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6-Thio-2'-deoxyguanosine (T-dGuo) has been reported to be both phosphorylated by deoxycytidine kinase and converted to 6-thioguanine by purine nucleoside phosphorylase (PNP). Combination of T-dGuo with an inhibitor of PNP would be expected to generate the 5'-triphosphate of T-dGuo and limit or prevent the formation of 6-thioguanosine triphosphate. Because the incorporation of 6-thioguanine into DNA is believed to be primarily responsible for the antitumor activity of the thiopurines, this treatment might result in enhanced activity against certain tumors, particularly those of T-cell origin. We have evaluated the metabolic basis of this strategy by examining the effects of 9-benzyl-9-deazaguanine (BDG), a potent inhibitor of PNP, on the metabolism of T-dGuo in CEM cells. The concentration of T-dGuo required to inhibit cell growth by 50% was approximately 50-fold greater in the presence of 8.0 microM BDG than in its absence. As expected, the addition of BDG to cells treated with T-dGuo prevented the metabolism of T-dGuo to 6-thio-guanine-containing ribo-nucleotides, but, unexpectedly, no 6-thio-2'-deoxyguanosine 5'-triphosphate was detected. In cells treated with T-dGuo plus BDG, the major phosphorylated metabolite was T-dGMP. These results indicated that even in the absence of PNP activity, T-dGuo cannot be phosphorylated directly to 6-thio-2'-deoxyguanosine 5'-triphosphate due to the inability of guanylate kinase to utilize T-dGMP as a substrate.

2-Chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-adenine (Cl-F-ara-A) has activity against the P388 tumor in mice on several different schedules. Biochemical studies with a chronic myelogenous leukemia cell line (K562) grown in cell culture have been done in order to better understand its mechanism of action. Cl-F-ara-A was a potent inhibitor of K562 cell growth. Only 5 nM inhibited K562 cell growth by 50% after 72 h of continuous incubation. The 5'-triphosphate of Cl-F-ara-A was detected by strong anion exchange chromatography of the acid-soluble extract of K562 cells incubated with Cl-F-ara-A. Competition studies with natural nucleosides suggested that deoxycytidine kinase was the enzyme responsible for the metabolism to the monophosphate. Incubation of K562 cells for 4 h with 50 nM Cl-F-ara-A inhibited the incorporation of [3H]thymidine into the DNA by 50%. Incubation with 0.1, 1, or 10 microM Cl-F-ara-A for 4 h depressed dATP, dCTP, and dGTP pools but did not affect TTP pools. Similar inhibition of deoxyribonucleoside triphosphate pools was seen after incubation with 2-chloro-2'-deoxyadenosine. Both Cl-F-ara-ATP and Cl-dATP potently inhibited the reduction of ADP to dADP in crude extracts of K562 cells (concentration producing 50% inhibition, 65 nM). The effect of Cl-F-ara-ATP on human DNA polymerases alpha, beta, and gamma isolated from K562 cells grown in culture was determined and compared with those of Cl-dATP and 9-beta-D-arabinofuranosyl-2-fluoroadenine triphosphate (F-ara-ATP). Cl-F-ara-ATP was a potent inhibitor of DNA polymerase alpha. Inhibition of DNA polymerase alpha was competitive with respect to dATP (Ki of 1 microM). The three analogue triphosphates were incorporated into the DNA by DNA polymerase alpha as efficiently as dATP. The incorporation of Cl-F-ara-AMP inhibited the further elongation of the DNA chain, similarly to that seen after the incorporation of F-ara-AMP. Extension of the DNA chain after the incorporation of Cl-dAMP was not inhibited as much as it was with either Cl-F-ara-AMP or F-ara-AMP. Cl-F-ara-ATP was not a potent inhibitor of DNA polymerase beta, DNA polymerase gamma, or DNA primase.(ABSTRACT TRUNCATED AT 400 WORDS)

The synthesis and isolation of two derivatives of 2-amino-1,3,4-thialdiazole(aminothiadiazole) are described. The derivatives are a nicotinamide adenine dinucleotide (NAD) analog prepared by an exchange reaction with NAD in the presence of nicotineamide adenine dinucleotide glycohydrolase and a presumed aminothiadiazole mononucleotide prepared by treatment of the NAD analog with nucleotide pyrophosphatase. Both derivatives are potent inhibitors of inosine 5'-phosphate (IMP) dehydrogenase obtained from leukemia L1210 cells. The NAD analog is a pseudoir-reversible inhibitor of the enzyme, noncompetitive with either IMP or NAD. The aminothiadiazole mononucleotide has a K1 of about 0.1 muM, is competitive with IMP, and is uncompetitive with NAD: the inhibition appears to be reversible by Ackermann-Potter analysis. A metabolite of [5-14C]aminothiadiazole is formed in L1210 cells in vivo to a level of 0.3 nmole/10(9) cells. Retention volume of the metabolite on a high-pressure liquid chromatography system is the same as that of the aminothiadiazole mononucleotide prepared as described above. These results suggest that IMP dehydrogenase is the site of action for aminothiadiazole metabolites as was indicated by earlier observations. There is no evidence that the NAD analog is formed in vivo. Nicotinamide prevented formation of the mononucleotide in vivo. Therefore, since formation and cleavage of the NAD analog apparently are not the route to the thiadiazole nucleotide, some other pathway for the metabolism of nicotinamide may be involved such as the action of a phosphoribosyltransferase or the sequential action of a nucleoside phosphorylase and a nucleoside kinase.

9-beta-D-Arabinofuranosyl-2-fluoroadenine (2-F-ara-A), a derivative of 9-beta-D-arabinofuranosyladenine (ara-A) that is resistant to deamination, selectively inhibits DNA synthesis and has activity against mouse leukemia L1210 comparable to that of ara-A plus the adenosine deaminase inhibitor, 2'-deoxycoformycin. To determine if these two nucleosides have similar modes of action, comparisons were made of their effects and those of their triphosphates on enzymes known to be inhibited by ara-A or 9-beta-D-arabinofuranosyladenine 5'-triphosphate. 9-beta-D-Arabinofuranosyl-2-fluoroadenine 5'-triphosphate was more effective than 9-beta-D-arabinofuranosyladenine 5'-triphosphate in inhibiting the reduction of adenosine 5'-diphosphate and cytidine 5'-diphosphate by ribonucleotide reductase from HEp-2 cells or L1210 cells. DNA polymerase alpha from L1210 cells was equally sensitive to 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate and 9-beta-D-arabinofuranosyladenine 5'-triphosphate, and DNA polymerase beta from L1210 cells was much less sensitive to both triphosphates. S-Adenosylhomocysteine hydrolase from L1210 cells was inactivated by 2-F-ara-A and ara-A, but higher concentrations of the fluoro derivative were required. These results are consistent with 2-F-ara-A and ara-A inhibition of DNA synthesis by inhibition of ribonucleotide reductase and DNA polymerase alpha.

8-Azainosine (8-aza-HR) is of interest because of its activity against experimental tumors. Metabolic studies in cell cultures were performed with 8-aza-HR and with the structurally related nucleoside, 8-azaadenosine (9-beta-D-ribofuranosyl-8-azaadenine) (8-aza-AR), which has a lower degree of antitumor activity than does 8-aza-HR. In H. Ep. 2 cells and in Ca755 cells, both 14C-labeled nucleosides were metabolized to nucleotides of 8-azaadenine (8-aza-A) and 8-azaguanine (8-aza-G) and incorporated into polynucleotides as 8-aza-A and 8-aza-G. 8-Aza HR was incorporated primarily as 8-aza-G, whereas 8-aza-AR was incorporated about equally as 8-aza-A and 8-aza-G. In H. Ep. 2 cells, the extent of incorporation of 8-aza-HR as 8-aza-G was about one-half that found when [14C]-8-aza-G was the precursor. In the H. Ep. 2/FA/FAR cell line, 8-aza-AR and 8-aza-HR were metabolized similarly, in that both were incorporated into polynucleotides principally as 8-aza-G; apparently, in this cell line which is deficient in adenosine kinase and adenine phosphoribosyltransferase, 8-aza-AR is metabolized by conversion to 8-aza-HR. A cell line (H. Ep 2/8-aza HR), which was resistant to 8-aza-HR but sensitive to 8-aza-AR and which retained hypoxanthine (guanine)-phosphoribosyltransferase activity, metabolized 8-aza-HR to only a small extent. However, in this cell-line, 8-aza-AR was more extensively metabolized and was incorporated primarily as 8-aza-A. The failure of these cells to convert 8-aza-AR or 8-aza-HR to 8-aza-G indicates that the basis for resistance may be a change in the substrate specificities of the enzymes of guanosine monophosphate synthesis such that these cells no longer effectively convert 8-azainosine monophosphate to 8-azaguanosine monophosphate. 8-Aza-AR was a potent inhibitor of purine synthesis de novo, but 8-aza-HR, at concentrations much higher than the inhibitory concentration of 8-aza-AR, did not inhibit this process. In H. Ep. 2 cells, 8-aza-HR blocked the conversion of orotic acid to uridine nucleotides and caused an accumulation of orotidine. This inhibition of pyrimidine biosynthesis apparently does not contribute significantly to the cytotoxicity of 8-aza-HR because uridine provided no degree of reversal of its inhibition of the growth of cell cultures.