Synthesis of new N‐(5,6‐methylenedioxybenzothiazole‐2‐yl)‐2‐[(substituted)thio/piperazine]acetamide/propanamide derivatives and evaluation of their AChE, BChE, and BACE‐1 inhibitory activities

In this study, the synthesis of N‐(5,6‐methylenedioxybenzothiazole‐2‐yl)‐2‐[(substituted)thio/piperazine]acetamide/propanamide derivatives (3a‐3k) and to investigate their acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and β‐secretase 1 (BACE‐1) inhibition activity were aimed. Mass, 1H NMR, and 13C NMR spectra were utilized to determine the structure of the synthesized compounds. Compounds 3b, 3c, 3f, and 3j showed AChE inhibitory activity which compound 3c (IC50 = 0.030 ± 0.001 µM) showed AChE inhibitory activity as high as the reference drug donepezil (IC50 = 0.0201 ± 0.0010 µM). Conversely, none of the compounds showed BChE activity. Compounds 3c and 3j showed the highest BACE‐1 inhibitory activity and IC50 value was found as 0.119 ± 0.004 µM for compound 3j whereas IC50 value was 0.110 ± 0.005 µM for donepezil, which is one of the reference substance. Molecular docking studies have been carried out using the data retrieved from the server of the Protein Data Bank (PDBID: 4EY7 and 2ZJM). Using in silico approach behavior active compounds (3c and 3j) and their binding modes clarified.


| INTRODUCTION
Dementia is a term used to describe several conditions that affect memory, thinking and the ability to carry out daily activities.A total of 55 million people worldwide have dementia.Nearly 10 million new cases are diagnosed each year.Alzheimer disease (AD) is the most common form of dementia, accounting for 60%-70% of all cases (Sang et al., 2022).AD is a progressive neurodegenerative condition characterized by age-related memory loss.It was first described by Dr. Alois Alzheimer in 1906 (Deture & Dickson, 2019).Due to the complexity of the disease, there is still a lack of understanding of the underlying mechanisms of AD (Erdogan et al., 2021).
The most elucidated pathogenesis of AD is the cholinergic deficiencies that cause the loss of memory and learning ability (Tok et al., 2022).The cholinergic hypothesis proposed that Alzheimer is caused by a lack of a key neurotransmitter called acetylcholine (ACh) in the neuronal gaps (Chen et al., 2022;Walczak-Nowicka & Herbet, 2021).Acetylcholinesterase (AChE) is an enzyme that hydrolyzes acetylcholine to choline and acetic acid (Saǧlık et al., 2020;Zhao et al., 2017).Inhibiting AChE increases acetylcholine levels by preventing its breakdown in brain tissue, thus improving cholinergic function in Alzheimer patients (Moss, 2020;Sahin et al., 2020;Srivastava et al., 2021).AChE inhibitors are the main class of drugs used to treat AD.Four out of the five drugs (tacrine, donepezil, galantamine, rivastigmine) approved by the Food and Drug Administration and available for the treatment of AD are AChE inhibitors (Marucci et al., 2021;Saǧllk, 2022;Uytun et al., 2022).
The drugs that are currently being used to treat AD are not as effective as hoped.In recent studies, researchers have focused on dual inhibition of the BACE-1 and AChE complexes to treat AD and other neurodegenerative disorders (Costanzo et al., 2016;Gabr & Abdel-Raziq, 2018;Rampa et al., 2015).
By combining the 5,6-dimethoxy residue in the structure of the drug donepezil, which has strong anticholinesterase activity, and the 2-aminobenzothiazole structures found in riluzole and sabeluzole molecules, which are known for their N-methyl-Daspartate receptor (NMDA) modulatory effects, 2-amino-5,6methylenedioxybenzothiazole derivatives were designed and the AChE, BChE and BACE inhibitory activities of the compounds were examined.From a similar perspective, 5,6-dimethoxybenzothiazolepiperazine acetamide derivatives (Figure 1,A) were synthesized and anticholinesterase activity was determined with high potency (IC 50 : 0.396-0.0462µM) on AChE (Demir Özkay et al., 2016).In another study, AChE inhibition with IC 50 : 0.023 µM was determined in the derivative containing nonsubstituted benzothiazole and dimethylaminoethyl residue (Figure 1,B) (Karaca et al., 2022).It was also determined that the benzothiazolacetamide derivative (Figure 1,C) containing 4-methylbenzyl exhibited anti-AChE activity with IC 50 : 0.061 µM in a different study (Özkay et al., 2012).Additionally, in a F I G U R E 1 Benzothiazole framework in some studies and 3a-3k compounds.The obtained compounds were analyzed.In the supporting information, the spectra obtained are given.And the list of compounds obtained is shown in Table 1.
When the 1 H-NMR results of the final synthesized compounds were examined, it was found that the results were in the range of 1.44-12.61ppm.In all compounds the peak belonging to the two hydrogens of the 1,3-dioxolo structure was observed in the range of 5.99-6.12ppm.In addition, it was determined that the hydrogens bonded to carbonyl were between 3.37 and 4.40 for COCH 2 and between 4.15 and 4.76 for COCHCH 3 .In addition, the peaks of the hydrogens of the benzothiazole ring were observed in the range of 7.29-7.56ppm.
When the 13 C-NMR results of the final synthesis compounds were examined, it was found that the results were in the range of 17.51-171.16ppm.In addition, the carbon atom attached to the carbonyl was found to be between 35.32 and 60.61 for COCH 2 and between 43.76 and 45.58 for COCHCH 3 .A range of 100.47-101.89ppm was observed for peaks belonging to carbon of 1,3-dioxolo structure.It was also observed that the peaks belonging to the carbons of the benzothiazole ring were found to range from 100.95 to 171.16 ppm.
Mass spectra of the synthesized result compounds were obtained using the APCI-MS method.The results showed that the molecular weights of the compounds are compatible with the M +H peak or M-H.However, the M+H peak could not be obtained because some compounds degraded during application.

| Evaluation of enzyme inhibition activity
In this study, the AChE, BChE, and BACE-1 activities of the synthesized

Docking study on AChE enzyme
To estimate the binding modes of the four active compounds (3b, 3c, 3f, and 3j), the compounds were docked to the active site of the AChE.
According to Figure 2, active compounds (3b, 3c, 3f, and 3j), binding forms and types on AChE are listed in Table 4.The 4EY7 crystal form of the enzyme has been chosen for the docking of the compounds.
According to the data provided, compound 3b interacted with Trp286, showed affinity to these residues with at least one interaction.
Commonly, active compounds interacted via π-π stacking.Only compound 3f did not show affinity to Trp86, but still this compound occupied the CAS region.All compounds formed H-bonds with Phe295 while compounds 3b and 3c also formed H-bonds with Arg296.For more information, compound 3c was used as a model for its analogs to investigate.

Docking study on BACE-1 enzyme
Molecular docking study against the BACE-1 inhibitor was performed for compound 3j, which showed the highest BACE-1 inhibitory activity among the final synthesized compounds.According to Figure 3, the highest active compounds (3j), binding forms and types on BACE-1 are listed in Table 5.The 2ZJM crystal form of the enzyme was selected for docking.According to the information obtained, it was observed that   (hydrophobic interaction and water-mediated hydrogen bond), Tyr124 (hydrogen bond, hydrophobic interaction, water-mediated hydrogen bond), Trp286 (hydrophobic interaction, water-mediated hydrogen bond), Phe295 (water-mediated hydrogen bond), Tyr337 (hydrogen interaction, hydrophobic interaction, water-mediated hydrogen bond), Phe338 (hydrophobic interaction, water-mediated hydrogen bond), and Tyr341 (hydrophobic interaction, watermediated hydrogen bond).
The interaction of compound 3c with Tyr341 was continuous and appeared to be largely hydrophobic bond and water-mediated hydrogen bonding.While Gly122 and Trp86 did not interact with compound 3c, Tyr124 did interact with compound 3c.However, most of the interaction of Tyr124 is via hydrogen bonding.When compound 3c interacts less with Tyr337, it interacts more with Asp74.Water-mediated hydrogen bonding is responsible for all the interaction with Asp74.When the interaction of Phe338 with compound 3c decreased, the interaction of Tyr337 increased.A decrease in the interaction of compound 3c with Ser203 was accompanied by an increase in the interaction of Trp286.

MDS of BACE-1
MDS study was performed on compound 3j, which showed the highest BACE-1 inhibitory activity among the final synthesized compounds.As described previously (in MDS results of AChE section), the stability of the complex was protected during the entire simulation according to Figure 5a-c.

| Results of ADME parameters and Lipinski's rule of five
The pharmacokinetic profiles of the synthesized compounds were calculated with the Swiss-ADME web-based program and whether they violated Lipinski's rule of five is shown in Table 6.The pre-calculation of ADME during drug discovery has been shown to significantly reduce the rate of pharmacokinetic-related drug failures at any clinical stage (Daina et al., 2017).The conditions required for compounds to be considered suitable for oral bioavailability are as follows MW ≤500 Da, logP <5, nHBD ≤5, nHBA ≤10, and TPSA <140 Å2 (Ya'u Ibrahim et al., 2020).
When the results were examined, it was found that the conditions of MW ≤500 Da, logP <5, nHBD ≤5, nHBA ≤10 were met for all compounds.In addition, TPSA was found to be in the range of 95.17-188.49Å2.Furthermore, all compounds except compounds 3b, 3c, 3h, and 3i were found to have suitable TPSA.
The log Kp value was found to be between −5.92 and −7.17.
This reaction mixture was placed in an ice bath and cooled to 0-10°C.
Bromine (0.82 mL, 0.016 mol) in 10 mL of acetic acid was then added dropwise to the reaction mixture.After the bromine addition was completed, the mixture was stirred at room temperature for a whole day.The precipitate was then collected and boiled in water for 8 h.
The resulting mixture was neutralized with ammonia solution in ice water.The resulting solids were washed with water and then dried (Karaca et al., 2022).
The end of the reaction was checked by TLC after 2 h of addition.After the solvent evaporated completely, the mixture was washed with water.

| AChE and BChE enzyme inhibition activities
The modified Ellman method was used to determine the anticholinesterase activity of the final synthesized compounds on AChE and BChE enzymes (Ellman et al., 1961).Solutions of AChE or BChE

| BACE-1 enzyme inhibition activities
The experimental procedure for BACE-1 inhibition was based on the protocol of the "Human β-Secretase (BACE-1) Inhibitor Screening Assay" kit (BioVision), which is based on the fluorometric method (Tok et al., 2022).

| Molecular docking studies and molecular dynamics studies
The in silico docking procedure was applied to understand potential interactions, which point out to us what's the relation between ligands and acetylcholinesterase and ß-secretase enzymes (PDBID: 4EY7 and 2ZJM, respectively).Herewith, it helps us to understand the behavior of how active compounds act in the active region of the enzyme.Considering in vitro enzyme tests, compounds 3b, 3c, 3f, and 3j were docked into AChE active pocket and BACE-1 binding cavity using structure-based in silico docking procedure.After docking studies, to understand environmental effects regarding time on the stability and behavior of ligand-protein complex, the most active compound on both enzymes, 3c and 3j, was used as a model for its analogs.The MDS method for at least 50 ns simulation time was applied the same as performed previously by our team (AL-Sharabi et al., 2023;Evren, 2022b;Naji et al., 2023).

F
I G U R E 3 2D and 3D molecular docking poses of 3j.TUTUŞ ET AL. | 7 of 16 2.4 | Structure-activity relationship evaluation The results we have obtained in our activity assays and in our molecular docking studies have shown that the mercaptoaryl or phenylpiperazine ring substituted benzothiazole ring system has AChE inhibitor activity as well as BACE-1 inhibitor activity.Most of the AChE inhibitory activity was found in compounds with a mercaptoaryl substituted benzothiazole ring system.In particular, compound 3c, characterized by 1-methyl-2-T A B L E 5 Summary of the interaction of 3j with the active pocket of the BACE-1 enzyme.stacking F I G U R E 4 Interaction plots of the MDS results for the enzyme complexes of 3c-acetylcholinesterase.(a) Diagram of the ligand properties, (b) RMSD plot of the ligand and of the protein throughout the simulation, (c) RMSF-plots of amino acids and their interactions, (d) Interaction between fraction and residue diagram, (e) Plot of the number of interactions-interaction types-time, and (f) Plot of the total interactionresidues-time.mercaptotetrazole substitution, showed remarkable AChE inhibitory activity.In addition, compound 3c has significant inhibitory activity against the enzyme BACE-1.Significant AChE inhibitory activity was observed with the 4-methyl-2-mercaptotriazole substituted compound 3b.On the other hand, changing the substitution to a ring with less nitrogen (e.g., compounds 3c and 3b) resulted in a slight decrease in AChE activity.Among all the phenylpiperazine substituted compounds that have been synthesized, only the 4-methoxyphenylpiperazine substituted derivative, compounds 3f showed AChE inhibitory activity.The addition of a methyl group to the acetamide/propanamide linker group, increases the AChE inhibitory activity, and the synthesized compound can also interact with the BACE-1 enzyme and inhibit the enzyme.In the activity test results of the AChE and BACE-1 enzyme, compound 3c (1-methyl-2-mercaptotetrazole substituted derivative) and 3j (1-methyl-2-mercaptoimidazole substituted derivative), showed a significant AChE and BACE-1 inhibitory activity.

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I G U R E 5 Interaction plots of the MDS results for the enzyme complexes of 3j-BACE-1.(a) Diagram of the ligand properties, (b) RMSD plot of the ligand and of the protein throughout the simulation, (c) RMSF-plots of amino acids and their interactions, (d) Interaction between fraction and residue diagram, (e) Plot of the number of interactions-interaction types-time, and (f) Plot of the total interaction-residues-time.

[
(substituted)thio/piperazine]acetamide/propanamide derivatives were synthesized in three steps starting from 3,4-(methylenedioxy)aniline.The structures of the synthesized compounds were confirmed by 1 H-NMR, 13 C-NMR, and mass spectrometry.The final synthesized compounds were tested for AChE, BChE and BACE-1 enzyme inhibition.Compounds 3b, 3c, 3f, 3g, and 3j showed high AChE inhibitory activity.Compound 3c (IC 50 = 0.030 ± 0.00 µM) showed the highest AChE inhibitory activity.Compound 3c and 3j showed high BACE-1 inhibitory activity.Compound 3j (IC 50 = 0.119 ± 0.004 µM) showed the highest BACE-1 inhibitory activity.In addition, it was determined that compounds 3c and 3j, which were among the compounds synthesized in this study, have both AChE and BACE-1 inhibitory activities.The results of molecular docking and molecular dynamics studies indicated that compound 3c was constantly interacting with the active site of the AChE enzyme.The binding of compound 3j ensured that the compound-BACE-1 enzyme complex remained stable throughout the simulation.The majority of the substances that showed AChE inhibitory activity were found to be compounds with a mercaptoaryl substituted benzothiazole ring system.In particular, compound 3c, which is characterized by the 1-methyl-2-mercaptotetrazol substitution, exhibited notable AChE inhibitory activity.Further, compound 3c has significant inhibitory activity against the enzyme BACE-1.Compound 3b substituted with 4-methyl-2-mercaptotriazole showed significant AChE inhibitory activity.In contrast, replacement of the substituent by a ring containing less nitrogen (e.g., compounds 3c and 3b) resulted in a slight decrease in AChE activity.Several previous studies have shown that benzothiazole derivatives have a high level of cholinesterase inhibitory activity.Based on the comprehensive information available, our study showed that the newly designed benzothiazoles exhibited inhibitory effects on both the AChE and BACE-1 enzymes.In the light of this information, we aim to synthesize a new AChE and BACE-1 inhibitor based on this structure in our future studies.
0.3 g of the corresponding N-(5,6-methylenedioxybenzothiazole-2-yl)-2-chloroacetamide/propanamide derivatives were taken in acetone.Equal moles of the appropriate mercapto or phenylpiperazine derivatives were then added.Then, equal mole of potassium carbonate was added and stirred at room temperature.

(
20 µL) prepared in 2% DMSO in the concentration range 10-1-10-6 mM and solutions of compounds (20 µL) were added to 140 µL phosphate buffer (pH 8 ± 0.1) and incubated at 25°C for 5 min.The reaction was initiated by the addition of 5-5-dithiobis(2-nitrobenzoic acid (DTNB) (20 µL) and acetylthiocholine iodide (ATC) (10 µL) to the mixture of enzyme and inhibitor.All solutions for this enzyme assay were stored at −20°C and were brought to room temperature just before the start of the experiments.After the solutions were mixed in the wells, they were incubated in an oven for 15 min at an absorbance value of 412 nm.The tests were carried out at a concentration of 10 −3 and 10 −4 M and the experiments were repeated four times.Lower concentrations were also tested for the determination of the IC 50 dose for some compounds. .