Design, Synthesis, and Biological Activity Evaluation of New Donepezil-Like Compounds Bearing Thiazole Ring for the Treatment of Alzheimer’s Disease

Alzheimer’s disease (AD) is a progressive and neurodegenerative disease that is primarily seen in the elderly population and is clinically characterized by memory and cognitive impairment. The importance of the disease has increased as a result of etiology of the disease having not yet been determined, an increase in patient population over the years, absence of radical treatment, high cost of treatment and care, and significant reduction in the quality of life of the patients, which have led researchers to direct more attention to this field. In a recent study, new indan-thiazolylhydrazone derivatives were designed and synthesized based on the chemical structure of the donepezil molecule, which is the most preferred and has the most appropriate response in the treatment of AD. The structures of the compounds were determined by 1H-NMR and 13C-NMR, and mass spectroscopic methods. Inhibition studies on the cholinesterase (ChE) enzymes and beta amyloid plaque inhibition test of the compounds were performed. Among the synthesized derivatives, compounds 2a, 2e, 2i, and 2l showed potent inhibitory activity on the AChE enzyme. Compound 2e was found to be the most active agent, with an IC50 value of 0.026 μM. The mechanism of AChE inhibition by compound 2e was studied using the Lineweaver-Burk plot, and the nature of inhibition was also determined to be mix-typed. Molecular docking studies were also carried out for compound 2e, which was found as the most potent agent within the AChE enzyme active site. Moreover, compounds 2a, 2e, 2i, and 2l displayed the ability to prevent beta amyloid plaque aggregation at varying rates. In addition, ADME (Absorption, Distribution, Metabolism, Elimination) parameters were evaluated for all synthesized compounds using the QikProp 4.8 software (Schrödinger Inc., NY, USA).


Introduction
The elderly population is increasing worldwide. Due to this increase, the possibility of occurrence of dementia or mental diseases will rise as well. The main cause of dementia in the elderly is Alzheimer's disease (AD) [1]. AD is a neurodegenerative disease that is manifested by the gradual degradation of the cognitive, memory, and executive functions of the central nervous system (CNS). The lower
The assay was performed in two steps. The first step was carried out using all of the synthesized compounds and reference agents, namely donepezil and tacrine, at concentrations of 1000 and 100 µM. The enzyme activity results of the first step are presented in Table 2. Next, the selected compounds that displayed more than 50% inhibitory activity at concentrations of 1000 and 100 µM were further tested, along with the reference agents, at concentrations of 10 to 0.001 µM. The IC 50 values of the test compounds and reference agents are presented in Table S1 (Supplementary Material). Table 2.
Inhibition (%) of the synthesized compounds, donepezil, and tacrine against acetylcholinesterase (AChE) and butyrylcholine (BChE) enzymes. According to the enzyme inhibition results, none of the synthesized compounds showed significant activity against the BChE enzyme. All compounds displayed selective inhibition to AChE. At a concentration of 1000 µM, all the compounds showed more than 50% inhibitory activity.

Compounds
Compounds 2a, 2e, 2i, and 2l passed the second step of the enzyme activity assay and their IC 50 values were calculated by performing an enzyme inhibition study at concentrations of 10 to 0.001 µM. The IC 50 values of compounds 2a, 2e, 2i, and 2l were calculated as 0.118, 0.026, 0.070, and 0.061 µM, respectively. Among these derivatives, compound 2e was found to be the most active agent in the series, with an IC 50 value of 0.026 µM. It was seen that compound 2e exhibited an inhibition profile similar to the reference drug, donepezil (IC 50 value = 0.021 µM).
Among the actively detected compounds, 2i and 2l carried dimethyl and dichloro substituents, respectively, at the orthoand parapositions of the phenyl ring. According to the enzyme inhibition results, it was thought that the substituents, especially at the orthoposition of the phenyl ring, positively contributed to the enzyme inhibition capacity. Moreover, compound 2e, which was determined to be the most potent derivative, had a nitro substituent at the paraposition of the phenyl ring. Due to the nitro substituent in this compound, it can be suggested that the electron withdrawing group made a very important contribution to enhancing inhibition potency against the AChE.

Kinetic Studies of Enzyme Inhibition
Enzyme kinetics studies were performed to determine the mechanism of the AChE inhibition using a procedure similar to that of the cholinesterase enzymes inhibition assay. Compound 2e, which was found to be the most potent agent, was included in these studies. In order to estimate the type of inhibition of this compound, linear Lineweaver-Burk graphs were used. Substrate velocity curves in the absence and presence of compound 2e were recorded. This compound was prepared at concentrations of IC 50 /2, IC 50 , and 2 × IC 50 for the enzyme kinetic studies. In each case, the initial velocity measurements were obtained at different substrate (ATC) concentrations, ranging from 600 to 18.75 µM. The secondary plots of the slope (K m /V max ) versus varying concentrations (0, IC 50 /2, IC 50 , and 2 × IC 50 ) were created to calculate the K i (intercept on the x-axis) value of this compound. The graphical analyses of the steady-state inhibition data for compound 2e are shown in Figure 2.
According to the Lineweaver-Burk plots, a graph with lines that do not intersect at the x-axis or the y-axis is formed for a mixed-type inhibition. Therefore, as shown in Figure 2, compound 2e was reversible and had mixed-type inhibitors with similar inhibition features to the substrates. K i values for compound 2e were calculated as 0.022 µM for inhibition of the AChE.
Irreversible enzymatic inhibition involves covalent interactions between the substrate and the enzyme. In contrast, there are non-covalent interactions, such as hydrophobic interactions, ionic bonds, and hydrogen bonds, involved in reversible inhibition. In this type of inhibition, inhibitors bind to the enzymes without forming any chemical bonds; thus, the enzyme-inhibitor complex could be separated quickly, because non-covalent interactions can form rapidly and break easily. Furthermore, reversible inhibitors have a lower risk of side effects than irreversible inhibitors, owing to their non-covalent binding ability. Consequently, compound 2e, whose inhibition type was determined to be reversible and mix-typed, has a pharmaceutical importance for being an inhibitor candidate of the AChE enzyme.

Inhibition of Beta Amyloid 1-42 (Aβ42) Aggregation
The accumulation of amyloid plaques that can be found in the brains of patients suffering from AD is another important reason as well as decreasing cholinergic transmission. Plaques are mainly composed of beta amyloid (Aβ) peptides. Recent studies have shown that two beta amyloid peptides (Aβ (1-40) and Aβ (1-42)) exist in brain tissues, cerebrospinal fluid (CSF), and plasma in patients suffering from AD. Therefore, screening of Aβ42 ligands that can prevent aggregation is critical for the development of potential therapeutic treatments. Hence, in this study, for the selected compounds, 2a, 2e, 2i, and 2l, which displayed potent inhibitory activity against the AChE enzyme, the inhibition of beta amyloid 1-42 (Aβ42) aggregation was evaluated using the beta amyloid 1-42 (Aβ42) ligand screening assay kit (BioVision, Milpitas, CA, USA). This assay kit is based on the fluorometric method and the assay procedure was applied according to the instructions of this kit. In the presence of an Aβ42 ligand, this reaction is impeded/abolished, resulting in a decrease or total loss of fluorescence. The percentage of inhibition of beta amyloid 1-42 (Aβ42) peptides aggregation are given in Figure 3 for compounds 2a, 2e, 2i, and 2l. These compounds were tested at concentrations of 100 and 10 µM, and the Aβ42 inhibitor included in the kit was used as the standard. All of the test compound concentrations were applied in quadruplicate in the plates.
bind to the enzymes without forming any chemical bonds; thus, the enzyme-inhibitor complex could be separated quickly, because non-covalent interactions can form rapidly and break easily. Furthermore, reversible inhibitors have a lower risk of side effects than irreversible inhibitors, owing to their non-covalent binding ability. Consequently, compound 2e, whose inhibition type was determined to be reversible and mix-typed, has a pharmaceutical importance for being an inhibitor candidate of the AChE enzyme.

Inhibition of Beta Amyloid 1-42 (Aβ42) Aggregation
The accumulation of amyloid plaques that can be found in the brains of patients suffering from AD is another important reason as well as decreasing cholinergic transmission. Plaques are mainly composed of beta amyloid (Aβ) peptides. Recent studies have shown that two beta amyloid peptides (Aβ (1-40) and Aβ (1-42)) exist in brain tissues, cerebrospinal fluid (CSF), and plasma in patients suffering from AD. Therefore, screening of Aβ42 ligands that can prevent aggregation is critical for the development of potential therapeutic treatments. Hence, in this study, for the selected compounds, 2a, 2e, 2i, and 2l, which displayed potent inhibitory activity against the AChE enzyme, the inhibition of beta amyloid 1-42 (Aβ42) aggregation was evaluated using the beta amyloid 1-42 (Aβ42) ligand screening assay kit (BioVision, Milpitas, CA, USA). This assay kit is based on the fluorometric method and the assay procedure was applied according to the instructions of this kit. In the presence of an Aβ42 ligand, this reaction is impeded/abolished, resulting in a decrease or total loss of fluorescence. The percentage of inhibition of beta amyloid 1-42 (Aβ42) peptides aggregation are given in Figure 3 for compounds 2a, 2e, 2i, and 2l. These compounds were tested at concentrations of 100 and 10 µM, and the Aβ42 inhibitor included in the kit was used as the standard. All of the test compound concentrations were applied in quadruplicate in the plates.
According to Figure 3, compounds 2a, 2e, 2i, and 2l showed more than 50% inhibition at a According to Figure 3, compounds 2a, 2e, 2i, and 2l showed more than 50% inhibition at a concentration of 100 µM. The percentage of inhibition of compounds 2a, 2e, 2i, and 2l at this concentration was as follows: 71.87%, 87.36%, 77.16%, and 82.51%, respectively. At a concentration of 10 µM, only compound 2e displayed more than 50% inhibition, namely 73.45%. These findings indicated that the related compounds had the ability to prevent beta amyloid plaque aggregation at varying rates, in addition to their AChE enzyme inhibitory potential.
Crystals 2020, 10, 637 7 of 17 compound concentrations were applied in quadruplicate in the plates. According to Figure 3, compounds 2a, 2e, 2i, and 2l showed more than 50% inhibition at a concentration of 100 µM. The percentage of inhibition of compounds 2a, 2e, 2i, and 2l at this concentration was as follows: 71.87%, 87.36%, 77.16%, and 82.51%, respectively. At a concentration of 10 µM, only compound 2e displayed more than 50% inhibition, namely 73.45%. These findings indicated that the related compounds had the ability to prevent beta amyloid plaque aggregation at varying rates, in addition to their AChE enzyme inhibitory potential.
Lipinski and Jorgensen defined physicochemical ranges for active compounds that are likely to be oral drugs. Based on the relationship between pharmacokinetic and physicochemical parameters, Lipinski's rule of five and Jorgensen's rule of three are used to determine the structural properties sought in a candidate compound that may be a drug [24,25]. Moreover, these rules are known as drug-like testing [26]. The calculated ADME parameters, including molecular weight (MW), number of rotatable bonds (RB), dipole moment (DM), molecular volume (MV), number of hydrogen donors (DHB), number of hydrogen acceptors (AHB), polar surface area (PSA), octanol/water partition coefficient (log P), aqueous solubility (log S), apparent Caco-2 cell permeability (PCaco), number of likely primer metabolic reactions (PM), percent of human oral absorption (%HOA), and the violations of the rules of three (VRT) and five (VRF) are presented in Table 3. It can be seen from this table that all of the parameters were within the reference ranges. In keeping with Jorgensen's rule of three and Lipinski's rule of five, the obtained compounds (2a-2l) were in accordance with the set parameters, as they did not cause more than one violation. Considering the results of the ADME parameter
Lipinski and Jorgensen defined physicochemical ranges for active compounds that are likely to be oral drugs. Based on the relationship between pharmacokinetic and physicochemical parameters, Lipinski's rule of five and Jorgensen's rule of three are used to determine the structural properties sought in a candidate compound that may be a drug [24,25]. Moreover, these rules are known as drug-like testing [26]. The calculated ADME parameters, including molecular weight (MW), number of rotatable bonds (RB), dipole moment (DM), molecular volume (MV), number of hydrogen donors (DHB), number of hydrogen acceptors (AHB), polar surface area (PSA), octanol/water partition coefficient (log P), aqueous solubility (log S), apparent Caco-2 cell permeability (PCaco), number of likely primer metabolic reactions (PM), percent of human oral absorption (%HOA), and the violations of the rules of three (VRT) and five (VRF) are presented in Table 3. It can be seen from this table that all of the parameters were within the reference ranges. In keeping with Jorgensen's rule of three and Lipinski's rule of five, the obtained compounds (2a-2l) were in accordance with the set parameters, as they did not cause more than one violation. Considering the results of the ADME parameter studies, the synthesized compounds had pharmacokinetic profiles that may be appropriate for clinical use.
Drugs that specifically target the CNS must first cross the BBB (blood brain barrier). Although the BBB is protective in nature, the use of drug candidates that affect the CNS in the clinical setting is unlikely if such drug molecules cannot penetrate it. For this reason, this feature must be explored earlier in the drug discovery process. Accordingly, it is of great importance to predict the BBB permeability of new compounds [27]. Therefore, the BBB permeability of the obtained compounds (2a-2l) was also evaluated using the QikProp 4.8 software [23]. Brain/blood partition coefficient (logBB) and apparent MDCK cell permeability (PMDCK) were calculated for this purpose. In keeping with the software estimates, the PMDCK values of < 25 and > 500 nm/s were determined as poor and great for non-active transport of the compounds. To evaluate the ability of a compound to pass through the BBB, logBB is another important parameter to consider with the recommended values between −3 and +1.2. The PMDCK and logBB values of the synthesized compounds are within the advised ranges as shown in Table 3. Therefore, it can be assumed that the synthesized compounds can exceed the BBB, which is very important for CNS-related drugs.
Considering the results of the ADME and BBB permeability studies, the synthesized compounds were determined to have pharmacokinetic profiles that may be appropriate for clinical use.

Molecular Docking
As mentioned in the cholinesterase enzymes inhibition assay, compound 2e was found to be the most active derivative in the series against the AChE enzyme. Hence, docking studies were carried out to evaluate its inhibition capability in silico. Obtaining more insight into the binding mode of compound 2e and an evaluation of the effects of the structural modifications on the inhibitory activity against the AChE enzyme was made possible as a result of the docking studies. These studies were carried out using the X-ray crystal structure of Homo sapiens AChE (hAChE PDB ID:4EY7) [12] retrieved from the Protein Data Bank server (www.pdb.org). The reason for choosing this X-ray crystal structure was that it was human in origin, had a high resolution, and contained the donepezil molecule as the ligand. First, the docking procedure was confirmed by performing the protocol with donepezil. Next, compound 2e was subjected to the same docking procedure. The rendered docking poses of the donepezil and compound 2e are provided in Figures 4 and 5. against the AChE enzyme was made possible as a result of the docking studies. These studies were carried out using the X-ray crystal structure of Homo sapiens AChE (hAChE PDB ID:4EY7) [12] retrieved from the Protein Data Bank server (www.pdb.org). The reason for choosing this X-ray crystal structure was that it was human in origin, had a high resolution, and contained the donepezil molecule as the ligand. First, the docking procedure was confirmed by performing the protocol with donepezil. Next, compound 2e was subjected to the same docking procedure. The rendered docking poses of the donepezil and compound 2e are provided in Figure 4 and Figure 5.
According to the X-ray crystallographic structure of AChE (PDB ID:4EY7), the enzyme active pocket consisted of two main binding sites, the CAS and PAS. The CAS contained Ser203, Glu334, His447, Trp86, Tyr130, Tyr133, Tyr337, and Phe338 amino acid residues; however, amino acids Tyr72, Asp74, Tyr124, Trp286, Phe295, and Tyr341 were located in the PAS [28][29][30][31]. Donepezil interacts with both the CAS and PAS. Therefore, it can be settled into the gorge concordantly as a result of its dual binding site (DBS) [32][33][34]. According to Figure 4, the benzyl moiety of donepezil has a π-π interaction with the indole of Trp86. Furthermore, the protonated nitrogen atom of piperidine forms cation-π interactions with the indole of Trp86 and the phenyl of Tyr337. Thus, the benzylpiperidine group of donepezil is strongly located in the CAS. The interactions of the 1-indanone ring are important for binding to the PAS. The 1-indanone constitutes a π-π interaction with the indole of Trp286. Moreover, there is a hydrogen bond formation between the carbonyl of the 1-indanone and the amine of Phe295. These identified interactions of donepezil were consistent with the data in the literature and the docking procedure was verified via these findings [12,[35][36][37].  Figure 5A presents the superimposition of donepezil and compound 2e in the active region of the AChE. It was clear that compound 2e bound to the active site of the AChE enzyme in a similar position as donepezil due to its dual binding sites. This compound mainly carries the lipophilic indan ring, 4-substituedphenyl-thiazole group, as a polar group, and a basic center. The docking poses indicated that lipophilic groups interacted with the PAS region, whereas the polar and basic groups bound to the CAS region.
The docking pose of compound 2e ( Figure 5B) revealed that the indan ring created a π-π interaction with the indole of Trp286. This interaction was the same as that with donepezil in relation According to the X-ray crystallographic structure of AChE (PDB ID:4EY7), the enzyme active pocket consisted of two main binding sites, the CAS and PAS. The CAS contained Ser203, Glu334, His447, Trp86, Tyr130, Tyr133, Tyr337, and Phe338 amino acid residues; however, amino acids Tyr72, Asp74, Tyr124, Trp286, Phe295, and Tyr341 were located in the PAS [28][29][30][31]. Donepezil interacts with both the CAS and PAS. Therefore, it can be settled into the gorge concordantly as a result of its dual binding site (DBS) [32][33][34]. According to Figure 4, the benzyl moiety of donepezil has a π-π interaction with the indole of Trp86. Furthermore, the protonated nitrogen atom of piperidine forms cation-π interactions with the indole of Trp86 and the phenyl of Tyr337. Thus, the benzylpiperidine group of donepezil is strongly located in the CAS. The interactions of the 1-indanone ring are important for binding to the PAS. The 1-indanone constitutes a π-π interaction with the indole of Trp286. Moreover, there is a hydrogen bond formation between the carbonyl of the 1-indanone and the amine of Phe295. These identified interactions of donepezil were consistent with the data in the literature and the docking procedure was verified via these findings [12,[35][36][37].
to the enzyme active site, docking studies were performed using Glide (Schrödinger, NY, USA), according to the per-residue interaction panel. Figure 5C,D presents the van der Waals and electrostatic interactions of compound 2e. As is shown, this compound had favorable van der Waals interactions with amino acids Tyr72, Trp86, Tyr124, Glu202, Trp286, Val294, Phe295, Phe297, Tyr337, Phe338, Tyr341, and Hid447, which are displayed in pink and red, as described in the Glide user manual [38]. Similarly, promising electrostatic contributions of compound 2e were determined with amino acids Asp74, Tyr124, Glu202, Phe295, and Arg296.

Chemistry
All the chemicals used in the synthesis studies were obtained from Merck (Darmstadt, Germany) or Sigma-Aldrich (St. Louis, MO, USA). A MP90 digital melting point apparatus (Mettler Toledo, OH, USA) was used to determine the melting points of the resulting compounds and was presented uncorrected. A Bruker 300 and 75 MHz digital FT-NMR spectrometer (Billerica, MA, USA) in DMSO-d6, respectively, was used to record the 1 H-NMR and 13 C-NMR spectra. In the NMR spectra, the splitting patterns were determined and recognized as follows: s: Singlet   Figure 5A presents the superimposition of donepezil and compound 2e in the active region of the AChE. It was clear that compound 2e bound to the active site of the AChE enzyme in a similar position as donepezil due to its dual binding sites. This compound mainly carries the lipophilic indan ring, 4-substituedphenyl-thiazole group, as a polar group, and a basic center. The docking poses indicated that lipophilic groups interacted with the PAS region, whereas the polar and basic groups bound to the CAS region.
The docking pose of compound 2e ( Figure 5B) revealed that the indan ring created a π-π interaction with the indole of Trp286. This interaction was the same as that with donepezil in relation to the PAS region. Moreover, it was seen that the thiazole ring in the middle of the structure was essential to attain strong binding. A hydrogen bond was observed between the nitrogen atom of the thiazole ring and the hydroxyl of Tyr124. This interaction highlighted the binding to the CAS region, as in the donepezil via the benzylamine moiety. The thiazole ring established two π-π interactions with the phenyl rings of Tyr337 and Tyr341. Thus, it can be said that the thiazole ring interacted with both the CAS and PAS, owing to these interactions with the Tyr337 and Tyr341 amino acid residues. In addition, the phenyl ring near the thiazole ring had a π-π interaction with the imidazole of Hid447. This interaction was a proof of binding to the PAS. Moreover, the nitro substituent of the phenyl ring was important for polar interaction. The oxygen atom of the nitro formed a hydrogen bond with the hydroxyl of Glu202.
Structurally, the main difference between compound 2e and the other derivatives was the nitro group at the paraposition of the phenyl ring. In this context, the docking studies supported the enzyme inhibition results. It was thought that the interactions related to the nitro group at the paraposition of the phenyl ring were important in terms of explaining its inhibitory activity against the AChE enzyme. It was seen that the presence of an electron withdrawing group, such as nitro, at this position was a positive contribution to the activity.
In order to analyze the contribution of the van der Waals and electrostatic interactions in binding to the enzyme active site, docking studies were performed using Glide (Schrödinger, NY, USA), according to the per-residue interaction panel. Figure 5C,D presents the van der Waals and electrostatic interactions of compound 2e. As is shown, this compound had favorable van der Waals interactions with amino acids Tyr72, Trp86, Tyr124, Glu202, Trp286, Val294, Phe295, Phe297, Tyr337, Phe338, Tyr341, and Hid447, which are displayed in pink and red, as described in the Glide user manual [38]. Similarly, promising electrostatic contributions of compound 2e were determined with amino acids Asp74, Tyr124, Glu202, Phe295, and Arg296.
The percentage of inhibition results and IC 50 values, which were calculated using a dose-response curve achieved by plotting the percentage of inhibition versus the log concentration using the GraphPad PRISM software version 5.0 (San Diego, CA, USA), were shown as the mean ± standard deviation (SD).

Kinetic Studies of Enzyme Inhibition
The AChE enzyme kinetics study was performed for compound 2e to determine the type of inhibition. Different concentrations (IC 50 , 2 × IC 50 and IC 50 /2) of compound 2e were prepared for this assay in addition to a substrate (ATC) at various concentrations (600, 300, 150, 75, 37.5, and 18.75 µM). The enzyme kinetics assay was carried out as in previous publications [7,[16][17][18][19][20][21][22]. Lineweaver-Burk plots were formed using Microsoft Office Excel 2013. The K i values of the compound were easily calculated from the second plot with a common intercept on the x-axis (corresponding to −K i ).

Inhibition of Beta Amyloid 1-42 (Aβ42) Aggregation
The test procedure was created based on the protocol of the beta amyloid 1-42 (Aβ42) ligand screening assay (BioVision, Milpitas, CA, USA), based on the fluorometric method.

Prediction of ADME Parameters and BBB Permeability
Physicochemical parameters were performed with the use of the QikProp 4.8 software [23] to predict pharmacokinetic profiles and BBB permeability of obtained compounds (2a-2l).

Molecular Docking
A structure based in silico procedure was applied to discover the binding modes of compound 2e to the hAChE enzyme active site. The crystal structures of hAChE (PDB ID: 4EY7) [12], which was crystallized with donepezil, was retrieved from the Protein Data Bank server (www.pdb.org).
The structures of ligands were built using the Schrödinger Maestro [40] interface and then were submitted to the Protein Preparation Wizard protocol of the Schrödinger Suite 2016 Update 2 [41]. The ligands were prepared by the LigPrep 3.8 [42] to assign the protonation states at pH 8.0 ± 1.0 and the atom types, correctly. Bond orders were assigned, and hydrogen atoms were added to the structures. The grid generation was formed using Glide 7.1 [38]. Flexible docking runs were performed with a single precision docking mode (SP).

Conclusions
In conclusion, a new series of indan-thiazolylhydrazone derivatives was designed, and the inhibition profiles of the cholinesterase enzymes were evaluated. None of the synthesized compounds displayed remarkable enzyme activity on the BChE enzyme. All of the compounds showed selectivity against the AChE enzyme. Among the obtained compounds, derivatives of 2a, 2e, 2i, and 2l were found to be most active agents. Compound 2e, which contained a 4-nitrophenyl ring, was determined to be the most effective inhibitor candidate, with an IC 50 value of 0.026 µM. As a result of the docking studies, it was believed that the 6-methoxyindan moiety and 4-substituedphenyl-thiazole ring were essential for inhibiting the AChE enzyme. These structures were responsible for binding to the CAS and PAS regions of the enzyme active sites. Thus, it was observed that compound 2e had a dual binding site feature by settling into the CAS and PAS, as did the donepezil molecule. Hence, all of the findings showed that the strategy of this study was correct and logical by structurally modifying the donepezil. These data provided the way to guide future studies.