Microtubule dynamic is exceptionally sensitive to modulation by small-molecule ligands. Our previous work presented the preparation of microtubule-targeting estradiol dimer (ED) with anticancer activity. In the present study, we explore the effect of selected linkers on the biological activity of the dimer. The linkers were designed as five-atom chains with carbon, nitrogen or oxygen in their centre. In addition, the central nitrogen was modified by a benzyl group with hydroxy or methoxy substituents and one derivative possessed an extended linker length. Thirteen new dimers were subjected to cytotoxicity assay and cell cycle profiling. Dimers containing linker with benzyl moiety substituted with one or more methoxy groups and longer branched ones were found inactive, whereas other structures had comparable efficacy as the original ED (e.g. D1 with IC50 = 1.53 µM). Cell cycle analysis and immunofluorescence proved the interference of dimers with microtubule assembly and mitosis. The proposed in silico model and calculated binding free energy by the MM-PBSA method were closely correlated with in vitro tubulin assembly assay.

Department of Chemistry of Natural Compounds University of Chemistry and Technology Prague Technická 5 166 28 Prague 6 Czech Republic

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacký University and University Hospital in Olomouc Hněvotínská 1333 5 779 00 Olomouc Czech Republic

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Click estradiol dimers with novel aromatic bridging units: synthesis and anticancer evaluation

StreaMD: the toolkit for high-throughput molecular dynamics simulations