Synthesis and Antiviral Activity of L-2′-Deoxy-2′-up-fluoro-4′-thionucleosides

Abstract

L-2′-Deoxy-2′-up-fluoro-4′-thionucleosides were efficiently synthesized from D-xylose via L-4-thioarabitol derivative as a key intermediate and evaluated for antiviral activities against HIV-1, HSV-1,2 and HBV.     

Synthesis and anti-HIV activity of 2′-deoxy-2′-fluoro-4′-C-ethynyl nucleoside analogs

Based on the favorable antiviral profiles of 4′-substituted nucleosides, novel 1-(2′-deoxy-2′-fluoro-4′-C-ethynyl-β-d-arabinofuranosyl)-uracil (1a), -thymine (1b), and -cytosine (2) analogs were synthesized. Compounds 1b and 2 exhibited potent anti-HIV-1 activity with IC₅₀ values of 86 and 1.34 nM, respectively, without significant cytotoxicity. Compound 2 was 35-fold more potent than AZT against wild-type virus, and also retained nanomolar antiviral activity against resistant strains, NL4-3 (K101E) and RTMDR. Thus, 2 merits further development as a novel NRTI drug.     

Nucleosides and Nucleotides. 232. Synthesis of 2′-C-Methyl-4′-thiocytidine: Unexpected Anomerization of the 2′-Keto-4′-thionucleoside Precursor

The synthesis of 2′-C-methyl-4′-thiocytidine (16) is described. Since the 2′-keto-4′-thiocytidine derivative 2β unexpectedly isomerized to 2α and the methylation of 2β proceeded predominantly from the less hindered α-face to give 7, the desired product 16 was synthesized via the Pummerer reaction of the sulfoxide 14 and N ⁴ -benzoylcytosine.     

Synthesis of 2′-Deoxy-2′ -Fluoro-D-Arabinopyranopyranosyl Nucleosides and Their 3′,4′-Seco analogues

Abstract

2′-Deoxy-2′-fluoro-D-arabinopyranosyl nucleosides were synthesized by condensation of 1,3,4-tri-O-benzoyl-2-deoxy-2-fluoro-D-arabinopyranose with the appropriate silylated bases in the presence of trimethylsilyl triflate. Scission of the 3′,4′-bond by periodate oxidation followed by sodium borohydride reduction resulted in the formation of the 3′,4′-seco analogues of the 2′-deoxy-2′-fluoro-D-arabinofuranosyl nucleosides.     

Resistance Profiles of (+) 2′-Deoxy-3′-oxa-4′-thiocytidine and (-) 2′-Deoxy-3′-oxa-4′-thio-5-fluorocytidine

Abstract

Resistant variants were selected in vitro against two novel nucleoside analogues, (+) dOTC and (-) dOTFC using the HIV-1 molecular clone HXB2D. The variants obtained displayed 6.5-fold and 10-fold resistance to these compounds, respectively. Cloning and sequencing of the RT genes of the selected viruses identified two mutations, M184I for (+) dOTC and M184V for (-) dOTFC. Results with mutated recombinant clones of HXB2D confirmed the importance of these mutations in MT-4 cells. The resistance profiles of clinical samples with wild-type or 3TC-resistant phenotypes were also studied; low to moderate levels of cross-resistance were observed against the novel compounds.     

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.     

Recruitment of divalent metal ions by incorporation of 4-thio-2′-deoxythymidine or 4-thio-2′-deoxyuridine into DNA

The modified nucleosides 4-thio-2-deoxyuridine (s⁴dU) and 4-thio-2-deoxythymidine (s⁴dT) are incorporated into dinucleosides, and s⁴dT is incorporated into a DNA hairpin loop to provide divalent metal ion binding sites. Binding of two different metal ions to these sites is studied, including Cd(II) as an NMR spectroscopy probe and Cu(II) as a reactive metal ion for DNA cleavage. Binding of Cd(II) to 4-thiouridine (s⁴U) and s⁴dT nucleosides, s⁴dU- and s⁴dT-containing dinucleosides, and a hairpin loop oligonucleotide containing s⁴dT is monitored by following the change in UV-vis absorbance of the thionucleosides at 340 nm and 21 °C in solutions containing 20.0–40 mM buffer, 1.00 M NaCl, and 15.0 mM BaCl₂. Cd(II) binds to the N(3) deprotonated form of s⁴dT with a binding constant (K=1.1×10⁴ M^–1) that is similar to that for Cd(II) binding to d(Tps⁴T) (K=9.2×10³ M^–1). Apparent binding constants (K_app) at pH 7.7 of Cd(II) to dinucleosides d(Gps⁴T), d(s⁴TpG), and d(Gps⁴U) are similar to those of their respective nucleosides s⁴U and s⁴dT, suggesting that neither the phosphate diester nor the second nucleoside has a major effect on Cd(II) binding. Binding of Cd(II) to s⁴U and d(Gps⁴U) is studied by use of ¹¹³Cd NMR and ¹H NMR spectroscopy, respectively. Binding strength and stoichiometry of the Cd(II) complex with d(Gps⁴U) as studied by ¹H NMR spectroscopy are similar to that obtained by UV-vis spectroscopy. Cd(II) binds strongly to s⁴dT in the loop portion of a DNA hairpin loop (K_app=2.7×10³ M^–1 at pH 7.7). However, the hairpin loop is moderately destabilized by Cd(II) binding, with a decrease in T_m of 14 °C in the presence of 10.0 mM Cd(II) as determined by optical melting experiments. Cu(II) oxidizes s⁴dT to form the disulfide of s⁴dT, limiting the usefulness of further studies with Cu(II).Electronic Supplementary Material Supplementary material is available in the online version of this article at .Abbreviations s⁴dU 4-thio-2-deoxyuridine - s⁴dT 4-thio-2-deoxythymidine - s⁴U 4-thiouridine     

Synthesis,evaluation of anti-HIV-1 and anti-HCV activity of novel 2′,3′-dideoxy-2′,2′-difluoro-4′-azanucleosides

A series of 2′,3′-dideoxy-2′,2′-difluoro-4′-azanucleosides of both pyrimidine and purine nucleobases were synthesized in an efficient manner starting from commercially available L-pyroglutamic acid via glycosylation of difluorinated pyrrolidine derivative 15. Several 4′-azanucleosides were prepared as a separable mixture of α- and β-anomers. The 6-chloropurine analogue was obtained as a mixture of N⁷ and N⁹ regioisomers and their structures were identified based on NOESY and HMBC spectral data. Among the 4′-azanucleosides tested as HIV-1 inhibitors in primary human lymphocytes, four compounds showed modest activity and the 5-fluorouracil analogue (18d) was found to be the most active compound (EC₅₀ = 36.9 μM) in this series. None of the compounds synthesized in this study demonstrated anti-HCV activity.     

Antileukemic Activities and Mechanism of Action of 2′-Deoxy-4′-methylcytidine and Related Nucleosides

Abstract

Antileukemic activity of several analogues containing 2′-deoxy-4′-methylcytidine and its araC counterpart were evaluated against murine leukemic P388 cells in vitro and in vivo. Both compounds showed significant cytostatic activity (both IC₅₀=0.4 μM) in vitro and the former compound administered intraperitoneally at a dose of 3 mg/kg/day × 5 showed high activity (T/C=175%) in vivo. The mechanism of action of these 5′-triphosphates on DNA polymerases in detail will be also described.     

Conformationally Locked Nucleoside Analogs. Synthesis of 2′-Deoxy-2′-C, 4′-C-Bridged Bicyclic Nucleosides

Abstract

1-α-Methylarabinose was converted, in three steps, to 2-deoxy-2-methyleneribose derivative 3, which was subjected to hydroboration to give 2-α-hydroxymethyl derivative 4 exclusively. 4 was converted to 2,4-bis(hydroxymethyl)ribose derivative 6 in four steps. Mesylation, detritylation, and ring closure, followed by hydrolysis of the mesyl group at O5, gave 3,6-dioxabicyclo[3,2,1]octane derivative 8. After acetylation, 8 was coupled with silylated 6-chloropurine to give desired α- and β-bicyclic-sugar nucleosides.     

Synthesis of 4-Substituted Pyrimidine 2′,3′-Dideoxynucleosides

Abstract

Reaction of 5′-0-(4,4′-dimethoxytriphenylmethyl)-3′-deoxythy-midine with triphenylphosphine/carbon tetrachloride, followed by deprotection of the 5′-hydroxyl group, afforded the 4-chloro derivative 3 from which some 4-substituted pyrimidin-2(1H)one-2′, 3′-dideoxyribosides were obtained by nucleo-philic substitution under very mild conditions.     

Efficient Synthesis of 2′-Amino-2′-deoxypyrimidine 5′-Triphosphates

Abstract

The synthesis of 2′-amino-2′-deoxypyrimidine 5′-triphosphates is described. The 2′-amino-2′-deoxyuridine 5′-triphosphate is obtained from uridine in four steps with 25% overall yield. The 2′-amino-2′-deoxycytidine 5′-triphosphate is obtained from uridine in seven steps with 13% overall yield.     

Efficient Large-Scale Synthesis of 5′-O-Dimethoxytrityl-N 4-Benzoyl-5-Methyl-2′-Deoxycytidine

An efficient process to synthesize 5′-O-dimethoxytrityl-N⁴-benzoyl-5-methyl-2 ′-deoxycytidine in high yield and quality is described. Final benzoylation was improved by developing a method to selectively hydrolyze benzoyl ester impurities. This inexpensive approach was scaled up to multi-kilogram quantities for routine use in oligonucleotide therapeutics.     

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.     

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.     

Microbial Synthesis of Purine 2′-Amino-2′-deoxyribosides

The microbial synthesis of some purine 2′-amino-2′-deoxyribonucleosides from purine bases and 2′-amino-2′-deoxyuridine is described. Various bacteria, especially Erwinia herbicola, Salmonella schottmuelleri, Enterobacter aerogenes and Escherichia coli, were able to transfer the aminoribosyl moiety of 2′-amino-2′-deoxyuridine to purine bases (transaminoribosylation) in the presence of inorganic phosphate. The optimum conditions for the reaction were pH 7.0 and 63°C. No reaction was observed in the absence of inorganic phosphate and the optimum concentration of it was around 30 mm. Adenine, guanine, 2-chlorohypoxanthine and hypoxanthine were transformed to the corresponding 2′-amino-2′-deoxyribonucleosides by the catalytic activity of the wet cell paste of Enterobacter aerogenes AJ 11125. The enzymatically synthesized purine 2′-amino-2′-deoxyribonucleosides were isolated and identified by physicochemical means. 2′-Amino-2′-deoxyadenosine strongly inhibited the growth of Hela cells in tissue culture, and the ED⁵⁰ was 2.5μ/ml.     

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.     

C1′ Acylated Derivatives of 2′-Deoxyuridine. Photolabile Precursors of 2′-Deoxyuridin-1′-yl

Abstract

ABSTRACT

C1′ acylated derivatives of 2′-dcoxyuiidinc (1a-c) were synthesised from 1-[3-deoxy-β-D-psieofiiraiiosylliii.acil (6). The acyl group is introduced via the C1′ aldehyde (11). Following nucleophilic addition, the ketones (1a-c) are obtained via periodinane oxidation and desilylation with NH₄F.     

SYNTHESIS OF 2′-C-METHYL-4′-THIO RIBONUCLEOSIDES

Starting from 2-C-methyl-ribonolactone, 1,2,3,5-tetra-O-acetyl-2-C-methyl-4-thioribofuranose was synthesized and condensed with heterocyclic bases to afford 2′-C-methyl-4′-thioribonucleosides.     

Convenient Synthesis of 4-C-Branched Lactones and 3′-C-Branched 2′,3′-Dideoxynucleosides

Abstract

The regio- and stereoselective photocatalysed addition of 2-propanol and cyclopentanol to (5S)-hydroxymethylfuran-2(5H)-one (1) gave 4-C-branched lactones 2 and 3 after selective silylations. The lactones 2 and 3 were radically deoxygenated affording lactones 4 and 5, respectively. As an example, compound 2 was transformed without purification of the intermediates into an anomeric mixtures of deprotected 3′-C-branched 2′, 3′-dideoxynucleosides 6 by the following reaction sequence: silylation, reduction, acetylation, coupling with silylated thymine and desilylation.