Cobalt-assisted route to rare carbocyclic C-ribonucleosides.

(Re)emerging RNA viruses have been major threats to public health in the past years, and from the few drugs available, nucleoside analogues are still at the cornerstone of the antiviral therapy. Among them, the synthesis of carbocyclic C-nucleosides is suffering from long syntheses and poor yields. Herein we report a concise stereoselective synthesis of rare carbocyclic C-nucleosides (11a-l) bearing non-canonical nucleobases through a cobalt-assisted-route as key step starting from the optically pure (-)-cyclopentenone 1. This approach paves the route for novel carbocyclic C-nucleoside discovery.

Circadian and chemotherapy-related changes in urinary modified nucleosides excretion in patients with metastatic colorectal cancer.

Urinary levels of modified nucleosides reflect nucleic acids turnover and can serve as non-invasive biomarkers for monitoring tumour circadian dynamics, and treatment responses in patients with metastatic colorectal cancer. In 39 patients, median overnight urinary excretion of LC-HRMS determinations of pseudouridine, was ~ tenfold as large as those of 1-methylguanosine, 1-methyladenosine, or 4-acetylcytidine, and ~ 100-fold as large as those of adenosine and cytidine. An increase in any nucleoside excretion after chemotherapy anticipated plasma carcinoembryonic antigen progression 1-2 months later and was associated with poor survival. Ten fractionated urines were collected over 2-days in 29 patients. The median value of the rhythm-adjusted mean of urinary nucleoside excretion varied from 64.3 for pseudouridine down to 0.61 for cytidine. The rhythm amplitudes relative to the 24-h mean of 6 nucleoside excretions were associated with rest duration, supporting a tight link between nucleosides turnover and the rest-activity rhythm. Moreover, the amplitude of the 1-methylguanosine rhythm was correlated with the rest-activity dichotomy index, a significant predictor of survival outcome in prior studies. In conclusion, urinary excretion dynamics of modified nucleosides appeared useful for the characterization of the circadian control of cellular proliferation and for tracking early responses to treatments in colorectal cancer patients.

Synthesis of a molecularly imprinted polymer to isolate glucosamine from plant extracts by an ionic-non-covalent dual approach.

OBJECTIVE: The objective of this study was to synthesize a novel glucosamine-imprinted sorbent based on ionic and non-covalent dual approach to purify glucosamine from chicory root extracts. METHODS: The synthesis of the molecularly imprinted polymer was optimized in terms of choice of monomers, porogen, cross-linker and initiator to have the best recognition as possible for targeted molecule. The sorbent obtained was characterized by nitrogen sorption (BET), scanning electron microscopy (SEM) and solid-phase extraction (SPE) to plot adsorption isotherms. The selectivity of polymer between glucosamine and interfering salt as ammonium sulphate was calculated. Extraction procedure was optimized in terms of loading, washing and elution solvents, to have the best recovery of glucosamine. Compounds were analysed by HPLC-UV after chemical derivatization. RESULTS: The results showed that the optimal conditions of extracting glucosamine on this new type of sorbent were as follows: percolation of plant extract in EtOH/aqueous HCl pH 3, washing of cartridge with water and elution of compound of interest with aqueous acetic acid solution at 5%. The recoveries of glucosamine were around 53% and 70%, from aqueous standard solution and aqueous chicory roots extracts, respectively, on the molecularly imprinted polymer. And, only 11% and 7% of the ammonium sulphate were recovered from standard solution and chicory roots extract, respectively. CONCLUSION: The use of the MIP as solid-phase extraction sorbent was able to extract preferentially glucosamine from structural analogues and ammonium salt. Assays on chicory roots extracts were carried out, and the MIP showed good results allowing the transfer methodology at semi-industrial scale for cosmetic companies. The optimized protocol of extraction of glucosamine allowed using only eco-friendly solvents, as ethanol, water and acetic acid.

Potential and perspectives of cyclonucleosides.

Cyclonucleosides are defined as analogs of natural nucleosides with an additional covalent bond between the nucleobase and the sugar moiety. They differ from classical nucleosides in more rigid structure and fixed conformation, which are responsible for unique properties and further applications. For instance, rigid structure can determine better interaction of the molecule with the acceptor, which is important in the design new bioactive of compounds. This class of nucleosides is known from the early fifties, when Todd et al. obtained cyclic salts of nucleosides. Although the formation of cyclic salts by purine nucleosides is quite common, the variety of cyclonucleosides is not only limited to this group. Up to now, various miscellaneous purine and pyrimidine cyclonucleosides and their analogs with great structural diversity were obtained; they differ from each other in position, length and type of linkage. Purine cyclonucleosides form a large group of artificially obtained derivatives. However, recently turned out cyclonucleosides also exist in nature. In fact, cyclopurine N(3),5-cycloxanthine was isolated from a marine sponge of genus Eryus sp. The aim of this review is to give an overview of the synthesis of some cyclonucleosides according to their structural types and to underline their biological activities. The article also refers to other relevant review articles that have covered particular areas of investigation or have dealt in depth with a single compound.

Analysis of intracellular didanosine triphosphate at sub-ppb level using LC-MS/MS.

An analytical procedure has been developed for the analysis of intracellular didanosine triphosphate (ddATP). An electrospray ionization tandem mass spectrometer (ESI-MS) was interfaced to liquid chromatography (LC) using a mobile phase CH3OH/H2O (25/75) containing 1% formic acid for the analysis of the 5'-triphosphate metabolite of the antiviral didanosine. In this procedure, ddATP was extracted from CEM-T4 cells, isolated using an exchange anion solid phase extraction procedure, enzymatically dephosphorylated and then analyzed by LC-MS/MS within a 1 min run time. The influence of several parameters (electrospray ionization interface, acidic modifiers of the mobile phase) has been studied. A calibration curve was generated and the linear regression analysis yielded a regression coefficient (r(2)) greater than 0.999. Using LC-MS/MS detection in single reaction monitoring mode (SRM), the limit of quantitation of ddA in CEM-T4 cells was 0.02 ng ml(-1). Furthermore, this procedure could be used to perform simultaneous detection of five nucleoside reverse transcriptase inhibitors, such as AZT, 3TC, ddA, ddC and d4T and make LC-MS/MS a method of choice for Therapeutic Drug Monitoring (TDM) in a clinical environment.

Simultaneous quantitation of nucleoside HIV-1 reverse transcriptase inhibitors by short-end injection capillary electrochromatography on a beta-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.

Analysis of anti-HIV nucleoside inhibitors by capillary electrophoresis-electrospray ionization mass spectrometry.

A method employing capillary electrophoresis (CE) with tandem mass spectrometry (MS) has been developed for the simultaneous determination, on one hand, of zidovudine (AZT) with stavudine (d4T), and on the other hand, of lamivudine (3TC) with a didanosine metabolite (ddA), four potent human immunodeficiency virus reverse transcriptase (RT-HIV) inhibitors. The influence of several parameters (pH and ionic strength of volatile formic acid-ammonia buffer) as well as the influence of magnesium cation upon electroosmotic flow, electrophoretic mobility and peak efficiency has been studied. The limit of detection (LOD) by this method is 2.5 ppb for AZT and 20 ppb for d4T, 2 ppb for ddA and 5 ppb for 3TC, respectively. This paper illustrates the current importance in CE-ESI/MS/MS technique as a complementary or substituted method to measure levels (at ng/mL) of anti-HIV drugs alone or in combination.

Synthesis of carbocyclic phosphononucleosides.

Synthesis of carbocyclic analogs of phosphononucleosides are described by two different methods (introduction of the heterocycle under Mitsunobu conditions or build-up of the base around a cyclopentylamine moiety).

Efficient synthesis of D-[1'-13C]-ribonucleosides for incorporation into oligo-RNA.

Syntheses of the monomer building blocks used for the solid-phase synthesis of specifically 1'-13C-labeled oligoribonucleotides from the D-[1-13C]ribose is presented. Procedure has been used for the selective formation of 2'-O-silylated ribonucleosides. After 5'-O-dimethoxytritylation, the synthesis of D-[1'-13C] ribonucleoside phosphoramidites has been achieved.

Synthesis of isotopically labeled D-[1'-13C]ribonucleoside phosphoramidites.

The preparation of fully protected labeled diisopropylamino-beta-cyanoethyl-[1'-13C]ribonucleoside phosphoramidites with regioisomeric purity is described. We demonstrated in this paper that a regioselective 2'-O-silylation, through a 3',5'-O-di-tert-butylsilanediyl protection, has been applied for the synthesis of [1'-13C]ribonucleoside phosphoramidite units. This method allowed us to obtain only the desired 2'-O-silyl-3'-O-phosphoramidites avoiding the undesired 3'-O-silyl-2'-O-phosphoramidite nucleosides isolated by standard procedures. This is a suitable procedure to RNA precursors with respect to the isotope-containing precursors.

Determination at ppb level of an anti-human immunodeficiency virus nucleoside drug by capillary electrophoresis-electrospray ionization tandem mass spectrometry.

Capillary electrophoresis coupled with tandem mass spectrometry was used to indirectly separate and quantify the active metabolite of the anti-human immunodeficiency virus (anti-HIV) didanosine drug. The influence of several parameters (pH and ionic strength of volatile formic acid-ammonia buffer) upon electroosmotic flow, electrophoretic mobility and peak efficiency of several nucleosides (A, dA, ddA, C) has been studied. This paper illustrates the current importance in CE-MS technique as a complementary or substituted method to the known HPLC-radioimmunoassay or HPLC-UV method to measure levels of anti-HIV drugs. The limit of detection for 2',3'-dideoxyadenosine by this method is 2 microg 1(-1) in a formic acid-ammonia buffer (pH 2.5, 10 mM ionic strength). This methodology could be used to perform simultaneous detection of two or more anti-HIV nucleosides, such as stavudine or didanosine in combination therapy.

Synthesis of 5'-thioalkyl, sulfoxide and sulfone pyrimidine nucleosides.

The preparation of 5'-thioalkyl, sulfoxide and sulfone pyrimidine nucleosides is [4-11] is described. The key steps of this synthesis are the nucleophilic displacements of a chlorine by a thioalkyl sodium salt or the direct introduction of the thioalkyl group under Mitsunobu conditions.

Phase I dose-escalation pharmacokinetics of AZT-P-ddI (IVX-E-59) in patients with human immunodeficiency virus.

3'-Azido-3'-deoxythymidilyl-(5',5')-2',3'-dideoxy-5'-inosinic acid (AZT-P-ddI, IVX-E-59, Scriptene) is a heterodimer composed of one molecule of 3'-azido-3'-deoxythymidine (zidovudine or AZT) and one molecule of 2',3'-dideoxyinosine (didanosine or ddI) linked through their 5' positions by a phosphate bond. AZT-P-ddI exhibits enhanced antiviral activity and selectivity in vitro compared with AZT and ddI alone. The pharmacokinetics of AZT-P-ddI were studied in 12 patients with human immunodeficiency virus (HIV) who had CD4+ cell counts higher than 200 cells/mm3. Isotopic preparations of (14C)-AZT-P-(3H)-ddI were administered intravenously (50 mg and 100 mg) to eight patients; 1 month later these patients were crossed over to oral administration (100 mg and 200 mg). A second group of patients (n = 4) received only a 450-mg oral dose of AZT-P-ddI. Plasma levels of unchanged AZT-P-ddI after intravenous infusion declined rapidly and were undetectable 0.75 hours after the end of infusion, whereas the parent compound was not detected after oral administration, indicative of a very rapid metabolism. The parent entity was enzymatically cleaved in vivo yielding the two constituent drugs AZT and ddI, which were subsequently subjected to their respective pharmacokinetic and metabolic processes. The beta-glucuronide derivative of AZT (GAZT) represented the major metabolite of AZT, but there were no detectable levels of the toxic metabolite 3'-amino-3'-deoxythymidine (AMT). A major and previously unrecognized in vivo metabolite of ddI, referred as ddI-M, was detected in plasma and urine. Analysis by high-field proton nuclear magnetic resonance and mass spectrometry led to the identification of ddI-M as being R(-)-dihydro-5-(hydroxymethyl)-2(3H)-furanone. The formation of AZT and ddI metabolites was increased after oral administration of AZT-P-ddI compared with the intravenous infusion, with an area under the concentration-time curve (AUC) ratio of metabolite to AZT and metabolite to ddI being 7.7 and 5.7 (oral) and 3.8 and 1.1 (intravenous), respectively. The newly identified ddI-M exhibited sustained plasma levels for extended time periods with an apparent elimination half-life (t1/2) of approximately 10 hours after oral administration of AZT-P-ddI. Recovery of radioactivity associated with 3H and 14C in urine was essentially complete within 48 hours after oral and intravenous administration of AZT-P-ddI. The oral bioavailability of AZT (64.7-67.3%) and ddI (33.6-42.9%) and the other pharmacokinetic parameters were consistent with previous data reported with each nucleoside analog alone or in combination therapy.

Inhibition of human immunodeficiency virus type 1 reverse transcriptase by the 5'-triphosphate beta enantiomers of cytidine analogs.

(-)-beta-L-2',3'-Dideoxycytidine (L-ddC) and (-)-beta-L-2',3'-dideoxy-5-fluorocytidine (L-FddC) have been reported to be potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) in vitro. In the present study, the 5'-triphosphates of L-ddC (L-ddCTP) and L-FddC (L-FddCTP) were demonstrated to competitively inhibit HIV-1 reverse transcriptase (RT), with inhibition constants (KiS) of 2 and 1.6 microM, respectively, when a poly(rI).oligo(dC)10-15 template primer was used; in comparison Ki values for beta-D-2',3'-dideoxycytidine 5'-triphosphate (D-ddCTP) and beta-D-2',3'-dideoxy-5-fluorocytidine 5'-triphosphate (D-FddCTP) were 1.1 and 1.4 microM, respectively. Use of the mutant RT at position 184 (substitution of methionine to valine [M184V]), which is associated with resistance to beta-L-2',3'-dideoxy-3'-thiacytidine (3TC) and beta-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC), resulted in significant increases (50- to 60-fold) in Ki values for L-ddCTP and L-FddCTP, whereas the elevation in Ki values for D-ddCTP and D-FddCTP was moderate (2-fold). L-ddCTP and L-FddCTP did not inhibit human DNA polymerases alpha and beta up to 100 microM. In contrast, D-ddCTP and D-FddCTP inhibited human DNA polymerase beta, with Ki values of 0.5 and 2.5 microM, respectively. By using sequencing analysis, L-ddCTP and L-FddCTP exhibited DNA chain-terminating activities toward the parental HIV-1 RT, whereas they were not a substrate for the mutant M184V HIV-1 RT.L-ddC and L-FddC did not inhibit the mitochondrial DNA content of human cells up to a concentration of 10 microM, whereas D-ddC and D-FddC decreased the mitochondrial DNA content by 90% at concentrations of 1 and 10 microM, respectively. All of these results suggest that further development of L-ddC, and L-FddC in particular, is warranted as a possible anti-HIV candidate.