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

Abstract

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, 1 (ORC-001), as protective against aminoglycoside antibiotic (AGA)-induced hair cell death. The 50% protection (HC₅₀) concentration conferred by 1 is 3.2 μM with protection against 200 μM neomycin approaching 100%. Compound 1 was sufficiently safe and drug-like to validate otoprotection in an in vivo 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 1 to yield 90 (ORC-13661). Compound 90 protects mechanosensory hair cells with HC₅₀ of 120 nM and demonstrates 100% protection in the zebrafish assay and superior physiochemical, pharmacokinetic, and toxicologic properties, as well as complete in vivo protection in rats.

Figure 1

Structure of 1.

Figure 2

Dose response functions for zebrafish neuromast hair cells treated with neomycin (200 μM) plus 1, 53, 76, 53, 87 or neomycin alone.

Figure 3

Dose response functions for zebrafish neuromast hair cells treated with neomycin (200 μM) plus 1, 67, 90, 99 or neomycin alone.

Figure 4

Crystal structure of 90.

Figure 5

Synthesis and resolution of chiral tropane analogues. a). NCCH2CO2Et, S8, morpholine, EtOH; b). p-ClC6H4N=C=O, TEA, THF; c). chiral HPLC separation.

Figure 6

Protection of hearing in Sprague-Dawley rats treated with kanamycin for 14 days (500 mg/kg/day s.c.) by concurrent i.p. administration of 1 (25 mg/kg/day). A. Mean (+1 SEM) ABR thresholds in rats recorded an additional 14 days after the 14-day drug treatment with 1 alone, kanamycin, or co-administration with kanamycin and 1. B. Mean (+1 SEM) threshold shifts in hearing from pre-treatment levels 14 days after the treatment for each group. Positive values indicate increasing levels of hearing loss.

Figure 7

ADME Profiling for Compound 90.

Figure 8

Protection of hearing in Fischer 344 rats treated with amikacin (320mg/kg/day s.c.) for 12 days with or without concurrent oral administration of 90 (5 mg/kg/day). Threshold shift from pre-treatment hearing levels at 2-weeks following treatment period.

Figure 9

Confocal images of the organ of Corti basal turn: representative examples from each treatment group. A. Control: saline treated, s.c. daily for 12 days. B. Amikacin treated, s.c. daily, 320 mg/kg/day for 12 days. C. Amikacin (Ami) + 90: Amikacin s.c. daily, 320 mg/kg/day + 90 p.o. daily, 5mg/kg for 12 days. Cochleae from animals were fixed with 4% paraformaldehyde and dissected. Organs of Corti were incubated with cell-type specific antibodies: anti-myosin 7A antibody (hair cells: green), anti-Sox2 antibody (supporting cell nuclei: red) and antineurofilament (NF) antibody (neuronal processes: blue). All animals were treated with drug for 12 days. Final post-treatment ABR testing was done after an additional 12 day period. Animals euthanized after ABR testing for histological examination.

Scheme 1

Gewald reaction to form 2-amino-thiophene carboxamides.