Synthesis, In Silico and In Vitro Evaluation of Cholinesterase and Tyrosinase Inhibition of Benzothiazinones for Cognitive Disorders

Abstract

Objective: Cognitive disorders, including Alzheimer’s disease (AD) are multifactorial conditions associated with cholinergic dysfunction and oxidative stress linked to abnormal melanogenesis. This study aimed to design, synthesise, and evaluate novel benzothiazinone derivatives for their dual inhibitory activity against acetylcholinesterase (AChE) and tyrosinase. Methods: Computational studies were performed using molecular docking on protein targets (PDB IDs: 6O4W, 5I38, and 1HD2), followed by MMGBSA for binding free-energy calculations, pharmacophore modelling and ADMET prediction. Designed compounds were synthesised via Schiff base intermediates and characterised using FTIR, ¹H NMR, ¹³C NMR, and mass spectrometry. In vitro AChE inhibition was evaluated using Ellman’s method, while tyrosinase inhibition was assessed by dopachrome formation at 492 nm. Antioxidant activity was determined using DPPH and hydrogen peroxide assays. All experiments were conducted in triplicate (n = 3), and results are expressed as mean±SD with 95% confidence intervals. Results: Docking studies revealed that compound 4a exhibited the highest binding affinities with scores of −6.72 kcal/mol (6O4W), −5.42 kcal/mol (5I38), and −4.48 kcal/mol (1HD2). In vitro, compound 4a demonstrated AChE inhibition (IC₅₀ = 6.05±1.03 µg/ml, n = 3; 95% CI: 3.49–8.61 µg/ml) and tyrosine inhibition (IC₅₀ = 23.00±0.98 µg/ml, n = 3; 95% CI: 20.56 – 25.44 µg/ml). Structure–activity relationship analysis indicated that substituents significantly influenced enzyme inhibition. Conclusion: Compound 4a emerged as a promising multifunctional lead compound with dual AChE and tyrosinase inhibitory activity, supported by both in silico and in vitro findings, highlighting its potential for further development in the management of cognitive disorders.