Tumor necrosis factor receptor-associated protein 1 (TRAP1), a mitochondrial HSP90 chaperone, is overexpressed in many cancers and supports tumor growth through regulation of energy metabolism and oxidative stress. We previously identified an allosteric site in the middle domain (MD) of TRAP1 using Mitoquinone (MitoQ), a mitochondria-targeted antioxidant with anticancer activity. In this study, novel MD-targeting TRAP1 inhibitors were designed and synthesized by modifying the linker and ubiquinone moiety of MitoQ. These compounds showed inhibitory activity against TRAP1 and represent a promising strategy for anticancer drug development.