Biological characterisation of natural products and derivatives in special consideration of high-content analysis methods

Viele Naturstoffe sind biologisch aktiv und finden in der Medizin und/oder als Hilfsmittel in der Forschung Verwendung. Dafür ist es essentiell, sie biologisch zu charakterisieren. In dieser Arbeit wurden verschiedene Naturstoffe und Naturstoffderivate auf ihre biologischen Wirkungen hin untersucht. Dazu gehörten unterschiedliche Tetramsäuren, Iridoide aus Pflanzen und von Myxobakterien produzierte Substanzen, die zunächst bezüglich ihrer wachstumshemmenden Wirkung an mehreren Säugerzelllinien und Mikroorganismen untersucht wurden. In einigen Fällen wurden weitere Untersuchungen wie Fluoreszenzfärbungen, Tests zur Actin-Polymerisation oder Zellzyklusanalysen durchgeführt. Als neuartige Methode zur Evaluierung von biologischen Wirkmechanismen konnte in der vorliegenden Arbeit erfolgreich eine High-Content Analyse (HCA) etabliert werden. Bei dieser HCA wurden phänotypische Veränderungen von Zellen, die durch die biologisch aktiven Substanzen hervorgerufen werden, über automatisierte Mikroskopie und softwaregestützte Bildanalyse quantifiziert. Mithilfe verschiedener statistischer Methoden konnten die phänotypischen Profile neuer Verbindungen mit den Profilen von Substanzen mit bekannter Wirkung verglichen werden, um so Hinweise auf den Wirkmechanismus der neuen Verbindung zu gewinnen. Zu den für die HCA optimierten Parametern gehörten die Auswahl und Konzentrationsbestimmungen der Referenzsubstanzen sowie geeigneter Antikörper/Farbstoffe und die Optimierungen praktischer Abläufe. Es wurden verschiedene statistische Analysemethoden evaluiert und die Funktionalität der HCA an zahlreichen Beispielen gezeigt. Mit der HCA steht ein neues Werkzeug bereit, um in Zukunft einzelne Verbindungen oder auch Substanzbanken charakterisieren zu können.Numerous natural products are biologically active and thus are used as drugs and/or as tools in research. Therefore deeper knowledge about their mode of action is essential. Diverse natural products or derivatives thereof have been biologically characterised for this thesis. These substances comprise various tetramic acids, iridoids from plants, and myxobacterial compounds. First, their anti-proliferative activity has been determined, using several mammalian cell lines as well as microorganisms. In some cases further examinations have been performed, including immunofluorescence, determination of actin polymerisation, and cell cycle analyses. High-content analysis (HCA) is a new strategy that could be utilised to gain further insight into the mode of action of small molecules. Such a method was successfully established as presented in this thesis. The HCA is based on phenotypic changes in cells that are induced by the biologically active compounds. These changes are measured and quantified using automated microscopy and appropriate image-analysis tools. By means of different statistical methods, compounds with an already known mode of action can be compared to the new entities, revealing information about the mode of action of the latter. In order to establish the HCA several parameters had to be ascertained. A set of reference compounds and appropriate antibodies/probes have been chosen and their concentrations have been determined. The optimisation also included numerous experimental steps. Different statistical methods have been compared and the efficacy/functionality of the HCA was demonstrated. The HCA is now available as a useful tool to characterise single substances or compound libraries

Biological Activity of Volatiles from Marine and Terrestrial Bacteria

The antiproliferative activity of 52 volatile compounds released from bacteria was investigated in agar diffusion assays against medically important microorganisms and mouse fibroblasts. Furthermore, the activity of these compounds to interfere with the quorum-sensing-systems was tested with two different reporter strains. While some of the compounds specific to certain bacteria showed some activity in the antiproliferative assay, the compounds common to many bacteria were mostly inactive. In contrast, some of these compounds were active in the quorum-sensing-tests. γ-Lactones showed a broad reactivity, while pyrazines seem to have only low intrinsic activity. A general discussion on the ecological importance of these findings is given

A selective 3-acylation of tetramic acids and the first synthesis of ravenic acid.

3-Acyltetramic acids, including delicate 3-oligoenoyl derivatives, such as the Penicillium metabolite ravenic acid, were prepared in two high-yielding steps. Reaction of tetramic acids with the ylide Ph(3)PCCO afforded exclusively the corresponding 3-acylylidenetetramic acids. These were amenable to Wittig olefinations with aliphatic, aromatic, saturated and unsaturated aldehydes after deprotonation with KOtBu. Due to its simplicity, selectivity and tolerance of pH-sensitive groups this method is superior to the established acylation protocols by Jones and Yoshii. It is also applicable to the synthesis of 3-acyltetronic acids. The new 3-oligoenoyl tetramic acids exhibited structure-dependent antimicrobial and cytotoxic activity

First Syntheses of Melophlins P, Q, and R, and Effects of Melophlins on the Growth of Microorganisms and Tumor Cells

The marine tetramic acid (=1,5-dihydro-4-hydroxy-2H-pyrrol-2-ones) derivatives melophlin P, Q, and R (1p-1r, resp.) were synthesized for the first time in only four steps. Together with the congenerous melophlins A-C and G, they were also tested for antimicrobial and cytotoxic effects. Melophlins B, C, P, Q, and R, which share a 5-Me residue, showed some antibacterial activity, mainly in Gram-positive bacteria. Melophlins B, C, and R, which have Me-branched 3-acyl side chains in common, inhibited the growth of cells of human KB-3-1 cervix carcinoma, A-498 kidney carcinoma, and U-937 leukemia with IC(50) values <10 muM. They were similar in activity to cisplatin. Melophlin Q, also Me-branched, was astoundingly specific in inhibiting A-498 kidney cancer cells, while melophlin P inhibited U-937 leukemia cells particularly well. The position of the Me branch is decisive for the magnitude of the antiproliferative effect of the melophlin couples B/C and R/Q

Novel peptidomimetic compounds containing redox active chalcogens and quinones as potential anticancer agents.

Many types of cancer cells are associated with a disturbed intracellular redox balance and oxidative stress (OS). Among the various agents employed to modulate the intracellular redox state of cells, certain redox catalysts containing quinone and chalcogen moieties have shown considerable promise. Passerini multicomponent reaction has been developed for the synthesis of agents combining two, three or even four redox centers in one molecule in a good yield. When incubated with cancer cells these agents inhibited cell proliferation and induced apoptotic cell death. Interestingly, some of these redox active compounds exhibited quite low toxicity with normal cells. The cause was obviously OS, which was reflected by significant decrease in reduced glutathione, subsequently cell cycle arrest and induction of apoptosis

Synthesis and biological properties of cylindramide derivatives: evidence for calcium-dependent cytotoxicity of tetramic acid lactams

To gain insight into the biol. properties of tetramic acid lactam cylindramide 1, the analogs 4a-d bearing a cyclopentane ring instead of the pentalene unit were prepd. by tandem conjugate addn./enolate trapping of cyclopentenone 10; a Sonogashira or Stille coupling, followed by a Julia-Kocienski olefination, macrolactamisation and Lacey-Dieckmann cyclisation were the key steps. The previous NMR structure of cylindramide 1, which was based on NOE and J coupling restraints, could be refined by including residual dipolar coupling data measured for a sample of cylindramide that was aligned in polyacrylonitrile (18%). Biol. screening of cylindramide 1 and its analogs 2-epi-1, 20 and 4 revealed promising antiproliferative activity against several tumor cell lines. It turned out that the activity is strongly correlated to the functionalised pentalene system. The configuration of the cyclopentane ring and an intact tetramic acid lactam with the correct configuration seem to play an equal role in the cytotoxicity. The antiproliferative activity was found to be calcium dependent. Phenotypic characterization of the mode of action showed vacuolisation and vesicle formation in the endoplasmic reticulum