Daunorubicin (DRB) and its two analogues containing a trisubstituted amidino group at the C-3' position of the daunosamine moiety have been compared regarding their cytotoxic activity, cellular uptake, subcellular localization and DNA damaging properties. An analogue containing in the amidino group a morpholine moiety (DRBM) as well as an analogue with a hexamethyleneimine moiety (DRBH), tested against cultured L1210 cells, exhibited lower cytotoxicity then DRB. The decrease of cytotoxic activity was not related to cellular uptake and subcellular localization of drugs. Although all tested drugs were active in the induction of DNA breaks and DNA-protein crosslinks, they differed in the mechanism of induction of DNA lesions. DRB produced DNA breaks mediated solely by topoisomerase II, whereas DRBM and DRBH induced two types of DNA breaks by two separate processes. The first is related to the inhibition of topoisomerase II and the second presumably reflects a covalent binding of drug metabolites to DNA. It is hypothesized that the replacement of the primary amino group (-NH(2)) at the C-3' position of the daunosamine moiety by a trisubstituted amidino group (-N=CH-NRR) may be a route to the synthesis of anthracycline derivatives with enhanced ability to form covalent adducts to DNA.

Hepatitis C virus (HCV) infections represent one of the major and still unresolved health problems because current therapy is effective in only 50-80% of cases, depending on viral genotype. A large group of amidinoanthracyclines, with decreased acute toxicity and cardiotoxicity compared to the parent antibiotics, was tested in a high-throughput fluorometric HCV helicase assay. Here, we report the selection of more than 50 potent inhibitors of helicase activity that inhibit the enzyme with IC(50) values in the range of 0.03- 10 mum; four of these compounds are the most potent inhibitors of helicase activity described in the literature. The activity of these inhibitors is highly dependent on their chemical structure, mainly on the substituent at the amidino carbon atom and on the orientation of the hydroxyl group at the 4 inch position of the daunosamine moiety. The most effective compounds act not solely via intercalation into the double-stranded DNA substrate, but also compete with the enzyme for access to the substrate, impeding formation of the active helicase complex. Selected amidinoanthracyclines were tested in the subgenomic HCV replicon system. These experiments confirmed the antiviral activity of two selected inhibitors (EC(50) values below 0.2 mum with selectivity indices of 19 and 33) and proved that they may be considered as potential anti-HCV drugs.

A broad spectroscopic characterization, using ultraviolet-visible (UV-vis) and Fourier transform infrared absorption as well as Raman scattering, of two commonly used anthracyclines antibiotics (DOX) daunorubicin (DNR), their epimers (EDOX, EDNR) and ten selected analogs is presented. The paper serves as a comprehensive spectral library of UV-vis, IR and Raman spectra of anthracyclines in the solid state and in solution. The particular advantage of Raman spectroscopy for the measurement and analysis of individual antibiotics is demonstrated. Raman spectroscopy can be used to monitor the in vitro uptake and distribution of the drug in cells, using both 488 nm and 785 nm as source wavelengths, with submicrometer spatial resolution, although the cellular accumulation of the drug is different in each case. The high information content of Raman spectra allows studies of the drug-cell interactions, and so the method seems very suitable for monitoring drug uptake and mechanisms of interaction with cellular compartments at the subcellular level.

Anthracycline antibiotics display genotoxic activity towards cancer cells but their clinical utility is limited by their cardiac and vascular toxicity. The aim of this study was to develop a Raman-based methodology to study the nuclear accumulation of anthracyclines in the endothelium. For this purpose bimodal confocal Raman and fluorescence imaging was used to monitor cellular composition changes as a result of anthracycline exposure on endothelial cells (EA.hy926), and nuclear drug accumulation, respectively. Simultaneously effects of anthracyclines on endothelium viability were investigated by caspases-3 and -7 and MTT assays. We demonstrated that nuclear accumulation of DOX and EDOX was similar; however, EDNR accumulated in endothelial nuclei at concentrations 10 times higher than DNR. In turn, epimers of DOX or DNR were both consistently less toxic on the endothelium as compared to their congeners as evidenced by MTT and caspase assays. In summary, bimodal Raman and fluorescence-based nucleus profiling proves to be a valuable tool to study structure-activity relationship of nuclear accumulation and toxicity of anthracyclines in endothelium.

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