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Paclitaxel is one of the most successful anti-cancer drugs, particularly in the treatment of breast cancer and ovarian cancer. For the investigation of the interaction between paclitaxel and MD-2 protein, and development of new antagonists for lipopolysaccharide, several C10 A-nor-paclitaxel analogs have been synthesized and their biological activities have been evaluated. In order to reduce the myelosuppression effect of the paclitaxel, several C3â ² and C4 paclitaxel analogs have been synthesized and their biological evaluation have been studied. / Master of Science

Today, the demand for speed in drug discovery is constantly increasing, particularly in the iterative processes of hit validation and expansion and lead optimization. Irradiation with microwaves (MWs) has been applied in the area of organic synthesis to accelerate chemical reactions and to facilitate the generation of new chemical entities since 1986. In the work presented in this thesis, the use of MW-mediated heating has been expanded to address three fields of drug discovery, namely hit expansion, chemical library generation and genomics. In the first project, potential inhibitors of malaria aspartic proteases were designed and synthesized, partly by MW-assisted organic chemistry, and evaluated with regard to their inhibitory efficacy on five malaria aspartic proteases and their selectivity over two human aspartic proteases. The synthetic work included the development of fast and convenient methods of MW-assisted formation of thiazolidines and epoxy esters. Some of the resulting structures proved to be efficacious inhibitors of the aspartic protease that degrades haemoglobin in all four malaria parasites infecting man. No inhibitor affected the human aspartic proteases. Expedient, two-step, single-operation synthetic routes to heterocycles of medicinal interest were developed in the second and third projects. In the former, the use of a versatile synthon, Ph3PCCO, provided α,β-unsaturated lactones, lactams and amides within 5–10 minutes. In the latter project, saturated lactams were formed from amines and lactones in 35 minutes, in the absence of strong additives. These two MW-mediated protocols allowed the reduction of the reaction time from several hours or days to minutes. In the fourth project, a fully automated MW-assisted protocol for the important enzyme-catalysed polymerase chain reaction (PCR) was established. In addition, the PCR reaction could be performed in unusually large volumes, 2.5 mL and 15 mL, with yields corresponding to those from conventional PCR. Good amplification rates suggested that the thermophilic enzyme, Taq polymerase, was not affected by the MW radiation.

No description available.

Infectious diseases cause substantial morbidity and mortality, exacerbated by increasing antibiotic resistance. In critically ill patients, recent studies indicate a substantial variability in β-lactam antibiotic levels when standardized dosing is applied. New methods for characterizing nosocomial outbreaks of bacterial infections are needed to limit transmission. The goals of this thesis were to investigate new strategies towards individualized treatment and control of bacterial infections.  In Paper I we confirmed high variability in β-lactam antibiotic levels among intensive care unit (ICU) patients from southeastern Sweden, where 45 % failed to reach treatment targets (100 % fT>MIC). Augmented renal clearance and establishing the minimum inhibitory concentration of the bacteria were important for evaluating the risk of not attaining adequate drug levels. In Paper II a rapid ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for simultaneous quantification of 11 commonly used antibiotics was developed and tested in clinical samples. Performance goals (CV<15%) were reached. A microbiological method for quantification of β-lactam antibiotics in serum was developed in Paper III. The method could be important for hospitals without access to an LC-MS method. Paper IV and Paper V investigated ligation-mediated qPCR with high resolution melt analysis (LMqPCR HRMA), for transmission investigation of extended spectrum β-lactamase (ESBL)-producing E. coli and other common bacterial pathogens. Results comparable to the reference method (PFGE) could be achieved within one day in a closed system and confirmed a nosocomial outbreak in Kalmar County. In Paper VI whole genome sequencing followed by bioinformatic analysis resolved transmission links within a nosocomial outbreak due to improved discriminatory power compared to LMqPCR HRMA. The high proportion of ICU patients with insufficient β-lactam drug levels emphasizes the need for individualized treatment by therapeutic drug monitoring (TDM). TDM is enabled by a highly sensitive method, such as UPLC-MS/MS, but if unavailable, also by a microbial method. Molecular typing methods used for transmission investigation can detect nosocomial outbreaks. LMqPCR HRMA can be used for screening purposes. For enhanced resolution, whole genome sequencing should be used, but always together with a rigorous epidemiological investigation.

(3R,5R)-Clavulanic acid is a clinically important inhibitor of Class A β-lactamases. Progress has been made in to establishing the steps of clavulanic acid biosynthesis leading to (3S,5S)-clavaminic acid. However, the mechanism by which (3S,5S)-clavaminic acid is converted to the penultimate intermediate (3R,5R)-clavaldehyde remains elusive. It is believed that the products of the later genes (orf10-orf18) of the clavulanic acid biosynthesis gene cluster are probably involved in this conversion. Part I of this thesis describes biochemical and structural studies carried out on OAT2, a member of N-terminal nucleophile (Ntn) hydrolase superfamily of enzymes. OAT2 has been characterised to be an ornithine acetyl transferase (OAT) and is involved in clavulanic acid biosynthesis. OAT2 catalyses the reversible transfer of the acetyl group between N-acetyl-L-ornithine and L-glutamate. It was found that this reaction is catalysed via the formation of an acyl-enzyme intermediate. Subsequent studies including mass spectrometry, 13C NMR spectroscopy, infrared spectroscopy, X-ray crystallography and molecular dynamics simulations, further confirmed the viability of the intermediate. This acyl-enzyme intermediate of OAT2 was found to be exceptionally stable at physiological pH, as compared to the acyl-enzyme intermediates involved in catalysis by hydrolytic enzymes including proteases, Ntn hydrolases and β-lactamses. The X-ray studies revealed possible reason for this unusual stability. The infrared studies revealed two conformations for the acyl-enzyme. Modeling (MDS) studies assigned one of these to the structure observed by X-ray and proposed the other one to result from a hydroxyl hydrogen 'flip' involving the oxyanion hole component Thr-111 resulting in a singly hydrogen bonded acyl-enzyme intermediate. α, β Subunit co-expression studies with OAT2 were used to investigate the autocatalytic cleavage step. In one case an interesting N-acyl enzyme species was observed. Part II of this thesis describes efforts carried out to characterise the ORF10 and ORF15 proteins of clavulanic acid biosynthesis. ORF10 was characterised to be an 'active' cytochrome P450 and ORF10 crystals were obtained in the presence spinach ferredoxin, highlighting the role of the ferredoxin interaction in assisting ORF10 crystallisation. ORF15 was shown to be a probable peptide transporter, which binds bradykinin as observed in the crystal structure.

This research developed validated QSPKR and PK-AS models for predicting human systemic PK properties of three, preselected, pharmacological classes of drugs, namely opioids, β-adrenergic receptor ligands (β-ARL) and β-lactam antibiotics (β-LAs) using pertinent human and animal systemic PK properties (fu,, CLtot, Vdss, fe) and their biologically relevant unbound counterparts from the published literature, followed by an assessment of the effect of different molecular descriptors on these PK properties and on the PK-AS slopes for CLtot and Vdss from two species (rat and dog). Lipophilicity (log (D)7.4) and molecular weight (MW) were found to be the most statistically significant and biologically plausible, molecular properties affecting the biologically relevant, systemic PK properties: For compounds with log (D)7.4 > -2.0 and MW < 350 D (e.g., most opioids and β-ARL), increased log (D)7.4 resulted in decreased fu and increased Vdssu, CLtotu and CLnonrenu, indicating the prevalence of hydrophobic interactions with biological membrane/proteins. As result, the final QSPKR models using log (D)7.4 provided acceptable predictions for fu, Vdssu, CLtotu and CLnonrenu. CLnonrenu and CLtotu. For both the datasets, inclusion of drugs undergoing extrahepatic clearance worsened the QSPKR predictions. For compounds with log (D)7.4 < -2.0 and MW > 350 D (e.g., β-LA), increased MW (leading to more hydrogen bond donors/acceptors) resulted in a decrease in fu, likely indicating hydrogen bonding interactions with plasma proteins. In general, it was more difficult to predict PK parameters for β-LAs, as their Vdssu approached plasma volume and CLrenu and CLnonrenu were low - as a result of their high hydrophilicity and large MW, requiring specific drug transporters for distribution and excretion. The PK-AS analysis showed that animal body size accounted for most of the observed variability (r2> 0.80) in systemic PK variables, with single species methods, particularly those using dog, gave the best predictions. The fu correction of PK variables improved goodness of fit and predictability of human PK. There were no apparent effects of molecular properties on the predictions. CLren, CLrenu, CLnonren, and CLnonrenu were the most difficult variables to predict, possibly due to the associated interspecies differences in the metabolism, renal and hepatobiliary drug transporters.

Ce travail de thèse a permis de développer un nouveau type de cycloaddition [3+2] des cétènes impliquant des aziridines. Les aziridines sont de bons précurseurs de dipôles 1,3-azotés zwittérioniques, via la coupure sélective de leur liaison C-N par activation avec un acide de Lewis. Nous avons montré qu'en présence d'un cétène et d'iodure de lithium, l'aziridine s'ouvre, et le dipôle 1,3 formé réagit avec le cétène pour offrir des gamma-lactames de manière très efficace. Cette nouvelle cycloaddition [3+2] s'étend à la transformation monotope d'une imine en gamma-lactame, stratégie compatible avec une large gamme d'imines aromatiques et de cétènes stables. Enfin, une synthèse monotope, catalytique et asymétrique de gamma-lactames énantioenrichis a été développée à partir d'oléfines par aziridination asymétrique avec des nitrènes. / This thesis work focuses on a new type of [3+2] cycloaddition of ketenes with aziridines. Aziridines are good precursors of zwitterionic 1,3-aza-dipoles, by selective C-N bond cleavage catalyzed by Lewis acid. We have found that ketenes react with the 1,3-dipole generated by addition of lithium iodide to the aziridine, efficiently providing the gamma-lactams. This new cycloaddition could be extended to a one-pot simple transformation of imines to gamma-lactams. The synthesis is compatible with a wide range of aromatic imines and stable ketenes. Finally, a one-pot catalytic asymmetric synthesis of enantioenrichied gamma-lactams have been developed starting from olefins, involving an asymmetric nitrene aziridination.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Em estudo anterior, as espécies de enterobactérias apresentando perfis variados de resistência aos antimicrobianos foram detectadas em 20% dos sítios com lesões periodontais de pacientes sadios do ponto de vista sistêmico. Tais cepas microbianas foram submetidas a investigações com o intuito de determinar à expressão de enzimas hidrolíticas para substratos diversos, a multirresistência aos agentes antimicrobianos e os mecanismos de resistência aos antimicrobianos da classe dos &#946; lactâmicos. A maioria das amostras expressou atividade de gelatinase (65%), caseinase (30%) e elastase (10%). Lipase, lecitinase e DNase foram observadas apenas para Serratia marcescens. A multirresistência (considerado como a resistência a pelo menos dois agentes antimicrobianos de famílias diferentes) foi observada em 56% das amostras isoladas. A maioria das cepas foi resistentes à ampicilina (93,75%) e amoxicilina/ácido clavulânico (81,25%). Investigações sobre a resistência aos antibióticos &#946;-lactâmicos mostraram que três amostras resistentes à cefalosporinas de 2 geração, apresentaram perfis plasmidiais de diferentes pesos moleculares. A expressão fenotípica de &#946;-lacatamases, foi detectada nas cepas de Enterobacter cloacae (PcOM46 e PcOM5) e S. marcescens (PcOM63). No entanto, na análise molecular, não foi possível confirmar a expressão fenotípica de diferentes &#946;-lactamases, com exceção do E. cloacae PcOM46, que apresentou amplificação para AmpC e blaTEM. Embora sensível à maioria dos antibióticos &#946;-lactâmicos (exceção feita à ampicilina e amoxicilina / ácido clavulânico), amostra de S. marcescens PcOM68 apresentou amplificação para o gene blaSHV. Os experimentos de conjugação não detectaram a transferência de plasmídios para uma cepa de Escherichia coli K12 sensívei aos &#946;-lactâmicos, o mesmo ocorreu nos procedimentos de transformação por eletroporação e por CaCl2, sugerindo uma resistência dependente de genes cromossomiais. A expressão de diferentes atividades enzimáticas, juntamente com a resistência aos antimicrobianos, aponta estes grupos de bactérias como agentes patogênicos potenciais capazes de contribuir para a patogênese e resposta à quimioterapia antimicrobiana nas doenças periodontais, além da disseminação sistêmica para outros locais do corpo, especialmente em indivíduos imunocomprometidos. A colonização prévia de lesões periodontais por espécies resistentes aos &#946;-lactâmicos, pode contribuir para a disseminação destes genes relacionados à resistência aos antimicrobianos em ambientes hospitalares. / In a previous study, the enterobacterial species were detected in 20% of systemically healthy patients presenting periodontal lesions, with different profiles of antimicrobial resistance. These microbial strains were investigated in view to determine the expression of hydrolytic enzymes for diverse substrates, multiresistance to antimicrobial agents, and the mechanisms of resistance to beta-lactam antibiotics. The enzymes related to bacterial virulence were detected phenotypically in 90% isolates. The proteolytic activity displayed by the isolates against gelatin, casein and elastin was detected in 65%, 30% and 10%, respectively. Lipase, lecithinase, and DNase were observed only for Serratia marcescens strains. Multi-resistant phenotypes (considered as the resistance to at least two antimicrobial agents from different families) were observed for 56% enterobacterial isolates. Most of the strains were resistant to ampicillin (93.75%) and amoxicillin/clavulanic acid (81.25%). Investigations concerned to the resistance to beta-lactam antibiotics demonstrated that three bacterial strains resistant to penicilins and 2nd generation cephalosporins, had detectable plasmids. The extended resistance to &#946;-lactams was detected phenotypically for E. cloacae PcOM46 and PcOM5) and S. marcescens (PcOM63) strains. However, the molecular detection of extended spectrum beta-lactamase failed to confirm the phenotypic expression of different &#946;-lactamases, with exception to the E. cloacae PcOM46 isolate, which presented amplification for both ampC and blaTEM. Although sensitive to most of the &#946;-lactam antibiotics (exception made to ampicilin and ampicilin/clavulanate), the strain S. marcescens PcOM68 was shown to amplify the blaSHV gene. Plasmid transference by both conjugation and transformation procedures failed to detect the resistance by a &#946;-lactam-sensitive strain (E. coli K12), suggesting a chromosomal dependent resistance. The expression of varied enzymatic activities along with the resistance to antimicrobial agents point these group of bacteria as potential pathogens that may contribute to both pathogenesis and antimicrobial management of periodontal diseases, and systemic dissemination to other body sites, specially in immunocompromised host. The previous colonization of periodontal lesions by &#946;-lactam resistant species may represent a threat for dissemination of genes related to antimicrobial resistance inside hospital environments.

Different catalytic approaches have been applied to new -lactams preparations. olefin metathesis has been successfully performed with 3--methylidene--lactams. It was verified that 3--methylidene--lactams olefin metathesis is applicable to preparation of biologically active -lactam of Ezetimibe-type.