Hsp90 C-terminal domain (CTD) inhibitors are promising novel agents for cancer treatment, as they do not induce the heat shock response associated with Hsp90 N-terminal inhibitors. One challenge associated with CTD inhibitors is the lack of a co-crystallized complex, requiring the use of predicted allosteric apo pocket, limiting structure-based (SB) design approaches. To address this, a unique approach that enables the derivation and analysis of interactions between ligands and proteins from molecular dynamics (MD) trajectories was used to derive pharmacophore models for virtual screening (VS) and identify suitable binding sites for SB design. Furthermore, ligand-based (LB) pharmacophores were developed using a set of CTD inhibitors to compare VS performance with the MD derived models. Virtual hits identified by VS with both SB and LB models were tested for antiproliferative activity. Compounds 9 and 11 displayed antiproliferative activities in MCF-7 and Hep G2 cancer cell lines. Compound 11 inhibited Hsp90-dependent refolding of denatured luciferase and induced the degradation of Hsp90 clients without the concomitant induction of Hsp70 levels. Furthermore, compound 11 offers a unique scaffold that is promising for the further synthetic optimization and development of molecules needed for the evaluation of the Hsp90 CTD as a target for the development of anticancer drugs.