The iminoquinone is an important moiety of a large number of antineoplastic drugs and plays a significant role in the nucleus of actinomycins, powerful, highly toxic, natural antibiotics that target DNA as intercalating agents. A series of polycyclic iminoquinonic compounds, 2-amino-3H-phenoxazin-3-one (1), 2-amino-1,9-diacetyl-3H-phenoxazin-3-one (2), 2-acetylamino-3H-phenoxazin-3-one (3), 3H-phenoxazin-3-one (4), 5H-pyrido[3,2-a]phenoxazin-5-one (5), and 5H-pyrido[3,2-a]phenothiazin-5-one (6), strictly related to the actinomycin chromophore, were synthesized for developing new anticancer intercalating drugs. The antiproliferative activity of these compounds, evaluated against representative human liquid and solid neoplastic cell lines, showed that 5 and its isoster 6 were the most active compounds inhibiting cell proliferation in a submicromolar range. Compound 5 was also evaluated against KB subclones (KBMDR, KB7D, and KBV20C), which overexpress the MDR1/P-glycoprotein drug efflux pump responsible for drug resistance. All the above KB subclones did not show altered sensitivity to the antiproliferative activity of 5. UV-vis and (1)H NMR spectroscopy experiments support the phenoxazinone 5/DNA binding. Molecular mechanics methods were used to build a three-dimensional model of the 5/[d(GAAGCTTC)]2 complex. Electrostatic interactions between the hydrogen of the positively charged pyridine nitrogen of 5 and the negatively charged oxygen atoms (O4' and O5') of the cytosine C5 residue together with stacking forces contribute to the high antiproliferative activity. The metal(II)-assisted synthesis procedure of 5 is described, and the formation mechanism is proposed.