Computational Docking Study of Oxazepine Based Compounds as Inhibitors of Acetylcholinesterase Enzyme in Alzheimer’s Disease

INTRODUCTION

A progressive loss of cognitive function and episodic memory is the hallmark of Alzheimer's Disease (AD), the most prevalent kind of dementia. In addition to verbal and visuospatial deficits, this results in behavioral issues like melancholy, aggressiveness and indifference. Extracellular plaques of insoluble β-amyloid peptide (Aβ) and Neurofibrillary Tangles (NFTs) containing hyperphosphorylated tau Protein (P-tau) in the cytoplasm of neurons are two important pathological characteristics seen in the brains of AD patients. Genetics is responsible for around 70% of the chance of acquiring AD. Moreover, acquired variables that raise the risk of AD include diabetes, obesity, dyslipidemia, hypertension and cerebrovascular illnesses (Silva et al., 2019).

Different pharmacological strategies are used to treat Alzheimer's disease, a progressive neurodegenerative ailment marked by cognitive decline and memory loss. Among the medication groups being investigated for their possible advantages in treating this illness are acetylcholinesterase inhibitors and benzodiazepines. Oxazepines are a class of drugs that act on the brain's neurotransmitter systems to modulate neuroprotective and cognitive-enhancing effects. Through its effects on the GABA and glutamate pathways, they may aid in mood stabilization and cognitive enhancement. On the other hand, acetylcholinesterase inhibitors work by inhibiting the enzyme that breaks down acetylcholine, an essential neurotransmitter for memory and learning. These medications improve cholinergic transmission and cognitive performance by blocking this enzyme, which raises acetylcholine levels in the brain. Among the common acetyl cholinesterase inhibitors are galantamine, rivastigmine and donepezil. All of these therapies work to reduce symptoms, delay the onset of the illness and enhance the quality of life for those who have Alzheimer's. Nevertheless, further study is necessary to completely comprehend their effectiveness and maximize their application in therapeutic contexts (Nordberg and Svensson, 1998; Abduljelil et al., 2022). Oxazepine derivatives were first introduced in 1965 and it is used in the relief of the psychoneuroses which is characterized by anxiety and tension. Oxazepines are seven membered heterocyclic compounds with oxygen and nitrogen (Wilson and Gisvold, 1966). Oxazepines can be prepared by multistep reactions. In the first step schiff base derivative is prepared from amino group of primary amine compounds reacts with an active carbonyl group of aldehydes or ketones. And within the second step condensation reaction takes place between imine or schiff base (-N=CH-) compounds and various anhydrides such as pthalic, maleic and succinic anhydride to form oxazepine compounds according to pericyclic reaction (Ruaa et al., 2012; Abdullatieef, 2010; Abdullatieef, 2010; Ayad et al., 2015; Nagham, 2013; Verm et al., 2015). Oxazepine and their derivatives have some important biological and pharmacological activities such as enzyme inhibitors (Moawad, 1989), analgesic (Muhammad et al, 2024), antidepressant, psychoactive drugs (Mesripour et al., 2023), anti-bacterial (Yao et al., 2021), anti-fungal (Serrano-Wu et al., 2002), hypnotic muscle relaxant (Aljohani et al., 2022), antagonistic (Qi et al., 2022), anti-epileptic (Siddiqui et al., 2020) and anti-inflammatory activity (Hussain et al., 2020; Vasudha et al., 2024).

MATERIALS AND METHODS

General scheme for synthesis of oxazepine derivative

STEP 1: PREPARATION OF SCHIFF BASES

Equimolar amounts of aldehyde derivatives (0.01 moles) and anthranilic acid (0.01 moles) were accurately weighed and dissolved in 25 mL of ethyl alcohol. A small amount of glacial acetic acid and sodium lauryl sulfate was added to the solution. The resulting mixture was refluxed for 2-4 hr. The solid product formed was vacuum-filtered, purified by recrystallization from ethanol and then dried.

STEP 2: PREPARATION OF OXAZEPINE DERIVATIVE

Equimolar amounts of Schiff base (0.01 moles) from step-1 and various anhydrides (0.01 moles) of phthalic anhydride (4), maleic (5) and succcinic anhydrides (6) were taken and dissolved in 20 mL of Dimethyl Formamide (DMF) and add 2-3 drops of glacial acetic acid and reflex it for 8-10 hr. The reaction time was determined by thin layer chromatography with mobile phase n-hexane: ethyl acetate (7:3). The resultant synthesized oxazepines were collected after filtration of the reaction mixture and purified by recrystallization using absolute alcohol and then washed, dried.

Synthesis of oxazepine derivatives

The synthetic procedures for oxazepine derivatives were taken from the existing literature and final products were designed in accordance with the scheme. Vast library of compounds was acquired and they underwent an in silico toxicity screening using the software TopKat (Prival, 2001) and then, utilizing SwissADME software, their ADME characteristics were anticipated (Daina et al., 2017; Daina et al., 2019). For molecular docking investigations, designed compounds with good ADME properties and predicted non-toxic and non-carcinogenic compounds were selected.

Molecular Docking

The Swiss Target Prediction software was used to choose the protein target from a library of compounds that were prepared in accordance with the previously outlined scheme. Swiss Target Prediction software predicted every possible combination for the target protein, including safe, non-toxic and non-carcinogenic ligands. Acetyl cholinesterase enzyme has been identified as a potential target to the majority of ligands.

ChemSketch

After drawing the ligands' 2D structures with ChemSketch, they were saved in .mol format. The ligand structures were translated from the .mol format to the .pdb format using the Avogadro tool (Hanwell et al., 2012). For the produced compounds that are safe, non-carcinogenic and have strong ADME properties, docking studies were carried out to assess binding poses and interactions.

The iGEMDOCK version 2.1

For docking, software was utilized. The acronym for Genetic Evolutionary Method for Molecular Docking is GEMDOCK. iGEMDOCK is the name of a graphical-automated drug design system for docking, screening and analysis. With reference to the protein's active site, this software determines the ligands' orientation and structure. To evaluate how the selected safe chemicals interact molecularly with the enzyme acetyl cholinesterase (PDB ID: 7e3h) (Figure 1) (Hsu, 2011).

Discovery Studio Visualizer (Biovia)

The ligand interactions were observed and examined using an accurate docking approach following the conventional docking protocol. The scoring function was utilized to evaluate the best docking solutions by integrating electrostatic, hydrogen bonding and van der Waals energies. Post-docking interaction profile analysis of the best poses for the top compounds revealed the interactions between the ligand and the target protein. These interactions were visualized and analyzed using Discovery Studio Visualizer (Biovia).

In silico toxicity prediction was utilized to identify safe and non-carcinogenic chemicals. These compounds were chosen for molecular docking in addition to common Acetyl choline esterase inhibitors such as Galantamine and Rivastigmine. Docking simulations were used to evaluate molecular interactions and binding affinities. Out of the top 10 compounds with the best binding energies, the top two compounds with the best binding energies and molecular interaction profile were selected for post-docking interaction study (Vijaya et al., 2023; Peitzika et al., 2023; Vijaya et al., 2023; Gudise et al., 2024).

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RESULTS

DISCUSSION

When compared to standard inhibitors like galantamine, which has a binding energy of -96.45 k.cal/mol and rivastigmine, which has a binding energy of -89.81 k.cal/mol, compound 8d and compound 9c have binding energies of -115.75 k.cal/mol and -113.08 k.cal/mol, respectively as depicted in Table 1, Figure 2. The binding energies of nearly all of the top 10 compounds have greater binding energies than standard Acetylcholineesterase inhibitors. Therefore, the binding energies of these two compounds are greater and hence selected for visualization.

The co-crystallized ligand and the top two ligands were compared for structural similarity during the visualization process. The number of conventional hydrogen bonds was displayed in a three-dimensional interaction model. The 2D interaction diagram clearly illustrated which amino acid residues were interacting with the ligands and their respective distances from the active site pocket which can be observed in Table 2. In this study focusing on acetylcholinesterase enzyme inhibitors, several ligands were investigated for their interactions with the enzyme through hydrogen bonding. The findings reveal distinct patterns among the inhibitors studied.

Firstly, Ligand 8d was observed to form a single conventional hydrogen bond with the amino acid Glycine at position 121 (GLY: 121) of the enzyme's active site. This interaction suggests a specific binding mode that potentially influences the inhibitor's efficacy. Conversely, Ligand 9C exhibited a more complex interaction profile, establishing three conventional hydrogen bonds with key residues on the enzyme: Serine 125 (SER:125), Tyrosine 124 (TYR:124) and Tyrosine 337 (TYR:337). Such multiple interactions likely contribute to a robust binding affinity, possibly enhancing the inhibitor's potency. The cocrystalized Ligand 2, on the other hand, formed two hydrogen bonds with Serine 203 (SER:203) and Tyrosine 337 (TYR:337). This binding pattern, shared with another ligand, suggests a common mode of interaction that may be critical for stabilizing the inhibitor within the enzyme's active site. Among the standard acetyl cholinesterase enzyme inhibitors studied, Rivastigmine demonstrated interactions with Threonine 75 (THR:75) and Leucine 76 (LEU:76) through two hydrogen bonds. These specific interactions highlight key residues involved in Rivastigmine's binding, potentially contributing to its effectiveness as an inhibitor. Interestingly, Galantamine did not form any hydrogen bonds with the enzyme under these experimental conditions, suggesting an alternative mode of interaction or binding mechanism that does not rely on direct hydrogen bonding interactions. Overall, these findings provide valuable insights into the molecular interactions between acetyl cholinesterase inhibitors and their target enzyme, shedding light on the structural basis for their inhibitory activity. Such detailed understanding is crucial for the rational design and optimization of future enzyme inhibitors aimed at treating conditions such as Alzheimer's disease.

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CONCLUSION

As the top ligands 8d and 9c were non-toxic, having better ADME, better binding affinities and common amino acid residue interactions with that of the standard Acetylcholine esterase inhibitors, they can be further synthesized and used for in vivo studies.

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