Analogues of the previously reported potent cytotoxic spiro[(dihydropyrazine-2,5-dione)-6,3'-(2',3'-dihydrothieno[2,3-b]naphtho-4',9'-dione)] derivatives (3, 3') were prepared to explore new structural requirements at the diketopiperazine domain for the cytotoxic activity. The in vitro activity was evaluated against the MCF-7 human breast carcinoma and SW 620 human colon carcinoma cell lines. The 4-[(2-N, N-dimethyl)amino]ethyl (6i), and the 4-(2-pyrrolydin)ethyl (6l) derivatives emerged as the most potent compounds of this series, with a cytotoxic activity comparable to that of doxorubicin. These compounds, in both racemic and pure enantiomeric forms, showed also a high efficacy in cell lines resistant to doxorubicin (MCF-7/Dx) and in cell lines that were highly resistant to treatment with doxorubicin, such as HEK-293 (kidney), M-14 (melanoma), and HeLa (cervical adenocarcinoma) human cell lines. In addition, the effects on growth and cell cycle progression in CaCo-2 cell line (colon adenocarcinoma) and DNA-binding properties were investigated.