ROCK2-IN-13 is a selective inhibitor of ROCK2. It functions by hindering ROCK2 kinase activity and reducing its nuclear expression, consequently disrupting the interaction between ROCK2 and transcription co-activators p300 and PGC 1α, thereby suppressing oncogene transcription. ROCK2-IN-13 activates FOXO1-driven PTEN expression, which in turn inhibits the PI3K/Akt pathway, induces G2/M cell cycle arrest, and promotes apoptosis (apoptosis). This compound effectively eliminates the nuclear transcription function of ROCK2 that maintains oncogenic signaling and restores the tumor-suppressive PTEN/FOXO1 axis. ROCK2-IN-13 is utilized in prostate cancer research.

ROCK2-IN-13 (compound 25) demonstrates significant antiproliferative activity in human prostate cancer PC-3 cells (30 μM for 24 hours), with an inhibition rate of 84.5%. It exhibits GI50 values of 3.7 μM in PC-3 cells and 3.9 μM in LNCaP cells, showing strong and consistent potency, while exhibiting notably low cytotoxicity to normal cells (GI50 of 55.0 μM for CCD841 cells and 41.8 μM for HEK-293 cells). In PC-3 cells, ROCK2-IN-13 reduces the expression of the oncogenic protein FOXA2 (HNF-3β) and the less expressed FOXO1. At concentrations of 1-10 μM, it induces a concentration-dependent decrease in FOXA2 mRNA and an increase in FOXO1 mRNA in PC-3 and LNCaP cells, resulting in reduced nuclear FOXA2 protein, enhanced nuclear accumulation of FOXO1, decreased cytoplasmic FOXO1 phosphorylation, and the inhibition of the PI3K/Akt axis through PTEN upregulation, thus increasing p27 expression and Bax/Bcl-2 ratio while decreasing cyclin D1 levels. ROCK2-IN-13 also reduces the proportion of cells in the G1 and S phases and induces significant accumulation in the G2/M phase in PC-3 cells (1-10 μM for 24 hours). It induces apoptosis in a concentration-dependent manner in PC-3 cells, with early and late apoptotic cells reaching 15.7% and 8.7%, respectively, at a 10 μM concentration. After 48 hours, ROCK2-IN-13 suppresses the PI3K/Akt signaling pathway in PC-3 cells mainly through PTEN activation rather than direct EGFR inhibition. Furthermore, ROCK2-IN-13 enhances the PGC-1α mRNA level and significantly decreases ROCK1 and ROCK2 mRNA expression (more prominently affecting ROCK2), reducing nuclear ROCK2 protein levels while maintaining cytoplasmic ROCK1 stability. It achieves this by recruiting RNA polymerase II and PGC-1α to the FOXO1 promoter region, enhancing its transcription, and promoting PTEN transcription through recruitment of RNA polymerase II and essential co-regulators like ROCK1/2, PGC-1α, and p300 to the PTEN promoter.

Compound ROCK2-IN-13 (compound 25), administered intraperitoneally at dosages of 30 and 50 mg/kg once daily for 26 days, significantly inhibits tumor growth in a prostate cancer xenograft model.