ZM484 is a potent dual inhibitor of p53-MDM2/TOP1, demonstrating antiproliferative and antitumor activities both in vivo and in vitro. By releasing CPT and an effective p53-MDM2 inhibitor, ZM484 efficiently upregulates p53 and MDM2 proteins while maintaining TOP1 inhibitory activity. It induces cell cycle arrest and apoptosis by modulating the expression of key apoptosis and cell cycle-related proteins (including caspase-3, Bcl-2, and Cyclin B1). ZM484 is applicable for colorectal cancer research.

ZM484 exhibits potent anti-proliferative activity against the cancer cell lines HCT116, SJSA-1, and A549, with IC50 values of 0.03, 0.97, and 0.11 μM, respectively. It shows minimal cytotoxicity towards non-cancerous MRC-5 cells within the concentration range of 1.56-50 μM over 72 hours. At 100 μM, ZM484 does not exhibit hemolytic activity after a 30-minute exposure. The compound maintains over 95% concentration stability at 50 μM for 24 hours, even in acidic buffer solutions with pH 2 or 5. Additionally, ZM484 (25-100 nM) significantly reduces the colony-forming capability of HCT116 cells over 8 days. It inhibits TOP1 activity in a concentration-dependent manner, effective at concentrations as low as 0.195 μM within 15 minutes. ZM484 also upregulates p53 and MDM2 protein levels in a concentration-dependent manner when administered at 50-100 nM over 4 hours. Furthermore, it effectively induces S phase and G2 phase cell cycle arrest in HCT116 cells at 10-40 nM over 24 hours. Lastly, ZM484 demonstrates strong, concentration-dependent pro-apoptotic effects in cancer cells, accompanied by a dose-dependent decrease in caspase-3 and Bcl-2 expression levels at concentrations ranging from 10-640 nM over 48 hours.

ZM484 (10 mg/kg, administered intraperitoneally every other day for 12 days) demonstrates significant tumor growth inhibition in the HCT116 xenograft model.