AbstractFour new lipophilic analogues of the natural pyrone pironetin have been prepared. The C9 side‐chain of the latter has been replaced in one analogue by a 4‐phenylbutyl chain and in the other three analogues by C13 or C16 aliphatic chains, all of them bearing two stereogenic centres. Their cytotoxic activities and interactions with tubulin have been investigated. It was found that all four are cytotoxic towards two either sensitive or resistant tumoral cell lines with similar IC50 values in each case, which indicates that, like the parent natural compound, they also display a covalent mechanism of action. However, one of them operates in all likelihood through a mechanism very similar to pironetin, whereas the other three seem to operate through a different mechanism.