Exploring Benzimidazole Chemistry: Synthesis, Biological Activity, and Molecular Docking Studies for Alzheimer's Treatment

Introduction

The heterocyclic ring benzoimidazole is created by combining imidazole with benzene containing the chemical formula C₇H₆N₂ and weighing 118.14 g/mol. 1H-benzimidazole is benzimidazole’s IUPAC name. The presence of two nitrogen-containing, five-membered planar rings that are amphoteric and have two equivalent tautomers makes up the imidazole ring, which is responsible for the tautomerization of benzimidazole. A lot of research is being done on nitrogen-containing heterocyclic in the hopes of finding new drugs to treat different diseases¹. Vitamin B12 was initially shown to include 5,6-dimethyl-1-(α-D-ribofuranosyl) benzimidazole in the early 1950s. Due to its ability to selectively target a wide range of enzymes and protein receptors, its fundamental structure is essential to medicinal chemistry research. Furthermore, other fields, such as anti-hypertensive drugs, frequently employ benzimidazole derivatives (such as Telmisartan, Candesartan), as well as antifungal medications (such as Carbendazim, Fuberidazole), as anti-histamine drugs (e.g. Leriserton, Climizole), as proton-pump inhibitor agents (e.g. Omeprazole, Pantoprazole) and as anthelmintic agents (e.g. Thiabendazole, Albendazole)¹

Globally, the prevalence of neurodegenerative illnesses is rising as the population ages. Alzheimer’s disease (AD) is a kind of dementia that affects roughly 70% of people; by 2050, there will be 100 million individuals worldwide. Memory loss, language impairment, and a progressive loss of cognitive function are the hallmarks of this neurodegenerative disease. One of the primary causes of memory loss in Alzheimer’s patients is thought to be a lack of cholinergic neurotransmission ². Acetylcholinesterase inhibitors (AChEI) are a useful tool for enhancing cholinergic transmission. When searching for novel, highly effective acetylcholinesterase inhibitors, it’s critical to possess several heterocyclic rings with strong inhibitory activity, such as galantamine, donepezil, and tacrine, which are medications used to treat Alzheimer’s disease. Certain compounds of imidazole’s, including benzimidazole, play a role in discovery of new derivatives with AChEI activity³.

Figure 1: illustrated some Benzimidazole as a core moiety in different categorical drug. Click here to View Figure

Overview of Alzheimer’s Disease

Also is Alzheimer first identified Alzheimer’s disease (AD) in 1907; it is recognized as a degenerative disorder of the central nervous system (CNS) characterized by early-onset senile dementia³. This type of dementia is the most common form and represents a severely advanced brain neurodegenerative illness. Alzheimer’s disease damages the area of the brain responsible for thought, language, and memory⁴. Several factors are thought to be important in the initiation and progression of AD, despite the fact that its etiology is unknown. Amyloid-β (Aβ) deposits and associated extracellular plaques, oxidative stress, low acetylcholine (ACh) levels, and neuro fibrillary tangles resulting from tau-hyper phosphorylation are thought to be the typical clinical symptoms⁵.

Alzheimer’s Disease (AD) is characterized by reduced cortical cholinergic neurotransmission. Enhancing the activity of cholinergic neurons is considered a key strategy for developing effective drugs to manage AD. This approach involves modulating the levels of the enzyme acetylcholinesterase (AChE) in the central nervous system. Acetylcholinesterase plays a vital role by rapidly breaking down the neurotransmitter acetylcholine, thereby terminating cholinergic transmission at the postsynaptic membrane⁶. Inhibiting AChE leads to the accumulation of acetylcholine in the synapses, which boosts acetylcholine effects, improves cholinergic nervous system functioning, and enhances cognitive abilities. Therefore, targeting AChE inhibition is crucial for controlling AD⁷.

As researchers further investigate the extensive possibilities of benzimidazole derivatives, it’s crucial to examine their structure-activity relationships, pharmacokinetics, and mechanisms of action in greater detail. This thorough knowledge will lay the foundation for a more strategic design and development of more potent and selective benzimidazole-based therapeutic agents, addressing a wide range of medical needs^[8]. Further research and exploration of this fascinating class of compounds hold immense promise for the development of novel therapeutic agents, ultimately contributing to the advancement of human health and well-being.

Synthesis

Phillips-Ladenburg reaction

The Phillips-Ladenburg reaction involves the reaction of benzene-1,2-diamine with a carboxylic acid in the presence of a dilute acidic environment, Scheme 1 resulting in the formation of desired derivatives.⁹

Scheme 1: Philips-Ladenburg Reaction Click here to View Scheme

The Weidenhagen Reaction

The Weidenhagen reaction produces compounds by reacting 1,2-diaminobenzene with aldehydes or ketones in water/alcohol, Scheme 2 using copper (II) salts as oxidizing agents to facilitate cyclization and oxidation, from the carbonyl compound.⁹

Scheme 2: Weidenhagen Reaction Click here to View Figure

Hoebrecker Synthesis:

Hoebrecker reduced 2-nitro-4-methylacetanilide to form benzimidazole.^10. Scheme 3

Scheme 3: Hoebrecker Synthesis Click here to View Scheme

Application of Benzimidazole derivatives

Benzimidazole as an Anti-Alzheimer agent

Ozkay Y et al. a study was carried out to evaluate the anticholinesterase characteristics of several benzimidazole derivatives due to their biological significance. The most potent derivatives were found to be compounds 4f and 4g, which displayed IC₅₀ values of 0.091 mM and 0.134 mM, respectively, in comparison to the reference medication donepezil¹¹.

Figure 2: Compound 4f and Compound 4g Click here to View Figure

Gurjar, A et al. study aimed to combat Alzheimer’s disease by investigating benzimidazole analogues targeting the key enzyme involved in Aβ peptide formation. They identified analogues 11 and 14 as the most promising candidates, exhibiting both BACE1 inhibition and neuroprotective effects, as well as aiding in memory retention compared to the standard drug donepezil¹².

Figure 3: Compound 11 and Compound 14 Click here to View Figure

Alim, Z et al. constructed ACH inhibitor as a target molecule in drug development studies for Alzheimer’s disease. They synthesized 4 benzimidazole acetamide derivatives, which have the inhibition effect against human erythrocyte carbonic anhydrase 1 and 2, as well as ACHE. They found that compound 9a, 9b, 10a, and 10b has good inhibitor activity. The IC₅₀ values for carbonic anhydrase 1 were 7.21 µm, 4.72 µm, 6.08 µm, and 8.23 µm. On the other hand, in comparison to the reference medication donepezil, the IC₅₀ values for compounds 9a, 9b, 10a, and 10b for carbonic anhydrase 2 were 8.64 µm, 7.07 µm, 4.12 µm, and 5.93 µm¹³.

Ozturk, O et al. constructed 22 novel benzimidazole derivatives to evaluate the BCHE inhibition activity and they found that compound 2j and 3f exhibit potent and selective BCHE inhibition with IC₅₀ value of 1.13 and 1.46 µm compared to the standard drug¹⁴.

Yoon, Y et al. created two novel classes of compounds with the fundamental structure of benzimidazole, intended to function as ACHE and BCHE inhibitors. Compound 5IIC was discovered to have the strongest inhibitory action; its IC₅₀ values for ACHE and BUCHE are 5.12 µm and 8.63 µm, respectively³.

Figure 4: Compound 9a, 9b, 10a and 10b Click here to View Figure

Figure 6: Compound 5II-c Click here to View Figure

Adalat, B et al. constructed 10 analogues of benzimidazole derivatives and they found that compound 1b, 1c, 1g, 2c, and 2e has the highest inhibitory activity against ACHE and BCHE with the IC₅₀ value 1.30, 0.60, 2.40, 1.50, and 0.60 µm compared to the standard drug donepezil¹⁵.

Figure 7: Compound 1b, 1c, 1g, 2c and 2e Click here to View Figure

Materials and Methods

Sar of Benzimidazole as an Anti-Alzheimer Agent

Through an extensive literature review, we carefully studied the chemical modifications to the benzimidazole ring system and their effects on the inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)., crucial for understanding potential therapeutic approaches for diseases like Alzheimer’s. It highlights

The strategic placement of a phenyl group at the first position and a benzyl group at the second position, coupled with either hydroxyl (-OH) or chlorine (-Cl) substituents at the fourth position on both rings, significantly boosts the inhibitory efficacy against the target enzymes³.

It is demonstrated that this inhibitory activity can be further enhanced by adding a 4-nitrophenyl group at the second position of the benzimidazole ring system and offering the possibility of additional hydrophobic group replacements at the fourth position on the phenyl ring.

This enhancement is attributed to the specific electronic and steric effects imparted by these substituents, which are meticulously optimized for maximum interaction with the enzyme’s active site¹⁶.

Additionally, substituting a dichlorophenol group at the second position and a methoxy group at the fifth position has proven to be a highly effective modification strategy. This combination not only increases the molecule’s affinity for the enzyme but also suggests the potential for further activity improvement through additional hydrophobic substitutions¹¹.

These findings, deeply rooted in the principles of medicinal chemistry and enzyme inhibition kinetics, following these points offer valuable insights into the design of more effective inhibitors for AChE and BuChE.

Design of the Molecules

Figure 8: SAR Click here to View Figure

The substitution of a phenyl group at the 1^stposition of the benzimidazole ring has been observed to enhance the activity towards acetylcholinesterase (ACHE)³.

The substitution of hydrophobic groups at the 2^nd position of the benzimidazole ring   has been demonstrated to enhance inhibitory activity towards both acetylcholinesterase (ACHE) and butyrylcholinesterase (BUCHE).

Notable examples include chlorobenzaldehyde, bromobenzaldehyde, N, N-dimethyl aniline, and (tert-butyl) benzaldehyde¹⁶.

The incorporation of either a methoxy group or an ethoxy group at the 5th position of the benzimidazole ring has been demonstrated to enhance inhibitory activity towards acetylcholinesterase (ACHE)¹¹.

Molecular Docking study

Molecular docking studies was carried out on the crystal structure of 4ey7 which was retrieved from PDB data bank.

Protein 4ey7

Classification: OXIDOREDUCTASE

Organism(s): Sinorhizobium meliloti 1021

Expression System: Escherichia coli

Mutation(s): No

Experimental Data

Method: X-RAY DIFFRACTION

Resolution: 1.80 Å

R-Value Free: 0.197

R-Value Work: 0.167

R-Value Observed: 0.168

Software Used and its Version

1-BIOVIA Discovery Studio Visualizer version 24.1.0.23298

2-UCSF Chimera version 1.17.3

Table 1: System Parameters

PC	DELl
Operating System	Windows11 Pro
Processor	Intel-Core
RAM	32GB
Hard disc	1TB

Pharmaco Kinetic Study

A pharmacokinetic study is a scientific investigation that examines how a drug is absorbed, distributed, metabolized, and excreted in the body over time. It involves measuring drug concentrations in biological fluids like blood or urine and uses this data to understand the drug’s behaviour in the body. The study helps determine the optimal dosage, frequency, and route of administration to ensure safety and efficacy.

Computational Pharmacokinetic Study was carried out by calculating ADME parameters using Swiss ADME¹⁷ and Protox-II online tool¹⁸.  The toxicity prediction study was conducted using the Protox-II online tool.

Toxic doses are often represented as LD₅₀ values in mg/kg of body weight.

The median lethal dose (LD₅₀) is the amount at which 50% of test subjects die after exposure to a chemical.

Table 2

SR NO.	Formula	Molecular Weight (g/mol)	Log                P	GI Absorption	Lipins    Ki	Bioavailbili-ty      score	Synthei-ctic Asscess-ibility	LD50 Value	Predicted Toxicity Data
I-1	C15H11BrN2O2	331.16	2.39	High	0	0.55	2.16	800mg/kg	Class-IV
I-2	C15H11BrN2O2	331.16	2.14	High	0	0.55	2.23	800mg/kg	Class-IV
I-3	C16H13BrN2O2	345.19	2.65	High	0	0.55	2.26	800mg/kg	Class-IV
I-4	C16H13BrN2O2	345.19	2.33	High	0	0.55	2.33	4500mg/kg	Class-V
I-5	C22H19BrN2O2	423.3	3.69	High	0	0.55	2.66	400mg/kg	Class-IV
I-6	C22H19BrN2O2	423.3	3.57	High	0	0.55	2.76	400mg/kg	Class-IV
I-7	C22H19BrN2O2	423.3	3.57	High	0	0.55	2.76	400mg/kg	Class-IV
I-8	C21H17BrN2O2	409.28	3.28	High	0	0.55	2.54	400mg/kg	Class-IV
I-9	C21H17BrN2O2	409.28	3.28	High	0	0.55	2.54	400mg/kg	Class-IV
I-10	C21H17BrN2O2	409.28	3.17	High	0	0.55	2.64	400mg/kg	Class-IV
I-11	C16H13ClN2O2	300.74	2.58	High	0	0.55	2.19	800mg/kg	Class-IV
I-12	C16H13ClN2O2	300.74	2.58	High	0	0.55	2.14	800mg/kg	Class-IV
I-13	C22H19ClN2O2	378.85	3.4	High	0	0.55	2.54	400mg/kg	Class-IV
I-14	C22H19ClN2O2	378.85	3.48	High	0	0.55	2.61	400mg/kg	Class-IV
I-15	C22H19ClN2O2	378.85	3.48	High	0	0.55	2.61	400mg/kg	Class-IV
I-16	C21H17ClN2O2	364.83	3.23	High	0	0.55	2.48	400mg/kg	Class-IV
I-17	C21H17ClN2O2	364.83	3.07	High	0	0.55	2.49	400mg/kg	Class-IV
I-18	C21H17ClN2O2	364.83	3.07	High	0	0.55	2.49	400mg/kg	Class-IV
I-19	C16H13N3O4	311.29	1.8	High	0	0.55	2.48	800mg/kg	Class-IV
I-20	C16H13N3O4	311.29	1.74	High	0	0.55	2.54	800mg/kg	Class-IV
I-21	C19H20N2O2	308.37	2.79	High	0	0.55	2.41	800mg/kg	Class-IV
I-22	C19H20N2O2	308.37	2.86	High	0	0.55	2.41	800mg/kg	Class-IV
I-23	C20H22N2O2	322.4	2.99	High	0	0.55	2.52	800mg/kg	Class-IV
I-24	C20H22N2O2	322.4	2.85	High	0	0.55	2.52	800mg/kg	Class-IV
I-25	C24H25N3O2	387.47	3.4	High	0	0.55	2.86	837mg/kg	Class-IV
I-26	C24H25N3O2	387.47	3.47	High	0	0.55	2.92	837mg/kg	Class-IV
I-27	C24H25N3O2	387.47	3.47	High	0	0.55	2.92	837mg/kg	Class-IV
I-28	C23H23N3O2	373.45	3.24	High	0	0.55	2.74	400mg/kg	Class-IV
I-29	C23H23N3O2	373.45	2.94	High	0	0.55	2.8	837mg/kg	Class-IV
I-30	C23H23N3O2	373.45	2.94	High	0	0.55	2.8	837mg/kg	Class-IV

Table 3: Interaction Table of the Synthesized Compounds

Sr.No	Name	H-bond interaction	Other Interaction
1.	Donepezil	ARG:247	VAL:239, THR:238, PRO:235, ARG:296, PRO:368, PRO:290, PRO:420
2.	Co-crystal ligand	TYR:510	VAL:408, ARG:525, GLU:431, VAL:429, ARG:521, ARG:522
3.	I-6	TRP:86	TRP:286, TYR:341, VAL:294, TYR:124, HIS:447
4.	I-15	ASN:87	TYR:124, HIS:447, TRP:86, TYR:341, TRP:286
5.	I-2	ARG:296	TRP:286, TYR:337, HIS:447, TYR:341, PHE:338, TYR:72
6.	I-12	ARG:296 PHE:295	HIS:447, TYR:337, TRP:86, PHE:338, TYR:341, TYR:72, TRP:286
7.	I-8	GLY:121	HIS:447, PHE:297, TRP:236, PHE:295, ASP:74, TYR:341, TRP:286, PHE:338

Docking interaction of standard drug Donepezil, Donepezil with co-crystal ligand and proposed derivatives

COMPOUND I-6:

Figure 9: COMPOUND I-6 Click here to View Figure

COMPOUND I-15

Figure 10: COMPOUND I-15 Click here to View Figure

COMPOUND I-2

Figure 11: COMPOUND I-2 Click here to View Figure

COMPOUND I-12

Figure 12: COMPOUND I-12 Click here to View Figure

COMPOUND I-8

Figure 13: COMPOUND I-8 Click here to View Figure

Conclusions

In conclusion, this research article presents an overview of benzimidazole derivatives with a particular focuson their anti-alzheimer potential. Benzimidazole  and its derivatives exhibit a wide range of pharmacological activities, including anti-viral, anthelmintic and antifungal effects.However,their anti-alzheimeractivity is particularly noteworthy.The synthesis and reactions of benzimidazole provide a platform for the generation of diverse derivatives with tailored properties. Through structure-activity relationship (SAR) studies, it has been observed that specific modificationsat different positionson the benzimidazole ring can significantly enhance their anti-alzheimer activity.

Ache and Buche enzymes are crucial for memory and cognition,as they facilitate the breakdown of Ach, disrupting communication between nerve cells and contributing to Alzheimer’s disease. Consequently, pharmaceutical efforts have concentrated on developing cholinesterase inhibitors to address cognitive disorders. Our research has centered on novel compounds synthesized from ring-fused benzimidazoles as key structures, aiming to target these enzymes for potential therapeutic interventions.In this study, we investigated the potential of ring-fused benzimidazoles as neuroprotective therapeutics targeting Ache and Buche enzymes implicated in Alzheimer’s disease. Through the synthesis of 30 novel compounds and subsequent in-silico studies, we demonstrated that these compounds exhibit promising binding affinity against Ache and Buche. These findings suggest that benzimidazole derivatives hold potential as effective treatments for cognitive disorders, offering a promising avenue for future pharmaceutical research in the field of neuroprotection.

Acknowledgement

I express my deepest gratitude to God for the strength, wisdom, and perseverance to complete this.I am profoundly grateful to my parents for their unwavering support, encouragement, and love, which have been the foundation of my academic endeavors. Special thanks to my guiding advisors for their expertise, mentorship, and constructive feedback. Particular gratitude goes to Mr. Neil B. Panchal for his exceptional guidance, profound insights, and encouragement throughout my research  work. Lastly, I sincerely thank my sisters for their continuous support and belief in my abilities, which have driven my achievements.

Conflict of Interest

We assert clearly that we have no conflicts of interest.

Funding Sources

There is no source of funding.

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