Novel 2-Amino-pyrano[3,2-c]quinoline-3-carbonitrile Derivatives Bearing Benzyloxy Phenyl Moiety as Butyrylcholinesterase Inhibitors: Design, Synthesis, In Vitro Evaluation, and Molecular Docking Studies

Background: Alzheimer’s disease (AD), the main form of dementia, is a multifactorial neurodegenerative disease, and several hypotheses have been proposed for its pathogenesis. Among them, cholinergic hypofunction is the main reason and plays a significant role in cognitive impairment. According to this theory, ChE inhibitors improve the performance of the cholinergic system and increase memory function. Thus, this study investigated a novel series of 2-amino-pyrano[3,2-c ]quinoline-3-carbonitrile derivatives bearing benzyloxy phenyl moiety as ChE enzyme inhibitors. Methods: The synthesized compounds 6a-o are divided into three series based on benzyloxy phenyl moiety. The structure of all compounds was identified by the NMR ( 1 H and 13 C) and IR spectra. Then, their inhibitory activities against ChE enzymes were evaluated by Ellman’s spectrophotometrical method. The kinetic and molecular docking studies were performed for compound 6l as the most potent butyrylcholinesterase (BChE) inhibitor. Results: The 2-amino-4-(4-((4-fluorobenzyl)oxy)-3-methoxyphenyl)-5-oxo-5,6-dihydro-4 H -pyrano[3,2-c ] quinoline-3-carbonitrile ( 6l ) demonstrated the best anti-BChE activity with a half maximal inhibitory concentration value of 1.00 ± 0.07. The kinetic and molecular docking studies confirmed that 6l is a mixed inhibitor and binds to both the anionic catalytic site and peripheral anionic site (PAS) of BChE. In silico study approved that the methoxy group on the middle phenyl ring has a significant role in interacting with the PAS of the enzyme. Conclusion: These findings indicated that 2-amino-pyrano[3,2-c ]quinoline-3-carbonitrile derivatives bearing benzyloxy phenyl moiety have therapeutic potential as BChE inhibitors in the last stages of AD.

and galantamine (Figure 1) have been approved for the treatment of AD (9,10).In patients with AD, a misbalance takes place between AChE and BChE so that the activity of AChE does not change or decrease, whereas BChE activity increases significantly.Therefore, it seems that the inhibition of BChE could also be considered a valuable therapeutic target for the treatment of AD (11).In recent years, medicinal chemists have represented great interest in designing and synthesizing a new hybrid molecule of two or more different pharmacophores that have different biological properties and structures (12).Thus, the hybridization method has grown to be a golden approach to drug discovery.Pyran and quinoline rings are valuable structures in medicinal chemistry with a wide range of biological functions such as anticancer, antimalarial, antibacterial, antifungal, antituberculosis, and anti-Alzheimer properties (13)(14)(15).These pharmaceutical and biological activities of pyran and quinoline rings, along with the presence of benzyl moiety in ChEIs (Figure 2) (16)(17)(18)(19)(20)(21), encouraged us to design novel hybrid molecules.As a result, in continuation of our research programs on the synthesis of ChEIs (16,22), this study aimed to synthesize and evaluate novel 2-amino-pyrano[3,2-c]quinoline-3carbonitrile derivatives bearing benzyloxy phenyl moiety as BChEIs.

Chemistry
The routes to synthesize compounds 6a-o are shown in Scheme 1.For the synthesis of compounds 6a-o, a series of substituted benzyloxy benzaldehydes has been considered according to our previous work (23).In this stage, 4-hydroxy benzaldehyde, 3-hydroxy benzaldehyde, and 3-methoxy-4-hydroxy (1a-c) benzaldehyde were reacted with various benzyl halide derivatives (2) in the presence of K 2 CO 3 as the base and dimethylformamide (DMF) as the solvent.Next, compounds 6a-o were obtained from the reaction of benzyloxy benzaldehydes (3a-o), malononitrile (4), and 4-hydroxyquinoline or 4-hydroxyquinoline-2(1H)-one (5) in the ethanol under reflux conditions at 80 °C in the presence of NH 4 OAc as a catalyst (24).

In Vitro Acetylcholinesterase and Butyrylcholinesterase Inhibition Assay
The in vitro ChE inhibitory activities of all synthesized compounds 6a-o were evaluated by modified Ellman's method (22) and compared with donepezil as the reference drug (Table 1).The percentage of inhibition and half maximal inhibitory concentration (IC 50 ) values were presented as the mean ± standard deviation (SD) of three independent experiments.
Based on the structure, the compounds can be divided into three series based on benzyloxy phenyl moiety, including 4-(benzyloxy) phenyl (6a-e), 3-(benzyloxy) phenyl (6f-j), and 4-(benzyloxy)-3-methoxyphenyl derivatives (6k-o).Based on the results (Table 1), all synthesized compounds had small activity against the AChE enzyme, and the results were expressed as a percentage of inhibition.Among the synthesized compounds, the compound of the third series (6k-o) represented good inhibition against the BChE enzyme.Moreover, the synthesized compounds of the first series 6a-e and the second series 6f-j had negligible activity against BChE.The compound 6e was the only active compound in the first series 6a-e.It seems that the presence of 3-methoxy in the middle ring of the molecule has an important rule for inhibitory activity against BChE.The docking studies also confirmed that the methoxy group could interact with the critical residue of the peripheral anionic site (PAS) of BChE.
The compound 6k without substitution and compound 6l with fluorine at the 4 th position of the benzyl ring demonstrated the best anti-BChE activities with IC 50 values of 1.00 ± 0.07 and 1.08 ± 0.09 μM, respectively.Introduction of the electron-withdrawing groups (EWGs), including fluorine, chlorine, and bromine, at the 4 th position of the benzyl ring led to the production of compounds 6l, 6m, and 6n with IC 50 values of 1.08 ± 0.09, 2.63 ± 0.11, and 11.08 ± 0.03 μM, respectively.These results indicated that the size of EWGs affects the anti-BChE activity so that with the increasing size of EWGs, inhibitory effects decrease and the orders of activities are 4-F > 4-Cl > 4-Br.
The introduction of 4-methyl as an electron-donating group on the benzyl ring created compound 6o with good BChE inhibitory activity (IC 50 = 4.23 ± 0.35μM).Based on these results, electronic effects did not significantly contribute to the inhibitory activity, and probably the size of substitutions on the benzyl group had an important role in anti-BChE activities.

Kinetic Studies of Butyrylcholinesterase Inhibition
To determine the mechanism of inhibition for compound (6l) as the most potent anti-BChE agent, a linear regression analysis was performed by Lineweaver-Burk plot.Based on Figure 3, with increasing inhibitor concentration, K m increases whereas V max decreases compared to the values for the uninhibited reaction, indicating a mixedtype inhibition.As a result, compound 8g could bind to both enzyme and enzyme-substrate, but with different affinities.The estimate of the inhibition constant (K i ) for mixed-type inhibition is the same as the IC 50 (22).

Molecular Docking
To study the interactions of the most potent anti-BChE compound 6l in the active site of BChE, a computational study was performed using the AutoDock 4.2 package with Discovery Studio 4.0 Client.Since compound 6l has a chiral centre at the 4-position of the pyran ring, both (R)-and (S)-enantiomers were used for docking studies.The superposed structure of S-chlorotacrine-tryptophan as a ligand of crystallography and both enantiomers of compound 6l in the active site are illustrated in Figure 4.All structures were inserted into the active site of BChE as a U-shape so that two ends of molecules occupied the catalytic anionic site (CAS) and acyl binding pocket.In addition, the middle of them created interaction with the key amino acids of PAS (25).
According to Figure 5, the 2-amino-5-oxo-5,6-dihydro-  showed that all synthesized compounds had small activity against AChE.However, the compound of the third series (6k-o) demonstrated significant inhibition against the BChE.The compound 6l represented the best anti-BChE activity with an IC 50 value of 1.00 ± 0.07.The kinetic and molecular docking studies confirmed that 6l is a mixed inhibitor and binds to both the CAS and PAS of BChE.Further, in silico studies indicated that the methoxy group on the middle phenyl ring has a significant role in interacting with the PAS of the enzyme.

Experimental
All chemicals were obtained from Merck and Sigma Companies and were used without further purification.Melting points were measured on a Stuart melting point smp3 apparatus.The nuclear magnetic resonance (NMR; 1 H and 13 C) and infrared (IR) spectra were obtained by using a Bruker 400-NMR and ALPHA Fourier-transform infrared spectrometer on KBr disks, respectively.The

Chemistry General Procedure for the Synthesis of Benzyloxy Aldehtdes Derivatives (3a-o):
4-Hydroxy benzaldehyde, 3-hydroxy benzaldehyde, and 3-methoxy-4-hydroxy benzaldehyde (vanillin) (1 mmol) were reacted with various benzyl halide (1.2 mmol) derivatives in the presence of K 2 CO 3 (1.5 mmol) and DMF (5 mL).After the reaction was completed, cold icewater was added to the mixture of the reaction.Benzyloxy aldehydes were obtained as white precipitates and used for the next stage without further purification (23).

General Procedure for the Synthesis of New Benzyloxy Pyrano[3,2-c]quinoline-3-carbonitrile Derivatives (6a-o)
The benzyloxy aldehydes (1 mmol) of the previous step, malononitrile 0.066 g (1 mmol), and 4-hydroxyquinolin-2(1H)-one 0.16g (1 mmol) were added to the roundbottomed flask in the presence of a catalytic amount of NH 4 OAc in ethanol (80 °C) as a solvent (5 mL) and refluxed for 12 hours.The progress of the reaction was followed by the thin layer chromatography technique.After the completion of the reaction, the mixture of reaction cold to room temperature and resulting precipitates were simply filtered and washed with 70% ethanol (24).

Kinetic Studies of AChE Inhibition
For estimates of the inhibition model and inhibition constant K i , the reciprocal plots of 1/V versus 1/[S] were 2-amino-pyrano[3,2-c]quinoline-3-carbonitrile derivatives as obtained using different concentrations of the substrate.For this purpose, experiments were performed similar to enzyme inhibition assay (22).The rate of enzymatic reaction was obtained with different concentrations of the inhibitor and in the absence of the inhibitor.For each experiment, the reaction was started by adding substrate, and progress curves were recorded at 405 nm over 2 minutes.Next, double reciprocal plots (1/v vs. 1/[s]) were made using the slopes of progress curves to obtain the type of inhibition.The slopes of these reciprocal plots were then plotted against the concentration of the compound in a weighted analysis, and K i was determined as the intercept on the negative x-axis.All rate measurements were performed in triplicate, and the data were analyzed by Microsoft Excel 2003.

Molecular Docking Study
Docking simulations were performed using AUTODOCK 4.2 software (http://autodock.scripps.edu/)(26).In this respect, the PDB structure of 6I0B was retrieved from the Brookhaven protein database (http://www.rcsb.org).Then, the water molecules and the inhibitor were removed, and the enzyme's pdbqt was prepared by AutoDock Tools (version 1.5.6)using default parameters.The three-dimensional structure of compound 6l was prepared by MarvineSketch 5.8.3, 2012, and then ligand.pdbqt was provided by AutoDock Tools (version 1.5.6).The AutoDock scoring grid box was approximately fixed between the CAS and PAS (for AChE, x-center: 134.75, y-center: 112.63, z-center: 40.79).The grid size was set to 60 × 60 × 60 points with a spacing value of 0.375 Å.The prepared compound was docked to the AChE template using a Lamarckian genetic algorithm of an initial population of 150 randomly placed individuals, the maximum number of 2.5 × 10 6 energy evaluations, the maximum number of 27 000 generations, and the number of 100 GA runs.A cluster analysis was performed on the docking results using a root mean square tolerance of 2.0, and the lowest energy conformation of the highest populated cluster was selected for analysis.Graphic visualizations were performed by Discovery Studio client software (version 2021).

Figure 1 .Figure 2 .
Figure 1.Structures of (a) Tacrine, (b) Rivastigmine, (c) Donepezil, and (d) Galantamine as AChEIs.Note.AChEI: Acetylcholinesterase inhibitor 4H-pyrano[3,2-c]quinoline-3-carbonitrile ring of (R)enantiomer was oriented toward the CAS and quinolone moiety created π-π stacking with amino acids Trp82 and Trp430.The NH 2 group made H-binding with Glu197 of the catalytic triad.The key amino acids Tyr332 and Phe329of PAS indicated hydrophobic interaction with OMe of the phenyl ring.The benzyl ring was directed to the acyl binding pocket of the enzyme and demonstrated alkyl-π and π-π interactions with Leu286 and Trp231, respectively.As depicted in Figure 6, the 2-amino-5-oxo-5,6dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrilering of (S)-enantiomer was inversely oriented to CAS compared to the (R)-enantiomer of compound 6l, while another part of structure made interactions similar to the (S)enantiomer.The quinolone ring showed π-π and π-alkyl interactions with amino acids Trp82, Tyr440, and Ala328 of CAS.The NH of quinolone indicated H-binding with His438 of the catalytic triad.The critical amino acids Tyr332, and Phe329 of PAS demonstrated a hydrophobic interaction with OMe of the middle phenyl ring and Asp70 showed H-binding with the 2-NH 2 group.The benzyl ring was oriented to the acyl binding pocket of the enzyme and revealed alkyl-π and π-π interactions with Leu286 and Trp231, respectively.Conclusion A new series of 2-amino-pyrano[3,2-c]quinoline-3-carbonitrile derivatives bearing benzyloxy phenyl moiety were designed, synthesized, characterized, and evaluated against AChE and BChE as potential agents for the treatment of AD.The synthesized compounds 6a-o are divided into three series based on benzyloxy phenyl moiety, namely, 4-(benzyloxy) phenyl (6ae), 3-(benzyloxy) phenyl (6f-j), and 4-(benzyloxy)-3methoxyphenyl derivatives (6k-o).The in vitro results

Figure 6 .
Figure 6.Interaction of (S)-Enantiomer (Green) of Compound 6l in the Active Site of BChE.Note.BChE: Butyrylcholinesterase.Hydrogen bonds are depicted by green dashed lines.The hydrophobic and π-π interactions are displayed as purple dashed lines

Table 1 .
The IC 50 Values of the Compounds