INACTIVATION OF S-ADENOSYL-L-HOMOCYSTEINE HYDROLASE WITH FLUORINATED ANALOGS OF 2′- AND 3′-DEOXY-5′-METHYLTHIOADENOSINE

Fluorinated analogs of 2′- and 3′-deoxy-5′-methylthioadenosine 1–4 caused irreversible inactivation of AdoHcy hydrolase. Based on the ESI-Mass spectra analysis of the inactivated enzyme with the fluorinated analog 1 a mechanism of inactivation is proposed.     

SYNTHESIS OF SOME 2′-FLUORO-2′-DEOXY-3′-C-ETHYNYL AND 3′-C-VINYL-β-D-LYXOFURANOSYL NUCLEOSIDES

1-(2-Fluoro-2-deoxy-β-D-arabinofuranosyl)uracil (5) and 1-(2-fluoro-2-deoxy-β-D-arabinofuranosyl)cytosine (6) were synthesized as reported earlier. Both of these compounds were converted into 2′-fluoro-2′-deoxy-3′-C-ethynyl and 3′-C-vinyl-β-D-lyxofuranosyl nucleosides (16–19) by a multistep sequence. All these new nucleosides were evaluated against seven human tumor cell lines in vitro.     

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).     

SYNTHESIS AND BIOLOGICAL ACTIVITY OF 2′-DEOXY-4′-THIO-PYRAZOLO[3,4-d]PYRIMIDINE NUCLEOSIDES

The coupling of 4-aminopyrazolo [3, 4-d]pyrimidine with the appropriate thio sugar gave a 3:1 ratio of α,β blocked 4-amino-1-(2-deoxy-4-thio-D-erythropentofuranosyl)- 1H pyrazolo[3,4-d]pyrimidine nucleosides. The mixture was deblocked, both the anomers were separated, and the β-anomer was readily deaminated by adenosine deaminase. The nucleosides have been characterized, and their anomeric configurations have been determined by proton NMR. All three nucleosides were evaluated against a panel of human tumor cell lines for cytotoxicity in vitro. The details of a convenient and high yielding synthesis of these nucleosides are described.     

SYNTHESIS AND IN VITRO ANTI-HCV ACTIVITY OF β-D- and L-2′-DEOXY-2′-FLUORORIBONUCLEOSIDES

Based on the discovery of β-D-2′-deoxy-2′-fluorocytidine as a potent anti-hepatitis C virus (HCV) agent, a series of β-D- and l-2′-deoxy-2′-fluororibonucleosides with modifications at 5 and/or 4 positions were synthesized and evaluated for their in vitro activity against HCV and bovine viral diarrhea virus (BVDV). The introduction of the 2′-fluoro group was achieved by either fluorination of 2,2′-anhydronucleosides with hydrogen fluoride-pyridine or potassium fluoride, or a fluorination of arabinonucleosides with DAST. Among the 27 analogues synthesized, only the 5-fluoro compounds, namely β-D-2′-deoxy-2′,5-difluorocytidine (5), had anti-HCV activity in the subgenomic HCV replicon cell line, and inhibitory activity against ribosomal RNA. As β-D-N4-hydroxycytidine (NHC) had previously shown potent anti-HCV activity, the two functionalities of the N4-hydroxyl and the 2′-fluoro were combined into one molecule, yielding β-D-2′-deoxy-2′-fluoro-N4-hydroxycytidine (12). However, this nucleoside showed neither anti-HCV activity nor toxicity. All the l-forms of the analogues were devoid of anti-HCV activity. None of the compounds showed anti-BVDV activity, suggesting that the BVDV system cannot reliably predict anti-HCV activity in vitro.     

3′-AZIDO-3′-DEOXY-5′-O-ISONICOTINOYLTHYMIDINE,A NEW PRODRUG OF ZIDOVUDINE. SYNTHESIS,SOLID STATE CHARACTERIZATION AND ANTI HIV-1 ACTIVITY

ABSTRACT

Synthesis, solid state characterization and anti HIV-1 activity of 3′-azido-3′-deoxy-5′-O-isonicotinoylthymidine (2), a new prodrug of zidovudine (AZT, 1), are described. Two solid forms of 2 prepared by crystallization from ethyl acetate-petroleum ether (form α) and from a melt sample of form α (amorphous form) were characterized by X-ray diffractometry, infrared spectroscopy, differential scanning calorimetry (DSC) and thermogravimetry (TGA) techniques. The novel nucleoside exhibited antiviral activity against standard and resistant strain panels of HIV-1 as well as cytotoxicity similar to that of AZT.     

SYNTHESIS OF NOVEL 3′-DEOXY-3′-C-HYDROXYMETHYL NUCLEOSIDES WITH CONFORMATIONALLY RIGID SUGAR MOIETY AS POTENTIAL ANTIVIRAL AGENTS

Based on the fact that the ring expanded 3′-C-hydroxymethyl analogue of oxetanocin A exhibited potent antiviral activity, two types of conformationally rigid 3′-C-hydroxymethyl derivatives in which 2′-hydroxyl group is linked to the 4′-position or to the 6′-position were synthesized starting from 1,2;5,6-di-O-isopropylidene-D-glucose, respectively.     

A NEW APPROACH TO THE SYNTHESIS OF 4′-CARBON-SUBSTITUTED NUCLEOSIDES: DEVELOPMENT OF A HIGHLY ACTIVE ANTI-HIV AGENT 2′, 3′-DIDEHYDRO-3′-DEOXY-4′-ETHYNYLTHYMIDINE

Oxidation of 3′-O-TBDMS-4′,5′-unsaturated thymidine 3 with dimethyldioxirane (DMDO) allowed the isolation of the epoxide 4. Upon reacting with organosilicon reagents in the presence of SnCl₄, 4 underwent stereoselective ring opening to give 4′-α-allyl (6), 4′-α-(2-bromoallyl) (7), 4′-α-(cyclopenten-3-yl) (8), and 4′-α-cyano (9) derivatives of thymidine. Reactions of the 3′-epimer 12 with organoaluminum reagents gave 4′-α-methyl (13), 4′-α-vinyl (14), and 4′-α-ethynyl (15) analogues. Compounds 13–15 were transformed into corresponding 2′,3′-didehydro-3′-deoxy derivatives. Evaluation of their ability to inhibit the replication of HIV in cell culture showed that 4′-ethynyl-d4T (19) is more potent and less toxic than the parent compound d4T.     

INHIBITION OF HUMAN TELOMERASE BY L-ENANTIOMERS OF NATURAL 2′-DEOXYRIBONUCLEOSIDE 5′-TRIPHOSPHATES

In order to clarify whether L-enantiomers of natural 2′-deoxyribonucleoside 5′-triphosphates (dNTPs) are recognized by human telomerase, a quantitative telomerase assay based on the ‘stretch PCR’ method was developed and used for kinetic analysis. Among the four L-dNTPs, L-dTTP and L-dGTP inhibited telomerase activity and the others showed slight or no inhibitory effect. Lineweaver-Burk plot analysis showed that the inhibition mode L-dGTP was competitive with dGTP.     

SYNTHESIS OF NOVEL D- AND L-3′-DEOXY-3′-C-HYDROXYMETHYL NUCLEOSIDE WITH EXOCYCLIC METHYLENE AS POTENTIAL RIBONUCLEOTIDE REDUCTASE INHIBITOR

D- and L-3′-Deoxy-3′-C-hydroxymethyl thymidine substituted with exocyclic methylene at 2′-position were synthesized, starting from D- and L-xylose as potential ribonucleotide reductase inhibitor, respectively, but they were found to be inactive against several tumor cell lines.     

SYNTHESIS OF A NEW 5′-O-TRIPHOSPHATE ANALOG OF 5-METHYL 2′-O-DEOXYCYTIDINE. PRELIMINARY IN VITRO LABELING FOR NON-RADIOACTIVE DETECTION OF DNA

We report the synthesis of the triphosphate of 5-methyl 4-N-[6-(p-bromobenzamido)hex-1-yl]-2′-O-deoxycytidine 3A . We also analyzed the formation of intramolecular H-bonds of 5-methyl 4-N-{n-[6-(p-bromobenzamido) caproyl amino]alk-1-yl}-2′-deoxycytidine compounds, and confirmed their presence by ¹H-NMR studies. In vitro DNA labeling with modified nucleotides is preliminarily evaluated.     

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 A NOVEL BRIDGED NUCLEIC ACID ANALOGUE, 5′-AMINO-3′,5′-BNA

An oligonucleotide P3′?N5′ phosphoramidate (5′-amino-DNA) attracts much attention because of its potential for application to DNA sequencing; however, its ability to hybridize with complementary strands is low. To overcome this drawback of the 5′-amino-DNA, we have designed and successfully synthesized a novel nucleic acid analogue having a P3′?N5′ phosphoramidate linkage and a constrained sugar moiety, 5′-amino-3′-C,5′-N-methylene bridged nucleic acid (5′-amino-3′,5′-BNA). The binding affinity of the 5′-amino-3′,5′-BNA towards complementary DNA and RNA strands was investigated by UV melting experiments. The melting temperature (Tm) of the duplex comprising the 5′-amino-3′,5′-BNA and its complementary strand was much higher than that of the duplex containing the corresponding 5′-amino-DNA.     

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 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.     

5′-Nucleotidase

Summary The distribution of 5-nucleotidase in several tissues of rat and mouse has been investigated. The enzyme is greatly specific in dephosphorylating 5-nucleotides. Two 5-nucleotidases seem to exist: one showing greatest activity at pH 5.0, while the other is most active at pH 7.0–7.5. The localization of these two enzymes is not identical. At the acid pH the deoxyribonucleotides are dephosphorylated faster than the ribonucleotides, while at the neutral pH the ribonucleotides are hydrolysed more rapidly. In contrast to the non-specific phosphatases the nucleotidases can be stimulated by magnesium and manganese ions. The acid nucleotidase can be considered as a lysosomal enzyme. The neutral nucleotidase can be involved in transport processes in capillaries and sinusoids. Other localizations of the enzyme suggest a role of the enzyme in the catabolism of nucleic acids.     

5′-Nucleotidase

Summary To get more insight in the function of 5-nucleotidase catabolic and anabholic processes were investigated in which 5-nucleotides are involved. The catabolism of adenosine-5-monophosphate was studied by investigating the reaction products obtained after incubation of homogenates of several organs of rat and mouse with adenosine-5-monophosphate and with adenosine. Two experimental tumours of the mouse were investigated in the same way. It was found that in tissues containing a high activity of 5-nucleotidase other enzymes involved in the catabolism of 5-nucleotides, such as nucleosidase, adenosine deaminase and adenosine-5-monophosphate deaminase could also be demonstrated.The anabolic processes in which 5-nucleotides are involved had been studied by investigating the incorporation of tritium-labeled thymidine in several tissues of the mouse. It appeared that in cells showing a high 5-nucleotidase activity no incorporation of radioactive thymidine could be found, while in cells showing incorporation of thymidine enzyme activity could not be demonstrated.A discussion is given about the possible role of 5-nucleotidase in the control of nucleic acid biosynthesis and in the catabolism of nucleic acids.Abbreviations used DNA deoxyribonucleic acid - RNA ribonucleic acid - AMP Adenosine-5-monophosphate - ADP Adenosine-5-diphosphate - ATP Adenosine-5-triphosphate - IMP Inosine-5-monophosphate - GMP Guanosine-5-monophosphate - GDP Guanosine-5-diphosphate - GTP guanosine-5-triphosphate - CMP Cytidine-5-monophosphate - CDP Cytidine-5-diphosphate - CTP Cytidine-5-triphosphate - UMP Uridine-5-monophosphate - UDP Uridine-5-diphosphate - UTP Uridine-5-triphosphate - TMP Thymidine-5-monophosphate - TDP Thymidine-5-diphosphate - TTP Thymidine-5-triphosphate - Ado Adenosine - Ad Adenine - Ino Inosine - Hypox Hypoxanthine - Xanth Xanthine - Xantho Xanthosine - Guano Guanosine - Gua Guanine - Ura Uracil - U Uridine - Cyt Cytidine - Cyto Cytosine - Thym Thymidine The corresponding deoxy-compounds have been indicated with the prefix d for instance dCMP, deoxycytidine-5-monophosphate.     

5′-Nucleotidase

Summary Determinations of pH activity curves and of Michaelis constants of 5-nucleotidases in organs of rat and mouse indicate the heterogenity of the enzyme in these tissues. Electrophoretic analyses of homogenates and cell component fractions reveal the presence of 5-nucleotidase isoenzymes in the investigated tissues. At the acid as well as at the neutral pH five isoenzymes were found. In addition three alkaline phosphatases were found in the rat; in the mouse four alkaline phosphatase isoenzymes could be demonstrated. The different combinations of 5-nucleotidase isoenzymes in the investigated tissues possibly indicate different functions of the isoenzymes. A discussion is given of the correlations between the electrophoretic results and the histochemical findings.