Molecular mechanism of binding of pyrrolo(1,4)benzodiazepine antitumour agents to deoxyribonucleic acid—: Anthramycin and tomaymycin

Abstract

The synthesis of 3,3-dimethyl-4-oxo-3,4-dihydroquinoline (16), 3,3-dimethyl-4-oxo-2-methoxy-1,2,3,4-tetrahydroquinoline (17). and of 1 1a-S-pyrrolo(1, 4)benzodiazepine (21) as models to study the mechanism of action of the pyrrolo(1, 4)benzodiazepine antitumour antibiotics is described. Both 16 and 21 readily add nucleophiles to the imine bond but only 21, like the parent antibiotics, readily produces covalent attachment to DNA. The extent of binding of the pyrrolo(1, 4)benzodiazepine antibiotics to DNA, measured by suppression of ethidium fluorescence, is proportional to the antibiotic concentration and is partly reversed by a heat-denaturation-renaturation cycle. The extent of binding of the pyrrolo(1, 4)benzodiazepines to DNA is also promoted by lower pH (range 4.7 to 9) and higher temperatures (range 0–51°), and the DNA-antibiotic complex is stable to dialysis. There is no evidence that these antibiotics intercalate into DNA, assayed by calf thymus topoisomerase, but they are more reactive toward relaxed PM2-DNA than to supercoiled DNA. Examination of DNA binding of the antitumour antibiotics and their analogues to DNAs of different base composition and separately in conjunction with sequence specific binding agents showed little base preference for the binding. Reaction of the pyrrolo(1, 4)benzodiazepines with DNA produces neither depurination, assayed with endonuclease VI, nor strand scission. A free or potential carbinolamine or imine function at the 10, 11 positions in a benzo(1, 4)diazepine nucleus is an absolute requirement for DNA binding or for reaction with nucleophiles. These results with the native antibiotics and their analogues, in particular the N-acetyl compound 7 favor a molecular mechanism of action by acid-promoted addition of biological nucleophiles to the 10, 11 conjugated imine closely analogous to that proposed for the antitumour agent maytansine.