Thirty-three benzophenanthridine alkaloid derivatives (1a–1u and 2a–2l) were synthesized, and their cytotoxic activities against two leukemia cell lines (Jurkat Clone E6-1 and THP-1) were evaluated in vitro using a Cell Counting Kit-8 (CCK-8) assay. Nine of these derivatives (1i–l, 2a, and 2i–l) with IC50 values in the range of 0.18–7.94 µM showed significant inhibitory effects on the proliferation of both cancer cell lines. Analysis of the primary structure–activity relationships revealed that different substituent groups at the C-6 position might have an effect on the antileukemia activity of the corresponding compounds. In addition, the groups at the C-7 and C-8 positions could influence the antileukemia activity. Among these compounds, 2j showed the strongest in vitro antiprolifera-tive activity against Jurkat Clone E6-1 and THP-1 cells with good IC50 values (0.52 ± 0.03 µM and 0.48 ± 0.03 µM, respectively), slightly induced apoptosis, and arrested the cell-cycle, all of which sug-gests that compound 2j may represent a potentially useful start point to undergo further optimization toward a lead compound. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.