STRUCTURE ELUCIDATION OF BIOACTIVE NATURAL PRODUCTS FROM MADAGASCAR MARINE ALGAE AND CYANOBACTERIA Eric Hajaniriana Andrianasolo Ph. D. 2005 AN ABSTRACT OF THE THESIS OF Eric Hajaniriana Andrianasolo for the degree of Doctor of Philosophy in Pharmacy presented on November 18, 2005 . Title: Structure Elucidation of Bioactive Natural Products from Madagascar Marine Algae and Cyanobacteria Abstract approved: William H. Gerwick This thesis is an investigation of the natural products deriving from marine algae and cyanobacteria and has resulted in the discovery of eleven new secondary metabolites. The structure elucidations of these new molecules were performed using a variety of spectroscopic techniques. Four new macrolides were isolated and characterized from the Madagascar marine cyanobacterium Geitlerinema sp. These ankaraholides are structurally similar to the potently cytotoxic swinholides and were found to have cytotoxicities ranging from 178 nM to 354 nM against human lung cancer (NCI-H460) and mouse neuro-2a cell lines. Since swinholide-type compounds were previously localized to the heterotrophic bacteria of sponges, these findings raise intriguing questions about their true metabolic source. Geitlerinema sp. was found to be particularly rich in chemistry, and also produced the new linear lipopeptide mitsoamide with unusual structural features including an aminal moiety, a homolysine residue and a polyketide unit (3,7- dimethoxy-5-methyl- nonanedioic acid) (DMNA). A collection of the red marine alga Portieria hornemannii from the south of Madagascar (Tolagniaro, Fort Dauphin), led to the isolation of the previously reported halogenated monoterpene, halomon, and the discovery of three new related metabolites. These molecules were found to inhibit DNA methyltransferase 1 (DNMT-1). As a result of efforts to identify bioactive agents from the marine cyanobacterium Lyngbya majuscula, tanikolide dimer, a novel SIRT2 inhibitor (IC 50 = 176 nM), and tanikolide seco-acid were isolated. The depside molecular structure of tanikolide dimer, which is likely a meso compound, was established by NMR, MS and chiral HPLC analyses. The structure of tanikolide dimer raises a number of intriguing configurational and biosynthetic questions for further study. The bioassay guided fractionation of a collection of the brown marine alga Dictyota sp . from Netherland Antilles Playa Fort , led to the identification of a novel HDAC inhibitor with a dolastane carbon skeleton. The novel molecule was also found to possess antimalarial activity. Other known HDAC inhibitors with interesting antimalarial activity have been reported previously, and based on this efficacy against malaria, HDAC appears to be a viable target for the development of antiparasitic agents. Copyright by Eric Hajaniriana Andrianasolo November 18, 2005 All Rights Reserved STRUCTURE ELUCIDATION OF BIOACTIVE NATURAL PRODUCTS FROM MADAGASCAR MARINE ALGAE AND CYANOBACTERIA by Eric Hajaniriana Andrianasolo A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented November 18, 2005 Commencement June 2006 Doctor of Philosophy thesis of Eric Hajaniriana Andrianasolo presented on November 18, 2005 . APPROVED: Major Professor, representing Pharmacy Dean of the College of Pharmacy Dean of the Graduate School I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. Eric Hajaniriana Andrianasolo, Author ACKNOWLEDGEMENTS I would like to thank my major advisor, Dr. William H. Gerwick, for his exceptional generous support, academic guidance and advice throughout the course of my graduate study at Oregon State University. My appreciation also goes to my committee members, Dr. Gary DeLander, Dr. Philip Proteau, Dr. Max Deinzer and Dr. Lawrence Curtis for their time and useful suggestions. I am grateful to Jeff Morre, Brian Arbogast and Lilo Barofsky (Department of Chemistry, OSU) for their extensive mass spectral work. I express my special thanks to Roger Kohnert for his valuable NMR assistance and council. I would also like to acknowledge the members of Gerwick group, past and present. I am indebted to my wife for her support, encouragement and patience throughout the entire process. Above all, thanks to Lord Jesus Christ for his indescribable gifts. CONTRIBUTION OF AUTHORS Cytotoxicity bioassays against human lung cancer and mouse neuro-2a were carried out by Dr. Douglas Goeger in our laboratory. Isolation and characterization of swinholide A from a Fijian cyanobacterium of the genus Symploca cf. was performed by Dr Harald Gross in our laboratory. Cyanobacterial Taxonomy was conducted by Mirjam Musafija-Girt in our laboratory. X-ray crystallography of Tanikolide seco-acid was carried out by Dr. Alexander Yokochi, Department of Chemistry, OSU. Microfilament disrupting assay and cytotoxicity using Sulforhodamine B (SRB) assays were carried out by Dr. Rachel M. Leal in the laboratory of Dr. Susan L. Mooberry, Southwest Foundation for Biomedical Research, San Antonio, Texas . DNMT-1, SIRT2 and HDAC inhibitory assays were carried out by Dennis France and Susan Cornell-Kennon, Novartis Institute for Biomedical Research, Summit, New Jersey. TABLE OF CONTENTS Page CHAPTER ONE: GENERAL INTRODUCTION HIGHLIGHTS OF MARINE NATURAL PRODUCTS ........................... 1 Introduction.............................................................................. 1 Marine natural products in general........................................... 2 Marine Natural Products as Anticancer Drugs ......................... 9 Bioactive Metabolites from Marine Invertebrates ..................... 19 Uncommon chemistry of cyanobacterial metabolites ............... 22 Bioactive metabolite symbioses............................................... 28 GENERAL THESIS CONTENTS......................................................... 32 REFERENCES.................................................................................... 34 CHAPTER TWO: ANKARAHOLIDES, GLYCOSYLATED DERIVATIVES OF SWINHOLIDES FROM THE MADAGASCAR MARINE CYANOBACTERIUM GEITLERINEMA SP. ABSTRACT......................................................................................... 41 INTRODUCTION................................................................................. 42 RESULTS AND DISCUSSION ............................................................ 43 EXPERIMENTAL ................................................................................ 80 REFERENCES.................................................................................... 84 CHAPTER THREE: HALOGENATED MONOTERPENES FROM THE MADAGASCAR RED MARINE ALGA PORTIERIA HORNEMANNII ABSTRACT......................................................................................... 86 INTRODUCTION................................................................................. 87 RESULTS AND DISCUSSION ............................................................ 89 EXPERIMENTAL ................................................................................ 123 REFERENCES.................................................................................... 125 TABLE OF CONTENTS (Continued) Page CHAPTER FOUR: TANIKOLIDE DIMER AND TANIKOLIDE SECO-ACID FROM THE MADAGASCAR MARINE CYANOBACTERIUM LYNGBYA MAJUSCULA : INTRIGUING STEREOCHEMICAL INSIGHTS AND IMPLICATIONS ABSTRACT......................................................................................... 127 INTRODUCTION................................................................................. 128 RESULTS AND DISCUSSION ............................................................ 130 EXPERIMENTAL ................................................................................ 151 REFERENCES.................................................................................... 155 CHAPTER FIVE: TRICYCLIC DITERPENES FROM BROWN MARINE ALGA DICTYOTA SP. ABSTRACT......................................................................................... 158 INTRODUCTION................................................................................. 159 RESULTS AND DISCUSSION ............................................................ 161 EXPERIMENTAL ................................................................................ 175 REFERENCES.................................................................................... 177 CHAPTER SIX: MITSOAMIDE, A CYTOTOXIC LINEAR LIPOPEPTIDE FROM THE MADAGASCAR MARINE CYANOBACTERIUM GEITLERINEMA SP. ABSTRACT......................................................................................... 180 INTRODUCTION................................................................................. 181 RESULTS AND DISCUSSION ............................................................ 183 EXPERIMENTAL ................................................................................ 215 REFERENCES.................................................................................... 217 CHAPTER SEVEN: CONCLUSIONS.............................................................. 220 BIBLIOGRAPHY ................................................................................. 223 LIST OF FIGURES Figure Page 2.1. 400 MHz 1 H NMR spectrum and 100 MHz 13 C spectrum of ankaraholide A (1) in CD 3OD ............................................................................................. 51 1 1 2.2. 400 MHz H-