UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Bioactive marine natural products Desjardine, Kelsey Lorne 2007

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-ubc_2007-317617.pdf [ 8.33MB ]
Metadata
JSON: 831-1.0228812.json
JSON-LD: 831-1.0228812-ld.json
RDF/XML (Pretty): 831-1.0228812-rdf.xml
RDF/JSON: 831-1.0228812-rdf.json
Turtle: 831-1.0228812-turtle.txt
N-Triples: 831-1.0228812-rdf-ntriples.txt
Original Record: 831-1.0228812-source.json
Full Text
831-1.0228812-fulltext.txt
Citation
831-1.0228812.ris

Full Text

BIOACTIVE MARINE NATURAL PRODUCTS by KELSEY LORNE DESJARDINE B.Sc. The University of Manitoba, 2001 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA September, 2007 © Kelsey Lome Desjardine, 2007 A B S T R A C T The chemical exploration of extracts from cultures of the marine bacterial isolate PNG-276 yielded the novel antibiotic tauramamide (2.13), a non-ribosomal peptide active against cultures of Enterococcus sp. and methicillin-resistant Staphylococcus aureus (MRSA). A study of extracts of the marine sponge Spirastrella coccinea yielded the novel macrolide methylspirastrellolide C (3.14), which is active against protein phosphatase 2A (PP2A). A third study examined sponge extracts active in a cannabinoid receptor assay, yielding two known compounds, an A-nor-steroid derivative (4.10) and bengamide A (4.11). Neither purified compound was active in the cannabinoid receptor assay, although in both cases this is the first report of these compounds being isolated from Stylissa massa and Hemiasterella aff. affinis sponges, respectively. 4.11 3.14 ii TABLE OF CONTENTS Abstract ii Table of Contents iii List of Tables vi List of Figures vii List of Abbreviations xiv Acknowledgements xvii Dedication xviii Chapter 1: Marine Natural Products Chemistry 1 1.1 Marine Natural Products Chemistry: A Multidisciplinary Approach 1 1.2 Drugs From The Sea 2 1.3 Marine Microorganisms as an Emerging Resource 7 1.4 Marine Sponges as a Productive Resource 10 1.5 Antibiotic Resistance 13 1.6 Cancer Chemotherapeutics 15 1.7 Cellular Signaling/Cannabinoid Receptors 18 1.8 Summary 19 Chapter 2: Tauramamide, A Novel Antibacterial Lipopeptide from the Marine Bacterial Isolate PNG-276 21 2.1 Introduction 21 2.2 Isolation and Characterization of Tauramamide Methyl Ester 29 2.2.1 Isolation of Tauramamide Methyl Ester 30 2.2.2 Structure Elucidation of Tauramamide Methyl Ester 31 2.2.3 LSIMS Information 48 2.2.4 Comments on Tauramamide Methyl Ester 49 2.3 Isolation and Characterization of Tauramamide Ethyl Ester 50 2.3.1 Isolation of Tauramamide Ethyl Ester 50 2.3.2 Structure Elucidation of Tauramamide Ethyl Ester 51 2.3.3 Comments on Tauramamide Ethyl Ester 60 2.4 Configurational Information 60 2.5 Synthesis of Tauramamide Ethyl Ester 62 2.6 Biological Activity of Tauramamide 67 2.7 Discussion and Conclusions 69 2.8 Experimental 72 2.8.1 General Experimental Procedures 72 2.8.2 Culture Conditions for PNG-276 73 2.8.3 Isolation of Tauramamide Methyl Ester 73 2.8.4 Tauramamide Methyl Ester Physical Data 74 2.8.5 Acid Hydrolysis of Tauramamide Methyl Ester 75 2.8.6 Preparation of FDAA Derivative Standards 75 2.8.7 Marfey's Analysis of Tauramamide Methyl Ester 76 2.8.8 Isolation of Tauramamide Ethyl Ester 76 2.8.9 Tauramamide Ethyl Ester Physical Data 77 2.8.10 Synthetic Tauramamide Physical Data 77 2.8.11 Synthetic Tauramamide Ethyl Ester Physical Data 77 Chapter 3: Methylspirastrellolide C, an Antimitotic Macrolide Isolated from the Marine Sponge Spirastrella coccinea 78 3.1 Introduction 78 3.2 Isolation and Characterization of Methylspirastrellolide C . . . 90 3.2.1 Isolation of Methylspirastrellolide C 90 3.2.2 Subsequent Isolation of Methylspirastrellolide C 92 3.2.3 Structure Elucidation of Methylspirastrellolide C 95 3.2.4 Relative Configuration of Methylspirastrellolide C 116 3.3 Biological Activity of Methylspirastrellolide C 126 3.4 Discussion and Conclusions 127 3.5 Experimental 129 3.5.1 General Experimental Procedures 129 iv 3.5.2 Initial Isolation of Methylspirastrellolide C 130 3.5.3 Second Isolation of Methylspirastrellolide C 131 3.5.4 Methylation of Spirastrellolide Mixture 132 3.5.5 Chromatography of Spirastrellolides 132 3.5.6 Methylspirastrellolide C Physical Data 133 Chapter 4: Cannabinoid Receptor Studies 134 4.1 Cannabinoids and Cannabinoid Receptors 134 4.2 A-nor-steroids 141 4.2.1 Isolation of A-nor-steroids from Stylissa massa (Carter, 1881) 141 4.2.2 Structure Elucidation of A-nor -C 2 7 A 2 2 Derivative 143 4.2.3 Biological Activity of A-nor-steroid 157 4.3 Bengamide A 158 4.3.1 Isolation of Bengamide A from Hemiasterella aft. affinis. 158 4.3.2 Structure Elucidation of Bengamide A 160 4.3.3 Biological Activity of Bengamide A 169 4.4 Future Directions with the CB Assay 170 4.5 Experimental 172 4.5.1 General Experimental Procedures 172 4.5.2 Isolation of A-nor-steroid 173 4.5.3 Derivatization of A-nor-steroid 173 4.5.4 A-nor-steroid Physical Data 174 4.5.5 Isolation of Bengamide A 174 4.5.6 Bengamide A Physical Data 175 Bibliography 176 Appendix I R O E S Y NMR spectrum of tauramamide methyl ester, recorded at 500 MHz in DMSO-afe, showing inter-residue correlations 187 Appendix II LSIMS spectrum of tauramamide methyl ester 188 v LIST OF TABLES Table 2.1 1D and 2D NMR data for tauramamide methyl ester (2.14), recorded at 500 MHz (1H) and 100 MHz ( 1 3C) in DMSO-cfe 38 Table 2.2 1D and 2D NMR data for tauramamide ethyl ester (2.15), recorded at 500 MHz (1H) and 100 MHz ( 1 3C) in DMSO-ofe 58 Table 2.3 Retention times (minutes) of FDAA-modified standards and derivatized tauramamide methyl ester acid hydrolysate 61 Table 2.4 Retention times (minutes) of FDAA-modified standards and derivatized tauramamide methyl ester acid hydrolysate from second hydrolysis 61 Table 2.5 Bioassay data (MIC) from both tauramamide (2.13) and tauramamide ethyl ester (2.15) 68 Table 3.1 1D and 2D NMR data for methylspirastrellolide C (3.14) recorded at 600 MHz (1H) and 150 MHz ( 1 3C) 104 Table 4.1 1D and 2D NMR data for A-nor-C2i^22 derivative (4.10) recorded at 600 MHz (1H) in C 6 D 6 149 Table 4.2 1D and 2D NMR data for bengamide A (4.11) recorded at 600 MHz (1H) and 150 MHz ( 1 3C) in CDCI 3 166 vi LIST OF FIGURES Figure 1.1 A selection of drugs from terrestrial sources 3 Figure 1.2 Structures of Ara-C, spongothymidine, spongouridine and ziconotide 4 Figure 1.3 Structures of ET-743, pseudopterosin A and E 5 Figure 1.4 Proportion of compounds in Marinlit database, grouped by phyla of origin 6 Figure 1.5 Structures of some drugs derived from terrestrial microorganisms 8 Figure 1.6 Structures of azamarone and actinofuranones A and B 9 Figure 1.7 Structure of guangamides A and B 10 Figure 1.8 Examples of compounds isolated from marine sponges 11 Figure 1.9 Structures of manoalide and discodermolide 12 Figure 1.10 Some major classes of antibiotics currently in use 14 Figure 1.11 Structures investigated for anticancer activity 17 Figure 1.12 Structure of desmethyleleutherobin 18 Figure 1.13 Structures of anandamide and (-)-A 9-tetrohydrocannabinoL 19 Figure 2.1 Marinomycin A, salinosporamide A and omuralide 22 Figure 2.2 Massetolide D, holyrine A and staurosporine 23 Figure 2.3 Loloatin A 24 Figure 2.4 Bogorol A 25 Figure 2.5 Basiliskamides A and B, and tupuseleiamides A and B 26 Figure 2.6 Schematic of peptide synthesis in a typical N R P S 27 Figure 2.7 Structure of tauramamide 30 Figure 2.8 Structure of tauramamide methyl ester 31 Figure 2.9 500 MHz 1 H NMR spectrum of tauramamide methyl ester recorded in DMSO-c/ 6 32 Figure 2.10 500 MHz 1 3 C NMR spectrum of tauramamide methyl ester recorded in DMSO-Oe 33 vii Figure 2.11 500 MHz C O S Y NMR spectrum of tauramamide methyl ester recorded in DMSO-d 6 34 Figure 2.12 500 MHz R O E S Y NMR spectrum of tauramamide methyl ester recorded in DMSO-d 6 35 Figure 2.13 500 MHz HMQC NMR spectrum of tauramamide methyl ester recorded in DMSO-cfe 36 Figure 2.14 500 MHz HMBC NMR spectrum of tauramamide methyl ester recorded in DMSO-c/ 6 37 Figure 2.15 Expansion of C O S Y NMR spectrum of tauramamide methyl ester, showing H a -NH correlations 39 Figure 2.16 C O S Y NMR spectrum, proton and carbon assignments, as well as select 2D correlations for arginine residue in tauramamide methyl ester 41 Figure 2.17 C O S Y NMR spectrum, proton and carbon assignments, as well as select 2D correlations for tryptophan residue in tauramamide methyl ester 43 Figure 2.18 C O S Y NMR spectrum, proton and carbon assignments, as well as select 2D correlations for leucine residue in tauramamide methyl ester 44 Figure 2.19 C O S Y NMR spectrum, proton and carbon assignments, as well as select 2D correlations for serine residue in tauramamide methyl ester 46 Figure 2.20 C O S Y NMR spectrum, proton and carbon assignments, as well as select 2D correlations for tyrosine residue in tauramamide methyl ester 47 Figure 2.21 Fragments observed in LSIMS spectrum of tauramamide methyl ester 49 Figure 2.22 Constitution of tauramamide ethyl ester 51 Figure 2.23 600 MHz 1 H NMR spectrum of tauramamide ethyl ester recorded in DMSO-d 6 52 viii Figure 2.24 150 MHz 1 3 C NMR spectrum of tauramamide ethyl ester recorded in DMSO-cfe 53 Figure 2.25 600 MHz C O S Y NMR spectrum of tauramamide ethyl ester recorded in DMSO-c/ 6 54 Figure 2.26 600 MHz R O E S Y NMR spectrum of tauramamide ethyl ester recorded in DMSO-afe 55 Figure 2.27 600 MHz HSQC NMR spectrum of tauramamide ethyl ester recorded in DMSO-cVe 56 Figure 2.28 600 MHz HMBC NMR spectrum of tauramamide ethyl ester recorded in DMSO-c/ 6 57 Figure 2.29 Structure of tauramamide ethyl ester showing selected inter-residue correlations 59 Figure 2.30 Structure of tauramamide with amino acid configurations shown 62 Figure 2.31 Synthetic scheme for tauramamide: synthesis of the first subunit 63 Figure 2.32 Synthetic scheme for tauramamide: synthesis of the second subunit, final assembly and deprotection 64 Figure 2.33 Comparison of synthetic and natural tauramamide ethyl ester 1 H NMR spectra (600 MHz, recorded in DMSO-d 6 ) 66 Figure 3.1 The eukaryotic cell cycle 78 Figure 3.2 Docetaxel and paclitaxel 79 Figure 3.3 Vincristine and vinblastine 80 Figure 3.4 Discodermolide 81 Figure 3.5 Eleutherobin 82 Figure 3.6 Structures of dolastatin 10, hemiasterlin, hemiasterlin A and B, and HTI-286 83 Figure 3.7 Spongistatin and SPIKET-P 84 Figure 3.8 Okadaic acid 85 Figure 3.9 Proposed role of protein phosphatase 2A (PP2A) in control of mitosis 86 ix Figure 3.10 Spirastrellolide A 87 Figure 3.11 Methylspirastrellolide C 88 Figure 3.12 Three areas of known relative configuration of spirastrellolide A 88 Figure 3.13 Structure of methylspirastrellolide B 89 Figure 3.14 Expansion of 600 MHz 1 H NMR spectra of methylspirastrellolide C from original extract recorded in C 6 D 6 92 Figure 3.15 Isolation scheme for methylspirastrellolide C 93 Figure 3.16 600 MHz 1 H NMR spectrum of methylspirastrellolide C recorded in C-6D 6 96 Figure 3.17 150 MHz 1 3 C NMR spectrum of methylspirastrellolide C recorded in C 6 D 6 97 ^  Figure 3.18 600 MHz C O S Y NMR spectrum of methylspirastrellolide C recorded in C 6 D 6 98 Figure 3.19 600 MHz HSQC NMR spectrum of methylspirastrellolide C recorded in C 6 D 6 99 Figure 3.20 600 MHz HMBC NMR spectrum of methylspirastrellolide C recorded in C 6 D 6 100 Figure 3.21 600 MHz R O E S Y NMR spectrum of methylspirastrellolide C recorded in C 6 D 6 101 Figure 3.22 600 MHz T O C S Y NMR spectrum of methylspirastrellolide C recorded in C 6 D 6 102 Figure 3.23 600 MHz H S Q C - T O C S Y NMR spectrum of methylspirastrellolide C recorded in C 6 D 6 103 Figure 3.24 C1-C17 fragment of methylspirastrellolide C with key correlations, 1 H and 1 3 C NMR assignments shown 106 Figure 3.25 Expansion of C O S Y spectrum, showing some correlations from C1-C17 in methylspirastrellolide C 106 Figure 3.26 Expansion of C O S Y spectrum, showing correlations from C7-C12 in methylspirastrellolide C 107 Figure 3.27 Expansion of T O C S Y spectrum of methylspirastrellolide C... 108 x Figure 3.28 Expansion of HMBC NMR spectrum of methylspirastrellolide C 110 Figure 3.29 C 1 7 -C31 fragment of methylspirastrellolide C with key correlations, 1 H and 1 3 C NMR assignments shown 110 Figure 3.30 Expansion of C O S Y NMR spectrum, showing some correlations from C25-C30 in methylspirastrellolide C 111 Figure 3.31 C 3 1 - C 3 5 fragment of methylspirastrellolide C with key correlations, 1 H and 1 3 C NMR assignments shown 112 Figure 3.32 C35-C47 fragment of methylspirastrellolide C with key correlations, 1 H and 1 3 C NMR assignments shown 113 Figure 3.33 Expansion of C O S Y NMR spectrum, showing correlations from C35-C47 in methylspirastrellolide C 114 Figure 3.34 Complete structure of methylspirastrellolide C with key correlations shown 115 Figure 3.35 A) R O E S Y NMR information, B) 1 H assignments and coupling constant data for the C 3 - C 1 3 segment of methylspirastrellolide C. C) An alternate configuration at C8 requires a R O E S Y NMR coupling between very distant protons, thus this C8 configuration is not likely 118 Figure 3.36 Expansions of the R O E S Y NMR spectrum, showing key correlations from C 3 - C 1 3 in methylspirastrellolide C 119 Figure 3.37 Summary of R O E S Y NMR data for C13-C21 segment of methylspirastrellolide C 121 Figure 3.38 R O E S Y NMR spectrum, showing key correlations from C 1 3 -C21 in methylspirastrellolide C 122 Figure 3.39 Summary of R O E S Y NMR data for C21-C24 segment of methylspirastrellolide C 123 Figure 3.40 R O E S Y NMR spectrum, showing key correlations from C27-C 3 8 in methylspirastrellolide C 124 Figure 3.41 Summary of R O E S Y NMR data for C 2 7 -C 3 8 segment of methylspirastrellolide C 125 Figure 4.1 Some compounds isolated from C. sativa 134 xi Figure 4.2 Structures of some CB agonists 137 Figure 4.3 Structures of some CB antagonists 138 Figure 4.4 Schematic for general G P C R bioassay 139 Figure 4.5 Structures of A - n o r - C 2 7 A 2 2 derivative and bengamide A 141 Figure 4.6 Stylissa massa, collected near Kavieng, Papua New Guinea... 142 Figure 4.7 600 MHz 1 H NMR spectrum of A - n o r -C 2 7 A 2 2 derivative recorded in C 6 D 6 144 Figure 4.8 600 MHz C O S Y NMR spectrum of A-/70/--C27A2 2 derivative recorded in C 6 D 6 145 Figure 4.9 600 MHz T O C S Y NMR spectrum of A - n o r - C 2 7 A 2 2 derivative recorded in C 6 D 6 146 Figure 4.10 600 MHz HSQC NMR spectrum of A - n o r - C 2 7 A 2 2 derivative recorded in C 6 D 6 147 Figure 4.11 600 MHz HMBC NMR spectrum of A - n o r - C 2 7 A 2 2 derivative recorded in C 6 D 6 148 Figure 4.12 A) Ring A partial spin system showing C O S Y NMR correlations, B) 1 H NMR assignments, and C) 1 3 C NMR assignments 151 Figure 4.13 A) A-B ring structure showing C O S Y and HMBC NMR correlations, B) 1 H NMR assignments, and C) 1 3 C NMR assignments 152 Figure 4.14 A) C-D ring structure showing key C O S Y and HMBC NMR correlations, B) 1 H NMR assignments, and C) 1 3 C NMR assignments 153 Figure 4.15 A) Completed A-nor-steroid structure, with key C O S Y and HMBC NMR correlations, B) 1 H NMR assignment of side chain, and C) 1 3 C NMR assignment of side chain 155 Figure 4.16 A) Key COSY, HMBC and R O E S Y NMR correlations in the side chain derivative, B) 1 H NMR assignments, and C) 1 3 C NMR assignments 156 Figure 4.17 Relative configuration of 4.10, with key R O E S Y NMR correlations 157 Figure 4.18 Hemiasterella aff. affinis sponge, collected in Milne Bay, Papua New Guinea 159 xii Figure 4.19 Structure of bengamide A 160 Figure 4.20 600 MHz 1 H NMR spectrum of bengamide A recorded in CDCI 3 161 Figure 4.21 150 MHz 1 3 C NMR spectrum of bengamide A recorded in CDCI3 162 Figure 4.22 600 MHz C O S Y NMR spectrum of bengamide A recorded in CDCI3 163 Figure 4.23 600 MHz HSQC NMR spectrum of bengamide A recorded in CDCI3 164 Figure 4.24 600 MHz HMBC NMR spectrum of bengamide A recorded in CDCI3 165 Figure 4.25 A) Fragment of bengamide A showing key C O S Y and HMBC NMR correlations, B) 1 H NMR assignments, and C) 1 3 C NMR assignments 167 Figure 4.26 Fragment of bengamide A showing key C O S Y and HMBC NMR correlations, B) 1 H NMR assignments, and C) 1 3 C NMR assignments 168 Figure 4.27 Fragment of bengamide A showing key C O S Y and HMBC NMR correlations, B) 1 H NMR assignments, and C) 1 3 C NMR assignments 169 Figure 4.28 Massadine, an alkaloid isolated from Stylissa massa 171 Figure 4.29 Structure of LAF-389, a synthetic bengamide A analogue.... 172 xiii LIST OF ABBREVIATIONS ° -degree(s) 1D -one-dimensional 2D -two-dimensional [ O C ] 2 5 D -specific rotation at wavelength of sodium D line at 25°C Ac -acetate AcOH -acetic acid Arg -arginine (three letter abbreviation) b -broad bs -broad singlet 1 3 C -carbon-13 °C -degrees Celsius C-18 -octadecylsilane calcd -calculated CB -cannabinoid CD2CI2 -deuterated dichloromethane C 6 D 6 -deuterated benzene CH -methine C H 2 -methylene C H 3 -methyl CH2CI2 -dichloromethane C O S Y -two-dimensional correlation spectroscopy 5 -chemical shift in parts per million d -doublet D -dextrorotatory dd -doublet of doublets dt -doublet of triplets DMSO -06 -deuterated dimethyl sulphoxide DNA -deoxyribonucleic acid Et -ethyl Et3N -triethylamine EtOAc -ethyl acetate EtOH -ethanol FDAA -Na-(2,4-dinitro-5-fluorophenyl)-L-alaninamide G -gram(s) 1 H -proton HCI -hydrochloric acid H 2 0 -water HMBC -two-dimensional heteronuclear multiple bond correlation spectroscopy HMQC -two-dimensional heteronuclear multiple quantum coherence spectroscopy HPLC -high-performance liquid chromatography hr -hour(s) HRESIMS -high-resolution electrospray ionisation mass spectrometry xiv HSQC -two-dimensional heteronuclear single quantum coherence spectroscopy Hz -hertz I -impurity J -coupling constant in hertz L -leucine (single letter abbreviation) L -tevorotatory Leu -leucine (three letter abbreviation) LRESIMS -low-resolution electrospray ionisation mass spectrometry m -multiplet M -molar concentration M + -molecular ion Me -methyl MeCN -acetonitrile MeOH -methanol mg -milligram(s) MHz -megahertz MIC -minimum inhibitory concentration mL -millilitre(s) mm -millimetre(s) mmol -millimol(s) mRNA -messenger ribonucleic acid MRSA -methicillin-resistant Staphylococcus aureus MS -mass spectrometry m/z -mass to charge ratio N -normal N 2 -nitrogen NaCl -sodium chloride nm -nanometre(s) NMR -nuclear magnetic resonance NP -normal phase N R P S -non-ribosomal peptide synthase "PrOH -1-propanol P C P -peptidyl carrier protein 4 ' -PP -4'-phospopantethiene ppm -parts per million PyBOP -benzotriazol-1 -yl-oxytripyrrolidinophosphonium hexafluorophosphate q -quartet R -arginine (single letter abbreviation) R O E S Y -rotating frame Overhauser enhancement spectroscopy RP -reversed-phase s -singlet, solvent (on spectrum) S -serine (single letter abbreviation) SAR -structure-activity relationship S C U B A -self-contained underwater breathing apparatus xv Ser -serine (three letter abbreviation) sp -species - sp 2 hybrid orbital sp 3 - sp 3 hybrid orbital t -triplet TFA -trifluoroacetic acid TLC -thin-layer chromatography T O C S Y -total correlation spectroscopy TOFMS -time-of-flight mass spectrometry Trp -tryptophan (three letter abbreviation) TXI -Triple Resonance Probe Tyr -tyrosine (three letter abbreviation) MS -microgram(s) -microlitre(s) uM -micromolar umol -micromole(s) UV -ultraviolet V R E -vancomycin-resistant Enterococci W -tryptophan (single letter abbreviation) w -water (on spectrum) Y -tyrosine (single letter abbreviation) xvi ACKNOWLEDGEMENTS First and foremost, I would like to thank my supervisor, Ray Andersen, without whose support and guidance this all would not have been possible. The enthusiasm you bring to the study of science inspired me to explore the different aspects of the field, culminating in this work. The opportunity to take part in collecting trips, both abroad and local, and the many teaching and diving opportunities that those trips entailed was truly a once in a lifetime experience. Many thanks to David Williams for all of the help in the lab, whether it was showing how to seal NMR tubes or get an instrument running. Thanks as well to Mike Leblanc for the help with everything in and out of the lab. Although cake on the ferry was always forbidden, and the hovels always of questionable quality, there are few divers I have learnt more from. The members of the Andersen lab made running a column in the basement of biosciences on a summer day a thing to look forward to, no small feat indeed. Particular thanks are owed to Chris Gray and Rob Keyzers for all of their help with this thesis, as well as their help making coffee time so enjoyable. To my friends and fellow chemists, Roger, Matt, Kate, Alban, Justin, Ana, Kaoru, Julie and Harry and the rest of lab for making life in the lab and under water so much fun, thank you all. My dive buddies, Kate, Matt, Roger, Justin, Alban and Julie deserve special thanks for sharing in our undersea adventures. Thanks to my parents, Dave and Geri, and my in-laws, Barney and Roberta for their love and support through grad school and after. And of course, my heartfelt thanks to Erin, who put up with fragrant dive gear, long hours at school, a student salary and six years of questionable beards, and made them the best six years of my life thus far. Lastly, I would like to thank everyone who has helped with these projects. In particular I would like to that Helen Wright, Tamsin Tarling and Tom Pfeifer for their help with the bioassays. Thanks as well to the friendly staff in NMR and Mass Spectrometry Services who were always helpful, as was everyone in Bio-services and at Bruker Canada. xvii DEDICATION For Erin xviii Chapter 1: Marine Natural Products Chemistry 1.1 Marine Natural Products Chemistry: A Multidisciplinary Approach "Why in the world do you want to go down into the sea?" is a riddle we are often asked by practical people. George Mallory was asked why he wanted to climb Mt. Everest, and his answer serves for us, too. "Because it is there," he said. We are obsessed with the incredible realm of oceanic life waiting to be known. The mean level of habitation on land, the home of all animals and plants, is a thin tissue shorter than a man. The living room of the oceans, which average twelve thousand feet in depth, is more than a thousand times the volume of the land habitat. J . Y. Cousteau 1 Marine natural products (MNP) chemistry is a relatively young science, made practical only after the widespread availability of the demand regulator driven Aqua Lung invented by Jacques-Yves Cousteau and Emile Gagnan in 1943, which allowed semi-autonomous exploration of the undersea realm. 2 However, a wide range of chemical structures have been identified in the relatively short time since its inception. 3 The many different types of studies carried out on marine organisms and their chemical constituents are representative of the different groups of scientists who study this vast subject. Scientists with biological backgrounds have directed studies into the chemical ecology or biosynthesis of secondary metabolites.4"6 Those with a chemistry background have focused upon novel chemical structures (chemical prospecting) or potential applications of these compounds as chemotherapeutics (drug 1 discovery). As is often the case, discoveries made in one stream of study frequently lead into another. The main goal of the research contained herein is that of drug discovery, although the natural biological role of compounds and their methods of biogenesis are also addressed. The diversity of biological activities and structural classes of these compounds is testament to the diversity of chemical structures and their concomitant biological functions in the marine environment. 1.2 Drugs From The Sea Natural products have historically been strong contributors to the pool of available drugs. 7 ' 8 Secondary metabolites are compounds produced by organisms which are arguably unnecessary for the base functioning of the organism.9 , 1" The vast structural diversity of secondary metabolites in nature makes them an ideal resource for the discovery of new drugs. 1 0 It is unbiased by human imagination or ease of synthesis (unlike combinatorial chemistry efforts), and thus is a rich resource for new chemistry. Research on the secondary metabolites of terrestrial organisms has been ongoing for many years, with a great number of successes (Figure 1.1). Although the natural product itself is occasionally the compound used therapeutically, more often it is a lead from which more pharmaceutical^ appropriate compounds are derived. Nevertheless, the drug is of natural origins. 1 This is in contrast to primary metabolites such as amino acids, nucleic acids, sugars and lipids, which are necessary for the functioning of all cells. 2 Figure 1.1 A selection of drugs from terrestrial sources. Morphine (1.1) isolated from Papaver somniferum; penicillin G (1.2), isolated from Penicillium notatum; salicin (1.3), isolated from Salix sp.; and paclitaxel (1.4), isolated from Taxus brevi folia Despite past successes in the terrestrial realm, the search for new and more potent drugs continues. In addition, novel molecular targets with therapeutic potential are continually being unveiled as biochemists study diseases in greater detail than was previously possible. In contrast to terrestrial studies, which have been ongoing for a great many years, marine studies have only been practical for the last 30 or 40 years. With the relative ease of collection of marine organisms since the advent of modern SCUBA, it has become possible for chemists to assemble libraries of crude extracts from marine organisms and, working in close collaboration with biochemists, to study these libraries with the objective of isolating and identifying new and interesting bioactive secondary metabolites. 1 1 This, in essence, is the study of marine natural products chemistry. A wide range of organic chemical structural classes have been observed in extracts of marine organisms, many of which have been found to be uniquely 3 marine in origin. 1 2 From this wide spectrum of structural classes a large number have been found to be bioactive. Some of these (or modifications thereof) have even been found to be active enough to warrant expensive and time consuming clinical trials. 1 3 Ara C (1.5) was developed following the discovery of spongothymidine (1.6) and spongouridine (1.7), and is used in the treatment of leukemia. 1 4 " 1 6 Ziconotide (1.8), marketed under the band name Prialt®, is an u> conotoxin MV-IIA derived from the Conus magus, a marine fish-hunting cone s n a i l . 1 7 , 1 8 This small (25 residue) peptide is currently used as an analgesic due to potent activity on voltage-gated C a 2 + channels. NHc N- HN O ^ N O HN' O c r ^N ' -OH r ^ - O W ^ J U O H HO - HO £ HO -OH OH OH 1.5 1.6 1.7 H 2 N-CKGKGAKCSRLMYDCCTGSCRSGKC-CONH 2 1.8 Figure 1.2 Structures of Ara-C (1.5), spongothymidine (1.6), spongouridine (1.7) and ziconotide (1.8) Ecteinascidin 743 (ET-743, Yondelis, 1.9) was isolated from the marine tunicate Ecteinascidia turbinata, and identified as possessing strong anti-tumor activity following an initial observation of cytotoxicity in the crude extracts. 1 9 The 4 structural complexity of 1.9 has led to a number of synthetic studies, whereas the biological activity has given rise to a number of biological studies, including advanced clinical trials. 2 1 It is currently approved for use for treatment of soft-tissue sarcomas, and is being studied for its effects on ovarian cancer. Finally, pseudopterosins A (1.10) and C (1.11) were isolated from the Caribbean sea whip Pseudopterogorgia elisabethae in the mid 1980s and were found to possess potent analgesic and anti-inflammatory activit ies. 2 2 , 2 3 They are currently used by Estee Lauder as a topical anti-inflammatory agent in cosmetics. 2 4 A great many more marine-derived compounds are currently in clinical and preclinical trials. 1 3 5 A survey of the literature published up to the end of June 2005 indicates several trends in marine natural products research.1" The most productive source of chemical structures is attributed to phylum Porifera (sponges), from which a number of interesting discoveries continue to be made today (Figure 1.4). There are a growing number of studies aimed at determining whether compounds isolated from sponges are in fact synthesized by the sponge, or by microorganisms living on or within the sponge host. 2 5 " 2 8 This question is far from resolved, although microorganisms appear to be a more important source than the relatively small number of compounds attributed to them in the literature indicate. In a typical research laboratory, while many projects may continue to focus on sponges, bacterial studies are becoming increasingly commonplace. 2 9 Figure 1.4 Proportion of compounds in Marinlit database, grouped by phyla of origin. Total 17068 structures. Search conducted using MarinLit Marine Literature Database, Version pc13.4. 6 1.3 Marine Microorganisms as an Emerging Resource Many of the terrestrial drugs currently in use have been isolated from microorganisms (penicillin G, (1.2); erythromycin (1.12), adriamycin (1.13), and vancomycin, (1.14) are a few of note). 3 0" 3 4 It is only in recent years, however, that the vast resource of marine microorganisms has become readily accessible, thus the study of the chemistry of these fascinating organisms has only been possible for a relatively short while. It is useful to note that the term marine microorganisms used in the discussion to follow refers to heterotrophic organisms (primarily bacteria and fungi). The chemistry of autotrophic marine microorganisms such as blue-green or red algae has been studied in much more detail than that of the heterotrophs, due in part to ease of isolation, relative prevalence, as well as ease of collection and cultivation. 3 5 , 3 6 In essence, one major difference between autotrophs and heterotrophs is that the latter derive their energy from nutrition, whereas the former derive their energy from photosynthesis. 7 Figure 1.5 Structures of some drugs derived from terrestrial microorganisms: Erythromycin (1.12), from Streptomyces erythreus, adriamycin (1.13), from Streptomyces peucetius, and vancomycin (1.14), from Streptomyces orientalis It has been often reported that there are greater than one hundred thousand microbial cells per mL of seawater, and recent studies indicate even higher concentrations of microbial diversity in unique biospheres such as deep sea vents . 3 7 ' 3 8 While genetic studies have constantly increased the number of identified unique species present in the ocean, the ability to culture these organisms has improved much more slowly. 3 9 However, recent studies indicate that major hurdles have been overcome in the culturing of these intriguing and chemically productive organisms. 4 0" 4 3 The research by Dr. William Fenical and Dr. Paul Jensen at Scripps Institute of Oceanography is testament to the possibilities of the culturing of 8 marine microorganisms. Azamarone (1.15) is one of many novel structures identified from their investigations of the secondary metabolites from marine bacteria. 4 4 In this case, the novel meroterpenoid was isolated from a saline culture of a Streptomyces strain CNQ776. The same culture was found to produce actinofuranones A and B (1.16 and 1.17, respectively), novel polyketides which, like 1.15, showed weak (20-40 uM) cytotoxicity against mouse splenocyte T-cells and macrophages. 4 5 Further studies on cultures of the newly discovered genera Salinospora and Marinospora by the same group has yielded a number of interesting structures (see Chapter 2 ) . 4 6 - 4 8 1.17 Figure 1.6 Structures of azamarone (1.15) and actinofuranones A (1.16) and B (1.17) Another approach to the cultivation of marine bacteria is to isolate organisms directly from the surface of marine invertebrates. A prime example of this is the isolation of guangamides A and B (1.18 and 1.19, respectively) from an unidentified fungus which was isolated from a marine lanthella sp. sponge 4 9 Compounds 1.18 and 1.19 were found to be weakly toxic against Staphylococcus epidermis and Enterococcus durans (MIC = 100 ug/mL). 9 Figure 1.7 Structure of guangamides A (1.18) and B (1.19) One appealing feature of the study of marine bacteria is that it provides an answer to the supply issue. Studies of marine invertebrate chemistry are often plagued by the low natural abundance of metabolites in the organism. By obtaining stable cultures of marine microorganisms, researchers can feasibly culture indefinite amounts of microbes, and thus potentially produce unlimited amounts of compound. One such culture is PNG-276, a remarkably productive microbe which has been the focus of much study in our laboratory. 5 0" 5 5 Further studies of this organism are discussed in Chapter 2. 1.4 Marine Sponges as a Productive Resource As mentioned previously, marine sponges have been a significant contributor to marine natural products chemistry. It should be noted that for the purposes of this discussion no differentiation will be made between compounds produced by the sponge and compounds possibly produced by symbiotic 10 microorganisms (vide supra). Unless the culture has been grown apart from the sponge, it will be treated as a sponge metabolite. Irrespective of their origin, a huge number of compounds have been isolated from sponges. Early investigations focussed upon fascinating multi-halogenated compounds such as the carbodiiminic dichloride 1.20, as well as peptides such as jaspamide (1.21) and cyclotheonamide A (1.22).56"58 Entire families of compounds were discovered in sponge extracts, such as the oroidin alkaloids, of which oroidin (1.23), dibromophakellin (1.24) and palau'amine (1.25) are just a few. 5 9 " 6 2 1.23 1.24 1.25 Figure 1.8 Examples of compounds isolated from marine sponges 11 One of the underlying themes of marine natural products chemistry is that diversity in chemical structures corresponds to a diversity of biological activity. This is particularly the case with compounds produced by marine sponges. Manoalide (1.26) was isolated from the sponge Luffariella variabilis collected in Palau, and found to possess potent anti-inflammatory activity. 6 3 ' 6 4 Discodermolide (1.27) is a potent cytotoxin isolated from the deep-water sponge Discodermia dissoluta found in the Bahamas, 6 5 the activity of which will be discussed in more detail in Chapter 3. Bioassays have been focussed primarily on anticancer activities, which is due both to the relative prevalence of funding in cancer research, and also the high occurrence of cytotoxic compounds in marine extracts and readily cultured cancer cells to test these extracts. 2 4 Nevertheless, a survey of the literature shows that antibiotic, antifungal, immune active (either activation or repression), antiviral, antiparasitic and antifouling activities are also common for marine natural products.1" With this in mind, it remains to outline the particular projects which are summarized herein. Figure 1.9 Structures of manoalide (1.26) and discodermolide (1.27) f The listed categories collectively account for 46% of reported activities, whereas cytotoxicity and anticancer activity accounts for the remaining 54% of reported activities (Total 11238 reported activities). Search conducted using MarinLit Marine Literature Database, Version pc13.4. 12 1.5 Antibiotic Resistance Antibiotic resistance is a problem that is rapidly approaching crisis proportions. A number of excellent reviews explore this subject, in particular a recent issue of Chemical Reviews66'74 and a special issue of Biochemical Pharmacology.75'8^ While it is not necessary to exhaustively report the history of antibiotic resistance, it is worthwhile to look at a few of the major developments in the field of antibiotic therapy. The discovery of the antibiotic producing fungus Penicillium and subsequent isolation of penicillin by Fleming and coworkers in 1940 ushered in the golden era of antibiotic usage . 3 0 , 8 2 A period of rapid expansion followed, culminating in the late 1960s with the discovery of many of the classes of antibiotics currently in use (Figure 1.10). Although there have been important developments in antibiotic research since then, there have in fact been few new chemical classes discovered. The majority of the research to develop new antibiotics has focused upon the development of new versions of the classic structural types. 8 3 13 Figure 1.10 Some major classes of antibiotics currently in use. Shown are penicillin G (1.2), erythromycin (1.12), vancomycin (1.14), sulfamethoxazole (1.28), chloramphenicol (1.29), tetracycline (1.30) and streptomycin (1.31) Exposure to antibiotics in the environment during the last 60 years has created an evolutionary selective pressure that favors microorganisms that are able to survive in the presence of antibiotics. 8 4 Coupled with the slowdown in discovery of new antibiotics, this has led to large increases in the severity and prevalence of outbreaks of antibiotic resistant bacteria. 8 5 There is an urgent need for the discovery and development of the next line of antibiotics. 8 6 , 8 7 14 This is an area of research ideally suited to marine natural products drug discovery. Whereas soil organisms are the principal source of most current antibiotics, marine microorganisms may well be a source of new antibiotics. Microorganisms in the marine environment maintain a complex balance with their competitors through the use of a number of techniques, one of which may be chemical warfare. 8 8 , 8 9 The compounds produced for this purpose could well be the next generation of antibiotics if isolated and characterized. 1.6 Cancer Chemotherapeutics Approximately 153,100 new cases of cancer and approximately 70,400 deaths from cancer are predicted in Canada in 2006. 9 0 Traditionally, natural products have contributed greatly to the search for new anti-cancer drugs. Classes of compounds such as the vinca alkaloids and the taxanes (see Chapter 3) are currently on the front line of cancer therapies. This strong contribution from terrestrial sources has extended into the field of marine natural products, with several successfulresearch programs underway, one in which the Andersen lab participates. 4 3 , 9 1 The efforts of several leading marine natural products groups have yielded a number of clinical candidates, several of which are in advanced clinical trials. 9 2 Bryostatin 1 (1.32), isolated from the bryozoan Bugula neritina has been studied in a number of human clinical trials for its unique and selective interaction with protein kinase C isozymes, although this activity seems to be 15 most useful when combined with other cytotoxic compounds. 1 3 , 9 3 Of course, the use of the natural product may be limited by supply, where it would be prohibitively expensive and/or destructive to collect the necessary amount of organisms to provide the compounds needed. As such, related synthetic compounds are often pursued due to the increased feasibility of using a synthetically attainable analogue. For example, the combined structures of the marine natural product psammaplin A (1.33) with information gleaned from trichostatin A (1.34) and trapoxin B (1.35) led to the development of NVP-LAQ824 (1.36), an anticancer clinical candidate having powerful histone deacetylase activity. 9 4" 9 6 Compound 1.36 is not clearly derived from any of compounds 1.33, 1.34 or 1.35, but is rather a synthetic derivative which possesses the pharmacophore of each of them. 16 H Figure 1.11 Structures investigated for anticancer activity One of the keys to a successful drug discovery program is the range of bioassays available to the chemists. Our laboratory has had a long and fruitful collaboration with the laboratory of Michel Roberge, also located at UBC. A particularly productive assay has been an antimitotic assay, which tests for compounds that arrest cells in mitosis using a TG -3 monoclonal antibody. 9 7 Using this assay, desmethyleleutherobin (1.37) and five related novel analogues, were identified from the octocoral Erythropodium caribaeorum.98 Further studies on antimitotic extracts of the Caribbean sponge Spirastrella coccinea, also identified using this assay, are outlined in Chapter 3. 17 O H 1.37 Figure 1.12 Structure of desmethyleleutherobin (1.37), a novel antimitotic diterpene identified using the antimitotic bioassay 1.7 Cellular Signaling/Cannabinoid Receptors A relatively recent area of study for our laboratory is a collaboration with the lab of Tom Grigliatti, also at UBC. A pilot screen of a subset of our marine extract library against insect cell cultures transfected with human G-protein-coupled receptors yielded a number of hits. The investigation of two of these hits is outlined in Chapter 4 . 9 9 " 1 0 1 This particular project focussed on two cannabinoid (CB) receptors, CB1 and C B 2 , and looked at both agonist and antagonist effects. Both CB1 and C B 2 are membrane bound G-protein-coupled receptors, although CB1, is concentrated primarily in the central nervous system, whereas C B 2 is concentrated in the immune system. 1 0 2 Although little has been done to research new CB active compounds, the CB receptors have been implicated in a number of disease states ranging from multiple sclerosis and cancer to schizophrenia and post-traumatic stress disorder. 1 0 3 They are also thought to be involved in pain management and appetite control. As such, compounds which activate or deactivate these 18 receptors could be of use for therapy or further study of these receptors and their function. The representative compounds which interact with C B receptors are anandamide (1.38) and (-)-A9-tetrahydrocannabinol (1.39). Compound 1.38 is an endocannabinoid, which is a molecule synthesized within the body that interacts with CB receptors. Compound 1.39 is one of a number of related cannabinoids found in the plant Cannabis sativa which interact with CB receptors in the human body. Beyond these two structure types, very few other small molecules have been found to interact with these receptors. The chemical diversity present in the marine environment is proposed to be a good place to start looking for new CB active compounds. Chapter 4 contains the results of the study of two sponge extracts using the CB assay. 1.38 1.39 Figure 1.13 Structures of anandamide (1.38) and (-)-A9-tetrahydrocannabinol (1.39), two CB active compounds 1.8 Summary One important area of marine natural products research involves the examination of the secondary metabolites of marine organisms for novel and 19 bioactive compounds. This natural source of chemical creativity has already yielded a number of effective compounds, and research is ongoing on a number of promising lead structures. The studies described herein focus on three specific areas of research. In the second chapter the isolation and structure elucidation of a new antibiotic lipopeptide from a marine bacterium is described. In the third chapter the isolation and structure elucidation of a novel antimitotic macrolide from a marine sponge is described. The structure elucidation of this compound helps solve questions on the relative configuration of a related analogue. The final chapter describes the preliminary investigation of two sponges which showed interesting activity in the cannabinoid receptor assay. 20 Chapter 2: Tauramamide, A Novel Antibacterial Lipopeptide from the Marine Bacterial Isolate PNG-276 2.1 Introduction Historically, the field of marine natural products had been dominated by compounds from sponges, coelenterates, bryozoans, molluscs, tunicates, echinoderms and a lgae. 1 0 4 It is reasonable to conclude that this is due to their complex chemistry and ecology, as well as their relative ease of collection. Conversely, marine microorganisms were only rarely examined in the early days, primarily due to the difficulty in culturing these organisms. A number of studies, however, began to indicate the possibility that some secondary metabolites thought to be biosynthesized by the host organism (e.g. the sponge) are in fact metabolites produced by the vast array of microorganisms living in and on the sponge. 1 0 5 Recent advances in sample collection and microorganism cultivation have allowed researchers to study these organisms in greater detail, accessing the huge potential of marine microbial compounds. Marine bacteria are a vast and largely untapped resource of chemical diversity. 1 0 6 " 1 0 8 Nevertheless, there are a growing number of success stories present in the literature. A large body of work on the culturing of chemically interesting marine microorganisms has been done by the research group of Dr. William Fenical, located at Scripps Institute of Oceanography, in La Jolla, California. Focusing largely upon the isolation and cultivation of marine actinomycetes, a number of new genera have been characterized, including the 21 marinomycin (2.1) producing genus Marinospora and salinosporamide A (2.2) producing genus Salinosporam,uo Compound 2.1 was found to possess both antimicrobial activity and selective cytotoxic activity against cancer cell lines, whereas 2.2 was found to be highly cytotoxic against cancer cells, specifically by inhibiting proteasome activity. The proteasome is a multiunit enzyme complex that is responsible for most protein degradation in the cell apart from the lysozome, and is thought to be a good target for drug discovery due to its role in degrading regulatory proteins. Compound 2.2 shares this activity with omuralide (2.3), which possesses similar beta lactone functionality, although 2.2 is significantly more active than 2.3.110 Figure 2.1 Marinomycin A (2.1), salinosporamide A (2.2) and omuralide (2.3) Our research laboratory has also had a number of successes in the pursuit of marine natural products derived from marine bacterial isolates. Massetolide D (2.4) was one of a family of analogues isolated from cultures of marine pseudomonads MK90e85 and MK91CC8 . 1 1 1 These compounds were 22 found to be active against both Mycobacterium tuberculosis and Mycobacterium avium-intracellulare (MICs 2.5-10 ug/mL). Holyrines A (2.5) and B were isolated from cultures of marine actinomycete N96C-47 . 1 1 2 The holyrines are intermediates in the biosynthesis of the antifungal and anticancer compound staurosporine (2.6).113"115 2.5 2.6 Figure 2.2 Massetolide D (2.4), holyrine A (2.5) and staurosporine (2.6) The marine isolate PNG-276 is a particularly chemically prolific organism cultured from the tissues of an unidentified tube worm collected off the coast of Loloata Island, Papua New Guinea. Initial bioassays of crude extracts from 23 cultures of this isolate indicated strong antibiotic activity against a range of human pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), Escherichia coli, and Candida albicans. The bioassay-guided fractionation of PNG-276 culture extracts yielded a series of cyclic decapeptide antibiotics, loloatins A (2.7) -D. 1 1 6" 1 1 8 Of particular note is their strong antibacterial activity against MRSA, V R E and penicillin-resistant Streptococcus pneumoniae (MIC = 0.5-4 ug/mL), and lesser activity against the yeast C. albicans (MIC = 8-16 ug/mL). They possess many common features of nonribosomal peptides including D-amino acids and unusual amino acids (eg. hydroxyproline in loloatin D). —. — n HO- oK N H 2 2.7 Figure 2.3 Loloatin A (2.7) Further analysis of PNG-276 extracts yielded bogorol A (2.8), a novel cationic peptide antibiotic. 1 1 9 ' 1 2 0 This compound exhibits strong activity against MRSA and V R E (MICs of 2.0 and 10 ug/mL, respectively). As with the loloatins, 24 it too possesses D-amino acids and unique amino acids (in this case ornithine, and 2-amino-2-butenoic acid), and thus is also likely nonribosomal'in origin. Unlike the loloatins, however, bogorol A is classified as a linear cationic peptide, and thus is thought to act primarily upon the cell membrane of target organisms. 1 2 1 The presence of N-H NOESY correlations in bogorol A between adjacent amino acid residues supports the proposal of an a-helix secondary structure common among other linear cationic peptides. Cationic peptides are found in nature as part of the innate immune system, and may be potential tools against antibiotic resistant organisms. 1 2 2 , 1 2 3 Figure 2.4 Bogorol A (2.8) Two antifungal compounds, basiliskamides A and B (2.9, 2.10), were subsequently isolated from cultures of PNG-276, as were two acyldipeptides, tupuseleiamides A and B (2.11, 2.12), with no known activity. 1 2 4 Basiliskamides A and B were both strongly active against C. albicans (MICs of 1.0 and 3.1 ug/mL, respectively). Further testing against fresh cultures of C. albicans 25 demonstrated that 2.9 was as active as amphotericin B, a common benchmark for antifungal activity. Whereas 2.9 and 2.10 are polyketide in origin, 2.11 and 2.12 are of mixed polyketide and peptide origin. Figure 2.5 Basiliskamides A and B (2.9 and 2.10), and tupuseleiamides A and B (2.11 and 2.12) A survey of the structural classes produced by PNG-276 indicates that the majority of compounds are produced via nonribosomal peptide synthesis (NRPS). In standard ribosomal peptide synthesis, genetic information is translated from DNA via RNA, and leads to the assembly of peptides and proteins based upon the genetic code in the DNA sequence. This employs the 20 standard amino acids, all of which are in the natural L configuration. N R P S occurs via a completely different process, however, on which a number of excellent reviews have been wr i t ten . 1 2 5 , 1 2 6 Instead of being assembled on the ribosome, as standard peptides, nonribosomal peptides are assembled on large, multiunit enzymes or multi-enzyme complexes (Figure 2.6). 26 Figure 2.6 Schematic of peptide synthesis in a typical N R P S (adapted from Schwarzer et a/ .) 1 2 6 A) The 4'-phosphopantetheine (4'PP) cofactor, bound covalently to the peptidyl-carrier protein (PCP) is, in its unloaded state, strongly attracted to the adenylation (A) domain, which selects the amino acid to be loaded. B) The loaded aminoacyl-S-4'PP is then attracted to the acceptor position of the upstream condensation-(C)-domain, and remains until the peptide bond is formed. C) After the peptide bond is formed, the peptidyl-S-4'PP becomes attracted to the donor position of the downstream C domain, setting the stage for the subsequent peptide bond to be formed. As it does not rely upon the 20 amino acids in the genetic code, unique amino acids can be added to the peptide as it is formed. Instead, amino acids are selected for each step by the adenylation domains of the N R P S enzyme (Figure 2.6). The enzyme, much like an assembly line, passes the growing peptide between condensation domains. The "code", as it were, is in the NRPS itself, not read off mRNA as occurs with traditional ribosomal biosynthesis. 27 The ability to incorporate novel amino acid substrates is not the only unique feature that NRPSs have at their disposal. Natural L amino acids can be epimerized to the D configuration on special epimerization (E) domains. Polyketide or fatty acid subunits can also be introduced due to the similarity between N R P S synthases and polyketide synthases. Small heterocyclic rings may be formed through cyclization of threonine, serine or cysteine residues on to the peptide backbone on heterocylizase (Cy) domains. Methylation of nitrogen on the amide backbone is also common, via S-adenosylmethionine (SAM) and a A/-methylase (N-Mt) domain. All of these modifications to the standard peptide features engender new functions and stability to proteolysis in the resulting products. It is reasonable to predict that studies of marine bacteria will continue to produce novel and potentially useful antibiotics. The relatively high numbers of bacteria present in the ocean ( >105 cells/mL seawater) 1 2 7 , coupled with their unique environment and genetic diversity present optimal conditions for producing a large variety of secondary metabolites. 1 0 8 Indeed, recent studies indicate that marine microbial numbers and diversity may be even higher than previously estimated. 1 2 8 One feature of complex mixtures of competing organisms is the presence of a number of strategies for gaining an advantage over competitors. These range from differing growth rates, differing nutrient requirements and methods for sequestering limiting nutrients, to the use of and defense against inhibitory metabolites in the environment. If even a small fraction of these organisms have adapted secondary metabolites to gain 28 advantage over their competitors, there should be a wealth of antibiotics to discover. With this in mind, our research group continues to study bacteria isolated from marine habitats for new antibiotics. 2.2 Isolation and Characterization of Tauramamide Methyl Ester The marine bacterial isolate PNG-276 was cultured from an unidentified tube worm collected at a depth of 15 m near Loloata Island, Papua New Guinea . 1 2 9 The initial rationale for this study of PNG-276 cultures was to isolate more of the basiliskamides (2.9 and 2.10) for an unrelated project. The culture conditions and extraction techniques were therefore based largely upon those used to isolate 2.9 and 2.10. Subsequent analysis of 1 H NMR of resultant C. albicans active fractions however identified tauramamide (2.13) as a novel and bioactive compound. This compound is named after Taurama point, which is adjacent to Loloata Island, Papua New Guinea, in the fashion of the loloatins, bogorols and tupuseleiamides. The isolation, and structure elucidation of this lipopeptide became the primary focus of the project. 29 2.2.1 Isolation of Tauramamide Methyl Ester PNG-276 was grown as confluent lawns on trays of solid tryptic soy agar supplemented with 1% NaCl to simulate marine conditions. Cells were collected by scraping the agar surface, and lyophilized to yield a brown powder (-30 g), prior to exhaustive MeOH extraction (3 X 250 mL). The combined methanol extract was concentrated in vacuo to yield a brown gum (3.8 g) and then partitioned between H 2 0 and EtOAc. The organic partition (1.0 g) was active against C. albicans. Lipophilic size exclusion chromatography (Sephadex LH-20®) in MeOH followed by subsequent partitioning size exclusion chromatography (Sephadex LH-20®) in 20:5:2 EtOAc /MeOH/H 2 0 yielded a number of fractions which were pooled based upon bioassay information, NMR and TLC characteristics. The most active fraction was then subjected to reversed-phase C-18 chromatography (Waters 2 g Sep-Pak®) eluting with a stepwise gradient from H 2 0 to MeOH. Bioassay, TLC and 1 H NMR were again used to pool the resultant fractions. The active material from this 30 chromatography (92.8 mg) was then separated by reversed-phase HPLC (40% M e C N / H 2 0 + 0.1 % TFA, CSC-lnertsil® ODS2 column) yielding tauramamide methyl ester (2.14, 7.4 mg, 0.02% dry cell weight). 2.2.2 Structure Elucidation of Tauramamide Methyl Ester Tauramamide A methyl ester was isolated as a clear pale yellow solid that gave a [M+H]+ ion at m/z 878.5181 in high-resolution electrospray ionization time-of-flight mass spectrometry (HRESI-TOFMS), indicating a molecular formula of C45H67N9O9 (C45H68N9O9 calculated for m/z 878.5185) requiring 17 degrees of unsaturation. Analysis of 1 H and 1 3 C 1D and 2D NMR (COSY, HMQC, HMBC, ROESY) data identified several amino acid fragments, which were assigned as follows. For clarity of discussion, the numbering scheme is shown in Figure 2.8. Each amino acid is numbered separately for greater ease of discussion. Arg [5 HNL ME / o . 8 Trp 2.14 Figure 2.8 Structure of tauramamide methyl ester (2.14) 31 Figure 2.9 500 MHz 1 H NMR spectrum of tauramamide methyl ester (2.14) recorded in DMSO-cfe 32 , 33 34 Figure 2.12 500 MHz R O E S Y NMR spectrum of tauramamide methyl ester (2.14) recorded in DMSO-d 6 35 Figure 2.13 500 MHz HMQC NMR spectrum of tauramamide methyl ester (2.14) recorded in DMSO-cfe 36 Figure 2.14 500 MHz HMBC NMR spectrum of tauramamide methyl ester (2.14) recorded in DMSO-c/ 6 37 Table 2.1 1D and 2D NMR data for tauramamide methyl ester (2.14), recorded at 500 MHz (1H) and 100 MHz ( 1 3C) in DMSO-Os c# " C . JH (J ) COSY ROESY HMBC (1H to i3C) Methyl Ester, ME 1 51.8 3.62 s, 3H - - R-CO Arginine, Arg, R CO 172.1 - - - -1 51.3 4.29 m R-NH, R2' R2, R2', R3, R-NH R-CO 2 27.5 H2 1.67 m H2' 1.77 m R1, R3 R1, R-NH R-CO 3 25.0 1.52 m, 2H R2, R2', R4 R1 -4 40.6 3.11 m, 2H ' R3, R-NH' R3 R5 5 156.7 - - - -NH - 8.39 bd (7.0) R1 W1,R1,R2 -NH' - 7.50 bs R4 - R5 Tryptophan, Trp, W CO - - - - -1 53.1 4.53 bs W-NH, W2, W2' R-NH, W2, W2', W5, W10 -2 27.8 H2 2.89 m H2' 3.15 m W1 W-NH, W1, W10 -3 109.8 - - - -4 127.1 - - - -5 118.4 7.60 d (7.7) - -6 118.0 6.96 dd (7.7, 7.2) W7 W1 W4 7 120.7 7.04 bd (7.2) W6, W8 - W5, W9 8 111.1 7.30 d (7.8) W7, W9 - W6, W10 9 136.0 - W8 W-NH' W5, W7 10 123.9 7.11 bs W-NH' W1, W2, W-NH' W3 NH - 8.14 bs W1 W1, L1, W2 -NH' - 10.76 bs W3 W9, W10 W4, W10 Leucine, Leu, L CO - - - - -1 51.7 4.20 m L-NH, L2 W-NH, L3, L4, L4' -2 40.5 1.06 m, 2H L1, L3 - -3 23.7 1.25 L2, L4, L4' L1 -4 22.8 0.71 d (6.3), 3H L3 L1 L3, L4' 4' 21.5 0.68 d (6.3), 3H L3 L1 L3, L4 NH - 7.75 bs L1 L1, S1 -Serine, Ser, S CO - - - - -1 55.1 4.22 m S-NH, S2, S2' L-NH -2 61.7 H2 3.47 m H2' 3.52 m S1, S2-OH S2-OH -2-OH - 4.83 bs S2, S2' S2, S2' -NH - 8.03 bs S1 S1, Y1 -Tyrosine, Tyr, Y CO - - - - -1 54.5 4.41 bs Y-NH, Y2, Y2' S-NH, Y2, Y2' -2 36.5 H2 2.64 m H2' 2.85 m Y1 Y1, Y4/Y8 -3 127.8 - - - -4,8 130.0 7.02 d (7.8), 2H Y5/Y7 Y1, Y2, Y2' Y4/Y8, Y6 5, 7 114.7 6.62 d (7.8), 2H Y4/Y8 Y6-OH Y3, Y5/Y7 6 155.7 - '- - -6-OH - 9.13 s - Y5/Y7 Y6, Y5/Y7 NH - 7.96 bs Y1 OA2 -7-methyloctanoamide, OA 1 175.0 - - - -2 35.1 2.01 m, 2H OA3 Y-NH OA1 3 25.1 1.36 m, 2H OA2, OA4 - OA1 4 28.7 1.07 m, 2H OA3, OA5 - -5 26.5 1.14 m, 2H OA4, OA6 - -6 38.0 1.07 m, 2H OA5, OA7 - -7 27.0 1.44 bm (6.7) OA6, 0A8/8' - -8 22.5 0.83 d (6.7), 3H OA7 - -8' 22.5 0.83 d (6.7), 3H OA7 - -38 Analysis of the NMR data suggested that the molecule was a peptide based upon the readily observable a-methines producing resonances at 5 4-5 in the proton spectrum, as well as the backbone amide N-H protons which produced resonances at 5 7.5-8.5 in the proton spectrum which were correlated in a pairwise fashion in the C O S Y spectrum (F igure 2.15). Discussion of the spectra will thus first examine each individual amino acid residue, followed by connection to the adjacent residue. 8.0 F i g u r e 2.15 Expansion of C O S Y NMR spectrum of tauramamide methyl ester (2.14), showing H a -NH correlations A proton which resonates at 5 4.29 (R1) showed a C O S Y correlation to an amide proton resonating at 5 8.39 (R-NH). Further sequential C O S Y correlations 39 from R1 to 5 1.67 (R2) and 5 1.77 (R2") establish the adjacent methylene protons. Both R2 and R2' subsequently correlate in the C O S Y spectrum to the methylene protons at 6 1.52 (R3), which in turn correlate to the methylene protons at 6 3.11 (R4). The R4 protons correlate in the C O S Y spectrum to 6 7.50 (R-NH'), which completes the side chain 1 H spin system. HMBC correlations from both 5 3.11 (R4) and 5 7.50 (R-NH') to 5 C 156.7 (R5), identified the guanidinium carbon, and HMQC correlations assigned all other carbon signals as 5 C 51.3 (R1), 5 C 27.5 (R2), 5 C 25.0 (R3) and 5 C 40.6 (R4), respectively. A weak HMBC correlation from 5 4.29 (R1) to a carbon at 5 C 172.1 (R-CO) indicated a carbonyl, which is established as a methyl ester by observation of an HMBC correlation from 5 3.62 to the same carbonyl at 5 C 172.1 (R-CO). ROESY correlations are consistent with the C O S Y assignments, confirming the presence of an arginine residue, which accounts for two degrees of unsaturation. A R O E S Y correlation from 5 8.39 (R-NH) to 5 4.53 (W1) connects the arginine spin system to the adjacent amino acid. A summary of the data used to elucidate the arginine residue can be found in Figure 2.16. 40 .A Figure 2.16 C O S Y NMR spectrum, proton and carbon assignments, as well as select 2D correlations for arginine residue in tauramamide methyl ester (2.14) The proton resonance at 5 4.53 (W1) showed a C O S Y correlation to a resonance at 5 8.14 (W-NH), as well as 5 2.89 (W2) and 5 3.15 (W2'). A R O E S Y correlation between 5 2.89 (W2) to 5 7.11 (W10) indicated an aromatic system proximate to the amino acid backbone. The proton resonance at 5 7.11 41 (W10) showed a C O S Y correlation to 5 10.76 (W-NH'), appropriate for an indole NH. This in turn showed a R O E S Y correlation to a proton resonance at 5 7.30 (W8), which correlated in the C O S Y spectrum to 5 7.04 (W7), and onwards in the C O S Y spectrum to 6 6.96 (W6), terminating with a correlation to 5 7.60 (W5). This spin system is also consistent with the presence of an indole. The protonated carbon signals were assigned using HMQC data as 5 C 123.9 (W10), 5 C 118.4 (W5), 5 C 118.0 (W6), 5 C 120.7 (W7) and 5 C111.1 (W8), respectively. An HMBC correlation from 5 6.96 (W6) assigned 5 C 127.1 (W4), similarly an HMBC correlation from 5 7.04 (W7) assigned 6 C 136.0 (W9). No protons correlated to 5 C 109.8 (W3) in the HMBC spectrum, however it could be assigned by process of elimination following assignment of the tyrosine residue {vide infra). The carbonyl of the tryptophan residue remains unassigned due to insufficient data in the HMBC spectrum, however, the number of carbonyl resonances in the 1 3 C spectrum (Figure 2.10) are consistent with the proposed structure. The completion of the assignment of the tryptophan residue accounts for seven degrees of unsaturation. A R O E S Y correlation from 6 8.14 (W-NH) to 6 4.20 (L1) connected the tryptophan residue to the next amino acid. A summary of the data used to elucidate the tryptophan residue can be found in Figure 2.17. 42 Figure 2.17 C O S Y NMR spectrum, proton and carbon assignments, as well as select 2D correlations for tryptophan residue in tauramamide methyl ester (2.14) The methine proton resonance at 5 4.20 (L1) showed a C O S Y correlation to 5 7.75 (L-NH), as well as 5 1.06 (L2). Subsequent C O S Y correlations from L2 to 5 1.25 (L3), and on from L3 to 5 0.71 and 5 0.68 (L4, L4') complete the leucine 1 H spin system. Protonated carbon resonances were assigned as 6 C 51.7 (L1), 43 5 C 40.5 (L2), 5 C 23.7 (L3), 5 C 22.8 (L4) and 5 C 21.5 (L4') using HMQC data, allowing assignment of the leucine residue, and accounting for one degree of unsaturation. A R O E S Y correlation from 5 7.75 (L-NH) to 5 4.22 (S1) connected the leucine residue to the following amino acid. A summary of the data used to elucidate the leucine residue is found in Figure 2.18. 3 3 _ A _ A / * ^ ^ A A J — — - L -Figure 2.18 C O S Y NMR spectrum, proton and carbon assignments, as well as select 2D correlations for leucine residue in tauramamide methyl ester (2.14) 44 The proton resonance at 5 4.22 (S1) showed a C O S Y correlation to 5 8.03 (S-NH), as well as 5 3.47 (S2) and 5 3.52 (S2'). C O S Y correlations were evident from 5 3.47 (S2) and 5 3.52 (S2') to a resonance at 5 4.83 (S-OH), completing the serine 1 H spin system. Protonated carbon signals were assigned as 6 C 55.1 (S1) and 6 C 61.7 (S2) using HMQC data, confirming the presence of a serine residue, and accounting for one degree of unsaturation. A R O E S Y correlation from 5 8.03 (S-NH) to 5 4.41 (Y1) connected the serine to the adjacent amino acid. A summary of the data used to elucidate the serine residue is found in Figure 2.19. 45 3.47 3.52 H-,0-H 4.83 O4.22H 8.03 6 1 . 7 ^ ^ ' ' i l H O - H C O S Y Figure 2.19 C O S Y NMR spectrum, proton and carbon assignments, as well as select 2D correlations for serine residue in tauramamide methyl ester (2.14) The proton resonance at 5 4.41 (Y1) showed a C O S Y correlation to 5 7.96 (Y-NH) as well as 5 2.64 (Y2) and 5 2.85 (Y2'). R O E S Y correlations from 5 2.64 (Y2) and 5 2.85 (Y2') to 5 7.02 (Y4/Y8) connected the aromatic spin system to the aliphatic spin system. A C O S Y correlation from 5 7.02 (Y4/Y8) to 5 6.62 (Y5/Y7) identified the vicinal protons while a R O E S Y correlation from 5 6.62 (Y5/Y7) to 5 9.13 (Y6-OH) completed the tyrosine spin system, which contains 46 an element of symmetry. Protonated carbon resonances were assigned as 6C 54.5 (Y1), 5C 36.5 (Y2), 5C 130.0 (Y4/Y8) and 5C 114.7 (Y5/Y7) using HMQC correlations. An HMBC correlation from 5 7.02 (Y4/Y8) to 5C 155.7 (Y6), and an additional HMBC correlation from 5 6.62 (Y5/Y7) to 5C 127.8 (Y3) established the quaternary carbon centres. The final amino acid is thus identified as tyrosine, which accounts for five degrees of unsaturation. A summary of the data used to elucidate the tyrosine residue is found in Figure 2.20. Figure 2.20 C O S Y NMR spectrum, proton and carbon assignments, as well as select 2D correlations for tyrosine residue in tauramamide methyl ester (2.14) 47 A R O E S Y correlation from 6 7.96 (Y-NH) to a methylene at 5 2.01 (OA2) connects the tyrosine residue an aliphatic system via an amide linkage. Subsequent C O S Y correlations into an aliphatic envelope in the 1 H NMR spectrum are due to a lipid chain, which terminates in a geminal dimethyl at 5 0.83 (OA8/OA8'), and requires one degree of unsaturation. The lack of functionality evident in the 2D spectra, coupled with the mass spectral evidence indicated the lipid to be 7-methyloctanoic amide (Figure 2.8). Careful inspection of the C O S Y spectrum, as well as comparison with the 600 MHz data from tauramamide ethyl ester yielded the proton and carbon assignments found in Table 2.1. The remaining signal (5 3.62, 5 C 51.8 (ME1)) is thus placed at the C-terminus of the peptide as a methyl ester, resulting in the complete constitution of tauramamide methyl ester (2.14), and contains the requisite 17 degrees of unsaturation (Figure 2.8). A summary of the R O E S Y data used to connect the amino acids in the chain is found in Appendix I. 2.2.3 LSIMS Information Given the peptide nature of 2.14, sequence information was sought from liquid secondary ion mass spectrometry (LSIMS), a comparatively soft ionization technique which can frequently yield sequence information in peptides.1" Full sequence information was not forthcoming, as the sample did not fragment as well as expected, however a few key ions were observed which were consistent with the proposed structure. The intact molecular species was observed both at f Measurement performed by UBC Mass Spectrometry Service. 48 m/z 878.2 [M]+ and m/z 879.2 [M+H] +. Cleavage of the serine-tyrosine amide bond yields a smaller fragment at m/z 327 [F1+Na]+ (Figure 2.21). Cleavage of the tyrosine residue adjacent to the benzylic carbon yields an intense fragment at m/z 197 [F2]+. The complete LSIMS spectrum of 2.14 can be found in Appendix II. Figure 2.21 Fragments observed in LSIMS spectrum of tauramamide methyl ester (2.14) 2.2.4 Comments on Tauramamide Methyl Ester Although the 2D NMR data of the 2.14 was sufficient to determine the constitution of tauramamide, there were still some gaps which existed in the data due to the relative paucity of information from the HMBC. More material was needed for complete bioassay and configurational analysis {vide infra), thus an extraction of a second bacterial culture was performed. Of particular interest in the structure of tauramamide methyl ester is whether or not the C-terminal ester is natural or an isolation artifact. Methyl HO. 327 [F1+Na] + 49 esters of this kind are occasionally formed as a result of repeated treatment with MeOH during extraction and chromatography. To test the hypothesis that the methyl ester was an artifact of the isolation conditions, the entire extraction was repeated on fresh cells using EtOH in place of MeOH during extraction and chromatography. Tauramamide ethyl ester was isolated from this second extract, illustrating that the methyl esters are in fact isolation artifacts and are not the true natural products themselves. 2.3 Isolation and Characterization of Tauramamide Ethyl Ester 2.3.1 Isolation of Tauramamide Ethyl Ester The isolation of the ethyl ester proceeded using an abbreviated method from that used to purify tauramamide methyl ester. Cells were grown as confluent lawns on tryptic soy agar supplemented to 1% NaCl to simulate marine conditions. Cells were collected by scraping the agar surface, and lyophilized (~28g) prior to exhaustive EtOH extraction (3 X 250 mL). The ethanolic extract was dried in vacuo to yield a brown gum and then partitioned between H 2 0 and EtOAc. The EtOAc soluble material was separated by lipophilic size exclusion chromatography (Sephadex LH-20®) in EtOH and fractions were pooled based upon 1 H NMR and TLC characteristics. The ethyl ester containing fraction (230.4 mg) was then subjected to reversed-phase C-18 HPLC (50% M e C N / H 2 0 + 0.1% TFA gradient to 70% M e C N / H 2 0 + 0.1% TFA over 40 minutes, C S C -50 Inertsil 0 D S 2 column) yielding tauramamide A ethyl ester (2.15, 4.1 mg, 0.015% dry cell weight). 2.3.2 Structure Elucidation of Tauramamide Ethyl Ester 1D ( 1H and 1 3 C) and 2D NMR (COSY, ROESY, HSQC, HMBC) experiments were performed and the data is summarized in Table 2.2. The availability of a 600 MHz spectrometer equipped with a cryoprobe was a definite advantage for data acquisition, as correlations not observed using the 500 MHz instrument were now readily observed using comparable amounts of material. The structure of 2.15 differs from that of 2.14 only by an extra methylene in the ester, and thus the NMR data are quite similar, so the discussion of the structural elucidation is abbreviated. All one bond 1 H - 1 3 C correlations were established using H S C Q data. For clarity of discussion, the numbering scheme used during the structural elucidation of 2.15 is found in Figure 2.22. Arg H 2 N ^ N H [5 HNL 8 EE ' 2.15 Trp Figure 2.22 Constitution of tauramamide ethyl ester (2.15) 51 52 53 54 o q o o CO E Figure 2.26 600 MHz R O E S Y NMR spectrum of tauramamide ethyl ester (2.15) recorded in DMSO -Og 55 Figure 2.27 600 MHz HSQC recorded in DMSO-c/e NMR spectrum of tauramamide ethyl ester (2.15) 56 o a. (Si O O ( Figure 2.28 600 MHz HMBC NMR spectrum of tauramamide ethyl ester (2.15) recorded in DMSO-cfe 57 Table 2.2 1D and 2D NMR data for tauramamide ethyl ester (2.15), recorded at 600 MHz (1H) and 150 MHz ( 1 3C) in DMSO-cfc c# "c "H COSY ROESY HMBC (1H to 1 3C) Ethyl Ester, EE 1 14.1 1.18 t (7.2), 3H EE2 EE2 EE2 2 60.5 4.09 m, 2H EE1 EE1 EE1, R-CO Arginine, Arg, R CO 172.0 - - - -1 51.8 4.25 bs R2, R-NH R2, R2', R3, R4, R-NH R-CO, R2 2 27.8 1.67 m 1.77 m R1, R3 R1, R-NH' R-CO, R1, R3, R4 3 25.1 1.53 m, 2H R2, R2', R4 R1, R-NH' R2, R4 4 40.3 3.11 m, 2H R3, R-NH' R1, R-NH' R2, R3, R5 5 156.6 - - - -NH - 8.43 d (7.7) R1 R1, W1 R-CO, R1 NH' - 7.49 m R4 R2, R2', R3, R4 R4 Tryptophan, Trp, W CO 171.7 - - - -1 53.1 4.54 bs W2, W2', W-NH W2, W2', W5, W10, W-NH, R-NH 2 27.6 2.88 m 3.16 m W1 W1, W10, W5, W-NH W1, W4, W10, W-CO 3 109.9 - - - -4 127.1 - - - -5 118.4 7.62 d (7.7) W6 W2', W1, W6 W3, W7, W9 6 118.1 6.96 dd (7.2, 7.7) W5, W7 W5 W4, W8 7 120.7 7.04 m W6, W8 W8 W5, W9 8 111.2 7.30 d (8.2) W7 W7, W-NH' W4, W6 9 136.0 - - - -10 123.9 7.11 d (1.5) W-NH' W1, W2, W-NH, W-NH' W3, W4, W9 NH - 8.17 bd (8.2) W1 W, W2, W10, L1, L-NH NH' - 10.77 s W10 W8, W10 W3, W4, W9, W10 Leucine, Leu, L CO 171.6 - - - -1 51.2 4.23 bs L2, L-NH L3, L4', W-NH -2 40.7 1.08 m, 2H L1, L3 L-NH L-CO 3 23.7 1.23 m L2, L4, L4' L1 -4 22.8 0.70 d (6.6), 3H L3 - -4' 21.5 0.67 d (6.7), 3H L3 L1 -NH - 7.76dJ£2]__ L1 L2, S1, S-NH, W-NH L1, S-CO Serine, Ser, S CO 169.7 - - - -1 55.0 4.24 bs S2, S2', S-NH S2, S2', S2-OH, S-NH, L-NH S-CO, S2 2 61.7 3.46 m 3.51 m S1 S1, S2-OH, S-NH S-CO 2-OH - 4.84 t (5.4) S2, S2' S1, S2, S2' S1, S2 NH - 8.05 d (7.7) S1 S1, S2, S2', S2-OH, L-NH, Y1 S1, Y-CO Tyrosine, Tyr, Y CO 171.7 - - - -1 54.5 4.42 bs Y2, Y2', Y-NH Y2, Y2', Y4/Y8, Y-NH, S-NH -2 36.6 2.63 m 2.84 m Y1 Y1, Y4/Y8, Y-NH Y1, Y3, Y4/Y8, Y-CO 3 127.9 - - - -4, 8 130.0 7.02 d (8.2), 2H Y5/Y7 Y1, Y2, Y2', Y5/Y7, Y-NH Y2, Y4/Y8, Y5/Y7, Y6 5, 7 114.7 6.61 d(8.2), 2H Y4/Y8 Y4/Y8, Y6-OH Y3, Y5/Y7, Y6 6 155.7 - - - -6-OH - 9.14 s - Y5/Y7 Y5/Y7, Y6 NH - 7.99 d (7.7) Y1 Y1, Y2, Y2', Y4/Y8, OA2 Y1, Y2, OA1 7-methyloctanoamide, OA 1 172.4 - - - -2 35.1 2.00 m, 2H OA3 OA3, Y-NH OA1, OA3, OA4 3 25.2 1.35 m, 2H OA2, OA4 OA2 OA1 4 28.8 1.06 m, 2H - - -5 26.5 1.14 m, 2H - - -6 38.3 1.08 m, 2H - - -7 27.4 1.45 m OA8, OA8' OA8, OA8' OA6, OA8, OA8' 8 22.5 0.83 d (6.7), 3H OA7 OA7 OA6, OA7 8' 22.5 0.83 d (6.7), 3H OA7 OA7 OA6, OA7 58 The data collected was completely consistent with the formation of the ethyl ester during isolation. The ethyl ester was readily evident by a 1 H signal at 5 1.18 (EE1) which showed a C O S Y correlation to a proton resonance at 5 4.09 (EE2). An HMBC correlation from a proton resonance at 6 4.09 (EE2) to a carbon resonance at 5 C 172.0 (R-CO) established the arginine residue at the C-terminus of the peptide. An HMBC correlation from 5 8.43 (R-NH) to a carbon resonance at 5 C 172.0 (R-CO) supports this assignment. Multiple R O E S Y and HMBC correlations were present which confirmed the peptide sequence as shown in Figure 2.29. It follows that the full constitution of tauramamide ethyl ester is 2.15. Figure 2.29 Structure of tauramamide ethyl ester (2.15) showing selected inter-residue correlations H 2 N NH / " ~ " \ HMBC R O E S Y 59 2.3.3 Comments on Tauramamide Ethyl Ester The isolation of the ethyl ester from the EtOH extract, coupled with the absence of the methyl ester indicates that the ester is formed during the isolation steps. It is thus likely that the free acid is the true natural product, and the esters are simply formed as a result of repeated exposure to alcohol during extraction and chromatography. This metabolite was reproducibly synthesized by P N G -276. 2.4 Configurational Information Marfey's analysis 1 3 0 of the 6M HCI hydrolysate of tauramamide methyl ester was performed to determine the configuration of the amino acids. A series of standards (both L- and D,L-mixtures) were reacted with Na-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (FDAA), and the derivatised standards analyzed by gradient HPLC to determine their relative retention t imes. 1 3 1 Injections of the derivatised acid hydrolysate, as well as coinjections with standards confirmed four of the five amino acid configurations as outlined in Table 2.3. No peak was evident for tryptophan in the original hydrolysate, so a second hydrolysis was performed on fresh tauramamide methyl ester, this time under milder (1/16 t h duration) hydrolysis conditions. Injections of the second hydrolysate, complete with coinjection of standards, provided the data necessary to determine the 60 configuration of the tryptophan residue. In all cases the FDAA-L conjugate eluted before the FDAA-D conjugate. Table 2.3 Retention times (minutes) of FDAA-modified standards and derivatised tauramamide methyl ester acid hydrolysate from first hydrolysis FDAA-Amino Acid L -FDAA D-FDAA Acid Hydrolysate Arginine 31.3 32.3 31.0 (L) Leucine 55.9 62.8 61.6 (D) Serine 35.1 36.9 35.4 (/_) Tyrosine 47.2 50.5 50.5 (D) Table 2.4 Retention times (minutes) of FDAA-modified standards and derivatised tauramamide methyl ester acid hydrolysate from second hydrolysis FDAA-Amino Acid L-FDAA D-FDAA Acid Hydrolysate Tryptophan 55.9 59.2 55.3 (/_) Marfey's analysis of tauramamide revealed the presence of two D amino acids (Leu and Tyr, Figure 2.30), further supporting the proposal that the compound is of nonribosomal origin. Certainly, the presence of non protein amino acids is not unusual for this organism, although it certainly adds to the novelty of tauramamide. The loloatins, bogorols and tupuseleiamides all feature D amino acids. It has been observed that D amino acids in oligopeptides give increased resistance to enzymatic proteolysis . 1 2 3 , 1 3 2 Although no studies in our lab have focussed on this issue to date, it would be interesting to assemble a combinatorial library of the various configurational isomers of tauramamide, and potentially the other PNG-276 compounds to examine the relationship between their structure and antibacterial activity. 61 H 2 I S L . N H Figure 2.30 Structure of tauramamide (2.13) with amino acid configurations shown 2.5 Synthesis of Tauramamide Ethyl Ester (2.15) A complete synthesis of tauramamide ethyl ester (2.15) was performed by Alban Pereira, a fellow graduate student in the Andersen Lab, in order to confirm the stereochemical assignment of tauramamide as well as to investigate the importance of the C-terminal free carboxylic acid to antibacterial activity. The synthetic scheme followed is outlined in Figures 2.31 and 2.32. The details of this synthesis will be reported elsewhere. 62 Figure 2.31 Synthetic scheme for tauramamide: synthesis of the first subunit 63 Figure 2.32 Synthetic scheme for tauramamide: synthesis of the second subunit, final assembly and deprotection 64 Using the synthetic schemes outline in Figures 2.31 and 2.32, samples of both the free acid (2.13) and ethyl ester (2.15) were prepared. They were then subjected to standard characterization techniques (MS, NMR, OR) and the resulting data compared with that of natural tauramamide ethyl ester (2.15). The natural and synthetic ethyl esters of tauramamide were identical by both 1 H NMR spectroscopy (Figure 2.33) and mass spectrometry comparison. The total synthesis of 2.15 confirms the structure proposed from analysis of the spectroscopic data of the isolated compound. The optical rotation data corroborates this assessment ([a] D 2 5 -31.9 (c = 0.8 MeOH) for the natural product, [a] D 2 5 -23.4 (c = 0.7 MeOH) for the synthetic) and confirms the assignment of tauramamide as containing D- leucine and tyrosine, and L-arginine, serine and tryptophan. 65 0 ) 11 <E Z c J3 (D ro co CD W ^ £ cu o ~ 3 O m O ^ ^ O X =5 - N o Synthetic CD O O —^ C L CD C L C/> <^ f—t-zr CD _ _ o"r =3" co i ! l—h c — \ 3 3 CL CD CD i 1 u UL4 u 11 .0 I I I I I 10 .0 I I I I I I I I 9.0 ' I M ' I I | I M I | I I I I | I I I I | I I I I | 8.0 7.0 6.0 5 .0 i I i i i i I i i i i i ' 4 .0 3.0 I i i i i | i i i i | i 2.0 i I i i i i i i i i i i i 1.0 0 .0 (ppm) Natural CD Ui i—t-CD LUIAJ14. fO I I I i l | I ' ' M 11 .0 1 1 I 1 1 10 .0 I I I I I I 9.0 8 .0 7.0 i | i i i i i i i i i | i i i i | 6.0 5.0 4 .0 3.0 I i ' ' i i i 2.0 i I i i i i | i ' i i I i ' i 1.0 0 .0 (ppm) 2.6 Biological Activity of Tauramamide Tauramamide was found to be antibacterial against MRSA, C. albicans, and Enterococcus sp. A number of bioassays were employed on both 2.15 and 2.13, the results of two of which are summarized in Table 2.5. The initial assay used to follow the activity during the isolation was the Alamar Blue™ assay, which uses a dye to measure the presence of proliferating pathogen ce l l s . 1 3 3 Dilutions of test compounds are placed into 96-well plates, and pathogen in growth medium is added. Examination of plates following an incubation period shows the presence of viable cells with a blue colour, and inhibition of growth with a pink colour. Compound 2.15 showed reasonable activity against MRSA using this method (MIC = 25 ug/mL), and slight inhibition of C. albicans at 200 ug/ml_. Compound 2.13 showed no inhibition of these two pathogens, although it was observed to precipitate out of solution in the wells. Both were active against Enterococcus sp. at 0.1 ug/mL. 67 Table 2.5 Bioassay data (MIC) from both tauramamide (2.13) and tauramamide ethyl ester (2.15) Bioassay Pathogen Vancomycin ug/mL Amphotericin B ug/ml_ 2.13 (ug/mL) 2.15 (ug/mL) Agar Inclusion MRSA 0.625 - 200 9.4 C. albicans - No endpoint 50 75 Alamar Blue C. albicans MRSA - - ppt ppt 200 25 Enterococcus - - 0.1 0.1 sp-More interesting was the data obtained from the agar inclusion bioassay. In this assay, small agar plates are made containing growth medium and pathogen of a standard concentration, and test compounds are diluted into multiple plates. Following incubation, plates are inspected for pathogen growth. The plate with the lowest concentration of compound that shows lack of growth gives the minimum inhibitory concentration. Standard antibiotics were also introduced as positive controls. Compound 2.15 was found to inhibit MRSA at a MIC of 9.4 ug/ml_, which, while ten-fold less active than vancomycin (0.625 ug/mL), is still of interest, and considerably more active than 2.13 (200 ug/mL). However, 2.13 was more active than 2.15 (50 ug/mL vs. 75 ug/mL) against C. albicans. In addition, making the sample up in solid phase medium seems to remove the problems associated with precipitation of 2.13 in the Alamar Blue™ assay. The high activity against Enterococcus sp. (0.1 ug/mL) is uniform for both 2.13 and 2.15. This is indicative of even higher potential activity, given the 68 precipitation of sample which occurs in the Alamar Blue™ assay. However, neither 2.13 and 2.15 were tested in the agar inclusion assay against Enterococcus sp. 2.7 Discussion and Conclusions The isolation of tauramamide esters from cultures of PNG-276 provides yet another example of a novel antimicrobial product from this organism. The isolation of both the methyl (2.14) and ethyl (2.15) esters of tauramamide from MeOH and EtOH extracts of PNG-276, respectively, indicates that the ester functionality is an artifact of the isolation, and thus the natural product is likely the free acid (2.13). Marfey's analysis of the amino acids indicated that two (leucine and tyrosine) of the five amino acids possess the D configuration. This, coupled with mixed biogenesis, indicates a non-ribosomal origin for the compounds. Independent synthesis of the free acid allowed for further biological testing to be done, resulting in the observation that the free acid is more active against C. albicans (MIC 50 ug/mL), whereas the ethyl ester is more active against MRSA (MIC 9.38 ug/mL). The different bioassays employed help to explain the results observed during the isolation. The initial focus of this project was upon finding the known antifungal basiliskamides (2.9 and 2.10) from PNG-276 cultures. Upon treatment with MeOH in the extraction process and chromatography, however, the C. albicans activity seemed to weaken although the MRSA activity remained strong. 69 This is consistent with the conversion of 2.13 to 2.14 during the procedure. As for a putative structure activity relationship shown by the relative activities of 2.13 and 2.15, it is difficult to say which functionalities are required without further study. It may be that the change in charge due to esterification allows a different interaction with a specific target, or perhaps the cell membrane as a whole. With the ester in place, perhaps 2.15 interacts with the cell membrane in a different way, due to the net positive charge of the compound, and either is able to be transported across the membrane or not, depending on the nature of the membrane. Given the differences between eukaryotic and prokaryotic membranes, the difference in the effect of esterification of 2.13 on the C. albicans and MRSA activities is not unusual. It is interesting to compare the structure of 2.13 with other metabolites obtained from the same organism. The lipid tail of 2.13 is identical to that present in tupuseleiamide A (2.11), although in that compound both the tyrosine and the serine are in the D configuration, and in the inverse order when compared to 2.13. The bogorol class of compounds possess D-leucine, and the loloatins have been found to contain D-tyrosine. All of these features, put together, provide the necessary building blocks to make the novel aspects of 2.13. The rest could be incorporated via normal metabolic pathways. It could be said that the species, in its ongoing process of experimentation and genetic manipulation, has come upon a particularly fruitful subset of molecular tools that can be used to produce a number of useful compounds. The discovery of 70 tauramamide is yet another testament to the versatility of these tools. It possesses features found in the bogorols, tupuseleiamides and loloatins. Given the highly modular nature of non-ribosomal and polyketide biosynthesis, it is entirely consistent to see classes of compounds which contain substructures from several disparate classes of compounds. In fact, this modularity of polyketide and NRPS is currently being used to create new natural p roducts . 1 2 6 , 1 3 4 As discussed earlier the N R P S can contain any number of domains, each of which will have an impact on the end product produced by the enzyme. Including or excluding a given domain will form a different product. In a sense, this is evidence of the wide array of biosynthetic pathways available to an organism. A mix and match method (like a biological version of combinatorial chemistry) is ideal for creating a large number of possible compounds. Over time, organisms which are successful at this might survive, while others may not. PNG-276 has certainly been shown to be one of these biosynthetically gifted organisms. It is also testament to the possibilities which exist in the realm of marine drug discovery. While much of the research on terrestrial microbes seems to be slowing, studies into marine microorganisms show great promise. 71 2.8 Experimental 2.8.1 General Experimental Procedures 2D-NMR spectra were recorded on either Bruker AMX-500 or AV-600 spectrometers. The AV-600 is equipped with a cryoprobe. 1 3 C NMR data for tauramamide methyl ester (2.14) was recorded on a Bruker AM400 spectrometer while all other 1 3 C NMR data was recorded on the AV-600 system. 1 H NMR chemical shifts were referenced to the residual DMSO -06 signal (5 2.50 ppm), and 1 3 C NMR chemical shifts were referenced to the DMSO-cfe solvent peak (6C 39.51 ppm). All NMR solvents were obtained from Cambridge Isotope Laboratories. All NMR data was processed using Bruker WINNMR® software. All chromatography was performed using HPLC grade solvents from Fisher Scientific with no further purification. Water was purified using a Millipore MQ filter system. Reversed-phase chromatography was performed using 10, 5 or 2 gram Waters C-18 Sep-Paks. HPLC isolation was performed using a Waters Breeze HPLC system consisting of a Waters 1525 Binary HPLC Pump and Waters 2487 Dual Wavelength Absorbance Detector interfaced to a PC, whereas the Marfey's analysis was performed using a Waters 600E system controller liquid chromatograph interfaced to a Waters 486 tunable absorbance detector and a Waters 994 programmable photodiode array detector. Optical rotations were measured with a J A S C O J-1010 spectrophotometer using a 10 mm cell with a sodium light (589 nm). High-resolution electrospray mass spectra were collected on a Micromass LCT mass spectrometer, whereas low-resolution 72 mass spectra were collected on a Bruker Esquire LC mass spectrometer. Liquid secondary ion mass spectrometry (LSIMS) was performed on a Kratos Concept IIHQ mass spectrometer using a thioglycerol matrix. Amino acid standards were obtained from Sigma-Aldrich, as was the FDAA (Na-(2,4-dinitro-5-fluorophenyl)-L-alaninamide, D7906). PNG 276 was identified as Brevibacillus laterosporus by 16S RNA analysis. 2.8.2 Culture Conditions for PNG 276 PNG-276 was cultured on 21 pans of solid tryptic soy agar supplemented with NaCl to a final concentration of 1%. Each pan was 24 cm X 37 cm X 0.5 cm deep, and the cultures were grown at room temperature for five days. Live cells were then scraped from the solid agar and lyophilized. 2.8.3 Isolation of Tauramamide Methyl Ester (2.14) For the general isolation scheme see section 2.2.1. Lyophilized cells (-30 g) were exhaustively extracted with MeOH (3 X 250 mL). The combined methanol extract was concentrated, in vacuo to yield a brown gum (3.8 g) and then partitioned between H 2 0 and EtOAc, both of which were concentrated in vacuo. The organic partition (1.0 g) showed activity against C. albicans. Initial lipophilic size exclusion chromatography (Sephadex LH-20®) in MeOH was followed by partitioning size exclusion chromatography (Sephadex LH-20®) in 73 20:5:2 EtOAc /MeOH/H 2 0 yielding a number of fractions which were pooled based upon bioassay information, NMR and TLC characteristics (Rf = 0.7 on normal phase silica (4:1:1 n-BuOH:MeOH:H 2 0, visualized with eerie sulfate/phosphomolybdic acid dip)). The most active fraction (140.9 mg) was then subjected to reversed-phase C-18 chromatography (Waters 2 g Sep-Pak®) as follows. Sample was dissolved in MeOH, and adsorbed in vacuo onto reversed phase C-18 silica. A step gradient of 10 mL volumes of 10% increments of H 2 0 :MeOH (ie. 10 mL of 100% H 2 0 , then 10 mL of 90 % H 2 0 :MeOH, etc.) was performed, with an additional 10 mL steps of 50% MeOH:DCM and 100% DCM used to flush the column, resulting in 36 fractions'. From this, TLC characteristics were again used to pool the fractions of interest. This sample (92.8 mg) was then separated by repeated injections on isocratic reversed-phase HPLC (40% MeCN/H20 + 0.1 % TFA, CSC-lnertsil® ODS2 column, 2 mL/min flow rate, dual wavelength (210 nm and 254 nm) uv detection) yielding tauramamide methyl ester (2.14, 7.4 mg, 0.02% dry cell weight). 2.8.4 Tauramamide Methyl Ester (2.14) Physical Data Clear, pale yellow glass (7.4 mg); [a] D 2 5 -14.6 (c=0.6 MeOH). For 1D and 2D NMR data see Table 2.1. HRESIMS: [M+H]+ m/z = 878.5181 (calcd for C45H67N909 ! 878.5185). 74 2.8.5 Acid Hydrolysis of Tauramamide Methyl Ester (2.14) Purified 2.14 (0.8 mg, 0.9 umol) was hydrolyzed in 0.5 mL of 6 N HCI, and sealed in a screw top vial. The mixture was heated to 108 °C with stirring for 16 hr. Upon cooling, the mixture was evaporated under N 2 , and repeatedly under same from H 2 0 (3 x 0.5 mL) to removed traces of HCI. A second hydrolysis was performed using purified 2.14 (0.2 mg, 0.2 umol) under identical conditions, but heated for 1 hr instead of 16 hrs. 2.8.6 Preparation of FDAA Derivative Standards Amino acid standards (2.0 umol) were dissolved in H 2 0 (40 uL). To this was added Marfey's reagent (Na-(2,4-dinitro-5-fluorophenyl)-L-alaninamide, 62.5 mM in Me 2 CO, 60uL), followed by N a H C 0 3 (1 M, 20 uL). The mixture was heated for 1 hr at 43 °C, after which HCI (2 N, 10 uL) was added. An additional aliquot of M e 2 C O (50 uL) was added to solubilize the samples. The acid hydrolysate of 2.14 was treated in the same fashion as the amino acid standards. 75 2.8.7 Marfey's Analysis of Tauramamide Methyl Ester (2.14) Repeated HPLC injections were performed using an Alltech Econosil C18 5pm HPLC column, and run using the following solutions. Solution A was prepared by adding Et 3 N (28 mL, 0.2 mol) to H 2 0 (3.572 L) while stirring. To this mixture, H 3 P O 4 was added drop-wise until the mixture was pH 3.0. MeCN (400 mL) was then added, resulting in a mixture of 9:1 triethylammonium phosphate (50 mM, ph 3.0)/MeCN. Solution B was HPLC grade MeCN. The HPLC conditions used were a linear gradient from 100 % Solution A to 60 % Solution A/40 % Solution B over 40 minutes at a flow rate of 1 mL/min, with uv detection at 210 nm. 2.8.8 Isolation of Tauramamide Ethyl Ester (2.15) Cells were cultured as before (Section 2.8.2). Cells were collected by scraping the agar surface, and lyophilized (~28g) prior to exhaustive EtOH extraction (3 X 250 mL). The ethanolic extract was dried in vacuo to yield a brown gum, and then partitioned between H 2 0 and EtOAc. The EtOAc soluble material was concentrated in vacuo, then dissolved in EtOH (5mL) and separated by lipophilic size exclusion chromatography (Sephadex LH-20®) in EtOH and fractions were pooled based upon 1 H NMR and TLC characteristics (Rf = 0.63 on reversed-phase C-18 silica, 100 % MeOH solvent, visualized with vanillin acid spray). The ethyl ester-containing fraction (230.4 mg) was then 76 subjected to reversed-phase C-18 HPLC (50% M e C N / H 2 0 + 0.1% TFA linear gradient to 70% M e C N / H 2 0 + 0.1% TFA over 40 minutes, CSC-lnertsil® ODS2 column, 2 mL/min flow rate, dual wavelength (210 nm and 254 nm) uv detection) yielding tauramamide A ethyl ester (2.15, 4.1 mg, 0.015% dry cell weight). 2.8.9 Tauramamide Ethyl Ester (2.15) Physical Data Clear, pale yellow glass (2.1 mg); [a] D 2 5 -31.9 (c = 0.8 MeOH). For 1D and 2D NMR data see Table 2.2. HRESIMS: [M+H]+ m/z = 892.5294 (calcd for C46H70N9O9, 892.5297). 2.8.10 Synthetic Tauramamide (2.13) Physical Data Clear, pale yellow glass (5.8 mg); [a] D 2 5 -51.8 (c = 0.9 MeOH). HRESIMS: [M+H]+ m/z = 864.4982 (calcd for C44H66N9O9, 864.4984). 2.8.8 Synthetic Tauramamide Ethyl Ester (2.15) Physical Data Clear, pale yellow glass (3.7 mg); [a] D 2 5 -23.4 (c = 0.7 MeOH). HRESIMS: [M+H]+ m/z = 892.5291 (calcd for C46H70N9O9, 892.5297). 77 Chapter 3: Methylspirastrellolide C, an Antimitotic Macrolide Isolated from the Marine Sponge Spirastrella coccinea 3.1 Introduction Mitosis is the step in the eukaryotic cell cycle where replicated chromosomes are distributed into two daughter cells. It is an attractive target for cancer therapy, and is the focus for a number of classes of cancer chemotherapeutics. 1 3 5 , 1 3 6 Although mitotic cancer cells can be targeted in a number of different ways, one of the classic methods is to target microtubule function. Microtubules are important structures in a mitotic cell that are vital for the proper separation and integrity of the DNA. 1 3 7 The majority of antimitotic drugs are compounds which target microtubule function. Figure 3.1 The eukaryotic cell cycle 78 The taxanes are a very well studied class of microtubule binding drugs, isolated initially from the bark of Taxus brevifolia.^38 The natural product paclitaxel (Taxol®, 1.4), along with the related synthetic analogue docetaxel (Taxotere®, 3.1) are the two commercially available taxanes employed in the treatment of ovarian, lung, and metastatic breast cancers, as well as the management of early breast cancer . 1 3 9 One of the many activities of the taxanes is to promote the assembly of microtubule polymers and stabilize the polymerized structure, thereby inhibiting the normal progression of mitosis, leading to apoptosis and cell death. Figure 3.2 Docetaxel (3.1) and paclitaxel (1.4) This is in contrast to the function of the vinca alkaloids, such as vincristine (3.2) and vinblastine (3.3), which were isolated from the leaf extract of Vinca rosea by two groups independently in the late 1950s , 1 4 0 ' 1 4 1 and were initially thought to act by inhibiting microtubule formation by binding specifically in the Vinca domain of tubulin. 1 4 2 It is now known, however, that they possess multiple types of activity, depending upon the drug concentration. At high concentrations 79 they indeed act as a microtubule depolymerizer, however, at low concentrations they stabilize microtubules. 1 4 3 As is common with many biological systems, the seemingly straightforward activity of a compound is in reality much more complicated. Figure 3.3 Vincristine (3.2) and vinblastine (3.3) Although the vinca alkaloids and the taxanes are some of the most prominent antimitotic agents currently known, a host of others have been discovered. In contrast to the plant compounds mentioned above, a large number of promising antimitotic agents have been isolated from marine sources. These will be discussed below, and grouped based upon their mode of action. Discodermolide (1.27) is a polyketide isolated from the marine sponge Discodermia dissoluta collected off Grand Bahama Island, and reported in 1990 as possessing both immunosuppressive and cytotoxic properties. 1 4 4 Subsequent studies indicated that 1.27 halts cells in the G2 or M phase of the cell cycle at low concentrations (IC50 3-80 nM), and have also established the absolute configuration of 1.27 through synthesis. 1 4 5 Compound 1.27 was found to bind on or near the same site on microtubules as paclitaxel, although with higher affinity, 80 and is thus thought of as a possible alternative or supplement to paclitaxel therapy. 1 4 6 A number of syntheses have been performed to make 1.27, its enantiomer, and structural analogues. 1 4 7 Novartis is currently conducting Phase I clinical trials with discodermolide. 1 4 8 1.27 Figure 3.4 Discodermolide (1.27) Eleutherobin (3.4) is a diterpene glycoside originally isolated from Eleutherobia sp., a soft coral, and subsequently isolated from the soft coral Erythropodium caribaeorum along with related diterpene g lycos ides . 1 4 9 , 1 5 0 Compound 3.4 was found to possess significant cytotoxicity against a range of cancer cell lines (IC50 10-15 nM) and it binds tubulin in a similar fashion to paclitaxel and discodermolide. Together with the development of paclitaxel analogues, eleutherobin has been used to help establish a common pharmacophore for tubulin stabilizing agents. 1 5 1 Clinical studies of 3.4 have been hampered by inadequate supply. 1 5 2 81 3.4 Figure 3.5 Eleutherobin (3.4) A second class of antimitotic agents are exemplified by dolastatin 10 (3.5) and hemiasterlin (3.6). Compound 3.5 is one of a number of interesting bioactive compounds isolated from the sea hare Dolabella auricularia found to be extremely cytotoxic against P388 leukemia cells (ED 5 0 0.1 ng /mL) . 1 5 3 , 1 5 4 Hemiasterlin was originally reported in 1994 from the sponge Hemiasterella minor collected in South Af r ica . 1 5 5 Subsequently, analogues hemiasterlin A (3.7) and B (3.8) were isolated, and the synthetic analogue HTI-286 (3.9) developed. 1 5 6 " 1 5 8 All were found to be potent cytotoxins active against a range of cancer cell lines. Both the hemiasterlins and dolastatin 10 are thought to act by binding tubulin in the vicinity of the Vinca binding site, and have been shown to competitively inhibit each other. 1 5 9 " 1 6 1 Dolastatin 10 has been evaluated in a number of clinical trials, but did not pass Phase II due to a low therapeutic i n d e x . 1 3 5 , 1 6 2 , 1 6 3 HTI-286 has passed Phase I clinical trials, and is in the process of being licensed for further studies. 1 6 4 82 I 3.6 Ri = Me R 2 = Me 3.7 Ri = H R 2 = H 3.8 RT = Me R 2 = H 3.9 Figure 3.6 Structures of dolastatin 10 (3.5), hemiasterlin (3.6), hemiasterlin A (3.7) and B (3.8), and HTI-286 (3.9) Spongistatin 1 (3.10) is the first of a group of nine related compounds isolated from the sponges Spongia sp. and Spirastrella spinispirulifera by the Pettit group in the early 1990s, and reported as exhibiting growth inhibition against a number of chemoresistant cell types in the US National Cancer Institute's panel of 60 human cancer cell l ines. 1 6 5 It was simultaneously reported by Kobayashi and co-workers from the Okinawan sponge Hyrtios a/frvm.1 6 6 Subsequent studies showed that 3.10 bound to tubulin in the Vinca domain, and inhibited tubulin assembly, thus manifesting antimitotic activity. 1 6 7 It was found that this binding is unlike both dolastatin 10 (3.5) and the vinca alkaloids, in that 83 a tubulin polymer is not formed upon treatment of cells with 3.10. It is not surprising that 3.10 interacts differently than 3.5, considering the marked difference in chemical structure between the two. Further analysis indicated that the unique activity of 3.10 may be attributed to the two spiroketal pyran moieties. A synthetic compound, SPIKET-P (3.11), exhibited similar activity to 3.10, and is currently under further investigation. 1 6 8 OH 3.10 3.11 Figure 3.7 Spongistatin (3.10) and SPIKET-P (3.11) Microtubule function is not the only process that, when targeted for chemotherapy, can lead to cancer cell death via mitotic interference. Okadaic acid (3.12) was originally isolated from the Japanese sponge Halichondria okadai and the Caribbean sponge Halichondria melanodocia as a cytotoxin, but was subsequently identified as antimitotic agent acting through inhibition of serine/threonine protein phosphatase activity. 1 6 9" 1 7 1 It has subsequently been 84 shown that okadaic acid (3.12) and structurally similar compounds are produced by the marine dinoflagellates Prorocentrum and Dinophysis s p . 1 7 2 , 1 7 3 3.12 Figure 3.8 Okadaic acid (3.12) Serine/threonine protein phosphatases are enzymes that catalyze the removal of phosphate from serine and threonine residues on phosphoproteins. 1 7 4 Reversible phosphorylation is an important process used by the cell to modify the biological activity of a number of structural and regulatory proteins. 1 7 5 Because of this important function, a number of studies now focus on finding inhibitors of protein kinases, enzymes which are responsible for adding phosphates to serine and threonine residues. Kinases outnumber the protein phosphatases by approximately 15:1 and were originally thought to be the main players in phosphorylation pathways, although it has been recently shown that both processes are important to cellular function. 1 7 5 The protein phosphatases can be grouped genetically into two families: the P P P family (PP1, PP2A and PP2B), and the P P M family (PP2C) . 1 7 4 Okadaic acid was found to inhibit the activity of PP2A most strongly, and PP2C not at a l l . 1 7 1 As PP2A has been 85 shown to play a central role in the cell cycle, it is thought that protein phosphatase inhibitors such as okadaic acid may become clinically u s e f u l . 1 7 6 , 1 7 7 Figure 3.9 Proposed role of protein phosphatase 2A (PP2A) in control of mitosis (adapted from Wera and Hemmings). PP2A controls activation of cyclin-dependant kinase 1 (cdkl) via inhibition of cdkl activating kinase (CAK). Cdk1 activation is central in the cellular entry into mitosis. PP2A also is implicated in controlling levels of cdc25, a protein phosphatase that, among other functions, can itself activate c d k l . Inhibition of PP2A results in the loss of these and other controls, and results in uncontrolled entry into mitosis. The structure of spirastrellolide A (3.13) was initially reported by our research group in 2003 as an antimitotic compound from the marine sponge Spirastrella coccinea, and was amended in 2004 following further mass spectral and configurational ana lys i s . 1 7 9 , 1 8 0 Interestingly, although it is structurally similar to the spongistatins, it does not appear to interact with purified tubulin. In fact, 3.13 drove cells directly from S phase into mitosis, similar to okadaic acid and 86 other serine/threonine phosphatase inhibitors. 1 7 6 It inhibited protein phosphatase 2A (PP2A) strongly (IC 5 0 = 1 nM) and protein phosphatase 1 (PP1) less effectively (IC50 = 50 nM). 1 8 0 No inhibition was observed for protein phosphatase 2C (PP2C). Figure 3.10 Spirastrellolide A (3.13) As well as 3.13, up to 15 other related macrolides were observed in the extract of S. coccinea. Unfortunately, limited supply and instability of the components initially prohibited analysis of the minor spirastrellolides. A recent recollection of S. coccinea permitted the analysis of these side fractions. In the following chapter the isolation and structural elucidation of methylspirastrellolide C (3.14), a novel antimitotic analogue of spirastrellolide A, is reported. 87 MeCUj 53 | f 49 15 19 Figure 3.11 Methylspirastrellolide C (3.14) Compound 3.14 differs from 3.13 in three areas. It lacks the C28 chlorine, the C15-C16 alkene, and is hydroxylated at C8. While interesting due to its unique composition, the elucidation of 3.14 also contributed a valuable piece of information to the study of the relative configuration of spirastrellolide A. Prior to this study, only the relative configuration of three segments of 3.13 were known (Figure 3.12). This has been the focus of a great deal of study, with several synthetic groups attempting to synthesize the possible combinations of these subunits in the pursuit of the complete relative configuration of 3.13.181"186 Figure 3.12 Three areas of known relative configuration of spirastrellolide A (3.13) 88 The relative configuration of these three areas of 3.13 was established using R O E S Y and 1 H - 1 H coupling constant data. To establish the relative configuration of C9-C-13, as well as that of C21-C23, acetonides were formed and analyzed. 1 8 0 The presence of the C8 alcohol in 3.14, compared to 3.13, allowed the complete assignment of the relative configuration of the C 3 - C 2 4 segment of the molecule using a similar analysis, without the formation of the acetonides. This is thus the first assignment of the C3-C21 relative configuration of the spirastrellolide core. It has been subsequently verified through the use of X-ray crystallographic techniques on a modified fragment of methylspirastrellolide B (3.15). 1 8 7 Using this recent information, the complete absolute configuration of 3.14 is proposed, with the exception of that of the C46 alcohol, which remains unknown. .OMe ,>vOH 'OH OMe Figure 3.13 Structure of methylspirastrellolide B (3.15) 89 3.2 Isolation and Characterization of Methylspirastrellolide C 3.2.1 Isolation of Methylspirastrellolide C1 Spirastrella coccinea was collected from reef walls at depth of 2-5 m near Capucin, Dominica. The sponge was extracted repeatedly with MeOH and resulting extracts were dried in vacuo. The resulting reddish gum was subjected to a number of solvent-solvent partitions (see Experimental), culminating in a CH 2 CI 2 extract which was evaporated to dryness to yield a potently antimitotic amorphous red solid (2.21 g). At this point, the CH 2 CI 2 extract was methylated to simplify the isolation process. The sample was treated with diazomethane generated in situ by addition of trimethysilyldiazomethane in hexanes to anhydrous MeOH in C 6 D 6 , and left stirring at room temperature for 16 hours. All reagents were evaporated, and the sample fractionated using normal phase step gradient silica gel chromatography (from 19:1 hexanes/EtOAc to MeOH). Two fractions, eluting with EtOAc (363.7 mg) and 1:9 MeOH/EtOAc (110.6 mg) were biologically active, and further separated using size exclusion chromatography (Sephadex LH-20, 4:1 MeOH/CH 2 CI 2 ) . From these two separations were obtained potently antimitotic fractions (135.1 and 39.2 mg, respectively), both of which contained a complex mixture of methylspirastrellolides. Methylspirastrellolide A and a number of analogues were obtained from the first fraction using a range of reversed-phase HPLC conditions. The second fraction yielded impure 1 Initial isolation work performed by Dr. D. E. Williams, Andersen Lab, University of British Columbia. 90 methylspirastrellolide C using reversed-phase HPLC (3:1 MeOH/H 2 0) , as well as trace quantities of other analogues. This sample was subjected to further reversed-phase HPLC (gradient MeCN/H 2 0) to yield pure methylspirastrellolide C (3.14, 1.8 mg). Initial inspection of the proton NMR spectrum of 3.14 was promising, although subsequent collection of 1D and 2D data showed that the sample resonances changed over a period of weeks when stored in an NMR tube in C 6 D 6 at -20 °C (Figure 3.14). This is likely due to small changes in the concentration of the sample, as well as absorption of small amounts of water from the surroundings. This complicated the analysis of data sets, as the same sample would yield slightly different 2D data over a period of several weeks week due to minute changes in sample composition. Fortunately, more sponge had been collected and extracted for studies on other analogues, allowing for the isolation of fresh 3.14 in greater quantities than before. 91 l t |Mlir i IIIIT»IITIIIIIIIII l ' I III |MMIITITpilI l l t l l |TIIT|ITTT I WirnTlTp -T i rr i r i t |TIM 1 rTTT ( TTITM '"r , , l n»«'r"n« IT p TT IJIT. T'] T 11 I f r i l I J . T I r | I I • I J 5.0 4.9 4,8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 -rpT-rrTTTTTJTTTTTTTT^^ 5.0 4.9 4.8 4.7 4.6 4 ,5 4.4 4.3 4.2 4.1 4 ,0 3.9 3.8 3.7 3.6 3.5 1 1 1 1 1 " 1 | i n m n> |n i | n n | ' "t" " H I m i I I I I | i i i i | i i i i | i n | M 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4,1 4.0 3.9 3.8 3.7 3.6 3.5 Figure 3.14 Expansion of 600 MHz 1 H NMR spectra of methylspirastrellolide C (3.14) from original extract recorded in C 6 D 6 . The region from 5 3.5 - 5 5.0 showed marked differences when viewed at 0 (A), 3 (B) and 6 (C) weeks. 3.2.2 Subsequent Isolation of Methylspirastrellolide C1 Spirastrella coccinea was collected from reef walls at depths of 2-5 m near Capucin, Dominica. Sponge (22 kg) was extracted repeatedly with MeOH, and the resulting extracts dried in vacuo. The resulting reddish gum was 1 Sponge workup prior to H P L C performed by Dr. K. Warabi, Andersen Lab, University of British Columbia. 92 partitioned between H 20 and EtOAc. The two layers were treated as follows (Figure 3.15). I Sponge (22 kg) Extract with M e O H (88 L total) Reddish Gum H20 (3.8 L) X 1 EtOAc (9.3 L) H20 n -BuOH (2.9 L) 1 1 1 Hexanes (0.45 L) 4:1 MeOH/H 2 0 (0.5 L) 4:1 MeOH/H 2 0(1 L) T 1 Hexanes (0.9 L) 4:2 M e O H / r W (1.21) 1 CH2CI2 (0.8 mL) 4:2 M e O H / H 2 0 (0.6 L) 1 CH2CI2 (0.4 mL) 1 Red Oil (0.74 g) Methylation, Normal Phase Column Chromatography Pooled Fractions (0.66 g) LH-20 Size Exclusion Chromatography Methylspirastrellolide Enriched Fraction Reversed Phase H P L C Methylspirastrellolide C (1.4 mg) Figure 3.15 Isolation scheme for methylspirastrellolide C (3.14) 93 The H 2 0 layer was extracted with n-BuOH, and the organic layer concentrated in vacuo prior to partition between hexanes and 4:1 MeOH/H 2 0 . The methanolic layer was adjusted to 4:2 M e O H / H 2 0 and extracted with CH 2 CI 2 . The original EtOAc layer was dried in vacuo and partitioned between 4:1 M e O H / H 2 0 and hexanes. The methanolic layer was adjusted to 4:2 MeOH/H 2 0 , and extracted with CH 2 CI 2 . The CH 2 CI 2 layers trom both original phases were combined and concentrated to yield a deep red oil (7.4 g). This was then methylated with trimethylsilyldiazomethane in anhydrous MeOH and toluene and stirred at room temperature for 18 hours. The reaction was quenched with acetic acid (6 mL, 0 °C), diluted with toluene, and evaporated to dryness. Methylation conditions are described in the Experimental at 3.5.4. The resulting mixture was subjected to stepwise normal-phase silica gel chromatography (hexanes to MeOH), from which fractions were pooled according to TLC characteristics (0.66 g). This mixture was then separated by size exclusion chromatography (LH-20, 4:1 MeOH/CH 2 CI 2 ) to yield a mixture of methylspirastrellolides (0.13 g). Reversed-phase HPLC (3:1 MeOH/H 2 0) produced pure methylspirastrellolide C (3.14, 4.4 mg), and related analogues. Due to the relative instability of 3.14 all 1D and 2D NMR data needed to be collected within a short period of time. A sample of 3.14 (1.4 mg) was isolated and all data was collected immediately, over a 72-hour period on a Bruker AV-600 with cryoprobe. 94 3.2.3 Structure Elucidation of Methylspirastrellolide C High-resolution electrospray mass spectrometric (HRESIMS) analysis of 3.14 indicated a molecular formula of C 5 3 H 8 6 0 i 8 , indicating 11 degrees of unsaturation. Low-resolution ESIMS in CD 3 OD yielded a pseudo-molecular ion of m/z 1040, whereas the corresponding measurement in C H 3 O H yielded a pseudo-molecular ion of m/z 1034, indicating the presence of 6 exchangeable protons. Inspection of the 1D and 2D NMR data obtained for 3.14 (Figures 3.16-3.23) allowed the elucidation of its structure as follows. All one bond C-H correlations were established by analysis of the HSQC spectrum, and T O C S Y and H S Q C - T O C S Y data were used minimally due to the large number of correlations and presence of superfluous data, and are thus reported only when utilized. A complete assignment of the 1 H , 1 3 C , COSY, HSQC, HMBC and R O E S Y spectra can be found in Table 3.1, as well as selected correlations from the H S Q C - T O C S Y and TOCSY. The numbering scheme for 3.14 is found in Figures 3.11, 3.16 and 3.17. 95 Figure 3.17 150 MHz 1 3 C NMR spectrum of methylspirastrellolide C (3.14) recorded in C 6 D 6 97 98 0 n « 0 I 9 t "ITI'IIHIIII I I T T i i —nprm r Figure 3.20 600 MHz HMBC NMR spectrum of methylspirastrellolide C (3.14) recorded in C 6 D 6 100 Figure 3.23 600 MHz H S Q C - T O C S Y NMR spectrum of methylspirastrellolide C (3.14) recorded in C 6 D 6 103 Table 3.1 1D and 2D NMR data for methylspirastrellolide C (3.14) recorded at 600 M H z ( 1 H ) a n d 1 5 Q M H z ( 1 3 C ) i n C 6 D 6 C # 1 3 C 'TTTJT COSY/TOCSY ~"R"6"JSY HMBC (1Hto 13C) 1 169.9 - - - -2 43.2 H2 2.20 bd (17.3 Hz) H3 C1, C3, C4 H2' 2.58 dd (17.3, 9.6 Hz) 3 74.4 3.82 bd (9.6 Hz) H2, H2', H4 H5, H7 C1, C2 4 32.2 1.04 H3, H5, H5', H6(W) C3, C5, C6 5 24.1 H5 1.30 H4, H4', H6, H6' H3, H7 H5' 1.58 6 27.4 H6 1.47 H7, H5, H5', H4(W) H8 C4, C7 H6' 1.63 7 78.3 3.97 bd (11.6 Hz) H8, H6, H6' H3, H5, C9-OH C3, C5, C8, C9 8 76.6 3.54 bdd (8.6, 7.7 Hz) H9, H7, C8-OH H6, H11 C6, C9, C10 9 69.1 4.22 H10, H10', H8, C9-OH H10' C11 10 42.7 H10 1.77 m H11, H9 H12 C8, C9, C11 H10' 2.37 H9, H12' 11 65.8 4.69 bdd (10.9, 8.8 Hz) H10, H10', H12, H12', H8 C13 C11-OH 12 42.3 H12 1.52 H11, H13 H10, H14 C10, C11, C13, C14 H12' 2.02 H10', H48 13 73.8 3.74 H13, H13', H14 C11-OH, H15, C17 H21 14 35.2 1.23 H13, H48, H15, H16 H12, H16' -15 29.6 1.38 H14, H16, H16' H13 C13, C16, C17 1.52 16 35.6 1.40 H15, H15' H18' C14/C18, C15, C17 1.50 H14 17 96.0 - - - -18 35.2 H18 1.23 H19, H19' H20 C17, C19, C20 H18' 1.61 H16 19 24.6 H19 1.69 m H18, H18', H20 C17, C18, C20, C21 H19' 1.97 H21 20 75.3 3.29 bt (9.8 Hz) H19, H19', H21 H18 C21, C49 21 69.7 4.27 bd (9.8 Hz) H20, H22 H13, H19' C17, C19, C21, C22, C23 22 69.6 4.20 t (10.7 Hz) H21, H23, C22-OH H24 C20, C23, C24 23 76.6 3.78 H22, H24, C23-OH C21, C24, C25, C50 24 34.4 2.28 H23, H25, H50 H22 C23 25 27.7 1.29 H24, H26 C24, C27 2.27 26 35.9 1.63 H25, H25' C25, C27, C28 27 70.9 3.69 m H26, H28, H28' H28', H38 C25, C26, C29, C31 28 38.8 H28 1.18 H27, H29 H30 C26, C30 H28' 1.97 H27, H29 29 73.9 3.75 H28, H28', H30, H30' H28' C30, C51 30 44.7 H30 1.40 H29, H29' H28 C28, C31, C32 H30'2.16 m H32 31 98.8 - - - -32 37.7 H32 1.44 H33, H33' H30', H34 C30, C31, C34 H32' 1.81 dt (13.2, 3.1 Hz) 33 24.5 H33 1.24 m H32, H32', H34 C31, C32, C35, C52 H33'2.07 dt (13.2, 3.1 Hz) 34 38.6 1.51 H52, H32, H32' H32, H36 C33, C35, C52 35 109.0 - - - -36 47.5 H36 2.01 d (15.6 Hz) H37 H34 C35, C37, C38 H36' 2.35 37 73.5 5.70 m H36, H36', H38 H38 C1, C35, C38, C39 38 83.6 4.29 m H37, H39, H39' H27, H37 C37, C39 39 31.2 H39 2.65 m H38, H40 C37, C38, C40, C41 H39' 3.09 m 40 126.3 5.68 m H39, H41 C39, C42 41 132.6 5.97 dt (15.3, 7.4 Hz) H40, H42, H42' C42, C43 42 31.4 H42 2.69 dt (15.2, 7.4 Hz) H41, H43 C40, C41, C43, C44 H42'2.79 dt (15.2, 7.4 Hz) 43 132.1 5.72 dd(10.7, 7.9 Hz) H42, H42', H44 H44 C41 44 124.8 5.44 dt (10.7, 7.4 Hz) H43, H45, H45' H43 C42, C45, C46 45 33.0 H45 2.35 H44, H46 C43, C44, C47 H45' 2.49 m 104 46 70.8 4.16 m H45, H45', C46-OH C44, C47 47 175.1 - - -48 18.4 0.80 d (6.6 Hz) H14 H12' C13, C14, C15 49 57.0 3.13s - H50 C20 50 20.0 1.30 d (6.9 Hz) H24 H49 C23, C24, C25 51 55.5 3.25 s - C29 52 17.0 1.09 d (6.7 Hz) H34, H33, H33', H32, H32' C33, C34, C35 53 52.1 3.31 s - C47 8-OH - 3.18 bd (7.7 Hz) H8 C7, C8, C9 9-OH - 4.13 bd (4.0 Hz) H9 H7, C11-OH C8, C9, C10 11-OH - 4.57 bd (2.8 Hz) H11 C9-OH, H13 C10, C11, C12 22-0H - 1.68 m H22 C21, C22, C23 23-OH - 4.43 bd (6.7 Hz) H23 C22, C23, C24 46-OH - 3.88 bd (7.2 Hz) H46 C45, C46, C47 The structure elucidation can be broken down into four sections of the molecule based upon contiguous 1 H NMR spin systems, and will be discussed as such. The first segment, from C1-C17 (Figure 3.24), initiates at an ester carbonyl at 5 C 169.9 (C1), which correlates in the HMBC spectrum to two protons at 5 2.20 (H2) and 5 2.58 (H2'). From here, correlations in the C O S Y spectrum between 5 2.58 (H2') and 5 3.82 (H3) establish the adjacent proton. It is then possible in the C O S Y spectrum to work around the tetrahydropyran ring from H4 to H6 (Figure 3.25). The protons at 5 1.63 and 5 1.47 (H6) correlate in the C O S Y to a broad doublet 5 3.97 (H7). An HMBC correlation from 5 3.97 (H7) to 6 C 74.4 (C3) connects C3 and C7 via an ether oxygen, establishing the tetrahydropyran ring. 105 29.6 35.6 Figure 3.24 C1-C17 fragment of methylspirastrellolide C (3.14) with key correlations, 1 H and 1 3 C NMR assignments shown Figure 3.25 Expansion of C O S Y NMR spectrum, showing some correlations from C1-C7 in methylspirastrellolide C (3.14) 106 A C O S Y correlation from 5 3.97 (H7) to 5 3.54 (H8) establishes the adjacent carbinol methine. From here C O S Y correlations can be observed from 5 3.54 (H8) to 5 4.22 (H9), and onward to 5 1.77 (H10) and 5 2.37 (H10'). The methylene protons at 6 2.37 (MO") and 5 1.77 (H10) correlate in the C O S Y spectrum into an additional carbinol methine at 5 4.69 (H11), which correlates into the adjacent methylene protons at 5 2.02 and 5 1.52 (H12). All carbinol methines in the C s - C i 2 chain showed C O S Y correlations to their respective alcohol protons (Figure 3.26). HSQC information completes the assignment of the corresponding carbon atoms, and HMBC evidence corroborates this data (Table 3.1). Figure 3.26 Expansion of C O S Y NMR spectrum, showing correlations from C 7 -C12 in methylspirastrellolide C (3.14) (ppm) H11 (ppm) 107 From the methylene protons at 5 2.02 and 5 1.52 (H12) a C O S Y correlation is observed into 5 3.74 (H13). This proton correlates into a quaternary carbon at 5C 96.0 (C17) in the HMBC spectrum, suggesting the presence of a spiroketal moiety. A weak correlation in the C O S Y spectrum connects the resonance at 5 3.74 to 5 1.23, necessitating the analysis of T O C S Y data to confirm this connection (Figure 3.27). Correlations in the T O C S Y spectrum complete the spin system, connecting H13 to a tertiary methine at 5 1.23 (H14), which in turn correlates to a methyl at 5 0.80 (H48) and a methylene at 5 1.52 and 5 1.38 (H15). This final methylene correlates to 5 1.50 and 5 1.40 (H16) in the C O S Y spectrum, and all protons on C15 and C16 correlate in the HMBC spectrum into the C17 quaternary centre, completing the ring and the first substructure (Figure 3.24). 3.88 3.84 3.80 3.76 3.72 3.68 3.64 (ppm) Figure 3.27 Expansion of T O C S Y NMR spectrum of methylspirastrellolide C (3.14) 108 Five additional protons correlate in the HMBC spectrum to 5 C 96.0, linking to the second fragment of methylspirastrellolide C (Figure 3.28). The proton at 5 4.27 (H21) is one of these, and also shows a C O S Y correlation to the proton at 5 3.29 (H20). This proton further correlates into protons at 5 1.97 and 6 1.69 (H19), which subsequently correlate into protons at 5 1.23 and 5 1.61 (H18). All of the protons on C18 and C19 correlate in the HMBC spectrum into 5 C 96.0 (C17), completing what is now clearly a spiroketal ring system. A second C O S Y correlation from 5 4.27 (H21) to 6 4.20 (H22) provides the link to the adjacent carbinol methine, which in turn correlates to a second carbinol methine at 5 3.78 (H23). A C O S Y correlation from 5 3.78 (H23) to an aliphatic methine at 5 2.28 (H29) links to the adjacent atom, which correlates to a broad peak consisting of both the methyl at 5 1.30 (H50) and a methylene proton at 5 1.29 (H25). The lack of dispersion in the proton spectrum in this region required that HMBC data be used to verify these assignments. 109 H23 <7-•C50^S)! <3 H23 H23-C24 O (ppm) Figure 3.28 Expansion of HMBC NMR spectrum of methylspirastrellolide C (3.14) 4.43 3.78 - X O H 4 2 7 H I H 4 . 2 0 2.16 1.40 JH H 3.69 1.23 M H H M 1 . 9 7 68 1.61 1.69 C17 - C23 C O S Y / T O C S Y H M B C ( 1 H t o 1 3 C ) Figure 3.29 C17-C31 fragment of methylspirastrellolide C (3.14) with key correlations, 1 H and 1 3 C NMR assignments shown Correlations in the HMBC spectrum from 5 3.78 (H23) to 5 C 20.0 (C50), 5 C 27.7 (C25) and 5 C 34.4 (C24) were observed (Figures 3.28 and 3.29). HSQC 110 information was used to identify the chemical shifts of the protons on these nuclei as 5 1.30 (H50), 5 2.27 (H25') and 6 1.29 (H25), and 5 2.28 (H24), respectively. With this information in hand, C O S Y correlations from 5 2.27 (H25") and 5 1.29 (H25) to 5 1.63 (H26) connect to the adjacent methylene in the chain. This in turn correlates to a methine at 5 3.69 (H27). C O S Y correlations from 5 3.69 (H27) to 5 1.97 (H28') and 5 1.18 (H28), and on to 5 3.75 (H29) and 5 2.16 (H30") and 5 1.40 (H30) complete the proton spin system (Figure 3.30). HMBC correlations from 5 2.16 (H30') and 5 1.40 (H30) and 5 3.69 (H27) to a quaternary carbon at 5C 98.8 (C31) suggest a second spiroketal system, and complete the C 1 7 - C 3 1 substructure (Figure 3.29). Alcohol protons on C22 and C23 were assigned through C O S Y correlations to H22 and H23, respectively. Figure 3.30 Expansion of C O S Y NMR spectrum, showing correlations from C25-C 3 0 i n methylspirastrellolide C (3.14) (ppm) (ppm) 111 The third ( C 3 1 - C 3 5 ) spin system was elucidated as follows. Two methylene protons at 5 1.81 (H32") and 5 1.44 (H32) show HMBC correlations into both 5 C 98.8 (C31) and 5 C 44.7 (C30), placing them nearest the C31 spiroketal. One set of methylene protons at 5 2.07 and 5 1.24 (H33) show HMBC correlations to both 5 C 98.8 (C31) and 5 C 109.0 (C35), placing them next along the chain. A final methine proton at 5 1.51 (H34) correlates in the HMBC to 6 C 109.0 (C35), linking to the adjacent spiroketal. A C O S Y correlation from 5 1.51 (H34) to 5 1.09 (H52) establishes a methyl on C34, and completes the C 3 r C 3 5 substructure (Figure 3.31). 1.09|_| 2 Q 7 1 2 4 \ H H H i si ^ 1'51 H ^ ^ C ^ H ^ l i e "H1.40 17.0 C O S Y / T O C S Y H M B C ( 1 H t o 1 3 C ) '31 C 3 5 Figure 3.31 C 3 1 - C 3 5 fragment of methylspirastrellolide C (3.14) with key correlations, 1 H and 1 3 C NMR assignments shown The final substructure originates at both the C35 spiroketal and the C1 carbonyl. An HMBC correlation from 5 5.70 (H37) to 5 C 169.9 (C1) links this fragment to the C i - C 1 7 fragment via an ester linkage. The proton at 5 5.70 (H37) shows C O S Y correlations to 5 2.35 and 5 2.01 (H36) (Figure 3.33), which along with 5 5.70 (H37) correlate in the HMBC into 5 C 109.0 (C35), thus linking to the 112 C17-C31 fragment. An additional C O S Y correlation from 6 5.70 (H37) to an oxymethine at 5 4.29 (H38) establishes the adjacent atom, and places C36 next to the C35 spiroketal. The presence of the final ring is inferred both by the requirement of a ketal at C35 (5C 109.0), the necessity of oxygen substitution at C38 (5C 83.6). The proton at 5 4.29 (H38) correlates to a pair of methylene protons at 5 3.09 and 5 2.65 (H39), which in turn correlate to an alkenyl methine at 6 5.68 (H40). C O S Y correlations are evident down the side chain to 5 5.97 (H41), 6 2.79 and 5 2.69 (H42), 5 5.72 (H43), 5 5.44 (H44), 5 2.49 and 5 2.35 (H45), and 5 4.16 (H46) (Figure 3.33). An HMBC correlation from 5 4.16 (H46) to a carbonyl at 5C 175.1 (C47) completes the side chain, yielding the C35-C37 fragment (Figure 3.32). Figure 3.32 C35-C47 fragment of methylspirastrellolide C (3.14) with key correlations, 1 H and 1 3 C assignments shown 113 Figure 3.33 Expansion of C O S Y NMR spectrum, showing correlations from C ; C 4 6in methylspirastrellolide C (3.14) 114 With the majority of the connectivity of methylspirastrellolide established, only a few more connections need to be made to complete the flat structure of 3.14. Protons from two methyl ethers are observed at 6 3.13 (H49) and 5 3.25 (H51), and correlate in the HMBC spectrum to 6 C 75.3 (C20) and 5C 73.9 (C29), respectively. Protons at 5 3.31 (H53) are likewise established as a methyl ester by the presence of an HMBC correlation to 5C 175.1 (C47). Two carbonyls, two double bonds, and the presence of a total of seven rings result in eleven degrees of unsaturation, as required by the high-resolution mass spectrometry measurements. A summary of key C O S Y and HMBC correlations can be found in Figure 3.34. Figure 3.34 Complete structure of methylspirastrellolide C (3.14) with key correlations shown 115 3.2.4 Relative Configuration of Methylspirastrellolide C The relative configuration of spirastrellolide A has been the focus of much study (vide supra).181-186 Previous data established the relative configurations in subunits of the molecule, however, gaps remained. Analysis of the R O E S Y spectrum of 3.14 in concert with 3 J coupling constant data allowed the connection of two of these subunits ( C 3 - C 7 and C 9 - C 2 4 ) The dihedral angle between two vicinal protons can be inferred from the magnitude of their 3 J coupling constant. 1 8 8 The orientation of the other substituents is then inferred by the presence of correlations in the R O E S Y spectrum. As the conformation of the C 7 - C 1 3 chain is relatively rigid, the relative configurations of the two can be connected using this 3 J and R O E S Y information. For the purpose of clarity, unless otherwise noted all correlations are from the R O E S Y spectrum. The C 3 - C 7 tetrahydropyran ring is established in a chair conformation by axial R O E S Y correlations between 5 3.97 (H7), 5 3.82 (H3) and 6 1.30 (H5), as well as axial correlations between 5 1.63 (H6') and 5 1.04 (H4). A weak W (four bond) coupling in the C O S Y spectrum between 5 1.47 (H6) and 5 1.04 (H4) supports their placement in equatorial orientations. H3 and H7 are established as cis in the ring (Figure 3.35) due to their diaxial relationship. H7 is observed in the 1 H NMR spectrum as a broad doublet ( 3 J = 11.6 Hz), trans to H6' (5 1.63). The small coupling constant between H7 and H8 places the angle between them close to 90°. H8 is a broad doublet of doublets ( 3 J = 8.6 and 7.7 Hz), coupled both to C8-OH (7.7 Hz), and H9 (8.6 Hz), which must be anti due to the large 116 coupling constant. A R O E S Y correlation between 5 1.47 (H6) and 5 3.54 (H8), as well as between 5 4.69 (H11) and 5 3.54 (H8) fixes the C8-C9 bond in such a way that the C8-OH is antiXo H7, and antiXo the C9-OH (Figure 3.36). A weak R O E S Y correlation between 5 3.97 (H7) and 5 4.13 (C9-OH) further establishes C8-OH anti to H7. A consideration of the alternative configuration at C8 would require a R O E S Y correlation between H11 and H8 that would not be possible given the extended chain between the two centres. 117 Figure 3.35 A) R O E S Y information, B) proton assignments and coupling constant data for the C 3 - C 1 3 segment of methylspirastrellolide C (3.14). C) An alternate configuration at C8 requires a R O E S Y coupling between very distant protons, thus this C8 configuration is not likely 118 Figure 3.36 Expansions of the R O E S Y NMR spectrum, showing key correlations from C 3 - C i 3 i n methylspirastrellolide C (3.14) 1 1 9 The relative configurations established above are consistent with previous investigations of the relative configuration of spirastrellolide A (3.13), which is thought to possess the same relative configuration throughout as 3.14. Multiple R O E S Y correlations are observed which support the staggered structure of the C7-C13 chain (see Figures 3.35, 3.36 and Table 3.1), which is proposed for spirastrellolide A . 1 8 0 In particular, the extensive R O E S Y correlations between the protons on C10 and the protons on C12 confirm their close proximity in space. A correlation between 5 4.13 (C9-OH) and 5 4.57 (C11-OH) conforms with their axial positions on the C 7 - C 1 3 chain (Figure 3.35), as observed from the acetonide of spirastrellolide A (3.13).180 A correlation in the R O E S Y spectrum between 6 3.54 (H8) and 5 4.69 (H11), as mentioned previously, establishes close proximity between H8 and H11. A further R O E S Y correlation between 5 3.74 (H13) and 6 4.57 (C11-OH) establishes H13 in an axial conformation as shown in Figure 3.35. Further support for placing H13 axial in the C13-C21 segment is found below. Finally, R O E S Y correlations between 5 0.80 (H48) and 5 2.02 (H12'), as well as between 5 1.23 (H14) and 5 1.52 (H12) confirm the equatorial position of the C12-C13 bond in relation to the C13-C21 structure. Analysis of R O E S Y data for the C13-C21 segment is consistent with that of 3.13.180 Diaxial R O E S Y correlations between 5 1.23 (H14) and 5 1.50 (H16'), as well as diaxial R O E S Y correlations between 6 1.38 (H15) and 6 3.74 (H13) place the ring in a chair conformation, with the C48 methyl and C12 alkyl chain trans and equatorial (Figure 3.37). Additional R O E S Y correlations between 5 3.74 (H13) and 5 4.27 (H21) as well as a weak correlation between 5 1.40 (H16) and 120 5 1.61 (H18') establish the relative contiguration of the spiroketal. Further diaxial R O E S Y correlations between 5 1.97 (H19') and 5 4.27 (H21), as well as between 5 1.23 (H18) and 5 3.29 (H20) set the second ring in a chair conformation, with the C21 alkyl chain and C49 methoxy group equatorial (Figure 3.38). Figure 3.37 Summary of R O E S Y data for C13-C21 segment of methylspirastrellolide C (3.14) 121 , 4.8 4.0 5.2 2.4 1.6 0.8 >m) Figure 3.38 R O E S Y NMR spectrum, showing key correlations from C13-C21 in methylspirastrellolide C (3.14) The C21-C24 segment of the molecule can be established as follows. H21 had previously been established as axial, and is observed as a broad doublet (9.8 Hz), antiXo H20. The small coupling to 5 4.27 (H21) places it gauche to 5 4.20 (H22). The proton signal at 6 4.20 (H22) is observed as a doublet of doublets, coupled to 5 1.68 (C22-OH) and 5 3.78 (H23), placing H23 antiXo H22. Finally, R O E S Y correlations between 5 2.28 (H24) and 5 4.20 (H22), as well as between 5 3.13 (H49) and 5 1.30 (H50) orient the chain in such a way that the relative configurations are as shown in Figure 3.39. These are in accordance with data obtained from the acetonide of 3.13. 122 ROESY Figure 3.39 Summary of R O E S Y data for C21-C24 segment of methylspirastrellolide C (3.14) The key R O E S Y correlation that establishes the relative configuration of the C 2 7 - C 3 9 subunit is observed between 5 3.69 (H27) and 5 4.29 (H38) (Figure 3.40). This places C30 and C36 cis on the C31-C35 ring, with H27 and H39 both facing into the bowl-like structure (Figure 3.41). Diaxial R O E S Y correlations between 5 1.40 (H30) and 6 1.18 (H28) place the C 2 7 - C 3 i ring in a chair conformation. Strong correlations between 5 3.69 (H27) and 5 3.75 (H29) to equatorial 5 1.97 (H28') support a cis orientation between C28 and C29. The C31-C35 ring is likewise in a chair conformation, as shown the diaxial R O E S Y correlation between 5 1.44 (H32) and 0" 1.51 (H34). The axial proton 5 1.44 (H32) and the equatorial proton 5 2.16 (H30') also show a correlation in the ROESY, further fixing the relative configuration of the C31 spiro centre. Likewise a strong R O E S Y correlation between pseudo-equatorial 5 2.01 (H36) and axial 5 1.51 (H34) fix the configuration of the C35 spiro centre. Protons resonating at 5 4.29 (H38) and 5 5.70 (H37) are shown to be cis by a correlation in the R O E S Y 123 spectrum, as is the pseudo-axial 5 2.35 (H36'). This analysis is consistent with that of 3.13, and is summarized in Figure 3.41. E S °0 <D OJ O 00 i£> ^ o — "S *t V in Figure 3.40 R O E S Y NMR spectrum, showing key correlations from C27-C38 in methylspirastrellolide C (3.14) 124 ROESY Figure 3.41 Summary of R O E S Y NMR data for C27-C38 segment of methylspirastrellolide C (3.14) The remaining relative configuration questions relate to the C38-C47 side chain of 3.14. The C43-C44 double bond was established as cis due to the existence of a R O E S Y correlation between 5 5.72 (H43) and 5 5.44 (H44). A similar correlation appears to exist in the R O E S Y spectrum between 5 5.68 (H40) and 5 5.97 (H41), although the difference in phase to other ROESY correlations proves it to be due to a T O C S Y correlation. The large coupling constant in 5 5.97 (H41) (3J = 15.3 Hz) is attributed to a trans coupling to 5 5.68 (H40). The configuration at the final chiral centre, at 5 4.16 (H46), is unassigned at this point. By connecting the relative configuration of C 3 - C 8 to the C 9 - C 2 4 segment, a large portion of the overall relative configuration is in hand. As mentioned 125 previously, this is in full agreement with the relative configurations determined for methylspirastrellolide A , 1 8 0 and those recently determined by X-ray crystallography of methylspirastrellolide B (3.15).187 The optical rotation of methylspirastrellolide C (3.14, [a] D 2 5 +17.6 (c=0.9 CH 2CI 2)) is also comparable to that obtained for methylspirastrellolide A (3.13, [a] D 2 5 +27 (c=0.16 CH 2CI 2)) and methylspirastrellolide B (3.15, [a] D 2 5 +44.7 (c=0.5 MeOH)). Given the similarities of both R O E S Y and optical rotation data, the absolute configuration of methylspirastrellolide C is assigned as 3R, 7S, 8S, 9S, 11S, 13R, 14S, 17S, 20S, 21S, 22S, 23S, 24S, 27R, 29S, 31R, 34S, 35R, 37S, 38S. 3.3 Biological Activity of Methylspirastrellolide C Methylspirastrellolide C (3.14) was tested against protein phosphatase 2A (PP2A), and found to be a potent (IC50 = 1nM) inhibitor, similar to methylspirastrellolide A . 1 8 0 Further structure-activity relationship (SAR) studies are underway to look at the effect of structural changes on the observed biological activity of the compounds, however extremely limited supply currently hampers these efforts. 126 3.4 Discussion and Conclusions With the isolation and elucidation of methylspirastrellolide C (3.14) complete, a third member is added to the number of known spirastrellolides. The elucidation is complicated by the complex nature of the NMR spectra observed, as well as the relative instability of the compounds upon isolation. The structural types thus elucidated present a common core, with typical polyketide variations on that core (hydroxylation and dehydration) as well as the less typical chlorination found in spirastrellolide A (3.13). The sole barrier to establishing the absolute configuration of the methyspirastrellolides is the configuration at C46. Although a number of techniques exist which would allow the determination of the stereochemistry of this center in the natural product, this has not been possible as of yet due to the extremely limited supply of compound. More likely, the synthetic groups currently studying the compound will synthesize the appropriate core, and append side chains with different stereochemistry. Detailed analysis of the resulting products and comparison with the natural products may reveal the correct configuration. Alternatively, the center may be important for the biological activity of the compound, and thus the two resultant diastereomers may exhibit different biological activities. Regardless, this center will likely be assigned in the near future. In addition to the three spirastrellolides already isolated, it appears from HPLC analysis that a number of other analogues are present in the 127 spirastrellolide-enriched fractions. The elucidation of another analogue simplifies the analysis of the remaining compounds, which in turn may shed some light upon SAR as their structures are elucidated and biological activities tested. There is much more to do with these fascinating compounds. Indeed the spirastrellolides are prime examples of a typical marine natural products research project. The study of a sponge which showed anti-mitotic activity led to the discovery of an interesting and highly active compound, spirastrellolide A (3.13). While biologists studied the compound and established its mode of action, our laboratory studied the structure with the aim of establishing the absolute configuration of the compound. Other research labs began to synthesize increasingly complex portions of the molecule, and comparing the synthesized products with data obtained from the natural product to see what fit and what did not. After a re-collection of the same sponge, a number of analogues of methylspirastrellolide A were isolated. Analysis of one (methylspirastrellolide C (3.14)) revealed the small, but important inclusion of a C8 alcohol, which allowed the relative configuration of the C3-C23 segment to be established. Degradation and derivatization of a third analogue (methylspirastrellolide B (3.15)) allowed for the growth of crystals and the determination of the absolute configuration of the macrolide core. Both methylspirastrellolides B (3.15) and C (3.14) are highly active against protein phosphatase 2A, as is spirastrellolide A (3.13). 128 3.5 Experimental 3.5.1 General Experimental Procedures All 1D and 2D NMR data was collected on a Bruker AV-600 spectrometer equipped with a cryoprobe ( 1H at 600 MHz, 1 3 C at 150 MHz). 1 H chemical shifts were referenced to the residual CeD 5H signal (5 7.16 ppm), and 1 3 C chemical shifts were referenced to the C 6 D 6 solvent peak (5C 128.39 ppm). All NMR solvents were obtained from Cambridge Isotope Laboratories. All chromatography was performed using HPLC grade solvents from Fisher Scientific with no further purification. Water was purified using a Millipore reverse osmosis filter system. Normal phase chromatography was performed using Silicycle Ultra Pure silica gel (230-400 mesh) under compressed air. HPLC isolation was performed using a Waters 600E system controller liquid chromatograph interfaced to a Waters 486 tunable absorbance detector and a Waters 994 programmable photodiode array detector. Optical rotations were measured with a J A S C O J-1010 polarimeter using a 10 mm cell. High-resolution mass spectra were collected on a Micromass LCT mass spectrometer, whereas low-resolution mass spectra were collected on a Bruker Esquire LC mass spectrometer. (Trimethylsilyl)diazomethane was obtained from Sigma-Aldrich (362832-5mL). 129 3.5.2 Initial Isolation of Methylspirastrellolide C Spirastrella coccinea was collected from reef walls at depth of 2-5 m near Capucin, Dominica. The sponge (19 kg) was extracted repeatedly with MeOH (3 X 20 L), and resulting extracts dried in vacuo. The resulting reddish gum was partitioned between H 2 0 (6 L) and EtOAc ( 5 X 1 L), and the EtOAc partition evaporated to yield a red oil (42 g). This was partitioned between hexanes (4 X 300 mL) and 4:1 M e O H / H 2 0 (1000 mL). The methanolic layer was adjusted to 2:1 M e O H / H 2 0 (to a total of 1200 mL) and extracted with C H 2 C I 2 (4 X 200 mL). The CH 2 CI 2 extracts were evaporated to dryness to yield a potently antimitotic amorphous red solid (2.21 g). At this point, the CH 2 CI 2 extract was methylated to simplify the isolation (see Experimental 3.5.4, vide infra). All reagents were evaporated under a stream of N 2 , and the sample fractionated using normal phase silica gel chromatography (step gradient from 19:1 hexanes/EtOAc to MeOH). Two fractions, eluting with EtOAc (363.7 mg) and 1:9 MeOH/EtOAc (110.6 mg) were both biologically active, and further separated using size exclusion chromatography (Sephadex LH-20, 4:1 MeOH/CH 2 CI 2 ) . From these two separations were obtained potently antimitotic fractions (135.1 and 39.2 mg, respectively) both of which consisted of a complex mixture of methylspirastrellolides as viewed by NMR. Methylspirastrellolide A and a number of analogues were obtained from the first fraction using a range of reversed-phase HPLC conditions. The second fraction yielded impure 130 methylspirastrellolide C using reversed-phase HPLC (3:1 MeOH/H 2 0) , as well as trace quantities of other analogues. This sample was subjected to further reversed-phase HPLC (gradient of 44% M e C N / H 2 0 to 50% M e C N / H 2 0 over 60 minutes) to yield pure methylspirastrellolide C (3.14, 1.8 mg). Small sample volume coupled with sample instability necessitated further sponge collection and extraction. 3.5.3 Second Isolation of Methylspirastrellolide C Spirastrella coccinea was collected from reef walls at depth of 2-5 m near Capucin, Dominica. Sponge (22 kg) was extracted repeatedly with MeOH (4 X 22 L), and resulting extracts dried in vacuo. The resulting reddish gum was partitioned between H 2 0 (3.8 L) and EtOAc (9.3 L). The two layers were treated as outlined in Figure 3.15. The H 2 0 layer was extracted with n-BuOH (2.9 L), and the organic layer concentrated in vacuo prior to partition between hexanes (450 mL) and 4:1 M e O H / H 2 0 (500 mL). The methanolic layer was adjusted to 4:2 M e O H / H 2 0 (600 mL), and extracted with CH 2 CI 2 (400 mL). The original EtOAc layer was dried in vacuo and partitioned between 4:1 M e O H / H 2 0 (1000 mL) and hexanes (900 mL). The methanolic layer was adjusted to 4:2 M e O H / H 2 0 (1200 mL), and extracted with CH 2 CI 2 (800 mL). The CH 2 CI 2 layers from both original phases were combined and concentrated to yield a deep red oil (7.4 g). This mixture was then methylated (vide infra). 131 3.5.4 Methylation of Spirastrellolide Mixture The deep red oil (7.4 g) was dissolved in anhydrous MeOH (40 mL) and toluene (160 mL), to which was added 30 mL of trimethylsilyldiazomethane (2.0 M in hexanes, 0 °C), and stirred at room temperature for 18 hours. The reaction was quenched with acetic acid (6 mL, 0 °C), diluted with toluene, and evaporated to dryness under a stream of N 2 followed by lyophilization to remove trace solvents. 3.5.5 Chromatography of Spirastrellolides The resulting mixture was subjected to stepwise normal-phase silica gel chromatography (hexanes to MeOH), from which fractions were pooled according to TLC characteristics (0.66 g). This mixture was then separated by size exclusion chromatography (LH-20, 4:1 MeOH/CH 2Cl2) to yield a mixture of methylspirastrellolides (0.13 g). Reversed-phase HPLC (Inertsil C-28 column, 3:1 MeOH/H 2 0 , 2 mL/min flow, uv detected at 203 nm) produced pure methylspirastrellolide C (3.14, 4.4 mg), and related analogues. Due to the relative instability of 3.14 all 1D and 2D NMR data was collected immediately. A sample of 3.14 (1.4 mg) was isolated and all data was collected over a 72 hour period on a Bruker AV-600 with cryoprobe. 132 3.5.6 Methylspirastrellolide C Physical Data Clear, pale yellow glass (1.8 mg); [a] D 2 5 +17.6 (c=0.9 CH 2 CI 2 ) . For 1D and 2D NMR data see Table 3.1. HRESIMS: [M+Na]+ m/z = 1033.5721 (calcd for CsaHseOisNa, 1033.5712). 133 Chapter 4: Cannabinoid Receptor Studies 4.1 Cannabinoids and Cannabinoid Receptors The cannabinoids are a group of naturally occurring compounds isolated from the Cannabis sativa L. plant, along with related terpenoids and flavonoids. C. sativa had been used medicinally for upwards of 5000 years, and was studied in great detail during a flurry of plant natural product research during the late 1800s and early 1900s. 1 8 9 " 1 9 1 The first natural cannabinoid isolated was cannabinol (4.1) in 1899. 1 9 2 It was acetylated and subjected to chemical degradation studies, although it was not tested for biological activity. Later studies resulted in the isolation of (-)-cannabidiol (CBD, 4.2), a cannabinoid now known to be a minor constituent of fresh C. sativa extracts. 1 9 3 The structure of 4.2 was determined much later using early NMR techniques. 1 9 4 The first report of a biologically active C. sativa compound was that of A 9-tetrahydrocannabinol (A 9 -THC, 1.39) in 1964. 1 9 5 A host of related compounds were subsequently isolated and characterized, on which a number of excellent reviews have been written. 1 9 6 " 1 9 8 Figure 4.1 Some compounds isolated from C. sativa 134 The increase in the number of studies on C. sativa during the mid-1960s and 70s can be attributed, in part, to the increase in its use as a recreational drug during that era. Studies focused on the psychoactive properties of C. sativa, and . 1.39 specifically, to the detriment of the study of the other pharmacological effects of the plant. 1 9 0 However, a number of potential roles have been proposed for 1.39 including appetite stimulation, nausea control and pain relief. 1 9 9 Compound 4.2 has been investigated for the management of cancer, acute schizophrenia, nausea, anxiety, epilepsy, glaucoma, inflammatory disorders, and for neuroprotection. 2 0 0 Of particular interest is the fact that 4.2 seems to not carry the psychotropic affects associated with 1.39. With the study of the plant cannabinoids (phytocannabinoids) and their effects on animal physiology came the natural questions regarding their mode of action. The search for receptors of the phytocannabinoid agonists produced a number of studies which established a dose-dependant, stereoselective response to phytocannabinoids, and eventually, cloning of the receptors themselves . 2 0 1 , 2 0 2 There are two primary receptor subtypes, C B i and C B 2 , located in the neuronal tissue of the nervous system and immune tissue, respectively. 2 0 3 Both possess 7-transmembrane spanning segments, similar to rhodopsin, and bind their agonists at the central core of the subunits. 2 0 4 Their mode of action is to inhibit adenylyl cyclase via associated G proteins, as well as modulating the function of potassium and calcium channels 2 0 1 ' 2 0 2 ' 2 0 5 , 2 0 6 135 Several other structural classes of compounds have been observed to be active on the C B receptors. All told, there are currently thought to be four classes of CB agonists. 2 0 4 The first type of agonist is the classical phytocannabinoid, A 9-tetrahydrocannabinol (A 9 -THC, 1.39), consisting generally of a dihydrobenzopyran-type structure, with an alkyl group at the C-3 aromatic position, and a hydroxyl group at the C-1 aromatic position (Figure 4.1). The second group is typically called the non-classical cannabinoids, as evidenced by C P 55,940 (4.3) and other synthetic analogues. These are typically more water-soluble and potent than classical cannabinoids, and thus have served as important tools in discovering and exploring the CB receptors. 2 0 7 The third class of C B agonists is the aminoalkylindoles, such as WIN 55,212-2 (4.4). These are structurally different from the classical and non-classical cannabinoids, and are thought to bind to a slightly different site on the receptor. 2 0 8 The fourth class of known CB agonists encompasses the eicosanoids, of which anandamide (1.38) was the first isolated from mammalian brain t issue. 2 0 9 This was a particularly interesting discovery, as it was the first of the five known endogenous CB agonists to be discovered. 136 1.38 Figure 4.2 Structures of some CB agonists. Shown are C P 55,940 (4.3), WIN 55,212-2 (4.4), and anandamide (1.38) The topic of CB receptor antagonists is slightly more complicated due to the complexity of the receptors themselves. SR141716A (4.5) potently binds CB i receptors, 2 1 0 whereas SR144528 (4.6) strongly binds C B 2 receptors. 2 1 1 Compound 4.5 has been observed to display both antagonist and inverse agonist activities, which is thought to be due to binding to two different sites on the CB-[ receptor. 2 1 2 This inverse agonism arises from the interaction between constitutively activated CB receptor and 4.5. Compounds 4.7 (6-bromopravadoline) and 4.8 (LY320135) also exhibit this mixed antagonist/inverse agonist activity, and are structurally similar to the aminoalkylindole agonists such as 4 . 4 . 2 1 3 , 2 1 4 Finally, 0-1184 (4.9) acts on CBi as low efficacy agonist, with little of the inverse agonist activity associated with the other CB antagonists. 2 1 5 A great deal of work has gone into looking at these interactions, but for the purpose of this study it is sufficient to note that there are a host of possible interactions between ligands and CB receptors, including both 137 antagonism and potential allosteric interactions. 2 1 6 A number of excellent reviews explore this subject in greater d e t a i l . 2 0 6 , 2 1 7 , 2 1 8 Figure 4.3 Structures of some CB antagonists. Shown are SR141716A (4.5), SR144528 (4.6), WIN54461 (4.7), LY320135 (4.8) and 0-1184 (4.9) Given the interesting activity shown in the endogenous cannabinoids, phytocannabinoids and synthetic compounds, it became clear that a wealth of information about these receptors could be gathered from the study of compounds which interact with them. The lab of Tom Grigliatti at UBC has studied the molecular biology of insects, and recently published a technique to study human G-protein-coupled receptors (GPCRs) in insect cell l ines . 2 1 9 " 2 2 1 To 138 advantages are noted for the use of insect cell lines: the cells do not possess the endogenous G P C R s found in mammalian cells (thus avoiding false positive results); and the G P C R s are properly expressed and modified post-translationally unlike those found in yeast cell lines (thus avoiding false negative results). The assay utilizes a light producing reaction to follow the interaction of an agonist or antagonist with the G P C R in question (Figure 4.4). Figure 4.4. Schematic for a general G P C R bioassay (from Knight et al.f2^ Human G P C R , G a human G protein subunit and jellyfish aequorin reporter are expressed in an insect cell line. Binding of agonist to G P C R produces changes in GTP-bound Ga, which is linked via phospholipase CB (PLCS) pathway to cellular C a 2 + levels. The aequorin photoprotein reports this change in C a 2 + levels with the creation of light and conversion of cellular 0 2 to C 0 2 . 139 A range of marine sponge extracts were tested in cells expressing a host of human G P C R s , including those expressing CBi and C B 2 . Several extracts from our library were identified as having activity in this CB bioassay. The first extract was 03-039, subsequently identified as derived from Stylissa massa (Carter, 1881). The crude methanolic extract was found to be active primarily as a C B 2 antagonist with lesser agonism and antagonism of both C B i and C B 2 . Bioassay guided fractionation of this extract yielded a complex mixture of A-nor steroids. Derivatization with m-bromophenyl isocyanate was performed on this mixture both to provide a chromophore for HPLC and to attempt recrystallization for X-ray analysis. After numerous unsuccessful attempts to grow crystals, repeated HPLC separation resulted in the purification and characterization of the 3-bromophenyl isocyanate derivative of 3S-hydroxymethyl-A-nor-5a-cholest-22E-ene (4.10, hereinafter A - n o r -C 2 7A 2 2 derivative). The second was extract 03-253, identified as from Hemiasterella aff. affinis. The crude methanolic extract was found to be a CB i antagonist. Bioassay guided fractionation of this extract yielded a mixture of bengamides, primarily bengamide A (4.11). Both purified compounds failed to show activity in the CB assay. 140 Br-H N Y O H O 4.10 O 4.11 Figure 4.5 Structures ot A-/70/--C27A derivative (4.10) and bengamide A (4.11) 4.2 A-nor-steroids 4.2.1 Isolation of A-nor-steroids from Stylissa massa (Carter, 1881) The orange cake sponge Stylissa massa (Carter, 1881) was collected at a depth of 10 m near Kavieng, Papua New Guinea (2° 36.71' S, 150° 42.56' E) on Sept. 11, 2003 (Figure 4.6). During an assay of our library of marine extracts it was identified as possessing a number of activities in the CB bioassay, including both C B i and C B 2 agonism and antagonism. 141 Figure 4.6 Stylissa massa, collected near Kavieng, Papua New Guinea A sample of the sponge was extracted repeatedly with MeOH. The organic extract was dried in vacuo to yield an orange paste (109.6 mg). A portion of this paste was then partitioned between H 2 0 and EtOAc, each of which was dried in vacuo. The organic fraction was subjected to normal phase silica gel chromatography (Hexanes/EtOac). TLC analysis resulted in the pooling of a steroid enriched fraction, which was then derivatized with m-bromophenyl isocyanate to both attempt crystallization for X-ray analysis and to provide a uv chromophore for HPLC analysis. Repeated H P L C separation of the resulting sample yielded increasingly pure steroid samples under carefully moderated conditions (MeOH/H 2 0, C18) giving 0.6 mg of partially pure steroid derivative, followed by a second separation (propanol/H 20, ODS2), ultimately providing a purified steroid derivative (0.1 mg). NMR and MS studies identified this sample as the A-nor -A 2 2 steroid derivative (4.10). 142 4.2.2 Structure Elucidation of A-nor-A Steroid Derivative Standard 1D and 2D NMR data sets were collected in C 6 D 6 on a Bruker 600 MHz spectrometer with a TXI probe. Spectra are shown in Figures 4.7-4.11. High-resolution mass spectrometry data indicated a molecular formula of C34H5oN02Br, requiring 10 degrees of unsaturation. A summary of the NMR data is found in Table 4.1. Only clearly discernable R O E S Y and T O C S Y correlations are tabulated. Due to a paucity of material, 1 3 C resonances were determined from HSQC and HMBC correlations. 143 144 Figure 4.10 600 MHz HSQC NMR spectrum of 4.10 recorded in C 6 D 6 147 Figure 4.11 600 MHz HMBC NMR spectrum of 4.10 recorded in C 6 D 6 148 Table 4.1 1D and 2D NMR data for A-nor-C27A derivative (4.10) recorded at 600 MHz (1H) in C 6 D 6 c# "C '•H {J) _ COSY ROESY TOCSY HMBC 1 39.5 1.01 H2, H2' C2 1.54 H2, H2' 2 27.7 1.50 H1, H1\ H3 C1, C3 1.96 m H1, H1', H3 H5 3 39.3 2.36 m H2, H2', H4, H4', H5 H5 H1, H1', H2, H2', H4, H4\ H5, H6, H6', H7, H7' 4 69.4 4.07 dd (10.4, 9.2) H3 H1, H1', H2, H2', H3, C2, C3, H4\ H5, H6, H7, H7' C5, C27 H1, H1', H2, H2', H3, 4.29 dd(10.4, 6.8) H3 H4, H5, H6, H7, H7' C2, C3, C5, C27 5 52.6 1.31 H3, H6, H6' H2', H3, H9 6 23.0 1.25 m H5, H7, H7' H19 1.56 H5, H7, H7' 7 33.1 0.79 m H6, H6', H8 H2\ H3, H5, H6, H6\ H7', H8, H11, H11', H6, H6\ H7, H8, H9, 1.70 m H6, H6', H8 H14 H11, H14, H15 8 35.8 1.22 m H7, H7\ H9, H14 H18, H19 9 55.8 0.66 m H8, H11, H11' H5, H12", H14 10 45.0 - - - - -11 23.7 1.33 H9, H12, H12' H18, H19 1.42 H9, H12, H12' 12 42.5 1.11 H11, H11' 1.94 H11, H11' H9, H14 H11, H12', H15, H17 13 40.5 - - - - -14 56.7 0.96 H8, H15, H15' H7', H9, H12' H12, H15', H16', H20 15 24.9 1.07 H14, H16, H16' 1.59 H14, H16, H16' 16 29.2 1.37 H15, H17 1.81 m H15, H15', H17 H8, H14, H15, H15', H16, H17, H20, H21 17 56.5 1.17 H16, H16', H20 18 12.7 0.70 s - H8, H11, H22, - C12, H21 C13,' C14, C17 19 14.7 0.69 s - H6, H8, H11 - C1, C5, C9, C10 20 40.8 2.12 m H17, H21, H22 H14, H15', H17, H21 C22 21 21.4 1.14 d (13.1) H20 H18 C17, C20 22 138.7 5.35 m (15.2) H20, H23 H18 H20, H21, H23, H24, C20, H24', H25, H26, H27 C24 23 126.7 5.38 m (15.2) H22, H24' H20, H21, H22, H24', C20, H25, H26, H27 C24 24 40.3 1.17 - C23 1.97 H23, H25 C22, C23 25 29.0 1.61 H24', H26, H27 H24', H26, H27 C23, C26, C27 26 22.6 0.93 d (1.9) H25 H23, H24, H24', H25 27 22.6 0.95 d (1.9) H25 H23, H24, H24', H25 28 153.1 - - - - -29 123.0 - - - - -30 116.5 7.27 bm H33 -NH - -31 140.3 - - - - -32 126.0 6.95 d (8.0) H33 H32, H33 C30, C34 33 130.5 6.69 t (8.0) H30, H32, H34 H32, H34 C29, C31 34 122.0 7.23 m H33 -NH H33, H34 C29, C30 -NH - 5.86 s - H30, H34 - C28 149 The structure elucidation of 4.10 is both complicated and simplified by the lack of functionality on the steroid nucleus. The lack of double bonds within the ring structure means that nearly all of the structure is a continuous spin system, allowing great use of extended 2D correlations. However, this lack of functionality also results in very little dispersion in the 1 H spectrum, resulting in few unique resonances from which one can access the steroid core. A total of two spin systems are present, with one encompassing the whole of the sterol subunit and the second in the aromatic derivatized alcohol side chain. The T O C S Y spectrum reveals valuable information on the relative connectivity in the aliphatic spin system, allowing a total assignment of the structure. The primary carbinol at C4 is observed by a characteristic pair of multiplets at 5 4.07 (H4) and 5 4.29 (H4') which correlate in the HSQC into 5 C 69.4 (C4). The protons at 5 4.07 (H4) and 5 4.29 (H4') correlate in the C O S Y to a multiplet at 6 2.36 (H3), which correlates in the HSQC to 5 C 39.3 (C3). Correlations between 5 2.36 (H3) and all of 5 1.50 (H2), 5 1.96 (H2') and 5 1.34 (H5) establish the protons on the adjacent centres, with HSQC correlations establishing their respective carbon signals at 6 C 27.7 (C2) and 5 C 30.3 (C5). C O S Y correlations from 5 1.50 (H2) and 6 1.96 (H2') to 5 1.01 (H1) and 6 1.54 (HT) establish the adjacent centre. Both 6 1.01 (H1) and 5 1.54 (HT) correlate in the HSQC spectrum to a signal at 5 C 39.5 (C1), completing all protonated centres on the A ring (Figure 4.12). At this point C O S Y correlations became difficult to distinguish due to the lack of dispersion, thus C O S Y data was used in concert with T O C S Y and HMBC data to work through the rest of the steroid. 150 A correlation in the C O S Y spectrum from 5 1.31 (H5) to both 5 1.25 (H6) and 5 1.56 (H6') established the adjacent centre in the B ring of the nucleus. Both 5 1.25 (H6) and 5 1.56 (H6") correlate in the C O S Y to 5 0.79 (H7) and 5 1.70 (H7') on the subsequent methylene, which in turn correlate in the C O S Y spectrum to a methine at 5 1.22 (H8). The observation of a correlation from 5 1.22 (H8) to at methine at 5 0.66 (H9) completes the protonated portion of the B ring. All protonated carbons were assigned from the HSQC spectrum as: 5 C 23.0 (C6), 5 C 33.1 (C7), 5 C 35.8 (C8) and 5 C 35.8 (C9), respectively. The remaining centre at C10 is the site of the C19 methyl group, and thus assigned by observation of HMBC correlations from the methyl singlet at 5 0.69 (H19) to 5 C 45.0 (C10). HMBC correlations were also observed from 5 0.69 (H19) to 5 C 39.5 151 (C1), 5 C 52.6 (C5) and 6 C 55.8 (C9), which allows the assembly of the completed A -B ring bicycle (Figure 4.13). A correlation in the C O S Y spectrum from 5 1.22 (H8) to 5 0.96 (H14) establishes the adjacent methine in the structure. Correlations from 5 0.66 (H9) to both 5 1.33 (H11) and 5 1.42 (H11') work around the other side of the C ring to establish the next centre. Both 5 1.33 (H11) and 5 1.42 (H11') correlate to the adjacent methylene at 5 1.11 (H12) and 5 1.94 (H12'), which completes the assignment of the protonated carbons in the C ring. HSQC correlations are again used to establish the protonated carbons as S c 23.7 (C11), 5 C 42.5 (C12) and 5 C 56.7 (C14). 152 Correlations from 6 0.96 (H14) to 5 1.07 (H15) and 5 1.59 (H15') in the C O S Y spectrum establish the adjacent methylene in the D ring. Both 5 1.07 (H15) and 5 1.81 (H15') in turn correlate in the C O S Y to 5 1.37 (H16) and 6 1.81 (H16'), establishing the next methylene. A subsequent correlation from 5 1.37 (H16) and 6 1.81 (H16') to 6 1.17 (H17) in the C O S Y spectrum completes the protonated centres on the D ring. The remaining quaternary centre at C 13 is again assigned by a correlation in the HMBC spectrum from the attached C18 methyl group. In this case, an HMBC correlation from 5 0.70 (H18) to 5C 40.5 (C13) assigns the centre, while additional HMBC correlations from 5 0.70 (H18) to 5C 42.5 (C12), 5C 56.7 (C14) and 5C 56.5 (C17) establish the adjacent centres. HSQC information is used to assign all protonated carbons (Figure 4.14). Figure 4.14 A) C-D ring structure showing key C O S Y and HMBC correlations, B) 1 H assignments, and C) 1 3 C assignments 153 A C O S Y correlation from 5 1.17 (H17) to 5 2.12 (H20) establishes the link from the core steroid to the side chain. The C21 methyl group is established by the presence of a C O S Y correlation from 5 2.12 (H20) to 5 1.14 (H21). A site of unsaturation is present on the side chain, as observed by a C O S Y correlation from 6 2.12 (H20) to 5 5.35 (H22), and onwards by a C O S Y correlation from 5 5.35 (H22) to 5 5.38 (H23). The 3 J coupling constant between H22 and H23 is 15.2 Hz, as measured from the 1 H spectrum, indicating a trans relationship between H22 and H23. From 5 5.38 (H23) additional C O S Y correlations establish the neighboring protons as 5 1.17 (H24) and 5 1.97 (H24'), which in turn correlate into a methine at 5 1.61 (H25). The side chain terminates in a geminal dimethyl as shown by two signals at 5 0.93 (H26) and 5 0.95 (H27), each of which integrates for 3 protons. These correlate in the C O S Y spectrum from 5 0.93 (H26) and o 0.95 (H27) to 5 1.61 (H25), establishing the final connection in the steroid side chain. HSQC correlations were used to assign the protonated carbons as 5C 40.8 (C20), 5C 21.4 (C21), 6 C 138.7 (C22), 5C 126.7 (C23), 5C 40.3 (C24), 5C 29.0 (C25) and 5C 22.6 (C26 and C27), respectively. 154 Figure 4.15 A) Completed A-nor-steroid structure, with key C O S Y and HMBC NMR correlations, B) 1 H NMR assignment of side chain, and C) 1 3 C NMR assignment of side chain The aromatic residue is connected to the steroid core by observation of HMBC correlations from 5 4.07 (H4) and 5 4.29 (H4') to an amide carbonyl at 5C 153.1 (C28). The amide NH is observed by a similar HMBC correlation from 5 5.86 (NH) to 5C 153.1 (C28). The presence of R O E S Y correlations from 5 5.86 (NH) to 6 7.27 (H30) and 6 7.23 (H34) place these aromatic protons closest to the amide bond. Correlations in the C O S Y between 5 7.23 (H34) and 5 6.69 (H33), which itself correlates to 5 6.95 (H32) completes the assignment of protonated centres in the aromatic ring. HSQC correlations establish the carbon centres at 5C 116.5 (C30), 5C 126.0 (C32), 5C 130.5 (C33) and 5C 122.0 (C34), 155 respectively. The final centres were assigned by the observation of HMBC correlations from 5 7.23 (H34) to 5 C 123.0 (C29) and from 5 6.69 (H33) to 5c 140.3 (C31). This places the bromine on C31 (Figure 4.16). 130.5 r 1 R O E S Y Figure 4.16 A) Key COSY, HMBC and R O E S Y NMR correlations in the side chain derivative, B) 1 H NMR assignments, and C) 1 3 C NMR assignments All of the above C O S Y correlations were supported by excellent data in the T O C S Y spectrum, as listed in Table 4.1. This proved invaluable during the initial structural elucidation. Less data was available in the R O E S Y spectrum, although 1,3-diaxial correlations support the placement of C18 and C19 in the axial position, with a chair-chair relative configuration of the B-C rings. A R O E S Y correlation between 5 0.70 (H18) and 5 2.12 (H20) supports the 156 placement of the side chain in the pseudoaxial position. Likewise, the observation of a R O E S Y correlation between 5 2.36 (H3) and 5 1.31 (H5) indicates that H3 and H5 are cis, and since H5 is axial, C4 should be pseudoequatorial. A summary of the R O E S Y information can be found in Figure 4.17. Due to the extremely small sample size no optical rotation measurements were made. Figure 4.17 Relative configuration of 4.10, with key R O E S Y NMR correlations 4.2.3 Biological Activity of A-nor-Steroid Initial bioassay results showed the full spectrum of CB activities in the 03-39 extract. However, as purification proceeded biological activity diminished in the resulting fractions. Due to small sample size the A - / W - C 2 7 A 2 2 derivative (4.10) was not bioassayed, however precursor fractions from which 4.10 was isolated, including one containing a mixture of non-derivatized A-nor-sterols showed no activity at relatively high concentrations (16.5 mM). This leads to one of two possible conclusions: either the biologically active compound was lost somewhere during the isolation, or the observed biological activity was a result of 157 a number of different compounds acting in concert on the CB receptor. A review of the literature shows some reference to membranolytic activity in sponge extracts. This is proposed to be due to A 7-sterols present in a similarly complex mixture to that found in this sample of Stylissa massa 2 2 2 Given the membrane-spanning properties of the CB receptors, it is possible to imagine this as a more general membrane receptor interaction. At the least, the A-nor-sterols are identified as potential nuisance compounds in this assay. This group of compounds has been the subject of some study. A great deal of work was performed on these and other related minor sterols from marine sponges by the laboratory of Carl Djerassi, particularly in the early 1980s. 2 2 3 " 2 2 5 It has been shown that these compounds are produced in the sponge by contraction of the A ring of exogenous steroids. While they have been observed in a number of sponges in the order Axinellida, this is to the author's knowledge the first report of A-nor-steroids from Stylissa massa, a sponge from the order Hal ichondria. 2 2 6 ' 2 2 7 4.3 Bengamide A 4.3.1 Isolation and Characterization of Bengamide A from Hemiasterella aff. affinis The orange fan sponge Hemiasterella aff. affinis was collected from a vertical wall at a depth of 15 m in Milne Bay, Papua New Guinea (10° 32.02' S, 150°39.07' E) on September 17, 2003 (Figure 4.18). During an assay of our 158 library of marine extracts, it was identified as possessing C B i antagonism in the CB bioassay. A sample of sponge was extracted repeatedly with MeOH. The methanolic extract was dried in vacuo to yield an orange paste. A sample of this paste (1.4 g) was then partitioned between H 2 0 and EtOAc, each of which was dried in vacuo. The EtOAc fraction was subjected to lipophilic size-exclusion chromatography (Sephadex LH-20®) in MeOH. The resulting fractions were submitted for bioassay, and one of the active fractions (81.7 mg) subjected to both normal and reversed-phase column chromatography with little success in purification as seen by TLC analysis. The resulting sample (34.8 mg) was 159 separated by reversed-phase C-18 HPLC (MeOH/H 2 0, 0 D S 2 column) to yield a series of bengamide containing fractions. The main fraction (5.9 mg) was found to be active as a CE^ agonist. Further HPLC purification was performed on this sample, yielding (2.7 mg) of relatively pure sample, as observed by TLC, NMR and MS analysis. NMR and MS studies identified this sample as bengamide A (4.11). 4.11 Figure 4.19 Structure of bengamide A (4.11) 4.3.2 Structure Elucidation of Bengamide A Standard 1D and 2D NMR data sets were collected on a sample of 4.11 in CDCI 3 on a Bruker 600 MHz spectrometer with a TXI probe. Spectra are found in Figures 4.20-4.24. High-resolution mass spectrometry data indicated a molecular formula of C31H56N2O8, requiring 5 degrees of unsaturation. A summary of NMR data is found in Table 4.2. 160 Figure 4.20 600 MHz 1 H NMR spectrum of bengamide A (4.11) recorded CDCI 3 161 162 O O O O Q O O o o .o o q o o «— IN -J" uO ID CO Figure 4.22 600 MHz C O S Y NMR spectrum of bengamide A (4.11) recorded CDCI 3 163 Figure 4.23 600 MHz HSQC NMR spectrum of bengamide A (4.11) recorded in CDCI 3 164 Table 4.2 1D and 2D NMR data for bengamide A (4.11) recorded at 600 MHz (1H) and 150 MHz ( 1 3C) in CDCI 3 c# 1 3 C COSY HMBC 1 22.4 0.98 d (3.3) H2 C3 2 31.0 2.30 m (6.5) H1, H3 C1, C3, C4, C15 3 142.2 5.77 dd (6.5, 15.4) H2, H4 C1, C2, C5, C15 4 125.6 5.43 dd (6.8, 15.4) H3, H5 C2, C5, C6 5 74.5 4.21 m H4, H6 C3, C4, C6 6 72.5 3.59 bs H5 7 73.0 3.81 bd (7.0) H8 C5, C8, C9 8 81.1 3.77 bd (7.0) H7 C6, C7, C31 9 172.6 - - -10 51.7 4.57 dd (6.1, 10.4) H11, H11\ Na C9, C11, C12, C16 11 29.1 1.72 ddd (2.5, 13.3, 24) H10, H12, H12' C10, C13, C16 2.14 bd (13.8) 12 33.2 1.94 ddd (3.7, 13.5, 24.5) H11, H11', H13 C10, C13, C14 2.19 bd (13.3) 13 71.0 4.63 m (10.5, 3.3) H12, H12', H14, H14' 14 45.3 3.29 bdd (8.0, 14.5) H13, Nb C12, C13, C16 3.36 ddd (5.3, 9.9, 14.7) 15 22.3 0.97 d (3.3) H2 C3 16 174.2 - - -17 173.1 - - -18 34.6 2.28 t (7.6) H19 C17, C19 19 25.1 1.58 m (7.1) H18, H20 C17, C18, C21-28 20 22.9 1.28 m H19, H21-28 21-28 29.9-29.3 1.32-1.18 Aliphatic envelope C19, C21-28, C29 29 32.1 1.24 m H21-28, H30 30 14.3 0.86 t (6.9) H29 C29 31 60.3 3.53 s - C8 Na - 7.98 d (6.1) H10 C9, C10, C16 Nb - 5.95 bs H14, H14' C10, C16 Compound 4.11 consists of three separate proton NMR spin systems. Each of these can be readily assembled using C O S Y data, and the three connected using HMBC correlations. Six methyl protons at 5 0.98 (H1) and 6 0.97 (H15) correlate in the C O S Y spectrum to a tertiary methine at 5 2.30 (H2), which then correlates into an alkenyl methine at 5 5.77 (H3). This correlates in the C O S Y to another alkenyl methine at 5 5.43 (H4), and the olefin configuration is established as trans to H3 due to the large 3 J coupling constant (15.4 Hz) between them. A C O S Y cross peak between 5 5.43 (H4) and 5 4.21 (H5) establishes the next proton in the chain. A C O S Y correlation between 5 4.21 (H5) and 6 3.59 (H6) establishes the subsequent centre, which weakly correlates in the C O S Y spectrum to the proton at 5 3.81 (H7). A final C O S Y correlation 166 from 5 3.81 (H7) to 5 3.77 (H8) completes the proton spin system (Figure 4.25). The HSQC spectrum was used to assign protonated carbons, and HMBC correlations used to confirm C O S Y information. HMBC correlations from both 5 3.81 (H7) and 5 3.77 (H8) to a carbonyl at 5 C 172.6 (C9) establish the adjacent centre in the chain. Three methoxy protons at 5 3.53 (H31) are placed on C8, as a strong HMBC correlation to 5 C 81.1 (C8) is observed. The chemical shifts of C5-7 establish them as being hydroxylated. OH{OMe A HMBC COSY B 0.98 2.30! H. A H H 5.43 H I H 4.21 O 3.810" HH3.53 H l_l 0.97 H H 5.77 60.3 H OMe u 1-1 N . 74.5 T73.0 VT[172.6* H . H I H 03.59 0 3 . 7 7 0 I H H7.98 I I H Figure 4.25 A) Fragment of bengamide A (4.11) showing key C O S Y and HMBC NMR correlations, B) 1 H NMR assignments, and C) 1 3 C NMR assignments The second spin system begins at a methine at 5 4.57 (H10), which correlated in the C O S Y spectrum into an adjacent methylene at 5 1.72 (H11) and 5 2.14 (H11'). Both 5 1.72 (H11) and 5 2.14 (H11') correlate in the C O S Y spectrum to the adjacent methylene at 5 1.94 (H12) and 5 2.19 (H12'), which in turn correlated in the C O S Y spectrum to an oxymethine at 6 4.63 (H13). A 167 correlation from 5 4.63 (H13) to a methylene at 5 3.29 (H14) and 5 3.36 (H14') established the adjacent methylene. A final correlation in the C O S Y spectrum between 5 3.29 (H14) and 5 5.95 (Nb) established a link to the amide bond. All of 5 4.57 (H10), 6 3.29 (H14) and 3.36 (H14') show strong HMBC correlations to a carbonyl at 5 C 174.2 (C16), which places the amide between C10 and C6. HSQC data was used to establish the carbon resonances of this fragment as 5 C 51.7 (C10), 5 C 29.1 (C11), 5 C 33.2 (C12), 5 C 71.0 (C13), 5 C 45.3 (C14) and 5 C 174.2 (C16), respectively. HMBC data was consistent with these assignments. Figure 4.26 A) Fragment of bengamide A (4.11) showing C O S Y and HMBC NMR correlations, B) 1 H NMR assignments, and C) 1 3 C NMR assignments The final fragment of bengamide A (4.11) was established by an HMBC correlation from 5 4.63 (H13) to a carbonyl at 5 C 173.1 (C17). Two methylene protons at 5 2.28 (H18) also correlated in the HMBC spectrum to 5 C 173.1 (C17), which places an ester between C13 and C17. C O S Y correlations from 5 2.28 A 7.98 H 4.57Q 168 (H18) proceed down the alkyl chain into an aliphatic envelope at 5 1.18-1.32. The chain terminates at 8 0.86 (H30) in a methyl triplet. HMBC C O S Y A B 19 21 23 25 27 29 rs 1-58 Q H H alkyl envelope (1.32-1.18) H H H H 2.28 1.28 alkyl envelope 29.9-29.3 1.24 H H 14.3 Figure 4.27 A) Fragment of bengamide A (4.11) showing C O S Y and HMBC correlations, B) 1 H assignments, and C) 1 3 C assignments Connecting the three fragments was relatively straightforward, yielding the planar structure of 4.11. Given that the optical rotation data is relatively close to the literature value ([a] D 2 5 + 51.3 (c =0.9 MeOH) measured, [a] D 2 5 + 30.3 (c =0.081 MeOH) literature value), 2 2 8 it was reasonable to propose the same absolute configuration as the previously isolated natural product (Figure 4.19). 4.3.3 Biological Activity of Bengamide A Throughout the bioassay-guided fractionation, samples were assayed against both CB ! and C B 2 , and tested for both agonism and antagonism, using the Grigliatti assay.1" The initial extract of 03-253 was active as a CB-\ antagonist, 1 All C B bioassays conducted by Tom Pfeifer in the laboratory of Tom Grigliatti. 169 however subsequent fractions were active as CB i agonists throughout the isolation, although at relatively weak concentrations (2.5 mM). In comparison, the natural agonist, anandamide, is used as a positive control at 0.3 mM. This CBi agonism was focussed in the bengamide containing fractions, although exhaustive purification of the major bengamide (bengamide A, 4.1) showed no response in the CB bioassay at high (16.5 mM) concentration. This is suggestive of non-specific interaction with the receptors, particularly in the presence of several different bengamides, or perhaps that the active compound is one of the minor bengamides. 4.4 Future Directions with the CB Bioassay While both compounds isolated in this project were known compounds, neither of the sponges that they were isolated from have been identified as sources of these compounds to date. The A-nor sterols, as exemplified by 4.10, are relatively rare steroids, and to date (to this author's knowledge) this is the first report of A-nor-sterols from Stylissa massa} Although they have been isolated from several sponges in the family Axinellidae, this seems to be the first report from the family Dictyonell idae. 2 2 3 , 2 2 4 In fact, the only compounds reported in the marine natural products literature from this sponge are oroidin alkaloids such as massadine (4.12), which are reported to have activity against geranylgeranyl transferase and some protein k inases. 2 2 9 < * Search run on MarinLit Marine Literature Database, Version pc13.4. 170 H 4.12 Figure 4.28 Massadine (4.12), an alkaloid isolated from Stylissa massa With regards to the isolation of bengamide A (4.11) from Hemiasterella aff. affinis, this is also the first reported isolation of a bengamide from this genus of sponge.1" In fact, all other isolations of bengamide A were from the Jaspis species of sponge. The bengamides were initially identified as antihelmenthic compounds, and subsequently identified as methionine aminopeptidase inhibitors, although in general they are found to be inhibitory to tumor growth. 2 3 0" 2 3 2 An anti-cancer Phase 1 trial was begun with a synthetic analogue of bengamide A, LAF-389 (4.13), although it was shortly withdrawn due to issues with toxicity and uncertainty of the molecular target. 2 3 3 Although purified bengamide A (4.11) was not found to be active in the CB assay against either CB i or C B 2 , it is possible that a component in the fractions from which compound 4.11 was isolated will be a related bengamide and found to possess the observed activity. Further studies of these minor components will need to be undertaken. T Search run on MarinLit Marine Literature Database, Version pc13.4. 171 Figure 4.29 Structure of LAF-389 (4.13), a synthetic bengamide A analogue 4.5 Experimental 4.5.1 General Experimental Procedures All 1D and 2D were recorded on a Bruker AV-600 spectrometer equipped with a cryoprobe or TXI probe. 1 H chemical shifts were referenced to the residual C 6 D 6 or CDCI 3 solvent peak (5 7.16 and 5 7.24 ppm, respectively), and 1 3 C chemical shifts were referenced to the C 6 D 6 or CDCI 3 solvent peak (5C 128.39 and 5 C 77.23 ppm, respectively). All NMR solvents were obtained from Cambridge Isotope Laboratories. All chromatography was performed using HPLC grade solvents from Fisher Scientific with no further purification. Water was purified using a Millipore reverse osmosis filter system. Reversed-phase chromatography was performed using 10, 5 or 2 gram Waters C-18 Sep-Paks®. Normal phase chromatography was performed using Silicycle Ultra Pure silica gel (230-400 mesh) under N 2 pressure. HPLC isolation was performed using a Waters 600E system controller liquid chromatograph interfaced to a Waters 486 tunable absorbance detector. Optical rotations were measured with a J A S C O J-1010 spectrophotometer using a 10 mm cell. Reagents for derivatization were obtained from Sigma-Aldrich. 172 4.5.2 I so la t ion of A-nor-steroid A sample of sponge (~ 100 g wet weight) was extracted repeatedly with MeOH (3 X 100 mL). The organic extract was dried in vacuo to yield an orange paste (109.6 mg). A portion of this paste was then partitioned between H 2 0 (75 mL) and EtOAc (3 X 100 mL), each of which was dried in vacuo. The EtOAc fraction was subjected to normal phase silica gel chromatography (100% Hexane to 100% EtOAc step gradient, washed with 10% MeOH/DCM and 100% MeOH) yielding 81 fractions. TLC analysis resulted in the pooling of a steroid enriched fraction (3.7 mg). This mixture of sterols was then derivatized with m-bromophenyl isocyanate in CCI 4 and monitored by TLC ( E x p e r i m e n t a l 4.5.3). Repeated HPLC separation of the resulting sample first yielded increasingly pure steroid samples under carefully moderated conditions (97% M e O H / H 2 0 on a Phenominex Gemini® 5p C18 11 OA column) giving 0.6 mg of partially pure steroid, followed by a second separation (30% propanol/70% H 2 0 to 100% propanol over 70 minutes, CSC-lnertsil® ODS2 column), ultimately providing a purified steroid derivative (0.1 mg). NMR and MS studies identified this sample as the A - n o r - C 2 7 A 2 2 derivative (4.10). 4.5.3 De r iva t i z a t i on of A-nor-steroid A mixture of A-nor-sterols (1.7 mg) was dissolved in CCU (1 mL) in a screw-top vial. To this was added 3-bromophenyl isocyanate (10.0 mg, pmol). 173 The reaction was sealed, and stirred at 65 °C for 3 hours. TLC analysis showed complete reaction, and CCU was removed under a stream of N 2 . The residue was lyophilized overnight prior to NP column chromatography. 4.5.4 A-nor-steroid Physical Data Clear, colorless glass (0.1 mg). For 1D and 2D NMR data see Table 4.1. HRESIMS: [M + Na] + m/z = 606.2900 (calcd for C 3 4 H 5 oN0 2 7 9 BrNa, 606.2923). 4.5.5 Isolation of Bengamide A A sample of sponge (-30 g wet weight) was extracted repeatedly with MeOH (3 X 100 mL). The methanolic extract was dried in vacuo to yield an orange paste. A sample of this paste (1.4 g) was then partitioned between H 2 0 (75 mL) and EtOAc (3 X 100 mL), each of which was dried in vacuo. The EtOAc fraction was subjected to lipophilic size-exclusion chromatography (Sephadex LH-20®) in MeOH. The resulting fractions were submitted for bioassay, and one of the active fractions (81.7 mg) subjected to both normal (CH 2 CI 2 to 50% MeOH in CH 2 CI 2 on S i0 2 ) and reversed-phase (50% M e O H / H 2 0 to MeOH on Waters 2g Sep-Pak®) column chromatography with little success in purification as seen by TLC analysis. The resulting pooled fractions (34.8 mg) were separated by reversed-phase C-18 HPLC (gradient from 80% M e O H / H 2 0 to 100% MeOH over 40 minutes, CSC-lnertsil® ODS2 column) to yield a series of bengamide 174 containing fractions. The main fraction (5.9 mg) was found to be active as a CB-\ agonist. Further HPLC purification (85 % M e O H / H 2 0 isocratic, CSC-lnertsil® ODS2 column) was performed on this sample, yielding (2.7 mg) of extremely pure sample. NMR and MS studies identified this sample as bengamide A (4.11). 4.5.6 Bengamide A Physical Data Clear, colorless glass (2.7 mg); [a] D 2 5 + 51.3 (c =0.9 MeOH). For 1D and 2D NMR data see Table 4.2. HRESIMS: [M+H]+ m/z = 607.3951 (calcd for CaiHseNsOsNa, 607.3934). 175 Bibliography (1) Cousteau, J . Y.; Dumas, F. The Silent World; Harper & Brothers: New York, 1953. (2) Cans, R. Nature (London, United Kingdom) 1997, 388, 334. (3) Blunt, J . W.; Copp, B. R.; Munro, M. H. G.; Northcote, P. T.; Prinsep, M. R. Natural Product Reports 2006, 23, 26-78. (4) Paul, V. J . ; Puglisi, M. P.; Ritson-Williams, R. Natural Product Reports 2006, 23,153-180. (5) Moore, B. S. Natural Product Reports 2005, 22, 580-593. (6) Moore, B. S. Natural Product Reports 2006, 23, 615-629. (7) Cragg, G. M.; Newman, D. J . ; Snader, K. M. Journal of Natural Products 1997, 60, 52-60. (8) Newman, D. J . ; Cragg, G. M.; Snader, K. M. Journal of Natural Products 2003, 66, 1022-1037. (9) Dewick, P. M. Medicinal Natural Products: A Biosynthetic Approach; 2nd ed.; John Wiley & Sons: New York, 2002. (10) Firn, R. D.; Jones, C. G. Natural Product Reports 2003, 20, 382-391. (11) Andersen, R. J . ; Williams, D. E. In Issues in Environmental Science and Technology 13: Chemistry in the Marine Environment Hester, R. E., Harrison, R. M., Eds.; Royal Society of Chemistry: Cambridge, 2000, pp 55-79. (12) Capon, R. J . European Journal of Organic Chemistry 2001, 633-645. (13) Newman, D. J . ; Cragg, G. M. Journal of Natural Products 2004, 67, 1216-1238. (14) Bergmann, W.; Feeney, R. J . Journal of the American Chemical Society 1950, 72, 2809. (15) Bergmann, W.; Feeney, R. J . Journal of Organic ChemistryWSA, 76,981-987. (16) Bergmann, W.; Feeney, R. J . Journal of Organic Chemistry 1955, 20, 1501-1507. (17) Olivera, B. M.; Rivier, J . ; Clark, C ; Ramilo, C. A.; Corpuz, G. P.; F.C., A.; Mena, E. E.; S.R., W.; Hillyard, D. R.; Cruz, L. J . Science 1990, 249, 257-263. (18) Myers, R. A.; Cruz, L. J . ; Rivier, J . E.; Olivera, B. M. Chemical Reviews 1993, 93, 1923-1936. (19) Rinehart, K. L ; Holt, T. G.; Fregeau, N. L ; Stroh, J . G.; Keifer, P. A.; Sun, F.; Li, L. H.; Martin, D. G. Journal of Organic Chemistry WW, 55, 4512-4515. (20) Endo, A.; Yanagisawa, A.; Abe, M.; Tohma, S.; Kan, T.; Fukuyama, T. Journal of the American Chemical Society 2002, 124, 6552-6554. (21) Fayette, J . ; Coquard, I. R.; Alberti, L.; Boyle, H.; Meeus, P.; Decouvelaere, A.-V.; Thiesse, P.; Sunyach, M.-P.; Ranchere, D.; Blay, J . -Y. Current Opinion in Oncology 2006, 18, 347-353. 176 (22) Look, S. A.; Fenical, W.; Jacobs, R. S.; Clardy, J . Proceedings of the National Academy of Sciences of the United States of America 1986, 83, 6238-6240. (23) Look, S. A.; Fenical, W.; Matsumoto, G. K.; Clardy, J . Journal of Organic Chemistry 1986, 51, 5140-5145. (24) Faulkner, D. J . Antonie van Leeuwenhoek 2000, 77, 135-145. (25) Kobayashi, J . i.; Ishibashi, M. Chemical Reviews 1993, 93, 1753-1769. (26) Faulkner, D. J . ; Harper, M. K.; Haygood, M. G.; Salomon, C. E.; Schmidt, E. W. In Drugs from the Sea; Fusetani, N., Ed.; Karger A G : Basel, 2000, pp 107-119. (27) Piel, J . Natural Product Reports 2004, 21, 519-538. (28) Piel, J . ; Butzke, D.; Fusetani, N.; Hui, D.; Platzer, M.; Wen, G.; Matsunaga, S. Journal of Natural Products 2005, 68, 472-479. (29) Jensen, P.; Fenical, W. In Drugs From The Sea; Fusetani, N., Ed.; Karger: Basel, 2000, pp 6-29. (30) Fleming, A. British Journal of Experimental Pathology 1929, 10, 226-236. (31) Flynn, E. H.; Sigal, M. V.; Wiley, P. F.; Gerzon, K. Journal of the American Chemical Society 1954, 76, 3121 -3131. (32) Arcamone, F.; Cassinelli, G.; Fantini, G.; Grein, A.; Orezzi, P.; Pol, C ; Spalla, C. Biotechnology and Bioengineering 1969, XI, 1101 -1110. (33) Fairbrother, R. W.; Williams, B. L LanceM 956, 271, 1177-1178. (34) Sheldrick, G. M.; Jones, P. G.; Kennard, O.; Williams, D. H.; Smith G. A. Nature 1978, 271, 223-225. (35) Shimizu, Y. Chemical Reviews 1993, 93, 1685-1698. (36) Shimizu, Y. Current Opinion in Microbiology 2003, 6, 236-243. (37) Porter, K. G.; Feig, Y. S. Limnol. Oceanogr. 1980s 25, 943-948. (38) Sogin, M. L ; Morrison, H. G.; Huber, J . A.; Welch, D. M.; Huse, S. M.; Neal, P. R.; Arrieta, J . M.; Herndl, G. J . Proceedings of the National Academy of Sciences of the United States of America 2006, 103, 12115-12120. (39) Pace, N. R. Science 1997, 276, 734-740. (40) Kaeberlein, T.; Lewis, K.; Epstein, S. S. Science 2002, 296, 1127-1129. (41) Bugni, T. S.; Ireland, C. M. Natural Product Reports 2004, 21, 143-163. (42) Fenical, W. Chemical Reviews 1993, 93, 1673-1683. (43) Fenical, W.; Jensen, P.; Kauffman, C ; Mayhead, S.; Faulkner, J . ; Sincich, C ; Rao, R.; Kantorowski, E.; West, L.; Strangman, W.; Shimizu, Y.; Li, B.; Thammana, S.; Drainville, K.; Davies-Coleman, M.; Kramer, R.; Fairchild, C ; Rose, W.; Wild, R.; Vite, G.; Peterson, R. Pharmaceutical Biology (Lisse, Netherlands) 2003, 41, 6-14. (44) Cho, J . Y.; Kwon, H. C ; Williams, P. G.; Jensen, P.; Fenical, W. Organic Letters 2006, 8, 2471 -2474. (45) Cho, J . Y.; Kwon, H. C ; Williams, P. G.; Kauffman, C. A.; Jensen, P. R.; Fenical, W. Journal of Natural Products 2006, 69, 425-428. 177 (46) Oh, D. -C; Williams, P. G.; Kauffman, C. A.; Jensen, P. R.; Fenical, W. Organic Letters 2006, 8, 1021-1024. (47) Kwon, H. C ; Kauffman, C. A.; Jensen, P. R.; Fenical, W. Journal of the American Chemical Society 2006, 128, 1622-1632. (48) Feling, R. H.; Buchanan, G. O.; Mincer, T. J . ; Kauffman, C. A.; Jensen, P. R.; Fenical, W. Angewandte Chemie, International Edition 2003, 42, 355-357. (49) Amagata, T.; Morinaka, B. I.; Amagata, A.; Tenney, K.; Valeriote, F. A.; Lobkovsky, E.; Clardy, J . ; Crews, P. Journal of Natural Products ASAP. (50) Barsby, T.; Kelly, M. T.; Gagne, S. M.; Andersen, R. J . Organic Letters 2001, 3, 437-440. (51) Barsby, T.; Kelly, M. T.; Andersen, R. J . Journal of Natural Products 2002, 65, 1447-1451. (52) Barsby, T.; Warabi, K.; Sorensen, D.; Zimmerman, W. T.; Kelly, M. T.; Andersen, R. J . Journal of Organic Chemistry 2006, 71, 6031-6037. (53) Barsby, T. A. New antibiotics from a marine isolate of Bacillus laterosporus; University of British Columbia: Vancouver, 2002. (54) Gerard, J . M.; Haden, P.; Kelly, M. T.; Andersen, R. J . Journal of Natural Products 1999, 62, 80-85. (55) Gerard, J . M. Antibiotic secondary metabolites of bacteria isolated from the marine environment; University of British Columbia: Vancouver, 1997. (56) Wratten, S. J . ; Faulkner, D. J . Journal of the American Chemical Society 1977, 99, 7367-7368. (57) Zabriskie, T. M.; Klocke, J . A.; Ireland, C. M.; Marcus, A. H.; Molinski, T. F.; Faulkner, D. J . ; Xu, C ; Clardy, J . C. Journal of the American Chemical Society 1986, 108, 3123-3124. (58) Fusetani, N.; Matsunaga, S.; Matsumoto, H.; Takebayashi, Y. Journal of the American Chemical Society 1990, 112, 7053-7054. (59) Forenza, S.; Minale, L ; Riccio, R.; Fattorusso, E. Journal of the Chemical Society D, Chemical Communications 1971, 18, 1129-1130. (60) Sharma, G. M.; Burkholder, P. R. Journal of the Chemical Society D, Chemical Communications 1971, 3, 151-152. (61) Kinnel, R. B.; Gehrken, H.-P.; Scheuer, P. J . Journal of the American Chemical Society 1993, 115, 3376-3377. (62) Mourabit, A. A.; Potier, P. European Journal of Organic Chemistry 2001, 2007, 237-243. (63) De Silva, E. D.; Scheuer, P. J . Tetrahedron Letters 1980, 21, 1611-1614. (64) Glaser, K. B.; Jacobs, R. S. Biochemical Pharmacology 1986, 35, 449-453. (65) Gunasekera, S. P.; Gunasekera, M.; Longley, R. E. Journal of Organic Chemistry 1990, 55, 4912-4915. (66) Fisher, J . F.; Meroueh, S. O.; Mobashery, S. Chemical Reviews 2005, 105, 395-424. (67) Kahne, D.; Leimkuhler, C ; Lu, W.; Walsh, C. Chemical Reviews 2005, 105, 425-448. 178 (68) Magnet, S.; Blanchard, J . S. Chemical Reviews 2005, 105, 477-497. (69) Katz, L ; Ashley, G. W. Chemical Reviews 2005, 105, 499-527. (70) Mukhtar, T. A.; Wright, G. D. Chemical Reviews 2005, 105, 529-542. (71) McDaniel, R.; Welch, M.; Hutchinson, C. R. Chemical Reviews 2005, 105, 543-558. (72) Lieber, S. A.; Marahiel, M. A. Chemical Reviews 2005, 105, 715-738. (73) Galm, U.; Hager, M. H.; Van Lanen, S. G.; Ju, J . ; Thorson, J . S.; Shen, B. Chemical Reviews 2005, 105, 739-758. (74) Brown, E. D.; Wright, G. D. Chemical Reviews 2005, 105, 759-774. (75) Alekshun, M. N.; Levy, S. B. Biochemical Pharmacology 2006, 71, 893-900. (76) Monaghan, R. L ; Barrett, J . F. Biochemical Pharmacology 2006, 71, 901-909. (77) Butler, M. S.; Buss, A. D. Biochemical Pharmacology 2006, 71, 919-929. (78) Pelaez, F. Biochemical Pharmacology 2006, 71, 981-990. (79) Rice, L. B. Biochemical Pharmacology 2006, 71, 991-995. (80) Singh, S. B.; Barrett, J . F. Biochemical Pharmacology 2006, 71, 1006-1015. (81) Pucci, M. J . Biochemical Pharmacology 2006, 77, 1066-1072. (82) Fleming, A. Pharmaceutical Journal 1940, 145, 162-172. (83) von Nussbaum, F.; Brands, M.; Hinzen, B.; Weigand, S.; Habich, D. Angewandte Chemie, International Edition 2006, 45, 5072-5129. (84) Rachakonda, S.; Cartee, L. Current Medicinal Chemistry 2004, 7 7, 775-793. (85) Whitney, C. G.; Farley, M. M.; Hadler, J . ; Harrison, L. H.; Lexau, C ; Reingold, A.; Lefkowitz, L.; Cieslak, P. R.; Cetron, M.; Zell, E. R.; Jorgensen, J . H.; Schuchat, A. New England Journal of Medicine 2000, 343, 1917-1924. (86) Leeb, M. Nature 2004, 437, 892-893. (87) Clardy, J . ; Fischbach, M. A.; Walsh, C. T. Nature: Biotechnology 2006, 24, 1541-1550. (88) Engel, S.; Jensen, P. R.; Fenical, W. Journal of Chemical Ecology 2002, 28, 1971-1985. (89) Paul, V. J . ; Puglisi, M. P. Natural Product Reports 2004, 21, 189-209. (90) C C S ; NCIC Canadian Cancer Statistics Toronto, Canada, 2006. (91) Ireland, C ; Aalbersberg, W.; Andersen, R.; Ayral-Kaloustian, S.; Berlinck, R.; Bernan, V.; Carter, G.; Churchill, A.; Clardy, J . ; Concepcion, G.; De Silva, D.; Discafani, C ; Fojo, T.; Frost, P.; Gibson, D.; Greenberger, L.; Greenstein, M.; Harper, M. K.; Mallon, R.; Loganzo, F.; Nunes, M.; Poruchynsky, M.; Zask, A. Pharmaceutical Biology (Lisse, Netherlands) 2003, 47, 15-38. (92) NCI NCI Cancer Bulletin 2004, 7, 3-4. 179 (93) Pettit, G. R.; Herald, C. L ; Hogan, F. In Anticancer Drug Development, Baguley, B. C , Kerr, D. J . , Eds.; Academic Press: San Diego, 2002, pp 203-235. (94) Quinoa, E.; Crews, P. Tetrahedron Letters 1987, 28, 3229-3232. (95) Arabshahi, L ; Schmitz, F. J . Journal of Organic Chemistry 1987, 52, 3584-3586. (96) Remiszewski, S. W. Current Medicinal Chemistry 2003, 10, 2393-2402. (97) Roberge, M.; Cinel, B.; Anderson, H. J . ; Lim, L. Y.; Jiang, X.; Xu, L ; Bigg, C. M.; Kelly, M. T.; Andersen, R. J . Cancer Research 2000, 60, 5052-5058. (98) Cinel, B.; Roberge, M.; Behrisch, H.; Ofwegen, L. v.; Castro, C. B.; Andersen, R. J . Organic Letters 2000, 2, 257-260. (99) Knight, P. J . K.; Pfeifer, T. A.; Grigliatti, T. A. Analytical Biochemistry 2003, 320, 88-103. (100) Knight, P. J . K.; Grigliatti, T. A. Journal of Receptors and Signal Transduction 2004, 24, 241 -256. (101) Knight, P. J . K.; Grigliatti, T. A. Archives of Insect Biochemistry and Physiology 2004, 57, 142-150. (102) Khanolkar, A. D.; Palmer, S. L ; Makriyannis, A. Chemistry and Physics of Lipids 2000, 108, 37-52. (103) Pertwee, R. G. British Journal of Pharmacology 2006, 747, S163-S171. (104) Faulkner, D. J . Natural Product Reports 2000, 77, 1-6. (105) Faulkner, D. J . ; Harper, M. K.; Haygood, M. G.; Salomon, C. E.; Schmidt, E. W. In Drugs from the Sea; Fusetani, N., Ed.; Karger A G : Basel, 2000, pp 107-119. (106) Fenical, W.; Jensen, P.; Kauffman, C ; Mayhead, S.; Faulkner, J . ; Sincich, C ; Rao, R.; Kantorowski, E.; West, L ; Strangman, W.; Shimizu, Y.; Li, B.; Thammana, S.; Drainville, K.; Davies-Coleman, M.; Kramer, R.; Fairchild, C ; Rose, W.; Wild, R.; Vite, G.; Peterson, R. Pharmaceutical Biology (Lisse, Netherlands) 2003, 47, 6-14. (107) Fenical, W. Chemical Reviews 1993, 93, 1673-1683. (108) Jensen, P.; Fenical, W. In Drugs From The Sea; Fusetani, N., Ed.; Karger: Basel, 2000, pp 6-29. (109) Kwon, H. C ; Kauffman, C. A.; Jensen, P. R.; Fenical, W. Journal of the American Chemical Society 2006, 725, 1622-1632. (110) Feling, R. H.; Buchanan, G. O.; Mincer, T. J . ; Kauffman, C. A.; Jensen, P. R.; Fenical, W. Angewandte Chemie, International Edition 2003, 42, 355-357. (111) Gerard, J . ; Lloyd, R.; Barsby, T.; Haden, P.; Kelly, M. T.; Andersen, R. J . Journal of Natural Products 1997, 60, 223-229. (112) Williams, D. E.; Bernan, V. S.; Ritacco, F. V.; Maiese, W. M.; Greenstein, M.; Andersen, R. J . Tetrahedron Letters 1999, 40, 7171-7174. (113) Onaka, H.; Asamizu, S.; Igarashi, Y.; Yoshida, R.; Furumai, T. Bioscience, Biotechnology and Biochemistry 2005, 69, 1753-1759. 180 (114) Howard-Jones, A. R.; Walsh, C. T. Journal of the American Chemical Society 2006, 128, 12289-12298. (115) Sanchez, C ; Brana, A. F.; Mendez, C ; Salas, J . A. ChemBioChem 2006, 7, 1231-1240. (116) Gerard, J . ; Haden, P.; Kelly, M. T.; Andersen, R. J . Tetrahedron Letters 1996, 37, 7201-7204. (117) Gerard, J . M. Antibiotic secondary metabolites of bacteria isolated from the marine environment, University of British Columbia: Vancouver, 1997. (118) Gerard, J . M.; Haden, P.; Kelly, M. T.; Andersen, R. J . Journal of Natural Products 1999, 62, 80-85. (119) Barsby, T.; Kelly, M. T.; Gagne, Si M.; Andersen, R. J . Organic Letters 2001, 3, 437-440. (120) Barsby, T.; Warabi, K.; Sorensen, D.; Zimmerman, W. T.; Kelly, M. T.; Andersen, R. J . Journal of Organic Chemistry 2006, 71, 6031-6037. (121) Friedrich, C. L ; Moyles, D.; Beveridge, T. J . ; Hancock, R. E. W. Antibacterial Agents and Chemotherapy 2000, 44, 2086-2092. (122) Bishop, J . L ; Finlay, B. B. Trends in Molecular Medicine 2006, 12, 3-6. (123) Hamamoto, K.; Kida, Y.; Zhang, Y.; Shimizu, T.; Kuwano, K. Microbiology and Immunology 2002, 46, 741 -749. (124) Barsby, T.; Kelly, M. T.; Andersen, R. J . Journal of Natural Products 2002, 65, 1447-1451. (125) Marahiel, M. A.; Stachelhaus, T.; Mootz, H. D. Chemical Reviews 1997, 97, 2651-2673. (126) Schwarzer, D.; Finking, R.; Marahiel, M. A. Natural Product Reports 2003, 20, 275-287. (127) Porter, K. G.; Feig, Y. S. Limnol. Oceanogr. 1980, 25, 943-948. (128) Sogin, M. L ; Morrison, H. G.; Huber, J . A.; Welch, D. M.; Huse, S. M.; Neal, P. R.; Arrieta, J . M.; Herndl, G. J . Proceedings of the National Academy of Sciences of the United States of America 2006, 103, 12115-12120. (129) Barsby, T. A. New antibiotics from a marine isolate of Bacillus laterosporus; University of British Columbia: Vancouver, 2002. (130) Marfey, P. Carlsberg Research Communications 1984, 49, 591 -596. (131) Williams, D. E.; Craig, M.; Holmes, C. F. B.; Andersen, R. J . Journal of Natural Products 1996, 59, 570-575. (132) Tugyi, R.; Uray, K.; Ivan, D.; Fellinger, E.; Perkins, A.; Hudecz, F. Proceedings of the National Academy of Sciences of the United States of America 2005, 702,413-418. (133) Ahmed, S. A.; Gogal, R. M. J . ; Walsh, J . E. Journal of Immunological Methods 1994, 7 70, 211 -224. (134) Hill, A. Natural Product Reports 2006, 23, 256-320. (135) Attard, G.; Greystoke, A.; Kaye, S.; De Bono, J . Pathologie Biologie 2006, 54, 72-84. (136) Pellegrini, F.; Budman, D. R. Cancer Investigation 2005, 23, 264-273. 181 (137) Jordan, M. A. Current Medicinal Chemistry - Anti-Cancer Agents 2002,2,1-17. (138) Wani, M. C ; Taylor, H. L ; Wall, M. E.; Coggon, P.; McPhail, A. T. Journal of the American Chemical Society 1971, 93, 2325-2327. (139) Ring, A. E.; Ellis, P. A. Cancer Treatment Reviews 2005, 31, 618-627. (140) Noble, R. L ; Beer, C. T.; Cutts, J . H. Biochemical Pharmacology 1958, 1, 347-348. (141) Johnson, I. S.; Wright, H. F.; Svoboda, G. H. Journal of Laboratory and Clinical Medicine 1959, 54, 830-837. (142) Bensch, K. G.; Malawista, S. E. Journal of Cell Biology1969, 40, 95-107. (143) Jordan, M. A.; Thrower, D.; Wilson, L. Journal of Cell Science 1992, 702,401-416. (144) Gunasekera, S. P.; Gunasekera, M.; Longley, R. E. Journal of Organic Chemistry 1990, 55, 4912-4915. (145) Hung, D. T.; Nerenberg, J . B.; Schreiber, S. L. Chemistry & Biology 1994, 1, 67-71. (146) Hung, D. T.; Nerenberg, J . B.; Schreiber, S. L. Journal of the American Chemical Society 1996, 7 75, 11054-11080. (147) Kalesse, M. ChemBioChem 2000, 7, 171-175. (148) Crews, P.; Gerwick, W. H.; Schmitz, F.; Bair, K. W.; Wright, A. E.; Hallock, Y. Pharmaceutical Biology 2003, 47 (Supplement), 39-52. (149) Lindel, T.; Jensen, P.; Fenical, W.; Long, B. H.; Casazza, A. M.; Carboni, J . ; Fairchild, C. R. Journal of the American Chemical Society 1997', 119, 8744-8745. (150) Cinel, B.; Roberge, M.; Behrisch, H.; Ofwegen, L. v.; Castro, C. B.; Andersen, R. J . Organic Letters 2000, 2, 257-260. (151) Ojima, I.; Chakravarty, S.; Inoue, T.; Lin, S.; He, L.; Horwitz, S. B.; Kuduk, S. D.; Danishefsky, S. J . Proceedings of the National Academy of Sciences of the United States of America 1999, 96, 4256-4261. (152) Faulkner, D. J . Antonie van Leeuwenhoek 2000, 77, 135-145. (153) Pettit, G. R.; Kamano, Y.; Herald, C. L.; Tuinman, A. A.; Boettner, F. E.; Kizu, H.; Schmidt, J . M.; Baczynskyj, L.; Tomer, K. B.; Bontems, R. J . Journal of the American Chemical Society 1987, 709, 6883-6885. (154) Pettit, G. R.; Singh, S. B.; Hogan, F.; Lloyd-Williams, P.; Herald, D. L.; Burkett, D. D.; Clewlow, P. J . Journal of the American Chemical Society 1989, 7 7 7, 5463-5465. (155) Talpir, R.; Benayahu, Y.; Kashman, Y.; Pannell, L.; Schleyer, M. Tetrahedron Letters 1994, 35. (156) Coleman, J . E.; De Silva, D.; Kong, F.; Andersen, R. Tetrahedron 1995, 57, 10653-10662. (157) Loganzo, F.; Discafani, C ; Annable, T.; Beyer, C ; Musto, S.; Hari, M.; Tan, X.; Hardy, C ; Hernandez, R.; Baxter, M.; Singanallore, T.; Khafizova, G. ; Poruchynsky, M.; Fojo, T.; Nieman, J . A.; Ayral-Kaloustian, S.; Zask, A.; Andersen, R.; Greenberger, L. Cancer Research 2003, 63, 1838-1845. 182 (158) Nieman, J . A.; Coleman, J . E.; Wallace, D. J . ; Piers, E.; Lim, L. Y.; Roberge, M.; Andersen, R. J . Journal of Natural Products 2003, 66, 183-199. (159) Bai, R.; Pettit, G. R.; Hamel, E. Journal of Biological Chemistry 1990, 265, 17141-17149. (160) Anderson, H. J . ; Coleman, J . E.; Andersen, R. J . ; Roberge, M. Cancer Chemotherapy and Pharmacology 1997, 39, 223-226. (161) Mitra, A.; Sept, D. Biochemistry 2004, 43, 13955-13962. (162) Margolin, K.; Longmate, J . ; Synold, T. W.; Gandara, D. R.; Weber, J . ; Gonzalez, R. Investigational New Drugs 2001, 19, 335-340. (163) Vaishampayan, U.; Glode, M.; Du, W.; Kraft, A.; Hudes, G.; Wright, J . Clinical Cancer Research 2000, 6, 4205-4208. (164) Ireland, C ; Aalbersberg, W.; Andersen, R.; Ayral-Kaloustian, S.; Berlinck, R.; Bernan, V.; Carter, G.; Churchill, A.; Clardy, J . ; Concepcion, G.; De Silva, D.; Discafani, C ; Fojo, T.; Frost, P.; Gibson, D.; Greenberger, L ; Greenstein, M.; Harper, M. K.; Mallon, R.; Loganzo, F.; Nunes, M.; Poruchynsky, M.; Zask, A. Pharmaceutical Biology (Lisse, Netherlands) 2003, 41, 15-38. (165) Pettit, G. R.; Cichacz, Z. A.; Gao, F.; Herald, C. L ; Boyd, M. R.; Schmidt, J . M.; Hooper, J . N. A. Journal of Organic Chemistry1993, 58, 1302-1304. (166) Kobayashi, M.; Aoki, S.; Sakai, H.; Kihara, N.; Sasaki, T.; Kitagawa, I. Tetrahedron Letters 1993, 34, 2795-2798. (167) Bai, R.; Taylor, G. F.; Cichacz, Z. A.; Herald, C. L ; Kepler, J . A.; Pettit, G. R.; Hamel, E. Biochemistry 1995, 34, 9714-9721. (168) Uckun, F. M.; Mao, C ; Jan, S.-T.; Huang, H.; Vassilev, A. O.; Navara, C. S.; Narla, R. K. Current Pharmaceutical Design 2001, 7, 1291-1296. (169) Tachibana, K.; Scheuer, P. J . ; Tsukitani, Y.; Kikuchi, H.; Van Engen, D.; Clardy, J . ; Gopichand, Y.; Schmitz, F. J . Journal of the American Chemical Society 1981, 103, 2469-2471. (170) Dounay, A. B.; Forsyth, C. J . Current Medicinal Chemistry'2002, 9, 1939-1980. (171) Bialojan, C ; Takai, A. Biochemistry Journal 1988, 256, 283-290. (172) Wright, J . L C ; Hu, T.; McLachlan, J . L ; Needham, J . ; Walter, J . A. Journal of the American Chemical Society 1996, 118, 8757-8758. (173) Norte, M.; Padilla, A.; Fernandez, J . J . Tetrahedron Letters 1994, 35, 1441-1444. (174) Cohen, P. T. W. Trends in Biochemical Science 1997, 22, 245-251. (175) Honkanen, R. E.; Golden, T. Current Medicinal Chemistry 2002, 9, 2055-2075. (176) McClusky, A.; Sim, A. T. R.; Sakoff, J . A. Journal of Medicinal Chemistry 2002, 45, 1151 -1175. (177) Millward, T. A.; Zolnierowicz, S.; Hemmings, B. A. Trends in Biochemical Science 1999, 24, 186-191. (178) Wera, S.; Hemmings, B. A. Biochemistry Journal 1995, 311, 17-29. (179) Williams, D. E.; Roberge, M.; Van Soest, R.; Andersen, R. J . Journal of the American Chemical Society 2003, 125, 5296-5297. 183 (180) Williams, D. E.; Lapawa, M.; Feng, X.; Tarling, T.; Roberge, M.; Andersen, R. J . Organic Letters 2004, 6, 2607-2610. (181) Pan, Y.; De Brabander, J . K. Synlett2006, 6, 853-856. (182) Paterson, I.; Anderson, E. A.; Dalby, S. M. Synthesis 2005, 19, 3225-3228. (183) Paterson, I.; Anderson, E. A.; Dalby, S. M.; Loiseleur, O. Organic Letters 2005, 7, 4121-4124. (184) Paterson, I.; Anderson, E. A.; Dalby, S. M.; Loiseleur, O. Organic Letters 2005, 7, 4125-4128. (185) Furstner, A.; Fenster, M. D. B.; Fasching, B.; Godbout, C ; Radkowski, K. Angewandte Chemie, International Edition 2006, 45, 5506-5510. (186) Furstner, A.; Fenster, M. D. B.; Fasching, B.; Godbout, C ; Radkowski, K. Angewandte Chemie, International Edition 2006, 45, 5510-5515. (187) Warabi, K.; Williams, D. E.; Patrick, B. O.; Roberge, M.; Andersen, R. J . Journal of the American Chemical Society 2007, 129, 508-509. (188) Karplus, M. Journal of the American Chemical Society 1963, 85, 2870-2871. (189) Fankhauser, M. In Cannabis and Cannabinoids: Pharmacology, Toxicology, and Therapeutic Potential; Grotenhermen, F., Russo, E., Eds.; Haworth Integrative Healing Press: New York, 2002, pp 37-51. (190) Pertwee, R. G. British Journal of Pharmacology 2006, 747, S163-S 1 7 1 . (191) Mechoulam, R.; Hanus, L. Chemistry and Physics of Lipids 2000, 108, 1-13. (192) Wood, T. B. Journal of the Chemical Society 1899, 75, 20-36. (193) Adams, R. Harvey Lectures Ser. 1941-1942, 37, 168-197. (194) Mechoulam, R.; Shvo, Y. Tetrahedron 1963, 19, 2073-2078. (195) Gaoni, Y.; Mechoulam, R. Journal of the American Chemical Society 1964, 86, 1646-1647. (196) Turner, C. E.; El Sohly, M. A.; Boeren, E. G. Journal of Natural Products 1980, 43, 169-234. (197) El Sohly, M. A. In Cannabis and Cannabinoids: Pharmacology, Toxicology, and Therapeutic Potential; Grotenhermen, F., Russo, E., Eds.; Haworth Integrative Healing Press: New York, 2002, pp 27-36. (198) Di Marzo, V.; Pop, E. In Cannabinoids; Di Marzo, V., Ed.; Kluwer Academic / Plenum Publishers: New York, 2004, pp 1-7. (199) Pop, E. In Cannabinoids; Di Marzo, V., Ed.; Kluwer Academic: New York, 2004, pp 8-31. (200) Pertwee, R. G. In Cannabinoids; Di Marzo, V., Ed.; Kluwer Academic: New York, 2004, pp 32-83. (201) Matsuda, L. A.; Lolait, S. J . ; Brownstein, M. J . ; Young, A. C ; Bonner, T. I. Nature 1990, 346, 561-564. (202) Pertwee, R. G. Pharmacology & Therapeutics 1997, 74, 129-180. (203) Howlett, A. C ; Shim, J. -Y. In Cannabinoids; Di Marzo, V., Ed.; Kluwer Academic: New York, 2004, pp 84-97. 184 (204) Hewlett, A. C ; Barth, F.; Bonner, T. I.; Cabral, G.; Casellas, P.; Devane, W. A.; Felder, C. C ; Herkenham, M.; Mackie, K.; Martin, B. R.; Mechoulam, R.; Pertwee, R. G. Pharmacology Review 2002, 54, 161-202. (205) Onaivi, E. S.; Ishiguro, H.; Zhang, P. W.; Lin, Z.; Akinshola, B. E.; Leonard, C. M.; Chirwa, S. S.; Gong, J . ; Uhl, G. R. In Endocannabinoids: The Brain and Body's Marijuana and Beyond; Onaivi, E. S., Sugiura, T., Di Marzo, V., Eds.; C R C Taylor & Francis: New York, 2006. (206) Demuth, D. G.; Molleman, A. Life Sciences 2006, 78, 549-563. (207) Devane, W. A.; Dysarz, F. A.; Johnson, M. R.; Melvin, L S.; Howlett, A. C. Molecular Pharmacology 34, 605-613. (208) Song, Z. H.; Bonner, T. I. Molecular Pharmacology 1996, 49, 891-896. (209) Devane, W. A.; Hanus, L.; Breuer, A.; Pertwee, R. G.; Stevenson, L. A.; Griffin, G.; Gibson, D.; Mandelbaum, A.; Etinger, A.; Mechoulam, R. Science 1992, 258, 1946-1949. (210) Rinaldi-Carmona, M.; Barth, F.; Heaulme, M.; Shire, D.; Calandra, B.; Congy, C ; Martinez, S.; Maruani, J . ; Neliat, G.; Caput, D.; Ferrara, P.; Soubrie, P.; Breliere, J . C ; LeFur, G. FEBS Letters 1994, 350, 240-244. (211) Rinaldi-Carmona, M.; Barth, F.; Millan, J . ; Derocq, J . -M . ; Casellas, P.; Congy, C ; Oustric, D.; Sarran, M.; Bouaboula, M.; Calandra, B.; Portier, M.; Shire, D.; Breliere, J . O ; LeFur, G. Journal of Pharmacology and Experimental Therapeutics 1998, 284, 644-650. (212) Bouaboula, M.; Perrachon, S.; Milligan, L.; Canat, X.; Rinaldi-Carmona, M.; Portier, M.; Barth, F.; Calandra, B.; Pecceu, F.; Lupker, J . ; Maffrand, J . -P . ; LeFur, G.; Casellas, P. Journal of Biological Chemistry 1997, 272, 22330-22339. (213) Eissenstat, M. A.; Bell, M. R.; DAmbra, T. E.; Alexander, E. J . ; SDaum, S. J . ; Ackerman, J . H.; Gruett, M. D.; Kumar, V.; Estep, K. G.; Olefirowicz, E. M.; Wetzel, J . R.; Alexander, M. D.; Weaver, J . D.; Haycock, D. A.; Luttinger, D. A.; Casiano, F. M.; Chippari, S. M.; Kuster, J . E.; Stevenson, J . I.; Ward, S. J . Journal of Medicinal Chemistry 1995, 38, 3094-3105. (214) Christopoulos, A.; Coles, P.; Lay, L.; Lew, M. J . ; Angus, J . A. British Journal of Pharmacology 2001, 132, 1281-1291. (215) Ross, R. A.; Gibson, T. M.; Stevenson, L. A.; Saha, B.; Crocker, P.; Razdan, R. K.; Pertwee, R. G. British Journal of Pharmacology 1999, 128, 735-743. (216) Price, M. R.; Baillie, G. L.; Thomas, A.; Stevenson, L. A.; Easson, M.; Goodwin, R.; McLean, A.; Mcintosh, L.; Goodwin, G.; Walker, G.; Westwood, P.; Marrs, J . ; Thomson, F.; Cowley, P.; Christopoulos, A.; Pertwee, R. G.; Ross, R. A. Molecular Pharmacology 2005, 68, 1484-1495. (217) Pertwee, R. G. Life Sciences 2005, 76, 1307-1324. (218) Pertwee, R. G. International Journal of Obesity 2006, 30, S13-S18. (219) Knight, P. J . K.; Grigliatti, T. A. Journal of Receptors and Signal Transduction 2004, 24, 241-256. (220) Knight, P. J . K.; Grigliatti, T. A. Archives of Insect Biochemistry and Physiology 2004, 57, 142-150. 185 (221) Knight, P. J . K.; Pfeifer, T. A.; Grigliatti, T. A. Analytical Biochemistry 2003, 320, 88-103. (222) Santalova, E. A.; Makarieva, T. N.; Gorshkova, I. A.; Dmitrenok, A. S.; Krasokhin, V. B.; Stonik, V. A. Biochemical Systematics and Ecology 2004, 32, 153-167. (223) Eggersdorfer, M. L ; Kokke, W. C. M. C ; Crandell, C. W.; Hochlowski, J . E.; Djerassi, C. Journal of Organic Chemistry 1982, 47, 5304-5309. (224) Bohlin, L ; Sjostrand, U.; Sodano, G.; Djerassi, C. Journal of Organic Chemistry 1982, 47, 5309-5314. (225) Malik, S.; Djerassi, C. Steroids 1989, 53, 271-284. (226) De Rosa, M.; Minale, L ; Sodano, G. Experientia 1975, 31, 408-410. (227) De Rosa, M.; Minale, L ; Sodano, G. Experientia 1976, 32, 1112-1113. (228) Quinoa, E.; Adamczeski, M.; Crews, P. Journal of Organic Chemistry 1986, 51, 4497-4498. (229) Nishimura, S.; Matsunaga, S.; Shibazaki, M.; Suzuki, K.; Furihata, K.; Van Soest, R. W. M.; Fusetani, N. Organic Letters 2003, 5, 2255-2257. (230) Quinoa, E.; Adamczeski, M.; Crews, P.; Bakus, G. J . Journal of Organic Chemistry 1986, 51, 4494-4497. (231) Towbin, H.; Bair, K. W.; DeCaprio, J . A.; Eck, M. J . ; Kim, S.; Kinder, F. R.; Morollo, A.; Mueller, D. R.; Schindler, P.; Song, H. K.; van Oostrum, J . ; Versace, R. W.; Voshol, H.; Wood, J . ; Zabludoff, S.; Phillips, P. E. Journal of Biological Chemistry 2003, 278, 52964-52871. (232) Thale, Z.; Kinder, F. R.; Bair, K. W.; Bontempo, J . ; Czuchta, A. M.; Versace, R. W.; Phillips, P. E.; Sanders, M. L ; Wattanasin, S.; Crews, P. Journal of Organic Chemistry 2001, 66, 1733-1741. (233) Crews, P.; Gerwick, W. H.; Schmitz, F.; Bair, K. W.; Wright, A. E.; Hallock, Y. Pharmaceutical Biology 2003, 41 (Supplement), 39-52. 186 Appendix I R O E S Y spectrum of tauramamide methyl ester (2.14), recorded at 500 MHz DMSO-cfe, showing inter-residue correlations o o o o cj tb cd 187 oo oo File Name Creation Date/Time File Type File Source File Title Operator Instrument C:\Maspec\Data\SL-12319(2).ms2 8/14/2003 at 11:32:22 La-Res data (Magnet) Acquired on MASPEC II system [1132/1199] SL-12319 in thioglycerol UBC MS CONCEPT IIHQ SCAN GRAPH. Flagging=Low Resolution M/z. Highlighting=Base Peak. <„„ Scan 89#8:44 - 98#9:37. Entries=208. Base M/z=237. 100% lnt.=3.20461. 100T 2 1 4 90-j 80 70 60-| 50-40-30-20-10-0 £• 50 100-90 80 70 60 50 40 30 20-10-0 105 197 136.1 237 295 321 )L|.lllIL4.l.!Ulll.l|._|il]l: 433 -14 In l.iil, 200 250 300 ^ 1 ' 1 ' I 350 400 878.2 I ' | 1 I 1 I 450 ' | 1 I ' I 500 550 600 650 700 i ' 'A ( .5, ' 750 800 Low Resolution M/z 910.2 998.2 I 1 I 1 : • I 850 900 950 1000 r~ > 0)-D co Q . CD _ O = CU —* CU CL CD CD .—t-zr CD CO I—I-CD p i 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0228812/manifest

Comment

Related Items