Mechanistic aspects of DNA damage by morpholinyl and cyanomorpholinyl anthracyclines

Morpholino and cyanomorpholino derivatives of the anthracycline antitumor antibiotics, Adriamycin (ADM) and daunomycin (DNM), differ from their parent compounds in potency, cross-resistance and other biological properties. We therefore investigated the DNA interactions of several derivatives of ADM and DNM, including iminomorpholinoadriamycin, oxazolocyanomorpholinoadriamycin, (OCADM), and cyanomethyladriamycin (CMeADM). This work complements previous studies on morpholinodaunomycin (MoDNM), morpholinoadriamycin (MoADM), and cyanomorpholinoadriamycin (CNMoADM). As described in this work, unscheduled DNA synthesis is induced by iminomorpholinoadriamycin, 5-iminocyanomorpholinoadriamycin (ICADM), OCADM, and CMeADM but not by ADM and DNM. In addition, we observed the induction of DNA single-strand breaks by ICADM, OCADM, and CMeADM in V79 cells; previous work has shown that ADM and DNM but not MoDNM induce single-strand breaks in these cells. DNA cross-links were induced by ICADM at high concentrations (greater than 50 nM) but not by MoADM, OCADM, and CMeADM as described herein. In previous investigations, we observed that CNMoADM and cyanomorpholinodaunomycin (CNMoDNM) but not ADM, DNM, and MoDNM induce DNA cross-links. CNMoADM and CNMoDNM are the most potent cytotoxic derivatives of ADM and DNM and have their cross-linking potential in common. The fact that CNMoADM and CNMoDNM (as well as ICADM at higher concentrations) all contain an alpha-cyanamide functionality and also yield cross-links suggests that this functionality is important for cross-linking. The CN moiety in [14C]N-MoADM was shown to be lost during the reaction with DNA and rat liver S9 (but not with buffer), which suggests that adduction via CN displacement is possible. On the other hand, OCADM and CMeADM both contain the alpha-cyanamine functionality but have a modified morpholino ring and do not yield cross-links. This suggests that an intact morpholino ring, which is metabolized to a reactive form, is necessary for cross-linking. A number of metabolites from MoDNM treated with rat liver S9 mix were isolated. These products, when analyzed by gas chromatography-mass spectrometry, are consistent with metabolism in the morpholino ring; however, definitive structural assignments could not yet be made.