Structure-activity relationships of pyrimidine nucleotides containing a 5'-α,ß-methylene diphosphonate at the P2Y6 receptor.

The Gq-coupled P2Y6 receptor (P2Y6R) is a component of the purinergic signaling system and functions in inflammatory, cardiovascular and metabolic processes. UDP, the native P2Y6R agonist and P2Y14R partial agonist, is subject to hydrolysis by ectonucleotidases. Therefore, we have synthesized UDP/CDP analogues containing a stabilizing α,ß-methylene bridge as P2Y6R agonists and identified compatible affinity-enhancing pyrimidine modifications. A distal binding region on the receptor was explored with 4-benzyloxyimino cytidine 5'-diphosphate analogues and their potency determined in a calcium mobilization assay. A 4-trifluoromethyl-benzyloxyimino substituent in 25 provided the highest human P2Y6R potency (MRS4554, 0.57 µM), and a 5-fluoro substitution of the cytosine ring in 28 similarly enhanced potency, with >175- and 39-fold selectivity over human P2Y14R, respectively. However, 3-alkyl (31-33, 37, 38), ß-d-arabinofuranose (39) and 6-aza (40) substitution prevented P2Y6R activation. Thus, we have identified new α,ß-methylene bridged N4-extended CDP analogues as P2Y6R agonists that are highly selective over the P2Y14R.

P2Y Receptors Sensitize Mouse and Human Colonic Nociceptors.

Activation of visceral nociceptors by inflammatory mediators contributes to visceral hypersensitivity and abdominal pain associated with many gastrointestinal disorders. Purine and pyrimidine nucleotides (e.g., ATP and UTP) are strongly implicated in this process following their release from epithelial cells during mechanical stimulation of the gut, and from immune cells during inflammation. Actions of ATP are mediated through both ionotropic P2X receptors and metabotropic P2Y receptors. P2X receptor activation causes excitation of visceral afferents; however, the impact of P2Y receptor activation on visceral afferents innervating the gut is unclear. Here we investigate the effects of stimulating P2Y receptors in isolated mouse colonic sensory neurons, and visceral nociceptor fibers in mouse and human nerve-gut preparations. Additionally, we investigate the role of Nav1.9 in mediating murine responses. The application of UTP (P2Y2 and P2Y4 agonist) sensitized colonic sensory neurons by increasing action potential firing to current injection and depolarizing the membrane potential. The application of ADP (P2Y1, P2Y12, and P2Y13 agonist) also increased action potential firing, an effect blocked by the selective P2Y1 receptor antagonist MRS2500. UTP or ADP stimulated afferents, including mouse and human visceral nociceptors, in nerve-gut preparations. P2Y1 and P2Y2 transcripts were detected in 80% and 56% of retrogradely labeled colonic neurons, respectively. Nav1.9 transcripts colocalized in 86% of P2Y1-positive and 100% of P2Y2-positive colonic neurons, consistent with reduced afferent fiber responses to UTP and ADP in Na(v)1.9(-/-) mice. These data demonstrate that P2Y receptor activation stimulates mouse and human visceral nociceptors, highlighting P2Y-dependent mechanisms in the generation of visceral pain during gastrointestinal disease.

Recent advances on patents of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors as antimalarial agents.

INTRODUCTION: Pyrimidine nucleotides are essential for the parasite's growth and replication. Parasites have only a de novo pathway for the biosynthesis of pyrimidine nucleotides. Dihydroorotate dehydrogenase (DHODH) enzyme is involved in the rate-limiting step of the pyrimidine biosynthesis pathway. DHODH is a biochemical target for the discovery of new antimalarial agents. AREA COVERED: This review discussed the development of patented PfDHODH inhibitors published between 2007 and 2023 along with their chemical structures and activities. EXPERT OPINION: PfDHODH enzyme is involved in the rate-limiting fourth step of the pyrimidine biosynthesis pathway. Thus, inhibition of PfDHODH using species-selective inhibitors has drawn much attention for treating malaria because they inhibit parasite growth without affecting normal human functions. Looking at the current scenario of antimalarial drug resistance with most of the available antimalarial drugs, there is a huge need for targeted newer agents. Newer agents with unique mechanisms of action may be devoid of drug toxicity, adverse effects, and the ability of parasites to quickly gain resistance, and PfDHODH inhibitors can be those newer agents. Many PfDHODH inhibitors were patented in the past, and the dependency of Plasmodium on de novo pyrimidine provided a new approach for the development of novel antimalarial agents.

Endogenous purinergic signaling is required for osmotic volume regulation of retinal glial cells.

Intense neuronal activity in the sensory retina is associated with a volume increase of neuronal cells (Uckermann et al., J. Neurosci. 2004, 24:10149) and a decrease in the osmolarity of the extracellular space fluid (Dmitriev et al., Vis. Neurosci. 1999, 16:1157). Here, we show the existence of an endogenous purinergic mechanism that prevents hypoosmotic swelling of retinal glial (Müller) cells in mice. In contrast to the cells from wild-type mice, hypoosmotic stress induced rapid swelling of glial cell somata in retinal slices from mice deficient in P2Y(1), adenosine A(1) receptors, or ecto-5'-nucleotidase (CD73). Consistently, glial cell bodies in retinal slices from wild-type mice displayed osmotic swelling when P2Y(1) or A(1) receptors, or CD73, were pharmacologically blocked. Exogenous ATP, UTP, and UDP inhibited glial swelling in retinal slices, while the swelling of isolated glial cells was prevented by ATP but not by UTP or UDP, suggesting that uracil nucleotides indirectly regulate the glial cell volume via activation of neuronal P2Y(4/6) and neuron-to-glia signaling. It is suggested that autocrine/paracrine activation of purinergic receptors and enzymes is crucially involved in the regulation of the glial cell volume.

Synthesis and evaluation of two series of 4'-aza-carbocyclic nucleosides as adenosine A2A receptor agonists.

The synthesis of two series of 4'-aza-carbocyclic nucleosides are described in which the 4'-substituent is either a reversed amide, relative to the carboxamide of NECA, or an N-bonded heterocycle. Using established purine substitution patterns, potent and selective examples of agonists of the human adenosine A(2A) receptor have been identified from both series. The propionamides 14-18 and the 4-hydroxymethylpyrazole 32 were determined to be the most potent and selective examples from the 4'-reversed amide and 4'-N-bonded heterocyclic series, respectively.

Design, synthesis and antiviral evaluation of novel acyclic phosphonate nucleotide analogs with triazolo[4,5-b]pyridine, imidazo[4,5-b]pyridine and imidazo[4,5-b]pyridin-2(3H)-one systems.

A new series of phosphonylated triazolo[4,5-b]pyridine (1-deaza-8-azapurine), imidazo[4,5-b]pyridine (1-deazapurine) and imidazo[4,5-b]pyridin-2(3H)-one (1-deazapurin-8-one) were synthesized from 2-chloro-3-nitropyridine and selected diethyl É·-aminoalkylphosphonates followed by reduction of the nitro group and cyclization. In the final step O,O-diethylphosphonates were transformed into the corresponding phosphonic acids. All synthesized compounds were evaluated in vitro for inhibitory activity against a broad variety of DNA and RNA viruses and their cytotoxic potencies were also established. Compound 12f showed marginal activity against cytomegalovirus Davis strain (EC50 = 76.47 µM) in human embryonic lung (HEL) cells while compounds 10g (EC50 = 52.53 µM) and 12l (EC50 = 61.70 µM) were minimally active against the varicella-zoster virus Oka strain in HEL cells. Compounds under investigation were not cytotoxic at the maximum concentration evaluated (100 µM).

Antisense gene inhibition by oligonucleotides containing C-5 propyne pyrimidines.

Phosphorothioate oligodeoxynucleotides containing the C-5 propyne analogs of uridine and cytidine bind RNA with high affinity and are potent antisense inhibitors of gene expression. In a cellular assay, gene-specific antisense inhibition occurred at nanomolar concentrations of oligonucleotide, was dose-dependent and exquisitely sensitive to sequence mismatches, and was correlated with the melting temperature and length of oligonucleotide. Activity was independent of RNA target site and cell type but was detectable only when the oligonucleotides were microinjected or delivered with cell-permeabilizing agents. These oligonucleotides may have important applications in therapy and in studies of gene function.

Antitrypanosomal effect of allopurinol: conversion in vivo to aminopyrazolopyrimidine nucleotides by Trypanosoma curzi.

The parasite Trypanosoma cruzi metabolizes allopurinol by a sequential conversion to allopurinol mononucleotide and aminopurinol mononucleotide. The latter is incorporated into RNA. This transformation of a widely used innocuous agent, allopurinol, into a toxic adenine analog appears to account for the antiprotozoan effect of allopurinol. These unique enzymatic activities appear to occur only in T. cruzi and the pathogenic lesihaminae. Allopurinol may serve as a model for the synthesis of similar antiprotozoan agents.

Structure-Activity Relationship of Purine and Pyrimidine Nucleotides as Ecto-5'-Nucleotidase (CD73) Inhibitors.

Cluster of differentiation 73 (CD73) converts adenosine 5'-monophosphate to immunosuppressive adenosine, and its inhibition was proposed as a new strategy for cancer treatment. We synthesized 5'- O-[(phosphonomethyl)phosphonic acid] derivatives of purine and pyrimidine nucleosides, which represent nucleoside diphosphate analogues, and compared their CD73 inhibitory potencies. In the adenine series, most ribose modifications and 1-deaza and 3-deaza were detrimental, but 7-deaza was tolerated. Uracil substitution with N3-methyl, but not larger groups, or 2-thio, was tolerated. 1,2-Diphosphono-ethyl modifications were not tolerated. N4-(Aryl)alkyloxy-cytosine derivatives, especially with bulky benzyloxy substituents, showed increased potency. Among the most potent inhibitors were the 5'- O-[(phosphonomethyl)phosphonic acid] derivatives of 5-fluorouridine (4l), N4-benzoyl-cytidine (7f), N4-[ O-(4-benzyloxy)]-cytidine (9h), and N4-[ O-(4-naphth-2-ylmethyloxy)]-cytidine (9e) ( Ki values 5-10 nM at human CD73). Selected compounds tested at the two uridine diphosphate-activated P2Y receptor subtypes showed high CD73 selectivity, especially those with large nucleobase substituents. These nucleotide analogues are among the most potent CD73 inhibitors reported and may be considered for development as parenteral drugs.

pyr RNA binding to the Bacillus caldolyticus PyrR attenuation protein - characterization and regulation by uridine and guanosine nucleotides.

The PyrR protein regulates expression of pyrimidine biosynthetic (pyr) genes in many bacteria. PyrR binds to specific sites in the 5' leader RNA of target operons and favors attenuation of transcription. Filter binding and gel mobility assays were used to characterize the binding of PyrR from Bacillus caldolyticus to RNA sequences (binding loops) from the three attenuation regions of the B. caldolyticus pyr operon. Binding of PyrR to the three binding loops and modulation of RNA binding by nucleotides was similar for all three RNAs. The apparent dissociation constants at 0 degrees C were in the range 0.13-0.87 nm in the absence of effectors; dissociation constants were decreased by three- to 12-fold by uridine nucleotides and increased by 40- to 200-fold by guanosine nucleotides. The binding data suggest that pyr operon expression is regulated by the ratio of intracellular uridine nucleotides to guanosine nucleotides; the effects of nucleoside addition to the growth medium on aspartate transcarbamylase (pyrB) levels in B. subtilis cells in vivo supported this conclusion. Analytical ultracentrifugation established that RNA binds to dimeric PyrR, even though the tetrameric form of unbound PyrR predominates in solution at the concentrations studied.

A-94964, a novel inhibitor of bacterial translocase I, produced by Streptomyces sp. SANK 60404. I. Taxonomy, isolation and biological activity.

Bacterial phospho-N-acetylmuramyl-pentapeptide translocase (translocase I: EC 2.7.8.13) is a key enzyme in peptidoglycan biosynthesis, and a known target of antibiotics. Here we report a novel nucleoside inhibitor against translocase I, A-94964, isolated from the culture broth of the strain Streptomyces sp. SANK 60404. A-94964 inhibited bacterial translocase I with IC50 value of 1.1 microg/ml, and showed antimicrobial activities against Staphylococcus aureus and Enterococcus faecalis with MIC of 100 and 50 microg/ml, respectively. A-94964 did not show cytotoxicity against mammalian cell lines.

Human DEAD-box ATPase DDX3 shows a relaxed nucleoside substrate specificity.

Human DDX3 (hDDX3) is a DEAD-box protein shown to possess RNA-unwinding and adenosine triphosphatase (ATPase) activities. The hDDX3 protein has been implicated in nuclear mRNA export, cell growth control, and cancer progression. In addition, a role of this protein in the replication of human immunodeficiency virus Type 1 and in the pathogenesis of hepatitis C virus has been recently proposed. Its enzymological properties, however, are largely unknown. In this work, we characterized its ATPase activity. We show that hDDX3 ATPase activity is stimulated by various ribo- and deoxynucleic acids. Comparative analysis with different nucleoside triphosphate analogs showed that the hDDX3 ATPase couples high catalytic efficiency to a rather relaxed substrate specificity, both in terms of base selection and sugar selection. In addition, its ability to recognize the L-stereoisomers of both 3' deoxy- and 2',3' dideoxy-ribose, points to a relaxed stereoselectivity. On the basis of these results, we hypothesize the presence of structural determinants on both the base and the sugar moieties, critical for nucleoside binding to the enzyme. Our results expand the knowledge about the DEAD-box RNA helicases in general and can be used for rational design of selective inhibitors of hDDX3, to be tested as potential antitumor and antiviral agents.

Furano- and pyrrolo [2,3-d] pyrimidine nucleosides and their 5'-O-triphospates: synthesis and enzymatic activity.

A series of bicyclic [2,3-d]furano- and pyrrolopyrimidine ribonucleosides were synthesized and converted chemically into corresponding 5'-O-triphosphates. Substrate properties of the triphosphates toward some RNA and DNA polymerases are reported.

Selective regulatory effects of purine and pyrimidine nucleotides on vacuolar transport of amino acids.

The release of amino acids from their vacuolar store was studied in situ, i.e. in cells with selectively permeabilized plasma membrane and functionally intact vacuoles. As we previously described [Roos et al., J. Biol. Chem. 272 (1997) 15849-15855], this transport process is regulated by extravacuolar adenylates at their physiological concentrations. We now show, using our test object Penicillium cyclopium, that not only purine but also pyrimidine nucleotides are involved in the control of efflux of vacuolar phenylalanine. At 0.1 mM adenosine or guanosine phosphates inhibit, whereas cytidine or uridine phosphates stimulate the rate of efflux. At 1 mM the same nucleotides have no measurable impact on efflux but abolish the effects of other nucleotides present at 0.1 mM. This argues for at least two interacting binding sites with different nucleotide affinities. The minimum structural requirement for any of the observed effects is a non-cyclic ribonucleoside monophosphate. In intact cells, cytosolic concentrations of ATP (representing purine nucleotides) and CTP (representing pyrimidine nucleotides) are 1-2 mM and 0.05-0.2 mM, respectively. ATP is therefore assumed to dominate transport control and allow optimum efflux (and uptake) rates. Short-time starvation of carbon and nitrogen adjusts CTP and ATP at levels that cause declining efflux rates. During prolonged starvation both nucleotides fall below their transport-controlling concentrations and thus allow increasing rates of efflux from the still maintained vacuolar pool. Hence, efflux control under nutrient limitation includes an interplay of purine and pyrimidine nucleotides which precisely regulates the release of vacuolar amino acids and enables flexible adjustment to either amino acid saving or cell survival.

Pyrimidine nucleotides impair phosphoribosylpyrophosphate (PRPP) synthetase subunit aggregation by sequestering magnesium. A mechanism for the decreased PRPP synthetase activity in hereditary erythrocyte pyrimidine 5'-nucleotidase deficiency.

The activity of phosphoribosylpyrophosphate (PRPP) synthetase (ATP: D-ribose-5-phosphate pyrophosphotransferase, EC 2.7.6.1) is decreased in the erythrocyte in hereditary pyrimidine 5'-nucleotidase (P5N) deficiency. Given the increased pyrimidine nucleotide content of the P5N-deficient erythrocyte, we evaluated the effects of prototypic pyrimidine nucleotides on the activity of PRPP synthetase. In normal hemolysate a 1.0 mM combination of cytidine tri-, di- and monophosphate (CTP/CDP/CMP) inhibited PRPP synthetase activity and changed the ribose 5-phosphate (R5P) saturation curve from a hyperbola to a biphasic shape. Untreated crude hemolysate from P5N-deficient erythrocytes showed a biphasic R5P kinetic curve. Since the activity of PRPP synthetase is dependent on its state of subunit aggregation, we examined PRPP synthetase subunit aggregation using gel permeation chromatography. P5N-deficient erythrocytes had a decreased absolute amount of aggregated PRPP synthetase and almost a total loss of disaggregated PRPP synthetase. Using normal hemolysate, 1 mM CTP/CDP/CMP interfered with the ability of 1.0 mM ATP and 2.0 mM MgCl2 to promote PRPP synthetase subunit aggregation. Increasing the MgCl2 to 6.0 mM overcame the inhibitory effect of CTP/CDP/CMP. Thus, the decreased PRPP synthetase activity of the P5N-deficient erythrocyte is due, at least in part, to the ability of the accumulated pyrimidine nucleotides to sequester magnesium and to interfere with the subunit aggregation of PRPP synthetase.

RNA synthesis in starfish embryos: developmental consequences of its inhibition by formycin.

Embryos of the starfish Asterina pectinifera were examined with regard to their ability to undergo the early events of embryonic development in the presence of formycin, an analogue of adenosine and a reported inhibitor of RNA synthesis. It was shown that in normal embryos the pool of ribonucleoside 5'-triphosphates increased during the period of blastula formation. The increase of the UTP pool was blocked nearly completely by 25 micrograms/ml formycin, and that of the CTP pool was inhibited partially by the same concentration of the drug. On the other hand, the pools of ATP and GTP were the same for both control and formycin-treated embryos. The development of embryos cultured in the presence of 25 micrograms/ml formycin stopped at the early blastula stage. Addition of 100 micrograms/ml each of uridine and cytidine to cultures of embryos that had been placed in 25 micrograms/ml formycin at the onset of blastulation allowed gastrulation to occur, suggesting that the developmental arrest produced by formycin is due primarily to the inhibition of pyrimidine nucleotide biosynthesis.

[Effects of exogenerous nucleotides on the apoptosis of intestinal epithelial cells IEC-6].

OBJECTIVE: To investigate the effects of exogenous nucleotides on apoptosis of a normal rat small intestinal epithelial cell line, IEC-6. METHODS: Cultured IEC-6 cells were treated by four kinds of monophosphate nucleotides and their mixture prepared according to their composition in human milk, then the cell apoptosis was determined by flow cytometry measurement, morphologic characterization, and electron-microscope observation. RESULTS: IEC-6 cells treated with AMP or GMP showed a apotosis peak in flow cytometry measurement, but only AMP produce typical apoptosis characteristics in electron-microscope observation. Pyrimidine nucleotides (UMP and CMP)and nucleotides mixture could not induce apoptosis. However, UMP could significantly eliminate the apoptosis-inducing effects of AMP or GMP. CONCLUSION: Purine nucleotides induce apoptosis of IEC-6, inducing effects of purine nucleotides. pyrimidine nucleotides UMP could abolish the apoptosis-inducing effects of purine nucleotides.

Novel acyclonucleotides: synthesis and antiviral activity of alkenylphosphonic acid derivatives of purines and a pyrimidine.

A series of phosphonoalkenyl and (phosphonoalkenyl)oxy derivatives of purines and a pyrimidine were synthesized. These compounds are the first reported acyclonucleotides which incorporate the alpha,beta-unsaturated phosphonic acid moiety as the phosphate mimic and include compounds in which the acyclic substituent is attached to N-9 of a purine or N-1 of a pyrimidine by either a nitrogen-carbon or a nitrogen-oxygen bond. The phosphonoalkenyl-substituted compounds 7a-c, 8a-c, 9, 10, and 12 were prepared either by Mitsunobu coupling of alcohols with purine or pyrimidine derivatives or by alternative alkylations of the heterocyclic bases. The (phosphonoalkenyl) oxy derivatives 7d-g, 8d-g, and 11 were synthesized by coupling of alcohols with 9-hydroxypurines or a 1-hydroxypyrimidine under Mitsunobu conditions. The novel acyclonucleotides were tested for activity against herpes simplex types 1 and 2 (HSV-1 and HSV-2), varicella zoster virus (VZV), cytomegalovirus (CMV), visna virus, and human immunodeficiency virus type 1 (HIV-1). Guanine derivatives were moderately to extremely cytotoxic, but the adenines were less toxic to cells. At the concentrations tested, (Z)-isomers in the unbranched series had no activity against herpes viruses or HIV-1. (E)-9-[(4-Phosphonobut-3-enyl) oxy]adenine (7d) displayed selective activity against HIV-1, (E)-2,6-diamino-9-(4-phosphonobut-3-enyl) purine (9) showed selective antiretrovirus activity, and (E)-9-[2-(hydroxymethyl)-4-phosphonobut-3-enyl]adenine (7c) showed selective antiherpesvirus (VZV and CMV) activity.

Structure-antiviral activity relationship in the series of pyrimidine and purine N-[2-(2-phosphonomethoxy)ethyl] nucleotide analogues. 1. Derivatives substituted at the carbon atoms of the base.

A series of dialkyl esters of purine and pyrimidine N-[2-(phosphonomethoxy)ethyl] derivatives substituted at position 2, 6, or 8 of the purine base or position 2, 4, or 5 of the pyrimidine base were prepared by alkylation of the appropriate heterocyclic base with 2-chloroethoxymethylphosphonate diester in the presence of sodium hydride, cesium carbonate, or 1,8-diazabicyclo[5,4, 0]undec-7-ene (DBU) in dimethylformamide. Additional derivatives were obtained by the transformations of the bases in the suitably modified intermediates bearing reactive functions at the base moiety. The diesters were converted to the corresponding monoesters by sodium azide treatment, while the free acids were obtained from the diester by successive treatment with bromotrimethylsilane and hydrolysis. None of the PME derivatives in the pyrimidine series, their 6-aza or 3-deaza analogues, exhibited any activity against DNA viruses or retroviruses tested, except for the 5-bromocytosine derivative. Substitution of the adenine ring in PMEA at position 2 by Cl, F, or OH group decreased the activity against all DNA viruses tested. PMEDAP was highly active against HSV-1, HSV-2, and VZV in the concentration range (EC50) of 0.07-2 microg/mL. Also the 2-amino-6-chloropurine derivative was strongly active (EC50 = 0.1-0. 4 microg/mL) against herpes simplex viruses and (EC50 = 0.006-0.3 microg/mL) against CMV and VZV. PMEG was the most active compound of the whole series against DNA viruses (EC50 approximately 0.01-0.02 microg/mL), though it exhibited significant toxicity against the host cells. The base-modified compounds did not show any appreciable activity against DNA viruses except for 7-deazaPMEA (IC50 approximately 7.5 microg/mL) against HIV-1 and MSV. The neutral (diisopropyl, diisooctyl) diesters of PMEA were active against CMV and VZV, while the corresponding monoesters were inactive. The diisopropyl ester of the 2-chloroadenine analogue of PMEA showed substantially (10-100x) higher activity against CMV and VZV than the parent phosphonate. Also, the diisopropyl and diisooctyl ester of PMEDAP inhibited CMV and VZV, but esterification of the phosphonate residue did not improve the activity against either MSV or HIV.

Synthesis of 2'-aminomethyl derivatives of N-(2-(phosphonomethoxy)ethyl) nucleotide analogues as potential antiviral agents.

A series of purine and pyrimidine N-(2-(phosphonomethoxy)ethyl) derivatives bearing aminomethyl, (dimethylamino)methyl, morpholinomethyl, and (trimethylammonio)methyl groups at the 2'-position were synthesized. The compounds were prepared by alkylation of the heterocyclic bases with appropriately substituted (aminoalkyl)oxiranes followed by condensation of the resulting intermediates with dialkyl ((p-tolylsulfonyl)oxy)methanephosphonate and subsequent treatment of the obtained diester with bromotrimethylsilane. 9-(3-Amino-2-(phosphonomethoxy)propyl)adenine (2a) proved active against varicella zoster virus (VZV), cytomegalovirus (CMV), and Moloney murine sarcoma virus (MSV) in the concentration range of 7-35 micrograms/mL. None of the other aminoalkyl derivatives demonstrated significant antiviral activity against herpes simplex virus type 1 and 2 (HSV-1 and HSV-2), VZV, (CMV), vaccinia virus (VV), MSV, and human immunodeficiency virus type 1 and 2 (HIV-1 and HIV-2).