Luigi A. Agrofoglio

Synthesis of 1,2,3-triazolo-carbanucleoside analogues of ribavirin targeting an HCV in replicon.

The synthesis of carbocyclic and phosphonocarbocyclic analogues of ribavirin, an anti-HCV inhibitor, are described. Those compounds were evaluated against HCV but also against other important viruses in order to determine their spectrum of antiviral activity. Compounds 6 and 13 displayed a moderate IC(50) against HIV-1 of 43.8 and 37 microM, respectively.

Preparation of Cyclonucleosides

2.2.1. Synthesis from Classical Nucleosides 1838 2.2.2. From an Antibiotic Nucleoside 1839 2.2.3. From Miscellaneous Nucleosides 1839 3. Synthesis of Pyrimidine Cyclonucleosides 1840 3.1. Halogen-Involved Methods 1840 3.1.1. During Halogenation Reactions 1840 3.1.2. During Dehalogenation Reactions 1842 3.2. Under Radical Conditions 1844 3.2.1. Formation of a 6,5′-O-Bridge 1844 3.2.2. Formation of a 6,1′-Bridge (Spironucleoside) 1844

Human and viral nucleoside/nucleotide kinases involved in antiviral drug activation: Structural and catalytic properties

Antiviral nucleoside and nucleotide analogs, essential for the treatment of viral infections in the absence of efficient vaccines, are prodrug forms of the active compounds that target the viral DNA polymerase or reverse transcriptase. The activation process requires several successive phosphorylation steps catalyzed by different kinases, which are present in the host cell or encoded by some of the viruses. These activation reactions often are rate-limiting steps and are thus open to improvement. We review here the structural and enzymatic properties of the enzymes that carry out the activation of analogs used in therapy against human immunodeficiency virus and against DNA viruses such as hepatitis B, herpes and poxviruses. Four major classes of drugs are considered: thymidine analogs, non-natural L-nucleosides, acyclic nucleoside analogs and acyclic nucleoside phosphonate analogs. Their efficiency as drugs depends both on the low specificity of the viral polymerase that allows their incorporation into DNA, but also on the ability of human/viral kinases to provide the activated triphosphate active forms at a high concentration at the right place. Two distinct modes of action are considered, depending on the origin of the kinase (human or viral). If the human kinases are house-keeping enzymes that belong to the metabolic salvage pathway, herpes and poxviruses encode for related enzymes. The structures, substrate specificities and catalytic properties of each of these kinases are discussed in relation to drug activation.

Novel Antiviral C5-Substituted Pyrimidine Acyclic Nucleoside Phosphonates Selected as Human Thymidylate Kinase Substrates

Acyclic nucleoside phosphonates (ANPs) are at the cornerstone of DNA virus and retrovirus therapies. They reach their target, the viral DNA polymerase, after two phosphorylation steps catalyzed by cellular kinases. New pyrimidine ANPs have been synthesized with unsaturated acyclic side chains (prop-2-enyl-, but-2-enyl-, pent-2-enyl-) and different substituents at the C5 position of the uracil nucleobase. Several derivatives in the but-2-enyl- series 9d and 9e, with (E) but not with (Z) configuration, were efficient substrates for human thymidine monophosphate (TMP) kinase, but not for uridine monophosphate-cytosine monophosphate (UMP-CMP) kinase, which is in contrast to cidofovir. Human TMP kinase was successfully crystallized in a complex with phosphorylated (E)-thymidine-but-2-enyl phosphonate 9e and ADP. The bis-pivaloyloxymethyl (POM) esters of (E)-9d and (E)-9e were synthesized and shown to exert activity against herpes virus in vitro (IC(50) = 3 μM) and against varicella zoster virus in vitro (IC(50) = 0.19 μM), in contrast to the corresponding inactive (Z) derivatives. Thus, their antiviral activity correlates with their ability to act as thymidylate kinase substrates.

Synthesis of new C5-(1-substituted-1,2,3-triazol-4 or 5-yl)-2′-deoxyuridines and their antiviral evaluation

The synthesis and antiviral evaluation of a series of C5-(1,4- and 1,5-disubstituted-1,2,3-triazolo)-nucleoside derivatives is described. The key steps of this synthesis are regioselective Huisgens 1,3-dipolar cycloaddition, using either copper-catalyzed azide-alkyne cycloaddition (CuAAC) or ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC) under microwave activation. Some compounds among the 5a-l series possess activity against herpes simplex viruses 1 and 2, varicella-zoster virus, human cytomegalovirus and vaccinia virus. Their cytostatic activities were determined against murine leukemia cells, human T-lymphocyte cells and cervix carcinoma cells. Compounds were also evaluated on a wide panel of RNA viruses, including Vesicular stomatitis virus, influenza viruses type A (H1N1 and H3N2) and B in MDCK cell cultures, parainfluenza-3 virus, reovirus-1, Sindbis virus and Punta Toro virus in Vero cell cultures and Vesicular stomatitis, Coxsackie B4 and respiratory syncytial virus, with no specific antiviral effect.

Simultaneous quantitation of nucleoside HIV-1 reverse transcriptase inhibitors by short-end injection capillary electrochromatography on a β-cyclodextrin-bonded silica stationary phase

As part of our on-going study of the analysis and quantitation of anti-HIV nucleosides, a capillary electrochromatography (CEC) method has been developed for the simultaneous quantitation of nucleoside HIV reverse transcriptase inhibitors (NRTIs), i.e. zidovudine (AZT), lamivudine (3TC), didanosine (ddA) and its administrated form (ddI), stavudine (d4T) and hivid (ddC). CEC on chiral stationary phase has mainly been dedicated to the separation of enantiomers. However, this paper explores an original application of a beta-cyclodextrin-bonded silica packed column, taking advantage of the internal hydrophobicity of the polysaccharide to separate the NRTIs. The influence of several parameters (pH buffer, ionic strength, acetonitrile content, temperature and voltage) has been investigated using the short-end injection technique to achieve baseline separation in a short-time analysis before quantitation.

Crystal structure of poxvirus thymidylate kinase: An unexpected dimerization has implications for antiviral therapy

Unlike most DNA viruses, poxviruses replicate in the cytoplasm of host cells. They encode enzymes needed for genome replication and transcription, including their own thymidine and thymidylate kinases. Some herpes viruses encode only 1 enzyme catalyzing both reactions, a peculiarity used for prodrug activation to obtain maximum specificity. We have solved the crystal structures of vaccinia virus thymidylate kinase bound to TDP or brivudin monophosphate. Although the viral and human enzymes have similar sequences (42% identity), they differ in their homodimeric association and active-site geometry. The vaccinia TMP kinase dimer arrangement is orthogonal and not antiparallel as in human enzyme. This different monomer orientation is related to the presence of a canal connecting the edge of the dimer interface to the TMP base binding pocket. Consequently, the pox enzyme accommodates nucleotides with bulkier bases, like brivudin monophosphate and dGMP; these are efficiently phosphorylated and stabilize the enzyme. The brivudin monophosphate-bound structure explains the structural basis for this specificity, opening the way to the rational development of specific antipox agents that may also be suitable for poxvirus TMP kinase gene-based chemotherapy of cancer.

Chemical synthesis of 13C labeled anti-HIV nucleosides as mass-internal standards

Abstract Synthesis of [ 13 C 5 ]-labeled anti-HIV nucleosides, e.g. d4T, ddI, ddA, is described. The methodology used has been optimized due to the very high cost of the starting compound. The key step of this approach was the stereoselective dehomologation of 1,2:5,6-di- O -isopropylidene-3-oxo-α- d -glucofuranose ( 2 ) with periodic acid and sodium borohydride, which gave optically pure ribose derivative as the exclusive product. Nucleoside derivatives 6a – c were obtained from ribosylation of 5 with persilylated nucleobases under Vorbruggen conditions. Deoxygenation of 9a – c under Corey-Winter conditions afforded the desired labeled nucleoside analogues 12a – c .

Molecularly imprinted polymer of 5-methyluridine for solid-phase extraction of pyrimidine nucleoside cancer markers in urine.

Normal and modified urinary nucleosides represent potential biomarkers for cancer diagnosis. To selectively extract modified nucleosides, we developed a molecularly imprinted polymer (MIP) of 5-methyluridine as selective material for molecularly imprinted solid-phase extraction (MISPE). The MIPs were obtained from vinyl-phenylboronate ester derivative of the template, acrylamide and pentaerythritol triacrylate co-polymer, and were tested in batch and cartridge experiments with aqueous samples. Our results indicated that the imprinted polymer was selective for pyrimidine nucleosides with a K(d) and a B(max) of 46 microM and 18 micromol/g, respectively. Finally, a MISPE of the most common pyrimidine nucleoside cancer markers in urine sample was realized.

Liquid chromatographic separation of phosphoramidate diastereomers on a polysaccharide-type chiral stationary phase

To improve the therapeutic potential of anti-HIV nucleoside analogues (d4T, AZT, 3TC and ddl), the delivery of the corresponding monophosphate from neutral, membrane-permeable prodrugs has been realised by the synthesis of lipophilic phosphoramidate triester prodrugs, such as the simple phenyl-L-alaninephosphate derivatives. However, the present non-stereoselective synthesis results in a mixture of 1:1 diastereomers, which differ from the configuration of the phosphorus atom asymmetric center. Since each diastereomer may have different biological activity and pharmacokinetic profile, analytical methods have to be developed for their separation. This work aims at showing the ability of a polysaccharide-type chiral stationary phase to resolve such diastereomers in reversed-phase high-performance liquid chromatography. The influence of operating parameters has been studied to optimise the separation; a thermodynamic approach has also been investigated to gain an insight in the retention mechanism of the prodrugs. Preliminary validation study (linearity, accuracy, repeatability) has yielded good results; in addition, the feasibility of HPLC-electrospray-mass spectrometry (HPLC-ESI-MS) coupling has been demonstrated and it is expected that this will lead to lower detection limits.