Activities of novel nonglycosidic epipodophyllotoxins in etoposide-sensitive and -resistant variants of human KB cells, P-388 cells, and in vivo multidrug-resistant murine leukemia cells.

Previous structure-activity studies of the antitumor compound etoposide (VP-16) have suggested that replacement of the glycoside moiety could afford therapeutically active analogues with different biochemical determinants for cellular accumulation and drug resistance. In the present report, 10 analogues of VP-16 in which the glycosidyl moiety was replaced with alkyl or arylamino substituents exhibited 5-10-fold better binding affinity for topoisomerase II/DNA complex in human KB cells. A similar increase in the binding affinity was observed in an isolated-nuclei model. The analogues displayed greater or comparable potency to VP-16 in cell growth-inhibition studies and were less affected by cell membrane-associated drug resistance mechanisms, as exemplified by overexpressions of P-glycoprotein multidrug-resistance gene or multidrug resistance-associated protein. Interestingly, in animal studies, analogues least affected by the membrane transport-deficiency phenotypes exhibited low therapeutic index values, thus suggesting that highly efficient modulation of cellular membrane transport defects could perturb the selectivity of antitumor agents for cancer cells. This report also suggests a new method of quantifying drug-induced protein-linked DNA breaks by graphically determining the apparent dissociation-inhibition constant (Kdi) for the inhibitors.

Oxidative coupling of mithramycin and hydroquinone catalyzed by copper oxidases and benzoquinone. Implications for the mechanism of action of aureolic acid antibiotics.

The copper oxidases human ceruloplasmin and Polyporous anceps laccase catalyze the oxidative coupling of mithramycin (1) and its aglycone chromomycinone (2) with p-hydroquinone to form new mithramycin-hydroquinone (3) and chromomycinone-hydroquinone adducts (4), respectively. Similar adducts could be formed by the nonenzymatic mimic of this reaction using benzoquinone and these aureolic acids in buffer solutions. FABMS of 3 indicated that the hydroquinone moiety was attached to the aureolic acid aglycone. Acid hydrolysis of 3 yielded a compound with the same chromatographic and spectroscopic characteristics as 4. Structure elucidation of 4 by NMR and MS revealed that the hydroquinone was attached to the C-5 position of the aglycone. NMR evidence indicated that 4 consisted of a mixture of ortho-substituted biphenyl rotamers. The mechanism of the copper oxidase catalyzed adduct formation reaction is presumed to involve radical formation through hydrogen removal at the 8-phenolic position, radical isomerization, and coupling with semiquinone radical also formed during enzymatic and nonenzymatic incubations. Identification of the covalent-hydroquinone adduct provides evidence that aureolic acid antibiotics can be metabolically converted to reactive radical intermediates, and it establishes the C-5 position of aureolic acid as an enzymatically reactive site. Unlike mithramycin, the mithramycin-hydroquinone adducts was inactive in the in vivo P388 leukemic antitumor test system.