Novel Secondary Metabolites from Selected British Columbian
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NOVEL SECONDARY METABOLITES FROM SELECTED BRITISH COLUMBIAN MARINE INVERTEBRATES by STEPHEN WILLIAM AYER A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Chemistry We accept this thesis as conforming d standard THE UNIVERSITY OF BRITISH COLUMBIA March 1985 © Stephen William Ayer, 1985 In presenting this thesis in partial fulfilment of the re• quirements for an advanced degree at the The University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his or her representatives. It is under• stood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Chemistry The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date: March 1985 Abstract Marine organisms show potential as sources for novel, biologically and pharmacologically active, secondary metabolites. Examination of three nudibranch and one bryozoan species for biologically active metabolites has led to the isolation and structural elucidation of nine new and two known secondary metabolites. The structures of all the compounds were determined by using a combination of spectral analysis, chemical interconversion, synthesis, and single-crystal X-ray diffraction analysis. The British Columbian dorid nudibranch Acant hodori s nanaimoensi s yielded three new sesquiterpenoids. The struc• tures of nanaimoal (6_1) , acanthodoral (64) , and isoacanthodoral (6J5) represent novel sesquiterpenoid carbon skeletons. The natural mixture of aldehydes 61, 64, and 65 exhibited antibacterial and antifungal activity. From Aldisa cooperi, two A*-3-ketosteroidal acids 23 and 24, and glycerol ether 25 were isolated. Acid 23 showed feeding deterrent activity against fish. The dendronotid nudibranch Meli be leonina gave 2,6-dimethy1-5-heptenal 53 and 2,6-dimethyl-5-heptenoic acid 5_4_. The aldehyde 5_3 was responsible for the "grapefruit like" odour of the nudibranch. The bryozoan Phidolopora pacifica was examined in an attempt to correlate the absence of surface fouling, in the field, with the presence of biologically active secondary metabolites. The purine alkaloids 179 and 180, which i i contain the rare naturally occurring nitro functionality, were responsible for much of the antifungal and antialgal activity of the crude extracts. Three nitrophenols 181, 189, and 209 were also isolated from P. pacifica. Nitrophenol 181 had been previously shown to inhibit chloroplast development both in green plants and in the unicellular algae Eugl ena sp. Table of Contents Abstract ii List of Figures vi List of Schemes vii List of Tables viii List of Appendices ix Acknowledgements x Dedication xi Abbreviations xii I . Introduction 1 A. Overview 1 B. Natural Products Chemistry 3 C. Primary and Secondary Metabolites 8 D. Chemical Ecology 11 II. Nudibranchs 16 A. Introduction 16 1. Gastropod Secondary Metabolites 16 2. Nudibranch Defense Mechanisms 21 B. Secondary Metabolites from the Dorid Nudibranch Aldisa cooperi (Robilliard and Baba, 1972) 25 1 1. Introduction 25 2. Isolation and Structure Elucidation 25 3. Biological Activities of Aldisa cooperi Metabolites 42 4. Discussion 43 C. Secondary Metabolites from the Dendronotid Nudibranch Meli be leonina (Gould, 1852) 47 1. Introduction 47 2. Isolation and Structure Elucidation 47 iv 3. Discussion 53 D. Secondary Metabolites from Acant hodoris nanaimoensis (O'Donoghue, 1921) 56 1 . Introduction 56 2. Nanaimoal 59 3. Synthesis of Nanaimoal's (p-Bromophenyl)urethane Derivative. Assignment of Structure 70 1 4. Assignment of the H NMR Spectrum of Nanaimoal using One and Two-Dimensional NMR Techniques 81 5. Isoacanthodoral 99 6. Acanthodoral 112 7. Biological Activities of A. nanaimoensis Secondary Metabolites 117 8. Discussion 117 III. Bryozoans 132 A. Introduction to the Bryozoans 132 B. Secondary Metabolites from Phidolopora pacifica (Robertson 1908) 149 1 . Discussion 163 IV. Experimental 171 V. Appendices 200 VI. Bibliography 204 v List of Figures 1. Phylogenetic classification of nudibranchs 17 2. Typical cryptobranch dorid nudibranch 20 3. Aldisa cooperi 26 4. Secondary metabolites from the dorid nudibranch Aldisa cooper i 27 5. "Un-natural" 20S marine steroids 37 6. Mel i be leonina 48 7. Secondary metabolites from the dendronotid nudibranch Meli be Ieoni na 49 8. Acanthodori s nanaimoensis 57 9. Secondary metabolites from Acanthodoris nanaimoensis 60 10. GC analysis of a crude chloroform extract of A. nanaimoensis 61 1 11. 400 MHz H NMR spectrum of nanaimoal (CDC1 ) ... 63 (61) 3 1 12. Model systems for the H NMR chemical shifts of the gem-dimethyl group in nanaimoal (6_1) 70 13. Nanaimoane carbon skeleton showing the numbering scheme 80 14. Pulse sequence for the homonuclear COSY NMR experiment 89 1 15. 400 MHz H NMR COSY/45 spectrum of nanaimoal's (p-bromophenyl)urethane derivative 91 16. Expansion and amplification of Figure 15 to show homoallylic couplings 92 17. Pulse sequence for 2D /-resolved NMR experiment .... 93 18. Partial 400 MHz *H NMR 2D /-resolved spectrum (symmetrized) of nanaimoal (61) 97 19. Slices of individual peaks shown at the top of Figure 18 to show multiplicities 98 20. Computer generated X-ray structure of isoacanthodoral's 2,4-dinitrophenylhydrazone derivative 96 110 vi 21. Computer generated X-ray structure of acanthodoral's (p-bromophenyl )urethane derivative 114 116 22. Phidolopora pacifica 150 23. A computer-generated perspective drawing of the final X-ray model of p-bromophenacylphidolopin 194 160 vi i List of Schemes 1. Interpretation of the HRMS of 3-oxo-4-cholenoic acid (23) 30 2. Interpretation of the HRMS of 3-oxo-4,22-choladienic acid (24) 33 3. Interpretation of the MS of diacetyl derivative 44 .. 41 4. Proposed mechanism for the microbial transformation of cholesterol into 17-ketosteroids 44 5. Interpretation of the mass spectral fragmentation of 2,6-dimethyl-5-heptenal (53) 52 6. Interpretation of the MS of nanaimoal (61) 62 7. Biogenetic arguments used in support of structure 61 for nanaimoal 67 8. Retrosynthetic analysis of the postulated structure for nanaimoal 72 9. Previous synthesis of the nanaimoane carbon skeleton 73 10. Interpretation of the MS of urethane 79 11. Interpretation of the MS of isoacanthodoral (65) .. 101 12. Biogenetic arguments leading to the consideration of 100 as the structure for isoacanthodoral 103 13. Acid catalyzed rearrangement of 98 107 14. Proposed biogenesis of isoacanthodoral (65) from acanthodoral (64) .". 109 15. Interpretation of the MS fragmentation of nitrophenol 189 154 16. Interpretation of the MS of desmethylphidolopin .... viii List of Tables 1. Comparison of the 'H NMR data for various A"-3-ketosteroids (CDC1 ) 29 3 13 2. C NMR data and spectral comparisons for the assignment of stereochemistry to 3-oxo-4-cholenoic acid (23) 36 3. 'H NMR data for various 20R and 20S steroids (CDC1 ) . 39 3 13 4. C NMR data for 2,6-dimethyl-5-heptenal (53) and citronellal (55) 51 1 5. H NMR data (400 MHz) for nanaimoal (61) and derivatives 83 6. Nudibranch sesquiterpenoids 119 7. Bryozoan metabolites 134 8. 'H NMR data (CDC1 , 80 MHz) and spectral comparisons for 3 nitrophenols isolated from Phidolopora paci fi ca 153 9. 'H NMR data and spectral comparisons for purine derivatives isolated from Phidolopora pacifica 158 ix List of Appendices 1. 400 MHz NMR spectrum of 75 201 2. 400 MHz NMR spectrum of 98 202 3. 400 MHz NMR spectrum of 114 203 x Acknowledgements I would like to gratefully acknowledge the guidance, encouragement, patience, and friendship of Dr. R.J. Andersen. Many people have influenced the outcome of this work and a number of individuals stand out. My wife Roxanne pro• vided unrelenting love, support, and encouragement for which I am deeply indebted. Mike LeBlanc competently introduced me to SCUBA diving, assisted with all the invertebrate collections, and performed the bioassays. Dr. 0. Chan and Ms. Marietta T. Austria provided friendly and helpful assistance with the 2D-NMR studies. David Behrens allowed me to reproduce his drawing of a typical cryptobranch dorid nudibranch and Ron Long kindly provided photographs of all the invertebrates studied. I thank Sandra Millen for helpful discussions on nudibranch biology. A number of people assisted with the collection of the marine organisms, and the spectral data of the compounds isolated from them. I thank the staff of the Bamfield Marine Station and the departmental NMR and MS laboratories for courteous and reliable assistance. xi To Jean, Roxanne, Genevieve, Dorothy, Bill, and Allen Abbreviat ions AQN = Acquisition DMSO = Dimethylsulfoxide EtOAc = Ethyl acetate g = Grease peak GC = Gas chromatography GC-MS = gas chromatography - mass spectrometry HPLC = High performance liquid chromatography HRMS = High resolution mass spectrum IR = Infrared MS = Low resolution mass spectrum 'H NMR = Proton nuclear magnetic resonance 13 C NMR = Carbon-13 nuclear magnetic resonance nOe = Nuclear Overhauser enhancement mp = Melting point RD = Relaxation delay RT = Room temperature S = Solvent signal SFORD = Single frequency off resonance decoupled SCUBA = Self-contained underwater breathing apparatus TLC = Thin layer chromatography U = Unknown impurity signal UV = Ultraviolet W = Water signal xi i i I. INTRODUCTION A. OVERVIEW The purpose of the research