New azole-derived hemiaminal ethers as promising acetylcholinesterase inhibitors: synthesis, X-ray structures, in vitro and in silico studies

Abstract A new class of azole-derived hemiaminal ethers is designed as acetylcholinesterase (AChE) inhibitors. The synthesized compounds exhibited remarkable inhibitory activity against acetylcholine. Chiral hemiaminals (3d and 3i) based on (R)-menthoxymethyl group exhibit excellent inhibition with IC50 values of 0.983 ± 1.41 and 1.154 ± 0.89 µM. Similarly, butoxymethyl derivatives 3a, 3f and 3h, also showed promising inhibition comparable to the standard drug, Donepezil. In silico studies were performed to understand the mode of interactions with the target proteins, where menthoxymethyl azoles 3d and 3i demonstrated the highest docking scores. Molecular dynamics simulations displayed the stable ligand–protein complex of 3i with effective binding interactions. The bioavailability and pharmacokinetic parameterssupported the suitability of these small molecule inhibitors to develop cost-effective drug leads for Alzheimer’s disease (AD). MTT assay substantiated the non-cytotoxic nature of the compounds. The synthesized compounds are extensively characterized by 1H NMR, 13C NMR and mass spectral data and SC-XRD. Communicated by Ramaswamy H. Sarma


Introduction
Alzheimer's disease (AD) is one of the major global health threats, accounting for 60-70% cases of dementia, and the related coginative disabilities in older age.Despite excessive investigations, the pathology and treatment of AD is not fully understood (Anstey et al., 2019;Breijyeh & Karaman, 2020;Dubois et al., 2021;Livingston et al., 2020;Weller & Budson, 2018).The most favored hypothesis for the disease initiation and progression is a low level of acetylcholine (ACh) in the brain.ACh is a neurotransmitter that modulates memory function, whereas cholinesterases (ChEs) are a class of enzymes which regulate the ACh level and cholinergic signaling.Hence, acetylcholinesterase inhibitors (AChE) play a fundamental role in the ACh's regulation, for restoring the neurotransmitter level in the brain.Many inhibitors have been developed and proven effective in treating the cognitive and functional symptoms of Alzheimer's disease (Bishara et al., 2015;Black et al., 2018;Elufioye et al., 2019;Gody� n et al., 2016;Hersi et al., 2017;McGleenon et al., 1999;Ramirez-Bermudez, 2012) such as tacrine (A), donepezil (B), rivastigmine (C) and galantamine (D) (Figure 1).Due to certain limitations associated with these drugs, there is a continuing need for the development of more effective treatment for AD.

Experimental
All synthetic experiments were carried out under an argon atmosphere with oven-dried glassware and magnetic stirring.All solvents were dried and distilled in accordance with the CONTACT Humaira Yasmeen Gondal hygondal@yahoo.com,humaira.yasmeen@uos.edu.pkInstitute of Chemistry, University of Sargodha, Sargodha, 40100, Pakistan Supplemental data for this article can be accessed online at https://doi.org/10.

Synthesis of hemiaminal ethers (3a-j)
To the stirred solution of azole (Benzotriazole or Benzimidazole, 10 mmol) in dichloromethane (10 ml), DIPEA (diisopropyl ethyl amine, 12 mmol) was added drop-wise.The solution was allowed to stir for 30 min at room temperature, followed by drop-wise addition of freshly prepared alkoxymethyl halides (12 mmol).The reaction was allowed to reflux until completion (4 h).The progress of the reaction was monitored by TLC.The product was purified by column chromatography using hexane/ethyl acetate solvents.

Acetylcholinesterase inhibition
In vitro inhibition potential of synthesized derivatives was determined by spectrophotometric method established by Ellman et al. (1961) with slight modification (Mumtaz et al., 2018).A mixture consisting of 60 lL phosphate buffer (KH 2 PO 4 /KOH, pH 7.7), 10 lL test compounds and 10 lL enzyme (0.015 unit/well for AChE was incubated at 37 � C for 10 min for pre-incubation.After this, 10 lL substrate, acetylthiocholine chloride, 1 mM was added to AChE reaction mixtures to start the reaction.Later, 10 lL of 0.5 mM DTNB was added as coloring reagent.After this incubation at 37 � C for 20 min was carried out and absorbance was measured at 405 nm using microplate readers.Donepezil was taken as a positive control.All the experiments were carried out in triplicate, and the results were calculated as percentage inhibition values.The compounds that exhibited inhibition > 50% against ChE were further checked for the IC 50 values by GraphPad PRISM (USA) (Le Berre et al., 2022).

Protein and pocket selection
The protein data bank was searched for the human AChE.Seven different acetylcholine esterases/carboxylesterases were downloaded from the RCSB protein data bank.The proteins were selected on the criteria that they should contain a co-crystallized ligand and should have good resolution.The pockets of all proteins were detected and evaluated through dpocket module of fpocket (Le Guilloux et al., 2009).Protein 2h7c was selected based on results of fpocket.The pockets identified by fpocket were also compared to pockets of the cocrystallized ligands to avoid docking of ligands in any ambiguous pocket.

Molecular docking
The structures of ligands were drawn and saved in pdb format using Discovery Studio Visualizer (Accelrys, 2008).The proteins were downloaded in pdb format from the RCSB protein data and prepared for docking in autodock tools (Huey et al., 2012).All the water molecules were removed, and hydrogen atoms were added to the proteins.Kollman charges were added to balance it, and the Gasteiger method was applied to calculate the partial charge available on the proteins.Finally, the prepared protein was saved in pdbqt format.Docking study was carried out using CB-dock2 (Liu et al., 2022) and auto Dock Vina (Huey et al., 2012;Trott & Olson, 2010), whereas results were visualized using Pymol (DeLano, 2002), ChimeraX (Goddard et al., 2018) and Discovery Studio Visualizer (Accelrys, 2008).

MD simulation
MD simulations were performed by GROMACS 2020 software by applying the CHARMM36 force field (Abraham et al., 2015).Protein and ligand topologies were prepared separately by extracting from the complex file generated by Pymol (DeLano, 2002).Protein topology was prepared by the pdb2gmx tool in the GROMAC and solvated in a decahedral box by the TIP3 solvent model.Six sodium ions were added to neutralize the system.After defining the box margins, energy minimization was carried out by the steepest descent algorithm consisting of 50,000 steps.The prepared system was then subjected to simulation process for 100 ps.GROMACS analysis tools were used to analyze the trajectory obtained from the MD simulation.The root means square deviation (RMSD) and the root means square fluctuations (RMSF) were determined by gmx rms and gmx rmsf tools.Radius of gyration (Rg) and hydrogen bond analysis was performed through the gyrate tool and gmx hbond tool.All the files were generated in the xvg extension, and graphs were formed on excel.The visualization and analysis of the trajectory for the evaluation of movement of the complex were performed by the VMD software version 2.0.2.

Chemistry
We designed a new class of azole-derived hemiaminal ethers as a potential candidate for AChE inhibition.Structurally diverse chiral and achiral alkoxymethyl groups were introduced to the biologically known azoles (benzimidazole and benzotriazole) to develop a lucid insight into the structureactivity relationship.We believe that the alkoxymethyl group can be involved in effective H-bonding, whereas benzazole moiety can interact through p-p interactions with the target proteins.From the perspective of the synthesis of the target   hemiaminals, the reported methods for the alkoxymethyl azaheterocycles demonstrate certain limitations concerning reaction parameters and substrate scope (Juliusz & Lucyna, 1994;Quan et al., 2011).Therefore, we decided to reinvestigate the reaction conditions for the N-alkoxymethylation of azole rings.After screening various bases and solvents at different temperatures, we have developed a general and expeditious method for the target compounds.Under the optimized conditions, the reaction of benzazoles ( 1) with alkoxymethyl halides (2) in the presence of DIPEA provided the corresponding hemiaminal ethers (3a-j) in high yields (Scheme 1).
Alkoxymethylation of benzimadazole provided exclusively single products (3f-j), while two isomers were obtained in the case of benzotriazole.Interestingly, all the minor N-2 isomers were oils, whereas the target N-1 isomers were obtained in the solid crystalline form.Therefore, major N-1 isomers (3a-e) were easily purified by column chromatography with isolated yields of 78-88%.All the synthesized compounds were characterized based on 1 H-NMR, 13 C-NMR and mass spectrometry data.Similarly, the structures of two benzotriazole derivatives (3b and 3e) were confirmed by single-crystal XRD.The 1 H-NMR spectra of all hemiaminals showed a distinctive singlet for methylene of O-CH 2 -N in the range of d H 4.6-6.0ppm.In addition, all these compounds showed signals in the aromatic region between d H 7.2-8.1 ppm belonging to benzazoles.Similarly, in 13 C-NMR, appearance of a low-field methylene signals at d C 74.6-79.8 is considered a characteristic signal of the hemiaminal moiety, whereas all other peaks were consistent with the molecular structures.

Single crystal-XRD analysis
SC-XRD analysis ultimately confirmed the structures of 3b and 3e.The structural parameters of 1-(isobutoxymethyl)-1Hbenzo[d] [1][2][3]triazole (3b) revealed that the compound is crystallized in the triclinic crystal system with space group P-1, having two molecules in the unit cell.The ORTEP diagram (Figure 2) of 3b and 3e illustrated the molecular structure consisting of benzotriazole ring (C1-C6, N1-N3) connected with the iso-butoxymethyl and fenchyloxymethyl groups respectively.

Hirshfeld surface analysis
Hirshfeld surface analysis was used to provide the quantitative knowledge of inter-molecular interaction based on  d norm property of the molecules and the distance of atom (internal, di and external, de) to generate the Hirshfeld surface.The visualization of the Hirshfeld surface is done by the adjusting the color scale range from À 0.06 (red) to 1.262 (blue) Å, in the crystal explorer program.The red color shows interatomic contact site and intermolecular interaction.

Hirshfeld surface analysis of iso-butoxymethylbenzotriazole 3b
Hirshfeld surface of iso-butoxymethylbenzotriazole i-BMBT 3b was generated to reveal the intermolecular interactions

Hirshfeld surface analysis of N-1 fenchyloxymethylated benzotriazole 3e
Similarly, Hirshfeld surface of N-1 fenchyloxymethylated benzotriazole 3e was generated to reveal the intermolecular interactions quantitatively (Khan et al., 2020).Based on the d norm property of molecule, red spots were not clearly observed on the generated surface, which indicated weak intermolecular interactions as observed in PLATON software Figure 6.The contribution of interaction to the overall crystal lattice measured by 2D finger print plot revealed the H … H (70.5%), N … H (16.3%), C … H (12.6%) and O … H (0.6%) (Figure 7).The increased contribution of H … H intermolecular interactions owed to the abundance of H atoms present in the molecule, which are responsible in the crystal packing.

AChE inhibition invitro studies
The synthesized compounds (3a-j, Scheme 1) were screened for AChE inhibition activity.All the newly synthesized   8).Smaller size and linear chained derivatives can be considered biomimetic of acetylcholine, a natural ligand of AChE.In addition, the alkoxy group can provide H-bonding while the azole rings are responsible for extra binding through p-p stacking with the enzyme resulting in the antagonistic behavior of our synthesized derivatives.

AChE inhibition in silico studies
To understand the mode of interactions of the newly synthesized alkoxymethyl benzazoles with the target proteins, in silico studies were performed.The protein data bank was searched for the human AChE, and seven different acetylcholine esterases/carboxylesterases of good resolution with the co-crystallized ligands were selected (Table 2).Pockets of all the proteins were evaluated through the dpocket module of fpocket (Le Guilloux et al., 2009).Human carboxylesterase protein (2h7c) was selected based on the results from the fpocket.Hence, the pockets identified were compared with the co-crystallized ligands to avoid ambiguities in the docking studies.Results obtained from the fpocket revealed similar binding sites for all the selected proteins.Multiple pockets were identified, and the details of the highest-scoring pockets are tabulated (Table S20, SI).Human carboxylesterase protein (2h7c) is a homo-6-mer comprised of 6 similar protein chains.It was observed that each chain had a similar binding pocket.Structure of the protein was arranged in such a way that two binding pockets were facing each other to develop a large triple binding site, each capable of binding two similar molecules at a time.Thus, the protein (2h7c) can bind six molecules simultaneously.The docking results indicated that all the synthesized compounds showed effective binding affinity with the esterase enzymes (Table 3).Outcomes from the individual docking studies were found in agreement with the results from the enzyme inhibition assay, where menthoxymethyl derivatives MMBM 3i and MMBT 3d demonstrated the highest esterase inhibition potential compared to the compounds (3e, 3j) with bicyclic alkoxy group that is fenchol.However, the excellent activity of smaller alkoxy groups like n-butyl (3a, 3f) in the enzyme assay may be attributed to their small size and structural resemblance with choline.The pocket analysis of the   Negative - The highest value obtained are bold.
esterases demonstrates that the esterases are a promiscuous drug target that can bind to several ligands due to the large pockets available by multiple chains.As our synthesized compounds are of small size, therefore multiple molecules of each ligand are expected to bind in the active site.In such cases, significant intermolecular interactions are considered responsible for the stability of the drug-protein complexes.
Based on the initial docking results, menthoxymethyl benzimidazole MMBM 3i was selected as a representative hit compound for further studies.Figure 9 shows two poses of the compound 3i with almost similar binding affinity in two different chains (2h7c).
The visualization of the docking results suggested that the most effective interactions observed between the ligand (MMBM 3i) and protein (2h7c) residues are hydrophobic, usually p-alkyl interactions.The pocket for the ligand was identified in the D chain of protein receptors.The amino acids involved in hydrophobic interactions are ILE:5359,   VAL:5254, MET:5364, LEU: 5304, LEU:5318, LEU:5388, LEU:5097 and ALA:5093.VAL:5146 and LEU:5363 showed pisigma bonding with benzene and imidazole ring with a distance of 4.01 and 4.62 Å, respectively.
A comparison with the reference compound (donepezil) demonstrated that our synthesized compound 3i exhibited comparable interactions with more interactive chains of protein (2hc7) (Figure 10).To evaluate the contribution of the benzene ring in the binding interactions, the docking of simple menthoxymethyl imidazole (MMIM) was carried out with the 2h7c protein.The compound showed low binding energy by interacting with only leucine (LEU) in three regions of chain C (Figure 11a).These results helped to infer that the hydrophobic interactions due to the aromatic regions are significantly involved in a stable binding complex of the synthesized derivatives with the target proteins (Figure 11b).
To figure out the role of azole, it was observed that both (3i and 3d) showed high docking scores with 2h7c (Table 4) and interacted similarly within the same pocket (Figure 12).
Results revealed that change in the ring from benzimidazole to benzotriazole does not meaningfully affect the interactions with the protein.Therefore, we extended our investigations on the menthoxymethyl benzimidazole MMBM 3i to evaluate its interactions with the other proteins of the human AChE.Our results illustrated that the hit compound 3i showed valuable interactions with the other selected proteins (Table 4), where the highest docking score of À 9.5 was found with the human AChE 6o4x.
The blind docking analysis by CB-dock 2 (Liu et al., 2022) suggested the five best-interacting pockets in the human AChE 6o4x for the MMBM 3i, where the maximum interactions were observed in chain A with the cavity volume of 1161 Å 3 .Further, the site specific docking of MMBM 3i was carried out using the dimensions of Grid box for this pocket.Interestingly, many interactions were found common  between protein 6o4x and 2h7c except the conventional hydrogen bonding with the amino acid TYR:124 (Figure 13).A comparison of docking results of MIMBM 3i with the reference compound (donepezil) is evident in the effective interactions of our synthesized compound (Figure 14).In the case of compound 3i, p-p interactions with TRP:286 and TYR:341, p-alkyl interactions with VAL:294, TRP:439, TYR:337 and TRP:86, and hydrogen bonding with TYR:124 were observed.On the other hand, donepezil was found to interact with TRP:286, PHE:338, VAL:294 and TYR:341 through p-p, p-alkyl and alkyl-alkyl interactions, and TYR:72 through hydrogen bonding.Encouraging results from the enzyme inhibitory assay and docking studies helped us to infer that the synthesized hemiaminal ethers have promising potential for AChE inhibition.Further, the molecular dynamic simulation was employed to assess the stability of the complex between hit compound 3i and the protein receptor (2h7c) under physiological conditions.Different parameters like RMSD, RMSF, Rg and hydrogen bonds (hbonds) data were calculated by processing MD data (Alhomrani et al., 2022) for the dynamic interactions within the 3i-2h7c complex.RMSD value (nm) was plotted against time (ps) to find the stability of the complex during the simulation process (Alhomrani et al., 2022).RMSD values for the 3i-2h7c complex were found in the range of 0.14-0.23 nm, suggesting the establishment of stable connectivity between the ligand and protein (Figure 15a).RMSF (root means square fluctuations) trajectory provided information about the fluctuations in the structure of protein residue and stability of the complex with ligand (3i) during the whole run.The plot of RMSF value (nm) for the protein backbone and the ligand was plotted against atom numbers in the protein residue.A similar pattern was observed for both ligand and protein, except for a slight fluctuation at 0.15 nm (Figure 15b).The RMSF value for the backbone and 3i was observed between 0.05-0.1 nm indicating well-structured  regions in the protein-ligand complex with less fluctuation and less distortion.Rg was plotted against time, where a constant value of 0.27 nm was obtained with minor changes, indicated a stable ligand-protein complex (Figure 15c).The visualization of the movement of the hit ligand (3i) concerning the protein displayed the ligand remains inside the protein pocket during the whole period, confirmed the effective interactions (Hasan et al., 2022).A snapshot demonstration of the movement of complex during the simulation is presented in Figure 16.
The pharmacokinetics predictions, ADMET (absorption, distribution, metabolism, excretion and toxicity) and physicochemical properties of compound 3i were carried out to evaluate its suitability as a drug.The compound has a molecular weight of 286.41, with a lipophilicity value of 3.84, and a molar refractivity of 88.04 (Table 5).Three H-bond acceptors and no H-bond donor site found in the molecule also supported the rules.Similarly, the topological polar surface area (TPSA) of 27.05 Å 2 indicated good blood-brain penetration ability of the molecule.Based on the ADMET analysis, compound 3i exhibited low to moderate toxicity with an estimated LD50 value of 1000 mg/kg.The compound was found inactive towards carcinogenicity, mutagenicity and cytotoxicity and can be categorized in class IV according to the globally harmonized system of classification of chemicals (Banerjee et al., 2018;Salih et al., 2022).

Conclusion
In conclusion, a new class of azole-based hemiaminals as AChE was synthesized.The products were characterized by NMR spectroscopy, Mass spectrometry and SC-XRD studies.
In vitro studies showed promising AChE inhibition ability of the newly synthesized compounds.Molecular docking studies displayed the effective binding of these compounds with different AChE proteins.The most active compound MMBM 3i was considered a hit for detailed in silico studies.Results from the docking and MD simulation manifested an excellent protein-ligand binding complex.Moreover, general drug-likeness parameters supported the candidacy of MMBM 3i as an ACh inhibitor.Considering simple and efficient synthesis, these new hemiaminal ethers have significant potential to develop cost-effective drugs for Alzheimer's disease (AD).
quantitatively, based on the d norm property of the molecule.The red spots are indication of the sites of close contacts Figure 4.The overall contribution of the intermolecular interactions in the crystal lattice is measured by 2D fingerprint plot (Figure 5), where the H … H (60.5%), N … H (19.2%), C … H (10.9%), O … H (4.7%), C … C (2.8%) and C … N (1.9%) contributions towards the lattice stability were observed.The significant interactions responsible for crystal packing were identified as H … H and N … H interactions due to the availability of the more exposed H atoms. On the other hand, the interactions involving the least exposed C atoms have the minimum contribution to the stabilization.

Figure 15 .
Figure 15.RMSD representation for 3i-2h7c complex (a) RMS fluctuation in the structure of ligand-protein backbone (b) Radius of gyration (c) during MD simulation.

Figure 16 .
Figure 16.Snapshot demonstration of the movement of 3i with the protein 2h7c during simulation.

Table 2 .
Details of the seven selected proteins.

Table 5 .
Representative parameters of the hit compound 3i based on in silico analysis.
Figure 17.Cytotoxicity evaluation of hemiaminal ethers in MTT assay.