The Natural Products Chemistry of the Red Algae Ptilota Filicina and Laurencia Obtusa
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AN ABSTRACT OF THE THESIS OF Albert Lopez for the degree of Master of Science in Pharmacy presented onMay 14, 1987. Title: The Natural Products Chemistry of the Red Algae Ptilota filicina and Laurencia obtusa Abstract approved: Redacted for Privacy Dr. William H. Gerwick The sea offers a wide variety of potentially useful resources. Marine algae have been a rich source of structurally unique natural products showing diverse biological properties. The current need to develop new antifungal, antibiotic, anticancer and antiviral drugs has prompted intense research efforts into the discovery, isolation and structure determination of these potential medicinal agents from seaweeds. Over the past two years a survey of the biomedicinal potential of seaweeds from the Oregon Coast has beenconducted. In this period we have made 125 survey collections representingsome 85 different species of algae. On the basis of antimicrobial bioassays and preliminary chemical characterization, 16 seaweeds have been identified for further study. This research has investigated, using standard chromatographic and spectrochemical techniques including 2D NMR spectroscopy, the natural products of the previously unstudied red alga Ptilota filicina J. Agardh (order: Ceramiales). P. filicina, which grows abundantly on rocks in the mid- intertidal zones along the central Oregon coast, yielded a unique series of w-3 fatty acids. Two of them were found to be stereoisomeric icosapentaenoic acids containing conjugated triene functionalities, while a third, ptilodene, was characterized as another novel 20-carbon fatty acid with oxidation sites at C-11 and C-16. Ptilodene showed slight antimicrobial activity to pathogenic gram-negative and gram-positive bacteria, and moderate inhibition to the dog kidney Na+/K+ ATPase. From a previous survey of marine algae from the Caribbean, the red seaweed Laurencia obtusa (Hudson) Lamouroux (order: Ceramiales) was identified for further investigation. L. obtusa is a recognized source of many novel natural products and occurs worldwide in tropical and subtropical marine habitats. Of the several collections made, the Isleta Marina collection (northeastern coast of Puerto Rico) was shown to possess two novel isomeric brominated sesquiterpenes, in addition to the previously known sesquiterpenes, elatol and obtusol. A minor component of this research was devoted to the isolation of hormothamnione, the first naturally occuring styrylchromone, from the tropical marine cyanophyte Hormothamnion enteromorphoides Grunow. Hormothamnione was found to be a potent cytotoxin to cancer cells in vitro and appears to be a selective inhibitor of RNA synthesis. The results obtained from our survey of Oregon seaweeds contradicts the generally accepted hypothesis that temperate-cold water algae, as versus tropical algae, are devoid of interesting secondary metabolites. The occurrence of novel w-3 icosapentaenoic acids is of current interest because of their structural similarity to 5Z, 8Z, 11Z, 14Z, 17Z- icosapentaenoic acid, arachidonic acid and related derivatives of these, many of whichpossess significant hormonal and bioregulatory functions in mammalian systems. The Natural Products Chemistry of the Red Algae Ptilota filicina and Laurencia obtusa by Albert Lopez A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Completed May 14, 1987 Commencement June 1988 APPROVED: Redacted for Privacy \-....' Professor of Pharmacy in charge of major Redacted for Privacy Dean of'Colleie of Pharmacy Redacted for Privacy Dean of Graduat chool A ('1 Date thesis is presented May 14, 1987 Typed by researcher for Albert Lopez ACKNOWLEDGEMENTS I am extremely grateful to my advisor Dr. William H. Gerwick for his friendliness, patience, support and assistance throughout my graduate studies. His constant guidance and encouragements stimulated my curiosity as well as my desire to do research. I would like to thank my committee members, Dr. George C. Constantine and Dr. Stephen J. Gould for their valuable advice and assistance. I acknowledge the Oregon State Sea Grant Program and the College of Pharmacy for their financial support during the length of my graduate studies. I wish to express my deepest appreciation to all my fellow graduate students (including my friends at Chemistry), faculty, secretarial staff and stockroom manager of Pharmacy for their help and friendship. I am deeply grateful to my parents, brothers and sister for their love, financial support and for believing in me. My special thanks to my darling Lorraine (Mi Prieta) for her patience, devotion, faith and love. Even though we were separated for hundreds and hundreds of miles, she always managed to be close to my heart. TABLE OF CONTENTS CHAPTER I: GENERAL INTRODUCTION 1 CHAPTER II: A SURVEY OF THE BIOMEDICINAL POTENTIAL 25 OF SEAWEEDS FROM THE OREGON COAST ABSTRACT 25 INTRODUCTION 26 MATERIALS AND METHODS 28 RESULTS AND DISCUSSION 32 CONCLUSIONS 34 CHAPTER III. TWO NEW ICOSAPENTAENOIC ACIDS FROM THE 59 TEMPERATE RED SEAWEED PTILOTA FILICINA J. AGARDH ABSTRACT 59 INTRODUCTION 60 EXPERIMENTAL 62 RESULTS AND DISCUSSION 65 CHAPTER IV. PTILODENE, A NOVEL EICOSANOID NA+/K+ ATPASE 77 INHIBITOR FROM THE MARINE ALGA PTILOTA FILICINA J. AGARDH ABSTRACT 77 INTRODUCTION 78 EXPERIMENTAL 80 RESULTS AND DISCUSSION 83 CHAPTER V TWO NEW CHAMIGRENE SESQUITERPENOIDS FROM THE 90 RED ALGA LAURENCIA OBTUSA (HUDSON) LAMOUROUX ABSTRACT 90 INTRODUCTION 91 EXPERIMENTAL 92 RESULTS AND DISCUSSION 95 BIBLIOGRAPHY 101 APPENDIX 112 LIST OF FIGURES Figure Page I.1 Taxonomic classification of Ptilota filicina 16 J. Agardh. 1.2 Taxonomic classification of Laurencia obtusa 17 (Hudson) Lamouroux. 1.3 Sesquiterpenoids isolated from L.obtusa. 18 1.4 Diterpenoids isolated from L. obtusa. 21 1.5 Triterpenoids isolated from L. obtusa. 22 1.6 C-15 acetogenins isolated from L obtusa. 23 11.1 Generalized extraction scheme. 35 III.1 Chemical structures of the isolated P. filicina 70 metabolites (1) and (3), together with their respective methyl ester derivatives. 111.2 1H-1H COSY of methyl 5(Z),7(E),9(E),14(Z), 71 17(Z)-icosapentaenoate (2) showing correlations between coupled protons (ca.7 mg of 2 in 0.4 ml CDC13 with 0.3% TMS,5 mm tube, 400, MHz). 111.3 1H-13C HETCOR of methyl5(E),7(E),9(E),14(Z), 72 17(Z)-icosapentaenoate (4) showing one bond coupling (1J) correlation between carbons and their respective protons (ca. 20 mg of 4 in 0.4 ml CDC13 with 0.3% TMS, 5 mm tube; IH NMR at 400 MHz, 13C NMR at 100 MHz). 111.4 Proposed biogenesis of conjugated triene fatty 73 acids of P. filicina. IV.1 The chemical structures of ptilodene (6) and its 86 acetate-methyl ester derivative (7). IV.2 1H-1H COSY of acetate-methylester derivative (7) 87 showing one bond coupling (IJ) correlation between carbons and their respective protons. IV.3 Proposed biogenesis for ptilodene (6). 88 V.1 Chemical structures of obtusadiene (8) and 97 isoobtusadiene (9) isolated from L. obtusa and of the p-Br benzoate derivative (10). V.2 Proposed biogenesis of natural products 98 (8) and (9). A.1 Chemical structures of hormothamnione (11) and 118 its triacetate derivative (12). A.2 A computer generated perspective drawing of the 119 final x-ray model of hormothamnione triacetate (12).The chromone fragment (atoms 01 to C8a) is planar, and the substituents on the fully substituted aromatic ring are all rotated out of the plane. The relevant torsional angles vary from 50° for C8-C7-031-C21 to 87° for C7-C8-032-C22. The acetate substituents on the 1,3,5,- substituted ring are also rotated by roughly 77°. LIST OF TABLES Table Page II.1 Summary of the bioactivity and TLC analysis 36 of seaweeds collected from the Oregon coast. 11.2 Summary of bioactivity and chemistry in 57 different phyla of algae from Oregon. 11.3 Seaweeds collected in mass from the coast of 58 Oregon as a result of screening efforts. III.1 NMR data of the two icosapentaenoic acids 76 isolated from P. filicina as their respective methyl ester derivatives (2) and (4). IV.1 IH NMR data of ptilodene (6) and its 89 acetate-methyl ester derivative (7). V.1 13C NMR data of the brominated sesquiterpenes 99 obtusadiene (8) and isoobtusadiene (9) isolated from L. obtusa. V.2 IH NMR data of the brominated sesquiterpenes 100 obtusadiene (8) and isoobtusadiene (9) isolated from L. obtusa. A.1 Inhibition of cell growth and macromolecule 120 biosynthesis in several cancerous human cell lines by hormothamnione (11). Preface My special thanks to Michelle Solem and Matthew Bernard for their participation and valuable assistance in the collection of macroalgae from the Oregon Coast, extraction, TLC analysis and biological testing of crude extracts as described in chapter II. I acknowledge both Ramon Davila and Richard Albors for their initial contribution towards the extraction and isolation of natural products from Ptilota filicina as described in chapter V. My contribution in the study of constituents from the marine cyanophyte Hormothamnion enteromorphoides, directed by Dr. William Gerwick, involved the initial extraction of the first survey collection of this blue-green algae and purification of the natural product hormothamnione as described in the appendix. We are extremely grateful to Dr. Jon Clardy and Gregory D. Van Duyne (Department of Chemistry, Cornell University) for providing the unequivocal assignment of hormothamnione by an X-ray crystallographic experiment. Finally we wish to acknowledge Dr. Adriana Baez and Wanda Ortiz for determining the