Novel Bioactive Secondary Metabolites from the Marine Cyanobacteriumlyngbya Majuscula

Home , Antillatoxin, Cylindrospermopsin, Dinoflagellate Blooms, Kalkitoxin, Nodularin

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

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. In addition, many marinecompounds have been found tobe useful as biochemical tools for exploringcellular processes at themolecular level. The discovery andisolation of potentiallyuseful bioactive natural products leads tothe next stage of drugdevelopment: structural characterizationand analysis.The area of molecular characterization has benefitedtremendously from advancesin digital electronics andmodern analysis instrumentsof incredible 2 power. In particular, nuclear magnetic resonancespectroscopy (NMR) is able to completely define thethree-dimensional structure of molecules with only microgramsof material.Currently, high- field and microprobe NMR technologiesprovide a means to investigate the structure of tracequantities of natural products.'"

Biomedical Potential of Marine NaturalProducts

The exploitation of resourcesfrom the sea has taken place over thousands of years.For centuries the people of Chinaand Japan have prepared an extract from theboiled fresh blades of the red alga Digenea simplex to use as aremedy for intestinal parasite infections of children."The consequent isolation and structure determination of the biologically activeconstituent gave the unusual amino acid, a-kainic acid(1, Figure I.1).12The origins of the antiviral drug Ara-A (2),which is used in combination with other agents to treat leukemia, canbe traced back to the serendipitous isolation of the arabinosylnucleosides spongothymidine (3) and spongouridine(4) from the sponge Cryptotethia cryta.The observation of beach flies killedby contact with the marine annelid Lumbriconereisheteropoda led to the discovery of an unusual sulfurcontaining toxic amine, nereistoxin (5) which served as a lead compoundfor the synthesis of a new insecticide, PADEN (6).13.14 3

COOH N HN

I ...... COOH HOH2C 0

1 114)) Kainic acid OH 2 Ara-A 0

HN H

0 HOH2C 0 HO

OH 3 OH Spongothymidine 4 Spongouridine

H3C GS

SCONH2 / HC( H3C N SCONH2 H3C./ 5 6 Nereistoxin PADEN

Figure I.1 Structures of SeveralMarine Natural Products and Derivatives with Biomedical Utility. 4

00 411 Br CI HO 8 e Halomon 7 Stypoldione

N H2N 0 10 Cribrostatin 1

12 Dolasatin 10

13 D idemn in -B OMe

Figure 1.2Structures of Biomedical PotentialMarine Natural Products. OH .OH

OH OH : OH OH HO,, ,OH 0 0 Me OH Me OH' "OH OH HO ..,,.,/.)1-,, N 0 H H

OH 0 HO M "OH /* 'OH E OH a OH OH Me s'OH OH

Structure of Palytoxin Figure 1.3 (11 6

Throughout the 1980s, biomedicalinvestigations of marine natural products focused on ionchannel effectors and central nervous systemmembrane-active toxins, anticancerand antiviral agents, tumor promoters,and anti-inflammatoryagents.' Stypoldione (7, Figure I. 2) , apotent fish toxin, wasisolated from the tropical brownalgae Stypopdium zonale.It was found to be cytotoxic by inhibitingmitotic spindle formationvia inhibition of microtubule polymerization,the mechanism of actionfor several clinically useful anticanceragents.16.17 Halomon (8) from a Philippine collection of thered alga Portieria hornemanniihas been selected by the NCI DecisionNetwork Committee forpreclinical drug development due to its extremeselectivity to brain, renal andcolon tumor cell lines."The deep-water spongeDercitus sp. collected in the Bahamas yielded thenovel aminoacridine alkaloiddercitin (9), which possesses in vitrocytotoxic activities in the lownanomolar concentrationrange.19Cribrostatin 1 (10) fromdeep-blue specimens of Cribrochaline sp.shows selective activityagainst all nine human melanoma celllines in the NCI'spanel.' Palytoxin (11, Figure I. 3) has notonly been found in Palythoa soft corals but also inwide variety of otherorganisms,' such as the seaweed Chondria armata,crabs belonging to be the genera Demania and Lophozozyinus, atriggerfish Melichtys vidua, and a file-fish Aluterascripta.22-24 More recently, polytoxinanalogs have been isolated from thedinoflagellate Ostreopsissiamensis, calling into question the biogeneticorigin of palytoxin.25It stimulates arachidonic acid metabolismand regulates the response to epidermal growth factor byactivating a sodium pump in asignal 7 transduction pathway using sodium as asecondary messenger. These activities make palytoxin auseful tool for probing cellular recognition processes!' The sea hares are herbivorousopisthobranch molluscs that concentrate and storeselected algal metabolites fromtheir diet. Dolabella auricularia, collectedin the Indian Ocean, has beenthe source of more than15 cytotoxic peptides, thedolastatins.The most active metabolite,dolastatin 10 (12, Figure 1.2) is oneof the most potent antineoplasticsubstances known.'Didemnin-B (13) extracted from the Caribbeantunicate, Trididemnum solidum, exhibits an impressive array ofin vivo antitumor,antiviral, and immunosuppressant activities andbecame the first marine compound to enter human cancerclinical trials as a purifiednatural product.n. 28

Marine Toxins

Marine toxin research has become acrucial part of marine natural products studies due totheir involvement in human intoxication, animal poisoningsand economic impact brought onby these types of incidents.In general, marine toxins arehighly targeted to specific biomolecular receptorsand have unique structural features not foundin terrestrial compounds.For example, one of the best knowntoxins, tetradotoxin (14, Figure1.4), isolated from the pufferfishand finally traced to symbioticbacteria (e.g., Shewanella alga), togetherwith saxitoxin (15) produced by a 8

+ NH2

14 Tetradotoxin 15 Saxitodn

16 Okadaic acid

OH H Me

**OH s 0 i H % H H .t. .oO H0 o

R2 17 Ciguatoxin R1 = CH(OH)CH2OH;R2 = OH

Figure 1.4 Structuresof Several Marine Toxins. OH

H3C H3C 0 CH3 OH

0 CH3

OH OH 0 O OH OH 0 0 011 0 0 OH OH CH3 OSO3Na OHO OH HO OH OH OH

Figure 1.5Structure of Maitotoxin 10

CHO

19A Brevetoxin-A

CHO

19B Brevetoxin-B

Figure 1.6Structures of Brevetoxin-AAnd Brevetoxin-B 11 number of dinoflagellate species (e.g., Alexandrium sppand Gymnodinium catenatum) as well as some strains ofthe fresh water cyano-bacterium Aphanizomenonflos-aquae,'"are involved frequently in fatal poisoning due to blockingthe sodium channel in excitable membranes.32-34Another class of shellfish toxins, well known as diarrhetic shellfish poisons [e.g.,okadaic acid (16) found in the dinoflagellate Prorocentrum lima35], have been shown to be a completely new type of phosphataseinhibitor.36,37 Ciguatoxin (17), a polyether compoundthat causes seafood poisoning in humansby ingestion of coral reef fish that becometoxic through their diet was found to be produced by the epiphyticdinoflagellate Gambierdiscus toxicus.38Maitotoxin (18, Figure I. 5) with thelargest molecular weight (3422Da) of any non-biopolymernatural product and its extremely potent bioactivity, has attractedmuch attention in the scientific community.Maitotoxin's lethality against mice (LD50 = 50 ng/ml) indicated that it might be the most potentnonproteineceous toxin in nature."The total structure of maitotoxin hasrecently been proposed on the basis of extensivespectroscopic analysis including high-field multi-dimensional NMRmethods." Along the Florida coast, the dinoflagellateGymnodinium breve causes massive fishmortality.'Brevetoxin A (19, Figure I. 6) is the most potent ichthyotoxin among thesuite of toxins produced by the microalga (its lethality againstzebrafish is reportedly 3 ppb42'

43). These toxins have proved to be valuabletools as activators of sodium channels." As these previous studies show, many of themarine toxins with unique structures and pharmacologicalproperties have been 12

20 Motuporin

21 Microcystin-LR OH

22 Aeruginopepsin

Figure 1.7Structures of Bioactive Natural Products From Cyanobacteria. 13

OMe

IOII N H 0 OH 0 OMe OMe 0 23 Tolytoxin

1 1

NH HNC 0

24 Westiellamide 25 Tantazole B

26 Tolyporpin

Figure 1.8 Structures of BioactiveNatural Products from Cyanobacteria. 14

27 6-cyano-5-methoxy-12-methylindolo(2,3-a)carbazole

28 Microcolin A

CH3

H2C

OCH3 29 Curacin A

Figure 1.9 Structures of BioactiveNatural Products from Cyanobacteria. 15 found to be very useful tools for biological,medical, pharmacological and ecological studie,'." as well asattractive targets for chemical as modification followed by structure-activityrelationship studies.',

$ioactive Natural Products FromMarine Cyanobacteria

The cyanobacteria, or blue-greenalgae, are prokaryotic photosynthetic organisms which havebeen identified as a rich source of unusualtoxins". 51 and biologically active lead compounds.'An early survey of marinecyanobacteria crude extracts showed 6% to becytotoxic to KB cancer cells withMICs < 30 ug/ml, 9% to possess antifungalactivity, and > 5% to possess antiviral activity to Herpes simplex typeII."Reports of the potent toxicity associated with cyanobacteriagrowing in nature suggest that cyanobacteria serve aspromising sources of cytotoxic, fungicidal, antiviral and antimicrobialactive compounds.5"4 Motuporin (20, Figure I.7), which is derived from symbiotic cyanobacteria, was isolated from the spongeTheonella swinhoei.55 Microcystin-LR (21) is the most commonmicrocystin cyclic heptapeptide from Microcystis, Anabaena,Oscillatoria, and Nostoc species.It displays an LDso of 50 µg /m1in mice.'' 57From recent reports, Microcystis aeruginosahas been shown to simultaneously produce a series of depsipeptidescalled aeruginopepsins (i.e. aeruginopepsin 917S-A, 22), which appear toenhance the toxic effects of themicrocystins.58 16

Tolypothrix, Scytonema, and Cylindrospermum species have been shown to contain cytostatic and antimitotic metabolites called sytophycins. Tolytoxin (23, Figure I. 7), as one member of them, exerts a selective inhibitory effect on certainhuman tissue/tumor types in comparative bioassays performed at the U. S.National Cancer Institute."Westiellamide (24), a bistratamide-related cyclic peptide isolated from Westiellopsis prolifica, was cytotoxic against the KB and LoVo cell lines at 2 Tantazole B (25) from Scytonema mirabile is cytotoxic to KB cells at doses ranging from 0.01 to 10 pig/m1,61 while tolyporphin (26) produced by Tolypothrix ondosa potentiates the cytotoxicity of adriamycin or vinblastine in a vinblastine-resistant tumor cell line (SK-VLB) at doses as low as1 4g/mi.62Cyanobacteria also appear to be a rich source of new antiviral compounds.For example, the Nostoc sphaericum metabolite, 6-cyano-5-methoxy-12-methylindolo[2,3-cdcarbazole (27) displays anti-HSV-2 activity." Two additional classes of potentially important marine natural products were isolated from Caribbean collections of Lyngbya majuscula.Microcolin A (28) suppresses the two-way murine mixed lymphocyte reaction at low nanomolar concentrations." Curacin A (29), a unique thiazoline-containing lipid isolated from a Curacao collection of Lyngbya majuscula, is an exceptionally potent antiproliferative agent that shows some selectivity for colon, renal, and breast cancer-derived cell lines.' 17

Description of Chapters

This thesis consists of a total offour chapters and an appendix. Following this general introduction,chapter II describes the discovery of a potent new toxicnatural product named kalkitoxin. Determination of the chemical structureof kalkitoxin was made possible through the extensiveinvestigation by NMR spectroscopy including 2D-NMR at two different temperatures.Chapter II also details the methods for the brineshrimp toxicity guided fractionation and bioactivity evaluationof kalkitoxin. The third chapter describes theisolation and structural elucidation of two new malyngamidesfrom the Curacao Lyngbya majuscula.The structure of these twomolecules were determined by using 2D-NMR methodologytogether with comparisons of other malyngamides.The stereochemistry of malyngamideJ was investigated by using NMR andcircular dichroic spectroscopic techniques. Chapter IV give a conclusion aboutthis investigation of Curacao Lyngbya majuscula and perspectiveof these three novel bioactive metabolites. 18

CHAPTER II KALKITOXIN FROM THE CYANOBACIERIUMLYNGBYA MAJUSCULA

Abstract

Lyngbya majuscula, a chemically andbiologically rich strain of cyanobacteria, has been intensivelyresearched for its biomedical potential.Brine shrimp toxicity assay-guidedfractionation of an organic extract of Lyngbya majusculafrom a Curacao collection led to the isolation of a potentbrine shrimp toxic compound.The structure of this new thiazolinecontaining lipid, named kalkitoxin, was deduced fromextensive spectroscopic investigation. Other biological properties of kalkitoxin such asichthyotoxicity and molluscicidal activity have also beeninvestigated. 19

Introduction

Cyanobacteria, or blue-green algae, are foundalmost everywhere that light and water are available.In freshwater environments, blooms of toxic cyanobacteria can poseserious dangers for wildlife and livestock." Alarge number of interesting metabolites have been isolated fromcyanobacteria. Their unique structures and biological activitieshave received increasing attention from chemists andpharmacologists."' Most of the investigated cyanobacteria arefreshwater or terrestrial in origin.Only a limited number of marine cyanobacteria have been searched for secondarymetabolites. Nevertheless, there are a number ofindications that cyanobacteria are important players in the production of interestingcompounds that are found widely in marine environments.From the brackish water species, Nodularia spumigena, the cyclic pentapeptidenodularin (1, Figure

II.1) which had caused problems in the BalticSea and New Zealand, was isolated.'This compound is closely related to microcystin(2), a potent hepatotoxinand protein phosphatase 2 and 2A inhibitor from the fresh-water cyanobacteriumMicrocystisaeruginosa.68 A marine strain of Nostoc linckia producedthe potent cytotoxin borophycin (3), the structure of which wasdetermined by X-ray crystallography.'Tjipanazole Al (4), a N-glycoside ofindolo-[2,3- c]carbazole isolated from Tolypothrixtjipanasensis, exhibited appreciable fungicidal activity intests against phytopathogenic fungi." From an Australian Cylindrospemopsisraciborskii culture, 20

43 H COOH CH3 OH 00.0 0 II 3 C4 N CH3 0 O %OHO Na+ .,OCH3 H H -o 0 0 H3C' HCH3 0 0 H COOH 3 Borophycin NH 1 Nodularin HN NH2

COON CH30 HNI%-/%11N-Tr )1. NH H3C 0 0 CH 4 OCH3 H3 0 Tjipanazole Al ... NH H CH3 H HN CH3 ei H3 CH3 NvAirIiiA' NCH3 0C0011

HN HN3'NH2 OH 63so 2 N NH HN NH H3C Microcystin-LR N 0 H 5 Cylindrospermopsin

Figure II.1 Structures of NaturalProducts from Cyanobacteria. 21 which was obtained following an outbreak ofhepatoenteritis on Palm Island in northern Queensland, cylindrospermopsin(5), a potent hepatotoxin, has beenisolated.' of the most important cyanobacterialnatural products have been isolated from Lyngbya majuscula, amarine filamentous form. Lyngbyatoxin A (6, Figure 11.2),together with debromoaplysiatoxin(7), were isolated from a toxic strain of Lyngbya majuscula collected in Hawaii and shown tobe the cause of the severe contact dermatitis known asswimmer's itch.'Both classes of compound have played a significantrole in the study of protein kinase C (PKC) due to their binding tothe phorbol ester receptor on PKC.72."Majusculamide A (8), an unusual fatty acid amide derivative, was reported from a deep waterLyngbya majuscula." Our investigation of marine algae foranticancer drug leads has resulted in the identification of several structurallydiverse cytotoxic metabolites from microalgal sources.Hormothamnin A (9), isolated from the northern Puerto RicanHormothamnion enteromorphoides, was found to be potentlyantimicrobial to Bacillus subtilus and cytotoxic to several cancercell lines.'Isolated from the Caribbean cyanobacterium Lyngbyamajuscula, barbamide (10, Figure 11.3), a chlorinated metabolitewith molluscicidal activity, together with antillatoxin (11) whichexhibits extremely potent ichthyotoxicity, were reportedrecently:16a'Utilizing toxicity to brine shrimp as a bioassay, aunique thiazoline containing lipid, curacin A (12), was discovered from the organic extractof a Curacao collection of Lyngbyamajuscula.'Curacin A is a potent 22

7 Debromoaplysiatoxin 6 Lyngbyatoxin A

N1-12

OCH3 8 Majusculamide A

9 Hormothamnin A

Figure 11.2 BioactiveMetabolites from Cyanobacteria 23

Cl CI Cl OCH3 CH3 N H

10 Barbamide 11 Antillatoxin

0 r''ri 0 H Il N

N.'''''N1 CH31 0 AE CH3 0

13 Microcolin A

CH3 H3C CH CH3O H3C A H3C N N I I CH3 CH3 CH3 0 A CH3 0 H3C OH

14 Microcolin C

Figure 11.3 Bioactive Metabolitesfrom Lyngbya majuscula. 24 antimitotic agent that inhibits microtubuleassembly and colchicine binding to tubulin (Figure 11.3).78.79A Venezuelan collection of Lyngbya majuscula yielded animmunosuppressive linear peptide microcolin A (13),64 while a Curacaocollection provided microcolin C (14).81 These lipopeptides exhibited potentcytotoxic activity against several cell lines in vitrosuch as A 549 (79% inhibition), SW-480 (97% inhibition), and HMEC(90% inhibition) performed by the Sandoz Corp.

Results and Discussion

Lyngbya majuscula from Playa Kalki,Curacao was collected at a depth of 20 ft.It was preserved in isopropanoland stored at -20 °C.The organic extract was producedfrom the homogenized alga in CH2C12/Me0H (2:1, v/v), allowed to soakaround 30 minutes, and filtered through cheese cloth.The algal material was placed in fresh solvent (CH2C12 /MeOH, 2:1 v/v),heated to a gentle boil up to 20 minutes, and filtered.This process was repeated two times.The filtrate was partitioned betweenCH2C12 and water, and the organic fraction was reduced in vacuo andstored in Et20 (4.32 g, dark green oil). The isopropyl alcohol preservedsample (1L) was filtered prior to extraction.This alcohol was removed fromthe filtrate by evaporation in vacuo and added tothe aqueous extract.The CH2Cl2 /MeOH extracted algalresidue was soaked in Me0H/H20 (3:1, v/v) overnight, filtered, and thealcohol was removed in vacuo. The 25 aqueous extract waspartitioned between sec-butanoland water. The sec-butanol fraction (0.6g, dark greenoil) was reduced in vacuo and storedin Me0H (Figure 11.4). This investigation focused on theorganic extract. Two- dimensional TLC analysis (EtOAc /hexane1:1; CHC13/Me0H 9:1) of the organic extract suggested the presenceof several UV-active secondary metabolites.The crude extract was highlytoxic to brine shrimp (100% killed at 10ggim1).82Hence, the brine shrimp toxicity assay was utilized toguide each fractionation of this extract (Figure 11.5).The first fractionation step utilizedgradient vacuum chromatography (4%-40% EtOAC/hexane) togive seven fractions after recombination.Among them, fraction C gave thehighest toxicity against brine shrimp (100%killed at 1 µg /ml, Figure II.6a). Further flash chromatography (4%-40%EtOAC/hexane) was applied to fraction C to give atotal of ten fractions.Brine shrimp toxicity assay II exhibited aselective toxicity in fraction C-4(Figure II.6b). The continued fractionationmonitored with the brine shrimp toxicity assay (column chromatography,40% EtOAC/hexane) led me to focus on fractionC-4-G (Figure II.6c).The final purification was performed by HPLC (40% EtOAC/hexane) toyield 12.8 mg of compound 15, named kalkitoxin(Figure II.6d, Figure 11.7). Kalkitoxin showed [a],), = +16° (c =0.07, CHC13). High resolution EIMS (70 eV) gave a major [M]+ ion atm/z 366.2696 analyzing for C21H381\120S (-0.8 mamu dev.). Theformula C211438N2OS indicated that kalkitoxin possessed four degrees ofunsaturation.From "C NMR analysis, two of these degrees weredue to double bonds, one due to carbonyl group, and the remaining one to aring system. Lyngbya majuscula (in 1 L) 1.partially defrost 2. filter off preservation alcohol 3. extract cold in 2:1 CH2Cl2 /MeOH reduce 4. gently heat it in 2:1 CH2Cl2/MeOH aqueous for 30 min, cool, filter partition 5. repeat #4 twice between CH2Cl2 / H2O

residue I add cJ2Cl2layer soak in 3:1 Me0H/H20 organic solvent layer H20 (discard) overnight, then filter 1

aqueouslayer) reduce at 40 °C, filter particulates, partition with algal reduce on rotovap at 40°C material sec-butanol i' Organic extract sec-butanol H20 i' (4.32 g) "Aqueousextract" (0.66g)

Figure 11.4 Extractionof Lyngbya majuscula. Organic Extract of Lyngbya majuscula (4.32g) Gradient Vacuum Chromatography EtOAc / Hexane 4%-100% Brine Shrimp I I I-1 Assay I D E F G (420 mg) Gradient Flash Chromatography EtOAc / Hexane 4%--100%

I I Brine Shrimp E-1 I 1 I I Assay II C1 C2 C3 C4 C5 C6 C7C8 C9 C10 (101.2 mg) Column Chromatography EtOAc / Hexane 40%

Brine Shrimp I I 1 Assay III C4A C4B C4C C4D C4E C4F C4G C4H C41 C4..) (26.3mg) HPLC 40% EtOAc / Hexane

Brine shrimp Assay IV 1 3 4 LKalkitoxin (12.8 mg)

Figure 11.5Bioguided Fractionation of Kalkitoxin 28

120% g 100% --o 10 vg/m1each fraction io80% 0 1 µg /ml eachfraction 60% 40% 20% ft * 0% 5 6 7 1 2 3 4 TLC Fraction number

Figure II.6aBrine Shrimp Assay I forBioguided Isolation of Kalkitoxin.

120% c 100% 0-0.5 ug/m1 each fraction 80% vs° 0--0.1 ug/ml each fraction 60% a, 40% 2 20%

0% 6 7 8 9 10 1 2 3 4 5 TLC Fraction number

Figure II.6bBrine Shrimp Assay IIfor Bioguided Isolation of Kalkitoxin. 29

120% ,s 100% 0 0.1 µg /ml eachfraction 0 co 80% 0-- 0.01 µg /mleach fraction 60% c40%

4'20% 0% 6 7 8 9 10 11 1 2 3 4 5 TLC Fraction number Isolation of Figure II.6cBrine Shrimp AssayIII for Bioguided Kalkitoxin.

120%

c 100% 0 4J 80% 0-- 0.1 µg/m1 eachfraction u_ 60% 0-- 0.01 µg /ml eachfraction

40%

20% 32 0% 2 3 4 1 HPLC Fraction number

of Figure II.6dBrine Shrimp AssayIV for Bioguided Isolation Kalkitoxin. 30

The structure elucidation of compound 15 wasmostly based on extensive 1D and 2D NMR experiments. A complicationof this structure elucidation was that at room temperature(298K) many of NMR signal were twinned in a 3:2 ratio (inD6-DMSO, Figure 11.9). That was due to the presence of two slowlyinterconverting amide conformers (Figure II.10).The fact that these twinned proton peaks changed ratios in different NMR solvents confirmedthat they arose from two conformations of this molecule (tableII.1).However, at high temperature (340K) these peaks coalesced tosinglets (Figure II.11).While the high temperature NMR experimentsprovided valuable information for this structureelucidation, there was a complicating factor in that at the high temperature someof '3C NMR signals were missing and some '11-'3C correlations werelost (Figure 11.12-13). Therefore, the structure elucidation was based onthe combination of the two sets of NMR experiments at298K and 340K. Data from 'H -'H COSY, HMQC, DEPT and'H NMR decoupling experiments (Figure 11.14-17) allowed deduction ofsix partial structures (a-f, Figure 11.8) for compound15.Partial structure b contained a tertiary amide group which caused the two conformations in this molecule.Partial structure f possessed a terminal olefin adjacent to a thiazoline ring.This partial structure assignment was also based on analysis of EIMSdata and comparisons of '3C NMR chemical shifts withmodel compounds.'' 85, 86 Partial structure e possessed a CH3-CH-CH2 groupingwhile partial structure a contained a high fieldC4 group with two methyls.The partial structure c had a high field C4 groupwith one methyl group. 31

2 16

4' 1 12

14 15

Figure 11.7 The Overall PlanarStructure of Kalkitoxin.

a c

CH3

e f d CH3 H

CHCH2 sS5 H -->----Ths / i NZ( Pr

Figure 11.8Six Partial Structures of Kalkitoxin. 32

HMBC data were used to connect these six partial structures to give the full structural assignment of kalkitoxin (Figure11.18-19). Notably, the proton at 54.90 in partial structure f and the methylene protons at 62.23 and 62.45 in partial structure e were both correlated to the quaternary carbon at 6168 of the thiazoline ring.Similarly, partial structures b and c were readily connected by observing long range coupling between the methyl protons 62.95/2.80 (at 298K) of amide b and the methylene carbon 647/44.8 of c as well as the carbonyl of the amide at 8174.8 andthe methylene protons at 63.32 of c.In partial structure d, the methylene protons at 61.10 showed clear correlations to the methine carbon 636.7 of partial structure e and the methine at 627.5 of partial structure c. Table 11.2 displays '11 and13C NMR data and coupling constants of kalkitoxin. Kalkitoxin was found to possess highly potent brine shrimp toxicity (LC50 = 28 ng/ml, Figure 11.20).This rapid in-house screen has been shown to have a good correlation (p = 0.036) with the9KB cytotoxicity assay.88,89 Also, kalkitoxin displayed modest molluscicidal activity to Biomphalaria glambrata (LC100 = 22 µg /ml, Figure II.21).84 This freshwater mollusc is an intermediate hostfor three species of parasitic trematode blood flukes (shistosomes). Over 200 million people in over 75 countries are infectedwith schistosomiasis(bilharzia).87Chronic infection may result in disturbances of the central nervous system, liver fibrosis, portal hypertension, and possibly liver cancer.The difficulty and costs 18

1 14

4 15

2 3 48 4b \12 P

0, 0

, 2.5 2.0 1.5 4'5 4.0 5 0 6.0 5.5 5. 0 PPM 298K. Figure 11.911-1 NMR Of Kalkitoxin InD6-DMSO At Room Temperature 34

Table II.1The ratio of N-methyl group resonances in the'H NMR spectra of the two conformers of kalkitoxin invarious NMR solvents

solvent chemical shift ratio

CDC13 2.95/2.90 1.27:1

C6D6 2.79/2.43 0.5 : 1

D6-acetone 3.05/2.85 1.58:1

D4-Methanol 2.95/2.80 1.2 :1

D6-DMS0 2.95/2.80 1.5 : 1

D4-Me0D/CDC13 3.06/2.94 1.22: 1

1:1

* at room temperature 298K.

0- 0- 2.80 H3C.

CH3 (in D6 -DMSO) 2.95

FigureII.10 Two slowly intercovertingtert-amide isomers present at room temperature 298K. 5 6

14 15

4.5 4.0 3 5 6I0 5.5 5.0 PPM

Figure II.11 1H NMROf Kalkitoxin In D6 -DMSO At340K. 2'

6 18 16 47 3' 14 13 10 118 5' 4'

I I- T 30 20 10 I 50 40 " 100 90 80 70 60 140 130 120 110 170 160 150 PPM

Figure 11.1213C NMR Of Kalkitoxin In D6 -DMSO At 298K. 90 80 70 60 50 40 30 20 10 170 160 150 140 130 120 110 100 PPM

Figure 11.13 "C NMR Of KaLkitoxinIn D6 -DMSO At 340K. ai4.k___AJAieil_

1.0 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 6.0 5.5 PPM

Figure II.1511-I-1H COSY Of Kalkitoxin In D6-DMSO At 340K. 16

!N. 1.0

8 1.5

2.0

0e 2.5 I lot c.)

3.0

ct,P 3.5

_ 4.0

4.5

5,0 . a O. 0 _ O co o P a -5.5

8 6.0 a 030 El

PPM 2.0 1.5 1.0 5.0 4.5 4.0 3.5 3.0 2.5 8.0 5.5 PPM In D6-DMS0 At 298K. Figure 11.14'11-11-1 COSY Of Kalkitoin Figure 11.16 HMQC Spectrum Of Kalkitoxin In D6 -DMSO At 298K. 1 2 4 13 16 PH p1'11 didi° .00 101 1A1 1

1.5 1.0 6,0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 PPM

Figure 11.17 HMQC Spectrum OfKalkitoxin In D6-DMS0 At 340K. Partial structures deduced from combination of 1H-1H COSY, HMQC, DEPT and1H decoupling data r) HMBC (Selected,1H--0-13C )

Figure 11.18 Partial Structures of KalkitoxinConnected by HMBC. 43

2.95/2.80 0.97

1.45 0.84 1.25 2.61

1.45 3.32 I 11..5257

0.83 0

1H chemical shifts

114.6 137.4

34.7/33.0 17.1/17.7

34.9 N<7.46.1 11.7

27.5 47/44.8 26.8

13C chemical shifts

Figure 11.191H and 13C NMR Assignment of Kalkitoxin (298K). 44

Table 11.2 and "C NMR Data* of Kalkitoxin Isolated From Lyngbya majuscula

C-atom "C (D6 -DMSO) (D6-DMS0) 8 8(mult, J in Hz)

1 114.6 5.25 d (17.3) 5.10 d (10.6) 2 137.4 5.95 ddd (17.3, 10.6,6.2) 3 77.6 4.904 m 4 37.8 3.46 dd (10.8, 8.7) 3.03 dd (11.0, 7.9) 5 168.0 6 37.5 2.23 bdd (9.0, 14.5) 2.45 m 7 36.7 1.84 m 8 33.4 1.571 m 9 39.3 1.10 m 10 27.5 1.45 m 1 1 34.9 1.45 m 1.25 m 12 47/44.8 3.32 dd (5.6, 12.8) 1 3 19.0 0.87 s 14 16.0 0.85 s 15 16.0 0.83 s 16 34.7/33.0 2.97/2.80s 1' 174.8 2' 36.1 2.61 dd (6.7, 13.4) 3' 26.8 1.25 m 1.57 m 4' 11.7 0.84 d 5' 17.1/17.7 0.97 d (6.9)

* Data reported at room temperature 298K. 45

120%

100%

80%

60%

40%

20%

0% 01 1 10 50 100 500 1000 Concentration (ng /mI)

Figure 11.20 Pure Kalkitoxin in Brine Shrimp Toxicity Assay.

120%

100%

80%

60%

40%

20%

1 5 10 25 100 500 Concentration (ug/ml)

Figure 11.21Molluscicidal Activity of Kalkitoxin. 46

120%

100%

80%

60%

40%

20%

0.01 0.1 1 5 10 100 1000 5000 Concentration (ng /mI)

Figure 11.22Ichthyotoxic Effects of Kalkitoxin. involved in treating these large groups of people in remote impoverished areas has prompted a concerted effort by the World Health Organization (W.H.O.) to look for new means of preventing infection by controlling the snail vector. Kalkitoxin showed extremely potent ichthyotoxicity to the goldfish (LC50 = 5 ng/ml, Figure 11.22)."This is almost the same potency as the well known and highly potent ichthyotoxic metabolite, brevetoin A produced by G. breve (although tested against different fish).42,43 As has occurred in the past, insect antifeedants, antitumor agents, plant growth inhibitors, and insecticides have been isolated from ichthyotoxic plants."These biological features of kalkitoxin suggest possible ecological interactions and inspire further investigation. 47

Biosynthetically, kalkitoxin may derive from an acetyl CoA joined with a decarboxylated cysteine residue and a malonyl CoA to form a thiazoline ring attached with a terminal olefin.The middle polyketide chain of kalkitoxin may derive from the consequent condensations of methyl malonyl CoA, SAM and acetyl CoA.The terminal C5 chain including the nitrogen of the amide is possibly from a decarboxylated isoleucine. Unfortunately, this research was not able to solve the stereochemistry of kalkitoxin due to the limited amount of sample.

Experimental Methods

Kieselgel 60 silica (40-63 1.tm) was used for flash chromatography, and Merck aluminum-backed TLC sheets (silica gel 60 F254) were used for thin layer chromatography.All solvents were distilled from glass prior to use. This Lyngbya majuscula was collected from Playa Kalki, Curacao (20 ft underwater) on 11 August 1994 and preserved in isopropyl alcohol and frozen until workup.The defrosted alga was homogenized in CH2C12 /MeOH (2:1, v/v).The algal residue was repetitively extracted with CH2C12 /MeOH (2:1, v/v) twice. The mixture was filtered and the solvents removed under vacuum to yield a residue which was partitioned between CH2C12 and H2O. The CH2C12 solution was collected. A total of 4.32 g dark-green oily crude organic extract was obtained. The bioguided fractionation of the crude extract was obtained by silica gel vacuum chromatography, flash chromatography, and column chromatography.Finally, a normal phase HPLC was applied to give kalkitoxin (15 12.8 mg), as a light yellow oil (Figure 11.7). The 'H and '3C NMR spectra of kalkitoxin show very clean base lines and The HPLC of kalkitoxin displayed a symmetric peak. These suggested kalkitoxin great purity (> 95%) Kalkitoxin(1 5).IR (CHCI3 v 2961, 2928, 2880, 1643, 1464, 1086, 1410, 1380 cm'; UV (MeOH) Xmax 250 nm (E = 2600);Optical rotation [a]p = +16° (c = 0.07, CHC13); HR EIMS (70 eV) m/z obs. [M]+ 366.2696 (16, -0.8 mmu dev.) C21H381\120S, [M-CH3]+ 351.2472 (5.5, 0.2 mmu dev), [M-C2H3]+ 339.2465 (5.7, -0.5 mmu dev), [M-CH2CH3]+ 337.2294 (2.5, -1.9 mmu dev), 281.2591 (7, -0.2 mmu dev), [M- 05H9O]+ 281.2049 (3.9, -0.2 mmu dev), 265.2271 (2.4, -0.8 mmu 49

dev), [M-C61-18NS]+ 240.2329 (25, 0.2 mmu dev), [M-C,H14NO]+ 238.1639 (5, 1.0 mmu dev), [M-C8H16NO]+ 224.1470 (1.9, -0.3 mmu dev), 212.9960 (2, 0.9 mmu dev), 195.1860 (2.3, -0.1 mmu dev), [M-C111122N0]+ 182.1003 (4.4, -0.05 mmu dev), 156.1750 (5.2, -0.2 mmu dev), [M-C13H26NO]+ 154.0683 (100, -0.8 mmu dev), 149.0244 (1.5, 0.5 mmu dev), 142.1237 (2, 0.5 mmu dev), 140.0531 (2.6, -0.3 mmu dev), 129.1155 (3.8, 0.1 mmu dev), [C6H9NS]+ 127.0459 (26, 0.3 mmu dev);'I-1 NMR and "C NMR data see Table 11.2. Brine Shrimp Bioassay.The brine shrimp toxicity assay was used to analyze levels of toxicity at different concentrations of sample and this assay was utilized to guide fractionation of the sample.Brine shrimp eggs were incubated in Instant Ocean® water at 28 °C for 24 hours before the assay to allow the eggs to hatch and mature. Approximately 90% of the eggs usually hatch after this incubation period.'A shallow rectangular dish filled with artificial seawater (Instant Ocean®, Aquarium Systems, Inc.) containing a plastic divider with several 2 mm holes was used to separate the unhatched eggs from the nauphali.The eggs were sprinkled into one compartment that was kept in the dark for the incubation period.After 24 hours, the phototropic nauphali were collected from the lighted compartment.Approximately 15 shrimp were added to each vial containing different concentrations of sample in 4.5 ml artificial seawater.Appropriate solvent controls were done in duplicate.The brine shrimp were added to each vial using a long stem pipette.After 24 hours at 28 °C, the shrimp were counted using a dissecting light microscope.The percentage of dead shrimp 50 relative to the total number of shrimp was recorded for LC measurement. Ichthyotoxicity Assay. A single goldfish Carassius auratus was added into a 50 ml beaker containing 40 ml of distilled H2O.The sample was dissolved in 40 gl EtOH. In order to get an even solution of the test compound, a microliter syringe was used to add the sample solution at different water levels.The fish was observed for a one hour period. End points were established asdeath (lack of breathing) for LC measurement. Molluscicidal Assay. The sample was dissolved in 40 tl EtOH and diluted to 20 ml with distilled water to make several different concentrations.Two Biomphalaria glambrata were placed in each beaker and the beakers covered with glass plate for the duration of the assay.The condition of the snails was evaluated after 24 hours. Snail were considered dead when no heart beat could be observed upon microscopic examination.Minimum lethal doses required to kill the snails (LC100) were recorded. 51

CHAPTER III

TWO NEW MALYNGAMIDES PROM THECYANOBACTERIUM LYNGBYA MAJUSCULA

Abstract

Two new secondary metabolites,malyngamide J and malyngamide L, have been isolated from aCuracao collection of the cyanobacterium Lyngbya majuscula.The structure elucidation of these new compounds was accomplishedby analysis of 1H NMR, "C NMR and ID NMR, along with comparisonswith known natural products. Stereochemistry and biological properties havebeen investigated. 52

Introduction

Cyanobacteria have been a source ofdiverse types of secondary metabolites.Fatty acids are one of the most common compositions in cyanobacteria, which arenormally present as C14 to C18 components.Malyngic acid, 9(S), 12(R), 13(S)- trihydroxyoctadeca-1 0(E),15(Z)-dienoicacid (16, Figure III.1) is a major fatty acid that occurs inboth shallow-water and deep-water varieties of Lyngbyamajuscula,91 while 9-methoxy-9- methylhexadeca-4(E),8(E)-dienoic acid (17)is present in moderate amounts in the deep-waterLyngbya majuscula." In contrast to the situationin eukaryotic algae, nitrogen. containing secondary metabolites are commonin cyanobacteria. Shallow-water varieties of Lyngbyamajuscula contain amides of the fatty acid 7(S)-methoxytetradec-4(E)-enoicacid (18), which in free form displayed antimicrobialactivity to the gram positivebacteria Staphylococcus aureus and Bacillussubtilus.On the other hand, deep-water collections of Lyngbyamajuscula provided amides of 7- methoxy-9-methylhexadec-4(E)-enoic acid(19)." Malyngamide A (20) and malyngamideB (21) are two such amides of 7(S)-methoxytetradec-4(E)-enoicacid (18), which have been found in several shallow-watervarieties of Lyngbya majuscula from Hawaii.9"Their structures were determinedby NMR analysis and chemical degradationstudies.Malyngamide C (22), a chlorine-containing amide of7(S)-methoxytetradec-4(E)-enoic acid, was isolated from ashallow-water Lyngbya majusculafound on the 53

OH OH 16

OH 17

Figure III.1 Secondary Metabolitesfrom Different Varieties of L. majuscula. 54 reefs of Fanning Island in the Line Islands.' The complete structure of 22, including absolute stereochemistry, was determined by spectral and chemical studies.From a deep-water collection of Lyngbya majuscula found on the pinnacles inEniwetok lagoon, malyngamides D (23, Figure 111.2) and E (24), twoclosely related amides of 7(S)-methoxy-9-methylhexadec-4(E)-enoicacid (19) have been found.Detailed spectral analysis and chemical degradation studies defined the structures and the ring stereochemistry." Malyngamide F (25) was reported from shallow water collections of a Caribbean Lyngbya majuscula.This secondary metabolite showed mild cytotoxicity (ID50 <30 µg /ml) againstKB cells in tissue culture.The structure elucidation was based on 2D NMR data and chemical interconversion.97Malyngamide G (26) was isolated from a cyanobacterium epiphyte on the brown mediterranean alga Cystoseira crinita.98Guided by ichthyotoxicity against goldfish, malyngamide I-1(27) was isolated from another Caribbean L. majuscula.The structure and the absolute stereochemistry of the cyclohexenone moiety was elucidated by spectroscopic analysis and exciton chirality in the circulardichroism spectrum.99The latest report of malyngamides was of malyngamide I (28), isolated from a Okinawan shallow water collectionof Lyngbya majuscula.This compound showed moderate toxicity towards brine shrimp (LD50 ca. 35 µg /m1) and goldfish (LD50 <10 µg/m1)."° 55

OCH3

H3C OCH3

CH3 0 26 Cl

OCH3 0 CH3 /\7.\.77./.)(NH 27

OCH3 0

CH3 28

Figure 111.2 Structures of VariousMalyngamides from L. majuscula. 56

Results and Discussion

For this work, a collection of Lyngbyamajuscula was made from Playa Kalki, Curacao in about20 ft of water.The sample was preserved in isopropanol and stored at-20 °C.The organic extract was obtained fromthe homogenized alga by soakingin CH2C12 /MeOH (2:1, v/v) for 30 minutesfollowed by filtration through cheese cloth.The algal material was placed infresh solvent (CH2C12/Me011, 2:1 v/v), heated to a gentle boilfor 20 minutes, and filtered.This process was repeatedtwice.The filtrate was partitionedbetween CH2C12 and water, and the organicfraction was reduced in vacuo and stored in Et20 (4.32 g, dark greenoil). The isopropyl alcohol preservedsample (1L) was filtered prior to extraction.This alcohol was removed byevaporation in vacuo and the residue was added tothe aqueous extract.The CH2C12 /MeOH extracted algalresidue was soaked in Me0H/H20(3:1, v/v) overnight, filtered, and thealcohol was removed in vacuo.The "aqueous extract" was partitionedbetween sec-butanol and water. The sec-butanol fraction (0.68 g,dark green oil) was reduced in vacuo and storedin Me0H (Figure 11.4). Two-dimensional TLC analysis of theorganic extract suggested the presence of several UV-activecompounds. The fractionation utilized gradient vacuumchromatography (EtOAc /hexane, 4 %- 100 %), gradient flashchromatography (Me0H/CHC13, 0%-2%),RP C- 18 plug chromatography (MeOH/H20, 80%-100%) and HPLC (Alltech, Lichrosorb Diol column 10u,Me0H/Et0Ac/hexane 1:3:16) to yield a colorless lipid (29). This was shown tobe a new member of the 57

Organic Extract of Lyngbyamajuscula from Curacao (4.32g) Gradient Vacuum Chromatography EtOAc / Hexane 4%--100%

1-1 I I I I A B C D E F G (70 mg)

I Gradient Flash Chromatography I I I Me0H / CHCI3 0%--2% 1 2 3 4 0% 0.5% 2%

I Reversed-phase RP C-18 chromatographyr----- I 1 1 2 3 4 Gradient Solvent Me0H / H2O 80% 90% 100% 100%

HPLC Me0H / EtOAc / Hexane 0.5 : 1.5 : 8

A B

malyngamide J 14.5mg

Figure 111.3The Isolation of Malyngamide J(29). 58 malyngamide family, and was therefore named malyngamideJ (Figure 111.3).Another less polar fraction was subjected tosilica gel column chromatography (EtOAc /hexane, 85%),gradient flash chromatography (Me0H/ CHC13, 0.1%-10%v/v) and HPLC (Alltech, Lichrosorb Diol column 10u, Me0H/Et0Ac/hexane1.5:9:120 v/v) to give a light-yellow oil (30, malyngamide L,Figure 111.4). Compound 29 showed optical rotation [43 +64°(c = 0.15,

CHC13).High resolution FAB MS (positive ion,3-nitrobenzyl alcohol) gave a major [M+H]+ion at m/z 608.3798 (0.0 mmu dev.) which was consistent with a molecular formula ofC33H53N09.This molecular formula indicated that compound 29 possessedeight degrees of unsaturation.Analysis of UV (MeOH, Xma, 240 nm c =5600), 111 NMR and "C NMR data (Table III.1) revealed the presenceof three double bonds, an amide carbonyl group, an a,0-unsaturated carbonyl, and three rings. By using COSY and HETCOR spectral data (Figure 111.5), several distinct spin systems could beassembled (Figure 111.6). Fragment a possessed a 7-methoxytetradec-4(E)-enonatemoiety, which is a common feature of malyngamides.The partial structure b placed two consecutive methylene groupscontiguous to an amide N atom by observation of HMBC correlationsbetween the methylene protons Ha,b-1 (83.43 , 83.37) andthe carbonyl carbon C-1' (8172.7). The deshielded methylene singlets Ha,,,-4(85.22, 85.28) showed no correlation by '11-11-1 COSY to any other groupand suggested a terminal olefin with a quaternary carbon inpartial structure c. 111-'14 COSY correlations gave the partial structure dwith a 59

Extract of Lyngbya majuscula from Curacao (4.32g)

EtOAc / Hexane 4%--100% Gradient Vacuum Chromatography

4% 25% 25% 50% 50% 75% 100% (100 mg) Column Chromatography EtOAc / Hexane 85%

1 10 4

Me0H / CHC13 0.1% -- 10% Gradient Flash Chromatography

I I I 1 1 2 3 4 0.1% 0.2% 0 5% 10% HpLc Me011 / EtOAc / Hexane 1.5 : 9 : 120

I I I-1 Fl 3 5 6 7 1 2

Malyngamide L 6.6 mg

Figure 111.4The Isolation of Malyngamide L (30). 60 deshielded methylene contiguous to four consecutive deshielded methines.The more deshielded methine C-12 (1H 54.48,"C 5103) at the end of this chain suggested an acetal carbon.In partial structure e, a long range coupling wasclearly observed by 11-1-111 COSY between the deshielded methyl 51.88 (C-11) andthe olefin proton 5 6.42 (H-8). This placed the methyl group onthe quaternary carbon (C-7) of the olefin, next to which were twosequential deshielded methine groups 54.68 (I1-9), 53.70 (H-10).The "C chemical shift of 563.8 for the C-10 methineCH 53.70) and a quaternary carbon at 562.3 (C-5) suggestedthe presence of an epoxide ring.The HMBC correlation between the 11-10 proton (53.70) and the C-5 quaternary carbon (562.3) confirmedthis assignment. These partial structures were connected byinterpretation of long- range correlations observedin the HMBC spectrum (Figure 111.7). The correlation cross-peak observed from Hab-1 toC-1' secured the amide linkage between partial structures a and b.The correlation seen from 11.,b-4 to C-5 andC-2 confirmed the connections between partial structure c, b and e of the cyclohexenone.The correlations between Ham-16 and C-12 completed asix-membered heterocyclic ring.The correlation observed from H-12 to C-9 andfrom H-9 to C-12 confirmed the linkage between the cyclohexenoneand dimethylated pentose sugar. The correlations seenfrom the H3-17 methoxyl group (53.6) to C-13 at 582.8 and from theH3-18 methoxyl to C-15 (578.9) placed these methoxyl groups onthe methine carbons C-13 and C-15.From these data, a full assignment 18 16' 9' 17 4' 5' 12 18A 10 13 11 8 MAO 16b

14 .5 0

43.1 _ 1.0

9 _ 1.5

0 2.0 O 0 2.5

O 3.0 0 3.5

- 4.0

O - 4.5

5.0

5.5 A 09

_ 6.0 0 s-00. 0 0 _ 6.5

PPM 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 .5 PPM

Figure 111.5 1H-1H COSY of MalyngamideJ (29). 62

H3C a

b C

d e

8 3.60 8 3.50 5 3.32 au-13 ocH3 ocH3

Figure 111.6 Partial Structures ofMalyngamide J by 1H-1H COSY and HETCOR. 63

Table 11.1 'H,'3C and HMBC NMR Data Of Malyngamide J (29)

C-atom '3C (CDC13) 'H (CDC13) for 1 HMBC 8 for 1 8 muti J in Hz 1', 3, 2 1 37.5 3.37 m 3.43 m 2 33.0 2.44 m 3, 4, 5 2.35 m 3 139.9 -- 4 117.6 5.22 s 5,2 5.28 s 5, 2, 3 5 62.3 6 194.2 7 136.1 8 136.9 6.42 bs 11, 10 9 70.4 4.68 bd (4.4) 7, 12, 5, 10 10 63.8 3.7 bs 8, 5, 9 11 16.5 1.88 s 6, 7, 8 12 103 4.48 d (7.4) 9, 17 17, 14, 12 13 82.8 3.02 dd (9.2, 7.4) 14 75.5 3.53 dd (10.6, 8.9) 13, 15 18, 14 15 78.9 3.25 dddm (5.0, 9.1,8.9) 16 63.4 3.15 bdd (8.9, 11.5) 14, 15, 12 4.04 dd (5.0, 11.5) 14, 12 13 17 58.9 3.6 s 15, 14 18 61.0 3.5 s 1' 172.7 - 2' 36.8 2.28 m 1', 3' 4' 3' 28.9 2.35 m 4' 5' 4' 131.2 5.48 m 3', 6' 5' 127.6 5.48 m 3', 6' 6' 36.6 2.18 m 5', 7' 7' 81.0 3.15 m 6', 15' 8' 33.4 1.43 m 9' 32.0 1.28 m 10' 28.9 1.28 m 11' 29.5 1.28 m 12' 30.0 1.28 m 13' 22.9 1.28 m 14' 14.3 0.89 t (6.7) 13', 12' 15' 56.7 3.32 s 7' NH 6.07 m 1' OH 2.78 bs 14, 15, 13 64

14'

O 17 H,co 12 O 13 14 15 16

3.32 5.25 OCH3 3.37 1.88 1.28 1.28 1.28 5.48 2.35 3.43 2.1: CH3 3 15 N 2A4 5.48 2.28 H 0.89 1.28 1.28 1.43 6.07 2.35 6.42

3.70 .68

3.6 Selected HMBC H3CO 4A8 3.02 3.53 325 1H NMR 4.04 HO 2.78 OCH3 3.50

56.7 117.6 OCH3 O 127.6 16.5 22.9 29.5 32.0 28.9 37.5 36.6 9\2 CH3 172.7 N 33.0 62.3 136.1 33.481 131.2 14.3 30.0 28.9 36.8 1 ;Z:g\,.136.9 70.4 0

13C NMR

63.4

OCH3 61

Figure 111.7 The Structureof Malyngamide J (29) with1H and 13CNMR Assignments by HMBCCorrelations. 65

Table 111.2 '1-1 and 13C NMR Date of Malyngamide L (30) from L. majuscula

C-atom 13C (CDC13) 1H (CDC13) 5 5 muti, J in Hz 1 44.0 3.94 d (6.1) 2 136.7 3 119.7 6.2 bs 4 136.5 5 198.6 6 38.6 2.54 ddd (6.5, 13.5) 7 22.8 2.08 ddd (6.5, 13.2, 10.3) 8 26.3 2.50 ddd (10.2, 4.3) 9 151.5 6.95 t (4.1) 1' 172.5 2' 36.6 2.24 dd (5.8, 7.5) 3' 29.5 2.32 m 4' 130.9 5.47 m 5' 127.9 5.47 m 6' 36.6 2.20 m 7' 80.9 3.15 dddd (5.8, 11.4) 8' 33.6 1.43 m 9' 32.0 1.28 m 1C' 25.5 1.28 m 11' 29.9 1.28 m 12' 30.0 1.28 m 13' 22.8 1.28 m 14' 14.3 0.88 mt 15' 56.7 3.34 NH 6.10 m

of the planar structure of malyngamide J (29) wasestablished. This is the first malyngamide-type natural product tobe found containing a sugar in the molecule. The relative stereochemistry of the dimethoxylated pentose sugar in compound 29 wasestablished through the analysis of the proton coupling constants and nOeobservations.The coupling constant between H-13 (53.02) andH-14 (53.53) was 9.2 Hz which indicated these two protons were diaxial (FigureIII.10).Similarly, the coupling constants 8.9 Hz between H-15and H-16a (53.15);10.6 Hz between H-14 and H-15 (53.25) gave theirdiaxial orientation, 66 while J15,16b = 5.0 Hz suggested that H-16b (84.04) is in the equatorial orientation.A cross peak between H-13 and H-15 in the NOESY confirmed their axial orientations.Although the observed coupling constant between H-12 and H-13 was 7.4 Hz, a nOecorrelation between H-16a, H-14 and H-12 (84.48) provided unambiguous evidence that H-12 was axial. Thus, an a-dimethoxylated xylose structure can be assigned.The absolute stereochemistry of the sugar was not investigated, but may be Dbased on the previously isolation of D-xylose from L. majuscula. A small coupling constant between H-9 and H-10 (1.7 Hz) required a dihedral angle between these protons to be around 60 °- 70°In combination with dihedral angle calculations from molecular modeling (Chem3D-Plus), two of the stereomeric models (A and B, Figure III.11) with dihedral angle 65° and 71° between 11-9 and H- 10 were best matched to the coupling constants based on Karplus's equations.Thus, a trans relationship between H-9 and H-10 was established. The absolute stereochemistry of the epoxycyclohexenone ring was determined by the observationof nOe between H-10 and H-4a, H-9 combined with the CD analysis.'Both diastereomers (A' and B', Figure 111.11) could reasonably display nOe between H-10 and H-4a. The negative first Cotton effect (MeOH Aa247 max = -12.5) in the CD spectrum can only be explained by conformation B', thus establishing the absolute stereochemistry of C-9, C-10 and C-5 to be R, S and S,respectively.Figure 111.12 shows the structures with stereochemistry of malyngamide J (29). 67

Dimethoxylated pentose ring of malyngamideJ (29) 0A 3.6 4.48 H3co3.02 12 O

3.53 3.15 4.04 HO 2.78 3.25 OCH3 3.50

Coupling constants

Figure Ill.10 Stereochemistryof Dimethoxylated Xylose Residue in Malyngamide J (29). 68

H 4 H

H H H H A B

Figure III.11Proposed CD and NOE of TwoMalyngamide J Configurations. 69

Compound 30 showed optical rotation [cc]D = -8.4° (c = 0.28, CHC13). HRFAB MS (positive ion, 3-nitrobenzyl alcohol) gave a [M+H]+ ion at m/z 424.2618 (0.0 mmu dev.) which was consistent with the molecular formula C24H38NO3C1.This molecular formula indicated six degrees of unsaturation.Examination of the UV (?max 223 nm, E = 8900), and "C NMR indicated the presence of an a, (3- unsaturated carbonyl (8198.6), an amide carbonyl (8172.5) and six olefinic carbons forming three double bonds (Table 111.2).Hence, this compound was monocyclic.Furthermore, the 'H NMR spectrum showed characteristics for a methoxylated fatty acid, a common feature of malyngamides.Analysis of the 'H -'H COSY (Figure 111.8) and 'I-1-13C HETCOR data and comparison with the spectral data of malyngamide F (25)' gave a full structural assignment of compound 30 (Figure 111.9). The previous studies of malyngamides from L. majuscula have shown that the stereochemistry of malyngamides at C-7' are always of th S configuration."From the same extract, the known metabolite malyngamide H (27) was isolated," which also contains a S configuration at C-7'.Biosynthetic considerations suggest that the 7-methoxytetradec-4-enoate moiety in malyngamide J (29) and malyngamide L (30) have the same S configuration at position C-7'. Malyngamide J showed brine shrimp toxicity with LC50 = 18 gg/m1 (ca.) and ichthyotoxicity with LC50 = 40 µg /ml, while malyngamide L showed brine shrimp toxicity with LC50 = 6 µg /m1 and ichthyotoxicity with LC50 = 7 µg /ml. 14'

4.0

5.0

6.0

7.9

I ' PPM 1.0 .5 0 !O 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 7.0 PPM

Figure 111.81H-1H COSY of Malyngmide L (30). 71

15'

13' 11' 9' 14' 12' 10'

3.34

0.88

56.7 OCH3 0 0 22.8 29.9 32.0 127.9 29.5 172.5 198.6 36.6N.... 136.7 38.6 80.9 N 3.94 14.3 30.0 25.5 33.6 130.9 2.24 H 119.7 22.8 13C NMR CI 26.3

Figure 111.9 The Structureof Malyngamide L (30) with1H and 13CData. ders

15(:) 73

Experimental

General Methods.Ultraviolet spectra were recorded on a Hewlett Packard 8452A diode arrayspectrophotometer, and infrared spectra (IR) were recorded on aNicolet 510P-RHS spectrophotometer.Nuclear magnetic resonance (NMR) spectra were recorded oneither Bruker AM 400 or Bruker AM 300 NMR spectrometer.All NMR chemical shifts are reported relative to an internaltetramethylsilane (TMS) standard and"C spectra referenced to the center line CDC13 at 77.25 ppm.CD measurements were obtained on a Jasco41A spectropolarimeter.Low resolution mass spectra (LRMS) wereobtained on a Varian MAT CH7 spectrometer, while high resolution massspectra (HRMS were obtained on a Kratos MS 50 TC. Highperformance liquid chromatography (HPLC) was performed using aM-6000 pump, U6K injector and either R 401 differentialrefractometer or a lambda- Max 480 lc spectrophotometer.TLC-grade (10-40 gm) silica gel was used for vacuum chromatography andKieselgel 60 silica (40-63 gm) was used for flashchromatography, and Merck aluminum-backed TLC sheets (silica gel 60 F254) were usedfor thin layer chromatography. All solvents were distilledfrom glass prior to use. Isolation of Malyngamide J (2 9).The fractionation of the crude extract was achieved by silicagel vacuum chromatography using a stepwise gradient from 4% to 100% (v/v)EtOAc in hexane (Figure

111.3).Two-dimensional TLC (EtOAc /hexane 1:1; CHC13/MeOH9:1) suggested the presence of severalUV-active secondary metabolites in fraction E, and its 'H NMR spectrumshowed interesting structural 74 features.Further fractionation was applied by gradientflash chromatography (MeOH /CHC13, 0%-2%) andcontinued with reversed-phase RP C-18 chromatography to remove mostof the pigments.The final purification was completed byHPLC (10-p.m Phenomenex Maxial Diol column,Me0H/Et0Ac/hexane 1/3/16) to yield malyngamide J (29, 14.5 mg,Figure 111.3). Isolation of Malyngamide L (3 0).The fractionation of the crude extract was followed by silica gel vacuumchromatography (EtOAc/hexane, 4%-100%, v/v).Two-dimensional TLC (EtOAc /hexane 1:1; CHC13/MeOH 9:1)suggested the presence of several UV-active compounds in fractionD (Figure 111.4).Further fractionation was provided by silica gelchromatography (85% EtOAc /hexane) followed by gradient flashchromatography (MeOH /CHC13, 0%-10%). The natural product wasthen isolated by using HPLC (10-pm Phenomenex MaxailDiol column, Me0H/Et0Ac/hexane 1.5/9/120) to yield a UV-activenatural product, named malyngamide L(30, 6.6 mg,Figure 111.4). Malyngamide J (2 9). UV (MeOH) 'max 240 nm(E = 5600); optical rotation [a]E, = +64° (c = 0.15,CHC13); CD (MeOH): DE = -12.5, +9.2 (Xmax 247, 217 nm); HR FABMS(positive ion, 3-nitrobenzyl alcohol) in/z obs. [M+H]+ 608.3798 (C33H54N09,0.0 mmu dev.); LR FABMS nilz obs. 608 (28), 448 (100), 416(26), 192 (17), 175 (25), 163 (15), 136 (13), 101 (24), 95(13), 87 (24), 81 (26), 75 (27), 69 (42), 59 (12), 55 (31), 45 (33);'11 NMR and 13C NMR data see Table 75

Malyngamide L (3 0). UV (MeOH) Xmax 223 nm (c = 8900); optical rotation [oc]D = -8.4° (c = 0.28, CHC13); HR FABMS(positive ion, 3-nitrobenzyl alcohol) m/z obs. [M+H]+ 424.2618(C24H39NO3C1, 0.0 mmu dev.); LR FABMS m/z obs. 424 (78),426 (32), 394 (20), 392 (57),388(8),307(17), 289 (15), 281 (14),220(15),186(10), 171(18),169(51),154(100), 150 (17), 138(30),136(82),133(10); NMR and "C NMR data see Table 111.2. Brine Shrimp Bioassay.See Chapter II. experimental. Ichthyotoxicity Assay.See Chapter II. experimental. 76

CHAPTER IV. CONCLUSION

Lyngbya majuscula has been recognized as achemically and biologically rich strain.This investigation gave a good representation showing the diversityof organic molecules produced by Lyngbya majuscula. From asingle collection at Playa Kalki, Curacao (11 August 1994), we haveisolated three novel bioactive secondary metabolites (Figure. IV).Although not investigated in this work, these metabolites mayplay an important ecological role in the survival of this species. Kalkitoxin has potent biologicalactivities which inspire us to further investigation of its biologicalproperties and mechanism of toxicities.Kalkitoxin also possesses a unique structurewith relatively simple chemical charactericsand small molecular weight. These features would be advantagesfor kalkitoxin to serve as a lead compound in drug developmentand as biochemical probes for the discovery of pharmacologicaland biochemical processes. The malyngamide class represents arich family of compounds, which offer a great deal ofinvestigative possibilities in areas such as algal physiology,chemo-defense, chemo-communication, and chemical ecology.These new malyngamides display very interesting chemical features.In particular, malyngamide J possesses a pentosering with a highly electro dense epoxycyclohexenone ring at one end,and a lipid portion at another end. A nitrogen-containingunit connects these two parts intothe molecule.These features impressed usbased on our knowledge and 77 experience about known drugs. Theseexciting features also motivated us to search and develop noveland sensitive techniques to best detect and uncovertheir full biological properties. Extract of Lyngbya majuscula from Playa Kalki, Curacao 4.32g Gradient Vacuum Chromatography EtOAc / Hexane 4%--100%

A B C F

NS 0 kalkitoxin 12.9 mg

O 0

malyngamide L 6.6mg

Figure IV.1The Chemical Diversity of Lyngbya majusculafrom Play Kalki, Curacao. 79

BIBLIOGRAPHY

1. Sondheimer, E.; Simeone, J. B. Chemical Ecology, 1970, Academic Press, New York

2. Dixon, S. E. Sea Technology, 1985, 19-23.

3. Youngken, H. W. J. Nat. Prod., 1969, 32, 407.

4. Scheuer, P. J. Sea Grant Quarterly, 1986, 8, 8.

5. Scheuer, P. J.,Marine Natural Products: Chemical and Biological Perspectives, 1978-1981, Vols. 1 to 4.

6. Faulkner, D. J.; Fenical, W. H., Eds., Marine NaturalProducts Chemistry, 1977, Plenum, New York.

7. Faulkner, D. J. Tetrahedron, 1977, 33, 1421

8. Crouch, R. C.; Martin, G. E. J. Nat. Prod., 1993, 55,1343.

9. Martin, G. E.; Crouch, R. C. J. Nat. Prod., 1991, 54, 1.

10. Murata, M.; Naoki, H.; Iwashita, T; Matsunaga, S.;Sakaki, M.; Yokoyama, A.; Yasumoto, T. J. Am. Chem. Soc., 1993,115, 2060.

11.Arasaki, S.; Arasaki, T. Low Calorie, High NutritionVegetables from the Sea to help you look and feelbetter, Japan Publications, Inc., Tokyo, 1983, p 196.

12.Scheuer, P. J. Med. Res. Rev., 1989, 9, 535.

13. Hashimoto, K. Marine Toxins and Other Bioactive Marine Metabolites, Japan Scientific Societies Press, Tokyo.1959, p 369.

14. Okaichi, T.; Hashimoto, Y. Physiological action ofnereistoxin, Nippon Suisan Gakkaishi, 1962, 28: 930-935. 80

15. Ireland C, Copp B, Faster M, McDondaldL, Radisky D, Swersey J., Marine biotechnology, Vol. 1:Pharmaceutical and bioactive natural products. New York,Plenum Press.1993, p 1-43.

16.Gerwick, W. H.; Fenical, W., Journalof Organic Chemistry, 1981, 46:22-27.

17.Jacobs R. S.; Culver, P.; Landon,R.; O'Brien, T.; White, S., Marine Biotechnology, Vol. 1,Pharmacological atudies of novel marine metabolites. p 7-99.

18.Fuller, R. W., et al. Journal ofMedicinal Chemistry, 1994, 37 : 4407-4411.

19.Burres, NS; Clement, J., CancerResearch, 1989, 49 :5267- 5274.

20.Pettit, G. R.; Collins, J. C.; Herald,D. L.; Doubek, D. L.; Boyd, M. R., Canadian Journal of Chemistry,1992, 70: 1170-1175.

21.Moore, R. E.; Scheuer, P. J. Science,1971, 172, 495.

22.Maeda, M.; Kodama, R.; Tanaka, T.;Yoshizumi, H.; Nomoto, K.; Takemoto, T.; Fujita, M. Symposium,Papers, 27th Symposium on the Chemistryof Natural Products, Hiroshima,1985, p 616.

23.Yasumoto, T.; Yasumura, D.; Ohizumi,Y.; Takahashi, M.; Alcala, A. C.; Agric. Biol. Chem., 1986,50, 163.

24.Hashimoto, K. Marine Toxins andOther Bioactivie Marine Metabolites, Japan Scientific SocietiesPress: Tokyo, 1979.

25.Usami, M.; Satake, M.; Ishida,S.; Inoue, A.; Kan, Y.; Yasumoto,Y. J. Am. Chem. Soc., 1995, 117,5389-5390.

26.Petti, G. R.; Singh, S. B.; Hogan,F.; Lloyd-Williams, P.; Herald, C. L.; Burkett, D. D. J. Am. Chem.Soc., 1989, 111: 5463-5465. 81

27.Rinehart, K. L.; Shield, L. S.; Cohen-Parsons,M. 1993, Antiviral Substances, in Attaway D., Zaborsky, 0.Eds. Marine bioactive natural biotechnology .Vol. 1: Pharmaceutical and products, New York: Plenum Press.

28.Davison, B. S. Chemical Reviews,1993, 93: 1771-1791.

29. Graneli, E.; Sundstrom, B.; Edler,L.; Anderson, D. M., Eds. Toxic MarinePhytoplankton I, New York, 1989.

30.Jachin, E.; Gentile, J. Science,1968, 162, 915.

31.Alam, M.; Shimizu, Y.; Ikawa,M.; Sasner, J. J., J. Environ. Sci. Health, 1978, A13, 493.

32.Yasunoto, T.; Yasumura, D.; Yotsu,M.; Michishita, T.; Endo, A.; Kotaki, J. Agric. Biol. Chem.,1986, 50, 793.

33.Simidu, U.; Kita-Tsukamoto, K.;Yasumoto, T.; Yotsu, M. Int. J. System.Bacteriol., 1990, 40, 331.

34.Noguchi, T.; Jeon, J. K.; Arakawa,O.; Sugita, H.; Deguchi, Y.; Shida, Y.; Hashimoto, K. J. Biochem.,1986, 99, 311.

35. (a) Murakami, Y.; Oshima, Y.;Yasumoto, T. Nippon Suisan Gakkaishi, 1982, 48, 69. (b) Kumagai,M.; Yanagi, T.; Murata, M.; Yasumoto, T.; Kat, M.; Lassua,R.; Rodoriguez-Vazquez, J. A. Agric. Biol. Chem., 1986, 50,2853-2857.

36.Tachibana, K.; Scheuer, P. J.; Tsukitani,Y.; Kikuchi, H.; Engen, D. V.; Schmitz, F. J. J. Am. Chem.Soc., 1981, 103: 2469.

37. Sassa, T.; Richter, W. W.; Uda,N.; Suganuma, M.; Suguri, H.; Yoshizwa, S.; Hirota, M.; Fujiki,H. Biochem. Biophys. Res. Commun., 1989, 159: 939.

38. (a) Yasumoto, T.; Nakajima,L.; Ohshima, Y.; Bagnis, R. Toxic Dinoflagellate Blooms; Taylor, D.L.; Seligner, H. Eds.; Elsevier: North-Holland, 1979, p 65. (b) Legrand,A. M.; Litaudon, M.; 82

Genthon, J. N.; Bagnis, R.; Yasumoto, T. J. Appl. Phycol., 1989, 1, 183.

39.Yokoyama, A.; Murata, M.; Oshima, Y.; Iwashita, T.; Yasumoto, T. J. biochem., 1988, 104, 6594.

40.Murata, M.; Naoki, H.; Iwashita, T.; Matsunaga, S.; Sasaki, M.; Yokoyama, A.; Yasumoto, T. J. Am. Chem. Soc., 1993,115, 2060.

41.Lin, Y. Y.; Risk, M.; Ray, S. M.; Van Engen, D.; Clardy, J.; Golik, J.; James, J. Am. Chem. Soc., 1981, 103, 6773-6775.

42. Shimizu, Y.; Gupta, S.; Norte, A.; Genenah, A.; Kobayashi, M. Proc. Int. Conf. on Toxic Dinofagellates, 1985, 3, 271.

43.Nakanishi, K. Toxicon, 1985, 23, 473-479.

44.Trainer, V. L.; Edwards, R. A.; Szmant, A. M.; Stuart, A. M.; Mende, T. J.; Baden, D. G. Brevetoxins: Unique Activators of Voltage-Sensitive Sodium ChannelsIn Marine Toxins: Origin, Structure, and Molecular Pharmacology (ACS Symposium Series 418), Hall, S. and Strichartz, G., Eds., American Chemical Society, Washington, DC, 1990, p 166.

45.Biolojan, C.; Takai, A. Biochem. J., 1988, 256, 283-290.

46.Haystead, T. A. J.; Sim, A. T. R.; Carling, D.; Honnor, R. C.; Tsukitani, Y. Nature, 1989, 227, 78.

47.Ragelis, E. P., Ed., Seafood Toxins, ACS Symposium Series 262; American Chemical Society: Washington, DC, 1984.

48.Hall, S.; Strichartz, G., Eds., Marine Toxins, ACS Symposium Series 418, American Chemical Society, Washington, DC, 1990.

49.Moore, R. E.; Patterson, G. M. L.; Carmichael, W. W. New pharmaceuticals from cultured blue-green algae, In Fautin, D. G. Ed., Biomedical Importance of Marine Organisms. Calif. Acad. Sci., 1988,13:: 143-150. 83

50.Moore, R. E. Marine Natural Products : Chemicaland Biological Perspectives, Vol. 4. Academic Press, New York,1981, p 1-52.

51.Rinehart, K. L.; Namikoshi, M.; Choi, B. W. J.Appl. Phycol., 1994, 6,159.

52.Patterson, G. M. L.; Larsen, L. K.; Moore, R.E. J. Appl. Phycol., 1994, 6,151.

53.Munro, M. H. G.; Luibrand, R. T..; Blunt, J.W. In Bioorganic MarineChemistry, Scheuer, P. J., Ed.; Spring-Verlag:Berlin Heidelberg, 1987, p 94-176.

54.Moore. R. E.; Patterson, G. M. L.;Carmichael, W. W. In Biomedical Importance of Marine Organisms;Fautin, D. G. Ed.; CaliforniaAcademy of Science, San Francisco, 1988, p143- 151.

55.DeSilva, E. D.; Willams, D E.; Andersen, R. J.;Klix, H.; Holmes, C. F. B.; Allen, T. M. Tetrahedron Lett.,1992, 33, 1561.

56.Rinehart, K. L.; Namikoshi, M.; Choi, B. W.J. Appl. Phycol., 1994, 6,159.

57.Krishnamurthy, T.; Carmichael, W. W.; Sarver,E. W. Toxicon, 1986, 24, 965.

58.Harada, K.; Mayumi, T.; Shimada, T.;Suzuki, M.; Kondo, F.; Park, H.D.; Watanabe, M. F. Tennen Yuki KagobutsuToronkai Koen Yoshishu, 1993, 35, 377, and referencestherein.

59.Boyd, Cardellina, Carmeli, Moore &Patterson, unpublished observations.

6 0 Prinsep, M. R.; Moore, R. E.; Levine, I. A.;Patterson, G. M. L. J. Nat. Prod., 1992, 55,140-142.

6 1 Carmeli, S.; Moore, R. E.; Patterson, G. M. L.;Corbett, T. H. J. Am. Chem. Soc., 1990, 112, 8195-8197. 84

62. Prinsep, M. R.; Caplan, F. R.; Moore, R. E.;Patterson G. M. L.; Smith, C. D. J. Am. Chem. Soc., 1992,114, 385-387.

63.Kniibel, G.; Larsen, L. K.; Moore, R.E.; Levine, I. A.; Patterson, G. M. L. J. Antibiot., 1990, 43,1236-1239.

64. Koehn, F. E.; Longley, R. E.; Reed, J.K. J. Nat. Prod., 1992, 55, 613-619.

65.Gerwick, W. H.; Proteau, P. J.; Nagle,D. G.; Hamel, E.; Blokhin, A.; Slate, D. L. J. Org. Chem.,1994, 59, 1243-1245.

66.Carmichael, W. W.; Mahmood, N. A.;Hyde, E. G. In Marine Toxins; Hall, S., Strichartz, G., Eds.;ACS Symposium Series, American Chemical Society: Washington,DC, 1990, p 87-106.

67. Rinehart, K. L.; Harada, K.; Namikoshi,M.; Chen, C.; Harvis, C. A.; Munro, M. H. G.; Blunt, J. W.;Mulligan, P. E.; Beasley, V. R.; Dahlem, A. M.; Carmichael, W. W.J. Am. Chem. Soc., 1988, 110. 8557.

68.Carmichael, W. W. in Handbook ofNatural Toxins; Tu, A. T. Ed.; Marcel Dekker: New York, 1988,Vol. 3. p 121-147.

69.Hemscheidt, T.; Puglisi, M. P.; Larsen,L. K.; Patterson, G. M. L.; Moore, R. E.; Rios, J. L.; Clardy, J.,J. Org. Chem., 1994, 59, 3467.

70.Bonjouklian, R.; Smitka, T. A.; Doolin,L. E.; Molloy, R. M.; Debono, M.; Shaffer, S. A.; Moore,R. E.; Steewart, J. B.; Patterson, G. M. L. Tetrahedron,1991, 47, 7739.

71.Ohtani, I.; Moore, R. E.; Runnegar,M. T. C., J. Am. Chem. Soc., 1992, 114, 7941.

72.Cardellina, J. H.; Marner, F. J.;Moore, R. E. Science, 1979, 204, 193.

73.Fujidi, H.; Sugimura, T.Advances in Cancer Research, 1987, 59, 223. 85

74.Moore, R. E. Pure Appl. Chem., 1982, 54, 1919.

75.Gerwick, W. H.; Jiang, Z. D.; Agarwal, S. K.;Farmer, B. T. Tetrahedron, 1992, 48, 2313.

76.Orjala, J.; Gerwick, W. H. J. Nat. Prod., 1996,59, 427.

77. Orjala, J.; Nagle, D. G.; Hsu, V. L.; Gerwick,W. H. J. Am. Chem. Soc. 1995, 117, 8281.

78.Hamel, E.; Blokhin, A. V.; Nagle, D. G.; Yoo,H-D.; Gerwick, W. H. Drug Dev. Res., 1995, 34, 110.

79.Blokhin, A. V.; Yoo, H-D.; Geralds, R.; Nagle, D.G.; Gerwick, W. H.; Hmael, E. Molecular Pharmacology,1995, 48, 523-531.

80. Marner, F. J.; Moore, R. E.; Hirotsu, K.;Clardy, J. J. Org. Chem., 1977, 42, 2815.

81.Yoo, H. D.; Gerwick, W. H. "MicrocolinC, a new cytotoxic lipopeptide from the blue-green alga Lyngbyamajuscula", manuscript in preparation.

82.Meyer, B. N.; Ferrigni, J. E.; Putnam, J. E.;Jacobson, L. B.; Nichols, D. E.; McLaughlim, J. L. Planta Med.,1982, 45, 31.

83.Hostettmann, K.; Kizu, H.; Tomimori, T. PlantaMed., 1982, 44, 34.

84. Backus, G. J.; Green, G. Science, 1974, 24,639.

85.Hawkins, C. J.; Levin, M. F.; Marshall, K. A.;Watters, D. J. J. Med. Chem., 1990, 33, 1634.

86.Jalal, M. A. F.; Hossain, M. B.; van derHelm, D.; Sanders-Loehr, J.; Actis, L. A.; Crosa, J. H. J. Am. Chem.Soc., 1989, 111, 292.

87.Marston A.; Hostettmann, H. Phytochemistry,1985, 24, 639 86

88.Anderson, J. E.; Goetz, C. M.;Mclaughlin, J. L. Phytochemical Analysis, 1991,2, 107 and Brine 89.McLaughlin, J. L. Crown GallTumors on Potato Discs Shrimp Lethality: TwoSimple Bioassays for HigherPlant Screening and Fractionation.In Methods in Plant Biochemistry, Hostettmann, K.Ed.' Academic Press Inc.,San Diego, 1991, pp. 8-32.

90.Marston, A.; Hostettmann,H. Mulluscicidal,Cercaricidal, Schistosomicidal and PiscicidalActivities, In Methods in Plant Biochemistry, Hostettmann,K. Ed., Academic PressInc., San Diego, 1991, pp. 153-78. Biological 91.Moore, R. E. Marine NaturalProducts: Chemical and Perspectives, Vol. IV, 1981,Academic Press Inc, p 34

92.Loui, M. S. M.; Moore,R. E. Phytochemistry,1979, 18, 1733. J. S.; 93.Cardellina II, J. H.; Dalietos,D.; Marner, F-J.; Mynderse, Moore, R.E. Phytochemistry,1978, 17, 2091. Chem. Soc., 94. Cardellina II, J. H.; Marmer,F-J.; Moore, R. E. J. Am. 1979, 101, 240. Mynderse, 95.Ainslie, R. D.; Barchi, J. J.;Kuniyoshi, M.; Moore, R. E.; J. S. J. Org. Chem.,1985, 50, 2859. 43, 22, 96.Mynderse, J. S.; Moore, R.E. J. Org. Chem., 1978, Vol. 4359. 1987, 97.Gerwick, W. H.; Reyes, S.;Alvrado, B. Phytochemistry, Vol. 26. 6, 1701.

98.Praud, A.; Valls, R.; Piovetti,L.; Banaigs, B. Tetrahedron Letters, Vol. 34, 34,5437. Vol. 58, 99.Orjala, J.; Nagle, D.; Gerwick,W. H. J. Nat. Prod., 1995, 5, 764. 87

100. Todd, J. S.; Gerwick, W. H. Tetrahedron Letters, 1995,Vol. 36, 43, 7837.

101. Harada, N.; Nakanishi, K., Circular DichroicSpectroscopy; Exiton Coupling in Organic Stereochemistry, UniversityScience Books, Mill Valley, Ca., 1983, pp 1-460. 88

APPENDIX RESULT 102.41

100.0--

95.0-

vxu J

80.0 --

i i II i i r i 1000 i i 15100 75.33 t i 2000 3000 2500 4000 3500 700.0 WAVENUMBERS

Figure A.2 IR Spectrumof 15 IN ONO 206K HNSC

cz. o as. 20

CZ 0. coo 40 lea 6°

111 60 0. 00 6,3 1:1114

100

0=1.0 120

f40 0 0 ad EX)

160 4

0100 I lop cO(:)0

'16 0 0 ED ... 160 PPM 2.5 2.0 5.0 4.5 4.0 3.5 3.0 6.0 5.5 PPM

Figure A.3 HMBC Spectrum of 15 inD6 -DMSO at 298K Figure A.4111 NMR Spectrum of 15 in C6D6 IN OMSO 340K

I 80

100

Is *41 120

140

160

si ' 11!"? °eel st1 1 °C;) 180

I ' I PPM 2.0 1.5 1.0 5.0 4.5 4.0 3.5 3.0 2.5 6.0 5.5 PPM

Figure A.S. HMBC Spectrum of15 in D6 -DMSO at 340K 80 70 60 50 40 30 20 170 160 150 140 130 120 110 100 90 PPM

Figure A.6DEPT (135) Spectrum of 15 in D6 -DMSO at 298K MNI1213F3 IN DMSO 2116K

1 7 ' r 1 40 30 20 10 170 160 150 140 130 120 110 100 PPM60 60 70 60 50

Figure A.7 DEPT (90) Spectrum of 15 in D6 -DMSO at298K IN OMSO 340K

00 70 60 b0 40 30 20 10 150 140 130 120 110 100 90 170 160 PPM

Figure A.8 DEPT (135) Spectrumof 15 in D6-DMS0 at 340K II

i 6.5 6.0 5!5 5.0 4.5 4.0 3.5 3.0 2.5 PPM

Figure A.9'H Decoupling Spectrum-1 of 15 in C6D6 di

3.5 3.0 2.5 2.0

Figure A.10'H Decoupling Spectrum-2 of 15 in C6D6 RESULT 103.01

100.0--

63.0-

80.0

I f I I i t t i F I 76.30 i I I T i 20.00 1500 1000 4000 3500 3000 2500 700.0 WAVENUMBERS

Figure A.11IR Spectrum of 29 100_

ea_

69 40_ 175 45 101 55 416 87

75 81 163 192 S9 9 136 147 I

, , , , -1 Y r11'1rIlijkI. , . 1.1 . , I,4 )1 , '' 0 11111 111, 11111111i11,111,111tiliniiillhillitdirlITA,J111111111, tilb-rdi l'ir'll'Iir"Thrt'tli"r -r let Y.1' t'. ( .,,-, i t 11111,1,1 400 300 350 100 200 250 50 IR

I00 448

ea_

608

434 592 624 464 476 576 t. I 0 " I 111'11 to I ti 'It II 650 7e0 550' seo 450 500

Figure A.12 LRFAB MassSpectrum of 29 15

10-

-5-

-10--

-15 iiiiiiiiiiIIIIIIIIIiiiiiiIIII320 330 340 350 300 310 200 210 220 230 240 250 260 270 280 290

Figure A.13 CD Spectrum of 29 91 11 9 9 9

50 40 30 20 10 100 90 80 70 60 150 140 130 120 110 200 190 180 170 160 PPM

Figure A.1413C NMR Spectrum of 29 MW//59FC.101 DATE 29-3-96 SF 400.134 SY 133.0 01 6400.000 SI 16384 TO 16384 SW 6024.96.735 HZ/PT PW 3.0 RD 2.000 AO 1.360 AG 20 NS 32 TE 298 FM 7600 02 0.0 DP 63L PO

L8 0.0 GB 0.0 CX 35.00 CY 17.00 F1 7.000P F574..3104 0P HZ /CM PPM/CM .186 SR 4390.25

f' Jif

2 )

30 2.5 2.0 1.5 1.0 5.5 5.0 4.5 4.0 3.5 5.5 6.0 PPM

Figure A.15'H NMR Spectrum of 29 20 O

9 0 40

100

I o '.0

,.. 120

140

180

0 160

1.0 PPM 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 PPM

Figure A.16 HMBC Spectrumof 29 II iil I II

1.0

Ia ill 1.5

2.0

2.5 I

3.0 14* 3.5 1 I .04 4.0 a 4.5

5.0

5.5 it 6.0

6.5

PPI4 80 60 40 2'0 140 120 100 200 180 160 PPI4

Figure A.17 BETCORSpectrum of 29 1 1

30 20 10 90 80 70 60 50 40 150 140 130 120 110 100 200 190 180 170 160 PPM

Figure A.18 DEPT (135 and90) Spectrum of 2 9 )

2.0 1.5 1.0 4.5 4.0 3.5 3 0 2.5 PPM

Figure A.19 NOE DifferenceSpectrum-1 of 29 1 I 1 1 1.5 1.0 4.5 4.0 3.5 3.0 2.5 2.0 6.5 6.0 5.5 5.0 PPM

Figure A.20 NOE Difference Spectrum-2of 29 1.0 .5 3.0 2.5 2.0 1.5 5.0 4.5 4.0 3.5 6.5 6.0 5.5 PPM

Figure A.21 NOE DifferenceSpectrum-3 of 29 1 4!5 4.0 3.5 3.0 2.5 2.10 PPM

Figure A.22 NOE DifferenceSpectrum-4 of 29 - 1.0

- 1.5

2.0

2.5

3.0

_ 3.5

- 4.0

4.5

- 5.0

- 5.5

- 6.0

- 8.5

1 PPM 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 PPM

Figure A.23 NOESY of 29 1 1 1 10' 60 70 60 50 401 301 220 160 150 140 130 120 110 100 90 200 190 1B 10 170 PPM

Figure A.2413C NMR Spectrum of 3 0 MWII70F3.201 DATE 26-4-96

SF 400.134 SY 133.0 01 6400.000 SI 16384 TD 16384 SW 6024.96 HZ/PT .7035 PM 3.0 RD 2.000 AG 1,360 AG 80 NS 32 TE 290 FM 7600 02 0.0 DP 63L PO

LB 0.0 GB 0.0 CX 35.00 CY 18,00 FI 7,492P F2 -.152P HZ/CM87.382 PPM/CM .218 SR 4394.37

r 3.0 2.5 2.0 1.5 1.0 .5 0.0 5.5 5.0 4.5 4.0 3.5 7.0 6.5 6.0 PPM

Figure A25 1H NMR Spectrumof 30 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 7 0 6.5 6.0 PPM

Figure A26 1H-'HCOSY of 30 1 - 1.0

- 2.0

_ 3.0

o o

4 _ 4.0

.. 5.0

_ 6.0

- 7.0 t

1 ; PPM 60 40 20 200 180 160 1140 120 100 80 PPM

Figure A.27 HETCOR Spectrum of 30 154

135

169

186

143 150 71 337 281 289 133 165 192

1111111 11 II j11 )11111i lill'itillAINIP1'1'1101.111WIli't111.1'k , r 1+11'1'1,.1.1111I ir r -,1% Ir11'ti it Nkk, Y , , 320 340 140 160 183 200 220 240 260 283 320

424

392

426

394

446 388 356 363 371 II 8 461 I 4°2 f t1111,-1, r f17 P 1 rt1,t11-tt II ih(111?(11(111(11 1,11111111111111111111 360 380 400 420 440 460 483 500 20 540 560

Figure A.28 LRFAB Mass Spectrum of 3 0