Synthesis of Benzimidazole–Based Analogs as Anti Alzheimer’s Disease Compounds and Their Molecular Docking Studies

We synthesized 10 analogs of benzimidazole-based thiosemicarbazide 1 (a–j) and 13 benzimidazole-based Schiff bases 2 (a–m), and characterized by various spectroscopic techniques and evaluated in vitro for acetylcholinesterase (AchE) and butyrylcholinesterase (BchE) inhibition activities. All the synthesized analogs showed varying degrees of acetylcholinesterase and butyrylcholinesterase inhibitory potentials in comparison to the standard drug (IC50 = 0.016 and 4.5 µM. Amongst these analogs 1 (a–j), compounds 1b, 1c, and 1g having IC50 values 1.30, 0.60, and 2.40 µM, respectively, showed good acetylcholinesterase inhibition when compared with the standard. These compounds also showed moderate butyrylcholinesterase inhibition having IC50 values of 2.40, 1.50, and 2.40 µM, respectively. The rest of the compounds of this series also showed moderate to weak inhibition. While amongst the second series of analogs 2 (a–m), compounds 2c, 2e, and 2h having IC50 values of 1.50, 0.60, and 0.90 µM, respectively, showed moderate acetylcholinesterase inhibition when compared to donepezil. Structure Aactivity Relation of both synthesized series has been carried out. The binding interactions between the synthesized analogs and the enzymes were identified through molecular docking simulations.


Introduction
Acetylcholinesterase (AchE) and butyrylcholinesterase (BchE) are enzymes that play an important role in the hydrolysis of acetylcholine into choline and acetic acid [1]. This results in the shortage of acetylcholine in areas such as cortex and hippocampus of brain, which are associated with high psychological functions [2]. These enzymes are responsible for Alzheimer's disease (AD)-a progressive and an irreversible brain disorder that causes disturbance of the cholinergic system of the brain. This disturbance may cause memory loss, disorientation, cognitive impairment, and difficulty in thinking and problem solving [3][4][5]. In the aging society, AD is the main cause of dementia. These enzymes help in the aggregation of neurotoxic beta amyloid, which causes neuronal cell apoptosis. One of the approaches for treating AD is to target both acetylcholinesterase and butyrylcholinesterase [6,7].
The AchE has two binding sites, a catalytic site that is responsible for acetylcholine hydrolysis and the peripheral site for beta amyloid interaction. The AchE interactions with protein give rise to a complex AchE-Aβ, which causes neurotoxicity. AchE is present in the brain, muscles, and cholinergic neurons while BchE is present in intestine, liver, kidneys, heart, lungs, and serum [8,9]. It plays a main role in the breakdown of ester containing compounds. Normally, AchE is dominant in the brain, while BchE function rises when acetylcholine function gradually decreases in the brain of a patient with Alzheimer's disease. Therefore, there is an intense need for a drug that can inhibit the activity of both AchE and BchE [10].
For the treatment of Alzheimer's disease, several drugs have been approved by the FDA such as donepezil, rivastigmine (Figure 1), tacrine, and galantamine [11]. However, hepatotoxicity, insufficient activity, and gastrointestinal disturbance limit the use and applicability of these drugs [12][13][14][15][16]. To overcome the side effect of synthetic ChEs inhibitors, researchers have shown a great interest in the isolation of these compounds as an alternative, and many non-toxic bioactive ChEs inhibitors have been isolated from natural sources [17][18][19]. Amongst these, donepezil and galantamine are selective to AchE, whereas tacrine and rivastigmine inhibit both AchE and BchE. The Benzimidazole nucleus possesses a wide spectrum of activities that ranges from antimicrobial activities to being used against the world most deadly diseases. It has gained importance in medicinal chemistry because of its affinity for different enzymes and protein receptors.

Introduction
Acetylcholinesterase (AchE) and butyrylcholinesterase (BchE) are enzymes that play an important role in the hydrolysis of acetylcholine into choline and acetic acid [1]. This results in the shortage of acetylcholine in areas such as cortex and hippocampus of brain, which are associated with high psychological functions [2]. These enzymes are responsible for Alzheimer's disease (AD)-a progressive and an irreversible brain disorder that causes disturbance of the cholinergic system of the brain. This disturbance may cause memory loss, disorientation, cognitive impairment, and difficulty in thinking and problem solving [3][4][5]. In the aging society, AD is the main cause of dementia. These enzymes help in the aggregation of neurotoxic beta amyloid, which causes neuronal cell apoptosis. One of the approaches for treating AD is to target both acetylcholinesterase and butyrylcholinesterase [6,7].
The AchE has two binding sites, a catalytic site that is responsible for acetylcholine hydrolysis and the peripheral site for beta amyloid interaction. The AchE interactions with protein give rise to a complex AchE-Aβ, which causes neurotoxicity. AchE is present in the brain, muscles, and cholinergic neurons while BchE is present in intestine, liver, kidneys, heart, lungs, and serum [8,9]. It plays a main role in the breakdown of ester containing compounds. Normally, AchE is dominant in the brain, while BchE function rises when acetylcholine function gradually decreases in the brain of a patient with Alzheimer's disease. Therefore, there is an intense need for a drug that can inhibit the activity of both AchE and BchE [10].
For the treatment of Alzheimer's disease, several drugs have been approved by the FDA such as donepezil, rivastigmine (Figure 1), tacrine, and galantamine [11]. However, hepatotoxicity, insufficient activity, and gastrointestinal disturbance limit the use and applicability of these drugs [12][13][14][15][16]. To overcome the side effect of synthetic ChEs inhibitors, researchers have shown a great interest in the isolation of these compounds as an alternative, and many non-toxic bioactive ChEs inhibitors have been isolated from natural sources [17][18][19]. Amongst these, donepezil and galantamine are selective to AchE, whereas tacrine and rivastigmine inhibit both AchE and BchE. The Benzimidazole nucleus possesses a wide spectrum of activities that ranges from antimicrobial activities to being used against the world most deadly diseases. It has gained importance in medicinal chemistry because of its affinity for different enzymes and protein receptors. Our research group has been continuously attempting to synthesize new heterocyclic moieties in search of lead therapeutics. Recently, we reported benzimidazole-based oxadiazole as an antialpha-glycosidase [20], benzimidazole-bearing bis-Schiff base as alpha glucosidase inhibitor [21][22][23], and 2-mercaptabenzimidazole as alpha amylase inhibitor [24]. Benzimidazole is a potent urease inhibitor [25]. Here, we report two new series of benzimidazole thiosemicarbazides and benzimidazole Schiff bases as novel Alzheimer inhibitors.

Synthesis of Benzimidazole Schiff Base
In first step, we synthesized benzimidazole analogs by treating 1-methyl-3,4-diaminobenzene (V) with ethyl-4-formyl benzoate (VI) in dimethyl formide (DMF) along with sodium meta bi-sulfite (Na 2 S 2 O 5 ) as a base under reflux condition for 4 to 5 h to yield intermediate (VII) as product. The intermediate (VII) was treated with benzene hydrate in ethanol and was refluxed for [4][5] h to yield intermediate (VIII). In the third step, the intermediate (VIII) was treated with various aromatic aldehydes in methanol in the presence of glacial acetic acid to synthesize our final desired products 2 (a-m) (Scheme 2, Table 2).

Synthesis of Benzimidazole Schiff Base
In first step, we synthesized benzimidazole analogs by treating 1-methyl-3,4-diaminobenzene (V) with ethyl-4-formyl benzoate (VI) in dimethyl formide (DMF) along with sodium meta bi-sulfite (Na2S2O5) as a base under reflux condition for 4 to 5 h to yield intermediate (VII) as product. The intermediate (VII) was treated with benzene hydrate in ethanol and was refluxed for [4][5] h to yield intermediate (VIII). In the third step, the intermediate (VIII) was treated with various aromatic aldehydes in methanol in the presence of glacial acetic acid to synthesize our final desired products 2 (a-m) (Scheme 2, Table 2).               We have synthesized ten analogs 1 (a-j) of benzimidazole-based thiosemicarbazide and evaluated them for ChE inhibition. All the analogs showed acetylcholinesterase inhibition of varying degrees ranging from 0.60 ± 0.05 µ M to 12.90 ± 0.20 µ M. All the analogs showed good inhibitory 18 We have synthesized ten analogs 1 (a-j) of benzimidazole-based thiosemicarbazide and evaluated them for ChE inhibition. All the analogs showed acetylcholinesterase inhibition of varying degrees ranging from 0.60 ± 0.05 µ M to 12.90 ± 0.20 µ M. All the analogs showed good inhibitory activities. The analogs 1c and 1g were the most active in the series. These analogs have chloro groups 21 We have synthesized ten analogs 1 (a-j) of benzimidazole-based thiosemicarbazide and evaluated them for ChE inhibition. All the analogs showed acetylcholinesterase inhibition of varying degrees ranging from 0.60 ± 0.05 µM to 12.90 ± 0.20 µM. All the analogs showed good inhibitory activities. The analogs 1c and 1g were the most active in the series. These analogs have chloro groups on the phenyl ring. Analog 1c having 2 chloro groups at position 2 and 3 of phenyl ring has an IC 50 value of 0.60 ± 0.05 µM, while in analog 1g, the chloro groups are at position 3 and 4 with the IC 50 value of 0.80 ± 0.50 µM. These results showed that enzyme inhibition activity mainly depends on the substitution pattern of chloro group on the phenyl ring. Similarly, analog 1b having floro group at position 4 on phenyl ring also showed a good inhibition with an IC 50 value of 1.30 ± 0.01 µM, while analog 1a having bromo group at position 2 of phenyl ring has an IC 50 value of 11.30 µM. This shows that inhibition activity not only depends on the position of substituent but also on the number of substituents attached to the phenyl ring. The analog 1h having one methyl group at position 4 has an IC 50 value of 3.50 ± 0.01 µM, while analog 1i having methyl group at 3 position has an IC 50 value of 7.30 ± 0.10 µM. These analogs also showed good inhibition. The difference in their inhibition values may be due to the difference in the position of methyl group on the phenyl ring. The analog 1d having 2 methyl groups-one at position 2 and the other at 6-has an IC 50 value of 5.60 µM. The analog 1f having nitro group at position 3 has an IC 50 value of 6.20 µM and analog 5 having nitro group at position 4 has an IC 50 value of 8.50 µM. The analog 1j having phenyl ring has an IC 50 value of 12.96 µM.
The analogs 1(a-j) showed varying degrees of butyrylcholinesterase inhibition ranging from 1.50 ± 0.10 to 29.10 ± 0.30 µM when compared to the standard drug donepezil having an IC 50 value of 4.5 ± 0.11 µM. The most active analogs among the series were 1b, 1c, 1e, 1f, and 1g that showed excellent butyrylcholinesterase inhibition with IC 50 values of 2.40 ± 0.10, 1.50 ± 0.10, 10.30 ± 0.20, 12.90 ± 0.20 µM, and 2.40 ± 0.10 µM, respectively. The analogs 1b, 1c, and 1g were the most active. These analogs have halogen atoms on different positions of the phenyl ring. This shows that the presence and position of halogen atoms on phenyl ring plays a major role in inhibition. Analog 1a showed moderate inhibition with the IC 50 value of 22.60 ± 0.50 µM. This analog has a bromine atom at position 2 on the phenyl ring. Analogs 1e and 1f have a nitro group on the phenyl ring. The slight difference in the inhibition value may be due to their different positions on the phenyl ring. Similarly, analogs 1h and 1i having a methyl group on the phenyl ring have an IC 50 value of 12.80 ± 0.20 µM and 13.40 ± 0.20 µM, respectively. Both these analogs have electron donating groups at different positions on the phenyl ring. The analog 1d showed a poor inhibition with an IC 50 value of 29.10 ± 0.30 µM. This analog has 2 methyl groups at position 2 and 6, respectively. Another least active analog was analog 1j having a phenyl ring with an IC 50 value of 24.40 ± 0.30 µM.

Acetylcholinesterase Inhibition Activity of Benzimidazole Schiff Bases
The synthesized benzimidazole Schiff base analogs were examined for AchE and BchE. All the compounds exhibited good to moderate activity when compared with the standard drug donepezil having an IC 50 value of AchE 0.016 ± 0.12 µM and BchE 4.5 ± 0.11 µM. Analog 2e was found to be the most active analog among the series, showing a good inhibition of both AchE and BchE with the IC 50 values of 0.60 ± 0.05 µM and 2.20 ± 0.10 µM, respectively. This analog has two chlorine atomsone at position 3 and the other at 4of the phenyl ring. Similarly, analog 2c also exhibited significant inhibition of AchE and BchE with an IC 50 value of 1.50 ± 0.10 µM and 4.10 ± 0.10 µM, respectively. This analog has two chlorine atoms-one at position 3 and the other at 4-and a hydroxyl group at position 2. The ChE inhibition data in Table 1 show that the substitution of chlorine atoms on the phenyl ring plays a significant role in inhibition. The next active analog among the series is analog 2h having a dimethyl amino group at position 4 on the phenyl ring. This analog also showed a significant inhibition of both AchE IC 50 = 0.90 ± 0.50 µM and BchE IC 50 = 2.20 ± 0.10 µM.
Similarly, the inhibitory potential of the compounds having hydroxyl group and methoxy group on phenyl ring was also good, e.g., analog 2l has a hydroxyl group at position 4 on the phenyl ring and a methoxy group at positions 3 and 5, and exhibited inhibition for AchE and BchE with IC 50 values of 2.30 ± 0.10 µM and5.15 ± 0.10 µM, respectively. Analog 2f having a hydroxyl group at position 3 and a methoxy group at position 4 showed a selective inhibition toward AchE with an IC 50 value of 4.20 ± 0.1 µM, and for BchE, the IC 50 = 7.60 ± 0.10 µM. Analog 2d having a hydroxyl group on the naphthalene ring also showed a good inhibition toward AchE, IC 50 = 3.10 ± 0.10 µM, and BchE, IC 50 = 6.20 ± 0.10 µM. This shows that the hydroxyl group plays an important role in inhibition. Similarly, analog 2a and 2b having a nitro group on the phenyl ring were more selective in inhibition toward AchE with IC 50 values 3.40 ± 0.10 µM and 8.50 ± 0.20 µM, while for BchE they showed a weak inhibition with IC 50 values of 7.50 ± 0.30 µM and12.10 ± 4.0 µM, respectively. The rest of the compounds of the series showed weak inhibitory activities.

Molecular Docking Studies
The inhibition concentration values of benzimidazole thiosemicarbazide derivatives 1 (a-j) and benzimidazole Schiff bases 2 (a-m) as effective acetylcholinesterase inhibitors are displayed in Table 3. The acetylcholinesterase inhibition by the synthesized derivatives may be influenced by the type, number, and positions of the substituted functional groups of their basic skeletons (Table 3). To rationalize the observed acetylcholinesterase inhibition by the synthesized derivatives, molecular docking has been carried out to determine the binding modes between the synthesized derivatives 1 (a-j) and 2 (a-m) from one side and the active residues of the acetylcholinesterase from another side. Table 3 gathers the calculated free binding energies of the stable complexes' ligand-acetylcholinesterase, the number of established intermolecular hydrogen bonding between the synthesized compounds and active site residues of acetylcholinesterase, and the closest residues to the docked compounds and their IC 50 values.
All the complexes formed between the synthesized derivatives 1 (a-j) and 2 (a-m) and the active residues of acetylcholinesterase displayed negative binding energies, which demonstrates that acetylcholinesterase inhibition by synthesized derivatives 1 (a-j) and 2 (a-m) is a thermodynamic favorable process ( Table 3). The docking of the best active benzimidazole thiosemicarbazides (1c, 1b and 1g), benzimidazole Schiff bases (2c, 2h and 2e), and donepezil into the active binding site of acetylcholinesterase are displayed in Figure 2.
Molecules 2020, 25, x FOR PEER REVIEW 9 of 17 higher acetylcholinesterase inhibition of 1e compared with 1f may refer to the number of hydrogen bonding that was established by the former with the active residues of acetylcholinesterase compared with the latter (Figure 3). As can be seen in Figure 3 and Table 3    For benzimidazole thiosemicarbazide derivatives, binding energies of the stable complexes vary with a maximal variation of 1.56 kcal mol −1 . In accordance with the MIC values, the best active compound 1c with the IC 50 value of 0.60 µM has the lowest binding energy of −13 kcal/mol. These variations are low enough to be considered as a strong descriptor in rationalizing the observed acetylcholinesterase inhibition. However, the number of hydrogen bonding, its distances, and the intermolecular interactions between the substitute groups of 1 (a-j) and the active residues may help in understanding the observed acetylcholinesterase inhibition. For instance, the compounds 1e and 1f differ by the position of the substituted nitro group at the aromatic ring, where in the former the nitro group is substituted at para position while in the latter it is substituted at meta position (Scheme 1). Experimentally, 1e shows a higher activity than 1f. Their corresponding complexes formed with acetylcholinesterase display similar binding energies with a variation less than 0.01 kcal/mol −1 . The higher acetylcholinesterase inhibition of 1e compared with 1f may refer to the number of hydrogen bonding that was established by the former with the active residues of acetylcholinesterase compared with the latter (Figure 3). As can be seen in Figure 3 and Table 3, in 1e, the nitro group at para position forms three hydrogen bonds with amino acids GLY B:122, ALA B:204, and GLY B:121 of acetylcholinesterase with distances 2.79, 3.00, and 3.23 Å, respectively. However, in 1f, the nitro group at meta position forms one hydrogen bonding with TYR B:124 with a distance of 2.85 Å.  thiosemicarbazides (1c, 1b, and 1g blue color representations), benzimidazole Schiff bases (2c, 2h, and 2e green color representations) and donepezil (brown color representation) into the active binding site of acetylcholinesterase. For benzimidazole Schiff bases, binding energies of the stable complexes vary with variations in the range 0-8.34 kcal/mol −1 with respect to the stable one. Similar to the benzimidazole thiosemicarbazide derivatives, the potency of benzimidazole Schiff bases to inhibit acetylcholinesterase is strongly related to the substituted functional groups, which may increase the stability of benzimidazole Schiff bases-acetylcholinesterase complexes. For instance, the compounds 2a and 2b differ by the position of the substituted nitro group at the aromatic, where in the former the nitro group is substituted at ortho position while in the latter it is substituted at meta position (Scheme 2). Experimentally, 2a shows a higher activity than 2b. The higher acetylcholinesterase inhibition of 2a compared with 2b may refer to the stability of the complex formed by the former with acetylcholinesterase, which displays a binding energy variation of 1.35 kcal/mol −1 compared to the complex formed with 2b. The stability of 2a-acetylcholinesterase may refer to the number of hydrogen bonds 2a established with the active residues of acetylcholinesterase compared with 2b ( Figure 4). As can be seen in Figure 4 and Table 3,   For benzimidazole Schiff bases, binding energies of the stable complexes vary with variations in the range 0-8.34 kcal/mol −1 with respect to the stable one. Similar to the benzimidazole thiosemicarbazide derivatives, the potency of benzimidazole Schiff bases to inhibit acetylcholinesterase is strongly related to the substituted functional groups, which may increase the stability of benzimidazole Schiff bases-acetylcholinesterase complexes. For instance, the compounds 2a and 2b differ by the position of the substituted nitro group at the aromatic, where in the former the nitro group is substituted at ortho position while in the latter it is substituted at meta position (Scheme 2). Experimentally, 2a shows a higher activity than 2b. The higher acetylcholinesterase inhibition of 2a compared with 2b may refer to the stability of the complex formed by the former with acetylcholinesterase, which displays a binding energy variation of 1.35 kcal/mol −1 compared to the complex formed with 2b. The stability of 2a-acetylcholinesterase may refer to the number of hydrogen bonds 2a established with the active residues of acetylcholinesterase compared with 2b ( Figure 4). As can be seen in Figure 4 and Table 3 inhibition of 2a compared with 2b may refer to the stability of the complex formed by the former with acetylcholinesterase, which displays a binding energy variation of 1.35 kcal/mol −1 compared to the complex formed with 2b. The stability of 2a-acetylcholinesterase may refer to the number of hydrogen bonds 2a established with the active residues of acetylcholinesterase compared with 2b ( Figure 4). As can be seen in Figure 4 and Table 3,

Conclusions
In this study, ten analogs of benzimidazole thiosemicarbazide 1 (a-j) and 13 analogs of benzimidazole Schiff bases 2 (a-m) were synthesized and characterized through 13 C NMR, 1 H, and HR-EIMS. All the synthesized analogs of the two series were examined for their acetylcholinesterase and butycholinesterase inhibitory potentials which exhibited varying degrees of biological activities. Amongst the synthesized analogs of series 1, a-j), 1b, 1c, and 1g having IC 50 values 1.30 ± 0.10, 0.60 ± 0.50, and 2.40 ± 0.10 µM, respectively, exhibited excellent acetylcholinesterase inhibition potential as compared to their inhibition potential to donepezil, and these analogs also showed excellent butycholinesterase inhibition with IC 50 values of 2.40 ± 0.10, 1.50 ± 0.10 µM, and 2.40 ± 0.10 µM as compared to their inhibition potential to donepezil. While other analogs of the series showed good to moderate activities (Table 1), the rest of the compounds of this series also showed moderate to weak inhibition. The analogs of the second series 2 (a-m) compounds 2c, 2e, and 2h having IC 50 values of 1.50 ± 0.10, 0.60 ± 0.05, and 0.90 ± 0.05 µM, respectively, showed excellent acetylcholinesterase inhibitory potentials and also the same analogs 2c, 2e, and 2h with IC 50 values of 4.10 ± 0.10, 2.20 ± 0.10, and 2.20 ± 0.30 µM showed that this series displayed excellent butycholinesterase inhibition when compared to the standard drug donepezil ( Table 2). The other analogs of this series showed good to moderate activity. SAR studies of these analogs was established through molecular simulation studies.

Conflicts of Interest:
The authors declare no conflict of interest.