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Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Nous avons réalisé la synthèse de thiazoles, sélénazoles, thiéno[2,3-d]thiazoles et thiéno[2,3-d][1,3]sélénazoles, ce dernier étant préparé facilement à partir de cyanamide et de sulfure de carbone. Nous avons de cette manière pu synthétiser des 4-amino-1,3-thiazoles et 1,3-sélénazoles substitués en position 2 et 5 en deux étapes. Appliqué ces hétérocycles, les 4-halogéno-1,3-thiazoles et 1,3-sélénazoles ont été synthétisés par la méthode de Doyle. La labilité des halogènes dans les dérivés précédents a permis de préparer de nouveaux thiéno[2,3-d]thiazoles et [1,3]sélénazoles. La détermination du potentiel antiprolifératif de ces composés a permis de mettre en évidence deux composés, présentant des IC50 de l’ordre du micromolaire sur les lignées cellulaires cancéreuses : MCF-7, PC-3, Hs683, U373, SKMEL-28 et A549. Un composé surtout a montré une activité d’inhibition de Na+/K+-ATPase et de l'oncogène Ras. Par ailleurs dans un second sujet, nous nous sommes intéressés à la mise au point d’une synthèse des deux isomères (alpha et bêta) de 4-butoxylvinyl-coumarines, -thiocoumarine et -2-quinolones par couplage de Heck. Nous avons ainsi pu montrer que suivant le substituant présent en position 4 des hétérocycles le couplage était régiosélectif. Ces dérivés butoxyvinyliques se caractérisaient par leur caractère diénique et nous avons étudié leur réactivité, stéréoséléctivité et régiosélectivité avec différents diénophiles dans des réactions de cycloaddition de Diels-Alder. Parmi tous les composés polyhétérocycliques ainsi préparés, nous avons identifié des composés tétracycliques à noyau quinonique qui présentent un potentiel anticancéreux par des valeurs d’IC50 sur l’inhibition des phosphatases CDC25 et sur plusieurs lignées de cellules tumorales / We performed the synthesis of thiazoles, selenazoles, thieno[2,3-d]thiazoles and thieno[2,3-d][1,3] selenazoles which are easily prepared from cyanamide and carbon disulfide. By this way, we have synthesized 4-amino-1,3-thiazoles et 1,3-selenazoles substituted in position 2 and 5 in two steps. Used these heterocycles, 4-halogeno-1,3-thiazoles et 1,3-selenazoles were synthesized by Doyle’s method. Lability of halogens in previous derivatives allowed to prepare new thieno[2,3-d]thiazoles and [1,3]selenazoles. Determination of the antiproliferative activity of these compounds has brought out two compounds, showing IC50 values in micromolar range on investigated cancer cell lines: MCF-7, PC-3, Hs683, U373, SKMEL-28, and A549. One particular compound showed a high activity of anti-Na+/K+-ATPase and anti-ROS. In addition in the second subject, we were interested in the development of the synthesis of two isomers (alpha and beta) of 4-butoxylvinyl-coumarins, -thiocoumarin and -2-quinolones by Heck coupling. We showed that, depending on the substituent in the position 4 of the heterocycle, the coupling was regioselective. These butoxyvinylic derivatives, characterized by dienic character, were studied for their reactivity, stereoselectivity and regioselectivity with several dienophiles in Diels-Alder cycloaddition. Among all the polyheterocyclic compounds prepared, we have identified the tetracyclic compounds with quinonic ring which have potential anticancer activity by inhibition of CDC25 phosphatase and on several tumor cell lines

Quinolones are a group of antibiotics that have gained significant popularity on a global scale since the end of the last century. This popularity was predominantly based on their proven potency, broad coverage against a wide range of bacteria, in addition to possessing a favorable pharmacologic profile. Whereas quinolone-associated adverse reactions are generally tolerable and self-limiting, some reactions have generated heightened concerns due to their serious nature, which have resulted in label changes or even market withdrawal in some instances. This thesis investigates the association between quinolone antibiotics and two adverse reactions of an acute and serious nature: acute liver failure and retinal detachment. Each adverse reaction is investigated through integrating evidence from three studies utilizing different designs based on data from different sources, with each source offering a unique perspective on this issue. The first study type (chapter 2 for acute liver failure ‘ALF’ and Chapter 5 for retinal detachment ‘RD’) analyzes spontaneous reports submitted to the US Food & Drug Administration (FDA) adverse event reporting system database. Chapters 3 and 6 systematically identified all relevant (published and unpublished) clinical trials for occurrences of ALF and RD, respectively, among trial participants. Finally, chapters 4 (ALF) and 7 (RD) involved case-control analysis of a major US database of electronic health records for nearly 70 million inpatients admitted to more than 500 hospitals between 2000 and 2016. The FAERS analysis revealed a positive ALF signal with ciprofloxacin and a marginal signal for RD with moxifloxacin. Examination of the evidence from clinical trials revealed only two cases of ALF, one associated with gemifloxacin and one with moxifloxacin. No cases of RD were reported in any of the identified clinical trials. Primary analyses of the Health Facts® data revealed no overall association between quinolones and the risk of ALF or RD. However, elevated risk was identified in some subgroups, including African Americans (ALF, RD), Caucasians (ALF), women (ALF, RD), men (ALF), those ≤60 years of age (ALF) or 56-70 years of age (RD), and those with no or few comorbidities (ALF). Evidence from analyses of data from spontaneous reports and clinical trials provided some evidence for an elevated risk of ALF or RD following the systemic administration of quinolone antibiotics. Some evidence of elevated risk was also identified in the case-control analyses of inpatient EHR records. Findings from our six epidemiologic studies are in line with current advisories by FDA and Health Canada.

Actuellement, les photochromes photoréversibles présentent un intérêt important en vue de leurs applications éventuelles comme interrupteurs optiques ou pour le traitement et le stockage de données. L’objectif principal de ce travail de thèse a été d’évaluer les 3-­-(2-­-benzylbenzoyl)-­-4-­-quinolones en tant que système photochromique photoréversible. Afin d’étudier la relation entre la structure chimique et le comportement photochromique de ce système, une série de nouvelles 3-­-(2-­-benzylbenzoyl)-­-4-­-quinolones à substitution variée a été préparée à l’aide de procédures connues ainsi qu’élaborées lors de ce travail. La photoréaction de ces nouveaux molecules étant très complexe, une méthode d’étude a été établie avec des 2-­-benzyl-­-3-­-benzoyl-­-4-­-quinolones photoénolisables au comportement plus simple. Leurs spectres d’absorption modélisés à l’aide de fonctions pekariennes ont été comparés avec les résultats des calculs quantiques. Le mécanisme selon lequel la réaction de décoloration se fait par l’ionisation des photoénols fortement acides via protonation des molécules du solvant a été établi. Enfin, la troisième partie est consacrée à l’étude des nouveaux dérivés de quinolones. D’après l’analyse de leurs spectres d’absorption, ainsi que les résultats des calculs quantiques, nous proposons un mécanisme où le transfert d’hydrogène photo-­-induit conduirait à la formation d’un intermédiaire biradicalaire, capable de se cycliser en dibenzo[b]acrydinones hydroxysubstituées. Un tel mécanisme expliquerait la forte influence qu’ont la nature des substituants en positions 1 et 2, ainsi que la température et la présence d’oxygène sur la régio-­- et stéréo-­-sélectivité de cette photoréaction. / Photoreversible photochromic compounds are currently of considerable interest from the point of view of their potential applications as molecular switches and for data storage and processing. The main target of the present investigation is to evaluate the scope and limitations of 3-­-(2-­-benzylbenzoyl)-­-4-­-quinolones as a photoreversible photochromic system. In order to investigate the relationship between quinolone chemical structures and photochemical behavior, a large series of new 3-­-(2-­-benzylbenzoyl)-­-4-­-quinolones with different substituents has been prepared using known as well as newly elaborated synthetic procedures. In the second part of the work a series of simpler photoenolizable 2-­-benzyl-­-3-­-benzoyl quinolones is studied by the means of fitting their UV-­-Vis absorption spectra and comparison with the results of quantum mechanical calculations at the TD DFT level. It was concluded that the mechanism of the reverse reaction involves ionization of the strongly acidic photoenols via protonation of the solvent molecules. The third part of the work includes investigation of the new quinolone derivatives. From the analyses of their absorption spectra and the results of quantum mechanical calculations, we propose a tentative mechanism, according to which the photoinduced hydrogen transfer yields a biradical, capable of cyclization into the hydroxy substituted dibenzo[b]acrydinones. This mechanism accounts for the observation that the nature of substituents in positions 1 and 2, the temperature and the presence of oxygen strongly affect regio-­- and stereoselectivity of the cyclization.

Plasmonics techniques, such as surface plasmon resonance (SPR) and surface-enhanced Raman scattering (SERS), have been widely used for chemical and biochemical sensing applications. One approach to excite surface plasmons is through the coupling of light into metallic grating nanostructures. Those grating nanostructures can be fabricated using state-of-the-art nanofabrication methods. Laser interference lithography (LIL) is one of those methods that allow the rapid fabrication of nanostructures with a high-throughput. In this thesis, LIL was combined with other microfabrication techniques, such as photolithography and template stripping, to fabricate different types of plasmonic sensors. Firstly, template stripping was applied to transfer LIL-fabricated patterns of one-dimensional nanogratings onto planar supports (e.g., glass slides and plane-cut optical fiber tips). A thin adhesive layer of epoxy resin was used to facilitate the transfer. The UV-Vis spectroscopic response of the nanogratings supported on glass slides demonstrated a strong dependency on the polarization of the incident light. The bulk refractive index sensitivities of the glass-supported nanogratings were dependent on the type of metal (Ag or Au) and the thickness of the metal film. The described methodology provided an efficient low-cost fabrication alternative to produce metallic nanostructures for plasmonic chemical sensing applications. Secondly, we demonstrated a versatile procedure (LIL either alone or combined with traditional laser photolithography) to prepare both large area (i.e. one inch2) and microarrays (μarrays) of metallic gratings structures capable of supporting SPR excitation (and SERS). The fabrication procedure was simple, high-throughput, and reproducible, with less than 5 % array-to-array variations in geometrical properties. The nanostructured gold μarrays were integrated on a chip for SERS detection of ppm-level of 8-quinolinol, an emerging water-borne pharmaceutical contaminant. Lastly, the LIL-fabricated large area nanogratings have been applied for SERS detection of the mixtures of quinolone antibiotics, enrofloxacin, an approved veterinary antibiotic, and one of its active metabolite, ciprofloxacin. The quantification of these analytes (enrofloxacin and ciprofloxacin) in aqueous mixtures were achieved by employing chemometric analysis. The limit of quantification of the method described in this work is in the ppm-level, with <10 % SERS spatial variation. Isotope-edited internal calibration method was attempted to improve the accuracy and reproducibility of the SERS methodology. / Graduate / 2018-02-17

Stenotrophomonas maltophilia é um bacilo Gram-negativo, não fermentador, considerado um microorganismo pouco virulento, relacionado principalmente a infecções associadas à assistência a saúde. A S. maltophilia apresenta um padrão de resistência intrínseca à maioria das classes de antibióticos. A droga de escolha para o tratamento das infecções por S. maltophilia é a sulfametoxazol/ trimetoprima (SMX/TMP). Entretanto, estudos atuais relatam o aumento da resistência a esse antibiótico, o que limita assim as opções para terapia efetiva. Outras opções de tratamento são o levofloxacino e a tigeciclina, porém, faltam estudos clínicos e in vitro dessas drogas. A proposta deste estudo foi avaliar os possíveis mecanismos de resistência a SMX/TMP e as quinolonas em isolados clínicos de pacientes internados no Instituto Central do Hospital das Clínicas e do Hospital A.C. Camargo. Foram avaliadas 106 amostras de S. maltophilia isoladas de pacientes adultos com infecção relacionada à assistência a saúde, internados no Instituto Central do Hospital das Clínicas da FMUSP e no Hospital de Câncer A.C Camargo durante o período de dezembro de 2008 a dezembro de 2010. A sensibilidade à SMX/TMP foi de 78,3%, para levofloxacino de 82% e 14,2% para cirpofloxacino, para minociclina de 100% e tigeciclina 91,6%. Foi realizado PCR para detecção dos genes sul1, sul2 e dfrA1 para avaliar a resistência à SMX/TMP, os genes int1 e iscr2 para avaliação da presença de elementos genéticos móveis e os genes gyrA, qnr, smeD, smeT e aac(6)-Ib-cr para avaliação da resistência as quinolonas. Quatorze amostras (13,2%) foram positivas para o gene sul1. Desses isolados, nove amostras apresentavam resistência ao SMX/TMP com CIM50 de 8 g/mL e CIM90 de 128 g/mL. Cinco amostras positivas para o gene sul1 foram sensíveis a SMX/TMP com CIM50 de 1 g/mL e CIM90 de 1 g/mL. A sequencia do integron1 da amostra com CIM >125 g/mL mostrou um tamanho aproximado de 4000 pb contendo os genes cassetes aac4 e aadA1 e a região qac/sul1. Uma amostra resistente a SMX/TMP foi positiva para o gene sul2 localizado na transposase-like ISCR 2. Observamos a presença de quatro novos qnr em cepas de S. maltophilia e a presença da enzima aac(6)-ib-cr em 4 amostras. 100% das cepas foram positivas para o gene do sistema de efluxo smeDEF e 12/38 amostras tiveram o gene smeT do sistema de efluxo smeDEF, ,porém não foi observada mutação nesse gene. Na sequencia de aminoácidos da girase A de 15 amostras resistentes a levofloxacino não observamos mutações relacionadas à resistência a quinolonas. As cepas resistentes a SMX/TMP apresentaram um padrão policlonal. Dezoito amostras resistentes ao levofloxacino apresentaram 14 perfis clonais, distribuídos em 10 clusters. S. maltophilia exibe múltiplos mecanismos de resistência, nesse estudo observamos um grande número de cepas com elementos genéticos móveis carregando o gene sul1 e outros genes de resistência. A S. maltophilia pode ser um importante reservatório de transmissão de genes de resistência / Stenotrophomonas maltophilia is a gram-negative, non-fermenter, considered a low virulent organism, mainly related to healthcare associated infections. S. maltophilia shows a pattern of intrinsic resistance to many classes of antibiotics. The drug of choice for the treatment of infections caused by S. maltophilia is SMX/TMP, however, current studies have reported increased resistance to this antibiotic, thus limiting the options for effective therapy. Among the treatment options appear tigecycline and levofloxacin, but clinical trials and studies in vitro to such drugs are lacking. The purpose of this study was to evaluate the possible mechanisms of resistance to SMX/TMP and quinolones in clinical isolates from patients admitted to the Institute\'s Central Clinical Hospital and the Hospital A.C Camargo. We evaluated 106 strains of S. maltophilia isolated from adult patients with healthcare associated infections, at the Instituto Central do Hospital das Clínicas and the Cancer Hospital AC Camargo in the period of December 2008 to December 2010. The sensitivity to SMX/TMP was 78.3%, 82% for levofloxacin, 14,2% for ciprofloxacin, minocycline 100% and for tigecycline 91.6%. PCR was performed for detection of gene sul1, sul2 and dfrA1 to evaluate the resistance to SMX/TMP, genes iscr2 int1 was performed to evaluate the presence of mobile genetic elements and genes gyrA, qnr, smeD , smeT and aac (6 \')-Ib-cr for evaluation of resistance to quinolones. Fourteen samples (13.2%) were positive for the gene sul1. In these isolates, nine samples showed resistance to SMX/TMP with MIC50 of 8 g/ml and MIC90 of 128 g/mL. Five strains were positive for sul1 gene and were susceptible to SMX/TMP with MIC50 of 1 g/ml and MIC90 of 1 g/mL. The sequence of the integron class 1 strain with an MIC> 125 g/mL showed an approximate size of 4000 bp containing the gene cassettes aadA1, aac4 and qac/sul1. A strain resistant to SMX/TMP was positive for the gene sul2 located on ISCR2 a transposase-like. We observed the presence of four new qnr in strains of S. maltophilia and the presence of the enzyme aac (6 \')-ib-cr in 4 samples. 100% of the strains were positive for the gene of the efflux system smeDEF and 12/38 samples had the gene smeT repressor of smeDEF efflux system, however there was no mutation in this gene. In the amino acid sequence of gyrase A of 15 strains resistant to levofloxacin did not observe mutations related to resistance to quinolones. Strains resistant to SMX/TMP had a polyclonal PFGE pattern. Eighteen strains resistant to levofloxacin showed 14 clonal profiles 14 divided into 10 clusters S. maltophilia displays multiple mechanisms of resistance. In this study, we observed a large number of strains with mobile genetic elements carrying the sul1 gene and other resistance genes. S. maltophilia is may be an important source for transmission of genes of resistance

Made available in DSpace on 2017-07-21T19:59:59Z (GMT). No. of bitstreams: 1 Denis Leandro Freitas.pdf: 2460536 bytes, checksum: 9c33d2a38cc2aba90b9f27c476e8d2c5 (MD5) Previous issue date: 2013-03-27 / Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná / The urinary tract infections (UTI) have high incidence in the population, mainly affecting women, the elderly and pregnant women. In most cases an UTI is caused by Enterobacteriaceae, especially Escherichia coli. The main antimicrobials used in the treatment of UTIs, are the quinolones and fluoroquinolones. Since its discovery in the 60s, such antimicrobials were improved and increasing its spectrum of activity, but also the micro-organisms has evolved to develop resistance to these drugs. The resistance comes down to two mechanisms: changes in targets of quinolones or decreased concentration of the drug within the cell. Quinolones are antimicrobial agents that target two enzymes involved in transcription and replication of bacterial DNA, DNA Gyrase, consisting of two subunits GyrA and two GyrB products of the genes gyrA and gyrB respectively and Topoisomerase IV, also composed of two ParC and two ParE subunits, encoded from genes parC and parE. Mutations in these genes can confer high-level resistance to these antimicrobials. Other mechanisms involve genes carried in plasmids such as qnr's, responsible for the production of proteins that protect the target enzymes of quinolones. The gene aac (6 ')-Ib-cr also present in plasmids can alter the chemical structure of certain fluoroquinolones interfering efficiency. The association of resistance to quinolones and β-lactams due to the presence of Extended Spectrum β-lactamases (ESBL) has been widely reported in the literature. The main objective of this study was to evaluate the patterns of resistance to quinolones and fluoroquinolones in 55 samples of uropathogenic E. coli collected in Curitiba, PR, Brazil. The resistance to nalidixic acid, norfloxacin, ciprofloxacin and ofloxacin was observed in 69.1% of the samples. Molecular analysis of the resistant strains showed mutations in gyrA (codons 83 and 87) and parC (codon 80). Also investigated was the presence of genes qnr's and aac (6 ')-Ib-cr and their association with ESBLs. Two of the 55 samples were positive for aac (6 ')-Ib-cr. The presence of this gene in Brazil was first reported in 2012 in Belo Horizonte, MG. No samples were positive for the qnr's genes. These results show a high incidence of resistance to quinolones and the ability to horizontally spread of acquired resistance between the bacterial strains. / As infecções do trato urinário (ITU) apresentam grande ocorrência na população, afetando principalmente mulheres, idosos e gestantes. Na maioria dos casos uma ITU é causada por enterobactérias, especialmente Escherichia coli. A principal escolha no tratamento das ITUs são as quinolonas e fluorquinolonas. Desde sua descoberta na década de 60, tais antimicrobianos foram aperfeiçoados aumentando seu espectro de ação, porém os micro-organismos também evoluíram desenvolvendo resistência a tais fármacos. A resistência se resume a alterações nos alvos das quinolonas e ou a diminuição da concentração da droga no interior da célula. Quinolonas são antimicrobianos que têm como alvo duas enzimas envolvidas na replicação e transcrição do DNA bacteriano, a DNA Girase, formada por duas subunidades GyrA e duas GyrB, produtos dos genes gyrA e gyrB respectivamente e a Topoisomerase IV, também composta de duas subunidades ParC e duas ParE, codificadas a partir dos genes parC e parE. Mutações nesses genes podem conferir alto nível de resistência a estes antimicrobianos. Outros mecanismos envolvem genes transportados em plasmídeos, como os qnr’s, responsáveis pela produção de proteínas capazes de proteger as enzimas alvo das quinolonas. O gene aac(6’)-ib-cr, também presente em plasmídeos, é capaz de alterar a estrutura química de certas fluorquinolonas interferindo na sua eficiência. A associação de resistência a quinolonas e β-lactâmicos devido à presença de β-lactamases de espectro ampliado (ESBLs), tem sido amplamente relatada na literatura. O principal objetivo desse trabalho foi avaliar os padrões de resistência a quinolonas e fluorquinolonas em 55 amostras de E. coli uropatogênica coletadas em Curitiba, PR. A resistência ao ácido nalidíxico, a norfloxacina, ciprofloxacina e ofloxacina, foi observada em 69,1% das amostras. A análise molecular das cepas resistentes revelou mutações em gyrA (códons 83 e 87) e em parC (códon 80). Também foi pesquisada a presença dos genes qnr’s e aac(6’)-ib-cr e a associação destes com ESBLs. Duas das 55 amostras foram positivas para aac(6’)-ib-cr. A presença desse gene no Brasil foi relatada pela primeira vez em 2012, em Belo Horizonte, MG. Nenhuma amostra foi positiva para os genes qnr’s. Estes resultados mostram uma elevada incidência de resistência às quinolonas e a capacidade de transferência horizontal de resistência adquirida entre as cepas bacterianas.

Stenotrophomonas maltophilia é um bacilo Gram-negativo, não fermentador, considerado um microorganismo pouco virulento, relacionado principalmente a infecções associadas à assistência a saúde. A S. maltophilia apresenta um padrão de resistência intrínseca à maioria das classes de antibióticos. A droga de escolha para o tratamento das infecções por S. maltophilia é a sulfametoxazol/ trimetoprima (SMX/TMP). Entretanto, estudos atuais relatam o aumento da resistência a esse antibiótico, o que limita assim as opções para terapia efetiva. Outras opções de tratamento são o levofloxacino e a tigeciclina, porém, faltam estudos clínicos e in vitro dessas drogas. A proposta deste estudo foi avaliar os possíveis mecanismos de resistência a SMX/TMP e as quinolonas em isolados clínicos de pacientes internados no Instituto Central do Hospital das Clínicas e do Hospital A.C. Camargo. Foram avaliadas 106 amostras de S. maltophilia isoladas de pacientes adultos com infecção relacionada à assistência a saúde, internados no Instituto Central do Hospital das Clínicas da FMUSP e no Hospital de Câncer A.C Camargo durante o período de dezembro de 2008 a dezembro de 2010. A sensibilidade à SMX/TMP foi de 78,3%, para levofloxacino de 82% e 14,2% para cirpofloxacino, para minociclina de 100% e tigeciclina 91,6%. Foi realizado PCR para detecção dos genes sul1, sul2 e dfrA1 para avaliar a resistência à SMX/TMP, os genes int1 e iscr2 para avaliação da presença de elementos genéticos móveis e os genes gyrA, qnr, smeD, smeT e aac(6)-Ib-cr para avaliação da resistência as quinolonas. Quatorze amostras (13,2%) foram positivas para o gene sul1. Desses isolados, nove amostras apresentavam resistência ao SMX/TMP com CIM50 de 8 g/mL e CIM90 de 128 g/mL. Cinco amostras positivas para o gene sul1 foram sensíveis a SMX/TMP com CIM50 de 1 g/mL e CIM90 de 1 g/mL. A sequencia do integron1 da amostra com CIM >125 g/mL mostrou um tamanho aproximado de 4000 pb contendo os genes cassetes aac4 e aadA1 e a região qac/sul1. Uma amostra resistente a SMX/TMP foi positiva para o gene sul2 localizado na transposase-like ISCR 2. Observamos a presença de quatro novos qnr em cepas de S. maltophilia e a presença da enzima aac(6)-ib-cr em 4 amostras. 100% das cepas foram positivas para o gene do sistema de efluxo smeDEF e 12/38 amostras tiveram o gene smeT do sistema de efluxo smeDEF, ,porém não foi observada mutação nesse gene. Na sequencia de aminoácidos da girase A de 15 amostras resistentes a levofloxacino não observamos mutações relacionadas à resistência a quinolonas. As cepas resistentes a SMX/TMP apresentaram um padrão policlonal. Dezoito amostras resistentes ao levofloxacino apresentaram 14 perfis clonais, distribuídos em 10 clusters. S. maltophilia exibe múltiplos mecanismos de resistência, nesse estudo observamos um grande número de cepas com elementos genéticos móveis carregando o gene sul1 e outros genes de resistência. A S. maltophilia pode ser um importante reservatório de transmissão de genes de resistência / Stenotrophomonas maltophilia is a gram-negative, non-fermenter, considered a low virulent organism, mainly related to healthcare associated infections. S. maltophilia shows a pattern of intrinsic resistance to many classes of antibiotics. The drug of choice for the treatment of infections caused by S. maltophilia is SMX/TMP, however, current studies have reported increased resistance to this antibiotic, thus limiting the options for effective therapy. Among the treatment options appear tigecycline and levofloxacin, but clinical trials and studies in vitro to such drugs are lacking. The purpose of this study was to evaluate the possible mechanisms of resistance to SMX/TMP and quinolones in clinical isolates from patients admitted to the Institute\'s Central Clinical Hospital and the Hospital A.C Camargo. We evaluated 106 strains of S. maltophilia isolated from adult patients with healthcare associated infections, at the Instituto Central do Hospital das Clínicas and the Cancer Hospital AC Camargo in the period of December 2008 to December 2010. The sensitivity to SMX/TMP was 78.3%, 82% for levofloxacin, 14,2% for ciprofloxacin, minocycline 100% and for tigecycline 91.6%. PCR was performed for detection of gene sul1, sul2 and dfrA1 to evaluate the resistance to SMX/TMP, genes iscr2 int1 was performed to evaluate the presence of mobile genetic elements and genes gyrA, qnr, smeD , smeT and aac (6 \')-Ib-cr for evaluation of resistance to quinolones. Fourteen samples (13.2%) were positive for the gene sul1. In these isolates, nine samples showed resistance to SMX/TMP with MIC50 of 8 g/ml and MIC90 of 128 g/mL. Five strains were positive for sul1 gene and were susceptible to SMX/TMP with MIC50 of 1 g/ml and MIC90 of 1 g/mL. The sequence of the integron class 1 strain with an MIC> 125 g/mL showed an approximate size of 4000 bp containing the gene cassettes aadA1, aac4 and qac/sul1. A strain resistant to SMX/TMP was positive for the gene sul2 located on ISCR2 a transposase-like. We observed the presence of four new qnr in strains of S. maltophilia and the presence of the enzyme aac (6 \')-ib-cr in 4 samples. 100% of the strains were positive for the gene of the efflux system smeDEF and 12/38 samples had the gene smeT repressor of smeDEF efflux system, however there was no mutation in this gene. In the amino acid sequence of gyrase A of 15 strains resistant to levofloxacin did not observe mutations related to resistance to quinolones. Strains resistant to SMX/TMP had a polyclonal PFGE pattern. Eighteen strains resistant to levofloxacin showed 14 clonal profiles 14 divided into 10 clusters S. maltophilia displays multiple mechanisms of resistance. In this study, we observed a large number of strains with mobile genetic elements carrying the sul1 gene and other resistance genes. S. maltophilia is may be an important source for transmission of genes of resistance

Conselho Nacional de Desenvolvimento Cientifico e Tecnológico / Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / Neste trabalho foram sintetizados os heterociclos 3-carbetoxi-4(1H)-quinolona 98a-98o contendo substituintes flúor, cloro, bromo, iodo, metil, metoxi e nitro nas posições 6 ou 7, em rendimentos de 72 a 87%. Estes foram sililados com N,O-bis(trimetilsilil)trifluoracetamida (BSTFA) sendo posteriormente submetidos à reação de acoplamento com 1-O-acetil-2,3,5-tri-O-benzoil-&#946;-D-ribofuranose (49), sob catálise do ácido de Lewis trimetilsililtrifluormetanossulfonato (TMSO-Tf), obtendo-se os respectivos ribonucleosídeos 3-carbetoxi-1-(2,3,5-tri-O-benzoil-&#946;-D-ribofuranosil)-4(1H)-quinolonas 99a-99o em rendimentos 65 a 89%. A reação de O-desbenzoilação dos nucleosídeos 99, utilizando-se solução metanólica de carbonato de sódio levou aos ribonucleosídeos inéditos 3-carbometoxi-1-&#946;-D-ribofuranosil-4(1H)-quinolona correspondentes (100) em rendimentos de 70 a 77%. Os ribonucleosídeos quinolônicos 100 (6-F, Cl, Br, Me e 7-F) foram utilizados como precursores na obtenção de derivados nucleosídicos bromo-O-acetilados do tipo 90. Nestas reações os produtos resultantes da bromoacetilação de 100 (6-Cl, Br, Me) foram obtidos como mistura de regioisômeros 2(3)-Br, 3(2)-acetato, na proporção de (3:1), (5:1) e (3:1), respectivamente. As reações com os nucleosideos 100 (6-F e 7-F) levaram `a formação dos derivados 2,3,5-tri-O-acetilados. Contudo, a reação de bromoacetilação do ácido 6-metil-1-&#946;-D-ribofuranosil-4(1H)-quinolona-3-carboxílico levou ao bromoacetato 90e como único produto, em rendimento de 65%. Estes derivados bromo-O-acetilados foram submetidos a reação de &#946;-eliminação redutiva promovida por metais empregando-se duas metodologias. A primeira utilizando-se liga de zinco-cobre em DMF como solvente, e a segunda usando-se lítio metálico em THF sob ultra-som, porém estas reações não produziram os produtos olefínicos desejados. Contudo, a &#946;-eliminação redutiva de 90e empregando-se liga de zinco-cobre sob ultra-som, levou ao 2`,3`-didesidrodidesoxinucleosídeo esperado. A reação de desproteção regiosseletiva de nucleosídeos 2,3,5-tri-O-benzoilados (99), empregando-se metóxido de sódio em THF, levou aos ribonucleosídeos inéditos 5-O-benzoilados 103a-103c, em bons rendimentos. Estes compostos foram utilizados em tentativas de obtenção de derivados 2,3-tiocarbonatos cíclicos e 2,3-bisxantatos, sem que tenham sido obtidos os produtos esperados. Na pesquisa em busca de novos heterociclos pirimidoquinolínicos, com potencial atividade antiviral, as quinolonas 98 foram reagidas com uréia ou tiouréia em solução etanólica de hidróxido de sódio. Porém, as condições reacionais empregadas não foram satisfatórias, ocorrendo apenas a hidrólise do grupamento éster da posicao C3 do anel quinolonico. O mesmo procedimento foi aplicado aos nucleosídeos quinolônicos 99, obtendo-se neste caso os ribonucleosídeos 100 nos quais todos os grupos éster dos nucleosídeos originais foram hidrolisados, em rendimentos de 59 a 76%, o que nos levou a estabelecer metodologia adequada de obtenção de nucleosideos ácidos do tipo 100. Estes nucleosídeos foram submetidos a teste de atividade biológica frente ao vírus HSV-1 obtendo-se, obtendo-se bons resultados de inibição viral a uma concentração de 50 M, destacando-se os nucleosídeos contendo substituintes cloro e metil na posição 6 do anel quinolônico, que apresentaram um percentual de inibição igual a 99%. Paralelamente, foram realizadas as reações de N-alquilação dos heterociclos quinolônicos 98 levando às quinolonas N-etiladas 108 em rendimentos de 74 a 84%. Posteriormente, estas foram submetidas a reação de hidrólise básica obtendo-se os ácidos carboxílicos N-etilados do tipo 109 em rendimentos de 62 a 80%. A quinolona 108f (6-OMe) foi submetida a reação com tiouréia na presença de carbonato de potássio levando à obtenção do heterociclo inétido 6-etil-9-metoxi-4-oxo-2-tiono-2,3,4,6-tetraidropirimido[5,4-c]quinolina 94 em 72% de rendimento. Para obtenção de novos heterociclos quinolônicos contendo substituintes triazólicos, foram sintetizados as aminoquinolonas 108o e 108p em rendimentos de 70 e 72%, respectivamente, por redução das nitroquinolonas 108g e 108n. Inicialmente, estas foram submetidas a reação de conversão na enamina desejada, que produziria posteriormente o núcleo triazólico. Entretanto, não se obteve em ambos os casos as enaminas em questão. Assim, foi sintetizado o aminoacrilato 104p, em 75% de rendimento, o qual foi entao reagido com diazomalonaldeído, levando à formação do núcleo triazólico, tendo no entanto ocorrido subsequente reação de condensação entre o grupo aldeídico do anel triazólico e o grupo amino do aminoacrilato 104p, obtendo-se como produto final a imina 119 em 75% de rendimento. Esta imina foi então submetida a reação de hidrólise ácida, obtendo-se o acrilato triazólico 118 em 65% de rendimento. / In the present work the 6 and 7 substituted 3-carboethoxy-4(1H)-quinolones 98a-98o (fluorine, chlorine, bromine, iodine, methyl, methoxy and nitro) were synthesized in 72-87% yields. These quinolones were silylated by using bis-(trimethyl)trifluoroacetamide (BSTFA). Glycosylation of these silylated heterocycles was accomplished by their treatment with 1-O-acetyl-2,3,5-tri-O-benzoyl-&#946;-D-ribofuranose (49) in the presence of Trimethylsilyltrifluoromethanesulphonate ( TMSO-Tf), providing the desired ribonucleosides 3-carboethoxy-1-(2,3,5-tri-O-benzoyl-&#946;-D-ribofuranosyl)- 4(1H)-quinolones 99a-99o, in 65-89% yields. The de-O-benzoylation reaction of 99, using methanolic sodium carbonate solution led to the new 3-carbomethoxy-1- &#946;-D-ribofuranosyl)- 4(1H)-quinolones 100 in 70-77% yields. The 6-F,Cl, Br, Me and 7-F-ribonucleosides 100 were used as precursors in the synthesis of their respective bromoacethylated derivatives 90. In these reactions, the bromoacetylation of 100 (6-Cl, Br and Me) yielded the corresponding mixture of regioisomeric bromoacetates 3(2)-Br, 2(3)-O-acetyl, being 2-O-acetyl-3-Br derivative the majoritary one ( 3:1, 5:1 and 3:1, respectively). This reaction when applied to 6-fluoro and 7-fluoro ribonucleosides 100a and 100f afforded the corresponding 2,3,5-tri-O-acetylated derivatives. However, the same reaction when using 6-methyl-1-&#946;-D-ribofuranosyl-4(1H)-quinolone-3-carboxylic acid (100m) as the starting material produced the new 6-methyl-1-(2,5-di-O-acetyl-3-bromo-3-deoxy-&#946;-D-ribofuranosyl-4(1H)quinolone-3-carboxylic acid (90e) as the only regioisomer in 65% yield. The &#946;-Elimination reactions with these bromo-O-acetylated nucleosides were attempted by two methodologies. The first one using zinc-copper alloy in DMF and the second one using lithium in THF under ultrasound. These reactions failed to give the olefinec products. However, when the pure bromoacetate 90e was reacted with zinc-copper alloy, in DMF, under ultrasound, the desired 2`.3`-didesydrodidesoxy ribonucleoside was obtained. Regioselective deprotection of 2,3,5-tri-O-benzoylated nucleosides 99 by using sodium methoxide in THF led to the new 5-O-benzoylated ribonucleosides 103a-103c in good yields. These substances were employed in attempts to obtain 2,3-cyclic thiocarbonates or to obtain 2,3-bisxanthates, unsuccessfuly. In the search for new heterocyclic nucleosides having pyrimidoquinoline ring, with potential antiviral activity, the quinolones 98 were reacted with urea or thiourea in sodium hydroxide solution leding only to the product in which the ester moiety of the starting material was hydrolyzed The same procedure was applied to the nucleosides 99 affording the ribonucleosides 100 in which all the ester groups of the protected ribonucleosides XV were hydrolyzed (59-76% yields). Additional efforts to obtain new pyrimidoquinoline derivatives were done. The quinolones 98 were alkylated with ethyl bromide in DMF producing the corresponding N-ethylquinolones 108 in 74-84% yields. Subsequently basic hydrolysis of the esther group at C3 led to carboxylic acids derivatives XXI in 62-80% yields. The condensation of 108f (R=6-OMe) with thiourea in the presence of sodium carbonate resulted in the isolation of the new heterocycle 6-ethyl-9-methoxy-4-oxo-2-thione-2,3,4,6-tetrahydropyrimido[5,4-c]quinoline (94), in 72% yield. In the search of new quinolonic derivatives containing the tryazolic nucleous as substituint the aminoquinolones 108o and 108p were synthesized in 70 and 72% yields respectively, by reduction of the nitroquinolones 108g and 108n. In the first approach the amines were reacted with 1,3-dicaronylcompounds aiming to obtain the corresponding enamino esters, which would produce the tryazolic ring. However, this reaction failed to give the desired product. In order to overcome this problem, the aminoacrylate 104p was synthesized (75% yield), and subsequently reacted with diazomalonaldehyde forming the tryazolic moiety, followed by a condensation reaction leading to the iminoderivative (119), in 75% yield, which was hydrolyzed, under acid conditions, giving the tryazolic acrylate 118, in 65% yield.