The CTD of Hsp90 contains several binding areas: the C-terminal ATP binding site, the MEEVD motif at the end of the CTD, and the primary dimerization interface of Hsp90. The C-terminal ATP binding site is only available upon occupation of the N-terminal ATP binding pocket and binds purine and pyrimidine nucleotides, while the N-terminal ATP binding site (NTD) is more specific for adenine derivatives. The MEEVD motif binds the TRP-domain of cochaperones such as Hop and immunophilins, which is formed by a four-helix bundle and is crucial for Hsp90 dimerization. Proteinprotein interactions in the interface of the four-helix bundle maintain the dimeric state of Hsp90. In this study, we present the development of the first-in-class small-molecule inhibitor of Hsp90, 5b, which was rationally designed to target the C-terminal dimerization interface. Based on our prior hotspot prediction and the prototype compound AX , we successfully performed a scaffold-hopping from the aminoxy peptide-backbone towards more drug-like tripyrimidones. 5b mimics α-helical side-chains that form hotspot residues located on H5 in the dimerization interface. To independently predict the binding site and mode of 5b, we performed extensive MD simulations, in which the inhibitor was allowed to diffuse freely around an MD-CTD construct of Hsp90α, similar to our analysis on AX binding and related studies. The results revealed the C-terminal dimerization interface as the most likely binding region of 5b, which was confirmed by effective binding energy computations, corroborating the hypothesis underlying the design of 5b. Following the high sequence-identity in the interface region, similar results were obtained for Hsp90β, suggesting that 5b does not exhibit isoform specificity.