Spiro[(dihydropyrazin-2,5-dione)-6,3’-(2’,3’-dihydrothieno[2,3-b]naphtho-4’,9’-dione)]-Based Cytotoxic Agents: Structure-Activity Relationship Studies on the Substituent at N4-Position of the Diketopiperazine Domain

Analogues of the 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.Preliminary studies on cell cycle progression in CaCo-2 cell line showed that these derivatives markedly prolonged the S phase of the cell cycle inducing delay of cell cycle progression in responsive cells and cellular proliferation inhibition. All these data revealed significant differences in the cytotoxic behavior of these compounds compared to doxorubicin. In addition, STD-NMR spectroscopy investigation performed on these compounds demonstrated that they interact with DNA with a dual binding mode: intercalative for the tricyclic planar core and external considering the side-chain moiety