Design and synthesis of ROCK2-selective inhibitors bearing 6-indazolamino-2-pyrrolidinylpyridine for the treatment of breast cancer metastasis.

Rho-associated coiled-coil kinase (ROCK) serves as a critical driver of cellular metastasis, demonstrating therapeutic potential against breast cancer cell metastasis. To further enhance the ROCK2 inhibitory activity of belumosudil (ROCK2 IC50 = 0.153 μM), a novel series of 6-indazolamino-2-pyrrolidylpyridine derivatives were designed and synthesized. Kinase-Glo assay identified cyclopentylamino analogues S9 (ROCK2 IC50 = 0.020 μM) as the most potent ROCK2 inhibitor with 41-fold selectivity over isoform ROCK1 (ROCK1 IC50 = 0.821 μM). Immunofluorescence imaging illustrated that S9 (5 μM) could disrupt the cytoskeletal organization of MDA-MB-231 cells, and the superior inhibitory potency of S9 against cancer cell metastasis in vitro was further verified with cell scratch assay. Mechanistic study suggested that such anti-metastatic effect of S9 was closely related to the suppression of STAT3 phosphorylation. Finally, the binding mode of S9 with ROCK2 well rationalized its superior potency structurally, suggesting the potential application of S9 in breast cancer metastasis treatment.