Phenothiazine-tetrazole hybrids as potent cholinesterase inhibitors: Integrated experimental and computational insights for Alzheimer's therapy

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by neuronal loss and cognitive decline. A key pathological feature of AD is the reduction of acetylcholine (ACh) levels resulting from the enzymatic activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Therefore, inhibition of these enzymes remains a well-established therapeutic strategy for AD management. In this study, a novel series of phenothiazine-tetrazole hybrids was designed and synthesized as potential cholinesterase inhibitors. Their inhibitory activities against AChE and BChE were evaluated in vitro, and IC₅₀ values were determined in the micromolar range. Among the tested derivatives, Compound E3a (IC₅₀ = 10.664 μM) and Compound E5b (IC₅₀ = 10.315 μM) exhibited the strongest AChE inhibition, whereas Compound D5b showed the highest activity toward BChE (IC₅₀ = 18.734 μM). Enzyme kinetic analysis revealed that E5b and E3a act as competitive AChE inhibitors, with Ki values of 1.313 ± 0.032 μM and 1.520 ± 0.033 μM, respectively. To elucidate the molecular basis of inhibition, molecular docking studies were performed, followed by 500 ns molecular dynamics simulations, MM-PBSA binding free energy calculations, and free energy landscape analyses. The computational results revealed that Compound E3a forms a more dynamically and thermodynamically stabilized complex with AChE, primarily driven by favorable van der Waals interactions. ADMETlab 3.0 predictions further indicated acceptable preliminary drug-like features, although lipophilicity, solubility, CYP liability, and toxicity-related endpoints require further optimization and experimental validation. Collectively, these findings highlight phenothiazine-tetrazole hybrids as promising scaffolds for the rational development of next-generation cholinesterase inhibitors for Alzheimer's therapy.