New drugs are needed for better cancer management. Clinical trials are currently underway to assess the use of flavonoids (natural polyphenols) as anticancer agents. Among them, myricetin has been shown to induce cell cycle arrest and apoptosis in pre-clinical cancer models. We hypothesised that myricetin-derived novel flavonoids designed to enhance this natural potential and improve on the drug-likeness limitations of myricetin might have increased potential for their application in the management of breast and ovarian cancer. The effect of a library of novel flavonoids was screened on 3 panels of breast and ovarian cancer cell lines, representing different molecular subtypes and phenotypes, to assess their potency. The second-generation bi-methoxylated analogue AO-1530-OMe (Oncamex) was identified as the most effective candidate in the library, with sub-micromolar concentrations exerting a strong antiproliferative effect across almost all models studied. Results suggested that changes in the hydroxylation profile, the addition of methoxylations and a decyl alkyl chain were some of the structure-activity relationships contributing to this improved efficacy. Plate assays showed 8 h treatment with Oncamex reduced cell viability and induced cytotoxicity and apoptosis, concomitant with caspase activation and PARP cleavage. Pre-incubation with an antioxidant partially blocked these effects, suggesting the possible involvement of ROS modulation in the mechanism of action of Oncamex. Fluorescence microscopy reported the quick and stable delivery of Oncamex to the mitochondria. Fluorescent probes showed that Oncamex can induce mitochondrial superoxide production at concentrations associated with its antiproliferative effects. Study of the electrochemical properties of Oncamex by cyclic voltammetry supported this. Differential gene expression analysis following a microarray experiment showed Oncamex induces changes in the expression of genes controlling cell cycle and apoptosis. Together with previous results, the findings from this analysis led to the postulation of a model for the mechanism of action of Oncamex: due to its enhanced reactivity and mitochondrial targeting, Oncamex can generate mitochondrial superoxide, leading to mitochondrial dysfunction, membrane permeabilisation and the activation of the JNK pathway and the transcription factor FOXO3, which together contribute to the induction of intrinsic apoptosis and the inhibition of proliferation. Further proliferation assays on cell culture models also reported enhanced effect of Oncamex when administered in combination with paclitaxel and TRAIL. These improved responses were observed in breast and ovarian cancer models, including cells lines characterised by their treatment-resistant phenotype. Cotreatment with Oncamex also improved the effect of tamoxifen on anti-oestrogen resistant LCC9 breast cancer cells. Results from preliminary in vivo studies in mice implanted with the MDA-MB-231 breast cancer xenograft were consistent with an antiproliferative effect of Oncamex (25mg/kg/day) in vivo, as treatment inhibited tumour growth and reduced the expression of the marker of proliferation Ki-67 without signs of systemic toxicity. Tissues from this experiment also allowed for preliminary in vivo validation of the proposed mechanism of action of Oncamex by immunohistochemistry. The in vivo cytostatic effect of Oncamex was confirmed in a second in vivo experiment, which also investigated the effect of Oncamex at higher doses or in combination with paclitaxel. In conclusion, the novel flavonoid Oncamex has shown a promising antiproliferative effect in pre-clinical models of breast and ovarian cancer, including models of treatment-resistant cancers. Preliminary in vivo studies have demonstrated a partial recapitulation of the effect of Oncamex. A mechanistic model has been proposed by which Oncamex induces intrinsic apoptosis through its redox reactivity and mitochondrial targeting. These results support the potential of this prototypic candidate, although possible work in the structure and formulation of this candidate and further study and validation of its mechanism of action is needed for its continued development as an anticancer agent.

Parte 1 - Estudos para a síntese do petrosinol O petrosinol é um produto natural obtido a partir de esponjas do gênero Petrosia sp. Trata-se de um poliacetileno que possui atividade anti-HIV. A síntese deste composto, ainda não descrita na literatura, mostrou-se difícil. No presente trabalho são descritas as rotas sintéticas testadas para a obtenção do petrosinol. Foram feitas abordagens, totalizando sete, que consistiram na síntese de sistemas dicarbonílicos α,β-insaturados, os quais se mostraram pouco reativos, que não permitiram a continuidade das rotas com esta abordagem, ou tentativas de construção de álcoois propargílicos, que neste caso, os intermediários de síntese se mostraram muito instáveis. A síntese do petrosinol necessita de mais estudos visando novas tentativas para se obter o sistema diol insaturado central presente na molécula, chave para a síntese total da mesma. Parte 2 - Estudos para a síntese da amamistatina B A amamistatina B faz parte do grupo de moléculas que são sideróforos, utilizadas por bactérias para o transporte de íons ferro. O estudo de sideróforos tem crescido muito nos últimos anos devido o interesse de se obter prófármacos que utilizem tais sideróforos como transportadores. No presente trabalho foram feitos estudos para obtenção de fragmentos úteis na síntese convergente da amamistatina B. Foram realizados estudos de obtenção de β-hidróxiácidos α,α-dissubstituidos usando-se como direcionadores quirais as oxazolidinonas. O uso de agentes oxidantes foi necessário na obtenção de nitronas, intermediários sintéticos úteis na obtenção de formamidas substuidas, e que podem ser usados na síntese da amamistatina B. / Part 1 - Studies on the synthesis of petrosinol - Petrosinol is a natural product isolated from the marine genus from sponge Petrosia sp. It is a poliacetylene which has citotoxic and anti-HIV activity. The synthesis of this compound is not described on literature yet and it is very difficult. This work describes synthetic routes which were tested to obtain petrosinol. There were made seven approaches to construct α,β-unsaturated dicarbonyl systems which showed little reactivity. It was not possible to continue the routes to use this approach to construct propargyllic alcohols because these are very unstable. The synthesis of the petrosinol needs some more studies aiming new attempts to obtain the central unsaturated diol system in the molecule, the key to the total synthesis of it. Part 2 - Studies on the synthesis of amamistatin B - Amamistatin B is part of the group of molecules called siderophores, used by bacteria for the transportation of iron. Studies on siderophores has grown a lot in the last years because of the interest on the obtention of prodrugs that use such sidrophores as transport. In the present work the main goal was the obtention of useful fragments for the convergent synthesis of amamistatin B. Studies for the obtention of α,α-disubstituted β-hidroxyacids were achieved using chiral directors such as oxazolidinones. The use of oxydizing agents for the obtention of nitrones, synthetic internmediates useful for the obtention of substituted formamides.

Não consta resumo na publicação / Abstracts not available.

A crescente resistência dos micro-organismos patogênicos aos fármacos já existentes gera intensa demanda por novos agentes terapêuticos. Em contrapartida, a eficiência na descoberta de compostos com novas estruturas químicas diminuiu nos últimos anos. Tendo em vista a vasta biodiversidade existente de micro-organismos, a redução da eficiência na descoberta de novos produtos naturais não indica que todos compostos existentes já foram descritos, mas sim que as metodologias para isolamento dos mesmos devem ser aperfeiçoadas e diversificadas, e novos nichos devem ser explorados. Esta tese compreende três capítulos, que trazem abordagens que podem ser utilizadas na busca por produtos naturais. O capítulo 1 aborda a aplicação de conhecimentos de biologia molecular na pesquisa de produtos naturais, através da metagenômica. O capítulo 2 aborda o conceito de química ecológica para estimular o metabolismo secundário, através da utilização de interações microbianas. O capítulo 3 aborda o uso de genome mining para entender a capacidade metabólica de uma linhagem bacteriana, bem como o uso de variações nos parâmetros da cultura para alterar o metabolismo desta. Metagenômica: a triagem anti-parasitária das bibliotecas metagenômicas detectou clones bioativos contra Leishmania major. As análises químicas e biológicas das culturas destes clones não permitiram a identificação dos compostos responsáveis pelas atividades observadas. Esta abordagem apresenta grandes desafios técnicos e tem passado por ajustes envolvendo sequenciamento e bioinformática para aumentar a taxa de sucesso em seu uso. Interações microbianas: esta metodologia mostrou-se promissora para a busca por compostos bioativos, tendo em vista que diversos pares exibiram nova atividade antibiótica, corroborando a hipótese que interações microbianas podem levar à expressão diferenciada do metabolismo secundário. Foi escolhida para caracterização química e biológica a interação entre uma actinobactéria rara (Krasilnikovia sp. T082) e uma actinobactéria endossimbionte de besouro (Streptomyces sp. SPB78). Esta interação é robusta e estimula a biossíntese de um antibiótico polar capaz de inibir o crescimento de uma bactéria multirresistente. Diversas técnicas foram testadas para o isolamento do composto indutor e do antibiótico induzido. Este processo foi desafiador devido ao caráter polar de ambos compostos e pelo fato da atividade antibiótica ser instável. Foi demonstrado que o antibiótico induzido é capaz de inibir o crescimento do micro-organismo indutor, sugerindo importância ecológica deste composto. A utilização desta abordagem metodológica permitiu a identificação de uma linhagem pertencente a um gênero de actinobactéria rara que nunca teve seu metabolismo secundário estudado (Krasilnikovia). Isto foi realizado através da investigação de seu genoma e também através do isolamento de compostos produzidos por esta linhagem. Esta linhagem demonstrou potencial para a produção de metabólitos secundários, apresentando pelo menos 21 potenciais clusters gênicos biossintéticos detectados pelo antiSMASH em seu genoma. Esta linhagem foi cultivada em dois meios de cultura (ISP2 e TSB) e diferentes metodologias de extração foram empregadas. O metabolismo secundário desta linhagem é expresso de maneira diferente de acordo com a metodologia de cultivo, evidenciando a importância da variação da composição do meio de cultura para o acesso da real capacidade metabólica de microorganismos. Foram identificadas algumas dicetopiperazinas, que são conhecidas por seu amplo espectro de atividades biológicas, e três compostos pertencentes a uma classe de peptídeos nãoribossomais não usuais com atividade antibiótica, que possuem estrutura química complexa e incomum para produtos naturais / Increasing drug resistance among microbial pathogens is a public health threat; therefore, new antibiotics are needed. On the other hand, the rate of discovering new compounds has diminished. Considering the wide biodiversity of microorganisms, reduced efficiency on the discovery of new natural products does not indicate that all existing compounds have been described, but that methods for isolation should be improved and diversified and new niches should be explored. This thesis comprises three chapters that demonstrate approaches that can be used in the search for natural products. Chapter 1 demonstrates the application of molecular biology tools on the search for natural products through metagenomics. Chapter 2 discusses the concept of chemical ecology for the stimulation of secondary metabolism by the use of microbial interactions. Chapter 3 discusses the use of genome mining to understand the metabolic capacity of a bacterial strain as well as the use of different culture parameters to alter bacterial metabolism. The anti-parasitic metagenomic screening on metagenomic libraries detected bioactive clones against L. major. Chemical and biological analysis of cultures of these clones did not permit identifying the compounds responsible for the observed activities. This approach faces diverse technical challenges and is currently being improved by the use of sequencing and bioinformatics analysis in order to increase its hit rate. Microbial interactions: this approach has shown to be promising in the search for bioactive compounds, considering that several pairs exhibited new antibiotic activity, supporting the hypothesis that microbial interactions can stimulate differential expression of secondary metabolism. It was chosen for chemical and biological characterization the interaction between a rare actinobacteria (Krasilnikovia sp. T082), which belongs to a genus that its secondary metabolism has not yet been studied in the literature, and an endosymbiont actinobacteria of beetle (Streptomyces sp. SPB78). This interaction is robust and stimulates the biosynthesis of a polar antibiotic capable of inhibiting the growth of multi-resistant bacteria. Several techniques have been tested for the isolation process of the inducer compound and the induced antibiotic. This process has been particularly challenging due to the polar character of both compounds, and because the antibiotic activity is unstable. It has been shown that the induced antibiotic is capable of inhibiting the growth of the inducer microorganism, suggesting an ecological importance of this compound. The use of this co-culture approach led to the identification of a strain belonging to a rare actinobacteria genus whose secondary metabolism has never been studied before (Krasilnikovia). This was accomplished through sequencing and analysis of its genome and also by isolating compounds produced by this strain (chapter 3). This strain has shown great potential for the production of secondary metabolites, exhibiting at least 21 biosynthetic gene clusters detected by antiSMASH in its genome, and only four of them showed high similarity to any known gene cluster. This strain was cultured in two different culture media (ISP2 and TSB), and different methods of extraction were used. The secondary metabolism of this strain is expressed differently according to the method of cultivation, showing the importance of variation in the composition of the culture medium to access the actual metabolic capacity of microorganisms. Some diketopiperazine were isolated, which are known for their wide spectrum of biological activities, and also three compounds belonging to a class of non-ribosomal peptides known for their high bioactivity, which have complex and unusual chemical structures for natural products

Fungos do gênero Colletotrichum são considerados um dos principais fitopatógenos do mundo, comprometendo diversas culturas e causando danos econômicos e sociais para diversos países, inclusive o Brasil. Apesar de medidas de controle já serem empregadas, estas nem sempre são eficazes, motivo pelo qual se faz necessária a busca por novas opções de controle que possam atuar no manejo integrado. Dentre estas, encontram-se os metabólitos secundários produzidos por fungos endofíticos, os quais estão envolvidos na produção de compostos de interesse biotecnológico, com aplicações em diversas áreas, inclusive a agronômica. Neste contexto, o presente trabalho de doutorado teve por objetivo explorar o potencial biológico e químico de metabólitos secundários produzidos por fungos endofíticos isolados de guaranazeiros da Amazônia, avaliando sua atividade antifúngica \"in vitro\" a Colletotrichum gloeosporioides, isolado da cultura do guaranazeiro, a C. acutatum e C. gloeosporioides, isolados da cultura do pimentão. Para tanto, 16 linhagens de fungos endofíticos foram avaliadas em ensaio biológico por cultivo pareado e pelo método de difusão em disco contra Colletotrichum spp. Observou-se que quatro linhagens se mostraram mais promissoras, as quais foram selecionadas para o estudo de bioprospecção. A partir do extrato bruto do endófito Aspergillus flavus (272) foi isolado o composto ativo asperfuran, que apresentou atividade inibitória aos três fitopatógenos, com CI50 < 100 &mu;g disco-1, semelhante ao fungicida comercial difenoconazol. A partir do extrato bruto de Xylaria sp. (214) foi isolado o composto ativo citocalasina D, que apresentou atividade inibitória a C. gloeosporioides isolado do guaranazeiro, com CI50 > 500 &mu;g disco-1. A partir do extrato bruto de Xylaria sp. (249) foi isolado o composto ativo ácido pilifórmico, que apresentou atividade inibitória a C. gloeosporioides isolado do guaranazeiro, com CI50 de 500 &mu;g disco-1 e à C. acutatum isolado o pimentão, com CI50 de 300 &mu;g disco-1. E, a partir do extrato bruto de Talaromyces aculeatus (507) foram isolados três compostos, da classe dos ésteres ftalicos, sendo um ativo. A fração antecessora destes três compostos apresentou atividade inibitória a C. gloeosporioides isolado do guaranazeiro, com CI50 > 500 &mu;g disco-1. A partir do extrato bruto de T. aculeatus também foi isolado um composto ativo parcialmente identificado e um composto ativo semi-purificado. A fração antecessora destes dois compostos apresentou atividade inibitória aos dois C. gloeosporioides, com CI50 > 300 &mu;g disco-1 e a C. acutatum, com CI50 em torno de 150 &mu;g disco-1. A atividade antifúngica destes compostos a fungos do gênero Colletotrichum está sendo relatada pela primeira vez neste estudo. / Colletotrichum fungi are considered one of the main pathogens in the world, affecting different cultures and causing social and economic damage to several countries, including Brazil. Despite control measures already being employed, these are not always effective, which is why the search for new control options that can act in the integrated management is necessary. Among these, the secondary metabolites produced by endophytes are involved in the production of compounds of biotechnological interest, with applications in several areas, including the agronomic. In this context, this study aimed to explore the biological and chemical potential of secondary metabolites produced by endophytic fungi isolated from the Amazonian guarana, evaluating their antifungal activity \"in vitro\" to Colletotrichum gloeosporioides, isolated from the culture of guarana and C. acutatum and C. gloeosporioides, isolated from the red pepper crop. For this purpose, 16 strains of endophytic fungi were evaluated in biological assay of dual culture and disk diffusion method against Colletotrichum spp. Four strains of these strains presented promising results which were selected for bioprospecting. From the crude extract of the endophyte Aspergillus flavus (272) was isolated the active compound asperfuran, which showed inhibitory activity to three pathogens, with IC50 <100 &mu;g disk-1, similar to the commercial fungicide difenoconazole. From the crude extract of Xylaria sp. (214) was isolated the active compound cytochalasin D, which showed inhibitory activity against C. gloeosporioides isolated from guarana (IC50> 500 &mu;g disk-1). From the crude extract of Xylaria sp. (249) was isolated the active compound piliformic acid, which showed inhibitory activity against C. gloeosporioides isolated from guarana (IC50 of 500 &mu;g disk-1) and C. acutatum isolated from red pepper (IC50 of 300 &mu;g disk-1). And from the crude extract of Talaromyces aculeatus (507) were isolated three compounds, of phthalate esters class, one of them with activity. The predecessor fraction of these three compounds showed inhibitory activity against C. gloeosporioides isolated from guarana (IC50> 500 &mu;g disk-1). From the crude extract of T. aculeatus it was also isolated a partially identified active compound and a semi-purified active compound. The predecessor fraction of these two compounds showed inhibitory activity to both C. gloeosporioides (IC50> 300 &mu;g disk-1) and to C. acutatum (IC50 around 150 &mu;g disk-1). The antifungal activity of the compounds isolated in this study is being reported for the first time against fungus of the genus Colletotrichum.

Este trabalho descreve os isolamentos e as determinações estruturais dos metabólitos secundários das diferentes partes dos frutos (pericarpos, arilos, tegumentos e amêndoas) de Virola elongata. 0 material foi coletado em Humaitá (AM) e no Km 96 da Rodovia Santarém-Cuiabá (PA). Os constituíntes químicos foram isolados por cromatografias de adsorção e tiveram suas estruturas identificadas ou determinadas por técnicas espectrométricas usuais (IV, UV, RMN e EM). As configurações absolutas dos metabólitos foram deduzidas a partir de medidas espectropolarimétricas, ou auxiliadas pela obtenção de derivados mais informativos. As substâncias foram agrupadas nas seguintes subclasses: lignanas furofurânicas [(+)-sesamina (LF-1), (+)-epifargesina (LF-2), (+)-eudesmina (LF-3), (+)-fargesina (LF-4), (+)-filigenina (LF-5) e (+)-epieudesmina (LF-6)]; lignanas tetraidrofurânicas [(+)-magnostelina-A (LT-1), (+)-magnostelina-C (LT-2), (±)-4\'-metil-5\'-metoxi-lariciresinol (LT-3), (±)-4,4\'-dimetil-5\'- metoxi-lariciresinol (LT-4) e (±)-4-4\'-dimetil-5\'-hidroxi-5-metoxi-lariciresinol (LT-5)]; Lignana diarilbutanólica [(-)-4,4\'-dimetil-5-metoxi-secolariciresinol (LD-1)]; e lignanas dibenzilbutirolactâmicas [(-)-hinokinina (DB-1), (-)-kusunokinina (DB-2), (-)-dimetilmatairesinol (DB-3), (-)-metiltujaplicatina (DB-4), (-)-dimetil-5-hidroximatairesinol (DB-5) e (-)-dimetil-5-metoxi-matairesinol (DB-6)]; neolignanas diarilbutânicas [ND-1, ND-2 e ND-3], neolignana ariltetralínica: galbulina (NA-1) e neolignanas ariltetralônicas [(-) e (+)-oxoisogalcatina (NA-2), (+)-hidroxi-isogalcatina (NA-3), diidroxi-oxoisogalcatinas epiméricas inéditas (NA-4 e NA-5), NA-6, NA-7, (+)-otobanona (NA-8), hidroxi-otobanonas epiméricas (NA-9 e NA-10)]; neolignanas secoariltetralínicas (ND-5 e seu acetato ND-6]; uma neolignana dimérica [NA-11] e ainda diversos policetídeos acilresorcinólicos e acifloroglucinôlicos [AR-1 a AR-8]. 0 padrão 3,4-dioxigenado nos anéis aromáticos é predominante na maioria dos lignóides isolados. 0 padrão 3,4,5-trioxigenado observado nos anéis aromáticos de lignanas dibenzilbutirolactônicas (DB-4 e DB-5), diarilbutanólica (LD-1) e tetraidrofurânicas (LT-3, LT-4 e LT-5) e, o padrão 2,4,5-trioxigenado observado para neolionanas ariltetralônicas (NA-6, NA-7), conferem para algumas o caráter inédito. A maior variação no padrão de substituição de esqueletos foi observada para neolignanas ariltetralônicas, o que motivou a elaboração de experimentos especíificos em RMN- 1H (ENO e uso de reagente de deslocamento) associados a análise dos dados de RMN- 13C para proposições de constituições e de configurações relativas. Foi constatada a presença das neolionanas NA-2, NA-9 e NA-10 nos extratos dos tegumentos e de seus enantiômeros nos extratos das amêndoas de V. elongata (PA). Algumas classes de substâncias isoladas foram correlacionadas biossinteticamente a partir de suas configurações absolutas. A quantificação aproximada das substâncias identificadas foi efetuada, através das quantidades isoladas e análises de espectros de RMN-1H de frações impuras. Nos pericarpos e arilos foi constatada a presença exclusiva e alternada de lignanas furofurâanicas e lignanas dibenzilbutirolactônicas entre os frutos de diferentes localidades. Essas lignanas, também presentes nos tegumentos, ocorrem em maior teor com maior diversidade de substituição nos anéis aromáticos, mas são completamente ausentes nos extratos das amêndoas, onde o perfil é baseado na presença de neolignanas ariltetralônicas e policetídeos. Os policetídeos com anéis aromáticos di- e triidroxilados foram isolados, principalmente, dos tegumentos e amêndoas. A análise do extrato de uma das amêndoas foi baseada na análise conjunta do espectro de RMN-1H, dos cromatogramas em camada delgada e dos dados obtidos por cromatografia gasosa acoplada a espectrometria de massas. A metodologia foi tentativamente aplicada á análise das diversas partes dos frutos de V. sebifera coletada em Roraima. Os dados obtidos sobre as estruturas e distribuição dos metabólitos são confrontados com os descritos na literatura para espécies taxonomicamente afins. / This work describes isolations and structural determinations of secondary metabolites in parts of fruits (pericarps, arils, teguments and kernels) from Virola elongata. Material was collected in Humaita (AM) and Km 96 of Santarém-Cuiabá Road (PA). The chemical constituents were isolated by chromatographic processes and their structures identified or elucidated by usual spectrometric techniques (IR, UV, NMR, and MS). Absolute configuration were deduced through spectropolarimetric measures, or based on data from derivatives. The compounds were classified in the following classes: furofuran lignans [(+)-sesamin (LF-1), (+)-epifargesin (LF-2), (+)-eudesmin (LF-3), (+)- fargesin (LF-4), (+)-phylligenin (LF-5) and (+)-epieudesmin (LF-6)]; tetrahydrofuran lignans [(+)-magnostelin-A (LT-1), (+)-magnostelin-C (LT-2), (±)-5\'-methox y -4\'-methyl-lariciresinol (LT-3), (±)-5\'-methoxy-4,4\'-dimethyl-lariciresinol (LT-4) and (±)-5\'-hydroxy-5-methoxy-4,4\'-dimethvl-lariciresinol (LT-5)]; diarylbutanol lignan [(-)-4,4\'-dimethvl-5-methoxy-secolariciresinol (LD-1)]; and dibenzylbutyrolactone lignans [(-)-hinokinin (DB-1), (-)-kusunokinin (DB-2), (-)-dimethylmatairesinol (DB-3), (-)-methylthujaplicatin (DB-4)]; diarylbutane neolignans (ND-1, ND-2 and ND-3); aryltetralin neolignans [galbulin (NA-1) and aryltetralone neolignans [(-)- and (+)-oxoisogalcatin (NA-2), (+)-hydroxyisogalcatin (NA-3), unpublished epimeric dihydroxy-isogalcatins (NA-4 and NA-5), (+)-otobanone (NA-8), epimeric hydroxy-otobanones (NA-9 and NA-10)]; secoaryltetralin neolignans [ND-5 and its acetate (NA-6).1; dimeric neolignan (NA-11) and several acylresorcinol and acylphloroglucinol polyketides (AR-1 to AR-8). The 3,4-dioxygenated pattern was observed in aromatic rings of the majority isolated lignans. The 3,4,5-trioxygenated pattern observed in dibenz y lbutyrolactone lignans (DB-4 e DB-5), diarylbutanol (LD-1), and tetrahydrofuran lignans (LT-3, LT-4, LT-5), and the 2,4,5-pattern observed in aryltetralone neolignans, confer to some of them the unusual character. The main variation in the substitution pattern was observed in aryltetralone neolignans, which needed more detailed study involving 1H-NMR experiments (NOE and LIS reagent) and refined analysis of 13C-NMR data. It was observed the presence of NA-2, NA-9 and NA-10 neolignans in teguments and its enantiomers in kernels of fruits from Virola elongata (PA). The biosynthetic relationships among several classes of lignoids are discussed under their absolute configurations. The near concentration of identified compounds was obtained from isolated quantities and analysis of 1H-NMR spectra of mixtures. In pericarps and arils was determined the exclusive and alternated presence of furofuran and dibenzylbutyrolactone lignans between fruits from different localities. These last two subclasses, also present in teguments, occur in higher concentrations and substitution diversifications in aromatic rings, but are completely absent in kernels whose profile is based on the presence of aryltetralone lignans. The poliketide with di- and trihydroxylated aromatic rings were isolated mainly from teguments and kernels. The analysis from kernels extract was performed by combined analysis involving 1H-NMR spectra, thin layer chromatography, and gas chromatography coupled to mass spectrometry. This methodology was tested to analyse the parts of fruits from Virola sebifera collected in Roraima. The obtained informations about structures of metabolites and their distributions in fruits was compared with those data described for taxonomically closed species.

This thesis describes synthetic studies towards the tricyclic terpene framework of the cytotoxic natural product eleutherobin, and the investigation of biocatalytic methodology towards late stage functionalisation of the structure. Chapter 1 provides an introduction to the biomedical significance of the natural product and to the various synthetic studies reported to date. Chapter 2 explores intramolecular Diels-Alder approaches to the construction of the nine- and six- membered rings present in the natural product. Chapter 3 describes alternative approaches to the eleutherobin core structure, leading to a formal synthesis of the natural product. The synthetic sequence developed is applied to the preparation of a small library of furanoid analogues of eleutherobin. Chapter 4 details the development of methodology for the one-pot preparation of (E)-4-methylhexa-3,5-dien-1-ol, and its use in an intermolecular Diels-Alder/lactonisation sequence to achieve enantioselective preparation of an important lactone building block. Chapter 5 describes studies on the mP450-mediated oxidation of various eleuthoside structures obtained. Finally, Chapter 6 details the construction of an electrochemical flow cell, and its use for the anodic oxidation of furfuryl alcohol derivatives.

The aim of this dissertation was to elaborate the exploration of biologically active secondary metabolites from the marine sponge Cacospongia cf. linteiformis collected from the Bahamas and the soft coral Briareum asbestinum collected from two different sites in Florida State, Boca Raton and Dry Tortugas. In chapter one, a review on previous chemical and biological studies of the marine sponge C. cf. linteiformis and soft coral B. asbestinum is provided. Particular attention is given to spongianolides and briarellins, two important classes of compounds isolated from C. cf. linteiformis and B. asbestinum, respectively, and their structural features and diverse bioactivities. In chapter two, the isolation and relative configuration determination of four epimeric sesterterpenoids, spongianolides E & F (18c, 18d, 19c, 19d) from C. cf. linteiformis collected from the Bahamas are discussed. Thanks to chemical modification (acetylation), diastereomeric 18c&18d and 19c&19d, respectively, were able to be isolated using chromatographic techniques for the first time, and then the relative configurations of 18c, 18d, 19c, 19d were determined based on NOESY NMR experiments. The bioactivity of mixture of compounds 18c, 18d, 19c, 19d were tested and it exhibited inhibition against Schnurri-3 (a regulator of postnatal bone mass). In chapter three, the isolation and structural elucidation of four new compounds, florellins A-D (49-52), from B. asbestinum collected off the coast of Boca Raton, FL are discussed. The molecular structures of these compounds were established by spectroscopic analysis. Compounds 49-52 are the first briarellins containing an acyl group at C-13, while 49 and 50 are the first briarellins possessing acylation at C-15. Florellins A–C (49-51) were screened and found cytotoxic against three human cell lines, BT474, WM266−4 and HEK293. In chapter four, the isolation and structural elucidation of four new compounds, florellins E-H (57-60), from B. asbestinum collected in Dry Tortugas, FL are discussed. The molecular structures of these compounds were established by spectroscopic analysis. Florellins F (58) and H (60) were screened against three human cell lines, BT474, WM266−4 and HEK293, but no cytotoxicity was exhibited. In chapter five, all the experimental procedures are described, including analytical instruments, animal materials, extraction and isolation processes, spectroscopic data and protocols of bioassays. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2017. / FAU Electronic Theses and Dissertations Collection

Made available in DSpace on 2014-06-11T19:22:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-12-05Bitstream added on 2014-06-13T19:27:31Z : No. of bitstreams: 1 vellosa_jcr_me_arafcf.pdf: 1352402 bytes, checksum: 1fb4f8a94f0db92c39104b3590886eff (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / A relevância da pesquisa de produtos naturais proporciona a descoberta de novos fármacos e o estudo de plantas que apresentem substâncias que possam agir sobre as diferentes espécies oxidantes geradas em nosso organismo torna-se de grande importância. No estudo da avaliação do potencial antioxidante das amostras deve-se considerar que: i) um composto deve ser testado em concentrações disponíveis in vivo e ii) ao avaliar os antioxidantes, deve-se utilizar pelo menos uma espécie relevante biologicamente. Deve-se perguntar como age o antioxidante, se ele age diretamente sobre a ERO ou ele inibe a sua geração e ainda, se ele age indiretamente regulando defesas antioxidantes endógenas. Foram avaliados extratos ou moléculas dos vegetais: Pterogyne nitens, Maytenus ilicifolia, Maytenus aquifolium, Salacia campestris, Piper gaudichaudianum, Piper crassinervium e Piper aduncum. A proposta deste trabalho é: i) avaliar a possibilidade de ação direta destas amostras sobre diferentes espécies reativas modelo e geradas em nosso organismo e ii) avaliar a inibição da mieloperoxidase, enzima envolvida em diferentes patologias. Observamos um grande potencial das plantas estudadas como fonte de compostos com ação antioxidante. Destacam-se as frações hidroalcoólicas e hexano-acetato da M. ilicifolia e M. aquifolium sobre o ânion superóxido. Além disso, identificou-se a existência de possíveis inibidores da MPO nos diferentes vegetais estudados, destacando-se quercetina (IC50=1,35mg/mL), 3-HexAcOEt (IC50=1,5mg/mL), 2-Hid (IC50=2,2mg/mL), 3-Hid (IC50=3,1mg/mL), S-3 (IC50=3,1mg/mL), 2-HexAcOEt (IC50=4,4mg/mL), 1C-BuOH (IC50=6,4mg/mL) e 4EtOH (IC50=8,9mg/mL). Por fim, caracterizou-se o tipo de inibição promovida pela S-3 (1,4-diidroxi-2-(3',7'-dimetil-1'-oxo-2'-E-6'-octadienil) benzeno) e pela quercetina como sendo do tipo mista. / Phagocytes, mainly polimorphonuclear neutrophils leucocytes, yield superoxide radical (O2 -), expending NADPH. By this process, many reactive species, such as HOCl, HO, ONOO -, are generated. These oxidants are made to fight microorganisms, but they are involved in many pathologies. Reactive species and free radicals generation are equilibrated by antioxidants. The search of new medicines turns natural products research an important option for discovering molecules with different biological activities. Natural products research is relevant for discovering new medicines and molecules able to fight oxidant species. In evaluating the antioxidant potential of substances, it is important considerate: i) a compound should be tested at concentrations achievable in vivo and ii) in assaying putative antioxidants, one should use biologically relevant ROS. It is important to ask if it works directly over the oxidant or it works by regulating endogenous antioxidants defenses. In this work, Pterogyne nitens, Maytenus ilicifolia, Maytenus aquifolium, Salacia campestris, Piper gaudichaudianum, Piper crassinervium and Piper aduncum were studied. The purpose of this work is: i) evaluate direct action of samples over different reactive species and ii) evaluate myeloperoxidase inhibition, an important enzyme involved in different pathologies. We observed that different evaluated plants are efficient sources of antioxidants. Moreover, different samples were able to inhibit myeloperoxidase, detaching quercetin (IC50=1,35mg/mL), 3-HexAcOEt (IC50=1,5mg/mL), 2-Hid (IC50=2,2mg/mL), 3-Hid (IC50=3,1mg/mL), S-3 (IC50=3,1mg/mL), 2-HexAcOEt (IC50=4,4mg/mL), 1C-BuOH (IC50=6,4mg/mL) and 4EtOH (IC50=8,9mg/mL). Besides, quercetin and S-3 (1,4-dihydroxi-2-(3',7'-dimethyl- 1'-oxo-2'-E,6'-octadienyl) benzene) were characterized as mixed MPO inhibitors.

Paclitaxel is an anticancer natural product with several biomedical applications produced by Taxus species, with a demand exceeding its supply. We have developed cambial meristematic cells (CMCs) from Taxus cuspidata as high yield source of paclitaxel. The biosynthesis of paclitaxel is predominantly under transcriptional control. Thus, the identification of transcriptional regulators of paclitaxel biosynthesis and their subsequent manipulation may enable further yield enhancement in Taxus CMCs. Previously, Roche 454 sequencing was employed to establish the transcriptome of T. cuspidata CMCs treated with the plant immune activator methyl jasmonate (MeJA). The bioinformatic analysis identified 19 jasmonate related transcription factors (TFs), based on their differential expression. Results of the Arabidopsis thaliana transient assay screen identified two MYB TFs that constitute positive regulators for paclitaxel genes, named MYB3 and MYB4. In this thesis, MYB3 and MYB4 showed in vitro binding to the cis-elements in ten promoters of paclitaxel genes using the electrophoretic mobility shift assay (EMSA). Then, a Taxus CMC protoplasts transient assay demonstrated that the expression of MYB3 and MYB4 trans-activated all tested genes. Further, MYB4 was found to activate the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) gene, key in the mevalonate pathway and precursor of paclitaxel biosynthesis. MYB3 and MYB4 were capable of auto-regulating their own transcription, constituting an important control point for paclitaxel biosynthesis. A possible mechanism for the early activation of MYB3 and MYB4 after MeJA elicitation is proposed. Finally, preliminary results on the expression of MYB3 and MYB4 in unelicited T. baccata CMC protoplasts indicate that their transient expression was sufficient to increase accumulation of paclitaxel and the precursor, 10-deacetyl baccatin III, highlighting their utility for paclitaxel production.