Design and Evaluation of Pyridine-Pyrazoline Based MAO Inhibitors: Synthesis, Molecular Docking, Molecular Dynamics, MM-GBSA, Principal Component Analysis, Free Energy Landscape, and <i>In Vitro</i> Cytotoxicity Studies

Parkinson’s disease is the second most prevalent neurodegenerative disorder, and current therapeutic strategies are limited by inadequate blood-brain barrier permeability and long-term adverse effects. The present study aimed to investigate the neuroprotective potential of novel pyridine-pyrazoline scaffolds through an integrated in silico and in vitro approach targeting Monoamine Oxidase (MAO). The designed compounds were synthesised via a multistep protocol involving Claisen-Schmidt condensation of 2-/4-pyridine aldehydes with p-substituted acetophenones to yield chalcones, followed by cyclisation with p-toluenesulfonyl hydrazine to obtain pyridine-pyrazolines derivatives. Physicochemical, pharmacokinetic and toxicity profiling indicated favourable drug-likeness. Molecular docking identified compound 8a (6.41 kcal/mol) as the top-ranked ligand against MAO, and molecular dynamics simulations with post-dynamic analyses confirmed the stability of the protein-ligand complex. Density functional theory (DFT) studies highlighted the critical contribution of the sulfonyl group, pyridine ring, and substituted phenyl ring to electronic distribution and binding interactions. In vitro evaluation reinforced the computational findings, with compound 8a (IC 50 =00.78±0.46 μM) demonstrating the highest MAO inhibitory and cell viability (IC 50 = 234.59±4.75μM) in SHSY-5Y cell line. Structure-activity relationship analysis revealed that the enhanced potency of 8a is attributed to the electron-withdrawing chloro substituent and improved electron delocalisation in the 4-pyridine scaffold. Overall, these findings identify compound 8a as a promising MAO inhibitor, supported by in vitro experimental validation. However, advanced mechanistic studies and in vivo investigation are required to fully establish the therapeutic potential.