Perophoramidine 1 is a halogenated natural product which contains two contiguous quaternary centres within its structure. In this thesis, approaches towards the synthesis of perophoramidine are described. In particular, the synthesis of the tetracyclic core structure and the formation of the quaternary centres have been examined. In Chapter 1, the natural product perophoramidine 1 is introduced and its isolation, structure and biological activity is discussed. The structurally related communesin family of natural products are also introduced before the literature published on both the biosynthesis and laboratory synthesis of perophoramidine 1, is reviewed. Finally the Westwood group's approach towards the synthesis of perophoramidine 1 is introduced with a summary of non-halogenated model system investigations previously carried out within the group being provided. Chapter 2 describes studies towards the synthesis of an appropriately halogenated indolo[2,3-b]quinoline core structure of perophoramidine 1. This then allowed methodology previously developed within the group on model system substrates to be applied to the formation of the first of the two quaternary centres required for the synthesis of perophoramidine 1. Chapter 3 describes the attempted formation of the second quaternary centre using an ester alkylation approach. After initial studies failed to generate the desired quaternary centre, non-halogenated model system studies were carried out in an attempt to develop an alternative approach. In Chapter 4, model system studies were continued with cyclic ether compounds investigated as potential intermediates towards the synthesis of perophoramidine 1. The results obtained in this chapter provided a novel route to the formation of the second quaternary centre and led to a redesigned approach towards perophoramidine 1 being developed. In Chapter 5, this redesigned approach was applied to the halogenated intermediates synthesised in Chapter 1. This led to the formation of the first halogenated intermediate synthesised within the group which contained the two contiguous quaternary centres required for the synthesis of perophoramidine 1.

The present investigation has involved a kinetic and mechanistic study of the thermal cyclization of 3-acetoxy-3-(2-pyridyl)-2-methylenepropanoate esters and related compounds to 2-substituted indolizines. Substrates for the kinetic study were prepared via the Baylis-Hillmann reaction of pyridine-2-carboxaldehydes with acrylate esters, acrylonitrile and methyl vinyl ketone. The resulting hydroxy compounds were then acetylated to afford the acetoxy derivatives, thermal cyclization of which gave the corresponding 2-substituted indolizines. The cyclization reactions was followed using 'H NMR spectroscopy and were shown to follow firstorder kinetics. The influence of the various substituents on the observed first-order rate constants has been examined and variable temperature studies have permitted evaluation of activation parameters for the formation of methyl indolizine-2-carboxylate and ethyl indolizine-2-carboxylate. An alternative route to 2-substituted indolizines via halogenated derivatives was explored and several halogenated 2-pyridyl derivatives were synthesised and their thermal cyclization to indolizines was attempted. Novel 5-methylindolizine-2-carboxamides were prepared as part of this investigation and dynamic NMR spectroscopy was used to study internal rotation about the amide N-CO bond in these compounds.

The crystalline compound resulting from thermal cyclization of the Baylis-Hillman product, methyl 3-hydroxy-2-methylene-3-(2-pyridyl)propanoate, has been identified as the indolizine derivative, methyl indolizine-2-carboxylate, and this approach involving the reaction of pyridine-2-carboxaldehydes and acrylate analogues has been established as a general route to 2-substituted indolizines. The ease of cyclization the Baylis-Hillman products to indolizines has been shown to increase by converting the hydroxy group to an acetoxy group, and a range of acetylated Baylis-Hillman products were prepared and cyc1ized to the corresponding 2-substituted indolizines, generally in good overall yield. In the reaction of pyridine-2-carboxaldehyde and methyl vinyl ketone, the intermediate cyclized readily and directly to the corresponding indolizine. One- and two-dimensional ¹H and ¹³C NMR analysis of the 2-substituted indolizine products has permitted complete assignment of all ¹H and ¹³C NMR signals, as well as the measurement of all coupling constants for these compounds. A kinetic and mechanistic study has been conducted on the Baylis-Hillman reaction using ¹H NMR spectroscopy. A range of substrates has been examined and the reaction has been found to be third-order overall. A mechanism involving an addition - elimination sequence is proposed, which fits the kinetic data and accounts for observed substituent effects. Reaction of N,N-dimethylacrylamide with pyridine-2-carboxaldehyde in the presence of the tertiary amine catalyst, DABCO, in chloroform, yielded an unexpected product which has been identified by single crystal X-ray diffraction analysis as 1-(2,2,2-trichloro-1-hydroxyethyl)pyridine. Attempted extension of the general indolizine route to the preparation of chromene systems by reacting salicylaldehyde with methyl acrylate in the presence of DABCO, also led to an unexpected, crystalline material, identified by single crystal X-ray diffraction analysis as the coumarin derivative, 3-[(2-formylphenoxy)methyl]coumarin.A series of chloroquine analogues have been prepared from indolizine-2-carboxylic acid, pyrrolo[I,2-a]quinoline-2-carboxylic acid and imidazo[I,2-a]pyridine-2-carboxylic acid by reaction with suitable amines in the presence of the coupling reagent 1, I' -carbonyldiimidazole. This route has been shown to be a vast improvement on earlier procedures and has provided access to both secondary and tertiary indolizine-2-carboxamides. A range of N,N-dialkylindolizine-2-carboxamides have been prepared by this route, and the influence of substituents on their N-CO rotational energy barriers has been determined using variable temperature ¹H and ¹³C NMR techniques. Intercalation with natural DNA by both chloroquine and the synthesized chloroquine analogues has been examined using UV spectrophotometry, and ¹H and ³¹P NMR spectroscopy. The pyrrolo[I,2-a]quinolines have been shown to be DNA intercalators with binding affinities similar to that of the known antimalarial intercalator, chloroquine. In a preliminary study the synthesis of a short oligonucleotide has been undertaken and changes have been observed in the ¹H and ³¹P NMR spectra of the oligonucleotide on addition of the intercalator, chloroquine.

Degradability of atrazine, cyanazine, and dicamba under methanogenic conditions was evaluated using serum bottle microcosms containing wetland soil inocula obtained from three different sites. Pesticides were monitored by high-performance liquid chromatography (HPLC) and the production of methane was measured with a gas chromatograph (GC). Dicamba was the most susceptible to degradation in the microcosms, followed by cyanazine. Atrazine was not degraded in the wetland soils. A dicamba-degrading methanogenic consortium was enriched from one of the initial wetland soil microcosms (Lawnes). Dicamba degradation was further examined using this consortium. Net methane production suggested that the aromatic ring was not degraded. Rates of dicamba degradation were enhanced with addition of 0.2 % yeast extract. Dicamba degradation was accomplished within 4 days compared to 22 days without yeast extract addition. The inability of the consortium to degrade the benzenoid ring was confirmed when no ¹⁴CO₂ was produced upon addition of [U-¹⁴C]dicamba to the cultures. Analysis of culture filtrate by HPLC revealed the presence of a possible metabolite that was aromatic in character. / Ph. D.

Indole-3-carbinol (I3C) inhibits chemically induced tumor formation in rodents and rainbow trout. This study examines the effect of I3C and its analog, indole-3-acetonitrile (I3N) on xenobiotic-metabolizing enzyme systems. The modulation of these enzyme systems have been shown to have significant effects on the interaction of chemical carcinogens and cellular constituents. Rainbow trout were fed 500, 1000 and 2000 ppm dietary levels of I3C and 50, 500 and 1000 ppm dietary levels of I3N for 8 days. β-napthoflavone (BNF), which is also an effective anticarcinogen in the trout, was fed at a 500 ppm dietary level and was used as a positive LM4b (a cytochrome P-450 isozyme) inducing control. Enzyme activities assayed were: ethoxyresorufin-O-deethylase (EROD), ethoxycoumarin-O-deethylase (ECOD), glutathione S-transferase (GST), and uridine diphosphoglucuronosyl transferase (UDPGT). Total cytochrome P-450 content was determined spectrophotometrically by the CO reduced method. The specific P-450 isozymes, LM2 and LM4b, were detected quantitatively using the western blot method. The BNF diet induced EROD and ECOD activities by an average of 17 fold and 5.5 fold, respectively. Total P-450 content was increased 2-fold; the P-450 isozyme LM4b was induced more than 5-fold, but LM2 content remained unchanged. This diet increased UDPGT activity 1.5-2-fold, but GST activity was not induced by dietary BNF. Neither I3C nor I3N induced the activity levels of the enzymes assayed at any administered dietary levels, which have previously shown to inhibit tumor formation and reduce formation of carcinogen-DNA adducts. Thus, the anticarcinogenic mechanism of I3C may proceed in trout by mechanisms other than enzyme induction. Further experiments on the effect of I3C and I3C acid condensation products (RXN) on in vitro AFB1-DNA binding resulted in a 40% and 48% inhibition of AFB1-DNA binding by I3C and RXN, respectively. Additions of RXN at levels much lower than those estimated to exist in vivo in hepatic tissue resulted in a significant reduction in AFB1-DNA formation suggesting that even small levels of RXN offers protection against the genotoxic effect of AFB1. However, in vitro additions of neither I3C nor RXN had an effect on DNA binding using AFBI-CI₂, an aflatoxin analog that does not require enzymatic activation. These results suggest that the primary mechanism for I3C inhibition of AFB1 induced carcinogenesis may proceed by inhibiton of formation of the ultimate electrophile, i.e. by reversible inhibition of cytochrome P-450. / Graduation date: 1989

This thesis describes investigations directed towards developing a novel synthetic route to the natural products perophoramidine and the communesins, with particular emphasis placed on the formation of the two vicinal all-carbon quaternary centres contained in these molecules. Chapter 1 introduces perophoramidine and the communesin group of natural products and explains how they are related to the calycanthaceous alkaloids. The isolation of perophoramidine and the communesins is outlined and their biosynthesis is discussed. Specific structural features of these natural products are highlighted before established synthetic strategies are reviewed. Chapter 1 concludes by proposing a novel synthetic route for the synthesis of perophoramidine and the communesins that involves a Claisen rearrangement in the indolo[2,3-b]quinoline system as a key step. Chapter 2 describes model studies on the proposed Claisen rearrangement in an attempt to form a quaternary centre in the indolo[2,3-b]quinoline system. These initial studies did not result in the generation of the desired quaternary centre. However, a detailed understanding of the reactions that occur leads to the design of a new model substrate. Chapter 3 describes studies on the revised model system that result in the formation of the desired quaternary centre using a Claisen rearrangement. The differences between the two systems are discussed before an investigation into the scope of the rearrangement is described. Chapter 3 concludes by describing an investigation into a protecting group strategy that would by required with this synthetic route. Chapter 4 describes investigations into the formation of the second vicinal quaternary centre using a model system. The synthetic routes investigated lead to two separate methods for the formation of the desired quaternary centre. Chapter 5 describes investigations into the effect a C-10 substituent has on the Claisen rearrangement. Additionally, an asymmetric version of the Claisen rearrangement is examined. Chapter 5 culminates in the preparation of an intermediate relevant to an asymmetric synthesis of the communesins.

Metabólitos secundários são produzidos por alguns grupos vegetais e são essenciais nas diferentes estratégias de adaptação às adversidades ambientais, atuando na proteção e comunicação das plantas, sendo responsivos a diversos fatores bióticos e abióticos. Entre as diversas categorias de metabólitos secundários, os alcalóides apresentam principal função relacionada à defesa contra herbívoros; atuam também na proteção contra patógenos e na interação química com outras plantas (alelopatia). Alcalóides monoterpenos indólicos (MIAs) são uma classe de alcalóides de origem biossintética mista, e apresentam propriedades farmacológicas conhecidas (e.g. MIAs de Catharanthus roseus e Rauwolfia serpentina). MIAs provenientes de algumas espécies de Psychotria do Sul do Brasil são descritos como agentes antioxidantes, antimutagênicos, ansiolíticos, antidepressivos, antipsicóticos e analgésicos, apresentando grande potencial farmacológico. N,β-D-glicopiranosil vincosamida (GPV) é o alcalóide majoritário de Psychotria leiocarpa (Rubiaceae – APG III), apresentando estrutura semelhante a alguns alcalóides bioativos de Psychotria da região, com a peculiaridade de ser N-glicosilado. No presente trabalho, foi avaliado o efeito de dano mecânico e aplicação de jasmonato sobre o acúmulo de GPV no contexto de um possível papel do alcalóide em respostas à herbivoria, além de propriedades antioxidantes do composto. O teor de GPV se manteve constante após a aplicação dos tratamentos, ao longo de todo o experimento. Portanto, a estratégia de acúmulo deste alcalóide segue o padrão de fitoanticipina. No ensaio de dano mecânico os teores de compostos fenólicos também foram monitorados e se mantiveram constantes. Ensaios de herbivoria utilizando dois modelos generalistas e um especialista, não constataram eficácia do GPV na proteção contra estes predadores. Ensaios de atividade contra oxigênio singleto, ânions superóxido, radicais hidroxil e peróxido de hidrogênio revelaram ampla atividade antioxidante, com alguns resultados similares ao controle positivo (Trolox, um análogo da vitamina E). Os resultados obtidos neste trabalho, juntamente com dados existentes da literatura para metabólitos correlatos, sugerem uma função fundamentalmente antioxidante de MIAs de Psychotria, atuando como um modulador de estresse oxidativo. / Some plants groups accumulate secondary metabolites, which may play a major role in different strategies to deal with environmental challenges, being responsive to several biotic and abiotic factors and functioning as protection and communication agents. Among secondary metabolites, alkaloids play a major role as anti-feedant agents and are also involved in pathogen protection and chemical interaction (allelopathy). Monoterpene indole alkaloids (MIAs) are derived from two distinct biosynthetic pathways and possess well known pharmacological properties (e.g. MIAs from Catharanthus roseus and Rauwolfia serpentina). MIAs from Southern Brazilian Psychotria have been characterized as antioxidant, antimutagenic, ansyolitic, antidepressive, antipsychotic and analgesic agents, therefore bearing relevant pharmacological potential. N,β-D-glucopyranosil vincosamide (GPV) is the major alkaloid from Psychotria leiocarpa (Rubiaceae - APG III) and its structure, besides being additionally glycosylated in the N indol ring, is similar to a few bioactive alkaloids from native Psychotria species. In the present work, the effects of wounding and jasmonate application on GPV accumulation, and also antioxidant properties, were evaluated in the context of a potential role of the alkaloid in herbivory responses. GPV content remained constant after treatments, at all times of exposure. Therefore, GPV seems to present a phytoanticipin-like accumulation pattern. In the mechanical wounding assay, phenolic compounds content was also monitored and remained constant. In two herbivory assay models, a generalist and a specialist, GPV was not efficient to prevent herbivore feeding. Singlet oxygen, superoxide anions, hydroxyl radicals and hydrogen peroxide assays showed GPV has broad antioxidant activity, in some cases with activity equivalent to the positive control (Trolox, a vitamin E analog). The results obtained in this work, together with published results from our research group, strongly suggest an antioxidant role for Psychotria MIA alkaloids, which may act as oxidative stress modulators.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2014-10-09T15:01:20Z No. of bitstreams: 2 Dissertação - Aline Pereira Moraes - 2013.pdf: 2375025 bytes, checksum: edebdd82c526094db969a19e855e1017 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2014-10-09T15:30:41Z (GMT) No. of bitstreams: 2 Dissertação - Aline Pereira Moraes - 2013.pdf: 2375025 bytes, checksum: edebdd82c526094db969a19e855e1017 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-10-09T15:30:41Z (GMT). No. of bitstreams: 2 Dissertação - Aline Pereira Moraes - 2013.pdf: 2375025 bytes, checksum: edebdd82c526094db969a19e855e1017 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-05-03 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The use of natural products and their synthetic derivatives has been a relevant strategy in the development of novel medicines. Phytochemical studies and synthesis of natural-product-based libraries are primordial in the search for therapeutic agents. Previous phytochemical studies of genus Psychotria (Rubiaceae) have resulted in the identification of polypyrrolidine indole and monoterpenoid indole alkaloids, which present a broad range of biological activities, such as antifungal and inhibition of monoamine oxidases (MAOs) A e B, that are related to neurodegenerative diseases. This study aims to evaluate the phytochemical composition of the aerial parts of Psychotria sp. collected in Brazilian Cerrado. Fractionation of the crude extract by column chromatography on silica gel and Sephadex led to isolation of three known indole alkaloids: bahienoside A, desoxycordifoline and desoxycordifolinic acid. Additionally, thiohydantoins and thioureas were synthesized from amino acids and R- (+)-limonene, in order to assess the cooperative effect of indole nucleus combined with terpene, thiourea and thiohydantoin units. Preliminary tests showed that desoxycordifoline, hydantoin (20) and thiohydantoin necrostatin-1 (14) inhibit the enzyme MAO-A higher than 80% at concentrations of 175 mM, 100 mM, 386 mM, respectively. Also, the polar extracts of the leaves of Psychotria sp. showed antioxidant activity with IC50 < 50 mg.mL-1 measured by DPPH free radical scavenging assay. / O uso de produtos naturais e seus derivados sintéticos tem sido uma relevante estratégia no desenvolvimento de novos medicamentos. Estudos fitoquímicos e derivatização de produtos naturais são fundamentais na busca por protótipos de fármacos. Estudos fitoquímicos anteriores do gênero Psychotria (Rubiaceae) resultaram na identificação de alcaloides indólicos monoterpênicos e pirroloindólicos. Essas classes de compostos são associadas a um amplo espectro de atividades biológicas, tais como antifúngica e de inibição de enzimas monoaminoxidases A e B (MAOs) relacionadas a doenças neurodegenerativas. Assim, esse trabalho teve como objetivo realizar o estudo fitoquímico das partes aéreas de Psychotria sp., presente no cerrado goiano. O fracionamento do extrato bruto por cromatografia em coluna em sílica gel e Sephadex resultou no isolamento de três alcaloides indólicos conhecidos: bahienosida A, desoxicordifolina e ácido desoxicordifolínico. Neste trabalho, tiohidantoínas e tioureias derivadas do R-(+)-limoneno e de aminoácidos, tais como o triptofano, foram sintetizadas, com o intuito de avaliar o efeito cooperativo do núcleo indólico combinado com as unidades terpênica, tioureia e tiohidantoína na ação antifúngica e de inibição das enzimas MAOs. Testes preliminares mostraram que desoxicordifolina, hidantoína (20) e tiohidantoína necrostatina-1 (14) inibem a enzima MAO-A acima de 80% nas concentrações de 175 μM, 100 μM, 386 μM, respectivamente. Ainda, os extratos polares das folhas de Psychotria sp mostraram atividade antioxidante com CI50 < 50 mg.mL-1 pelo método de captura dos radicais DPPH.

Faschingbauer J.: Cytotoxic and cholinesterase inhibitory activity of extracts from selected species of the Centaurea L. genus. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany, Hradec Králové, 2019. During the screening of biologically active secondary metabolites of plants carried out at the Department of Pharmaceutical Botany FAF UK, selected taxa of the genus Centaurea (Asteraceae) were investigated. This study is focused on a basic phytohemical research of extracts prepared from Centaurea cyanus, Centaurea jacea, Centaurea scabiosa, Centaurea pseudophrygia, Centuarea stoebe, Centaurea solstitialis a Centaurea benedicta. Extracts were prepared for evidence of the proof reactions of TLC and MS analysis (EI, ESI) to clarify a potential presence of alkaloids. EtOAc and ethanol extracts were evaluated for potential inhibitory activity against human erythrocyte acetylcholinesterase (AChE) and plasma butyrylcholinesterase (BChE) and cytotoxicity against selected 9 tumor lines. C. cyanus alkaloid extract had interesting cholinesterase activity which selectively inhibited BChE (IC50 BChE = 22.62 ± 3.62 μg / ml, IC50 AChE = 221.50 ± 44.56 g / ml). Other EtOAc extracts of selected Centaurea species were considered inactive (IC50 > 100 μg/ml)....

As doenças neurodegenerativas tais como as doenças de Alzheimer, Parkinson e desordens cerebrovasculares constituem-se em uma das principais causas de morbidade e de mortalidade na vida adulta. Além disso, o desequilíbrio entre os sistemas de geração e de proteção antioxidante celulares, chamado de estresse oxidativo, desempenha um papel importante nos danos neuronais causados pelos processos isquêmicos, provocando alterações funcionais em macromoléculas e promovendo a lipoperoxidação de membranas. Substâncias com dupla atividade anticolinesterásica e antioxidante vêm sendo consideradas como uma nova abordagem terapêutica para o tratamento farmacológico da doença de Alzheimer, incentivando a investigação e o estudo de produtos naturais para o desenvolvimento de fármacos novos e eficientes. Nesse estudo empregamos um modelo in vitro de fatias hipocampais de ratos, submetidas à privação de oxigênio e glicose (POG) e os métodos de avaliação da toxicidade dos alcalóides empregados foram a liberação da enzima lactato desidrogenase (LDH) citosólica e redução do MTT (viabilidade mitocondrial). Os alcalóides boldina e vincamina promoveram um aumento de 40 % na liberação de LDH nas fatias que sofreram POG na concentração de 100 μM, além de aumentos significativos na liberação desta enzima também nas fatias controles. Psicolatina e reserpina também tiveram efeitos neurotóxicos. Foi verificado que a POG em fatias hipocampais promove uma diminuição nas medidas do potencial antioxidante total (TRAP) e reatividade antioxidante total (TAR), de 63 % e 16,5 %, respectivamente, além de causar um aumento nos níveis de malonodialdeído liberado pelas fatias, detectado pelo ensaio de espécies reativas ao ácido tiobarbitúrico (TBA-RS). Entretanto, este efeito foi revertido pela presença de boldina nas concentrações de 10 μM e de 50 μM. Este mesmo alcalóide, com reconhecida atividade antioxidante, também demonstrou ser um seqüestrador de radicais peroxila mais potente que o padrão Trolox. Além disso, os alcalóides indólicos monoterpênicos coronaridina, venalstonina, andrangina, vincadiformina e voacristina, além da boldina, exibiram potentes atividades antioxidante e anticolinesterásica em ensaios autobiográficos in vitro. / Neurodegenerative disorders, such as Alzheimer, Parkinson and cerebrovascular diseases are one of the major causes of morbidity and mortality in the middle aged and the elderly. Also, the imbalance between the activity of free radicals generation and scavenging systems, called oxidative stress, plays a important role in the neuronal damages caused by ischemia, leading to functional alterations in macromolecules and promoting lipoperoxidation in membranes. Acetylcholinesterase inhibitors and antioxidant compounds have been extensively investigated as new pharmacological strategies for the symptomatic treatment of Alzheimer disease. In this way, natural products are potentially important in an attempt to develope newer and safer drugs. In the present study, we selected the in vitro model of oxygen and glucose deprivation (OGD) in hippocampal slices and the methods used to assess the neurotoxicity of the alkaloids were cellular lactate dehydrogenase (LDH) release and reduction of MTT salt (mitochondrial activity). Both alkaloids boldine and vincamine 100 μM promoted a 40 % increase in LDH release in POG slices, as well as significant increases in the activity of this enzyme in control slices. Psychollatine and reserpine had also neurotoxic effects. It was also verified that OGD decreased the measurements of total antioxidant potential (TRAP) in 63 % and the total antioxidant reactivity (TAR) levels in 16.5 %, as well as an increase in the malondialdehyde levels by slices, which was detected by thiobarbituric acid-reactive substances (TBA-RS). However, this effect was prevented by the presence of boldine 10 μM and 50 μM. This alkaloid is a known antioxidant and it displayed a potent scavenger activity for peroxyl radicals, when compared with Trolox. Another finding is that the monoterpene indole alkaloids coronaridine, venalstonine, andrangine, vincadifformine, voacristine and also boldine exhibited both potent antioxidant and acetylcholinesterase inhibitor activities in in vitro autobiographic assays.