Identification and characterization of in vitro metabolites of 2-[2'- (dimethylamino)ethyl]-1,2-dihydro-3h-dibenz[de,h]isoquinoline-1,3-dione (azonafide)

Azonafide (2-[2'-(dimethylamino)ethyl]-1,2-dihydro-3H-dibenz[de, h]isoquinoline-1,3-dione) is the parent of a new series of anthracene- containing antitumor agents. Its structure is based on amonafide but lacks a primary amine and has an anthracene chromophore rather than a naphthalene chromophore. Using a rat liver cytosol incubation and HPLC/MS detection, we have identified four metabolites resulting from in vitro metabolism of azonafide. These alkyl-modified derivatives include a mono- and a di-N'- desmethyl metabolite, an N'-oxide metabolite, and a carboxylic acid metabolite. Purified samples of these metabolites were analyzed for cytotoxic activity using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium vital dye (mitochondrial reductase) assay and for inhibition of topoisomerase II (TOPO II) using a cell-free enzymatic system. Each metabolite had decreased cytotoxicity relative to azonafide with the following relative potencies in descending order the mono-N'-desmethyl metabolite, di-N'- desmethyl metabolite, the N-oxide metabolite, and the carboxylic acid metabolite. Similarly, the N'-desmethyl metabolites retained TOPO II inhibitory activity but with lower potency then azonafide. The N-oxide and carboxylic acid metabolites did not inhibit TOPO II at 0.05 and 0.5 μg/ml, respectively. Thus, metabolism of azonafide by rat liver cytosol represents a detoxification pathway rather than a bioactivation scheme for this DNA intercalator.