Ces dernières années, différents épisodes d’intoxication ont touché les coquillages sur le littoral français et tout particulièrement les huîtres dans le Bassin d’Arcachon. Afin de venir en aide à la profession ostréicole, le Conseil Régional d’Aquitaine a mis en place un vaste projet de recherche. Au sein de ce programme, nous avons proposé d’étudier la faisabilité d’un immunocapteur, basé sur la reconnaissance anticorps/antigène, capable de détecter la présence de phycotoxines dans l’eau de mer. Les synthèses de molécules (haptènes), présentant une analogie structurale avec la partie commune des phycotoxines de la famille des ASP ont été réalisées à l'ISM. Après couplage de ces molécules à une protéine, des anticorps montrant une bonne reconnaissance des ASP ont été obtenus. La mise au point de l'immunocapteur a été effectuée en collaboration avec deux autres Unités de Recherche : le LIP (Univ. Bx II) pour l'immunologie et l'IMS (Univ. Bx I) pour la lecture et la transcription du signal sur le capteur. / In the last few years, oysters from the Arcachon Bay have been touched by several toxic episodes. To help oyster industry, an important research program was funded by the Conseil Régional d’Aquitaine. In this project, we proposed to study the feasibility of an immunosensor, based onantibody/antigen reaction, able to detect phycotoxins in seawater. Haptens showing astructural analogy with the common part of ASP toxins were synthesized at ISM. Aftercoupling of haptens with a protein carrier (BSA), antibodies showing a good ASP recognition were obtained. The transducer, a Love wave acoustic device, was developed by IMS (Univ.Bx I) and the antibodies used for specific biorecognition were characterized by LIP (Univ. BxII).

Total synthesis of the pyrrolizidine alkaloid NP25302: (+)-NP25302 is an unusual vinylogous urea containing pyrrolizidine alkaloid shown to exhibit cell adhesion inhibition. It was envisaged that this natural product could be accessed by a novel 5-endo-dig cyclisation to construct the pyrrolizidine core, and a Curtius rearrangement to install the vinylogous urea motif. This methodology was first tested on a model system, furnishing nor-NP25302 from L-proline in 12 steps and 9% overall yield. The total synthesis of (±)-NP25302 was completed in 9 steps and 26% overall yield from ethyl 2-nitropropionate using similar methodology. Studies into the stereospecificity of the Au(I)-catalysed cyclisation of monoallylic diols: During the synthesis of (+)-isoaltholactone in the Robertson group, the key Au(I)-catalysed cyclisation was observed to occur with some stereospecificity. Further investigations were therefore conducted into the stereochemical outcome of this reaction using stereodefined allylic alcohols, and from the combined results a mnemonic was proposed to predict the stereochemistry of the products of this reaction. Studies into the total synthesis of ascospiroketals A and B: Investigations were conducted into the total synthesis of the recently isolated natural products ascospiroketals A and B. A second generation synthesis was used to construct advanced intermediates 1 and 2.

A number of privileged starting materials based on aryl halide frameworks have emerged that allow access to a variety of different heterocyclic scaffolds through judicious choice of reaction conditions. This work describes efforts to develop and extend the utility of two of these general heterocycle precursors - ortho-(haloalkenyl)aryl halides A and α-(ortho-haloaryl) ketones B - in conjunction with cascade reactions involving the construction of key carbon-heteroatom bonds via palladium or copper catalysis. Chapter 1 entails an overview of the development of palladium- and copper-catalysed carbon-heteroatom bond forming processes. The application of these processes in heterocycle synthesis using ortho-(haloalkenyl)aryl halide and ortho-haloacetanilides/ α-(ortho-haloaryl) ketone precursors is also described. Chapter 2 focuses on the development of a two-step synthesis of cinnolines using ortho-(haloalkenyl)aryl halides via intermediate protected dihydrocinnoline derivatives C. Chapter 3 demonstrates how the inherent reactivity of protected dihydrocinnoline derivatives C can be harnessed to provide access to functionalised products. A brief target synthesis of a pharmaceutically-relevent cinnoline is also described. Chapter 4 details attempts to develop a novel synthesis of benzothiophenes D from both ortho-(haloalkenyl)aryl halide and α-(ortho-haloaryl) ketone precursors.

Bromodomains (BRDs) are protein modules which bind to acetylated lysines on histones and transcriptional regulating proteins. BRD-containing proteins are involved in a large variety of critical cellular processes and their misregulation, or mutation of the genes encoding for them, has been linked to pathogenesis in humans. The generation of chemical probes (potent, selective and cell permeable small molecules) in cellular experiments to investigate the biological role of the BRDs is thus desirable. A chemical probe for the CREB (cyclic-AMP response element binding protein) binding-protein (CBP) and E1A binding protein (p300) BRDs was developed, starting from a low molecular weight, weak and non-selective dimethylisoxazole benzimidazole compound. Parallel synthesis was used to optimise the initial hit into a weak, but selective CBP inhibitor. Further modification of the two N-1 and C-2 moieties of the benzimidazole scaffold, led to highly potent and selective CBP inhibitors. Structure-guided design was then applied to optimise the selectivity of the series for CBP over the first domain of bromodomain-containing protein 4 BRD4(1). A strategy to reduce the flexibility of the N-1 and C-2 ethylene linker groups through the incorporation of conformational constraints led to inhibitors with increased selectivity. The optimal compound was highly potent for the CBP and p300 BRDs (K_d 21 nM and 32 nM, respectively) and selective over BRD4(1) (40-fold and 27-fold, respectively). On-target cellular activity was observed in a fluorescence recovery after photobleaching (FRAP) assay (0.1 μM), a p53 reporter gene assay (IC₅₀ 1.5 μM) and a Förster resonance energy transfer (FRET) assay (5 μM). A weak indolizine bromodomain-containing protein 9 (BRD9) inhibitor was used as the starting point for the development of a BRD9/BRD7 chemical probe. Analogues were synthesised via [3+2] cycloadditions. An optimised compound was found to be highly potent (68 nM) and selective over BRD4(1) (34-fold). On-target cellular activity was observed in a FRAP assay (5 μM). Efforts were made to improve the cellular activity through the introduction of an ionisable centre to aid solubility. A selection of piperazine analogues were shown to be potent and selective, and these compounds warrant further investigation of their selectivity and cellular activity. Overall, the work has led to the first potent and selective inhibitors of the CBP/p300 and BRD9 BRDs. It also highlights the role of structural analysis in the development of inhibitors that modulate protein-protein interactions.

Chapter 1 is an overview of the key developments in rhodium-catalysed hydroacylation. The main focus of this chapter is the use of various chelation strategies for the stabilisation of key rhodium-acyl intermediates. In addition, the more recent emergence of regioselective hydroacylation processes has been highlighted. Chapter 2 discloses the branched-selective intermolecular hydroacylation of 1,3-dienes and S-chelating aldehydes to afford synthetically useful 1,5-dione products. The evaluation of a number of different phosphine ligands for this process identifies a correlation between ligand bite angle and reaction regioselectivity. Chapter 3 discusses the development of a linear-selective hydroacylation process for previously challenging alkyne substrates. This, in combination with a complementary branched-selective process, provides a ligand-controlled regioselectivity switch between the branched and linear pathways. Finally, Chapter 4 details efforts towards the development of multicomponent, tandem processes through exploitation of our synthetically useful branched hydroacylation adducts.

The aim of this project was to develop new catalytic methods to control asymmetry in electrocyclic reactions, and to apply these methods to generate complex molecules. Initial efforts were directed towards the catalysis of anionic 8π electrocyclizations (Chapter 2 and Figure i). 8π electrocyclization was not achieved due to issues with alkene geometry and anion stability. Our efforts were then directed towards using phase-transfer catalysis to generate complex polycyclic compounds via a cascade electrocyclization-1,4-addition (Chapter 3 and Figure ii). Pyrrolidines and indolizidines were generated in excellent yield from simple starting-materials with high levels of stereocontrol. Finally, we investigated the catalysis of a 6π [1,6] electrocyclization to generate dihydroquinolones (Chapter 4 and Figure iii). A novel BINOL-derived copper(II) catalyst was developed, and afforded dihydroquinolones directly from their amine and aldehyde precursors with good yields and enantioselectivities.

This thesis details the application of two synthetic methodologies, developed by the Donohoe group, to the synthesis of the ABC fragment of pectenotoxin-4, a macrolide marine natural product that consists of 19 stereogenic centres, three tetrahydrofuran rings, one spiroketal and one bicyclic ketal embedded within a 26-membered macrocycle. Pivotal to the developed synthetic route was the utilisation of an unprecedented cascade osmium catalysed oxidative cyclisation for the construction of two THF rings (the BC ring system). After successfully developing a model system for the synthesis of the AB anomeric 6,5 spiroketal, which involved the employment of a hydride shift initiated oxo carbenium ion formation followed by intramolecular spiroketalisation, the developed system was then applied to the fully elaborated synthesis of the ABC fragment. The synthesis of the ABC fragment of pectenotoxin-4 was completed in 20 linear steps, with an overall yield of 3.3%.

Rhodium-catalysed hydroacylation provides a highly atom economic synthesis of ketone products from the combination of aldehydes and multiple bond systems by C-H bond activation. This work evaluates the combination of intermolecular hydroacylation for the synthesis of classical heterocycle precursors and their dehydrative cyclisation to give rise to a range of substituted heterocyclic compounds. Chapter 1 outlines recent developments in the chemistry of hydroacylation. Particular attention is paid to the various chelation strategies employed in intermolecular hydroacylation. Chapter 2 discusses some relevant and recent developments in the field of pyridine and pyrrole synthesis. Having established that β-sulphur chelation controlled hydroacylation can be used to synthesise pyridines in Chapter 3; attention was turned to hydroacylation of propargyl amines in Chapter 4. The methodology was expanded to provide a synthesis of γ-amino enones. The hydroacylation reaction and cyclisation is combined in a procedure that utilises thermal Boc-deprotection and cyclisation to give a range of highly-substituted pyrroles. The regioselectivity of the hydroacylation of propargyl amines is investigated in Chapter 5 by application of statistical Design of Experiments methodology. Optimised conditions were identified with minor improvements in the selectivity of the reaction.

The development of new methods for carbon-carbon bond formation is a challenging topic at the heart of organic chemistry. Over the past ten years a number of methods for the catalytic asymmetric 1,4-addition of organometallic reagents such as Grignard, organozinc and organoaluminium reagents have been reported. However these reagents suffer from many limitations, including the need for cryogenic temperatures, which prevent their widespread use. Here we have developed a new asymmetric method: the copper-catalysed enantioselective 1,4-addition of alkylzirconium compounds, generated in situM/em>, from alkenes. A general introduction into the formation of carbon-carbon bonds and catalytic asymmetric 1,4-addition reactions is first given. We then focus our attention on hydrometallation reactions and their current use in the addition of alkenyl and alkyl groups in asymmetric 1,4-addition reactions. In Chapter two, we introduce the development of our methodology. We found that by using copper complex <b>(S,S,S)-A</b>, high enantioselectivities can be achieved (up to 96% ee), in the presence of a broad range of functional groups which are often not compatible with comparable methods using pre-made organometallic reagents. The method gives good enantioselectivity at room temperature, in a wide range of solvents, using readily available alkenes. Chapter three discusses the expansion of our method to the 1,4- and 1,6-addition to complex steroids. Modified conditions were then found to enable the addition to &beta;-substituted enones, to form quaternary centres. This is followed by the successful addition to &alpha;,&beta;-unsaturated lactones, another difficult substrate class. All these results gave excellent selectivity. In summary, we have developed a new reaction which offers an alternative to current methods reported in the literature. This robust reaction can tolerate a variety of functional groups and we hope that this will aid in the synthesis of important molecules.

High aerobic glycolytic capacity is correlated with poor prognosis and increased tumour aggressiveness. 6Phosphofructo-1-kinase catalyses the first irreversible step of glycolysis, and is activated by fructose-2,6-bisphosphate, a product of the kinase activity of four bifunctional isoenzymes, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFK-2/FBPase-2:PFKFB1-4). These are potential anti-tumour targets, but their individual and collective role requires further investigation. This thesis had three aims; to validate the PFK-2/FBPase-2 isoenzymes as anti-cancer targets, to investigate the requirement for isoenzyme-specific targeting, and to initiate assay development, enabling future identification of novel inhibitors. A panel of cancer cell lines was examined and PFKFB3 and PFKFB4 were confirmed to be the most strongly induced isoenzymes in hypoxia, regulated by HIF-1&alpha;. Basal and hypoxic relative PFKFB3/PFKFB4 expression varied markedly, and three cell lines with varying expression ratios (MCF-7, U87, PC3) were selected for further study. siRNA knockdown of each isoenzyme individually, markedly reduced 2D and 3D cell growth. The effect of PFKFB3 knockdown was consistently more pronounced, particularly in hypoxia. Double PFKFB3/PFKFB4 knockdown was significantly less effective than PFKFB3 knockdown alone. Direct antagonism of PFKFB3 and PFKFB4 on F-2,6-BP concentration was observed, with PFKFB3 exhibiting high kinase activity, as anticipated, and PFKFB4 exhibiting high bisphosphatase activity. The degree of antagonism was dependent on the relative PFKFB3/PFKFB4 expression ratio. Extensive efforts were made to examine the wider metabolic effect of PFKFB3/PFKFB4 on flux towards glycolysis or the pentose phosphate pathway (PPP), including using metabolite, lipid droplet, ¹³C NMR and mass spectrometry assays. No significant change in metabolic flux was detected, the evidence presented therefore suggesting the impact of the antagonistic effects of the isoenzymes on [F-2,6-BP] extends beyond regulation of metabolic flux alone. This study concluded that the most effective therapeutic strategy will be one that involves a PFKFB3-specific inhibitor, preferably hypoxia-targeted. Accordingly, steps were taken to validate and optimise a robust medium-throughput assay system.