The molecular chaperone heat shock protein 90 (HSP90) is a promising target for cancer therapy, as it assists in the stabilization of cancer-related proteins, promoting cancer cell growth, and survival. A novel series of HSP90 inhibitors were discovered by structure–activity relationship (SAR)-based optimization of an initial hit compound 11ahaving a 4-(4-(quinolin-3-yl)-1H-indol-1-yl)benzamide structure. The pyrazolo[3,4-b]pyridine derivative, 16e(TAS-116), is a selective inhibitor of HSP90α and HSP90β among the HSP90 family proteins and exhibits oral availability in mice. The X-ray cocrystal structure of the 16eanalogue 16ddemonstrated a unique binding mode at the N-terminal ATP binding site. Oral administration of 16edemonstrated potent antitumor effects in an NCI-H1975 xenograft mouse model without significant body weight loss.