Estradiol dimers (EDs) possess significant anticancer activity by targeting tubulin dynamics. In this study, we synthesised 12 EDs variants via copper-catalysed azide-alkyne cycloaddition (CuAAC) reaction, focusing on structural modifications within the aromatic bridge connecting two estradiol moieties. In vitro testing of these EDs revealed a marked improvement in selectivity towards cancerous cells, particularly for ED1-8. The most active compounds, ED3 (IC50 = 0.38 μM in CCRF-CEM) and ED5 (IC50 = 0.71 μM in CCRF-CEM) demonstrated cytotoxic effects superior to 2-methoxyestradiol (IC50 = 1.61 μM in CCRF-CEM) and exhibited anti-angiogenic properties in an endothelial cell tube-formation model. Cell-based experiments and in vitro assays revealed that EDs interfere with mitotic spindle assembly. Additionally, we proposed an in silico model illustrating the probable binding modes of ED3 and ED5, suggesting that dimers with a simple linker and a single substituent on the aromatic central ring possess enhanced characteristics compared to more complex dimers.

Department of Biology of Cytoskeleton Institute of Molecular Genetics of the Czech Academy of Sciences Praha 4 Czech Republic

Department of Chemistry of Natural Compounds University of Chemistry and Technology Prague Praha 6 Czech Republic

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacký University Olomouc Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Praha 6 Czech Republic

Laboratory of Experimental Medicine Institute of Molecular and Translational Medicine University Hospital Olomouc Olomouc Czech Republic

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