Background: Necrotizing pancreatitis is a life-threatening presentation of acute pancreatitis. The mortality of 20–80% initially depends on the persistence of organ failure and systemic inflammatory response syndrome (SIRS) and, in the later course of the disease, on secondary infection of the necrosis. The questions whether prophylactic antibiotics aiming to prevent this infection should be administered and which antibiotic is the best to use, as well as the problem of fungal infection under antibiotic treatment are still intriguing and insufficiently solved. Methods: A search of the literature using PubMed was carried out, supplemented by a review of the programmes of the Digestive Disease Week (DDW) and the United European Gastroenterology Week (UEGW). Results: Despite the widely practised prophylactic antibiotic administration in severe pancreatitis, no evidence for the benefit of this strategy exists. One of the drawbacks might be a tendency for disastrous fungal infection under prophylactic antibiotics. Bacterial translocation from the gut in the second week after the onset of symptoms is the major source for infection of pancreatic necrosis and provides a clear indication for antibiotic treatment. However, routine fine-needle aspiration for a calculated antibiotic therapy cannot be recommended, and all other tests offer only indirect signs. Important factors such as enteral versus parenteral feeding and the method of necrosectomy are mostly neglected in the trials but seem to be essential for the outcome of the patient. Conclusions: Even though most meta-analyses including the newer double-blind, placebo-controlled trials on prophylactic antibiotics showed no beneficial effects in the prevention of infection of necrosis and/or outcome of the patients, this strategy is still widely used in clinical routine. Since nearly all trials published so far show systematic problems (i.e. inaccurate definition of the severity of the disease, poor statistical testing, and neglect of differences in the route of nutrition), there is a need for randomized controlled prospective trials with exact definitions of the disease. / Hintergrund: Die nekrotisierende Pankreatitis weist eine Mortalität von 20–80% auf. Initial ist vor allem das Ausmaß des Organversagens entscheidend für die Prognose des Patienten. In der zweiten Krankheitswoche stellt dann die sekundäre Infektion der Nekrosen durch die Translokation von Darmkeimen das entscheidende Problem dar. Zur Vermeidung einer solchen Infektion werden klinisch sehr häufig Breitspektrumantibioktika prophylaktisch eingesetzt. Dies wird aber zunehmend kritisch diskutiert, und es existieren kontroverse Empfehlungen. Methoden: Eine Literaturrecherche unter Einbeziehung von PubMed und der Programme der Digestive Disease Week (DDW) und der United European Gastroenterology Week (UEGW) wurde durchgeführt. Ergebnisse: Die meisten Studien können den prophylaktischen Einsatz von Antibiotika bei der schweren Pankreatitis nicht rechtfertigen. Einige Studien belegen vielmehr eine Selektion resistenter Keime und vor allem auch eine erhöhte Rate von schwer therapierbaren Pilzinfektionen unter einer solchen Therapie. Daher sollte erst nach dem Nachweis einer Nekroseinfektion mit einer Antibiotikatherapie begonnen werden, wobei keine Routine-Feinnadelpunktion der Nekrose zum Keimnachweis durchgeführt werden sollte. Es stehen daher nur indirekte, meist bildgebende Verfahren für den Infektionsnachweis zur Verfügung. Entscheidende Faktoren wie die enterale Ernährung und die Methode der Nekrosektomie wurden bisher bei den meisten Studien vernachlässigt, scheinen aber essenziell für das Behandlungsergebnis des Patienten zu sein. Schlussfolgerungen: Die meisten publizierten Studien weisen eine sehr heterogene Definition der Erkrankung, uneinheitliche Behandlungsprotokolle und Ungenauigkeiten bei der statistischen Testung auf. Gerade entscheidende Faktoren wie die enterale Ernährung werden größtenteils komplett vernachlässigt. Es besteht daher ein Bedarf für randomisierte placebokontrollierte Studien, die diese Probleme berücksichtigen und suffiziente Schlussfolgerungen zur Antibiotikatherapie der schweren Pankreatitis zulassen.

Many organic compounds are released to the environment and can be harmful to living organisms. These compounds are often persistent and toxic. Some are mutagens, carcinogens, endocrine disruptors or they can cause an increase in bacterial resistance. They tend to accumulate in nature and their transformation is a long-term process. Therefore, various remediation techniques are needed for decontamination. Remediation and bioremediation processes depend on many factors which should be critically evaluated. This dissertation thesis studies the relationship between bioavailability, biodegradation and toxicity of polychlorinated biphenyls, polycyclic aromatic hydrocarbons and fluoroquinolone antibiotics. These compounds of different origin, character and properties were degradated by ligninolytic fungi. Desorption behaviour of pollutants from historically contaminated sites, degradation potential of ligninolytic fungi, ongoing degradation mechanisms, transformation products and their toxicity were studied as important factors for evaluation of mycoremediation and its environmental impact. The results show that determination of bioaccessible fraction by sequential supercritical fluid extraction is very useful for precise prediction of biodegradability of pollutants. The evidence that ecotoxicity and...

Cystic fibrosis (CF) is the most common autosomal recessive genetic disorder in Caucasians. Lower respiratory tract of CF patients is colonized by specific bacteria, often leading to chronic infection and lung tissue damage. In this thesis we characterized 338 isolates of S. aureus from 92 Czech CF patients isolated in 2011-2013. Using spa typing and PFGE we detected high clonal heterogenity of this collection with the exception of MRSA strains (resistant to oxacillin; 5% prevalence) which were clonally related. The prevalence of S. aureus MLSB resistance in our collection was high (69 %), which is a serious problem due to common usage of these antimicrobials in clinical practice. A half of the MLSB resistant strains lacked any known determinant of this resistance (ermA, ermC, ermT, msrA). Sequencing of the ribosomal genes revealed a high number of S. aureus strains carrying target site mutations resulting in MLSB resistance (37 %). This is new important information about the staphylococcal strains associated with chronic infections in Czech Republic. Focusing on mutability of analysed strains, we also detected several strains with point mutations or deletions in their mutator genes mutS a mutL. Hypermutability could be responsible for the high rate of ribosomal mutations and for the presence of...

The variety of bacteria and their genomes sometimes causes conservation of homologue molecules to be displayed not in sequence but in secondary and tertiary structures. In the case of the regulatory 6S RNA, sequence homologues have been found in over 100 bacterial species so far. However, none were found in the genus Streptomyces. The unique genome of these soil- dwelling bacteria, known for their capacity to produce antibiotics, has a high G/C content and diverges substantially from distantly related bacteria. Yet in the non-coding 6S RNA it is the secondary structure that is crucial for its function. The 6S RNAs trap sigma factors by mimicking target promoter sequences in order to help with switching sets of expressed genes during developmental transitions. 6S-like RNA genes in Streptomyces coelicolor have been computationally predicted by comparison of in silico modelled secondary structures of known 6S RNAs. The aim of this thesis was the verification of these 6S-like RNA predictions. The experimental approach was based on RNA co-immunoprecipitation (RNA CoIP), as well as RT- PCR from RNA samples. The outcomes of this project are the detection of six novel ncRNA transcripts with possible 6S-like RNA functions, which also served as the wet-lab verification of the in silico prediction technique...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Study program: Specialist on Laboratory Methods Autor: Bc. Marcela Vízková Supervisor: PharmDr. Ondřej Janďourek, Ph.D. Title: In vitro screening of novel potentially active antibacterial compounds III Since the discovery of penicillin, antibiotics have become part of modern therapeutic approaches. But the global spread of resistance makes their future uncertain. It is necessary to find new antibacterial substances useful in practice. As resistance is a global threat, the theoretical part deals with this issue. In addition to resistance, the theoretical part also briefly describes the antibiotics used, including new antibiotics, which were recently registered or likely to be registered. For selected groups, their mechanism of action is described in detail. Sensitivity should be determined to correctly indicate the antibiotic treatment. For this reason, the theoretical part also describes methods by which the sensitivity of a microbe to an antibiotic can be determined qualitatively or quantitatively. The theoretical part does not neglect the history of the development of antibacterial substances and familiarization with antibiotic policy in the Czech Republic. This diploma thesis is based on...

Antibiotics are substances that inhibit the growth of microorganisms and are widely used in modern medicine. High consumption of antibiotics correlates with their elevated occurence in sewage systems, from where they are further released into the environment. The threat of their occurrence in the environment is in triggering the formation and spread of antibiotic resistance. This thesis focuses on the removal of selected antibiotics and partly on the antibiotic resistance from wastewater by advanced oxidation processes (AOPs). AOPs are based on the creation of highly reactive hydroxyl radicals, which are able to oxidize even highly persistent substances. The thesis focuses on the purification of effluents from municipal wastewater treatment plants (WWTP), which are identified as the main source of pharmaceutical pollution in the environment, by AOPs mainly based on ozonation. These processes were tested under laboratory conditions on model water as well as under real conditions as the tertiary stage of the treatment at Brno-Modřice WWTP under various operating states. As the results of the performed experiments show, it could be concluded that the studied AOPs are capable of effective degradation of studied antibiotics from the treated medium. Under real conditions, the antibiotics sulfamethoxazole, trimethoprim, azithromycin and clarithromycin were monitored. Output concentrations in each operational state were achieved to be below the limit of detection for all four antibiotics. The application of AOPs based on ozonation as the tertiary step of the treatment also had a positive effect on the reduction of microbial contamination and antibiotic resistance. Within the pilot plant experiments, a reduction of up to 4 orders of magnitude of E.coli, coliform microorganisms and a reduction of their resistant strains in the range of 1.4 - 4.0 logs were observed, compared to the effluent from the WWTP.

Water pollutants, such as pharmaceuticals, became an important public health issue over the last years for their extensive presence in the aquatic ecosystem. Among several pollutants, antibiotics are especially worrying because of their potential to induce antimicrobial resistance in microorganisms. The inability of wastewater treatment plants (WWTPs) to effectively remove these pollutants makes necessary to find alternative methods for their elimination. Photocatalysis may become an alternative process since it allows rapid and efficient removal, transforming the initial compound into harmless products. It is a promising method because it uses nanomaterials that are highly photocatalytically active, photo-stable, and non-toxic. Anticipating the need for safe and more efficient water treatment methods, the scope of this thesis concerns the synthesis of different photocatalytic materials, as well as their characterization, determination of their photocatalytic properties, and respective reusability. In this context, the polymeric nanocomposites were produced by electrospinning and solvent casting, and the photocatalytic magnetic particles by co-precipitation and sol-gel. Their different morphologies and characteristics explain their different photocatalytic properties. Some of these materials overcome the limitations of the already existing materials regarding reusability and photocatalytic properties. A direct comparison of these materials in the literature proves difficult, as the experimental conditions, such as irradiation and types of photoreactors, are different among the different research groups. This thesis overcomes such limitations and therefore provides insights into the relative performance of different immobilization alternatives tested under identical conditions. The first task in this thesis is to provide evidence for the presence of pharmaceuticals in wastewater and the ability of the photocatalysts that were later intended to be immobilized to degrade them. In particular, it concerned pharmaceuticals detected on the wastewater effluent from Kaditz, Dresden, Germany. It was analyzed the degradation of 14 pharmaceuticals with initial concentrations higher than 0.3 µg L–1. Suspended commercial nanoparticles of titanium dioxide (TiO2) P25 and zinc oxide (ZnO) were used as photocatalysts. It led to a considerable degradation of the analyzed pharmaceuticals by both catalysts. ZnO nanoparticles degraded 95 % of these pharmaceuticals after 40 min under ultraviolet radiation (UV), while TiO2 took more than six times longer to reach the same degradation level. Systems using suspended photocatalysts have been shown to successfully degrade pharmaceuticals. The technique, however, has some disadvantages. In particular, it adds an additional and expensive filtration or sedimentation step to remove the photocatalyst at the end of the process. Moreover, without a commercial-scale recycling process, these types of methods prove to be cost-ineffective. In light of the need to reuse photocatalysts, this work focuses on the immobilization of photocatalytic nanoparticles, such as ZnO, TiO2, TiO2/graphene oxide (GO), and tungsten oxide (WO3) and on the posterior use in the degradation of a model pollutant. The photocatalysts were immobilized by solvent casting in poly(vinylidene difluoride-co-trifluoroethylene) (PVDF-TrFE), and by electrospinning in PVDF-TrFE and poly(methyl methacrylate) (PMMA) and their reuse was tested. Polymers are common materials suitable to be in contact with water. Therefore, these materials can be applied as valid catalysts support tools to remove organic contaminants from water. In this context, ZnO showed high toxicity towards Vibrio fischeri and consequently it should not be used, as it might have potential environmental impacts and biological effects. The TiO2 nanocomposites produced by electrospinning showed improved surface area and higher porosity compared to the solvent casting method, which is important for water percolation. In addition to the benefits of immobilization, TiO2/GO particles immobilized in the PVDF-TrFE electrospun achieved higher degradation rates under simulated sunlight. It increased the photocatalytic degradation when compared with the nanocomposites prepared with pristine TiO2, in UV and simulated sunlight. Therefore, it allows for further savings in operation costs by removing the necessity of UV lamps. However, immobilization systems have the disadvantage of losing surface area when compared to the traditional suspension systems. These studies indicate that magnetic nanoparticles are a suitable approach to address this issue, as they act as an immobilized form of the catalyst but offer high surface area, similar to the suspended systems. The prepared magnetic nanoparticles exhibited high photocatalytic activity and high reusability since the magnetic nanoparticles can be easily recovered by means of an external magnetic field and further reused. It was observed that with these materials and exposure to UV radiation or simulated sunlight, the studied compounds were degraded. UV radiation, the support, and the photocatalysts per se provide no significant degradation of the tested compounds. In conclusion, the produced nanomaterials offer an ecologically promising and efficient method to mitigate environmental pollution, by-passing many of the current issues that prevent the application of the nanomaterials for water treatment. This method may be combined with conventional water treatment systems providing a cost-efficient technique to handle the degradation of organic pollutants in aqueous systems under visible light or UV. / Die Präsenz von Pharmazeutika in aquatischen Ökosystemen wurde in den letzten Jahren zu einem wichtigen Thema der öffentlichen Gesundheit. Unter anderem sind Antibiotika besonders besorgniserregend wegen ihres Potenzials, in Mikroorganismen Resistenzen zu verursachen. Von Kläranlagen können diese Schadstoffe nicht wirksam entfernt werden, deshalb müssen alternative Methoden für deren Beseitigung gefunden werden. Photokatalyse hat in diesem Zusammenhang das Potenzial der Alternative zu herkömmlichen Prozessen, da sie eine schnelle und effiziente Entfernung ermöglicht und die Ausgangsstoffe in harmlose Produkte umwandelt. Sie ist eine vielversprechende Methode, da sie Nanomaterialien verwendet, die photokatalytisch hochaktiv, lichtstabil und ungiftig sind. Die vorliegende Arbeit beschäftigt sich mit der Synthese unterschiedlicher photokatalytisch aktiver Materialien, der Charakterisierung ihrer photokatalytischen Eigenschaften, sowie ihrer Wiederverwendbarkeit. In diesem Zusammenhang wurden polymere Nanokomposite durch Elektrospinning und Solvent-Casting, sowie photokatalytisch aktive, magnetische Partikel durch Co-Precipitation und Sol-Gel-Technik, hergestellt. Es stellte sich heraus, dass Unterschiede in der Morphologie und in anderen Merkmalen die verschiedenen photokatalytischen Eigenschaften dieser Materialien erklären können. Einige dieser Materialien zeigten deutliche Verbesserungen gegenüber bereits Vorhandenen hinsichtlich Wiederverwendbarkeit und photokatalytischer Eigenschaften. Ein direkter Vergleich mit Literaturdaten erwies sich oft als schwierig, da die experimentellen Bedingungen, wie z.B. Bestrahlungsstärke und Art des Photoreaktors der verschiedenen Forschungsgruppen unterschiedlich waren. Die vorliegende Arbeit stellt eine bessere Vergleichbarkeit her, indem sie alle erzeugten Materialien unter identischen Bedingungen testet. Der erste Teil dieser Arbeit beschäftigt sich damit, die Anwesenheit von Arzneimitteln im Abwasser nachzuweisen und außerdem die Aktivität der Photokatalysatoren, die später eingesetzt werden sollen, zu testen. Hier handelt es sich insbesondere um Arzneimittel, die im Abwasser aus der Kläranlage Kaditz, Dresden, gefunden wurden. Es wurde der Abbau von 14 Arzneimitteln mit Anfangskonzentrationen von mehr als 0.3 μg L-1 analysiert. Als Photokatalysatoren wurden suspendierte kommerzielle Nanopartikel aus Titandioxid (TiO2) P25 und Zinkoxid (ZnO) eingesetzt. Es wurde ein deutlicher Abbau der analysierten Arzneimittel durch beide Katalysatoren festgestellt. ZnO-Nanopartikel reduzierten die Arzneimittelkonzentration in 40 min um 95% unter UV-Strahlung (UV), während bei TiO2 zum Erreichen des gleichen Abbaugrades die 6-fache Zeit nötig war. Es wurde gezeigt, dass Systeme mit suspendierten Photokatalysatoren Arzneimittel erfolgreich abbauen können. Nachteilig ist jedoch der anschließend notwendige, teure Filtrations- oder Sedimentationsschritt zur Entfernung des Photokatalysators. Darüber hinaus erwiesen sich Methoden ohne kommerziell umsetzbares Recyclingverfahren als ökonomisch ineffizient. Angesichts der Notwendigkeit, Photokatalysatoren wiederzuverwenden, konzentriert sich die vorliegende Arbeit auf die Immobilisierung von photokatalytischen Nanopartikeln, wie z.B. ZnO, TiO2, TiO2/Graphenoxid (GO) oder Wolframoxid (WO3) und auf die spätere Verwendung für den Abbau eines Modell-Schadstoffs. Die Photokatalysatoren wurden durch Solvent-Casting in Poly(vinyliden-difluorid-co-trifluorethylen) (PVDF-TrFE) und durch Elektrospinning in PVDF-TrFE und Poly(methylmethacrylat) (PMMA) immobilisiert. Anschließend wurde ihre Wiederverwendbarkeit getestet. Diese Polymere sind handelsübliche Materialien, die für den Wasserkontakt geeignet sind. Daher können diese als Binder für Katalysatoren zur Entfernung organischer Verunreinigungen aus Wasser genutzt werden. ZnO zeigte dagegen eine hohe Toxizität gegenüber Vibrio fischeri, weshalb ein Einsatz in wässrigem Medium wegen potenzieller Umweltauswirkungen nicht geeignet erscheint. Die durch Elektrospinnen hergestellten TiO2-Nanokomposite zeigten im Vergleich mit den durch Solvent-Casting hergestellten eine verbesserte Oberfläche mit höherer Porosität, was für die Wasser-Perkolation wichtig ist. Vergleicht man die untersuchten Polymerfilm-Komposite hinsichtlich ihrer Abbaugeschwindigkeiten unter simuliertem Sonnenlicht und UV-Licht, dann erreichten die TiO2/GO-Partikel, die durch Elektrospinning in PVDF-TrFE immobilisiert wurden, die höchste Geschwindigkeit. Die Möglichkeit des Einsatzes von Sonnenlicht anstelle von UV-Lampen führt zu Kosteneinsparung.

Kritiskt sjuka patienter med infektioner är en börda för sjukvården och 70 % av alla patienter på intensivvårdsavdelningar är ordinerade antibiotika. Antibiotika binder till proteiner i blodet, men enbart den icke-proteinbundna (fria) fraktionen kan diffundera över kapillära membran och binda till receptorer. Standardproteinbindningsgrad för olika antibiotika har utvecklats från studier på friska frivilliga och doseringen av läkemedlen är anpassade därefter. Den totala koncentrationen av antibiotika i patienters blod är vanligen representativ för den farmakologiska effekten. Dock kan vissa sjukdomar påverka proteinbindningsgraden vilket resulterar i en större eller mindre mängd fria antibiotika i blodcirkulationen. Det här kan i sin tur resultera i toxicitet eller otillräcklig effekt av läkemedlet. Syftet med det här projektet var att utveckla en analytisk metod för att bestämma den fria koncentrationen av Beta-Lactam antibiotikan cefotaxim, flukloxacillin, kloxacillin och piperacillin i plasma. En metod utvecklades med ultrafiltrering för extraktion av den fria fraktionen och högupplösande vätskekromatografi och tandem masspektrometri, UHPLC-MS/MS, för kvantifiering av analyterna. Metoden validerades delvis enligt den Europeiska Läkemedelsmyndighetens riktlinjer för bioanalytisk metodvalidering. / Infections in critically ill patients are a problem for the healthcare system and at any one time, 70 % of all intensive care unit (ICU) patients are treated with antibiotics. Antibiotics bind to proteins in the blood, but only unbound drug can diffuse over capillary membranes and bind to the targeted receptor. Standard protein binding percentages for antibiotics have been developed from studies on healthy volunteers and dosing regimens for patients are adapted accordingly. The determination of the total concentration of antibiotics in patients’ blood samples is, based on the standard percentages, ordinarily representative for the pharmacological effect of the antibiotic. However, certain conditions that are common in critically ill patients can alter protein binding percentages, resulting in a larger or smaller unbound fraction. This in turn can result in toxicity or therapeutic failure. The aim of this project was to develop an analytical method for the determination of the unbound concentration of the Beta-Lactam antibiotics cefotaxime, flucloxacillin, cloxacillin and piperacillin in plasma. A method was successfully developed using ultrafiltration for the extraction of unbound analytes and ultra high performance liquid chromatography tandem mass spectrometry, UHPLC-MS/MS, for their quantification. The method was partly validated according to the European Medicines Agency’s guidelines on bioanalytical method validation.

Background: In preterm infants with premature rupture of membranes, antibiotic treatment is frequently started but rates of early onset sepsis are lower. In line with national guidelines, a stratified approach in the decision to start antibiotic treatment using maternal history, clinical impression and biomarkers has been implemented in our level III neonatal center and its results are evaluated. - Methods: Retrospective cohort study of all preterm newborns with rupture of membranes at least 1 h prior to delivery admitted to our tertiary neonatal intensive care unit. Data on antibiotic exposure, mortality and major neonatal complications were extracted from the electronic patient charts to evaluate the effects and safety of our stratified approach. - Results: Four hundred fifty-six infants met the inclusion criteria. 120 (26%) received primary antibiotics whereas 336 (74%) did not. Of those receiving primary antibiotics, 13 (11%) had a blood culture positive sepsis, 46 (38%) met the criteria of clinical sepsis and in 61 (51%) sepsis was ruled out and antibiotics were stopped after 48-96 h. All infants with blood culture positive sepsis were identified and treated within the first 24 h of life using this approach. None of the 336 infants who were not started on antibiotics primarily needed antibiotic therapy within the first 5 days of life. There were no deaths or major neonatal complications in the group that did not receive empiric antibiotics. - Conclusions: Our stratified approach for preterm infants with premature rupture of membranes allows a safe reduction of antibiotic exposure even in this high risk population. As a result, only 25% of high risk preterm newborns are treated with antibiotics of which more than half receive less than 5 days of treatment. To treat one infant with blood culture positive sepsis, only 9 infants receive empiric antibiotics.

In the course of this work, compounds of the natural product class of polycyclic polyprenylated acylphloroglucinols (PPAPs) were analysed. They consist of a polycyclic core structure decorated with carbonyl groups and unsaturated sidechains. The modular synthesis platform developed and optimised in the work group, facilitated the creation of a 23 compound PPAP library. This initiated the study of antibacterial properties. Further research into the derivatisation of PPAPs paved the way to a synthetic toolbox for the synthesis of new derivatives. Now a follow-up structure-activity relationship (SAR) study, aided by a novel C acylation strategy developed in the group, expanded the library with 25 new unnatural type B PPAPs. Antibiotic efficacies with minimum inhibitory concentrations (MICs) in the nanomolar range against methicillin-resistant Staphylococcus aureus (MRSA) were observed, identifying new lead compounds with significant therapeutic windows. Transitioning to cancer research, the anticancer activities of PPAPs across leukaemia, glioblastoma (GB), and prostate cancer (PCa) were investigated. A 3 phenylpropanoyl head group turned out to be crucial for proapoptotic efficacies. Optimisation efforts yielded different potent compounds for each cancer type with proapoptotic half maximal inhibitory concentration (IC50) values in the low micromolar and antiproliferative IC50 values in the nanomolar range. Flavonoid-like PPAP scaffolds were explored for enhanced biological activities through the combination of anticancer activities of PPAPs with flavonoids. A one-step synthesis method produced eleven flavonoid-like PPAPs with promising anticancer potential, particularly against GB. They exhibited strong proapoptotic effects with low toxicity on healthy cells. To expand the covalent combination of different active substances, further conjugation strategies were employed for the synthesis of PPAP-containing drug-drug conjugates (DDCs). This resulted in several PPAP–temozolomide- (TMZ) and PPAP–doxorubicin (DOX) conjugates. PPAP–TMZ showed significant anticancer potential against GB, while PPAP–DOX demonstrated substantial antileukemic activity. Challengingly, both DDCs also exhibited significant toxic effects on healthy peripheral blood mononuclear cells (PBMCs). Therefore, targeted drug delivery of PPAPs utilising PPAP hybrid drugs was investigated to enhance the selectivity of PPAP treatments. This involved the successful combination of PPAPs with linkers for antibody-drug conjugates (ADCs). To evaluate the coupling potential of the synthesised linker with monoclonal antibodies, it was reacted with cysteine. The successful reaction showed the potential of future couplings with real antibodies. For PCa specifically, prostate specific membrane antigen (PSMA)-targeting PPAPs were explored. This protein is solely expressed by PCa cells, therefore being an ideal target protein. Employing chemical structures from existing pharmaceuticals led to the design of different PSMA-targeting PPAPS. While in silico screenings indicated extraordinarily high affinity to the PSMA binding site, biological tests revealed very low anticancer activities. As the binding affinity was too strong, the PPAPs were presumably not released in the cell, which could explain the low anticancer efficacies. For this reason, cleavable linker systems were investigated as part of further research. In conclusion, this research significantly advanced the understanding of diverse PPAP derivatives, demonstrating their potential in antibacterial and anticancer applications. The outlined synthesis strategies, SAR studies, and conjugation approaches provide a foundation for future developments. Further optimisations and collaborations are needed to bring PPAPs a step closer to applications as medical treatment option.:Table of Contents Acknowledgements III Table of Contents V List of Abbreviations IX Abstract (English) XIV Abstract (German) XVII I Theoretical Part 1 Introduction 1 1.1 Medical need in modern society 1 1.2 Natural products in drug development 1 1.3 Polycyclic polyprenylated acylphloroglucinols (PPAPs) 2 1.3.1 Classifications 2 1.3.2 Biosynthesis 4 1.3.3 Biological activities 6 1.3.4 PPAP-synthesis strategies 7 1.4 Flavonoids in drug discovery 10 1.5 Hybrid drugs in cancer treatment 11 1.5.1 Antibody-drug conjugates 11 1.5.2 Drug-drug conjugates 12 1.5.3 Prostate cancer and prostate-specific membrane antigen (PSMA) 13 2 Objectives 16 2.1 Extending the PPAP library and improving biological activities 16 2.2 Examination and improvement of anticancer efficacy with unexplored PPAP scaffolds 17 2.3 Improving target selectivity for PCa using PSMA-targeting PPAPs 17 3 Expanding the PPAP library 18 3.1 Evaluation of the existing library 18 3.2 Synthesis of the PPAP22 core (PPAP26, 31) 21 3.3 Acylation of the C3 position (head group) 24 3.3.1 Established acylation method using acyl cyanides 24 3.3.2 Cyanide free acylation by PESLALZ 25 3.4 Structure-activity relationship (SAR) study of PPAP78 (50) derivatives (PPAP generation 2) 26 3.4.1 Designing the SAR study 26 3.4.2 Synthesis of PPAPs for the SAR study 28 3.4.3 Evaluation of antibacterial activity 29 3.4.4 Conclusion 32 3.5 Evaluation of anticancer activity 33 3.5.1 Synthesis of the used PPAPs 33 3.5.2 Blood cancer (leukaemia) 36 3.5.3 Brain cancer (glioblastoma) 39 3.5.4 Prostate cancer (PCa) 42 3.6 Exploring synthesis methods towards new PPAP amides 46 3.6.1 PPAP amide synthesis via isocyanates 46 3.6.2 PPAP amides from carbamates 49 3.7 Summary of the library expansion 51 4 Novel flavonoid-like PPAP scaffold 54 4.1 Exploring approaches to flavonoid-like PPAPs 55 4.1.1 Flavanones in one step from PPAP26 (31) 55 4.1.2 Conversion of flavanones to flavones 58 4.1.3 Flavone and aurone synthesis with propiolic acids 59 4.1.4 First biological activity screening 62 4.1.5 Synthesis of substituted propiolic acids 63 4.1.6 Elaborating the substitution scope with dimedone test substrates 65 4.2 Investigating flavone-like PPAPs 66 4.2.1 Synthesis of flavone-like PPAPs 66 4.2.2 Biological activity investigations 68 4.3 Exploring the synthesis of isoflavanone-like PPAPs 71 4.4 Summary of flavonoid-like PPAPs 74 5 Studies towards PPAP hybrid drugs 76 5.1 PPAP drug-drug conjugates 76 5.1.1 PPAP–TMZ conjugates 77 5.1.2 PPAP–DOX conjugates 92 5.1.3 Summary of PPAP drug-drug conjugates 96 5.2 PPAP antibody-drug conjugates 97 5.2.1 Synthesis of a linker with a Val–Cit recognition sequence 99 5.2.2 Synthesis and coupling of simplified linkers 102 5.2.3 Testing of cysteine coupling potential 106 5.2.4 Summary of PPAP antibody-drug conjugates 107 5.3 Using PSMA to increase PCa specificity of PPAPs 107 5.3.1 In silico studies for PSMA-targeting PPAPs 108 5.3.2 Synthesis of PSMA-targeting PPAPs 115 5.3.3 Biological activity of PSMA-targeting PPAPs 217 – 220 118 5.3.4 Studies towards PSMA-targeting PPAPs with cleavable hydrazide linkers 121 5.3.5 Summary of PSMA-targeting PPAPs 127 6 Summary of results 129 II Experimental Part 7 General Remarks 137 7.1 Depiction of structures 137 7.2 Substance names 137 7.3 Solvents and common chemicals 137 7.4 Analytical methods 138 7.5 Biological tests 140 7.6 Docking studies with Schrödinger© 146 8 General synthesis procedures 147 9 Syntheses expanding the PPAP library 150 9.1 Synthesis of PPAP22-core (PPAP26, 31) 150 9.2 PPAP derivatisation at C3 161 9.2.1 Standard acyl derivatisation 161 9.2.2 Standard PPAP amide synthesis 197 9.2.3 Studies towards a new PPAP amide synthesis 201 9.3 PPAP salts 205 10 Synthesis of flavonoid-like PPAPs 209 10.1 Synthesis of substituted propiolic acids 209 10.1.1 Acetylene precursors 209 10.1.2 Substituted propiolic acids 211 10.2 Synthesis of flavonoid-like dimedone test-substrates 216 10.3 Synthesis of flavonoid-like PPAPs 223 10.3.1 Aurone-like PPAPs 223 10.3.2 Flavanoid- and chalcone-like PPAPs 224 10.3.3 Flavone-like PPAPs 227 10.3.4 Dihydrocoumarin-like PPAPs 240 11 Synthesis of PPAP hybrids 242 11.1 PPAP drug-drug conjugates 242 11.1.1 PPAP-Temozolomide conjugates 242 11.1.2 PPAP-Doxorubicin conjugates 267 11.2 PPAP-antibody conjugates 276 11.2.1 Linker synthesis 276 11.2.2 Conjugate synthesis and modification 284 11.3 PSMA-targeting PPAPs 293 11.3.1 Synthesis of linkable PSMA-targeting units (PSMA-TUs) 293 11.3.2 Synthesis of a linkable PPAP amide 300 11.3.3 Linking PSMA-TUs to PPAPs 301 11.3.4 Deprotection of tBu-esters 312 III Appendix 12 X-Ray crystal structures 317 13 Exact structures of docked ligands 341 14 References 345 Curriculum vitae 355 List of publications 356 / Im Zuge dieser Arbeit wurden Verbindungen der Naturstoffklasse der polyzyklischen polyprenylierten Acylphloroglucinole (PPAPs) untersucht. PPAPs besitzen eine polyzyklische Kernstruktur, die mehrere Carbonylgruppen aufweist und mit ungesättigten Seitenketten dekoriert ist. Die in der Arbeitsgruppe entwickelte und optimierte, modulare Syntheseplattform ermöglichte die Erstellung einer ersten PPAP-Bibliothek mit 23 Verbindungen. Damit wurde die großflächige Untersuchung der antibakteriellen Aktivitäten von PPAPs eingeleitet. Weitere Erforschungen zur Derivatisierung von PPAPs ebneten den Weg zu einem synthetischen Werkzeugkasten für die Erzeugung neuer Derivate. Eine nachfolgende Struktur-Aktivitäts-Beziehungsstudie (engl. structure-activity relationship, SAR), die durch eine neuartige, in der Gruppe entwickelte C-Acylierungsstrategie ermöglicht wurde, erweiterte die Bibliothek um 25 neue nicht natürlich vorkommende Typ B PPAPs. Hierbei konnten antibiotische Wirksamkeiten mit minimalen Hemmkonzentrationen (MICs) im nanomolaren Bereich gegen Methicillin-resistenten Staphylococcus aureus (MRSA) erreicht werden und neue Leitverbindungen mit großen therapeutischen Fenstern wurden identifiziert. Die krebshemmende Wirkung von PPAPs wurde an den verschiedenen Krebsarten Leukämie, Glioblastom (GB) und Prostatakrebs (PCa) untersucht. Hierbei erwies sich eine 3 Phenylpropanoyl-Kopfgruppe als entscheidend für die proapoptotische Wirksamkeit. Strukturoptimierungen zeigten, dass für jede Krebsart jeweils unterschiedliche Verbindungen potente Wirkungen mit proapoptotischen halbmaximal inhibierenden Konzentrationen (IC50) im niedrigen mikromolaren und antiproliferativen IC50-Werten im nanomolaren Bereich aufwiesen. Flavonoid-ähnliche PPAP-Gerüste wurden im Hinblick auf verstärkte biologische Aktivitäten durch die Kombination der krebshemmenden Aktivitäten von PPAPs mit Flavonoiden untersucht. Es konnte eine einstufige Synthesemethode etabliert werden, mit welcher elf Flavonoid-ähnliche PPAPs mit vielversprechendem Krebs-bekämpfungspotenzial, insbesondere gegen GB, hergestellt wurden. Sie zeigten eine starke proapoptotische Wirkung bei geringer Toxizität gegenüber gesunden Zellen. Um die kovalente Verknüpfung verschiedener Wirkstoffe miteinander zu erweitern, wurden zusätzliche Konjugationsstrategien für die Synthese von PPAP-haltigen Wirkstoff-Wirkstoff-Konjugaten (engl.: drug-drug conjugates, DDCs) eingesetzt. Dies führte zu mehreren PPAP–Temozolomid- (TMZ) und PPAP–Doxorubicin (DOX) Konjugaten. PPAP–TMZ zeigte eine signifikante krebshemmende Wirksamkeit hinsichtlich GB, während PPAP–DOX eine beträchtliche antileukämische Aktivität zeigte. Problematisch ist, dass beide DDCs auch erhebliche toxische Wirkungen auf gesunde periphere mononukleare Blutzellen (PBMCs) zeigten. Aus diesem Grund wurden Untersuchungen zur Verbesserung der Selektivität von PPAP-Behandlungen unter Zuhilfenahme von Wirkstoffhybriden für den gezielten Wirkstofftransport (engl.: targeted drug delivery) durchgeführt. Dazu wurden PPAPs mit Linkern für Antikörper-Wirkstoff-Konjugate (engl.: antibody-drug conjugate, ADCs) verknüpft. Um das Kopplungspotenzial des hergestellten Linkers mit Antikörpern zu testen, wurde dieser mit Cystein umgesetzt. Die erfolgreiche Reaktion zeigte das Potenzial von zukünftigen Kopplungen mit echten Antikörpern. Speziell für PCa wurden gegen das prostataspezifische Membranantigen (PSMA) gerichtete PPAPs erforscht. Dieses Protein wird ausschließlich von PCa-Zellen exprimiert und stellt daher eine ideale Zielstruktur dar. Die Verwendung chemischer Strukturen aus bereits eingesetzten Arzneimitteln führte zur Entwicklung verschiedener PSMA-gerichteter PPAPs. Während in-silico-Screenings auf eine außergewöhnlich hohe Affinität zur PSMA-Bindungsstelle hinwiesen, ergaben biologische Tests nur eine sehr geringe krebshemmende Wirkung. Aufgrund der zu starken Bindungsaffinität wurden die PPAPs in der Zelle vermutlich nicht freigesetzt, was die niedrige krebshemmende Wirkung erklären könnte. Deshalb wurden im Rahmen weiterer Forschung spaltbare Linkersysteme untersucht. Insgesamt hat diese Forschungsarbeit das Verständnis für verschiedene PPAP-Derivate erheblich verbessert und ihr Potenzial für antibakterielle und krebsbekämpfende Anwendungen aufgezeigt. Die skizzierten Synthesestrategien, SAR-Studien und Konjugationsansätze bilden eine Grundlage für zukünftige Weiterentwicklungen dieses Forschungsprojektes. Weitere Optimierungen und Kooperationen sind notwendig, um PPAPs einen Schritt näher an die Anwendung als medizinische Behandlungsoption zu bringen.:Table of Contents Acknowledgements III Table of Contents V List of Abbreviations IX Abstract (English) XIV Abstract (German) XVII I Theoretical Part 1 Introduction 1 1.1 Medical need in modern society 1 1.2 Natural products in drug development 1 1.3 Polycyclic polyprenylated acylphloroglucinols (PPAPs) 2 1.3.1 Classifications 2 1.3.2 Biosynthesis 4 1.3.3 Biological activities 6 1.3.4 PPAP-synthesis strategies 7 1.4 Flavonoids in drug discovery 10 1.5 Hybrid drugs in cancer treatment 11 1.5.1 Antibody-drug conjugates 11 1.5.2 Drug-drug conjugates 12 1.5.3 Prostate cancer and prostate-specific membrane antigen (PSMA) 13 2 Objectives 16 2.1 Extending the PPAP library and improving biological activities 16 2.2 Examination and improvement of anticancer efficacy with unexplored PPAP scaffolds 17 2.3 Improving target selectivity for PCa using PSMA-targeting PPAPs 17 3 Expanding the PPAP library 18 3.1 Evaluation of the existing library 18 3.2 Synthesis of the PPAP22 core (PPAP26, 31) 21 3.3 Acylation of the C3 position (head group) 24 3.3.1 Established acylation method using acyl cyanides 24 3.3.2 Cyanide free acylation by PESLALZ 25 3.4 Structure-activity relationship (SAR) study of PPAP78 (50) derivatives (PPAP generation 2) 26 3.4.1 Designing the SAR study 26 3.4.2 Synthesis of PPAPs for the SAR study 28 3.4.3 Evaluation of antibacterial activity 29 3.4.4 Conclusion 32 3.5 Evaluation of anticancer activity 33 3.5.1 Synthesis of the used PPAPs 33 3.5.2 Blood cancer (leukaemia) 36 3.5.3 Brain cancer (glioblastoma) 39 3.5.4 Prostate cancer (PCa) 42 3.6 Exploring synthesis methods towards new PPAP amides 46 3.6.1 PPAP amide synthesis via isocyanates 46 3.6.2 PPAP amides from carbamates 49 3.7 Summary of the library expansion 51 4 Novel flavonoid-like PPAP scaffold 54 4.1 Exploring approaches to flavonoid-like PPAPs 55 4.1.1 Flavanones in one step from PPAP26 (31) 55 4.1.2 Conversion of flavanones to flavones 58 4.1.3 Flavone and aurone synthesis with propiolic acids 59 4.1.4 First biological activity screening 62 4.1.5 Synthesis of substituted propiolic acids 63 4.1.6 Elaborating the substitution scope with dimedone test substrates 65 4.2 Investigating flavone-like PPAPs 66 4.2.1 Synthesis of flavone-like PPAPs 66 4.2.2 Biological activity investigations 68 4.3 Exploring the synthesis of isoflavanone-like PPAPs 71 4.4 Summary of flavonoid-like PPAPs 74 5 Studies towards PPAP hybrid drugs 76 5.1 PPAP drug-drug conjugates 76 5.1.1 PPAP–TMZ conjugates 77 5.1.2 PPAP–DOX conjugates 92 5.1.3 Summary of PPAP drug-drug conjugates 96 5.2 PPAP antibody-drug conjugates 97 5.2.1 Synthesis of a linker with a Val–Cit recognition sequence 99 5.2.2 Synthesis and coupling of simplified linkers 102 5.2.3 Testing of cysteine coupling potential 106 5.2.4 Summary of PPAP antibody-drug conjugates 107 5.3 Using PSMA to increase PCa specificity of PPAPs 107 5.3.1 In silico studies for PSMA-targeting PPAPs 108 5.3.2 Synthesis of PSMA-targeting PPAPs 115 5.3.3 Biological activity of PSMA-targeting PPAPs 217 – 220 118 5.3.4 Studies towards PSMA-targeting PPAPs with cleavable hydrazide linkers 121 5.3.5 Summary of PSMA-targeting PPAPs 127 6 Summary of results 129 II Experimental Part 7 General Remarks 137 7.1 Depiction of structures 137 7.2 Substance names 137 7.3 Solvents and common chemicals 137 7.4 Analytical methods 138 7.5 Biological tests 140 7.6 Docking studies with Schrödinger© 146 8 General synthesis procedures 147 9 Syntheses expanding the PPAP library 150 9.1 Synthesis of PPAP22-core (PPAP26, 31) 150 9.2 PPAP derivatisation at C3 161 9.2.1 Standard acyl derivatisation 161 9.2.2 Standard PPAP amide synthesis 197 9.2.3 Studies towards a new PPAP amide synthesis 201 9.3 PPAP salts 205 10 Synthesis of flavonoid-like PPAPs 209 10.1 Synthesis of substituted propiolic acids 209 10.1.1 Acetylene precursors 209 10.1.2 Substituted propiolic acids 211 10.2 Synthesis of flavonoid-like dimedone test-substrates 216 10.3 Synthesis of flavonoid-like PPAPs 223 10.3.1 Aurone-like PPAPs 223 10.3.2 Flavanoid- and chalcone-like PPAPs 224 10.3.3 Flavone-like PPAPs 227 10.3.4 Dihydrocoumarin-like PPAPs 240 11 Synthesis of PPAP hybrids 242 11.1 PPAP drug-drug conjugates 242 11.1.1 PPAP-Temozolomide conjugates 242 11.1.2 PPAP-Doxorubicin conjugates 267 11.2 PPAP-antibody conjugates 276 11.2.1 Linker synthesis 276 11.2.2 Conjugate synthesis and modification 284 11.3 PSMA-targeting PPAPs 293 11.3.1 Synthesis of linkable PSMA-targeting units (PSMA-TUs) 293 11.3.2 Synthesis of a linkable PPAP amide 300 11.3.3 Linking PSMA-TUs to PPAPs 301 11.3.4 Deprotection of tBu-esters 312 III Appendix 12 X-Ray crystal structures 317 13 Exact structures of docked ligands 341 14 References 345 Curriculum vitae 355 List of publications 356