Introdução e objetivos: Tem sido demonstrado que um moderado desacoplamento mitocondrial em células hepáticas pode reverter a hipertrigliceridemia, a doença de fígado gorduroso e a resistência à insulina. A dissipação da energia conservada no espaço intermembranas mitocontrial, como ocorre no desacoplamento, aumenta o uso de substratos energéticos e também podem reduzir a geração mitocondrial de espécies reativas de O2 (EROs). Duas estratégias de desacoplamento mitocondrial foram estudadas neste trabalho: a primeira consistiu em reduzir a velocidade da via de síntese de triacilgliceróis por meio da triacsin C (inibidor da Acil CoA Sintetase - ACS), e dessa forma aumentar os ácidos graxos livres (AGL) como substratos das proteínas desacopladoras; a segunda foi verificar se clusianona (composto natural das raízes de Clusia congestiflora), análogo estrutural do desacoplador químico nemorosoma, é capaz de promover o desacoplamento químico. Resutados e discussão: A triacsin C, em concentrações até 1 ?M, não apresentou efeito tóxico em mitocôndrias isoladas de fígado e nem em hepatócitos primários. Nesses últimos, aumentou o consumo de oxigênio nos estados de respiração basal e de máxima velocidade respiratória. Além disso, foi verificado um aumento da expressão do fator de transcrição PGC1- alfa e de ?-hidroxiacil CoA desidrogenase (HAD), uma enzima da beta-oxidação de ácidos graxos. A clusianona aumentou o consumo de O2 no estado de repouso, diminuiu o potencial de membrana, reduziu a produção de EROs e preveniu o inchamento mitocondrial induzido por Ca2+ de forma dose dependente, porém menos potente que a nemorosona. Conclusões: Nossos resultados indicam que a triacsin C acelerou o metabolismo mitocondrial, a oxidação de ácidos graxos e a biogênese mitocondrial; a clusianona foi caracterizada como um desacoplador eficaz da fosforilação oxidativa mitocondrial, provavelmente envolvendo um mecanismo protonofórico devido as suas propriedades químicas. Dessa forma, ambas as estratégias estudadas se mostram com potencial terapêutico no tratamento de doenças como esteatose hepática, hipertrigliceridemia e obesidade. / Introduction and Objectives: It has been shown that a mild mitochondrial uncoupling in livers can reverse hypertriglyceridemia, fatty liver disease and insulin resistance. The dissipation of energy stored in mitochondrial intermembrane space as heat, as in uncoupling, increases the use of energy substrates and may also reduce the mitochondrial generation of reactive O2 species (ROS). Two strategies to induce mitochondrial uncoupling were studied in this work: the first consisted of slowing down the route of triacylglycerols synthesis by triacsin C (acyl CoA synthetase inhibitor), and thus increasing the free fatty acids (FFA) as substrates for uncoupling proteins; the second was to determine whether clusianone (natural compound from the roots of Clusia congestiflora), a structural analogue of chemical uncoupler nemorosoma, is capable of promoting the chemical uncoupling. Results and discussion: The triacsin C, in concentrations up to 1 ?M, showed no toxic effect on liver mitochondria and primary hepatocytes. In hepatocytes triacsin C increased oxygen consumption in the states of basal respiration and maximum respiratory rate. In addition, there was an increase in the expression of the transcription factor PGC1 - ? and ? - hydroxyacyl CoA dehydrogenase (ADH), an enzyme of ?- oxidation of fatty acids. The clusianone increased O2 consumption in resting state, decreased membrane potential, reduced the production of ROS and prevented the mitochondrial swelling induced by Ca2+ in a dose dependent manner, but less potent than nemorosone. Conclusions: Our results indicate that triacsin C accelerated mitochondrial metabolism, fatty acid oxidation and mitochondrial biogenesis; the clusianone was characterized as an effective uncoupler of mitochondrial oxidative phosphorylation, probably involving a protonoforic mechanism due to its chemical properties. Therefore, both strategies have therapeutic potential in the treatment of diseases such as liver steatosis, hypertriglyceridemia and obesity.

Introdução e objetivos: Tem sido demonstrado que um moderado desacoplamento mitocondrial em células hepáticas pode reverter a hipertrigliceridemia, a doença de fígado gorduroso e a resistência à insulina. A dissipação da energia conservada no espaço intermembranas mitocontrial, como ocorre no desacoplamento, aumenta o uso de substratos energéticos e também podem reduzir a geração mitocondrial de espécies reativas de O2 (EROs). Duas estratégias de desacoplamento mitocondrial foram estudadas neste trabalho: a primeira consistiu em reduzir a velocidade da via de síntese de triacilgliceróis por meio da triacsin C (inibidor da Acil CoA Sintetase - ACS), e dessa forma aumentar os ácidos graxos livres (AGL) como substratos das proteínas desacopladoras; a segunda foi verificar se clusianona (composto natural das raízes de Clusia congestiflora), análogo estrutural do desacoplador químico nemorosoma, é capaz de promover o desacoplamento químico. Resutados e discussão: A triacsin C, em concentrações até 1 ?M, não apresentou efeito tóxico em mitocôndrias isoladas de fígado e nem em hepatócitos primários. Nesses últimos, aumentou o consumo de oxigênio nos estados de respiração basal e de máxima velocidade respiratória. Além disso, foi verificado um aumento da expressão do fator de transcrição PGC1- alfa e de ?-hidroxiacil CoA desidrogenase (HAD), uma enzima da beta-oxidação de ácidos graxos. A clusianona aumentou o consumo de O2 no estado de repouso, diminuiu o potencial de membrana, reduziu a produção de EROs e preveniu o inchamento mitocondrial induzido por Ca2+ de forma dose dependente, porém menos potente que a nemorosona. Conclusões: Nossos resultados indicam que a triacsin C acelerou o metabolismo mitocondrial, a oxidação de ácidos graxos e a biogênese mitocondrial; a clusianona foi caracterizada como um desacoplador eficaz da fosforilação oxidativa mitocondrial, provavelmente envolvendo um mecanismo protonofórico devido as suas propriedades químicas. Dessa forma, ambas as estratégias estudadas se mostram com potencial terapêutico no tratamento de doenças como esteatose hepática, hipertrigliceridemia e obesidade. / Introduction and Objectives: It has been shown that a mild mitochondrial uncoupling in livers can reverse hypertriglyceridemia, fatty liver disease and insulin resistance. The dissipation of energy stored in mitochondrial intermembrane space as heat, as in uncoupling, increases the use of energy substrates and may also reduce the mitochondrial generation of reactive O2 species (ROS). Two strategies to induce mitochondrial uncoupling were studied in this work: the first consisted of slowing down the route of triacylglycerols synthesis by triacsin C (acyl CoA synthetase inhibitor), and thus increasing the free fatty acids (FFA) as substrates for uncoupling proteins; the second was to determine whether clusianone (natural compound from the roots of Clusia congestiflora), a structural analogue of chemical uncoupler nemorosoma, is capable of promoting the chemical uncoupling. Results and discussion: The triacsin C, in concentrations up to 1 ?M, showed no toxic effect on liver mitochondria and primary hepatocytes. In hepatocytes triacsin C increased oxygen consumption in the states of basal respiration and maximum respiratory rate. In addition, there was an increase in the expression of the transcription factor PGC1 - ? and ? - hydroxyacyl CoA dehydrogenase (ADH), an enzyme of ?- oxidation of fatty acids. The clusianone increased O2 consumption in resting state, decreased membrane potential, reduced the production of ROS and prevented the mitochondrial swelling induced by Ca2+ in a dose dependent manner, but less potent than nemorosone. Conclusions: Our results indicate that triacsin C accelerated mitochondrial metabolism, fatty acid oxidation and mitochondrial biogenesis; the clusianone was characterized as an effective uncoupler of mitochondrial oxidative phosphorylation, probably involving a protonoforic mechanism due to its chemical properties. Therefore, both strategies have therapeutic potential in the treatment of diseases such as liver steatosis, hypertriglyceridemia and obesity.

<p>Three methodology studies and two total synthesis endeavors are presented. First, a study of Lewis acid and hydrogen bond mediated soft enolization of thioesters and their addition to imines in the Mannich reaction is reported. MgBr2*OEt2 and Hunig's base are used in concert with bulky thioesters and aromatic aldehydes to generate syn-b-aminothioesters with moderate diastereoselectivity and yield. Next, a biomimetic organocatalytic Mannich reaction is presented using a chiral cinchona alkaloid to effect the enantioselective addition of an imines to thioesters with high yield and diastereoselectivity and enantioselectivities up to 88:12.</p><p>The direct addition of enolizable aldehydes to a-iodo thioesters to produce b-hydroxy thioesters enabled by reductive soft enolization is reported. The transformation is operationally simple and efficient and has the unusual feature of giving high syn-selectivity, which is the opposite of that produced in the aldol addition with (thio)esters under conventional conditions. This method is tolerant to aldehydes and imines that contain acidic a-protons, as well as electrophiles containing other acidic protons and base-sensitive functional groups.</p><p>The development of a strategy for the asymmetric synthesis of a large portion of the polycyclic polyprenylated acyl phloroglucinols via N-amino cyclic carbamate hydrazones, and its application to the first asymmetric total synthesis of both (+)- and (-)-clusianone is discussed. The clusianones are synthesized with an er of 99:1 and their anti-HIV activity is found to be 1.53 and 1.13 &#61549;M, respectively. A library of clusianone-like compounds is synthesized and their biological activity has been probed.</p><p>Finally, efforts towards the total synthesis of brasilicardin A are reported. An appropriate model system was synthesized, and conditions were established using a pinene-based aldol reaction to synthesize the b-methoxy-a-amino ester side chain of the molecule. Next, efforts toward the synthesis of the anti-syn-anti- perhydro-phenanthrene core are discussed.</p> / Dissertation

Polycyclic polyprenylated acylphloroglucinols (PPAPs) are structurally complex natural products with promising biological activities. These compounds have interesting anticancer and anti-HIV properties as well as other biological activities making them highly attractive synthetic targets. We report a stereodivergent, asymmetric total synthesis of (−)-clusianone in six steps from commercial materials. We have implemented a challenging cationic cyclization forging a bond between two sterically encumbered quaternary carbon atoms. Mechanistic studies point to the unique ability of formic acid to mediate the cyclization forming the clusianone framework. We also present a biosynthesis-inspired, diversity-oriented synthesis approach for rapid construction of PPAP analogs via palladium-catalyzed dearomative conjunctive allylic alkylation (DCAA). These efficient palladium-catalyzed protocols construct the [3.3.1]-bicyclic PPAP core in a single step from their stable aromatic precursors. The first syntheses of 13,14-didehydroxyisogarcinol and garcimultiflorone A stereoisomers are reported in six steps from a commercially available phloroglucinol. Lewis acid-controlled, diastereoselective cationic oxycyclizations enabled asymmetric syntheses of (−)-6-epi-13,14-didehydroxyisogarcinol and (+)-30-epi-13,14-didehydroxyisogarcinol. A similar strategy enabled production of the meso-derived isomers (±)-6,30-epi-13,14-didehydroxyisogarcinol and (±)-6,30-epi-garcmultiflorone A. A convenient strategy for gram scale synthesis of these stereoisomers was developed utilizing diastereomer separation at a later stage in the synthesis that minimized the number of necessary synthetic operations to access all possible stereoisomers. Finally, we report cationic rearrangements of dearomatized acylphloroglucinols leading to the formation of unprecedented PPAP scaffolds. A novel type A [3.3.1]-bicyclic PPAP was produced as a major product and the structure confirmed by X-ray crystallographic analysis. A novel [3.3.1]-bicyclolactone was also produced utilizing an alternative substrate. Efforts will be described to determine the scope of these rearrangements and type A-selective cyclizations. / 2018-08-09T00:00:00Z

Polycyclic polyprenylated acylphloroglucinols (PPAPs) are plant- (Guttiferae) derived natural products. They have fascinating bicyclo[3.3.1]nonane-2,4,9-trione or [3.2.1]nonane-2,4,8-trione cores decorated with prenyl or geranyl groups. More than 200 PPAPs have been isolated, but only a few of them have been synthesized, although most of the synthesized PPAPs are of type A and have an exo substituent at C (7). Here, we are trying to make a type B 7-endo PPAP, 7-epi-clusianone. The synthetic plan involves an alkynylation–aldol strategy to construct the bicyclic core. Having established the bicyclic core, the synthesis presents a new challenge: the oxidation of a very hindered 2-alkenone to the β-hydroxy 2-alkenone.

A biodiversidade de plantas brasileiras é uma fonte muito rica de moléculas bioativas, dentro da proposta da busca por novas drogas antitumorais, avaliamos neste estudo o potencial antiproliferativo do composto 7-epi-clusianona. Foram utilizadas duas linhagens celulares derivadas de tumor de mama humana, Hs 578T e MCF-7, cultivadas em monocamada e como esferoides. O IC50 após 48 horas de tratamento das células é de 20 &mu;M para Hs 578T e 6 &mu;M para MCF-7. A análise do ciclo celular mostrou que o composto é capaz de reter as células em fase G1/G0 em ambas as linhagens em 2D, mas não em 3D. O composto é capaz de induzir as células a senescência celular, como mostrado pelo ensaio de detecção de &beta;-galactosidase. Esses dados indicam que o composto 7-epi-clusianona é uma molécula promissora, que demonstrou potencial antitumoral em células de tumor de mama. A cultura tridimensional se mostrou mais resistente ao tratamento com 7-epi-clusianona, portanto estudos mais abrangentes são necessários para melhor entendimento dos efeitos do composto sobre esse tipo de cultura. / Brazilian flora is considered one of the most diverse in the world and natural products are some of the important sources of new antitumoral compounds. The aim of this study was to evaluate the antiproliferative potential of 7-epi-clusianone. Two cell lines derived from human breast tumor were used, Hs 578T and MCF-7, cultured in monolayer and as spheroids. The IC50 after 48 hours of treatment is 20 &mu;M to Hs 578T cells and 6 &mu;M to MCF-7 cells. Cell cycle analysis showed induction of cell cycle arrest in G1/S phase in cells cultured in monolayers, but not in spheroids. The amount of cells in senescence after the treatment with 7-epi-clusianone is higher than the control group, as seen by the senescence &beta;-galactosidase staining assay. These data suggest that 7-epi-clusianone is a promising molecule against breast cancer cells. We show that 3D culture was more resistant to treatment than 2D culture, therefore more comprehensive studies are needed to better understand the effects of 7-epi-clusianone on this kind of culture.

A biodiversidade de plantas brasileiras é uma fonte muito rica de moléculas bioativas, dentro da proposta da busca por novas drogas antitumorais, avaliamos neste estudo o potencial antiproliferativo do composto 7-epi-clusianona. Foram utilizadas duas linhagens celulares derivadas de tumor de mama humana, Hs 578T e MCF-7, cultivadas em monocamada e como esferoides. O IC50 após 48 horas de tratamento das células é de 20 &mu;M para Hs 578T e 6 &mu;M para MCF-7. A análise do ciclo celular mostrou que o composto é capaz de reter as células em fase G1/G0 em ambas as linhagens em 2D, mas não em 3D. O composto é capaz de induzir as células a senescência celular, como mostrado pelo ensaio de detecção de &beta;-galactosidase. Esses dados indicam que o composto 7-epi-clusianona é uma molécula promissora, que demonstrou potencial antitumoral em células de tumor de mama. A cultura tridimensional se mostrou mais resistente ao tratamento com 7-epi-clusianona, portanto estudos mais abrangentes são necessários para melhor entendimento dos efeitos do composto sobre esse tipo de cultura. / Brazilian flora is considered one of the most diverse in the world and natural products are some of the important sources of new antitumoral compounds. The aim of this study was to evaluate the antiproliferative potential of 7-epi-clusianone. Two cell lines derived from human breast tumor were used, Hs 578T and MCF-7, cultured in monolayer and as spheroids. The IC50 after 48 hours of treatment is 20 &mu;M to Hs 578T cells and 6 &mu;M to MCF-7 cells. Cell cycle analysis showed induction of cell cycle arrest in G1/S phase in cells cultured in monolayers, but not in spheroids. The amount of cells in senescence after the treatment with 7-epi-clusianone is higher than the control group, as seen by the senescence &beta;-galactosidase staining assay. These data suggest that 7-epi-clusianone is a promising molecule against breast cancer cells. We show that 3D culture was more resistant to treatment than 2D culture, therefore more comprehensive studies are needed to better understand the effects of 7-epi-clusianone on this kind of culture.