Synthesis,In Vitro Anti-HIV Activity,and Biological Stability of 5′-O-Myristoyl Analogue Derivatives of 3′-Fluoro-2′,3′-Dideoxythymidine (FLT) as Potential Bifunctional Prodrugs of FLT

Abstract

A group of 5′-O-myristoyl analogue derivatives of FLT (2) were evaluated as potential anti-HIV agents that were designed to serve as prodrugs to FLT. 3′-Fluoro-2′,3′-dideoxy-5′-O-(12-methoxydodecanoyl)thymidine (4) (EC₅₀ = 3.8 nM) and 3′-fluoro-2′,3′-dideoxy-5′-O-(12-azidododecanoyl)thymidine (8) (EC₅₀ = 2.8 nM) were the most effective anti-HIV-1 agents. There was a linear correlation between Log P and HPLC Log retention time for the 5 ′-O-FLT esters. The in vitro enzymatic hydrolysis half-life (t½), among the group of esters (3–8) in porcine liver esterase, rat plasma and rat brain homogenate was longer for 3′-fluoro-2′,3′-dideoxy-5 ′-O-(myristoyl)thymidine (7), with t½ values of 20.3, 4.6 and 17.5 min, respectively.     

SYNTHESIS of 2′-DEOXY-2′-FLUOROGUANYL-(3′,5′)-GUANOSINE

ABSTRACT

The protected analogue of 2-amnio-6-chloropurine arabinoside (3b) was subjected to reaction with diethylaminosulfur trifluoride (DAST) and subsequently treated with NaOAc in Ac₂O/AcOH to give N ²,O ^3′,O ^5′-triacetyl-2′-deoxy-2′-fluoroguanosine (5a). After deacetylation of the sugar moiety and protection of 5′-OH by a 4,4′-dimethoxytrityl group, this nucleoside component was converted to 2′-deoxy-2′-fluoroguanyl-(3′,5′)-guanosine (6c, GfpG).     

Two new anti-plasmodial flavonoid glycosides from Duranta repens

The CHCl₃-soluble fraction of the whole plant of Duranta repens showed anti-plasmodial activity against the chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium falciparum, with IC₅₀ values of 8.5?±?0.9 and 10.2?±?1.5?μg/mL, respectively. From this fraction, two new flavonoid glycosides, 7-O-α-d-glucopyranosyl-3,4′-dihydroxy-3′-(4-hydroxy-3-methylbutyl)-5,6-dimethoxyflavone (1) and 7-O-α-d-glucopyranosyl(6′′′-p-hydroxcinnamoyl)-3,4′-dihydroxy-3′-(4-hydroxy-3-methylbutyl)-5,6-dimethoxyflavone (2), along with five known flavonoids, 3,7,4′-trihydroxy-3′-(4-hydroxy-3-methylbutyl)-5,6-dimethoxyflavone (3), 3,7-dihydroxy-3′-(4-hydroxy-3-methylbutyl)-5,6,4′-trimethoxyflavone (4), 5,7-dihydroxy-3′-(2-hydroxy-3-methyl-3-butenyl)-3,6,4′-trimethoxyflavone (5), 3,7-dihydroxy-3′-(2-hydroxy-3-methyl-3-buten-yl)-5,6,4′-trimethoxyflavone (6), and 7-O-α-d-glucopyranosyl-3,5-dihydroxy-3′-(4′′-acetoxy-3′′-methylbutyl)-6,4′-dimethoxyflavone (7), have been isolated as anti-plasmodial principles. Their structures were deduced by spectroscopic analysis including 1D and 2D NMR techniques. The compounds (1–7) showed potent anti-plasmodial activities against D6 and W2 strains of Plasmodium falciparum, with IC₅₀ values in the range of 5.2–13.5?μM and 5.9–13.1?μM, respectively.     

Nucleic Acid Related Compounds. LXXXI. Efficient General Synthesis of Purine (Amino,Azido, and Triflate)-Sugar Nucleosides

Abstract

Treatment of 3′,5′-O-(tetraisopropyldisiloxanyl)adenosine and its arabino epimer with trifluoromethanesulfonyl chloride/DMAP gave the 2′-triflates in high yields. Displacements (LiN₃/DMF) and deprotection gave 2′-azido-2′-deoxyadenosine and its arabino epimer which were reduced with Bu₃SnH/AIBN/DMAC/benzene (or Staudinger reduction) to give 2′-amino-2′-deoxyadenosine and its epimer. Oxidation of 2′,5′-bis-O-(tert-butyldimethylsilyl)adenosine, stereoselective reduction, triflation, azide displacement, deprotection, and reduction gave 3′-amino-3′-deoxyadenosine.     

Synthesis of (E)-5-(2-cIOdovinyl)-3′-0-(1-Methyl-1,4-Dihydropyridyl-3 -Carbonyl)-2′-Fluoro-2′-Deoxyuridine (Ivfru-Cds) for Brain Targetted Delivery of Ivfru,an Antiviral Nucleoside

Abstract

(E)-5-(2-lodovinyl)-2′-fluoro-3′-0-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-2′-deoxyuridine (11) was synthesized for future evaluation as a lipophilic, brain-selective, pyrimidine phosphorylase-resistant, antiviral agent for the treatment of Herpes simplex encephalitis (HSE). Treatment of (E)-5-(2-iodovinyl)-2′-fluoro-2′-deoxyuridine (6) with TBDMSCI in the presence of imidazole in DMF yielded the protected 5′-O-t-butyldimethylsilyl derivative (7). Subsequent reaction with nicotinoyl chloride hydrochloride in pyridine afforded (E)-5-(-2-iodovinyl)-2′-fluoro-3′-O-(3-pyridylcarbonyl)-5′-O-t-butyldimethylsily-2′-deoxyuridine (8). Deprotection of the silyl ether moiety of 8 with n-Bu₄N⁺F^? and quaternization of the resulting 3′-O-(3-pyridylcarbonyl) derivative 9 using iodomethane afforded the corresponding 1-methylpyridinium salt 10. The latter was reduced with sodium dithionite to yield (E)-5-(2-iodovinyl)-2′-fluoro-3′-O-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-2′-deoxyuridine (11).     

P-CHIRAL OLIGONUCLEOTIDES. 2D ROESY NMR ASSIGNMENT OF ABSOLUTE CONFIGURATION AT PHOSPHORUS AND CONFORMATIONAL ANALYSIS OF 5′-O-MONOMETHOXYTRITYL-(2′-O-DEOXYRIBONUCLEOSIDE) 3′-O-[O-(4-NITROPHENYL)]METHANEPHOSPHONATES

ABSTRACT

Fast and simple methodology for the assignment of the absolute configuration at the phosphorus atom in diastereomerically pure R_P and S_P 5′-O-monomethoxytrityl-2′-O-deoxynucleoside 3′-O-(O-4-nitrophenyl)methanephosphonate (3) was established. The method utilizes 2D ROESY NMR and can be used for the stereochemical analysis of other P-chiral mononucleotides. Configurational analysis shows that the major conformation of the sugar residue in 3 is of the S (South) type. This study will facilitate synthesis of stereoregular methylphosphonate oligonucleotide analogues via the transesterification method.     

Purification of 5′-O-Trityl-on Oligoribonucleotides. Investigation of Phosphate Migration During Purification and Detritylation.

Abstract

Synthetic oligoribonucleotides (RNA) are efficiently prepared with 2′-O-tert-butyldimethylsilyl nucleoside 3′-O-phosphoramidites with labile base-protection; A_dmf or A_Pac, G_dmf, C_ibu, U. After cleavage from the polystyrene support, the exocyclic amine protecting groups are removed with conc. NH₄OH: ethanol/3:1 by heating at 55°C for 3–5 h. The 2′-O- silyl protecting groups are removed with tetra-n-butylammonium fluoride in THF or more conveniently with neat triethylamine trihydrofluoride. To gain the advantages of increased capacity on reverse phase HPLC and the convenience of cartridge based purification (OPC, Oligonucleotide Purification Cartridge), the 5′ trityl was left on the RNA as the final protecting group to be removed. The mild conditions which are effective for trityl removal are shown to preserve 3′-5′ phosphate linkage integrity in RNA. The absence of phosphate migration is demonstrated by model studies, utilizing N⁴ -isobutyryl-5′-O-DMT-3′-O-TBDMS-2′-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite) as a control monomer and digestion by 3′-5′ selective P1 nuclease and alkaline phosphatase and HPLC analysis. Oligoribonucleotides were analyzed by Microgel capillary electrophoresis, anion-exchange HPLC, and the enzymatic digest/HPLC method.

     

Synthesis of Methylene-Bridged Analogues of Nicotinamide Riboside,Nicotinamide Mononucleotide and Nicotinamide Adenine Dinucleotide

Abstract

5-O-tert-Butyldimethylsilyl-1,2-O-isopropylidene-3(R)-(nicotinamid-2-ylmethyl)-α-D-ribofuranose (11a) and ?3(R)-(nicotinamid-6-ylmethyl)-α-D-ribofuranose (11b) were prepared by condensation of 5-O-tert-butyldimethylsilyl-1,2-O-isopropylidene-α-D-erythro-3-pentulofuranose (10) with lithiated (LDA) 2-methylnicotinamide and 6-methylnicotinamide, respectively, and then deprotected to give 1,2-O-isopropylidene-3-(R)-(nicotinamid-2-ylmethyl)-α-D-ribofuranose(12a) and 1,2-O-isopropylidene-3(R)-(nicotinamid-6-ylmethyl)-α-D-ribofuranose (12b). Benzoylation as well as phosphorylation of compounds 12 afforded the corresponding 5-O-benzoate (13b) and 5-O-monophosphates (14a and 14b). Treatment of 13b with CF₃COOH/H₂O caused 1,2-de-O-isopropylidenation with simultaneous cyclization to the corresponding methylene-bridged cyclic nucleoside - 3′,6-methylene-1-(5-O-benzoyl-β-D-ribofuranose)-3-carboxamidopyridinium trifluoro-acetate (8b) - restricted to the “anti” conformation. In a similar manner compounds 14a and 14b were converted into conformationally restricted 2,3′-methylene-1-(β-D-ribofuranose)-3-carboxamidopyridinium-5′-monophosphate (9a - “syn”) and 3′,6-methylene-1-(β-D-ribofuranose)-3-carboxamido -pyridinium-5′monophosphate (9b - “anti”) respectively. Coupling of derivatives 12a and 12b with the adenosine 5′-methylenediphosphonate (16) afforded the corresponding dinucleotides 17. Upon acidic 1,2-de-O-isopropylidenation of 17b, the conformationally restricted P¹-[6,3′-methylene-1-(β-D-ribofuranos-5-yl)-3-carboxamidopyridinium]-P²-(adenosin-5′-yl)methylenediphosphonate 18b -“anti” was formed. Compound 18b was found to be unstable. Upon addition of water 18b was converted into the anomeric mixture of acyclic dinucleotides, i. e. P¹-[3(R)-nicotinamid-6-ylmethyl-D-ribofuranos-5-yl]-P²-(adenosin-5′-yl)-methylenediphosphonate (19b). In a similar manner, treatment of 17a with CF₃COOH/H₂O and HPLC purification afforded the corresponding dinucleotide 19a.

     

Studies on Synthesis and Structure of O-β-D-Ribofuranosyl(1″→2′)-ribonucleosides and Oligonucleotides♣

Abstract

Minor nucleosides found in several eukaryotic initiator tRNAs_i ^Met, O-β-D-ribofuranosyl(1″→2′)adenosine and -guanosine (Ar and Gr), as well as their pyrimidine analogues, were obtained from N-protected 3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)ribonucleosides and 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose in the presence of tin tetrachloride in 1,2-dichloroethane. A crystal structure has been solved for 2′-O-ribosyluridine. The 3′-phosphoramidites of protected 2′-O-ribosylribonucleosides were prepared as the reagents for 2′-O-ribofuranosyloligonucleotides synthesis. O-β-D-Ribofuranosyl(1″→2′)adenylyl(3′→5′)guanosine (ArpG) was obtained and its structure was analysed by NMR spectroscopy.

     

Efficient Synthesis of 2′-Bromo-2′-Deoxy-3′,5′-O-TPDS-pyrimidine Nucleosides by Boron Trifluoride Catalyzed Reaction of O2,2′-Anhydro-(1-β-D-Arabinofuran0Syl)pyrimidine Nucleosides with Lithium Bromide

Abstract

Efficient syntheses of 2′-bromo-2′-deoxy-3′,5′-O-TPDS-uridine (5a) and 1-(2-bromo-3,5-O-TPDS-β-D-ribofuranosyl)thymine (5b) from uridine and 1-(β-D-ribofuranosyl)thymine are described, respectively. The key step is a treatment of 3′,5′-O-TPDS-O²,2′-anhydro-1-(β-D-ardbinofuranosyl)uracil (4a) and -thymine (4b) with LiBr in the presence of BF₃-OEt₂ in 1,4-dioxane at 60°C to give 5a and 5b in 98%, and 96% yield, respectively.

     

A Novel Method for the Synthesis of ddA and F-ddA Via Regioselective 2′-O-Deacetylation of 9-(2,5-DI-O-Acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)adenine

Abstract

Regioselective 2′-O-deacetylation of 9-(2,5-di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)adenine (1) is achieved by treatment of 1 with β-cyclodextrin (β-CyD) / aq. NaHCO₃ or N₂H₄·H₂O / EtOH. The 9-(5-O-Acetyl-3-bromo-3-deoxy-β-D-xylo-furanosyl)adenine (2) obtained is a common intermediate for the synthesis of 2′,3′-dideoxy-adenosine (ddA) (7) and 9-(2-fluoro-2,3-dideoxy-β-D-threo-pentofuranosyl)-adenine (F-ddA) (9).     

Synthesis,Cytostatic Activity and Inhibition of Ribonucleotide Reductase by 5′-Phosphoramidates and 5′-Diphosphates,of 2′-O-Allyl-arabinofuranosyl Nucleosides

Abstract

The diphosphates of a series of 2′-O-allyl-1-β-D-arabinofuranosyl derivatives, previously obtained by us, have been prepared and tested for their inhibitory activity in an in vitro assay using R1 and R2 subunits of the purified recombinant mouse ribonucleotide reductase (RNR). 2′-O-Allyl-araU diphosphate proved to be inhibitory, with an IC₅₀ of 100 μM. The 5′-phosphoramidate pronucleotide of 2′-O-allyl-araU was also prepared and tested for inhibition of tumor cell proliferation.     

Chemical-Enzymatic Synthesis of 3′-Amino-2′, 3′-dideoxy-β-D-ribofuranosides of Natural Heterocyclic Bases and Their 5′-Monophosphates

Abstract

Treatment of O², 3′-anhydro-5′-O-trityl derivatives of thymidine (1) and 2′-deoxyuridine (2) with lithium azide in dimethylformamide at 150 °C resulted in the formation of the corresponding isomeric 3′-azido-2′, 3′-dideoxy-5′-O-trityl-β-D-ribofuranosyl N¹- (the major products) and N³-nucleosides (3/4 and 5/6). 3′-Amino-2′, 3′-dideoxy-β-D-ribofuranosides of thymidine [Thd(3′NH₂)], uridine [dUrd(3′NH₂)], and cytidine [dCyd(3′NH₂)] were synthesized from the corresponding 3′-azido derivatives. The Thd(3′NH₂) and dUrd(3′NH₂) were used as donors of carbohydrate moiety in the reaction of enzymatic transglycosylation of adenine and guanine to afford dAdo(3′NH₂) and dGuo(3′NH₂). The substrate activity of dN(3′NH₂) vs. nucleoside phosphotransferase of the whole cells of Erwinia herbicola was studied.     

Synthesis of Chlorodideoxynucleosides Using Tris(2,4,6-tribromophenoxy)-dichlorophosphorane (BDCP)

Abstract

5′-Chloro-5′-deoxy-N,3′-O-dibenzoylthymidine (3a), 5′-chloro-5′-deoxy-N⁴, 3′-O-dibenzoyldeoxycytidine(3b), 5′-chloro-5′-deoxy-N⁶,3′-O-dibenzoyldeoxyadenosine(3c), N-benzoyl-1-(3-chloro-2,3-dideoxy-5-O-trityl-ß-D-xylofuranosyl)thymine (5a) and N⁶-benzoyl-9-(3-chloro-2,3-dideoxy-5-O-trityl-ß-D-xylofuranosyl)adenine (5b) have been synthesized in very high yields using a new efficient reagent, tris(2,4,6-tribrom-ophenoxy)dichlorophosphorane (BDCP). The reaction time was greatly reduced to 5–8 min. NOE data suggested an inversion of configuration at C₃-position and thus an SN2 mechanism has been proposed for the chlorination reaction.

     

CycloSaligenyl Pronucleotides of 5-Iodo and 5-Trifluoromethyl-1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluorobenzene Mimics of Thymidine: Synthesis and Evaluation of this Pronucleotide Monophosphate Delivery System for Compounds with Potential Anticancer Activity

Abstract

A group of unnatural 1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluorobenzenes possessing a 5-I or 5-CF₃ substituent, that were originally designed as thymidine mimics, were coupled via their 5′-OH group to a cyclosaligenyl (cycloSal) ring system having a variety of C-3 substituents (Me, OMe, H). The 5′-O-cycloSal-pronucleotide concept was designed to effect a thymidine kinase-bypass, thereby providing a method for the intracellular delivery and generation of the 5′-O-monophosphate for nucleosides that are poorly phosphorylated. The 5′-O-cycloSal pronucleotide phosphotriesters synthesized in this study were obtained as a 1:1 mixture of two diastereomers that differ in configuration (S _P or R _P) at the asymmetric phosphorous center. The (S _P)- and (R _P)-diastereomers for the 5′-O-3-methylcycloSal- and 5′-O-3-methoxycycloSal derivatives of 1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-iodobenzene were separated by silica gel flash column chromatography. This class of cycloSal pronucleotide compounds generally exhibited weak cytotoxic activities in a MTT assay (CC₅₀ values in the 10^?3 to 10^?4 M range), against a number of cancer cell lines (143B, 143B-LTK, EMT-6, Hela, 293), except for cyclosaligenyl-5′-O-[1′-(2,4-difluoro-5-iodophenyl)-2′-deoxy-β-D-ribofuranosyl]phosphate that was more potent (CC₅₀ values in the 10^?5 to 10^?6 M range), than the reference drug 5-iodo-2′-deoxyuridine (IUDR) which showed CC₅₀ values in the 10^?3 to 10^?5 M range.     

The 1,1-Dianisyl-2,2,2-trichloroethyl Moiety as a New Protective Group for the Synthesis of Dinucleostde Trifluoromethylphosphonates

Abstract

The new 1,1-Dianisyl-2,2,2-trichloroethyl moiety (DATE) is used as an acid and base stable protective group for nucleosides. 5′-O-DATE-thymidine and 3′-O-acetyl-thymidine are phosphorylated with CF₃P(NR₂)₂ to the corresponding thymidine trifluoromethylphosphonous amidites. These building blocks are coupled with appropriate protected thymidines to a dinucleotide trifluoromethylphosphonate.

     

Synthesis of 6-Methyluridine via Palladium-Catalyzed Cross-Coupling Between A 6-Iodouridine Derivative and Tetramethylstannane

Abstract

6-Methyluridine can be synthesized from 5′-O-(tert-butyl-dimethylsilyl)-6-iodo-2′,3′-O-isopropylideneuridine via palladiumcatalyzed cross-coupling with Me₄Sn followed by deprotection. Application of this method for the synthesis of 6-phenyluridine was also carried out.     

Nucleosides and Nucleotides. 139. Stereoselective Synthesis of (2′S)-2′-C-Alkyl-2′-deoxyuridines

Abstract

A synthetic method for (2′S)-2′-C-alkyl-2′-deoxyuridines (9) has been described. Catalytic hydrogenation of 1-[2-C-alkynyl-2-O-methoxalyl-3,5-O-TIPDS-β-D-arabino-pentofuranosyl]uracils (5) gave 1-[2-C-(2-alkyl)-2-O-methoxalyl-3,5-O-TIPDS-β-D-arabino-pentofuranosyl]uracils (4) as a major product, which were then subjected to the radical deoxygenation, affording (2′S)-2′-alkyl-2′-deoxy-3′,5′-O-TIPDS-uridines (7) along with a small amount of their 2′R epimers.

     

Synthesis of Homologated Halovinyl Derivatives from Aristeromycin and Their Inhibition of Human Placental S-Adenosyl-L-homocysteine Hydrolase¶

Abstract

Moffatt oxidation of 2′,3′-O-isopropylidenearisteromycin (1a) and treatment of the 5′-carboxaldehyde with [(p-tolylsulfonyl)methylene]triphenylphosphorane gave the homologated vinylsulfone 2. Treatment of 2 with tributylstannane/AIBN gave the (E/Z)-vinylstannanes which were converted into the E and Z fluoro- and iodovinyl analogs. Chain extension via the 5′-cyano-5′-deoxy derivative 10a gave the 6′-carboxaldehyde of homoaristeromycin. S-Adenosyl-L-homocysteine hydrolase was strongly inhibited by the fluorovinyl, 5b, and iodovinyl, 4b and 7b, compounds, and time-dependent kinetics were observed [1–2 μM (K_i) and 0.1–0.2 min^?1 (k _inact)]. The mechanism of inactivation was shown to involve addition of water at the vinyl 5′ or 6′ carbons with elimination of halide.

     

Nucleosides VI: A Novel and Convenient Synthesis of Purine S-Cyclonucleosides Via Mitsunobu Reaction

Abstract

Two representative S-cyclonucleosides, 8,5′-anhydro-2′, 3′-O-isopropylidene-8-mercaptoadenosine (3) and 8,2′-anhydro-3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-8-mercaptoguanosine (8), were prepared in good yields by dropwise addition of one equivalent each of triphenylphosphine and DEAD in DMF into a mixture of 2′,3′-O-isopropylidene-8-mercaptoadenosine (2) or 3′,5′-O-(tetra-iso-propyldisiloxane-1,3-diyl)-8-mercaptoguanosine (7), respectively, in DMF. Treatment of compound 2 with two equivalents each of triphenylphosphine and DEAD in DMF afforded N-[8,5′-anhydro-2′,3′-O-isopropylidene-8-mercaptopurin-6-yl]triphenylphospha-λ5-azene (4) in 87% yield.