There are many natural peptides with multiple N-methylamino acids that exhibit potent attractive biological activities. N-methylation of a peptide bond(s) is also one of the standard approaches in medicinal chemistry of bioactive peptides, to improve the potency and physicochemical properties, especially membrane permeability. In this study, we investigated a facile synthesis process of N-methylated peptides via simultaneous N-methylation of several peptide bonds in the presence of peptide bonds that were not to be methylated. As a model study, we investigated the synthesis of the antiproliferative depsipeptide, IB-01212. We used a pseudoproline to protect the non-methylated peptide bond during a simultaneous N-methylation with MeI-Ag2O. Using further manipulations including a dimerization/cyclization process, IB-01212 and its derivatives were successfully synthesized. A preliminary structure-activity relationship study demonstrated that the symmetric structure contributed to the potent cytotoxic activity of IB-01212. (C) 2014 Elsevier Ltd. All rights reserved.