Rational Design of 6‐Amino‐2‐Piperazinylpyridine‐Based ROCK2 Inhibitors With Anti‐Breast Cancer Metastatic Efficiency

ABSTRACT Rho‐associated coiled coil kinase (ROCK) plays a pivotal role in regulating actin cytoskeleton remodeling and cellular motility, establishing it as a promising therapeutic target for metastatic breast cancer. Guided by the lead compound belumosudil, a novel series of ROCK2 inhibitors based on a 6‐amino‐2‐piperazine pyridine scaffold was designed and synthesized. The Kinase‐Glo assay identified compound 14r as the most potent analog, showing an IC50 value of 82.6 nM, which represented a 1.2‐fold improvement over belumosudil. Furthermore, the morphology of MDA‐MB‐231 cells treated with 14r was significantly changed in immunofluorescent staining analysis. Simultaneously, cell scratch experiments showed that 14r inhibited the migration of MDA‐MB‐231 cells in a dose‐dependent manner. Additionally, molecular docking studies elucidated the binding mode of 14r within the ROCK2 ATP pocket, corroborating its nanomolar‐level inhibitory activity. In conclusion, 14r showed great potential for further optimization to suppress breast cancer metastasis.