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In comparative tests of the capabilities of various analogs of doxorubicin and daunorubicin to augment oxygen consumption in microsome preparations from P388 mouse leukemia cells and from rat liver cells, we found statistically significant positive correlations between the Km values for microsomes from the two sources for parent compounds and analogs containing morpholinyl, cyanomorpholinyl- or imino-substitutions. Km values ranged from 0.015 to 1.3 mM, or about 90-fold for all analogs tested. Thus, rat liver microsomes are predictive for this characteristic in P388 microsomes. Examinations of the relationships between Km values from either source and ED50 values for cytotoxicity of the compounds against the P-388 cells also showed statistically significant positive correlations. However, the ED50 values encompassed a range from 0.00020 to 0.22 microM, or about 1000-fold. Therefore, additional mechanism(s) of action besides toxic oxygen radical formation must explain the extremely high cytotoxicity of the cyanomorpholinyl anthracyclines compared with their parent drugs.

The synthesis, characterization, and biochemical evaluation of 1-beta-D-ribofuranosylnaphtho[2,3-d]imidazole-4,9-dione (3), 2-beta-D-ribofuranosylnaphtho[2,3-d]pyrazole-4,9-dione (6), and 2-beta-D-ribofuranosylnaphthol[2,3-d]triazole-4,9-dione (9) are reported. These quinone nucleosides and the corresponding quinone heterocycles were tested as inhibitors of purine nucleotide biosynthesis in Ehrlich ascites cells. The nucleosides 3 and 9 and naphtho[2,3-d]imidazole-4,9-dione were effective inhibitors of hypoxanthine phosphoribosyltransferase.

A general reaction of glycosyl cyanides with liquid hydrogen sulfide in the presence of 4-dimethylaminopyridine to provide the corresponding glycosylthiocarboxamides is described. These glycosylthiocarboxamides were utilized as the precursors for the synthesis of 2-D-ribofuranosylthiazole-4-carboxamide and 2-beta-D-ribofuranosylthiazole-5-carboxamide (23). The structural modification of 2-beta-D-ribofuranosylthiazole-4-carboxamide (12) into 2-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)thiazole-4-carboxamide (15), 2-beta-D-ribofuranosylthiazole-4-thiocarboxamide (17), and 2-(5-deoxy-beta-D-ribofuranosyl)thiazole-4-carboxamide (19) is also described. These thiazole nucleosides were tested for in vitro activity against type 1 herpes virus, type 3 parainfluenza virus, and type 13 rhinovirus and an in vivo experiment was run against parainfluenza virus. They were also evaluated as potential inhibitors of purine nucleotide biosynthesis. It was shown that the compounds (12 and 15) which possessed the most significant antiviral activity were also active inhibitors (40-70%) of guanine nucleotide biosynthesis.

2-beta-D-Ribofuranosylselenazole-4-carboxamide, a selenazole analog of the antitumor agent Tiazofurin, is severalfold more cytotoxic to murine tumor cells in culture than Tiazofurin. Like Tiazofurin, the cytotoxicity of the selenazole analog is reversed by exogenous guanosine, and both nucleosides specifically inhibit IMP dehydrogenase activity in cultured P388 cells. The dose-dependency for this inhibition correlates with the relative cytotoxicities of both drugs, indicating that a more potent inhibition of IMP dehydrogenase by the selenazole analog is primarily responsible for its increased cytotoxicity. The specific inhibition of the isolated enzyme by potential metabolites of the selenazole analog is discussed.

Starting with AICA ribonucleoside the following nucleosides were prepared. Methyl 5-amino-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)imidazole-4-carboxylate (5) was converted into methyl 5-chloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)imidazole-4-carboxylate (6) via diazotization in the presence of cuprous chloride. Similarly, 5-amino-1-(2,3,5-tri-O-acetyl-beta-D-ribofuanosyl)imidazole-4-carbonitrile (9) was converted into 5-chloro-, 5-bromo-, and 5-iodo-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)imidazole-4-carbonitrile derivatives. These 5-halogenated imidazole nucleosides were treated with several nucleophiles such as ammonia, hydroxylamine, and hydrogen sulfide to provide, respectively, 5-haloimidazole-4-carboxamide, 5-haloimidazole-4-carboxamidoxime, and 5-haloimidazole-4-thiocarboxamide ribonucleosides. 5-Chloro- or 5-bromo-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)imidazole-4-carbonitrile was treated with potassium hydrosulfide to yield 5-mercapto-1-beta-D-ribofuranosylimidazole-4-thiocarboxamide (16). The catalytic reduction of 5-chloro- or 5-bromo-1-beta-D-ribofuranosylimidazole-4-carboxamidoxime provided 1-beta-D-ribofuranosylimidazole-4-carboxamidines as their hydrochloride and hydrobromide salts, respectively. These nucleosides were tested for in vitro antiviral, antifungal, and antibacterial activity. The 5-halo analogues of 1-beta-D-ribofuranosylimidazole-4-carboxamide showed significant antiviral activity whereas compound 16 was found inhibitory to fungi.

Treatment of doxorubicin with formaldehyde and NaCN afforded the N-(cyanomethyl) derivative as a stable alpha-cyanoamine with but moderate antitumor activity in mice, although it was prototypal to the intensely potent alpha-cyanomorpholine derivative. 2-Methoxyacetaldehyde and NaCN afforded the N-(2-methoxy-1-cyanoethyl) derivative as an open-chain analogue of the cyanomorpholine. This analogue underwent rapid hydrolysis to doxorubicin and appeared to act as a prodrug, giving increased antitumor efficacy although with decreased potency. N-(Carboxymethyl)daunorubicin was a highly water-soluble but inactive analogue, synthesized by N-alkylation with ethyl iodoacetate and saponification. The similar N-alkylation of N-(cyanomethyl) daunorubicin demonstrated the combining of N-alkyl chains having different functional substituents.

The susceptibility of 3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin to hydrolysis at pH 7, 4, and 2 has been compared with that of the typically stable morpholine analogue. At pH 7, 74% of the cyanomorpholine was unchanged after 24 h at room temperature, but at pH 2 only 10% remained. Products identified were aglycon (8%) and N-(2-hydroxyethyl)doxorubicin (7%). Most of the losses were to unidentified polar products not eluted from HPLC. Authentic hydroxyethyl was synthesized from doxorubicin by reductive alkylation with glycolaldehyde. Antitumor potency was comparable to that of doxorubicin rather than of cyanomorpholine.

The antiviral activity of the synthetic nucleoside, Virazole (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), against measles virus in Vero cell cultures was substantially reversed by xanthosine, guanosine, and to a slightly lesser extent by inosine. Virazole 5'-phosphate was subsequently found to be a potent competitive inhibitor of inosine 5'-phosphate dehydrogenase (IMP:NAD(+) oxidoreductase, EC 1.2.1.14) isolated from Escherichia coli (K(m) = 1.8 x 10(-5) M) with a K(i) of 2.7 x 10(-7) M. Guanosine 5'-phosphate (GMP) was a competitive inhibitor of this enzyme with a K(i) of 7.7 x 10(-5) M. Virazole 5'-phosphate was similarly active against IMP dehydrogenase isolated from Ehrlich ascites tumor cells, with a K(i) of 2.5 x 10(-7) M. The K(m) for this enzyme was 1.8 x 10(-5) M, and the K(i) for GMP was 2.2 x 10(-4) M. These results suggest that the antiviral activity of Virazole might be due to the inhibition of GMP biosynthesis in the infected cell at the step involving the conversion of IMP to xanthosine 5'-phosphate. This inhibition would consequently result in inhibition of the synthesis of vital viral nucleic acid.