In the present work we examined novel doxorubicin (DOX) analogues with a formamidino group containing a cyclic amine moiety such as morpholine (DOXM) and hexamethyleneimine (DOXH). Both analogues were compared with DOX for their cytotoxicity, DNA damaging properties, cellular uptake and lipid peroxidation activity. We also have made an attempt to find a biochemical explanation for lower cardiotoxicity of DOXM and DOXH in vivo. As some cellular oxidoreductases such as NADH dehydrogenase, NADPH cytochrome P450 reductase and xanthine oxidase are believed to contribute in the metabolic activation of anthracyclines and to participate in the generation of reactive oxygen species by these drugs we have studied the ability of DOX and its analogues to stimulate NADH and NADPH oxidation in a cell-free systems containing mentioned above oxidoreductases.

The anthracycline derivatives studied exhibited high cytotoxic activity toward L1210 cells. The most cytotoxic drug was DOX (ED₅₀ = 0.018 μM), its derivative DOXM exhibited very similar activity, whereas DOXH was about 5 times less active then DOX.

The differences in cytotoxicity do not correlate with the uptake of anthracyclines by treated cells. The most cytotoxic drugs DOX and DOXM vary significantly in cellular uptake. DOXH which is the least cytotoxic drug was accumulated by L1210 cell with very similar efficiency like observed for DOX.

The anthracycline tested differ in their capacity to cause DNA breaks. DOX induced DNA breaks in the manner typical for DNA topoisomerase II poisons. Its effect on DNA was reduced by ICRF-187 which is a catalytic inhibitor of topoisomerase II. ICRF-187 had little effect on DNA breaks induced in L1210 cells by DOXM and DOXH.

The rate of NADH oxidation catalyzed by NADH dehydrogenase in the presence of anthracyclines was different for all tested compounds. DOX was the most active oxidizer whereas its analogues DOXM and DOXH induced NADH oxidation at significantly lower rate and this effect was particularly pronounced for DOXM. Similar results were obtained in the case NADPH oxidation catalyzed by NADPH cytochrome P450 reductase but in this assay only DOXM was evidently less active then the parent anthracycline. In the xanthine system introduction of the formamidino group containing morpholine ring to the anthracycline molecule produced different effect then observed in other oxidoreductases assays. It was found that DOXM was clearly the most potent oxidizer and the rate of NADPH oxidation in the presence of this drug was higher by about 70% when compared with other anthracyclines.

DOX and DOXH produced lipid peroxidation in rat cardiomyocytes with very similar yield, whereas DOXM was significantly less active.

CONCLUSIONS: 1 - topoisomerase II is not primary cellular target for DOXM and DOXH; 2 - the presence of heterocyclic rings in daunosamine moiety changes affinity of some cellular oxidoreductases to anthracyclines and their capacity to produce lipid peroxidation.

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1. Cytotoxic and proapoptotic in vitro activity of two novel flavone complexes of ruthenium(II)
2. DNA Damage and Lipid Peroxidation in L1210 Cells by Formamidino Derivatives of Doxorubicin