Effects of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine on K562 cellular metabolism and the inhibition of human ribonucleotide reductase and DNA polymerases by its 5'-triphosphate.

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)

Synthesis and biological evaluations of certain 2-halo-2'-substituted derivatives of 9-beta-D-arabinofuranosyladenine.

The synthesis of a series of 2-chloro- or 2-fluoro-9-(2-substituted-2-deoxy-beta-D-arabinofuranosyl)adenines (4g-n) is described. New compounds were prepared from either 2-chloroadenosine or 2-fluoroadenosine by first blocking the 3'- and 5'-hydroxyls as the tetraisopropyldisiloxane derivatives. Activation of O-2' by formation of a triflate followed by nucleophilic displacement allowed introduction of various groups in the proper configuration at C-2'. Fluoride ion treatment then produced the deblocked nucleosides. All of the new compounds were evaluated as cytotoxic agents against L1210 and H.Ep.-2 cells and as antiviral agents against herpes simplex viruses 1 and 2 and vaccinia virus in culture.

5'-deoxy-5'-methylthioadenosine phosphorylase--IV. Biological activity of 2-fluoroadenine-substituted 5'-deoxy-5'-methylthioadenosine analogs.

5'-Deoxy-5'-methylthioadenosine phosphorylase (MTAPase) phosphorolyzes 5'-deoxy-5'-methylthioadenosine (MTA) generated during polyamine biosynthesis to adenine and 5-methylthioribose-1-phosphate. Two doubly-substituted, 2-fluoroadenine-containing analogs of MTA, 5'-deoxy-2-fluoroadenosine (5'-dFAdo) and 5'-deoxy-5'-iodo-2-fluoroadenosine (5'-IFAdo), were synthesized and studied as substrates of MTAPase: their reaction with this enzyme resulted in the liberation of the cytotoxic base, 2-fluoroadenine, as well as potentially cytotoxic analogs of 5-methylribose-1-phosphate. The activities of these MTA analogs were compared to that of the singly-substituted analog, 5'-deoxy-5'-methylthio-2-fluoroadenosine (5'-MTFAdo). The cytotoxic action of these MTA analogs depended primarily on their conversion to 2-fluoroadenine-containing nucleotides, as a cell line that contains both MTAPase and adenine phosphoribosyltransferase (APRT) activity (HL-60 human promyelocytic leukemia) readily converted these MTA analogs to 2-fluoroadenine-containing nucleotides (especially 2-fluoroadenosine triphosphate) and was highly sensitive to the growth-inhibitory effects of all three compounds (IC50 values in the 10(-8) M range), whereas cell lines lacking MTAPase (CCRF-CEM human T-cell leukemia) or APRT (HL-60/aprt1 cells) did not form analog nucleotides and were relatively insensitive to these compounds (IC50 values in the 10(-5) M range). The doubly-substituted analogs were not more growth inhibitory than 5'-MTFAdo in wild type HL-60 cells as the potent effects of 2-fluoroadenine may mask the activity of the 5-methylthioribose-1-phosphate analogs generated in the reaction of these compounds with MTAPase. 5'-dFAdo and 5'-IFAdo also were irreversible inhibitors of S-adenosylhomocysteine hydrolase, which may explain in part the weak but observable growth inhibitory action of these compounds against MTAPase-deficient cell lines.

9-(2-Deoxy-2-fluoro-beta-D-arabinofuranosyl)guanine: a metabolically stable cytotoxic analogue of 2'-deoxyguanosine.

The synthesis of 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)guanine (1b) from 1,3-di-O-acetyl-5-O-benzoyl-2-deoxy-2-fluoro-D-arabinofuranose (2a) and 2,6-dichloropurine in six steps using an enzymatic deamination as the last step is reported. The target compound was found to be stable to purine nucleoside phosphorylase cleavage and was cytotoxic in two cell lines, one a T-cell line. Incubation of L1210 cells with 1b results in an inhibition of DNA synthesis as judged by the reduced incorporation of labeled thymidine into DNA, while RNA and protein syntheses were unaffected.

2-Fluoroformycin and 2-aminoformycin. Synthesis and biological activity.

Syntheses of 2-fluoroformycin [7-amino-5-fluoro-3-(beta-D-ribofuranosyl)pyrazolo[4,3-d]pyrimidine] (2b) and 2-aminoformycin [5,7-diamino-3-(beta-D-ribofuranosyl)pyrazolo[4,3-d]pyrimidine] (2c) are described. Cytotoxicity data are given for 2b and 2c alone as well as with added pentostatin. Kinetic parameters for adenosine deaminase are also provided. 2-Fluoroformycin, although a much poorer substrate for adenosine deaminase than formycin A, is not nearly as cytotoxic to cells in culture.

Synthesis and biological evaluation of 2-fluoro-8-azaadenosine and related compounds.

The synthesis of 2-fluoro-8-azaadenosine (6e) and 2-amino-8-azaadenosine (6d) is described. Condensation of 9H-2,6-bis(methylthio)-8-azapurine (4) with 2,3,5-tri-O-acetyl-D-ribofuranosyl chloride (5) produces a mixture of 6a (9-beta-D-ribofuranosyl) and 7a (8-beta-D-ribofuranosyl). Standard functional group manipulation, including a modified Schiemann reaction to introduce the fluorine, allows preparation of 6d and 6e from the major isomer 6a. By a similar series of reactions the minor component 7a was converted to 7d and 7e, with the ribose ring attached at N-8 of the 8-azapurine ring system. Structure proofs utilized UV and 1H and 13C NMR data. Compounds 6b-e,g and 7b-f were evaluated in the H.Ep.-2 cell culture screen, and compounds 6c-e and 7d were evaluated in the P388 mouse leukemia screen. Adenosine deaminase data are also presented for some compounds.

Identification of metabolites of 9-beta-D-arabinofuranosyl-2-fluoroadenine, an antitumor and antiviral agent.

Analysis of blood from a dog given a 400 mg/m2 dose of 9-beta-D-arabinofuranosyl-2-fluoroadenine (2-F-araA) led to the identification of parent drug and a major metabolite, 9-beta-D-arabinofuranosyl-2-fluorohypoxanthine. 2-Fluoroadenine, a toxic derivative of 2-F-araA, was not detected in blood within the limits of detection, suggesting that parent drug was absorbed and distributed without systemic exposure to this toxic derivative. Parent drug, 2-fluoroadenine, and 9-beta-D-arabinofuranosyl-2-fluorohypoxanthine were identified in urine of dog, monkey, and mouse.

2-Substituted derivatives of 9-alpha-D-arabinofuranosyladenine and 9-alpha-D-arabinofuranosyl-8-azaadenine.

2-Substituted derivatives of 9-alpha-D-arabinofuranosyladenine were prepared via the fusion of tetra-O-acetyl-alpha-D-arabinofuranose with 2,6-dichloropurine followed by stepwise displacement of the chloro groups. A different approach, the reaction of 2,3,5-tri-O-benzoly-D-arabinofuranosyl bromide with 2,6-bis(methylthio)-8-azapurine in refluxing toluene in the presence of molecular sieve followed by stepwise reaction of the blocked nucleoside with methanolic ammonia and then other nucleophiles, gave 2-substituted derivatives of 9-alpha-D-arabinofuranosyl-8-azaadenine; In contrast to the parent compounds, none of these 2-substituted derivatives showed significant antiviral activity.

Nucleosides of 2-azapurines. 7 H-Imidazo[4,5-d]-1,2,3-triazines. 2.

A number of nucleosides of 2-azaadenine (4-amino-7H-imidazo[4,5-d]-1,2,3-triazine) were prepared by a previously described route, and some of these were deaminated by means of adenosine deaminase. Alternatively, nucleosides of 2-azahypoxanthine (7H-imidazo[4,5-d]-1,2,3-triazin-4(3h)-one) were prepared from hypoxanthine nucleosides by a 2-azahypoxanthine (7H imidazo[4,5]-1,2,3-triazin-4(3H)-one) were prepared from hypoxanthine nucleosides by a ring-opening and reclosure sequence. The cytotoxicity of these compounds to human epidermoid carcinoma No. 2 cells in culture and to certain resistant sublines thereof was determined. 2-Azaadenine nucleosides chan be metabolized to nucleotides, the cytotoxic agents, by two pathways, but the activity of the 2-azahypoxanthine nucleosides appears to result only from cleavage back to 2-azahypoxanthine.