26 p.-6 fig.2 tab. ; 12-Aza-epothilones (azathilones) incorporating quinoline side chains and bearing different N12-substituents have been synthesized via highly efficient RCM-based macrocyclizations.Quinoline-based azathilones with the side chain N-atom in the meta-position to the C15 atom in the macrocycle are highly potent inhibitors of cancer cell growth in vitro. In contrast, shifting the quinoline nitrogen to the position para to C15 leads to a ca. 1000-fold loss in potency. Likewise,the desaturation of the C9-C10 bond in the macrocycle to an E double bond produces a substantial reduction in antiproliferative activity. This is in stark contrast to the effect exerted by the same modification in the natural epothilone macrocycle. The conformation of a representative azathilone bound to / -tubulin heterodimers was determined based on TR-NOE measurements and a model for the posture of the compound in its binding site on -tubulin was deduced through a combination of STD measurements and CORCEMA-ST calculations. The tubulin-bound, bioactive conformation of azathilones was found to be overall similar to that of epothilones A and B. ; This work was supported by an institutional grant from the ETH Zürich (A.J., K-H.A.,J.G.), by MICIN through grant CTQ-2006-10874-C002-01 (J.J.B.), by FPI through grant BES-2007-16429 (L.N.),by the Spanish MINECO through a “Ramon y Cajal” postdoctoral contract and project grants CTQ2012-32025, SAF 2013-48271-C2-IR, and SAF 2010-22198-C02-01 (A.C.), and by grants BIO2013-42984-R from Ministerio de Economia y Competitividad, and grant S2010/BMD-2457 BIPEDD2 from Comunidad Autónoma de Madrid ; Peer reviewed