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...

Author: Lucie Jánská Název: Alkaloids of family Amaryllidaceae: genus Zephyranthes Diploma thesis Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmacognosy 2018, 75 p. The aim of this diploma thesis was to summarize all knowledge about alkaloids izolated from Zephyranthes plants of Amaryllidaceae family. It contains a detail overview of botanical charactericts of phytochemically studied plants of the genus Zephyranthes. Also the overview of alkaloids with bilogical activity was described. Within the genus Zephyranthes 10 species were studied phytochemically and 89 alkaloids were isolated from this plants. This alkaloids are divided in several structural groups. The lycorine-, haemanthamine-, galanthamine- and pancratistatine-type alkaloids occure the most frequently. Acetylcholinesterase-inhibitory, anticancer and antimalarial activity of the alkaloids was described like the most important. The most significant acetylcholinesterase- inhibitory activity was observed in alkaloids from galanthamine sctructural type. Anticancer activity was found the most in lycorine-, pancratistatine- and haemanthamine- type alkaloids. The most notable antimalarial activity was observed in lycorine- and heamanthamine- type alkaloids. Keywords Zephyranthes, Amaryllidaceae,...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany Candidate: Kamila Lipovská Supervisor: doc. Ing. Lucie Cahlíková, Ph.D. Title of Diploma thesis: Preparation of lycorine derivatives and their biological activity The plants of the Amaryllidaceae family are one of the most important sources of biologically active alkaloids. Lycorine, a phenanthridine alkaloid, isolated from various species of the Amaryllidaceae plant family, has attracted considerable attention due to its promising biological activities. Specifically, its anticancer properties have been evaluated in vitro and in vivo in various preclinical models of human cancers. Further biological effects manifested by lycorine are: antiviral, antibacterial, antifungal, antiplasmodial, anti-oxidant, anti-inflammatory and insect antifeedant effects, as well as ascorbic acid biosynthesis and RNA inhibitory activity. So far, lycorine was used for preparation of many derivatives by modification of different functional groups in its molecule, and screened for a various biological activities such as anticancer activity, inhibition of cholinesterases, antiplasmodial, antitrypanosomal, antiviral and anti-Trichomonas vaginalis activity. The present work deals with the preparation of lycorine derivatives and their...

Author: Adéla Hodulová Title: Alkaloids of Amaryllidaceae family, genus Nerine Diploma thesis Charles University, Faculty of pharmacy in Hradec Králové, Department of Pharmaceutical botany 2018, 51 p. The aim of this diploma thesis was to summarize up the findings about alkaloids which where isolated from genus Nerine plants of Amaryllidaceae family. There was introduced a botanical characteristic of the phytochemical studied species of the genus Nerine. The most studied species was Nerine bowdenii. All the knowledges about mentioned alkaloids were sum up. Ten species have been phytochemical studied out of the genus Nerine until now. In total, 40 alkaloids were isolated from these plants. Alkaloids which where isolated from the Nerine are divided into several structural types. Anticancer, acetylcholinesterase-inhibitory of the alkaloids were described. The biological activity is connected with their stucture. The most significant anticancer activity was observed in alkaloids from crinine- and lycorine type . Acetylcholinesterase-inhibitory activity was pronounced the most in crinine-type alkaloids. Keywords: Nerine, Nerine bowdenii, Amaryllidaceae, alkaloids