Synthesis and evaluation of 5-amino-1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine and certain related nucleosides as inhibitors of purine nucleoside phosphorylase.

The 5-amino and certain related derivatives of the powerful purine nucleoside phosphorylase (PNPase) inhibitor 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine (TCNR,3) have been prepared and evaluated for their PNPase activity. Acetylation followed by dehydration of 5-chloro-1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (4a) gave 5-chloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)-1,2,4-triazole-3- carbonitrile (5). Ammonolysis of 5 furnished 5-amino-1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine (5-amino-TCNR, 6), the structure of which was assigned by single-crystal X-ray analysis. Acid-catalyzed fusion of methyl 5-chloro-1,2,4-triazole-3-carboxylate (7a) with 5-deoxy-1,2,3-tri-O-acetyl-D-ribofuranose (8) gave methyl 5-chloro-1-(2,3-di-O-acetyl-5-deoxy-beta-D-ribofuranosyl)- 1,2,4-triazole-3-carboxylate (9a) and the corresponding positional isomer 9b. Transformation of the functional groups in 9a afforded a route to 5'-deoxyribavirin (9i). Compound 9a was converted in four steps to 5-amino-1-(5-deoxy-beta-D-ribofuranosyl)-1,2,4-triazole-3- carboxamidine (5'-deoxy-5-amino-TCNR, 9g). Similar acid-catalyzed fusion of 1,2,4-triazole-3-carbonitrile (7b) with 8 and ammonolysis of the reaction product 9h gave yet another route to 9i. Treatment of 9h with NH3/NH4Cl furnished 1-(5-deoxy-beta-D-ribofuranosyl)- 1,2,4-triazole-3-carboxamidine (5'-deoxy-TCNR, 9k). The C-nucleoside congener of TCNR (3-beta-D-ribofuranosyl- 1,2,4-triazole-5-carboxamidine, 12) was prepared in two steps from 3-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)- 1,2,4-triazole-5-carbonitrile (10) by conventional procedure. 5-Amino-TCNR (6) displayed a more potent, high-affinity inhibition than TCNR, with a Ki of 10 microM. In contrast, 5'-deoxy-5-amino-TCNR (9g) was a significantly less potent inhibitor of PNPase, compared to 5'-deoxy-TCNR (Ki = 80 and 20 microM, respectively). Neither the C-nucleoside congener of TCNR (12) nor that of ribavirin were found to inhibit inosine phosphorolysis.

Inhibition of pyrimidine metabolism in myeloid leukemia cells by triazole and pyrazole nucleosides.

Two triazole nucleosides, 1 (3-beta-D-ribofuranosyl-1,2,4-triazole-5-carboxamide) and 2 (2-beta-D-ribofuranosyl-1,2,3-triazole-4,5-dicarboxamide), and a pyrazole nucleoside, 3 (1-beta-D-ribofuranosylpyrazole-3,4-dicarboxamide), were found to inhibit pyrimidine nucleotide biosynthesis in the human myeloid leukemia cell line, K562. Cells treated with these inhibitors released orotate in quantities of 8-35 nmol/10(5) cells/day. Treatment with these compounds caused the K562 cells to accumulate in the S phase of the cell cycle and induced the cells to synthesize hemoglobin.

Potent and specific inhibitors of mammalian phosphoribosylpyrophosphate (PRPP) synthetase.

The monophosphates of the exocyclic amino ribonucleosides, 4-amino- and 4-methoxy-8-(D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine, are potent and specific inhibitors of human erythrocyte and B-lymphoblast PRPP synthetase. The inhibition by MRPP monophosphate is competitive (Ki = 35 microM with the PRPP synthetase cofactor, Pi (Km = 2 mM). The nucleosides are phosphorylated to the active metabolite by adenosine kinase and these nucleoside monophosphates accumulate in the cell. beta-ARPP is a substrate, albeit poor, for adenosine deaminase and solutions of the beta-anomer of this nucleoside and its monophosphate anomerize over time to give alpha- and beta-mixtures. beta-MRPP is more resistant to adenosine deaminase and anomerization of the nucleoside and its monophosphate is negligible. The effect of treatment of cells with the nucleosides is a time-dependent and nearly universal reduction in the nucleotide content which appears to result from a reduction in the availability of PRPP for dependent metabolic pathways. In studies with the WI-L2 lymphoblasts, some of these pathways, de novo and salvage (hypoxanthine and guanine) synthesis of purine nucleotides, are more sensitive to a restriction of PRPP availability than others, i.e. de novo pyrimidine synthesis. The nucleosides have shown promise as therapeutic agents in a mouse leukemia evaluation system but may also have future use in unravelling the complex regulation of PRPP synthetase and the dependent nucleotide synthesis pathways.

Nucleoside and nucleotide modulation of genetic expression--a new approach to chemotherapy.

Unlike conventional enzymes, receptors that activate G proteins do not catalyze the direct formation or cleavage of covalent bonds but act instead as a catalyst for the exchange of GTP vs GDP, which results in major conformational changes in the alpha subunit of G proteins and dissociation and selective binding of the alpha subunit which provokes direct enzyme activation eventually resulting in stimulation of protein kinase A, B or C. Each of these kinases can phosphorylate specific DNA binding proteins which allow new portions of DNA to be read and expressed. Such a series of events can act as switches to control cellular genetic expression resulting in cellular proliferation, differentiation or hormonal secretion of growth factors (Scheme I). Examples of nucleosides and nucleotides which appear to exert their therapeutic effects via G protein control of cellular proliferation resulting in differentiation are tiazofurin, selenazofurin, and 8-chloro-cAMP which have been synthesized and studied in our laboratories. The clinical application of these nucleosides in cancer treatment is presently underway and offers a viable alternative to chemotherapy with highly cytotoxic agents. The use of these derivatives result in down-regulation of the G protein regulatory pathways responsible for rapid cell division. Alternatively, a series of guanosine analogs prepared in our laboratories, 8-bromoguanosine, 8-mercaptoguanosine, 7-methyl-8-oxoguanosine and 7-thia-8-oxoguanosine, all activate various aspects of the immune response by up-regulation of G protein regulatory pathways in various lymphocyte derived cells. Guanosine-like nucleosides which function in this manner could have major clinical application as antitumor, antiviral and antimetastatic agents providing the desired specificity can be achieved. Specific immune enhancement of the aged might be an attainable goal if suitable orally active guanosine derivatives with high specificity can be achieved. The G protein regulatory pathways for modulation of genetic expression in specific cell types provide a major modern approach to new chemotherapeutic agents.

Spectrophotometric determination of acadesine (AICA-riboside) in plasma using a diazotization coupling technique with N-(1-naphthyl)ethylenediamine.

A simple spectrophotometric assay has been developed for determining concentrations of the new cardioprotective agent, acadesine (AICA-riboside), in human plasma. The method is adapted from the Bratton-Marshall (BMA) procedure for detection of primary aromatic amines. The assay was developed to measure concentrations of the drug in plasma during i.v. infusion to patients undergoing coronary artery bypass graft (CABG) surgery. The limit of quantitation of the assay is 0.25 microgram/ml using a 0.25 ml sample of plasma. Use of 96-well titer plates and reader can rapidly process many samples at one time. The colorimetric assay correlates well (r = 0.98) with a previously described high performance liquid chromatographic (HPLC) procedure in terms of range of quantitation, limit of quantitation, and precision.

Spectrophotometric determination of the cardioprotective drug GP531 in human plasma.

A simple spectrophotometric assay has been developed for determining concentrations of the new cardioprotective agent, GP531, in human plasma. The method is adapted from the Bratton-Marshall assay (BMA) for the detection of primary aromatic amines. The assay can be used to measure plasma concentrations of the drug during i.v. infusion administration to patients. The limit of quantitation of the assay is 1 microgram/ml using a 0.8 ml sample ultrafiltrate of plasma. The colorimetric assay correlates well with a previously described high performance liquid chromatographic (HPLC) procedure.

A novel non-radioactive method for detection of nucleoside analog phosphorylation by 5'-nucleotidase.

Cytosolic 5'-nucleotidase has been implicated in the phosphorylation of certain nucleosides of therapeutic interest. In vitro, IMP and GMP serve as the optimal phosphate donors for this nucleoside phosphotransferase reaction. Existing assays for nucleoside phosphorylation effected by 5'-nucleotidase require a radiolabeled nucleoside as the phosphate acceptor and separation of the substrate-nucleoside from product-nucleotide has been accomplished either by a filter binding method or HPLC. However, detection of the phosphorylation of unlabeled nucleoside by HPLC is difficult since the ultraviolet absorbance of the phosphate donor, IMP, frequently obscures the absorbance of newly formed nucleotide. The use of ribavirin 5'-phosphate (RMP, 1,2,4-triazole-3-carboxamide riboside 5-monophosphate) as the phosphate donor obviates this difficulty since this triazole heterocycle does not significantly absorb at the wavelengths used to detect most nucleoside analogs. Using this procedure, a 5'-nucleotidase activity from the 100,000 x g supernatant fraction of human T-lymphoblasts deficient in adenosine kinase, hypoxanthine-guanine phosphoribosyltransferase, and deoxycytidine kinase, was characterized with regard to structure-activity relationships for certain inosine and guanosine analogs.

A sensitive assay for the aminoimidazole-containing drug GP531 in plasma using liquid chromatography with amperometric electrochemical detection: a new class of electroactive compounds.

Aminoimidazole-containing compounds have been found to be electroactive and can be detected by amperometric electrochemical detection (ECD) with a high degree of sensitivity. A liquid chromatography (LC) method using ECD was developed for measuring plasma concentrations of the aminoimidazole-containing drug GP531, a potent adenosine-regulating agent. Plasma samples were extracted with 2-propanol and analyzed by LC under isocratic conditions using a mobile phase of methanol-sodium phosphate (pH 6.3; 3.3 mM) (32:68, v/v). The potential of the glassy carbon working electrode was set at +800 mV. The limit of quantitation was 12.5 ng ml-1 of GP531 using 100 microliters of plasma. The method was used to define the pharmacokinetics of GP531 in monkey following i.v. administration.

Design, synthesis, and structure-activity relationships of the first highly potent, selective, and bioavailable adenosine 5'-monophosphate deaminase inhibitors.

Structure-activity studies have been performed to optimize the potency of this novel series of AMPDA inhibitors. Conformational rigidification of the N-3 sidechain resulted in substantial effect on the potency. Addition of the hydrophobic groups provided further benefit. The most potent compound identified, 4g (Ki = 3 nM), bears little structural resemblance to AMP and exhibits a remarkable improvement (10(3) and 10(5)) in binding affinity relative to the original lead and AMP, respectively. The application of prodrug strategy achieved a large improvement (benzyl ester 5d) in oral bioavailability, resulting in compounds that should be useful in evaluating the role of AMPDA in normo- and pathophysiological states.

Thyroid hormone beta receptor activation has additive cholesterol lowering activity in combination with atorvastatin in rabbits, dogs and monkeys.

BACKGROUND AND PURPOSE: Thyroid hormone receptor (TR) agonists are in clinical trials for the treatment of hypercholesterolaemia. As statins are the standard of clinical care, any new therapies must have adjunctive activity, when given in combination with statins. As already known for the statins, the cholesterol lowering effect of TR activation involves increased expression of the low-density lipoprotein receptor. Using animal models, we tested whether TR activation would have additive cholesterol lowering activity in the presence of effective doses of a statin. EXPERIMENTAL APPROACH: We evaluated the activity of a liver-targeted prodrug, MB07811, of a novel TH receptor beta agonist, MB07344, as monotherapy and in combination with atorvastatin in rabbits, dogs and monkeys. KEY RESULTS: In rabbits, MB07344 (i.v.) decreased total plasma cholesterol (TPC) comparable to that achieved with a maximally effective dose of atorvastatin (p.o.). The addition of MB07344 to atorvastatin resulted in a further decrease in TPC. Similarly, the addition of MB07811 (p.o.) to atorvastatin treatment decreased TPC beyond the level achieved with either agent as monotherapy. In dogs and monkeys, atorvastatin and MB07811 were administered as monotherapy or in combination. Consistent with the rabbit studies, the combination treatment caused a greater decrease in TPC than either MB07811 or atorvastatin administered as monotherapy. CONCLUSIONS AND IMPLICATIONS: We conclude that the effects of MB07811 and atorvastatin in lowering cholesterol are additive in animals. These results would encourage and support the demonstration of similarly improved efficacy of combination versus monotherapy with such agents in the clinic.

Characterization of chemical and enzymatic acid-labile phosphorylation of histone H4 using phosphorus-31 nuclear magnetic resonance.

Phosphorus-31 nuclear magnetic resonance (31P NMR) is used to investigate acid-labile phosphorylation of histone H4. 31P NMR detects phosphorylated histidine residues in in vitro enzymatically phosphorylated H4. The source of kinase is nuclei from either regenerating rat liver or Walker-256 carcinosarcoma. When regenerating rat liver is the source, 31P NMR spectroscopy on the denatured phosphorylated protein exhibits a resonance at 5.3 ppm relative to an 85% orthophosphoric acid external reference. This peak corresponds well with the chemical shift of standard pi-phosphohistidine scanned under similar conditions. Sodium dodecyl sulfate (NaDodSO4)--polyacrylamide gel electrophoresis confirms acid lability. When the source of kinase is Walker-256 carcinosarcoma, the 31P NMR spectrum contains a resonance at 4.9 ppm which corresponds well with standard tau-phosphohistidine run under the same conditions. Chemical phosphorylation of H4 has been accomplished by using dipotassium phosphoramidate which specifically phosphorylated the imidazole moiety of histidine at neutral pH. NaDodSO4--polyacrylamide gel electrophoresis confirms acid lability, and high-pressure liquid chromatography of protein hydrolysates yields phosphohistidine. 31P NMR of chemically phosphorylated H4 in a structured state reveals two peaks at 4.8 and 7.3 ppm with line widths of 9 and 55 Hz, respectively. These resonances indicate that both histidine residues of H4 (His-18 and His-75) are phosphorylated, the latter relatively immobile and the former relatively free in solution. 31P NMR studies on chemically phosphorylated peptide fragments of H4, namely, H4(1-23) and H4(38-102), confirm this model of H4 structure.

High-performance liquid chromatography of acid-stable and acid-labile phosphoamino acids.

A high-performance liquid chromatographic system has been developed which permits the separation of both acid-stable and acid-labile phosphoamino acids. An anion-exchange resin and two buffers of different ionic strength and near neutral pH are used. A low-ionic-strength buffer is used for the separation of N-omega-phosphoarginine and N-epsilon-phospholysine, while the higher-ionic-strength buffer permits the clear separation of tau-phosphohistidine, omicron-phosphoserine and omicron-phosphothreonine. An in-stream fluorometric detection system using omicron-phthalaldehyde permits the rapid analysis of samples containing as little as 25 pmoles of phosphoamino acid. This method has been applied to the detection of tau-phosphohistidine from alkaline digests of chemically phosphorylated calf thymus histone 4 and bovine myelin basic protein.

A simple preparation of N-phosphorylated lysine and arginine.

An improved specific synthesis of N-epsilon-phospholysine is described. Phosphorylation under basic conditions of the copper chelate of L-lysine with phosphorus oxychloride and subsequent removal of the copper ion affords N-epsilon-phospholysine as the sole phosphorylated product. Similar treatment of L-arginine yields N-omega-phosphoarginine as the sole product which is identical to that produced enzymatically.

Colvalent linkage of phospholipid to myelin basic protein: identification of phosphatidylinositol bisphosphate as the attached phospholipid.

Evidence presented demonstrates a covalent attachment of a phospholipid to bovine myelin basic protein. Partial characterization of the phospholipid moiety was performed on myelin basic protein obtained from 32P-phosphorylated whole myelin that was first delipidated by two ether/ethanol (3:2 v/v) extractions, ether extraction, and acetone extraction and then purified by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The myelin basic protein was precipitated with aqueous acetone and treated with proteases. Treatment with carboxypeptidase Y or trypsin for several hours released a lipophilic fragment, which was purified by reverse-phase high-performance liquid chromatography to yield two "lipopeptides". Such lipopeptides were obtained from both the major and minor myelin basic proteins of rat and bovine brain. Treatment with either mild base or phospholipase C removes the lipophilic character of the peptide fragment. The lipophilic fragment is a substrate for phospholipase D, but it does not comigrate on thin-layer chromatography with any 32P-labeled lipid obtained from myelin incubated with [gamma-32P]ATP. Polyphosphoinositides were shown to be released by mild acid treatment of myelin basic protein that had been extracted with organic solvent and then purified by SDS-polyacrylamide gel electrophoresis. Along with the fact that inositol monophosphate was identified in the partial acid hydrolysate of the lipopeptide, we have concluded that polyphosphoinositide (phosphatidylinositol 4-phosphate and/or phosphatidylinositol 4,5-bisphosphate) was the original phospholipid portion of the lipopeptide.

Covalent linkage of phospholipid to myelin basic protein: identification of serine-54 as the site of attachment.

The peptide portion of the lipopeptide isolated from bovine myelin basic protein contained glycine, lysine, and serine in a 2:1:1 molar ratio as determined by amino acid analysis. The N-terminus of the peptide was determined to be glycine. The tetrapeptide Gly53-Ser-Gly-Lys56 was the only segment of myelin basic protein that matched the above two characteristics. This tetrapeptide is highly conserved among the myelin basic proteins sequenced so far. After the selective degradation of the lipopeptide, phosphoserine was identified in the acid hydrolysate, thus indicating that Ser-54 of myelin basic protein in bovine brain is the site of attachment of polyphosphoinositide. Interestingly, serine-54 of myelin basic protein can be phosphorylated by the endogenous protein kinase myelin. However, myelin basic protein phosphorylated by the catalytic subunit of an exogenous soluble protein kinase failed to produce radioactively labeled lipopeptide. Hence the endogenous enzymes of myelin are thought to be involved in the formation of the covalent linkage between polyphosphoinositide and myelin basic protein. The conservation in sequence suggests a possible important structural role for the "phospholipidation" of myelin basic protein.