Two series of quinolinquinone derivatives, 2'H-spiro[imidazolidine-4,3'-thieno[2,3-g]quinoline]-2,4',5,9'-tetraones (2a-n) and 2H-spiro[thieno[2,3-g]quinoline-3,5'-[1,2,4]triazinane]-3',4,6',9-tetraones (3a-e), were designed and synthesized using the previously described ethyl 3-amino-4,9-dioxo-2,3,4,9-tetrahydrothieno[2,3-g]quinoline-3-carboxylate (1) as a starting material. All compounds were evaluated for their antiproliferative activity against a panel of representative liquid and solid human tumor cell lines and exhibit IC(50) values in the micromolar/submicromolar range. Series 2 displayed higher cytotoxicity than did series 3. The nature of the substituents on both imidazoline and triazinane N1 nitrogen markedly affected the activity profile of these series. Spectrophotometric and fluorescence measurements as well as unwinding assays performed on the most cytotoxic compounds, 2c, 2g, and 2k, showed that they are nonintercalative DNA agents and inhibit the catalytic activity of Topo II in a concentration-dependent mode. 2g was the most active Topo II inhibitor with activity levels comparable to those of VP-16.