Antiviral Activity of Some 2'-Deoxyuridine 5-Arylethynyl Derivatives

5-(3-Perylenylethynyl)-2'-deoxyuridine was prepared by crosslinking 5-iodo-2'-deoxyuridine derivatives with 3-ethynylperylene followed by deprotection. 5-(1-Perylenylethynyl)-, 5-(3-perylenylethynyl)-, and 5-[4-(2-benzoxazolyl)phenylethynyl]-2'-deoxyuridine were found to inhibit in Vero cells the replication of type 1 herpes simplex virus and its drug-resistant strains.     

Measurement of 5-fluoro-2'-deoxyuridine serum levels by gas chromatography chemical ionization mass spectrometry

Serum levels of 5-fluoro-2'-deoxyuridine in cancer treated patients were measured by gas chromatography mass spectrometry under chemical ionization conditions; 1-(2-deoxy-beta-D-lyxofuranosyl)-5-fluorouracil (3'-epi-5-fluoro-2'-deoxyuridine) was used as an internal standard. The drug and internal standard were quantitatively isolated from the serum sample by a mini-column anion exchange method and the extract permethylated using potassium-tert-butoxide in dimethylsulphoxide and methyl iodide. The derivatized nucleosides were then re-extracted from the reaction mixture and analysed on a glass capillary column coated with Superox-4. The column was coupled directly to the chemical ionization source of the mass spectrometer; NH3 was used as the reagent gas. The gas chromatographic effluent was monitored at m/z 289, the [MH]+ ion of the N,O-permethyl derivatives of 5-fluoro-2'-deoxyuridine and the internal standard. Recovery of 5-fluoro-2'-deoxyuridine from serum in the 0-1 microgram ml-1 concentration range averaged 93 +/- 2% (SD); a linear detector response was observed up to 50 ng 5-fluoro-2'-deoxyuridine ml-1. At the 10 ng ml-1 level, a within-run assay precision of 10% (CV) (n = 5) was found, while a detection limit of about 1 ng 5-fluoro-2'-deoxyuridine ml-1 of serum was attained. The method was applied to the measurement of disappearance curves of 5-fluoro-2'-deoxyuridine in the serum of treated patients.     

Perylene attached to DNA through stiff or flexible linker: duplex stability and FRET

Two fluorescent nucleosides, 5-(perylen-3-ylethynyl)-2'-deoxyuridine and 5-[(perylen-3-yl)methoxypropyn-1-yl]-2'-deoxyuridine, were incorporated into synthetic oligodeoxyribonucleotides and spectral properties of the conjugates and their duplexes were studied.     

Synthesis of N,N-dialkylaniline-2'-deoxyuridine conjugates for DNA-mediated electron transfer studies

Syntheses of two analogs of deoxyuridine with N,N-dialkylaniline chromophores are reported. 5-[3-(N-methylphenylamino)propanoyl]-2'-deoxyuridine (1) and 5-[2-(4-N,N-dimethylaminophenyl)ethyl)]-2'-deoxyuridine (2) are prepared by palladium-mediated coupling. Preparation of 2 was facilitated by in situ transient O4-trimethylsilyl protection during alkynylation which suppressed secondary cyclization of the coupling adduct.     

A comparison of the conformations adopted by some 5-bromovinyl-2''-deoxyuridines and a correlation with their antiviral properties: an X-ray study.

Crystal structures of (Z)-5-(2-bromovinyl)-2'-deoxyuridine, 3',5'-di-O-acetyl-(E)-5-(2-bromovinyl)-2'-deoxyuridine and 3',5'-di-O-p-chlorobenzoyl-5-(2-dibromovinyl)-2'-deoxyuridine are compared with each other and with that of the most potent antiviral agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (E-BVDU) reported earlier. A comparison of the conformation of 3',5'-di-O-acetyl-pyrimidine nucleoside structures in which intermolecular hydrogen bond network formation is minimized, with those of their parent compounds has shown that the greatest change in rotation about the glycosyl bond and in the sugar ring pucker is exhibited by E-BVDU. Upon acylation this molecule changes from C2'-endo/C3'-exo conformation to C3'-endo/C4'-exo conformation. The relevance of these structures upon the biological activity of the nucleosides and in particular to their ability to be a substrate for thymidine kinase is discussed.     

Synthesis, conversion and biological activity of 5-substituted 2'-deoxyuridines modified by carbohydrates]

5-Benzyloxymethyl(Bom)-2'-deoxyuridine and its alpha-anomer were used as the key compounds for syntheses of thymidine analogues or 3'-derivatives. Anomeric 5-Bom-2'-deoxyuridines were synthesized from 5-Bom-uracil and 2-deoxy-3,5-di-O-p-toluyl-alpha-D-ribo-furanosyl chloride by means of the silyl method. 5-Bom-2'-deoxyuridine was transformed successively to 3',5'-di-O-mesyl derivative, 2,3'-anhydro-1-(2-deoxy-5-O-p-toluyl-beta-D-xylofuranosyl)-5-Bom-uracil and 3'-azido-2',3'-dideoxy-5-Bom-uridine. Treatment of the last with SnCl4 in methylene dichloride--methanol led to 3'-azido-2',3'-dideoxy-5-methoxymethyluridine. Under the same conditions the 5-methoxymethyl derivative was obtained from 3',5'-di-O-p-toluyl-5-Bom-2'-deoxyuridine. Interaction of 1-(2-deoxy-alpha-D-ribofuranosyl)-4-Bom-uracil with SnCl4 in methylene dichloride as well as the hydrogen transfer hydrogenolysis in the presence of cyclohexene and Pd(OH)2/C in ethanol led to 1-(2-deoxy-alpha-D-ribofuranosyl)-5-hydroxymethyluracil. Only 3'-azido-2',3'-dideoxy-5-Bom-uridine showed a cytotoxic activity against CaOv cells in vitro: in 10(-5)-10(-4) M concentrations it inhibits the thymidine incorporation into DNA by 78.8-95.1%. Elucidation of antitumor activity in vivo showed that this nucleoside inhibits growth of solid tumours, Ca755 and LLC, by 79 and 79-83%, respectively, but has no therapeutic effect against lympholeukemia P388.     

New reagents and methods for the synthesis of internal and 3'-labeled DNA

The syntheses of two new nucleoside phosphoramidites containing a hydroxyl functionality masked by a levulinate protecting group are presented; N(4)-(2-(ethylene glycol-2-levulinate)ethyl)-5-methyl-5'-(4,4'-dimethoxytrityl)-3'-O-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxycytidine 1 and 5-(N-(6-O-levulinoyl-1-aminohexyl)-3(E)-acrylamido)-5'-(4,4'-dimethoxytrityl)-3'-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxyuridine 3. Optimization of solid-phase-supported synthetic parameters for incorporation of these into DNA, removal of the levulinate group by exposure to dilute hydrazine, and subsequent attachment of dye labels is described. Synthesis of the known compound 5-(N-(6-trifluoroacetylaminohexyl)-3(E)-acrylamido)-5'-(4,4'-dimethoxytrityl)-3'-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxyuridine 2 (1), containing a masked amine at the end of an alkyl chain attached at the 5 position, was also revisited using new techniques developed for 3.     

Murine mammary FM3A carcinoma cells transformed with the herpes simplex virus type 1 thymidine kinase gene are highly sensitive to the growth-inhibitory properties of (E)-5-(2-bromovinyl)-2'-deoxyuridine and related compounds

Murine mammary carcinoma (FM3A TK-/HSV-1 TK+) cells, which are thymidine kinase (TK)-deficient but have been transformed with the herpes simplex virus type 1 (HSV-1) TK gene are inhibited in their growth by (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) and (E)-5-(2-bromovinyl)-2'-deoxycytidine (BVDC) at 0.5, 0.5 and 0.8 ng/ml, respectively; i.e., a concentration 5000 to 20 000-fold lower than that required to inhibit the growth of the corresponding wild-type FM3A/0 cells. Hence, transformation of tumor cells with the HSV-1 TK gene makes them particularly sensitive to the cytostatic action of BVDU and related compounds.     

5-Fluorouracil derivatives. III. A simple method to prepare 5-fluoro-2'-deoxyuridine derivatives

3',5'-Diacyl-2'-bromo-5-fluoro-2'-deoxyuridine (4) was obtained by the reaction of 5, 6-dihydro-6-hydroxy-5-fluorouridine (2) and acyl bromide. Because the route from uridine (1) to 2, the route from 4 to 3',5'-diacyl-5-fluoro-2'-deoxyuridine (5), and the route from 5 to 5-fluoro-2'-deoxyuridine (FUDR, 6) are known reactions, the three step synthesis from uridine to 5 and four step synthesis from uridine to FUDR have been accomplished.     

5-bromo-2''-deoxyuridine-stimulated calcium ion- or magnesium ion-dependent ecto-(adenosine triphosphatase) activity of cultured hamster cardiac cells.

1. Treatment of hamster heart cells in primary culture with 5-bromo-2'-deoxyuridine resulted in the greatly increased activity of a particulate Ca2+- or Mg2+-dependent ATPase (adenosine triphosphatase). 2. 5-Bromo-2'-deoxyuridine exerted these effects only when it was incorporated into cellular DNA, and then in a concentration-dependent manner. 3. Serially replated cells contained less of the activity (expressed as a function of total cell protein) than did the primary cultures, but the stimulation caused by 5-bromo-2'-deoxyuridine addition was much greater. 4. The affected enzyme was apparently localized in the plasma membrane of the cells with its active centre exposed to the outer environment [ecto-(ATPase) dependent on Ca2+ or Mg2+].5. The activity was unaffected by treatment with p-chloromercuriphenylsulphonate, ouabain andverapamil. 6. Ecto (5'-nucleotidase) activity was not increased by 5-bromo-2'-deoxyuridine treatment of cells, and ecto-(p-nitrophenyl phosphatase) activity was only slightly enhanced.     

Delivery of floxuridine derivatives to cancer cells by water-soluble organometallic cages

The self-assembly of 2,4,6-tris(pyridin-4-yl)-1,3,5-triazine (tpt) triangular panels with p-cymene (pPr(i)C(6)H(4)Me) ruthenium building blocks and 2,5-dioxydo-1,4-benzoquinonato (dobq) or 5,8-dioxydo-1,4-naphthoquinonato (donq) bridges, in the presence of a pyrenyl-nucleoside derivatives (pyreneR), affords the triangular prismatic host-guest compounds [(pyrene-R)?Ru(6)(pPr(i)C(6)H(4)Me)(6)(tpt)(2)(dobq)(3)](6+) ([(pyrene-R)?1](6+)) and [(pyrene-R)?Ru(6)(pPr(i)C(6)H(4)Me)(6)(tpt)(2)(donq)(3)](6+) ([(pyrene-R)?2](6+)), respectively. The inclusion of six monosubstitutedpyrenyl-nucleosides (pyrene-R1 = 5'-(1-pyrenyl butanoate)-2'-deoxyuridine, pyrene-R2 = 5-fluoro-5'-(1-pyrenyl butanoate)-2'-deoxyuridine, pyrene-R3 = 5'-{N-[1-oxo-4-(1-pyrenyl)butyl]-glycyl}-2'-deoxyuridine, pyrene-R4 = 5-fluoro-5'-{N-[1-oxo-4-(1-pyrenyl)butyl]-glycyl}-2'-deoxyuridine, pyrene-R5 = 5-fluoro-5'-{N-[1-oxo-4-(1-pyrenyl)butyl]-phenylalanyl}-2'-deoxyvuridine, pyrene-R6 = 5-fluoro-5'-{N-[1-oxo-4-(1-pyrenyl)butyl]-phenylalanyl}-2'-deoxyuridine) has been accomplished. The carceplex nature of [(pyrene-R)?1](6+) with the pyrenyl moiety firmly encapsulated in the hydrophobic cavity of the cage with the nucleoside groups pointing outward was confirmed by NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS), while the host-guest nature of [(pyrene-R)?2](6+) was studied in solution by NMR techniques. In contrast to the floxuridine compounds used in the clinic, the host-guest complexes are highly water-soluble. Consequently, the cytotoxicities of these water-soluble compounds have been established using human ovarian A2780 and A2780cisR cancer cells. All the host-guest systems are more cytotoxic than the empty cages alone [1][CF(3)SO(3)](6) (IC(50) = 23 μM) and [2][CF(3)SO(3)](6) (IC(50) = 10 μM), the most active compound [pyrene-R4?1][CF(3)SO(3)](6)being 2 orders of magnitude more cytotoxic (IC(50) = 0.3 μM) on these human ovarian cancer cell lines (A2780 and A2780cisR).     

Green biosynthesis of floxuridine by immobilized microorganisms

This work describes an efficient, simple, and green bioprocess for obtaining 5-halogenated pyrimidine nucleosides from thymidine by transglycosylation using whole cells. Biosynthesis of 5-fluoro-2'-deoxyuridine (floxuridine) was achieved by free and immobilized Aeromonas salmonicida ATCC 27013 with an 80% and 65% conversion occurring in 1 h, respectively. The immobilized biocatalyst was stable for more than 4 months in storage conditions (4 °C) and could be reused at least 30 times without loss of its activity. This microorganism was able to biosynthesize 2.0 mg L(-1) min(-1) (60%) of 5-chloro-2'-deoxyuridine in 3 h. These halogenated pyrimidine 2'-deoxynucleosides are used as antitumoral agents.     

Gas-liquid chromatographic analysis of fluoropyrimidine nucleosides and fluorouracil in plasma and urine

A gas-liquid chromatographic method employing on-column alkylation and a nitrogen-sensitive detector was developed for the analysis of 5-fluoro-2'-deoxyuridine, 5-fluorouridine, and 5-fluorouracil in plasma and urine. Samples (0.72 ml) containing the fluoropyrimidine and internal standard (5-chloro-2'-deoxyuridine for nucleoside analyses and 6-methyluracil for 5-fluorouracil analyses) were prepared for gas-liquid chromatography by sequential cation-exchange and anion-exchange column chromatography. Recoveries of fluoropyrimidines were 71-95% over the concentration ranges studied. The dried eluate from the anion-exchange column was dissolved in p-tolyltrimethylammonium hydroxide in methanol before gas-liquid chromatographic analysis. Columns packed with either 3% SP-2100 on Supelcoport or 3% OV-1 on Gas-Chrom Q were suitable for nucleoside analyses; a column packed with 0.75% Carbowax 20M-5% KOH on Chromsorb G was used for 5-fluorouracil analyses. The fluoropyrimidine nucleosides were well separated from each other and from the potentially interfering endogenous compounds 2'-deoxyuridine and uridine; 5-fluorouracil was well separated from uracil. Linear standard curves (peak area ratio method) were obtained for plasma containing 0.025 to 20 micrograms FdUrd (0.1 to 81 microM) or 0.05 to 1.0 microgram FUrd (0.2 to 3.8 microM), and for urine containing 0.2 to 1.0 microgram (0.8 to about 4 microM) of the nucleosides. Standard curves for 5-fluorouracil (1.5 to 7.9 microM) and 2'-deoxyuridine (0.9 to 4.4 microM) were also linear. A measurable amount of 5-fluorouracil, equivalent to 4 to 7% of the 5-fluoro-2'-deoxyuridine injected, was formed from the nucleoside on the gas-liquid chromatographic column, requiring correction of 5-fluorouracil concentrations measured in the presence of 5-fluoro-2'-deoxyuridine.     

Supported synthesis and functionnalization of 2'-deoxyuridine by Suzuki-Miyaura cross-coupling

The synthesis and modification of 2'-deoxyuridine has been realized under Suzuki-Miyaura palladium-catalyzed cross-coupling conditions. Using Pd(PPh(3))(4) and Na(2)CO(3), 5-iodo-2'-deoxyuridine bound to solid support is coupled with various boronic acids to give 5-(hetero)aryl-2'-deoxyuridine. Pd(PPh(3))(4) palladium catalyzed was found to be superior to Pd(OAc)(2) and (NHC)Pd(allyl)Cl for Suzuki-Miyaura palladium-catalyzed reactions.     

Selective inhibition of the proliferation of herpes simplex virus type 1 thymidine kinase gene-transformed murine mammary FM3A carcinoma cells by (E)-5-(2-bromovinyl)-2'-deoxyuridine and related compounds

Mouse mammary carcinoma FM3A cells deficient in thymidine kinase were transformed by a cloned gene for herpes simplex virus type 1 thymidine kinase. Among several anti-herpetic nucleoside analogues, (E)-5-(2-bromovinyl)-2'-deoxyuridine, (E)-5-(2-iodovinyl)-2'-deoxyuridine and (E)-5-(2-bromovinyl)-2'-deoxycytidine inhibited the growth of the transformed cells at concentrations 5000- to 20000-fold lower than those required to inhibit the growth of the corresponding wild-type cells. The selective inhibitory action of these compounds was due to a specific phosphorylation by the viral thymidine kinase. From the transformed cells, thymidine-auxotrophic mutants that are deficient in thymidylate synthase were isolated. These mutant cell lines should prove useful in elucidating the mechanism of action of the antiherpetic nucleoside analogues.     

Antisense oligodeoxynucleotides: synthesis, biophysical and biological evaluation of oligodeoxynucleotides containing modified pyrimidines.

6-Azathymidine, 6-aza-2'-deoxycytidine, 6-methyl-2'-deoxyuridine, and 5,6-dimethyl-2'-deoxyuridine nucleosides have been converted to phosphoramidite synthons and incorporated into oligodeoxynucleotides (ODNs). ODNs containing from 1 to 5 of these modified pyrimidines were compared with known 2'-deoxyuridine, 5-iodo-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, 5-fluoro-2'-deoxyuridine, 5-bromo-2'-deoxycytidine, and 5-methyl-2'-deoxycytidine nucleoside modifications. Stability in 10% heat inactivated fetal calf serum, binding affinities to RNA and DNA complements, and ability to support RNase H degradation of targeted RNA in DNA-RNA heteroduplexes were measured to determine structure-activity relationships. 6-Azathymidine capped ODNs show an enhanced stability in serum (7- to 12-fold increase over unmodified ODN) while maintaining hybridization properties similar to the unmodified ODNs. A 22-mer ODN having its eight thymine bases replaced by eight 6-azathymines or 5-bromouracils hybridized to a target RNA and did not inhibit RNase H mediated degradation.     

Structurally diverse 5-substituted pyrimidine nucleosides as inhibitors of Leishmania donovani promastigotes in vitro

The following structurally diverse 5-substituted-2'-deoxyuridine nucleosides displayed potent in vitro antileishmanial activity: 5-formyl, 5-(2,2,-dicyanovinyl)-, 5-(2-cyano-2-ethoxycarbonylvinyl), 5-(2-cyano-2-methoxycarbonylvinyl)-, 5-(2-amino-3-cyano-5-oxo-5,6,7,8-tetrahydro-4H-chromen-4-yl)- and related congeners, and the 5-(3-methyl-5-oxo-1-phenyl-4,5-dihydro-4H-pyrazol-4-ylidene) group.     

Nucleoside conjugates. 8. The preparation of 5-fluoro-2'-deoxyuridine conjugates of corticosteroids

Three 5'-(steroid-21-phosphoryl)-5-fluoro-2'-deoxyuridines (VI-VIII) have been prepared and characterized by uv, ir, 1H-nmr, elemental analysis, chemical and enzymatic hydrolyses. These new compounds are 5-fluoro-2'-deoxyuridine conjugates of cortisol (VI), cortico-sterone (VII), and prednisolone (VIII). Besides the physical and analytical data, all of the conjugates were demonstrated to be enzymatically hydrolyzed to the corresponding steroid and 5-fluoro-2'-deoxyuridine 5'-monophosphate (III), and the latter was further shown to be hydrolyzed to 5-fluoro-2'-deoxyuridine (II) by phosphodiesterase I, 5'-nucleotidase, and acid phosphatase. However, they were shown to be resistant to hydrolysis by bacterial alkaline phosphatase.     

Crystal structure of varicella zoster virus thymidine kinase

Herpes virus thymidine kinases are responsible for the activation of nucleoside antiviral drugs including (E)-5-(2-bromovinyl)-2'-deoxyuridine. Such viral thymidine kinases (tk), beside having a broader substrate specificity compared with host cell enzymes, also show significant variation in nucleoside phosphorylation among themselves. We have determined the crystal structure of Varicella zoster virus (VZV, human herpes virus 3) thymidine kinase complexed with (E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-monophosphate and ADP. Differences in the conformation of a loop region (residues 55-61) and the position of two alpha-helices at the subunit interface of VZV-tk compared with the herpes simplex virus type 1 (human herpes virus 1) enzyme give rise to changes in the positioning of residues such as tyrosine 66 and glutamine 90, which hydrogen bond to the substrate in the active site. Such changes in combination with the substitution in VZV-tk of two phenylalanine residues (in place of a tyrosine and methionine), which sandwich the substrate pyrimidine ring, cause an alteration in the positioning of the base. The interaction of the (E)-5-(2-bromovinyl)-2'-deoxyuridine deoxyribose ring with the protein is altered by substitution of tyrosine 21 and phenylalanine 139 (analagous to herpes simplex virus type 1 histidine 58 and tyrosine 172), which may explain some of the differences in nucleoside sugar selectivity between both enzymes. The altered active site architecture may also account for the differences in the substrate activity of ganciclovir for the two thymidine kinases. These data should be of use in the design of novel antiherpes and antitumor drugs.     

Mechanism of ribonucleotide reductase from herpes simplex virus type 1. Evidence for 3' carbon-hydrogen bond cleavage and inactivation by nucleotide analogs

Isotope effects of 2.5, 2.1, and 1.0 were measured on the conversion of [3'-3H]ADP, [3'-H]UDP, and [5-3H] UDP to the corresponding 2'-deoxynucleotides by herpes simplex virus type 1 ribonucleotide reductase. These results indicate that the reduction of either purine or pyrimidine nucleotides requires cleavage of the 3' carbon-hydrogen bond of the substrate. The substrate analogs 2'-chloro-2'-deoxyuridine 5'-diphosphate (ClUDP), 2'-deoxy-2'-fluorouridine 5'-diphosphate, and 2'-azido-2'-deoxyuridine 5'-diphosphate were time-dependent inactivators of the herpes simplex virus type 1 ribonucleotide reductase. Incubation of [3'-3H]ClUDP with the enzyme was accompanied by time-dependent release of 3H to the solvent. Reaction of [beta-32P]ClUDP with the reductase resulted in the production of inorganic pyrophosphate. These results are consistent with the enzyme-mediated cleavage of the 3' carbon-hydrogen bond of ClUDP and the subsequent conversion of the nucleotide to 2-methylene-3(2H)furanone, as previously reported with the Escherichia coli ribonucleotide reductase (Harris, G., Ator, M., and Stubbe, J. A. (1984) Biochemistry 23, 5214-5225; Ator, M., and Stubbe, J. A. (1985) Biochemistry 24, 7214-7221).