Mechanism of Antiviral Activity of 5-Ethyl-2′-Deoxyuridine

Abstract

5-Ethyl-2′-deoxyurldine (EDU) is phosphorylated to a much greater extent by herpes simplex virus (HSV)-infected Vero cells than by mock-infected cells. Within the infected cells, EDU is preferentially incorporated into viral DNA and more inhibitory to viral than cellular DNA synthesis     

5-Ethyl-2′-deoxyuridine: Absence of effects on the chromosomes of human lymphocytes and fibroblasts in culture

Summary In vitro studies have been performed with 5-ethyl-2-deoxyuridine (EDU) concerning its incorporation into the cell nuclear material and its effects on the chromosome morphology of cultivated human lyphocytes and skin fibroblasts. This compound is presumably incorporated in the DNA without visible chromosome aberrations as is seen with many other pyrimidine analogues. A slight inhibition of the cell growth was noted at high concentration (120g/ml) of EDU. A similar degree of cell growth inhibition was found with corresponding doses of deoxythymidine.

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Zusammenfassung Es wurden in vitro-Untersuchungen hinsichtlich des Äthyldeoxyuridineinbaus (ÄDU) und dessen Einfluß auf menschliche Lymphocyten- und Fibroblastenchromosomen durchgeführt. Diese Substanz wird in die DNS eingebaut und führt nicht zu sichtbaren Chromosomenaberrationen wie bei einigen anderen Pyrimidinanaloga. Eine leichte Hemmung des Zellwachstums wurde mit 120 g/ml ÄDU festgestellt. Eine ähnliche Hemmung des Zellwachstums ließ sich auch mit gleicher Deoxythymidinkonzentration nachweisen.

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Supported by the Deutsche Forschungsgemeinschaft.     

Effect of 2-Amino Substitution on the Antiviral Effects of 5-Ethyl-2′-Deoxyuridine and (E)-5-(2-bromovinyl)-2′-Deoxyuridine and Their Incorporation into DNA

Abstract

The 2-amino derivatives of 5-ethyl-2′-deoxyuridine (EDU) and (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) have been synthesized and evaluated for anti-herpesvirus activity. They were at least 1000-fold less effective against herpes simplex virus replication than the parent compounds EDU and BVDU. The 5′-triphosphates of the 2-amino substituted EDU, BVDU and thymidine derivatives were also synthesized and examined on their substrate/inhibitor properties against different DNA polymerases. None of the compounds proved markedly inhibitory to HSV-1 DNA polymerase or cellular DNA polymerase a. Nor were they incorporated into the growing DNA chain.     

Syn- and anti-conformations of 5′-deoxy- and 5′-O-methyl-uridine 2′,3′-cyclic monophosphate

Two uridine 2′,3′-cyclic monophosphate (cUMP) derivatives, 5′-deoxy (DcUMP) and 5′-O-methyl (McUMP), were studied by means of quantum chemical methods. Aqueous solvent effects were estimated based on the isodensity-surface polarized-continuum model (IPCM). Gas phase calculations revealed only slight energy differences between the syn- and anti-conformers of both compounds: the relative energies of the syn-structure are −0.9 and 0.2 kcal mol^-1 for DcUMP and McUMP, respectively. According to the results from the IPCM calculations, however, both syn-conformers become about 14 kcal mol^-1 more stable in aqueous solution than their corresponding anti-structures. Additionally, the effects of a countercation and protonation on DcUMP were studied, revealing that the syn-structure is also favored over the anti-one for these systems.     

Deuteration and Tritiation of 2′-Deoxyribonucleosides in the 5′ and 4′ Positions

Abstract

The deuterations of 2′-deoxyguanosine in the 4′ and 5′ positions have been described elsewhere (1). The starting material is the 5′-aldehyde formed by mild oxidation with N,N-dicyclohexyl carbodiimide in dimethyl sulphoxide of the fully protected nucleoside with free 5′-alcoholic function. The 5′4euteration was achieved by reduction with deuterated sodium borohydride. Incorporation of deuterium in the 4′-position was achieved v i a an enhanced keto-enol tautomerim by heating the aldehyde in 50/50 D20/pyridine, with subsequent reduction of the aldehyde with NaBH4. The 6-furanoid form was isolated from the I-lyxo by-product by reverse phase HPLC. Applied to pyrimidine 2′-deoxyribonucleosides, this method was shown to give deuterated 2′-deoxycytidine and thymidine in good yield.     

Synthesis and Incorporation of 5″-Amino- and 5′-Mercapto-5′-Deoxy-2′-O-Methyl Nucleosides Into Hammerhead Ribozymes

Abstract

Novel 5′-amino-5′-deoxy-2′-O-methyl uridine, guanosine and adenosine 3′-O-phosphoramidites 5, 11, and 20, as well as protected 5′-mercapto-5′-deoxy-2′-O-methyl uridine 3′-O-phosphoramidite 23 were synthesized from 2′-O-methyl nucleosides. These analogs were incorporated at the 5′-ends of hammerhead ribozymes to evaluate achiral bridging 5′-N- phosphoramidates and 5′-S-phosphorothioates as alternatives for non- bridging phosphorothioates commonly used for end stabilization against nucleases. Oligonucleotide synthesis and deprotection conditions were optimized for better yields of these modified ribozymes.     

Synthesis and antibacterial activity of 5′-tetrachlorophthalimido and 5′-azido 5′-deoxyribonucleosides

Reported is an efficient synthesis of adenyl and uridyl 5′-tetrachlorophthalimido-5′-deoxyribonucleosides, and guanylyl 5′-azido-5′-deoxyribonucleosides, which are useful in solid-phase synthesis of phosphoramidate and ribonucleic guanidine oligonucleotides. Replacement of 5′-hydroxyl with tetrachlorophthalimido group was performed via Mitsunobu reaction for adenosine and uridine. An alternative method was applied for guanosine which replaced the 5′-hydroxyl with an azido group. The resulting compounds were converted to 5′-amino-5′-deoxyribonucleosides for oligonucleotide synthesis. Synthetic intermediates were tested as antimicrobials against six bacterial strains. All analogs containing the 2′,3′-O-isopropylidine protecting group demonstrated antibacterial activity against Neisseria meningitidis, and among those analogs with 5′-tetrachlorophthalimido and 5′-azido demonstrated increased antibacterial effect.     

Conformation and Antiherpes Activity of 3′- and 5′-Azido and Amino Analogs of 5-Methoxymethyl-2′-Deoxyuridine

Abstract

The molecular conformations of 3′- and 5′-azido and amino derivatives of 5-methoxymethyl-2′-deoxyuridine, 1, were investigated by nmr. The glycosidic conformation of 5-methoxymethyl-5′-amino-2′,5′-dideoxy-uridine, 5 had a considerable population of the syn form. The 5′-derivatives show a preference for the S conformation of the furanose ring as in 1. In contrast, the 3′-derivatives show preference for the N conformation. For 5-methoxymethyl-3′-amino-2′,3′-dideoxyuridine, 3, the shift towards the N state is pH dependent. The preferred conformation for the exocyclic (C4′,C5′) side chain is g⁺ for all compounds except 5 which has a strong preference for the t rotamer (79%). Compounds 1, 3 and 5 inhibited growth of HSV-1 by 50% at 2, 18 and 70 μg/ml respectively, whereas 2 and 4 were not active up to 256 μg/ml (highest concentration tested). The compounds were not cytotoxic up to 3,000 μM.     

Synthesis and Properties of 2′4′- and 3′-5′-Linked Ribodinucleoside Monophosphates Containing 2-Aminoadenosine and Uridine

Abstract

Self complementary diribonucleoside monophosphates containing 2-aminoadenosine (n²A) and uridine (U) residues, (2′-5′) n²ApU (1), (3′-5′) n²ApU (2), (2′-5′) Upn²A (3) and (3′-5′) Upn²A (4), were synthesized by condensation of suitably protected nucleoside and nucleotide units using dicyclohexylcarbodiimide (DCC). The dimers, (3) and (41, were also obtained from uridine 2′,3′-cyclic phosphate and unprotected 2-aminoadenosine using 2,4,6-triisopropylbenzenesulfonyl chloride (TPS-Cl) as the condensing agent. The conformational properties of these dimers were examined by UV, CD and NMR spectroscopy. The results reveal that the 2′-5′ isomers take a stacked conformation, which contains a larger base-base overlap and is more stable against thermal perturbation with respect to the 3′-5′ isomers. The n²ApU isomers have more stacked structure than the Upn²A isomers.     

Synthesis of 5′-Fluoro-5′-deoxy-and 5′-Amino-5′-Deoxytoyocamycin and Sangivamycin and Some Related Derivatives

Abstract

A series of 5′-substituted analogs of toyocamycin were prepared by condensation of silylated 4-amino-6-bromo-5-cyanopyrrolo[2,3-d]pyrimidine with protected 5-azido-5-deoxy- or 5-fluoro-5-deoxyribofuranose followed by debromination and deblocking. Alternatively, 5′-azido-5′-deoxytoyocamycin was prepared by azidation of toyocamycin. Conversion of the 5-nitrile function of the toyocamycin derivatives into a carboxamide or a thiocarboxamide gave the corresponding analogs of sangivamycin or thiosangivamycin while reduction of the 5′-azido-5′-deoxy nucleosides provided 5′-amino-5′-deoxy derivatives.     

Synthesis of 2′-Deuterio and 3′-Deuterio Cytidine 5′-Diphosphate

Abstract

2′-²H- and 3′-²H-CDP were synthesized from 5′-MMT-3′-O-TBDMS and 2′,5′- O-diTBDMS cytidine derivatives, respectively, by oxidation followed by acidic removal of 5′-protection, reduction with [NaBD(OAc)₃] and finally displacement of a tosyl group by pyrophosphate.     

Synthesis of 3′-Amino and 5′-Amino Hydantoin 2′-Deoxynucleosides

Abstract

3′-Amino and 5′-amino derivatives of hydantoin 2′-deoxynucleosides have been prepared from the corresponding 3′-phthalimido and 5′-azido nucleosides, respectively, which in turn were prepared by condensation of appropriate sugars with 5-benzylidenehydantoin. The amino nucleosides were tested for their potential activity against HIV and HSV.     

The 5′-Nor Aristeromycin Analogues of 5′-Deoxy-5′-Methylthioadenosine and 5′-Deoxy-5′-Thiophenyladenosine

To extend the potential of 5′-noraristeromycin (and its enantiomer) as potential antiviral candidates, the enantiomers of the carbocyclic 5′-nor derivatives of 5′-methylthio-5′-deoxyadenosine and 5′-phenylthio-5′-deoxyadenosine have been synthesized and evaluated. None of the compounds showed meaningful antiviral activity.     

Synthesis and Properties of Oligonucleotide Chimeras Containing 5′-Amino-2′-deoxy-2′-fluoroarabinonucleosides

Abstract

Oligonucleotide analogues comprised of 2′-deoxy-2′-fluoro-β-D-arabinose units joined via P3′-N5′ phosphoramidate linkages (2′F-ANA^5′N) were prepared for the first time. Among the compounds prepared were a series of 2′OMe-RNA-[GAP]-2′OMe-RNA ‘chimeras’, whereby the “GAP” consisted of DNA, DNA^5′N, 2′F-ANA or 2′F-ANA^5′N segments. The chimeras with the 2′F-ANA and DNA gaps exhibited the highest affinity towards a complementary RNA target, followed by the 5′-amino derivatives, i.e., 2′F-ANA > DNA > 2′F-ANA^5′N > DNA^5′N. Importantly, hybrids between these chimeras and target RNA were all substrates of both human RNase HII and E.coli RNase HI. In terms of efficiency of the chimera in recruiting the bacterial enzyme, the following order was observed: gap DNA > 2′F-ANA > 2′F-ANA^5′N > DNA^5′N. The corresponding relative rates observed with the human enzyme were: gap DNA > 2′F-ANA^5′N > 2′F-ANA > DNA^5′N.     

5′-Phosphonates of Ribonucleosides and 2′-Deoxyribonucleosides: Synthesis and Antiviral Activity

Abstract

5′-Phosphonates of natural 2′-deoxynucleosides and ribonucleosides were synthesized by condensation of 3′-O-acylated 2′-deoxynucleosides or 2′,3′-substituted (2′,3′-O-isopropylidene, 2′,3′-O-methoxymethylene or 2′,3′-O-ethoxymethylene) ribonucleosides. As condensing agents, either N,N′-dicyclohexylcarbodiimide or 2,4,6-triisopropylbenzenesulphonyl chloride were used. Nucleoside 5′-ethoxycarbonylphosphonates were converted into corresponding nucleoside 5′-aminocarbonylphosphonates by action of ammonia in methanol or aqueous ammonia. 5′-Hydrogenphosphonothioates of thymidine and 3′-deoxythymidine were obtained by reaction of phosphinic acid in the presence of pivaloyl chloride with 3′-O-acetylthymidine or 3′-deoxythymidine, respectively, followed by addition of powedered sulfur. 5′-O-methylenephosphonates of thymidine and 2′-deoxyadenosine were prepared by intramolecular reaction of corresponding 3′-O-iodomethylphosphonates under basic conditions. All compounds were tested for inhibition of several viruses, including HSV-2 and CMV, but showed no activity. A few compounds insignificantly inhibited HIV-1 reproduction. Thymidine 5′-hydrogenphosphonate neutralized anti-HIV action of 3′-azido-3′-deoxythymidine (AZT) and it indirectly showed that even some nucleoside 5′-phosphonates could be partly hydrolyzed in cell culture to corresponding nucleosides.

5′-Phosphonates of modified 2′-deoxynucleosides in which one group in a phosphate residue is substituted for hydrogen, alkyl or other groups, have shown to be potent biologically     

Biological Activity and Phosphorylation of 2′-Azido-2′-Deoxyuridine and 2′-Azido-2′-Deoxycytidlne

Abstract

2′-Azido-2′-deoxyuridine and 2′-azido-2′-deoxycytidine were evaluated for their inhibitory activity against ribonucleotide reductase and for subsequent cell growth inhibition. Their mono-and di-phosphates were synthesized and their inhibitory activities against the reductase were also determined in a permeabilized cell system, along with the two nucleosides. The results of the present study identify the first phosphorylation step involved in the conversion of the two azidonucleosides to the corresponding diphosphates to be rate-limiting in the overall activation.     

Synthesis and Biological Evaluation of Some 5-Nitro- and 5-Amino Derivatives of 2′-Deoxycytidine, 2′,3′-Dideoxyuridine,and 2′,3′-Dideoxycytidine

Abstract

In this article, we describe the synthesis of 5-nitro-1-(2-deoxy-α-D-erythro-pentofuranosyl)cytosine (4α), 5-nitro-1-(2-deoxy-β-D-erythro-pentofuranosyl)cytosine (4β), 5-amino-1-(2-deoxy-α-D-erythro-pentofuranosyl)cytosine (5α), 5-nitro-1- (2-deoxy-β-D-erythro-pentofuranosyl)cytosine (5β), 5-nitro-1-(2,3-dideoxy-β- D-ribofuranosyl)uracil (6β), 5-amino-1-(2,3-dideoxy-α,β-D-ribofuranosyl)uracil (7), 5-nitro-1-(2,3-dideoxy-α,β-D-ribofuranosyl)cytosine (8) and 5-amino-1-(2,3-dideoxy-β-D-ribofuranosyl)cytosine (9β). The prepared compounds were tested for their activity against HIV and HBV viruses, but they did not show significant activity.     

Synthesis and Antitumor Properties of Some Neutral Triesters of 5-Fluoro-2′-deoxyuridine-5′-monophosphate and 3′,5′-Cyclic Monophosphate

Abstract

Several new prodrugs of 5-fluoro-2′-deoxyuridine 5′-monophosphate and 3′,5′-cyclic monophosphate were synthesized and their antitumor activities were evaluated in vitro.     

3′-3′,3′-5′ and 5′-5′ TpT-Amides: Synthesis,Characterization and Alkaline Hydrolysis

Abstract

A simple procedure is described for the preparation of the title compounds 1, 8 and 9. 3′-3′ or 3′-5′ or 5′-5′ TpT was reacted with a twofold molar excess of TPS in anhydrous DMF, at room temperature, for 5 min, followed by a 1 min in situ treatment of the reaction mixture with excess 7.0 N NH₄OH, at 0°C. The alkaline hydrolysis of 1, 8 and 9 proceeds without the assistance of 3′- and 5′-hydroxyl groups resulting in equimolar mixtures of thymidine (4) and thymidine 3′-phosphoramidate (6) (for the 3′-3′ isomer) or thymidine 5′-phosphoramidate (7) (for the 5′-5′ isomer) or 6 and 7 in equal quantities (for the 3′-5′ isomer).