Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Monika Moravcová Supervisor: Assoc. Prof. Přemysl Mladěnka, Pharm.D., Ph.D. Title of thesis: Preparation of a Novel Method for Screening of Cobalt Chelators Cobalt as a structural part of the vitamin B12 is an essential microelement for living organisms including humans. However, its excess is associated with pathological conditions. Cobalt poisoning can be caused for example by exposure to cobalt metal dust during the production of hard metals or follow the corrosion of metal hip prosthesis. Patients intoxicated by cobalt can develop different manifestations including neurological impairment, hypothyroidism or cardiomyopathy. The aim of this work is to prepare a standardized, rapid, cheap but precise method for the screening of cobalt chelators. For this purpose, spectrophotometric detection using 1-nitroso-2-nafphhol-3,6-disulfonic acid disodium salt as the indicator was used. Firstly, it was found that the addition of cobalt ions led to a clear bathochromic shift of the maximum absorbance of the indicator. The relationship between the absorbance and cobalt concentration was highly linear from 470 to 560 nm at all 4 tested pH conditions (4.5, 5.5, 6.8 and 7.5). The sensitivity of the method...

A series of acene-fused porphyrins and 1,10-phenanthroline-fused porphyrins were synthesized and characterized via NMR spectroscopy and mass spectrometry. The acene-fused porphyrins exhibit unique optoelectronic properties, most notably they exhibit highly red-shifted absorption bands. The 1,10-phenanthroline-fused porphyrins are of interest for their ability to bond to as variety of metals to form chelation complexes.

A growing issue in groundwater studies concerns the distinction between anthropological and geological metal contaminants in soil. Current extraction methods, such as hot acid extraction, result in the digestion of the sample matrix which may obscure the origin of the contaminant. Since metals from anthropological sources are most often adsorbed to the surface of the soil sediment, the dissolution of the geological matrix is not necessary to release the contaminat metals. The resolution to this problem may lie in the use of chelating agents, where the adsorbed metals can be removed from the sediments without disrupting the geological matrix. This would allow the surface bound metal contaminants to be differentiated from the geological metals which are a natural part of the soil's composition. The basis of this study is a comparison of a hot acid extraction method vs. a chelation extraction method for the determination of metals in soils. Statistical comparisons on the precision and accuracy of the these methods on a reference soil, salt amended soil, and sewage sludge amended soil is discussed. / Master of Science

En cas d’accidents nucléaires (Tchernobyl, Fukushima) ou d’exposition à de l'uranium appauvri dans des zones de conflit, la décontamination est nécessaire pour réduire au mieux les conséquences de l’ingestion de radionucléides. Après une contamination externe ou interne, les actinides solubilisées sont distribués dans les organes cibles (squelette, foie, tissus, reins, etc.) via la circulation sanguine. Compte tenu de cette dispersion, la chélation de ces radionucléides par des ligands biologiques est une méthode efficace de décorporation pour favoriser l'excrétion de ces métaux déposés et ainsi réduire les risques pour la santé. En raison du faible taux de distribution dans les organes cibles (os, foie, reins) de l'acide diéthylènetriaminepentaacétique (DTPA), des agents de chélation ont été synthétisés et testés in vitro ou in vivo. Dans ce projet, plusieurs ligands polyaminophosphonates, (conçus à l'origine pour être des agents de contraste), ont été synthétisés selon leurs propriétés de bio-distribution, de leurs groupes fonctionnels, de leur potentiel site de coordination et de leur lipophilie. Des études structurales et thermodynamiques ont ensuite été effectuées sur les complexes entre l'uranium (VI) et l’europium (III) (comme analogue de l’américium (III) et curium (III)) et les ligands polyaminophosphonates. La sphère de coordination de ces cations a été observée par spectroscopie UV-visible, TRLFS, FT-IR et la spectroscopie d’absorption X (EXAFS). L'étude de l’affinité a été réalisée par spectroscopie UV-visible. Enfin, les spectroscopies UV-visible et TRLFS ont été utilisées d’une part pour tester la stabilité du complexe ligand/uranyle en présence d’un ion métallique et d’autre part pour étudier le système ternaire : ion uranyle/ligand/calmoduline. Ces résultats ont permis de mieux comprendre les mécanismes de chélation et d’évaluer l'affinité chimique de ces ligands polyaminophosphonates pour l'uranium (VI) et l’europium (III). Cela devrait ainsi aider à la conception de nouveaux agents de chélation de plus en plus efficaces du point de vue de la décorporation. / For exposed person who suffers from contamination from nuclear accidents (Chernobyl, Fukushima) or depleted uranium in war zones, decontamination is required to reduce the sequence damage of radionuclide intake. After an external or internal contamination, the solubilized actinides could be distributed to the target organs (skeletal, liver, kidneys tissues, etc.) via the bloodstream. Considering the dispersion, fate and health effect of the actinides, chelation therapy is an effective decorporation method to promote the excretion of deposited actinides to reduce the health risk. Due to the defect on weak distribution rate to the target organs (bone, liver, kidneys) of diethylenetriaminepentaacetic acid (DTPA) which currently used in clinics, plenty chelation agents were synthesized and tested in vitro or in vivo. In this project, several polyaminophosphonates ligands, a series ligand originally designed for MRI contrast and SPECT agents, were synthesized according to the properties of ligand bio-distribution, functional groups, coordination site and lipophilic. Then the structural and thermodynamic studies were done with the complexes between metal ion such as uranium(VI) and europium(III) (as americium/curium(III) analogue), and polyaminophosphonates ligands. The sphere of coordination of these cations was observed by UV-visible spectroscopy, TRLFS, FT-IR and Extended X-Ray Absorption Fine Structure (EXAFS). The affinity study was done with UV-visible spectroscopy. Finally, the UV-visible spectroscopy and TRLFS were used to test the stability of uranyl ligand complex with competition metal ion in biological conditions and to reveal the interactions between the ternary system, uranyl ion/ligand/calmodulin. These results allow to better understand the chemical affinity and possible chelation mechanism of the polyaminophosphonates ligands for the above actinides and therefore to promote the design of new chelation agents.

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Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical botanic and ekology Candidate: Nikol Vavřichová Supervisor: Ing. Kateřina Macáková Ph.D. Title of diploma thesis: Iron - chelating effects of flavonoid metabolites - small polyphenolic substances (2015/2016) Iron is a trace element which is necessary for function of an organism. There is no specific excretion mechanism for iron, therefore any deflection causes deficiency or abundance. Iron abundance leads to hemochromatosis. An application of iron chelators is a treatment of choice for hemochromatosis. There are three clinically used chelators for treatment of iron overload: deferoxamine, deferasirox and deferiprone. These drugs are inappropriate for therapy (side effects, dosage form). Therefore there is a place for serching for new types of medicine for treatment of hemochromatosis. Flavonoids are natural substances with positive influence on a human organism. The most investigated effects are antioxidant and chelating activity. As flavonoids are metabolised in a human body, there is an arising question if flavonoid metabolites are also able to chelate iron ions. For this work we chose derivates of benzoic acid: benzoic acid, 3 - hydroxybenzoic acid, 4 - hydroxybenzoic acid, 2,4 - dihydroxybenzoic...

Dočekalová L., Iron-chelating activity of selected alkaloids III, Diploma thesis, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical botanic and ecology, Hradec Králové, 2016, 60 pp. Iron is important trace element necessary for human organism. When excess it develops free radicals and structures of organism are harmed. It can lead to organ failure. Excess is solved with chelators, the most used is deferoxamine. But his terapeutic regimen is disadvantageous and it has a lot of side effects. That's reason for searching for substances with better features. This paper focuses on studium of iron-chelating activity of selected alkaloids from family Amaryllidaceae. Concretely, it's galanthamine, undulatine, buphanisine, caranine, 1-O- acetylbulbisine, homolycorine, tazettine, galanthamine, chlidanthine, ambelline, haemanthamine, haemanthidine, hamayne, 9-O-demethylgalanthine and lycoramine. As standard for comparison of activity was used deferoxamine. Activity was measured by spectrophotometer. As indicator was used ferrozine. Chelatation was measured for ferrous ionts and for total iron, where hydroxylamine as reduction agent was used. Ferrous ionts was measured also with pH alteration. Highest activity reached deferoxamine in all executed measurements. In...

Iron is the most abundant transition metal found in living systems and plays a crucial role in DNA biosynthesis. To accommodate higher replication rates, cancer cells require higher amounts of iron compared to non-neoplastic counterparts. This higher demand for iron renders cancer cells susceptible to iron deprivation, and exposure to iron chelators leads to growth arrest and cell death. Iron chelation strategies employing a wide variety of iron-binding scaffolds are currently under investigation for use in cancer treatment. Although these chelation approaches are effective against several cancer cell types, their use is limited due to toxicity ascribed to indiscriminate metal sequestration and induction of oxidative stress. Prochelation strategies in which the chelating unit remains inactive until triggered by a disease-specific event are expected to increase the specificity of chelation-based therapeutics. Chapter 1 provides an overview of chelation and prochelation based therapies as well as disulfide-based approaches in the design of prodrugs. In Chapter 2, the reduction activation mechanism of disulfide-masked thiosemicarbazone prochelators is described. Whereas disulfide-masked prochelators do not bind iron, reduction of the disulfide bond upon cellular uptake produces active chelators that readily bind intracellular iron. These systems are not active extracellularly; rather, they target the intracellular labile iron pool. We found that the antiproliferative activity of these disulfide-masked prochelators is dependent on the intracellular redox environment, with enhanced toxicity in more reducing conditions. The iron complexes resulting from exposure of cultured cells to the chelation systems were detected intracellularly by electron paramagnetic resonance in intact frozen cells. The compounds in our first series do not engage in intracellular redox chemistry and do not cause oxidative stress. In Chapter 3, the synthesis and characterization of a larger series of disulfide-masked prochelators featuring several classes of tridentate ligands is described. We investigated the iron-binding efficacy of the corresponding chelators, their ability to induce oxidative stress and their cell-cycle effects. We found that these prochelator systems, regardless of the identity of the donor set of atoms, do not result in the intracellular generation of oxidative stress. We also found that treatment of cultured cancer cells with prochelators results in cell-cycle arrest at G1/0 in non-synchronized cells and G2/M in G2-synchronized cells. In addition, we found that all classes of prochelators exhibit antiproliferative effects likely through induction of apoptosis. In Chapter 4, the syntheses and biological evaluations of disulfide-masked prochelators that feature carbohydrate targeting units are described. The sugar conjugates present increased aqueous solubility, compete as effectively as D-glucose for transporter-mediated cellular uptake, and are 6 to 11-fold more selective towards colorectal cancer compared to an aglycone that does not contain a targeting unit. The design of more potent prochelator systems, as well as the design of systems with improved selectivity and aqueous solubility are discussed in Chapter 5.

Charles University, Faculty of Pharmacy in Hradec Králové Department of: Pharmaceutical Botany Consultant: Ing. Kateřina Macáková, Ph. D. Student: Markéta Šilhová Title of Thesis: Interactions of alkaloids with transition metals II. Copper is an important component of the human body. It is involved in the right functioning of organ systems and is also a part of many important body enzymes. It is necessary to maintain a balanced amount of copper in the body in order to avoid excess or deficiency, which can lead to various diseases. The aim of this diploma thesis was to determine the copper chelating and copper reducing effects of isoquinoline alkaloids berberine chloride, canadine, corydaline, sculerine, sinactine, stylopine, tetrahydropalmatine, allocryptopine, protopine, corycavamine and cryptopine. In experimental measurements the determination of chelation of copper ions with hemytoxyline and the determination of chelation and reduction of copper ions using disodium salt of bathocuproindisulfonic acid was performed. The highest reducing activity was exhibited by scoulerine, whose structure in comparison with other alkaloids contains hydroxyl groups and which has been exhibited in the past to inhibit the growth of tumour cell. The lowest reducing activity was measured for protopine alkaloids and...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany Candidate: Radka Rzepecká Supervisor: Ing. Kateřina Macáková, Ph.D. Title of Thesis: Interactions of alkaloids with transition metals I. Copper is one of the essential trace elements that are necessary for the proper functioning of the organism. Copper is a significant component of many enzymes that affect various metabolic processes in the human body. It is important that the level of copper in the body is regulated because its deficit or excess lead to a variety of pathological conditions. Alkaloids are secondary plant metabolites that are distinguished by numerous biological activities. In this thesis the copper-chelating and copper-reducing activity of eleven isoquinoline alkaloids was measured: boldine, isocorydine, (+)-bulbocapnine, (+)-corydine, glaucine, (-)-sinoacutine, (-)-californidine, (-)-escholtzine, platycerine, (-)-fumaricine, and (+)- parfumine. Alkaloid activity was measured at four (patho) physiological pH values by a verified spectrophotometric method using two indicators: bathocuproinedisulfonic acid disodium salt and hematoxylin. Based on the results, structure-effect relationships were derived. The results show that none of the tested substances was able to chelate copper ions. In...