Effects of the chiral isomers of erythro- and threo-9-(2-hydroxy-3-nonyl)adenine on purine metabolism in sarcoma 180 cells

The effects of the chiral isomers of erythro- and threo-9-(2-hydroxy-3-nonyl)adenines (EHNA and THNA) on purine metabolism in Sarcoma 180 cells have been determined. At concentrations of 10–80 μM [10- to 1000-fold greater than their K_i values with adenosine deaminase (ADA)], all isomers inhibited purine salvage and biosynthesis de novo. Although (+)-EHNA, the most potent ADA inhibitor, exerted the greatest effects, there was no direct correlation between the potency of ADA inhibition and the secondary effects on purine metabolism, e.g. (+)-EHNA is about 2-fold more inhibitory than (?)-EHNA in blocking purine base incorporation but about 250-fold more potent as an inhibitor of ADA (K_i of (+)-EHNA = 500nM[Bessodes et al., Biochem. Pharmac.31, 879 (1982)]). All the isomers inhibited the incorporation of radiolabeled purine bases (adenine, guanine and hypoxanthine) and nucleosides (guanosine and inosine) into acid-soluble nucleotides and of glycine into 5′-phosphoribosyl-formylglycineamide. Unlike the results of Henderson et al. [Biochem. Pharmac.26, 1967 (1977)] with Ehrlich ascites cells, the incorporation of adenosine into nucleotides was only slightly inhibited in Sarcoma 180 cells. (+)-EHNA did not inhibit the activities of 5-phosphoribosyl-1-pyrophosphate (PRPP) synthetase, purine phosphoribosyltransferases or nucleotide kinases in cell extracts. Accumulation of PRPP was inhibited only under conditions that fostered rapid synthesis.     

Regulation of phosphoribosylpyrophosphate synthetase by endogenous purine and pyrimidine compounds and synthetic analogs in normal and leukemic white blood cells

Phosphoribosylpyrophosphate (PRPP) is essential for the formation of both purine and pyrimidine nucleotides as well as for the active nucleotide form of some chemotherapeutic agents. The formation of PRPP is catalyzed by by enzyme PRPP synthetase, and many different compounds are known to affect the activity of this enzyme. This report examines the effects of endogenous purine and pyrimidine nucleotides, nucleosides, and several analogs of these compounds on the activity of PRPP synthetase from different types of normal and leukemic white blood cells (i.e. normal lymphocytes, normal granulocytes, phytohemagglutinin-stimulated lymphocytes, and acute and chronic leukemic cells). Our results show that the effect varied with each individual compound, and the magnitude of the effect was dependent on the source of the enzyme. Since it appears possible to differentially affect PRPP synthetase activity from the different types of leukemic cells, this enzyme may be a potential target site in the chemotherapy of leukemia.     

Pathways for alpha-D-ribose utilization for nucleobase salvage and 5-fluorouracil activation in rat brain

Recently, interest has increased in the use of alpha-D-ribose (Rib) as a naturally occurring nutriceutical for enhancement of cardiac and muscular performance. Most likely the elevation of available PRPP, following Rib administration, plays a key role in the salvage of purine nucleobases, thus, accelerating the restitution of ATP pool. In addition, administration of Rib improves some of the neurological symptoms in patients with adenylosuccinase deficiency. In this paper, we show that rat brain extract can catalyze the Rib-mediated salvage of both adenine and uracil, as well as the activation of the pyrimidine pro-drug, 5-fluorouracil (5-FU). The results strongly support that the pentose may be converted to both PRPP and Rib1-P for the salvage of the adenine and uracil, respectively. Most likely two-reaction pathway, composed of ribokinase and PRPP synthetase, is responsible of the PRPP formation, needed to salvage adenine to adenine nucleotides. A two-reaction pathway, composed of ribokinase and phosphopentomutase, appears to be responsible of the Rib1-P formation, needed to salvage uracil to uracil-nucleotides and to activate 5-FU to cytotoxic 5-FU-ribonucleotides. alpha-D-2'-Deoxyribose (deoxyRib) has a negligible effect on both the salvage of natural nucleobases to deoxyribonucleotides and on the activation of 5-FU to cytotoxic 5-FU-deoxynucleotides.     

Antitumor and antiviral activity of synthetic alpha- and beta-ribonucleosides of certain substituted pyrimido[5,4-d]pyrimidines: a new synthetic strategy for exocyclic aminonucleosides

A novel and direct synthesis of the antiviral and antitumor agent 4-amino-8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine (ARPP, 8) and its alpha-anomer (11) has been developed. Treatment of 2,4,6,8-tetrachloropyrimido[5,4-d]pyrimidine (1) with 2,3-O-isopropylidene-D-ribofuranosylamine gave an anomeric mixture of 2,4,6-trichloro-8-(2,3-O-isopropylidene-beta- and -alpha-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidines (3 and 4) in a ratio of 1.0:0.7. A nucleophilic displacement of the 4-chloro group of 3 and 4 with NH3 furnished 4-amino-2,6-dichloro-8-[(2,3-O-isopropylidene-beta-D-ribofuranosyl)amino ] pyrimido[5,4-d]pyrimidine (6) and its alpha-anomer (9), respectively. Catalytic hydrogenation of 6 and 9, followed by deisopropylidenation gave ARPP (8) and the alpha-anomer 11, respectively. Similarly, 3 and 4 have been transformed to 4-methoxy-8-(beta-D-ribofuranosylamino)pyrimido-[5,4-d]pyrimidine (MRPP, 14) and its alpha-anomer (17). Application of this procedure to 3 with NH2Me or NHMe2 resulted in the synthesis of 4-(methylamino)- and 4-(dimethylamino)-8-(beta-D-ribofuranosylamino)pyrimido [5,4-d]pyrimidine (24 and 27, respectively). A synthesis of 8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidin-4(3H)-one (21) has also been accomplished from 3 in three steps. Selective hydrogenation of 6 furnished 4-amino-6-chloro-8-[(2,3-O-isopropylidene-beta-D-ribofuranosyl)amino] pyrimido[5,4-d]pyrimidine (36), the structure of which was established by single-crystal X-ray diffraction analysis. Deisopropylidenation of 36 gave 6-chloro-ARPP (37). Extended treatment of 36 with NH3 furnished 4,6-diamino-8-[(2,3-O-isopropylidene-beta-D-ribofuranosyl)amino]pyrimido [5,4-d]pyrimidine (34), which on deisopropylidenation gave 6-amino-ARPP (35). An unambiguous synthesis of 34 and 36 has also been accomplished by the reaction of 4,6,8-trichloropyrimido[5,4-d]pyrimidine (28) with 2, followed by the treatment with NH3. Nucleophilic displacement studies with 1, 6, and 28 indicated the reactivity of the halogens in these compounds is in the order of 8 greater than 4 greater than 6 greater than 2. The structures of 3 and 9 have been assigned on the basis of 1H NMR data and further confirmed by single-crystal X-ray diffraction analysis. The exocyclic aminonucleosides synthesized during this study were tested for their activity against several RNA and DNA viruses in vitro and against L1210, WI-L2, and LoVo/L in cell culture. The effect of these compounds on the de novo nucleic acid biosynthesis has been studied. Compound 14 (MRPP) exhibited enhanced activity against L1210 in vivo, when compared to ARPP (8).     

Nucleoside transport inhibitors: structure-activity relationships for pyrimido[5,4-d]pyrimidine derivatives that potentiate pemetrexed cytotoxicity in the presence of α1-acid glycoprotein

Membrane transport of nucleosides or nucleobases is mediated by transporters including the equilibrative nucleoside transporters (ENTs), and resistance to antitumor antimetabolite drugs may arise via salvage of exogenous purine or pyrimidine nucleosides or nucleobases by ENT transporters. The therapeutic utility of dipyridamole (3), a potent ENT inhibitor, is compromised by binding to the serum protein α(1)-acid glycoprotein (AGP). Derivatives and prodrugs of the ENT inhibitor 4,8-bis[(3,4-dimethoxybenzyl)amino]-2,6-bis[(2-hydroxypropyl)amino]pyrimido[5,4-d]pyrimidine (6, NU3108) are described, with improved in vivo pharmacokinetic properties and reduced AGP binding relative to dipyridamole. The mono- and diglycine carbamate derivatives were at least as potent as 6 and showed no reduction in potency by AGP. In a [(3)H]thymidine incorporation assay, employing COR-L23 cells, the diastereoisomers of 6 (IC(50) = 26 nM) exhibited activity comparable with 3 (IC(50) = 15 nM). The monophenyl carbamate and mono-4-methoxyphenyl carbamate exhibited the best ENT-inhibitory activity in the COR-L23 assay (IC(50) = 8 and 4 nM, respectively). All of the new prodrugs were also highly effective at reversing thymidine/hypoxanthine rescue from pemetrexed cytotoxicity in the COR-L23 cell line.     

Regulation of pyrimidine biosynthesis in cultured L1210 cells by 3-deazauridine

The effect of 3-deazauridine on the synthesis of uracil nucleotides by de novo and salvage pathways was investigated in intact cultured L1210 cells. De novo pyrimidine biosynthesis, as measured by sodium [14C]bicarbonate incorporation into uracil nucleotides, was inhibited 40-85% at intracellular 3-deazauracil nucleotide concentrations of 1-6 nmoles/10(6) cells. The inhibition was not due to an increase in the size of the uracil nucleotide pool since this pool was only 97-66% of control level at 3-deazauracil nucleotide concentrations of 1-6 nmoles/10(6) cells. Furthermore, intracellular 3-deazauracil nucleotide concentrations of 0.5 to 5.2 nmoles/10(6) cells inhibited the salvage of [14C]uridine by 25-75%. The data indicate that 3-deazauridine may potentiate its own inhibition of CTP synthetase by reducing the concentration of competing uracil nucleotides by inhibiting de novo pyrimidine biosynthesis and pyrimidine salvage. It is postulated that the biochemical mechanism by which 3-deazauridine inhibits uracil nucleotide synthesis is by acting as a fraudulent allosteric regulator of carbamyl phosphate synthetase II and uridine/cytidine kinase.     

Functional differences in nucleoside and nucleobase transporters expressed on the rabbit corneal epithelial cell line (SIRC) and isolated rabbit cornea

The purpose of this study was to investigate the expression of nucleoside/nucleobase transporters on the Statens Seruminstitut rabbit corneal (SIRC) epithelial cell line and to evaluate SIRC as an in vitro screening tool for delineating the mechanism of corneal permeation of nucleoside analogs. SIRC cells (passages 410–425) were used to study uptake of [³H]thymidine, [³H]adenine, and [³H]ganciclovir. Transport of [³H]adenine and [³H]ganciclovir was studied across isolated rabbit cornea. Uptake and transport studies were performed for 2 minutes and 120 minutes, respectively, at 34°C. Thymidine uptake by SIRC displayed saturable kinetics (K _m=595.9±80.4μM, and V _max=289.5±17.2 pmol/min/mg protein). Uptake was inhibited by both purine and pyrimidine nucleosides but not by nucleobases. [³H]thymidine uptake was sodium and energy independent but was inhibited by nitrobenzylthioinosine at nanomolar concentrations. Adenine uptake by SIRC consisted of a saturable component (K _m=14.4±2.3μM, V _max=0.4±0.04 nmol/min/mg protein) and a nonsaturable component. Uptake of adenine was inhibited by purine nucleobases but not by the nucleosides or pyrimidine nucleobases and was independent of sodium, energy, and nitrobenzylthioinosine. [³H]ganciclovir uptake involved a carrier-mediated component and was inhibited by the purine nucleobases but not by the nucleosides or pyrimidine nucleobases. However, transport of [³H]adenine across the isolated rabbit cornea was not inhibited by unlabeled adenine. Further, corneal permeability of ganciclovir across a 100-fold concentration range remained constant, indicating that ganciclovir permeates the cornea primarily by passive diffusion. Nucleoside and nucleobase transporters on rabbit cornea and corneal epithelial cell line, SIRC, are functionally different, undermining the utility of the SIRC cell line as an in vitro screening tool for elucidating the corneal permeation mechanism of nucleoside analogs.     

Purine de novo synthesis as the basis of synergism of methotrexate and 6-mercaptopurine in human malignant lymphoblasts of different lineages

Methotrexate (MTX) causes an inhibition of purine de novo synthesis (PDNS), resulting in increased intracellular availability of 5-phosphoribosyl-1-pyrophosphate (PRPP) in human malignant lymphoblasts with an active PDNS. Normal bone marrow cells and peripheral blood lymphocytes lack this capacity. The increased levels of PRPP can be used for enhanced incorporation of 6-mercaptopurine (6MP), indicating a potential time-, sequence- and dose-dependent synergism of both drugs. The effects of 0.02 microM and 0.2 microM MTX on the PDNS of MOLT-4 (T-), RAJI (B-) and KM-3 (non-B-non-T-) human malignant lymphoblasts were studied with respect to PRPP levels, aminoimidazolecarboxamide ribonucleosidemonophosphate (AICAR) levels and the incorporation of labeled glycine into purine metabolites. These results were correlated with the activity of the PDNS (labeled glycine incorporation) and the purine salvage pathway (labeled hypoxanthine incorporation) in untreated cells. Inhibition of PDNS by 0.02 microM MTX was complete in KM-3 cells with a moderately active PDNS and salvage pathway. RAJI cells, with a relatively low PDNS and high salvage pathway, demonstrated an incomplete, but increasing inhibition of PDNS, whereas inhibition of PDNS in MOLT-4 cells with both pathways active was minimal and recovered in time. Treatment with 0.2 microM MTX resulted in a complete inhibition of PDNS in all cell lines. After treatment with MTX an enhanced incorporation of labeled hypoxanthine and 6MP was noticed, confirming the potential rescue from MTX cytotoxicity by hypoxanthine and a potential synergism of MTX and 6MP on cytotoxicity. The enhanced incorporation of 6MP was more obvious in RAJI and KM-3 cells in comparison with MOLT-4 cells. These data demonstrate the important role of both the activities of the PDNS and the purine salvage pathway in malignant lymphoblasts of different subclasses with respect to the synergism of MTX and 6MP.     

Effects of uridine diphosphoglucose (UDPG) infusion on 5-phosphoribosyl pyrophosphate (PRPP) levels of mouse tissues

Uridine diphosphoglucose (UDPG) has been shown to have tissue-specific effects that have proved to be of clinical value in the treatment of some liver ailments. In an effort to determine something about the mechanism of action, we investigated the effect of UDPG on the levels of 5-phosphoribosyl pyrophosphate (PRPP) and PRPP synthetase in mouse liver, spleen and transplanted tumors. Three strains of mice were studied with and without tumors under various experimental conditions. Balb/c mice were infused with UDPG intraperitoneally at levels of 0.16 g/kg/day (0.28 mmole) to 1.6 g/kg/day (2.8 mmoles) for 5 days. At the low dose rate the PRPP level in the liver was found to increase 3-fold. A slight increase was noted in the activity of PRPP synthetase. However, when the UDPG was infused at a level of 2.8 mmoles/kg/day, the increases in both the synthetase and PRPP were inhibited. Both CRF1 and CD8 mice were less sensitive to the effects of UDPG per se. However, the high level of PRPP in the tumors they carried was greatly affected by the UDPG infusion. The tumor-specific inhibition of PRPP suggests that this action might prove to be useful combination therapy with inhibitors of purine and pyrimidine nucleotide synthesis in various rescue regimens. UDPG was found to enter cells intact before it was cleaved into glucose phosphate and UMP. The fact that UDPG was also found in the membrane fraction suggests that either there is a specific transport mechanism or UDPG exerts its action via interaction with the cell membrane.     

Mechanism of antiuric action of 4-oxy- and 4-thiopyrazolopyrimidines

Among the pyrazolopyrimidine derivatives used in hyperuricemia, thiopurinol [4-mercaptopyrazolo-(3,4-d) pyrimidine] exhibits different therapeutic effects than those of allopurinol [4-hydroxypyrazolo-(3,4-d) pyrimidine] and oxypurinol [4,6-dihydroxypyrazolo-(3,4-d) pyrimidine] which leads us to assume that its hypouricemic mechanism is different. The action of the three derivatives has been compared: (a) as xanthine oxydase inhibitors: thiopurinol activity is intermediate between that of the two others, (b) as subtrates of the phosphoribosyl transferase system: the nucleotide of thiopurinol has been isolated and identified, (c) as natural substrate HGPRT inhibitors (guanine and hypoxanthine). Finally, their action on PRPP consumption and on its intracellular level has been specified. In the absence of precise data on the concentrations realized in vivo with the therapeutic doses used, the interpretation of enzymatic kinetic data does not appear significant. It seems, however, that allopurinol and thiopurinol reduce de novo synthesis of purine ribonucleotides by depressing the PRPP intracellular level.     

Inhibition of two enzymes in de novo purine nucleotide synthesis by triciribine phosphate (TCN-P)

We previously reported that triciribine (tricyclic nucleoside, TCN, NSC-154020), after phosphorylation in cultured CCRF-CEM human leukemic lymphoblasts inhibited de novo purine nucleotide synthesis, GTP more than ATP [Moore et al. Biochem. Pharmac. 38, 4037 (1989)]. To determine the enzymes inhibited, triciribine phosphate (TCN-P, NSC-280594) was tested in dialyzed extracts of the cells. A new assay for glycinamide ribotide (GAR) synthesis was based on incorporation of [14C]glycine into GAR as a ribose-containing compound retained on boronyl gel columns. Glutamine, phosphoribosyl pyrophosphate (PRPP), ATP and glycine were required for the two-step sequence of glutamine:amidophosphoribosyltransferase (EC 2.4.2.14) and phosphoribosylamine-glycine ligase (EC 6.3.4.13). When PRPP was near the normal intracellular concentration (0.1 mM), 1.2 mM TCN-P inhibited GAR synthesis by 71-95%. To permit separate assay of the ligase step, 6-diazo-5-oxo-L-norleucine was used to inhibit amidophosphoribosyltransferase and phosphoribosylamine (PRA) was supplied in situ by chemical reaction of ribose-5-phosphate and ammonia (as ammonium acetate). The ligase was not inhibited by TCN-P. Thus, TCN-P inhibits amidophosphoribosyltransferase; it acts as an analog of the purine nucleotides which regulate this first committed step of de novo purine biosynthesis by an allosteric feedback mechanism. The measured intracellular concentration (0.1 mM) of PRPP was not changed in cells treated with TCN. IMP dehydrogenase (EC 1.1.1.205), the first de novo step committed to guanosine nucleotide synthesis, was also tested. It was inhibited by TCN-P, competitively with IMP, 66% at 1.2 mM TCN-P and 8 microM IMP. The degree of inhibition of these two enzymes was sufficient to account for the effects on purine nucleotide biosynthesis observed in intact cells treated with TCN.     

Synthesis and cytotoxicity of 4-amino-5-oxopyrido[2,3-d]pyrimidine nucleosides

A number of nucleoside analogues have been either used clinically as anticancer drugs or evaluated in clinical studies, while new nucleoside analogues continue to show promise. In this article, we report synthesis and cytotoxicity of a series of new pyrido[2, 3-d]pyrimidine nucleosides. 2-Amino-3-cyano-4-methoxypyridine was converted, in two steps, to 4-amino-5-oxopyrido[2,3-d]pyrimidine. A variety of 1-O-acetylated pentose sugar derivatives were condensed with silylated 4-amino-5-oxopyrido[2,3-d]pyrimidine, followed by protection, to afford a series of 4-amino-5-oxopyrido[2, 3-d]pyrimidine nucleosides. Further derivatizations provided an additional group of pyrido[2,3-d]pyrimidine nucleosides. These nucleosides were evaluated for in vitro cytotoxicity to human prostate cancer (HTB-81) and mouse melanoma (B16) cells as well as normal human fibroblasts (NHF). A number of compounds (1a,b, 2a-c,f, 3f+4d) showed significant cytotoxicity to cancer cells, with 4-amino-5-oxo-8-(beta-D-ribofuranosyl)pyrido[2,3-d]pyrimidine (1b) being the most potent proliferation inhibitor (EC(50): 0.06-0.08 microM) to all types of cells tested. However, a selective inhibition to the cancer cells was observed for 4-amino-5-oxo-8-(beta-D-xylofuranosyl)pyrido[2,3-d]pyrimidine (2b), which is a potent inhibitor of HTB-81 (EC(50): 0.73 microM) and has a favorable in vitro selectivity index (28).     

Purine and pyrimidine salvage pathways in Leishmania donovani

Leishmania donovani, grown in culture, salvaged radiolabeled purine bases which were distributed into adenine and guanine ribonucleotides and into the RNA of these cells. De novo synthesis of purines in L. donovani does not occur [J. J. Marr, R. L. Berens and D. J. Nelson, Biochim. biophys. Acta544, 360 (1978)]. [8-¹⁴C]Adenine was rapidly deaminated to hypoxanthine via the action of an adenine aminohydrolase (EC 3.5.4.2). [8-¹⁴C]Guanine was also rapidly deaminated by guanase (EC 3.5.4.3) to form xanthine in these cells. Therefore, the formation of nucleotides of hypoxanthine and xanthine are the first committed steps of purine salvage in L. donovani. While purines are efficiently conserved by this parasite, the salvage of pyrimidines is not so dramatic. [2-¹⁴C]Orotic acid was converted to OMP and then incorporated into the pyrimidine nucleotides and into RNA, indicating the existence of the later steps of de novo pyrimidine synthesis. [6-¹⁴C]Thymidine was salvaged by L. donovani, being incorporated into the thymine deoxyribonucleotides and into DNA. The major pathway of thymidine metabolism in this parasite, however, was cleavage of the deoxyriboside linkage to form thymine, probably via the action of a thymidine phosphorylase (EC 2.4.2.4).     

Selective abolishment of pyrimidine nucleoside kinase activity of herpes simplex virus type 1 thymidine kinase by mutation of alanine-167 to tyrosine

Herpes simplex virus type 1 (HSV-1) encodes a thymidine kinase (TK) that markedly differs from mammalian nucleoside kinases in terms of substrate specificity. It recognizes both pyrimidine 2'-deoxynucleosides and a variety of purine nucleoside analogs. Based on a computer modeling study and in an attempt to modify this specificity, an HSV-1 TK mutant enzyme containing an alanine-to-tyrosine mutation at amino acid position 167 was constructed. Compared with wild-type HSV-1 TK, the purified mutant HSV-1 TK(A167Y) enzyme was heavily compromised in phosphorylating pyrimidine nucleosides such as (E)-5-(2-bromovinyl)-2'-deoxyuridine and the natural substrate dThd, whereas its ability to phosphorylate the purine nucleoside analogs ganciclovir (GCV) and lobucavir was only reduced approximately 2-fold. Moreover, a markedly decreased competition of natural pyrimidine nucleosides (i.e., thymidine) with purine nucleoside analogs for phosphorylation by HSV-1 TK(A167Y) was observed. Human osteosarcoma cells transduced with the wild-type HSV-1 TK gene were extremely sensitive to the cytostatic effects of antiherpetic pyrimidine [i.e., (E)-5-(2-bromovinyl)-2'-deoxyuridine] and purine (i.e., GCV) nucleoside analogs. Transduction with the HSV-1 TK(A167Y) gene sensitized the osteosarcoma cells to a variety of purine nucleoside analogs, whereas there was no measurable cytostatic activity of pyrimidine nucleoside analogs. The unique properties of the A167Y mutant HSV-1 TK may give this enzyme a therapeutic advantage in an in vivo setting due to the markedly reduced dThd competition with GCV for phosphorylation by the HSV-1 TK.     

Inhibition by methotrexate of the stable incorporation of 5-fluorouracil into murine bone marrow DNA

As a consequence of the inhibition of de novo purine synthesis by methotrexate (MTX) there is an increase in 5-phosphoribosyl-1-pyrophosphate (PRPP) concentration. In cells where 5-fluorouracil (FUra) is activated via orotate phosphoribosyltransferase (OPRtase), increased PRPP results in greater conversion of FUra to nucleotides. In the murine CD8F1 breast tumor system, MTX markedly enhances the antitumor activity of FUra, increasing both the activation of FUra to FUMP and the incorporation of FUTP into RNA. However, in contrast to reported tumor tissue culture studies, MTX pretreatment in vivo prevents the stable incorporation of FUra into CD8F1 bone marrow DNA. Pretreatment with MTX (300 mg/kg) 2.5 hr prior to [3H]FUra (100 mg/kg), with a 2-hr labeling, reduced the level of FUra in DNA from 921 pmol to 66 pmol/mg of DNA. Without MTX pretreatment, 59% of the total incorporation of FUra into nucleic acids was into DNA when FUra was administered. After MTX the percentage of incorporation into DNA was reduced to 9%. Additionally, the ratio of [3H]FUra to 32P in DNA when both were given simultaneously was reduced by greater than 90%, suggesting that MTX must be specifically blocking the incorporation of FUra rather than nonspecifically preventing its incorporation by inhibiting DNA synthesis. In contrast, MTX failed to reduce the formation of DNA containing fluorouracil residues from FdUrd. To test whether MTX prevents the initial incorporation of FUra into DNA, or acts to enhance removal by the DNA glycosylase repair system, DNA was prelabeled in vivo with [3H]FUra, and MTX or MTX plus dThd was then administered. The level of FUra in bone marrow DNA was not reduced by subsequent treatment with MTX, or MTX plus dThd, indicating that MTX does not enhance the removal of FUra from DNA. The level of total free fluorodeoxynucleotides formed from FUra was reduced by two-thirds following MTX pretreatment, suggesting that the action of MTX in preventing the stable incorporation of FUra into DNA was to reduce the availability of FdUTP.     

Stimulation of nucleoside efflux and inhibition of adenosine kinase by A1 adenosine receptor activation

Adenosine is produced intracellularly during conditions of metabolic stress and is an endogenous agonist for four subtypes of G-protein linked receptors. Nucleoside transporters are membrane-bound carrier proteins that transfer adenosine, and other nucleosides, across biological membranes. We investigated whether adenosine receptor activation could modulate transporter-mediated adenosine efflux from metabolically stressed cells. DDT1 MF-2 smooth muscle cells were incubated with 10 microM [3H]adenine to label adenine nucleotide pools. Metabolic stress with the glycolytic inhibitor iodoacetic acid (1AA, 5 mM) increased tritium release by 63% (P < 0.01), relative to cells treated with buffer alone. The IAA-induced increase was blocked by the nucleoside transport inhibitor nitrobenzylthioinosine (1 microM), indicating that the increased tritium release was primarily a purine nucleoside. HPLC verified this to be [3H]adenosine. The adenosine A1 receptor selective agonist N6-cyclohexyladenosine (CHA, 300 nM) increased the release of [3H]purine nucleoside induced by IAA treatment by 39% (P < 0.05). This increase was blocked by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (10 microM). Treatment of cells with UTP (100 microM), histamine (100 microM), or phorbol-12-myristate-13-acetate (PMA, 10 microM) also increased [3H]purine nucleoside release. The protein kinase C inhibitor chelerythrine chloride (500 nM) inhibited the increase in [3H]purine nucleoside efflux induced by CHA or PMA treatment. The adenosine kinase activity of cells treated with CHA or PMA was found to be decreased significantly compared with buffer-treated cells. These data indicated that adenosine A1 receptor activation increased nucleoside efflux from metabolically stressed DDT1 MF-2 cells by a PKC-dependent inhibition of adenosine kinase activity.     

P2-purinoceptors regulate surfactant secretion from rat isolated alveolar type II cells.

Rat isolated alveolar Type II cells were utilized to examine the effect of purine and pyrimidine analogues on secretion of pulmonary surfactant. ATP potently stimulated [3H]-phosphatidylcholine ([3H]-PC) secretion in a time- and dose-dependent manner. The effect of ATP was noted by one hour of exposure and persisted for three hours. The EC50 (concentration producing 1/2 the maximal response) for ATP-induced [3H]-PC secretion was 100 nM. ADP was also a potent secretagogue for surfactant secretion, but AMP and adenosine had no significant effect on surfactant secretion at concentrations less than or equal to 250 microM. The EC50 for ADP-induced [3H]-PC secretion was 250 nM. Other purine and pyrimidine nucleotides (ITP, GTP, CTP, TTP) were examined for their effect on [3H]-PC secretion. All purine and pyrimidine triphosphates examined significantly augmented [3H]-PC secretion, but were much less potent than ATP. The EC50s were ITP = 10 microM; GTP = 100 microM; CTP = 250 microM; TTP = 100 microM. Neither 8-phenyltheophylline (10 microM, a P1-purinoceptor antagonist), propranolol (100 microM, a beta-adrenoceptor antagonist), nor indomethacin (10 microM, a prostaglandin synthetase inhibitor) inhibited ATP-induced [3H]-PC secretion from isolated Type II cells. These data provide evidence for regulation of surfactant secretion from alveolar Type II cells by a P2-purinoceptor.     

Synthesis and biological evaluation of certain C-4 substituted pyrazolo[3,4-b]pyridine nucleosides

A series of C-4 substituted pyrazolo[3,4-b]pyridine nucleosides have been synthesized and evaluated for their biological activity. Successful synthesis of various C-4 substituted pyrazolo[3,4-b]pyridine nucleosides involves nucleophilic displacement by a suitable nucleophile at the C-4 position of 4-chloro-1H-pyrazolo[3,4-b]pyridine (5), followed by glycosylation of the sodium salt of the C-4 substituted pyrazolo[3,4-b]pyridines with a protected alpha-halopentofuranose. Use of this methodology furnished a simple and direct route to the beta-D-ribofuranosyl, beta-D-arabinofuranosyl, and 2-deoxy-beta-D-erythro-pentofuranosyl nucleosides of C-4 substituted pyrazolo[3,4-b]pyridines, wherein the C-4 substituent was azido, amino, methoxy, chloro, or oxo. The regiospecificity of these glycosylations was determined on the basis of UV data and the anomeric configuration was established by 1H NMR analysis. Conclusive structural assignment was made by a single-crystal X-ray diffraction study of three compounds, 15, 31, and 42, as representatives of ribo-2'-deoxy-, and aranucleosides, respectively. The stereospecific attachment of all three alpha-halogenoses appears to occur by a Walden inversion (SN2 mechanism) at the C-1 carbon of the halogenose by the anionic N-1 of pyrazolo[3,4-b]pyridine. All deprotected nucleosides were tested against various viruses and tumor cells in culture. The effects of these compounds on de novo purine and pyrimidine nucleotide biosynthesis was also evaluated. Among the compounds tested, 4-chloro-1-beta-D-ribofuranosylpyrazolo[3,4-b]pyridine (16) and 1-beta-D-ribofuranosyl-4,7-dihydro-4-oxopyrazolo[3,4-b]pyridine (19) were found to be moderately cytotoxic to L1210 and WI-L2 in culture.     

Systematic synthesis and biological evaluation of alpha- and beta-D-xylofuranosyl nucleosides of the five naturally occurring bases in nucleic acids and related analogues

The alpha- and beta-D-xylofuranosyl analogues of the naturally occurring nucleosides, as well as other D-xylofuranosyl derivatives, have been the subject of a systematic synthesis and examination of their biological, i.e. antiviral antimetabolic, and cytostatic properties. The beta anomers were prepared by glycosylation of purine and pyrimidine aglycons with peracylated 1-O-acetyl-alpha-D-xylofuranoses, followed by removal of the blocking groups. The alpha anomers were obtained by a multistep synthesis with use of 2-amino- or 2-mercapto-alpha-D-xylofuran[1',2':4,5]-2-oxazoline as starting material. The xylofuranosyl nucleosides were tested for their activity against a variety of RNA and DNA viruses and for inhibition of cell growth and macromolecule synthesis. Three compounds, 9-(beta-D-xylofuranosyl)adenine, 9-(beta-D-xylofuranosyl)guanine, and 1-(beta-D-xylofuranosyl)cytosine, showed marked biological activity.     

Permeation and salvage of dideoxyadenosine in mammalian cells

Transmembrane equilibration of 2',3'-dideoxyadenosine (ddAdo) was measured by rapid kinetic techniques in deoxycoformycintreated P388 and L1210 mouse leukemia cells and human erythrocytes, at 25 degrees. It was only about 10% as rapid as that of other purine nucleosides that are known substrates for the nucleoside transporters of these cells. ddAdo entry was nonsaturable up to a concentration of 1 mM and was not inhibited by other nucleosides or two nucleoside transport inhibitors, dipyridamole and nitrobenzylthioinosine. Thus, ddAdo permeation was mainly nonmediated. It was relatively rapid because of the high lipid solubility of ddAdo. ddAdo entered the cells at least 100 times more rapidly than dideoxycytidine but less rapidly than trideoxythymidine, with an even greater lipophilicity than ddAdo. ddAdo was not phosphorylated in human erythrocytes, but there was some phosphorylation in deoxycoformycin-treated P388 and L1210 cells. In situ conversion of 10 microM ddAdo to ddATP, however, was slow and ceased after 5-10 min at 25 degrees or 37 degrees. Cessation of net uptake was not due to turnover of dideoxy-ATP or deamination of dideoxy-AMP. The results suggest that ddAdo salvage in the absence of deamination is limited by feedback inhibition of its phosphorylation, perhaps by deoxycytidine kinase. Permeation into the cells was not rate limiting to ddAdo salvage. In P388 and L1210 cells that had not been treated with deoxycoformycin, ddAdo was salvaged at least 100 times more efficiently than in deoxycoformycin-treated cells and converted to nucleoside triphosphates, but the end-products and pathways of salvage have not been resolved entirely. Salvage of ddAdo required deamination but was not primarily via dideoxyinosine----hypoxanthine----IMP, as is the case for 2'-deoxyadenosine salvage, because [3H]ddAdo salvage was only little inhibited by unlabeled hypoxanthine, whereas it was strongly inhibited by 2'-deoxyadenosine, adenosine, and adenine.