Isolation and structure elucidation of bioactive secondary metabolites from marine spong [Elektronische Ressource] = Isolierung und strukturelle Identifizierung von biologisch aktiven Naturstoffen aus marinen Schwämmen / vorgelegt von Wafaa Hassan
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Isolation and structure elucidation of bioactive secondary metabolites from marine spong [Elektronische Ressource] = Isolierung und strukturelle Identifizierung von biologisch aktiven Naturstoffen aus marinen Schwämmen / vorgelegt von Wafaa Hassan

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Isolation and Structure Elucidation of BioactiveSecondary Metabolites from Marine Sponges(Isolierung und strukturelle Identifizierung von biologischaktiven Naturstoffen aus marinen Schwämmen)Inaugural - Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultätder Heinrich-Heine-Universität DüsseldorfVorgelegt vonWafaa Hassanaus Sharkia, Ägypten Düsseldorf, 2004Gedruckt mit Genehmigung der Mathematischen-Naturwissenschaftlichen Fakultät der Heinrich-Heine Universität, DüsseldorfEingereicht am :Referent : Prof. Dr. Peter ProkschKoreferent : Dr. Rainer Ebel Tag der Prüfung : 13. 01. 04IIErklärungHiermit erkläre ich ehrenwörtlich, daß ich die vorliegende Dissertation (Isolierungund strukturelle Identifizierung von biologisch aktiven Naturstoffen aus marinenSchwämmen) selbständig angefertigt und keine anderen als die angegebenen Quellenund Hilfsmittel benutzt habe. Ich habe diese Dissertation in gleicher oder ähnlicherForm in keinem anderen Prüfungsverfahren vorgelegt.Düsseldorf, 06.11.2003Wafaa HassanIIIAcknowledgementsI wish to express my deep feeling of gratitude, great indebtedness and sincereappreciation to Professor Dr. Peter Proksch for his kind supervision, guidance,directions, comments, valuable support and revisions through the supervision andpresentation of the thesis.I am deeply indebted to grateful with deep sincere appreciation to Dr.

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Published 01 January 2004
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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, 2004Gedruckt 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
IIErklärung
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
IIIAcknowledgements
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.
IVDeep 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.
VTo my Family
VITable 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 ma ............................................ 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 chroma........................................................................................ 18
2.4.3. Semi-preparative HPLC..........................................................................................18
2.4.4. Analytical HPLC................................................................................................... 19
2.4.5. Vacuum liquid chromatography............................................................................. 19
VII2.4.6. 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 me Axinyssa aplysinoides............................ 22
2.5.3.1. Isolation of secondary me Axinella damicornis.............................23
2.5.3.2. Isolation of secondary metabolites from Axinella damicornis............................ 24
2.5.4. Isolation of secondary me 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, new compound). .............................. 39
3.1.1.2. Structure elucidation of naamine G (3, new compound)...............................46
3.1.1.4. Structure elucidation of kealiinine A (4, new compound) ............................. 53
3.1.1.5. Structure elucidation of kealiinine B (5, new com 59
3.1.1.6. Structure elucidation of kealiinine C (6, new compound) .............................. 65
3.1.2. Biological activities................................................................................................ 66
3.1.2.1. Cytotoxic activity ............................................................................................ 66
3.1.2.2. Anti-microbial activity .................................................................................... 67
3.2.3. Brine shrimp assay ................................................................................................. 67
VIII3.2. Secondary metabolites isolated from the sponge Axinyssa aplysinoides................. 69
3.2.1. Structure elucidation of the isolated compounds ................................................... 72
3.2.1.1. Structure elucidation of (6R,7S)-7-amino-7, 8-dihydro- α-bisabolene (7, known
compound).........................................................................................................71
3.2.1.2. Structure elucidation of (6R,7S)-7-ami α-bisabolene urea
(8, known compound) .................................................................................................. 76
3.2.1.3. Structure elucidation of epipolasin A (9, known compound) ......................... 79
3.2.1.4. Structure elucidation of [E-(-4-Hydroxystyryl ) trimethyl ammonium chloride]
(10, known compound) ............................................................................................... 86
3.2.2. Biological activity of the secondary metabolites from Axinyssa aplysinoides ...... 88
3.2.2.1. Cytotoxic activity ............................................................................................ 88
3.2.2.2. Brine shrimp assay .......................................................................................... 88
3.2.2.3. Antimicrobial activity ..................................................................................... 89
3.3. Secondary metabolites from Axinella damicornis .................................................... 90
3.3.1. Structure elucidation of the isolated compounds ................................................... 93
3.3.1.1. Structure elucidation of 3,4-dibromopyrrole-2-carbamide (11, new
compound).................................................................................................................... 94
3.3.1.2. Structure elucidation of 3,4-dibromopyrrole-2-carboxylic acid (12, new as a
natural product) ............................................................................................................ 98
3.3.1.3. Structure elucidation of 2-bromoaldisin (13, known compound) ............... 101
3.3.1.4. Structure elucidation of 3-bromoaldisin (14, new natural product) ........... 104
3.3.1.5. Structure elucidation of 2,3 dibromoaldisin (15, new compound).............. 109
3.3.1.6. Structure elucidation of oroidin (16, known compound) ............................. 113
3.3.1.7. Structure elucidation of hymenidin (17, known compound)......................... 118
3.3.1.8. Structure elucidation of dibromocantharelline (18, known compound) ...... 119
3.3.1.9. Structure elucidation of Z-hymenialdisine (19, known compound) ............. 124
3.3.1.10. Structure elucidation of hymenin (20, known compound).......................... 125
3.3.1.11. Structure elucidation of stevensin (21, known com....................... 126
3.3.1.12. Structure elucidation of 4, 5 dibromopyrrole-2-carbamide (22, known
compound).................................................................................................................. 129
3.3.1.13. Structure elucidation of 4,5-dibromopyrrole-2-carboxylic acid (23, known
compound) 131
3.3.1.14. Structure elucidation of 4-bromopyrrole-2-carbamide (24, known
compound) 132
3.3.1.15. Structure elucidation of 4,5-dibromopyrrole-2-methylcarboxylate ........... 133
(25, known compound) ............................................................................................. 133
3.3.1.16. Structure elucidation of longamide and manzacidin A (26 and 27, known
compounds) ................................................................................................................ 139
IX3.3.2. The results of biological assays............................................................................ 141
3.3.2.1. Cytotoxic activity .......................................................................................... 141
3.3.2.2: Anti-microbial assay ..................................................................................... 142
3.3.2.3. Brine shrimp assay ........................................................................................ 142
3.3.2.4. Fish-Feeding Assay with Blennius sphinx .................................................... 144
3.3.2.5. Protein kinase inhibition test......................................................................... 145
3.4. Metabolites isolated from the sponge Hamigera hamigera.................................... 146
3.4.1. Structure elucidation of the isolated compounds ................................................. 148
3.4.1.1. Structure elucidation of hamigeroxalamic acid (28, new compound) ......... 148
3.4.1.2. Structure elucidation of hamigeramine (29, new compound) ...................... 152
3.4.1.3. Structure elucidation of hamiguanasinol (30, new com.................... 157
3.4.1.4. Structure elucidation of 22-dehydrocampesterol (31, known compound).. 162
3.4.1.5. Structure elucidation of 27-nor-22-dehydrocampesterol (32, known
compound).................................................................................................................. 164
3.4.1.6. Structure elucidation of 24-methylcholesta-5,22-dien-3 β-ol (33, known
compound) 165
3.4.1.7. Structure elucidation of indole-3-carboxyaldehyde (34, known compound)174
3.4.1.8. Structure elucidation of hamigeramide (35, new compound) .................. 176
3.4.2. Biological activities.............................................................................................. 181
3.4.2.1. Cytotoxic activity .......................................................................................... 181
3.4.2.2. Brine shrimp assay ........................................................................................ 182
3.4.2.3. Antimicrobial assay....................................................................................... 182
4. Discussion.......................................................................................................................... 182
4.1. Imidazole alkaloids isolated from Leucetta chagosensis ........................................ 182
4.2. Biologically active metabolites from the sponge Axinyssa aplysinoides................ 183
4.3. Biologically active metaAxinella damicornis .................. 186
4.4. Different sets of metabolites isolated from the sponge Hamigera hamigera ...... 190
5. Summary........................................................................................................................... 193
6- References......................................................................................................................... 195
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