Multipotent Cholinesterase Inhibitors for the Treatment of Alzheimer’s Disease: Synthesis, Biological Analysis and Molecular Docking Study of Benzimidazole-Based Thiazole Derivatives

Twenty-four analogues of benzimidazole-based thiazoles (1–24) were synthesized and assessed for their in vitro acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory potential. All analogues were found to exhibit good inhibitory potential against cholinesterase enzymes, having IC50 values in the ranges of 0.10 ± 0.05 to 11.10 ± 0.30 µM (for AChE) and 0.20 ± 0.050 µM to 14.20 ± 0.10 µM (for BuChE) as compared to the standard drug Donepezil (IC50 = 2.16 ± 0.12 and 4.5 ± 0.11 µM, respectively). Among the series, analogues 16 and 21 were found to be the most potent inhibitors of AChE and BuChE enzymes. The number (s), types, electron-donating or -withdrawing effects and position of the substituent(s) on the both phenyl rings B & C were the primary determinants of the structure-activity relationship (SAR). In order to understand how the most active derivatives interact with the amino acids in the active site of the enzyme, molecular docking studies were conducted. The results obtained supported the experimental data. Additionally, the structures of all newly synthesized compounds were elucidated by using several spectroscopic methods like 13C-NMR, 1H-NMR and HR EIMS.


Chemistry
In this study, well-known methodology was adopted for the synthesis of benzimidazolebased thiazole derivatives  from readily accessible precursors such thiosemicarbazide, various phenacyl bromides and 2-marcaptobenzimidazole.
Initially, 2-bromoacetophenone (II) was reacted with 2-marcapto benzimidazole (I) in EtOH and Et 3 N (catalyst), and the reaction mixture was refluxed for 2-3 h to yield the first intermediate (III) [44]. An equivalent amount of intermediate (III) and thiosemicarbazide were further treated in EtOH and CH 3 COOH (glacial) and the solution was refluxed for about 4 h to yield a second intermediate product (IV). The reaction mixture was cooled to room temperature when it was finished, and the precipitate solid that resulted was then filtered and washed with n-hexane. The reaction's progress had been monitored by using TLC plate. Finally, intermediate (IV) underwent cyclization with stirring overnight with a different substituted 2-bromoacetophenone in EtOH and Et 3 N (catalyst) to yield benzimidazole-based thiazole analogues  in moderate to good yield (Scheme 1). The solvent was removed after being cooled to 25 • C and the resultant solid residue was then cleaned by washing with n-hexane and re-crystallized from ethyl acetate. The precise structures of all newly synthesized analogues were determined using several spectroscopic methods, including 13 C-NMR, 1 H-NMR and HR EIMS. The 1 H NMR spectrum of analogue 1 was recorded in DMSO-d 6 on a Bruker 600 MHz instrument. The peak for the hydroxyl proton (OH) was observed at δ H 10.11 (s, 1H, -OH). The most downfield singlets of two -NH protons, one for benzimidazole (NH) and another amino (NH) proton between the thiazole and benzimidazole rings, were resonated at δ H 13.25 and 11.81, respectively. The molecule comprises three aromatic rings labeled as A, B C. Among the ring B protons, doublets appeared at δ H 7.79 for two protons H-2 and H-6 (Ar-H), while another two protons of this ring H-3 H-5 (Ar-H) were resonated at δ H 7.88 (d, J = 8.88 Hz, 2H, Ar-H) as doublets, respectively. On the other hand, two aromatic protons labeled as H-3" and H-6"(Ar-H) of ring C resonated at δ H 8.28 (s, 1H, H-3") and 8.10 (s, 1H, H-6") as singlets. Besides this, a triplet was observed for two aromatic protons of benzimidazole at δ H 7.43 (t, J = 7.92 Hz, 2H, H-5/H-6). However, thiazole-H was resonated at δ H 7.33 as a singlet. Moreover, two more aromatic protons of benzimidazole, namely H-4 and H-7, were also resonated as doublets at δ H 7.27 (d, J = 7.68Hz, 1H, H-4) and 7.04 (d, J = 7.56Hz, 1H, H-7), respectively. Furthermore, a singlet was also observed for two active methylene protons (-CH 2 -) attached between sulfur and aromatic ring B at δ H 2.34 (s, 2H, -S-CH 2 ).
As for compound 1, the 13 C NMR signal δ C-13 at 175.81 was attributed to a thiazole carbon (C) present between sulfur and nitrogen atoms, while two peaks at δ C-13 139.85 (C-thiazole) and 110.66 (C-thiazole) were observed for the remaining two carbons of the thiazole ring. The peak at δ C-13 138.96 (C) was observed for the benzimidazole carbon-bearing substitution, while two bridged carbons of benzimidazole were recorded at δ C-13 137.27 (C). The carbon involved in the doublet with nitrogen (C=N) was resonated at δ C-13 143.52 (C).The carbons of the aromatic phenyl rings B and C involving substitutions were resonated at δ C-13 133.67 (C-OH), 132.00 (C-NO 2 ), 127.83 (C), 126.25 (C-Cl) and 125.92 (C-Br). The peak corresponding to carbons of aromatic rings B and C without substitutions were resonated at δ C-13 122.93 (CH-3 and CH-5 ), 123.69 (CH), 120.66 (CH-2 and CH-6 ) and 119.01 (CH), respectively. The peaks corresponding to the remaining four carbons of benzimidazole were observed at δ C-13 123.7 (CH), 123.5 (CH), 115.8 (CH) and 115.3 (CH), respectively. The peak at δ C-13 38.5 (-CH 2 -) corresponded to active methylene groups attached to a sulfur atom.

In Vitro Evaluation of AChE and BChE Inhibition by Novel Benzimidazole-Based Thiazoles
All the newly synthesized derivatives of benzimidazole-based thiazole  were evaluated as potential inhibitors of AChE and BChE, and IC 50 was determined. All analogues showed moderate to good inhibitory potential against both AChE and BChE. The IC 50 values ranged from 0.10 ± 0.05-11.10 ± 0.30 µM (for AChE) and 0.20 ± 0.050 µM-14.20 ± 0.10 µM (for BChE). Some of these compounds were shown to be more potent inhibitors than the standard drug Donepezil (IC 50 = 2.16 ± 0.12 µM and 4.5 ± 0.11 µM respectively) ( Table 1). The synthesized analogue was divided into four parts: benzimidazole ring A, thiazole portion, phenyl rings B & C, in order to better understand the structure-activity relationship (SAR). The nature, electron-withdrawing or -donating effects, number(s) and position of the substituent(s) on both phenyl rings B and C respectively were measured to be the key determinants of the structure-activity relationship ( Figure 4).

R2
Benzimidazole ring A C Ring C

R2
Benzimidazole ring A C Ring C

R2
Benzimidazole ring A C Ring C

R2
Benzimidazole ring A C Ring C

R2
Benzimidazole ring A A C Ring C

Structure-Activity Relationship of acetylcholinesterase and butyrylcholinesterase activities
The derivative 21 (IC50 = 0.10 ± 0.05µM and 0.20 ± 0.05 µM), bearing di-Cl groups at meta-and para-positions of both phenyl rings B and C correspondingly, was emerged as most effective inhibitors of both targeted enzymes (AChE & BuChE). Additionally, the analogue 16 (IC50 = 0.20 ± 0.050 µM for AChE) and 0.50 ± 0.050 µM for BuChE), which had a hydroxy group at the para-position of phenyl ring C and a nitro group at the orthoposition of phenyl ring B was found to be the 2nd most potent inhibitor of both AChE & BuChE enzymes,. The different types, number (s), electron-donating or -withdrawing effect, positions of substituent(s) on the both phenyl rings B and C may account for the effective inhibitory potentials of analogues 16 and 21 ( Figure 5).

Structure-Activity Relationship of acetylcholinesterase and butyrylcholinesterase activities
The derivative 21 (IC 50 = 0.10 ± 0.05µM and 0.20 ± 0.05 µM), bearing di-Cl groups at metaand para-positions of both phenyl rings B and C correspondingly, was emerged as most effective inhibitors of both targeted enzymes (AChE & BuChE). Additionally, the analogue 16 (IC 50 = 0.20 ± 0.050 µM for AChE) and 0.50 ± 0.050 µM for BuChE), which had a hydroxy group at the para-position of phenyl ring C and a nitro group at the orthoposition of phenyl ring B was found to be the 2nd most potent inhibitor of both AChE & BuChE enzymes. The different types, number (s), electron-donating or -withdrawing effect, positions of substituent(s) on the both phenyl rings B and C may account for the effective inhibitory potentials of analogues 16 and 21 ( Figure 5). By comparing compounds 3-6 ( Figure 6), we noticed that there is a difference in the inhibitory potential of these compounds in regard to the position of the substituent(s). This may be caused by the type, position, and number(s) of the substituent(s) on ring C. Analogue 11 (dichloro group at metaand para-positions on ring C, IC 50 = 0.40 ± 0.050 µM and 1.10 ± 0.10 µM) was found to be superior to analogues 7 (nitro group at ortho on ring C, IC 50 = 2.90 ± 0.10µM and 3.50 ± 0.10 µM) and 8 (nitro group at meta position on ring C, IC 50 = 6.30 ± 0.10 µM and 7.90 ± 0.10 µM) (Figure 7). By comparing compounds 13-16 (Figure 8), we noticed that there is a difference in the inhibitory potentials of these compounds in regard to the position of the substituent(s). This could be due to the nature of the substituent(s) on ring C. By comparing compounds 17-19 and 21-22 (Figure 9), we noticed that there is a difference in the inhibitory potentials of these compounds in regard to the position of the substituent(s). This may be caused by the type, position, and number(s) of the substituent(s) on ring C. Overall, it was found that the nature, electron-donating or -withdrawing effect, number and position of the substituent(s) on both rings B and C may considerably affect the inhibitory potentials of the synthesized analogues.
Galantamine is a phenanthrene that induces reversible inhibition of AChE-BuChE; Donepezil is a piperidine that causes reversible AChE inhibition, highly specific; and huperzine A is a pyridine that causes reversible AChE and specific inhibition. Under ideal test circumstances, each AChEI is ranked in the following order by its inhibitory efficacy (IC 50 ) against the AChE activity: physostigmine (0.67 nM) > rivastigmine (4.4 nM) > Donepezil (6.7 nM) > TAK-147 (12 nM) > tacrine (77 nM) > ipidacrine (270 nM). Based on study conducted by Eisai scientists, derivatives with 4-aminopyridine such as ipidacrine and tacrine did not exhibit any selectivity, benzylpiperidine derivatives including TAK-147 and Donepezil exhibited high selectivity for AChE over BuChE, while the carbamate derivatives exhibited moderate selectivity. AChE inhibition by Donepezil is 40-500 times more effective than by galantamine, according to more recent studies. Galantamine leaves the brain more quickly than Donepezil does. Galantamine and Donepezil both inhibit brain AChE to a similar degree, according to their respective Ki values, which are 7.1-19.1 and 0.65-2.3 g/g in different species, respectively, the doses of needed for galantamine is 3-15 times greater than those of Donepezil [46].

Docking Study
The main objective of molecular docking study was to learn more about how newly afforded compounds bind to enzymes (i.e., both AChE and BuChE). Based on the residing co-crystal of each crystallographic structure, all of the compounds were docked. Each compound received a total of thirty conformations before the docking process. For additional protein-ligand interaction (PLI) profiling, the top-ranked conformations were chosen.
The docking results showed that all of the compounds were located in the active sites of both enzymes in the proper orientation. In general, we found that all the compounds had different substitution groups at their two ends, while the third end remained the same for all compounds, i.e., the electron-donating groups (also known as activated groups) and electron-withdrawing (also known as deactivated groups) at different positions. Additionally, we noticed that most active compounds held both the activated and deactivated groups at their two ends, which had a strong magnitude of activation. Surprisingly, the PLI profiles along with the in-vitro data revealed that compounds 16 and 21, which contain activated and deactivated substituting groups over the benzene ring, had the highest inhibitory potential levels of the entire series. For example, compounds 16 and 21 have nearly identical activity levels against AChE but are ranked 1st 21 and 2nd 16 against BuChE, respectively. Numerous significant interactions with catalytic residues were found in the comprehensive PLI profiles of both compounds against both targets. These interactions may have a significant impact on the improvement of the enzymatic activity of both enzymes. It was shown by PLI profile that analogue 21 adopted numerous key interactions with catalytic residues of AChE including the residues Phe330, Phe331, Tyr334, Asp72, Trp84, Tyr121 and Trp279 ( Figure 10A), while this analogue against BuChE exhibited several important interactions such as Tyr332, Phe329, Ala328, Trp82, Asp70, Gly116, Gly117, Trp231, Ser287 and Leu286 ( Figure 10B). The attached di-Cl on both ends of the compound may be the cause of its high potential, where the Cl that withdraw most of the electronic density from benzene ring, resulting in a partial positive charge over the benzene ring, causing this benzene ring to try to re-gain the stability via adopting several key interactions with active side residues, thereby enhancing the enzymatic activity. Overall, we observed that the compounds holding the substituted group, which had a strong magnitude of either withdrawal or donation, showed best potential against both targeted enzymes. Similarly, the PLI profile shown by analogue 16 against AChE revealed several key interactions with the active site residues, including the residues Trp84, Glu199, Tyr121, Trp279, Asp285, Tyr334 and Asp72 ( Figure 11C), while against the BuChE, this compound adopted several key interactions with Trp231, Leu286, Pro285, Phe329, Asp70, Ile69, Ala328 and Trp82 ( Figure 11D). The elevated potential of this compound may be a result of the strong electron-donating and electron-withdrawing groups that are connected to it, i.e., hydroxyl and nitro group, where the -OH-substituting group donates some its electronic density with high potential to the 6c-ring and then further this electronic density cascades to other important moieties of the compounds; hence, In this manner, the overall potency of compound as an inhibitor against both targeted enzymes is high. In addition, the PLI profile revealed several key interactions, particularly with the side of the attached substituted groups or nearby moieties. Additionally, the PLI profile shown by analogue 15 against AChE adopted several key interactions with the active site residues, including the residues Trp70, Trp84, Glu199, Phe331, Tyr334, Trp279 and Tyr70 ( Figure 12E), while against the BuChE, this compound adopted several key interactions with Thr120, Asp70, Trp82, His438, Phe329, Trp231, Leu286 and Gly117 ( Figure 12F). The elevated potential of this analogue 17 might have been due to the attached electron-withdrawing groups, such as di-Cl and nitro groups on both ends of analogue, where both di-Cl and -NO 2 groups withdraw most of the electronic density from the benzene rings, resulting in a partial positive charge over the benzene ring, and further this benzene ring tries to re-gain the stability via adopting several key interactions with active side residues, thereby enhancing the enzymatic activity.  16 both ends of analogue, where both di-Cl and -NO2 groups withdraw most of the electronic density from the benzene rings, resulting in a partial positive charge over the benzene ring, and further this benzene ring tries to re-gain the stability via adopting several key interactions with active side residues, thereby enhancing the enzymatic activity. The calculated binding energies, number of hydrogen bonds, and number of closest residues surrounding the selected docked analogues into the active sites of both AChE and BuChE enzymes are shown in Table 2. The calculated binding energies, number of hydrogen bonds, and number of closest residues surrounding the selected docked analogues into the active sites of both AChE and BuChE enzymes are shown in Table 2.

General Information
All of the solvents and chemicals, with a purity range of 97 to 99%, were acquired from Sigma Aldrich. Using DMSO as the solvent, the NMR spectra were recorded using a Bruker Ultra Shield Plus NMR spectrometer. By using TMS as reference standard, the chemical sifts values were measured. The high-resolution mass spectra (electron impact, 60 eV) were run on a MAT-311A instrument (Germany). For visualization of the chromatograms, a UV lamp (Schimazdu, Germany) with a wavelength of 254/365 nm was used.

General Procedure for the Synthesis of Benzimidazole-Bearing 1,3-Thiazole Scaffolds (1-24)
The intermediate (III) was obtained by reacting phenacyl bromide (II, 1 mmol) with 2-marcapto benzimidazole (I, 1 mmol) in EtOH (10 mL) and Et 3 N (few drops) and stirred for 2-3h under reflux condition [44]. An amount of equivalent intermediate (III) and thiosemicarbazide were further treated in ethanol (10mL) and CH 3 COOH (glacial) and the solution was refluxed for about 4 h to yield the second intermediate product (IV). Finally, intermediate (IV) underwent cyclization with stirring overnight with different substituted 2-bromoacetophenone in ethanol (10mL) Et 3 N (few drops) to yield benzimidazole-based thiazole analogues  in moderate to good yield. The solvent was removed after being cooled to room temperature and the resultant solid residue was then cleaned by being washed with n-hexane and re-crystallized from ethyl acetate.

Molecular Docking Protocol
The MOE software programme was used in molecular docking to determine how synthesized analogues interact with both targeted enzyme (AChE & BuChE) to triangulate the outcomes from in vitro and in silico analysis. Using the PDB codes 1ACL for AChE and 1P0P for BuChE, the RCSB protein databank's crystal structures for both targets were retrieved. The crystallographic structures and all synthesized analogues were protonated using the default MOE-Dock module parameters, resulting in analogue structures and optimized enzyme. After this, a docking study was conducted using the optimized enzyme and analogues structures. Our earlier investigations contain all of the detailed information about the docking process [47][48][49].

Acetylcholinesterase Activity Assay Protocol
The inhibition of AChE and BChE was determined using a method described earlier [50,51]. Briefly, the stock solutions (1 mg/mL) of test analogues were prepared using DMSO. The working solutions (1-100 µg/mL) were prepared using serial dilutions (a serial dilution means a series of diluted solutions, e.g., 0.1 mg/mL, 0.2 mg/mL and so on; the solutions contained 5% DMSO and 95% water). The various concentrations of test compounds (10 µL) were pre-incubated with sodium phosphate buffer (0.1 M; pH 8.0; 150 µL) and AChE (0.1 U/mL; 20 µL) for 15 min at 25 • C. The reaction was initiated via the addition of DTNB (1 mM; 10 µL) and AChEI (1 mM; 10 µL). The mixture of reaction was mixed using a cyclomixer and incubated for 10 min at 25 • C. The absorbance was measured using a microplate reader at a 410 nm wavelength against the blank reading containing 10 µL DMSO instead of the test compound (the solution contained 5% DMSO and 95% water). Utilizing the formula given in Equation (1), the percentage of inhibition was computed, and the IC 50 was determined under the positive control of Donepezil (0.01-100 g/mL). % Inhibition = (Absorbance of control-Absorbance of compound)/Absorbance of control × 100 IC 50 is the concentration of a drug or inhibitor required to inhibit 50% of an enzyme's activity which was calculated by constructing a non-linear regression graph between