Molecular Striptease of a Thiadiazolopyrimidine Hit Yields a Streamlined, Artifact-Free NADPH Mimic for NOX Inhibition.

The convergence of oxidative stress and inflammation drives neurodegeneration and cancer, positioning NADPH oxidases (NOXs) as critical therapeutic targets. Starting from the polyfunctional thiadiazolopyrimidine hit 1, we systematically truncated its peripheral arms to map minimum pharmacophoric requirements. While extensive clipping compromised activity, strategic optimization yielded the streamlined analogue 5. In silico, 5 acted as a competitive NADPH mimic; in cell-free assays, it maintained multi-isoform potency, inhibiting NOX1 and NOX5 at low-micromolar concentrations. In rat brain subcellular fractions, 1 and 5 demonstrated concentration-dependent neuroprotective and antioxidant efficacy (1-10 μM) by suppressing lipid peroxidation and preserving mitochondrial and synaptosomal viability. Notably, chemical profiling proved that 5 successfully stripped away the pro-oxidant liabilities and radical-scavenging artifacts inherent to 1. In cancer, 1 displayed some antiproliferative activity, mainly in hematological malignancies. Compound 5, although aqueous solubility issues limited its cellular antiproliferative performance, represents a specific, artifact-free architectural starting point for future selective NOX inhibitor development.