Based on the marine natural products piperafizine B, XR334, and our previously reported compound4m, fourteen novel 3,6-diunsaturated 2,5-diketopiperazine (2,5-DKP) derivatives (1,2,4–6,8–16), together with two known ones (3and7), were designed and synthesized as anticancer agents against the A549 and Hela cell lines. The MTT assay results showed that the derivatives6,8–12,and14had moderate to good anticancer capacities, with IC50values ranging from 0.7 to 8.9 μM. Among them, compound11,with naphthalen-1-ylmethylene and 2-methoxybenzylidene functions at the 3 and 6 positions of 2,5-DKP ring, respectively, displayed good inhibitory activities toward both A549 (IC50= 1.2 μM) and Hela (IC50= 0.7 μM) cancer cells. It could also induce apoptosis and obviously block cell cycle progression in the G2/M phases in both cells at 1.0 μM. The electron-withdrawing functions might not be favorable for the derivatives with high anticancer activities. Additionally, compared to piperafizine B and XR334, these semi-N-alkylated derivatives have high liposolubilities (>1.0 mg mL−1). Compound11can be further developed, aiming at the discovery of a novel anticancer candidate.