Biological evaluation of 96 analogs and synthetic intermediates of the naphthyridinomycin-type antitumor antibiotic Dnacin led to the identification of several low-micromolar inhibitors of dual specificity phosphatases, specifically Cdc25A 1 , Cdc25B 2 , and VHR, as well as the tyrosine phosphatase PTP1B. While the parent Dnacins are potent DNA cleavage agents, most of the analog structures, even those that retained significant phosphatase inhibitory activities, did not lead to plasmid DNA cleavage. Thus, the DNA-targeting and the phosphatase-inhibitory activities of Dnacins can be assigned to different pharmacophores.