DISSERTATION Titel der Dissertation Tropical Chemodiversity in Selected Angiosperm Families Verfasser Mag. rer. nat. Johann Schinnerl angestrebter akademischer Grad Doktor der Naturwissenschaften (Dr.rer.nat.) Wien, 2013 Studienkennzahl lt. Studienblatt: A 091 438 Dissertationsgebiet lt. Studienblatt: Botanik Betreuerin: Ao. Univ. Prof. Dr. Karin Valant-Vetschera Table of contents Abstract 1 Zusammenfassung 2 Co-authorship statement 3 Aims and outline 4 General Introduction: Plant secondary metabolites (SM) 8 Chapter 1: studied Plants and their SM 10 1.1. Stemonaceae 10 1.1.1 Stemona 10 1.1.2. Stichoneuron 14 1.2. Stemona alkaloids and their presumed biosynthesis 17 1.2.1. Proposed biosynthesis of the pyrrolo[1,2-α]azepine core structure 17 1.2.2. Formation of Pyrido[1,2-α]azepines 19 1.3. Rubiaceae 23 1.3.1.Ronabea emetica 23 1.3.2. Palicourea acuminata 24 1.3.3. Chassalia curviflora 24 1.3.4. Rudgea cornifolia 25 1.4. Tryptamine-Iridoid Alkaloids and Iridoids 25 Chapter 2: Pandanus Alkaloids in Stemonaceae: Finding of a Plausible Biogenetic Origin of Stemona Alkaloids 39 Chapter 3: Structural Relationships of Stemona Alkaloids: Assessment of Species-Specific Accumulation Trends for Exploiting Their Biological Activities 44 Chapter 4: Iridoids as chemical markers of false ipecac (Ronabea emetica), a previously confused medicinal plant 53 Chapter 5: Various types of tryptamine-iridoid alkaloids from Palicourea acuminata (= Psychotria acuminata) 60 Chapter 6: Alstrostines in Rubiaceae: Alstrostine A from Chassalia curviflora var. ophioxyloides and a novel derivative, Rudgeifoline from Rudgea cornifolia 66 Chapter 7: Ecological relevance of Stemona- and Tryptamine-Iridoid-Alkaloids 72 7.1. Stemona Alkaloids 72 7.2. Tryptamine-Iridoid Alkaloids and Iridoids 74 Conclusion and Outlook 83 Curriculum Vitae 86 List of figures Fig. 1. Leaves and flowers from Stemona tuberosa 12 Fig. 2. Leaves and flowers from Stemona phyllantha 13 Fig. 3. Habitus of Stichoneuron caudatum 15 Fig. 4. Habitus of Stichoneuron calcicola 16 Fig. 5. Pyrrolo[1,2-α]azepine and pyrido[1,2-α]azepine core structures 17 Fig. 6. Formation of the Iminium ion 18 Fig. 7. Formation of pyrido[1,2-α]azepines 19 Fig. 8. Biosynthesis connections between pyrrolo[1,2-α]azepine and pyrido[1,2-α]azepines 20 Fig. 9. Alternative biosynthesis leading to the pyrido[1,2-α]azepine core 21 Fig. 10. Formation of Pandanus alkaloids from Pandanamine 22 Fig. 11. Biosynthesis of Strictosidine 26 Fig. 12. Various Tryptamine-iridoid alkaloids derived from Strictosidine 26 Fig. 13. Chemical structures of common Iridoids in Rubiaceae 27 Fig. 14. Formation of Iridoids from IPP and DMAPP 28 Fig. 15. Chemical structures of Stemofoline-, Oxystemokerrine- and Tuberostemonine-derivatives 73 Fig. 16. Chemical structures of Stemona Alkaloid-N-oxides 74 Fig. 17. Chemical structures of Tryptamine-Iridoid Alkaloids 76 Fig. 18. Chemical structures of bioactive Iridoids 77 Acknowledgements I express my honest gratitude to the following people: My supervisor Prof. Dr. Karin Valant-Vetschera for her encouragement and support, and the valuable discussions during all stages of this thesis. Prof. Dr. Harald Greger for his teaching and priceless discussions throughout the last years. Prof. Dr. Lothar Brecker from the Department of organic Chemistry for the precious collaboration. Susanne Felsinger for performing the NMR measurements of the sometimes tricky samples. Tina Hehenberger, Silvia Pointinger, Adriane Raninger, Christian Gilli, Andrea Zraunig, Marina Bachratá, Stefan Mikulicic, Markus Hofbauer, David Lyon, Tshering Doma Bhutia, Grace Djoufack, Sonja Leißer, Birgit Gschweidl, Andreas Berger, Celine Zahradnik, Sigrid Steins, Rupert Kainradl, Florian Ehrlich and all the other not mentioned Diploma and Bachelor students for the good atmosphere in the Lab. Srunya Vajrodaya, Netnapis Khewkhom and Sumet Kongkiatpaiboon for a good collaboration, and all the nice places in Thailand which I could visit with their help. Pajaree Inthachub for the great pictures and drawings. Carolin Rebernig, Khatere Emadzade, Silvia Ulrich, Gudrun Kohl and Michi Sonnleitner for the good moments, we have shared. Elisabeth Grabner and Heidi Hochwallner for reading the manuscript. And at the end my family for always being a great help and support. Abstract Abstract The underlying hypothesis of this work is the expected correlation between phytochemical diversification, as expressed by the accumulation of secondary metabolites (SM), and systematic relationships in plant groups. Characteristic SM profiles and specific compounds may prove to be good chemical characters (=chemical markers) within plant families or orders. To verify this hypothesis, comparative phytochemical studies on alkaloids within different tropical plant families were carried out. We selected two families, the monocotyledonous Stemonaceae and some genera of the dicotyledonous Rubiaceae. The major accumulation tendencies of alkaloids found in Stemonaceae comprise structural types showing a pyrrolo- or pyrido[1,2-α]azepine core structure, which can be grouped into the three main derivatives, the stichoneurines, the protostemonines and the croomines. Most of the investigated Stemona species accumulate protostemonine-type alkaloids. However, Stemona tuberosa and S. phyllantha can clearly be distinguished from the other studied Stemona species by the accumulation of stichoneurine-type alkaloids. This structural type of alkaloids occurs only in these both species. Croomine derivatives occur scattered in analyzed Stemona species. Stichoneuron caudatum and S. halabalensis accumulate stichoneurine-type alkaloids. By contrast, the recently described Stichoneuron calcicola accumulates the Pandanus alkaloid Pandanamine as the major alkaloid. As for Rubiaceae, we focused on the presence of iridoids and tryptamine-iridoid alkaloids (= monoterpene-indole alkaloids) which proved to show characteristic major accumulation tendencies in the studied genera and species. Ronabea emetica and R. latifolia accumulate iridoids, whilst tryptamine-iridoid alkaloid derivatives are representative for Palicourea acuminiata. A variation of this type of alkaloids could also be detected in the leaves of Chassalia curviflora var. ophioxyloides and also in the leaves of Rudgea cornifolia. The isolated and structurally elucidated alkaloids named Alstrostine A and Rudgeifoline have two Secologanin glycoside moieties, in contrast to the Lagamboside, isolated from P. acuminata, which have one glycoside moiety directly linked to the nitrogen of the indole- ring. Our results confirm that most of the isolated SM can be regarded as good chemical characters, supporting generic and specific delimitation in the studied groups. 1 Zusammenfassung Zusammenfassung Die dieser Arbeit zugrundeliegende Hypothese ist der erwartete Zusammenhang zwischen phytochemischer Stoffausstattung und taxonomischer Verwandtschaft auf Familien- oder Gattungsebene. Das phytochemische Profil von einer Klasse von Sekundärmetaboliten umfasst sehr oft nur einige strukturell sehr ähnliche Derivate und ist zumeist charakteristisch für ein Taxon. Um diese Hypothese zu verifizieren wurden die Alkaloidprofile von Arten aus der paläotropisch verbreiteten Familie der Stemonaceae aus der Gruppe der Monokotyledonen sowie einige neotropische Arten der Rubiaceae aus der Gruppe der Dikotyledonen untersucht. Die Pflanzenfamilie der Stemonaceae ist durch das Auftreten von speziellen Alkaloiden, den Stemona Alkaloiden, gekennzeichnet. Diese Stoffklasse umfasst Substanzen mit entweder einer Pyrrolo[1,2-α]- oder einer Pyrido[1,2-α]-azepin Struktur. Innerhalb der Gattung Stemona zeigen diese Alkaloide ein auffallendes Verbreitungsmuster, das sich auch in der Phylogenie widerspiegelt. Innerhalb der Gattung Stichoneuron stellt die Art Stichoneuron calcicola durch das Anreichern von Pandanamin einen Sonderfall dar, da die beiden anderen bisher untersuchten Arten Stichoneuron caudatum und S. halabalensis Alkaloide vom Stichoneurin-Typ akkumulieren. Das Pandanamin ist aus Pandanus amaryllifolius bekannt und das Auftreten in den Stemonaceae weist auf enge verwandtschaftliche Beziehungen zu der Familie der Pandanaceae hin. Die Untersuchungen an verschiedenen neotropischen sowie paläotropischen Arten aus der Psychotria Verwandtschaft (Rubiaceae) zeigten einerseits die Tendenz Tryptamin- Iridoid Alkaloide, auch bekannt als Monterpen-Indol Alkaloide (MIA), und andrerseits Iridoide anzureichern. Die untersuchten Arten aus der Gattung Ronabea, R. emetica und R. latifolia, akkumulieren Iridoide als typische Sekundärstoffe. Im Gegensatz dazu weisen die anderen untersuchten Arten aus dieser Pflanzenfamilie Tryptamin-Iridoid Alkaloide als typische Sekundärstoffe auf. Innerhalb dieser Stoffklasse tritt jedoch eine Differenzierung im Verhältnis Tryptamin- zu Iridoidanteil von 1:1 bis 1:2 auf. Die erhaltenen Resultate zeigen, dass einige der isolierten Sekundärstoffe als chemische Marker geeignet sind und die Resultate von phylogenetischen Studien weitgehend unterstützen. 2 Co-authorship statement Co-authorship statement Chapter 2 (Paper 1): My contribution to the publication Pandanus Alkaloids in Stemonaceae: Finding of a Plausible Biogenetic Origin of Stemona Alkaloids, published 2009 in Journal of Natural Products, was mainly the experimental work. Chapter 3 (Paper 2): My contribution to the publication Structural Relationships of Stemona Alkaloids: Assessment of Species-Specific Accumulation Trends for Exploiting Their Biological Activities, published 2011 in Journal