Usnic acid has anti-cancer activity, however, low solubility and toxicity limit the potential. To investigate biological activity of usnic acid derivatives, enantiopure derivatives were synthesised by reacting usnic acid with ethylenediamine, which yielded one dimer product ((+)-1), and two tetra cyclic compounds ((+)-2 and (-)-2). The products were characterised with NMR, and evaluated in vitro in human colon cancer cell line Caco-2 by cell count, phase-contrast microscopy, MTT-assay, measurement of DNA content and cell cycle distribution. All compounds tested showed cytostatic effect in Caco-2 cells, but each compound had a distinct cellular effect. Compound (+)-1 showed anti-proliferative activity by increasing the percentage of cells in S-phase with 25% compared to the control. Compounds (+)-2 and (-)-2 induced paraptosis, but only compound (+)-2 modulated cell cycle distribution by accumulating cells in G2/M-phase by 47% and reduced DNA content by 60%. All compounds express interesting cellular and potential anti-proliferative activity.