Although Hsp90 inhibitors can inhibit multiple tumorigenic pathways in cancer cells, their anticancer activity has been disappointingly modest. However, by forcing Hsp90 inhibitors into the mitochondria with mitochondrial delivery vehicles, they were converted into potent drugs targeting the mitochondrial Hsp90 paralog TRAP1. Here, to improve mitochondrial drug accumulation without using the mitochondrial delivery vehicle, we increased freely available drug concentrations in the cytoplasm by reducing the binding of the drugs to the abundant cytoplasmic Hsp90. After analyzing X-ray cocrystal structures, the purine ring of the Hsp90 inhibitor 2 (BIIB021) was modified to pyrazolopyrimidine scaffolds. One pyrazolopyrimidine, 12b (DN401), bound better to TRAP1 than to Hsp90, inactivated the mitochondrial TRAP1 in vivo, and it exhibited potent anticancer activity. Therefore, the rationale and feasible guidelines for developing 12b can potentially be exploited to design a potent TRAP1 inhibitor.

Derivatives of 4‐hydroxy‐1‐alkyl‐2‐oxo‐1,2‐dihydrothieno[2,3‐b:4,5‐b′]dipyridine‐3‐carbonylglycine were developed as a novel hypoxia‐inducible factor prolyl hydroxylase domain (PHD) inhibitor. The chemical space of the tricyclic 4‐hydroxypyridinyl glycines was examined thoroughly during our optimization study. One of our most potent compounds 12ar exhibits superior enzymatic activity to the known PHD inhibitors that are in the late stage of clinical studies. The functional efficacy of our PHD inhibitors was confirmed via the increased level of erythropoietin (EPO) expression in a dose‐dependent manner in vitro.