Orientador: Marcos Hikari Toyama / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T09:14:55Z (GMT). No. of bitstreams: 1 Fagundes_FabioHenriqueRamos_D.pdf: 9330688 bytes, checksum: 3849f7f19f623ba248060731c3752b2e (MD5) Previous issue date: 2010 / Resumo: Mionecrose e edema são dois dos efeitos de fosfolipases A2 secretórias (sPLA2s) de serpents do genero Bothrops, BjVIII é uma nova Lys49 PLA2 miotóxica isolada do veneno de Bothrops jararacussu que exibe efeitos atípicos na agregação plaquetária humana e aumenta a secreção de insulina. Nestes estudos, demonstramos que BjVIII aumenta a liberação de insulina das células beta do pâncreas e induz a agregação plaquetária. Utilizando degradação de Edman e seqüenciamento de aminoácidos de novo por massa, nós determinamos a seqüência primária completa de BjVIII. Esta análise revelou uma mudança do aminoácido G35K na BjVIII, que difere da seqüência de BthTX-I, PrTX-I e PrTX-II e podem estar relacionados com as diferenças observadas na atividade biológica entre BjVIII e outras Lys49 sPLA2. Estes resultados indicam que além da região C-terminal e B-wing de PLA2, o laço de ligação do cálcio na BjVIII deve ser considerada uma importante região envolvida nos efeitos farmacológicos das isoformas Lys49 sPLA2 do gênero Bothrops. Para entender melhor o modo de ação da BjVIII, estudos cristalográficos foram iniciados. Duas formas de cristais foram obtidos, ambos contendo duas moléculas na unidade assimétrica (ASU). Dados de difração de radiação síncrotron foram coletados a 2,0 °A e 1,9 °A de resolução para os cristais que pertencem ao grupo espacial P212121 (a = 48:4 ° A, b = 65:3 ° A, c = 84:3 ° A) e grupo espacial P3121 (a = b = 55:7 º A, c = 127:9 ° A), respectivamente. Nós apresentamos uma caracterização cristalográfica detalhada de BjVIII. A estrutura foi resolvida em dois grupos de espaço diferentes, onde uma densidade de elétrons inesperada foi supostamente modelada no sítio ativo como uma molécula de ácido lisofosfatidico ao qual foi atribuído o efeito atípico de agregação plaquetária. Além disso, os estudos de bioinformática e comparações moleculares com outras PLA2s mostraram a conservação evolutiva dos mecanismos catalíticos e possibilitou a identificação do mesmo sitio miotóxico recentemente propostos para Lys49-PLA2s. Estrutura cristalográfica de BjVIII e análises do modelo realizadas em nossos estudos têm contribuído para uma melhor compreensão da ação farmacológica de fosfolipases A2. As cumarinas, são compostos químicos encontrados em muitas plantas e sintetizados em laboratórios, tem valor clínico, como precursor de vários fármacos anticoagulantes e antiinflamatórios. Nós caracterizamos os efeitos de duas cumarinas sintéticas, etil 2-oxo-2Hcromeno- 3-carboxilato (EHCC) e ácido 7-hidroxi-2-oxo-2H-cromeno-3-carboxílico (HHCC), sobre a estrutura, e as propriedades biológica e farmacológicas de BjVIII. Os resultados de espectroscopia de fluorescência intrínseca e estudos de dicroísmo circular indicaram que ambos os compostos induzem uma desestruturação parcial da proteína. A análise de aminoácidos revelou que o tratamento com EHCC (BjVIII-EHCC) e HHCC (BjVIII- HHCC) induzem a modificação de aminoácidos da BjVIII. Além disso, observamos uma redução do edema, mionecrose, agregação plaquetária, estimulação da secreção de insulina e atividade antibacteriana induzida por BjVIII após o tratamento com essas cumarinas. Tomados em conjunto, nossos resultados indicam que EHCC e HHCC induzem uma modificação estrutural irreversível na BjVIII que leva a uma diminuição nas atividades farmacológicas e biológicas induzidas por BjVIII nativa. / Abstract: Myonecrosis and edema are two of the effects of the secretory phospholipases A2 (sPLA2s) of Bothrops jararacussu snake venom, BjVIII is a new myotoxic Lys49-PLA2 isolated from Bothrops jararacussu venom that exhibits atypical effects on human platelet aggregation and increases insulin secretion. In these studies, we demonstrate BjVIII also enhances insulin release from pancreatic beta cells and induces platelet aggregation. Using both Edman degradation and de novo amino acid sequencing by mass, we determined the complete primary sequence of BjVIII. This analysis showed a G35K amino acid change in BjVIII, which differs from the sequences of BthTx-I, PrTx-I, and Prtx-II and may relate to the observed differences in biological activities among BjVIII and other Lys49 sPLA2. These results indicate that besides the C-terminal region and B-wing of PLA2, the calcium binding loop in BjVIII should be considered an important region involved in the pharmacological effects of Lys49 sPLA2 isoforms from the Bothrops genus. To better understand the mode of action of BjVIII, crystallographic studies were initiated. Two crystal forms were obtained, both containing two molecules in the asymmetric unit (ASU). Synchrotron radiation diffraction data were collected to 2.0 °A resolution and 1.9 °A resolution for crystals belonging to the space group P212121 (a = 48:4 ° A, b = 65:3 ° A, c = 84:3 ° A) and space group P3121 (a = b = 55:7 ° A, c = 127:9 ° A), respectively. We present a detailed crystallographic characterization of BjVIII. Structure was solved in two different space groups where an unexpected electron density in the active site was putatively modeled as a lysophosphatidic acid molecule to which has been attributed the atypical platelet aggregation effect. In addition, bioinformatics studies and molecular comparisons with other PLA2s have shown the evolutionary conservation of the catalytic machinery and enabled the identification of the same myotoxic site recently proposed for Lys49-PLA2S. BjVIII crystallographic structures and model analyses performed in our studies have contributed to a better understanding of the pharmacological action of phospholipases A2. Coumarin, a chemical compound found in many plants and synthesized in chemical laboratories, has clinical value as the precursor for several experimental anticoagulants and anti-inflammatory drugs. We characterized the effects of two synthetic coumarins, ethyl 2-oxo- 2H-chromene-3-carboxylate (EHCC) and 7-hydroxy-2-oxo-2H-chromene-3-carboxylic acid (HHCC), on the structural, biological, and pharmacological properties of BjVIII. The spectroscopic results from intrinsic fluorescence and circular dichroism studies indicated that both compounds induced partial protein unfolding. Amino acid analysis revealed that treatment with EHCC (BjVIII-EHCC) and HHCC (BjVIII-HHCC) induced amino acid modification of BjVIII. Furthermore, we observed a reduction of BjVIII-induced edema, myonecrosis, platelet aggregation, stimulation of insulin secretion, and antibacterial activity following treatment with these coumarins. Taken together, our results indicate that EHCC and HHCC induce an irreversible structural modification in the folding that leads to a decrease in pharmacological and biological activities induced by native BjVIII. / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

A formação de complexos antígeno-anticorpo ou imunocomplexos (ICs) na circulação e sua eliminação faz parte dos mecanismos de defesa imune humoral do ser humano. Em algumas patologias, como lupus eritematoso sistêmico, artrite reumatóide e vasculite auto-imune, ocorre um desequilíbrio nesse processo, que leva à deposição dos ICs nos tecidos e ao desencadeamento de uma reação inflamatória. Esta, por sua vez, envolve o recrutamento e ativação de neutrófilos, que têm importante participação na patogênese dessas doenças. A ativação dos neutrófilos pelos ICs, via receptores para a porção Fc de IgG (FcR) e receptores de complemento (CR), desencadeia diversas funções efetoras, tais como fagocitose, desgranulação e o metabolismo oxidativo, com a produção de espécies reativas de oxigênio (EROs). Estas funções estão envolvidas na digestão dos ICs, na morte de microorganismos, e na regulação do processo inflamatório. Entretanto, nas doenças mediadas por ICs, os neutrófilos ativados liberam grandes quantidades de enzimas e EROs para o meio extracelular, contribuindo para a lesão dos tecidos do hospedeiro e a amplificação do processo inflamatório. Neste trabalho foi avaliado o efeito modulatório de vinte derivados de 3-fenilcumarina nas funções de neutrófilos estimulados por ICs de ovalbumina (OVA) e IgG anti-OVA. Além disso, foi feita a investigação mecanismos de ação dessas substâncias e a análise da relação estrutura-atividade. O metabolismo oxidativo dos neutrófilos ativados por ICs foi medido por ensaio de quimioluminescência dependente de lucigenina ou de luminol (QLlucPMN e QLlumPMN, respectivamente). Observou-se que as 3-fenilcumarinas contendo o grupo substituinte 3,4-metilenodioxi e o grupo substituinte 6,7-orto-diidroxi (C13) ou 6,7-orto-diacetoxi (C13a), bem como a 3-fenilcumarina 6,7,3,4-tetraacetoxilada (C24a), apresentaram atividade inibitória maior que a quercetina (QUER) sobre a QLlucPMN e a QLlumPMN. Para as demais substâncias avaliadas, que foram tão ou menos ativas que a QUER, as características estruturais relacionadas à inibição da QLlucPMN foram um pouco diferentes daquelas relacionadas à inibição da QLlumPMN. Além disso, as 3-fenilcumarinas estudadas e a QUER não apresentaram efeito tóxico sobre os neutrófilos, avaliado pela liberação de lactato desidrogenase e pelo ensaio de exclusão ao corante Azul de Tripan, nas condições empregadas. Para as três 3-fenilcumarinas que apresentaram maior efeito inibitório sobre o metabolismo oxidativo dos neutrófilos (C13, C13a e C24a), o aumento do tempo de pré-tratamento levou a uma tendência de redução do efeito inibitório da substância C24a, mas não influenciou na atividade biológica das substâncias C13 e C13a. Essas três substâncias não interferiram na capacidade fagocítica das células, avaliada por microscopia eletrônica de transmissão. Para todas as 3-fenilcumarinas foi avaliada também a capacidade antioxidante frente ao radical livre 2,2-difenil-1-picril-hidrazil e o efeito inibitório dessas substâncias sobre a quimioluminescência produzida pela reação horseradish peroxidase-H2O2-luminol (QLHRP). Foi observado que a QUER e as 3-fenilcumarinas contendo o grupo substituinte orto-diidroxi (C13, C23, C24) tiveram atividade antioxidante e inibiram a QLHRP, mas suas análogas acetoxiladas (C13a, C23a, C24a), bem como as demais substâncias avaliadas, foram significativamente menos ativas nesses modelos experimentais não celulares. O conjunto de resultados deste trabalho sugere que as atividades biológicas das 3-fenilcumarinas estudadas foram dependentes de suas estruturas químicas, e pequenas modificações nestas podem levar a alterações significativas na magnitude de seus efeitos biológicos. Além disso, tanto a lipofilicidade das substâncias quanto a sua capacidade antioxidante parecem ser relevantes para a modulação eficiente do metabolismo oxidativo dos neutrófilos e da conseqüente lesão tecidual. / Formation and clearance of circulating antigen-antibody complexes or immune complexes (ICs) take part in the humoral immune defense mechanisms. In some diseases, as systemic lupus erythematosus, rheumatoid arthritis and auto-immune vasculitis, an imbalance of this process occurs, leading to the ICs deposition within tissues and triggering an inflammatory reaction. The last one involves the recruitment and activation of neutrophils, which have an important role in the pathogenesis of such diseases. Neutrophil activation by ICs, via receptors for the Fc portion of IgG (FcgR) and complement receptors (CR), triggers a sort of effector functions, such phagocytosis, degranulation and the oxidative metabolism, which produces reactive oxygen species (ROS). These functions are involved in the ICs digestion, microbial killing and the inflammatory process regulation. However, the activated neutrophils release large amounts of enzymes and ROS to the extracellular milieu, contributing to the tissue damage and amplification of the inflammatory process in the IC-mediated diseases. In this work, we evaluated the modulatory effect of twenty 3-phenylcoumarin derivatives in the neutrophil functions stimulated by ICs of ovalbumin (OVA) and IgG anti-OVA. In addition, the mechanisms of action of these compounds were investigated, and the structure-activity relationship was analyzed. The IC-activated neutrophil oxidative metabolism was measured by lucigeninor luminol-dependent chemiluminescence assay (CLlucPMN and CLlumPMN, respectively).It was observed that the 3-phenylcoumarins bearing a 3,4-methylenodioxy and the 6,7-orto-dihydroxy (C13) or the 6,7-orto-diacetoxy (C13a) group, as well as the 6,7,3,4-tetraacetoxylated 3-phenylcoumarin (C24a), inhibited CLlucPMN and CLlumPMN more than quercetin (QUER). Regarding the other evaluated compounds, whose inhibitory effects were similar to or lower than QUER, the structural features related to the CLlucPMN inhibition were different from those related to the CLlumPMN inhibition. Moreover, the studied 3-phenylcoumarins and QUER had no toxic effects on neutrophils, as evaluated by lactate dehydrogenase release and Trypan Blue exclusion, under the assessed conditions. With respect to the three 3-phenylcoumarins that had the highest inhibitory effects on the neutrophil oxidative metabolism (C13, C13a and C24a), the increase of the cell pre-treatment period showed a tendency to decrease the inhibitory ability of compound C24a, but did not influence the biological activity of compounds C13 and C13a. These three compounds did not interfere in the neutrophil phagocytic ability, as evaluated by transmission electron microscopy. The antioxidant activity against the 2,2-diphenyl-1-picrylhydrazyl free radical and the inhibitory effect in the chemiluminescence generated by the horseradish peroxidase-H2O2-luminol reaction (CLHRP) were evaluated for all 3-phenylcoumarins. It was found that QUER and those 3-phenylcoumarins bearing the orto-dihydroxy group (C13, C23, C24) had antioxidant activity and inhibited the CLHRP. However, their acetoxylated analogues (C13a, C23a, C24a) and the other evaluated compounds were significantly less active on these cell-free experimental models. Taken together, the results of the present work suggest that the biological activities of the 3-phenylcounarins here investigated were dependent on their chemical structures, and small changes on the molecule can lead to significant changes on the magnitude of their biological effects. Moreover, both lipophilicity and antioxidant capacity of these compounds seem to be relevant to an efficient modulation of the neutrophil functions and the consequent tissue damage.

Os neutrófilos são células fagocíticas do sistema imune inato com mecanismos especializados de digestão de patógenos, complexos imune e detritos celulares, que são mediados principalmente por espécies reativas de oxigênio (EROs) e enzimas proteolíticas. Entretanto, a ativação maciça de neutrófilos leva a uma liberação exacerbada de enzimas e EROs para o meio extracelular, o que pode ultrapassar a capacidade de defesa tecidual, composta por antioxidantes e antiproteinases, e lesar o tecido, bem como amplificar o processo inflamatório observado em algumas doenças inflamatórias, autoimunes e infecciosas. O envolvimento de neutrófilos na fisiopatologia de tais doenças tem atraído o interesse na pesquisa de novas substâncias com propriedades antioxidantes e imunomodulatórias. Neste trabalho, foi avaliado o efeito modulatório de onze derivados de cumarinas hidroxiladas e acetoxiladas sobre duas funções efetoras de neutrófilos humanos (produção de EROs e desgranulação), bem como a citotoxicidade dessas substâncias. Além disso, a relação estrutura-atividade foi analisada. Para tais investigações, o sangue venoso foi coletado de voluntários saudáveis e os neutrófilos foram isolados pelo método da gelatina. O metabolismo oxidativo dos neutrófilos foi desencadeado por zimosan opsonizado com soro humano normal (ZIops) ou forbol-12-miristato-13-acetato (PMA), e a resposta celular foi avaliada pelos ensaios de quimioluminescência dependente de lucigenina (QLluc) ou de luminol (QLlum). Para a realização desses ensaios, foram padronizadas as seguintes condições experimentais: concentração das sondas luminol e lucigenina; concentração do solvente dimetilsulfóxido; tempo de leitura da QLuc e QLlum. Posteriormente, a atividade antioxidante das cumarinas frente ao radical 2,2-difenil-1-picrilhidrazil (DPPH) foi avaliada espectrofotometricamente em 510 nm. Além disso, foi avaliado o efeito das cumarinas na desgranulação dos neutrófilos induzida por n-formil-metionil-leucil-fenilalanina (fMLP), utilizando-se a enzima elastase como marcador, bem como o efeito dessas substâncias na atividade da elastase liberada pelos neutrófilos. Ambos os ensaios foram realizados através da quantificação de p-nitroanilina liberada após a quebra de um substrato específico para essa enzima, em 405 nm. A toxicidade das cumarinas sobre os neutrófilos foi avaliada pela exclusão do azul de tripan e pela medida da liberação de lactato desidrogenase. Observou-se que, tanto para as células estimuladas por ZIops quanto por PMA: (i) a maioria das cumarinas inibiu a QLluc e a QLlum de maneira dependente da concentração, sendo que as mais ativas (C, D) possuíam grupos orto-diidroxi; (ii) quatro cumarinas (A, B, F, G) inibiram a QLluc, mas aumentaram a QLlum; (iii) a cumarina não-substituída (K) não teve efeito modulatório significativo sobre a QLlum ou QLluc; (iv) ordem de efeito inibitório das demais substâncias (E, H, I, J) foi dependente do número e posição dos grupos substituintes, bem como do tipo de sonda quimioluminescente (luminol ou lucigenina) e do estímulo utilizado. Verificou-se também que três cumarinas (C, D, H) tiveram atividade antioxidante significante frente ao radical livre DPPH. Além disso, quatro das substâncias avaliadas (A, B, E, G) inibiram a desgranulação dos neutrófilos, mas nenhuma delas (A - K) interferiu na atividade da elastase. A análise do conjunto de resultados obtidos sugere que o número e posição dos grupos hidroxil e acetil no esqueleto cumarínico foram importantes para a modulação do metabolismo oxidativo de neutrófilos humanos, sendo que, dependendo do tipo de EROs medida, tais características estruturais podem levar a um efeito anti- ou pró-oxidante. Além disso, o efeito modulatório das cumarinas sobre as funções efetoras dos neutrófilos foi dependente o tipo de estímulo utilizado, e não foi mediado pela toxicidade dessas substâncias, nas condições ensaiadas. Sendo assim, os resultados obtidos podem auxiliar no entendimento dos requisitos estruturais das cumarinas necessários para uma modulação eficiente das funções efetoras dos neutrófilos envolvidas em doenças inflamatórias. / Neutrophils are phagocytic cells from the innate immune system with highly developed mechanisms for intracellular digestion of pathogens, immune complexes and cell debris, which are mainly mediated by reactive oxygen species (EROs) and proteolytic enzymes. However, massive neutrophil activation lead to release of large amounts of enzymes and EROs to the extracellular milieu, that may overpower the tissue defense systems, composed of antioxidants and antiproteinases, and damage the tissue, as well as contribute to the amplification of the inflammatory process found in some inflammatory, autoimmune and infectious diseases. The involvement of neutrophils in the physiopathology of such diseases has attracted the interest in the search of new compounds with antioxidant and immunomodulatory properties. In this work, we evaluated the modulatory effect of eleven hydroxylated and acetoxylated coumarin derivatives in two effector functions of human neutrophils (EROs production and degranulation) as well as the cytotoxic effects of these compounds. In addition, the structure-activity relationship was analyzed. Venous blood was collected from healthy volunteers and neutrophils were isolated by the gelatin method to perform our experiments. The neutrophil oxidative metabolism was triggered by normal human serum-opsonized zymosan (ZIops) or phorbol-12-myristate-13-acetate (PMA), and the cellular response was evaluated by the lucigenin (QLluc)- or luminol (QLlum)-amplified chemiluminescence assays. In order to conduct this study, the following experimental conditions had to be established: concentration of the chemiluminescence probes luminol and lucigenin; concentration of the solvent dimethylsulfoxide; the QLluc and QLlum reaction time. Afterwards, the antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) was evaluated spectrophotometrically at 510 nm. Moreover, the effect of coumarins in the n-formyl-metionyl-leucyl-phenylalanine (fMLP)-induced neutrophil degranulation was evaluated by using elastase as marker, and the effect of these compounds in the elastase activity were also evaluated. Both assays were performed by measuring the elastase-mediated p-nitroaniline release from a specific substrate (405 nm). Toxicity of coumarins to the neutrophils was evaluated by trypan blue exclusion and measurement of lactate dehydrogenase release. Considering both, ZIops and PMA-stimulated neutrophils, we observed that: (i) most of coumarins inhibited the QLluc and the QLlum in a concentration-dependent manner, being the orto-dihydroxylated (C, D) the most active ones; (ii) four coumarins (A, B, F, G) inhibited the QLluc but increased the QLlum; (iii) the unsubstituted coumarin (K) had no significant modulatory effect on QLlum or QLluc; (iv) the rank order of inhibitory effect among the other compounds (E, H, I, J) was dependent on the number and position of substituents, as well as the type of chemiluminescent probe (luminol or lucigenin) and stimulus used. In addition, we observed that three coumarins (C, D, H) had a significant antioxidant activity against DPPH, and four compounds (A, B, E, G) inhibited the neutrophil degranulation, but none of the tested coumarins (A - K) interfered in the elastase activity. Taken together, our results suggest that the number and position of the hydroxyl and acetyl groups in the coumarin moiety were important to modulate the human neutrophil oxidative metabolism. Depending on the type of EROs measured, such structural features can lead to an anti- or pro-oxidant effect. Furthermore, the modulatory effect of coumarins on the neutrophil effector functions was dependent on the type of stimulus used, but it was not mediated by toxicity of these compounds to the neutrophils, under the assessed conditions. Therefore, the results described herein may be helpful to understand the structural requirements of coumarins to reach an efficient modulation of the neutrophil effector functions involved in inflammatory diseases.

A series of the Baylis-Hillman adducts have been obtained by reacting protected O-benzylated and unprotected substituted salicylaldehydes with methyl acrylate or tertbutyl acrylate, respectively, using DABCO as catalyst. Treatment of the Baylis-Hillman adducts with HCl in a mixture of acetic acid and acetic anhydride afforded the corresponding 3-(chloromethyl)coumarin derivatives with yields of up to 94%. Similar use of HI afforded the corresponding 3-(iodomethyl)coumarins but, depending on the reaction time, the reduced 3-methyl analogues could also be obtained. Arbuzov reactions of the 3-(halomethyl)coumarin derivatives have been undertaken to afford 4-phosphorylated and 1’-phosphorylated derivatives, regioselectivity being dependent on the halide-leaving group. The 3-(chloromethyl)coumarin derivatives have been subjected to nucleophilic (SN) attack by benzylamine to give the corresponding 3- [(benzylamino)methyl]coumarin derivatives in yields of up to 74%. Further treatment of the 3-[(benzylamino)methyl]coumarin derivatives with chloroacetyl chloride afforded the chloroacetamide derivatives, which exhibit hindered rotation about the amine C(O)-N bond. The acetamide derivatives have also been subjected to Arbuzov reaction conditions to afford the phosphorylated derivatives in yields of up to 86%. In a preliminary modelling study, hydrolysed analogues of the synthesized phosphorylated derivatives have been docked into the active site of the HIV-1 protease enzyme using the Cerius-2 Ligandfit software module to provide an insight into potential receptor-ligand hydrogen bonding interactions.

In dieser Arbeit wurde die gegenseitige Beeinflussung von mizellaren Strukturen und Photodimerisierung von Cumarinderivaten untersucht. Als Mizellbildner wurden das kationische Cetyltrimethylammoniumbromid (CTAB) und das nicht-ionische Triton X-100, als Solubilisate Cumarinderivaten verwendet. Es wurden rheologische, thermodynamische und photochemische Untersuchungen durchgeführt. Die Viskosität der beiden Tensidsysteme steigt mit zunehmender Solubilisatkonzentration je nach Cumarinderivat unterschiedlich stark an. Dabei zeigten 6-Alkylcumarine als Solubilisate die größten "rheologischen Effekte". Die Photodimerisierung der solubilisierten Cumarine führte zu einem weiteren Anstieg oder zu einer Abnahme der Viskosität der CTAB- und Triton X-100-Lösungen - "photorheologischer Effekt". In Abhängigkeit von der Konzentration werden Strukturänderungen der mizellaren Aggregate induziert, die teilweise zu drastischen Änderungen der makroskopischen Eigenschaften der Tensidlösungen-Lösung führen. Die Photodimerisierung fungiert dabei als "large-response-trigger". Die Kettenlänge der solubilisierten 6-Alkylcumarine beeinflusst das Fießverhalten der CTAB-Lösungen und Triton X-100-Lösungen. Mit zunehmender Kettenlänge steigt die Viskosität der beiden Tensidlösungen zunächst an, um dann bei noch längeren Ketten wieder abzunehmen. In beiden Tensidsystemen liegt der Maximalwert bei 6-Oktylcumarin. Der Photodimerisierung beeinflusst die Viskosität der beiden Tensid-Systeme unterschiedlich. In CTAB-Lösungen in Anwesenheit von kürzeren Alkylcumarinen steigt die Viskosität mit der Photodimerisierung weiter. Ab Pentylcumarin nimmt die Viskosität nach der Photodimerisierung jedoch ab. Im Triton X-100 System ist der photorheologische Effekt dagegen stets positiv. Thermodynamische Untersuchungen zeigen eine Korrelation mit dem rheologischen Effekt: Je größer die Viskosität, desto negativer sind Enthalpie- und Entropiedifferenzen bei der Bildung der mizellaren Aggregate. Die Ergebnisse legen als Ursache für die Viskositätseffekte Unterschiede in der Ausdehnung der Hydratwasserschicht der Mizellen nahe. Bei der photochemischen Untersuchungen wurden mit Hilfe von NMR-Spektroskopie und Kristallstrukturanalyse die isomeren Produkte der Photodimeriserung von Cumarinderivaten charakterisiert und die Produktverteilung unter verschiedenen Bedingungen ermittelt. In CTAB-Lösung konnte eine Möglichkeit Steuerung der Selektivität der Produktbildung gefunden werden: Lange 6-Alkylsubstituenten (ab 6-Propylcumarin) steuern zu Anti- und zu KK-Dimeren, während bei kurzen Ketten bzw. beim unsubstituierten Cumarin mehr Syn- und KS-Dimere gebildet werden. Bei dem im Detail untersuchten 6-Methylcumarin steigt die relative Quantenausbeute mit fallender Temperatur, abnehmender Konzentration und mit Zugabe des Triplettsensibilisators Benzophenon. In Gegenwart des Sensibilisators entsteht vermehrt das Anti-KK-Dimer, was darauf schließen lässt, dass dieses aus dem Triplett-Zustand gebildet wird. In ionischer mizellarer Lösung CTAB bilden sich bevorzugt Syn-Dimere. In unpolaren Lösungsmitteln entsteht nur Anti-KK, während in polaren Lösungsmitteln auch kleine Mengen Syn-Dimere gebildet werden. Die Lewis-Säure BF3 beschleunigt die Photodimerisierung deutlich und steuert zu Syn-KS.

3-bromophenyl 6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylate (IK9) was recently reported to be a potent inhibitor of cancer cell invasion and angiogenesis. It markedly reduced in vitro invasion of human HT1080 fibrosarcoma cells through collagen-coated porous membranes (Boyden chamber assay) and in vivo tumour growth in athymic nude mice. It was furthermore able to decrease angiogenesis ex vivo in a rat aortic ring assay and in vivo in a choroidal neovascularisation mice model. It nevertheless presents some water solubility and stability problems, which should be taken into account for further investigations. In the first part of the project, we synthesized original IK9 derivatives, modulated at the 3- and 6-positions, by introducing functional groups able to improve water solubility and metabolic stability. Their anti-invasive potency was screened in the Boyden chamber assay and the generated results highlighted some structure-activity relationships. A second part of the project was devoted to the elucidation of the actually unknown mechanism of action of IK9. Anti-invasive or anti-proliferative effects against endothelial cells, main actors of the angiogenic process, were not emphasised. We showed that IK9 acts likely not as an inhibitor of receptor tyrosine kinases (EGFR, PDGFR and VEGFR). The compound generates a weak decrease of mRNA coding for metalloproteinases (MMPs) 2 and 9, and on the other hand a substantial diminution of MMP 2 and 9 secretions by HT1080 fibrosarcoma cells. In conclusion, the consideration of anti-invasive properties together with the worked out solubility and stability profiles highlights several series, notably 6-hydroxycoumarins, 6-hydroxymethylcoumarins and coumarin-3-sulfonamides, whose interest as potential successors to IK9 is undeniable. Le 6-acétoxyméthyl-2-oxo-2H-1-benzopyrane-3-carboxylate de 3-bromophényle (IK9) est un dérivé coumarinique décrit comme inhibiteur puissant de linvasion tumorale et de langiogenèse. Il inhibe linvasion des cellules HT1080 de fibrosarcome humain in vitro à travers une membrane poreuse recouverte dune couche de collagène (test en « chambres de Boyden ») et la croissance tumorale in vivo chez des souris athymiques nues. Il est par ailleurs capable de bloquer langiogenèse, à la fois dans un modèle ex vivo danneaux daorte de rats et dans un modèle in vivo de néovascularisation choroïdale chez la souris. Il présente pour autant une problématique dhydrosolubilité et de stabilité, dont il faudra tenir compte lors dinvestigations futures. Dans une première partie du projet, nous avons synthétisé des dérivés originaux de lIK9, modulés en position 3 et 6 du noyau coumarinique, en introduisant des fonctions susceptibles daugmenter lhydrosolubilité et la stabilité métabolique des molécules obtenues. Leur pouvoir anti-invasif a été évalué dans le test en « chambres de Boyden », ce qui nous a permis de mettre en évidence différentes relations structure-activité. Une deuxième partie du projet fut consacrée à létude du mécanisme daction de lIK9, qui est non identifié jusquà présent. Un effet anti-invasif ou anti-prolifératif envers les cellules endothéliales, actrices principales du processus dangiogenèse, na pu être observé. LIK9 nagit vraisemblablement pas en tant quinhibiteur de plusieurs récepteurs de type tyrosine kinase (EGFR, PDGFR et VEGFR). Le composé engendre une légère baisse de lexpression de lARNm codant pour les métalloprotéases matricielles (MMPs) 2 et 9 mais par contre entraîne une diminution substantielle de la sécrétion des MMPs 2 et 9 par les cellules HT1080 de fibrosarcome humain. En conclusion, la prise en compte simultanée de lactivité anti-invasive, de lhydrosolubilité et de la stabilité met en avant plusieurs séries de dérivés, notamment des 6-hydroxycoumarines, des 6-hydroxyméthylcoumarines et des coumarine-3-sulfonamides, dont lintérêt en tant que successeurs potentiels de lIK9 est indéniable.

Made available in DSpace on 2014-07-29T16:11:54Z (GMT). No. of bitstreams: 1 DISSERTACAO MARCIO ANDRE DE PAULA FARMACIA.pdf: 930244 bytes, checksum: 84ee19ed6dd9d6a0b3f3414561eb6a1b (MD5) Previous issue date: 2009-08-26 / Celtis iguanaea (Jacq.) Sargent is a small postage plant belonging to the Ulmaceae family, it has very flexible branches, usually in a crossed and overcoated form armed with spines. The leaves are short-petioled, ovals and have sizes that vary from 5-13 cm long and 3-7 cm wide. The tea obtained through it leaves is popularly used for body pain, rheumatism, chest pain, asthma, cramps, poor digestion, and as diuretic. Samples were collected in Campestre-GO, a exsiccates identified and deposited in the Herbarium of UFG under number 40110. It was performed macro and microscopic analysis and part of the botanical material was dried and ground to testing for moisture, total ash and acid insoluble ash, phytochemical screening and obtaining the aqueous extract of leaves of esporão-de-galo (EAEG). The EAEG was obtained by infusion of 3% of the powder of the leaves. Swiss mice (male adult, weighing between 25 to 35 g) were pre-treated orally with EAEG (70, 200 and 600 mg / kg), vehicle (water filtered 10 mL / kg) and ranitidine (50 mg / kg) before induction of gastric lesions by indomethacin (30 mg / kg sc), ethanol 75% (v / v) and by restraint stress and hypothermia. The effects of the extract on the volume, pH and total acidity of gastric secretion were evaluated by the method of pyloric ligation, and the treatments were performed by intraduodenal administration (id). It also assessed the influence of intestinal transit EAEG in animals with previous treatments made orally.. In macroscopic analysis were confirmed common characteristics of the species. In the microscopic analysis it was observed structures, such as: short and long trichomes, unicellular and pluricellular, epidermal cells of various sizes, cells containing druses, prismatic crystals and cystoliths. In phytochemical prospecting was detected the presence of mucilage and secondary metabolites as coumarins and flavonoids. The efficiency of the extraction process was 20%. In models of ulcers induced by different agents: indomethacin, ethanol and stress in doses of 70, 200 and 600 mg/kg, there was reduction in the number of injuries caused by these agents. When the EAEG was administered by intraduodenal route, there was reduction in the volume of total and free acidity of gastric secretion in mice with ligation of pylorus. In assessing the influence of EAEG on intestinal transit was observed increase only with the dose increased to 600 mg/kg. The results suggest that the EAEG contains active ingredients gastro protectors that do not reduce intestinal motility and may justify the popular use of the plant to gastritis and ulcers / A Celtis iguanaea (Jacq.) Sargent é uma planta de pequeno porte pertencente à família Ulmaceae, possui ramos muito flexíveis, geralmente em forma de zigue-zague, compridos e armados de espinhos. As folhas são curto-pecioladas, ovado-oblongas ou ovadas e possui tamanhos que podem variar de 5-13 cm de comprimento e 3-7 cm de largura. O chá das folhas desta planta é usado popularmente para dores no corpo, reumatismo, dores no peito, asma, cólicas, mádigestão e como diurético. Amostras das folhas foram coletadas em Campestre-GO, identificadas e uma exsicata depositada no Herbário da UFG sob o nº 40110. Foram realizadas análises macro e microscópicas e parte do material botânico foi seco e triturado para a realização dos testes de umidade, cinzas totais e insolúveis em ácido, triagem fitoquímica e obtenção do extrato aquoso das folhas do esporão-degalo (EAEG). O EAEG foi obtido por infusão a 3% do pó das folhas. Camundongos Swiss (machos, adultos, pesando entre 25 35 g) foram pré-tratados oralmente com EAEG (70, 200 e 600 mg/kg), veículo (água filtrada 10 mL/kg) e ranitidina (50 mg/kg), antes da indução das lesões gástricas por endometacina (30 mg/kg s.c.), etanol 75% (v/v) e estresse por contenção e hipotermia. Os efeitos do extrato sobre o volume, pH e acidez total da secreção gástrica foram avaliados pelo método da ligadura pilórica, sendo que os tratamentos foram realizados por via intraduodenal (i.d.). Avaliou-se também a influência do EAEG no trânsito intestinal dos animais, com tratamentos prévios realizados por via oral. Na análise macroscópica observouse características comuns da espécie. Na análise microscópica observou-se estruturas, tais como: tricomas tectores curtos e longos, unicelulares e pluricelulares, células epidérmicas de tamanhos variados, células contendo drusas, cristais prismáticos e cistólitos. Na prospecção fitoquímica foi detectada a presença de mucilagem, cumarinas e flavonóides. O rendimento do processo extrativo para obtenção do EAEG foi de 20%. Nos modelos de úlceras induzidas pelos diferentes agentes: indometacina, etanol e estresse nas doses de 70, 200 e 600 mg/kg, observou-se redução do número de lesões provocadas por esses agentes. Quando o EAEG foi administrado pela via intraduodenal, observou-se diminuição do volume, da acidez livre e total da secreção gástrica em camundongos com ligadura de piloro. Na avaliação da influência do EAEG sobre o trânsito intestinal observou-se aumento somente com a dose de 600 mk/kg. Os resultados encontrados neste trabalho permitem sugerir que o EAEG contêm princípios ativos gastroprotetores que não reduzem a motilidade intestinal podendo justificar o uso popular da planta para tratar gastrites e úlceras

The objective of this study was to develop new synthetical routes to natural and industrial products utilizing ketene cycioaddition reactions. The cycioaddition of diphenylketene with α,β-unsaturated imines yields (2+2) cycioaddition products, g-lactams. However, electron donating groups, such as dimethylamine, in the 4-position of the α,β-unsaturated imines result in (4+2) cycloaddition products, ∂-lactams. Dichloroketene reacted with α,β-unsaturated imines to yield (4+2) cycloaddition products, g-lactams. Large substituents in the 4-position of a, ^-unsaturated imines resulted in a (2+2) cycioaddition product, β-lactam. The ∂-lactams derived from dichloroketene are easily dehydrochlorinated to the corresponding 2-pyridornes.

Pelargonium sidoides DC. (Geraniaceae) root extracts are used in the Eastern Cape Province of South Africa as a traditional medicine for the treatment of respiratory tract and gastro-intestinal infections. Ethanolic extracts are used globally as herbal treatments for bronchitis, asthma and as an immune system booster. Despite documented exploitation of wild populations by illegal harvesters, this species has not been awarded a protected status. The high level of harvest in the years preceding this study prompted this investigation of the prospects for sustainable root harvest through wild harvest and greenhouse cultivation. A novel method was developed for the purification of umckalin, a bioactive constituent in root extracts, such that the root umckalin concentrations of wild and cultivated plants could be quantified by HPLC. As part of the cultivation experiments, the concentration of umckalin in roots was measured for plants across part of the species’ distribution range in the Eastern Cape Province. This survey revealed that root umckalin concentrations were inversely related to the average annual rainfall of the collection site (r² = 0.94, p = 0.007) and directly related to soil pH (r² = 0.97, p = 0.002). Thus, the possibility of inducing high umckalin concentrations in greenhouse-cultivated plants was investigated by subjecting plants to rapid and prolonged water stress treatments. Two leaf applied hormone treatments (cytokinin and gibberellin) and a root competition treatment with a fast growing annual (Conyza albida) were also investigated based on the potential function of umckalin in P. sidoides plants. These five treatments did not significantly affect root umckalin concentrations compared to well-watered controls. The results of further experiments suggested that umckalin production may have been influenced by the geographical origin and genetics of plants rather than environmental variation. Following wild harvest experiments, the regrowth of replanted shoots from which a standard proportion of the root was harvested showed that water availability affected shoot survival but not root regrowth rate. Regrowth rates were low, questioning the viability of wild harvest. In contrast, greenhouse cultivated plants showed ca. six times greater growth rates, supporting the cultivation of roots to supply future market demand.

This master thesis deals with the use of microfluidics for the purpose of microfluidic enzymatic reactor for drug screening. At first it considers the issue from a theoretical point of view – describes microfluidics as a newly developing and promising field of production of microfluidic devices, materials, biomedical applications and advantages and disadvantages of microfluidics overall. Furthermore, it focuses on an area of analytical utilization of enzymes within enzyme reactors. In the first part of the experimental section, conditions for the testing of enzymes of xenobiotics metabolism in the liver were optimized, namely the model of coumarin metabolism via the spectrofluorimetry method. The second part of the experimental work dealt with optimization of the fabrication conditions of microfluidic chips from OSTE (off-stoichiometry Thiol Ene) via the soft lithography method. Subsequently, the functionality of the produced chips was tested. Based on the results of both parts of the experimental work, an evaluation was carried out to assess the suitability of their interconnection for future research – screening of microsomal enzyme activity and model biotransformation of drugs within the channels of the fabricated devices.