Design, Synthesis, and In Vitro Evaluation of Pinostrobin-Based Hydrazones as Potent α‑Glucosidase Inhibitors.

Twenty-four pinostrobin hydrazone derivatives (TN01-TN24) were synthesized through two continuous steps from pinostrobin, hydrazone, and aldehyde derivatives, well characterized and screened for inhibitory activity against yeast α-glucosidase (Saccharomyces cerevisiae). To further expand the chemical space, a QSAR-guided approach was employed, leading to the identification of four additional candidates (TN25-TN28). Eleven compounds exhibited >80% inhibition at 50 μM, outperforming acarbose, with most displaying IC50 values below 10 μM. Among them, TN27 showed the highest potency with an IC50 of 0.85 μM. Enzyme kinetic analysis confirmed a competitive inhibition mechanism, yielding a dissociation constant (Ki) of 3.56 μM. Molecular docking suggested favorable binding of TN27 within the active site of yeast α-glucosidase from Saccharomyces cerevisiae. To elucidate its binding mechanism, 500 ns molecular dynamics simulations were conducted in three independent replicas. Analysis of converged trajectories enabled end-point binding free energy calculations using MM/GBSA and QM-MM/GBSA methods, along with per-residue energy decomposition. These analyses revealed stable binding patterns driven by hydrophobic interactions and persistent hydrogen bonding with key catalytic residues. Collectively, the integrated experimental and multiscale computational results establish TN27 as a potent α-glucosidase inhibitor and demonstrate an effective strategy for rational lead identification and mechanistic validation.