Synthesis and anticancer activity of some 5-fluoro-2′-deoxyuridine phosphoramidates

Two series of novel 4-chlorophenyl N-alkyl phosphoramidates of 3′-O-(t-butoxycarbonyl)-5-fluoro-2′-deoxyuridine (3′-BOC-FdU) (9a–9j) and 5-fluoro-2′-deoxyuridine (FdU) (10a–10j) were synthesized by means of phosphorylation of 3′-BOC-FdU (4) with 4-chlorophenyl phosphoroditriazolide (7), followed by a reaction with the appropriate amine. Phosphoramidates 9a–9j were converted to the corresponding 10a–10j by removal of the 3′-t-butoxycarbonyl protecting group (BOC) under acidic conditions. The synthesized phosphoramidates 9a–9j and 10a–10j were evaluated for their cytotoxic activity in five human cancer cell lines: cervical (HeLa), nasopharyngeal (KB), breast (MCF-7), liver (HepG2), osteosarcoma (143B) and normal human dermal fibroblast cell line (HDF) using the sulforhodamine B (SRB) assay. Two phosphoramidates 9b and 9j with the N-ethyl and N-(methoxy-(S)-alaninyl) substituents, respectively, displayed remarkable activity in all the investigated cancer cells, and the activity was considerably higher than that of the parent nucleoside 4 and FdU. Among phosphoramidates 10a–10j compound 10c with the N-(2,2,2-trifluoroethyl) substituent showed the highest activity. Phosphoramidate 10c was more active than the FdU in all the cancer cell lines tested.     

Studies directed toward the asialoglycoprotein receptor mediated delivery of 5-fluoro-2′-deoxyuridine for hepatocellular carcinoma

The anticancer nucleoside 5-fluoro 2′-deoxyuridine-5′-phosphate (5-FdU-P) was attached via an amide chain linker to a triantennary GalNAc cluster as a means to deliver the drug to hepatic cells that recognize the amino sugar units.     

Development of ethynyl-2′-deoxyuridine chemical probes for cell proliferation

A common method of evaluating cellular proliferation is to label DNA with chemical probes. 5-Ethynyl-2′-deoxyuridine (EdU) is a widely utilized chemical probe for labeling DNA, and upon incorporation, EdU treatment of cells is followed by a reaction with a small molecule fluorescent azide to allow detection. The limitations when using EdU include cytotoxicity and a reliance on nucleoside active transport mechanisms for entry into cells. Here we have developed six novel EdU pro-labels that consist of EdU modified with variable lipophilic acyl ester moieties. This pro-label:chemical probe relationship parallels the prodrug:drug relationship that is employed widely in medicinal chemistry. EdU and EdU pro-labels were evaluated for their labeling efficacy and cytotoxicity. Several EdU pro-label analogues incorporate into DNA at a similar level to EdU, suggesting that nucleoside transporters can be bypassed by the pro-labels. These EdU pro-labels also had reduced toxicity compared to EdU.     

A Convenient Synthesis of N,N′-dibenzyl-2,4-diaminopyrimidine-2′-deoxyribonucleoside and 1-Methyl-2′-Deoxypseudoisocytidine

The syntheses of N,N′-dibenzyl-2,4-diaminopyrimidine-2′-deoxyribonucleoside and 1-methyl-2′-deoxypseudoisocytidine via Heck coupling are described. A survey of the attempts to use the Heck coupling to synthesize N,N′-dibenzyl-2,4-diaminopyrimidine-2′-deoxyribonucleoside is provided, indicating a remarkable diversity in outcome depending on the specific heterocyclic partner used.     

Synthesis of a Novel Aldehyde: 4-O-Methyl-5-formylmethyl- 2′-deoxyuridine

Abstract

The synthesis of the blocked nucleoside 3′,5′-di-O-p-toluoyl-4-O-methyl-5-formylmethyl-2′-deoxyuridine (19) was accomplishied in eleven steps from gamma-butyrolactone. This aldehyde, which should facilitate the synthesis of nucleosides containing ¹⁸F, was converted to the corresponding blocked dithianyl nucleoside (21), and also to 5-(2,2-difluoroethyl)-substituted derivatives of 2′-deoxyuridine and 2′-deoxycytidine.     

Studies on the hydrolysis of 3-methyl-2′-deoxycytidine in aqueous solution A synthesis of 3-methyl-2′-deoxyuridine

The hydrolysis of 3-methyl-2′-deoxycytidine in aqueous solution has been investigated. Varying proportions of 3-methylcytosine, 3-methyluracil and 3-methyl-2′-deoxyuridine are formed depending upon conditions of pH and temperature. All three hydrolytic products are formed at pH 6.8 and 90°C. At pH 2, depyrimidination of 3-methylcytosine occurs as the only hydrolysis product. When the pH is increased to 12, 3-methyl-2′-deoxycytidine on heating at 90°C is completely deaminated to 3-methyl-2′-deoxyuridine with few side products formed. This reaction serves as the basis for a convenient synthesis of 3-methyl-2′-deoxyuridine. The 300 MHz spectra of 3-methyl-2′-deoxycytidine and 3-methyl-2′-deoxyuridine indicate that the sugar ring in these compounds is predominantly in ²E conformation.     

New Synthesis of 5-Carboxy-2′-deoxyuridine and Its Incorporation into Synthetic Oligonucleotides

Abstract

5-Carboxy-2′-deoxyuridine is a methyl oxidation product of thymidine. It can be formed by the menadione-mediated photosensitization of thymidine in aerated aqueous solution. Here in we present a new four-step synthesis of the 5-carboxy-2′-deoxyuridine phosphoramidite building block based on the alkaline hydrolysis of 5-trifluoromethyl-2′-deoxyuridine. The phosphoramidite derivative has been incorporated at defined sites into oligonucleotides using the solid phase synthesis approach.     

Synthesis,Anti-HIV Activity and Stability Studies of 3′-Azido-2′,3′-dideoxythymidine-5′-fluorophosphate

Abstract

The synthesis, in vitro anti-HIV activity, and stability studies of AZT 5′-fluorophosphate (F-AZTMP) are reported. The present results demonstrate that such compound is a bioprecursor of its parent 5′-mononucleotide (AZTMP) but its biotransformation does not allow its selective intracellular delivery. Moreover, several attempts were carried out in order to improve the biological activity of this compound by the use of a SATE prodrug strategy.     

Studies on the Synthesis of N′-Acetyl AZA-Analogues of Ganciclovir—Unexpected Liability of N′-(2-Hydroxyethyl)-Azanucleosides Under Basic Conditions

The O′-pivaloyl diesters of N′-acetyl-azanucleosides were obtained from N-[1,3-di(pivaloyloxy)prop-2-yl]-N-(pivaloyloxymethyl)acetamide and a silylated nucleobase under Vorbrüggen's conditions. Unexpectedly, de-pivaloylation of the diesters under basic conditions afforded the corresponding nucleobase and N-acetylserinol. Mechanistic investigations showed that these products result from the following cascade of spontaneous transformations initiated by the mono de-pivaloylation of the starting diesters. N′-Deacetylation of the resultant mono-esters via the intramolecular N-O acetyl migration is the key step of the cascade; the corresponding NH-azanucleosides in the form of O-acetyl-O′-pivaloyl diesters are formed. Fragmentation of these diester intermediates gives the nucleobase and O-acetyl-O′-pivaloylserinol. Conversion of the latter to N-acetylserinol involves the selective O-N acetyl migration followed by de-pivaloylation of the resulting N-acetyl-O-pivaloylserinol.     

Concise Synthesis of Triazole-Linked 5′-Peptide-Oligonucleotide Conjugates by Click Chemistry

A concise synthesis of oligonucleotide 5′-peptide-conjugates via copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition in aqueous solution is described. Synthesis of reagents was accomplished by on-column derivatization of corresponding peptides and oligonucleotides. This method is well suited for the preparation of peptide–oligonucleotide conjugates containing 1,2,3-triazole linkage between the 5′-position of an oligonucleotide and the N-terminus of a peptide.     

Carborane-linked 2′-deoxyuridine 5′-O-triphosphate as building block for polymerase synthesis of carborane-modified DNA

5-[(p-Carborane-2-yl)ethynyl]-2′-deoxyuridine 5′-O-triphosphate was synthesized and used as a good substrate in enzymatic construction of carborane-modified DNA or oligonucleotides containing up to 21 carborane moieties in primer extension reactions by DNA polymerases.     

Synthesis of 2′-O-p-Nitrophenylethylsulfonyl-Ribonucleosides

Abstract

The appropriate protection of the 2′-OH group in ribonucleosides is the crucial point of oligoribonucleotide synthesis. The use of the tetrahydropyranyl (1), 4-methoxytetrahydropyranyl (2), tert. butyldimethylsily1 (3) and o-nitrobenzyl group (4) respectively has its limitations in the acid sensitivity or the problems arising during the deprotection steps. A more stable blocking group is needed, which is stable towards acid and base treatment but can be cleaved selectively under different conditions.     

Aminolysis of 2′-Deoxyinosine Aryl Ethers: Nucleoside Model Studies for the Synthesis of Functionally Tethered Oligonucleotides

Abstract

Several O⁶-aryl-2′-deoxyinosines were synthesized and found to undergo conversion to N⁶-substituted-2′-deoxyadenosines upon treatment with aqueous amines. The kinetics for reaction of these nucleosides with various amines suggests that O⁶-phenyl- and O⁶-(p-nitrophenyl)-2′-deoxyinosine are suitable “convertible nucleoside” precursors for the site-specific introduction of functionally tethered 2′-deoxyadenosines into DNA.     

Evaluation of Lactam Protection for Synthesis of 2′-O-Alkylated Uridines

Two different classes of protection for the uridine lactam function have been evaluated. These are benzoyl protections and different acetal functions. In particular the triisopropylsiloxymethyl protection is a most promising lactam protecting group for use in synthesis of 2′-O-alkyl-uridines.     

Synthesis of Suitably-Protected Phosphoramidites of 2′-Fluoro-2′-Deoxyguanosine and 2′-Amino-2′-Deoxyguanosine for Incorporation Into Oligoribonucleotides

Abstract

A novel synthesis of 2′-fluoro-2′-deoxyguanosine employing DAST as the fluorinating agent is presented. The preparation of its phosphoramidite as well as that of 2′-amino-2′-deoxyguanosine is also described.     

A Convenient Synthesis of N4,O3′, O5′-Triacetyl-2′-Ketocytidine

Abstract

A convenient synthesis of the title compound in four steps from cytidine is reported. Key transformations include differentiation of the 2′ position as N⁴,O^3′,O^5′-triacetyl-2,2′-anhydrocytidine, opening to the arabino derivative, and oxidation of the 2′ position with the Dess-Martin reagent.     

Novel Method for the Synthesis of 2′ -Phosphorylated Oligonucleotides

We have developed a new method for the preparation of oligodeoxyribonucleotides and oligo(2′-O-methylribonucleotides) that contain a 2′-phosphorylated ribonucleoside residue, and optimized it to avoid 2′ -3′ -isomerization and chain cleavage. Structures of the 2′ -phosphorylated oligonucleotides were confirmed by MALDI-TOF MS and enzymatic digestion, and the stability of their duplexes with DNA and RNA was investigated. 2′-Phosphorylated oligonucleotides may be useful intermediates for the introduction of various chemical groups for a wide range of applications.     

Facile Synthesis of 8, 2′-S-Cyclopurine Nucleoside

Abstract

This paper describes the synthesis of 8,2 -anhydro-8-mercapto-9-(β-D-arabinofuranosyl)purine (8,2′-S-cyclopurinenucleoside, 1) via the shorter route from 3′,5′-di-O-acectyl-8,2′-S-cycloadenosine (6) and by direct reductive deamination with n- pentyl nitrite in tetrahydrofuran (THF) and deacetylation. The preparation of 8,2′-S-cycloadenosine (2) was achieved in good yield by the cyclization of the protected 8-mercaptoadenosine with triphenylphosphine and diethyl azodicarboxylate (DEAD) in THF at room temperature, under Mitsunobu reaction conditions.     

The Synthesis of Bicyclic N4-Amino-2′-deoxycytidine Derivatives

Abstract

A number of bicyclic N⁴-amino-2′-deoxycytidine derivatives have been prepared. Their ambivalent hydrogen bonding potential makes them of interest for mutagenesis studies, and for incorporation into oligonucleotides for probes and primers.     

Diastereo- and Enantioselective Synthesis of 1′-C-Branched N,O-Nucleosides

Abstract

A synthetic approach towards 1′-C-branched N, O-nucleosides is reported, based on 1,3-dipolar cycloaddition of ethoxycarbonylnitrone. The asymmetric version of the process exploits the presence of a chiral auxiliary at the carbon atom of nitrone and leads to β-D and β-L nucleosides in good yields.