Introduction:Protein p97 is an extensively investigated AAA ATPase with various cellular activities, including cell cycle control, ubiquitin–proteasome system, autophagy, and NF-κB activation.Method:In this study, we designed, synthesized and evaluated eight novel DBeQanalogs as potential p97 inhibitorsin vivoandin vitro.Results:In the p97 ATPase inhibition assay, compounds6and7showed higher potency than the known p97 inhibitors, DBeQ and CB-5083. Compounds4-6dramatically induced G0/G1 phase arrest in the HCT116 cells, and compound7arrested the cells in both G0/G1 and S phases. Western blots showed elevated levels of SQSTM/p62, ATF-4, and NF-κB in HCT116 cells with the treatment of compounds4–7, confirming their role in inhibiting the p97 signaling pathway in cells. In addition, the IC50of compounds4–6against HCT116, RPMI-8226, and s180 proliferation were 0.24–6.9 µM with comparable potency as DBeQ. However, compounds4–6displayed low toxicity against the normal human colon cell line. Thus, compounds6and7were proved to be potential p97 inhibitors with less cytotoxicity.In vivostudies using the s180 xenograft model have demonstrated that compound6inhibited tumor growth, led to a significant reduction of p97 concentration in the serum and tumor, and indicated non-toxicity on the body weight and organ-to-brain weight ratios except for the spleen at the dose of 90 μmol/kg/day for 10 days. Furthermore, the present study indicated that compound6may not induce s180 mice myelosuppression often observed in the p97 inhibitors.Conclusion:Compound6displayed high binding affinity to p97, great p97 ATPase inhibition, selective cytotoxicity, remarkable anti-tumor effect, and upregulated safety, which improved the clinical potential of p97 inhibitors.