interactions of novel morpholine and hexamethylene derivatives of anthracycline antibiotics with dna doxorubicin dox daunorubicin drb epidoxorubicin edox and their analogues with a 3nh2 group in daunosamine form a covalent bond with a 2nh2 group of guanine via a methylene group from formaldehyde ch2o it is assumed that a schiff base type intermediate is formed between ch2o and the 3nh2 group in the reaction this reaction is supposed to occur in the cell new analogues of anthracyclines with formamidine functionality bound to c3 of daunosamine and containing the bulky morpholine drbm doxm and edoxm or hexamethyleneimine rings attached are studied in our laboratory these substituents decrease the association of the drugs to dna and potentially hinder the formation of schiff baseintermediates our experiments indicate that the formation of the covalent complexes by drb dox and edox under these conditions is confirmed by a high enhancement 1740x of the inhibition of overall rna synthesis by e coli rna polymerase on t7 dna drbm and doxm exhibit a lower enhancement of the inhibition by ch2o 713x the other analogues show a 163x increase of inhibition hence their covalent binding is lower than that of the parent compounds these conclusions are confirmed by spectrophotometric estimations following removal of noncovalently associated drugs electrophoretic analysis of drugdna complexes formed in the presence of ch2o indicates that drbm and doxm as their parent compounds induce labile crosslinks in dna comparison of the results obtained at the subcellular level with cytotoxicity estimations indicates that there is a correlation between cytotoxicity of the anthracyclines on l1210 cells and transcriptional template activity of drugdna complexes formed in the presence of ch2o r 064 n 9 these data confirm a notion that covalent attachment of anthracyclines to dna is an essential event leading to cytotoxicity