Sampangine is an azaoxoaporphine alkaloid with interesting biological activities. Elucidating the mode of action of sampangine is a topic of continuous research. Recently reported cell-based data have indicated heme-dysfunction and subsequent reactive oxygen species production as being responsible for the biological activity of the natural product. By using an in vitro biochemical assay the ability of sampangine to produce reactive oxygen species was confirmed. The production of reactive oxygen species occurred upon mild chemical reduction of sampangine in absence of any cellular components. In an additional structure-activity-relationship study, utilizing synthesized analogs of sampangine, we identified the 1,4-iminoquinone scaffold as the key motif for the observed reactive oxygen species production. To assess the ability of sampangine to induce DNA damage, the direct binding of sampangine to calf thymus-DNA was measured using UV-Visible spectroscopy. No DNA binding was observed when sampangine was tested against calf thymus -DNA up to a ratio of 1:100. This observation rules out the direct involvement of sampangine in DNA binding and damage.