A Tetrahydrobiopterin-DisplacingPotent Neuronal NitricOxide Synthase Inhibitor with an Unprecedented Binding Mode

Nitric oxide synthase (NOS) is a pivotal enzyme that regulates various physiological processes, and the dysregulation of neuronal NOS (nNOS) is implicated in neurodegenerative diseases. In our efforts to leverage existing X-ray crystallography data to develop novel aminoquinoline–pyridine hybrid inhibitors and evaluate their inhibitory activities and interactions with NOS isoforms, we identified compounds <b>8</b> and <b>9</b> as potent human nNOS inhibitors (<i>K</i>_i = 38 and 22 nM, respectively). Notably, compound <b>8</b> displayed an unprecedented binding mode, displacing the essential cofactor tetrahydrobiopterin (H₄B). Furthermore, compound <b>9</b> exhibited excellent selectivity, with a 900-fold preference for human nNOS over human eNOS, making it one of the most potent and selective aminoquinoline-based nNOS inhibitors reported to date. Herein we present our inhibitor design rationale, the synthesis, and the biochemical/physical evaluation of binding along with X-ray crystallographic studies with three NOS isoforms, providing detailed insights into the observed potency and selectivity of these inhibitors.