The f)-lactam or azetidinone 4-membered heterocyclic nng system has been synthesised by many different synthetic strategies and these are well documented in the literature. Many of these strategies give substituted azetidinone rings displaying defined stereochemistry which are used as the building block for many modern day antibiotics. It was therefore intended to pursue a method of azetidinone ring formation using cheap and readily available starting materials employing a [2+2] cycloaddition reaction which, according to the literature, had only a few examples and was seen to be an area ripe for research. Taking urethane in anhydrous THF and metalating with n-butyl lithium, followed by the subsequent addition of phenyl vinyl sulfone, it was envisaged that the two would react in such a way to give the -l-membered azetidinone ring. It was found however that the carbon-nitrogen bond was formed but subsequent cyclisation did not follow and therefore no azetidinone was formed. Although azetidinone ring was not detected the novel compound N,N-bis-(phenylsulfonylethyl) urethane was isolated. This compound was of interest since it was hoped that it could provide a new route to substituted pyridines. Various methods were undertaken in an attempt to cyclise this molecule via the generation of a-sulfonyl carbanions and reactions with a variety of electrophiles. During our experiments no cyclised products were detected. TIle replacement of phenyl sulfonyl group with triphenylphosphonium bromide provided a new hope for azetidinone synthesis since its ability to stabilise a-sulfonyl carbanions and promote reaction with electrophiles was well established. Experiments of this nature failed to produce an azetidinone. TIle next stage of research was to investigate a new route to the production of quinolone antibiotics. Quinolones, particularly those which are furnished with a fluorine at C-6 position, are the new generation of antibiotics which exhibit a high activity and selectivity towards pathogens which have become resistant to older antibiotics such as penicillin. Needless to say their importance in the pharmaceutical industry has increased dramatically over recent years and research into the improvement of synthetic strategies towards the total synthesis of these molecules has attracted much commercial interest. Meth-Cohn showed that quinolones can be formed in excellent yield by the reaction of N-alkylformanilides in phosphorus oxychloride and activated acid chlorides e.g. methyl malonyl chloride. This approach is examined regarding the synthesis of both novel and known quinolone antibiotics (norfloxacin and ciprofloxacin). Work on this topic has produced encouraging results with the synthesis of important antibacterial intermediates.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O perfil farmacocinÃtico de duas diferentes formulaÃÃes de norfloxacino - comprimido revestido de 400 mg - como formulaÃÃo teste versus a formulaÃÃo referÃncia, Floxacin comprimido revestido de 400 mg (Merck Sharp & Dohme) foi avaliado em 32 voluntÃrios saudÃveis de ambos os sexos. A pesquisa consistiu de um estudo aberto, aleatorizado, cruzado, com 2 tratamentos, 2 perÃodos (duas sequÃncias) com intervalo mÃnimo de sete dias entre os internamentos. Amostras de sangue foram coletas em intervalos regulares para construÃÃo das curvas de concentraÃÃo plasmÃtica versus tempo. As concentraÃÃes de norfloxacino foram analisadas por Cromatografia LÃquida de Alta EficiÃncia acoplada à Espectrometria de Massa (LC-MS/MS). A mÃdia geomÃtrica do Norfloxacino teste/Norfloxacino referÃncia e o intervalo de confianÃa (IC) de 90% para Cmax e ASCo-t Ãltimo foram 103,9020% (89,5247-120,5881%) e 99,3030% (87,8186-112,2892%) respectivamente. Considerando que as formulaÃÃes estudadas apresentaram razÃo entre as mÃdias de Cmax e ASC0-tÃltimo entre 80 e 125%, e considerando um intervalo de confianÃa de 90% como proposto pelo Food and Drug Administration e ANVISA, conclui-se que as duas formulaÃÃes de norfloxacino apresentaram biodisponibilidades semelhantes tanto na taxa quanto na extensÃo de absorÃÃo. / The pharmacokinetic profile of two different formulations of Norfloxacin tablets - 400 mg; as test formulation versus FloxacinÂ, Norfloxacin reference formulation (- 400 mg; Merck Sharp & Dohme) was evaluated in 32 healthy volunteers of both sexes. The research was consisted of an open, randomized, crossover study, designed with two treatments, two periods (two sequences) with a minimum interval of seven days (washout) between the admissions. Blood samples were collected at regular intervals to construct the plasma concentration versus time curves. The concentrations of Norfloxacin were analyzed by High Performance Liquid Chromatography coupled to Mass Spectrometry (HPLC-MS/MS). The geometric mean of test Norfloxacin / reference Norfloxacin and confidence interval (CI) of 90% for Cmax and AUC0-t last were 103.9020% (89.5247 to 120.5881%) and 99.3030% (87.8186 - 112.2892%) respectively. Whereas the studied formulations showed the ratio between the mean Cmax and AUC0-t last between 80 and 125%, considering a confidence interval of 90%, as proposed by the Food and Drug Administration and ANVISA, it appears that the two formulations of Norfloxacin showed similar bioavailability in rate and in extent of absorption.

Orientador: Gilbert Bannach / Banca: Adriano Buzutti de Siqueira / Banca: Luiz Carlos da Silva Filho / Resumo: Cocristais farmacêuticos podem ser definidos como sólidos cristalinos multicomponentes (dois ou mais componentes), sendo estes um API (do inglês "active pharmaceutical ingredients", ou seja, uma molécula com atividade farmacológica) e um coformador, os quais são unidos por interações não covalentes, mais comumente ligações de hidrogênio. O coformador deve ser uma molécula segura para consumo (uma vitaminas, um antioxidantes ou outro API, por exemplo) e deve conter grupos funcionais que possibilitem a interação com o API. Os cocristais são um artificio eficiente para solucionar problemas de solubilidade encontrados nos fármacos classes II e IV (segundo o Sistema de Classificação Biofarmacêutica). O método mecanoquímico permite reprodutibilidade na síntese dos cocristais. Neste trabalho realizou-se a síntese mecanoquímica do API Curcumina com os coformadores ácido ascórbico, ácido cítrico, cafeína e sacarina, e do API Norfloxacino com os coformadores riboflavina, ácido salicílico e acido succínico. Observou-se que a riboflavina apresenta comportamento térmico diferente do relatado na literatura, mostrando inicialmente uma etapa de decomposição térmica, seguido de um evento endotérmico referente à fusão, eventos confirmados por imagens do DSC-Fotovisual. O Norfloxacino sofre influencia pelo processo de moagem, a qual induz a transferência de prótons e interação do composto com moléculas de água, condição característica do norfloxacino, já relatado na literatura. O Norfloxacino f... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Pharmaceutical cocrystals can be defined as multicomponent crystalline solids (two or more components), these being an active pharmaceutical ingredients (API) and a coformer, which are joined by non-covalent interactions, most commonly hydrogen bonds. The coformer must be a safe molecule for consumption and must contain functional groups that allow interaction with the API. Cocrystals are an efficient device for solving solubility problems found in Class II and IV drugs (according to the Biopharmaceutical Classification System). The mechanochemical method allows reproducibility in the synthesis of cocrystals. In this work the mechanochemical synthesis of API Curcumina with the ascorbic acid, citric acid, caffeine and saccharin coformers and the Norfloxacino API with riboflavin, salicylic acid and succinic acid coformers were performed. It was observed that riboflavin exhibits thermal behavior different from that reported in the literature, initially showing a thermal decomposition stage, followed by an endothermic event related to the fusion, events confirmed by DSC-Photovisual images. Norfloxacin is influenced by the milling process, which induces the proton transfer and interaction of the compound with water molecules, a characteristic condition of norfloxacin, already reported in the literature. Norfloxacin formed cocrystals with the riboflavin, salicylic acid and succinic acid coformers, all in 1:1 proportions by mechanochemical method, solvent-assisted grinding using 15 uL of chloroform solvent. Curcumin did not form cocrystals with the coformers studied, however curcumin formed physical mixtures with the ascorbic acid coformator, eutectic compounds with excess reagents with the saccharin coformer, as well as the caffeine coformer, and physical mixtures and a eutectic compound such as citric acid coformer, all by mechanochemical method using 15 uL of solvent ethanol. / Mestre

Shiga Toxin produzierende Escherichia coli (STEC) sind wichtige Erreger von Lebensmittelinfektionen und gelten als Hauptverursacher für die Ausbildung einer hämorrhagischen Colitis und dem lebensbedrohlichen hämolytisch-urämischen Syndrom. Als Hauptvirulenzfaktor und wichtigstes Charakteristikum der STEC wird die Fähigkeit angesehen, Shiga Toxine (Stx) zu produzieren. Die genetische Information für deren Produktion ist im Genom lambdoider Prophagen kodiert. Eine Antibiotikatherapie bei STEC-assoziierten Infektionen wird sehr kontrovers diskutiert, da sowohl die Produktion der Stx als auch die Freisetzung der Bakteriophagen in vitro durch antibiotisch wirkende Substanzen stimuliert werden kann. Der enterohämorrhagische E. coli O157:H7 Stamm EDL933 beherbergt insgesamt 18 lambdoide Prophagen und prophagenähnliche Elemente, darunter den Stx1-kodierenden Phagen CP-933V und den Stx2-kodierenden Phagen BP-933W. Ziel der vorliegenden Arbeit war es, den Einfluss des Gyrasehemmers Norfloxacin auf das Gesamttranskriptom von EDL933 mit Hilfe der DNA-Microarraytechnologie und unter Verwendung des MWG E. coli O157 Arrays umfassend zu untersuchen und insbesondere die Expression der Prophagengene zu analysieren. Hierfür wurde der E. coli O157:H7 Stamm EDL933 mit 200 ng/ml Norfloxacin inkubiert, Gesamt-RNA isoliert und diese mittels reverser Transkription in cDNA synthetisiert, wobei ein Einbau von fluoreszenzmarkierten Nukleotiden erfolgte. Diese cDNA wurde mit den auf dem E. coli O157 Array befindlichen Oligonukleotiden hybridisiert. Die Auswertung der Fluoreszenzintensitäten ermöglicht eine Analyse der Genexpression. Infolge der Induktion von EDL933 mit 200 ng/ml Norfloxacin zeigten 118 Spots eine Hochregulation und 122 eine Deregulation von Genen an. Bei genauerer Betrachtung resultierte auf Grund der Inkubation mit Norfloxacin eine erhöhte Expression von 52 Genen der Stx-kodierenden Phagen CP-933V und BP-933W. Insbesondere erfolgte eine erhöhte Regulation von Genen der späten Region des BP-933W. Das stxA2-Gen wurde dabei im Vergleich zur nicht-induzierten Kultur 158-fach stärker exprimiert. Im Falle des Stx1-Phagen wiesen nur einige Gene der frühen Region eine gesteigerte Genaktivität auf. Auffallend war die Hochregulation einzelner Gene der nicht Stx-kodierenden Phagen. Gene des Primärstoffwechsels, u. a. Gene der Aminosäurebiosynthese, des Energiehaushaltes und der Zellteilung zeigten eine verminderte Genaktivität nach Induktion. Diese Ergebnisse weisen darauf hin, dass die verwendete Konzentration von Norfloxacin große Auswirkungen auf das Gesamttranskriptom des untersuchten Stammes haben und insbesondere die Genexpression von zehn im Genom des EDL933 befindlichen Prophagen erhöht wurde. Die durch die Microarrayversuche erhaltenen Expressionsraten wurden durch Quantifizierung der cDNA mittels RT Real-Time PCR Untersuchungen überprüft. Zusammenfassend ist festzustellen, dass Norfloxacin neben der antibiotischen Wirkung in der hier verwendeten geringen Konzentration multiple Effekte auf E. coli O157:H7 ausübt und die Auswirkungen in Zukunft noch detaillierter untersucht werden müssen. Die DNA-Microarraytechnologie und der kommerziell erhältliche E. coli O157 Array ermöglichen diese umfassenden Analyse des Transkriptionsprofils. Im Rahmen dieser Arbeit konnte diese Technologie für Untersuchungen der Genexpression von E. coli O157 etabliert und validiert werden. / Infection with Shiga toxin producing Escherichia coli (STEC) is a serious cause of bloody diarrhea and sporadic cases and outbreaks of food-related diseases such hemorrhagic colitis and the hemolytic uremic syndrome (HUS) worldwide. The use of antibiotics in therapy of STEC-associated diseases has been discussed controversially. Release of phage-encoded Shiga toxins is the major virulence factor of enterhemorrhagic Escherichia coli (EHEC). The genome of the EHEC strain E. coli O157:H7 EDL933 contains 18 prophages or prophages elements, including the Stx1- and Stx2-encoding phages CP-933V and BP-933W. Stx-production and Stx-prophage induction can be stimulated by certain antibiotics, e.g. mitomycinC or UV light. The aim of this study was to investigate the influence of a low concentration of the gyrase inhibitor norfloxacin on the whole transcriptom of E. coli O157:H7 strain EDL933 and particularly on the gene expression of prophages using the DNA-microarraytechnology and the commercial available MWG E. coli O157 Array. To determine this, E. coli O157:H7 cultures were incubated with 200 ng/ml norfloxacin. Total RNA was isolated and labelled with fluorescence dyes during reverse transcription. Following this, the labelled cDNA was hybridized with the commercial E. coli O157 Arrays and the fluorescence intensities were measured, analysed and evaluated with appropriate software. Results of this study have indicated that a low concentration of norfloxacin have profound effects on the trancriptome of E. coli O157:H7. Under the conditions applied (200 ng/ml norfloxacin) and an incubation time of 120 minutes, 118 spots indicated a transcriptional upregulation and 122 spots a transcriptional downregulation of E. coli O157:H7 genes present on the array. In detail, 85 spots could be ascribed to EDL933 phage genes. Fifty-two of them could be assigned to the Shiga toxin encoding phages CP-933V and BP-933W, the others belonged to the non-Stx encoding phages or prophages elements existing in the EDL933 genome. Conspicuous, genes present in the late region of the BP-933W prophage were induced most strongly, up to 158-fold in the case of stxA2. Only some genes present in the early region of the Stx1 encoding phage CP-933V were induced upon induction with norfloxacin. Notably, only some genes of the non-Stx phages of EDL933 appeared to be induced after induction. The additional upregulated genes were related to recombination and stress functions and to E. coli O157:H7 RIMD0509952 genes. The majority of downregulated genes belonging to primary metabolism, such as amino acid biosynthesis, cell division and energy metabolism. In conclusion, an induction of E. coli O157:H7 strain EDL933 with 200 ng/ml norfloxacin has profound effects on the transcriptome of E. coli O157:H7, in particular on the global gene expression of more then ten prophages. The DNA-microarray technology and especially the E. coli O157 Array offer a modern tool for analysis of transcription profiles of the serious pathogen EHEC O157 in response to stress, e.g. antibiotics. In the context of this work, the DNA-microarraytechnology could be established and validated to provide the opportunity for further studies about the global effects on the whole transcriptome of E. coli O157.

Shiga Toxin produzierende Escherichia coli (STEC) sind wichtige Erreger von Lebensmittelinfektionen und gelten als Hauptverursacher für die Ausbildung einer hämorrhagischen Colitis und dem lebensbedrohlichen hämolytisch-urämischen Syndrom. Als Hauptvirulenzfaktor und wichtigstes Charakteristikum der STEC wird die Fähigkeit angesehen, Shiga Toxine (Stx) zu produzieren. Die genetische Information für deren Produktion ist im Genom lambdoider Prophagen kodiert. Eine Antibiotikatherapie bei STEC-assoziierten Infektionen wird sehr kontrovers diskutiert, da sowohl die Produktion der Stx als auch die Freisetzung der Bakteriophagen in vitro durch antibiotisch wirkende Substanzen stimuliert werden kann. Der enterohämorrhagische E. coli O157:H7 Stamm EDL933 beherbergt insgesamt 18 lambdoide Prophagen und prophagenähnliche Elemente, darunter den Stx1-kodierenden Phagen CP-933V und den Stx2-kodierenden Phagen BP-933W. Ziel der vorliegenden Arbeit war es, den Einfluss des Gyrasehemmers Norfloxacin auf das Gesamttranskriptom von EDL933 mit Hilfe der DNA-Microarraytechnologie und unter Verwendung des MWG E. coli O157 Arrays umfassend zu untersuchen und insbesondere die Expression der Prophagengene zu analysieren. Hierfür wurde der E. coli O157:H7 Stamm EDL933 mit 200 ng/ml Norfloxacin inkubiert, Gesamt-RNA isoliert und diese mittels reverser Transkription in cDNA synthetisiert, wobei ein Einbau von fluoreszenzmarkierten Nukleotiden erfolgte. Diese cDNA wurde mit den auf dem E. coli O157 Array befindlichen Oligonukleotiden hybridisiert. Die Auswertung der Fluoreszenzintensitäten ermöglicht eine Analyse der Genexpression. Infolge der Induktion von EDL933 mit 200 ng/ml Norfloxacin zeigten 118 Spots eine Hochregulation und 122 eine Deregulation von Genen an. Bei genauerer Betrachtung resultierte auf Grund der Inkubation mit Norfloxacin eine erhöhte Expression von 52 Genen der Stx-kodierenden Phagen CP-933V und BP-933W. Insbesondere erfolgte eine erhöhte Regulation von Genen der späten Region des BP-933W. Das stxA2-Gen wurde dabei im Vergleich zur nicht-induzierten Kultur 158-fach stärker exprimiert. Im Falle des Stx1-Phagen wiesen nur einige Gene der frühen Region eine gesteigerte Genaktivität auf. Auffallend war die Hochregulation einzelner Gene der nicht Stx-kodierenden Phagen. Gene des Primärstoffwechsels, u. a. Gene der Aminosäurebiosynthese, des Energiehaushaltes und der Zellteilung zeigten eine verminderte Genaktivität nach Induktion. Diese Ergebnisse weisen darauf hin, dass die verwendete Konzentration von Norfloxacin große Auswirkungen auf das Gesamttranskriptom des untersuchten Stammes haben und insbesondere die Genexpression von zehn im Genom des EDL933 befindlichen Prophagen erhöht wurde. Die durch die Microarrayversuche erhaltenen Expressionsraten wurden durch Quantifizierung der cDNA mittels RT Real-Time PCR Untersuchungen überprüft. Zusammenfassend ist festzustellen, dass Norfloxacin neben der antibiotischen Wirkung in der hier verwendeten geringen Konzentration multiple Effekte auf E. coli O157:H7 ausübt und die Auswirkungen in Zukunft noch detaillierter untersucht werden müssen. Die DNA-Microarraytechnologie und der kommerziell erhältliche E. coli O157 Array ermöglichen diese umfassenden Analyse des Transkriptionsprofils. Im Rahmen dieser Arbeit konnte diese Technologie für Untersuchungen der Genexpression von E. coli O157 etabliert und validiert werden. / Infection with Shiga toxin producing Escherichia coli (STEC) is a serious cause of bloody diarrhea and sporadic cases and outbreaks of food-related diseases such hemorrhagic colitis and the hemolytic uremic syndrome (HUS) worldwide. The use of antibiotics in therapy of STEC-associated diseases has been discussed controversially. Release of phage-encoded Shiga toxins is the major virulence factor of enterhemorrhagic Escherichia coli (EHEC). The genome of the EHEC strain E. coli O157:H7 EDL933 contains 18 prophages or prophages elements, including the Stx1- and Stx2-encoding phages CP-933V and BP-933W. Stx-production and Stx-prophage induction can be stimulated by certain antibiotics, e.g. mitomycinC or UV light. The aim of this study was to investigate the influence of a low concentration of the gyrase inhibitor norfloxacin on the whole transcriptom of E. coli O157:H7 strain EDL933 and particularly on the gene expression of prophages using the DNA-microarraytechnology and the commercial available MWG E. coli O157 Array. To determine this, E. coli O157:H7 cultures were incubated with 200 ng/ml norfloxacin. Total RNA was isolated and labelled with fluorescence dyes during reverse transcription. Following this, the labelled cDNA was hybridized with the commercial E. coli O157 Arrays and the fluorescence intensities were measured, analysed and evaluated with appropriate software. Results of this study have indicated that a low concentration of norfloxacin have profound effects on the trancriptome of E. coli O157:H7. Under the conditions applied (200 ng/ml norfloxacin) and an incubation time of 120 minutes, 118 spots indicated a transcriptional upregulation and 122 spots a transcriptional downregulation of E. coli O157:H7 genes present on the array. In detail, 85 spots could be ascribed to EDL933 phage genes. Fifty-two of them could be assigned to the Shiga toxin encoding phages CP-933V and BP-933W, the others belonged to the non-Stx encoding phages or prophages elements existing in the EDL933 genome. Conspicuous, genes present in the late region of the BP-933W prophage were induced most strongly, up to 158-fold in the case of stxA2. Only some genes present in the early region of the Stx1 encoding phage CP-933V were induced upon induction with norfloxacin. Notably, only some genes of the non-Stx phages of EDL933 appeared to be induced after induction. The additional upregulated genes were related to recombination and stress functions and to E. coli O157:H7 RIMD0509952 genes. The majority of downregulated genes belonging to primary metabolism, such as amino acid biosynthesis, cell division and energy metabolism. In conclusion, an induction of E. coli O157:H7 strain EDL933 with 200 ng/ml norfloxacin has profound effects on the transcriptome of E. coli O157:H7, in particular on the global gene expression of more then ten prophages. The DNA-microarray technology and especially the E. coli O157 Array offer a modern tool for analysis of transcription profiles of the serious pathogen EHEC O157 in response to stress, e.g. antibiotics. In the context of this work, the DNA-microarraytechnology could be established and validated to provide the opportunity for further studies about the global effects on the whole transcriptome of E. coli O157.

A ocorrência de fármacos no meio ambiente têm despertado o interesse de pesquisadores, uma vez que podem causar efeitos adversos à comunidade biótica. O norfloxacino (NOR) é um fármaco amplamente empregado no tratamento de infecções bacterianas tanto em humanos como em animais, e devido as suas propriedades físico-químicas, tem sido alvo de estudos envolvendo o seu monitoramento e efeitos toxicológicos em micro-organismos, principalmente em ambientes aquáticos. Neste contexto, o presente trabalho teve como objetivo a investigação da biotransformação/biodegradação do fármaco NOR por fungos derivados de ambiente marinho. Primeiramente, foi realizada uma triagem a partir de 7 cepas fúngicas, das quais 4 foram selecionadas - Penicillium raistrickii CBMAI 931, Cladosporium sp. CBMAI 1237, Aspergillus sydowii CBMAI 1241 e Penicillium raistrickii CBMAI 1235 - para avaliar a influência da adição do fármaco na inoculação dos mesmos. Os experimentos foram realizados na presença do fármaco (0,1 mg mL-1), e em caldo nutritivo (malte 2% em água do mar artificial) por 35 dias (32°C, 130 rpm). A adição do fármaco no dia, e após a inoculação, não influenciou no crescimento dos fungos. No entanto, a formação de produtos de biotransformação foi observada para o experimento com adição do NOR no dia da inoculação, os quais foram identificados por LC-QqTOF, baseando-se na similaridade entre as massas obtidas experimentalmente e teórica, assim como os produtos já reportados na literatura. A porcentagem de biodegradação do fármaco foi determinada para o experimento com adição do fármaco após a inoculação, para os fungos: P. raistrickii CBMAI 931 (34,07%) e A. sydowii CBMAI 1241 (58,91%). Para os experimentos realizados em meio mineral (59,35%) e na presença de um consórcio de fungos (57,05%), não foram observadas diferenças significativas. Não foi possível elucidar a estrutura do produto isolado na presença do fungo A. sydowii CBMAI 1241, devido a sua baixa concentração e a possibilidade de conjugação com substâncias endógenas. Um método analítico foi desenvolvido e validado para a determinação da porcentagem de biodegradação do fármaco nos experimentos com os fungos marinhos. / The occurrence of drugs on the environment has called attention to researchers, since they can cause adverse effects to the biotic community. The norfloxacin (NOR) is a compound widely used for treatment of serious bacterial infections in human and animals. Due to the physicochemical properties of this compound it has been focus of studies concerning about its monitoring and toxicological effects on microorganisms, mainly in aquatic environment. Thus, in the present study the biotransformation/biodegradation of NOR by marine-derived fungi was investigated. Firstly, it was performed a screening with 7 strain of marine fungi, in a which 4 were selected - Penicillium raistrickii CBMAI 931, Cladosporium sp. CBMAI 1237, Aspergillus sydowi CBMAI 1241 and Penicillium raistrickii CBMAI 1235 - to evaluate the influence of NOR addition in the inoculation. The experiments were carried out in the presence of NOR (0,1 mg mL-1) in nutritive broth (malt 2% in artificial sea water) for 35 days (32°C, 130 rpm). The NOR addition on the first day and after inoculation, did not affect the fungal growth. Nevertheless, the formation of biotransformation products was observed to the experiment with addition on the first day. These products were identified by LC-QqTOF, based on the similarity between experimental and theoretical mass, as the products already reported on the literature. The percentage of drug biodegradation was determined for the fungi P. raistrickii CBMAI 931 (34,07%) and A. sydowi CBMAI 1241 (58,91%) for the experiment carried out with NOR addition after inoculation. For the experiments performed in mineral medium (59,35%) and in the presence of fungal consortium (57,05%) no differences were observed for the biodegradation. It was not able to elucidate the structure of isolated product, in the presence of A. sydowii CBMAI 1241, due to its low concentration and probable conjugation with endogenous substances. The analytical method was developed and validated to determine the percentage of drug biodegradation in the experiments with marine fungi.

FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Foram desenvolvidos e validados trÃs mÃtodos robustos para a determinaÃÃo de amoxicilina, norfloxacino, oxcarbazepina (OXC) e 10,11-dihidro-10-hidroxicarbamazepina (MHD) em plasma, utilizando cromatografia lÃquida de alta eficiÃncia em fase reversa (RP-HPLC) com detecÃÃo ultravioleta, fluorescÃncia e espectrometria de massa, respectivamente. Os mÃtodos envolveram extraÃÃo lÃquido-lÃquido, com diclorometano (amoxicilina), acetonitrila (amoxicilina e norfloxacino), Ãter etÃlico-diclorometano (60:40 v/v, oxcarbazepina), utilizando cefadroxil, ciprofloxacino e d10-carbamazepina como padrÃes interno (PI). SeparaÃÃes cromatogrÃficas foram realizadas utilizando colunas Gemini C18 5 Âm (150 X 4,6 mm), Synergi RP-MAX 4 Âm (150 X 4,6 mm) e Luna C18 Âm (150 X 4,6 mm), com sistemas de eluiÃÃo constituÃdos por mistura de tampÃo fosfato de 0,01 M (pH 3,5)/acetonitrila (95:5 v/v), acetonitrila-tampÃo fosfato (85:15, v/v) e acetonitrila-Ãgua (50:50 v/v) + 20 mM Ãcido acÃtico, respectivamente. As curvas de calibraÃÃo foram lineares, nas faixas de 0,5 a 40 Âg/mL, 30 a 3500 ng/mL, 20 a 5250 ng/ml e 40 a 10500 ng/ml. As recuperaÃÃes nas concentraÃÃes de 1,5, 15 e 30 Âg/ml foram de 59,4%, 60,5% e 67,1% para amoxicilina; 90, 1400 e 2800 ng/mL foram de 103,5%, 100,2% e 100,2% para norfloxacino, 60, 2000 e 4000 ng/ml foram de 105,4, 89,2 e 92,8% para OXC e 120, 4000 e 8000 ng/ml foram de 88,4, 88,7 e 90,6% para MHD, respectivamente. Os mÃtodos validados incluÃram avaliaÃÃo de precisÃo e exatidÃo intra e interlote, assegurando que estes estavam dentro de limites admissÃveis. Os mÃtodos foram entÃo aplicados com sucesso em estudos de bioequivalÃncia, administrando 500 mg ou 400 mg das formulaÃÃes referÃncia/teste de amoxicilina e norfloxacino, e em estudos farmacocinÃticos com formulaÃÃo de oxcarbazepina suspensÃo (6%), em voluntÃrios sadios. / A robust method for the determination of amoxicillin, norfloxacin, oxcarbazepine (OXC) and its active metabolite, 10,11-dihydro-10-hydroxycarbamazepine (MHD) in human plasma, using reversed-phase high-performance liquid chromatography (RP-HPLC) with ultraviolet, fluorescence and mass spectrometry detection, respectively, have been developed and valited. The methods involve precipitation of plasma protein with dichloromethane (amoxicillin), acetonitrile (amoxicillin and norfloxacin) and diethyl etherâdiclhoromethane (60:40 v/v, oxcarbazepine), using cefadroxil, ciprofloxacin and deuterade carbamazepine (d10-carbamazepine) as internal standard (IS). Chromatographic separations were performed on a column Gemini C18 5 Âm (150 X 4.6 mm), Synergi MAX-RP 4 Âm (150 X 4.6 mm) and Luna C18 5 Âm (150mm X 4.6 mm) with an elution system consisting of a mixture of 0.01 M buffer phosphate (pH 3.5)/acetonitrile (95:05 v/v), phosphate bufferâacetonitrile (85:15, v/v) and acetonitrile/water (50:50 v/v) + 20mM acetic acid, respectively. The calibration curve was linear, in the range of 0.5 to 40 Âg/mL, 30 to 3500 ng/mL, 20 to 5250 ng/mL and 40 to 10,500 ng/mL. The recoveries at concentrations of 1.5, 15 and 30 Âg/mL were foram 59.4%, 60.5% and 67.1% for amoxicillin; 90, 1400 and 2800 ng/mL were 103.5%, 100.2% and 100.2% for norfloxacin, 60, 2000 and 4000 ng/mL were 105.4, 89.2 and 92.8% for OXC and 120, 4000 and 8000 ng/mL were 88.4, 88.7 and 90.6% for MHD, respectively. The statistical evaluation of the developed method was conducted by examining within-batch and between-batch precision data, which were within the required limits. The methods were successfully applied in bioequivalence studies given 500 or 400-mg of the reference formulation / test amoxicillin and norfloxacin, and pharmacokinetic studies with formulation of oxcarbazepine suspension (6%) in healthy volunteers.

A ocorrência de fármacos no meio ambiente têm despertado o interesse de pesquisadores, uma vez que podem causar efeitos adversos à comunidade biótica. O norfloxacino (NOR) é um fármaco amplamente empregado no tratamento de infecções bacterianas tanto em humanos como em animais, e devido as suas propriedades físico-químicas, tem sido alvo de estudos envolvendo o seu monitoramento e efeitos toxicológicos em micro-organismos, principalmente em ambientes aquáticos. Neste contexto, o presente trabalho teve como objetivo a investigação da biotransformação/biodegradação do fármaco NOR por fungos derivados de ambiente marinho. Primeiramente, foi realizada uma triagem a partir de 7 cepas fúngicas, das quais 4 foram selecionadas - Penicillium raistrickii CBMAI 931, Cladosporium sp. CBMAI 1237, Aspergillus sydowii CBMAI 1241 e Penicillium raistrickii CBMAI 1235 - para avaliar a influência da adição do fármaco na inoculação dos mesmos. Os experimentos foram realizados na presença do fármaco (0,1 mg mL-1), e em caldo nutritivo (malte 2% em água do mar artificial) por 35 dias (32°C, 130 rpm). A adição do fármaco no dia, e após a inoculação, não influenciou no crescimento dos fungos. No entanto, a formação de produtos de biotransformação foi observada para o experimento com adição do NOR no dia da inoculação, os quais foram identificados por LC-QqTOF, baseando-se na similaridade entre as massas obtidas experimentalmente e teórica, assim como os produtos já reportados na literatura. A porcentagem de biodegradação do fármaco foi determinada para o experimento com adição do fármaco após a inoculação, para os fungos: P. raistrickii CBMAI 931 (34,07%) e A. sydowii CBMAI 1241 (58,91%). Para os experimentos realizados em meio mineral (59,35%) e na presença de um consórcio de fungos (57,05%), não foram observadas diferenças significativas. Não foi possível elucidar a estrutura do produto isolado na presença do fungo A. sydowii CBMAI 1241, devido a sua baixa concentração e a possibilidade de conjugação com substâncias endógenas. Um método analítico foi desenvolvido e validado para a determinação da porcentagem de biodegradação do fármaco nos experimentos com os fungos marinhos. / The occurrence of drugs on the environment has called attention to researchers, since they can cause adverse effects to the biotic community. The norfloxacin (NOR) is a compound widely used for treatment of serious bacterial infections in human and animals. Due to the physicochemical properties of this compound it has been focus of studies concerning about its monitoring and toxicological effects on microorganisms, mainly in aquatic environment. Thus, in the present study the biotransformation/biodegradation of NOR by marine-derived fungi was investigated. Firstly, it was performed a screening with 7 strain of marine fungi, in a which 4 were selected - Penicillium raistrickii CBMAI 931, Cladosporium sp. CBMAI 1237, Aspergillus sydowi CBMAI 1241 and Penicillium raistrickii CBMAI 1235 - to evaluate the influence of NOR addition in the inoculation. The experiments were carried out in the presence of NOR (0,1 mg mL-1) in nutritive broth (malt 2% in artificial sea water) for 35 days (32°C, 130 rpm). The NOR addition on the first day and after inoculation, did not affect the fungal growth. Nevertheless, the formation of biotransformation products was observed to the experiment with addition on the first day. These products were identified by LC-QqTOF, based on the similarity between experimental and theoretical mass, as the products already reported on the literature. The percentage of drug biodegradation was determined for the fungi P. raistrickii CBMAI 931 (34,07%) and A. sydowi CBMAI 1241 (58,91%) for the experiment carried out with NOR addition after inoculation. For the experiments performed in mineral medium (59,35%) and in the presence of fungal consortium (57,05%) no differences were observed for the biodegradation. It was not able to elucidate the structure of isolated product, in the presence of A. sydowii CBMAI 1241, due to its low concentration and probable conjugation with endogenous substances. The analytical method was developed and validated to determine the percentage of drug biodegradation in the experiments with marine fungi.

Este trabalho tem como objetivo sintetizar e caracterizar um novo complexo mononuclear de rutênio (III) e enrofloxacina (enro, fármaco antibacteriano da família das fluoroquinolonas), [Ru(enro)3].nH2O. Foram testadas várias rotas sintéticas e apenas a partir de uma delas obteve-se o composto desejado. O produto foi caracterizado pelas técnicas espectroscópicas de absorção na região do UV-visível e do infra-vermelho. Através desta última técnica foi possível determinar o modo pelo qual a enrofloxacina se coordena ao íon rutênio: a coordenação ocorre de modo bidentado através do oxigênio da piridona e do oxigênio do grupamento carboxilato. Outro objetivo deste trabalho foi investigar a interação do complexo mononuclear de rutênio (III) e norfloxacina, [Ru(nor)3].nH2O, com a albumina de soro humano (HSA), através da técnica de luminescência. Mais especificamente pelo estudo da supressão da luminescência dos resíduos de triptofano, aplicando-se o modelo de tratamento da supressão bimolecular de Stern-Volmer. O estudo de supressão de fluorescência mostrou, por meio de espectros de emissão da HSA, que com o aumento da concentração do complexo [Ru(nor)3].nH2O na solução de HSA, ocorre uma redução gradual da luminescência da HSA, devido a alterações da conformação da proteína, que sugerem alteração do microambiente próximos aos resíduos de triptofano. A partir do tratamento dos dados pode-se determinar tanto K_sv quanto a constante cinética do processo de supressão, que mostraram uma dependência com a temperatura sugerindo como mecanismo predominante de supressão o mecanismo dinâmico. Porém essa conclusão foi revista a partir da determinação dos tempos de vida do estado excitado da HSA, e pode-se concluir que o mecanismo predominante à temperatura ambiente é o mecanismo estático, porém com o aumento da temperatura ocorre a predominância do mecanismo do tipo dinâmico. Através da determinação dos parâmetros termodinâmicos, concluiu-se que as interações entre a HSA e o complexo são espontâneas, e forças de van der Waals e ligações de hidrogênio estão envolvidas na ligação entre a HSA e o supressor. / This work aims to synthesize and characterize a new mononuclear ruthenium (III) complex and enrofloxacin (enro, antibacterial drug of the fluoroquinolone family), [Ru(enro)3].nH2O. Several synthetic routes were tested, but only from one of them it was obtained the desired compound. The product was characterized by spectroscopic techniques of absorption in UV-visible and infra-red regions. Through this last technique, it was possible to determine the coordination mode of enrofloxacin to the ruthenium ion: the coordination occurs in a bidentate way through the pyridone oxygen and the oxygen of the carboxylate group. Another aim of this study was to investigate the interaction of mononuclear ruthenium (III) complex and the norfloxacin, [Ru(nor)3].nH2O, with the human serum albumin (HSA), through the technique of luminescence. More specifically, by the study of the quenching of luminescence of tryptophan residues, by applying the Stern-Volmers model of treatment of bimolecular suppression. The fluorescence quenching study showed, through the emission spectra of HSA, that increasing the complex concentration in HSA solution, there is a gradual reduction of the luminescence of HSA, due to the conformational changes of the protein that suggests the change of microenvironment near tryptophan residues. From the data processing it is possible to determine both K_sv and the kinetic constant of the suppression process, which showed temperature dependence, suggesting as the predominant mechanism of quenching the dynamic mechanism. However, this conclusion has been revised from the determination of the lifetimes of the excited state of HSA, and it can be concluded that the predominant mechanism at room temperature is the static mechanism, but with the temperatures increase, it occurs the predominance of the dynamic type mechanism. By determining the thermodynamic parameters, it was concluded that the interactions between HSA and the complex are spontaneous, and Van der Waals forces and hydrogen bonds are involved in the binding between HSA and suppressor.

This thesis aimed to investigate the features and properties of the ground states, transient species and photoproducts involved in the photophysical and photochemical processes for four kinds of drug compounds: lomefloxacin (LF), norfloxacin (NF), tiaprofenic acid (TPA), and flurbiprofen (Fp). The investigation used femtosecond transient absorption (fs-TA), nanosecond transient absorption (ns-TA), UV/Vis absorption spectra (UV/Vis), nanosecond transient resonance Raman (ns-TR2) and nanosecond time-resolved resonance Raman spectroscopy (ns-TR3), as well as density functional theory (DFT) calculations. Although many previous investigations have indicated that photo-defluorination or photo-decarboxylation reactions may account for the phototoxicity for these compounds, detailed information on the mechanisms remains unclear. In this thesis, the photo-defluorination reaction of LF was explored in neutral water at pH 7.2. The fs-TA results revealed that the lowest lying excited singlet state species (S1) partially decayed into the ground state through fluorescence emission and partially underwent cleavage of the carbon-fluorine bond at position 8 to generate into a singlet aryl cation. Subsequently, intersystem crossing (ISC) allowing the transformation from singlet cation to triplet carbene was observed. Finally, a cyclization reaction with the N-ethyl chain took place for the triplet carbene to generate the final product. The mechanism underlying NF phototoxicity involves a photo-defluorination reaction in neutral water (pH=7.2). The fs-TA spectra indicated that the S1 underwent efficient ISC to swiftly transform into lowest excited triplet (T1) The ns-TA gained under nitrogen-saturated condition observed a new transient species produced from T1 that was proposed to be a transient species derived from the photo-defluorination reaction involving a SN2Ar* mechanism. The photo-defluorinated product ultimately experienced an ISC process to produce the final product. The photo-decarboxylation mechanism of TPA was studied in a neutral phosphate buffered solution (PBS). The fs-TA data revealed that S1 went through an efficient ISC to rapidly transform into T1 that then undergoes a photo-decarboxylation reaction to produce a triplet biradical species (denoted as TB3). The ns-TA and ns-TR3 results supplied evidence of the protonation process of TB3 that produces the neutral species (denoted as TBP3) that then decayed through ISC to give rise to the singlet TBP species, which underwent further reaction to make the final product (DTPA). The photo-decarboxylation reaction of Fp was explored in pure acetonitrile (MeCN). The second excited singlet (S2) went through internal conversion (IC) to decay to S1. Intriguingly, three different pathways for S1 decay co-exist. One pathway is fluorescence emission and the second is an ISC process. The third pathway is the homolysis of the carbon α bond reaction that proceeds to generate two radical species, one being a carboxyl species and the other being the residual, denoted as FpR that was liable to be oxidized under an oxygen-saturated condition to yield a new radical species with the addition of one oxygen molecule which is denoted as FOR that then experienced intramolecular hydrogen transfer (IHT) and dehydroxylation (DHO) to produce the final product. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy