Phenotypic Optimization of Urea–Thiophene Carboxamides To Yield Potent, Well Tolerated, and Orally Active Protective Agents against Aminoglycoside-Induced Hearing Loss

Hearing loss is a major public health concern with no pharmaceutical intervention for hearing protection or restoration. Using zebrafish neuromast hair cells, a robust model for mammalian auditory and vestibular hair cells, we identified a urea–thiophene carboxamide, <b>1</b> (ORC-001), as protective against aminoglycoside antibiotic (AGA)-induced hair cell death. The 50% protection (HC<sub>50</sub>) concentration conferred by <b>1</b> is 3.2 μM with protection against 200 μM neomycin approaching 100%. Compound <b>1</b> was sufficiently safe and drug-like to validate otoprotection in an <i>in vivo</i> rat hearing loss model. We explored the structure–activity relationship (SAR) of this compound series to improve otoprotective potency, improve pharmacokinetic properties and eliminate off-target activity. We present the optimization of <b>1</b> to yield <b>90</b> (ORC-13661). Compound <b>90</b> protects mechanosensory hair cells with HC<sub>50</sub> of 120 nM and demonstrates 100% protection in the zebrafish assay and superior physiochemical, pharmacokinetic, and toxicologic properties, as well as complete <i>in vivo</i> protection in rats.