Taxanes and other tubulin-targeting medications are essential for treating advanced malignancies, especially in patients undergoing less aggressive chemotherapy. However, their clinical efficacy is often limited by significant off-target toxicity and adverse side effects. In this study, the synthesis and characterisation of novel steroidal A-ring-fused isoxazoles, which were obtained through iodine-mediated oxidative cyclization of dihydrotestosterone (DHT)-derived α,β-unsaturated oximes, are reported. According to mechanistic studies, the most potent compounds induced mitotic arrest and disrupted cytoskeletal integrity at low micromolar concentrations. The lead compound, 2j, notably increased the rate of tubulin polymerisation in vitro and stabilised polymerised tubulin in the cells, leading to a G2/M block of the cell cycle. Molecular docking studies indicated that 2j is bound preferably to the taxane site on tubulin, forming conserved interactions. MicroScale Thermophoresis was used to further study this binding and showed a nanomolar KD for 2j. The fact that 2j maintained its activity in docetaxel-resistant prostate cancer cells, demonstrating its ability to circumvent resistance pathways linked to existing therapies with taxane-like drugs, supports its clinical relevance. Therefore, our results encourage additional research and development for its potential therapeutic use in cancer treatment, particularly in resistant cases.

Department of Experimental Biology Faculty of Science Palacký University Olomouc Olomouc Czech Republic

Department of Molecular and Analytical Chemistry University of Szeged Szeged Hungary

Institute of Pharmacodynamics and Biopharmacy University of Szeged Szeged Hungary

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