Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and toxicology Student: Lucie Rozkydalová Supervisor of Diploma thesis: Prof. PharmDr. František Štaud, PhD. Specialized supervisor: Pr. Pierre Cuq PharmD. PhD., Laure-Anaïs Vincent Title of diploma thesis: Differential resistance of melanoma to vinca-alkaloids Malignant melanoma (MM) represents the most dangerous and very aggressive skin tumor with fast development of drug resistance which is the main obstacle in successful treatment of MM. According to previous studies (microarray data analysis), KIT gene, which plays key role in melanoma pathophysiology, was chosen as one of the potential causes of failure of treatment by vinca alkaloids (VAs) because of its complete underexpression in melanoma CAL1 resistant cells (CAL1R-VAs) in comparison with parental cells (CAL1-wt). Moreover, KIT also interacted with NF-κB and cyclin D1-2 proteins involved in chemoresistance of melanoma - inside molecular network built using IPA software. Although KIT underexpression in resistant CAL1 R-VAs cell lines were confirmed (qRTPCR), KIT repression using specific siRNA transfection did not show any effect on in vitro sensibility of CAL1-wt cells to VAs. It signifies that KIT is not directly involved in melanoma resistance...

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany and Ecology Candidate: Bc. Blanka Kaluzsná Supervisor: Prof. RNDr. Lubomír Opletal, CSc. Title of Diploma Thesis: Biological activity of plant metabolites XXIII. alkaloids of Corydalis cava (l.) Schweigg. & Körte and their effect on acetylcholinesterase and butyrylcholinesterase An ether extract which contained tertiary basic alkaloids was prepared from the dry bulb of Corydalis cava (L.) SCHWEIGG. & KÖRTE. A subfaction labeled 2/B was obtained by column chromatography and crystallization. Three alkaloids were isolated by preparative TLC and they were identified by GC/MS and 1 H a 13 C NMR analyzes as (+)-canadine, (+)-tetrahydropalmatine and domestine. Isolated alkaloids were tested by spectrophotometric Ellman method for inhibitory activity against human erythrocyte acetylcholinesterase (HuAChE) and plasma butyrylcholinesterase (HuBuChE). In comparison with the refernce substances, from these isolated alkaloids only (+)-canadine showed an relatively high inhibitory activity against HuAChE (12,4 µM). (+)-Tetrahydropalmatine and (+)-domestine had no significant inhibitory activity in this direction, against both HuAChE and HuBuChE. Key words: Corydalis cava, (+)-canadine, (+)-tetrahydropalmatine,...

Vítovcová, A.: Biological activity of secondary plants metabolites IV. Alkaloids of Vinca minor L. Diploma thesis, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany and Ecology, Hradec Králové 2016. Extract of Vinca minor L. was separated to fractions in column chromatography. Flash chromatography, preparative TLC and crystalisation led to isolation of two alkaloids from fraction number two and number five. Alkaloids were identified by GC/MS as (+)-vincaminorine and vincorine. These alkaloids were tested for their inhibition activity towards cholinesterase (AChE and BuChe). Obtained activities IC50 were compared with standards (galanthamine, huperzine A). The most interesting activity against galanthamine (AChE 1,710 ± 0,065 µM, BuChE 42,30 ± 1,30 µM) and huperzine A (AChE 0,033 ± 0,001 µM, BuChE > 1000 µM) showed vincorine (AChE > 1000 µM, BuChE 9,75 ± 0,45 µM). His activity toward BuChE is higher than the activity of both standards. Inhibition activities of (+)-vincaminorine (AChE 746,5 ± 84,13 µM, BuChE 684,32 ± 70,66 µM) are negligible. Key words: Alzheimerʼs disease, cholinesterase, Apocynaceae, Vinca minor L., indole alkaloids

1 ABSTRACT Kučerová J.: Biological activity of secondary plants metabolites VI. Alkaloids of Vinca minor L. Diploma thesis, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of pharmaceutical botany and ecology, Hradec Králové 2016, 67 pages. Isolation of alkaloids was done from extract of Vinca minor, family Apocynaceae. Individual substances obtained were examined for their ability to inhibit human acetylcholinesterase and butyrylcholinesterase. The basic extract was after an initial extraction with 95% ethanol pre-purified by extraction with chloroform. This chloroform extract was divided further by column chromatography on a total of 531 fractions. The fractions obtained were, based on the similarities identified by thin layer chromatography, interconnected in a final number of 17 fractions. The mobile phase for column chromatography were mixtures of chloroform and medical gasoline, chloroform and ethanol, and ethanol itself. For further testing were used fractions number 2 and 5. From the fraction 2 was by preparative thin layer chromatography and subsequent crystallization isolated substance number 1. After analysis of this substance by GC/MS was found that it is a (+) - vincaminorein. Further was examined it's biological activity towards cholinesterases, which found a...

The pyrrolidine and piperidine ring systems are present in a variety of different classes of amphibian alkaloids. We have found the iridium catalyzed N-heterocylization reaction of diols with amines to be very useful for the construction of novel pyrrolidine, piperidine and piperazine derivatives. The scope and utility of the iridium catalyzed N-heterocyclization reaction for the construction of novel anuran scaffolds using amino diols and triols are presented. Studies directed towards the total synthesis of 4,6-disubstituted quinolizidine and (±)-epiquinamide are discussed. The second study is focused on the selective conversion of terminal dienes to primary diols. This conversion has always had problems with regioselectivity and low yields due to polymer formation with carbon chains having more than 7 carbon atoms. An improvement in the yield and regioselectivity was observed with disiamylborane prepared in situ using 2-methyl-2-butene and BH3•DMS. The scope of this method with 7, 8 and 9 carbon chains and different alcohol protecting groups for synthesis of triols is presented.

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.

The thesis deals with the creation of the biosynthetic pathways of selected alkaloids of opium poppy. Then is following the design primers for selected genes and optimization of PCR reaction of these genes for amplification and sequencing of the longest fragments. The PCR reaction was optimized for the 7OMT, TNMT and CODM genes. These genes are involved in biosynthetic pathways of morphine, codeine, papaverine, noscapine and sanguinarine. Each gene was split in half. The primers were designed separately for each half of the gene. Altogether were designed 13 pairs primers. 5 pairs primers were optimized by gradient PCR and gel electrophoresis. These primers were used for sequencing analysis.

Senecio brasiliensis, conhecida popularmente como maria-mole, é uma das principais causas de intoxicação em animais de produção, principalmente em eqüinos e bovinos. A toxicidade desta planta ocorre devido à presença dos alcalóides pirrolizidínicos (APs), os quais sofrem biotransformação no fígado gerando como metabólitos tóxicos os pirróis. Além disso, esses compostos tóxicos podem ser transferridos para o homem através de produtos comestíveis de origem animal contaminados ou pelo uso na medicina popular. Até o momento, não há relatos a respeito de seus efeitos tóxicos sobre a prole de animais expostos durante a gestação. Assim, o objetivo deste trabalho foi avaliar os possíveis efeitos tóxicos da exposição pré-natal ao S. brasiliensis. Ratas Wistar fêmeas prenhes receberam por gavagem, do 6° até o 20º dia de gestação, diferentes doses de S. brasiliensis (3, 6 e 9 mg/Kg/dia). Durante o período de gestação foi avaliado o peso materno, consumo de água e de ração; ainda, nas progenitoras se avaliou o comportamento materno e materno agressivo. Na prole avaliaram-se os parâmetros do desenvolvimento físico e reflexológico; quando adultos avaliou-se aspectos comportamentais, hematológicos, bioquímicos, anatomopatológico e níveis de neurotransmissores. Os resultados mostraram diminuição no consumo de ração e no ganho de peso nos diferentes grupos experimentais, de forma dose-dependente. Ratas tratadas com a maior dose de S. brasiliensis apresentaram prejuízo no comportamento materno e materno agressivo. Os filhotes provenientes de ratas que receberam as doses de 6 e 9 mg/Kg apresentaram atraso para o início do desenvolvimento físico e reflexológico. Na prole adulta masculina proveniente da maior dose experimental observou-se aumento da atividade motora no campo aberto, bem como aumento na frequência de entrada e no tempo gasto nos braços abertos do labirinto em cruz elevado. Na natação forçada observou-se aumento no tempo de escalada na prole feminina proveniente da maior dose, enquanto na prole masculina adivinda dos grupos de 6 e 9 mg/Kg notou-se diminuição no tempo de natação. Na avaliação do comportamento estereotipado observou-se aumento deste comportamento em fêmeas advindas do grupo de maior dose experimental. Ainda, na prole adulta, foram osbervadas alterações hematológicas e bioquímicas; a análise histológica revelou aumento de células multinucleadas em animais provenientes dos grupos de 6 e 9 mg/Kg e com relação análise dos neurotransmissores, foram observadas alterações a nível estriatal. Estes resultados indicam que a exposição durante a gestação ao S. brasiliensis causa toxicidade materna acompanhada de prejuízo em ambos comportamento, materno e materno agressivo. Com relação à prole, houve prejuízo no desenvolvimento físico e reflexológico, e na idade adulta foram observadas alterações comportamentais e hematológicas, bem como algumas alterações bioquímicas e anatomapatológicas. / Senecio brasiliensis popularly known as \"Maria Mole\" (=lazy Mary), is a principal cause of poisoning in livestock, mainly in horses and cattle. The toxicity of this plant is caused by pyrrolizidine alkaloids (PAs) that are metabolized by hepatic enzymes to very toxic pyrrole metabolites. In addition, these compounds can be transferred to humans through animal products or using this plant as popular medicine. There are no reports about its toxic effects on the offspring. Thus, the aim of this study was evaluate the possible toxic effects of prenatal exposure to S. brasiliensis on rat offspring. Pregnant Wistar rats received different doses of S. brasiliensis (3, 6 and 9 mg/kg, by gavage, from 6th to 20th pregnancy day. During the gestational period were evaluated the maternal weight gain and water and food intakes, as well in dams were evaluated maternal and maternal aggressive behavior. In offspring were evaluated physical and reflexologic development and, when adult, the offspring were evaluated for behavioral aspects, haematological, biochemical, anatomopathological parameters, and neurotransmitters levels. The results showed decreased a dose-dependent decrease in food intake and weight gain of dams. Dams treated with the highest S. brasiliensis dose showed impairment in maternal and maternal aggressive behavior. The offspring exposed to 6 and 9 mg/Kg of S. brasiliensis showed delay at the beginning of the physical and reflexologic development. In adult male offspring the highest dose was observed increased on open field motor activity and the frequency of entries and spent time on open arms of the elevated plus-maze. In forced swimming test was observed increase on climbing time female offspring exposed to highest dose and decrease swimming time in male offspring from 6 mg/kg and 9 mg/kg doses. On stereotypic behavior test, only the female offspring exposed to the highest dose showed increase of this behavior. The adult offspring showed few haematological and biochemical alterations, and the study histophatology demonstrated increased of hepatic multinucleated cells in animals exposed to both 6 and 9 mg/kg groups ;on the neurotransmitters levels alterations only at striatum. These results suggest that the S. brasiliensis exposure during the pregnancy cause maternal toxicity and impairment in both maternal and aggressive maternal behavior. The offspring showed damage in physical and reflexologic development, while in adulthood was observed behavioral and changes and some haematological, biochemical and anatomopathological alterations.

O presente trabalho descreve 3 abordagens divergentes para obtenção do esqueleto bicíclico presente nos alcaloides indolizidínicos. A primeira abordagem consiste no desenvolvimento de um novo método de preparação de diazocetonas α,β-insaturadas a partir da reação de Horner-Wadsworth-Emmons (HWE) entre diazofosfonato e aldeídos. As dizocetonas α,β-insaturadas obtidas foram utilizadas como bloco de construção do esqueleto cabocíclico indolizidínico, onde o intermediário chave foi obtido através do rearranjo de Wolff. A segunda estratégia consiste no desenvolvimento do acoplamento redutivo entre derivados α-aminocarbonílicos e acrilato de metila mediado por SmI2, onde em apenas duas etapas foram obtidos os intermediários avançados da síntese da (-)-pumiliotoxina 251D e da (+/-)-epiquinamida. A terceira estratégia utiliza como etapa chave a reação de Wittig/HWE intramolecular para preparação do intermediário bicíclico contendo o sistema α,β-insaturado que pode ser utilizado na síntese divergente dessas substâncias. / Herein were described three diverted oriented approaches for the construction of the bicyclic scaffold of indolizidines alkaloids, that figures between one of the most important classes of natural products. In the first approach, a new method to prepare α,β - unsaturated diazoketones was described using the Horner-Wadsworth-Emmons (HWE) reaction between diazophosphonate and aldehydes. The unsaturated diazoketones were used as powerful plataforms to construct the indolizidine carbocyclic scaffold, enploying the Wolff rearrangement as the key step. The second approach was the development of a reductive coupling between α-aminocarbonyl derivatives and methyl acrylate, mediate by SmI2, from this approach, the well-known advanced intermediate for the synthesis of (-)-pumiliotoxin 251D e of the (+/-)-epiquinamide was obtained in only two steps. The third approach uses the intermolecular Wittig/HWE reaction as the key step in the construction of a bicyclic intermediate containing an α,β -unsaturated moiety that could be used for a diverted oriented approach in the indolizidine alkaloids synthesis.

Resultados recentes mostraram efeito ansiolítico do extrato hidroalcoólico bruto de Erythrina mulungu (EM) em ratos submetidos aos modelos do labirinto em T elevado (LTE) e da transição claro-escuro (TCE). O objetivo do presente trabalho foi realizar um estudo fitoquímico biomonitorado com a intenção de se verificar a participação dos alcalóides eritrínicos na atividade ansiolítica do extrato. Foi observado que o extrato bruto de EM apresentou efeito ansiolítico nas medidas de esquiva inibitória (100, 200 e 400 mg/Kg) e fuga (400 mg/Kg) dos braços abertos do LTE e na medida de tempo gasto pelos animais no compartimento iluminado (100 e 200 mg/Kg) no TCE. A partir do extrato hidroalcoólico de EM foi isolado um novo alcalóide eritrínico, a 11-hidroxi-eritravina, e dois já registrados na literatura, a eritrartina e a eritravina. Os testes com o LTE mostraram que a eritrartina, a eritravina (3 e 10 mg/Kg) e a 11-hidroxi-eritravina (10 mg/Kg), apresentaram efeito ansiolítico na medida de esquiva inibitória dos braços abertos. No TCE foi observado efeito ansiolítico com a eritravina (3 e 10 mg/Kg) e com a 11-hidroxi-eritravina (10 mg/Kg) na medida de tempo gasto pelos animais no compartimento iluminado. No TCE, a 11-hidroxi-eritravina (3 mg/Kg) também apresentou efeito ansiolítico aumentando o número de transição entre os dois compartimentos do modelo. Os efeitos ansiolíticos provocados pelos três alcalóides foram independentes de qualquer alteração locomotora, pois nenhum dos compostos estudados e, tampouco, o extrato bruto alterou o comportamento avaliado na arena. Desta forma, os resultados do presente trabalho mostraram que os alcalóides eritrartina, eritravina e 11-hidroxi-eritravina são responsáveis pelo efeito ansiolítico observado com o extrato bruto, o que explica a ampla utilização popular das plantas do gênero Erythrina como \"calmante\". / One new erythrinian alkaloid derivative 11-OH-erythravine (3) and the known erythravine (2), and erythrartine (1) from Erythrina mulungu were isolated, and their structures were determined by spectral analysis. The relative stereochemistry of the new alkaloid was determined mainly by 1H NMR experiments, including NOESY spectrometry. Furthermore, the anxiolytic properties of the hydroalcoholic crude extract (CE) and of the alkaloids were evaluated using the elevated plus-maze test (EPM), the elevated T-maze test (ETM) and the light-dark transition model (LDTM) for mice. The CE showed anxiolytic-like effects in the ETM, i.e., the doses 100, 200, and 400 mg/kg (po) of CE impaired the inhibitory avoidance of the open arms of the maze. In the LDTM, CE (100 and 200 mg/kg) increased the time spent by the animals in the illuminated compartment, an anxiolytic-like effect. Since CE did not alter the anxiety related responses in mice exposed to the EPM, we did not use this model to test the anxiolytic-like effects of the erythrinian alkaloids. Interestingly, the compounds 1, 2, and 3 also attenuated anxiety in the ETM, an effect that was confirmed in the LDTM with the alkaloids 2 and 3. Taken in account, these results strongly suggest that these erythrinian alkaloids are the compounds responsible for the anxiolytic properties attributed to the crude extract and seem to supports the folk medicinal use as a natural anxiolytic.