The heat shock protein (Hsp90) is an abundant, cytosolic molecular chaperone that modulates the folding, stabilization, and maturation of over 400 client proteins in eukaryotes. It is involved in essential processes such as signal transduction, cell cycle progression, and transcription regulation. In cancer cells, Hsp90 is overexpressed, involved in uncontrolled proliferation and anti-apoptotic effects, and, that way, essential for the malignant transformation and progression of several cancer types. Thus, cancer cells are more dependent on Hsp90 activity than normal cells. In various cancers, multiple signal transduction-promoting oncoproteins are clients of Hsp90. Hence, inhibiting the activity of Hsp90 is a promising strategy for the development of anticancer therapy. Several Hsp90 inhibitors (Hsp90i) have been developed recently, however adverse effects including dose-limiting ocular and cardiac toxicity and poor patient stratification have precluded their clinical approval. Most of the 19 Hsp90 inhibitors (Hsp90i) studied in clinical trials so far (except RTA 901) target the N-terminal ATP binding site (NTD) and are termed N-terminal inhibitors. A common pitfall of Hsp90 NTD-targeting inhibitors is the induction of a pro-survival heat shock response (HSR). The HSR is a stress response mechanism mediated by heat-shock factor 1 (HSF-1), which leads to the expression of other heat shock proteins (HSPs) including Hsp27, Hsp40 and Hsp70 as a rescue mechanism upon Hsp90 inhibition and eventually weakens cytotoxic effects of Hsp90i. In addition, Hsp90 NTD-targeting inhibitors potentially inflict cytotoxicity through mechanisms that involve targets other than Hsp90, (off-target effects). The off-target effect hypothesis is also supported by the drastic differences between cytotoxicity concentrations of Hsp90 NTD-targeting inhibitors vs. their binding affinity to Hsp90. Furthermore, there are two major cytosolic isoforms of Hsp90 (Hsp90α and Hsp90β) expressed in humans. Hsp90α is an inducible isoform, overexpressed in several cancer types, whereas the Hsp90β isoform is expressed constitutively. Thus, targeting Hsp90 with isoform-specific inhibitors can afford a therapeutic window. Most of the previously studied Hsp90i exhibit pan-inhibitory activity (i.e., targeting all isoforms). However, the Hsp90α and Hsp90β isoforms share a high degree of similarity (SI Figure ), making it challenging to develop isoform-selective inhibitors. Hsp90 is a flexible homodimer, and each monomer consists of three major functional domains: Nterminal domain (NTD), middle domain, and C-terminal domain (CTD). Hsp90's activity depends on the binding and hydrolysis of ATP at the NTD and on its dimerization via the CTD. The middle domain (MD) that connects the NTD and the CTD mediates the binding of clients and cochaperons. The CTD is connected to the MEEVD motif, which interacts with the subset of tetratricopeptide repeat (TPR) domain-containing cochaperones. To our knowledge, inhibiting Hsp90 dimer formation by targeting the CTD dimerization interface constitutes a so far unexplored mode of action (MOA) of small-molecule Hsp90i. In contrast to Hsp90i targeting the N-terminal ATP binding site, C-terminal inhibitors do not generally induce HSR. The most important classes of C-terminal inhibitors are: (1) inhibitors binding to the C-terminal ATP-binding site (e.g., novobiocin and analogs), (2) modulators of the Hsp90-CDC37 interaction (e.g., celastrol, induces HSR ), (3) modulators of the Hsp90-p23 interaction (e.g., gedunin), (4) modulators of the Hsp90-HOP interaction (e.g., LB76), and ( ) aminoxyrone (AX), the first non-peptidic inhibitor of the C-terminal dimerization of Hsp90. Following a strategy recently introduced by us to identify PPI inhibitors, we initially identified hot spot residues in the CTD dimerization interface that accounted for most of the binding affinity and identified the first peptidic inhibitors shown to bind to the CTD of Hsp90. Furthermore, we developed AX, the first peptidomimetic Hsp90 CTD dimerization inhibitor, which is a promising lead candidate effective against BCR-ABL1+ TKI-resistant leukemic cells. Based on these experiences, here, we report the rational design, chemical synthesis, binding mode model, biochemical affinity, and biological in vitro evaluation of the first-in-class small molecule inhibitor (5b) of Hsp90 CTD dimerization based on a tripyrimidonamide scaffold.