Tasci_2026_ACS.Omega_11_43129

Alzheimer's disease (AD) benefits from multitarget-directed ligands (MTDLs) that can enhance cholinergic transmission while attenuating monoamine-oxidase-linked oxidative stress. Here, we report a benzoxazolinone-based propionyl thiosemicarbazide series, synthesized and fully characterized by infrared (IR) spectroscopy, (1)H nuclear magnetic resonance (NMR), and high-resolution mass spectra (HRMS). The compounds showed consistent submicromolar inhibitory activity across AChE, BChE, MAO-A, and MAO-B in vitro. Several AChE potencies approached the reference donepezil, and selected BChE activities were within an order of magnitude of tacrine. Notably, 4bk' (5-Me/benzyl) inhibited three targets (IC(50): 0.029 +/- 0.001 microM for AChE, IC(50): 0.071 +/- 0.003 microM for BChE, IC(50): 0.048 +/- 0.002 microM for MAO-B), and 4af' (5-Cl/phenyl) showed a balanced profile (IC(50): 0.025 +/- 0.001 microM for AChE, IC(50): 0.056 +/- 0.002 microM for BChE, IC(50): 0.095 +/- 0.003 microM for MAO-A), 4ac' (5-Cl/propyl) combined potent AChE and MAO-B inhibition (IC(50): 0.035 +/- 0.001 microM for AChE, IC(50): 0.045 +/- 0.002 microM for MAO-B), whereas 4ag' (5-Cl/4'-Cl-phenyl) was strongly MAO-B-selective (IC(50): 0.041 +/- 0.001 microM for MAO-B). Antioxidant capacity was pronounced for para-substituted analogues. The efficient compounds 4bk', 4af', 4ac', and 4ag' presented quite low toxicity on healthy cells (cell survival % was above %70 for 4bk', 4af', and 4ac', while it was around 64% for 4ag') even when they were applied at 100 times higher concentrations than their IC(50) values, which indicates that they are safe at effective doses. Moreover, assessment of the compounds at a concentration of 10 microM demonstrated no cytotoxic effects on either healthy BV-2 microglial cells or H9c2 rat myoblastoma cells. Docking and 100 ns molecular dynamics (MD) simulations (AChE: 4EY7; MAO-B: 2V5Z) supported stable binding for dual-active representatives (RMSD -1.5-2.8 A). In silico ADME (QikProp) indicated compliance with Lipinski's Rule of Five and Jorgensen's Rule of Three. Collectively, this scaffold is tunable from MAO-B-selective to balanced MTDL profiles suitable for further AD-relevant optimization.