Making Epothilones Fluoresce: Design, Synthesis, and Biological Characterization of a Fluorescent N12-Aza-Epothilone (Azathilone)

A green fluorescent 12-aza-epothilone (azathilone) derivative has been prepared through the attachment of the 4-nitro-2,1,3-benzoxadiazole (NBD) fluorophore to the 12-nitrogen atom of the azamacrolide core structure. While less potent than natural epothilones or different N12-acylated azathilone derivatives, NBD-azathilone (3) promotes tubulin assembly, inhibits cancer cell proliferation in vitro and arrests the cell cycle at the G2/M transition. Most significantly, the binding of 3 to cellular microtubules (MTs) could be directly visualized by confocal fluorescence microscopy. Based on competition binding experiments with laulimalide-stabilized MTs in vitro, the N12-Boc substituted azathilone 1, Epo A, and NBD-azathilone (3) all interact with the same tubulin-binding site. Computational studies provided a structural model of the complexes between β-tubulin and 1 or 3, respectively, in which the NBD moiety of 3 or the BOC moiety of 1 directly and specifically contribute to MT binding. Collectively, these data demonstrate that the cellular effects of 3 and, by inference, also of other azathilones are the result of their interactions with the cellular MT network.