Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 121-140).

Dynamic combinatorial chemistry (DCC) is a powerful tool to identify new ligands for biological targets. In the technique, library synthesis and hit identification are neatly combined into a single step. A labile functionality between fragments allows the biological target to self-select binders from a dynamic combinatorial library (DCL) of interconverting building blocks. The scope of suitable reversible reactions that proceed under thermodynamic control in physiological conditions has been gradually expanded over the last decades, however DCC has thus far failed to gain traction as a technique appropriate for drug discovery in the pharmaceutical industry. The constraints placed on library size by validated analytical techniques, and the effort-intensive reality of this academically elegant concept have not allowed DCC to develop into a broad-platform technique to compete with the high-throughput screening campaigns favoured by medicinal chemists. This thesis seeks to develop DCL analysis techniques, in an effort to increase the library size and accelerate the analysis of DCC experiments. Using a 19F-labelled core scaffold, we constructed a DCL that could be monitored non-invasively by 19F NMR. Building on NMR techniques developed by fragment screening and non-biological DCC campaigns, the method was developed to circumvent the undesired equilibrium-perturbing side effects arising from sample-consuming analytical methods. The N-acylhydrazone (NAH) DCL equilibrated rapidly at pH 6.2 using 4-amino-L-phenylalanine (4-APA) as a novel, physiologically benign, nucleophilic catalyst. The DCL was designed to target b-ketoacyl-ACP synthase III (FabH), an essential bacterial enzyme and antibiotic target. From the 5-membered DCL, a single combination was identified as a privileged structure by our 19F NMR method. The result correlated well with an in vitro assay, validating 19F NMR as a tool for DCL screening. During the 19F NMR study we identified an established antimicrobial compound, 4,5- dichloro-1,2-dithiole-3-one (HR45), to have potential as a core scaffold from which to develop future DCLs targeting FabH. Despite the potentially tractable chemistry of HR45 for DCC, lack of knowledge around the inhibitory mechanism of the compound prevented us from proceeding. Thus, we used mass spectrometry, NMR and molecular modelling to show that HR45 acts by forming a covalent adduct with S. aureus FabH. The 5-chloro substituent directs attack from the nucleophilic thiol side chain of the essential active site cysteine-112 residue via a Michael-type addition elimination mechanism. Although interesting, this mechanism disfavoured the use of HR45 as a core scaffold for NAH exchange in a DCC campaign. Electrospray ionisation mass spectrometry (ESI-MS) is a powerful technique that allows for larger DCLs by eliminating the size-limitations imposed by the need for spectral or chromatographic resolution of DCL members. We developed a 4-APAcatalysed NAH library targeting the pyridoxal 5’-phosphate (PLP) dependent enzyme 7,8-diaminopelargonic acid synthase (BioA), an essential enzyme in the biotin biosynthesis pathway. We exploited the aldehyde moiety of PLP to form an NAH DCL with a panel of hydrazides, and used the BioA isozymes from M. tuberculosis (Mtb) and E. coli to template the library. A combination of buffer exchange and denaturing ESI-MS allowed us to conduct a DCC experiment with a 29-member DCL. Hits from the DCC experiment correlated well with differential scanning fluorimetry (DSF) results. Of these hits, 5 compounds were selected for further study. In vivo activity was displayed by 2 compounds against E. coli and the ESKAPE pathogen A. baumannii. The identification of compounds with antibacterial activity from a DCL further validates ESI-MS as a platform technology for drug discovery.

La découverte de petites molécules capables de moduler les systèmes biologiques présente un intérêt majeur dans l'étude des mécanismes cellulaires et la mise au point de nouvelles méthodes thérapeutiques. Bien que les techniques de criblage haut débit soient régulièrement utilisées, il y a un vrai besoin de réduire le coût et le temps associés à la découverte de ligands, afin de valider la fonction de nombreuses cibles potentielles dans notre protéome ou ceux d' organismes pathogènes. A cette fm, l'émergence de technologies basées sur l'encodage de chimiothèques par des acides nucléique offre une alternative répondant à ces critères. Nous avons développé un système permettant une synthèse rapide de bibliothèques de structures variées,conjuguées à des codes PNA (acide peptidique nucléique) uniques, ainsi qu'une technologie de criblage basée sur la sélection par affinité, qui permet une étude rapide de l'interaction avec une protéine-cible et par conséquent l'identification de nouveaux ligands. Plusieurs chimiothèques ont déjà été synthétisées et criblées, et compte tenu de la stabilité chimique remarquable du PNA, nous avons également développé une nouvelle gamme de réactions compatibles avec la synthèse PNA-encodée, la voie étant maintenant ouverte pour la génération de chimiothèques plus complexes et pour l'étude de cibles biologiques très variées. / The discovery of srnall molecules capable of modulating biological systems is of major interest for the understanding of cellular mechanisms as weil as for the drug discovery process. In spite of established high throughput techniques routinely used, there is a clear need to reduce the time and cost •associated to ligand discovery, in order to validate the function of numerous potential targets in our proteome or the one of pathogens. In this perspective, the emergence of technologies based on nucleic acid encoding of chemical libraries presents an alternative that fulfills these criteria. We have developed a system enabling the rapid synthesis of libraries containing various structures, conjugated to unique PNA (peptide nucleic acid) tags, a weil as a screening technique based on affinity selection that allows for the rapid study of the interaction witb a target protein and the consequent identification of new ligands. Several libraries have already been synthesized and screened, and based on the remarkable chemical stability of PNA, we have also developed a new palette of reactions compatible with PNA-encoded synthesis, the path now being open for the generation of more complex libraries, and the study of various biological targets

Cette thèse en partenariat avec Total Petrochemicals, porte sur deux grands axes: la préparation de nouveaux catalyseurs pour la polymérisation d'éthylène et l'analyse des propriétés des polyéthylènes obtenus. Face au grand nombre de combinaisons à tester et à la diversité de polymères produits, des méthodes de travail haut-débit sont indispensables pour la comparaison et la distinction de produits d'intérêt. Ainsi, le développement d'une nouvelle méthode de préparation de catalyseurs et de polymérisation en parallèle a permis de comparer directement et visuellement plusieurs complexes catalytiques suivant leur activité. Des méthodes d'analyse en parallèle ont également conduit à la comparaison de polyéthylènes produits suivant leur densité, leur température de fusion et leurs masses moléculaires. La détermination des masses moléculaires en parallèle a nécessité la conception d'un prototype de chromatographie planaire et le développement d'une nouvelle méthode d'analyse. L'ensemble de ces travaux en haut-débit a fourni un grand nombre d'informations. Le traitement statistique de cette banque de données permet d'apporter un outil de comparaison multi-paramètres et d'aide à la compréhension des relations catalyseur/propriétés de polymères. / This thesis in partnership with Petrochemicals Total, is composed of two main axis: preparation of new catalysts for ethylene polymerization and analysis of the properties of polyethylenes obtained. Face to a large number of combinations being tested and the diversity of produced polymer, high-throughput methods are essential for the comparison and the distinction of products of interest. Thus, the development of a new method for the preparation of catalysts and for polymerization in parallel leads to compare directly and visually several catalytic complexes according to their activity. Methods of analysis in parallel also led to the polyethylene comparison produced according to their density, melting point and molecular masses. The determination of the molecular masses in parallel required the design of a planar prototype of chromatography and the development of a new method of analysis. The whole of this work in highthroughput provided a large number of information. The statistical processing of this data bank brings a tool of multivariate comparison and helps to the comprehension of the relations catalyst/polymer properties.

No description available.

Widespread antimicrobial resistance, particularly in Gram-negative pathogens, is a serious threat facing the global community. Aminoglycosides are inactivated by enzymes such as aminoglycoside N-acetyltransferase-3 (AAC(3)) and O-nucleotidyltransferase-2” (ANT(2”)), while the New Delhi metallo-b- lactamase-1 (NDM-1) degrades carbapenems. Inhibition of these enzymes should result in bacteria becoming once again susceptible to aminoglycosides and carbapenems. This thesis describes the development of inhibitors to these enzymes, in an effort to rescue the utility of aminoglycoside and carbapenem drug classes through adjuvant therapy. High-throughput screening of protein kinase libraries identified two AAC(3)-Ia inhibitors with a common 3-benzylidene-2-indolinone core. New methods for purification of AAC(3)-Ia and monitoring its activity were developed. A chemical library was built around this scaffold and assessed for SAR. It was found that the initial hit (Z)-methyl 3-(3,5-dibromo-4-hydroxybenzylidene)-2- oxoindoline-5-carboxylate was the most active against AAC(3)-Ia, and alterations to either the 3,5-dibromo-4-hydroxybenzyl warhead or methyl ester substituent resulted in a decrease in activity. Previous whole-cell screening had identified two protein kinase inhibitors with a biphenyl isonicotinamide scaffold as inhibitors of ANT(2”)-Ia. A convergent parallel synthesis was developed, involving Suzuki and amide couplings and protecting group strategies. This methodology was used to assemble a focused chemical library for SAR analysis. Stepwise removal of extraneous complexity from the initial hits yielded a selective ANT(2”)-Ia inhibitor which demonstrated in vivo synergy with gentamicin. Aspergillomarasmine A (AMA) is a natural product with activity against NDM-1. Several derivatives of AMA have been synthesized to assess SAR, but the specific contributions of individual carboxylic acids have yet to determined due to difficulties accessing position 6. A synthetic approach was developed via reductive amination using Garner’s aldehyde as a serine equivalent. This strategy was used to synthesize an AMA analog with a hydroxyl group in place of the carboxylic acid in position 6. Additionally, an imine-promoted isomeric resolution was discovered. / Thesis / Doctor of Philosophy (PhD) / Antibiotics, such as aminoglycosides and carbapenems, are losing their effectiveness against bacteria responsible for deadly diseases. This is often due to resistance enzymes such as aminoglycoside N-acetyltransferase-3 (AAC(3)) and O- nucleotidyltransferase-2” (ANT(2”)), which inactivate aminoglycosides, and the New Delhi metallo-b-lactamase-1 (NDM-1), which destroys carbapenems. If these enzymes are blocked, the antibiotics should work against bacteria again. In order to develop compounds that will inhibit these enzymes, sets of similar compounds are made and tested. Patterns of what chemical groups improve or worsen inhibitory activity are noted and used to make another set of compounds in an iterative process. This thesis describes the development of inhibitors of AAC(3)-Ia and ANT(2”)-Ia by this process. Additionally, a specific compound was made to test if a particular chemical group has a role in inhibiting NDM-1.

The complex natural product paclitaxel (Taxol®), first isolated from Taxus brevifolia, is a member of a large family of taxane diterpenoids. Paclitaxel is extensively used for the treatment of solid tumors, particularly those of the breasts and ovaries. In order to obtain additional information about the mechanism of action of paclitaxel and the environment of the paclitaxel-binding site, several fluorescent analogs of paclitaxel were synthesized, and their biological activities have been evaluated. For the investigation of possible synergistic effects, concurrent modifications on selected positions have been performed and their biological evaluation were studied. / Ph. D.

L’imagerie moléculaire est aujourd’hui un outil non-invasif essentiel pour le diagnostic de nombreuses pathologies. Les traceurs technétiés sont actuellement les plus répandus car le 99mTc est facilement disponible, abordable et présente des caractéristiques idéales pour l’imagerie. Néanmoins, le développement de traceurs efficaces nécessite un long et coûteux processus d’optimisation souvent empirique. Dans ce contexte, nous avons entrepris le développement d’une plateforme technétiée conçue pour présenter au sein de sa structure de nombreux sites potentiels de fonctionnalisation et compatible avec une approche combinatoire.Dans un premier temps, un ensemble de 12 ligands N3X (X = N, O, S) a été préparé. Chacun d’entre eux présente dans sa structure un motif triazole introduit par chimie-click et intervenant dans la complexation du métal par un de ses atomes d’azote. Nous avons ensuite évalué l’aptitude de ces ligands à chélater le cœur oxotechnétium dans des conditions douces (5 min, température ambiante) compatible avec une utilisation en milieu hospitalier. Le complexe TriaS-99mTc a été formé quantitativement et sa stabilité en plasma murin a été étudiée. Il s’est révélé stable à plus de 90% dans le plasma murin après 6h d’incubation. L’étude in vivo de ce complexe a par la suite révélé une élimination efficace du milieu circulant par la voie urinaire avec une dégradation minoritaire.A titre d’illustration, nous avons ensuite engagé la structure TriaS dans deux approches distinctes pour le développement de traceurs de la néoangiogenèse tumorale en ciblant l’intégrine αvβ3. D’une part, dans le cadre d’une approche intégrée, plusieurs complexes fonctionnalisés, mimes de RGD, ont été obtenus. Dans chaque cas, l’adjonction de groupements fonctionnels n’a pas affecté l’efficacité de la chélation. En outre la stabilité en plasma est maintenue à un niveau très correct. D’autre part, nous avons développé une approche bifonctionnelle dans laquelle le motif c(RGDfK) joue le rôle de molécule ciblante. Dans ce cas, un motif variable (ici un PEG) peut être introduit par chimie combinatoire pour moduler la solubilité, la biodistribution, et l’excrétion des traceurs. / Molecular imaging is an essential non-invasive tool usable for diagnosis and characterisation of many diseases. Technetium-based tracers are the most popular ones due to disponibility, cost and radiochemical properties of 99mTc. Nevertheless, effective tracers development requires a long, expensive, and mainly empirical optimisation process. This context prompted us tu carry on the development of a new technetium structure which exhibits lots of potential functionalisation spots compatible with a combinatorial approach. We synthesised 12 N3X (X = N, O, S) different ligands. Each of them includes a triazole moiety, (formed via a click-chemistry reaction), which is involved in the metal complexation that implies one of its nitrogen atoms. Then we evaluated their ability to readily form oxotechnetium complexes in conditions that are compatible with medical use in hospital. One complex was formed in quantitative yields and its stability in mice plasma was investigated. A complex called TriaS-99mTc, stable to more than 90% after 6h incubation, was selected. In vivo study of TriaS-99mTc revealed an efficient blood clearance via the urinary excretion pathway with very low degradation. As an application, we used this structure for the development of tracers that target integrin αvβ3, a known biomarker of tumor neoangiogenesis. First, we synthesised functionnalised TriaS-based integrated complexes. Fonctionnal modification of TriaS by addition of side chains and substituents did not affect its ability to chelate oxotechnetium quantitatively. In addition, its stability in mice plasma was satisfactory. We also developped a bifonctionnal approach using c(RGDfK) peptide as the targeting biomolecule. In this way, a variable moiety (herein a PEG moiety) can be inserted in the structure through click-chemistry in order to modulate tracers solubility, biodistribution and excretion.

De nombreux glycoclusters multivalents des calixarènes, des pillararènes ou des fullerènes ont été synthétisés au sein de notre laboratoire et ont montré d'excellentes affinités pour diverses lectines grâce à leur multivalence et au « glycoside cluster effect ». Nous avons cherché à approfondir ces résultats en ajoutant un degré de dynamisme à ces molécules. Pour cela, nous avons appliqué les concepts de la chimie combinatoire dynamique où des briques moléculaires s'auto-assemblent via des liaisons réversibles pour générer à l'équilibre thermodynamique une chimiothèque d'oligomères. Des briques moléculaires dithiophénols glycosylés sont capables de s'auto-assembler via la formation de ponts disulfures. Leurs propriétés ont été investiguées en chimie combinatoire dynamique et la distribution d'espèces résultant de l'équilibration a montré la formation exclusive des cyclotrimères et cyclotétramères, ou dyn[3]- et dyn[4]arènes. La répétition de l'expérience en présence d'une lectine modèle (ConA) a mené à l'amplification des homodyn[3]- et homodyn[4]arènes. Ces derniers ont été isolés par HPLC semi-préparative et leurs affinités pour ConA ont été mesurées en ITC dans le domaine du nanomolaire. Une extension de cette méthodologie aux lectines LecA et LecB de Pseudomonas aeruginosa est en cours / Several glycoclusters based on calixarenes, pillararenes or fullerenes have been synthesized in our laboratory. They exhibited strong affinities for several lectins through their multivalence and the “glycoside cluster effect”. The prupose of this study was to add a dynamic part to these molecules. We therefore applied the concept of dynamic combinatorial chemistry in which building blocks are able to self-assemble through reversible bonds to generate a library of oligomers. Dithiophenols bearing carbohydrate epitopes can self-assemble through the formation and exchange of disulfide bonds. Their properties in dynamic combinatorial chemistry were studied and the species distribution at the thermodynamic equilibrium revealed the selective formation of cyclotrimers and cyclotetramers named dyn[3]- and dyn[4]arenes. The equilibration in the presence of ConA, used as a model lectin, have led to the amplification of homodyn[3]- and homodyn[4]arenes. These glycodyn[n3,4]arenes have been isolated and their affinities toward ConA measured by ITC in the nanomolar range. Extension of this methodology toward the lectins LecA and LecB of Pseudomonas aeruginosa is in progress

According to a strategy based on an Aza-Wittig reaction type, an efficient methodology in solution toward the synthesis of libraries of 2-aminoimidazolones with high molecular diversity has been developed. The strategy is readily adapted to the solid support. The regioselectivity in the heterocyclisation reaction between the corresponding carbodiimides and different primary amines for the synthesis of 2-aminoimidazolones has been studied. A correlation between the regioisomer obtained and the stereoelectronic properties of the primary amines used was stablished. The structural elucidation of there 2-aminoimidazolones was unambiguously established based on spectroscopic properties and X-Ray diffraction data / D’acord amb una estratègia basada en la reacció Aza-Wittig, s’ha desenvolupat una metodologia eficient en dissolució, fàcilment adaptable a la síntesi en fase sòlida per l’obtenció de llibreries de 2-aminoimidazolones tipus 2 i 3 amb elevada diversitat molecular. S’ha estudiat la regioselectivitat en les reaccions d’heterociclació per l’obtenció de 2-aminoimidazolones a partir de les corresponents carbodiimides 1 i diferents amines primàries, relacionant els regioisòmers obtinguts amb les propietats estereoelectròniques de les diferents amines utilitzades. Les estructures de 2-aminoimidazolones obtingudes s’han determinat inequívocament en base a les seves dades espectroscòpiques i per difracció de raigs X