Mode of action of 2-amino-6-chloro-1-deazapurine

Abstract

2-Amino-6-chloro-1-deazapurine is of interest as a purine analog with demonstrated in vivo activity against mouse leukemia L1210. That the active form of this agent is a nucleotide and that the nucleotide is formed by the action of hypoxanthine (guanine) phosphoribosyltransferase were shown by the facts that (a) L1210 cells deficient in hypoxanthine phosphoribosyltransferase were insensitive to the analog; (b) hypoxanthine, but not adenine, prevented the formation of the analog nucleotide by enzyme preparations containing activities of both hypoxanthine and adenine phosphoribosyltransferases; and (c) the cytotoxicity of the analog was prevented by hypoxanthine. The ribonucleoside of this analog was not toxic to cell cultures and hence is not phosphorylated or cleaved to the base. In intact HEp-2 cells and L1210 cells, the analog was metabolized to the nucleoside 5′-phosphate which accumulated to concentrations as high as 1000 nmoles/10 9 cells; no di- or triphosphates were detected. In HEp-2 cells, the analog reduced the pools of purine nucleotides with some accumulation of IMP. The toxicity of minimal inhibitory concentrations of the analog to HEp-2 cells could be prevented or reversed by 4(5)-amino-5(4)-imidazolecarboxamide (AIC); the toxicity of higher concentrations could be prevented or reversed by a combination of adenine and guanosine but not by AIC. The analog inhibited the incorporation of formate into purine nucleotides and into macromolecules at concentrations that had no effect on utilization of hypoxanthine; at higher concentrations the incorporation of hypoxanthine was inhibited. Low concentrations also inhibited the utilization of uridine and thymidine. The incorporation of hypoxanthine and AIC into guanine nucleotides, but not adenine nucleotides, was inhibited. These results indicate two sites of inhibition of the biosynthesis of purine nucleotides, the more sensitive one being on an early step of the pathway and the less sensitive one on the IMP-GMP conversion. That the blockade of de novo synthesis probably was at the site of feedback inhibition was indicated by the fact that the analog inhibited the accumulation of formylglycinamide ribonucleotide in azaserine-treated cells but did not inhibited the synthesis of 5′-phosphoribosyl 1-pyrophosphate. Comparative studies were performed with the related analog, 2-amino-6-chloropurine, which has been reported to produce a similar dual blockade of the purine pathway. This purine was less toxic than its 1-deaza analog; it produced a modest decrease in adenine nucleotides but increased pools of guanine nucleotides. IMP dehydrogenase from L1210 cells was not inhibited by the nucleotides of 2-amino-6-chloropurine or 2-amino-6-chloro-1-deazapurine at 1 mM comcentrations. The nucleotide of 2-amino-6-chloropurine was dechlorinated by AMP deaminase; the apparent K m was about the same as that of AMP and the V max was 21% that of AMP. The nucleotide of 2-amino-6-chloro-1-deazapurine was not a substrate. Conversion of the nucleotide of 2-amino-6-chloropurine to GMP by the action of AMP deaminase explains both the lower toxicity of 2-amino-6-chloropurine as compared to its 1-deaza analog and its capacity to increase pools of guanine nucleotides. Thus, of the two compounds the 1-deaza analog may be the more interesting as an antitumor agent because of the greater metabolic stability of its nucleotide.