Pyrrolizinones represent an interesting class of compounds with varied degrees of structural complexity and pharmacological activity. Among these, 9H-pyrido[2,3-b]pyrrolizin-9-one, tripentone analogs, recently reported by us, showed significant antiproliferative activity against human tumor cell lines, inducing apoptosis and not affecting viability of Caco-2 differentiated in normal intestinal-like cells. Considering their interesting biological activity, their 5H-pyrido[3,2-b]pyrrolizin-5-one analogs were efficiently synthesized in good to excellent yields (61-91%). All tripentone derivatives were tested to assess their cytotoxicity against two human tumor cell lines, HCT-116 (human colorectal carcinoma) and MCF-7 (human breast cancer). The most active derivatives, with IC50 ranging from 0.11 to 16.11 μM, did not affect viability of Caco-2 differentiated in normal intestinal-like cells, suggesting tumor cells as the main target of their cytotoxic action. The same compounds, further investigated, showed that they did not exert necrotic effects, while induced a clear shift of viable cells towards early apoptosis confining the cells in the mitotic phases.