Synthesis and Evaluation of Novel S-alkyl Phthalimide- and S-benzyl-oxadiazole-quinoline Hybrids as Inhibitors of Monoamine Oxidase and Acetylcholinesterase

New S-alkyl phthalimide 5a–f and S-benzyl 6a–d analogs of 5-(2-phenylquinolin-4-yl)-1,3,4-oxadiazole-2-thiol (4) were prepared by reacting 4 with N-bromoalkylphthalimide and CF3-substituted benzyl bromides in excellent yields. Spectroscopic techniques were employed to elucidate the structures of the synthesized molecules. The inhibition activity of newly synthesized molecules toward MAO-A, MAO-B, and AChE enzymes, was also assessed. All these compounds showed activity in the submicromolar range against all enzymes. Compounds 5a and 5f were found to be the most potent compounds against MAO-A (IC50 = 0.91 ± 0.15 nM) and MAO-B (IC50 = 0.84 ± 0.06 nM), while compound 5c showed the most efficient acetylcholinesterase inhibition (IC50 = 1.02± 0.65 μM). Docking predictions disclosed the docking poses of the synthesized molecules with all enzymes and demonstrated the outstanding potency of compounds 5a, 5f, and 5c (docking scores = −11.6, −15.3, and −14.0 kcal/mol against MAO-A, MAO-B, and AChE, respectively). These newly synthesized analogs act as up-and-coming candidates for the creation of safer curative use against Alzheimer’s illness.


Introduction
Alzheimer's disease (AD) is a complex neurological disturbance associated with memory loss and language skills, as well as behavioral and psychological changes [1]. According to the Alzheimer's Association report, about 0.47 billion people globally suffer from AD, and the number of patients will exceed 1.50 billion in 2050 [2]. AD is a multifactorial pathological condition with several causes. Among these, the deposition of beta-amyloid fibers, the death of neural cells, and high levels of monoamine oxidase (MAO) and acetylcholinesterase (AChE) enzymes occur [3,4].
The MAO enzyme presents in the exterior membrane of mitochondria [5] and metabolizes a variety of dietary amines besides neurotransmitters [6]. There are two known variants of the MAO enzyme, MAO-A and MAO-B, which have 70% similarity in protein structure and can be found in different parts of the body, liver, and brain [7]. MAO-B catalyzes beta-phenylethylamine and benzylamine, while MAO-A preferably catalyzes serotonin, noradrenaline, and adrenaline [5]. The inhibition of these enzymes plays a key role in AD [8,9]. In addition to MAOs, the AChE enzyme is a target for AD management [10,11]. The inhibition of these neural enzymes (AChE and MAOs) possess neuroprotective effects and decreases oxidative stress [12], leading to an increased level of neurotransmitters in the pre-synaptic cleft [13]. A diversity of dual-inhibiting molecules has developed by merging the moieties for MAOs and AChE in one compound [14]. Different research groups have identified various chemical classes bearing benzylamine [15,16], coumarin [17], oxazole, triazole [18], indole [19], quinolinone [20], isoindoline [21], and oxadiazole [22], as potent inhibitors of the targeted enzymes. Among those classes, quinoline has gained much attention as a privileged scaffold for multi-targeting enzymes (MAOs and AChE).
Towards discovering new compounds for MAO and AChE enzymes inhibition, herein we are reporting the synthesis, structure elucidation, molecular modeling, and enzyme inhibition of novel S-alkyl phthalimide-and S-benzyl-oxadiazole-quinoline hybrids.

Inhibitory Activity and SAR
In the synthesized compounds, the oxadiazole ring was substituted with quinoline on the left side. Besides, the oxadiazole ring was replaced with an isoindoline on the right side. The position of different functional groups on the basic pharmacophore ((2-phenylquinolin-4-yl)-1,3,4-oxadiazol-2-yl)) ring was studied to get specific information about the identification and selectivity of compounds to inhibit MAOs and AChE enzymes. Two series of compounds (5a-f and 6a-d) showed activity in low μM to nM ranges on the inhibition of MAOs and AChE enzymes (Table 1). Clorgyline and deprenyl were utilized as reference ligands for MAO-A and MAO-B, respectively. At the same time, donepezil Scheme 1. Synthesis of S-alkyl phthalimide-and S-benzyloxadiazole-quinoline hybrids (5a-f and 6a-d).
The target molecules 5a-f and 6a-d were purified by recrystallization, and the structures of novel molecules were authenticated with the help of complementary analytical tools, including 13 C-NMR, 1 H-NMR, and FT-IR spectroscopic techniques ( Figure S1). Compound 5a's 1H-NMR analysis revealed typical peaks in the aromatic and aliphatic areas. The singlet for the two protons at δ = 5.54 ppm represented methylene proton (S-CH 2 -N) Pharmaceuticals 2023, 16, 11 4 of 21 surrounded by sulfur and nitrogen atoms. In compounds 5b-f, a triplet for the methylene protons of the S-CH 2 group was found between δ = 3.45 and δ = 3.26 ppm, whereas the methylene protons of N-CH 2 were found between δ = 3.77 and δ = 3.56 ppm. The methylene groups between N-CH 2 and S-CH 2 of compounds 5c-f ranged from δ = 2.19 to δ = 1.32 ppm.
The protons present in the benzylic S-CH 2 group of compounds 6a-d were observed as a singlet that ranged from δ = 4.69 to δ = 4.85 ppm. The singlet for one proton on the adjacent carbon atoms of the quinoline ring was observed between δ = 8.65 and δ = 8.06 ppm. All the protons present in the aromatic rings of the quinoline and phthalimide in compounds 5a-f were found in the range of δ 9.05-7.13 ppm, whereas aromatic protons present in the aromatic ring of quinoline and S-benzyl in compounds 6a-d were observed in the range of δ 9.05-7.35 ppm.
Similarly, 13 C NMR of compounds 5a-f and 6a-d showed peaks for relevant carbon atoms at appropriate positions. Carbon atoms present in the carbonyl group of phthalimide moiety in compounds 5a-f were observed above δ 164 ppm. The carbon atoms OCN and SCN of the 1,3,4-oxadiazole in compounds 5a-f and 6a-d ring were observed between δ 164 and δ 155 ppm. All the carbon atoms present in the aromatic rings were found in the range of δ 149-118 ppm. The bridging carbon atom (SCN) present in 5a was observed at δ 39.13 ppm. The two bridging carbon atoms (SCCN) present between S and N atoms in compound 5b were observed at δ 37.13 and 30.14 ppm. The carbon atoms linked to the N atom were found in the range of δ 38-35 ppm. The carbon atoms linked to S atom were found in the range of δ 32-30 ppm. Other carbon atoms of the alkyl chain between N and S atoms in 5c-f were observed below δ 30 ppm. The benzylic carbon of compounds 6a-d was observed between δ 35.98 and δ 33.51 ppm.

Inhibitory Activity and SAR
In the synthesized compounds, the oxadiazole ring was substituted with quinoline on the left side. Besides, the oxadiazole ring was replaced with an isoindoline on the right side. The position of different functional groups on the basic pharmacophore ((2phenylquinolin-4-yl)-1,3,4-oxadiazol-2-yl)) ring was studied to get specific information about the identification and selectivity of compounds to inhibit MAOs and AChE enzymes. Two series of compounds (5a-f and 6a-d) showed activity in low µM to nM ranges on the inhibition of MAOs and AChE enzymes (Table 1). Clorgyline and deprenyl were utilized as reference ligands for MAO-A and MAO-B, respectively. At the same time, donepezil was utilized as a positive AChE control. Compounds 5a and 5f showed activity in submicromolar ranges against MAO-A and MAO-B (IC 50 = 0.91 ± 0.15 and 0.84 ± 0.06 µM, respectively). For AChE enzyme inhibition, compound 5c demonstrated the most active compound (IC 50 = 1.02 ± 0.65 µM).

Kinetic Studies
To determine the method of targeting MAO-A, MAO-B, and AChE enzymes, kinetic experiments for the most potent molecules (5a, 5f, and 5c) were carried out ( Figure 3). Different test chemicals and substrate concentrations were utilized in the kinetic experiments. Lineweaver-Burk plots were employed to determine the kind of inhibition, and they were used to track how the inhibitor affected K m and V max by plotting the reciprocal reaction rate against the reciprocal substrate concentrations. As depicted in Figure 3, the investigated molecules displayed a pure competitive type of inhibition as the V max of enzymes was unaffected by differing doses of the test compounds while the K m increased.

Kinetic Studies
To determine the method of targeting MAO-A, MAO-B, and AChE enzymes, kinetic experiments for the most potent molecules (5a, 5f, and 5c) were carried out ( Figure 3). Different test chemicals and substrate concentrations were utilized in the kinetic experiments. Lineweaver-Burk plots were employed to determine the kind of inhibition, and they were used to track how the inhibitor affected Km and Vmax by plotting the reciprocal reaction rate against the reciprocal substrate concentrations. As depicted in Figure 3, the investigated molecules displayed a pure competitive type of inhibition as the Vmax of enzymes was unaffected by differing doses of the test compounds while the Km increased.

Docking Studies
To investigate the docking pose of the most promising molecules with the binding pocket of the targeted enzymes, molecular docking predictions were executed. The assessment of the AutoDock4.2.6 software with the utilized settings was first carried out in accordance with the accessible experimental data. The co-crystallized ligands -namely, harmine, safinamide, and donepezil-with the MAO-A, MAO-B, and AChE were redocked and compared to the experimentally resolved structures (PDB codes: 2Z5X, 2V5Z, and 4EY7, respectively) ( Figure 4). As depicted in Figure 4, the portended docking poses resembled the native structures, having 0.23, 0.27, and 0.24 Å RMSD in relation to the cocrystallized conformations of harmine, safinamide, and donepezil, respectively ( Figure 4). Summing up, the employed docking protocol would be utilized to predict the correct docking pose of inhibitors with the targeted enzymes.

Docking Studies
To investigate the docking pose of the most promising molecules with the binding pocket of the targeted enzymes, molecular docking predictions were executed. The assessment of the AutoDock4.2.6 software with the utilized settings was first carried out in accordance with the accessible experimental data. The co-crystallized ligands -namely, harmine, safinamide, and donepezil-with the MAO-A, MAO-B, and AChE were re-docked and compared to the experimentally resolved structures (PDB codes: 2Z5X, 2V5Z, and 4EY7, respectively) ( Figure 4). As depicted in Figure 4, the portended docking poses resembled the native structures, having 0.23, 0.27, and 0.24 Å RMSD in relation to the co-crystallized conformations of harmine, safinamide, and donepezil, respectively ( Figure 4). Summing up, the employed docking protocol would be utilized to predict the correct docking pose of inhibitors with the targeted enzymes.
Utilizing the docking protocol, the binding scores and modes of 5a, 5f, and 5c with MAO-A, MAO-B, and AChE enzymes, respectively, were anticipated ( Figure 5). As illustrated in Figure 5, the investigated compounds demonstrated good inhibition affinity toward the inspected enzymes, with docking scores of -11.6, -15.3, and -14.0 kcal/mol with MAO-A, MAO-B, and AChE, respectively. The monitored possibility of inspected compounds towards MAO-A, MAO-B, and AChE might be ascribed to their capacity to exhibit numerous H-bonds, hydrophobic, vdW, and π-based interactions with the substantial residues within the binding pockets of these enzymes. Utilizing the docking protocol, the binding scores and modes of 5a, 5f, and 5c with MAO-A, MAO-B, and AChE enzymes, respectively, were anticipated ( Figure 5). As illustrated in Figure 5, the investigated compounds demonstrated good inhibition affinity toward the inspected enzymes, with docking scores of -11.6, -15.3, and -14.0 kcal/mol with MAO-A, MAO-B, and AChE, respectively. The monitored possibility of inspected compounds towards MAO-A, MAO-B, and AChE might be ascribed to their capacity to exhibit numerous H-bonds, hydrophobic, vdW, and π-based interactions with the substantial residues within the binding pockets of these enzymes. When the results were compared with the co-crystallized conformations of harmine, safinamide, and donepezil, many common interactions were observed ( Figure 5). The presence of a quinoline ring formed the π-alkyl interactions with ILE180, LEU337, ILE335, LEU164, and ILE316 amino acids ( Figure 5). Similarly, the 1,3,4-oxadiazol and isoindoline-,3-dione ring exhibited hydrogen bond, π-π stacked, and π-amide interactions with ASN141, TYR435, TYR124, TYR405, TYR398, TYR337, and TYR341 ( Figure 5). When the results were compared with the co-crystallized conformations of harmine, safinamide, and donepezil, many common interactions were observed ( Figure 5). The presence of a quinoline ring formed the π-alkyl interactions with ILE180, LEU337, ILE335, LEU164, and ILE316 amino acids ( Figure 5). Similarly, the 1,3,4-oxadiazol and isoindoline-,3-dione ring exhibited hydrogen bond, π-π stacked, and π-amide interactions with ASN141, TYR435, TYR124, TYR405, TYR398, TYR337, and TYR341 ( Figure 5).

Drug-Like and ADMET Characteristics
The drug-like and ADMET features of the potent molecules were computed and compared with clorgyline, deprenyl, and donepezil as controls ( Table 2). A compound's hydrophilicity is indicated by Log P; if the value is negative, the compound is hydrophilic. All of the compounds in Table 2 are lipophilic. The total number of OH and NH atoms equals the nHBD, while the total number of N and O atoms resembles the nHBA. As listed in Table 2, the nHBA and nHBD are in the optimal ranges (nHBD < 5 and nHBA < 10). The ideal lipophilicity for BBB penetration for medications having CNS activity is a Log D ≤ 2. A Log D of more than 4 is considered as not suitable for a CNS medication. In vivo intestinal Pharmaceuticals 2023, 16, 11 9 of 21 medication absorption is demonstrated by CaCo-2 permeability values. The ideal range of values should be more than −5.15 Log unit. All compounds showed normal values of CaCo-2 permeability. The higher HIA value, the higher intestinal absorption will be. All studied molecules pointed out an appropriate HIA value compared to the controls. Because of their lipophilic nature, all of our compounds can cross BBB. In metabolism and excretion, all investigated compounds were inhibitors of CYP2C19 (Table 2). A drug's bodily clearance rate is categorized as high (>15), moderate (5-15), or low (<5). All the newly synthesized compounds had a low clearance rate. The AMES toxicity was evaluated. All compounds manifested low toxicity. Therefore, it can be suggested that all potent derivatives have a good ADMET profile compared to the controls. Further investigation is required to improve the toxicity profile of compounds.

Density Function Theory (DFT) Calculations
Further insight into the features of the investigated compounds was traced using a plethora of quantum mechanical calculations. In the first place, geometrical optimization was executed at the B3LYP/6-31G level of theory, and the obtained structures are illustrated in Figure 6. Afterwards, frequency computations were exerted, outlining that the optimized structures were true minima with no observable imaginary frequency values. Upon the optimized compounds, single-point energy and Frontier molecular orbitals (FMO) calculations were performed, and the global reactivity parameters were evaluated. Diagrams of HOMO and LUMO distributions are displayed in Figure 7. Table 3 enrolls energies of the optimized compounds (E opt ), the highest occupied molecular orbital (E HOMO ), and the lowest unoccupied molecular orbital (E LUMO ), along with the energy gap (E gap ), global hardness (η), global softness (σ), polarizability (α), and dipole moment (µ) of the studied compounds. Table 3. Energies of the optimized compounds (E opt , in au), the highest occupied molecular orbital (E HOMO , in eV), and the lowest unoccupied molecular orbital (E LUMO , in eV), along with energy gap (E gap , in eV). Computed values of global hardness (η), global softness (σ), polarizability (α), and dipole moment (µ) of the studied compounds.   In the context of FMO theory, the molecule with less and high negative values of EHOMO and ELUMO, respectively, had superior nucleophilic nature. For ∆Egap, less positive values ensured the noticeable ability of the inspected compound to donate electrons.

Compound
. The most reactive compound would be the one that has the smallest energy gap. The most kinetically stable compound showed a more favorable energy gap compared to the In the context of FMO theory, the molecule with less and high negative values of E HOMO and E LUMO , respectively, had superior nucleophilic nature. For ∆E gap , less positive values ensured the noticeable ability of the inspected compound to donate electrons.
The most reactive compound would be the one that has the smallest energy gap. The most kinetically stable compound showed a more favorable energy gap compared to the others. As evident in Figure 7, HOMO and LUMO distributions were noticed with lower and higher concentrations, respectively, around the phthalimide group in 5a-f compounds. While both distributions were found over the 5-(2-phenylquinolin-4-yl)-1,3,4-oxadiazole-2thiol (4) in the 6a-d compounds.
According to data listed in Table 3, compound 5a would be highly reactive with favorable nucleophilic nature. This observation could be ascribed to the least negative E HOMO (-0.221 eV), highest negative E LUMO (-0.112 eV), and least positive E gap (0.108 eV) values. In comparison, the reversed affirmations were generally noticed in the case of compound 6d, which in turn was addressed as the most kinetically stable compound. The computed values of the global reactivity parameters ensured the FMO affirmations.
According to data listed in Table 3, compound 5a would be highly reactive with favorable nucleophilic nature. This observation could be ascribed to the least negative EHOMO (-0.221 eV), highest negative ELUMO (-0.112 eV), and least positive Egap (0.108 eV) values. In comparison, the reversed affirmations were generally noticed in the case of compound 6d, which in turn was addressed as the most kinetically stable compound. The computed values of the global reactivity parameters ensured the FMO affirmations.

Materials and Apparatus
All commercially obtained chemicals and reagents were employed to accomplish the targeted synthesis. Solvents of analytical grade were employed as provided. Uncorrected

Materials and Apparatus
All commercially obtained chemicals and reagents were employed to accomplish the targeted synthesis. Solvents of analytical grade were employed as provided. Uncorrected melting points were evaluated in open capillaries by a Gallenkamp melting point instrument (MP-D). Thin-layer chromatography was employed to analyze all the reactions. It was executed on Merck pre-coated plates (silica gel 60 F254, 0.25 mm), and was used to visualize the reactions utilizing fluorescence quenching under UV light (254 nm). A Bruker AV-300 spectrometer was applied to measure the 1 H-NMR and 13 C-NMR spectra (300 MHz). ATR was used to record IR spectra on a Shimadzu Fourier Transform Infrared spectrophotometer model (Attenuated Total Reflectance).

General Procedure
A modified multistep synthetic procedure was followed to access 5a-f and 6a-d. In a typical experimental procedure, 2-phenylquinoline-4-carboxylic acid (1) (30 mmol) was esterified in 20 mL of methanol using catalytic amounts of sulfuric acid (0.5 mL) at reflux temperature for 4 h. The mixture was neutralized with 50 mL of saturated NaHCO 3 solution and extracted three times with 30 mL of ethyl acetate. The organic phase was filtered after being dried over anhydrous sodium sulfate. The solvent was then extracted to get crude methyl 2-phenylquinoline-4-carboxylate in quantitative yield.
A solution of 2-phenylquinoline-4-carbohydrazide (3) (20 mmol) in 10 mL of methanol and 3 equivalent KOH dissolved in 30 mL of methanol was inserted. After 10 min, carbon disulfide (30 mmol) was slowly inserted, and the whole reaction mixture was subjected to reflux for 12 h. The reaction mixture was concentrated, brought to room temperature, and then added to ice water. With dilute HCl, the pH of the solution was brought down to 2. Warm water was used to wash the precipitated 5-(2-phenylquinolin-4-yl)-1,3,4-oxadiazole-2-thiol (4) before it was recrystallized from methanol.
Acetone was used to dissolve 5-(2-phenylquinolin-4-yl)-1,3,4-oxadiazole-2-thiol (4) (3 mmol), and potassium carbonate (4 mmol) was added. N-bromoalkylphthalimide (4 mmol) was added to the reaction mixture after it had been agitated at room temperature for 10 min. The reaction mixture was then mixed once more at room temperature for 6 h. To obtain pure 5a-f, the crude was recrystallized from methanol after the solvent was removed.
Acetone was utilized to dissolve 5-(2-Phenylquinolin-4-yl)-1,3,4-oxadiazole-2-thiol (4) (3 mmol), and potassium carbonate (4 mmol) was added. Substituted benzyl bromide (4mmol) was added to the reaction mixture after it had been agitated at room temperature for 10 min. The reaction mixture was then mixed once more at room temperature for 6 h. To get pure 6a-d, the crude was recrystallized from methanol. visualize the reactions utilizing fluorescence quenching under UV light (254 nm). A Bruker AV-300 spectrometer was applied to measure the 1 H-NMR and 13 C-NMR spectra (300 MHz). ATR was used to record IR spectra on a Shimadzu Fourier Transform Infrared spectrophotometer model (Attenuated Total Reflectance).

General Procedure
A modified multistep synthetic procedure was followed to access 5a-f and 6a-d. In a typical experimental procedure, 2-phenylquinoline-4-carboxylic acid (1) (30 mmol) was esterified in 20 mL of methanol using catalytic amounts of sulfuric acid (0.5 mL) at reflux temperature for 4 h. The mixture was neutralized with 50 mL of saturated NaHCO3 solution and extracted three times with 30 mL of ethyl acetate. The organic phase was filtered after being dried over anhydrous sodium sulfate. The solvent was then extracted to get crude methyl 2-phenylquinoline-4-carboxylate in quantitative yield.
A solution of 2-phenylquinoline-4-carbohydrazide (3) (20 mmol) in 10 mL of methanol and 3 equivalent KOH dissolved in 30 mL of methanol was inserted. After 10 min, carbon disulfide (30 mmol) was slowly inserted, and the whole reaction mixture was subjected to reflux for 12 h. The reaction mixture was concentrated, brought to room temperature, and then added to ice water. With dilute HCl, the pH of the solution was brought down to 2. Warm water was used to wash the precipitated 5-(2-phenylquinolin-4-yl)-1,3,4oxadiazole-2-thiol (4) before it was recrystallized from methanol.
Acetone was used to dissolve 5-(2-phenylquinolin-4-yl)-1,3,4-oxadiazole-2-thiol (4) (3 mmol), and potassium carbonate (4 mmol) was added. N-bromoalkylphthalimide (4 mmol) was added to the reaction mixture after it had been agitated at room temperature for 10 min. The reaction mixture was then mixed once more at room temperature for 6 h. To obtain pure 5a-f, the crude was recrystallized from methanol after the solvent was removed.

Monoamine Oxidase
According to the previously reported protocol, the synthesized compounds' action on the monoamine oxidase (MAO-A and MAO-B) enzymes was tested. The enzyme was produced just 15 to 20 min before, at a cold room temperature. Accordingly, clorgyline (60 nM) or deprenyl (300 nM) were used to permanently inhibit MAO-A and MAO-B activity. White 96-well plates were utilized for the test. The assay volume was 100 µL, having 60 µL buffer (pH 7.4) and 10 µL test compound (0.1 mM, 10% DMSO), followed by adding enzyme 10 µL (26 µg of protein for MAO-A and 5.0 µg for MAO-B). For MAO-A and MAO-B, the mixture was incubated for 20 and 15 min, respectively. The mixture was then given 10 µL of the substrate and 10 µL of newly prepared Amplex red. Accordingly, the final concentrations of clorgyline and deprenyl were utilized to calculate the activities of non-MAO-A and MAO-B. Using a fluorescence plate reader (BMG Labtech GmbH, orten berg Germany), the change in fluorescence was identified. The compounds that showed inhibition of either MAO-A or MAO-B activity of >50% underwent additional testing to determine their IC 50 values. The non-linear curve fitting tool PRISM 5.0 (GraphPad, San Diego, CA, USA) was employed to compute IC 50 values.

Acetylcholinesterase
All substances were put through a little modified version of Ellman's test to gauge the effectiveness of their ability to inhibit acetylcholinesterase (AChE). Released thiocholine reacts with chromogenic reagent 5,5-dithio-bis (2-nitrobenzoic) acid to produce a colorful product (DTNB). At a concentration of 2.5 units/mL, the enzyme solutions were prepared. The assay volume was 100 µL, having 60 µL buffer and 10 µL test compound (0.1 mM, 10% DMSO), pursued by inserting enzyme 10 µL (0.04 U/well). The mixture was incubated for 10 min. 10 µL of the substrate and 10 µL of DTNB were added to the mixture. After 30 min, the production of the yellow anion was recorded at 405 nm.

Enzyme Preparation
The X-ray structures of MAO-A (PDB code: 2Z5X [87]), MAO-B (PDB code: 2V5Z [88]), and AChE (PDB code: 4EY7 [89]) were opted as templates for all docking predictions. The enzymes were prepared by excluding all ions, heteroatoms, water molecules, and ligands. The Modeller software was applied to create all missing residues [90]. The investigated enzymes' protonation states were examined using the H++ website [91]. All missing hydrogen atoms were inserted.

Inhibitor Preparation
The 3D molecular structures of the inspected molecules 5a-f and 6a-d were manually constructed. Before any computation, all investigated compounds were firstly minimized using the MMFF94S force field implemented inside the SZYBKI software [92,93]. The charges of all investigated compounds were computed utilizing the Gasteiger-Marsili method [94].

Docking Calculations
The AutoDock4.2.6 software was employed to execute all docking computations [95]. Based on the AutoDock protocol, the pdbqt file for the examined enzymes was generated utilizing the MGL (molecular graphics laboratory) tools 1.5.7 [96]. In general, the docking parameters of the AutoDock4.2.6 software were maintained at their default settings. The GA (number of genetic algorithms) run variables was 250. The eval (maximum number of energy evaluations) was 25,000,000. The grid box size was 50 Å × 50 Å × 50 Å. For all docking engines, the cartesian coordinates of the grid center were located at the center of the binding pockets of the investigated enzymes. The AutoGrid4.2.6 program was used to create the grid maps with a spacing of 0.375 Å. All molecular interactions were visualized by the Discovery Studio module of Biovia software [97].

ADMET Properties
For the purpose of identifying significant compounds that exhibit drug-likeness for current in silico studies, excellent and better experimental ADMET (absorption, distribution, metabolism, excretion, and toxicity) features are required. The physicochemical properties of the compounds under study, including MW, nHBA, nHBD, and Log P were estimated. Moreover, the properties in accordance with absorption and distribution included volume of distribution, HIA, Caco2 permeability, and BBB. Metabolism-associated properties were estimated using CYP2C19 inhibitor. Excretion included clearance. Toxicity was evaluated based on AMES toxicity. All the parameters were computed using the online server ADMET lab 2.0 [98].

DFT Studies
To thoroughly investigate the molecular structures of the molecules under study, density functional theory (DFT) calculations were executed at the B3LYP/6-31G level using Gaussian09 software [99]. The geometries of the studied compounds were first optimized and further submitted to frequency computations to ensure if the obtained structures were true minima or not. The distributions and energies of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) were unveiled by means of the Frontier molecular orbitals (FMO). To well-characterize the features of the studied compounds, a diversity of global reactivity descriptors, including global hardness (η), global softness (σ), polarizability (α), and dipole moment (µ), was assessed.

Conclusions
A series of 2-methyl-5-(2-phenylquinolin-4-yl)-1,3,4-oxadiazole were synthesized with various substituents and analyzed against monoamine oxidase (MAO-A and MAO-B) and acetylcholinesterase (AChE) enzymes. All molecules showed promising inhibition in the lower µM range against targeted enzymes. Compounds 5a and 5f were the most promising MAO-A and MAO-B inhibitors (IC 50 = 0.91 ± 0.15 and 0.84 ± 0.06 nM, respectively). While compound 5c exhibited the most efficient acetylcholinesterase inhibition (IC 50 = 1.02 ± 0.65 µM). Furthermore, 5a-f and 6a-d were in silico investigated towards MAO-A, MAO-B, and AChE as anti-Alzheimer treatments using the AutoDock4.2.6 software. According to docking scores results, 5a, 5f, and 5c demonstrated the promising docking scores against the investigated enzymes. Moreover, the crucial function played by the investigated enzymes in Alzheimer's remediation proposes that promising molecules may act as potent novel chemical entities in identifying multi-target-directed inhibitors.