Synthesis and biological evaluation of 5'-C-methyl nucleotide prodrugs for treating HCV infections.

Nucleotide prodrugs are of great clinical interest for treating a variety of viral infections due to their ability to target tissues selectively and to deliver relatively high concentrations of the active nucleotide metabolite intracellularly. However, their clinical successes have been limited, oftentimes due to unwanted in vivo metabolic processes that reduce the quantities of nucleoside triphosphate that reach the site of action. In an attempt to circumvent this, we designed novel nucleosides that incorporate a sterically bulky group at the 5'-carbon of the phosphoester prodrug, which we reasoned would reduce the amounts of non-productive PO bond cleavage back to the corresponding nucleoside by nucleotidases. Molecular docking studies with the NS5B HCV polymerase suggested that a nucleotide containing a 5'-methyl group could be accommodated. Therefore, we synthesized mono- and diphosphate prodrugs of 2',5'-C-dimethyluridine stereoselectively and evaluated their cytotoxicity and anti-HCV activity in the HCV replicon assay. All four prodrugs exhibited anti-HCV activity with IC50 values in the single digit micromolar concentrations, with the 5'(R)-C-methyl prodrug displaying superior potency relative to its 5'(S)-C-methyl counterpart. However, when compared to the unmethylated prodrug, the potency is poorer. The poorer potency of these prodrugs may be due to unfavorable steric interactions of the 5'-C-methyl group in the active sites of the kinases that catalyze the formation of active triphosphate metabolite.

2',3'-Dideoxyuridine triphosphate conjugated to SiO2 nanoparticles: Synthesis and evaluation of antiproliferative activity.

A conjugate of triphosphorylated 2',3'-dideoxyuridine (ddU) with SiO2 nanoparticles was obtained via the CuAAC click chemistry between a γ-alkynyl ddU triphosphate and azido-modified SiO2 nanoparticles. Assessment of cytotoxicity in human breast adenocarcinoma MCF7 cells demonstrated that ddU triphosphate conjugated to SiO2 nanoparticles exhibited a 50% decrease in cancer cell growth at a concentration of 183 ±â€¯57 µg/mL, which corresponds to 22 ±â€¯7 µM of the parent nucleotide, whereas the parent nucleoside, nucleotide and alkynyl triphosphate precursor do not show any cytotoxicity. The data provide an example of remarkable potential of novel conjugates of SiO2 nanoparticles with phosphorylated nucleoside analogues, even those, which have not been used previously as therapeutics, for application as new anticancer agents.

Synthesis and structure-activity relationship of uracil nucleotide derivatives towards the identification of human P2Y6 receptor antagonists.

P2Y6 receptor (P2Y6-R) is involved in various physiological and pathophysiological events. With a view to set rules for the design of UDP-based reversible P2Y6-R antagonists as potential drugs, we established structure-activity relationship of UDP analogues, bearing modifications at the uracil ring, ribose moiety, and the phosphate chain. For instance, C5-phenyl- or 3-NMe-uridine-5'-α,ß-methylene-diphosphonate, 16 and 23, or lack of 2'-OH, in 12-15, resulted in loss of both agonist and antagonist activity toward hP2Y6-R. However, uridylyl phosphosulfate, 19, selectively inhibited hP2Y6-R (IC50 112 µM) versus P2Y2/4-Rs. In summary, we have established a comprehensive SAR for hP2Y6-R ligands towards the development of hP2Y6-R antagonists.

Exploring the role of the 5-substituent for the intrinsic fluorescence of 5-aryl and 5-heteroaryl uracil nucleotides: a systematic study.

Derivatives of UMP (uridine monophosphate) with a fluorogenic substituent in position 5 represent a small but unique class of fluorophores, which has found important applications in chemical biology and biomolecular chemistry. In this study, we have synthesised a series of derivatives of the uracil nucleotides UMP, UDP and UTP with different aromatic and heteroaromatic substituents in position 5, in order to systematically investigate the influence of the 5-substituent on fluorescence emission. We have determined relevant photophysical parameters for all derivatives in this series, including quantum yields for the best fluorophores. The strongest fluorescence emission was observed with a 5-formylthien-2-yl substituent in position 5 of the uracil base, while the corresponding 3-formylthien-2-yl-substituted regioisomer was significantly less fluorescent. The 5-(5-formylthien-2-yl) uracil fluorophore was studied further in solvents of different polarity and proticity. In conjunction with results from a conformational analysis based on NMR data and computational experiments, these findings provide insights into the steric and electronic factors that govern fluorescence emission in this class of fluorophores. In particular, they highlight the interplay between fluorescence emission and conformation in this series. Finally, we carried out ligand-binding experiments with the 5-(5-formylthien-2-yl) uracil fluorophore and a UDP-sugar-dependent glycosyltransferase, demonstrating its utility for biological applications. The results from our photophysical and biological studies suggest, for the first time, a structural explanation for the fluorescence quenching effect that is observed upon binding of these fluorophores to a target protein.

Prebiotic synthesis: phosphorylation in aqueous solution.

Formation of uridine-5'-phosphate from uridine and inorganic phosphate in aqueous solution is effected by the following condensing agents: cyanogen, cyanoformamide, cyanate, cyanamide, thioformate, ethylisocyanide, and a watersoluble carbodiimide. The yields are always small, even when a large excess of condensing agent is used. The cyclization of uridine-3'-(2')-phosphate occurs under the same conditions but in much greater yield.

Cyanovinyl phosphate: a prebiological phosphorylating agent?

Cyanovinyl phosphate may be prepared by the addition reaction of phosphate to cyanoacetylene. Kinetic studies have established that cyanovinyl phosphate undergoes a slow pseudo-first-order hydrolysis in aqueous solution. Cyanovinyl phosphate converts uridine to uridine monophosphate and phosphate to pyrophosphate.

Molecular recognition in the P2Y(14) receptor: Probing the structurally permissive terminal sugar moiety of uridine-5'-diphosphoglucose.

The P2Y(14) receptor, a nucleotide signaling protein, is activated by uridine-5'-diphosphoglucose 1 and other uracil nucleotides. We have determined that the glucose moiety of 1 is the most structurally permissive region for designing analogues of this P2Y(14) agonist. For example, the carboxylate group of uridine-5'-diphosphoglucuronic acid proved to be suitable for flexible substitution by chain extension through an amide linkage. Functionalized congeners containing terminal 2-acylaminoethylamides prepared by this strategy retained P2Y(14) activity, and molecular modeling predicted close proximity of this chain to the second extracellular loop of the receptor. In addition, replacement of glucose with other sugars did not diminish P2Y(14) potency. For example, the [5'']ribose derivative had an EC(50) of 0.24muM. Selective monofluorination of the glucose moiety indicated a role for the 2''- and 6''-hydroxyl groups of 1 in receptor recognition. The beta-glucoside was twofold less potent than the native alpha-isomer, but methylene replacement of the 1''-oxygen abolished activity. Replacement of the ribose ring system with cyclopentyl or rigid bicyclo[3.1.0]hexane groups abolished activity. Uridine-5'-diphosphoglucose also activates the P2Y(2) receptor, but the 2-thio analogue and several of the potent modified-glucose analogues were P2Y(14)-selective.

Synthesis and Anti-HCV Activities of 4'-Fluoro-2'-Substituted Uridine Triphosphates and Nucleotide Prodrugs: Discovery of 4'-Fluoro-2'- C-methyluridine 5'-Phosphoramidate Prodrug (AL-335) for the Treatment of Hepatitis C Infection.

We report the synthesis and biological evaluation of a series of 4'-fluoro-2'- C-substituted uridines. Triphosphates of the uridine analogues exhibited a potent inhibition of hepatitis C virus (HCV) NS5B polymerase with IC50 values as low as 27 nM. In an HCV subgenomic replicon assay, the phosphoramidate prodrugs of these uridine analogues demonstrated a very potent activity with EC50 values as low as 20 nM. A lead compound AL-335 (53) demonstrated high levels of the nucleoside triphosphate in vitro in primary human hepatocytes and Huh-7 cells as well as in dog liver following a single oral dose. Compound 53 was selected for the clinical development where it showed promising results in phase 1 and 2 trials.

Synthesis and potency of novel uracil nucleotides and derivatives as P2Y2 and P2Y6 receptor agonists.

The phosphate, uracil, and ribose moieties of uracil nucleotides were varied structurally for evaluation of agonist activity at the human P2Y(2), P2Y(4), and P2Y(6) receptors. The 2-thio modification, found previously to enhance P2Y(2) receptor potency, could be combined with other favorable modifications to produce novel molecules that exhibit high potencies and receptor selectivities. Phosphonomethylene bridges introduced for stability in analogues of UDP, UTP, and uracil dinucleotides markedly reduced potency. Truncation of dinucleotide agonists of the P2Y(2) receptor, in the form of Up(4)-sugars, indicated that a terminal uracil ring is not essential for moderate potency at this receptor and that specific SAR patterns are observed at this distal end of the molecule. Key compounds reported in this study include 9, alpha,beta-methylene-UDP, a P2Y(6) receptor agonist; 30, Up(4)-phenyl ester and 34, Up(4)-[1]glucose, selective P2Y(2) receptor agonists; dihalomethylene phosphonate analogues 16 and 41, selective P2Y(2) receptor agonists; 43, the 2-thio analogue of INS37217 (P(1)-(uridine-5')-P(4)-(2'-deoxycytidine-5')tetraphosphate), a potent and selective P2Y(2) receptor agonist.

Stereospecific synthesis of sugar-1-phosphates and their conversion to sugar nucleotides.

As Leloir glycosyltransferases are increasingly being used to prepare oligosaccharides, glycoconjugates, and glycosylated natural products, efficient access to stereopure sugar nucleotide donor substrates is required. Herein, the rapid synthesis and purification of eight sugar nucleotides is described by a facile 30 min activation of nucleoside 5'-monophosphates bearing purine and pyrimidine bases with trifluoroacetic anhydride and N-methylimidazole, followed by a 2 h coupling with stereospecifically prepared sugar-1-phosphates. Tributylammonium bicarbonate and tributylammonium acetate were the ion-pair reagents of choice for the C18 reversed-phase purification of 6-deoxysugar nucleotides, and hexose or pentose-derived sugar nucleotides, respectively.

Synthesis and structure-activity relationships of uracil nucleotide derivatives and analogues as agonists at human P2Y2, P2Y4, and P2Y6 receptors.

A series of UTP, UDP, and UMP derivatives and analogues were synthesized and evaluated at the human pyrimidinergic P2Y receptor subtypes P2Y2, P2Y4, and P2Y6 stably expressed in 1321N1 astrocytoma cells. Substituents at N3 of UTP were poorly tolerated by P2Y2 and P2Y4 receptors. In contrast, a large phenacyl substituent at N3 of UDP was well tolerated by the P2Y6 receptor, yielding a potent and selective P2Y6 receptor agonist (3-phenacyl-UDP, EC50=70 nM, >500-fold selective). The most potent and selective P2Y2 receptor agonist of the present series was 2-thio-UTP (EC50=50 nM, >or=30-fold selective vs P2Y4 and P2Y6). All modifications at the uracil base of UTP led to a decrease in potency at the P2Y4 receptor. A beta,gamma-dichloromethylene modification in the triphosphate chain of 5-bromo-UTP was tolerated by all three receptor subtypes, thus opening up a new strategy to obtain ectonucleotide diphosphohydrolase- and phosphatase-resistant P2Y2, P2Y4, and P2Y6 receptor agonists.

Efficient Synthesis of 5-Carboxy-2'-Deoxypyrimidine Nucleoside 5'-Triphosphates.

An efficient P(V)-N activation method for the synthesis of 5-carboxy-2'-deoxyuridine and 5-carboxy-2'-deoxycytidine triphosphates directly from the corresponding phosphoropiperidate precursors has been developed.

Polymer-linked 6-azauridine 5'-monophosphate, a resin of high bioaffinity to orotidine-5'-phosphate decarboxylase.

Condensation of 6-azauridine with ethyl levulinate, followed by saponification or phosphorylation, leads to 2',3'-O-[1-(2-carboxyethyl)ethylidene]-6-azauridine and its 5'-monophosphate. The latter was coupled to 6-aminohexylagarose via its carboxylic group. Using the same synthetic route, agarose-linked uridine 5'-monophosphate has been prepared. Both polymers show specific binding toward orotidine-5'-monophosphate decarboxylase. The immobilized inhibitor (6-azauridine 5'-monophosphat) binds the enzyme more strongly than the immobilized uridine 5'-monophosphate. Both resins have been used to separate orotidine-5'-monophosphate decarboxylase from orotidine-5'-monophosphate pyrophosphorylase.

Thymidylate synthetase inhibitors. Synthesis of N-substituted 5-aminomethyl-2'-deoxyuridine 5'-phosphates.

A series of substituted 5-aminomethyl-2'-deoxyuridines was synthesized as analogues of 5-thymidylyltetrahydrofolic acid, a proposed intermediate in the thymidylate synthetase catalyzed reaction. 1-(3,5-Di-O-p-toluoyl-2-deoxy-beta-D-ribofuranosyl)-5-chloromethyluracil (3) was treated with the appropriate amine to give the ester protected 5-aminomethyl nucleoside. Removal of the ester groups was accomplished with anhydrous potassium carbonate in methanol to afford the free beta-nucleoside. In this way 5-(2-dimethylaminoethylaminomethyl)-2'-deoxyuridine (5a), 5-dimethylaminomethyl-2'-deoxyuridine (5b), 5-N-mehtylpiperazinylmethyl-2'-deoxyuridine (5c), and 5-pyrrolidinylmethyl-2'-deoxyuridine (5d) were prepared. Compounds 5a,b,d were converted to the respective 5'-phosphates 6a,b,d. All three compounds were subtrate competitive inhibitors of thymidylate synthetase purified from Escherichia coli, calf thymus, and Ehrlich ascites tumor cells. The most active compound was 6a with KI's of 6,3.1, and 14 micronM observed for the respective enzymes.

A potent multisubstrate analogue inhibitor of human thymidylate synthetase.

The synthesis of an 8-deazafolate analogue of the intermediate in the methylation of 2'-deoxyuridylate is described. Alkylation of diethyl 5,6,7,8-tetrahydro-8-deazafolate with 3'-O-acetyl-5-(bromomethyl)-2'-deoxyuridine 5'-[bis-(trichlorethyl) phosphate], followed by removal of the trichloroethyl groups with a Zn/Cu couple and mild saponification, gave the target inhibitor N-[4-[[[2-amino-3,4,5,6,7, 8-hexahydro-4-oxo-5-[(2'-deoxyuridin-5-yl)methyl]-pyrido[3,2-d] pyrimidin-6-yl]methyl]amino]benzoyl]-L-glutamic acid 5'-monophosphate. The free nucleoside and the 5'-(methyl phosphate) diester were similarly prepared. Each of these reactions yielded a pair of diastereoisomers about C-6 of the reduced deazafolate in approximately a 1:1 ratio. These diastereoisomeric mixtures were evaluated as inhibitors of thymidylate synthetase derived from human tumor (HeLa) cells. The 5'-monophosphate was a potent inhibitor, competitive with respect to both 2'-deoxyuridylate (Ki = 0.06 microM) and tetrahydrofolate (Ki = 0.25 microM). In contrast, the nucleoside and the nucleotide methyl ester were poorer inhibitors by more than 3 orders of magnitude, attesting to the importance of the anionic function at the nucleoside 5'-position in the affinity of an inhibitor for the enzyme active site.

Synthetic inhibitors of Escherichia coli, calf thymus, and Ehrlich ascites tumor thymidylate synthetase.

In a study of active site binding the inhibition of thymidylate synthetase derived from Escherichia coli, calf thymus, and Ehrlich ascites tumor was examined using eight inhibitors. 5-Substituted 2'-deoxyuridine 5'-phosphate analogues used in this study are the hydroxymethyl, methoxymethyl, benzyloxymethyl, formyl, acetyl, allyl, and two potential active site alkylating substituents: 2,3-oxypropyl and the azidomethyl analogues. All compounds were competitive with the substrate, 2'-deoxyuridine 5'-phosphate; the most potent inhibitor was 5-formyl-dUMP (Ki = 0.1, 0.09, and 0.08 muM for the respective enzyme). The 5-hydroxymethyl, 5-benzyloxymethyl, and 5-azidomethyl derivatives of dUMP showed some differential inhibition; these compounds were two to three times more active against the ascites tumor enzyme than against the thymus enzyme.

Synthesis and antitumor and antiviral properties of 5-alkyl-2'-deoxyuridines, 3',5'-cyclic monophosphates, and neutral cyclic triesters.

A series of 5-alkyl-2'-deoxyuridine 3',5'-cyclic monophosphates (5-R-cdUMP's, R = Et, i-Pr, n-Pr, n-Bu, n-Pent, n-Hex, n-Oct) was prepared and tested in culture systems as antitumor and antiviral agents in comparison to the 5-alkyl-2'-deoxyuridines (5-R-dUrd's) themselves. Only the 5-Et- and 5-n-Bu-cdUMP showed appreciable cytostatic activities against murine L1210 and human lymphoblast Raji cells (ID50 range: 28-82 micrograms/mL). 5-Et-dUrd itself was much more active (ID50 = 1.6 and 2.9 micrograms/mL). The 5-i-Pr-, and 5-n-Bu-dUrd's were inactive, but activity increased again for groups with chain lengths of five carbons or greater. 5-Et-cdUMP and 5-Et-dUrd had greatly reduced activities against deoxythymidine kinase deficient (TK-) L1210 and Raji cells. 5-Et-cdUMP evidently is not an efficient prodrug source of the corresponding 5'-monophosphate where the TK- cells are concerned. Of the 5-R-cdUMP's, 5-Et-cdUMP displayed reasonably good antiviral potency against herpes simplex types 1 and 2 (MIC50, mostly 7-70 micrograms/mL) and vaccinia virus (MIC, 70 micrograms/mL). The activity was nonetheless 10- to 100-fold less than that for 5-Et-dUrd. The other 5-R-dUrd's generally showed decreasing antiviral activity with increasing 5-R chain length. Methyl and/or benzyl neutral triesters of certain 5-R-cdUMP's were inactive as antivirals and largely inactive against tumor cells in culture. In contrast to the 5'-monophosphates, the 5-R-cdUMP's failed to inhibit thymidylate synthetase from L1210 cells.

Synthesis and antiviral activity of novel 5-(1-cyanamido-2-haloethyl) and 5-(1-hydroxy(or methoxy)-2-azidoethyl) analogues of uracil nucleosides.

A new class of 5-(1-cyanamido-2-haloethyl)-2'-deoxyuridines (4-6) and arabinouridines (7, 8) were synthesized by the regiospecific addition of halogenocyanamides (X-NHCN) to the 5-vinyl substituent of the respective 5-vinyl-2'-deoxyuridine (2) and 2'-arabinouridine (3). Reaction of 2 with sodium azide, ceric ammonium nitrate, and acetonitrile-methanol or water afforded the 5-(1-hydroxy-2-azidoethyl)-(10) and 5-(1-methoxy-2-azidoethyl)-2'-deoxyuridines (11). In vitro antiviral activities against HSV-1-TK(+) (KOS and E-377), HSV-1-TK(-), HSV-2, VZV, HCMV, and DHBV were determined. Of the newly synthesized compounds, 5-(1-cyanamido-2-iodoethyl)-2'-deoxyuridine (6) exhibited the most potent anti-HSV-1 activity, which was equipotent to acyclovir and superior to 5-ethyl-2'-deoxyuridine (EDU). In addition, it was significantly inhibitory for thymidine kinase deficient strain of HSV-1 (EC(50) = 2.3-15.3 microM). The 5-(1-cyanamido-2-haloethyl)-2'-deoxyuridines (4-6) all were approximately equipotent against HSV-2 and were approximately 1.5- and 15-fold less inhibitory for HSV-2 than EDU and acyclovir, respectively. Compounds 4-6 were all inactive against HCMV but exhibited appreciable antiviral activity against VZV. Their anti-VZV activity was similar or higher to that of EDU and approximately 5-12-fold lower than that of acyclovir. The 5-(1-cyanamido-2-haloethyl)-(7,8) analogues of arabinouridine were moderately inhibitory for VZV and HSV-1 (strain KOS), whereas compounds 10 and 11 were inactive against herpes viruses. Compounds 5 and 6 also demonstrated modest anti-hepatitis B virus activity against DHBV (EC(50) = 19.9-23.6 microM). Interestingly, the related 5-(1-azido-2-bromoethyl)-2'-deoxyuridine (1n) analogue proved to be markedly inhibitory to DHBV replication (EC(50) = 2.6-6.6 microM). All compounds investigated exhibited low host cell toxicity to several stationary and proliferating host cell lines as well as mitogen-stimulated proliferating human T lymphocytes.

Inhibition of HIV-1 replication in cultured cells with phosphorylated dideoxyuridine derivatives encapsulated in immunoliposomes.

Among the 2',3'-dideoxynucleoside 5'-triphosphates containing a physiological base, 2',3'-dideoxyuridine 5'-triphosphate (ddUTP) has been reported to be among the most powerful inhibitors of human immunodeficiency virus (HIV) reverse transcriptase (RT) in cell-free systems. However, in contrast to other dideoxynucleosides, 2',3'-dideoxyuridine (ddU) is inactive in treatment of HIV-infected cells in culture, since it is a poor substrate for cellular nucleoside kinases. This problem cannot be overcome by the use of phosphorylated ddU because such compounds are unable to cross cell membranes. To promote entry and thus bypass the limiting steps of intracellular phosphorylation, we have encapsulated mono- and tri-phosphorylated ddU in liposomes coupled to monoclonal antibodies (immunoliposomes). We investigated antiviral effects in two human T cell lines (MT-4, CEM). We observed that ddU nucleotides remain phosphorylated for several weeks after encapsulation in immunoliposomes, and potent antiviral activity is obtained when these drugs are delivered into infected cells by cell-specific antibodies (ED50 < or = 1 microM on CEM). In contrast, no inhibition was observed with non-targeted liposomes containing phosphorylated ddU, or with empty liposomes, whether targeted or not.

Inhibitory effects of nucleoside 5'-alkylphosphates on sexual agglutination in Saccharomyces cerevisiae.

Among various nucleoside 5'-alkylphosphates, uridine 5'-hexadecylphosphate (UMPC16) and adenosine 5'-hexadecylphosphate (AMPC16) inhibited the sexual agglutination between a and alpha haploid cells of Saccharomyces cerevisiae. The effect of AMPC16 accompanied severe growth inhibition of the yeast cells but it was not observed with UMPC16. Sexual agglutination was not inhibited by the presence of UMPC16 or AMPC16 when the yeast cells had been pretreated with the mating pheromone. UMPC16 was characterized as a specific inhibitor of sexual agglutination without direct influence on the agglutinin function, being distinguishable from any of those ever known.