Optimisation of pyrazolo[1,5-a]pyrimidin-7(4H)-one derivatives as novel Hsp90 C-terminal domain inhibitors against Ewing sarcoma

Ewing sarcoma is the second most prevalent paediatric malignant bone tumour. In most cases, it is driven by the fusion oncoprotein EWS::FLI1, which acts as an aberrant transcription factor and dysregulates gene expression. EWS::FLI1 and a large number of downstream dysregulated proteins are Hsp90 client proteins, making Hsp90 an attractive target for the treatment of Ewing sarcoma. In this article, we report a new structural class of allosteric Hsp90 C-terminal domain (CTD) inhibitors based on the virtual screening hit TVS24, which showed anti -proliferative activity in the SK -N-MC Ewing sarcoma cell line with an IC50 value of 15.9 +/- 0.7 mu M. The optimised compounds showed enhanced anticancer activity in the SK -N-MC cell line. Exposure of Ewing sarcoma cells to the most potent analogue 11c resulted in depletion of critical Hsp90 client proteins involved in cancer pathways such as EWS::FLI1, CDK4, RAF-1 and IGF1R, without inducing a heat shock response. The results of this study highlight Hsp90 CTD inhibitors as promising new agents for the treatment of Ewing sarcoma.