Martin Napierkowski, Tom Schöne, Siva Sankar Murthy Bandaru, Jo Judernatz, Lukas Schulig, Louis Schmidt, Carola Schulzke, Patrick J. Bednarski

• The biological properties of pentathiepins have been intensively studied in recent years. Although the proposed mechanism of action requires activation by intracellular thiols, the dependence of activity on the stability of pentathiepins towards glutathione (GSH) has not been directly investigated. Here, we determined the structure‐related stability of six different pentathiepins with four different scaffolds in the presence of GSH by using reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and UV‐vis spectroscopy over a wide range of GSH concentrations. We found significant differences in compound stability depending on the pentathiepin scaffold; these differences were reflected in their cytotoxic activities. However, we found no substantial differences in their inhibition of glutathione peroxidase 1 (GPx‐1). While the intact pentathiepin ring is necessary for the antiproliferative activity of pentathiepins, the depletion of intracellular GSH content with dl ‐buthionine‐( S , R )‐sulfoximine (BSO) led to a significant increase in cytotoxicity of the tested substances. In view of the increased cytotoxicity following artificial GSH depletion, this calls into question the sole role of GSH in the intracellular activation mechanism.