Alkylierte polyzyklische aromatische Kohlenwasserstoffe werden in vielen Matrizes wie Fahrzeugabgasen und Tabakrauch und auch als Kontaminanten in Nahrungsmitteln neben rein aromatischen Kongeneren gefunden. Alkylierte PAK können über die Alkylseitenkette über benzylische Hydroxylierung und nachfolgende Sulfonierung katalysiert über Sulfotransferasen (SULT) zu reaktiven Schwefelsäureestern umgesetzt werden. Die SULT-vermittelte Bioaktivierung zu einem genotoxischen Schwefelsäureester wurde für den benzylischen Alkohol 1-Hydroxymethylpyren des Hepatokanzerogens 1-Methylpyren in früheren Arbeiten gezeigt. In der vorliegenden Arbeit wurde überprüft, ob die benzylischen Alkohole weiterer alkylierter PAK über Sulfonierung zu genotoxischen Schwefelsäureestern umgesetzt werden. Hierzu wurde eine Gruppe von 17 Modellsubstanzen ausgewählt, um die Ableitung von Struktur-Aktivitäts-Beziehungen zu ermöglichen. Das genotoxische Potenzial authentischer benzylischer Schwefelsäureester der Modellsubstanzen wurde zunächst in vitro über DNA-Adduktbildung im zellfreien System und Mutagenität im Salmonella-Rückmutationstest untersucht. Die Sulfate zeigten große Reaktivitätsunterschiede in Abhängigkeit von der Struktur des aromatischen Systems und der Position der Alkylseitenkette, wobei die Endpunkte DNA-Adduktbildung und Mutagenität gut korrelierten. Des Weiteren wurde der Salmonella-Mutagenitätstest mit den benzylischen Alkoholen der untersuchten alkylierten PAK und gentechnisch veränderten S. typhimurium-Stämmen, die SULT-Formen des Menschen heterolog exprimieren, durchgeführt. Bis auf die Alkohole 2- und 4-HMP zeigten alle untersuchten benzylischen Alkohole deutliche mutagene Effekte in einem oder mehreren humane SULT exprimierenden Stämmen. Die durchgeführten in vitro-Versuche zeigten das Potenzial der benzylischen Metabolite alkylierter PAK für genotoxische Wirkungen. Nachfolgend musste geklärt werden, welche Relevanz die beobachteten Effekte für die komplexere in vivo-Situation haben. Nach Verabreichung verschiedener benzylischer Schwefelsäureester und Alkohole an männliche Ratten konnten DNA-Addukte in den untersuchten Organen detektiert werden, was im Fall der Schwefelsäureester deren systemische Bioverfügbarkeit und im Fall der benzylischen Alkohole deren Umsatz durch SULT der männlichen Ratte zeigte. Da im Gegensatz zum Menschen die SULT-Expression in der Ratte auf die Leber fokussiert ist, musste ein Großteil des Umsatzes zu genotoxischen Sulfaten in der Leber stattgefunden haben. DNA-Addukte wurden jedoch auch in extrahepatischen Organen gefunden, was über einen hepatischen Export der gebildeten reaktiven Sulfate und deren Transport über den Blutkreislauf zu diesen Geweben erklärt werden kann. Für die weiterführenden in vivo-Studien wurden die benzylischen Alkohole 1-HMP und 1-HM-8-MP ausgewählt, die trotz großer struktureller Ähnlichkeit toxikodynamische Unterschiede zeigten. Zur Untersuchung der Bedeutung des SULT-vermittelten Toxifizierungsweges als auch konkurrierender detoxifizierender oxidativer Stoffwechselprozesse, wurden für 1-HMP und 1-HM-8-MP in vivo-Inhibitionsstudien mit SULT-Inhibitoren und für 1-HM-8-MP auch mit ADH/ALDH-Inhibitoren durchgeführt. Eine Vorbehandlung mit dem SULT-Hemmstoff Pentachlorphenol führte zu einer Reduktion der DNA-Adduktniveaus in Organen 1-HMP- und 1-HM-8-MP-behandelter Tiere. Die Verabreichung von Quercetin hatte keine Auswirkung auf die DNA-Adduktniveaus. Die Hemmung der DNA-Adduktbildung bei Verabreichung von Pentachlorphenol verdeutlichte jedoch, dass benzylische Alkohole alkylierter PAK in vivo über Sulfonierung bioaktiviert werden. Eine Vorbehandlung mit dem ADH-Inhibitor 4-Methylpyrazol und dem ADH-Substrat Ethanol führte zu erhöhten DNA-Adduktniveaus in Organen 1-HM-8-MP-behandelter Tiere. Den gleichen Effekt, jedoch in geringerem Ausmaß, hatte auch die Vorbehandlung mit dem ALDH-Inhibitor Disulfiram. Dies deutet darauf hin, dass oxidative Modifikationen an der Seitenkette des 1-HM-8-MP einen Detoxifizierungsmechanismus darstellen. Nach Verabreichung benzylischer Metabolite alkylierter PAK wurden oftmals hohe Adduktniveaus in der Niere detektiert. Als mögliche Ursache hierfür wurde eine Transporter-vermittelte renale Sekretion reaktiver Sulfate postuliert, die über Vorbehandlung mit Probenecid vor Verabreichung von 1-HMP und 1-HM-8-MP überprüft wurde. Der Haupteffekt der Probenecid-Behandlung wurde jedoch nicht in der Niere, sondern in der Leber beobachtet, die stark erhöhte Adduktniveaus zeigte. Eine mögliche Erklärung hierfür ist die Hemmung des Exportes in der Leber gebildeter reaktiver Sulfate über Inhibition hepatischer organischer Anionentransporter. / Alkylated polycyclic aromatic hydrocarbons are found besides purely aromatic congeners in numerous matrices like car engine exhausts and tobacco smoke and as contaminants in foods. Alkylated PAH can be converted at the alkyl side chain to reactive sulfuric acid esters via benzylic hydroxylation and subsequent sulfonation catalysed by sulfotransferases (SULT). The SULT-mediated bioactivation to a genotoxic sulfuric acid ester was shown for the benzylic alcohol 1-hydroxymethylpyrene of the hepatocarcinogen 1-methylpyrene in previous studies. In the thesis at hand it was studied if the benzylic alcohols of further alkylated PAH are converted to genotoxic sulfuric acid esters via sulfonation. For this purpose a group of 17 model substances was chosen to allow for deduction of structure activity relationships. The genotoxic potential of authentic benzylic sulfuric acid esters of the model substances was initially investigated in vitro via DNA adduct formation in a cell free system and mutagenicity in the Salmonella reverse mutation test. The sulfates showed large differences in reactivity depending on the structure of the aromatic system and the position of the alkyl side chain whereupon the endpoints DNA adduct formation and mutagenicity correlated well. Furthermore, the Salmonella mutagenicity test was carried out with the benzylic alcohols of the alkylated PAH studied and S. typhimurium strains genetically engineered for the heterologous expression of human SULT forms. Except for the alcohols 2- and 4-HMP all benzylic alcohols studied showed clear mutagenic effects in one or more SULT-expressing strains. The studies performed in vitro demonstrated the potential of benzylic metabolites of alkylated PAH for genotoxic effects. Consecutively, the relevance of the observed effects for the more complex in vivo situation had to be clarified. After administration of different benzylic sulfuric acid esters and alcohols to male rats DNA adducts were detected in the organs studied, in case of the sulfuric acid esters showing their systemic bioavailability and in case of the benzylic alcohols demonstrating their conversion to the corresponding reactive benzylic sulfuric acid esters by SULT of the male rat. Since in contrast to man SULT expression in the rat is focused on the liver, a large part of the conversion to genotoxic sulfates must have been taken place in the liver. However, DNA adducts were also found in extrahepatic tissues which can be attributed to a hepatic export of the reactive sulfates formed and their transport to these tissues via circulation. For the continuative in vivo studies the benzylic alcohols 1-HMP and 1-HM-8-MP were chosen that demonstrated toxicodynamic differences in spite of their great structural resemblance. To investigate the importance of the SULT-mediated toxification pathway as well as competing detoxifying oxidative metabolic pathways, in vivo inhibition studies with SULT inhibitors were performed for 1-HMP and 1-HM-8-MP and with ADH/ALDH inhibitors also for 1-HM-8-MP. A pretreatment with the SULT inhibitor pentachlorophenol led to a reduction of DNA adduct levels in organs of animals treated with 1-HMP and 1-HM-8-MP. Administration of quercetin had no impact on the DNA adduct levels. However, inhibition of DNA adduct formation at administration of pentachlorophenol demonstrated that benzylic alcohols of alkylated PAH are bioactivated via sulfonation in vivo. A pretreatment with the ADH inhibitor 4-methylpyrazole and the ADH substrate ethanol led to increased DNA adduct levels in organs of animals treated with 1-HM-8-MP. The same effect but to a lesser extent was caused by a pretreatment with the ALDH inhibitor disulfiram. This indicates that oxidative modifications at the side chain of 1-HM-8-MP represent a detoxification mechanism. After administration of benzylic metabolites of alkylated PAH often high DNA adduct levels were detected in kidney. A transporter-mediated renal secretion was postulated as possible cause which was investigated using a pretreatment with probenecid before administration of 1-HMP and 1-HM-8-MP. However, the main effect of the treatment with probenecid was not observed in kidney but in liver that showed strongly increased adduct levels. A possible explanation for this effect is the inhibition of the export of reactive sulfates formed in liver via inhibition of hepatic organic anion transporters.

Small molecule Zn(II) complexes containing N- and S- donor environments may serve as appropriate models for mimicking Zn protein sites, and thus, their reactions with heavy metal ions such as Pt(II) and W(0) may provide insight into possible adduct formation and zinc displacement. To study such possible interactions between zinc finger proteins and platinum-bound DNA, the ZnN2S2 dimeric complex, N,N?-bis(2- mercaptoethyl)-1,4-diazacycloheptane zinc (II), [Zn-1?]2, has been examined for Znbound thiolate reactivity in the presence of Pt(II) nitrogen ? rich compounds. The reactions yielded Zn/Pt di- and tri- nuclear thiolate-bridged adducts and metalexchanged products, which were initially observed via ESI-mass spectrometry (ESI-MS) analysis of reaction solutions, and ultimately verified by comparison to the ESI-MS analysis, 195Pt NMR spectroscopy, and X-ray crystallography of directly synthesized complexes. The isolation of Zn-(?-SR)-Pt-bridged [(Zn(bme-dach)Cl)(Pt(dien))]Cl adduct from these studies is, to our knowledge, the first Zn-Pt bimetallic thiolatebridged model demonstrating the interaction between Zn-bound thiolates and Pt(II). Additional derivatives involving Pd(II) and Au(III) have been explored to parallel the experiments executed with Pt(II). The [Zn-1?]2 was then modified by cleavage with Na+[ICH2CO2]- to produce (N- (3-Thiabutyl)-N?-(3-thiapentaneoate)-1,4-diazacycloheptane) zinc(II), Zn-1?-Ac or ZnN2SS?O, and 1,4-diazacycloheptane-1,4-diylbis(3-thiapentanoato) zinc(II), Zn-1?-Ac2 or ZnN2S?2O2, monomeric complexes (where S = thiolate, S? = thioether). The [Zn-1?]2 di- and Zn-1?-Ac mono-thiolato complexes demonstrated reactivity towards labile-ligand tungsten carbonyl species, (THF)W(CO)5 and (pip)2W(CO)4, to yield, respectively, the [(Zn-1?-Cl)W(CO)4]- complex and the [(Zn-1?-Ac)W(CO)5]x coordination polymer. With the aid of CO ligands for IR spectral monitoring, the products were isolated and characterized spectroscopically, as well as by X-ray diffraction and elemental analysis. To examine the potential for zinc complexes (or zinc-templated ligands) to possibly serve as a toxic metal remediation agents, Zn-1?-Ac and Zn-1?-Ac2 were reacted with Ni(BF4)2. The formation of Zn/Ni exchanged products confirmed the capability of ?free? Ni(II) to displace Zn(II) within the N-, S-, and O- chelate environment. The Zn/Ni exchanged complexes were analyzed by ESI-MS, UV-visible spectroscopy, IR spectroscopy of the acetate regions, and X-ray crystallography. They serve as foundation molecules for more noxious metal exchange / zinc displacement products.

A significant focus of nucleic acids research is on the reactivity of electrophilic species with DNA to form addition products (adducts). Phenols are known to be able to form adducts at the C8 site of deoxyguanosine (dG). This dissertation studies the oxygen (O)-linked class of phenolic dG adducts for their hydrolytic stability as well as their structural impact on the DNA duplex. To determine the effect of C8 O-linked phenolic dG adducts on glycosidic bond stability spectrophotometric determination of hydrolysis kinetics was performed. The kinetics establish the adducts to be less stable than native dG in acid, but surprisingly stable under physiological conditions. Then to assess the modified duplex structure, a C8 O-linked phenolic dG adduct was incorporated into a DNA duplex. Thermal melting analysis establish the adduct as having a destabilizing effect on the regularly paired duplex and the conformational analysis suggests the phenolic lesion to be weakly mutagenic. / NSERC

Submitted by Maike Costa (maiksebas@gmail.com) on 2017-06-27T14:23:29Z No. of bitstreams: 1 arquivototal.pdf: 5840129 bytes, checksum: 587ce0612688b158f82e9c8c528f38e7 (MD5) / Made available in DSpace on 2017-06-27T14:23:29Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 5840129 bytes, checksum: 587ce0612688b158f82e9c8c528f38e7 (MD5) Previous issue date: 2016-08-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / This work was performed in order to synthesize and bioavaliar the activity of new adducts Morita-Baylis-Hillman (AMBH) as potential drug candidates. The AMBH were synthesized from the twin drug approach (approach twin drugs) and bioavaliados against Leishmania promastigote form donovanii, a kind of visceral leishmaniasis and more severe disease, which has a drug used for the treatment accompanied by high toxicity. As Michael acceptor to be used in Morita-Baylis-Hillman (MRBH) was synthesized diacrylate ethylene glycol (50) from the esterification reaction between ethylene glycol (65) and acrylic acid (66). The first MRBH was investigated between two equivalent 2-nitrobenzaldehyde (57) and one equivalent of diacrylate 50, in acetonitrile as solvent in the presence of DABCO, yielding two products: an adduct 67 and an adduct homodimeric 42. In investigations of experimental parameters the MRBH, DMF, DABCO and room temperature proved to be the most favorable conditions for the formation of adducts homodimeric, these being obtained in yields of 35-94% and reaction times between 24 and 20 days, isolated by liquid / liquid and via flash chromatography. Homodimers and other bioavaliados AMBH results were satisfactory to excellent IC50 for homodimeric adducts (IC50 126.20 to 0,50M). All homodimeric AMBH had higher bioactivity the corresponding AMBH, showing the success of the twin drugs approach against promastigote species of leishmania donovanii, reaching the impressive result, in the case of 49 homodimer be 393.1 times more active than the corresponding AMBH 56, being 1.24 more active than the anfoterinica B, and no reported toxicity exposure in red blood cells of human blood (iS> 400 against iS = 18.73 amphotericin B). These results show that 49 homodimer is a promising molecule in the search for new drug candidates. / Este trabalho foi realizado com o objetivo de sintetizar e bioavaliar a atividade de novos Adutos de Morita-Baylis-Hillman (AMBH) como potenciais candidatos a fármacos. Os AMBH foram sintetizados a partir da abordagem drogas gêmeas (twin drugs approach) e bioavaliados contra a forma promastigota Leishmania donovanii, uma espécie da forma visceral e mais grave da doença, a qual possui o fármaco utilizado para o tratamento acompanhado de grande toxicidade. Como aceptor de Michael para ser utilizado na reação de Morita-Baylis-Hillman (RMBH), foi sintetizado o diacrilato do etileno glicol (50) a partir da reação de esterificação entre o etileno glicol (65) e o ácido acrílico (66). A primeira RMBH investigada foi entre dois equivalentes 2-nitrobenzaldeído (57) e um equivalente do diacrilato 50, em acetonitrila como solvente na presença de DABCO, obtendo-se dois produtos: um aduto 67 e um aduto homodimérico 42. Nas investigações dos parâmetros experimentais da RMBH, o DMF, o DABCO e a temperatura ambiente mostraram ser as condições mais favoráveis para a formação dos adutos homodiméricos, sendo esses obtidos com rendimentos entre 35-94% e em tempos reacionais entre 24h e 20 dias, isolados por extração líquido/líquido e via cromatográfia flash. Os homodímeros e os demais AMBH bioavaliados tiveram resultados de satisfatórios a excelentes de CI50 para os adutos homodiméricos (CI50 126,20 a 0,50M). Todos os AMBH homodiméricos tiveram bioatividade superior aos AMBH correspondentes, evidenciando o sucesso da abordagem de drogas gêmeas contra a espécie promastigota da leishmania donovanii, chegando ao impressionante resultado, no caso do homodímero 49, ser 393,1 vezes mais ativo que o AMBH correspondente 56, sendo 1.24 mais ativo que a anfoterinica B, além de não apresentar toxicidade na exposição em glóbulos vermelhos do sangue humano (IS > 400, contra IS = 18,73 da anfotericina B). Estes resultados evidenciam que homodímero 49 é uma molécula promissora na busca de novos candidatos a fármacos.

Submitted by ANA KARLA PEREIRA RODRIGUES (anakarla_@hotmail.com) on 2017-08-04T17:21:10Z No. of bitstreams: 1 arquivototal.pdf: 15443881 bytes, checksum: a81c2afaef7e1fccdeb2543bc340d5a1 (MD5) / Made available in DSpace on 2017-08-04T17:21:10Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 15443881 bytes, checksum: a81c2afaef7e1fccdeb2543bc340d5a1 (MD5) Previous issue date: 2015-08-10 / Nitroaromatic compounds derived from Morita-Baylis-Hillman reaction (RMBH) have been tested in the treatment of most neglected diseases such as malaria, Chagas disease and leishmaniasis. An important experimental observation is the relation between biological activity (measured by IC50) and reduction potential of these compounds (estimated by the cathodic and anodic peak potentials determined by electroanalytical techniques), the latter directly connected to the reduction of the nitro group (-NO2 ). For this reason, electrochemical methods have been used in order to mimic the enzymatic bioreduction of these compounds, as reported by Vasconcellos et al. (J. Braz. Chem. Soc. 23:894, 2012). The objective of this work was to develop a computational protocol to predict the reduction potential in aprotic media to support the molecular modeling of new compounds with desired pharmacological activity. The developed direct protocol (for aprotic solvents) consists of performing DFT calculations with B98, PBE1PBE or M06-2X functionals with 6-31+G(d,p) basis set and C-PCM solvation method (with standard cavitation method UFF/VdW). The results show that it is possible to predict the experimental variation of the reduction potential of at least 70 % of confidence (in a range of experimental data of only 140 mV) with absolute average errors less than 45 mV (much less than the experimental uncertainty of the absolute reaction potential of hydrogen electrode, approximately 400 mV) and standard deviation of about 35 mV (inferior to 1,0 kcal/mol). The application of direct protocol for a series of 65 uncorrelated molecules, whose reduction potentials vary in a range of more than 6 V, provided a model with more than 99% of predictive power. From the application of the protocol to a series of 40 molecules, for which experimental results are not available, it was possible to predict that some of these structures may have more favorable potentials to bioreduction process than the systems used in the calibration step, which makes them candidates for new drugs. / Compostos nitroaromáticos derivados da reação de Morita-Baylis-Hillman (RMBH) vêm sendo testados no tratamento de doenças extremamente negligenciadas, tais como malária, doença de Chagas e leishmanioses. Uma importante observação experimental consiste na relação entre a atividade biológica (medida pelo IC50) e o potencial de redução (estimado pelos potencias de pico anódico e catódico determinados por técnicas eletroanalíticas) destes compostos, este último diretamente ligado à redução do grupo nitro (-NO2). Por esta razão, métodos eletroquímicos têm sido utilizados com o intuito de simular a biorredução enzimática destes compostos, como reportado por Vasconcellos e colaboradores (J. Braz. Chem. Soc. 23:894, 2012). O objetivo deste trabalho foi o de desenvolver um protocolo computacional para a predição de potenciais de redução em meio aprótico para auxiliar a modelagem molecular de novos compostos com a atividade farmacológica desejada. O protocolo direto desenvolvido (para solventes apróticos) consiste na realização de cálculos DFT com os funcionais B98, PBE1PBE ou M06-2X, com o conjunto de funções de base 6-31+G(d,p) e método de solvatação C-PCM (com o método de cavitação padrão UFF/VdW). Os resultados mostram que é possível prever a variação experimental do potencial de redução com pelo menos 70 % de confiança (em uma faixa de valores experimentais de apenas 140 mV) e erros médios absolutos inferiores a 45 mV (muito inferior à incerteza experimental do potencial de redução absoluto do eletrodo de hidrogênio, que é de cerca de 400 mV) e desvio-padrão de cerca de 35 mV (inferior a 1,0 kcal/mol). A aplicação do protocolo direto a uma série de 65 moléculas não-correlacionadas, cujos potenciais de redução variam em uma faixa de mais de 6 V, forneceu um modelo com mais de 99 % de poder preditivo. A partir da aplicação do protocolo a uma série de 40 moléculas, para as quais ainda não estão disponíveis resultados experimentais, foi possível prever que algumas destas estruturas podem possuir potenciais mais favoráveis ao processo de biorredução que os estudados na etapa de calibração, o que as tornam candidatos à novos fármacos.

Made available in DSpace on 2015-05-14T13:21:06Z (GMT). No. of bitstreams: 1 parte1.pdf: 1561006 bytes, checksum: 92830d9ef59f992edaa0a7616cdb4412 (MD5) Previous issue date: 2011-03-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / This study was conducted in order to develop the synthesis of new Morita-Baylis-Hillman adducts of (MBHA) water soluble (29-34) with potential parasitic activity, using glycerol (1) as raw material. Two synthetic routes were carried out producing twelve new MBHA (23 - 34), six of which have hydrophilic characteristics 29-34 (main target of the work) and six more hydrophobic 23 28 (as synthesis intermediates), and these intermediaries are also important for comparative studies of structure and biological activity (SAR). Starting from glycerol (1), we synthesize the 2,2-dimethyl-1,3-dioxolan-4-yl)methyl acrylates (21, 94%) and from this, two synthetic routes were investigated to produce the 29- 34 adducts. On Route 1, the adducts 29-34 were produced in one-step (54 - 82%) from the Michael acceptor 2,3-dihydroxypropyl acrylate 22, which was prepared in 100% yield from 21. In route 2, MBHA intermediates 23 - 28 were prepared directly from 21 (90 - 100%) and these adducts were subsequently processed in adducts 29-34 (70 - 90%). In the synthesis of adduct 32 was observed the formation of the Michael addition product 47 and Indolizines unprecedented 48 as co-products of synthesis and characterized only by CGMS during the synthetic route 2. All syntheses in this work were developed in industry standards convenient. The activation reaction by microwave irradiation (MW) was widely used in most steps of this synthetic work, leading to high chemical yields and reduced reaction times. / Este trabalho foi realizado no intuito de desenvolver a síntese de novos adutos de Morita-Baylis-Hillman (AMBH) hidrossolúveis (29-34) com potenciais atividades antiparasitárias, usando o glicerol (1) como matéria-prima. Duas rotas sintéticas foram desenvolvidas conduzindo a doze AMBH inéditos (23 - 34), dos quais seis possuem as características hidrofílicas 29-34 (alvo principal do trabalho) e as outras seis mais hidrofóbicas 23 - 28 (como intermediários de síntese), sendo que estes intermediários são também importantes para estudos comparativos entre estrutura e atividades biológicas (SAR). Partindo do glicerol (1), sintetizamos o acrilato de 2,2-dimetil-1,3-dioxalila (21, 94%) e a partir deste, duas rotas sintéticas foram investigadas para os adutos-alvo 29- 34. Na rota 1, os adutos 29-34 foram produzidos em uma única etapa sintética (54 - 82%) a partir do aceptor de Michael acrilato de 2,3- dihidroxipropila 22, que foi preparado em 100% de rendimento a partir de 21. Na rota 2, os AMBH intermediários 23 28 foram preparados em 90 - 100% diretamente de 21 e estes adutos foram subseqüentemente transformados nos adutos 29-34 (70 - 90%). Na síntese do aduto 32 foi observado a formação do produto de adição de Michael 47 e da indolizina inédita 48 como co-produtos de síntese e caracterizados apenas por CGMS, durante a rota sintética 2. Todas as sínteses neste trabalho foram desenvolvidas em padrões convenientes a indústria. A ativação reacional por irradiação de microondas (MO) foi amplamente utilizada na maioria das etapas sintéticas deste trabalho, conduzindo aos altos rendimentos químicos e aos tempos reacionais reduzidos.

Made available in DSpace on 2015-05-14T13:21:18Z (GMT). No. of bitstreams: 1 Arquivototal.pdf: 11661227 bytes, checksum: e157f4398306766375669b2e52ab9310 (MD5) Previous issue date: 2012-11-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / In this work, we describe the investigation of the Morita-Baylis-Hillman reaction in microwave reactor, under closed vessel conditions to prepare 40 adducts, six novel, using 30 aromatic aldehydes and isatin as electrophiles and acrylonitrile (62a) , methyl acrylate (2), hydroxyethyl acrylate (62b), 2,2-dimethyl-1 ,3-dioxalyl acrylate (62c) and 2,3-dihydroxypropyl acrylate (62d) as Michael acceptors catalyzed by 1, 4-diazabicyclo [2.2.2] octane (DABCO). Most adducts were synthesized efficiently in short reaction times and excellent yields (70-99%) using this reaction protocol. However, this synthetic methodology showed limitations for less reactive substrates due to formation of byproducts and in some cases the formation of the desired adduct was not obtained. The application of low temperatures and / or conventional room temperature protocol led to the adducts preparation in a chemoselective manner. We proved that the formation of 2-hydroxy (4-bromophenyl) methyl acrylate (43) is reversible at 120 ° C, which explains the restriction observed in the preparation of certain adducts using heating by microwave irradiation and successfully protocol employing temperature of 0º C. Preliminary evaluation of cytotoxic activity against cell lines of human leukemia HL-60 was performed, where the nitrilated isatin derivatives adducts (56a and 56b) were the most active. Preliminary studies of antimitotic activity in embryonic cells of sea urchin (Echinometra lucunter) performed with nitrilated adducts containing pyridine rings (49, 47, 20), nitro group (16, 38, 15) and bromine atom attached to the aromatic ring (42) revealed that except 47, all investigated adducts strongly inhibited stages of embryonic development of the first cleavage and morula. Moreover, experimental observations showed no inhibition of microtubule organization, induction of cell necrosis and toxicity of the adducts, suggesting that the blocking mechanism of action of protein synthesis and / or DNA. / Neste trabalho, nós descrevemos a investigação da reação de Morita-Baylis-Hillman em reator de micro-ondas, sob condições de vaso fechado para preparação de 40 adutos, sendo seis inéditos, utilizando 30 aldeídos aromáticos e a isatina como eletrófilos e acrilonitrila (62a), acrilato de metila (2), acrilato de hidroxietila (62b), acrilato de 2,2-dimetil-1,3-dioxalila (62c) e acrilato de 2,3-diidroxipropila (62d) como aceptores de Michael catalisada por 1,4-diazabiciclo [2.2.2] octano (DABCO). A maioria dos adutos foram sintetizados de forma eficiente em curtos tempos reacionais e excelentes rendimentos (70-99%) usando este protocolo reacional. No entanto, esta metodologia sintética apresentou limitações para substratos menos reativos, devido a formação de subprodutos e em alguns casos a não formação do aduto desejado. A aplicação de baixa temperatura e / ou protocolo convencional a temperatura ambiente conduziram a preparação dos adutos de forma quimiosseletiva. Provamos que a reação de formação do acrilato de 2-hidroxi(4bromofenil)metila (43) é reversível a 120° C, fato que explica a limitação observada na preparação de alguns adutos usando aquecimento por irradiação de micro-ondas e o sucesso no emprego de protocolo a temperatura de 0º C. A avaliação preliminar da atividade citotóxica contra linhagem de células de leucemia humana HL-60 foi realizada, onde os adutos nitrilados derivados da isatina (56a e 56b) foram os mais ativos. Estudos preliminares da atividade antimitótica em células embrionárias de ouriço do mar (Echinometra lucunter) realizados com adutos nitrilados contendo anéis piridínicos (49, 47, 20), grupo nitro (16, 38, 15) e átomo de bromo ligados ao anel aromático (42) revelaram que, exceto 47, todos os adutos investigados inibiram fortemente os estágios de desenvolvimento embrionário de primeira clivagem e mórula. Além disso, observações experimentais não demonstraram inibição da organização dos microtúbulos, indução de necrose celular e toxicidade por parte dos adutos, sugerindo como mecanismo de ação o bloqueio da síntese protéica e/ou de DNA.

Made available in DSpace on 2015-05-14T13:21:44Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 4637140 bytes, checksum: f9c50e9a2115f5a805442d163ed54f1e (MD5) Previous issue date: 2012-12-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The Morita-Baylis-Hillman Adducts (MBHA) is a class of molecules studied by our research group on synthetic, theoretical and bioactivity aspects. In this work, we present Quantitative Structure-Activity Relationship (QSAR) models involving 32 aromatic MBHA. Initially, the most stable conformations of thirty-two MBHA were investigated by theoretical methods, which were used to construct models. For this study, were obtained potential energy curves using AM1 semi-empirical method, considering rotational degrees of freedom (sigma bonds). From these curves, the less energy conformation to each molecule was selected and optimized at B3LYP/6- 31+G(d) level, considering solvent effects through Polarizable Continuum Model (PCM). Proton Nuclear Magnetic Ressonance data are in agreement with the conformational study. Intramolecular Hydrogen Bonds (IHB) are presents in the most of the studied compounds, according to structural characterization and QTAIM calculations. Curiously, compounds that showed hydrogen bonds involving the nitro and hydroxyl groups have the best values of biological activity (IC50). An explanation is based on redox mechanism of action of nitrocompounds. NBO (Natural Bond Orbital) charges and LUKO (Lowest Unoccupied Kohn-Sham Orbitals) analysis at the ortho-nitro group are in agreement with these analyses. Considering quantum calculations and structural observations, four descriptors were selected a priori and submitted to a QSAR study using PLS (Partial Least Squares) and MLR (Multiple Linear Regression) modeling. A second QSAR approach was made from the another set of descriptors obtained through the online platform E-DRAGON, which were submitted to a variable selection method. The quality parameters obtained for models indicate that both are robust and predictive. / Os Adutos de Morita-Baylis-Hillman (AMBH) compreendem uma classe de moléculas investigadas por nosso grupo de pesquisas nos aspectos sintéticos, teóricos e de bioatividade. Neste trabalho, apresentamos modelos de Relação Quantitativa entre a Estrutura Química e a Atividade Leishmanicida (QSAR) envolvendo 32 AMBH aromáticos. Deste modo, inicialmente foram investigadas as conformações mais estáveis de cada composto através de métodos teóricos, as quais foram utilizadas na construção dos modelos. Foram obtidas curvas de energia potencial utilizando o método semi-empírico AM1, considerando graus de liberdade rotacionais (ligações sigma). A partir destas curvas, a conformação de menor energia para cada molécula foi selecionada e otimizada ao nível B3LYP/6-31+G(d), considerando os efeitos do solvente aquoso usando PCM ( Polarizable Continuum Model ). Dados espectroscópicos de Ressonância Magnética Nuclear de prótons corroboraram o estudo conformacional. Ligações de Hidrogênio Intramoleculares (LHI) se mostraram presentes na maioria das moléculas estudadas, conforme caracterização estrutural e cálculos QTAIM ( Quantum Theory Atoms in Molecules ). Curiosamente, os compostos que apresentaram Ligações de Hidrogênio envolvendo o grupo nitro (NO2) e a hidroxila (OH) possuem melhores valores de atividade biológica (menor IC50). Uma explicação está baseada no mecanismo de ação redox de nitrocompostos. Observação das cargas NBO ( Natural Bond Orbitals ) e análise dos orbitais de fronteira LUKO ( Lowest Unoccupied Kohn-Sham Orbitals ) ao nível do grupo orto-nitro estão de acordo com estas análises. Considerando os cálculos quânticos, bem como observações estruturais, quatro descritores foram selecionados a priori e submetidos a um estudo QSAR ( Quantitative Structure- Activity Relationships ) utilizando modelagem PLS ( Partial Least Squares ) e MLR ( Multiple Linear Regression ). Uma segunda abordagem QSAR foi realizada a partir de outro conjunto de descritores obtidos através da plataforma online E-DRAGON, os quais foram submetidos ao método de seleção de variáveis OPS ( Ordered Predictor Selection ). Os parâmetros de qualidade obtidos para os modelos indicam que ambos são robustos e preditivos.

A presente tese apresenta: I - Uma revislo bibliográfica sobre os adutos de Diels-Alder entre benzoquinonas 2-mono-substituídas ou benzoquinona não-substituída e ciclopentadieno; II - Uma visão mecanística, de literatura, sobre as reações de ciclo-adição; III - A síntese, por nós efetuada, de uma série de adutos de Diels-Alder entre ciclopentadieno e benzoquinonas 2-mono-substituídas por cloro, bromo, ou ainda, substituintes contendo nitrogênio, oxigênio, enxofre ou selênio; IV - O estudo que empreendemos sobre a fotociclização dos adutos acima mencionados. Na obtenção dos adutos, foram empregados dois métodos distintos: a reação de Diels-Alder e a substituição do halogênio por nucleófilos, nos adutos halogenados. Nove adutos inéditos foram isolados e caracterizados. A estereoquímica dos treze adutos preparados foi esclarecida por RMN de 13C e de 1H. O estudo da fotociclização dos adutos visava dois aspectos: sintético, para a obtenção de compostos-gaiola, e mecanístico. Foram isolados e caracterizados sete novos compostos-gaiola. Foi sugerido o mecanismo das referidas fotociclizações, através das determinações dos rendimentos quânticos e dos resultados de voltametria cíclica. Foi, também, fornecida uma possível explicação para a falta de reatividade fotoquímica de alguns adutos. / This thesis contains: I - A literature review about cyclopentadiene Diels-Alder adducts of benzoquinone and its 2-mono-substituted derivatives; II - A mechanistic overview of cycloaddition reactions; III - The synthesis of a series of Diels-Alder adducts of cyclopentadiene and 2-mono-substituted (by chloro-, bromo- or groups containing nitrogen, oxigen, sulfur or selenium) benzoquinones; IV - The investigation about the photocyclization of the same adducts. Two different methods were employed for the obtention of these adducts: either the Diels-Alder reaction, or the substituition of the halogen by nucleophiles, in the halo-containing adducts. Nine previously unreported adducts were isolated and characterized. The sterochemistry of the thirteen adducts prepared was determined through 13C and 1H NMR. The investigation about the photocyclization of the adducts had two main objectives: the synthesis of the corresponding cage-compounds and the elucidation of the photocyclization\'s mechanism. Seven new cage-compounds, previously unreported, were obtained and characterized. Quantum yield determinations and cyclic voltammetry data led to the proposition of a mechanism for the photocyclization reactions. An explanation for the lack of photoreactivity presented by some adducts was also furnished.

A produção de radicais de carbono \"in vivo\" durante a biotransformação da hidrazina foi demonstrada por ressonância para magnética eletrônica, utilizando o método do captador de spin. Eritrócitos de rato também oxidaram a hidrazina, formando radicais de carbono e nitrogênio, além de espécies reativas de oxigênio. Todas estas espécies, possivelmente formadas \"in vivo\", são potencialmente causadoras de dano a macromoléculas. Podem, por exemplo, iniciar reações secundárias formando radicais de componentes celulares, como ocorreu com a hemoglobina que foi oxidada a radicais tiil-hemoglobina em eritrócitos tratados com hídrazina. Radicais de carbono formados durante a biotransformação da hidrazina em animais expostos provêm necessariamente de substâncias endógenas e podem ser direta ou indiretamente responsáveis pela modificação ( alquilação ) de bases no DNA \"in vivo\". A hidrazona do formaldeído é descrita na literatura como um intermediário da alquilação induzida por hidrazina \"in vivo\". Células L 1210, catalase ou oxihemoglobina de rato foram capazes de formar radicais de carbono durante a oxidação da hidrazona do formaldeído. A oxidação da hidrazona do formaldeído pela catalase foi estudada \"in vítro\" e os radicais de carbono formados, identificados como radicais metila. A base modificada C8 -metil-guanina foi formada em animais expostos, como demonstrado por cromatografia líquida de alta eficiência associada à detecção eletroquímica, sugerindo que ocorreu alquilação do DNA por radicais metila durante a biotransformação da hidrazina \"in vivo\". / The production of carbon-centered radicais during hydrazine biotransformation \"in vivo\" was demonstrated by electron paramagnetic resonance ( EPR ) spin trapping technique. Rat red blood cells also oxidized hydrazine, forming carbon and nitrogen centered radicais, besides oxygen reactive speties. Ali these species, possibly formed \"in vivo\", are potentially harmful to macromolecules. For example, they can initiate secondary reactions in which the radicais from cell components are formed, as it occurred with hemoglobin, forming thiyl-hemoglobin radicais in the red blood cells treated with hydrazine. Carbon-centered radicais produced during the biotransformation of hydrazine in exposed animais must be derived from endogenous sources and may be directly or indirectly responsible for the modificaton ( alkylation ) of DNA bases \"in vivo\". The formaldehyde hydrazone is reported in the literature as an intermediate of hydrazine-induced alkylation \"in vivo\". L1210 cells, catalase and rat hemoglobin were able to produce carbon-centered radicais during the oxidation of the formaldehyde hydrazone. The oxidation of formaldehyde hydrazone by catalase was studied \"in vitro\" and the generated carbon-centered radicais were identified as methyl radicais. The modified base C8 -methylguanine was formed in exposed animais, as demonstrated by high performance liquid chromatography with electrochemical detection, suggesting that DNA alkylation by methyl radicais occurred during hydrazine biotransformation \"in vivo.\"