Medicarpin and Homopterocarpin Isolated from Canavalia lineata as Potent and Competitive Reversible Inhibitors of Human Monoamine Oxidase-B

Thirteen compounds were isolated from the Canavalia lineata pods and their inhibitory activities against human monoamine oxidase-A (hMAO-A) and -B (hMAO-B) were evaluated. Among them, compounds 8 (medicarpin) and 13 (homopterocarpin) showed potent inhibitory activity against hMAO-B (IC50 = 0.45 and 0.72 µM, respectively) with selectivity index (SI) values of 44.2 and 2.07, respectively. Most of the compounds weakly inhibited MAO-A, except 9 (prunetin) and 13. Compounds 8 and 13 were reversible competitive inhibitors against hMAO-B (Ki = 0.27 and 0.21 µM, respectively). Structurally, the 3-OH group at A-ring of 8 showed higher hMAO-B inhibitory activity than 3-OCH3 group at the A-ring of 13. However, the 9-OCH3 group at B-ring of 13 showed higher hMAO-B inhibitory activity than 8,9-methylenedioxygroup at the B-ring of 12 (pterocarpin). In cytotoxicity study, 8 and 13 showed non-toxicity to the normal (MDCK) and cancer (HL-60) cells and moderate toxicity to neuroblastoma (SH-SY5Y) cell. Molecular docking simulation revealed that the binding affinities of 8 and 13 for hMAO-B (−8.7 and −7.7 kcal/mol, respectively) were higher than those for hMAO-A (−3.4 and −7.1 kcal/mol, respectively). These findings suggest that compounds 8 and 13 be considered potent reversible hMAO-B inhibitors to be used for the treatment of neurological disorders.


Introduction
Parkinson's disease (PD) is one of the most common neurodegenerative diseases in the elderly, regardless of gender, race, or social status, and affects about 1.5 to 2.0% of people over 60 years of age and 4% of people over 80 years of age [1]. The main cause of PD is loss of dopaminergic neurons in the substantia nigra, and PD patients show major motor symptoms such as tremor and stiffness along with anxiety, depression, and dementia. Increasing dopamine (DA) level and targeting the DA-related serotonin system have been expected as anticipating areas for the treatment of PD [2]. DA levels within substantia nigra pars compacta (SNpc) neurons are maintained through synthesis of DA, synaptic vesicle loading, uptake from the extracellular space, and catabolic degradation. DA catabolism, on the other hand, begins with oxidative deamination, a reaction mediated by mitochondrial monoamine oxidase (MAO, EC 1.4.3.4) that also produces H2O2 and ammonia [3]. Therefore, increasing the concentration of DA through MAO inhibitors can be expected to relieve the symptoms by maintaining the lost DA concentration in PD patients.
Alzheimer's disease (AD), a leading cause of dementia, is a progressive neurodegenerative disease [4]. AD affects more than 50 million people, and its main symptoms are memory and cognitive declines [5]. Various inhibitors have been being studied for the drugs of AD treatment. As one of the inhibitors, MAO inhibitors are used to treat neurological and psychiatric disorders by increasing levels of neurotransmitter such as DA, serotonin (or 5-hydroxytryptamine, 5-HT), and norepinephrine, thereby reducing MAO activity, which can cause neurological disorders [6]. On the other hand, cholinesterase (ChE) inhibitors have been studied as important tools for the treatment of AD. Acetylcholine (ACh) is a neurotransmitter in the central and peripheral nervous systems, and thus, a decrease in ACh by acetylcholinesterase (AChE) has been identified as a major cause of AD, and therefore many researches about ChE inhibitors are ongoing [7]. In addition, aggregation of beta-amyloid (Aβ) produced by beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) was also studied as a cause of AD [8,9].
More specifically, MAO catalyzes the oxidative deamination of monoamines during neurotransmission with two isoforms, namely, MAO-A and MAO-B, in mitochondrial outer membranes [10]. MAO is a pharmacological target because it is involved in catecholamine and 5-HT inactivation pathways. In fact, selective reversible inhibitors of MAO-A have been targeted for anti-depression treatment, while selective reversible inhibitors of MAO-B are targets of therapeutic agents for AD and PD. Typically, selegiline, rasagiline, pargyline, and clorgyline are used as selective MAO inhibitors to reduce the symptoms of neurodegenerative and neurological diseases [11,12]. On the other hand, it was reported that MAO is also associated with the formation of amyloid plaques, which is a main cause of AD [13]. In addition, serious side effects of non-selective MAO inhibitors have been reported [14]. For this reason, selective MAO-B inhibitors based on diverse scaffolds have been actively researched (for reviews see [15][16][17]) and recently reported [18,19]. AChE (EC 3.1.1.7) shows therapeutic efficacy by increasing synaptic ACh levels in the cerebral cortex of AD patients, thereby improving cholinergic transmission [20]. As an AChE inhibitor, tacrine was used as a first drug for AD treatment. However, tacrine has been found to have a severe hepatotoxicity and is not currently used. Since then, donepezil, rivastigmine, and galantamine have been used as AChE inhibitors for therapeutic agents [11]. Like AChE, butyrylcholinesterase (BChE) is a serine hydrolase that rapidly affects the hydrolysis of the neurotransmitter ACh. The main catalytic activity of BChE is the hydrolysis of the neurotransmitters such as ACh and butyrylcholine (BCh) with a preference of BCh [21,22]. BChE is also involved in the development of the nervous system, detoxification, hydrolysis of drugs such as cocaine, heroin and aspirin, fat metabolism, and the interaction and functional modification of other proteins such as polyproline and trypsin [23]. In addition, inhibition of BACE1 related to the accumulation of Aβ for the treatment of AD patients is regarded as one of the important factors. Although many studies about BACE1 inhibitors have been reported, no one has been successful [24]. However, recently aducanumab is found to be effective to reduce Aβ plaques approved as a drug for AD by FDA [25]. Therefore, it is judged that BACE1 inhibitors are deserved to be extensively studied to reduce Aβ plaques.
Furthermore, multitargeting therapeutic strategies have been developed to target MAO-B, AChE, and/or BACE1 [26,27]. It has been reported MAO and AChE inhibitors may improve cognitive functions and alleviate symptoms in AD by elevating levels of monoamines and choline esters [28,29].
During our on-going efforts to identify potent compounds in an herbal extract library, rhamnocitrin isolated from the leaves of Prunus padus var. seoulensis was found to potently and selectively inhibit human MAO-A (hMAO-A) [30], and calycosin and 8-Omethylretusin isolated from Maackia amurensis were identified as selective human MAO-B (hMAO-B) inhibitors [31]. In addition, ellagic acid isolated from Castanopsis cuspidata var. sieboldii showed inhibition of hMAO-B and BACE1 as a multi-target inhibitor [32].
Canavalia lineata is a tendril perennial plant that blooms from June to August and bears fruit in October, rarely growing on the beach. C. lineata grows on Jeju Island in Korea and is also distributed in China, Japan, and Taiwan. It is mainly used as feed for livestock, but because of its toxicity, it was also used for abortion in the past [33]. A few physiological activities of C. lineata have been reported about anti-inflammation [34], and proteinase inhibition [35]. However, no study has been reported about MAO inhibitory activity of C. lineata. In the present study, we isolated thirteen compounds from the pods of C. lineata by activity-guided screening for hMAO inhibition, and evaluated their inhibitory effects on hMAO-A, hMAO-B, AChE, BChE, and BACE1, including kinetic studies and molecular docking simulation.

MAO Inhibitory Activities
A primary screening for MAO inhibition was performed using MeOH extract of C. lineata pods, and its EtOAc and BuOH fractions. The MeOH extract showed high MAO-B inhibition with residual activity of 53.5%. However, inhibition of MAO-A, AChE, BChE, and BACE1 inhibitions were weak (residual activities of 76.1, 78.2, 98.9, and 76.1%, respectively). After second additional extraction, MAO-B residual activity of EtOAc fraction (41.4%) was higher than that of BuOH fraction (85.9%) ( Table 1). During the isolation of compounds from the extracts, thirteen compounds were identified and evaluated for their hMAO-A, hMAO-B, AChE, BChE, and BACE1 inhibitory activities. Four compounds showed MAO-A residual activities of less than 50%, and two compounds showed MAO-B residual activities of less than 10% ( Table 2). In IC 50 determination, compound 8 (medicarpin) showed the highest inhibitory activity against MAO-B with an IC 50 value of 0.45 µM (Table 2, Figure S16), followed by 13 (homopterocarpin, IC 50 = 0.72 µM) (Table 2, Figure  S17). However, compound 12 (another pterocarpan) showed 7.47 times lower MAO-B inhibition (IC 50 (Table 2). However, most of the compounds showed low inhibitory activity against AChE, BChE, and BACE1 (Table 1).

Analysis of the Reversibility of hMAO-A and hMAO-B Inhibitions
Reversibilities of hMAO-A and hMAO-B inhibitions by compounds 8 and 13 were investigated by dialysis. Inhibition of hMAO-A by 13 was substantially recovered from 25.4% (A U ) to 75.8% (A D ), and these values by 13 were similar to those by the reversible inhibitor toloxatone (22.7 to 84.6%). Little recovery was observed for the irreversible inhibitor clorgyline (23.7 to 32.1%) (Figure 3). Inhibitions of hMAO-B by 8 and 13 were substantially recovered from 29.8% (A U ) to 72.7% (A D ), and from 32.0% to 73.5%, respectively, and these values were similar to those observed for the reversible inhibitor lazabemide (31.6 to 83.3%) ( Figure 4). Besides, the irreversible inhibitor pargyline did not show activity recovery at all (34.4 to 29.1%). These results indicated compound 8 was a reversible inhibitor of hMAO-B and 13 was a reversible inhibitor of hMAO-A or hMAO-B.
hibitor clorgyline (23.7 to 32.1%) (Figure 3). Inhibitions of hMAO-B by 8 and 13 were substantially recovered from 29.8% (AU) to 72.7% (AD), and from 32.0% to 73.5%, respectively, and these values were similar to those observed for the reversible inhibitor lazabemide (31.6 to 83.3%) ( Figure 4). Besides, the irreversible inhibitor pargyline did not show activity recovery at all (34.4 to 29.1%). These results indicated compound 8 was a reversible inhibitor of hMAO-B and 13 was a reversible inhibitor of hMAO-A or hMAO-B.

Analysis of Inhibitory Patterns
Enzyme kinetics of compounds 8 and 13 were investigated using Lineweaver-Burk plots. Plots of hMAO-B inhibition by 8 were linear and intersected the y-axis ( Figure  5A,B), indicating compound 8 is a competitive inhibitor of MAO-B. Secondary plots of the slopes of Lineweaver-Burk plots against inhibitor concentration showed the Ki value of 8 was 0.197 ± 0.004 µM ( Figure 5B). Similarly, compound 13 was found to be a competitive inhibitor of hMAO-A or hMAO-B with Ki values of 0.617 ± 0.023 ( Figure 6A,B) and 0.212 ± 0.008 µM ( Figure 6C,D), respectively. These Ki values of 8 and 13 for MAO-B were slightly higher than those of the potent fluorinated chalcones f1 and f2 (0.027 and 0.020 µM, respectively) [46], 1-methyl, 5-phenyl substituted thiosemicarbazones MT5 (6.58 µM) [47]. These results indicate compounds 8 was a potent competitive inhibitor of hMAO-B and compound 13 was an effective competitive inhibitor of hMAO-A and a more effective competitive inhibitor of hMAO-B.

Analysis of Inhibitory Patterns
Enzyme kinetics of compounds 8 and 13 were investigated using Lineweaver-Burk plots. Plots of hMAO-B inhibition by 8 were linear and intersected the y-axis ( Figure 5A,B), indicating compound 8 is a competitive inhibitor of MAO-B. Secondary plots of the slopes of Lineweaver-Burk plots against inhibitor concentration showed the K i value of 8 was 0.197 ± 0.004 µM ( Figure 5B). Similarly, compound 13 was found to be a competitive inhibitor of hMAO-A or hMAO-B with K i values of 0.617 ± 0.023 ( Figure 6A,B) and 0.212 ± 0.008 µM ( Figure 6C,D), respectively. These K i values of 8 and 13 for MAO-B were slightly higher than those of the potent fluorinated chalcones f1 and f2 (0.027 and 0.020 µM, respectively) [46], 1-methyl, 5-phenyl substituted thiosemicarbazones MT5 (6.58 µM) [47]. These results indicate compounds 8 was a potent competitive inhibitor of hMAO-B and compound 13 was an effective competitive inhibitor of hMAO-A and a more effective competitive inhibitor of hMAO-B.

Cytotoxicity Test
The effects of 8 and 13 on the viabilities of MDCK, HL-60, and SH-SY5Y cells were investigated using the CCK-8 assay. Compounds 8 and 13 showed no effects on the viabilities of MDCK (normal cell line) or HL-60 (cancer cell line) cells at 50 µM, the highest concentration tested (Figure 7A,B). However, 8 and 13 showed moderate toxicity (74.05% and 71.12% viability, respectively) to SH-SY5Y (neuroblastoma) cells at 30 µM, which is 66.7-and 41.7-times higher, respectively, than IC50 values of 8 and 13 ( Figure 7C). These results suggest that 8 and 13 are non-toxic to the cells tested with a moderate toxicity to

Cytotoxicity Test
The effects of 8 and 13 on the viabilities of MDCK, HL-60, and SH-SY5Y cells were investigated using the CCK-8 assay. Compounds 8 and 13 showed no effects on the viabilities of MDCK (normal cell line) or HL-60 (cancer cell line) cells at 50 µM, the highest concentration tested (Figure 7A,B). However, 8 and 13 showed moderate toxicity (74.05% and 71.12% viability, respectively) to SH-SY5Y (neuroblastoma) cells at 30 µM, which is 66.7-and 41.7-times higher, respectively, than IC 50 values of 8 and 13 ( Figure 7C). These results suggest that 8 and 13 are non-toxic to the cells tested with a moderate toxicity to SH-SY5Y cells.

Molecular Docking Simulation
Major compounds 8 and 13, two pterocarpans, were docked to hMAO-A and hMAO-B. Compound 12 was also included in docking simulation, because it was a pterocarpan derivative, though it was not a potent hMAO-B inhibitor. The docking simulations showed that the chemicals were located properly within the active region of hMAO-A (PDB: 2Z5X) and hMAO-B (PDB ID:4A79) (Figure 8). The compounds were docked in similar locations hMAO-A or hMAO-B, based on the surrounded amino acid residues: the

Molecular Docking Simulation
Major compounds 8 and 13, two pterocarpans, were docked to hMAO-A and hMAO-B. Compound 12 was also included in docking simulation, because it was a pterocarpan derivative, though it was not a potent hMAO-B inhibitor. The docking simulations showed that the chemicals were located properly within the active region of hMAO-A (PDB: 2Z5X) and hMAO-B (PDB ID:4A79) (Figure 8). The compounds were docked in similar locations hMAO-A or hMAO-B, based on the surrounded amino acid residues: the compounds were located near FAD cofactor in hMAO-A, whereas they were located far from the FAD site ( Figure 8). Three compounds showed higher binding affinities to hMAO-B than hMAO-A, representing they are selective hMAO-B inhibitor. Compound 8 showed the highest binding affinity (−8.7 kcal/mol) for hMAO-B as compared to other docking complexes, and formed a hydrogen bond interaction with Cys172 residue at a distance of 3.328Å ( Figure 8A,B, Table 3). Compound 13 also displayed similar results, but relatively lower binding affinities for both of MAO-A (−7.1 kcal/mol) and hMAO-B (−7.7 kcal/mol) by hydrogen bond interactions with Tyr444 (hMAO-A at a distance of 3.588 Å) and Cys172 (hMAO-B at a distance of 3.333 Å), respectively ( Figure 8E,F, Table 3). Compound 12, which differs in only substituent of the B-ring of compound 13, was predicted to have no hydrogen bond interaction with hMAO-A and hMAO-B and showed lower binding affinities (−2.4 and −7.5 kcal/mol, respectively) to both hMAO-A and hMAO-B than 8 and 13 ( Figure 8C,D, Table 3). hMAO-B has relatively narrow 'gate' (defined by Ile199 and Tyr326) in the cavity, although hMAO-B has high sequence identity ≥70% to hMAO-A [48]. In this aspect, it might be suggested that 3-hydroxy group in 8 was more favored to hMAO-B than 3-methoxy group in 13, and 8,9-methylenedioxy group in 12 decreased the affinity to hMAO-B compared to 9-methoxy group in 8 and 13 ( Figure 8C,D). In addition, docking scores using AutoDock Vina are calculated based on considerations of various factors such as hydrogen bonding, electrostatic bonding, van der Waals forces, and dissolvent effects [49]. These scores obtained in this study were coincided well to the IC 50 values of the compounds determined experimentally. These results suggest that compound 8 would be the most selective inhibitor against hMAO-B and compound 13 have effective inhibition activity against both hMAO-A and hMAO-B.

In Silico Pharmacokinetics of Medicarpin (8) and Homopterocarpin (13)
The pharmacokinetics of compounds 8 and 13 using SwissADME web tool showed these two compounds had high GI absorption and BBB permeability (Table 4). However, drug suitability of two compounds should be investigated in further study, because these compounds showed CYP450 inhibition including the important enzyme CYP3A4, although cytotoxicity to three cell lines tested above was low and some of the drugs show CYP3A4 inhibition properties [50]. Lipinski violation is judged by counting the number of violations in the Lipinski's rule, i.e., MW < 500, cLog P o/w < 5.00, HBD < 5, HBA < 10, TPSA < 140 Å 2 , and RB < 10. For Lipinski parameters of these two compounds, no violations of Lipinski's rule of five were predicted (Table 5). These results suggest that compounds 8 and 13 can used as central nervous system (CNS) drugs.
decreased the affinity to hMAO-B compared to 9-methoxy group in 8 and 13 ( Figure  8C,D). In addition, docking scores using AutoDock Vina are calculated based on considerations of various factors such as hydrogen bonding, electrostatic bonding, van der Waals forces, and dissolvent effects [49]. These scores obtained in this study were coincided well to the IC50 values of the compounds determined experimentally. These results suggest that compound 8 would be the most selective inhibitor against hMAO-B and compound 13 have effective inhibition activity against both hMAO-A and hMAO-B.   (8) and Homopterocarpin (13) The pharmacokinetics of compounds 8 and 13 using SwissADME web tool showed these two compounds had high GI absorption and BBB permeability (Table 4). However, drug suitability of two compounds should be investigated in further study, because these

General Experimental Procedures
NMR spectroscopic data were recorded by using JEOL ECZ500R [ 1 H NMR at 500 MHz, 13

Enzyme Assays
hMAO-A and -B activities were determined using a continuous spectrophotometric method, as described previously [53,54]. The K m values of hMAO-A for kynuramine and hMAO-B for benzylamine were 0.043 and 0.14 mM, respectively. The concentrations of kynuramine (0.06 mM) and benzylamine (0.3 mM) used were 1.4-and 2.1-times K m values, respectively. AChE and BChE activities were assayed as described by Ellman et al. [55], with slight modification [40]. Reactions were performed using AChE and BChE in the 0.5 mL reaction mixtures including 0.5 mM substrate (ATCI and BTCI, respectively) and color reagent (DTNB). Absorbance measurements were continuously monitored for 10 min at 412 nm. Reaction rates are expressed as changes in absorbance per min. [56]. Inhibitory activities of AChE and BChE were measured after preincubating enzyme with inhibitors for 15 min before adding DTNB and each substrate. BACE1 assay was measured by the activity detection kit using a fluorescence spectrometer (FS-2, Scinco, Seoul, Republic of Korea), for 2 h at 37 • C with 7-methoxycoumarin-4-acetyl-[Asn670, Leu671]-amyloid β/A4 protein fragment 667-676-(2,4-dinitrophenyl)Lys-Arg-Arg amide trifluoroacetate as a substrate [57].

Kinetics of Enzyme Inhibition
Inhibitions of hMAO-A, hMAO-B, AChE, BChE, and BACE1 by the thirteen compounds were initially investigated at a concentration of 10 µM, and then IC 50 values of the compounds and the reference inhibitors (toloxatone and clorgyline for hMAO-A, lazabemide and pargyline for hMAO-B, donepezil for AChE and BChE, and quercetin for BACE1) were determined. Kinetic parameters, inhibition types, and K i values of the most potent hMAO-B inhibitors (compounds 8 and 13) were analyzed, as described previously [31]. The kinetics of MAO-B inhibition by 8 and 13 were investigated at five different substrate concentrations (0.03-0.6 mM) and in the absence or presence of each inhibitor at three concentrations of~0.5, 1.0, and 2.0 times of each IC 50 value [30]. The kinetics of MAO-A inhibition by 13 was also investigated as above, except at 0.0075-0.12 mM of substrate concentrations. Inhibitory types and K i values were determined using Lineweaver-Burk plots and secondary plots of their slopes, respectively.

Analysis of Inhibitor Reversibility
The reversibilities of hMAO-A or hMAO-B inhibitions by compounds 8 and 13 were investigated by dialysis, as previously described [53], at~2 times of IC 50 concentrations. Reversible and irreversible reference inhibitors were also included in the experiment at 2 times of IC 50 concentrations. After preincubating compounds or reference inhibitors with hMAO-A and hMAO-B, residual activities for undialyzed and dialyzed samples were measured. Relative values for undialyzed (A U ) and dialyzed (A D ) activities were then calculated and compared with non-inhibitor treated controls. Reversibilities were determined by comparing the A U and A D values of inhibitors with those of references.

Cell Viability
MDCK, HL-60, and SH-SY5Y cells were cultured in DMEM, RPMI 1640, or MEM, respectively [58], supplemented with heat-inactivated 10% FBS, 100 U/mL Penicillin/ Streptomycin solution, and 50 µM 2-ME in a humidified atmosphere at 37 • C with 5% CO 2 with media change every 2 days, using 75 cm 2 T-flask, and were grown until 80% confluent [58]. Passages were used between 10 and 30. Following trypsinization, MDCK (1 × 10 4 ), HL-60 (5 × 10 4 ), and SH-SY5Y (5 × 10 4 ) cells were seeded in 96-well plates and treated with 1, 3, 10, 30 and 50 µM of each inhibitor. The plates were then incubated for 24 h at 37 • C with 5% CO 2 , and cell viability was determined by the CCK-8 and by measuring the optical density at 450 nm using a microplate reader (Versa Max, Molecular Devices, Sunnyvale, CA, USA). The cell viability was expressed as % by comparing the absorbance of the sample to that of the control [59]. Statistical differences between groups were analyzed by ANOVA using IBM SPSS statistics 27 and probability values (p-values) less than 0.05 were marked as significant values.

Docking Simulations of the Compounds with MAO-A and MAO-B
AutoDock Vina was used to simulate docking of the chemicals to MAO-A and MAO-B [47]. The binding sites of the chemicals were selected to the binding pocket of 7-methoxy-1-methyl-9H-beta-carboline (HRM) co-crystallized with hMAO-A (PDB ID: 2Z5X), and the binding pocket of (5R)-5-{4-[2-(5-ethylpyridin-2-yl)ethoxy]benzyl}-1,3-thiazolidine-2,4dione (P1B) co-crystallized with hMAO-B (PDB ID: 4A79). To define these binding pockets, the grid boxes for docking were centered to 40.582, 26.931, and −14.540 for hMAO-A, and 50.730, 157.601, and 30.131 for hMAO-B, with dimensions of 15 × 15 × 15 Å. The following steps were carried out to prepare the chemicals for the docking simulation: creation of 2D structures of the chemicals [60], conversion of the 2D structures into 3D structures [61], and energy minimization using the ChemOffice program (http://www.cambridgesoft.com, 23 February 2021) [61]. Docking simulations of MAO-A and MAO-B with the derivatives were performed using Chimera [62]. Based on the docking results, possible hydrogen bonding interactions were checked with relaxation constraints of 0.6 Å and 20.0 • [62]. Amino acids with overlapped volume of van der Waals more than −0.4 Å with any atoms in the chemicals were defined as favorable residues.

Conclusions
Thirteen compounds were isolated from the methanolic extract of C. lineata including EtOAc and BuOH fractions, and identified as one flavonol, two flavanones, five isoflavones, two syringaresinols, and three pterocarpans. Compounds medicarpin (8) and homopterocarpin (13) were reversible and competitive inhibitor against hMAO-B (IC 50 = 0.45 and 0.72 µM, respectively), with high selectivity (SI = 44.2) and low selectivity (SI = 2.07), respectively. Structurally, the group 3-OH at A-ring of 8 increased hMAO-B inhibitory activity, compared to 3-OCH 3 group at the A-ring of 13. In addition, 9-OCH 3 group at the B-ring of 8 and 13 showed higher MAO-B inhibition than 8,9-methylenedioxy group at the B-ring of 12. Compounds 8 and 13 were non-or less toxic to the normal and cancer cells, including neuroblastoma cells. Molecular docking simulation revealed that 8 or 13 both bonded to Cys172 of hMAO-B with a binding affinity of −8.7 and −7.7 kcal/mol, respectively. However, compound 12 was not predicted to have hydrogen bond to MAO-B. These findings suggest that compounds 8 and 13, a novel potent and reversible hMAO-B inhibitor, should be considered potential candidate agents for the treatment of neurological disorders.