Le principal objectif de cette thèse, au caractère interdisciplinaire, concernait l'étude de l'éponge Biemna laboutei de Madagascar, connue pour sa toxicité (causant notamment des dermatites). Les travaux entrepris comprenaient en premier lieu, l'étude chimique de cette éponge incluant l'extraction, l'isolement et l'identification des métabolites secondaires par différentes techniques chromatographiques (CLMP, CLHP…) et spectroscopiques (UV-visible, HRMS, RMN 1D et 2D…). Dix-huit alcaloïdes appartenant à la classe rare des hétérocycles tricycliques (5,6,8b)-triazaperhydroacénaphtylène (i.e. ptilocaulines, mirabilines, nétamines) ont été isolés et identifiés. Douze de ces alcaloïdes tricycliques guanidiniques, de structures nouvelles, ont été nommées nétamines H-S. Ces composés ont été classés en fonction des insaturations de leur noyau tricyclique : type pyrimidine, insaturés en Δ8,8a et insaturés en Δ8a,8b. Pour deux des composés isolés, les nétamines I (58) et J (59), une étude configurationnelle reposant sur la comparaison de spectres UV et DCE, expérimentaux et théoriques (théorie de la fonctionnelle et de la densité), a permis la détermination de leurs configurations absolues : 5aS, 7R, 8R pour la nétamine I et 5aS, 7R, 8S pour la nétamine J. La valorisation des molécules isolées a ensuite été envisagée via d'une part la réalisation d'une étude chimiotaxonomique et d'autre part, l'évaluation de leurs activités biologiques (cytotoxicité, activité antipaludique, …). La nétamine M (62) a présenté une activité cytotoxique sur les cellules cancéreuses KB (CI50 = 1,0 μg/mL) et les nétamines O (64), Q (66) et K (60) se sont montrées actives contre le parasite Plasmodium falciparum, responsable du paludisme avec respectivement une CI50 de 4,66 ; 2,53 et 0,62 μg/mL. Enfin, au cours de ces travaux de thèse, ont été explorées trois nouvelles stratégies de synthèse conduisant au squelette des alcaloïdes guanidiniques tricycliques, suivant une approche biomimétique. / The main purpose of this interdisciplinary thesis was to study Biemna laboutei, a sponge from Madagascar, known to have toxic properties (dermatitis-producing). The chemical investigation of this sponge including extraction, isolation and identification of secondary metabolites was first undertaken using several chromatographic (HPLC, MPLC…) and spectroscopic (UV-visible, HRMS, NMR 1D et 2D…) techniques. Eighteen alkaloids belonging to the rare class of tricyclic (5,6,8b)-triazaperhydroacenaphtylene heterocycles (i.e. ptilocaulins, mirabilins, netamines) were isolated and identified. Twelve new alkaloids from this group of guanidine derivatives were named netamine H-S. These compounds were grouped on the basis of unsaturation and double bond regiochemistry, with pyrimidine, Δ8,8a and Δ8a,8b heterocycles. For two compounds, netamine I (58) and J (59), a joint theoretical (Density functional theory) and experimental study of UV and ECD spectra allowed the determination of their absolute configuration: 5aS, 7R, 8R for netamine I and 5aS, 7R, 8S for netamine J. The chemotaxonomic meaning of these alkaloids was discussed. Their biological activities were also evaluated. Netamine M (62) exhibited a cytotoxic activity towards KB cells (IC50 = 1,0 μg/mL) while netamine O (64), Q (66) and K (60) were active against the malaria parasite Plasmodium falciparum with IC50 value of 4,66; 2,53 and 0,62 μg/mL respectively. Finally, this work was also dedicated to the biomimetic synthesis of the tricyclic guanidine skeleton. Three new synthesis routes were explored.

<div>Enantioselective syntheses of hexacyclic aspidoalbidine alkaloids (+)-fendleridine and (+)-acetylaspidoalbidine are described. These syntheses feature an asymmetric decarboxylative allylation and photocyclization of a highly substituted enaminone. Also, the synthesis highlights the formation of a C19-hemiaminal ether via a reduction/condensation/intramolecular cyclization cascade with the C21-alcohol. The present synthesis provides convenient access to the aspidoalbidine hexacyclic alkaloid family in an efficient manner.</div><div>A copper-catalyzed cross-coupling is described. Use of Cu(I) salts in the presence of allyl bromides and organostannyl furans were found to undergo catalytic turnover under ambient conditions and afford the coupled products in good to great yields. Model substrate screening led to conditions used in the concise formal synthesis of FR901464 analogues. Optimization of the described coupling step led to suppression of undesired isomers and byproducts affording the desired diene coupled product in high yield, stereo-, and regioselectivity on a multigram scale. Novel protection of the resulting diene moiety as an unconventional protecting group, and a facile four-step single column chromatographic stereoselective sequence are also reported.</div>

The thesis deals with the natural substances in context with the cancer disease. The natural substances have a positive effect on the human organism and they are able to influence the viability and the growth of the cancer cells. The main mechanical device is to influence the mechanisms needed to start the apoptosis of the cancer cells and stopping further proliferation. The cancer cell lines utilization in the cancer disease is discussed in the thesis too. The thesis states common methods of determining the natural substances cytotoxicity. For the experimental part of the thesis it was chosen the MTT test method and the xCELLigence system for monitoring in real time. The mechanical device of the tested substance capsaicin in application on the prostate cell lines, tumorous PC3 and nontumorous PNT1A influence will be observed within the experimental part of the thesis.

MSc (Chemistry) / Department of Chemistry

We describe the regioselective prenylation of 3-bromocarbazole by palladium(0)-catalysed cross coupling with a prenylstannane or a prenylboronate. The procedure is applied to the synthesis of precursors for biologically active carbazole alkaloids.

Iron-mediated oxidative cyclisation provides an efficient approach to pyrano[3,2-a]carbazole alkaloids. Thus, improved routes to girinimbine and murrayacine as well as the first total syntheses of O-methylmurrayamine A and 7-methoxymurrayacine are reported. Asymmetric epoxidation of girinimbine led to (−)-trans-dihydroxygirinimbine and the assignment of its absolute configuration. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

While the molecular defect that cause sickle cell disease has well been established, the cause of vaso-occlusive crisis remains elusive and largely debated upon. Majority of studies have linked the painful episodes to polymerization of sickle hemoglobin following its deoxygenation. The variability of the disease symptoms among patients, compounds efforts for a holistic therapy. Hydroxyurea, a stimulator of Hb F induction and a widely used treatment, has ameliorated the complication of SCD but it is only effective in 50% of the patients. Expression of Hb F lowers the content of Hb S in blood and hence reduces oxidative stress caused by Hb S denaturation. Sickle cell disease severity depends on several factors. Most importantly, the ability of red cell to sickle dominates all other determinants. While deoxygenation of sickle hemoglobin may be inevitable, the duration with which the red cell remains in the deoxygenated state can be manipulated. Deoxygenation is a transient process that when compared to the time taken to develop the long filaments of deoxyhemoglobin to causes severe sickling, the red cell would have been cycled back to the lungs and re-oxygenated to restore the healthy conditions of the cell. In fact, if sickle cells would flow as fast as healthy erythrocytes, the detrimental impacts of sickling such as vaso-occlusive crisis, would not be a concern for this disease. Unfortunately, the unstable sickle hemoglobin undergoes denaturation through auto-oxidation, which imposes oxidative stress to the cells. The oxidative stress inhibits erythrocytes tyrosine phosphatases, a course which subsequently impair their constitutive action against the tyrosine kinases. In the end, a net tyrosine phosphorylation state in the red cell membrane proteins, most notably the transmembrane protein band 3, succeeds. Band 3 tyrosine phosphorylation abrogates the protein’s interaction with ankyrin and spectrin-actin cytoskeleton, hence the cytoskeleton loses its major anchorage to the membrane thus engendering membrane destabilization. A destabilized erythrocyte sheds membrane fragments in form of microvesicles/microparticles and discharges free hemoglobin into the extra cellular matrix. In consequence, the microparticles power initiation of coagulation cascade through activation of thrombin, while free Hb inflicts inflammation, scavenges nitric oxide which is necessary for vasodilation and induces further oxidative stress within the microvasculature, and activates expression of adhesion receptors on the endothelium. Taken together, these events culminate in entrapment of red cells (not naming leucocytes and platelets) in the microvasculature, blockade of blood vessels and further damage of erythrocytes through prolonged deoxygenated state thus terminating in tissue injury, strokes, and organ damage, amid vaso-occlusive episodes which always require hospitalization and extensive medical care for survival. Band 3 tyrosine phosphorylation and membrane weakening is not unique just to SCD, but also a druggable target for malaria. Malaria, a disease that is touted as the evolutionary cause of sickle cell disease, surprisingly thrives through the same mechanism. Briefly, malaria parasite consumes hemoglobin for its DNA synthesis, and in the process generate reactive oxygen species from denatured hemoglobin that feeds into the oxidative stress which triggers band 3 tyrosine phosphorylation. In this case however, a destabilized membrane offers perfect conditions for merozoites’ (malaria daughter parasites) egress/exit out of the cell to begin infecting other red cells. Ultimately, the ensuing anemia and organ dysfunction leads to patient’s death. Treatment of diseased cells with imatinib and other Syk inhibitors effectively reversed membrane weakening. A stabilized membrane not only survives longer in circulation to alleviate SCD symptoms but also traps and starves malaria parasite leading to termination of the parasitic infection. With band 3 tyrosine phosphorylation at center stage, this dissertation explores the above events in an effort to unveil a novel therapy for sickle cell and malaria diseases. First, the therapeutic strategy regarding SCD is discussed in detail beginning with non-transfused patients and ending in additional mechanistic study on inactivation of the principal erythrocyte’s protein tyrosine phosphatase 1 B, PTP1B. The dissertation then provides an initial proof of concept on efficacy of imatinib in treatment of malaria as a monotherapy and its efficacy when used in a triple combination therapy with the standard of care treatment. Finally, I outline an alternative possible mechanism of action of quinine against malaria.

The first topic presented in the dissertation thesis is determination of isoprostanes as markers of oxidative stress and other compounds affected by presence of oxidative stress. Isoprostanes iPF2-III, iPF2-VI, iPF2-VI, astaxanthin and polyunsaturated fatty acids (PUFA), especially arachidonic acid (AA) were monitored in Atlantic salmon eggs (Salmo salar). Methods for the determination of these compounds have been developed and optimized using chromatographic separation coupled to conventional or mass spectrometric detection. Freshly laid eggs, eyed embryos and non-viable eggs were used to test a general hypothesis that egg viability can be affected by susceptibility to oxidative stress, either through the specific fatty acid concentration and/or the antioxidant capacity of the eggs. Levels of isoprostanes and arachidonic acid (AA) were significantly higher in non-viable eggs than in control (eyed embryos) as well as relative abundance of PUFA. While no difference of isoprostanes was found between freshly laid and control those from the Atlantic stock except iPF2-VI which was observed under the LOQ in the control. Higher levels of PUFA and AA in comparison with the control were observed in the freshly laid eggs. However, the only statistically significant difference was observed in the amount of astaxanthin. Different levels of PUFA and astaxanthin may be related to their biochemical consumption during the development of eggs. This work evaluated potential effect on the viability of eggs Salmo salar due to the presence of oxidative stress. The monitoring of mycotoxins in food and feed was the subject of the second topic. Mycotoxins are secondary metabolites produced by fungi. They are ubiquitous undesirable natural contaminants that are toxic for humans and animals. Today are known more than 500 mycotoxins. However, only few of them are regulated by the European Union. The European Food Safety Authority (EFSA) was asked by the European Commission to provide a scientific opinion on other mycotoxins for which statutory limits could be developed. In this study is proposed simultaneous screening allowing fast, reliable and sensitive approach, identification and quantification of 17 mycotoxins in food and feed sample. The method includes both mycotoxins regulated by the EU and selected mycotoxins required by the EFSA (aflatoxins, deoxynivalenol, nivalenol, zearalenone, fumonisin, ochratoxin A, T-2 toxin, HT-2 toxin, enniatins and beauvericin). Analytes are isolated by the modified QuEChERS method. For separation and target mycotoxins detection, ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC –MS/MS) was employed. The method also allows determination of ergot alkaloids (ergocornine, ergosine, ergocryptine, ergocristine and their respective epimers). The developed method was used either for monitoring mycotoxins and ergot alkaloids in feed and raw materials and barley and malt prepared from it.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctorate of Philosophy. / Senecio serratuloides is widely used for wound healing in South Africa but minimal information regarding its efficacy is available. Furthermore toxic pyrrolizidine alkaloids may be present. The following investigation sought firstly to evaluate the efficacy and safety of Senecio serratuloides in a porcine wound model; secondly to assess for a potential mechanism and finally isolate and identify fractions in in-vitro assays. Assessment of Efficacy and Safety Materials and Methods: Deep partial thickness and full thickness wounds were created on 9 pigs. Treatment included an occlusive dressing (negative control), activated carbon, or the Senecio preparation. Wounds were monitored using photographic documentation, pH measurement and histological analysis (skin thickness and collagen content). Toxicity was monitored on blood and liver samples. Results and Discussion: Efficacy of Senecio serratuloides was established with a significantly thicker epidermis, maximal at day 7 post-operative, 2 days before the controls. Effects on collagen content was negligible with no toxicity detected. Mechanistic investigation Materials and Methods: Wound fluid was analysed for IL-10, IL-12, IL-1β, IL-6, IL-8, TNF-α using flow cytometry based assays. Tyrosine phosphorylation and cellular proliferation was assessed using dual immunofluorescence staining. Results and Discussion: IL-1β levels were significantly greater in the Senecio treatment. Tyrosine phosphorylation increased to day 9 post-operative where it stabilised in all groups. In the same period, cellular proliferation was sustained in the Senecio treated wounds but not in the controls. Keratinocyte proliferation was identified as the target for in-vitro assays. Extraction, Isolation and Partial Identification using In-vitro Proliferation Assays. Materials and Methods: The plant was fractionated using solid phase extraction cartridges. Keratinocytes were grown under standard conditions in 96-well plates. Cellular proliferation was assessed spectrophotometrically using a resazurin dye technique. Active fractions were analysed using gas chromatography and mass spectrometry. Results and Discussion: Identified fractions increased the rate of proliferation by 300- 400%. Potential lead compounds were identified. Importantly, pyrrolizidine alkaloids could not be detected. Conclusion Senecio serratuloides is efficacious in treating deep partial thickness wounds without inducing liver toxicity. Sustained keratinocyte proliferation linked to tyrosine phosphorylation may be an underlying mechanism. Although successful, in-vitro detection of active fractions requires further characterisation.

Il y a plus de cinquante ans, la pactamycine a été isolée en tant qu’agent antitumoral potentiel. Il a été réalisé plus tard qu’il s’agissait en fait d’un agent antibactérien capable d’inhiber la synthèse de protéines lors du procédé de traduction. Récemment, il a même été démontré que certains de ses analogues possèdent des propriétés antiprotozoaires prometteuses. La présente thèse détaille la première synthèse totale de la pactamycine, entreprise au sein du groupe Hanessian, ainsi que la préparation d’une sélection d’analogues testés pour leurs propriétés biologiques. En outre, la daphniglaucine C appartient à une vaste famille de composés naturels isolés des feuilles du daphniphyllum au cours des dix dernières années. Bien que relativement peu d’information soit connue par rapport à l’activité biologique de la daphniglaucine C, la synthèse de celle-ci représente certainement un défi intéressant pour un chimiste organicien. Au passage, nos efforts vers la synthèse totale du composé cible auront permis d’explorer l’emploi de plusieurs méthodes en vue de la formation de centres quaternaires. De plus, un réarrangement réductif atypique, catalysé au palladium à partir d’alcools allyliques non-activés, a été étudié et employé afin de générer une sélection de pyrrolidines polysubstituées. / Although pactamycin was first isolated as a potential antitumoral drug, further studies highlighted its capacities in inhibiting protein synthesis, and thus its potency as an antibacterial agent. Furthermore, it was recently discovered that some of its analogs display promising antiprotozoal activity. The present thesis reports and details the first total synthesis of pactamycin, pursued in the Hanessian lab over the last few years, as well as the preparation of a selection of analogs thereby tested for their biological properties. Daphniglaucin C belongs to a large family of natural compounds isolated from the leaves of daphniphyllum over the last decade. Although relatively little is known as to the biological activity of daphniglaucin C, its synthesis poses an obvious and interesting challenge for organic chemists. En route towards its total synthesis, the use of several methods for the formation of quaternary centers was explored. Moreover, an atypical reductive allylic transposition, catalyzed by palladium from unactivated allylic alcohols, was studied and used to generate a variety of polysubstituted pyrrolidines.