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.

Conflict of interest statement

Notes

The authors declare the following competing financial interest(s): Part of the research reported was funded by Oricula Therapeutics. G.J., V.E.G., D.W.R., E.WR and J.A.S are founders of Oricula Therapeutics.

Figures

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 with1 alone, kanamycin, or co-administration with kanamycin and1. 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 of90 (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.