiperazine were reflected in several central nervous system (CNS) acting drugs, such as clozapine, loxapine, olanzapine, trifluoperazine, and thiothixene ( Figure 1) [20]. Furthermore, N-methylpiperazine was introduced in a number of anticancer kinase inhibitors, to improve water solubility and/or target affinity, such as bosutinib, ponatinib, nintedanib, brigatinib, and gilteritinib [21]. Moreover, recent studies revealed that the replacement of one or both nitrogen atoms in the piperazine ring with different structural motifs could significantly improve MAO-B, and AChE inhibitions [22][23][24]. Based on this perspective, insertion of N-methylpiperazine into the proper scaffolds of certain MAO-B inhibitors would be expected to afford dual MAO-B/AChE inhibitions. Further, chalcone, 1,3-diphenylprop-2-en-1-one, represents a substantial flexible scaffold for the design of selective MAO-B and/or AChE inhibitors [25], where the existence of propenone (α-β unsaturated ketone) and three rotatable bonds in chalcones can furnish various binding orientations with multiple targets [26]. Recent studies pointed out that conjugation of certain FDA-approved drugs, such as rivastigmine and donepezil, with chalcone scaffold led to generation of multifunctional MAO-B/ChE inhibitors, such as compound I with potent neuroprotective properties for AD [15]. Installing different electron-pulling and pushing groups on the aryl/heteroaryl rings of chalcones can modulate the electrophilic character of the Michael acceptor propanone as exemplified by chalcones II and III [27,28], where the introduction of lipophilic halogens (F, Cl, Br)/electron donation groups (-OMe, -N(Me) 2 ) onto the phenyl B ring of chalcones afford highly selective MAO-B inhibitors such as compound IV [29,30]. On the other hand, the existence of proper alkylamino moieties on the ketone linked ring A (i.e., compound V) was found to be favorable for achieving AChE inhibitory activity [31,32].
As part of our continued endeavors to develop novel chemical entities as potent MAO-B/or AChE inhibitors, we have recently reported compounds PC-10 and PC-11 as piperazine featuring chalcones with promising selective MAO-B inhibitors ( Figure 2) [33]. Compounds PC-10 and PC-11 showed IC 50 values of 0.65 and 0.71 µM, respectively, against MAO-B, with selectivity index (SI) of 48.3 and 49.2 over MAO-A. Furthermore, kinetics study of PC-10 and PC-11 revealed their competitive mode of MAO-B inhibition with inhibition constant (K i ) of 0.63 and 0.53 µM, respectively. However, these molecules exerted modest activity towards both AChE and BChE with IC 50 values of 26.3-28.0 and 36.2-36.4 µM, respectively. Motivated by such findings, as well as the aforementioned considerations highlighting the significance of terminal methyl group (Figure 2), we pursued further structural modifications on compounds PC-10 and PC-11 aiming at design of dual MAO-B/AChE inhibitors with improved potency. Thus, in the current study, seventeen N-methylpiperazine chalcones were designed, synthesized, and biologically tested against MAO-A/B and AChE/BChE. We expected that insertion of the small bulky methyl group on the terminal amine of piperazine would impart AChE inhibitory activity for the target molecules. In addition, diverse mono-and di-substituted phenyl rings, with various lipophilic characters, were investigated to construct a reliable structure activity relationship (SAR) study. To the best of our knowledge, this is the first report harnessing N-methylpiperazine moiety to afford dual MAO-B/AChE chalcone inhibitors.

Synthesis of Compounds
As illustrated in Scheme 1, the synthesis of the target 4-methylpiperazine-containing α,β-unsaturated ketones 2a-q was commenced with preparation of 4-methylpiperazine acetophenone 1 as the key precursor. Heating of 4-fluoroacetophenone with 4-methylpiperazine at 140 • C under neat conditions afforded compound 1 in 84% yield [34]. The Claisen-Schmidt condensation of 1 was with equimolar amounts of various mono/di-substituted benzaldehydes in 40% alcoholic NaOH at room temperature, and furnished the target molecules 2a-q in good yield. The 1 H NMR charts of the final compounds showed the 4-methylpiperazine ring protons N-CH 2 resonating at 3.42 and 2.57 ppm as triplets, as well as N-CH 3 protons as a singlet at 2.36 ppm. The large coupling constant (J = 15.6 Hz) value of H α and H β enone protons confirmed the trans (E) configuration of the 4-methylpiperazine chalcones. The spectra were provided in Supplementary (Figures S1-S3 Chalcone is a well-known privileged scaffold as a MAO inhibitor, particularly selective towards MAO-B [35]. From this background, a plethora of structurally diverse chalcone derivatives has been synthesized by our team, and their MAO-A/B inhibition profiles have been reported [33,36,37]. The results revealed that the best members in those chalcone derivatives had IC 50 spanning from 0.0021 to 0.65 µM against MAO-B [33,36,37]. In this study, a set of seventeen N-methyl-piperazine chalcone derivatives were synthesized and their inhibitory activities were evaluated against the target enzymes. In the preliminary inhibition assay at 10 µM concentration, all compounds exerted <50% of residual activity for MAO-B, except 2j, whereas all compounds showed >50% of residual activity for MAO-A, except 2p (Table 1). Compound 2k (3-trifluoromethyl-4-fluorinated derivative) elicited the highest selective inhibition against MAO-B with IC 50 of 0.71 µM and selectivity index (SI) of 56.34, followed by the 2-fluoro-5-brominated derivative 2n (IC 50 = 1.11 µM, SI = 16.04) (Table 2, Figure S4). Furthermore, most of the tested chalcone derivatives displayed <50% of residual activity for AChE at 10 µM, except 2g, 2i, 2j, and 2l, whereas most of compounds showed >50% of residual activity for BChE at 10 µM, except 2c, 2e, and 2n-p. Among them, 2b had the highest AChE inhibition with IC 50 of 2.26 µM and SI (ChE) of 5.92, followed by 2d with IC 50 of 2.38 µM, and SI (ChE) of 10.38. Concerning BChE, 2o stood out as the most potent member with IC 50 of 1.19 µM. Remarkably, all the compounds exerted selective dual MAO-B/AChE inhibition, except 2o, disclosing that N-methyl-piperazine chalcone derivatives could be expected as multi-target inhibitors for AD.
Referring to the disubstituted derivatives 2i-q, it was noticed that the existence of a four-halogen moiety (F/Cl) adjacent to meta-trifluoromethyl is optimal for MAO-B inhibition. For example, compound 2k bearing 3-trifluoromethyl-4-fluorophenyl showed superior potency against MAO-B (IC 50 = 0.71 µM) compared to its 3,5-positional isomer 2i (IC 50 = 2.33 µM). Upon comparing the 4-halogenated-3-trifluoromerthylphenyl derivatives 2k and 2l, it was found that 2k is relatively more potent than its corresponding 4-chloro congener 2l over MAO-B, AChE, and BChE. Such a finding is in agreement with the recent report of Rullo et al., which underscored the importance of fluorinated motifs in achieving sound MAO-B inhibition [38]. Worth noting is that replacing fluorine in 2i (IC 50 = 2.33 µM) with a trifluoromethyl group (2j) led to dramatic drop in MAO-B inhibition (IC 50 = 15.70 µM). In addition, the introduction of a 3-methoxy group, in place of the 3-trifluoromethyl motif of 2k, afforded the chalcone 2m with significantly reduced potency for MAO-B (IC 50 = 6.76 µM), yet with reasonable inhibition towards AChE and BChE. Upon inspection of the activity of 2,5-disubstituted chalcones 2n and 2o, it was observed that compound 2o, the positional isomer of 2l, displayed a distinct multi-target inhibitory effect over MAO-B, AChE, and particularly BChE (IC 50 = 1.19 µM). As an exception to the noticed dual selective MAO-B/AChE inhibition of this set of 4-methylpiperazine, the 2,4-difluoro member 2p elicited moderate MAO-A potency (IC 50 = 6.67 µM) with a low SI value of 3.06. In view of these biochemical assay results and as illustrated in Figure 3, it is evident that both the nature and position of the substituent installed on terminal phenyl ring possess critical roles in modulating the inhibitory potency towards MAO-A/B and AChE/BChE.

Inhibition Pattern Analysis of 2k and 2n
A kinetic study of the most active compounds 2k and 2n against MAO-B was performed to analyze the inhibition patterns using Lineweaver-Burk plots, and their Ki values were determined using their secondary plots. The Lineweaver-Burk plots of 2k and 2n, matched in Y-axis, showed that Ki values were 0.21 ± 0.03 µM and 0.28 ± 0.01 µM, re-spectively, which points out that both compounds are competitive inhibitors of MAO-B ( Figure 4). Since 2k and 2n displayed AChE inhibition with IC 50 of 8.10 and 4.32 µM, respectively, kinetics for AChE were also performed ( Figure 5). 2k and 2n were found to be mixed type inhibitors for AChE, showing contact points in second quadrants with K i values of 1.44 ± 0.33 and 0.82 ± 0.08 µM, respectively.
In reversibility tests of 2k and 2n for MAO-B using the dialysis method, their residual activities were efficiently recovered from 14.63% and 26.47% before dialysis, respectively, to 80.26% and 74.83% after dialysis, respectively ( Figure 6). In references, pargyline, an irreversible MAO-B inhibitor, displayed no recovery of the residual activity, i.e., 24.81% to 24.88%, whereas lazabemide, a reversible MAO-B inhibitor, showed efficient recovery of the residual activity, i.e., from 20.53% to 98.78%. From these results, it is concluded that 2k and 2n are reversible MAO-B inhibitors. In a previous study, piperazine chalcone derivatives were also analyzed for their inhibitory activities against MAO-B [33], and p-fluorinated piperazine chalcone derivative PC10 showed the highest reversible competitive inhibition for MAO-B with an IC 50 value of 0.65 µM (K i = 0.50 µM). On the other hand, 2k, the most potent MAO-B inhibitor in this study, also showed reversible competitive inhibition with IC 50 of 0.71 µM (K i = 0.21 µM), followed by 2n (IC 50 = 1.11 µM, K i = 0.28 µM). Interestingly, 2k and 2n elicited effective inhibition against AChE with IC 50 values of 8.10 and 4.32 µM, respectively, in contrast to PC10 which showed a moderate inhibition with IC 50 of 28.0 µM [33]. 2k and 2n showed mixed type inhibition for AChE with K i values of 1.44 and 0.82 µM, respectively; however, there were no kinetics data of PC10 against AChE [33]. We previously reported about AChE inhibition of morpholine-based chalcones, which have a similar structure to the piperazine chalcone; however, the lead compounds MO5 and MO9 exerted competitive and non-competitive inhibition, not mixed-type, for AChE with K i values of 2.52 µM and 7.04 µM, respectively, which are remarkably lower than those of 2k and 2n [39]. These findings suggest that N-methyl piperazine chalcones possess comparable or better K i values for MAO-B than their corresponding piperazine chalcones, as well as possessing higher affinities with multi-target inhibitions for targeting AD. , and 2n (2.54 µM) before dialysis for 6 h. The residual activities were determined before and after dialysis to confirm the reversibility pattern using enzyme assay described in Materials and Methods.

Cytotoxicity Study of 2k and 2n
The cytotoxicity profile of 2k and 2n on the cellular viability of VERO normal cells was investigated at ten different concentrations (0.0102-200 µM), using MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. As depicted in Figure 7, compounds 2k and 2n were non-toxic to normal VERO cells with IC 50 values of 19.93 µM and 62.04 µM, respectively, pointing out their suitability as potential therapeutic candidates.  Docking simulations of the best two members 2k and 2n with MAO-A (pdb code: 2Z5X), MAO-B (pdb code: 4A79), and AChE (pdb code: 6O4W) were performed using AUTODOCK-VINA [40]. The catalytic sites of MAO-A, MAO-B, and AChE are highlighted, as depicted in Figure 8A, 8B, and 8C, respectively. Six combinations of docking simulations were performed (MAO-A-2k and MAOA-2n in Figure 8D, MAO-B-2k and MAO-B-2n in Figure 8E, and AChE-2k and AChE-2n in Figure 8F). The docked compounds were clustered based on their position on the protein. Each top lowest energy conformation in the catalytic region of the three proteins is tabulated in Table 3. Each lowest energy compound near the catalytic region is sketched, with their docking energies in Figure 8D-F. Among the generated conformations, compound 2k has the lowest energy with MAO-B (−11.6 kcal/mol), indicating the tightest binding which is consistent with the experimental IC 50 value. That tight binding stems from the existence of several hydrogen bonding interactions (blue dash lines), as shown in Figure 9. The main key residues are THR196, GLN206, and TYR435, and the crucial interacting functional groups in 2k are CF 3 and two nitrogen atoms in piperazine. Because the side chain of THR196 can rotate freely and stay in three conformers, Gauche+ (60 • of torsional angle in N-CA-CB-OG1), Gauche-(−60 • ), and trans (180 • ), the hydrogen bonds of the donor, OG1 in THR196 could enhance its binding affinity toward the -CF 3 of 2k. The nitrogen atom in the backbone of THR196 can also engage in hydrogen bonds with the -CF 3 . Two terminus atoms in the side chain (-NE 2 for GLN206 and -OH for TYR435) can share binding interactions toward the two nitrogen atoms in the piperazine of 2k. Although the number of hydrogen bonds in MAO-B-2k is four hydrogen bonds, the MAO-A-2k also has four hydrogen bonds. However, MAO-B-2k possesses lower binding energy and the number of π-π interaction is two, as shown in Figure 8, which is higher than one in MAO-A-2k. Two π-π interactions are found between (1) TYR326 and the central planar ring, and (2) TRP119 and the fluorine aromatic ring. In particular, the first π-π interaction between TYR326 and the central ring of 2k plays an important role in inhibitory selectivity of MAO proteins. The MAO-B has TYR326, and the corresponding residue in MAO-A is ILE335. The structural difference confirms that the inhibitor selectivity of MAO-A and MAO-B is caused by π-π interaction [41]. The docking of compounds 2k and 2n on AChE had higher binding energies than the MAO series. However, AChE showed a higher number of hydrogen bonds and the same number of π-π interactions. Therefore, both compounds 2k and 2n could have tighter binding on AchE than the MAO-A protein. As revealed from the docking simulation results, compound 2k was the most favorable inhibitor in MAO-B in terms of the lowest binding energy and higher number of hydrogen bonds along with π-π interactions, and these results are in good agreement with the IC 50 or K i experiment results. In AChE inhibition, K i results were not in accordance with the results of the docking data; 2n (K i = 0.82 uM) bound more tightly on AChE than 2k (K i = 1.44 uM), but differences in the docking data of AChE were smaller than those of MAO-B. This discrepancy might come from the difference in inhibition modes; AChE inhibition by 2k and 2n was mixed type with a different degree, while MAO-B inhibition was competitive. In addition, docking data were calculated based on a combination of various factors such as hydrogen bonding, electrostatic bonding, van der Waals forces, dissolvent effects, and the flexibilities of compounds [42]. Therefore, mixed inhibition degrees and microelement environments of the binding sites might influence the experimental K i values, though docking values were not exactly in line with the K i values.  a Each first lowest energy compound in the catalytic region is chosen. The result is tabulated in terms of number of compounds existing on the pocket (No. of Cpds), average energy on the pocket, the number of docking compounds on the same pocket, lowest energy with cluster rank with Parentheses, free energy, number of Hydrogen bonding, and number of π-π interaction/aromatic rings. Energy unit is kcal/mol. The free energy is the colony energy [43] calculated by E-kT log(N), where E is the lowest energy, N is the number of compounds in the catalytic region. The k is Boltzmann constant and T is room temperature, 300 K. The free energy considers both energetic and entropic contribution.

Pharmacokinetic Prediction of 2k and 2n
As a part of our study, ADME prediction for compounds 2k and 2n was performed. 2k and 2n were predicted to have high gastrointestinal absorption, blood-brain barrier (BBB) permeability, and no substrates of P-glycoprotein (P-gp). 2k inhibited cytochrome P450 (CYP) 2C19, 2D6, and 3A4, whereas 2n inhibited CYP 2C19, 2C9, and 2D6 (Table 4). Furthermore, 2k and 2n were predicted to have no violations for Lipinski rules (Table 5) [44]. Accordingly, 2k and 2n would be expected to possess favorable pharmacokinetic properties for the treatment of neurodegeneration disorders.

Chemistry
General: All solvents and reagents were obtained from commercial suppliers and were used directly without further purification. The reaction progress was monitored on a TLC plate (Merck, silica gel 60 F 254 , Darmstadt, Germany). Melting points were measured using OptiMelt MPA100 melting point apparatus and were uncorrected. FT-IR spectra were scanned using Frontier IR SP8000 MIR DTGS / KBr / Al / Ext beams with Spotlight 200i microscopy (PerkinElmer Inc., MA, USA). 1 H and 13 C NMR spectra were recorded on a Bruker Advance 400 MHz spectrometer, using deuterated chloroform. Chemical shifts (δ) are given in parts per million (ppm) upfield from tetramethylsilane (TMS) as an internal standard, and s, d, t, and m are presented as singlet, doublet, doublet triplet and multiplet, respectively. Coupling constants (J) are reported in hertz (Hz). High resolution mass spectra (HRMS) were recorded on a Waters Acquity UPLC/Synapt G2 QTOF MS or JMS 700 (Jeol, Tokyo, Japan) mass spectrometer.

1-(4-(4-Methylpiperazin-1-yl)phenyl)ethan-1-one (1) [34]
A mixture of 1-(4-fluorophenyl)ethan-1-one (2.5 g, 18 mmol) and 4-methylpiperazine (9 g, 90 mmol) was stirred and heated in a sealed tube at 140 • C for 12 h. The reaction mixture was cooled down to room temperature and poured into ice water. The precipitated solid was filtered under vacuum and dried to afford the title compound as a pure yellow solid, 3.5 g (84.2% yield). 1 H and 13 C NMR data are consistent with the reported in literature.

General Procedure for Synthesis of Compounds 2a-q
To a solution of compound 1 (250 mg, 1.15 mmol) and the appropriate aromatic aldehyde (1.15 mmol) in 95% ethanol (5 mL), 40% alcoholic sodium hydroxide (1 mL) was added. The reaction mixture was stirred at rt for 0.5-4 h, and the formed precipitate was filtered, washed with cold ethanol, dried and crystallized from ethanol to afford the target compounds, as a yellow amorphous powder, in their pure form.  187.06, 154.32, 140.39, 139.76, 132.61, 130.85,  128.55, 127.54, 125.21, 118.54, 113.43, 112.99, 54.71  MAO assays were performed as described previously [45], i.e.,~0.09 U/mL of MAO-A and MAO-B were reacted with 0.06 mM kynuramine and 0.3 mM benzylamine, respectively, in 50 mM sodium phosphate (pH 7.2). The absorbances of reaction mixtures of MAO-A and MAO-B were observed at 316 nm and 250 nm, respectively, for 30 min under kinetic mode of the spectrophotometer (OPTIZEN, K-Lab, Dajeon, Republic of Korea). For inhibition assay, the compounds were dissolved in DMSO and added into the reaction mixture. The final concentration of DMSO in the reaction mixture was less than 1% to avoid solvent disturbance.
For measuring cholinesterase (ChE) activity, the Ellman method [46] was employed with a slight modification. The AChE and BchE with~0.02 U/mL were reacted with 0.5 mM ATCI and BTCI, respectively, followed by addition of 0.5 mM DTNB. The absorbance of the reaction mixture was continuously observed at 412 nm for 15 min under the kinetic mode of the spectrophotometer. In the inhibition assay, the enzyme and the compound were preincubated in reaction buffer for 15 min, followed by adding substrates and DTNB.

Kinetics and Reversibility Analysis
To analyze the inhibitory patterns of compounds 2k and 2n, which showed the most potent MAO-B inhibition with the lowest IC 50 , kinetics and reversibility tests were conducted [47]. In kinetics for MAO-B, 0.038, 0.075, 0.15, 0.3, and 0.6 mM of benzylamine were used to construct Lineweaver-Burk plots, and the compounds were added with concentrations of~1/2× IC 50 , IC 50 , and 2× IC 50 for a secondary plot to obtain K m and K i values. In the kinetics for AChE, 0.0625, 0.125, 0.25, 0.5, 1.0 mM of ATCI were used in the presence of DTNB, and the compounds were added to be~1/2× IC 50 , IC 50 , and 2× IC 50 concentrations for the secondary plot to obtain K m and K i values.
Reversibility of the compounds for MAO-B was measured using the dialysis method. The compounds were treated with~1.5× IC 50 for 2k and~2× IC 50 for 2n, respectively, with the reaction mixture without substrate. The reaction mixture was placed in a dialysis tube (DiaEasyTM Dialyzer (800 µL) MWCO 6-8 kDa, Biovision, CA, USA) and dialyzed for 6 h. After that, the 0.3 mM of benzylamine was added into the reaction mixture for measuring enzyme activity. The relative activity to the control (without inhibitors) before and after dialysis was calculated, respectively. The lazabemide and pargyline were used as standard reversible and irreversible MAO-B inhibitors, respectively.

Materials
The reference compound staurosporine was purchased from Sigma-Aldrich (Saint Louis, MO, USA). Compounds 2k and 2n were dissolved in DMSO as 50 mM stock. CellTiter 96 ® Non-Radioactive Cell Proliferation Assay (MTT) reagent was purchased from Promega (Cat# G4000). The normal Vero cell line was purchased from ATCC. Normal Vero cells were cultured with EMEM medium plus 10% FBS. In total, 100 µg/mL penicillin and 100 µg/mL streptomycin were added to the culture media. Cultures were maintained at 37 • C in a humidified atmosphere of 5% CO 2 and 95% air.

Method
Compounds 2k and 2n and staurosporine were diluted with DMSO in 10-dose and 3-fold serial dilution starting at 50 mM (test compounds) and 10 mM (staurosporine). The test compounds (100 nL) and staurosporine (25 nL) were delivered from the source plate to the wells of 384-well cell culture plates by Echo 550 Liquid Handler. Culture medium (25 µL) containing 2000 of normal Vero cells was added to the 384-well cell culture plates, and the plates were incubated at 37 • C in a humidified atmosphere of 5% CO 2 and 95% air for 72 h. MTT Dye Solution (8 µL) was added to each well, and the plates were incubated at 37 • C in a CO 2 incubator for 6 h. After incubation, the formazan was produced in the cells. Solubilization/Stop Solution (30 µL) was added to each well and the plates were incubated for another 1 h at 37 • C in a CO 2 incubator to dissolve the formazan crystals. The absorbance of each sample was measured at 590 nm using Envision 2104 Multilabel Reader (PerkinElmer, Santa Clara, CA, USA), and the cell viability was determined based on the quantification of the color intensity in each culture well (duplicate assay mode). The IC 50 curves were plotted, and the IC 50 values were calculated using the GraphPad Prism 4 program based on a sigmoidal dose-response equation.

Molecular Docking
Docking simulation of three enzymes, MAO-A, MAO-B, and AChE with compounds 2k and 2n were performed using AUTODOCK-VINA [40]. The protein structures of MAO-A, MAO-B, and AChE are PDB (Protein Data Bank) ID of 2Z5X, 4A79, and 6O4W, respectively. MAO-B and AChE have a homogenous complex with two chains, A and B. We used two programs; the first program predicts a binding site, and the second program, Pck, is a pocket search program, not a binding site. Because the pocket and the binding site are not identical, we cannot find another binding site working under allosteric inhibition when the docking simulation runs only in the binding site. The first program is for understanding the enzyme, and the second one is for general docking procedure. For the docking simulation, the first chain, A, was chosen. The binding sites of two proteins were determined using a binding pocket program, DPSP (dockable pocket site prediction: as an in-house program). It searches and collects all of the ligands existing on similar sequence structures, and obtains pockets available on the MAO and AChE proteins. Compounds 2k and 2n were sketched, energy minimized, and converted to the general structure format, i.e., PDB, using the Marvin program (ChemAxon; http://www.chemaxon.com, accessed on 1 July 2022). For the docking simulation, the pockets in these proteins were searched utilizing Pck pocket detection program (http://schwarz.benjamin.free.fr/Work/Pck/home.htm, accessed on 1 July 2022), where it detects the amino acids forming pockets and measures their volumes. Twelve, thirteen, and fourteen pockets over 100 Å 3 of volume were found in MAO-A, MAO-B, and AChE, respectively. These pockets consist of 168, 158, and 101 amino acids for MAO-A, MAO-B, and AChE. Compounds 2k and 2n were put on Carbon α atom positions in these pocket residues, and the docking simulations were run ten times with different random seeds, i.e., a total of 1680, 1580, and 1010 simulations were carried out for MAO-A, MAO-B, and AChE, respectively. The box size with a length of 15 Å was used to prevent the compounds from drifting from the center of the pocket residue. With these docking poses, the clustering based on the center of mass was performed to group these docking conformations using CHARMM (Chemistry at HARvard Macromolecular Mechanics) [48]. The compounds were ranked by the lowest energy of the group, the largest number of the group, and the lowest energy conformation in the group. The molecular structures were drawn by VMD (visual molecular dynamics) visualizer program (https://www.ks.uiuc.edu/Research/vmd/, accessed on 1 July 2022). Hydrogen bond and π-π interaction analysis between each compound and proteins were calculated based on a geometric analysis. First, the acceptor and donor atoms of hydrogen bonds were assigned and then their Cartesian coordinates were obtained. Second, all of the pairs between acceptor and donor atoms were arranged to measure geometric distance and angle. The used hydrogen bonding criteria is the distance below 4.5 Å between the acceptor and donor atoms. The π-π interaction forms between two close parallel aromatic rings. First, the aromatic rings were sought, and then the atoms forming the aromatic rings were found. The centroid of these atoms was calculated and the Cartesian coordinate was obtained. The distance between the two centroids was measured, in the case that both aromatic rings are close and parallel.

Conclusions
In this report, we describe the synthesis of new N-methyl-piperazine chalcones, and their MAOs and AChE inhibition profiles. Most of the tested compounds exerted potent MAO-B inhibition with considerable SI along with moderate AChE inhibition. The fluorinated substituted derivatives 2k and 2n inhibited MAO-B with K i values of sub-micromolar range. The kinetics study of 2k and 2n disclosed their competitive reversible MAO-B inhibitory pattern. A cell-based assay of 2k and 2n against normal Vero cells revealed their minimized cytotoxicity. A docking simulation predicted that 2k showed the lowest energy for MAO-B (−11.6 kcal/mol) with four hydrogen bonds and two π-π interactions. In silico ADME/Tox prediction studies of 2k and 2n pointed out their favorable drug-like properties. Overall, compounds 2k and 2n could serve as promising MAO-B/AChE inhibitors for the treatment of NDD.