Design, Synthesis, and Evaluation of Novel ROCK Inhibitors for Glaucoma Treatment: Insights into In Vitro and In Vivo Efficacy and Safety

The inhibition of Rho-associated coiled-coil kinase (ROCK) has emerged as a promising strategy for reducing intraocular pressure (IOP) and treating glaucoma. Here, we report the synthesis and evaluation of novel ROCK inhibitors, D25 and R3, which were designed to optimize selectivity, efficacy, and ocular bioavailability. D25 potently inhibited ROCK1/2 with IC₅₀ values of 47.2 nM and 33.8 nM, respectively, surpassing Netarsudil. Compound R3 had weaker ROCK inhibition but demonstrated favorable lipophilicity (logP) and good selectivity to ROCKs, which enhances its potential and safety for ocular delivery. In human trabecular meshwork (HTM) cells, R3 showed lower cytotoxicity than Netarsudil and effectively mitigated oxidative damage, enhanced cellular integrity, and reduced inflammatory cytokine secretion. In rabbit models, D25 significantly lowered IOP, outperforming (S)-Netarsudil. R3 exhibited weaker IOP-lowering efficacy but better selectivity. D25 is a promising glaucoma treatment candidate, with R3 as a safer alternative for further optimization.