A new class of antitumor agents named azonafides has been developed. It contains the dibenz[de,h]isoquinoline-1,3-dione nucleus, which intercalates into DNA, and a side chain with a basic amino group. Its design is based on amonafide and certain anthracene antitumor agents, and molecular modeling studies indicate that the parent compound, azonafide, should bind more strongly than amonafide to DNA because of greater overlap with the DNA base pairs and greater lipophilicity. This expectation is realized in a greater effect on DNA transition melt temperature and increased cytotoxicity to tumor cells. Analogs of azonafide containing a variety of groups on all of the nuclear positions and in the side chain were synthesized and screened in a panel of tumor cells designed to select compounds with high potency against human tumor cells resistant to standard antitumor agents. Compounds with these properties and low toxicity to fetal rat heart myocytes in culture were evaluated further against leukemias and subcutaneous B16 melanoma in mice. The best compound in these in vivo assays, 6-ethoxyazonafide, was tested against human tumors in SCID mice and found active against breast carcinoma, multiple myeloma, and leukemia. Quantitative structure-activity studies revealed some statistically significant correlations between potency in tumor cells and DNA transition melt temperature increases. Correlations also were found between potency and physicochemical properties including substitutent size (large substituents decreased potency) and lipophilicity. These correlations support the intercalation binding model. A study on the comparative properties of selected azonafides found that compounds containing a second side chain with a basic nitrogen were more cytotoxic to CHO cells and more highly concentrated in their nuclei than azonafides without this feature, but they caused fewer single strand breaks and protein-DNA cross links.