Design, Synthesis, and Biological Evaluation of Novel Activators of Human Caseinolytic Protease P with a Pyrazololactam Scaffold

Based on D9, a previously reported small-molecule agonist of hClpP, a class of novel hClpP activators with a pyrazololactam scaffold was designed and synthesized. Detailed structure–activity relationship studies (SAR) for this class of compounds led to the identification of compound 24, which potently activated recombinant hClpP in a proteolysis assay with submicromolar potency and effectively inhibited cell growth in a broad panel of cancer cell lines with IC₅₀ values of 0.1–1 μM. Mechanism studies indicated that compound 24 can potently bind to cellular hClpP, effectively promote the formation of the hClpP tetradecamer, efficiently induce the degradation of hClpP substrates, robustly upregulate the expression of ATF4, and strongly induce apoptosis in Molm13 and MDA-MB-231 cells. More importantly, compound 24 has promising PK and safety profiles, and showed potent antitumor activity in a murine MDA-MB-231 xenograft model.