Synthesis and preliminary in vivo evaluation of new [18F]fluoro-inositols as Positron Emission Tomography radiotracers.

This study describes the synthesis and radiosynthesis of eight new [18F]fluoro-inositol-based radiotracers in myo- and scyllo-inositol configuration. These radiotracers are equipped with a propyl linker bearing fluorine-18. This fluorinated arm is either on a hydroxyl group, i.e. O-alkylated inositols, or on the cyclohexyl backbone, i.e. C-branched derivatives. To modulate lipophilicity, inositols were synthesized in acetylated or hydroxylated form. Automated radiosynthesis was performed on the AllInOne module and the radiotracers were produced in good radiochemical yields (15-31.5% dc). Preliminary in vivo preclinical evaluation of these eight [18F]fluoro-inositols as Positron Emission Tomography (PET) imaging agents in a breast tumour-bearing mouse model was performed and compared with [18F]-2-fluoro-2-deoxy-d-glucose ([18F]FDG). Amongst the different inositols, [18F]myo-2 showed the highest tumour uptake 2.34±0.39%ID/g, revealing the potential of this tracer for monitoring breast cancer.

Synthesis and revised stereochemical assignment of C-allyl glucopyranosides and derivatives.

α- and ß-C-allylglucopyranosides and hydroxy-, bromo- and azido-derivatives were prepared through allylation at C-1 of methyl 2,3,4,6-tetra-O-benzyl-D-glucopyranoside or 1-O-acetyl-2,3,4,6-tetra-O-benzyl-D-glucopyranose and subsequent chemical modifications of the alkene on the anomeric arm. However, we picked out some discordance between some recent published studies and our results. After a thorough work of characterization and NMR analysis, we unambiguously confirmed α and ß stereochemistry of the two series of compounds and fully described for the first time ß-C-propyl alcohol, bromide and azide of 2,3,4,6-tetra-O-benzyl-D-glucopyranose.

Diastereoselective synthesis of new O-alkylated and C-branched inositols and their corresponding fluoro analogues.

Efficient routes were developed for the diastereoselective synthesis of new O-alkylated and C-branched inositols and their corresponding fluoro analogues. The key steps of the synthesis were the easy accessibility of different types of arms in term of configuration (myo and scyllo), the linking method and length, which could modulate the biological properties. These inositol derivatives, bearing an arm terminated either with a hydroxy group or a fluorine atom, could be interesting candidates for diastereoisomeric intermediates and biological evaluations, especially for PET imaging experiments.

Development of 6-[(18) F]fluoro-carbohydrate-based prosthetic groups and their conjugation to peptides via click chemistry.

This work describes the development of new 6-[(18) F]fluoro-carbohydrate-based prosthetic groups equipped with an azido arm that are able to participate in copper(I)-catalyzed cycloadditions for (18) F labeling of biomolecules under mild conditions. The radiolabeling in high radiochemical yields (up to 68 ± 6%) of these different prosthetic groups is presented. The flexibility of the azido arm introduced on the carbohydrate moieties allows efficient click reactions with different alkyne functionalized peptides such as gluthation or Arg-Gly-Asp derivatives in order to prepare glycopeptides. The radiosyntheses of (18) F-labeled glycopeptides proceed in high radiochemical yields (up to 76%) in an automated process with excellent radiochemical purity. The addition of a sugar moiety on peptides should enhance the bioavailability, pharmacokinetic, and in vivo clearance properties of these glycopeptides, compared with the unlabeled native peptide, and these properties are highly favorable for positron emission tomography imaging. A high uptake of (18) F-ß-gluco-c(RGDfC) is shown by positron emission tomography imaging in a subcutaneous abscess model in the rat, revealing the potential of this tracer to monitor integrin expression as a part of inflammation and/or angiogenesis processes.

Enzymatic production of bioactive docosahexaenoic acid phenolic ester.

Docosahexaenoic acid (DHA) is increasingly considered for its health benefits. However, its use as functional food ingredient is still limited by its instability. In this work, we developed an efficient and solvent-free bioprocess for the synthesis of a phenolic ester of DHA. A fed-batch process catalyzed by Candida antarctica lipase B was optimised, leading to the production of 440 g/L vanillyl ester (DHA-VE). Structural characterisation of the purified product indicated acylation of the primary OH group of vanillyl alcohol. DHA-VE exhibited a high radical scavenging activity in acellular systems. In vivo experiments showed increased DHA levels in erythrocytes and brain tissues of mice fed DHA-VE-supplemented diet. Moreover, in vitro neuroprotective properties of DHA-VE were demonstrated in rat primary neurons exposed to amyloid-ß oligomers. In conclusion, DHA-VE synergized the main beneficial effects of two common natural biomolecules and therefore appears a promising functional ingredient for food applications.

Oxidative stability of DHA phenolic ester.

Docosahexaenoic acid vanillyl ester (DHA-VE) was synthesized from docosahexaenoic acid ethyl ester (DHA-EE) and vanillyl alcohol by a solvent-free alcoholysis process catalysed by Candida antarctica lipase B. Oxidative stability of pure DHA-VE and the crude reaction medium consisting of 45% DHA-VE and 55% DHA-EE were compared with that of DHA-EE under various storage conditions. Oxidation progress was followed by determination of conjugated dienes and FTIR measurements. Analyses showed that DHA-EE was rapidly oxidised under all storage conditions in comparison with DHA-VE and crude reaction medium, whatever the temperature and the storage time. The grafting of vanillyl alcohol appeared as a powerful way to stabilize DHA against oxidation. Thanks to their stability, both DHA-VE and the crude reaction medium, allowing the production of the ester, offer huge potential as functional ingredients.

'Click' glycosylation of peptides through cysteine propargylation and CuAAC.

'Click' glycosylation of cysteine-containing peptides were carried out in good yield by Copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC). For that peptides were functionalized though direct propargylation of the cysteine residue allowing their use in CuAAC with suitable free or protected azido sugars of gluco, manno and galacto configuration. Among these free and protected glycopeptides a series of 'glycoRGD' peptides were obtained and submitted to in vitro platelet aggregation tests, showing that the pseudoglycosylation of the adhesion sequence lowers the IC50 value and thus could improve the in vivo pharmacokinetic properties.

The 1,3-dipolar cycloaddition reaction of chiral carbohydrate-derived nitrone and olefin: towards long-chain sugars.

The thermal and microwave-activated 1,3-dipolar cycloadditions of several α,ß-unsaturated esters derived from d-mannose and chiral nitrones derived from threitol have been studied as a model reaction en route to eleven carbon long chain carbohydrates. Very high facial selectivity is observed for the chiral nitrones whereas the olefin facial selectivity varies with the substrate. The presence of a dioxolane ring α to the olefinic bond is beneficial to the facial selectivity of the olefin whereas a pyranose ring is not. The combination of a d-mannose derivative and a l-threitol-derived nitrone is a matched pair suitable for the synthesis of long chain sugars with nine contiguous chiral centres. Finally complete facial selectivity was observed with exo-glycals which gave a single cycloadduct.

Formation of septanoses from hexopyranosides via 5,6-exo-glycals.

Methyl D-hexo-5-ulosides are obtained in high yield by dihydroxylation of 5,6-exo-glucal compounds. The bicyclic structure (1,6-anhydropyrano-5-ulose) of the products is adopted in solution and in solid state in a (4)C(1) conformation. This methodology has been used to prepare 1,6-anhydro-L-idopyrano-5-uloses. Further manipulation of the 1,6-anhydro bridge allowed the preparation of the yet unknown septano-5-uloses in moderate to high yields.

Kinetic study of the rearrangement of deuterium-labeled 4'-O-methylnorbelladine in Leucojum aestivum shoot cultures by mass spectrometry. Influence of precursor feeding on amaryllidaceae alkaloid accumulation.

Alkaloids from plants of the family Amaryllidaceae have important pharmacological properties and can be regarded as derivatives of the common precursor 4'-O-methylnorbelladine (6) via intramolecular oxidative phenol coupling. Their biosynthetic pathway, particularly in Leucojum aestivum, has not yet been totally elucidated. Therefore, shoot cultures of this plant were subcultured in medium containing the labeled precursor 4'-O-methyl-d(3)-norbelladine (3) at various concentrations (0.05, 0.10, and 0.20 g/L) and were incubated for various periods of time (15, 30, and 40 days). The aim of this work was to study the influence of this precursor on both labeled and native alkaloid accumulation. Biotransformation into galanthamine (1) and lycorine (2) in shoot cultures was demonstrated using HPLC coupled to mass spectrometry. A maximal amount of 0.16% of 1 referred to the dry weight was obtained at day 15 in shoots fed with 0.10 g/L of precursor. In addition, a 20.5% dry weight of 2 was reached after 40 days of feeding with 0.20 g/L of precursor.

New method for the study of Amaryllidaceae alkaloid biosynthesis using biotransformation of deuterium-labeled precursor in tissue cultures.

Biotransformation of deuterated-4'-O-methylnorbelladine into alkaloids galanthamine and lycorine in tissue cultures of Leucojum aestivum was demonstrated using HPLC coupled to mass spectrometry. GC-MS screening was also carried to investigate other native and deuterated alkaloids. A total of six labeled alkaloids were identified indicating that 4'-O-methyl-d(3)-norbelladine is incorporated into three different groups of Amaryllidaceae alkaloids that are biosynthesized by three modes of intramolecular oxidative phenol coupling.

Synthesis and solution conformation of homo-beta-peptides consisting of N-mannofuranosyl-3-ulosonic acids.

The synthesis and solution conformation of homo-oligomers of beta-aminoacids, beta-N-mannofuranosyl-3-ulosonic acids, have been studied by NMR, MD simulation, and circular dichroism. These oligomers feature a spirocyclic disubstitution and a N,O-acetal functionality at the beta-carbon of the backbone, an unprecedented situation in the realm of beta-peptides. Our study shows that tetramer 10 and hexamer 11 adopt a characteristic secondary structure. In the hexamer 11, NMR investigations coupled with MD simulations suggest the preference for a double C(8) turn forming conformation.

Irreversible inhibition of glucose-6-phosphate dehydrogenase by the coenzyme A conjugate of ketoprofen: a key to oxidative stress induced by non-steroidal anti-inflammatory drugs?

Oxidative damage by non-steroidal anti-inflammatory drugs (NSAIDs) has been considered relevant to the occurrence of gastro-intestinal side-effects. In the case of chiral arylpropionate derivatives like ketoprofen (KPF), this mechanism has been evidenced for the R-enantiomer, especially when chiral inversion was observed, and lets us suppose the involvement of CoA conjugates. Glucose-6-phosphate dehydrogenase (G6PD) is the crucial enzyme to regenerate the GSH pool and maintain the intracellular redox potential. This enzyme is known to be down-regulated by palmitoyl-CoA thioester. We hypothesised then that G6PD is the target of carboxylic NSAIDs, via their CoA metabolites. We used molecular docking to localise a putative site in the human G6PD then we chose the Yeast orthologue, as the most suitable species to study experimentally the precise molecular interaction. KPF-CoA was effectively shown to bind covalently to the unique cysteine residue of the yeast enzyme. Binding was found to occur in the same site as palmitoyl-CoA. It was decreased in the presence of an allosteric inhibitor of G6PD, phospho(enol)pyruvate, and was not detected with G6PD of Leuconostoc mesenteroides, which does not possess the allosteric site. This site is distinct from the catalytic site, and probably allosteric, explaining the observed non-competitive inhibition of its activity by KPF-CoA. KPF-CoA was shown to induce the production of reactive oxygen species in Caco-2 cells, where its inhibition of G6PD activity was observed.

D-myo-inositol 1-phosphate as a surrogate of D-myo-inositol 1,4,5-tris phosphate to monitor G protein-coupled receptor activation.

Phospholipase C beta (PLC-beta)-coupled G protein-coupled receptor (GPCR) activities traditionally are assessed by measuring Ca2+ triggered by D-myo-inositol 1,4,5-trisphosphate (IP3), a PLC-beta hydrolysis product, or by measuring the production of inositol phosphate using cumbersome radioactive assays. A specific detection of IP3 production was also established using IP3 binding proteins. The short lifetime of IP3 makes this detection very challenging in measuring GPCR responses. Indeed, this IP3 rapidly enters the metabolic inositol phosphate cascade. It has been known for decades that lithium chloride (LiCl) leads to D-myo-inositol 1-phosphate accumulation on GPCR activation by inhibiting inositol monophosphatase, the final enzyme of the IP3 metabolic cascade. We show here that IP1 can be used as a surrogate of IP3 to monitor GPCR activation. We developed a novel homogeneous time-resolved fluorescence (HTRF) assay that correlates perfectly with existing methods and is easily amenable to high-throughput screening. The IP-One assay was validated on various GPCR models. It has the advantage over the traditional Ca2+ assay of allowing the measurement of inverse agonist activity as well as the analysis of PLC-beta activity in any nontransfected primary cultures. Finally, the high assay specificity for D-myo-inositol 1 monophosphate (IP1(1)) opens new possibilities in developing selective assays to study the functional roles of the various isoforms of inositol phosphates.

Elucidation of the mechanism of inhibition of cyclooxygenases by acyl-coenzyme A and acylglucuronic conjugates of ketoprofen.

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the cyclooxygenase (COX) isoforms which accounts for their clinical effects. The differential inhibition of COX-1 and COX-2 is not sufficient to explain the absence of a correlation between in vitro and in vivo effects, especially for 2-aryl-propionates, thus indicating the participation of metabolites. Conjugates to glucuronic acid and to coenzyme-A are mainly produced, and have been shown to be chemically reactive. Therefore, we studied the interaction of the ketoprofen metabolites with the COX enzymes. After incubation with bovine pulmonary artery endothelial cells (BPAEC), COX-1 was inhibited stereoselectively by S-ketoprofen acylglucuronide, and more significantly by CoA-thioester. After washing-out the medium, COX-1 activity was essentially recovered, indicating a reversible inhibition. In LPS-stimulated J774.2 cells, COX activity (mainly inducible COX-2) was inhibited reversibly and stereospecifically by S-ketoprofen glucuronide, whereas it disappeared totally and was not recovered after incubation with CoA-thioester. Correspondingly, inhibition of purified COX-2 with this compound was observed to be rapid and irreversible. Using an anti-ketoprofen antibody, COX immunoprecipitated from cells exhibited adduct formation for COX-2 but not for COX-1. This was observed after incubation with CoA-thioester, and, surprisingly, also with glucuronide. Molecular docking gave support to explain this discrepancy: the glucuronide was found to establish a strong interaction with Y115 located in the membrane binding domain, whereas the thioester was preferentially bound to the active site of the enzyme. Overall, our results suggest a contribution of CoA-thioester metabolites of carboxylic NSAIDs to their pharmacological action by irreversibly and selectively inhibiting COX-2.

A short route to enantiomerically pure benzophenanthridinone skeleton: synthesis of lactone analogues of narciclasine and lycoricidine.

Condensation of functionalized o-toluamide anions on a carbohydrate-derived lactone, followed by intramolecular aldol cyclization, provides enantiomerically pure 2-arylcyclohexenones. Different approaches for the stereoselective transformation of the carbonyl group of these key intermediates into an amino group were unsuccessful. However 1,4-addition of thiolate and concomitant ring closure to isocoumarine provided a useful method for the transformation of the tertiary amide function. Opening of the isocoumarin with ammonia provided the corresponding amide and recovery of the enone system. Subsequent reductive amination of this cyclohexenone was found to depend on the nature of the protecting groups and led to the protected form of 4-epi- and -iso-narciclasine. Oxo analogues of narciclasine and epi-narciclasine and lycoricidine were also obtained after reduction of the enone and subsequent lactonization. They showed no biological activity as antitumor agents.

Synthesis and biological testing of Acyl-CoA-ketoprofen conjugates as selective irreversible inhibitors of COX-2.

Ketoprofenoyl-CoA thioester 3 was synthesized by coupling ketoprofen to coenzyme A using the mixed anhydride method. Diastereoisomeric compounds 3a and 3b corresponding to the enantiomers of ketoprofen, were obtained in optically pure form by preparative HPLC. A non-acylating analogue, rac-3-(3-benzoylphenyl)-2-oxo-butanoyl-CoA (7) was also prepared. The biological evaluation suggested that 3a and 3b are reversible inhibitors of COX-1 and irreversible inhibitors of COX-2. Compound 7 appears to be a poor but selective inhibitor of COX-1.

D-myo-inositol-1,4,5-trisphosphate and adenophostin mimics: importance of the spatial orientation of a phosphate group on the biological activity.

Three different routes for the synthesis of heterocyclic analogues of the second messenger D-myo-inositol-1,4,5-trisphosphate (InsP(3)) and the natural adenophostins, starting from allyl D-xyloside are described. The two diastereoisomers at C-2 of new compounds, which we named xylophostins, were obtained. The preliminary biological studies shows that the presence of the adenine residue has a beneficial effect on the affinity for the receptor. The low potency of one of the two diastereoisomeric compounds shows that the configuration of the carbon bearing the non-vicinal phosphate group is an important requirement for a high affinity to the receptor. These results provide evidence for the existence of a binding pocket for the adenine ring nearby the InsP(3) binding site. The consequence of these stabilizing interactions should be to place the phosphate group in a suitable position to perfectly mimic InsP(3) in the more active diastereoisomer. Obviously, in the other diastereoisomer, the phosphate cannot accommodate the same orientation, thus explaining the low affinity. The existence of such a binding pocket for adenine is in line with the high potency of adenophostins.