1. Metabolites of the antimycobacterial agent 4-deoxo-3,4-[2-spiro-(N-isobutyl-4-piperidyl)]-(1H)-imidazo-(2,5-dihydro )- rifamycin S (rifabutin) were isolated from human urine after administration of a single oral dose of the drug. Some of these metabolites were identified by direct inlet mass spectrometry, 1H-n.m.r. spectrometry and, in two cases, by chromatographic comparison with reference compounds. 2. Unchanged drug, 25-O-deacetyl rifabutin and four other metabolites were identified in human urine. 25-O-Deacetyl rifabutin was the main urinary metabolite, other metabolites were characterized as oxidized, and oxidized-deacetylated derivatives. 3. Routes of metabolic transformation were: (a) deacetylation at position 25, (b) oxidation of methyl groups 31 or 32 or at the piperidine nitrogen, and (c) combination of these.

2D 31P/1H correlation spectroscopy has been used to assign individual phosphates in the self-complementary DNA fragment d(CGTACG), which is present in solution as a B-DNA type double helix. 31P chemical shift variation with temperature and with the base sequence has been studied, and evidence is given of a steric clash between purines in the minor groove, affecting the chemical shift of the innermost TpA phosphate. For this hexanucleotide there is a good correlation of "P shifts with both the helical twist (C,) and roll angle (Z2) Calladine-Dickerson sum functions. Different types of linear correlations, also including data from other nucleotides, have been performed. The results show that it is more appropriate to consider the change in chemical shifts as it occurs during melting rather than the actual "P shifts. An interesting correlation was also found between "P chemical shifts and P,H-3' coupling constants.

3lp N]V[R spectroscopy has been used to study the intercalation of the anthracyclines doxorubicin 1, daunorubicin 2, 4-demethoxydaunorubicin 3, morpholinodoxorubicin 4, methoxymorpholinodoxorubicin 5 and 9-deoxydaunorubicin 6 with the DNA fragment d(CGTACG)2. The individual phosphate resonances of the oligonucleotide were assigned in the free as well as in the intercalated species. The 3~p chemical shitt vadations allowed us to identify the intercalation sites, which resulted to be the same for all compounds i e. between the terminal CG base-pairs of the helix (two molecules of drug per duplex). The binding coustants, the dissociation rete constants and AG ~ values have been determined in different conditions of ionic strength and temperature. The kinetic constant (ko~) of the slow step of the anthracycline/duplex intercalation process has been directly measured by NOE exchange techniques. Binding constants depend on the ionic strength and on the self-association process so greatly, that their use to study by NMR anthracycline/DNA interactions is questionable. On the contrary, the kof r are not affected by these phenomena and presentan interesting trend for 1~, thus showing that the average lifetime of the drug in the intercalation site appears to be important for determining the cytotoxicity and the antimitotic activity.

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