AN ABSTRACT OF THE THESIS OF Min Wu for the degree of Master ofScience in Pharmacy presented on September 13, 1996. Title:Novel Bioactive Secondary Metabolites from the Marine CyanobacteriumLyngbya majuscula Abstract ved: Redacted for Privacy William H, Gerwick Marine algae have been recognized as arich resource of new and unusual organic molecules withdiverse biological properties. The current need to develop new antifungal,anticancer, antibiotic and antiviral drugs has led to an intenseresearch effort into the discovery, isolation and structure determinationof potential medicinal agents from marine algae. In the past two years, I have participatedin a drug discovery program designed forantitumor, antifungal and other agents of potential pharmaceutical utility from themarine cyanobacterium Lyngbya majuscula. This research utilized modernchromatographic and spectrochemical techniques including2D NMR spectroscopy. Brine shrimp toxicity guided the fractionationthat led to the discovery of the biologically activecompound kalkitoxin from a Curacao Lyngbya majuscula extract.The structure of this new thiazoline ring-containing lipid wasdetermined spectroscopically by interpretation of 2D-NMR experiments,including heteronuclear multiple quantum coherence (HMQC),heteronuclear multiple-bond coherence spectroscopy (HMBC) and'H-'H COSY at room temperature and elevated temperature.Kalkitoxin shows modest molluscicidal toxicity, good brine shrimptoxicity and extremely potent ichthyotoxicity. From the same extract of Lyngbyamajuscula, I also isolated two other secondary metabolites,malyngamide J and malyngamide L. The structures of these new compounds,including stereochemistry, were determined by spectroscopictechniques including 2D-NMR experiments and bycomparison with other known malyngamides. Novel Bioactive Secondary Metabolitesfrom the Marine Cyanobacterium Lyngbya majuscula by Min Wu A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Completed September 13, 1996 Commencement June 1997 Master of Science thesis of Min Wupresented on September13, 1996 APPROVED: Redacted for Privacy Major Professor, representingPharmacy Redacted for Privacy Dean of College of Pharmacy Redacted for Privacy Dean of Grad ate School I understand that my thesiswill become part of the permanent collection of Oregon StateUniversity libraries. My signature below authorizes release of my thesis to anyreader upon request. Redacted for Privacy Min Wu, Author ACKNOWLEDGEMENTS I am extremely grateful to my advisorDr. William H. Gerwick for his patience, guidance, inspirationand support throughout my graduatestudies. I would like to thank my committeemembers, Dr. Kevin Gable, Dr. George H. Constantine and Dr. GeorgeS. Bailey for their valuable advice and assistance. I acknowledge the following peoplefor their technical assistance in my graduate research: BrianArbogast for his providing the high quality mass spectra andhelpful suggestion; Rodger L. Kohnert for his assistance to setNMR experiments. Jeannie Lawrence for her generous help inobtaining CD data. I would like to thank my lab colleaguesfor their help, friendship and encouragement. I especiallythank Namthip Sitachitta, Mary Roberts and Brian Marqezfor critically reading the manuscript. I am deeply grateful to my parentsfor their love, support and confidence to me. TABLE OF CONTENTS Page CHAPTER I: GENERAL INTRODUTION 1 Biomedical Potential Marine Natural Products 2 Marine Toxins 7 Bioactive Natural Products From Marine Cyanobacteria 15 Descriptions of Chapters 17 CHAPTER II: KALKITOXIN FROM THE CYANOBAC 1 ERIUM LYNGBYA MAJUSCULA 1 8 Abstract 1 8 Introduction 1 9 Results and Discussion 2 4 Experimental Methods 4 7 CHAPTER III: TWO NEW MALYNGAMIDES FROM THE CYANOBACTERIUM LYNGBYA MAJUSCULA 5 1 Abstract 5 1 Introduction 5 2 Results and Discussion 5 6 Experimental 7 3 CHAPTER IV CONCLUSION 7 6 Page BIBLIOGRAPHY 7 9 APPENDIX: Spectral Data 8 8 LIST OF FIGURES Figure Page I.1 Structures of Several Marine Natural Productsand Derivatives with Biomedical Utility 3 I.2 Structures of Biomedical Potential MarineNatural Products 4 1.3 Structure of Palytoxin 5 1.4 Structures of Several Marine Toxins 8 1.5 Structure of Maitotoxin 9 I.6 Structures of Brevetoxin-A and Brevetoxin-B 10 I.7 Structures of Bioactive Natural Products from Cyanobacteria 12 1.8 Structures of Bioactive Natural Productsfrom Cyanobacteria 1 3 1.9 Structures of Bioactive Natural Products from Cyanobacteria 1 4 II. 1 Structures of Natural Products from Cyanobacteria 2 0 11.2 Bioactive Metabolites from Cyanobacteria 2 2 11.3 Bioactive Metabolites from Lyngbya majuscula 2 3 11.4 Extraction of Lyngbya majuscula 2 6 11.5 Bioguided Fractionation of Kalkitoxin 2 7 II. 6aBrine Shrimp Assay I for Bioguided Isolationof Kalkitoxin 2 8 LIST OF FIGURES (Continued) Figure Page II.6bBrine Shrimp Assay II for Bioguided Isolationof Kalkitoxin 28 I1.6cBrine Shrimp Assay III for Bioguided Isolationof Kalkitoxin 2 9 II.6dBrine Shrimp Assay IV for Bioguided Isolationof Kalkitoxin 2 9 11.7 The Overall Planar Structure of Kalkitoxin 31 11.8 Six Partial Structures of Kalkitoxin 31 11.9 'H NMR of Kalkitoxin in D6-DMS0 at 298K 3 3 II.10 Two slowly interconverting tert-amideisomers present at room temperature 298K 3 4 II.11'H NMR Of Kalkitoxin In D6 -DMSO at 340K 3 5 11.12"C NMR Of Kalkitoxin In D6 -DMSO at 340K 3 6 11.13"C NMR Of kalkitoxin In D6 -DMSO at 298K 3 7 11.14'H -'H COSY Of kalkitoxin In D6 -DMSO at 298K 3 8 11.15'H -'H COSY Of kalkitoxin In D6 -DMSO at 340K 3 9 11.16 HMQC Spectrum Of Kalkitoxin In D6 -DMSO at298K 4 0 11.17 HMQC Spectrum Of Kalkitoxin In D6-DMSO at 340K 41 11.18Partial Structures of Kalkitoxin Connected by HMBC 4 2 11.19'H and '3C NMR Assignment of Kalkitoxin 4 3 LIST OF FIGURES (Continued) Figure Page 11.20Pure Kalkitoxin in Brine ShrimpToxicity Assay 4 5 11.21Mollucicidal Activity of Kalkitoxin 45 11.22Ichthyotoxic Effects of Kalkitoxin 47 III. 1Secondary Metabolites from DifferentVarieties of L. majuscula 53 111.2 Structures of Various Malyngamidesfrom L. majuscula 55 111.3 The Isolation of Malyngamide J 57 111.4The Isolation of Malyngamide L 59 111.5 'H -1H COSY of Malyngamide J 61 111.6Partial Structures of Malyngamide J by'H -'H COSY and BETCOR 62 111.7The Structure of Malyngamide J(29) with 11-1 and '3C NMR Assignments by HMBC Correlations 64 111.8 'H -'H COSY of Malyngamide L 70 111.9The Structure of Malyngamide L (30)with 'H and 13C NMR Data 71 II1.10 Stereochemistry of DimethoxylatedXylose Residue in Malyngamide J 67 III.1 1 Proposed CD and NOE ofTwo Malyngamide J Configurations 68 111.12 Structures of Malyngamides Jand L with Stereochemistry 7 2 LIST OF FIGURES (Continued) Figure Page, IV.1 The chemical diversity of Lyngbyamajuscula from Playa Kalki, Curacao 78 LIST OF TABLES Table Page II.1 The ratio of N-methyl group resonancesin the 'H NMR spectra of the twoconformers of kalkitoxin in various NMR solvents 34 11.2 'H and "C NMR Data of KalkitoxinIsolated from Lyngbya majuscula 4 4 III. 1 and "C NMR Data ofMalyngamide J (29) Isolated from L. majuscula 6 3 111.2'H and "C NMR Data of Malyngamide L(30) from L. majuscula 6 5 LIST OF APPENDIX FIGURES Figures Page A.2 IR Spectrum of 15 8 9 A.3 HMBC Spectrum of 15 in D6 -DMSO at 298K 9 0 A.4 41 NMR Spectrum of 15 in C6D6 91 A.5 HMBC Spectrum of 15 in D6-DMS0 at 340K 9 2 A.6 135 DEPT Spectrum of 15 in D6 -DMSO at 298K 9 3 A.7 45 DEPT Spectrum of 15 in D6-DMS0 at 298K 9 4 A.8 135 DEPT Spectrum of 15 in D6 -DMSO at 340K 9 5 A.9 135 DEPT Spectrum of 15 in D6 -DMSO at 340K 9 6 A.10'H Decoupling Spectrum of 15 at 8 5.95 in C6D6 9 7 A.11IR Spectrum of 29 9 8 A.12LRFAB Mass Spectrum of 29 9 9 A.13CD Spectrum of 29 100 A.14'3C NMR Spectrum of 29 1 0 1 A.15'H NMR Spectrum of 29 10 2 A.16 HMBC Spectrum of 29 103 A.17HETCOR Spectrum of 29 104 A.18DEPT (135 and 90) Spectrum of 29 105 LIST OF APPENDIX FIGURES(Continued) Figure Page A.19NOE Difference Spectrum-1 of 29 106 A.20NOE Difference Spectrum-2 of 29 107 A.21NOE Difference Spectrum-3 of 29 108 A.22NOE Difference Spectrum-4 of 29 1 09 A.23NOESY of 29 110 A.2413C NMR Spectrum of 30 111 A.251H NMR Spectrum of 30 112 A.26 114-1H COSY of 30 113 A.27HETCOR Spectrum of 30 114 A.28LRFAB Mass Spectrum of 30 115 LIST OF ABBREVIATIONS COSY 'H -'H Chemical Shift Correlation Spectrometry CD Circular Dichroic Spectroscopy DEPT Distortion less Enhancement by PolarizationTransfer DMSO Dimethylsulfoxide EIMS Electron Impact Mass Spectrometry EtOAc Ethyl Acetate FABMS Fast Atom Bombardment Mass Spectrometry FT Fourier Transform FTIR Fourier Transformed Infrared Spectroscopy HETCOR Heteronuclaer Correlation Spectroscopy HMBC Heteronuclear Multiple Bond Correlation Spectroscopy HMQC Heteronuclear Multiple Quantum Coherence HPLC High-Performance Liquid Chromatography HRMS High Resolution Mass Spectrometry IPA Isopropyl Alcohol IR Infrared or Infrared Spectroscopy MS Mass Spectrometry NCI National Cancer Institute NMR Nuclear Magnetic Resonance NOE Nuclear Overhauser Effect TLC Thin Layer Chromatography TMS Tetramethylsilane UV Ultravioler or Ultraviolet Spectrometry MARINE NOVEL BIOACTIVE SECONDARYMETABOLITES FROM THE CYANOBACTERIUM LYNGBYAMAJUSCULA CHAPTER I. GENERALINTRODUCTION Terrestrial plants andanimals represent biologicallyimportant entities and have beenutilized to treat humandiseases since plants antiquity. Studies of the secondarymetabolites of terrestrial and animals were begunin the 1800's.' However,until the last three decades of this century,there have beenincreasingly intensive efforts towardexploring the marineenvironment for useful biomedicinalagents.'Especially over the last decade, advances in diving technologyhave opened up vast areasof unexplored marine environmentsand habitatsto marine natural product scientists.The marine environmentis an exceptional reservoir of bioactivenatural products, many ofwhich exhibit structural features not foundin terrestrial naturalproducts.