Microtubule-targeting agents that bind at the colchicine-site of tubulin are of particular interest in antitumoral therapy due to their dual mechanism of action as antimitotics and vascular disrupting agents. Cyclohexanediones derivatives have been described as a new family of colchicine-domain binders with an association constant to tubulin similar to that of colchicine. Here, the high-resolution structures of tubulin in complex with cyclohexanediones TUB015 and TUB075 were solved by X-ray crystallography. A detailed analysis of the tubulin-TUB075 interaction by means of computational affinity maps allowed the identification of two additional regions at the binding site that were addressed with the design and synthesis of a new series of cyclohexanediones with a distal 2-substituted benzofurane. These new compounds showed potent antiproliferative activity with IC50 values in the nM range, arrested cell cycle progression at the G2/M phase and induced apoptosis at sub μM concentrations. Moreover, they caused the destruction of a preformed vascular network in vitro and inhibited the migration of endothelial cells at non-toxic concentrations. Finally, these compounds displayed high affinity for tubulin as substantiated by a K b value of 2.87 × 108 M-1 which, to the best of our knowledge, represents the highest binding constant measured to date for a colchicine-domain ligand.