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.     

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 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.     

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.     

Concise and Efficient Synthesis of 3′-O-Tetraphosphates of 2′-Deoxyadenosine and 2′-Deoxycytidine

We describe concise and efficient synthesis of biologically very important 3′-O-tetraphosphates namely 2′-deoxyadenosine-3′-O-tetraphosphate (2′-d-3′-A4P) and 2′-deoxycytidine-3′-O-tetra-phosphate (2′-d-3′-C4P). N⁶-benzoyl-5′-O-levulinoyl-2′-deoxyadenosine was converted into N⁶-benzoyl-5′-O-levulinoyl-2′-deoxyadenosine-3′-O-tetraphosphate in 87% yield using a one-pot synthetic methodology. One-step concurrent deprotection of N⁶-benzoyl and 5′-O-levulinoyl groups using concentrated aqueous ammonia resulted 2′-d-3′-A4P in 74% yield. The same synthetic strategy was successfully employed to convert N⁴-benzoyl-5′-O-levulinoyl-2′-deoxycytidine into 2′-d-3′-C4P in 68% yield.     

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.     

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).     

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.     

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 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.     

Sub-cellular trafficking and functionality of 2′-O-methyl and 2′-O-methyl-phosphorothioate molecular beacons

Molecular beacons (MBs) have shown great potential for the imaging of RNAs within single living cells; however, the ability to perform accurate measurements of RNA expression can be hampered by false-positives resulting from nonspecific interactions and/or nuclease degradation. These false-positives could potentially be avoided by introducing chemically modified oligonucleotides into the MB design. In this study, fluorescence microscopy experiments were performed to elucidate the subcellular trafficking, false-positive signal generation, and functionality of 2′-O-methyl (2Me) and 2′-O-methyl-phosphorothioate (2MePS) MBs. The 2Me MBs exhibited rapid nuclear sequestration and a gradual increase in fluorescence over time, with nearly 50% of the MBs being opened nonspecifically within 24 h. In contrast, the 2MePS MBs elicited an instantaneous increase in false-positives, corresponding to ∼5–10% of the MBs being open, but little increase was observed over the next 24 h. Moreover, trafficking to the nucleus was slower. After 24 h, both MBs were localized in the nucleus and lysosomal compartments, but only the 2MePS MBs were still functional. When the MBs were retained in the cytoplasm, via conjugation to NeutrAvidin, a significant reduction in false-positives and improvement in functionality was observed. Overall, these results have significant implications for the design and applications of MBs for intracellular RNA measurement.     

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     

4′-Aza-3′,5′-dihomothymidine and Derivatives

Abstract

A series of 3′-branched 4′-azanucleoside analogues have been prepared. These compounds comprise three asymmetric atoms, two carbons and one nitrogen. They constitute nucleoside analogues imparted with a “flickering configuration”, the nitrogen inversion replacing a D-L epimerization of their natural congeners. The 1′,3′-cis and 1′,3′-trans isomers have been separated and their configuration established by ¹H NMR and the X-ray diffraction structure of one crystalline example. The configurations of the frozen invertomers were assessed by low temperature ¹H NMR experiments assisted by molecular mechanics simulations. None of these compounds exhibited any significant in vitro antiviral activity.     

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.     

2,2′-Anhydro-4′-Thionucleosides: Precursors for 2′-Azido- and 2′-Chloro-4′-thionucleosides and for a Novel Thiolane to Thietane Rearrangement

Abstract

2,2′-Anhydro-4′-thio-β-and α-nucleosides 9 and 10 have been prepared by an in situ 4-thio-1,2-glycal addition route. They undergo ring-opening by azide or chloride ion to give, after deprotection, the 2′-substituted-4′-thionucleosides 13 and 14, whereas reactions with cyanide or fluoride sources lead to the unsaturated nucleosides 17 or 18, depending upon conditions. An unexpected and clean rearrangement to the thietane 23 occurs on treatment of uracil derivative 20 with DAST.     

The occurrence of the syn-C3′ endo conformation and the distorted backbone conformations for C4′-C5′ and P-O5′ in oligo and polynucleotides

Abstract

The nucleoside constituents of nucleic acids prefer the anti conformation (1). When the sugar pucker is taken into account the nucleosides prefer the C2′endo-anti conformation. Of the nearly 300 nucleosides known, about 250 are in the anti conformation and 50 are in the syn-conformation, i.e., anti to syn conformation is 5:1. The nucleotide building blocks of nucleic acids show the same trend as nucleosides. Both the deoxy-guanosine and ribo- guanosine residues in nucleosides and nucleotides prefer the syn-C2′endo conformation with an intra-molecular hydrogen bond (for nucleosides) between the O5′- H and the N3 of the base and, a few syn-C3′endo conformations are also observed. Evidence is presented for the occurrence of the C3′endo-syn conformation for guanines in mis-paired double helical right-handed structures with the distorted sugar phosphate C4′-C5′ and P-O5′ bonds respectively, from g+ (gg) and g- to trans. Evidence is also provided for guanosine nucleotides in left-handed double-helical (Z-DNA) oligo and polynucleotides which has the same syn-C3′endo conformation and the distorted backbone sugar-phosphate bonds (C4′-C5′ and P- O5′) as in the earlier right-handed case.     

Synthesis of 2′-Deoxyformycin B and 2′-Deoxyoxoformycin B

Abstract

3-β-D-Ribofuranosylpyazolo[4,3-d]pyrimidines (formycins)¹ modified in the heteroaromatic moiety are of biological interest as analogues of adenosine and guanosine, and have been the objects of intensive synthetic chemical effort by several groups.^2-9 2′-Deoxynucleosides^2c,2d,7b,13 and other analogties of the formycins modified in the sugar moiety^10-12 are also of potential interest, but have been less extensively studied. Examples of the 2′-deoxyribonucleoside type known to date include the 2′-deoxy-6-thioguanosine analogue 1, the 2′-deoxyadenosine (dAdo) analogue 2 (2′-deoxyformycin A),^10,13 and the 2-chloro-2′-deoxyadenosine analogue 3.^7b Compound 2 was found to be 10-15 times more potent than 2′-deoxyadenosine as an inhibitor of the growth of S49 cells, a murine lymphoma line of T-cell origin.¹³ Activity depended on 5′- phosphorylation, since mutants lacking the enzymes adenosine kinase (AK) and deoxycytidine kinase (dCK) were insensitive to the drug. Furthermore, activity was comparable in the presence and absence of an AK inhibitor, suggesting that 2, unlike dAdo, may be a poor substrate for adenosine deaminase. That 5′-phosphorylation of 2 was mediated by AK rather than dCK was indicated by the fact that miitants lacking only dCK retained sensitivity. This contrasted with the behavior of dAdo, which is known to be n substrate for both AK and dCK.¹⁴     

3′-Deoxy-3′-Hydroxyamino-β-D-Xylofuranosyluracil and Derivatives Thereof

Abstract

The title compound was prepared by reduction of the oxime of the 3′-ketouridine. Condensation with aldehydes gave a series of nitrones whose reduction afforded “second generation” hydroxylamines, some of which showing antiviral activity. The nitroxide free radicals formed upon oxidation of hydroxylamines gave good e.s.r. spectra useful for configurational and conformational assignments.