The ustiloxins are a family of heterodetic cyclopeptides that are potent antimitotic agents. Some modifications have been made for the total synthesis of ustiloxin D based on the ethynyl aziridine ring-opening of phenol derivatives. Change of the iso-propyl group at 6-position of ustiloxin D to other alkyl groups resulted in new analogues of ustiloxin D. Among them, the 6-t-Leu-Ustiloxin showed the best biological activity for the inhibition of tubulin assembly.