Abstract

A novel non-competitive p97 ATPase inhibitor with a scaffold of diphenylmethyl moiety. • Exploration of the structure-activity relationship of new compounds. • 10a exhibited inhibitory effects against 3D patient-derived colorectal organoid. • 10a suppressed tumor growth in a mouse MC38 colorectal cancer model (55% inhibition) The AAA+ ATPase p97/VCP is a central regulator of protein homeostasis and has emerged as an attractive anticancer target. However, first-generation ATP-competitive inhibitors have faced clinical setbacks due to off-target toxicity and sensitivity to ATP concentrations. Herein, we report the design, synthesis, and systematic structure–activity relationship (SAR) study of a novel series of diphenylmethyl-based non-competitive p97/VCP inhibitors derived from the allosteric hit MSC1094308. SAR optimization revealed that conversion of the amide linker to a secondary amine, coupled with the introduction of a tetrahydropyrido[3,4-b]indole scaffold and fluorine substitution on the biphenylmethyl group, dramatically enhanced p97/VCP inhibitory activity. The lead compounds, 10a and 10b, exhibited potent non-competitive inhibition (IC 50 = 1.04 μM and 17 nM, respectively) and maintained efficacy independent of ATP concentration. Microscale thermophoresis (MST) confirmed strong binding affinity of 10a to p97/VCP (Kd = 14.99 μM), and limited proteolysis-mass spectrometry (LiP-MS) identified p97/VCP as a direct cellular targ et of 10a. Mechanistically, 10a induced mitochondrial membrane depolarization, leading to concurrent regulation of both apoptotic (caspase-3, PARP cleavage) and autophagic (LC3-II, p62) pathways. In vitro, 10a demonstrated broad-spectrum antiproliferative activity across multiple cancer cell lines and completely suppressed the growth of patient-derived colorectal cancer organoids. In an MC38 colorectal cancer xenograft mouse model, 10a achieved 55% tumor growth inhibition with manageable toxicity. Collectively, this study identifies 1