Bioactive Compounds in the Chemical Defence of Marine Sponges
Total Page：16
File Type：pdf, Size：1020Kb
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 63 Bioactive Compounds in the Chemical Defence of Marine Sponges Structure-Activity Relationships and Pharmacological Targets ERIK HEDNER ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6192 UPPSALA ISBN 978-91-554-6971-9 2007 urn:nbn:se:uu:diva-8218 Dissertation presented at Uppsala University to be publicly examined in B7:113a, BMC, BMC, Uppsala, Friday, October 19, 2007 at 13:15 for the degree of Doctor of Philosophy (Faculty of Pharmacy). The examination will be conducted in English. Abstract Hedner, E. 2007. Bioactive Compounds in the Chemical Defence of Marine Sponges. Structure-Activity Relationships and Pharmacological Targets. Acta Universitatis Upsaliensis. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 63. 54 pp. Uppsala. ISBN 978-91-554-6971-9. Marine invertebrates, in particular sponges, represent a source of a wide range of secondary metabolites, many of which have been attributed various defensive capabilities against environmental stress factors. In this thesis sponge-derived low-molecular peptide-like compounds and associated analogs are investigated for bioactivity and pharmacological targets. The compound bromobenzisoxazolone barettin (cyclo[(6-bromo-8-(6-bromo-benzioxazol -3(1H)-one)-8-hydroxy)tryptophan)]arginine) was isolated from the sponge Geodia barretti and its ability to inhibit larval settlement of the barnacle Balanus improvisus was determined. With an EC50 value of 15 nM, this compound’s antifouling effect was higher than those of the previously reported brominated dipeptides from Geodia barretti, i.e., barettin and 8,9-dihydrobarettin; moreover, this antifouling effect was demonstrated to be reversible. However, the compound lacked affinity for 5-HT1-7 receptors, whereas barettin possessed specific affinity to 5-HT2A, 5-HT2C and 5-HT4, while 8,9-dihydrobarettin interacted with 5-HT4. In an attempt to evaluate structure-activity relationships synthesized analogs with barettin and dipodazine scaffolds were investigated for antifouling activity. The analog benso[g]dipodazine, with an EC50 value of 34 nM, displayed the highest settlement inhibition. The studies of the structure-activity relationships of sponge-derived compounds were extended to cover analogs of agelasines and agelasimines originally isolated from sponges of the genus Agelas. Synthesized (+)-agelasine D and two structurally close analogs were investigated for cytotoxic and antibacterial activity. The profound cytotoxicity and broad spectrum antibacterial activity found prompted a further investigation of structure-activity relationships in 42 agelasine and agelasimine analogs and several characteristics that increased bioactivity were identified. In conclusion this work has produced new results regarding the potent bioactivity of compounds derived from the sponges Geodia barretti and Agelas spp. and increased SAR knowledge of the fouling inhibition, cytotoxicity and antimicrobial activity of these compounds. Keywords: 5-hydroxytryptamine, Agelas, agelasine, agelasimine, antibacterial, antifouling, barettin, bromobenzisoxazolone barettin, cytotoxic, Geodia barretti, marine, secondary metabolite, sponge Erik Hedner, Department of Medicinal Chemistry, Division of Pharmacognosy, Box 574, Uppsala University, SE-75123 Uppsala, Sweden © Erik Hedner 2007 ISSN 1651-6192 ISBN 978-91-554-6971-9 urn:nbn:se:uu:diva-8218 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8218) Distributor: Uppsala University Library, Box 510, SE-751 20 Uppsala www.uu.se, [email protected] “Haraka haraka haina baraka” There is no benefit in hurrying (Swahili saying) List of Papers This doctoral thesis is based on the following papers, referred to in the text by their Roman numerals (IV): I. Hedner, E., Sjögren, M., Andersson, R., Göransson, U., Hodzic, S., Jonsson, P. R. and Bohlin, L. Antifouling activity of a novel dibro- minated cyclopeptide from the sponge Geodia barretti. In manu- script. II. Sjögren, M., Johnson, A-L., Hedner, E., Dahlström, M., Shirani, H., Göransson, U., Bergman, J., Jonsson, P. R. and Bohlin, L. (2006) Antifouling activity of synthesized peptide analogs of the sponge metabolite barettin. Peptides 27: 20582064. III. Hedner, E., Sjögren, M., Frändberg, P.-A., Johansson, T., Görans- son, U., Dahlström, M., Jonsson, P. R., Nyberg, F. and Bohlin, L. (2006) Brominated cyclodipeptides from the marine sponge Geodia barretti as selective serotonin 5-HT2 ligands. Journal of Natural Products 69: 14211424. IV. Vik, A., Hedner, E., Charnock, C., Samuelsen, Ø., Larsson, R., Gundersen, L.-L. and Bohlin, L. (2006) (+)-Agelasine D: improved synthesis and evaluation of antibacterial and cytotoxic activities. Journal of Natural Products 69: 381386. V. Vik, A., Hedner, E., Charnock, C., Tangen, L., Samuelsen, Ø., Lars- son, R., Bohlin, L. and Gundersen, L.-L. (2007) Antimicrobial and cytotoxic activity of agelasine and agelasimine analogs. Bioorganic and Medicinal Chemistry 15: 40164037. All papers were written by the first author with comments and suggestions given by the co- authors. In I and III EH was responsible for all analyses and the major part of the laboratory work. Settlement inhibition in II was planned by MS. Synthesis and NMR analysis in IV-V were performed by AV. Antimicrobial activity in IV-V was measured by ØS. Cytotoxicity in IV-V was measured by EH. Table of contents 1. Introduction.................................................................................................9 1.1 Natural product chemistry....................................................................9 1.2 Marine natural products......................................................................11 1.3 Marine bioprospecting........................................................................12 1.4 Secondary metabolites and host defence............................................12 1.5 Biofouling...........................................................................................14 1.6 Sponge physiology and biology .........................................................15 1.7 Geodia barretti...................................................................................15 1.8 Agelas species ....................................................................................17 2. Aims of the study......................................................................................19 3. Brominated peptides from Geodia barretti (IIII)....................................20 3.1 Isolation and structure characterization..............................................20 3.2 Barettin and dipodazine analogs.........................................................22 3.3 Larval settlement assay ......................................................................23 3.4 Serotonin receptor affinity..................................................................26 4. Agelasine and agelasimine analogs (IVV)..............................................28 4.1 Analog structures................................................................................28 4.2 Cytotoxicity in the FMCA assay........................................................29 4.3 Antimicrobial activity ........................................................................32 5. Discussion.................................................................................................34 5.1 Antifouling activity of bromobenzisoxazolone barettin (I)................34 5.2 Antifouling and barettin analogs (II)..................................................35 5.3 Geodia barretti metabolites as 5-HT receptor ligands (III) ...............35 5.4 Biological activity of agelasine D analogs (IV) .................................37 5.5 SAR studies of agelasine and agelasimine analogs (V) .....................37 5.6 Chemical defence compounds – potential applications......................39 6. Conclusion and future perspectives ..........................................................40 7. Populärvetenskaplig sammanfattning .......................................................42 8. Acknowledgements...................................................................................44 9. References.................................................................................................47 Abbreviations and conventions 5-HT 5-hydroxytryptamine/serotonin ACHN renal adenocarcinoma cell line AcN acetonitrile Arg arginine Br bromine CEM/s leukemia cell line DKP diketopiperazine DMSO dimethylsulphoxide EC50 effective concentration (50%) ESI-MS electrospray ionization mass spectrometry FDA fluorescein diacetate FMCA fluorometric microculture cytotoxicity assay FSW filtered seawater GPCR G-protein coupled receptor HEK293 human embryonic kidney cells HPLC high performance liquid chromatography IC50 inhibitory concentration (50%) MDR multidrug resistance MIC minimal inhibitory concentration MS mass spectrometry NCE new chemical entity NCI National Cancer Institute NMR nuclear magnetic resonance PBS phosphate buffered saline Pro proline RP reversed phase RPMI 8226/s myeloma cell line SAR structureactivity relationship SI survival index sp./spp. species (singular/plural) TFA trifluoroacetic acid Trp tryptophan U-937 GTB lymphoma cell line 1. Introduction The term “pharmacognosie” was used for the first time, in 1811, in Lehrbuch der Materia Medica by Johann Adam Schmidt (Sandberg and Corrigan 2001). The word is derived from the two Greek words pharmakon and gno- sis, which mean “drug” and “knowledge”,