Microtubule dynamics is one of the major targets for new chemotherapeutic agents. This communication presents the synthesis and biological profiling of steroidal dimers based on
estradiol,
testosterone and
pregnenolone bridged by 2,6-bis(azidomethyl)pyridine between D rings. The biological profiling revealed unique properties of the
estradiol dimer including cytotoxic activities on a panel of 11 human cell lines, ability to arrest in the G2/M phase of the cell cycle accompanied with the attenuation of
DNA/
RNA synthesis. Thorough investigation precluded a genomic mechanism of action and revealed that the
estradiol dimer acts at the cytoskeletal level by inhibiting
tubulin polymerization. Further studies showed that
estradiol dimer, but none of the other structurally related dimeric
steroids, inhibited assembly of purified
tubulin (IC50, 3.6 μM). The
estradiol dimer was more potent than
2-methoxyestradiol, an endogenous metabolite of 17β-estradiol and well-studied microtubule polymerization inhibitor with antitumor effects that was evaluated in clinical trials. Further, it was equipotent to
nocodazole (IC50, 1.5 μM), an
antimitotic small molecule of natural origin. Both
estradiol dimer and
nocodazole completely and reversibly depolymerized microtubules in interphase U2OS cells at 2.5 μM concentration. At lower concentrations (50 nM),
estradiol dimer decreased the microtubule dynamics and growth life-time and produced comparable effect to
nocodazole on the microtubule dynamicity. In silico modeling predicted that
estradiol dimer binds to the
colchicine-binding site in the
tubulin dimer. Finally, dimerization of the
steroids abolished their ability to induce transactivation by
estrogen receptor α and
androgen receptors. Although other
steroids were reported to interact with microtubules, the
estradiol dimer represents a new structural type of
steroid inhibitor of
tubulin polymerization and microtubule dynamics, bearing
antimitotic and cytotoxic activity in
cancer cell lines.