Characterization of Covalent DNA Binding of Morpholino and Cyanomorpholino Derivatives of Doxorubicin

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Abstract
Background : The doxorubicin analogues cyanomorpholino doxorubicin (MRA-CN) and morpholino doxorubicin (MRA) were synthesized in an attempt to avoid the cardiotoxicity and drug resistance of doxorubiricn therapy. MRA-CN forms interstrand DNA cross-links without requiring microsomal metabolic activitation in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to form a product that alkylates DNA, but MRA requires metabolic activation Alkyalation produces DNA cross-links, which are associated with potentiation of the cytotoxicity of some drugs. Purpose: Our purpose was to study the DNA binding of MRA-CN and MRA with and without metabolic activation in order to better understand the mechanisms for cross linking DNA. Methods: We used ( 3 MRA and ( 3 H) MRA-CN, with the 3 H labeled at C-2 and C-6 of the morpholino ring. MRA (10 n M) was incubated with human liver microsomes with or without NADPH to measure DNA binding. In addition, a filter elution assay was used to determine the nature and extent of drug binding to DNA in the human ovarian carcionma cell line ES-2. We studied the appearence of interstrand crosslinks versus total DNA adducts in pBR322 plasmid DNA incubated with 100 n M MRA-CN in cell free medium and then subjected to denaturation and agarose gel electropohoresis. Results: Regardless of the extracellular concentration of the drug (1– 85% of intracellular MRA-CN was covalently bound to DNA, and the total amount to DNA, and the total amount of drug bound to DNA was proportional to extracellular drug concentration. No covalent binding of MRA to DNA was found in cells exposed to 10 n M MRA alone for 2 hours. In contrast, 10% of the intracellular drug was bound to DNA iof the cells were exposed to MRA preincubated with human liver microsomes and NADPH. The percentages of plasmide containing at least one interstrand cross-links rose from 35% at 15 minutes to 92% at 2 hours. We estimate that eight molecoules of MRA-CN were adducted per molecule of p BR322 DNA (or one drug adduct per 545 base pairs), with a minimum of 12% of the adducts forming interstrand cross links. Conclusions: These result suggest that the carbons at positions 2 and 6 of the morpholino ring of both MRA-CN and the activated metabolite of MRA are retained in the drug-DNA adduct. They also indicate that the formation of interstrand DNA cross links by MRA-CN is preceded by formation of drug adducts to a single strand of DNA. (J Natl Cancer Inst 1587–1592, 1992)

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