Biochemical properties of the nucleoside of 3-amino-1,5-dihydro-5-methyl-1,4,5,6,8-pentaazaacenaphthylene (NSC-154020).

Abstract A study of 3-amino-1, 5-dihydro-5-methyl-l-β- d -ribofuranosyl-1, 4, 5,6,8- pentaazaacenaphthylene (NSC-154020), a “tricyclic” nucleoside with activity against certain experimental tumors, was undertaken to determine if it differed in biochemical properties from structurally related7- deazapurine nucleosides with established biological activity, such as sangivamycin. In cultured L1210 cells, [ 14 C-methyl]-NSC-154020 was converted to a single metabolite with the properties of a 5'-monophosphate as shown by (a) similarity to AMP in migration on paper chromatograms and in retention time when subjected to high pressure liquid chromatography (h.p.l.c.) on an ion exchange column and (b) conversion to a compound with the properties of NSC-154020 upon treatment with alkaline phosphatase or 5'-nucleotidase. In cultured H.Ep.-2 cells, the principal metabolite of NSC-154020 was also the monophosphate. H.Ep.-2 cells contained in addition a variable amount of a second metabolite which also had the retention time (on h.p.l.c. analysis) of a monophosphate and which was converted by the action of alkaline phosphatase or 5'-nucleotidase to a compound that migrated like NSC-154020 upon chromatography in three solvent systems. This second metabolite is as yet unidentified. In crude extracts of L1210 cells, addition of adenosine or 6-(methylthio)purine ribonucleoside decreased the phosphorylation of NSC-154020. NSC-154020 was a substrate for adenosine kinase 110-fold purified from H.Ep.-2 cells; the K m was 215 μM and the V max was 1.8 times greater than that of adenosine. No 14 C from labeled NSC-154020 was found in the polynucleotides of either H.Ep.-2 cells or L1210 cells grown for 24 hr in the presence of the labeled nucleoside. Several different studies failed to reveal any selective sites of action for NSC-154020. In cultured L1210 cells it inhibited synthesis of DNA, RNA and protein and reduced ribonucleotide pools, but with little selectivity. The incorporation of [ 14 C]formate into polynucleotides was inhibited more severely than that of hypoxanthine; this is evidence for a blockade of purine synthesis de novo , an effect also produced by many other analogs of purines and nucleosides. Sangivamycin produced generally similar inhibitions of incorporation of formate and hypoxanthine. However, the cytotoxicity of NSC-154020 and sangivamycin to L1210 cells was not prevented or reversed by 5-amino-4-imidazolecarboxamide (AIC), adenine, guanine, hypoxanthine, uridine, or AIC + uridine; therefore, inhibition of de novo synthesis of purine and pyrimidine nucleotides is not a primary site of action of these compounds. Although the loci of action of NSC-154020 are not yet defined, the fact that it is not metabolized to polyphosphates indicates that its mechanism of action probably differs significantly from those of the related compounds, tubercidin and sangivamycin, which are converted to polyphosphates and incorporated into RNA and DNA.