Benzimidazole-Based Schiff Base Hybrid Scaffolds: A Promising Approach to Develop Multi-Target Drugs for Alzheimer’s Disease

A series of benzimidazole-based Schiff base derivatives (1–18) were synthesized and structurally elucidated through 1H NMR, 13C NMR and HREI-MS analysis. Subsequently, these synthetic derivatives were subjected to evaluation for their inhibitory capabilities against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). All these derivatives showed significant inhibition against AChE with an IC50 value in the range of 123.9 ± 10.20 to 342.60 ± 10.60 µM and BuChE in the range of 131.30 ± 9.70 to 375.80 ± 12.80 µM in comparison with standard Donepezil, which has IC50 values of 243.76 ± 5.70 µM (AChE) and 276.60 ± 6.50 µM (BuChE), respectively. Compounds 3, 5 and 9 exhibited potent inhibition against both AChE and BuChE. Molecular docking studies were used to validate and establish the structure–activity relationship of the synthesized derivatives.


Introduction
Alzheimer's dementia (AD) is a well-known neurodegenerative condition affecting the aging population [1][2][3].AD is characterized by a gradual progression within the brain, initiating years prior to the manifestation of visible symptoms [4].Early clinical indications of AD also include memory issues with names, events, or conversations from recent interactions.Some of the later symptoms include decreased communication, disorientation, confusion, strange behavior, poor judgment, swallowing, walking difficulties, and deterioration in speech [5].While a comprehensive understanding of the underlying causes and origins of the disease is still in progress, the histopathological alterations observed in the brains of patients encompass the presence of extracellular amyloid β-protein (Aβ) accumulation within amyloid plaques [6], as well as the formation of intraneuronal neurofibrillary tangles [7].AD is not currently managed in any way.The treatments for AD that are currently available only offer symptomatic alleviation; they cannot alter the course of the disease [8].Aβ and τ-protein development, which lowers levels of brain acetylcholine (ACh), is related to increased acetylcholinesterase (AChE) enzyme activity and the loss of muscarinergic neurons [9].Numerous drugs intended for Alzheimer's disease patients have been medically approved, such as Donepezil and galantamine.These drugs are designed to selectively target AChE.In contrast, compounds like rivastigmine and tacrine have been identified as BuChE and AChE inhibitors (Figure 1) [10].
Pharmaceuticals 2023, 16, x FOR PEER REVIEW 2 of 20 AD that are currently available only offer symptomatic alleviation; they cannot alter the course of the disease [8].Aβ and τ-protein development, which lowers levels of brain acetylcholine (ACh), is related to increased acetylcholinesterase (AChE) enzyme activity and the loss of muscarinergic neurons [9].Numerous drugs intended for Alzheimer's disease patients have been medically approved, such as Donepezil and galantamine.These drugs are designed to selectively target AChE.In contrast, compounds like rivastigmine and tacrine have been identified as BuChE and AChE inhibitors (Figure 1) [10].It has been demonstrated that benzimidazole and its analogs have a variety of biological profiles, including those that act with antihypertensive [11,12], anti-Alzheimer [13], antimicrobial, antiviral [14], anti-diabetic [15,16], and anticancer characteristics [17].Some bioactive drugs with intriguing biological profiles, such as astemizole, albendazole, bendamastin, candesartan, enviradine, omeprazole, and benzimidazole, are useful in medicinal chemistry due to their heterocyclic nature (Figure 2) [18].Schiff base and its scaffolds have been reported to have numerous significant biological profiles due to their interesting biological activities.In addition, heterocyclic scaffolds with the Schiff base moiety were known to demonstrate a broad spectrum of therapeutic and biological potentials, including anti-helminthic [19], antimicrobial [20], antitumor [21], and antiviral agents (Figure 3) [22].It has been demonstrated that benzimidazole and its analogs have a variety of biological profiles, including those that act with antihypertensive [11,12], anti-Alzheimer [13], antimicrobial, antiviral [14], anti-diabetic [15,16], and anticancer characteristics [17].Some bioactive drugs with intriguing biological profiles, such as astemizole, albendazole, bendamastin, candesartan, enviradine, omeprazole, and benzimidazole, are useful in medicinal chemistry due to their heterocyclic nature (Figure 2) [18].
Pharmaceuticals 2023, 16, x FOR PEER REVIEW 2 of 20 AD that are currently available only offer symptomatic alleviation; they cannot alter the course of the disease [8].Aβ and τ-protein development, which lowers levels of brain acetylcholine (ACh), is related to increased acetylcholinesterase (AChE) enzyme activity and the loss of muscarinergic neurons [9].Numerous drugs intended for Alzheimer's disease patients have been medically approved, such as Donepezil and galantamine.These drugs are designed to selectively target AChE.In contrast, compounds like rivastigmine and tacrine have been identified as BuChE and AChE inhibitors (Figure 1) [10].It has been demonstrated that benzimidazole and its analogs have a variety of biological profiles, including those that act with antihypertensive [11,12], anti-Alzheimer [13], antimicrobial, antiviral [14], anti-diabetic [15,16], and anticancer characteristics [17].Some bioactive drugs with intriguing biological profiles, such as astemizole, albendazole, bendamastin, candesartan, enviradine, omeprazole, and benzimidazole, are useful in medicinal chemistry due to their heterocyclic nature (Figure 2) [18].Schiff base and its scaffolds have been reported to have numerous significant biological profiles due to their interesting biological activities.In addition, heterocyclic scaffolds with the Schiff base moiety were known to demonstrate a broad spectrum of therapeutic and biological potentials, including anti-helminthic [19], antimicrobial [20], antitumor [21], and antiviral agents (Figure 3) [22].Schiff base and its scaffolds have been reported to have numerous significant biological profiles due to their interesting biological activities.In addition, heterocyclic scaffolds with the Schiff base moiety were known to demonstrate a broad spectrum of therapeutic and biological potentials, including anti-helminthic [19], antimicrobial [20], antitumor [21], and antiviral agents (Figure 3) [22].Keeping in mind the biological significance of benzimidazole [23][24][25] and Schiff base [26][27][28] derivatives, in this study, we synthesized benzimidazole-based Schiff base hybrid scaffolds to further explore the AChE and BuChE inhibition in search of lead molecules (Figure 4).Keeping in mind the biological significance of benzimidazole [23][24][25] and Schiff base [26][27][28] derivatives, in this study, we synthesized benzimidazole-based Schiff base hybrid scaffolds to further explore the AChE and BuChE inhibition in search of lead molecules (Figure 4).Keeping in mind the biological significance of benzimidazole [23][24][25] and Schiff base [26][27][28] derivatives, in this study, we synthesized benzimidazole-based Schiff base hybrid scaffolds to further explore the AChE and BuChE inhibition in search of lead molecules (Figure 4).

Chemistry
The synthesis of benzimidazole-based Schiff base compounds (1-18) was carried out through a multi-step procedure.In the initial step, carbon disulfide was gradually mixed with a solution of benzene-1,2-diamine (I) and stirred in a mixture of ethanol and water (1:1).Following this, KOH was introduced, and the resulting solution was refluxed for a period of 5 h.This led to the formation of 2-mercaptobenzimidazole, serving as substrate (II).Then, the intermediate (II) underwent further reflux with phenacyl bromides in the presence of ethanol and triethylamine for approximately 8 h.Subsequently, the surplus solvent was evaporated, yielding the formation of S-substituted benzimidazole, identified as intermediate (III).

Chemistry
The synthesis of benzimidazole-based Schiff base compounds (1-18) was carried out through a multi-step procedure.In the initial step, carbon disulfide was gradually mixed with a solution of benzene-1,2-diamine (I) and stirred in a mixture of ethanol and water (1:1).Following this, KOH was introduced, and the resulting solution was refluxed for a period of 5 h.This led to the formation of 2-mercaptobenzimidazole, serving as substrate (II).Then, the intermediate (II) underwent further reflux with phenacyl bromides in the presence of ethanol and triethylamine for approximately 8 h.Subsequently, the surplus solvent was evaporated, yielding the formation of S-substituted benzimidazole, identified as intermediate (III).

Acetylcholinesterase and Butyrylcholinesterase Inhibition Studies
The in vitro enzymatic activities of benzimidazole-based Schiff base derivatives (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) were examined to assess their impact on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE).Results are shown in Table 1 synthesized Schiff base derivatives also effectively inhibited the targeted enzymes and even showed stronger inhibitory potency than Donepezil.synthesized Schiff base derivatives also effectively inhibited the targeted enzymes and even showed stronger inhibitory potency than Donepezil.synthesized Schiff base derivatives also effectively inhibited the targeted enzymes and even showed stronger inhibitory potency than Donepezil.synthesized Schiff base derivatives also effectively inhibited the targeted enzymes and even showed stronger inhibitory potency than Donepezil.synthesized Schiff base derivatives also effectively inhibited the targeted enzymes and even showed stronger inhibitory potency than Donepezil.synthesized Schiff base derivatives also effectively inhibited the targeted enzymes and even showed stronger inhibitory potency than Donepezil.synthesized Schiff base derivatives also effectively inhibited the targeted enzymes and even showed stronger inhibitory potency than Donepezil.synthesized Schiff base derivatives also effectively inhibited the targeted enzymes and even showed stronger inhibitory potency than Donepezil.synthesized Schiff base derivatives also effectively inhibited the targeted enzymes and even showed stronger inhibitory potency than Donepezil.synthesized Schiff base derivatives also effectively inhibited the targeted enzymes and even showed stronger inhibitory potency than Donepezil.synthesized Schiff base derivatives also effectively inhibited the targeted enzymes and even showed stronger inhibitory potency than Donepezil.synthesized Schiff base derivatives also effectively inhibited the targeted enzymes and even showed stronger inhibitory potency than Donepezil.synthesized Schiff base derivatives also effectively inhibited the targeted enzymes and even showed stronger inhibitory potency than Donepezil.synthesized Schiff base derivatives also effectively inhibited the targeted enzymes and even showed stronger inhibitory potency than Donepezil.synthesized Schiff base derivatives also effectively inhibited the targeted enzymes and even showed stronger inhibitory potency than Donepezil.

Structure-Activity Relationship (SAR) for Acetylcholinesterase and Butyrylcholinesterase Inhibition Studies
To determine and simplify SAR for AChE and BuChE inhibition, the Schiff base derivatives (1-18) were divided into two groups based on the nature and position of substituents as R and R 1 groups.Its group comprises compounds 1-8, where R 1 represents OH at the 2 position and NO2 at the 4 position of the phenyl ring (Figure 5).  IC 50 = 226.30± 10.10 µM (AChE), IC 50 = 273.70± 13.80 µM (BuChE)), bearing the CH 3 group at the 4 and 2 positions, while compound 6 was bearing the 2-Br and 4-NO 2 group, respectively.In compound 7 (IC 50 = 313.60 ± 12.80 µM (AChE), IC 50 = 325.80± 13.70 µM (BuChE)), bearing the 2-Br group, a decrease in inhibitory activity was observed as compared to other compounds, but these derivatives were more active than the standard drug Donepezil.Now, we switch toward the second group of synthesized derivatives that comprises compounds 9-18, based on the R 1 group, where R 1 represents the 3-NO 2 group at the phenyl ring, as depicted in Table 1.
Based on the SAR analysis, it can be inferred that benzimidazole-based Schiff base derivatives carrying 2-OH and 4-NO 2 substitutions on the phenyl ring (referred to as R 1 ) showcased favorable inhibitory potential.However, alterations in the substituents located at the phenyl ring (referred to as R) also played a role in influencing variations in inhibitory activity.To reinforce these findings, a docking study was performed, shedding light on the interactions occurring between the synthesized derivatives and the active sites of the said enzymes.

Molecular Docking
A docking analysis was conducted on the most potent analogs employing multiple software tools, including AutoDock (version 1.5.7), as well as Pymol and Discovery Studio Visualizer (DSV), for visualizing the 2D and 3D molecular structures within the protein complex.To establish a meaningful correlation among in vitro and in silico studies, we conducted docking analyses on the most potent scaffolds against AChE and BuChE.The purpose of this was to elucidate the interactions among the active scaffolds (scaffolds 3, 5, and 9) and the active sites of the enzymes, which play a pivotal role in their functioning.These interactions were further substantiated by examining protein-ligand interactions (PLI), as outlined in detail in Table 2. Notably, the outcomes of these interactions, depicted in Figures 6-11, underscore the significance of these scaffold molecules in augmenting the enzymatic activities of AChE and BuChE.
conducted docking analyses on the most potent scaffolds against AChE and BuChE.The purpose of this was to elucidate the interactions among the active scaffolds (scaffolds 3, 5, and 9) and the active sites of the enzymes, which play a pivotal role in their functioning.These interactions were further substantiated by examining protein-ligand interactions (PLI), as outlined in detail in Table 2. Notably, the outcomes of these interactions, depicted in Figures 6-11, underscore the significance of these scaffold molecules in augmenting the enzymatic activities of AChE and BuChE.Table 2.The interaction details between ligands and active residues of targeted enzymes, along with docking scores.
Active Analogues Distance (A°) Types of Interactions Receptor Docking Score   13 C NMR spectra were recorded using a Bruker FT-NMR spectrometer operating at the digital frequencies of 600 and 150 MHz (Bioscience, Bruker, Billerica, MA, USA).Mass spectra were acquired utilizing a Shimadzu LCMS-IT-TOF system (Kyoto, Japan) employing the electrospray ionization (ESI) technique.The purity of synthesized derivatives was checked through thin-layer chromatography using silica gel 60 F254 (Merck, KGaA, Darmstadt, Germany).The carbon disulphide (1 mmol) and benzene-1,2-diammine (I, 1 mmol) were taken in EtOH-H 2 O (10 mL).The mixture was agitated for a duration of 6 h, and the precipitates obtained were then subjected to a drying process and underwent recrystallization to yield an intermediate product (II).Then, an equimolar amount of substrate (II) and different substituted phenacyl bromide were refluxed and stirred in ethanol (10 mL) and triethylamine (catalyst) to afford S-substituted benzimidazole (III).

Acetylcholinesterase and Butyrylcholinesterase Inhibition Assay
The study was carried out following the guidelines outlined in our previously published study [29].

Molecular Docking Protocol
The study was carried out following the guidelines outlined in our previously published study [30][31][32].
These findings underscore the potential of benzimidazole-Schiff-base scaffolds as AChE and BuChE inhibitors, holding potential for advancing innovative treatments for Alzheimer's disease.Noteworthy analogues, particularly 3, 5, and 9, exhibit significant potency and hold potential as lead compounds in the quest for pioneering anti-Alzheimer agents.

Figure 3 .
Figure 3. Bioactive drugs with a Schiff base skeleton in their core structure.

Figure 4 .
Figure 4. Rationale study of the present work.

Figure 3 .
Figure 3. Bioactive drugs with a Schiff base skeleton in their core structure.

Pharmaceuticals 2023 , 20 Figure 3 .
Figure 3. Bioactive drugs with a Schiff base skeleton in their core structure.

Figure 4 .
Figure 4. Rationale study of the present work.Figure 4. Rationale study of the present work.

Figure 4 .
Figure 4. Rationale study of the present work.Figure 4. Rationale study of the present work.

Figure 6 .
Figure6.Protein-ligand interaction (PLI) analysis for the most potent compound 3 with the targeted AChE enzyme, along with its 3D (left) and 2D (right) representations.Figure6.Protein-ligand interaction (PLI) analysis for the most potent compound 3 with the targeted AChE enzyme, along with its 3D (left) and 2D (right) representations.

Figure 6 . 20 Figure 7 .
Figure 6.Protein-ligand interaction (PLI) analysis for the most potent compound 3 with the targeted AChE enzyme, along with its 3D (left) and 2D (right) representations.Figure 6. Protein-ligand interaction (PLI) analysis for the most potent compound 3 with the targeted AChE enzyme, along with its 3D (left) and 2D (right) representations.Pharmaceuticals 2023, 16, x FOR PEER REVIEW 9 of 20

Figure 7 .
Figure 7. Protein-ligand interaction (PLI) analysis for the most potent compound 3 with the targeted BuChE enzyme, along with its 3D (left) and 2D (right) representations.Figure 7. Protein-ligand interaction (PLI) analysis for the most potent compound 3 with the targeted BuChE enzyme, along with its 3D (left) and 2D (right) representations.

Figure 7 .
Figure 7. Protein-ligand interaction (PLI) analysis for the most potent compound 3 with the targeted BuChE enzyme, along with its 3D (left) and 2D (right) representations.

Figure 8 .
Figure 8. Protein-ligand interaction (PLI) analysis for the 2nd most potent compound 5 with the targeted BuChE enzyme, along with its 3D (left) and 2D (right) representations.Figure 8. Protein-ligand interaction (PLI) analysis for the 2nd most potent compound 5 with the targeted BuChE enzyme, along with its 3D (left) and 2D (right) representations.

Figure 9 .
Figure 9. Protein-ligand interaction (PLI) analysis for the 2nd most potent compound 5 with the targeted BuChE enzyme, along with its 3D (left) and 2D (right) representations.Figure 9. Protein-ligand interaction (PLI) analysis for the 2nd most potent compound 5 with the targeted BuChE enzyme, along with its 3D (left) and 2D (right) representations.

Figure 9 .
Figure 9. Protein-ligand interaction (PLI) analysis for the 2nd most potent compound 5 with the targeted BuChE enzyme, along with its 3D (left) and 2D (right) representations.

Figure 10 . 20 Figure 11 .
Figure 10.Protein-ligand interaction (PLI) analysis for the 3rd most potent compound 9 with the targeted AChE enzyme, along with its 3D (left) and 2D (right) representations.Figure 10.Protein-ligand interaction (PLI) analysis for the 3rd most potent compound 9 with the targeted AChE enzyme, along with its 3D (left) and 2D (right) representations.Pharmaceuticals 2023, 16, x FOR PEER REVIEW 11 of 20

Figure 11 .
Figure 11.Protein-ligand interaction (PLI) analysis for the 3rd most potent compound 9 with the targeted BuChE enzyme, along with its 3D (left) and 2D (right) representations.

Table 2 .
The interaction details between ligands and active residues of targeted enzymes, along with docking scores.