Isolation and Structure Elucidation of Bioactive Secondary Metabolites from Marine Sponges (Isolierung und strukturelle Identifizierung von biologisch aktiven Naturstoffen aus marinen Schwämmen) Inaugural - Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Vorgelegt von Wafaa Hassan aus Sharkia, Ägypten Düsseldorf, 2004 Gedruckt mit Genehmigung der Mathematischen-Naturwissenschaftlichen Fakultät der Heinrich-Heine Universität, Düsseldorf Eingereicht am : Referent : Prof. Dr. Peter Proksch Koreferent : Dr. Rainer Ebel Tag der Prüfung : 13. 01. 04 II Erklärung Hiermit erkläre ich ehrenwörtlich, daß ich die vorliegende Dissertation (Isolierung und strukturelle Identifizierung von biologisch aktiven Naturstoffen aus marinen Schwämmen) selbständig angefertigt und keine anderen als die angegebenen Quellen und Hilfsmittel benutzt habe. Ich habe diese Dissertation in gleicher oder ähnlicher Form in keinem anderen Prüfungsverfahren vorgelegt. Düsseldorf, 06.11.2003 Wafaa Hassan III Acknowledgements I wish to express my deep feeling of gratitude, great indebtedness and sincere appreciation to Professor Dr. Peter Proksch for his kind supervision, guidance, directions, comments, valuable support and revisions through the supervision and presentation of the thesis. I am deeply indebted to grateful with deep sincere appreciation to Dr. Rainer Ebel for his continuous help and guidance especially in the interpretation of the NMR data of the isolated compounds. I am deeply indebted and very grateful with deep sincere appreciation to Dr. Ru Angelie Edrada for her encouragement and unlimited help during the course of this work. I would also wish to thank Dr. Wray for the measurement of the NMR spectra and his assistance in the interpretation of my data and also Dr. Peter (Institute für Anorganische und Strukturchemie) for NMR measurement in HHU Duesseldorf. I would like to express my deep thanks to Dr. R. van Soest (Zoological Museum, Amsterdam) for the identification of the sponges. I am greatly thankful to Dr. U. Matthiesen, Dr. Berg, Prof. Dr. Gräfe (HKI für Naturstofforschung, Jena), and Dr. P. Tommes (HHU) for the mass spectral measurements. I am greatly thankful to Dr. Monsier Lozach at CNRS Station Biologique (France) for doing the protein kinase screening assays for the brominated alkaloids. I am greatly thankful to the Government of Egypt for the scholarship. IV Deep thanks for my supervisors in the master thesis, Prof. Dr. Afaf Abdel Ghani, Prof. Dr. Taha Sarg and Prof. Dr. Abdel Monem Ateya for their recommendations and teaching me. I am greatly thankful to my colleagues at the Institute of Pharmaceutical Biology, (Heinrich Heine University) for their help and encouragement. I would like to acknowledge Carsten Thoms, for guiding me during my first months in the research team. I would like to thank Ziyad Baker and Mostafa Abdelgawwad for their help and support during my stay in Germany. Special debt of gratitude and deepest thank to my husband, my children for their help, encouragement and for providing an excellent atmosphere for doing my work. Finally grateful thank to my family and to all who contributed by one way or another for the realisation of the present work. V To my Family VI Table of contents 1. Introduction .......................................................................................................................... 1 1.1. The significance of the study ....................................................................................... 1 1.1.1. The biological importance of marine natural products ........................................... 2 1.1.1.1. Antiviral and antitumour marine natural products ............................................ 2 1.1.1.2. Protein kinase inhibition activity of marine natural products ........................... 3 1.1.1.3. Antimalarial marine natural products................................................................ 4 1.1.1.4. Anthelmintic activity of marine natural products ............................................. 6 1.1.1.5. Reversing multidrug resistance (MDR) activity .............................................. 6 1.1.1.6. Immunosuppressive activity.............................................................................. 6 1.2. The importance of marine natural products in agriculture.......................................6 1.3. The importance of marine metabolites for the source organisms .......................... 9 1.3.1. Chemical defence against fouling and spatial competition................................. 9 1.3.2. Chemical defence against fish............................................................................. 9 1.4. The current status of marine natural products research ......................................... 9 1.5. The aim of the present study ...................................................................................... 11 2. Materials and methods....................................................................................................... 12 2.1. Animal materials ....................................................................................................... 12 2.1.1. Leucetta chagosensis.............................................................................................. 14 2.1.2. Axinyssa aplysinoides............................................................................................ 14 2.1.3. Axinella damicornis............................................................................................... 14 2.1.4. Hamigera hamigera ............................................................................................... 14 2.2 Chemicals used ............................................................................................................. 16 2.2.1. General laboratory chemicals................................................................................. 16 2.2.2. Solvents .................................................................................................................16 2.3. Equipments used ........................................................................................................ 16 2.3.1. HPLC equipment............................................................................................... 17 2.4. Chromatographic methods......................................................................................... 17 2.4.1. Thin layer chromatography ................................................................................... 17 2.4.2. Column chromatography........................................................................................ 18 2.4.3. Semi-preparative HPLC..........................................................................................18 2.4.4. Analytical HPLC................................................................................................... 19 2.4.5. Vacuum liquid chromatography............................................................................. 19 VII 2.4.6. Flash chromatography............................................................................................ 20 2.5. Schemes of isolation....................................................................................................21 2.5.1. Isolation of secondary metabolites from Leucetta chagosensis..............................21 2.5.2. Isolation of secondary metabolites from Axinyssa aplysinoides............................ 22 2.5.3.1. Isolation of secondary metabolites from Axinella damicornis.............................23 2.5.3.2. Isolation of secondary metabolites from Axinella damicornis............................ 24 2.5.4. Isolation of secondary metabolites from Hamigera hamigera............................... 25 2.6. Structure elucidation of the isolated compounds ..................................................... 26 2.6.1. Mass spectrometry (MS) ........................................................................................ 26 2.6.2. Nuclear magnetic resonance spectroscopy (NMR)................................................ 27 2.6.3. The optical activity................................................................................................. 27 2.7. Bioassay........................................................................................................................ 28 2.7.1. Fish-feeding assay.................................................................................................. 28 2.7.2. Brine shrimp assay ................................................................................................. 31 2.7.3. Antimicrobial activity ............................................................................................ 31 2.7.4. Cytotoxicity test......................................................................................................32 2.7.5. Protein kinase screening assays..............................................................................33 3. Results ................................................................................................................................. 34 3.1. Imidazole alkaloids from Leucetta chagosensis ........................................................ 34 3.1.1. Structure elucidation of the isolated compounds ................................................... 37 3.1.1.1. Structure elucidation of naamine A (1, known compound) ........................... 37 3.1.1.3. Structure elucidation of naamine F (2,