Student Number : 0000127R - MPharm dissertation - School of Pharmacy and Pharmacology - Faculty of Health Sciences / Traditional medicines form a significant part of the lives of many people around the world and in South Africa almost 60 % of people consult traditional healers in addition to the modern medical services available. Plants form a significant part of traditional healing and hence, selected species of a traditionally used plant genus, Hermannia, were chosen for biological and chemical investigation to determine a scientific basis for the traditional use of these plants. A phytochemical investigation was carried out, firstly using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) and then isolation and identification of compounds from various Hermannia species. TLC analysis indicated significant similarities between the various species with only H. saccifera displaying chemical anomalies. This was further corroborated by the HPLC analysis although very conservative profiles were produced. Isolation of compounds from H. saccifera yielded a novel labdane compound, E-17, 19-diacetoxy - 15 - hydroxylabda - 7,13 - diene, as well as two flavones, 5,8- dihydroxy-6,7,4’- trimethoxyflavone and cirsimaritin which have previously been isolated. In addition, two commonly found compounds, lupeol and β- sitosterol were isolated from H. cuneifolia and H. salviifolia respectively. This is the first report on the isolation and identification of all five compounds from Hermannia species. Antimicrobial activity was assessed using two methods i.e. minimum inhibitory concentrations as well as the death kinetics assay. Minimum inhibitory concentrations were determined using four Gram-positive and two Gram-negative bacteria as well as two yeasts. All species investigated indicated antimicrobial activity with H. saccifera showing good activity against S. aureus and B. cereus. E-17, 19-diacetoxy - 15 - hydroxylabda - 7,13 - diene isolated from H. saccifera indicated activity (MIC = 23.6 μg/ml against S.aureus) although the activity was less than that of the crude extract (MIC = 19.5 μg/ml), thus, demonstrating that there are a number of compound contributing to the promising activity of the crude extract. This was further corroborated by the bioautograms developed of the H. saccifera extract. Time-kill studies on H. saccifera against S. aureus indicated that at concentrations of 0.1, 0.25 and 0.5 % bacteriostatic activity was observed while at 0.75% the extract achieved complete bactericidal activity after 240min. Free radical scavenging activity was assessed using the 2,2-diphenyl-1-picrylhydrazy (DPPH) and 2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) assays. Ten of the twelve species indicated good activity with H. cuneifolia demonstrating the most promising activity (IC50 = 10.26 μg/ml for DPPH and 10.32 μg/ml for ABTS). Two of the isolated compound, 5,8- dihydroxy-6,7,4’- trimethoxyflavone and cirsimaritin displayed insignificant activity. The 5-lipoxygenase assay was used to assess the anti-inflammatory activity of Hermannia species. All species exhibited intermediate activity with the exception of H. cuneifolia (IC50 = 15.32 μg/ml). In addition, four isolated compounds, 5,8- dihydroxy-6,7,4’- trimethoxyflavone, cirsimaritin, lupeol and β-sitosterol showed moderate inhibition of the enzyme indicating that while these compounds do contribute to the activity of the extracts they are not individually responsible for any significant activity. Antimalarial activity was assessed using the titrated hypoxanthine incorporation assay while toxicity was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell proliferation assay. Only three species indicated any good antimalarial activity i.e. H. saccifera, H. muricata and mostly H. trifurca (IC50 = 25.30, 28.17 and 18.80 μg/ml respectively). However, the activity of H. saccifera and H. trifurca are probably due to a general cytotoxicity as these species exhibited a low safety index. All other species appear safe for use. Several Hermannia species have indicated in vitro biological activity in a number of assays which is related to their use in traditional medicines to treat a number of disease states. Hence, a scientific basis, albeit in vitro, has been established for the use Hermannia species in traditional healing.

Mesmo antes do advento da escrita, o homem já buscava na natureza a cura para suas doenças. Dentre as diferentes classes de produtos naturais usados para esse fim, destaca-se a dos alcalóides, por exibir relevante diversidade estrutural e farmacológica, e que tem como fonte importante o gênero Aspidosperma, do qual foram isolados alcalóides que têm sido usados nas terapias atuais. Nesse contexto, o presente trabalho teve como objetivo investigar os alcalóides obtidos a partir de diferentes partes de duas espécies ainda pouco exploradas: Aspidosperma ramiflorum e Aspidosperma tomentosum quanto à sua composição e atividades antifúngica, antimicrobiana e antitumoral. Foram identificados através de Cromatografia à Gás acoplada à Espectrometria de Massas (CG-EM) e Ressonância Magnetica Nuclear os alcalóides beta-ioimbina, nos pericarpos e arilos de A. ramiflorum, uleína, nas folhas, ramos e sementes de A. tomentosum, além de quebrachamina e razinilama, nos arilos e sementes dessa espécie. Adicionalmente, com base na literatura e nos resultados das análises por CG-EM verificou-se em A. tomentosum a presença de desmetileno-oxa-uleína, nas folhas, de eburnamonin-19-ona nos ramos e de di-hidro-razinilama, de aspidospermidina e também de di-hidro-razinilama e de 1-acetil-17-metóxi-aspidospermidina nas sementes e arilos dessa mesma espécie. Nenhum dos extratos de alcalóides totais ou alcalóides isolados apresentou atividade antibacteriana relevante. Verificou-se atividade antitumoral em extratos de alcalóides dos pericarpos de A. ramiflorum e dos ramos, folhas e arilos de A. tomentosum. Observou-se ainda atividade antifúngica em todos os extratos de alcalóides ensaiados, além de atividade específica contra isolados de Criptococcus neoformans resistentes ao fluconazol para os alcalóides quebrachamina e beta-ioimbina. O extrato de alcalóides totais das folhas de A. tomentosum, além da atividade frente a essas leveduras, também se mostrou ativo frente a duas espécies de Candida (C. krusei e C. parapsilosis). Concluiu-se a partir dos resultados obtidos que as duas espécies estudadas podem ser consideradas fontes promissoras de alcalóides que poderão ser utilizados como modelos para novos fármacos antifúngicos e antitumorais. / Even before the advent of writing, man already searched the nature for the cure of his illnesses. Among the different classes of natural products used for this purpose, monoterpenoid indole alkaloids exhibit large structural and pharmacological diversities and these compounds are commonly found in the Aspidosperma genus. In this context, the present work had as main goals to investigate the alkaloids from different parts of two poorly studied species, Aspidosperma ramiflorum and Aspidosperma tomentosum, regarding their composition and antibacterial, antifungal, and antitumoral activities. Gas Chromatography coupled to Mass Spectrometry (GC-MS) and Nuclear Magnetic Ressonance analysis allowed the identification of the following alkaloids: beta-yohimbine, in the pericarp and arils of A. ramiflorum; uleine, in leaves, branches and seeds of A. tomentosum; quebrachamine and rhazinilam, in the arils and seeds of the same species. Moreover, based on literature data and the results of the GC-MS analysis was characterized in A. tomentosum the presence of oxo-uleine, in the leaves and stems, eburnamonine, in the stems, and rhazinilam, aspidospermidine, dihydrorhazinilam and 1-acetyl-17-methoxy-aspidospermidine like alkaloids in the seeds and arils. Neither crude alkaloid extracts nor isolated alkaloids presented a relevant antibacterial activity. An antitumoral activity could be detected in crude alkaloids extracts from pericarps of A. ramiflorum and from stems, leaves and arils of A. tomentosum. On the other hand, antifungal activity was observed in all crude alkaloid extracts assayed, besides a specific activity of the alkaloids quebrachamine and beta-yohimbine against fluconazol resistant Criptococcus neoformans. Additionally, it was observed that crude alkaloid extract from leaves of A. tomentosum was also active against two Candida species (C. krusei e C. parapsilosis), as well as to the C. neoformans isolate. From these results we can conclude that the two studied species has to be considerated as promising sources for antifungal and antitumor alkaloids to be used as models to design new drugs.

O presente trabalho enfocou o isolamento, a análise da atividade biológica e a caracterização dos componentes majoritários do Photogem® através de Cromatografia Líquida de Alta Eficiência (CLAE), espectroscopia eletrônica na região do ultravioleta-visível (UV-Vis) e espectrometria de massa. O Photogem® é um derivado de hematoporfirina usado como fotossensibilizador em Terapia Fotodinâmica (TFD). O derivado de hematoporfirina é composto por uma mistura de monômeros, dímeros e oligômeros mas as estruturas químicas destes componentes ainda não estão bem caracterizadas. Empregou-se extração em fase sólida para limpeza e fracionamento dos componentes do Photogem®. As três frações principais foram separadas de acordo com suas respectivas polaridades. Em seguida, testes preliminares de atividade biológica foram desenvolvidos com as respectivas frações do Photogem® em células tumorais (HEp-2) e normais (VERO). A fração 2 apresentou citotoxicidade cinco vezes maior do que o Photogem® tanto nas células tumorais quanto nas células normais. Os componentes majoritários do Photogem® foram isolados por meio de cromatografia líquida de alta eficiência (CLAE) e analisados por espectroscopia eletrônica na região do ultravioleta-visível (UV-Vis) e por espectrometria de massa. O componente que corresponde à fração 1 pode ser atribuído à hematoporfirina IX em função do seu espectro de absorção eletrônica (banda de Soret em 396nm e bandas Q em 497, 531, 568 e 619nm), seu coeficiente de absortividade molar ((1,30±0,07)x105 cm-1 mol-1 L) e seu espectro de massa (íon pseudomolecular em m/Z 599Da). É importante mencionar que este trabalho proporcionou a elaboração de um procedimento acessível à maioria dos laboratórios brasileiros no que se refere a purificação, separação, isolamento e caracterização das frações constituintes do Photogem®, sendo uma contribuição relevante para o avanço da TFD no Brasil. As etapas da metodologia completa são comentadas e discutidas em detalhes. / The present work focused the isolation, the biological activity analysis and the characterization of the major components of Photogem® through High Performance Liquid Chromatography (HPLC), UV-Vis electronic absorption spectroscopy (UV-Vis) and mass spectrometry. Photogem® is a hematoporphyrin derivative used as photosensitizer in Photodynamic Therapy (PDT). The hematoporphyrin derivative is composed by a mixture of monomers, dimers and oligomers but the chemical structures of these components are not well-characterized yet. It was employed solid phase extraction to clean-up and fractionation of the Photogem® components. Three principal fractions was separated as function of their respective polarities. Subsequently, initial biological tests were developed with the respective Photogem® fractions in tumoral (Hep-2) and normal (VERO) cells. Fraction 2 presented citotoxicity approximately 5-fold higher than Photogem® as in tumoral as in normal cells. The major components of Photogem® were isolated by High Performance Liquid Chromatography (HPLC) and analyzed by UV-Vis electronic absorption spectroscopy (UV-Vis) and mass spectrometry. The component that corresponds to fraction 1 can be assigned to hematoporphyrin IX as function of its electronic absorption spectrum (Soret band in 396nm and Q bands in 497, 531, 568 and 619nm), its molar absortivity coefficient ((1,30?0,07)x105 cm-1 mol- 1 L) and its mass spectrum (pseudomolecular ion in m/z 599Da). It is important to mention that this work provided the elaboration of an accessible procedure to the most of the brazilian laboratories regarding purification, separation, isolation and characterization of the constituent fractions of Photogem®, being a relevant contribution to the advancement of PDT in Brazil. The steps of the complete methodology are commented and discussed in details.

Author: Jana Öhlschlegelová Title: Alkaloids of family Amaryllidaceae: genus Hippeastrum Diploma thesis Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany 2019, 74 p. Keywords: Hippeastrum, Amaryllidaceae, alkaloids, antiproliferative activity, Alzheimer's disease, cholinesterase inhibitors, galanthamine The aim of this diploma thesis was to unify current findings about alkaloids isolated from selected plants of the Hippeastrum genus in the Amaryllidaceae family. The fytochemical characteristics of the examined species was introduced, and a group of alkaloids which were isolated from these plant species was composed. Also, the biological activity was evaluated. Up to date, at least 13 plant species of the Hippeastrum genus were examined from the fytochemical perspective. Out of these species examined, 56 different alkaloids with defined structure were isolated. The isolated alkaloids are divided into several groups based on their structure. Namely, these are lycorine, homolycorine, crinine, galanthamine, narciclasine, tazettine, haemanthamine and montanine structural types. Also, alkaloids which differed structurally from these basic types were found in several plants studied. In the substances gained, the antiproliferative activity, inhibitory activity...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany Candidate: Ludmila Homolková Supervisor: doc. Ing. Lucie Cahlíková, Ph.D. Title of diploma thesis: Derivatives of Amaryllidaceae alkaloid haemanthamine as potential drugs Modern research has shown that Amaryllidaceae alkaloids represent a rich reservoir of potential small molecules exhibiting several medicinal properties through various mechanisms. Among the many Amaryllidaceae alkaloids, galanthamine has been given a great amount of attention due the fact that it possesses potent acetylcholinesterase inhibition activity, and is distributed worldwide for the treatment of Alzheimer's disease. One of the interesting compounds is haemanthamine, β-crinine-type of Amaryllidaceae alkaloids, which displays significant in vitro cytotoxic activity against several different types of cancer cell lines. The object of this diploma thesis was to prepare several derivatives of alkaloid haemanthamine, and try to find relationship between structure and biological effect. The present work deals with the preparation of haemanthamine derivatives, and their biological activity connected to the treatment of cancer and Alzheimer's disease. Twelve aromatic ester of haemanthamine derivatives were prepared. The chemical structures...

Bethoxazin is a broad spectrum industrial biocide with commercial applications as a material preservative; however its mechanism of action has not been investigated. In this study, the chemical reactivity of bethoxazin towards biologically important nucleophiles was assessed with UV-Vis spectroscopy. Bethoxazin reacted with molecules containing free sulfhydryl groups such as glutathione and human serum albumin but not with amino, acetate or phenol containing compounds. Bethoxazin was shown to potently inhibit the growth of the K562 human cancer cell line with an IC50 value in the micromolar range. The sulfydryl fluorescent label ThioGlo-1 was used to investigate the biological effects of bethoxazin in K562 cells and explore its mechanism of action. Bethoxazin inhibited the formation of covalent adducts in K562 cells between the free sulfhydryl group of biomolecules and ThioGlo-1, implying that bethoxazin reacts with molecules containing free sulfhydryl groups. Likewise, when glutathione was depleted in K562 cells, by buthionine sulfoximine, high concentrations of bethoxazin were able to inhibit the formation of covalent adducts between sulfhydryl biomolecules and ThioGlo-1. The growth inhibition assay (MTS) was used to investigate the effect of continuous bethoxazin treatment versus wash out in K562 cells. The MTS assay revealed a reduction in the potency of bethoxazin due to the wash out effect, suggesting that the growth inhibition effects of bethoxazin are likely not due to glutathione depletion. A two-colour flow cytometry analysis of bethoxazin treated K562 cells for eight hours demonstrated that bethoxazin provokes necrosis induced cell death in K562 cells. Taken together, these experimental results demonstrate that the reaction of bethoxazin with proteins containing an accessible sulfhydryl group is more likely to be the mechanism of action of the cell growth inhibition effects rather than glutathione depletion.

Neurodegenerative disorders, including Alzheimer's and Parkinson's disease, and the development of neuroprotective agents have received significant research attention in recent years. Development of novel imaging techniques to study the biological mechanisms involved in the progression of these disorders have become an area of research interest. The design of novel small molecule imaging probes in combination with modem imaging techniques may provide information on neuroprotective binding site• interactions and would assist in the design of novel biological assay methods. Techniques to visualize physiological or pathophysiological changes in proteins and living cells have become increasingly important in biomedical sciences, especially fluorescent techniques. Fluorescent ligands in combination with sophisticated fluorescent imaging technologies are useful tools to analyze and clarify the roles of biomolecules in living cells, affording high spatial and temporal resolution. This study is based on the development of polycyclic fluorescent ligands, which may be used in the study of receptor-ligand and/or enzyme-ligand interactions, utilizing these fluorescently labeled ligands in combination with fluorescent imaging techniques. Fluorescent conjugates with high affinity for the• N-methyl-D-aspartate (NMDA) receptor, voltage gated calcium channels (VGCC) and/or the nitric oxide synthase (NOS) enzyme were designed and synthesised with the aim to directly measure binding of these novel molecules to receptors and/or enzymes. The first goal was to develop fluorescent ligands that exhibit similar inhibitory activity on NOS compared to the well-known selective neuronal NOS inhibitor 7-nitroindazole (7-NI). Polycyclic compounds, including amantadine and pentacycloundecane derivatives, were conjugated to fluorescent moieties that resemble the structure of 7-NI. It was thought that the lipophilic nature of the polycyclic compounds would increase the activity of the fluorescent moieties by facilitating increased blood brain barrier permeability and penetration through cell membranes. This would also potentially increase the selectivity of the novel conjugated compounds as selective neuronal NOS inhibitors, similar to 7-NI. The results from the NOS inhibition studies indicated that the novel fluorescent conjugates (5-14) inhibited the NOS enzyme at micromolar concentrations. Although none of the novel fluorescent polycyclic compounds were found to be more potent than 7-NI (IC50 = 0.11 11M), the indazole pentacyclorindecane (5), the coumarin-adamantane (7), the dansyl-adamantane (8), and the cyanoisoindole-adamantane (11) conjugates, exhibited IC5o values below 1 uM. These compounds could possibly be used as molecular probes in the development of high-throughput screening or competitive NOS displacement assays. Further studies on isoform selectivity will elaborate on the potential of these compounds as fluorescent molecular probes. The aforementioned fluorescent derivatives were further developed resulting in a series of novel fluorescent polycyclic conjugates with potent NOS inhibition indicating the potential of these compounds as neuroprotective agents. Due to the polycyclic structure's inherent inhibitory activity towards the NMDA receptor and VGCC we evaluated these derivatives as possible multifunctional neuroprotective agents acting on various neuroprotective targets. In the biological studies it was observed that four adamantane fluorescent compounds (7, 8, 10, 11) exhibited a high degree of inhibitory activity against the NOS enzyme and NMDA receptor and blocked VGCC. The fluorescent compounds were further able to scavange detrimental neurodegenerative free radicals. In silica studies also predicted a high degree of oral bioavailability and that these novel compounds should be effectively transported across the blood brain barrier. Taking the positive findings on the inhibition of the NMDA receptor and VGCC activity of the novel fluorescent polycyclic ligands into account we focused on the expansion of this series. This resulted in the synthesis of a series of fluorescent derivatives utilizing adamantane-3-aminopropanol as an intermediate to extend the chain length between the adamantyl and fluorescent moieties, to potentially reduce sterical hindrance and increase activity. These novel adamantane-3-aminopropanol fluorescent ligands were also evaluated for inhibition of the NMDA receptor and VGCC. The coumarin-, dansyl- and cyanoisoindole adamantane-3-aminopropanol fluorescent conjugates (15, 16, 19) displayed significant VGCC inhibition, with the dansyl (16) and di-nitrobenzene (20) fluorescent derivatives exhibiting NMDA receptor antagonistic activity. All these compounds showed improved activity when compared to known NMDA receptor and VGCC inhibitors in this class. Generally it was observed that the increased chain length analogues had improved VGCC inhibition and NMDA receptor activity when compared to their directly• conjugated counterparts. This led to the conclusion that an increase in chain length might indicate deeper immersion into the NMDA receptor and VGCC which may be necessary for stronger interaction with their putative binding sites. The dansyl analogue, N-[3-(1-adamantylamino)propyl]-5- dimethylaminonaphthalene-1-sulfonamide (16), was further used as a fluorescent NMDA receptor ligand in a fluorescent competition assay, utilizing known NMDA receptor inhibitors to demonstrate the possible applications of these novel fluorescent analogues and their benefit over the use of hazardous and expensive radioligand binding studies. Further investigation on the application of these derivatives, especially on the NOS enzyme and the NMDA receptor, will develop their potential as fluorescent ligands in the study of neurodegeneration and may also yield novel therapeutic agents against neurodegenerative disorders. / PhD (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2012

Neurodegenerative disorders, including Alzheimer's and Parkinson's disease, and the development of neuroprotective agents have received significant research attention in recent years. Development of novel imaging techniques to study the biological mechanisms involved in the progression of these disorders have become an area of research interest. The design of novel small molecule imaging probes in combination with modem imaging techniques may provide information on neuroprotective binding site• interactions and would assist in the design of novel biological assay methods. Techniques to visualize physiological or pathophysiological changes in proteins and living cells have become increasingly important in biomedical sciences, especially fluorescent techniques. Fluorescent ligands in combination with sophisticated fluorescent imaging technologies are useful tools to analyze and clarify the roles of biomolecules in living cells, affording high spatial and temporal resolution. This study is based on the development of polycyclic fluorescent ligands, which may be used in the study of receptor-ligand and/or enzyme-ligand interactions, utilizing these fluorescently labeled ligands in combination with fluorescent imaging techniques. Fluorescent conjugates with high affinity for the• N-methyl-D-aspartate (NMDA) receptor, voltage gated calcium channels (VGCC) and/or the nitric oxide synthase (NOS) enzyme were designed and synthesised with the aim to directly measure binding of these novel molecules to receptors and/or enzymes. The first goal was to develop fluorescent ligands that exhibit similar inhibitory activity on NOS compared to the well-known selective neuronal NOS inhibitor 7-nitroindazole (7-NI). Polycyclic compounds, including amantadine and pentacycloundecane derivatives, were conjugated to fluorescent moieties that resemble the structure of 7-NI. It was thought that the lipophilic nature of the polycyclic compounds would increase the activity of the fluorescent moieties by facilitating increased blood brain barrier permeability and penetration through cell membranes. This would also potentially increase the selectivity of the novel conjugated compounds as selective neuronal NOS inhibitors, similar to 7-NI. The results from the NOS inhibition studies indicated that the novel fluorescent conjugates (5-14) inhibited the NOS enzyme at micromolar concentrations. Although none of the novel fluorescent polycyclic compounds were found to be more potent than 7-NI (IC50 = 0.11 11M), the indazole pentacyclorindecane (5), the coumarin-adamantane (7), the dansyl-adamantane (8), and the cyanoisoindole-adamantane (11) conjugates, exhibited IC5o values below 1 uM. These compounds could possibly be used as molecular probes in the development of high-throughput screening or competitive NOS displacement assays. Further studies on isoform selectivity will elaborate on the potential of these compounds as fluorescent molecular probes. The aforementioned fluorescent derivatives were further developed resulting in a series of novel fluorescent polycyclic conjugates with potent NOS inhibition indicating the potential of these compounds as neuroprotective agents. Due to the polycyclic structure's inherent inhibitory activity towards the NMDA receptor and VGCC we evaluated these derivatives as possible multifunctional neuroprotective agents acting on various neuroprotective targets. In the biological studies it was observed that four adamantane fluorescent compounds (7, 8, 10, 11) exhibited a high degree of inhibitory activity against the NOS enzyme and NMDA receptor and blocked VGCC. The fluorescent compounds were further able to scavange detrimental neurodegenerative free radicals. In silica studies also predicted a high degree of oral bioavailability and that these novel compounds should be effectively transported across the blood brain barrier. Taking the positive findings on the inhibition of the NMDA receptor and VGCC activity of the novel fluorescent polycyclic ligands into account we focused on the expansion of this series. This resulted in the synthesis of a series of fluorescent derivatives utilizing adamantane-3-aminopropanol as an intermediate to extend the chain length between the adamantyl and fluorescent moieties, to potentially reduce sterical hindrance and increase activity. These novel adamantane-3-aminopropanol fluorescent ligands were also evaluated for inhibition of the NMDA receptor and VGCC. The coumarin-, dansyl- and cyanoisoindole adamantane-3-aminopropanol fluorescent conjugates (15, 16, 19) displayed significant VGCC inhibition, with the dansyl (16) and di-nitrobenzene (20) fluorescent derivatives exhibiting NMDA receptor antagonistic activity. All these compounds showed improved activity when compared to known NMDA receptor and VGCC inhibitors in this class. Generally it was observed that the increased chain length analogues had improved VGCC inhibition and NMDA receptor activity when compared to their directly• conjugated counterparts. This led to the conclusion that an increase in chain length might indicate deeper immersion into the NMDA receptor and VGCC which may be necessary for stronger interaction with their putative binding sites. The dansyl analogue, N-[3-(1-adamantylamino)propyl]-5- dimethylaminonaphthalene-1-sulfonamide (16), was further used as a fluorescent NMDA receptor ligand in a fluorescent competition assay, utilizing known NMDA receptor inhibitors to demonstrate the possible applications of these novel fluorescent analogues and their benefit over the use of hazardous and expensive radioligand binding studies. Further investigation on the application of these derivatives, especially on the NOS enzyme and the NMDA receptor, will develop their potential as fluorescent ligands in the study of neurodegeneration and may also yield novel therapeutic agents against neurodegenerative disorders. / PhD (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2012

A pesquisa de substâncias biologicamente ativas, advinda principalmente de plantas, tem levado a descoberta de moléculas clinicamente benéficas. A família Amaryllidaceae é conhecida por sintetizar alcalóides biologicamente ativos, principalmente do grupo tetraisoquinolínico. Estudos têm demonstrado que a atividade biológica destes vegetais está relacionada com a presença deste grupo de metabólitos secundários. No Rio Grande do Sul, é encontrado o gênero Hippeastrum, pertencente a família Amaryllidaceae. Neste trabalho, realizou-se a investigação preliminar de alcalóides dos extratos de Hippeastrum papilio, coletada na região serrana do estado. Foram isoladas 12 substâncias, nas quais 9 apresentam carcaterísticas químicas de alcalóides de Amaryllidaceae. Estudos preliminares de CLAE-EM e CG-EM apontam para a existência de alcalóides de núcleos licorina e haemantamina, além de núcleo galantamina, em menor quantidade. Estas análises também mostram a possibilidade de estruturas inéditas. Os extratos enriquecidos em alcalóides de Hippeastrum papilio demonstraram uma grande capacidade de inibição da enzima acetilcolinesterase, pelo método da bioutografia em cromatoplaca. Além disso, foi realizada a análise comportamental do alcalóide montanina, previamente isolada de Hippeastrum vittatum Este alcalóide foi avaliado na tarefa de reconhecimento de objeto, com administração intrahipocampal, testado 24 horas pós-treino (memória de longa duração). Obteve-se curva dose-resposta para o alcalóide, com dose pró-cognitiva estatisticamente significante de 1μg/μl. Esta mesma dose foi avaliada na mesma tarefa para obtenção de curva temporal. Montanina demonstrou atividade pró-cognitiva na mesma tarefa e testada 24 horas pós-treino, em administração imediatamente pós-treino, 180 minutos pós-treino, mas não em 360 min pós-treino. O alcalóide montanina também foi avaliado quanto ao perfil de inibição da enzima acetilcolinesterase, utilizando método de ultravioleta. Obteve-se inibição da enzima em concentrações de 10 μM, 100 μM e 1 mM. Os resultados obtidos neste trabalho apontam para a necessidade de continuação da investigação química de Hippeastrum papilio, motivada pelo grande potencial de inibição da enzima acetilcolinesterase e por possibilidade de isolamento de estruturas inéditas. Além disso, foi confirmado um grande potencial pró-cognitivo do alcalóide montanina, demonstrado comportamentalmente, além de inibir a enzima acetilcolinesterase, atividade esta necessária e importante para que uma substância seja candidata à terapia da Doença de Alzheimer. / A search for biologically active substances in plants has led to the discovery of clinically beneficial molecules. The family Amaryllidaceae is known to synthesize biologically active alkaloids, principally of the tetraisoquinolinic group. Studies have shown that the biological activity of these plants is due to the presence of these secondary metabolites. The Hippeastrum genus, belonging to the Amaryllidaceae family, is found in Rio Grande do Sul. A preliminary investigation of alkaloids from extracts of Hippeastrum papilio, collected in the mountain region of the Rio Grande do Sul was done in this work. Twelve substances were isolated, in which nine have chemical profile of Amaryllidaceae alkaloids. Preliminary studies with HPLC-MS and GC-MS showed the existence of lycorine and haemantamine type alkaloids, as well as galantamine type alkaloids in a lower quantity. These analysis also showed a possibile presence of unknown structures. The extracts, enriched in alkaloids of Hippeastrum papilio, demonstrated a great ability to inhibit the acetylcholinesterase enzyme by a rapid thin layer chromatography bioautographic method. Furthermore, it was done a behavior analysis of montanine, previously isolated from Hippeastrum vittatum This alkaloid was evaluated in recognition object task with intrahippocampal administration and tested for 24 hours postrainig (long term memory). There was found a doseeffect curve of montanine with cognitive-enhanced dose at 1μg/μl. This dose was evaluated in the same task for temporal-curve. Montanine showed procognitive activity in immediately postraining administration and 180 minutes postraining but not with 360 min postraining in the same task. The acetylcholinesterase inhibition profile of montanine alkaloid was also evaluated by a ultraviolet method. The enzyme was inhibited in concentrations of 10 μM, 100 μM and 1 mM of montanine. The results obtained in the present study show a need for further chemical investigation on Hippeastrum papilio due to its large potential for inhibition of the acetylcholinesterase and isolation of potentially new structures. Moreover, these results confirm the great pro-cognitive potential of montanine, behaviorally demonstrated, as well as its ability to inhibit the acetylcholinesterase enzime, which makes the alkaloid a candidate substance for the treatment of Alzheimer's Disease.

6 Abstract Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany and Ecology Candidate: Jana Marková Supervisor: doc. Ing. Lucie Cahlíková, Ph.D. Title of Diploma thesis: Synthesis of haemanthamine derivatives Based on previous studies, the haemanthamine alkaloid showing remarkable antitumorous activity was chosen as the subject of this thesis. The purpose of the thesis was preparation of its more active derivatives, namely esters. Preparation concerns the following derivatives: 11- O- butanoylhaemanthamine, 11-O-(3,4-dimethoxybenzoyl)-haemanthamine, 11-O-(4- trifluoromethoxybenzoyl)-haemanthamine, 11-O-isobutanoyhaemanthamine. These were created by the reaction of haemanthamine and the relevant acilating agent (acylchloride, anhydride) in the environment of anhydrous pyridine or tetrahydrofuran with adding dimethylaminopyridine as a catalyst. Most of the products were obtained in the form of colourless oil, one of the substances crystallized into white amorphous crystals. However, during an attempt of dioxolane ring opening, the preparation of 3-demethylhaemanthamine derivative proved unsuccessful. Prepared derivatives were mostly identified by EI-MS and NMR. All the substances were gained in sufficient outcomes in the scale of 43 - 81 %. All the compounds were...