A microextração em fase líquida com membranas cilíndricas ocas (HF-LPME) é uma técnica bastante interessante de preparação de amostras, uma vez que com pequenas quantidades de solventes orgânicos é possível a extração dos analitos presentes em matrizes complexas. Sendo assim, essa técnica foi empregada para extração da venlafaxina (VEN) e seus metabólitos em fração microssomal de fígado de ratos e plasma, visando o desenvolvimento de métodos para análise enantiosseletiva desses analitos. Esses métodos foram então empregados em um estudo in vitro de biotransformação da VEN e em um estudo piloto de disposição cinética em ratos e humanos. A VEN é um fármaco quiral empregado no tratamento da depressão, cujas propriedades farmacocinéticas e farmacodinâmicas são estereosseletivas. Após a otimização das condições de extração por HF-LPME, foram obtidas recuperações de 12-60%. O método empregado no estudo in vitro de biotransformação da VEN foi desenvolvido usando a coluna ChiralpaK AD®, fase móvel composta por hexano : 2-propanol (95:5, v/v) + 0,025% de dietilamina (DEA) e detecção por absorção no UV. A coluna ChiralpaK AD-H® e a fase móvel composta por metanol : etanol (70:30, v/v) + 0,025% de DEA foram empregadas para análise da VEN e seus metabólitos em plasma. Para esse método, empregou-se a detecção por espectrometria de massas visando à obtenção de menores limites de quantificação. O método empregado para a determinação da VEN e de seus metabólitos em fração microssomal de fígado de ratos foi linear no intervalo de 200 a 5000 ng mL-1 e o método empregado para a determinação da VEN e de seus metabólitos em amostras de plasma foi linear no intervalo de 5 a 500 ng mL-1. Os métodos analíticos desenvolvidos para determinação da VEN e seus metabólitos nas matrizes biológicas foram aplicados em estudos de biotransformação in vitro e em estudos de disposição cinética em duas espécies (ratos e humanos). O objetivo destes estudos foi avaliar a correlação da biotransformação entre as diferentes espécies avaliadas e comparar os resultados obtidos nos estudos in vitro com os verificados nos estudos in vivo. Os resultados dos estudos empregando fração microssomal de fígado de ratos são semelhantes aos obtidos no estudo de disposição cinética em ratos, com formação mais pronunciada da Ndesmetilvenlafaxina (N-VEN) e com produção prioritária do enantiômero (-)-(R). Comparando-se os estudos de disposição cinética em ratos e humanos, observa-se enantiosseletividade na biotransformação da VEN com a (-)-(R)-VEN sendo preferencialmente biotransformada. Entretanto, em humanos observa-se que a (-)- (R)-O-desmetilvenlafaxina ((-)-(R)-O-VEN) é formada em maior proporção enquanto que, em ratos, a (-)-(R)-N-VEN é preferencialmente formada. / Hollow Fiber-Liquid Phase Microextraction (HF-LPME) is a very interesting technique for sample preparation, since it uses small amounts of organic solvents to extract drugs present in complex matrices. Thus, this technique was employed for the extraction of venlafaxine (VEN) and its metabolites from rat liver microsomal fraction and plasma, aiming the development of methods for the enantioselective analysis of these analytes. These methods were then applied to study the in vitro biotransformation and the kinetic disposition of VEN in rats and humans. VEN is a chiral drug used in the treatment of depression, whose pharmacokinetic and pharmacodynamic properties are stereoselective. After the optimization of the HFLPME conditions, recoveries of 12-60% were obtained. The method employed in the in vitro biotransformation study of VEN was developed using a ChiralpaK AD® column, mobile phase consisting of hexane : 2-propanol (95:5, v/v) + 0.025% of diethylamine (DEA) and UV detection. The ChiralpaK AD-H® column and the mobile phase consisting of methanol : ethanol (70:30, v/v) + 0.025% of DEA were employed for the analysis of VEN and its metabolites in plasma. In order to obtain lower quantification limits, mass spectrometry detection was used in this method. The method used for the determination of VEN and its metabolites in rat liver microsomal fraction was linear over the concentration range of 200 to 5000 ng mL-1 whereas the method used for the determination of VEN and its metabolites in plasma was linear in the range of 5 to 500 ng mL-1. The developed methods for the determination of VEN in biological matrices were applied to an in vitro biotransformation study and to kinetic disposition studies in two species (rats and humans). The objective of these studies was to evaluate the correlation of the biotransformation between different species and to compare the results of the in vitro and in vivo studies. The results obtained using rat liver microsomal fraction were similar to those obtained in the rat kinetic disposition study, with more pronounced formation of N-desmethylvenlafaxine (NVEN) and major production of the (-)-(R) enantiomer. Comparing the kinetic disposition studies in rats and humans, the enantioselectivity in the biotransformation of VEN with (-)-(R)-VEN being preferentially biotransformed was observed for both species. However, (-)-(R)-O-desmethylvenlafaxine ((-)-(R)-O-VEN) is preferentially formed in humans whereas the major metabolite in rat plasma is (-)-(R)-N-VEN.

Abstract not available

<p>The main objective of this thesis was to investigate the in vivo relevance of membrane transporters and cytochrome P450 (CYP) 3A4-mediated metabolism in the intestine and liver for the bioavailability of drugs in humans after oral administration.</p><p>In the first part of the thesis, the main transport mechanisms involved in the intestinal absorption and bioavailability were investigated for fexofenadine, a minimally metabolized drug, which is a substrate for P-glycoprotein (P-gp) and members of organic anion transporting polypeptide (OATP) family. Jejunal perfusion studies revealed that co-perfusion with verapamil increased the bioavailability of fexofenadine by decreasing the first-pass liver extraction as the low intestinal permeability was unchanged by the transport inhibitors studied. The mechanism behind the interaction probably involves inhibition of OATP-mediated sinusoidal uptake and/or P-gp-mediated canalicular secretion of fexofenadine. Results from the Caco-2 model supported that the intestinal absorption of fexofenadine is mainly determined by the low passive permeability of the drug, even though fexofenadine clearly is a P-gp substrate. </p><p>In the second part of the thesis, the effect of repeated oral administration of the P-gp and CYP3A4 inducer St. John’s wort on the in vivo intestinal permeability and presystemic metabolism of the dual P-gp and CYP3A4 substrate verapamil was investigated in a jejunal perfusion study. St. John’s wort decreased the bioavailability of the enantiomers of verapamil by inducing the CYP3A4-mediated presystemic metabolism, probably mainly in the gut. It was also concluded that induction of efflux transporters, such as P-gp, does not affect the intestinal transport or the gut wall extraction of high permeability substrates like verapamil. Data from Caco-2 cells with induced CYP3A4-activity supported these findings. The plasma levels of the enantiomers of norverapamil also decreased despite an increased formation, which was attributed to induction of CYP3A4 and/or other metabolic routes. </p>

This thesis describes the use of 2H-azirines, three-membered unsaturatednitrogen-containing heterocycles, as reactive intermediates ina number of Lewis acid promoted alkylations and Diels-Alderreactions providing synthetically useful aziridines. In order to carry out this investigation a new generalprocedure for the ring closure of vinyl azides, forming theresultant 3-substituted-2H-azirines, was developed applying low boiling solventsin closed reaction vessels at elevated temperatures. The addition of organolithium reagents in the presence ofcommercially available chiral ligands, to the 3-(2-naphthyl)-2H-azirine was studied, which gave the correspondingaziridines. Several Lewis acids were shown to catalyze the normalelectron-demand Diels-Alder reaction between 3-alkyl-,3-aromatic-, and 3-ester-substituted 2H-azirines and various dienes. These reactions gave theexpected cycloadducts in moderate yields. Using a chiral auxiliary high diastereoselectivity wasobtained in the addition of alkyl radicals to a8-phenylmenthyl-substituted 2H-azirine-3-carboxylate. The alkyl radicals weregenerated from the corresponding trialkyl borane and molecularoxygen. Hydroborations and transmetallations were used toprepare these trialkylboranes. Catalytic amounts of CuClincreased the diastereoselectivity in the radical additionreactions. Attempts were made to explain how the coordination of aLewis acid to the azirine nitrogen atom affects thereactivity/stability of the azirine. DFT calculations and NMRexperiments involving Lewis acid-azirine complexes wereperformed. Keywords:Enantioselective, diastereoselective, vinylazide, 2H-azirines, aziridines, Lewis acid, chiral ligand,chiral auxiliary, organolithiums, Diels-Alder reaction, alkylradicals, triethylborane.

The ability to generate chiral building blocks is of paramount importance to organic chemists. This problem presents itself most notably at the interface of chemistry and biology, where molecules of only a single enantiomer can induce function to many biological systems. In this context, recent developments in the field of organocatalysis, most notably the employment of chiral N-heterocyclic carbenes (NHCs) have shown much promise. Our group has recently shown that one possible chiral NHC catalyzed Stetter cyclization product of a conjugated triene, a highly functionalized cyclopentenone, contains both a chiral center and an adjacent conjugated diene. This structure can be easily elaborated to a bicyclic structural motif present in some biologically active natural products from the ginkgolide family, and is difficult to access by other means. The synthesis of novel vinyl stannanes and other coupling substrates involved in the development of the aforementioned reaction discovery are described in this report.

Aminoalcohols are very important compounds used in various asymmetric transformations as chiral ligands or chiral auxiliaries. In this thesis, four novel heterocyclic amine substituted chiral 1,4-aminoalcohols were synthesized. In the synthetic strategy, amide esters were synthesized from (2S, 3R)-3-methoxycarbonylbicyclo[2.2.1]hept-5-ene-2-carboxylic acid by DCC coupling method. Subsequent reduction of these amide esters lead to target 1,4-aminoalcohols. The activities of these novel chiral 1,4-aminoalcohols were tested in enantioselective diethylzinc addition, Mukaiyama aldol and Diels-Alder reactions. The enantioselectivities were measured by HPLC. All the products were identified by H NMR and C NMR spectroscopy

The thesis entitled “Enantioselective synthesis of bio-active bicyclic acetals, cyclic ethers and lactones” demonstrates the utility of chiral pool tartaric acid as the source in the synthesis of natural products. The results are discussed in three chapters; 1) Enantioselective synthesis of bio-active bicyclic acetals, 2) Enantioselective synthesis of bio-active cyclic ethers and 3) Enantioselective synthesis of bio-active lactones. A brief introduction is provided in each chapter to keep the present work in proper perspective. Compounds (in bold) and references (in superscripts) are sequentially numbered differently for each chapter and references are given as foot notes. Experimental procedures are given differently for each chapter and placed at the end of chapter. Scanned 1H and 13C NMR spectras are given with description of signals. Chapter 1 describes the enantioselective synthesis of bicyclic acetal containing insect pheromones. First part of this chapter deals with the enantiodivergent synthesis of both enantiomers of hydroxy-exo-brevicomin and 2-hydroxy-exo-brevicomin starting from a single chiral compound, bis-Weinreb amide derived from L-(+)-tartaric acid. Controlled addition of Grignard reagent to bis-Weinreb amide followed by diastereoselective reduction of the resultant ketone was employed as the key step for the enantiodivergent synthesis of hydroxy-exo-brevicomin and 2-hydroxy-exo-brevicomin. In the second part, enantioselective synthesis of exo-brevicomin, iso-exo-brevicomin and formal synthesis of frontalin comprising similar framework is demonstrated, utilizing á -benzyloxy aldehydes derived from L-(+)-tartaric acid as chiral building block. Second Chapter describes the enantioselective synthesis of bio-active cyclic ethers, disparlure, centrolobine and isolaurepan. Employing á-benzyloxy aldehydes derived from L-(+)-tartaric acid as the chiral building block, synthesis of both enantiomers of insect pheromone disparlure is achieved involving the diastereoselective addition of allyltributyl tin to the á-benzyloxy aldehyde and cross metathesis of the resultant homoallylic alcohol with 4-methyl-1-pentene. Formal synthesis of centrolobine and isolaurepan are accomplished. Pivotal step involved in the synthesis of centrolobine is iron(III) mediated cyclization of 1,5-diol derived from L-(+)-tartaric acid, while Lewis acid mediated reductive cyclization of the hydroxy ketone derived from á-benzyloxy aldehyde is the key step in the synthesis of isolaurepan. Third chapter in the thesis deals with the enantioselective synthesis of bio-active lactones muricatacin, 6-acetoxy-5-hexadecanolide and boronolide. Utilizing á-benzyloxy aldehyde as the building block, synthesis of five and six membered lactones, muricatacin and 6-acetoxy-5-hexadecanolide were accomplished via the diastereoselective addition of 3-butenylmagnesium bromide and allyltributyl tin to á-benzyloxy aldehyde, respectively. Stereoselective formal synthesis of boronolide was described, starting from D-(–)-tartaric acid. Key reaction sequence includes the elaboration of ã-hydroxy amide obtained by a combination of controlled Grignard addition and diastereoselective reduction from bis- Weinreb amide derived from D-(–)-tartaric acid.

<p>This thesis describes the use of 2<i>H</i>-azirines, three-membered unsaturatednitrogen-containing heterocycles, as reactive intermediates ina number of Lewis acid promoted alkylations and Diels-Alderreactions providing synthetically useful aziridines.</p><p>In order to carry out this investigation a new generalprocedure for the ring closure of vinyl azides, forming theresultant 3-substituted-2<i>H</i>-azirines, was developed applying low boiling solventsin closed reaction vessels at elevated temperatures.</p><p>The addition of organolithium reagents in the presence ofcommercially available chiral ligands, to the 3-(2-naphthyl)-2<i>H</i>-azirine was studied, which gave the correspondingaziridines.</p><p>Several Lewis acids were shown to catalyze the normalelectron-demand Diels-Alder reaction between 3-alkyl-,3-aromatic-, and 3-ester-substituted 2<i>H</i>-azirines and various dienes. These reactions gave theexpected cycloadducts in moderate yields.</p><p>Using a chiral auxiliary high diastereoselectivity wasobtained in the addition of alkyl radicals to a8-phenylmenthyl-substituted 2<i>H</i>-azirine-3-carboxylate. The alkyl radicals weregenerated from the corresponding trialkyl borane and molecularoxygen. Hydroborations and transmetallations were used toprepare these trialkylboranes. Catalytic amounts of CuClincreased the diastereoselectivity in the radical additionreactions.</p><p>Attempts were made to explain how the coordination of aLewis acid to the azirine nitrogen atom affects thereactivity/stability of the azirine. DFT calculations and NMRexperiments involving Lewis acid-azirine complexes wereperformed.</p><p><b>Keywords:</b>Enantioselective, diastereoselective, vinylazide, 2<i>H</i>-azirines, aziridines, Lewis acid, chiral ligand,chiral auxiliary, organolithiums, Diels-Alder reaction, alkylradicals, triethylborane.</p>

Enantioselective indicator displacement assays (eIDAs), was transitioned to a high-throughput screening protocols, for the rapid determination of concentration and enantioselectivity (ee) of chiral diols and α-hydroxycarboxylic acid. To improve the design of our previously established receptor based on o-(N,N-dialkylaminomethyl)arylboronate scaffolds for eIDAs. The rigidity of the receptor, which pertinent from the formation of an intramolecular N-B dative bond was investigated. o-(Pyrrolidinylmethyl)phenylboronic acid its complexes with bifunctional substrates such as catechol, [alpha]-hydroxyisobutyric acid, and hydrobenzoin was studied in detail by x-ray crystallography and ¹¹B NMR. Our structural study predicts that the formation of an N-B dative bond, and/or solvolysis to afford a tetrahedral boronate anion, depends on the solvent and the complexing substrate present. To simplify the operation of eIDAs, we introduced an analytical method, which utilize a dual-chamber quartz cuvette, which reduces the number of spectroscopic measurements from two to one and introduced artificial neural networks (ANNs) which simplifies data analysis. In a second example a high-throughtput screening protocol for hydrobenzoin was developed. The method involves the sequential utilization of what we define herein as screening, training, and analysis plates. Several enantioselective boronic-acid based receptors were screened using 96-well plates, both for their ability to discriminate the enantiomers of hydrobenzoin and to find their optimal pairing with indicators resulting in the largest optical responses. The best receptor/indicator combination was then used to train an ANN to determine concentration and ee. To prove the practicality of the developed protocol, analysis plates were created containing true unknown samples of hydrobenzoin generated by established Sharpless asymmetric dihydroxylation reactions, and the best ligand was correctly identified. The system was extended to pattern recognition for the rapid determination of identity, concentration, and ee of chiral vicinal diols. A diverse enantioselective sensor array was generated with three chiral boronic acid receptors and pH indicators. The optical response produced by the sensor array, was analyzed by two pattern recognition algorithms: principal component analysis (PCA) and ANNs. The PCA plot demonstrated good chemoselective and enantioselective separation of the analytes, and ANNs was used to accurately determine the concentration and ee of five unknown samples. / text

Ce mémoire décrit le développement d’une nouvelle méthodologie d’expansion de cycle irréversible à partir de N-alkyl-3,4-déhydroprolinols pour former des N-alkyl tétrahydropyridines 3-substituées en passant par un intermédiaire aziridinium bicyclique. Cette méthode permet l’introduction d’un vaste éventail de substituants à la position 3 et tolère bien la présence de groupements aux positions 2 et 6, donnant accès à des pipéridines mono-, di- ou trisubstituées avec un excellent diastéréocontrôle. De plus, il est démontré que l’information stéréogénique du 3,4-déhydroprolinol de départ est totalement transférée vers le produit tétrahydropyridine. Additionnellement, une méthodologie fut dévelopée pour la préparation des produits de départ 3,4-déhydroprolinols en forme énantiopure, avec ou sans substituants aux positions 2 et 5, avec un très bon stéréocontrôle. Le premier chapitre présente un résumé de la littérature sur le sujet, incluant un bref survol des méthodes existantes pour la synthèse de pipéridines 3-substituées, ainsi qu’une vue d’ensemble de la chimie des aziridiniums. L’hypothèse originale ainsi que le raisonnement pour l’entreprise de ce projet y sont également inclus. Le second chapitre traite de la synthèse des N-alkyl-3,4-déhydroprolinols utilisés comme produits de départ pour l’expansion de cycle vers les tétrahydropyridines 3-substituées, incluant deux routes synthétiques différentes pour leur formation. Le premier chemin synthétique utilise la L-trans-4-hydroxyproline comme produit de départ, tandis que le deuxième est basé sur une modification de la réaction de Petasis-Mannich suivie par une métathèse de fermeture de cycle, facilitant l’accès aux précurseurs pour l’expansion de cycle. Le troisième chapitre présente une preuve de concept de la viabilité du projet ainsi que l’optimisation des conditions réactionnelles pour l’expansion de cycle. De plus, il y est démontré que l’information stéréogénique des produits de départs est transférée vers les produits. iv Au quatrième chapitre, l’étendue des composés pouvant être synthétisés par cette méthodologie est présentée, ainsi qu’une hypothèse mécanistique expliquant les stéréochimies relatives observées. Une synthèse énantiosélective efficace et divergente de tétrahydropyridines 2,3-disubstituées est également documentée, où les deux substituants furent introduits à partir d’un intermédiaire commun en 3 étapes. / This thesis describes the development of a novel methodology of irreversible ring expansion from N-alkyl-3,4-dehydroprolinols to N-alkyl-3-substituted tetrahydropyridines through a bicyclic aziridinium ion intermediate. This method allows a wide variety of substituents at the 3-position, and also permits substitution at the 2- and 6-positions of the tetrahydropyridine giving mono-, di- or tri-substituted piperidines with excellent diasterocontrol. Complete transfer of the stereogenic information of the 3,4- dehydroprolinol to the tetrahydropyridine product is demonstrated. Also, a methodology was developed to prepare the 3,4-dehydroprolinol starting materials in enantiopure form, with the possibility of substitution at the 2- and 5-positions with excellent diasterocontrol. The first chapter presents the literature background, including a brief summary of methodologies for the synthesis of 3-substituted piperidines, and an overview of aziridinium ion chemistry. Also presented is the original hypothesis of the project, and our reasoning for undertaking this project. The second chapter describes the synthesis of N-alkyl-3,4-dehydroprolinols used as precursors for the ring expansion to 3-substituted tetrahydropyridines, including two different synthetic routes. The first route route converts L-trans-4-hydroxyproline to enantioenriched N-benzyl-3,4-dehydroprolinol in 6 steps. The second synthetic route was developed using a variant of the Petasis-Mannich reaction and a ring closing metathesis,making the precursors more readily available and simple to synthesize. The third chapter presents the proof of concept of the viability of the project and optimization studies. Moreover, the transfer of stereogenic information to the resulting product is demonstrated. The fourth chapter demonstrates the broad scope of the ring expansion and mechanistic insight is given based on the relative configuration of the products. An expedient divergent enantioselective synthesis of a 2,3-disubstituted tetrahydropyridine is also shown, with both substituents being chosen from a common intermediate in 3 steps.