Les stilbénoïdes, sont des composés phénoliques majoritairement issus du règne végétal. La Vigne par l’intermédiaire du vin et du raisin est la principale source alimentaire de stilbènes. La mise en évidence de leur rôle dans les mécanismes de défense des plantes et leurs activités biologiques, y compris sur l’Homme, en font un sujet d’étude en plein essor. L’un des objectifs de cette thèse a été de développer un ensemble de stratégies à la fois analytiques et préparatives utilisant la Chromatographie de Partage Centrifuge (CPC) pour l’étude et l’obtention de ces molécules. Dans un premier temps, nous avons développé une approche de couplage entre cette technique et un spectromètre à Résonance Magnétique Nucléaire (RMN) par l’intermédiaire d’un système d’Extraction sur Phase Solide automatisé (EPS). Dans un second temps, nous avons mis au point un ensemble de méthodes et d’approches séparatives permettant d’obtenir ces composés. Nous avons, grâce au développement d’une méthodologie de dosage de solvants par spectrométrie RMN, étudié une gamme dérivée d’une gamme très utilisée en CPC, l’ARIZONA. Nous avons montré que ces systèmes dérivés, peuvent être utilisés au travers de stratégies d’élution telles que des pas et des gradients afin d’optimiser les séparations. L’une des finalités de notre travail est d’offrir des méthodes permettant d’obtenir ces composés dans des quantités et des qualités suffisantes pour pouvoir constituer une chimiothèque interne au laboratoire pouvant prétendre à s’intégrer dans la Chimiothèque Nationale. Une dernière partie fait état du développement d’outils informatiques, dont la création d’une base de donnée Libre pour les chercheurs en Substances Naturelles. / Stilbenoids are phenolic compounds mostly found in the vegetable kingdom. Vine through wine and grape is the main source of stilbenes in the human diet. The involvement of these compounds in plants resistance mechanisms and their diverse biological activities, including on the human health are continuously highlighted. Making this topic a fast-growing one. One of the objectives of this thesis has been to develop a whole set of analytical and preparative strategies using Centrifugal Partition Chromatography (CPC) in order to study and obtain these molecules. Over a first phase, we developed a hyphenated approach between this technique and Nuclear Magnetic Resonance (NMR) through an automated Solid Phase Extraction (SPE) system. Then, we developed a set of separative methods and approaches with the aim of obtaining these compounds. Then, we carried out the development of a solvent quantifications methodology using NMR spectrometry. This allowed us to study a spin-off scale of a widely-used solvent systems collection called ARIZONA. We showed that these systems are good candidates for different elution strategies using steps and gradients with the aim to optimize separations. One of our purpose was to provide methods for effective and efficient purification of these compounds. This would allow, together with a lab-scale compounds library, their integration into the Chimiothèque Nationale, a nationwide chemical library. Last part accounts for computer tools development, including the creation of a Free database system for Natural Substances researcher.

Stilbenoids possess a wide range of biological properties such as, anticancer, antiplatelet aggregation, antiestrogenic, antibacterial, antifungal and antiatherogenic, etc. Owing to these therapeutic values, a great deal of attention attracted in the synthesis of derivatives of stilbenes. During the course of the study, G6 a novel stilbenoid was discovered, through high throughput screening, to be a potent inhibitor of mutated JAK2-V617F. The mutated JAK2 variant has been implicated in various myeloproliferative disorders (MPDs) including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) has been targeted by therapeutics. Chapter 2 describes the synthesis of analogs of the stilbenoid G6 and N-substituted stilbenes bisoxazines by utilizing Mcmurry reaction and Mannich condensation methods. The main emphasis of this work is to develop novel stilbenoids as inhibitors of JAK2-V617F mutated Jak2 enzyme in Human erythroleukemia cells (HEL) since this mutation is discovered in the majority of patients with myeloproliferative disorders (MPDs). Using Mcmurry reaction, five novel trans-hydroxystilbenes have been synthesized from carbonyl compounds. Subsequently using Mannich coupling with five secondary amines and five primary amines, 25 novel stilbenoids and 9 novel N-substituted stilbene bisoxazines have been synthesized. In HEL cell assay, 8 stilbenoid analogues have been identified as potent inhibitors of Jak2 enzyme. Chapter 3 describes the modification of ketamine structurally for the synthesis of novel analogues to study for their agonist activity at GABAA receptors and antagonist activity at NMDA receptors. Ligand gated ion channels like GABAA and NMDA receptors are membrane-embedded proteins at synaptic cleft which controls intercommunication among neurons and plays an important role in motor control activity, learning. GABAA receptors are responsible for inhibitory action potentials while NMDA receptors are responsible for excitory action potentials. Ketamine, known as dissociative anesthetic, produces profound analgesia at low doses to a unique cardiovascular stimulation and a cataleptic state at higher doses with dose dependent side effects like vivid dreams, disruptions of cognitive functions. The main emphasis of this work is the synthesis of novel analogues of ketamine by transforming carbonyl group in ketamine to imine functionality with small to bulkier groups and to identify an analogue of ketamine which is highly potent in its activity at the both GABAA and NMDA receptors and improved clinical actions. Studies of analogues activity against GABAA subtypes α6Β2δ, α1Β2γ2 receptors and NMDA subtypes NR1/2A, NR1/2B, NR1/2D receptors have been described. Chapter 4 describes the formal synthesis of (±)-Rolipram and the chemoenzymatic synthesis of -aryl--lactone, a Rolipram analogue. The key steps, Pd catalyzed arylation of diethylmalonate and the efficient use of selective acylation of 1, 3-diol entails the formal synthesis of (±)-Rolipram. The regioselective deacylation of Β-aryl-1, 4-diacetate by lipase Pseudomonas Sepacia entails the formation of Β-aryl-γ-lactone. The efficient use of various methods including halogen exchange, Heck arylation of diethylmaleate and lactonization for the synthesis of Β-aryl-γ-lactone have been discussed. The present work provides an efficient and general route to γ-lactones.

Les maladies du bois de la vigne, par exemple le syndrome de l’esca et l’eutypiose, sont dues à des champignons qui se développent dans le xylème et dégradent les tissus ligneux. Elles sont en recrudescence depuis une vingtaine d’année dans tous les vignobles du monde. A ce jour, aucun traitement curatif n’est disponible alors que ces maladies engendrent généralement la mort de la plante. L’étude des relations hôte-parasite et notamment celle des réponses de défense de plante, est nécessaire pour la compréhension et le développement de nouvelles stratégies de lutte. Parmi les molécules de défense de la vigne, les stilbènes sont connus pour leur forte activité antifongique.Notre travail, basé sur des approches biochimiques et moléculaires apporte de nouvelles données sur le rôle potentiel des stilbènes en tant que molécules de défense contre les maladies du bois. Nous avons identifié et quantifié les stilbènes majoritaires des tissus ligneux de V. vinifera, évalué la sensibilité des agents de maladie du bois à l’effet antifongique de ces stilbènes et suivi leur éventuelle induction suite à l’inoculation d’agents de maladie du bois. Nous avons souligné l’importance des oligomères de stilbènes dont l’activité antifongique est élevée et montré qu’ils sont fortement induits dans le bois inoculé par un champignon pathogène de la famille des Botryosphaeriaceae. La comparaison des réponses de défenses entre des cépages tolérants et sensible, a montré que les cépages tolérants répondent globalement plus fortement et plus rapidement que le cépage sensible à une élicitation par un filtrat de milieu de culture d’un agent de l’esca. / Grapevine wood diseases, as esca syndrome and eutypiosis, are worldwide afflictions. They are due to xylem inhabiting fungi that cause the decay of woody tissues and finally the death of the plant. No curative treatment has been successfully developed until now. Studying grapevine-wood disease pathogen interaction is a prerequisite to the development of protection strategies. Phenolics, particularly stilbenoids, are antimicrobial molecules involved in the resistance of grapevine to fungal pathogens. Our work, based on biochemical and molecular approaches, reveals new data on the possible role of stilbenoids in grapevine defence against trunk diseases. We identified and quantified major stilbenoids of woody canes from V. vinifera. These compounds, especially stilbenoid oligomers, displayed in vitro antifungal activity against trunk disease agents and were induced in cuttings inoculated with an agent of the Botryosphaeriaceae family. We also compared the defence response of susceptible or tolerant cultivars to wood decay based on an elicitation assay with a culture filtrate of an esca pathogen. Defence responses of tolerant cultivars were more effective than those of the susceptible cultivar.

Ce travail porte sur la valorisation de sarments de vigne comme source de polyphénols bioactifs. Après purification d’oligomères du resvératrol et hémi synthèse d’analogues du resvératrol, l’activité antifongique de ces molécules a été testée. Le 3,5-diméthoxyresvératrol a montré des activités intéressantes sur douze espèces du genre Candida. Pendant le stockage des sarments, une forte augmentation en E-resvératrol et E-picéatannol a lieu de façon thermo dépendante et l’expression des gènes PAL, C4H et STS participent à la biosynthèse de novo du E-resvératrol. Une infection par le mildiou au vignoble pendant la période de croissance modifie à la fois la composition et la répartition spatiale des stilbénoïdes dans les sarments. L’analyse métabolomique ciblée par UPLC-MS couplée à une analyse PLS-DA permet de discriminer les sarments selon leur génotype et de déterminer des métabotypes. La distance biochimique observée correspond à la distance génétique inter cépage. Ces résultats démontrent le potentiel antifongique des stilbènoïdes et permettent d’identifier quelques facteurs clés influençant la composition phytochimique des sarments de vigne. / This work aims at grape cane valorization as a source of bioactive polyphenols. After purifying E-resveratrol oligomers and obtaining E-resveratrol analogues through semi-synthesis, the antifungal activity of the compounds was evaluated. The 3,5-dimethoxyresveratrol exhibited interesting activity against twelves Candida species. During post-pruned grape cane storage, a strong and temperature dependent increase in E-resveratrol and E-piceatannol was observed and the expression of PAL, C4H, 4CL and STS genes contributed to a de novo biosynthesis of E-resveratrol. Downy mildew infection in vineyard during the growing season modified both the composition and the spatial distribution of stilbenoids in grape canes. UPLC-MS-based targeted metabolomics coupled to multivariate statistical analysis discriminates grape canes according to their genotypic origin and determines metabotypes. The observed biochemical distances between genotypes corresponded to genetic distances. Finally, results highlight the antifungal potential of stilbenoids and several key factors affecting the phytochemical composition of grape canes

Les polyphénols naturels forment des complexes non-covalents dans lesquels le π-stacking et les liaisons hydrogène jouent un rôle clé dans la stabilisation. Les calculs DFT incluant la dispersion (DFT-D), la description des processus d'agrégation non-covalente de produits naturels devient fiable. Dans ce travail, les méthodes DFT-D sont appliquées à i) la compréhension de la biosynthèse stéréo- et régio-sélective des oligostilbenoïdes, ii) la prédiction de l'agrégation des antioxydants naturels au sein de la membrane bicouche lipidique, qui pourrait rationaliser la synergie de la vitamine E, la vitamine C et polyphénols dans leur action antioxydante, et iii) la modulation des propriétés optiques de dérivés de chalcones. / Natural polyphenols form non-covalent complexes in which π-stacking and H-bonding play a key stabilizing role. The dispersion-corrected DFT calculations have paved the way towards reliable description of aggregation processes of natural products. In this work, these methods are applied at i) understanding of stereo- and regio-selective oligostilbenoids biosynthesis; ii) predicting natural antioxidant aggregation within lipid bilayer membrane, which may allow rationalizing the synergism of vitamin E, vitamin C and polyphenols in their antioxidant action; and iii) modulating optical properties of chalcone derivatives.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Lenka Linhartová Supervisor: Prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Molecular mechanisms of intractions - interactions of constitutive androstane receptor with selected stilbene compounds Constitutive androstane receptor (CAR), member of nuclear receptors family, is a major regulator of gene expression of phase I and II enzymes metabolizing endobiotics and xenobiotics. Changes in its activity can lead to pharmacokinetic drug interactions, ineffective treatment or higher toxicity of drugs simultaneously administered with CAR ligands. Recently another effects of this receptor, especially in homeostasis of bile acids, lipids and glucose have been discovered and CAR is now considered as a potential drug target for the treatment of metabolic diseases. Stilbenes represent a small group of plant polyphenols with typical 1,2-diphenylethylene nucleus. The most famous member is resveratrol, which has attracted great attention thanks to its antioxidant, anti-inflammatory, antiproliferative and cardioprotective effects. Others stilbene compounds such as pterostilben, piceatannol or pinosylvin have shown similar health beneficial effects as well. The aim of this diploma thesis was...

Nous avons porté une attention particulière à 2 marqueurs chimiotaxonomiques de la famille botanique des orchidées, les stilbénoïdes et dérivés glucosyloxybenzyliques d’acides organiques, et les avons recherchés dans deux espèces tropicales. L’investigation phytochimique de Cyrtopodium paniculatum (Ruiz & Pav.) Garay a permis l’isolement de 35 stilbénoïdes dont 12 nouvellement décrits. L’évaluation de leur activité cytotoxique in vitro sur la lignée cellulaire cancéreuse UG-87 montre que seuls les stilbénoïdes dimères semblent aptes à induire une perturbation de l’intégrité cellulaire. Nous avons également exploré la composition chimique d’Arundina graminifolia (D.Don) Hochr. De ses parties aériennes, 9 stilbénoïdes (dont 2 nouvelles structures ont été isolés) ainsi que des 7 nouveaux dérivés hydroxybenzyliques de l’acide (R) 2-benzylmalique, les arundinosides. De ses parties souterraines, plus de 40 arundinosides ont également pu être identifiés. Nous avons finalement évalué le potentiel cytoprotecteur de stilbénoïdes et arundinosides d’ A. graminifolia sur lignée cellulaire PC12, sans qu’aucune activité relevée n’ait été concluante. / The aim of the study was focused on two chemotaxonomic markers of the family Orchidaceae, namely stilbenoids and the glucosyloxybenzyl derivatives of organic acids. We specifically explored these two chemical families in two tropical orchid species. The chemical investigation of Cyrtopodium paniculatum (Ruiz & Pav.) Garay led to the isolation of 35 stilbenoids, including 12 newly described compounds. Their cytotoxic activity on UG-87 cancer cell line was evaluated. The result obtained demonstrated that stilbenoids dimers were the only compounds capable of disturbing the cellular integrity. In parallel, we explored the chemical composition of Arundina graminifolia (D.Don) Hochr. From its aerial parts, 9 stilbenoids (including 2 newly described ones) together with 7 new (R) glucosyloxybenzyl 2-benzylmalate derivatives, the arundinosides, were isolated. From its underground parts, over 40 arundinoside-like compounds have also been identified. The cytoprotective evaluation of stilbenoids and arundinosides against beta-amyloid induce toxicity on PC12 cells was evaluated, however, no significant result was obtained from the biological evaluation.

Stilbene phytoalexins such as resveratrol, 1, and the arachidins, including arachidin-1,2, are naturally synthesised by peanut (Arachis hypogaea) plants. The peanut phytoalexins are polyphenolic compounds consisting of a stilbene backbone, with a number of derivatives also possessing a prenyl moiety. These distinctive phytoalexins have gained attention, as they exhibit various biological activities, for instance arachidin-1, 2, has been reported to be more potent than resveratrol, 1, in the inhibition of lipopolysaccharide-induced expression of cyclooxygenase-2 (COX-2) and COX-2 mRNA, in vitro at doses that were low in cytotoxicity. Additionally the various arachidins have recently been shown to exhibit their anti-inflammatory properties, through the inhibition of a number of inflammatory mediator pathways. In this work, various routes into the synthesis of arachidin-1, 2, are described, via use of the Horner-Wadsworth-Emmons (HWE) reaction. Three different methodologies were explored, the first approach involving silyl ether (TIPS or TBDMS) protected benzaldehydes, proved unsuccessful due to cleavage of the silyl ether protecting groups, in basic and/or acidic conditions. This led to an alternative approach, whereby formation of the stilbene backbone proceeded via the regioselective demethylation of an acetal in the presence of sodium metal, subsequent electrophilic substitution using iodomethane and finally acetal hydrolysis of the acetal, gave the isolated aldehyde in moderate yield (52 %). Coupling of the aldehyde with the substituted benzylphosphonate, via the HWE reaction gave the desired trans-stilbene in good yield (86 %), however incorporation of the prenyl side chain proved to be challenging via the Wohl-Ziegler bromination. Further adaptation of the aforementioned route, whereby alkylation using diethyl iodomethylphosphonate, enabled the incorporation of the prenyl moiety and the subsequent construction of the trans-stilbene backbone, gave the 4-(3-methyl-but-1- enyl)-3,5,3',4'-tetramethoxystilbene, 3, albeit in poor yield (47 %). The final step involving demethylation using BBr3 gave arachidin-1, 2, also in poor yield (30 %), nevertheless this approach has been proved to be a successful route for the total synthesis of arachidin-1, 2, however optimised studies are required in order to obtain the desired compound in quantitative yields. Synthetic analogues of resveratrol, 1, are also known for their biological activities, including anti-inflammatory and chemopreventative properties. Recently, the anti-proliferative activity of a number of stilbenesulfonamides, against the National Cancer Institute's 60 (NCI-60) human tumour cell line has been reported. Furthermore, the anti-inflammatory effects of novel heterocyclic methylsulfone and sulfonamide analogues, via inhibition of the COX-2 protein have also been published, however both synthetic routes described require a total of six or seven steps, from the sulfanilamide and are limited to the synthesis of primary sulphonamides (SO2NH2). In this work, an efficient three step synthesis has been designed and successfully implemented, proceeding via chlorosulfonation of diethyl benzylphosphonate, to form the sulfonyl chloride intermediate. Aminolysis of the sulfonyl chloride intermediate was then performed, using a range of primary, secondary and cyclic alkyl amines, as well as aromatic amines; including ammonia, dimethylamine, morpholine and diphenylamine. Finally, formation of the stilbene backbone with various substituted aldehydes, via the HWE reaction offered a short, versatile and alternative route to the synthesis of novel primary, secondary and tertiary trans-stilbene benzenesulfonamides and heterocyclic analogues, in yields of 42 - 100 %. The activity of a selection of the synthesised stilbene benzenesulfonamides was evaluated against the human lung adenocarcinoma epithelial cell line (A549). Amongst the compounds tested, analysis of the data showed that the novel analogue, 4, was found to be the most potent compound, with a GI50 of 0.1 μM. Comparison with the previously published data found analogue, 4, to be approximately 500-fold more potent than the lead compound resveratrol, 1, (GI50 = 51.64 μM) and approximately twice as potent than 5-fluorouracil (GI50 = 0.189μM), a chemotherapy drug used to treat various forms of cancer 8. Overall, these results demonstrate that the total synthesis of trans-arachidin-1, 2, can be achieved via a five step methodology. A versatile route to the synthesis of novel stilbene benzenesulfonamides has also been successfully achieved, amongst the compounds synthesised one appears to show promising anticancer activity, and warrants further investigation (i.e. in vitro studies using other cancer cell lines, and the synthesis of additional compounds using analogue, 4, as a lead compound).

L’analyse de la composition chimique de deux orchidées de la sous-tribu des Aeridinae, Aerides rosea Lodd. ex. Lindl. & Paxton et Acampe rigida (Buch.-Ham. ex Sm.) P. F. Hunt, a été menée en ayant recours à des techniques de couplages et grâce à une stratégie de déréplication. Dix dérivés phénanthréniques ont ainsi été identifiés dans les tiges d’A. rosea, dont deux nouvellement décrits. Quatre stilbénoïdes, trois dérivés d'acides phénoliques et quatre esters d'acide cinnamique ont été identifiés dans les tiges d’A. rigida. Nous avons également effectué des dosages de traceurs dans différents échantillons de deux représentants de la tribu des Vandeae : Vanda teres (Roxb.) Lindl. et Vanda coerulea Griff. ex. Lindl., afin de mettre en évidence d’éventuelles variations de composition en fonction des facteurs environnementaux et du stade de croissance végétative. Enfin, nous avons mis au point des conditions d’isolement préparatif par chromatographie de partage centrifuge afin de purifier des marqueurs biologiques préalablement identifiés dans les tiges de Vanda teres : trois glucospyranosyloxybenzyl - malates ainsi que leur précurseur biosynthétique. / Phytochemical analyses of the composition of two orchids Aeridinae subtribe, Aerides rosea Lod . ex . Lindl . & Paxton and Acampe rigida (Buch. -Ham . Ex Sm.) P. F. Hunt were conducted using the state-of-the-art hyphenated techniques and a developed dereplication strategy. Ten phenanthrene derivatives have been identified in the stems of A. rosea, including two newly described structures. Four stilbenoids, three phenolic acids derivatives and four cinnamic acid esters were identified in the stems of A. rigida. We also performed quantitative assays of chemical tracers in different samples of two representative species from the Vandeae tribe: Vanda teres (Roxb.) Lindl. and Vanda coerulea Griff. ex. Lindl., in the aim to highlight possible variations in their composition, depending on environmental factors and vegetative growth stage. Finally, we have developed conditions for preparative isolation using centrifugal partition chromatography to purify biomarkers previously identified in the stems of Vanda teres: three glucosyloxybenzyl malates derivatives and their biosynthetic precursor.