Os alcalóides ergolínicos são uma categoria de compostos que possuem um esqueleto tetracíclico [6,5,6,6] derivados da ergolina. Do ponto de vista farmacológico, estes compostos são uma classe de produtos naturais importantes, já que exibem uma grande variedade de atividade biológica. Estas moléculas têm sido alvos sintéticos devido ao seu complexo esqueleto tetracíclico e as propriedades supracitadas, o que resultou no desenvolvimento de sínteses totais ao longo do tempo. O ácido lisérgico é o representante notável da família dos alcaloides ergolínicos, uma vez que desde o ano de 1956 até 2013 foram desenvolvidas treze sínteses totais do mesmo. Por isso, o nosso grupo de pesquisa propõe uma rota sintética para a construção de um intermediário indólico-piperidinico, que será usado para realizar a síntese total estereosseletiva do ácido lisérgico em um trabalho de pesquisa futuro. A estratégia para a síntese do intermediário baseia-se na reação de Horner-Wadsworth-Emmons (HWE) e na reação de inserção N-H intramolecular a partir de um aminoaldeído derivado do L-triptofano. A rota sintética inicia-se a partir da elaboração do reagente de olefinação de HWE em três etapas: reação de Michaelis Becker, hidrogenólise catalítica e formação do diazofosfonato. A construção do aminoaldeído requer cinco etapas: esterificação, proteção com Boc, desproteção seletiva, redução do éster e oxidação de Swern. O reagente de olefinação e o aminoaldeído reagem através da reação HWE, fornecendo a diazocetona α, β -insaturada com configuração preferencial Z (Z:E=10:1). Finalmente a olefina Z reage mediante a reação de inserção N-H intramolecular, para fornecer o intermediário indólico-piperidínico. Adicionalmente, desenvolveu-se outra rota para construir o intermediário mencionado através da construção de um derivado de 4-nitroindol, porém esta não foi reprodutível. A síntese do intermediário indólico-piperidínico foi feita em sete etapas, partindo do L-triptofano com rendimento global de 14.9 %. / The ergot alkaloids are a class of compounds which have the tetracyclic skeleton [6,5,6,6] found in the ergoline molecule. These compounds are an important class of natural products that have wide biological activities. They have also been important synthetic targets due to their challenging tetracyclic skeleton as well as due to the previously mentioned biological properties. Lysergic acid is the main representative of the family of ergot alkaloids. Since 1956, thirteen total syntheses have been developed for this alkaloid. Considering the importance of lysergic acid and of the ergot alkaloids, our research group decided to propose a synthetic route to construct an advanced indole-piperidinic intermediate, which may be used to perform the total synthesis of lysergic acid and derivatives in a future work. The strategy to the synthesis of this advanced intermediate is based on the Horner-Wadsworth-Emmons reaction (HWE) and the intramolecular N-H insertion reaction, starting from a L-tryptophan aminoaldehyde derivative. The synthetic route started with the elaboration of the HWE olefination reagent in three steps: Michaelis Becker reaction, hydrogenolysis and diazophosphonate formation. In continuation, the construction of the aminoaldehyde required five steps: esterification, Boc protection, selective deprotection, ester reduction and Swern oxidation. The olefination reagent and the aminoaldehyde reacted by the HWE reaction furnishing an α, β -unsaturated diazoketone with Z configuration (Z:E = 10:1). Finally the Z isomer reacted by means of an intramolecular N-H insertion reaction to provide the indole-piperidine intermediate. Additionally, we developed another route to construct a 4-nitro-indole intermediate, but this was not reproducible. The synthesis of the indole-piperidine intermediate was carried out in seven steps starting from L-tryptophan, with an overall yield of 14.9%.

Baicalein is known to be a potent antifungal agent and induces programmed cell death in Candida albicans. In the present study, we found that baicalein also inhibited the growth of C. krusei isolates. The minimal inhibitory concentrations of baicalein against eight C. krusei isolates were 1.35--2.70 microg/ml. One-hour exposure to baicalein elicited a consistent and moderate post-antifungal effect on the C. krusei isolates. Further flow cytometric study demonstrated a depolarization of mitochondrial membrane potential. However, both the levels of reactive oxygen species and DNA fragmentation were not significantly changed after baicalein treatment in C. krusei. It can be concluded that the antifungal activity of baicalein was mitochondria-dependent in both C. krusei and C. albicans, but the antifungal mechanism was different. Reactive oxygen species may not play a direct role and baicalein does not initiate programmed cell death or apoptosis in C. krusei. The structure-activity relationship study showed that the three hydroxyl groups in baicalein were essential for its antifungal potency. / Candidiasis has become a serious infection with very high mortality and morbidity in the world if not providing effective treatments. However, due to clinical limitation and resistance of the current antifungal agents, there is an urgent need to search for novel antifungals. In this study, after screening a compound library (n=400) for antifungal activity, three members (metergoline, purpurin and baicalein) were chosen for further study. Their antifungal characteristics and the antifungal mechanisms were investigated. / Metergoline, a serotonin receptor antagonist, was found to have potent antifungal activity against the intrinsically fluconazole-resistant human fungal pathogen Candida krusei. The minimal inhibitory concentration and minimal fungicidal concentration of metergoline against C. krusei were 4 microg/ml and 8 microg/ml respectively. Metergoline induced post-antifungal effect. Significant synergism was found in combination of metergoline with amphotericin B by a checkerboard assay, which may be due to the perturbation of cell permeability and increase in the intracellular accumulation of antifungal agents. Metergoline also inhibited extracellular phospholipase production in C. krusei. To gain insights into the mechanisms, intracellular changes that accompany apoptosis were examined by flow cytometry and spectrophotometry. The results showed an increase in the level of reactive oxygen species, depolarization of mitochondrial membrane potential, phosphatidylserine externalization, and positive terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labelling in the metergoline-treated C. krusei . Taken together, we conclude that metergoline may promote apoptosis in C. krusei through reactive oxygen species production and perturbation in mitochondrial homeostasis, implying its antifungal potential to treat candidiasis. / The antifungal activity of purpurin, a natural red anthraquinone pigment in madder root (Rubia tinctorum L.), was evaluated against Candida isolates by a broth microdilution assay. The minimal inhibitory concentrations of purpurin against Candida species isolates were 1.28--5.12 microg/ml. Mechanistic studies indicated that purpurin inhibited energy-dependent efflux pumps of Candida isolates. Furthermore, purpurin demonstrated a depolarization of mitochondrial membrane potential, suggesting a possible linkage of the antifungal mechanism of purpurin to Candida apoptosis. / Kang, Kai. / Adviser: Fong Wing Ping. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 98-123). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Eliška Holinská Supervisor: Prof. MUDr. Radomír Hrdina, CSc. Title of diploma thesis: Pharmacoteraphy of headache in adults Headache is one of the most common health problem that encounters almost everyone during the life. Depending on the cause, headaches can be divided on the primary headaches, which are the subject of the diploma thesis, and secondary headaches, which are caused by other diseases. The primary headaches are migraine, tension-type headache, cluster headache and primary chronic daily headache. Headache is not a life-threating condition but it can significantly reduce the quality of life, particularly the chronic forms of headache. The determination of the right diagnosis is essential for the choice of appropriate therapy. For primary headache are typical negative test results, there are no structural lesions or signs of organic brain damage. Diagnostics is comlicated and it is primarily based on a carefully processed medical history. In the therapy of primary headache are used both pharmacological and non-pharmacological methods, optimaly in combination. Pharmacological treatment consists of preventive and acute therapy. The preventive treatment is given to reduce the...

The first topic presented in the dissertation thesis is determination of isoprostanes as markers of oxidative stress and other compounds affected by presence of oxidative stress. Isoprostanes iPF2-III, iPF2-VI, iPF2-VI, astaxanthin and polyunsaturated fatty acids (PUFA), especially arachidonic acid (AA) were monitored in Atlantic salmon eggs (Salmo salar). Methods for the determination of these compounds have been developed and optimized using chromatographic separation coupled to conventional or mass spectrometric detection. Freshly laid eggs, eyed embryos and non-viable eggs were used to test a general hypothesis that egg viability can be affected by susceptibility to oxidative stress, either through the specific fatty acid concentration and/or the antioxidant capacity of the eggs. Levels of isoprostanes and arachidonic acid (AA) were significantly higher in non-viable eggs than in control (eyed embryos) as well as relative abundance of PUFA. While no difference of isoprostanes was found between freshly laid and control those from the Atlantic stock except iPF2-VI which was observed under the LOQ in the control. Higher levels of PUFA and AA in comparison with the control were observed in the freshly laid eggs. However, the only statistically significant difference was observed in the amount of astaxanthin. Different levels of PUFA and astaxanthin may be related to their biochemical consumption during the development of eggs. This work evaluated potential effect on the viability of eggs Salmo salar due to the presence of oxidative stress. The monitoring of mycotoxins in food and feed was the subject of the second topic. Mycotoxins are secondary metabolites produced by fungi. They are ubiquitous undesirable natural contaminants that are toxic for humans and animals. Today are known more than 500 mycotoxins. However, only few of them are regulated by the European Union. The European Food Safety Authority (EFSA) was asked by the European Commission to provide a scientific opinion on other mycotoxins for which statutory limits could be developed. In this study is proposed simultaneous screening allowing fast, reliable and sensitive approach, identification and quantification of 17 mycotoxins in food and feed sample. The method includes both mycotoxins regulated by the EU and selected mycotoxins required by the EFSA (aflatoxins, deoxynivalenol, nivalenol, zearalenone, fumonisin, ochratoxin A, T-2 toxin, HT-2 toxin, enniatins and beauvericin). Analytes are isolated by the modified QuEChERS method. For separation and target mycotoxins detection, ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC –MS/MS) was employed. The method also allows determination of ergot alkaloids (ergocornine, ergosine, ergocryptine, ergocristine and their respective epimers). The developed method was used either for monitoring mycotoxins and ergot alkaloids in feed and raw materials and barley and malt prepared from it.