Part 1: Transition-metal-catalyzed direct transformations of aromatic C-H bonds are emerging as valuable tools in organic synthesis. These reactions are attractive because of they allow for inherently efficient construction of organic building blocks by minimizing the pre-activation of substrates. Of these processes, direct arylation has recently received much attention due to the importance of the biaryl core in medicinal and materials chemistry. Also, alkyne hydroarylation has garnered interest because it allows for the atom-economical synthesis of functionalized alkenes directly from simple arenes and alkynes. Described in this thesis are number of advancements in these areas. First, palladium catalyzed direct arylation of azine N-oxides using synthetically important aryl triflates is described. Interesting reactivity of aryl triflates compared to aryl bromides was uncovered and exploited in the synthesis of a compound that exhibits antimalarial and antimicrobial activity. Also reported is the efficient, direct arylation enabled (formal) synthesis of six thiophene based organic electronic materials in high yields using simple starting materials. Additionally, the site-selective direct arylation of both sp2 and sp3 sites on azine N-oxide substrates is described. The arylation reactions are carried out in either a divergent manner or a sequential manner and is applied to the synthesis of the natural products, Papaverine and Crykonisine. Mechanistic investigations point towards the intimate involvement of the base in the mechanism of these reactions. Next, the rhodium(III)-catalyzed hydroarylation of internal alkynes is described. Good yields are obtained for a variety of alkynes and arenes with excellent regioselectivity for unsymmetrically substituted alkynes. Mechanistic investigations suggest that this reaction proceeds through arene metalation with the cationic rhodium catalyst, which enables challenging intermolecular reactivity. Part 2: Access to single enantiomer compounds is a fundamental goal in organic chemistry and despite remarkable advances in enantioselective synthesis, their preparation remains a challenge. Kinetic resolution of racemic products is an important method to access enantioenriched compounds, especially when alternative methods are scarce. Described in this thesis is the resolution of tertiary and secondary alcohols, which arise from ketone and aldehyde aldol additions. The method is technically simple, easily scalable, and provides tertiary and secondary alcohols in high enantiomeric ratios. A rationale for the unique reactivity/selectivity associated with (1S,2R)-N-methylephedrine in the resolution is proposed. Organocatalysis is a rapidly developing, powerful field for the construction of enantioenriched organic molecules. Described here is a complimentary class of organocatalysis using simple aldehydes as temporary tethers to perform challenging formally intermolecular reactions at room temperature. This strategy allows for the enantioselective, intermolecular cope-type hydroamination of allylic amines with hydroxyl amines. Also, interesting catalytic reactivity for dichloromethane is revealed.

<p>Numerous methodologies for efficient formation of carbon-nitrogen bonds have been developed over the decades due to the widespread importance of nitrogen containing compounds in pharmaceuticals and bulk commercial chemicals. Among many methods, hydroamination, especially, has attracted enormous attention because of its atom-economical characteristic to synthesize amine moieties. As a result, numerous publications have been reported relating the hydroamination reaction using various metal catalysts. However, the hydroamination of unactivated alkenes still remains a challenge task because of the low reactivity of the CC double bond. Recent development of superior gold(I) catalysis in many organic transformations stimulated us to develop efficient gold(I)-catalyzed methods for enantioselective intra- and intermolecular hydroamination of unactivated alkenes. </p><p>A gold(I)-catalyzed system for enantioselective intramolecular hydroamination of unactivated alkenes has been developed. For the effective gold(I)-catalyzed method, various gold(I)-catalysts have been synthesized and tested. Among the catalysts, bis(gold) complexes containing an axially chiral bis(phosphine) ligand catalyze the enantioselective intramolecular hydroamination of unactivated alkenes with carboxamide derivatives, most effectively. The method was effective for both carbamates and ureas to form pyrrolidine derivatives with up to 85 % ee.</p><p>The first enantioselective intermolecular hydroamination of unactivated alkenes was realized by a gold(I)-catalyzed method. The gold(I) catalyst system adds cyclic ureas to unactivated 1-alkenes to produce corresponding enantiomerically enriched hydroamination product in good yield with enantioselectivity up to 78 % ee. </p><p>Polymer-embedded ligands have been synthesized to demonstrate proofs of concepts for fluxional mechanocatalysis. We applied a certain shear stress using a rheometer in the course of palladium-catalyzed asymmetric allylic alkylation to examine catalytic reactivity change under the mechanical force.</p> / Dissertation

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2004. / Includes bibliographical references (leaves 206-216). Also available in electronic version. Access restricted to campus users.

This thesis describes the combination of non-stabilized nucleophiles and prochiral/racemic electrophiles in transition metal catalyzed asymmetric transformations. These enantioselective reactions have tremendous potential for the formation of chiral building blocks and new structural motifs that can be found in a variety of natural products and their derivatives. The first part of the thesis focuses on the synthetic approach towards anti-cancer active diterpenoid structures. The two key steps involve a Cu-catalyzed asymmetric conjugate addition of alkylzirconocenes to enones and an intramolecular oxidative cyclisation. Particular investigations into the cyclisation are made with organocatalysis, transition metal catalysis and electrochemistry for the formation of these tricyclic scaffolds. In the second part this work builds on the Rh-catalyzed asymmetric Suzuki-Miyaura coupling of benzeneboronic acids and cyclic allyl chlorides, which has been developed in our group. Here, the main point is to use more challenging coupling partners, such as heteroaromatic boronic acids, which are coupled to racemic cyclic allyl halides. The utility of this method is demonstrated by performing further transformations and an asymmetric synthesis of the natural product (+)- isoanabasine. The last chapter describes the development of a new asymmetric Hiyama coupling of arylsiloxanes with racemic cyclic allyl chloride. Attempts are made to generate substrates that are not accessible via the asymmetric Suzuki - Miyaura reaction. After extensive optimisation a variety of arylsiloxanes is generated and tested with the best conditions to prove its utility in comparison to the asymmetric Suzuki-Miyaura coupling.

Chemical probes are necessary for elucidating the biochemical roles of proteins. Bromodomains are protein-interaction modules found in a family of proteins implicated in the epigenetic regulation of transcription; however, the individual roles remain unknown for many bromodomain proteins, without potent and selective ligands available to assist in their study. From lead compounds, a structure-based drug discovery program was to be explored with the use of biophysical assays and appropriate chemical methods to expediate development of probes against a number of these proteins. A fragment lead against BRD4 was developed into PNZ5, a potent (K_D 5 nM) BRD4 probe with a high ligand efficiency. Although enantioselective syntheses and the use of an alternative synthetic route were unsuccessful, PNZ5 showed cytotoxic activity against gastric cancer cell lines that had proved resilient to existing anticancer agents. Optimisation of a lead compound against BRD9 resulted in the development of LP99, the first reported BRD7/9 probe, that was potent (BRD9 K_D 99 nM, BRD7 K_D 909 nM), selective amongst bromodomain proteins and active in cells. An enantioselective synthesis was performed using chiral organocatalyts and LP99 was used to identify a previously unknown role of BRD7/9 in the regulation of inflammatory processes. Research is ongoing to assess further biochemical roles of these proteins with LP99. Arising from a more potent lead against BRD9, a series of structurally related compounds were synthesised to explore SAR around this ligand, however no improvement on the affinity of the lead was realised. Finally, based on disclosed lead structures against PCAF, a series of compounds were synthesised to replicate their activity. A number of important binding interactions were assessed and a lead structure was identified (K_D 1 &mu;M). Development is ongoing to progress this lead into the first reported PCAF probe.

Neste trabalho, avaliou-se o potencial do uso de oxidantes quirais em oxidações enantiosseletivas de compostos orgânicos de boro. É de conhecimento geral que compostos orgânicos de boro, especialmente ésteres e ácidos borônicos são facilmente oxidados por hidroperóxidos em meio básico. No entanto, são escassos na literatura exemplos destas reações de modo enantiosseletivo. A fim de realizar as reações mencionadas, sintetizou-se os hidroperóxidos quirais TADOOH ({(4R,5R)-5-[(hidroperoxidifenil)metil]-2,2-dimetil-1,3-dioxolan-4il}difenilmetanol) e o hidroperóxido quiral derivado de carboidrato, 2,3-dideoxi1-O-oxidanil-4,6-di-O-pivaloil-&#945;-D-eritro-hex-2-enopiranose (di-O-PivOOH). Estes compostos apresentaram resultados interessantes na literatura em oxidações enantiosseletiva de sulfetos orgânicos, em epoxidações de alcenos e em oxidações de Baeyer-Villiger. Inicialmente o potencial oxidativo de ambos hidroperóxidos, bem como a seletividade destes, foi avaliado frente a diversos ésteres borônicos, sendo que somente o TADOOH apresentou resultados promissores. (Ver esquema no PDF) Observou-se uma melhor seletividade do TADOOH frente a ésteres borônicos que possuíam grupos carbonílicos em sua estrutura. Ao submeter o &#946;-boronil-éster, 3-fenil-3-(4,4,5,5-tetrametil-1,3,2-dioxaborolan-2-il)propanoato de etila, à oxidação com o TADOOH em THF utilizando NaOH como base, a -30°C por 1 hora, obteve-se o respectivo álcool com 40% de e.e. Cálculos de DFT para o estado de transição na oxidação dos ésteres borônicos com o TADOOH foram realizados em colaboração com o grupo do Prof. Dr. Ataualpa Albert Carmo Braga. Estes cálculos demonstraram que o estado de transição é estabilizado por uma ligação de hidrogênio não clássica entre o oxigênio da carbonila e umas das ligações C-H dos grupos fenila do TADOOH. Além dos estudos relatados, a reconhecida metodologia de Sharpless na epoxidação assimétrica de alcoóis alílicos foi adaptada para a oxidação enantiosseletiva de ésteres borônicos. Ao trocar o ligante derivado de éster tártarico, normalmente utilizado nas epoxidações de Sharpless, por (-)-efedrina observou-se uma moderada seletividade deste sistema frente ao pinacol l-fenietilboronato. Investigações mais detalhadas demonstraram que a presença do Ti(IV) não era necessária, sendo que a (-)efedrina era a responsável pela ativação e indução quiral nesta reação. / In this work, it was investigated the potential use of chiral oxidants in organic boron compound oxidation. It is known in the literature, that organic boron compounds can be easily oxidized by hydroperoxides. However, an enantioselective approach in literature is scarce. In order to perform these reactions, hydroperoxide TADOOH ({(4R,5R)-5[(hydroperoxydiphenyl)methyl]-2,2-dimethyl-l,3-dioxolan-4-yl}diphenylmethanol) and carbohydrate derived hydroperoxide, 2,3-dideoxy-1-O-oxidanyl-4,6-di-O-pivaloyl-&#945;-D-erythro-hex-2-enopyranose (di-O-PivOOH), have been synthesized. These compounds showed interesting results in several enantioselective oxidations, as like, organic sulfides oxidation, alkenes epoxidation and Baeyer-Villiger oxidations. The oxidative potential of both hydroperoxides, as well as their selectivity, were evaluated against several boronic esters. Only TADOOH has shown promissing results for further studies. (See Scheme on PDF). Boronic esters containing a carbonyl moiety showed better selectivities with TADOOH, for example, the reaction of &#946;-boronyl-ester, ethyl 3-phenyl-3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)propanoate, gave the correponding alcohol with 40% e.e. DFT calculations for the transition state in the oxidation of the boronic esters with TADOOH were carried out in collaboration with the group of Prof. Dr. Ataualpa Albert Carmo Braga. These calculations have shown that the transition state is stabilized by a non-classical hydrogen bond between the carbonyl oxygen and one of the C-H bonds of the TADOOH phenyl groups. In addition to the studies, the well-known Sharpless protocol for asymmetric epoxidation of allylic alcohols was adapted in the enantioselective oxidation of boronic esters. By replacing the tartaric ester-derived, commonly used in the Sharpless experiments, for (-)-ephedrine moderate selectivity was observed with pinacol 1-phenylethyl boronate. Further investigations showed that the presence of Ti (IV) was not necessary, and (-)-ephedrine was responsible for the activation and chiral induction in this reaction.

A oxibutinina é um fármaco quiral empregado na forma de racemato que, após administração por via oral, sofre biotranformação hepática pronunciada levando à formação de N-desetiloxibutinina. Este metabólito apresenta atividade anticolinérgica semelhante a da oxibutinina, contribuindo com o efeito farmacológico e, também, com os efeitos adversos. Em relação às propriedades farmacocinéticas, alguns estudos prévios indicam que a biotransformação é estereosseletiva. Assim, propôs-se o desenvolvimento e validação de um método para análise dos enantiômeros do fármaco e seu metabólito em fração microssomal de fígado de ratos. O método foi desenvolvido empregando a cromatografia líquida de alta eficiência (HPLC) com detecção em 262 nm; a separação dos enantiômeros do fármaco e metabólito foi efetuada em uma coluna Chiralpak AD, empregando hexano: isopropanol: etanol (95:4:1, v/v/v) com 0,3% de dietilamina como fase móvel, na vazão de 0,9 mL min-1. A microextração em fase líquida (LPME) foi empregada como técnica de preparação das amostras e o método foi otimizado empregando planejamento fatorial. A seguinte condição final de extração foi selecionada: tempo de extração de 45 min, nenhuma adição de metanol ou NaCl, agitação da amostra a 4500 rpm, membrana de 6 cm de comprimento, fase doadora em pH 8,0 e ácido trifluoracético 0,1 mol L-1 como fase aceptora. O método mostrou ser linear na faixa de concentração de 312 - 5000 ng mL-1 para os enantiômeros da oxibutinina e 250 - 5000 ng mL-1 para os enantiômeros do metabólito. As recuperações foram de 61 e 55% para a (R)-oxibutinina e (S)-oxibutinina, respectivamente e de 70 e 72% para a (R)-N-desetiloxibutinina e (S)-N-desetiloxibutinina, respectivamente Obteve-se precisão com coeficientes de variação inferiores a 15% e exatidão com erros relativos menores que 15%. O método foi aplicado em um estudo de biotransformação in vitro empregando a fração microssomal de fígado de ratos. As constantes cinéticas foram determinadas e verificou-se uma pequena diferença de afinidade da enzima pelos enantiômeros da oxibutinina (Km= 9,3 nmol L-1 e 7,9 nmol L-1 para a (R)-oxibuinina e a (S)-oxibutinina, respectivamente), com maior afinidade à (S)-oxibutinina em função do menor valor de Km. / Oxybutynin is a chiral drug used as a racemate which, after oral administration, suffers pronounced liver biotransformation leading to the formation of N-desethyloxybutynin. This metabolite shows anticholinergic activity similar to the oxybutynin, contributing to the pharmacological effect and also with the adverse effects. Regarding the pharmacokinetic properties, some studies indicate the stereoselective biotransformation. Thus, it was proposed the development and validation of an enantioselective method for analysis of oxybutynin and its metabolite in rat liver microsomal fraction. The method was developed using high-performance liquid chromatography with detection at 262 nm; the separation of drug and metabolite enantiomers was performed on a Chiralpak AD column employing hexane: isopropanol: ethanol (95:4:1, v/v/v) plus 0.3 % diethylamine as the mobile phase, at a flow rate 0.9 mL min-1. Liquid phase microextraction was used for preparation of the samples and the method was optimized using factorial design; the following condition was established: extraction time of 45 min, no methanol and NaCl in donor phase, agitation of the sample at 4500 rpm, membrane of 6 cm in length, donor phase pH 8.0 and trifluoracetic acid 0.1 mol L-1 as aceptor phase. The method was linear over the range of 312 - 5000 ng mL-1 for oxybutynin enantiomers and over the range of 250 - 5000 ng mL-1 for the metabolite enantiomers. The recoveries were 61 and 55% for (R)-oxybutynin and (S)-oxybutynin, respectively and, for (R)-N-desethyloxybutynin and (S)- N-desethyloxybutynin 70 and 72%, respectively. Within-day and between-day assay precision and accuracy were lower than 15%. The method was applied to an in vitro biotransformation study using rat liver microsomal fraction. The kinetic constants were determined and there was a small difference in affinity of the enzyme for oxybutynin enantiomers (Km=9.3 nmol L-1 and 7.9 nmol L-1 for (R)-oxybutynin and (S)-oxybutynin, respectively), with higher affinity to the (S)-oxybutynin according to the lower value of Km

Neste trabalho, a microextração em fase líquida com membrana cilíndrica oca (HF-LPME), a microextração líquido-líquido dispersiva (DLLME) e a microextração em fase sólida na configuração de um filme delgado (SPME/TFME) foram empregadas como técnicas de microextração para a preparação de amostras microssomais no estudo de metabolismo in vitro de fármacos quirais com diferentes características ácido-base. Para análise empregou-se a cromatografia líquida de alta eficiência (HPLC) com detector por absorção no UV e a cromatografia líquida acoplada à espectrometria de massas (LC-MS-MS). O fármaco neutro isradipina (ISR) foi o primeiro a ser abordado. Um método estereosseletivo empregando HFLPME- HPLC foi desenvolvido para a determinação dos enantiômeros da ISR e seu principal metabólito (um derivado piridínico da isradipina - PDI) em fração microssomal isolada de fígado de ratos. Os analitos foram extraídos de 1 mL de meio microssomal utilizando o procedimento HF-LPME na configuração duas fases, tendo o solvente acetato de hexila como fase aceptora. Pela primeira vez, o PDI e os enantiômeros da ISR foram resolvidos numa mesma corrida cromatográfica. Para esta separação foi utilizada uma coluna Chiralpak AD® e hexano:2-propanol:etanol (94/04/02, v/v/v) como fase móvel, na vazão de 1,5 mL min-1. A ISR e o PDI foram detectados em 325 nm e o padrão interno, oxibutinina, foi detectado em 225 nm. A validação do método mostrou valores de recuperação de 23% para o PDI e 19% para cada enantiômero da ISR, 50 ng mL-1 como limite de quantificação (LOQ) para todos os analitos e linearidade entre 50-5000 ng mL-1 e 50-2500 ng mL-1 para o PDI e cada enantiômero da ISR, respectivamente. O método desenvolvido e validado foi aplicado em um estudo de metabolismo in vitro, utilizando microssomas hepáticos de ratos, mostrando que o (+)-(S)-ISR foi preferencialmente metabolizado. Do mesmo modo que para a ISR, um método analítico empregando HF-LPME-HPLC no modo três-fases foi desenvolvido para a análise estereosseletiva concomitante do bufuralol (BF) e dos seus principais metabólitos 1\'- oxobufuralol (1\'-Oxo-BF) e 1\'-hidroxibufuralol (1\'-OH-BF) em preparações microssomais. As análises por HPLC foram conduzidas empregando uma coluna Chiralcel OD-H® com fase móvel composta por hexano:2-propanol:metanol:dietilamina (97,5/2,0/0,5/0,5, v/v/v/v), na vazão de 1,5 mL min-1 e detecção em 248 nm e 273 nm. As condições otimizadas da HFLPME foram: n-octanol como solvente orgânico, ácido acético 0,2 mol L-1 como fase aceptora, fase doadora com pH ajustado em 13 e agitação de 1500 rpm por 30 min. O método foi validado e apresentou valores de recuperação entre 63 - 69% para os analitos e mostrou-se linear entre 100 - 5000 ng mL-1 para cada enantiômero do 1\'-Oxo-BF e 100 - 2500 ng mL-1 para cada estereoisômero do 1\'-OH-BF (r > 0,99), com LOQ de 100 ng mL-1 para todos os analitos. O método desenvolvido e validado foi aplicado em um estudo de metabolismo in vitro do BF utilizando microssomas hepáticos de ratos, que mostrou formação predominante do (S)-1\'-Oxo-BF e do (R,R)-1\'-OH-BF. Um segundo fármaco básico abordado neste trabalho foi a ranolazina (RNZ). Para a análise enantiosseletiva da RNZ e de um de seus metabólitos (desmetil ranolazina - DRNZ) em fração microssomal isolada de fígado de ratos, foi desenvolvido um método estereosseletivo empregando a DLLME-LC-MS-MS. Os analitos foram extraídos de 0,5 mL de meio microssomal utilizando o procedimento DLLME, tendo o clorofórmio como solvente extrator e acetona como solvente dispersante. Pela primeira vez os enantiômeros da RNZ e DRNZ foram resolvidos numa mesma corrida cromatográfica. ii Para esta separação foi utilizada uma coluna Chiralcel OD-H® e hexano:etanol (60/40, v/v) e 0,05% de dietilamina como fase móvel, na vazão de 1,0 mL min-1. A validação do método mostrou valores de recuperação em torno dos 55 e 45% para os enantiômeros da RNZ e DRNZ, respectivamente. Os LOQ foram de 25 ng mL-1 para cada enantiômero da RNZ e 10 ng mL-1 para cada enantiômero da DRNZ. A linearidade foi estabelecida entre 10-1000 ng mL-1 e 25-2500 ng mL-1 para cada enantiômero da DRNZ e RNZ, respectivamente. O método desenvolvido e validado foi aplicado em um estudo de metabolismo in vitro utilizando microssomas hepáticos de ratos, mostrando que a metabolismo da RNZ foi enantiosseletivo. Finalmente, um método analítico empregando a SPME/TFME-LC-MS-MS foi desenvolvido para a análise simultânea do fármaco anfótero repaglinida (RPG) e dois dos seus principais metabólitos, a 2-despiperiril-2-amino-repaglinida (DA-RPG) e a 2-despiperidil-2-(5- carboxipentilamina)-repaglinida (DC-RPG) em fração microssomal isolada de fígado humano. A SPME/TFME foi conduzida com o auxílio do equipamento Multi Sampler SPME nas seguintes condições: pré-condicionamento dos blades com 1 mL de uma solução metanol:água (50/50, v/v) por 30 min, 60 min de extração e 90 min de dessorção com 1 mL de fase móvel. A análise por LC-MS-MS foi feita empregando uma coluna cromatográfica C18 em modo reverso, com fase móvel composta por acetonitrila:água (50/50, v/v) e 0,1% de ácido acético, na vazão de 0,5 mL min-1. A validação do método mostrou valores de recuperação acima dos 80% para todos os analitos. O LOQ foi de 2 ng mL-1 para todos os analitos, sendo o método linear no intervalo 2-1000 ng mL-1 para a RPG e 2-500 ng mL-1 para a DA-RPG e DC-RPG. Os analitos foram estáveis durante todo o protocolo analítico e de metabolismo. O método validado foi aplicado em um estudo de metabolismo in vitro utilizando microssomas hepáticos de humanos, mostrando que o perfil metabólico da RPG e a taxa de formação da DA-RPG e DC-RPG é alterada em função da concentração de proteínas microssomais no meio de incubação e também em função do tempo de incubação. Além disso, os resultados mostrama possibilidade de haver diversos outros metabólitos que não foram monitorados. / In the present work, the microextraction techniques hollow-fiber liquid phase microextraction (HF-LPME), dispersive liquid-liquid microextraction (DLLME) and solid phase microextraction based on thin film (SPME/TFME) were used as sample preparation techniques to study the in vitro metabolism of chiral drugs with distinct acid-base characteristics. High-performance liquid chromatography (HPLC) with UV detection and liquid chromatography coupled to mass spectrometry (LC-MS-MS) were used for the analyses. An enantioselective liquid chromatographic method using two-phase hollow- fiber liquid-phase microextraction (HF-LPME-HPLC) was developed for the determination of isradipine (ISR) enantiomers and its main metabolite (pyridine derivative of isradipine - PDI) in microsomal fraction isolated from rat liver. The analytes were extracted from 1 mL of microsomal medium using a two-phase HF-LPME procedure with hexyl acetate as the acceptor phase, 30 min of extraction and sample agitation at 1500 rpm. For the first time, ISR enantiomers and PDI were resolved. For this separation, a Chiralpak AD® column with hexane:2-propanol:ethanol (94/04/02, v/v/v) as mobile phase at a flow rate of 1.5 mL min-1 were used. The column was kept at 23 °C ± 2. The drug and metabolite detection was performed at 325 nm and the internal standard oxybutynin was detected at 225 nm. The recovery rates were 23% for PDI and 19% for each ISR enantiomer. The method presented quantification limits (LOQ) of 50 ng mL-1 and it was linear over the concentration range of 50-5000 ng mL-1 and 50-2500 ng mL-1 for PDI and each ISR enantiomer, respectively. The validated method was employed to an in vitro metabolism study of ISR using rat liver microsomal fraction, showing that (+)-(S)-ISR is preferentially metabolized. In the same way, a three-phase HF-LPME-HPLC method for the stereoselective determination of bufuralol metabolites, 1\'-oxobufuralol (1\'-Oxo-BF) and 1\'-hydroxybufuralol (1\'-OH-BF), in microsomal preparations is described for the first time. The HPLC analysis was carried out using a Chiralcel OD-H® column with hexane:2-propanol:methanol (97.5/2.0/0.5, v/v/v) plus 0.5% diethylamine as the mobile phase, and UV detection at 248 and 273 nm. The HF-LPME optimized conditions involved: n-octanol as the organic solvent, 0.2 mol L-1 acetic acid as the acceptor phase, donor phase pH adjusted to 13, sample agitation at 1500 rpm and extraction for 30 min. By using this extraction procedure, the recovery rates were in the range of 63- 69%. The method was linear over the concentration range of 100-5000 ng mL-1 for each enantiomer of 1\'-Oxo-BF and of 100-2500 ng mL-1 for each stereoisomer of 1\'-OH-BF. The quantification limits were 100 ng mL-1 for all analytes. The validated method was used to assess the in vitro metabolism of bufuralol using rat liver microsomal fraction that demonstrated predominant formation of (S)-1\'-Oxo-BF and (R,R)-1\'-OH-BF. A second basic drug addressed in this thesis is ranolazine (RNZ). For the chiral analysis of RNZ and one of its metabolites (desmethyl ranolazine - DRNZ) in microsomal fraction isolated from rat liver, an analytical enantioselective method using DLLME-LC-MS-MS was developed. The analytes were extracted from 0.5 mL of microsomal medium by DLLME. Chloroform was the extractor solvent and acetone the dispersive solvent. The enantiomers of RNZ and DRNZ were analyzed simultaneously for the first time using a Chiralcel OD-H® column and hexane:ethanol (60/40, v/v) plus 0.05% diethylamine as mobile phase at a flow rate of 1.0 mL min-1. Method validation showed recoveries in the order of 55 and 45% for the enantiomers iv of RNZ and DRNZ, respectively. The LOQs were 25 ng mL-1 for each RNZ enantiomers and 10 ng mL-1 for each DRNZ enantiomers. Linearity was established between 10-1000 ng mL-1 and 25-2500 ng mL-1 for each DRNZ and RNZ enantiomers, respectively. The validated method was employed to an in vitro metabolism study of RNZ using rat liver microsomal fraction, showing that the metabolism of RNZ is enantioselective. Finally, an analytical method was developed employing SPME/TFME-LC-MS-MS for the simultaneous analyses of the anphoteric drug repaglinide (RPG) and two of the its main metabolites 2-despiperidyl- 2-amino repaglinide (DA-RPG) and 2-despiperidyl-2-(5-carboxypentylamine) repaglinide (DC-RPG) in human microsomal fraction. The SPME/TFME procedure was carried out with the support of \"Multi Sampler SPME\" under the following conditions: conditioning of the blades with 1 mL of methanol:water (50/50, v/v) for 30 min, 60 min for extraction and 90 min for desorption with 1 mL of mobile phase. The LC-MS-MS analyses were performed using a C18 column under reversed phase conditions, with the mobile phase of acetonitrile:water (50/50, v/v) plus 0.1% acetic acid at a flow rate of 0.5 mL min-1. The method validation showed recoveries over 80% for all analytes. The LOQ was 2 ng mL-1 for all analytes, and the method was linear over the concentration range of 2 e 1000 ng mL-1 for RPG and of 2 a 500 ng mL-1 for DA-RPG and DC-RPG. The validated method was used to assess the in vitro metabolism profile of RPG by using human liver microsomes. These studies showed that the rate of formation of DA-RPG and DC-RPG depends on both microsomal protein concentration in the incubation medium and the incubation time. Furthermore, the results highlighted the possibility of formation of several other metabolites which have not been monitored.

A fexofenadina (FEX) tem sido o fármaco de primeira escolha no tratamento sintomático de manifestações alérgicas, por ser um anti-histamínico dos receptores H1 de 2ª geração não sedativo. É o metabólito ativo e quiral da terfenadina (TERF), medicamento cuja produção e comercialização foram suspensas em função dos eventos adversos apresentados. Fungos têm se apresentado como uma alternativa promissora na produção de compostos com atividade biológica. Dessa forma, o objetivo desse projeto foi avaliar a capacidade de fungos em biotransformar enantiosseletivamente a terfenadina em seu metabólito ativo, a fexofenadina empregando fungos como agentes catalisadores. Para a análise enantiosseletiva da fexofenadina foi desenvolvido um método de separação cromatográfica empregando a coluna quiral Lux® cellulose-1, fase móvel constituída de água: metanol (35:65, v/v) + 0,3% trietilamina + 0,4% ácido acético, vazão de 0,5 mL min-1, com detecção em 220nm. Duas microtécnicas de preparação de amostras foram avaliadas na extração dos analitos do meio de cultura: a microextração liquido-liquido dispersiva (DLLME) e a microextração em fase liquida empregando membranas cilíndricas ocas (HF-LPME). Entre essas, a DLLME foi a microtécnica de escolha, pois forneceu melhores resultados tais como, maior valor de recuperação, cromatogramas sem picos de possíveis interferentes, maior rapidez e facilidade de preparação das amostras. As condições otimizadas da DLLME foram: clorofórmio (300 ?L) como solvente extrator, isopropanol (300 ?L) como solvente dispersor. Após a formação do ponto nuvem, as amostras foram submetidas à agitação por vórtex durante 15 segundos e centrifugação durante 10 minutos a 3000 rpm. As recuperações foram de 43% para ambos enantiômeros. O método se mostrou linear na faixa de concentração 2.0 - 15.0 ?g mL-1 para cada enantiômero da FEX (r > 0,990). O limite de quantificação foi de 2 ?g mL-1 para os enantiômeros da FEX. Dentre os sete fungos estudados (Papulaspora immersa Hotson SS13, Penicillium crustosum VR4, Mucor rouxii, Nigrospora sphaerica SS67, Fusarium oxysporum SS50, Cunninghamella echinulata var. elegans ATCC 8688A e Cunninghamella elegans NRRL 1393 ATCC 10028B) somente o fungo Fusarium oxysporum SS50 e Cunninghamella echinulata var. elegans ATCC 8688A apresentaram potencial para biotransformação da terfenadina em fexofenadina nas condições de incubação empregadas nesse trabalho. / Fexofenadine (FEX) has been the drug of choice for the symptomatic treatment of allergic manifestations, being an antihistamine H1 receptor 2nd generation non-sedating. It is the active and chiral metabolite of terfenadine (TERF), a drug whose production and marketing was suspended as a result of adverse events. Fungi have been presented as a promising alternative for the production of compounds with biological activity. Thus, the goal of this project was to evaluate the ability of fungi to biotransform asymmetric terfenadine to its active metabolite, fexofenadine using fungi as agents catalysts. For enantioselective analysis of fexofenadine a method for chromatographic separation was developed employing a chiral column Lux® cellulose -1, mobile phase water : methanol (35:65,v/v) + 0.3% triethylamine + 0.4% acetic acid, flow rate of 0.5 mL min-1, with detection at 220nm. Two sample preparation microtechnology were evaluated in the extraction of analytes from the culture medium: the dispersive liquid-liquid microextraction (DLLME) and hollow fiber liquid phase microextraction (HF- LPME). Between the two, the DLLME was the microtechnic chosen because it provided better results such as higher recovery values, chromatograms with no possible interfering peaks, greater speed and ease of sample preparation. The optimized conditions of DLLME were: chloroform (300 ?L) as extractor solvent, isopropanol (300 ?L) as disperser solvent. After the formation of the cloud point, the samples were subjected to agitation by vortexing for 15 seconds and centrifuging for 10 minutes at 3000 rpm. The recoveries were 43 % for both enantiomers. The method was linear in the concentration range from 2.0 -15.0 ?g mL-1 for each enantiomer of FEX (r > 0.990). The limit of quantification was 2 ?g mL-1 for the enantiomers of FEX. Among the seven fungi studied (Papulaspora immersa Hotson SS13, Penicillium crustosum VR4, Mucor rouxii, Nigrospora sphaerica SS67, Fusarium oxysporum SS50, Cunninghamella echinulata var. elegans ATCC 8688A e Cunninghamella elegans NRRL 1393 ATCC 10028B), only the fungi Fusarium oxysporum SS50 e Cunninghamella echinulata var. elegans ATCC 8688A showed potential for biotransformation of terfenadine in fexofenadine in the incubation conditions employed in this work.

Zopiclona (ZO) é um hipnótico não-benzodiazepínico da classe ciclopirrolonas, indicada para o tratamento da insônia. A ZO é um fármaco quiral administrada como uma mistura racêmica; no entanto, a sua atividade farmacológica está principalmente relacionada com o enatiômero (+)-(S)-ZO, também conhecido como eszopiclona. A ZO é extensivamente metabolizada e os metabólitos principais são a N-desmetil zopiclona (N-Des) e zopiclona-N-óxido (N-Ox). A N-Ox também é uma impureza encontrada na matéria prima. Além dessa impureza, outras oriundas do processo de síntese ou devido à degradação também podem ser encontradas: impureza B (RP29307), impureza C (2-amino-5-cloropiridina, ACP ou RP26695) e RP 48497. Sendo assim, o objetivo desse estudo foi desenvolver métodos de análise enantiosseletiva da ZO, metabólitos e impurezas em formulações farmacêuticas e materiais biológicos. Um método empregando a cromatografia líquida de alta eficiência com detecção por espectrometria de massas (LC-MS-MS) foi desenvolvido e validado para a quantificação simultânea da ZO e de seus metabolitos em plasma de ratos. Os analitos foram isolados das amostras por extração líquido-líquido e separados na coluna CHIRALPAK AD-RH, empregando a fase móvel constituída por etanol:metanol:acetonitrila (50:45:5, v/v/v) mais 0,025 % de dietilamina, na vazão de 1,0 mL min-1. A moclobemida foi utilizada como padrão interno. O método desenvolvido foi linear no intervalo de concentração plasmática de 7,5-500 ng mL-1. As recuperações médias absolutas foram de 74,6 e 75,7; 61,6 e 56,9; 72,5 e 70,7 % para os enantiômeros da ZO, N-Des e N-Ox, respectivamente, e 75,9 % para o padrão interno. A precisão e a exatidão apresentaram resultados dentro de níveis aceitáveis (<15 %). A aplicação do método em um estudo piloto de disposição cinética da ZO em ratos mostrou que os níveis de (+)-(S)-ZO foram sempre superiores aos de (-)-(R)-ZO. Concentrações mais elevadas também foram observadas para (+)-(S)-N-Des e (+)-(S)-N-Ox, confirmando a disposição cinética estereosselectiva da ZO. Outro método desenvolvido utilizando LC-MS-MS permitiu a determinação da ZO no cérebro de ratos. O tratamento das amostras foi realizado empregando a extração em fase sólida, obtendo-se recuperações elevadas de 89,6 e 91,7 % para cada enantiômero. Os enantiômeros foram separados em uma coluna CHIRALPAK AD, com a fase móvel constituída por acetonitrila:etanol:metanol (60:20:20, v/v/v), na vazão de 1,3 mL min-1. A moclobemida também foi utilizada como padrão interno. O método validado mostrou linearidade no intervalo de 0,29-344,8 ng g-1, com limite de quantificação de 0,29 ng g-1. Pôde-se observar que os níveis de (+)-(S)-ZO, o enantiômero mais ativo, foram sempre superiores aos de (-)-(R)-ZO. Finalmente, um terceiro método foi desenvolvido para análise enantiosseletiva da ZO e das suas impurezas N-Ox e ACP em comprimidos, empregando a eletroforese capilar com detecção UV (CE-UV). Os analitos foram extraídos dos comprimidos utilizando acetonitrila e foram separados em um capilar não revestido de sílica fundida (50 um, 42 cm de comprimento efetivo, 50 cm de comprimento total), utilizando tampão fosfato de sódio 80 mmol L-1, pH 2,5, contendo 5 mmol L-1 de ?-ciclodextrina carboximetilada. Os analitos e o padrão ii interno (trimetropina) foram detectados em 305 e 200 nm, respectivamente. Uma tensão de 27 kV foi aplicada e a temperatura do capilar foi mantida em 25 °C. Todos os analitos foram analisados em até 8 min e as curvas analíticas foram lineares no intervalo de concentração de 0,4-0,8 mg mL-1 para cada enantiômero da ZO, 0,8-1,6 ?g mL-1 para ACP e 0,4-0,8 ?g-1 mL para cada enantiômero da N-Ox. Os coeficientes de variação e erros relativos obtidos na avaliação da precisão e exatidão foram inferiores a 2% para todos os analitos. Este método validado foi utilizado para estudar a degradação e racemização da ZO sob condições de stress. As duas impurezas avaliadas foram formadas nas condições de estresse mas a racemização não foi observada. / Zopiclone (ZO) is a non-benzodiazepine hypnotic drug of the cyclopyrrolone class, indicated for the treatment of insomnia. ZO is a chiral drug administered as a racemic mixture, however its pharmacological activity is mainly related to (+)-(S)-ZO, also known as eszopiclone. It is extensively metabolized and the main metabolites are N-desmethyl zopiclone (N-Des) and zopiclone-N-oxide (N-OX). N-Ox is also found as an impurity in the raw material. Other impurities, coming from the synthetic procedure or due to degradation can also be found: impurity B (RP29307), impurity C (2-amine-5-chloropyridine, ACP or RP26695) and RP 48497. Therefore, the aim of this study was the development of methods for the enantioselective analysis of ZO, metabolites and impurities in pharmaceutical formulations and biological materials. A high-performance liquid chromatographic method with triple quadrupole mass spectrometry detection (LC-MS-MS) was developed and validated for the simultaneous quantification of ZO and its metabolites in rat plasma samples. The analytes were isolated from rat plasma by liquid-liquid extraction and separated using a CHIRALPAK AD-RH column, and ethanol:methanol:acetonitrile (50:45:5, v/v/v) plus 0.025 % diethylamine as mobile phase, at a flow-rate of 1.0 mL min-1. Moclobemide was used as internal standard. The developed method was linear over the concentration range of 7.5-500 ng mL-1. The mean absolute recoveries were 74.6 and 75.7; 61.6 and 56.9; 72.5 and 70.7 % for ZO enantiomers, for N-Des enantiomers and for N-Ox enantiomers, respectively, and 75.9 % for the internal standard. Precision and accuracy were within acceptable levels of confidence (<15 %). Method application in a pilot study of ZO kinetic disposition in rats showed that the levels of (+)-(S)-ZO were always higher than those of (-)-(R)-ZO. Higher concentrations were also observed for (+)-(S)-N-Des and (+)-(S)-N-Ox. Another method was developed for the determination of ZO in rat brain using LC-MS-MS. The sample treatment procedure was carried out employing solid-phase extraction yielding recoveries of 89.6 and 91.7 % for each ZO enantiomer. The ZO enantiomers were resolved on a CHIRALPAK AD column with a mobile phase consisting of acetonitrile:ethanol:methanol (60:20:20, v/v/v) at a flow rate of 1.3 mL min-1. Moclobemide was used as internal standard. The validated method showed linearity in the range of 0.29 - 344.8 ng g-1, with quantification limit of 0.29 ng g-1. It could be observed that the levels of (+)-(S)-ZO were always higher than those of (-)-(R)-ZO. Finally, a third method was developed for the enantioselective analysis of ZO and its impurities N-Ox and ACP in tablets using capillary electrophoresis with UV detection (CE-UV). The analytes were extracted from the tablets using acetonitrile and were separated in an uncoated fused-silica capillary (50 ?m, 42 cm effective length, 50 cm total length) using 80 mmol L-1 sodium phosphate buffer, pH 2.5, and 5 mmol L-1 carboxymethyl-?-cyclodextrin as running buffer. The analytes and the internal standard (trimethoprim) were detected at 305 and 200 nm, respectively. A tension of 27 kV was applied and the capillary temperature was maintained at 25 ºC. All analytes were analyzed within 8 min and linear calibration curves over the iv concentration range of 0.4-0.8 mg mL-1 for each ZO enantiomer, 0.8-1.6 ?g mL-1 for ACP and 0.4-0.8 ?g mL-1 for each N-Ox enantiomer were obtained. The coefficients of correlation obtained for the linear curves were greater than 0.99. The intra-day and inter-day accuracy and precision were lower than 2% for all analytes. This validated method was employed to study the degradation and racemization of ZO under stress conditions. The results obtained showed that ACP and N-Ox were both formed under the stress conditions used, but racemization was not observed.