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, <b>D25</b> and <b>R3</b>, which were designed to optimize selectivity, efficacy, and ocular bioavailability. <b>D25</b> potently inhibited ROCK1/2 with IC₅₀ values of 47.2 nM and 33.8 nM, respectively, surpassing Netarsudil. Compound <b>R3</b> had weaker ROCK inhibition but demonstrated favorable lipophilicity (log<i>P</i>) and good selectivity to ROCKs, which enhances its potential and safety for ocular delivery. In human trabecular meshwork (HTM) cells, <b>R3</b> showed lower cytotoxicity than Netarsudil and effectively mitigated oxidative damage, enhanced cellular integrity, and reduced inflammatory cytokine secretion. In rabbit models, <b>D25</b> significantly lowered IOP, outperforming (<i>S</i>)-Netarsudil. <b>R3</b> exhibited weaker IOP-lowering efficacy but better selectivity. <b>D25</b> is a promising glaucoma treatment candidate, with <b>R3</b> as a safer alternative for further optimization.