Molecular Pharmacology, Vol 16, 981-996, Copyright © 1979 by the American Society for Pharmacology and Experimental Therapeutics

Metabolism and Metabolic Effects of 8-Aza-6-Thioinosine and its Rearrangement Product, N--D-Ribofuranosyl-[1,2,3]thiadiazolo[5,4-d]-pyrimidin-7-amine

¹ Kettering-Meyer Laboratory, Southern Research Institute, 2000 Ninth Avenue South, Birmingham, Alabama 35205

8-Aza-6-thioinosine (8-aza-MPR), synthesized as an analogue of 6-thioinosine (MPR) and expected to have improved metabolic properties, was found, as predicted, to be a substrate for adenosine kinase (EC 2.7.1.20) but not for purine nucleoside phosphorylase (EC 2.4.2.1). In contrast MPR is known to be converted to its nucleotide via the free base. Both 8-aza-MPR and its rearrangement product, N--D-ribofuranosyl[1,2,3]thiadiazolo[5,4-d]pyrimidin-7-amine (TPR), were cytotoxic; the 50% inhibitory concentrations for H.Ep. #2 cells were 1.8 µM and 0.14 µM, respectively. Like 8-aza-MPR, TPR was a substrate for adenosine kinase but not for purine nucleoside phosphorylase. H.Ep. #2 cells grown in the presence of 8-aza-MPR contained three new nucleotides: the 5'-monophosphate of 8-aza-MPR and the 5'-monophosphates of the - and -anomers of TPR. Cells grown in the presence of TPR contained only the latter two metabolites. These nucleotides result from the phosphorylation of 8-aza-MPR and TPR, the rearrangement of 8-aza-MPR and its phosphate to TPR and TPR phosphate, and the anomerization of TPR phosphate. No di- or tri-phosphates were detected with either precursor. The total amount of nucleotides derived from TPR was of the order of 2.5 µmoles/10⁹ cells, an amount 10-15 fold greater than the amount of nucleotides derived from 8-aza-MPR. There was no detectable incorporation of [³⁵S] from [³⁵S]-labeled-8-aza-MPR or TPR into polynucleotides. There was little or no desulfuration of 8-aza-MPR as determined by the absence of 8-aza-GTP, the principal soluble metabolite of 8-azainosine. Both 8-aza-MPR and TPR inhibited the synthesis of RNA and DNA but not of protein. Both reduced selectively the pools of guanine nucleotides in H.Ep. #2 cells. In addition 8-aza-MPR, like 8-azainosine, caused an accumulation of orotidine. The cytotoxicity of TPR to H.Ep. #2 cells was prevented by hypoxanthine; the cytotoxicity of 8-aza-MPR was prevented by a combination of hypoxanthine and uridine but not by either agent alone. These results indicate that a primary blockade produced by TPR was at some step of the synthesis of purine nucleotides, whereas 8-aza-MPR inhibited synthesis of both purine nucleotides (as a result of its conversion to TPR phosphates) and pyrimidine nucleotides (as a result of its conversion to 8-aza-MPR phosphate). Although both 8-aza-MPR and TPR are new biologically active nucleoside analogues, TPR is of particular interest because of its unusual structure and the evidence that it functions as an adenosine analogue despite the fact that its ribosyl moiety is attached not to a heterocyclic ring but to a primary amino group.

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ACKNOWLEDGMENTS We thank Mr. T. C. Herren, and Mr. W. J. White for radioassays and Dr. D. L. Hill and Dr. R. W. Brockman for helpful discussions.