Discovery of novel ROCK inhibitors RX-021 and RX-044 with intraocular pressure-lowering effect for glaucoma treatment.

Through systematic optimization of lead D25, we identified two novel ROCK inhibitors, RX-021 and RX-044. Maintaining DFG interactions while optimizing linker flexibility was critical for potency. RX-044 showed excellent ROCK1/2 inhibition (IC50 = 10.01 and 9.68 nM), favorable kinase selectivity, and no cytotoxicity in HTM cells. In a mouse ocular hypertension model, RX-021 achieved superior IOP reduction (5.38 ± 1.51 mmHg at 4 h) versus (S)-Netarsudil, with sustained 24 h efficacy and reversible HTM cell effects. Both compounds provided significant retinal neuroprotection, preserving retinal ganglion cell survival, restoring electroretinography responses, and ameliorating histopathological changes. Slit-lamp exams confirmed that initial ocular irritation subsided with extended dosing. These findings establish RX-021 and RX-044 as promising novel ROCK inhibitors with enhanced IOP-lowering efficacy and good retinal protective effects for glaucoma therapy.