Zalypsis is a new synthetic alkaloid tetrahydroisoquinoline antibiotic that has a reactive carbinolamine group. This functionality can lead to the formation of a covalent bond with the amino group of selected guanines in the DNA double helix, both in the absence and in the presence of methylated cytosines. The resulting complex is additionally stabilized by the establishment of one or more hydrogen bonds with adjacent nucleotides in the opposite strand as well as by van der Waals interactions within the minor groove. Fluorescence-based thermal denaturation experiments demonstrated that the most favorable DNA triplets for covalent adduct formation are AGG, GGC, AGC, CGG and TGG, and these preferences could be rationalized on the basis of molecular modeling results. Zalypsis-DNA adducts eventually give rise to double-strand breaks, triggering S-phase accumulation and apoptotic cell death. The potent cytotoxic activity of Zalypsis was ascertained in a 24 cell line panel. The mean IC(50) value was 7nM and leukemia and stomach tumor cell lines were amongst the most sensitive. Zalypsis administration in four murine xenograft models of human cancer demonstrates significant tumor growth inhibition that is highest in the Hs746t gastric cancer cell line with no weight loss of treated animals. Taken together, these results indicate that the potent antitumor activity of Zalypsis supports its current development in the clinic as an anticancer agent.