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No. of bitstreams: 2 Dissertação - Lara Barroso Brito - 2016.pdf: 19284674 bytes, checksum: 73aa3e2fc34b886b960712220045839d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-03-23 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Pharmaceuticals can be introduced directly into the environmental by household disposal or pharmaceutical industry waste and indirectly through the excretion of humans and animals. Antibiotics are considered emerging contaminants because they are typically present at very low levels in the environmental and their human or ecological health effects are unclear. β-lactams represent more than 70% of antibiotics consumed in Brazil. Thus, in this context, this work evaluated the environmental impact of antibiotic amoxicillin (AMX) in different organisms, considering its presence in water. For that, we used the phytotoxicity test with seeds of tomato (Lycopersicon esculentum), cucumber (Cucumis sativus) and lettuce (Lactuca sativa), brine shrimp toxicity assay (Artemia salina), and embryo-larval toxicity test zebrafish (D. rerio), considering the lethal and sublethal effects and biomarkers determinations. AMX showed no toxicity to seeds of tomato, cucumber and lettuce in relation to seed germination and root elongation endpoints. For microcrustacean A. salina, AMX did not induce significant mortality after 24 h and 48 h exposure (LC50 > 100 mg/L) and it was classified as non-toxic (not categorized) according to the Globally Harmonized System (GHS). AMX also did not cause significant mortality in embryos and larvae zebrafish during 168 h of exposure. It had no significant effect on embryos hatching and larvae equilibrium. However, AMX significantly increased the larvae size at 6.25, 12.5 and 25 mg/L. Catalase (CAT), glutathione S-transferase (GST) and lactate dehydrogenase (LDH) activities in embryos and larvae of zebrafish were inhibited at 12.5 mg/L of AMX, 6.25, 12.5, 25, 50 and 100 mg/L of AMX and 1.5, 3.0, 6.25 and 12.5 mg/L of AMX, respectively. Therefore, AMX showed no significant acute toxicity to tested organisms, but it induced sublethal effects on larvae zebrafish in concentrations greater than those found in the aquatic environment, indicating that long-term chronic exposures must be investigated. / Os fármacos e insumos farmacêuticos podem ser introduzidos no ambiente de forma direta, ou seja, através do descarte doméstico ou por efluentes da indústria farmacêutica, e ainda indiretamente, por meio da excreção humana e animal. Os antibióticos são considerados contaminantes emergentes, uma vez que são detectados em concentrações muito baixas no ambiente e seus efeitos sobre o ambiente e saúde humana permanecem incertos. Os β- lactâmicos representam mais de 70% dos antibióticos consumidos no Brasil. Assim, dentro deste contexto, este trabalho propôs avaliar o impacto ambiental do antibiótico amoxicilina (AMX) em diferentes organismos, considerando a sua presença nos recursos hídricos. Para tanto foram utilizados os ensaios de fitotoxicidade com sementes de tomate (Lycopersicon esculentum), pepino (Cucumis sativus) e alface (Lactuca sativa) e de toxicidade aguda com Artemia salina, assim como o teste com o estágio embriolarval de zebrafish (Danio rerio), considerando os efeitos letais, subletais e atividade de biomarcadores enzimáticos. A AMX não foi tóxica para as sementes de tomate, pepino e alface, não apresentando diferenças estatísticas significativas para os parâmetros de germinação das sementes e do desenvolvimento das raízes. Para o microcrustáceo A. salina, a AMX não induziu mortalidade significativa com valores de CL50 maior que 100 mg/L para 24 h e 48 h de exposição, sendo classificada como não tóxica (não categorizada) de acordo com o Globally Harmonized System (GHS). A AMX não provocou mortalidade significante nos embriões e larvas de zebrafish durante as 168 h de exposição, assim como não alterou significativamente a eclosão dos embriões e o equilíbrio de larvas de zebrafish. Entretanto, o antibiótico aumentou significativamente o tamanho das larvas desse peixe nas concentrações de 6,25; 12,5 e 25 mg/L. As atividades da catalase (CAT), da glutationa Stransferase (GST) e da lactato desidrogenase (LDH) de embriões e larvas de peixezebra foram inibidas significativamente nas exposições à 12,5 mg/L de AMX, 6,25; 12,5; 25; 50 e 100 mg/L de AMX e 1,5; 3,0; 6,25 e 12,5 mg/L de AMX, respectivamente. Portanto, o antibiótico AMX não apresenta toxicidade aguda relevante para os organismos testados, mas causa alguns efeitos subletais em larvas de zebrafish em concentrações superiores às encontradas no ambiente aquático, o que indica a necessidade de se investigar exposições a esse composto a longo prazo.

L’objectif de ce travail a consisté en le développement de nouveaux processus multi-composant radicalaires et leur application en synthèse organique. Des carbo-alcénylation d'oléfines ont ainsi été réalisées avec de nouveaux précurseurs de radicaux, des oléfines diverses, en présence de Z-diphénylsulfonyléthylène comme accepteur terminal. Les conditions de la réaction ont été optimisées, en introduisant notamment la diphénylsulfonylhydrazine comme amorceur de radicaux sous irradiation UV, et substitut au couteux DTBHN. Des conditions sans étain ont également été étudiées avec l’objectif de remplacer le réactif (Bu3Sn)2 par des radicaux silylés. Le tris (triméthylsilyl)silylthiopropene a ainsi été testé avec succès en tant qu'agent de propagation des chaînes radicalaires. A l’issue de ce travail méthodologique, nous avons développé une stratégie de synthèse de l'Eucophylline, un alcaloïde isolé de Leuconotis griffithii, dont le squelette tétracyclique a été élaboré sur la base d’une réaction de carbo-oximation radicalaire d’oléfine. Ce processus multicomposant, suivi d’une réduction de la fonction oxime et d’une lactamisation offre une voie d’accès rapide au motif bicyclo[3.3.1]lactame, intermédiaire-clé de la synthèse. Une réaction de type Friedländer entre ce lactame et un ortho-aminobenzonitrile a permis d’accéder au squelette tetrahydrobenzo[1,8]naphthyridine de l'Eucophylline. La synthèse du composé modèle a enfin été complétée par l’introduction du substituant vinylique par un couplage de Heck. / The aim of this work was to develop new radical multi-component processes and their application in organic synthesis. Carbo-alkenylation processes were thus performed with new radical precursors, different olefins, in the presence of Z-diphenylsulfonylethylene as a terminal acceptor. Reaction conditions have also been optimized, including the diphenylsulfonylhydrazine as a radical initiator under U.V. irradiation, and substitute to the costly DTBHN. Tin-free conditions were also screened with the goal of replacing (Bu3Sn)2 with silyl radicals. Tris(trimethylsilyl)silylthiopropene was tested with success as a radical chain carrier. After this methodology studies, we developed a strategy toward the synthesis of Eucophylline, an alcaloid isolated from Leuconotis griffithii, which tetracyclic skeleton was elaborated based on a carbo-oximation of olefin. This multicomponent process, followed by a reduction of the oxime function and a lactamization offered a fast access to the bicyclo[3.3.1]lactam, a key-intermediate in the synthesis. A Friedländer-type reaction between this lactam and an ortho-aminobenzonitrile allowed an access to the Eucophylline tetrahydrobenzo[1,8]naphthyridine skeleton. The synthesis of the model compound was finally completed with the introduction of the vinylic substituent through a Heck coupling.

Récemment, l'activation C-H catalysée par des métaux de transition est devenue un outil performant pour construire des liaisons carbone-carbone et carbone hétéroatome à partir de liaisons C-H omniprésentes dans les molécules organiques. Bien que l'activation des liaisons C-H aromatiques ait été largement étudiée ces dernières années, celle des liaisons C-H aliphatiques représente encore un domaine faiblement exploré. Notre équipe s'est depuis plusieurs années intéressée au développement méthodologique de l'activation C(sp3)-H et à son application en synthèse de produits naturels et molécules bioactives. Dans la continuité des récents travaux sur la version asymétrique de cette réaction, cette thèse décrit le développement et la synthèse de nouveaux ligands du type Binepine. Ces ligands chiraux et monodentates nous ont permis de réaliser la synthèse d'indanes chiraux possédant un centre asymétrique quaternaire, de manière hautement énantio- et diastéréosélective. Cette réaction présente comme avantages l'utilisation d'une faible charge catalytique et d'une température de réaction inférieure à 100 °C, sans aucun additif. Le champ d'application de la réaction inclut notamment l'activation des liaisons C-H d'un groupement méthylène, donnant ainsi accès à des systèmes fusionnés, tricycliques. La construction de molécules non-aromatiques via une alcénylation C-H intramoléculaire a été récemment décrite et s'avère très prometteuse pour la synthèse de produits naturels saturés. Dans la continuité de ces travaux innovants, nous avons développé la synthèse de γ-lactames à partir de bromoalcènes acycliques. Cette nouvelle réaction permet de construire de manière simple et efficace des hétérocycles a cinq chainons de façon modulaire, donnant ainsi la possibilité d'envisager des nouvelles déconnections rétrosynthétiques, complémentaires des méthodes déjà établies. Cette nouvelle méthode a pu être appliquée à la synthèse totale de l'alcaloïde marin Plakoridine A, dont la structure centrale cyclique a été synthétisée en quatre étapes avec un rendement global de 37% / Recently, transition-metal-catalyzed C-H activation has emerged as a powerful tool to transform stable C-H bonds into carbon-carbon or carbon-heteroatom bonds. While the activation of aromatic C-H bonds has seen a tremendous development, less effort has been devoted to the more challenging activation of aliphatic C-H bonds. Our group has a long-standing interest in the development of C(sp3)-H activation reactions and their application in the synthesis of natural products and bioactive compounds. In line with previous efforts to develop an asymmetric C(sp3)-H activation, the herein presented work details the synthesis of new Binepine ligands. These monodentate, chiral ligands enabled us to realize a highly dia- and enantioselective C(sp3)-H activation reaction allowing the construction of chiral quaternary carbon centers. Strong points of this robust method are the low catalyst loading, the low reaction temperature and the absence of additives. The substrate scope includes the rare activation of methylene C-H bonds leading to fused tricyclic carbocycles and heterocycles. The construction of non-aromatic molecules through intramolecular C-H alkenylation was recently disclosed and has great potential for the construction of saturated natural products. Based on seminal work, we have developed the synthesis of valuable γ- lactams from acyclic bromoalkenes. This new methodology offers a powerful way to build simple, five-membered N heterocycles in a modular fashion. Notably, it enables a new retrosynthetic disconnection which is complementary to conventional approaches. Finally, we set out to showcase its utility as key step in the total synthesis of the pyrrolidine alkaloid Plakoridine A. The cyclic core structure was accessed in four steps and 37% overall yield

<p>Antibiotic treatment causes an ecological disturbance on the human microflora. Four commensal bacteria: E. coli, enterococci, a-streptococci and coagulase-negative staphylococci, from patients with extensive, high antibiotic usage were investigated with regard to resistance pattern and mutation frequency. Among 193 investigated strains it was found that high antibiotic usage selected for resistant bacteria and enriched for bacteria with a small but significantly increased mutation frequency. </p><p>The relative biological fitness cost of resistance in <i>Staphylococcus epidermidis</i> was assessed in a human in vivo model where the indigenous flora was present. In vitro data of the bacterial growth rate correlated well to in vivo fitness assayed in the competition experiments on skin. </p><p>An in vitro kinetic model was shown to be a useful tool to establish the pharmacokinetic and pharmacodynamic (PK/PD) indices for efficacy of antibiotics. It was confirmed that the time, when the concentration exceeds the minimal inhibitory concentration (MIC), correlates with efficacy for b-lactam antibiotics. To achieve maximal killing for penicillin-resistant pneumococci, with an MIC of 2 mg/L, the peak concentration was also of importance. </p><p>Suboptimal dosing regimen facilitates selection of resistance. Penicillin-resistant pneumococci were easily selected in a mixed population with penicillin-sensitive, -intermediate and -resistant pneumococci in an in vitro kinetic model. The selection of the resistant strain was prevented when the benzylpenicillin concentration exceeded the MIC for approximately 50% of 24 h.</p>

<p>Toxic products, such as endotoxin from the gram-negative and exotoxin from the gram-positive bacteria, are the most important initiators of the inflammatory host response in sepsis. In addition to antibacterial treatment, numerous attempts have been made to interfere with the exaggerated proinflammatory cascade initiated by the toxins. As most antitoxic and anti-inflammatory agents have shown no clear efficacy, an attractive alternative has been to prevent or minimise their release. Therefore, it was of interest to further study the antibiotic-induced release of toxins after exposure to antibiotics used for the treatment of the most severe infections, especially if protein synthesis inhibitors could reduce the release induced by PBP 3-specific β-lactam antibiotics.</p><p>There were significant reductions in endotoxin release from gram-negative bacteria when the combination of the PBP 3-specific β-lactam antibiotic, cefuroxime, and the protein synthesis inhibitor, tobramycin, was compared with cefuroxime alone. Increasing doses of tobramycin reduced endotoxin release and increased the killing rate. In a kinetic <i>in vitro</i> model the endotoxin release from <i>E.coli</i> was higher after the second dose of cefuroxime. Nevertheless, it was reduced after addition of tobramycin.</p><p>No binding of tobramycin to endotoxin was observed, either <i>in vivo</i> or <i>in vitro</i>. In a porcine sepsis model, a possible anti-inflammatory effect of ceftazidime and tobramycin, expressed as late cytokine inhibition, was seen.</p><p>The protein synthesis inhibitor, clindamycin, released less streptococcal pyrogenic exotoxin A (SpeA) from a group A streptococcus strain than penicillin, and addition of clindamycin to penicillin resulted in less toxin production than penicillin alone. The SpeA production was dependent on the bacterial number at the start of treatment. Higher doses of penicillin also led to less SpeA. </p><p>The choice of antibiotic class and dose may be important in the severely ill septic patient in whom an additional toxin release could be deleterious. A combination of a β-lactam antibiotic and a protein synthesis inhibitor seems beneficial but further investigations are needed.</p>

<p>This thesis describes the crystal structures of two proteins in the context of combatting bacterial infections. The GA module is a bacterial albumin-binding domain from a surface protein expressed by pathogenic strains of the human commensal bacterium <i>Finegoldia magna</i>. The structure of the GA module in complex with human serum albumin (HSA) provides insights into bacterial immune evasion, where pathogenicity is acquired by the bacterial cell through the ability to coat (and disguise) itself with serum proteins. The structure shows binding of the GA module to HSA in the presence of fatty acids, and reveals interactions responsible for the host range specificity of the invading bacterium. The complex resulting from binding of the GA module to HSA readily forms stable crystals that permit structural studies of drug binding to HSA. This was exploited to study the specific binding of the drug naproxen to the albumin molecule.</p><p>Antibiotics play a major role in controlling infections by attacking invading bacteria. The enzyme deacetylcephalosporin C acetyltransferase (DAC-AT) catalyses the last step in the biosynthesis of the beta-lactam antibiotic cephalosporin C, one of the clinically most important antibiotics in current use. The enzyme uses acetyl coenzyme A as cofactor to acetylate a biosynthetic intermediate. Structures of DAC-AT in complexes with reaction intermediates have been determined. The structures suggest that the acetyl transfer reaction proceeds through a double displacement mechanism, with acetylation of a catalytic serine by the cofactor through a suggested tetrahedral transition state, followed by acetyl transfer to the intermediate through a second suggested tetrahedral transition state. The structure of DAC-AT yields valuable information for the continued study of cephalosporin biosynthesis in the context of developing new beta-lactam compounds.</p>

New methods have been developed for the synthesis of bicyclic β-lactams and 2-pyridinones by combining acyl Meldrum’s acids and Δ2-thiazolines. The 2-pyridinones were synthesised both in solution using conventional heating or microwave assisted heating as well as by solid supported chemistry. The compounds (pilicides) were designed to interfere with the assembly of pili in uropathogenic E. coli by inhibiting the periplasmic chaperones. The affinity of the pilicides to the chaperones was investigated with surface plasmon resonance technique (Biacore) and with relaxation-edited 1H NMR spectroscopy experiments. Finally, the pilicides were investigated for their ability to inhibit pili formation in uropathogenic E. coli in a hemagglutination assay, where members of the 2-pyridinone family proved to be able to cause depiliation.

Toxic products, such as endotoxin from the gram-negative and exotoxin from the gram-positive bacteria, are the most important initiators of the inflammatory host response in sepsis. In addition to antibacterial treatment, numerous attempts have been made to interfere with the exaggerated proinflammatory cascade initiated by the toxins. As most antitoxic and anti-inflammatory agents have shown no clear efficacy, an attractive alternative has been to prevent or minimise their release. Therefore, it was of interest to further study the antibiotic-induced release of toxins after exposure to antibiotics used for the treatment of the most severe infections, especially if protein synthesis inhibitors could reduce the release induced by PBP 3-specific β-lactam antibiotics. There were significant reductions in endotoxin release from gram-negative bacteria when the combination of the PBP 3-specific β-lactam antibiotic, cefuroxime, and the protein synthesis inhibitor, tobramycin, was compared with cefuroxime alone. Increasing doses of tobramycin reduced endotoxin release and increased the killing rate. In a kinetic in vitro model the endotoxin release from E.coli was higher after the second dose of cefuroxime. Nevertheless, it was reduced after addition of tobramycin. No binding of tobramycin to endotoxin was observed, either in vivo or in vitro. In a porcine sepsis model, a possible anti-inflammatory effect of ceftazidime and tobramycin, expressed as late cytokine inhibition, was seen. The protein synthesis inhibitor, clindamycin, released less streptococcal pyrogenic exotoxin A (SpeA) from a group A streptococcus strain than penicillin, and addition of clindamycin to penicillin resulted in less toxin production than penicillin alone. The SpeA production was dependent on the bacterial number at the start of treatment. Higher doses of penicillin also led to less SpeA. The choice of antibiotic class and dose may be important in the severely ill septic patient in whom an additional toxin release could be deleterious. A combination of a β-lactam antibiotic and a protein synthesis inhibitor seems beneficial but further investigations are needed.

This thesis describes the crystal structures of two proteins in the context of combatting bacterial infections. The GA module is a bacterial albumin-binding domain from a surface protein expressed by pathogenic strains of the human commensal bacterium Finegoldia magna. The structure of the GA module in complex with human serum albumin (HSA) provides insights into bacterial immune evasion, where pathogenicity is acquired by the bacterial cell through the ability to coat (and disguise) itself with serum proteins. The structure shows binding of the GA module to HSA in the presence of fatty acids, and reveals interactions responsible for the host range specificity of the invading bacterium. The complex resulting from binding of the GA module to HSA readily forms stable crystals that permit structural studies of drug binding to HSA. This was exploited to study the specific binding of the drug naproxen to the albumin molecule. Antibiotics play a major role in controlling infections by attacking invading bacteria. The enzyme deacetylcephalosporin C acetyltransferase (DAC-AT) catalyses the last step in the biosynthesis of the beta-lactam antibiotic cephalosporin C, one of the clinically most important antibiotics in current use. The enzyme uses acetyl coenzyme A as cofactor to acetylate a biosynthetic intermediate. Structures of DAC-AT in complexes with reaction intermediates have been determined. The structures suggest that the acetyl transfer reaction proceeds through a double displacement mechanism, with acetylation of a catalytic serine by the cofactor through a suggested tetrahedral transition state, followed by acetyl transfer to the intermediate through a second suggested tetrahedral transition state. The structure of DAC-AT yields valuable information for the continued study of cephalosporin biosynthesis in the context of developing new beta-lactam compounds.

Antibiotic treatment causes an ecological disturbance on the human microflora. Four commensal bacteria: E. coli, enterococci, a-streptococci and coagulase-negative staphylococci, from patients with extensive, high antibiotic usage were investigated with regard to resistance pattern and mutation frequency. Among 193 investigated strains it was found that high antibiotic usage selected for resistant bacteria and enriched for bacteria with a small but significantly increased mutation frequency. The relative biological fitness cost of resistance in Staphylococcus epidermidis was assessed in a human in vivo model where the indigenous flora was present. In vitro data of the bacterial growth rate correlated well to in vivo fitness assayed in the competition experiments on skin. An in vitro kinetic model was shown to be a useful tool to establish the pharmacokinetic and pharmacodynamic (PK/PD) indices for efficacy of antibiotics. It was confirmed that the time, when the concentration exceeds the minimal inhibitory concentration (MIC), correlates with efficacy for b-lactam antibiotics. To achieve maximal killing for penicillin-resistant pneumococci, with an MIC of 2 mg/L, the peak concentration was also of importance. Suboptimal dosing regimen facilitates selection of resistance. Penicillin-resistant pneumococci were easily selected in a mixed population with penicillin-sensitive, -intermediate and -resistant pneumococci in an in vitro kinetic model. The selection of the resistant strain was prevented when the benzylpenicillin concentration exceeded the MIC for approximately 50% of 24 h.