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Chemical investigation of the antarctic marine invertebrates _synoicum adareanum_ and _artemisina plumosa_

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Title:
Chemical investigation of the antarctic marine invertebrates _synoicum adareanum_ and _artemisina plumosa_
Physical Description:
Book
Language:
English
Creator:
Noguez, Jaime
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla
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Subjects

Subjects / Keywords:
Organic chemistry
Natural producgts
Bioassay
Tunicate
Sponge
Dissertations, Academic -- Chemistry -- Doctoral -- USF   ( lcsh )
Genre:
non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: Chemical Investigation of the Antarctic Marine Invertebrates Synoicum adareanum and Artemisina plumosa Despite the lack of attention that marine organisms have received in comparison to terrestrial organisms, marine life have recently been found to represent a valuable source for novel bioactive compounds. Cold water marine habitats are home to a plethora of organisms that have the ability to produce secondary metabolites that exhibit a great deal of diversity in both their chemical structures and biological activities. The chemical investigation of these unique and relatively unstudied ecosystems is necessary to gain insight into the dynamics between predators and prey, while also making a significant impact in the field of drug discovery. This dissertation reports a small portion of the progress made in our laboratory towards the exploration of Antarctic marine organisms and includes a detailed account of the chemical investigation of the Antarctic marine invertebrates Synoicum adareanum and Artemisina plumosa. These circumpolar organisms have recently been found to produce a number of secondary metabolites and have since been evaluated for their biological activities against Leishmania parasites, melanoma cells, and mammalian vacuolar ATPase. The results of this research and their implications towards future research endeavors will be discussed in the upcoming chapters.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2010.
Bibliography:
Includes bibliographical references.
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Mode of access: World Wide Web.
System Details:
System requirements: World Wide Web browser and PDF reader.
Statement of Responsibility:
by Jaime Noguez.
General Note:
Title from PDF of title page.
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Document formatted into pages; contains X pages.
General Note:
Includes vita.

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ABSTRACT: Chemical Investigation of the Antarctic Marine Invertebrates Synoicum adareanum and Artemisina plumosa Despite the lack of attention that marine organisms have received in comparison to terrestrial organisms, marine life have recently been found to represent a valuable source for novel bioactive compounds. Cold water marine habitats are home to a plethora of organisms that have the ability to produce secondary metabolites that exhibit a great deal of diversity in both their chemical structures and biological activities. The chemical investigation of these unique and relatively unstudied ecosystems is necessary to gain insight into the dynamics between predators and prey, while also making a significant impact in the field of drug discovery. This dissertation reports a small portion of the progress made in our laboratory towards the exploration of Antarctic marine organisms and includes a detailed account of the chemical investigation of the Antarctic marine invertebrates Synoicum adareanum and Artemisina plumosa. These circumpolar organisms have recently been found to produce a number of secondary metabolites and have since been evaluated for their biological activities against Leishmania parasites, melanoma cells, and mammalian vacuolar ATPase. The results of this research and their implications towards future research endeavors will be discussed in the upcoming chapters.
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Chemical Investigation of the Antarctic Marine Invertebrates Synoicum adareanum and Artemisina plumosa by Jaime Heimbegner Noguez A dissertation submitted in partial fulfillment Of the requirements for the degree of Doctor of Philosophy Department of Chemistry College of Arts and Sciences University of South Florida Major Professor: Bill Baker, Ph.D. Edward Turos, Ph.D. Roman Manetsch, Ph.D. Abdul Malik, Ph.D. Date of Approval: March 26, 2010 Keywords: organic chemistry, natural products, bioassay, tu nicate, sponge Copyright 2010, Jaime Heimbegner Noguez

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Dedication This dissertation is dedicated to my hus band, Emilio, who stood beside me every step of the way. His strength and love helped get me through the hard times and made the good times even more precious. I would also like to dedicate this work to my parents for helping me to realize my dream and providing me with the means to make it come true. Without their love, support, and firm belief in the importance of education this work would not have been possible. And last but certainly not least, I would like to dedicate this work to my sisters and friends, who always provided a sympathetic ear or much needed laugh, and helped me to keep things in perspective when life seemed overwhelming.

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Acknowledgments First and foremost, I must thank my advi sor Dr. Bill Baker, for allowing me to be a part of his incredible res earch program. He has always inspired me, challenged me, and perhaps most importantly helped me to re alize my potential. Without his support and guidance this dissertation woul d hardly be possible. I thank the Florida Center of Excellence for Biomolecular Identification a nd Targeted Therapeutics for awarding me with a Thrust scholarship to help fund some of this research. I would also like to than k my committee members for their encouragement and taking the time to help mold me from a young woman with big dreams into a successful scientist with the tools to achieve them. Th anks to Dr. Dennis Kyle and the members of his laboratory as well as Leigh West for their assistance with the leishmania and cytotoxicity assays. I am grateful for your patience and teaching me the molecular biology techniques pertinent to my researc h. I owe many thanks to Dr. Xie and the members of his laboratory at UT Southwestern Medical Ce nter for testing our samples for v-ATPase activity. And to Dr. Edwin Rivera for his help over the years with the NMR data acquisition that my rese arch relied heavily on. And finally, I must thank all of the gr aduate students, particularly the other members of the Baker lab, for making my time at the University of South Florida so enjoyable. Whether it be sharing jokes and f unny stories to help the hours pass in the lab or drinks after a long week of work, you always found a way to remind me of how fortunate I was to be surrounded by such a great group of people.

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i Table of Contents List of Figures iv List of Tables vi List of Schemes vii List of Abbreviations viii Abstract x Chapter One. Natural Products as Drug Leads 1.1 Natural Products as Therapeutic Agents 1 1.2 The Impact of Natural Products on the Pharmaceutical Industry 2 1.3 Drugs from the Sea 8 1.4 Cold Water Chemistry 13 1.5 Research Objectives 18 Chapter Two. Chemical Investigation of the Antarctic Tunicate Synoicum adareanum 2.1 Introduction 2.1.1 The Chemistry of Cold Water Tunicates 19 2.1.2 Secondary Metabolites from the Synoicum genus 24 2.2 Chemical Investigation of the Antarctic Tunicate Synoicum adareanum 27 2.2.1 The Palmerolides 28 2.2.2 Ring System A Palmerolides 29

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ii 2.2.2.1 Stereochemical Assignment of Palmerolides D-G 34 2.2.3 Ring System B Palmerolides 38 2.2.3.1 Stereochemical Assignment of Palmerolide B and H 41 2.2.4 Ring System C Palmerolides 46 2.2.4.1 Stereochemical A ssignment of Palmerolides C and K 49 2.3 Structure Activity Relationship Studies of Palmerolide A 2.3.1 Bioactivity of the Palmerolides 52 2.3.2 Structure Activity Relationship Studies of Palmerolide A via Synthesis 56 2.3.3 Structure Activity Relationship Studies of Palmerolide A via Derivatization 2.3.3.1 Preparation of Palmerolide A Analogs 58 2.3.3.2 Biological Evaluation of Palmerolide A Analogs 63 Chapter Three. Further Investigatio n into the Chemical Composition of S.adareanum 3.1 Introduction to Glycosphingolipids 67 3.2 Isolation of Glycosphingolipids from S. adareanum 68 3.3 Structure Elucidation of Glycosphingolipids from S. adareanum 71 Chapter Four. Chemical Investigation of the Antarctic, Orange Encrusting Sponge Artemisina plumosa 4.1 Introduction to Leishmania 79 4.2 Bioassay-guided fractionation of Artemisina plumosa 82

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iii Chapter Five. Conclusion 88 Chapter Six. Experimental 90 6.1 General Experimental Procedures 90 6.2 Biological Material 91 6.3 Extraction of S.adareanum and Isolation of Secondary Metabolites 91 6.4 Acetylation of Glycosphingolipids from S. adareanum 94 6.5 Preparation of Palmerolide A Analogs 95 6.5.1 Preparation of 107 95 6.5.2 Preparation of 108, 109, 110 96 6.5.3 Preparation of 111 and 112 99 6.7 Cytotoxicity Assay 101 6.8 Leishmania Assay 102 List of References 103 Appendices 118 Appendix A: NMR data tables 119 Appendix B: Selected 1D and 2D NMR data 125 Appendix C: Mass Spectral Data 242 Appendix D: Bioassay Data 254 About the Author End Page

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iv List of Figures Figure 1. Synoicum adareanum collected at Palmer Station, Antarctica 28 Figure 2. Comparison of palmerolide D and palmerolide A 13C NMR chemical shifts 31 Figure 3. Comparison of palmerolide E and palmerolide A 13C NMR chemical shifts 32 Figure 4. Comparison of palmerolide F and palmerolide A 13C NMR chemical shifts 33 Figure 5. Comparison of palmerolide G and palmerolide A 13C NMR chemical shifts 33 Figure 6. Moshers depiction of th e MTPA plane of an MTPA ester 34 Figure 7. Palmerolide F ( R ) MTPA diester in d6-DMSO 35 Figure 8. Palmerolide F ( S) MTPA diester in d6-DMSO 36 Figure 9. Comparison of palmerolide B and palmerolide A 13C NMR chemical shifts 40 Figure 10. Comparison of palmerolide B and palmerolide H 13C NMR chemical shifts 40 Figure 11. Comparison of palmerolide H and palmerolide D 13C NMR chemical shifts 40 Figure 12. Newman projection of palm erolide B C-7/C-8 stereocenters 44

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v Figure 13. Comparison of palmerolide C and palmerolide A 13C NMR chemical shifts 47 Figure 14. Comparison of palmerolide Cand palmerolide K 13C NMR chemical shifts 48 Figure 15. Comparison of palmerolide K and palmerolide E 13C NMR chemical shifts 48 Figure 16. Newman projection of palmerolide C C-8/C-9 and C-9/C-10 stereocenters 50 Figure 17. 1H NMR spectrum of palmerolide A hydrogenation product 58 Figure 18. 1H NMR spectrum of palmerolide A C-7 p-bromobenzoate 59 Figure 19. 1H NMR spectrum of palmerolide A C-10 p-bromobenzoate 60 Figure 20. 1H NMR spectrum of palmerolide A C-7/C-10 p-bromobenzoates 60 Figure 21. 1H NMR spectrum of palmerolide A C-11 alcohol 62 Figure 22. 1H NMR spectrum of palmerolide A C-3 alcohol 62 Figure 23. Basic structural un its of glycosphingolipids 67 Figure 24. 1H NMR spectrum of glycosphingolipid 113a in d6-DMSO 71 Figure 25. Comparison of -galactopyranoside and -glucopyranoside 74 Figure 26. The most characteristic frag ment ions in the APCI-MS data of 113a 74 Figure 27. 1H NMR spectrum of glycosphingolipd 114a-b in d6-DMSO 77 Figure 28. 1H NMR spectrum of ster oid series 1 in CDCl3 83 Figure 29. 1H NMR spectrum of ster oid series 2 in CDCl3 83 Figure 30. 1H NMR spectrum of ster oid series 3 in CDCl3 84 Figure 31. Steroid nuclei and side chains found in Artemisina plumosa 86

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vi List of Tables Table 1. Stereochemical analysis ( ) of palmerolides A-G using Moshers method 36 Table 2. 3J H,H (Hz) analysis of key palm erolide stereocenters in palmerolides A-G 38 Table 3. 3J H,H (Hz) analysis of key palm erolide stereocenters in palmerolides A, B, H 41 Table 4. 3J H,H and 2,3J C,H (Hz) values for anti and gauche orientations in acyclic systems 42 Table 5. 3J H,H and 2,3J C,H (Hz) values for palmerolides B and H 43 Table 6. Stereochemical analysis ( ) of palmerolide B us ing Moshers method 45 Table 7. 3J H,H (Hz) analysis of key palm erolde stereocenters in palmerolides A and C 48 Table 8. 3J H,H and 2,3J C,H (Hz) values for palmerolide C 49 Table 9. Stereochemical analysis ( ) of palmerolide C us ing Moshers method 50 Table 10. 3J H,H (Hz) analysis of key palm erolde stereocenters in palmerolides C and K 50 Table 11. Bioactivity data for the palmerolides 52 Table 12. Bioactivity data for palmerolide A analogs 61 Table 13. Calculated coupling constants of sugar protons 71

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vii List of Schemes Scheme 1. The Extraction of Synoicum adareanum 69

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viii List of Abbreviations Ac2O acetic anhydride [ ] specific rotation = 100 / lc CDCl3 deuterated chloroform CD3OD deuterated methanol CH2Cl2 dichloromethane C-18 octadecyl bonded silica chemical shifts (NMR) DEPT distortionless enhancement by polarization transfer DMAP dimethylamino pyridine d6-DMSO deuterated dimethylsulfoxide EtOAc ethylacetate EtOH ethanol the molar extinction coefficient in UV spectroscopy gCOSY gradient correla tion spectroscopy (NMR) gHMQC gradient heteronuclear multiple quantum coherence (NMR) gHMBC gradient heteronuclear multiple bond connectivity (NMR) gHSQC gradient heteronuclear si ngle quantum correlation (NMR) gHSQMBC gradient heteronuclear single quantum multiple bond correlation (NMR) H2O water

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ix HR ESIMS high resolution electros pray ionization mass spectrometry HPLC high performance liquid chromatography IC50 inhibitory concentration for half of the population IR infrared J coupling constant nJC,H n-bond hydrogen to carbon correl ation (n = 2,3 or 4) nJH,H n-bond hydrogen to hydrogen correlation (n = 2,3 or 4) LR ESIMS low resolution electrospray ionization mass spectrometry LR APCIMS low resolution atmospheric pressu re chemical ionization mass spectrometry max the wavelength at which maximum absorption occurs MeCN acetonitrile MeOH methanol MgSO4 magnesium sulfate MS mass spectrometry MTPA-Cl -methoxy-(trifluoromethyl)phe nylacetyl chloride m/z mass/charge ratio in mass spectrometry NaOMe sodium methoxide NMR nuclear magnetic resonance Pal palmerolide Pd palladium ROESY rotating-frame overhauser enhancement spectroscopy (NMR) SRB sulforhodamine B UV ultraviolet

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x Chemical Investigation of the Antarctic Marine Invertebrates Synoicum adareanum and Artemisina plumosa Jaime Heimbegner Noguez ABSTRACT Of the small percentage of organisms chemically investigated over the years as potential sources of natural products, much less is known about those from the marine realm. Despite the lack of attention they have received in comparison to terrestrial organisms, marine life have recently been found to represent a valuable source for novel bioactive compounds. Cold water marine habitats are home to a plethora of organisms that have the ability to produce secondary metabolites that exhibit a great deal of diversity in both their chemical structures and biological activities. The chemical investigation of these unique and relatively unstudied ecosystems is necessary to gain insight into the dynami cs between predators and prey, while also making a significant impact in the field of drug discovery. Our laboratory has focused on the chemical investigation of invertebrate s from the waters of Antarctica in search of bioactive secondary metabolites that can be used for the treatment of human pathogens. This dissertation reports a sm all portion of the progress made in our laboratory towards the exploration of Antarc tic marine invertebra tes. The chemical investigation of the circumpolar colonial tunicate Synoicum adareanum and the orange, encrusting sponge Artemisina plumosa will be discussed in detail in the following chapters.

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CHAPTER ONE: Natural Products as Drug Leads 1.1 Natural Products as Therapeutic Agents Although a significant amount of research has been done in an effort to better understand nature only a small fr action of the worlds diversity has been explored to date in terms of bioactivity and drug potential. Of the 250,000 speci es of higher plants that exist approximately 5-10% of them have been chemically investigated,1 along with an even lower percentage of the 200,000+ inverteb rate and algal species contained in the worlds oceans.2 Investigation of these organisms ha s led to the isolation and structure elucidation of bioactive sec ondary metabolites that can be used for the treatment of a number of human ailments.3 The use of natural products, typically plan ts, as a source of therapeutic agents has continued for thousands of years and their uses have evolved from traditional medicines into modern drugs. One of the best examples of this is the evolution of Aspirin into the most widely used medication in the world. The therapeutic use of willow bark dates back more than a thousand years to as early as 400 BC and was ch ewed on or steeped into a tea in order to reduce fever, inflammation, and pain. In the early 1800s the active ingredient, salicylic acid ( 1 ), derived from the metabolism of salicin ( 2), was isolated and by the mid 1800s derivatization of the compound had begun.4 By the late 1800s the synthesis of a more 1

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stable and less-irritating fo rm of the natural product, acetyl salicylic acid ( 3), had been achieved by acetylation and was marketed as Aspirin by Bayer in 1899.3 Aspirin is still being used today to as an analgesic, anti-pyretic, a nd anti-inflammatory agent but since its discovery it has also been found to be an effective preven tative treatment against heart attacks and strokes.3 OH O HO HO O OH OH O OH OH O O O HO 1 2 3 1.2 The Impact of Natural Produc ts on the Pharmaceutical Industry In 2000, approximately 60% of the drugs in clinical trials against cancer were of natural origins.1 And in 2001, eight of the thirty top selling drugs were natural products or their derivatives and they totaled $16 billion in sales.1 Furthermore, a survey made by the National Cancer Institute in 2003 revealed that 61% of the 877 small-molecule chemical entities introduced as drugs worldwide during 19812002 could be traced to or were inspired by natural products.5 The 61% was further broken down to include: natural products (6%) and their deriva tives (27%), synthetic comp ounds with pharmacophores derived from natural products (5%), and synthetic compounds designed to mimic a natural product (23%).4 These statistics make it clear that natural products have undeniably played an important role in our pursuit of the discovery of new drugs. The inherent characteristics of natura l products as being relatively small (<2000 Da) and offering incomparable structural divers ity ensures that they will continue to be 2

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considered one of the major sources of ne w drugs in the future. For years, natural products have been inspiring chemists with th eir rich structural diversity and complexity proving that even the most creative scientis t cannot outdo nature. The endiyne antibiotic calicheamicin6 ( 4) derived from the bacteria Micromonospora echinospora and the ion channel blocker zetekitoxin AB7 ( 5) isolated from the Panamanian golden frog Atelopus zeteki are prime examples of some of the unimaginably complex compounds designed by nature. OCH3 OCH3 I O OH OCH3 O HO S O O O OH O O O OH H N O HO NHCO2CH3 SSSCH3 O H3CO H3CH2CHN 4 HN N N H N NH O NHOH O H HN N O O OH OSO3H OH OH 5 In addition to their uses as drugs in their unmodified state, natural products will also contribute to the search for new drugs by acting as chemical s caffolds that can be 3

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used to synthesize more complex mol ecules and by indicating new modes of pharmacological action that allow comp lete synthesis of novel analogs.1 Pharmaceutical companies can also use the structural s caffolds of published natural products of pharmacological interest as templates for the identification of similar molecules in their existing chemical libraries, or as st arting points for chemical modification.8 A few natural products that have been used as chemical scaffolds for current drugs are compactin9 ( 6), rapamycin10 ( 7), and taxol11 ( 8). Compactin, also known as mevastatin, is a hypolipidemic agent or iginally isolated from the mold Penicillium citrinum .8 Since the compounds discovery in the 1970s a number of cholesterol-lowering derivatives have been synthesized including atorvastatin (9 ), marketed by Pfizer under the trade name Lipitor, and its competitor cerivastatin ( 10) marketed under the trade names Baycol and Lipobay by Bayer. O HO O H O O O HO OMe N O O O O H3CO OH O HO MeO O O NH OH O O O O O OH H HO O O O O 6 7 8 4

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N CO2H HO OH F NH O N O F OH CO2H HO H 9 10 The natural product rapamycin is an immunosuppressant and antifungal drug produced by the bacterium Streptomyces hygroscopicus that was originally isolated from a soil sample from Easter Island.9 Rapamycin is also known as sirolimus and is marketed under the trade name Rapamune by Wyeth as an immunosuppressant drug used to prevent rejection in organ transplantation. The syntheti c derivatives everolimus (Certican, Afinitor, 11 ) and temsirolimus (Torisel, 12 ) are being marketed as immunosuppressant agents as well by the pharmaceutical companies Novartis and Wyeth, respectively. Another sirolimus deri vative recently renamed ridaforolimus ( 13), formerly known as deforolimus, is being co-developed by Merck and ARIAD and is in phase III clinical trials for metastatic soft-tissue and bone carcinomas responsive to chemotherapy. 5

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N O O O O O HO O O O OH O O O OH 11 N O O O O O H O O OH OH O OH O O O O HO 12 6

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N O O O OH O O H O O O O P O OH O O H 13 Docetaxel ( 14) is a semi-synthetic analog of taxol ( 8) which was isolated from the rare Pacific yew tree Taxus brevifolia .10 Taxol was commercially developed by BristolMeyers Squibb as an anti-mitotic used in cancer chemotherapy, followed by the introduction of its synthetic analog docet axel by Sanofi-Aventis under the trademark Taxotere for the same treatment. The abovementioned compounds are prime examples of the pivotal role natural products play in the discovery and design of modern drugs. These drugs merely highlight some of the natu ral product scaffolds that continue to yield efficacious treatments over the years and improve the quality of life of billions of people. O NH OH O O O OH O OH H HO O O O O 14 7

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1.3 Drugs from the Sea Of the small percentage of organisms ch emically investigated over the years, much less is known about marine organisms than other sources of natural products. Despite the lack of attention that they have been given in comparison to terrestrial organisms, marine organisms have been f ound to represent a valuable source for novel bioactive compounds.1 There are well over 17,000 published structures of marine natural products and hundreds of new compounds ar e still being discovered every year.12 These bioactive metabolites are often pr oduced by soft-bodied, sessile, or slowmoving marine invertebrates th at usually lack morphological defense structures such as spicules, spines, or a protective shell.7,13 In order for these vul nerable organisms to protect themselves they have evolved the ability to synthesize thes e toxic or distasteful compounds, to sequester them from their diet or to obtain protection through symbiotic marine microorganisms.7,13 Ecological research has s hown that these bioactive compounds function as chemical weapons that ha ve evolved into highl y potent inhibitors of physiological processes in the prey, predator s, or competitors of the marine organisms that use them for defense.7,13 The wealth and variety of bioactive metabolites that are isolated from these marine organisms reflects the ecological importance of these constituents for the inverteb rates that elaborate them.14 The aforementioned chemical substances ha ve been of interest in biomedical studies since many of them have been shown to be effective in the treatment of human diseases. A few ex amples are the tunicate Ecteinascidia turbinata which yields the anticancer agent ecteinascidin 743 (ET-743) 15, 16 ( 15 ) and the cone snail Conus magus 8

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which yields the analgesic ziconotide17 ( 16). ET-743 exhibits potent activity against many soft tissue carcinomas and is being marketed by PharmaMar under the trade name Yondelis.18 The marine-derived drug is the first treatment for myxoid liposarcoma to be released on the market in 30 years. Yondelis is currently being studi ed for the treatment of prostate cancer, breast cancer, and childhood sarcomas. Ziconotide is a non-opioid drug derived from the toxin of the piscivorous marine snail Conus magus and is currently being marketed by Elan as an isotonic solution under the brand name Prialt.17, 19 Prialt is prescribed for the ameliorati on of severe chronic pain in pa tients that are resistant to common analgesics. O S HO H3CO O H O O N N CH3 OCH3 CH3 HO OAc H3C H OH NH CH3 15 9

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H2N N H H N N H H N O O O O H2N H2N N H H N O O H2N S S NH O NH H N N H H N N H H N O O O O O S OH O HO NH NH S O O O S OH HN O NH H2N NH HO NH HN N H O O O OH H N N H H N NH O O O O S N H H2N NH OH NH2 NH O S H2N O HO O 16 With a number of well established marine natural products on the drug market and an even greater number of bioactive marine meta bolites in various stages of clinical trials, the viability of the marine realm as a s ource of new drugs is undeniable. Aplidine20 ( 17 ), bryostatin 121 ( 18), dolastatin 1022 ( 19 ), and kahalalide F 23 ( 20 ) are all marine-derived drugs produced by a variety of organisms and ar e currently in the sec ond phase of clinical trials. The colonial, Mediterranean tunicate Aplidium albicans produces the cyclic depsipeptide aplidine ( 17 ), also known as dehydrodidemnin B.20 The compound was recognized as a multi-factorial apoptosis inhi bitor and was granted orphan drug status in 2004 by the United States Food and Drug Admini stration for the treatment of multiple myeloma and acute lymphoblastic leukemia.18 10

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N O O O NH NH N O N O O O N O NH O O O O OH O O 17 Bryostatin 1 ( 18) is a complex polyketide elaborated by the bryozoan Bulgula neritina found in the Gulf of California.21 This secondary metabolite was granted orphan status in 2001 for the treatment of esophageal cancer. Bryostatin 1 has been found to be a potent activator of protein kinase C (PKC) and shows promise in human clinical trials for its potentially useful synergistic acti on with other chemotherapeutic agents.24 O O H O O O O H OH O O OH O H O O O H OH HO O 18 The anti-tumor agent dolastatin 10 ( 19) was originally isolated from the sea hare Dolabella auricularia and later found in a marine cynobacterium of the species Symploca 11

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as well.19,25 Preclinical trials on dolastatin 10 reveal ed that it was a cytotoxic peptide that exhibited potent microtubule-inhibiting and apoptotic e ffects against small lung cell cancer lines and the DU-145 human prostate cancer cell line both in vivo and in vitro .26 It is currently being studied in a Phase II cl inical trial with pa tients diagnosed with hormone-refractory metastatic prostate adenocarcinoma.26 N H N N N O O O O H N O O N S 19 The cyclic depsipeptide kahalalid e F is produced by the green algae Bryopsis pennata but can be found in larger quantities in the Hawaiian sea mollusc Elysia rufescens which feeds on the algae.23 Kahalalide F is one member of a family of dehydroaminobutyric containing cyc lic peptides isolated from Elysia rufescens and shows promise as a treatme nt for prostate cancer.18 The mechanism of action of this marine metabolite is mostly unknown and it ha s been classified as a National Cancer Institute-COMPARE negative compound, indicating the cytotoxicity it exhibits is likely related to a unique mode of action.18 12

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H2N NH O N H O NH HN O O O HNO NH O H N O O NH O N O NH O HN O HO NH O NH O 20 1.4 Cold Water Chemistry It is a surprising statistic that fewer than 3% of the marine natural products reported originate from organisms collected in polar habitats. This lo w percentage can be attributed to reasons as dive rse as accessibility, the unpleasant climate, and arguments of low biodiversity. As a result, the majority of the marine organisms that have been chemically investigated over the years have been collected from tropical and temperate waters. The obvious outcome is that a grea ter number of natura l products are being reported from organisms living in warmer ha bitats, overshadowing the equal potential of organisms living in cold climates to produce such compounds. The argument of low biodiversity with implications of low chemical diversity amongst cold-water marine organism is prematur e in both aspects. In fact, recent studies of cold-water habitats such as the sea floor of the Southern Ocean suggest that predation 13

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and competition drives chemical diversity in the same manner observed for temperate climates.27,28 Psychrophiles thriving in these extreme climates have proven to be a source of chemically diverse natural products that exhibit equally diverse biological activities against human diseases.29 The bright red sponge Kirkpatrickia variolosa is an inhabitant of the Antarctic benthos and produces a series of pigments known as the variolins ( 21-24 ).30,31 These metabolites have a very unusual pyridopyrrol opyrimidine ring system that has no precedence in either terrestrial or marine natural products. The variolins exhibited potent cytotoxicity towards the P388 cell line and further studies re vealed their mechanism of action as inhibitors of cyc lin-dependent kinase (CDK).32 N N N N N H3C NH2 O OH NH2 N N N N N NH2 R NH2 N N N N N NH2 OH NH2 CH3 23 22 R=OH 24 R=H 21 Mixirins A, B, and C ( 25-27) are iturin class acylpeptides produced by the bacterium Bacillus sp. isolated from sea mud near the North Pole.33 All of the mixirins were found to be potent inhibito rs of HCT-116 human colon tumor cells.33 14

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N H H N NH NH H N NH N H N O R O H2N O O OH O O H2N O O O O H2N HO O O NH2 25 R = C12H25 26 R = C9H19 27 R = (CH2)7CH(CH3)(C2H5) An undescribed Gram-positiv e bacterium originally isol ated from a slurry of sterile seawater and sediment from a 980 m sediment core from the North Pacific was found to produce a family of unusual antiviral and cytotoxic macrocyclic lactones: macrolactins A-F ( 28 33 ), macrolactinic acid ( 34), and isomacrolactinic ( 35) acid.34 Macrolactin A was the most bioactive compound of the se ries exhibiting selective antibacterial activity, inhibition of B16F20 murine melanoma cancer cells in in vitro assays, significant inhibition of mammalian Herpes simplex viruses (types I and II), as well as protection of T-lymphoblast cells against human HIV viral replication34 and of neuronal cells.35 15

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O O HO R2O OR1 O O HO HO O O O CO2H OH OH OH O 28 R1 = R2 = H 29 R1 = -glucosyl, R2 = H 30 R1 = H, R2 = -glucosyl 31 O O HO O OH O O HO O OH 32 33 O HO HO OH OH 15 HO 34 35 15-keto-16,17-dihydro 16

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Desmarestia menziesii is an Antarctic brown alga that elaborates the quinone derivative menzoquinone (36),36 and the chromenol derivatives 37 and 38 .37 Menzoquinone was fou nd to be the most biologically relevant of the group having displayed growth inhibition of the fo llowing microbes: methicillin-resistant Staphylococcus aureus (8 mm), methicillin-sensitive Staphylococcus aureus (6 mm), and vancomycin-resistant Enterococcus faecium (7 mm).37 O O O OH 36 O OH H O 37 O HO CO2H 38 17

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These organisms depict only a small por tion of the biodiver sity and chemical diversity waiting to be explored in cold water marine habitats. Cryophiles are an overlooked and untapped natural source of th erapeutic agents and will hopefully gain more interest as viable candidates in the near future. The pot ential for finding new drugs from the sea is infinite and deserv es a second look from the pharmaceutical companies diverging from natural products chemistry. 1.5 Research Objectives Cold water marine habitats are home to a number of organisms that have the ability to produce secondary metabolites that e xhibit a great deal of diversity not only in their chemical structures but in their biological activities as well. The chemical investigation of these unique and relatively unstudied ecosystems is necessary to gain insight into the dynamics between predators and prey, while also making a significant impact in the field of drug discovery. This dissertation reports a small portion of the progress made in our research laboratory towards the chemical investigation of marine Antarctic invertebrates as well as the ecological roles they play in the Antarctic benthos. 18

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CHAPTER TWO: Chemical Investigation of the Antarctic Tunicate Synoicum adareanum 2.1 Introduction 2.1.1 The Chemistry of Cold Water Tunicates Tunicates have been studied far less than other marine invertebrates in terms of their chemistry, especially in cold-water hab itats. The minimal chemical investigation of ascidians endemic to these frigid climates can possibly be attributed to their hostile collection conditions and relative abundance in a given ecosystem. Secondary metabolites discovered from cold-water tunicate s have been diverse in both structure and bioactivity and emerge primarily from coloni al tunicates as oppos ed to solitary. A few examples of cold-water tunicat es that have been explored chemically in recent years are Aplidium meridianum, Aplidium cyaneum Aplidum glabrum Dendrodoa grossularia Clavelina lepadiformis and Eudistoma sp. The ascidian Aplidium meridianum collected from the South Atlantic (South Georgia Islands, 100 m) produces a series of brominated 3-(2-aminopyrimidine)indoles named the meridianins ( 3945).38 The meridianins were all found to prevent cell proliferation and induce cell apoptosis and th e majority of them also demonstrated 19

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inhibition of CDKs, GSK-3, PKA and other kinases in the low micromolar range.39 This series of compounds has also been isolated from an Antarctic coll ection of the related tunicate Synoicum sp. found near Palmer Station.40 N H R1 R4 R3 R2 N N H2N R1 R2 R3 R4 39 OH H H H 40 OH H Br H 41 H Br H H 42 H H Br H 43 OH H H Br 44 H Br Br H 45 H H H H Aplicyanins A-F ( 4651 ) are bromoindole derivatives isolated from Aplidium cyaneum collected by trawling at th e Weddell Sea in Antarctica.41 Cytotoxic activity against HT-29 (colon), A-549 (lung), and MDA-MB-231(breast) tumor cell lines at submicromolar concentrations was exhibited by aplicyanins B, D, and F along with antimitotic activity.41Aplicyanins A and C, however, were inactive at all concentrations and E displayed only mild cytotoxicity.41 The biological data suggests that the presence of the acetyl moiety on the N-16 position of this group of compounds is vital in order to maintain any appreciable activity. 20

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N R3 Br H N HN N R2 R1 46 R1=R2=R3=H 47 R1= Ac, R2=R3=H 48 R2= OMe, R1=R3=H 49 R1= Ac, R2= OMe, R3=H 51 R1= Ac, R2=OMe, R3=Br The diprenylquinones glabruquinone A (52), also known as desmethylubiquinone Q2, and its minor isomer glabruquinone B ( 53) were found in the Far-Eastern ascidian Aplidium glabrum.42 These polyprenylated quinones showed activity in the anchorage-independent transformation assay against mouse JB6 P+ Cl 41 cells that had been transformed with an epidermal growth factor as well as anticancer activity against HCT-116, MEL-28, and HT-460 human tumor cells with IC50 values of 12.7, 17.5 and 50.5 M respectively.42 O O CH3O CH3O 1' 52 53 2'cis The first oxadiazinone alkaloid found in nature, alboinon ( 54), was isolated from a Baltic Sea collection of the solitary tunicate Dendrodoa grossularia .43 This sea squirt has also been found to elaborate the indole alkaloids grossularines-1( 55 ) and -2 ( 56), dendrodoine ( 57), imidazalone 58 and the indole alkaloid 59.44-46 Dendrodoine 21

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displayed moderated cytoxicity for L1210 leukemia cells (ID50=10 g/mL) and no activity was reported for the other metabolites. N H N NO O N N H N NH N N(CH3)2 N H O 54 55 N H N NH N N(CH3)2 O OH N H S N N O N(CH3)2 56 57 N H NN N(CH3)2 O N H NH N O N(CH3)2 O 58 59 Clavelina lepadiformis collected in the North Sea produces the alkaloids lepadin A ( 60 ), 6164 and the pentachlorooctatriene 65 .47 Lepadin A displayed a wide range of in vitro activity toward muri ne leukemia P388 (ED50, 1.2 g/mL) and glioblastoma/astrocytoma (U 373, 3.7 g/mL), along with breast (MCF7, 2.3 g/mL), ovarian (HEY, 2.6 g/mL), colon (LoVo, 1.1 g/mL) and lung (A549, 0.84 g/mL) cancers.47 Alkaloids 61 and 64 also displayed significant in vitro cytotoxicity against human cancer cell lines .47 22

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N H H H OR2 R1 N 60 R1 = H2, R2 = C(O)C 61 R1 = H2, R2 = H H2OH63 62 R1 = O, R2 = C(O)CH2OH Cl Cl Cl Cl Cl N ergoline alkaloids, pibocin A and B ( 66 67 ), were found in ascidians nlike cin B 65 64 The belonging to the genus Eudistoma collected in the Northern Sea of Japan.48,49 U pibocin A, pibocin B displays moderate ly cytotoxicity against mouse Ehrlich carcinoma cells, which may attributed to the substituted indole nitrogen. Pibo incorporates a unusual N Omethylindole group that has previously been found in terrestrial plants exclusively. N N H Br H R 66 R = OCH3 67 R = H 23

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The unique chemistry and broad range of biological activities em erging from these urther 2.1.2 Secondary Metabolites from the Synoicum genus Ascidians from the genus Synoicum have proven to be an outstanding source of novel m shores g because it is a, cold-water ascidians provides ample justifi cation for their conti nued study. Little is known about the ecosystems that these types of organisms live in due to the harsh environments and at times inaccessability. The intricate relationships that exist between predator and prey in these under-studied cold-wat er habitats warrants f investigation in order to gain more insight into the chemistry that evolves from them. arine natural products. The few tunicate s that have been chemically investigated from the Synoicum genus have yielded over a dozen new metabolites, some with appreciable biological activity. Samples of Synoicum castellatum collected off the of various Australian islands were found to c ontain the previously discovered compounds quinine ( 68), hydroquinone ( 69), and chromene ( 70) as well as a novel tetrahydrocannabinol derivative (71 ).50 Compound 71 is rather interestin the first derivative of tetrahydr ocannabinol to be isolated from a marine source and it exhibits mild cytotoxicity against P388 mu rine leukemia, A-549 human lung carcinom HT-29 human colon carcinoma and CV1 monkey kidney fibroblast cells.50 O O OH OH 69 68 24

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O HO O H HO H Rubrolides A-H ( 73) are a series of metabolisolated from the tunicate Ritterella rubra and were reported to possess anti bacterial and phosphatase-inhibiting activity.51 Shortly thereafter rubrolides I-N ( 84 89 ) were reported from the red colonial tunicate Synoicum blochmanni collected from Tarifa Island, Spain.52 Of the six new nitrogenous metabolites, rubrolides I, K, L, and M exhibited significant cytotoxicity. Bioassay-guided fractiona tion of a collection of Synoicum n. sp. from New Zealand led to the isolation of rubrolide O ( 90), which exists as a mixture of E / Z isomers.53 Rubrolide O is the first anti-inflammatory rubrolide reported. 71 70 68 tes i O O Br OR B r Br RO Z Br RO O O X OR X Y R'O Z Y 76 Rubrolide A, R=R=Z=H, X=Y=Br 77 Rubrolide B, R=R=H, X=Y=Br, Z=Cl 78 Rubrolide C, R=R=Z=Y=H, X=Br 82 Rubrolide G, R=Z=H 83 Rubrolide H, R=H, Z=C l 79 Rubrolide D, R=R=Z=X=H, Y=Br 80 Rubrolide E, R=R=X=Y=Z=H 81 Rubrolide F, R=Me, R=X=Y=Z=H 25

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O O Br OH B r Y HO X O O OH Y HO X Br 84 Rubrolide I, X = Cl, Y = Br 85 Rubrolide J, X = H, Y = Br 86 Rubrolide L, X = Cl, Y = H 87 Rubrolide K, X = Cl, Y = Br 88 Rubrolide M, X = Cl, Y = H 89 Rubrolide N, X = Br, Y = Cl O O OH B r HO Cl Br Br O O Br HO Cl Br HO B r 90Z Rubrolide O 90E Rubrolide O A series of weakly cytotoxic tetraphenolic, bis-spiroketals named prunolides A-C ( 91-93) were isolated from Synoicum prunum collected from North Stradbroke Island in Queensland, Australia.54 Rubrolide A ( 76), originally discovered in Ritterella rubra ,51 was isolated along with the structurally sim ilar prunolides, which are believed to arise from oxidative dimerization of a rubrolide precursor. O O O O O X HO X HO X X OH X OH X Y Y Prunolide A ( 91 ) X = Br, Y = Br Prunolide B ( 92 ) X = Br, Y = H Prunolide C ( 93 ) X = H Y = H 26

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Chemical investigation of the ascidian Synoicum macroglossum collected from the Indian Ocean near Tamilnadu, I ndia, led to the isolation of the carboline guanidine derivative, tiruchanduramine ( 94).55 Tiruchanduramine showed promising inhibitory activity against glucocidase with an IC50 of 78.2 g/mL N H N N H O N H NH NH 94 The chemical literature for the genus Synoicum reveals that it yi elds a number of bioactive secondary metabolites with a broa d range of biological activities. Further investigation into the chemistry produced by organisms belonging to this genus will undoubtedly continue to unveil new structurally intriguing compounds that will find their way into the drug discovery pipeline as plausible candidates. 2.2 Chemical Investigation of the Antarctic Tunicate Synoicum adareanum Synoicum adareanum (Figure 1) is a sea squirt na tive to the waters of Antarctica and can be found in abundance near the U.S. research station (P almer Station) on the Antarctic Peninsula. This col onial tunicate grows as a series of fist-sized colonies on a common base to form an assemblage of co lonies, and can be collected from 15 to 796 meters in depth. Chemical inves tigation of this marine invertebrate has revealed that its natural product diversity is broad, including polyketide macrolides (structurally similar to the antibiotic erythromycin)56, glucoshpingolipids (structurally related to fats)57, and 27

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steroids (structurally related to cholesterol)58, all of which have very different structural features as well as functions within the organism. Figure 1. Synoicum adareanum collected at Palmer Stati on, Antarctica (Photograph supplied by Bill J. Baker, University of South Florida) 2.2.1 The Palmerolides A series of polyketide macrolides known as the palmerolides have been isolated from S. adareanum by previous members of our laboratory and have generated a great deal of interest due to their cytotoxicity and selectivity ag ainst melanoma cells along with their low toxicity. The palmerolides are co mposed of a nineteen membered ring with pendant hydroxyl and carbamate groups in varying positions. A single side chain is attached to the ring that varies in length and functionality. All compounds in the series have been found to contain one of three different ring systems and one of five different side chains. Palmerolides A ( 95), B ( 96), and C ( 97) were the first compounds isolated in the series, each possessing one of the three unique ri ng systems. Their planar structures were determined using a series of spectroscopic techniques including the two dimensional 28

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nuclear magnetic resonance (NMR) expe riments gCOSY, gHSQC, and gHMBC in addition to mass spectrometry. The determinati on of the absolute configurations however required a combination of both wet and dry ch emical techniques, some of which is the subject of this dissertation. O O O NH2 O H N O O O H N O O NH2 O HO O O S O O O H N O O OH O O NH2 HO OH 21 1' 1 19 25 1' 21 19 1 25 1' 21 19 1 25 OH 95 96 97 2.2.2 Ring System A Palmerolides Palmerolide A ( 95) is the most abundant second ary metabolite produced by the tunicate and was the first of the series to be fully characterized including stereochemistry.56 The use of X-ray crystallography for the determination of the absolute configurations of palmerolide As five asymmetric centers was not possible due to the inability to produce a high quality crystal. Instead the commonly used wet chemical technique known as modified Moshers method was used in conjugation with the more advanced two dimensional NMR spectro scopic experiments ROESY and HETLOC. After publication it was noticed that the NMR data of the Moshers esters was transposed. 29

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Degradative and synthetic efforts later confir med the misinterpretation of the original Moshers data and revealed the absolute configurations as 7 S, 10 S, 11S, 19R and 20R .59-63 Palmerolide A has four analogs (palmerolides D ( 98), E ( 99), F ( 100), and G ( 101)), all containing the same macrolide ring core but posse ssing different side chains. The absolute configurations of the stereocen ters were determined, as described below, using the corrected structure of palm erolide A as the basis for comparison. O O O NH2 O H N O OH HO O O NH2 O O OH O 99 HO 95 R= 98 :R= 100:R= 101 :R= ,21Z 21 H 1' 23 1 19 25 1" 2' 2' 2' 2' 25 7 7 11 11 Palmerolide D ( 98) differs from palmerolide A ( 95 ) starting at the carboxamide portion of the side chain. Just as was th e case with palmerolide A, gHMBC correlations could be observed between a carbonyl at 162.9 (C-1), a methylene group at 40.3 (C4), and a methyl group at 25.1 (C-8) to H-2 ( 5.81.) These correlations were indicative of the presence of a trisubstituted olefin in which C-2 ( 119.7) was a participant. The C-2/C-3 tris ubstituted olefin was assigned as Z based on ROESY correlation between H-2 and H3-8 and was found to be c onjugated to an amide. The presence of gHMBC correlations from H3-8 ( 1.76) to C-2, C-3 ( 152.7), and C-4 30

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also supported the existence of this conjugated ol efin. It is at this poi nt in the side chain where the differences between palmerolid es A and D begin. In palmerolide A the carboxamide chain ends at this conjugated tris ubstituted olefin wher eas it is elongated in palmerolide D by three carbons. The exte nded side chain was assigned by gHMBC correlations from C-2, C-3, C-5 ( 143.0), and C-7 ( 22.0) to H2-4 ( 3.34). The planar structure could then be completed by correlations from H3-7 ( 1.61) to methylene C-4, quaternary C-5 and terminal methylene, C-6 ( 111.9). The proposed planar structure was confirmed by high resolution mass spectrometric analysis with an [M + 1]+ peak at m/z 625.3864 ( mmu 1.1 for C36H53N2O7). A comparison of the contiguous carbon backbone of palmerolides A and D is illustrated in Figure 2. The 13C NMR spectrum shows no more than 2 ppm ( = pal A pal D) when compared to palmerolide A over the contiguous carbon backbone (Fi gure 2), supporting the assignment of palmerolide D as a homolog of palmerolide A. Figure 2. Comparison of palmerolide D and palmerolide A 13C NMR chemical shifts by position number ( = Pal A Pal D) The 1H NMR spectrum of palmerolide E ( 99) was distinct in comparison to the other palmerolides because it contained a signal indicative of an aldehyde group. In addition, analysis of the NMR data reveal ed a significant reduction in the carbon and proton count than palmerolide A, and was later confirmed by mass spectral analysis ( m/z 512.2634 for C27H39NO7Na, calc. 512.2624). Although there were far fewer proton and carbon signals, the 1H NMR spectrum of palmerolide E retained many of the macrolide 31

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NMR signals originally obs erved in palmerolide A ( 95) (see, for example, Figure 3). Further analysis of the 2D NMR data facilita ted the determination of the macrocycle as identical to those of palmerolides A and D, and can be appreciated from Figure 3 which displays a minimal difference in values of the carbons comprising the macrolide core. With all of the carbons in the macrolide core accounted for it became evident that the side chain pendant at C-19 could account for the missing substructures. Assignment of the side chain began with correlations in the gCOSY and gHMBC spectra establishing that C-19 was adjacent to a C-20 methine, and could be further extended by correlations to the familiar C-21/C22 olefin and its attached vinyl methyl group found in both 95 and 98. The structure of palmerolid e E began to stray from the others at this point in that the C-21/C22 olefin was found to be conjugated to the aforementioned aldehyde func tion. Correlations in the gHMBC data between the aldehyde proton, H-23 ( 9.40) and both C-21 ( 154.9) and C-22 ( 138.7) terminated the side chain. Thorough analysis of all data obt ained for the compound led to the conclusion that palmerolide E is not the initially e xpected hydrolysis product but rather 24norpalmerolide. Figure 3. Comparison of palmerolide E and palmerolide A 13C NMR shifts Palmerolides F ( 100) and G ( 101) were found to be isomeric with palmerolide A based on the HR ESIMS data ( m/z : 100 607.3359; 101, 607.3350; calc. 607.3359 for C33H48N2O7Na). The carbon backbone of both compounds was established by 2D NMR analysis in the same manner as described fo r previous palmerolides. Carbons C-1 through 32

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C-24, comprising the macrolide core and the side chain up to the amide moiety, were found to be identical to palmerolide A in both constitution and olefin geometry. The 1H NMR spectrum of palmerolide F made it very easy to distinguish the carboxamide substructure as different from that in palmerolide A (95) by the presence of exomethylene signals of H2-4 ( 4.79 and 4.82). Analysis of the 13C NMR spectrum confirmed this observation. Work ing inward from the terminus of the side chain, the protons of the olefinic methyl ene C-4 could be corr elated to the vinyl methyl C-5 ( 22.2) and the vinyl methylene C-2 ( 44.5) using gHMBC data. Correlations observed from C-2 to the carboxamide carbonyl, C-1 ( 167.4), was the last piece of data necessary to establish the carboxamide group as 3-methyl-3-butenoyl. Figure 4. Comparison of palmerolide F 13C NMR shifts to those of palmerolide A Palmerolide G, another isomer of palmero lide A, differed only in the geometry of the C-21/C-22 olefin. The configuration of the olefin was demonstrated as Z based on ROESY data showing a correlation between H-23 ( 6.19) to H-20 ( 2.65) and from H21 ( 5.01) to H3-27 ( 1.76). The difference graphs (Figur es 4 and 5) for palmerolides F and G show little variation in the chemical shifts of the carbon b ackbone from that of palmerolide A, which is to be expected since they are isomers. Palmerolide G displays the lowest values overall being that it differs from palmerolide A only in the geometry of the C-21/C-22 olefin, affecting only the Figure 5. Comparison of palmerolide G 13C NMR shifts to those of p almerolide A 33

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chemical shifts of neighboring carbons as is demonstrated in Figure 5. Palmerolide F displays minimal differences in chemical shifts in the macrolide portion of the molecule with larger variations in the side chain due to the isomeriza tion of the C-2/C-3 internal alkene to a C-3/C-4 terminal alkene. 2.2.2.1 Stereochemical Assignme nt of Palmerolides D-G Modified Moshers method 64 is a wet chemical technique used for the configurational analysis of secondary alcohols and is considered to be an invaluable tool amongst natural products and synthetic chemists concerned with the absolute configurations of organic compounds. This technique entails the derivatization of secondary alcohols as the respective ( R )and ( S)-methoxytrifluoromethylphenylacetate (MTPA) esters Proton nuclear magne tic resonance data must be obtained and the 1H chemical shift differences that arise as a result of the diamagnetic effect of the benzene ring recorded. The values for the surrounding protons can then be calculated using the formula = S R Using this data a model can be made to reveal the absolute configuration of th e chiral center by placing all positive values on the right side of the MTPA plane and all negative values on the left side as demonstrated below.64 O CF3 O Ph OMe H (OMe) (Ph) ( R )-MTPA ( S )-MTPA HX HY HZ HC HB HA MTPAplane Figure 6. Moshers depiction of the MT PA plane of an MTPA ester64 34

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Modified Moshers method was used to determine the absolute configurations of the two alcohols on C-7 and C-10 for all four ring system A palmerolides (D-G, 98101). The ( R )and ( S )-MTPA palmerolide diesters were made by the reaction of each palmerolide with Hunigs base, dimethylam inopyridine (DMAP), and an excess of the respective Moshers chloride in dry dichloro methane. The crude reaction mixtures were separated using reversed pha se high pressure liquid chromatography and afforded the pure diester, which eluted at a much lower polarity than the natural product. Successful formation of the Moshers esters was confir med by the presence of aromatic and methoxy signals in the 1H NMR spectrum. NMR data was obtained for both ( R )and ( S)diester products of each palmerolide and the chemical shift differences analyzed. Figures 7 and 8 show the 1H NMR spectra obtained for the ( R )and (S)-MTPA diesters of palmerolide F as an example. 0 9 8 7 6 5 4 3 2 1 Ch i l Shif ( ) Figure 7. Palmerolide F ( R )-MTPA diester in d6-DMSO at 500 MHz 35

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10 9 8 7 6 5 4 3 2 1 Ch i l Shift ( ) Figure 8. Palmerolide F ( S)-MTPA diester in d6-DMSO at 500 MHz. The values for the proton chemical sh ifts relevant to the C-7 and C-10 stereocenters were calculated for palmero lides D-G and are reported in Table 1 along with the model constructed from them. The corrected values obtained for palmerolide A are also included. Application of m odified Moshers method revealed the configurations to be 7 S and 10 S in all ring system A palmerolides. 36

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Table 1. Stereochemical analysis ( ) of Palmerolides A-G ( 53, 5659) using Moshers method[a] H H HH H H H H O H R H O MTPA O H M T PA 5 6 7 8 9 10 11 12 variable + Pal A Pal D Pal E Pal F Pal G H-5a --10 -10 -20 -H-5b -150 ----130 H-6a -20 -30 -30 -40 -30 H-11 +110 +130 +140 +90 +140 H-12 +180 +170 +150 +130 +120 [a] All values multiplied by 1000 The absolute configurations of the C-11, C-19, and C-20 stereocenters were determined based on simulation of the 1H NMR signals. Since palmerolides D-G all have the same ring system as palmerolide A a comparison of the coupling constants of the relevant protons could secure the absolute configurations. It was anticipated that ring analogs having the same absolute configur ation as palmerolide A at a particular stereocenter would have the same coupling constants while diastereomers would have different coupling constants. This supposition was based on the knowledge that each proton has a unique 1H NMR signal inherent to its environment, the splitting pattern being indicative of the number of spin sy stem couplings and the configuration being indicative of their location in space. Table 2 illustrates a comparison of palmerolide As coupling constants to those extracted via 1H NMR simulation for palmerolides D-G. All 37

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of the coupling constants for the respective ster eocenters were found to be within 1 Hz of those of palmerolide A, strongly supporting the hypothesis that their stereochemistry would be the same since they are ring analogs. This enabled the stereochemistry of all ring system A palmerolides (A, D, E, F, and G) to be assigned as 7 S, 10S, 11S, 19R 20R Table 2. 3JH,H (Hz) analysis of key palmerolide stereocenters Pal A Pal D Pal E Pal F Pal G J10 11 4.8 5.0 5.0 5.1 4.8 J11 12 10.6 10.6 10.7 10.7 10.3 J18a 19 1.3 1.4 1.4 1.7 1.5 J18b 19 11.2 11.0 11.0 12.0 12.0 J19 20 10.0 10.0 9.9 9.7 10.0 J20 21 8.0 8.0 7.0 7.3 8.0 J20.26 6.6 6.6 6.9 7.0 6.6 2.2.3 Ring System B Palmerolides The B ring system is unique in that it is the only one of the three distinct palmerolide cores that posse sses a sulfate group. The presence of the sulfate group significantly increases the polarity of the compounds containing this macrocycle in comparison to the previously described palmer olides and appears to be responsible for their lack of stability in dimethyl sulfoxide (DMSO). Palmerolides B ( 96 ) and H ( 102) are the only two palmerolides found so far that contain the B ring sy stem. Despite their unique macrolide cores these compounds contain side chains originally observed in the rings system A analogs. Palmerolide B has th e same side chain as palmerolide A, and palmerolide H shares the same extended si de chain as the previously described 38

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palmerolide D. Their planar structures were determined using the same techniques used to elucidate the ring system A palmerolides with the exception of the sulfate group. Mass spectral analysis of palmerolides B and H using ESI-MS in positive ion mode gave the parent ions [M+1] 567.3 m/z and 607.3 m/z respectively, suggesti ng the sulfate group eliminated during the mass spectral analysis. Running the samples using ESI-MS in negative ion mode gave the re spective parent ions [M-1]663.3 m/z and 703.3 m/z establishing the presen ce of a sulfate group by mass spectrometry. O O H N O O NH2 O O O S O O O O H N O O NH2 O O O S O O 1 25 19 21 1' 1'' 1 19 21 1' 25 1'' HO HO 8 8 96 102 The ring system B macrocycle was constructed starting at the C-1 ( 168.2) ester carbonyl which showed gHMBC correlations to the olefinic protons H-2 ( 5.70) and H-3 ( 6.74), indicating the same -unsaturated ester seen in ring system A. The C-4 ( 34.0), C-5 ( 26.1) and C-6 ( 31.8) methylene groups were assigned based on gHMBC correlations to H-3 and their positions co rroborated by gCOSY data as was done with palmerolide A. The H-7 ( 4.57) oxymethine showed gCOSY coupling to the H-6 ( 1.55, 1.08) methylene protons and anot her oxymethine located on C-8 ( 72.1). Coupling between H-7 and a quaternary carbonyl ( 159.8) suggested the presence of the carbamate group as seen in palmerolide A on C-11, and was confirmed by ma ss spectral analysis. The C-9 ( 133.4)/C-10 ( 131.0) olefin was found to be flanked by the H-7 and H-11 39

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( 4.64) oxymethines, which were subsequently determined to be attached to carbons bearing hydroxyl and sulfate groups, respectivel y. This functional gr oup motif is similar to that seen in the ring system A palmerolid es but the order of oxyge nation is different in that the two adjacent oxygenated moieties are closer to the beginning of the macrocycle (C-7/C-8) as opposed to the middle (C-10/C11). The connectivity of methylene groups C-12 ( 1.79, 1.53) and C-13 ( 1.96, 1.21) could be established by both gCOSY and gHMBC correlations. Another disubstituted olefin was place between C-14 ( 133.3) and C-15 ( 128.0) and was determined to be trans based on the coupling constants ( J14,15=14.5 Hz) of methines H-14 ( 5.38) and H-15 ( 6.01). The macrocycle was further extended by gHMBC correlations from H-15 to a conjugated trisubstituted alkene positioned between C-16 ( 129.8) and C-17 ( 132.8), as well as the vinyl methyl C-25 ( 16.7). In addition, vinylic methyl H3-25 ( 1.58) could be correlated to C-15, C-17, and C-18 ( 45.2). Ring closure was established by gCOSY correlations from non-equivalent methylene H2-18 ( 2.15, 1.99) to oxymethine H-19 ( 4.82), Figure 9. Comparison of palmerolide B 13C NMR shifts to those of p almerolide A Figure 10. Comparison of palmerolide B 13C NMR shifts to those of p almerolide H Figure 11. Comparison of palmerolide H 13C NMR shifts to those of p almerolide D 40

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followed by gHMBC correlations to C-20 ( 2.68) of the side chain and the C-1 ( 168.2) ester carbonyl. Figure 9 illustrates a comparis on of the carbon backbones of palmerolides B and A. The graph cleary depicts the diffe rences in the macrolide cores by the large values for C-4 through C-12, and that they posse ss the same side chain by the negligible differences in the chemical shifts of the side chain carbons.The assignment of palmerolide H as having the same ring system as palmerolide B is illustrated in Figure 10 and the same extended side chain as palmerolide D is illustrated in Figure 11. 2.2.3.1 Stereochemical Assignment of Palmerolides B and H Since a formal synthesis of palmero lide B has not been completed it was necessary to modify the appro ach used to determine the abso lute configurations of the stereocenters from that used with th e ring system A compounds. Proton NMR simulation could still be used to determine the threo configurations of the C-19 and C-20 stereocenters by comparison of the J -coupling constants with those of palmerolide A (Table 3). The extracted J -values indicate that palmerolides B and H have the same ( R R )-configuration at these stereocenters as palmerolide A. Table 3. 3JH,H (Hz) analysis of key palmerolide stereocenters in palmerolides A, B, and H Pal A Pal B Pal H J19 2010.0 9.7 9.7 J20 218.0 7.9 7.9 J20.266.6 6.6 6.6 41

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The relative stereochemistry of the C-7 a nd C-8 stereocenters was determined via another popular method of J -based configuration analys is using the advanced twodimensional NMR experiment gHSQMBC.65 As is indicated by its title, this experiment is a combination of the two dimensional heteronuclear NMR experiments gHSQC (gradient heteronuclear single quantum cohe rence) and gHMBC (gra dient heteronuclear multiple bond coherence). The experiment illustrates heteronuclear single bond correlations as well as heteronculear mu ltiple bond correlations within three bond proximity. In addition, this experiment can al so be used to extr act two and three bond heteronuclear coupling constants which can re veal the relative position of two adjacent groups in space. This method was developed by Murata who demonstrated a correlation between the values/magnitude of three bond homonuclear (3JH,H), two bond heteronuclear (2JC,H), and three bond hetereonuclear (3JC,H) spin-coupling consta nts to the relative position of two adjacent oxygenated groups.66 Muratas method works because in systems with conformational changes, such as macrocycles, the coupling constants are observed as a weighted average of those due to each conformer. Although vicinal protonproton couplings (3JH,H) can be extracted easily from a proton spectrum, these values alone are not enough for configuration analysis because they cannot distinguish between two gauche rotamers. The additional in formation that can be gained from 2,3JC,H coupling constants, however, may dramatically expand the utility of the coupling constants by providing a distinction between the two gauche rotamers. Table 4 contains the values and magnitudes of the coupling constants outlin ed by Murata for the anti and gauche orientations in acyclic systems.66 42

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Table 4. 3JH,H and 2,3JC,H Values (Hz) for anti and gauche orientations in acyclic systems 3JH H 2JC H 3JC H oxygenation Anti large Gauche small Gauchealarge Antib small Anti large Gauche small di 7-10 0-3 -4 to -6 2-0 5-7 1-3 a,b Oxygen functions on relevant carbons are gauche and anti to their vicinal protons respectively Palmerolides B and H fall under the category of di-oxygenated systems (located on C-7 and C-8) and the spin coupling values extracted from the NMR data can be found in Table 5. The application of Muratas met hod reveals that the relative stereochemistry of the C-7 carbamate and C-8 hydroxyl groups of both palmerolide B and H are gauche as illustrated in Figure 12. It should be noted that although the arrangement of the secondary alcohol and carbamate groups are in different positions on the rings relative to those of palmerolide A, the relative stereochemistry of these moieties remains the same. Table 5. 3JH,H and 2,3JC,H values for palmerolides B and H Spin Cou p lin g s Palmerolide B Palmerolide H 3JH,H H7-H8 ~2.0 Hz (small) ~2.0 Hz (small) 2JC,H H7-C8 ~ -4.0 Hz (large) ~ -5.0 Hz (large) 3JC,H H8-C6 ~ 1.5 Hz (small) ~ 2.5 Hz (small) 43

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O C6 H7 C8H8 OH C9 H2N O Figure 12. Newman projection of palmerolide B C-7/C-8 stereocenters In order to elucidate the absolute confi gurations of the C-7 and C-8 stereocenters modified Moshers method was attempted. Un fortunately, due to the presence of the sulfate group on C-11 preparation of the Moshers esters was elusive. Several attempts at mild acid hydrolysis of the sulfate group proved unsuccessful and other methods found in the chemical literature seemed too abrasive for such a complex molecule with so many labile moieties. Palmerolide B was reacted with sodium methoxide in an attempt to hydrolyze the sulfate group so that Moshers method could subsequently be used to elucidate the absolute configur ation of the three resulting secondary alcohols. Separation of the crude reaction mixture by reversed phase chromatography using a gradient from 50% water/methanol to 100% methanol indi cated the formation of a compound that was significantly less polar than palmerolid e B according to its retention time. 1H NMR data was obtained on this fraction and the com pound appeared to be very similar to palmerolide B based on a superficial compar ison against previously acquired data. A more in depth analysis of the compound reveal ed that it had undergone hydrolysis of the C-7 carbamate group as well as elim ination of the C-11 sulfate group ( 103 ). ESI mass spectral analysis gave a parent ion [M+1] of 524.3 m/z confirming the proposed structure, 44

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and no ionization was observed when run in negative mode which ultimately confirmed the absence of th e sulfate group. O O H N O OH HO 103 Following removal of the sulfate group via base hydrolysis, the respective C-8 MTPA Moshers monoesters of compound 103 were made and full NMR data sets acquired. Analysis of the data revealed the absolute configuration of C-8 to be in the S configuration (Table 6). The absolute conf iguration of C-7 could subsequently be assigned as S based on the prior application of Mura tas method. Tables 3 and 5 above show comparisons of the key coupling constants for palmerolides B and H which suggests that their stereochemistry is the sa me. The successful application of Moshers method on the palmerolide B analog 103 allowed for the stereochemical assignment of palmerolides B and H as 7 S, 8S, 19R 20R 45

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Table 6. Stereochemical analysis ( ) of palmerolides B (96 ) using Moshers method (all values multiplied by 1000) H H OH MTPA H OH H H 6 7 8 9 10 + 1H position Pal B H-4 +80 H-6 +40 H-7 +10 H-9 -10 H-10 -10 H-11 -10 Further studies will have to be performe d in order to elucidate the configuration of the sulfate bearing C-11 stereocenter. Since the base catalyzed hydrolysis leads primarily to the C-11 dehydrated product, s ynthetic analysis app ears to be the most logical avenue to pursue. There are a number of syntheses described for palmerolide A which could potentially be modified to enab le a relatively quick synthetic route to palmerolide B. Comparison of the synthetic palmerolide B diastereomers to the natural product would allow for the relatively quick determination of the C-11 configuration. 2.2.4 Ring System C Palmerolides Palmerolides C ( 97) and K ( 104) are the only compounds fo und so far that contain the ring system C macrolide core. Despite that the molecular weight of palmerolide C was determined to be the same as palmer olide A via mass spectrometry, and that the 1H NMR data of the side chain protons wa s identical, it was evident from the 1H NMR 46

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spectrum that this compound had undergone a si gnificant rearrangement of the structural features within the macrolide core (Figur e 13). Although it shares the same oxygenated moieties as the ring system A analogs, these groups are compacted to the C-8, C-9, and C-10 carbons of the backbone as opposed to be ing separated by the familiar disubstituted olefin. O O O O NH2 O O H N O O O O NH2 1 19 21 25 1' 1" 1 21 25 19 1"OH OH OH OH 104 97 The -unsaturated ester found in the other palmerolide rings systems was confirmed in the C ring system as well by gHMBC correlations from olefinic H-2 ( 5.73) to the C-1 ( 166.9) ester carbonyl and C-3 ( 149.8) methine. Vinyl proton H-3 ( 6.77) showed gHMBC correlations to methylenes C-4 ( 31.7) and C-5 ( 32.2) which further extended the carbon backbone. Aliphatic H-5 ( 1.93, 1.84) could be correlated to a disubstituted alkene positioned between C-6 ( 131.8) and C-7 ( 131.1) through both gCOSY and gHMBC data. A vicinal diol on C-8 ( 72.8) and C-9 ( 75.6) was identified by gCOSY correlations from oxymethine H-8 to vinyl H-7 ( 5.58) as well as oxymethine H-9 ( 3.56). A third Figure 13. Comparison of palmerolide C 13C NMR shifts to those of p almeroli d e A 47

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oxymethine could be placed on C-10 ( 74.2) based on gCOSY correlations from fellow oxymethine H-9 and aliphatic H2-11 ( 1.49, 1.30) to H-10 ( 4.56). This was confirmed by gHMBC signals from aliphatic H2-12 ( 1.95) to C-10. A correlation to an ester-type carbonyl moiety was observed for H-10 as well, which upon mass spectral analysis was found to be the familiar carbamate seen in the other palmerolide ring systems. A series of methylene groups could be placed from C-11 ( 28.7) to C-13 ( 30.1), and the macrocycle closed the same as the A and B ring systems with the trans C-14 ( 132.3) /C-15 ( 127.2) disubstituted olefin co rrelating to the C-16 ( 128.8) /C-17 ( 132.5) trisubstituted olefin. H3-25 ( 1.69) demonstrated gHMBC correla tions to C-16, C-17, and C-18 ( 44.1) consistent with its assignment as a vinyl methyl. The 20-membered macrocycle was closed by gHMBC correlations from H-19( 4.85) to C-1, C-17 and C-18. Palmerolide C was found to have the same side chain as pa lmerolides A and is illustrated in Figure 13 by the minimal chemical shift differences from C-19 onward. Figure 14 demonstrates that palmerolides K shares the same ring system as palmerolide C, while Figure 15 shows that it contains the same truncated side chain as palmerolide E. Figure 14. Comparison of palmerolide C 13C NMR shifts to those of p almerolide K Figure 15. Comparison of palmerolide K 13C NMR shifts to those of p almerolide E 48

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2.2.4.1 Stereochemical Assignment of Palmerolides C and K Stereochemical assignment of palmerolides C ( 96 ) and K ( 104 ) required a combination of the techniques used to determin e the absolute configur ations of the ring system A and B palmerolides. Proton NMR simulation and extraction of coupling constants could be used to determine the configurations of C-19 and C-20 as was done with the other palmerolides (Table 7). Extraction of the J -values indicated that the relative configurations of th e C-19 and C-20 sterocenters ar e threo. By analogy with the other palmerolides, the absolute stereochemistry is assumed to be 19 R and 20R Table 7. 3JH,H (Hz) analysis of key palmerolide stereocenters in palmerolides A and C Pal A Pal C J19,2010.0 9.8 J20,218.0 7.6 J20.266.6 6.7 The relative stereochemistry of the C-8/C-9 diol in palmerolide C was determined using gHSQMBC data and Murata s method. Configuration analysis using the extracted J -coupling constants suggested that the hydroxyl groups were in the anti conformer (Figure 16, Table 8). The same type of analysis was conducted to determine the relative stereochemistry of the C-9 hydroxyl and C-10 carbamate groups. The data indicated that the C-9/C-10 stereocenters we re in the gauche conformer (Table 8). 49

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C8 H9 OH C10H10 OCONH2 C11 H8 HO C7 C9H9 OH C10 A B Figure 16. Newman projections of palmerolide C C-8/C-9 ( A ) and C-9/C-10 ( B ) stereocenters Table 8. 3JH,H and 2,3JC,H Values for palmerolide C C-8/C-9 Analysis C-9/C-10 Analysis Spin Couplings Palmerolide C Spin Couplings Palmerolide C 3JH,H H8-H9 ~7.0 Hz 3JH,H H9-H10 ~2.0 Hz 2JC,H H9-C8 ~ -5.5 Hz 2JC,H H10-C9 ~ -5.0 Hz 3JC,H H9-C7 H8-C10 ~ 3.0 Hz ~ 3.0 Hz 3JC,H H10-C8 H9-C11 ~ 7.0 Hz ~ 2.5 Hz Modified Moshers method was used initially to assign the absolute configurations of the C-9 and C-10 stereocenters. Synthesis of the Moshers diester was performed but assignment of the stereo chemistry remained elusive since the values were all the same sign. This effect can occu r when vicinal diols ar e derivatized and the resulting esters are unable to position themse lves far enough away from each other due to restricted rotation around the bond. The solu tion to this problem was to make the respective R and S monoesters of the C-8 hydroxyl group in order to figure out the absolute configuration of C-8. The values for the surrounding proton chemical shifts along with the model constructed from them can be found in Table 9. Assignment of C-8 as being in the S configuration via Moshers method allowed for the indirect assignment 50

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of the other stereocenters as 9 R and 10S based on the prior application of Muratas method. A comparison of the coupling constant s of palmerolides C and K (Table 10) proved the absolute stereochemistry of both compounds to be 8 S, 9R 10S, 19R 20R Table 9. Stereochemical analysis ( ) of palmerolides C (97 ) using Moshers method (all values multiplied by 1000) H H OH MTPA H OH H OR 6 7 8 9 10 + 1H position Pal C H-6 -90 H-7 -100 H-9 +110 H-10 +40 Table 10. 3JH,H (Hz) analysis of key palmerolide stereocenters in palmerolides C and K Pal C Pal K JH8 9 2.0 2.0 JH9 10 2.0 2.0 JH19 209.8 9.6 JH20 217.6 8.0 JH20.266.7 6.5 51

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2.3 Structure Activity Relation ship Studies on Palmerolide A 2.3.1 Bioactivity of the Palmerolides The discovery of palmerolide A generate d a great deal of interest due to the potent cytotoxicity and selectiv ity exhibited in the National Ca ncer Institutes (NCI) sixty cancer cell line panel along with its low toxi city. Palmerolide A displayed impressive activity against the UACC-62 melanoma cell line with an IC50 of 18 nM.56 The NCIs COMPARE database found that palmerolide A had a dose re sponse profile against the cancer cell lines that was very similar to those exhibited by the natural products bafilomycin A1 ( 105)65 isolated from the bacteria Streptomyces griseus, and salicylahalamide A ( 106 )66 isolated from the sponge Haliclona sp. These compounds act as potent inhibitors of mammalian vacuol ar-ATPase and prompted the assay of palmerolide A for such activity.67,68 Palmerolide A was found to act as a potent inhibitor as well with an IC50 of 2 nm but lacked the neur otoxicity observed with compounds 105 and 106. MeO O O HO OMe O OH OH OH O O O NH2 O HO H N O OH 95 105 OH O O OH HN O 106 52

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All of the palmerolides were evalua ted for cytotoxicity against UACC-62 melanoma cells via an in house assay as well as for their ability to inhibit mammalian vATPase (Table 11, conducted by X. S. Xie, University of Texas Medical Center, Dallas). The range in bioactivity exhibi ted by the palmerolides is indicative of a delicate balance between substructures within the side chai n and the macrolide core. This order of reactivity of the macrolide cores can best be observed in the bioactivity data collected for palmerolides A, B, and C, which all possess the same side chain but have differing ring systems. The data suggest that the A ring sy stem is the most bioactive overall against v-ATPase and UACC-62 melanoma cells with palmerolide A exhibiting IC50 values of 2 nm and 0.024 M, respectively. Palmerolide B was found to be the second most active against v-ATPase with an IC50 of 0.023 M but the least active of the three against UACC-62 cells at 0.25 M. Despite that palm erolide C is approximately 10-fold less potent against v-ATPase than palmerolide B with an IC50 of 0.150 M, it is more active against UACC-62 cells by 2-fold at 0.11 M These findings indicate that the arrangement of the functional groups in the macrolide core do in fact influence the bioactivity of the compound, but also implies that different substruc tures within the ring may be responsible for the activities exhibited against UACC-62 and v-ATPase. 53

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Table 11. Bioactivity data for palmerolides (IC50 values in M) Compound vATPase (M) Melanoma (M) v-ATP:c y totoxicit y ratio mammalian UACC-62 PalmerolideA 0.002 0.024 0.083 PalmerolideB 0.023 0.250 0.092 Palmerolide C 0.150 0.110 1.364 Palmerolide D 0.025 0.002 12.500 Palmerolide E 10.000 5.000 2.000 Palmerolide F 0.063 0.758 0.083 Palmerolide G 0.007 1.207 0.006 Palmerolide H 0.021 0.019 1.105 Palmerolide K 10.000 >10.000 > 1.000 The A ring system also proved to be the most potent when comparing palmerolides E and K. Palmerolides E and K both possess the truncated side chain but differ in that palmerolide E possess the A ring system and palmerolide K contains the C ring system. Although they both lack activ ity against v-ATPase with equivalent IC50 values of 10 M, palmerolide E is more activ e than palmerolide K exhibiting inhibitory activity against UACC-62 cells at 5 M as opposed to >10 M. This is also the case for palmerolides D and H both of which contain the extended side chain. Palmerolide D which possesses the A ring system proves to be more potent in terms of cytotoxicity than palmer olide H which bears the B ring system, demonstrating IC50 values of 0.002 M and 0.019 M, respectively. When compared by their ability to inhibit v-ATPase, however, they were equipotent for the most part with respective IC50 values of 0.025 M and 0.021 M. This data suggest that although the arrangement of the macrolide core influences overall bioactivity, it may have a greater impact on cytotoxicity than v-ATPase activity. Perhaps more importantly this set of data 54

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suggests that substructures within the si de chain exert more influence on overall bioactivity than those in the macrolide core. By performing a bioactivity comparison of all ring system A palmerolides, it is confirmed that substructures within the side chain most definitely influence the efficacy of the compounds as well. Palmerolide D (0.002 M), possessing the extended side chain, proves to be the most cytotoxic memb er of the series against melanoma followed by palmerolide A (0.024 M) > palmerolide F (0.758 M ) > palmerolide G (1.207 M ) and the least cytotoxic of the group being palmerolide E (5 M). The carboxamide moiety in the side chain app ears to have a large impact on th e bioactivity of this set of compounds overall since palmerolide E, lacking the entire carboxamide moiety, is devoid of activity against both UAC C-62 cells and v-ATPase. Although the C-2/C-3 trisub stituted alkene does not appear to influence the cytotoxicity of these palmerolides the da ta suggest it may have some influence on vATPase inhibition. Palmerolide F, which lack s the abovementioned trisubstituted olefin, is more cytotoxic than palmerolide G whic h possesses it. Interestingly, palmerolides A (0.002 M), D (0.025 M), and G (0.007 M), all of which contain the C-2/C-3 olefin are very potent v-ATPase inhibitors whereas palmerolide F (0.063 M) is significantly less potent. It is also interesting to no te that the isomeri zation of the C-21/C-22 trisubstituted olefin from the E -geometry to Z significantly affects the cytotoxicity of palmerolide G while barely affecting v-ATPa se inhibiting ability. This data not only suggests that substructures with in the side chain influence th e overall bioactivity but also implies that different parts of the side chai n are responsible for the activities exhibited against UACC-62 and v-ATPase. 55

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Mother Nature did an exceptional job of illustrating the relationship between the structure and biological activity of the palmerolides. Bioassa y of all of the compounds in the series allowed for the elucidation of th e most pertinent structural features and provided a good starting point for further stru cture activity relationship studies (SAR). Collectively, this data establishes that th e carboxamide moiety in the side chain is necessary for retaining overall bioactivity, but suggests that modificiation of substructures within the side chain and/or ring could possibly allow for the decoupling of v-ATPase activity from cytotoxicity. Palm erolides are less neurotoxic than other vATPase inhibitors (unpublished da ta), leading us to believe th at they bind more than one cellular target. The ratios shown in Table 11 were calculated in an effort to help prove or disprove the theory that the palmerolides ma y be binding multiple cellular targets. It was anticipated that if some of the palmerolides lost considerable v-ATPa se inhibiting activity while gaining significant cytotoxicity, particul arly relative to palmerolide A, then the polymodal mechanism of action would be apparent in the ratios. If this were the case then a reciprocal ratio to th at of palmerolide A would be expe cted. The closest example of this was found in palmerolides A and D, though th e data is still inconclusive. Additional SAR studies could provide more insight in to the presumed polymodal mechanism of cytotoxicity in the palmerolides. 2.3.2 Structure Activity Relationship St udies of Palmerolide A via Synthesis In a recent publication, Nico laou and coworkers shared their molecular design and chemical synthesis of several palmerolide A analogs.71 The biological evaluation of these compounds against a panel of cancer cells, including the UACC-62 melanoma cell line, 56

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provided the first structure ac tivity relationship studies to be performed on palmerolide A. In summary, their studies encompassed general structural m odifications of the macrolide ring, the side chain, and the stereo chemistry of palmerolide A. They observed that replacement of the C-11 carbamate w ith a hydroxyl group results in only a 5-fold decrease in activity, indicati ng the important role the carb amate functionality plays in palmerolide As activity against the UACC-62 cell line. Another important observation noted by Nico laou et al. was that the deletion of the C-10 hydroxyl moiety significantly reduces the activity of palmerolide A, while deletion of the C-7 hydroxyl appears to have no effect. This finding warrants further investigation of these types of deoxygenated analogs in or der to optimize the minimal natural product scaffold required to retain bi ological activity. It wa s also discovered that complete inversion of the molecules stereoch emistry results in a 100-fold decrease in activity but inversion of a fe w stereocenters does not render it inactive. This finding is interesting because there are many instan ces in which mis-assignment of a single stereocenter in an active na tural product can make the synthetic product devoid of pharmacological activity. The efforts of Nicolaou and coworkers toward the biological evaluation of synthetic palmerolide A analogs was extremely helpful because it enabled access to analogs that would not be possible th rough derivatization of the natural product. 57

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2.3.3 Structure Activity Relationship Studies on Palmerolide A via Natural Product Derivatization 2.3.3.1 Preparation of Palmerolide A analogs Six analogs ( 107112) of palmerolide A were made through derivatization of the natural product. The first analog, 107 was made by hydrogenation of all alkenes in the molecule using H2 and palladium on activated carbon ( 10% Pd). The product was verified by acquisition of a full NMR data set and ma ss spectrometry. The absence of all alkenes was evident by the lack of olefinic signals in both the 1H (Figure 17) and 13C NMR spectra. 8 7 6 5 4 3 2 1 0Figure 17. 1H NMR spectrum of palmerolide A hydrogenation product ( 107) O O O NH2 O HO H N O OH in d6-DMSO, 500 MHz 58

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Analogs 108 and 109 are the C-7 and C-10 p-bromobenzoyl monoesters of palmerolide A. Both esters were made by the use of Hunigs base, 4-bromobenzoyl chloride and N N -dimethylaminopyridine. The disappearance of the C-7 and C-10 hydroxyl protons were indications that the re spective esters were formed as was the appearance of aromatic prot ons (Figure 18-19). A shift of the oxymethines on the carbons bearing the esters was observed in both ca ses as well as a visi ble difference in the olefinic region for the C-10 monoe ster. Formation of the C-7/C-10 p-bromobenzoyl diester 110 was achieved by increasing the reacti on time and the product confirmed by disappearance of both hydroxyl protons in addition to doubling of the aromatic signals (Figure 20). 9 8 7 6 5 4 3 2 1 Ch i l Shift ( ) O O O NH2 O HO H N O O O Br Figure 18. 1H NMR spectrum of palmerolide A C-7 p-bromobenzoate ( 108) in d6-DMSO, 500 MHz 59

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9 8 7 6 5 4 3 2 1 Ch i l Shift ( ) O O O NH2 O O H N O OH O Br Figure 19. 1H NMR spectrum of palmerolide A C-10 p-bromobenzoate ( 109) in d6-DMSO, 500 MHz 0 9 8 7 6 5 4 3 2 1 Ch i l Shift ( ) O O O NH2 O O H N O O O Br O Br Figure 20. 1H NMR spectrum of palmerolide A C-7/C-10 p-bromobenzoates ( 110 ) in d6-DMSO, 500 MHz 60

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Palmerolide A was reacted with one equivalent of sodium methoxide to yield two major compounds, 111 and 112. Analog 111 is the C-11 carbamate hydrolysis product of palmerolide A and was verified by the disa ppearance of the carbamate signal in the carbon NMR spectrum as well as by the upf ield shifts of H-10 and H-11 in the 1H NMR spectrum (Figure 21). Analog 112, the unexpected side produ ct, is the result of the dehydration of the C-11 carbamate group and th e conjugate addition of hydroxide to the C-3 position by what was thought to be a dr y solution of sodium methoxide generated in situ The addition of hydroxide to the C-3 position as opposed to methoxide made it evident that water was present while trying to generate sodium methoxide from a solution of sodium hydroxide and dry methanol. Th e dehydration of the C-11 carbamate group was noted by the disappearance of the broad proton signal around 6.5 ppm (-NH2) as well as the absence of a carbon resonanc e at approximately 157 ppm in the 13C NMR spectrum. The conjugate addition of hydroxide was verified by the very obvious loss of the H-2 and H-3 olefin ic protons in the 1H NMR spectrum along with the appearance of an additional resonance around 3.3 ppm that integrated to one proton (Figure 22). 61

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0 9 8 7 6 5 4 3 2 1 Ch i l Shift ( ) Figure 21. 1H NMR spectrum of palmerolide A C-11 alcohol ( 111) O O OH HO H N O OH in d6-DMSO, 500 MHz 10 9 8 7 6 5 4 3 2 1 Ch i l Shif ( ) O O HO H N O OH OH Figure 22. 1H NMR spectrum of palmerolide A C-3 alcohol ( 112) in d6-DMSO, 500 MHz 62

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2.3.3.2 Biological Evaluation of Palmerolide A Analogs Each analog was evaluated for its cytot oxicity using a whole cell-based assay against UACC-62 melanoma cells (Table 12.). They were also evaluated for their ability to inhibit mammalian v-ATPase by the use of an in vitro enzyme-based assay which utilizes the bovine brain Vpump and the dissociated V0 sector (Table 12). Compound 107, the fully hydrogenated palmerolide A product, was found to be void of activity against both UACC-62 and v-ATPase. This overa ll loss of activity is most likely caused by the removal of the features that provide th e molecule its structural rigidity and much of its electronegativity. Indescriminate saturation of the alkenes in 107 prohibits us from determining which olefins are integral for retaining activity. Further SAR studies performing selective saturation of the ring olefins versus the side chain olefins could help narrow down which of the olefin(s) have the most bearing on the bioactivity. Compound vATPase (M) Melanoma (M) v-ATP:cytotoxicity ratio mammalian UACC-62 107 4.000 >10.000 > 0.400 108 0.002 0.600 0.003 109 0.014 0.309 0.045 110 2.640 >10.000 > 0.264 111 0.009 0.489 0.019 112 0.026 0.850 0.030 Table 12. Bioactivity data for palmerolide A analogs (IC50 values) 63

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Derivative 108 the C-7 p-bromobenzoyl ester, was found to be an equipotent inhibitor of v-ATPase in comparis on to palmerolide A exhibiting an IC50 value of 2 nM but a significant decrease in cytotoxicity with an IC50 of 0.60 M. This data indicates that the C-7 hydroxyl is not integral for retaining v-ATPase activity but does suggest that it may affect cytotoxicity. Unfortunately, it cannot be determined whether this observed decrease in cytotoxicity is a result of the significance of the C-7 hydroxyl or rather a decrease in cell membrane permeability due to the addition of a less polar substituent at the C-7 position. Since the assay for v-ATPase activity is enzyme-based the ability of the compound to cross the cell membrane is not an issue, whereas, the cytotoxicity assay requires the drug to first permeate the cell memb rane before it can be evaluated for its ability to kill the cell. Furthermore, derivative 109, the C-10 p-bromobenzoyl ester, was incrementally less potent in v-ATPase inhibition (IC50 =14 nM) than 108 while roughly proportionally more cytotoxic (IC50= 0.31 M). This suggests that the C-10 hydroxyl is integral for vATPase inhibition but does not clarify if the observed loss in activity is a result of masking the alcohol group so that the receptor binding is diminished or if the steric bulk of the benzoyl group restricts entry into the r eceptor. The cytotoxicity data implies that although it is still unclear whether or not cel l permeability is an issue, the C-10 hydroxyl is not as critical as the C7 hydroxyl for retaining activity. Although it appears as though the C-7 hydroxyl group is significant for retaining cytotoxicity and the C-10 hydroxyl group is si gnificant for retaining v-ATPase inhibiton, it is not possible to di stinguish from these limited examples whether or not the observed differences in activity are due to: 1) poor availability as a re sult of decreased cell 64

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permeability, 2) masking the alcohol groups so that the receptor binding is diminished, or 3) the steric bulk of the benzoyl group rest ricts entry into the receptor. Besides being sterically conge sted, derivative 110 is no doubt encumbered by significantly poorer solubility which accounts for the overall loss in activity. Hydrolysis of the carbamate group on palmerolide A ( 111) demonstrated little effect on v-ATPase inhibition (IC50= 9 nM) while significantly decreasing cytotoxicity (IC50= 0.49 M). The reduced activity of this analog against the UACC-62 melanoma cell line has been previously reported by Nicolaou, but until recently no additional data has been acquired evaluating its inhi bitory activity against v-ATPAse.68 The mild effect demonstrated on the v-ATPase activity by removal of the carbamate moiety suggests that the carbamate moiety is not necessary to re tain inhibition. The significant decreased in cytotoxicity, however, can suggest that 111 is less adept at crossing the cell membrane or that more than one receptor interaction is responsible for cytotoxi city. Since the removal of the carbamate does not result in a signifi cant difference in the compounds polarity the membrane permeability is expected to be minimally impacted by the structural difference. In compound 112, the removal of the -unsaturated ester by the conjugate addition of hydroxide had a greater impact than anticipated on the bioactivity. Diminished cytotoxicity (IC50=0.84 M) due to dehydration of the carbmate moiety was to be expected based on the e ffect observed for derivative 111 but it was interesting to note a decrease in v-ATPase inhbition as well (IC50=26 nM). Drugs containing Michael acceptors such as -unsaturated ester are notorious for undergoing alkylation in their metabolism which could explain the decrease in overall activity provided that cell 65

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66 membrane permeability is not a issue. Taking th e cytotoxicity data for the other analogs into consideration, it is quite possible that th eir bioactivity is achi eved through a two-step process. The palmerolide must first bind to its molecular target then a nucleophile attacks the C-C double bond of the -unsaturated ester which forms a covalent attached that activates the signals leading to cell death. Evaluation of the six palmerolide A analogs provided insightful data about the substructures necessary to retain bioactivity against both UACC-62 melanoma cell as well as mammalian v-ATPase, however, no conn ection was able to made between their cytotoxicity and their ability to inhibit the v-ATPase enzyme. The bioactivity ratios calculated in Table 12 for the palmerolide A an alogs were just as in conclusive as those calculated for the other palmerolides in te rms of confirming their polymodal mechanism of action. There are still many questions to be answered regarding the efficacy of palmerolide A as a potential drug but great strides have been made in determining the minimal chemical scaffold necessary to reta in such activity. The SAR studies discussed in this dissertation along with those r ecently published by Nicolaou and coworkers provide a good starting point for further inve stigation. We are hopeful that at the very least, palmerolide A can be used as inspiration from nature in the form of a chemical scaffold for future drug candidates.

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CHAPTER THREE: Glycosphingolipids from Synoicum adareanum 3.1 Introduction to Glycosphingolipids Glycosphingolipids are a group of amphipa thic membrane lipids comprised of two main structural units: a sugar and a cer amide, and are important components of the cell membranes of plants and animals (F igure 21). The hydrophilic saccharide portion consists of either a simple or complex carbohydrate, and the hydrophobic ceramide portion consists of an am ide-linked fatty acid chain bound to a sphingosine base. Compounds from this class have demonstrated a broad range of bioactivity which is postulated to arise from their am phipathic nature. Immunomodulating,72,73 cytotoxic,74-76 antimicrobial,76-78 antifungal,77 antiviral and ionophor ic activity for Ca2+ ions,79,80 and are among their recently discovered biological activities. OH O HN R (CH2)nO OH HO HO O (CH2)n OH C B A R=H,OH Figure 23. Basic structural units of glycosphingolipids (a) saccharide, (b) fatty acid, (c) sphingosine base 67

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The isolation of glycoshi ngolipids and their derivativ es from marine sources including sponges,81-83 tunicates,84,85 sea stars,86-90 and sea anemones91,92 has been reported although many do not disclose bioactiv ity. Due to the amphipathicity of these compounds and their propensity to exist as homologues, their separation and structure elucidation has proven arduous. 3.2 Isolation of Glycosphingolipids from Synoicum adareanum In our chemical investigation of the Antarctic tunicate Synoicum adareanum a number of glycosphingolipids have been isolated from the more polar fractions of the organic extract (Scheme 1). Freeze-dried t unicate was extracted three times over a seventy-two hour period with a 1:1 methanol:dichloromethane solution to generate a crude organic extract, replacing with just enough fresh solv ent to cover the organism every twenty-four hours. The organism was extracted in the same manner using a 1:1 water:methanol solution to produce a crude a queous extract. The organic extracts were combined and concentrated in vacuo The crude organic extract was dissolved in a mixture of 1:1 water:ethyl acetate and tran sferred into a separatory funnel to be partitioned. The aqueous layer was rinsed severa l times with ethyl acetate and the organic layers combined. The combined organic laye rs were subsequently dried over anhydrous magnesium sulfate. Filtrati on was used to remove the drying agent and the organic partition was concentrated in vacuo This extract was subjected to flash chromatography using silica gel with an ethyl acetate:methanol gradient so lvent system resulting in ten fractions, with the glycoshingolip ids eluting at the more polar end of the gradient around 8-10% MeOH/EtOAc. These fractions were purified by repeated high 68

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pressure/performance liquid chromatography (HPLC) using both normal phase silica gel and reversed phase C-18 columns to afford a crude glycolipid mixture. Scheme 1. The extraction of Synoicum adareanum FreezeDriedTunicate(0.523kg) Extractedw/1:1CH2Cl2/MeOH(~67g)Extractedw/1:1MeOH/H2O(~130g) PartitionedwithEtOAc/H2O DriedEtOAclayer andconc. invacuo (19.41g) Conc.aqueouslayer invacuo (47.62g) RanonSiO2columnwithanincreasing polaritygradientofEtOAc/MeOH 100%EtOAc(~4711mg) 1%MeOH/EtOAc(~1588mg) 100%MeOH(~110mg) 2%MeOH/EtOAc(~171mg) 50%MeOH/EtOAc(~8.2mg) 4%MeOH/EtOAc(~156mg) 6%MeOH/EtOAc(~380mg) 8%MeOH/EtOAc(~132mg) 10%MeOH/EtOAc(~1609mg) 20%MeOH/EtOAc(~10.7mg) Partitionedwith n -BuOH/H2O Driedn-BuOHlayer andconc. invacuo (~33g) Conc.aqueouslayer invacuo (~100g) glycosphingolipids 69

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The crude mixture was acetylated with acetic anhydride and pyridine then purified by repeated HPLC on silica columns using n-hexane:ethyl acetate and n-hexane: isopropanol.90 Two fractions, each of which contained a mixture of peracetylated glycosphingolipids, were obtained followed by subsequent deacetylation with sodium methoxide in methanol to afford the natural products.90 A reversed phase HPLC separation gave rise to two series of homologous glycosphingolipids, 113ad and an inseparable mixture of 114ab O H HO H HO H H OH H O OH (CH2)6 OH NH O (CH2)n OH 1 2 3 4 5 6 7 8 9 10 11 CH3 CH3 1" 2" 18" 18 1' 2' 3 4' 5' 6' 113a n = 15 113b n = 16 113c n = 17 113d n = 18 O H HO H HO H H OH H O OH (CH2)y OH NH O (CH2)x OH 1 2 3 4 5 6 CH3 1" 2" 1' 2' 3' 4' 5' 6' CH3 114a x + y = 24 114b x + y = 25 70

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3.3 Structure Elucidation of Glycosphingolipids from S. adareanum Analysis of the NMR data acquired for 113a revealed that it contained distinct signals characteristic of a sugar, a fa tty acid chain, and an amide li nkage strongly suggesting that the compound was a glycosphingolipid (Figure 22). The structure of 113a was elucidated starting at the amide nitrogen ( 7.41), which could be correlated to the attached carbonyl ( 175.7) and alpha hydroxyl-bearing C-2 ( 71.6) by gHMBC. A series of gCOSY correlations allowed for the assignment of methylenes C-3 ( 35.2) and C-4 ( 25.4), which appeared to be the beginning of a fatty acid chain. The chain extended from C-5 ( 29.5) onward and consisted of an unknown number of coincident methylenes ( 29.5) with the proton chemical shift 1.24 and ended with the C-18 methyl ( 0.84). The length of the alkyl chain remained to be estab lished and would later be determined by mass spectrometry. 7 6 5 4 3 2 1 PPO H HO H HO H H OH H O OH (CH2)6 OH NH O (CH2)15 OH 1 2 3 4 5 6 7 8 9 10 11 CH3 CH3 1" 2" 18" 18 1' 2' 3' 4' 5' 6' M 71 Figure 24. 1H NMR spectrum of glycosphingolipid 113a in d6-DMSO, 500 MHz

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The C-2 ( 53.5) carbon of the shingosine base was confirmed to be on the other side of the amide linkage based on gHMBC correlations to the C-1 amide carbonyl ( 175.7) and gCOSY correlations to the amide proton ( 7.41). A oxymethine ( 70.6) could be placed adjacent to C-2 by gCOSY followed by a trans disubstituted olfefin ( JH,H= 15.6 Hz) located between C-4 ( 131.1) and C-5( 130.0). Two methylenes were found to separate the C-4/C-5 olefin from a C-8 ( 129.2)/C-9 ( 133.0) methyl branched, trisubstituted olefin whic h was conjugated to another trans disubstituted olefin (JH,H= 15.6 Hz) positioned between C-10 ( 135.4) and C-11( 127.1). The trisubstituted olefin was assigned as having E geometry based on a ROESY correlation between H2-7 ( 2.13) and H3-19 ( 1.64). The remainder of the sphingosine moiety consisted of a seven carbon alkyl chain, with clear gHMBC and gCOSY corre lations to each of the carbons allowing for their assignment without th e use of mass spectroscopic data. A search of the chemical literature revealed this 4,8,10triunsaturated, 9-methyl bran ched sphingoid base to be common among the sphingolipids isolated from marine invertebrates. The sugar moiety was determined to be attached to the ceramide by gHMBC correlations from H-1 ( 4.12) to C-1 ( 69.3) and C-2 ( 53.5). The anomeric proton was identified from its gHSQC correla tion to the anomeric carbon at 104.2 ppm. After the anomeric proton was identified the other 1H and 13C NMR signals of the sugar could be assigned using the gCOSY and gHSQC data. The gluco configuration of the sugar as well as its anomeric configuration was establishe d based on a comparison of the ring proton coupling constants (Table 22) to the literature values of previously identified glycospingolipids.90,93 72

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OH O H H HO C H O HO H HO C Coupling Angle ( ) J-value (Hz) axial/axial 180 8-14 Hz axial/equatorial 60 1-7 Hz equatorial/equatorial 60 1-7 Hz = 60 = 180 -form form Table 22. Calculated coupling constants of sugar protons A comparison of the chemical shifts of th e sugar protons in deuterated pyridine to those of the previously isolated oreacerebrosides,90 which are known to contain either a glucopyranose or galactopyranose residue, suggested that compound 113a was a glucosphingolipid. Further analysis by comparison of the vicinal proton coupling constants confirmed this assignment. A large coupling constant ( J1,2 = 7.7 Hz) was observed for the vicinal coupling of H-1 and H-2 of the su gar, indicating that the two protons were axial and therefore establishing that the sugar was in the -conformation (Table 22). The vicinal coupling of H-2 and H-3 was also large ( J2,3 = 8.5 Hz), which showed that H-1, H-2 and H3 were all axial. A large J -coupling value ( J3,4 = 9 Hz) between H-3 and H-4 confirmed that th e saccharide was in fact a glucopyranose residue, because a small J3,4 coupling constant would have been observed if a galactopyranose residue was present due to the equatorial H-4 (Figure 23). 73

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O OH H CH2OH OH H H HO H OH H 1 2 3 4 5 6O H HO CH2OH OH H H HO H OH H 1 2 3 4 5 6 galactopyranoside glucopyranoside Figure 25. Comparison of -galactopyranoside and -glucopyranoside Analysis of the APCI MS data showed an [M+Na]+ pseudomolecular ion for compound 113a of 776 m/z and a parent ion [M + 1]+ of 754 m/z along with insightful fragmentation. An [M-162]+ peak of 591 m/z was observed due to the cleavage of the sugar unit as well as an [M-470]+ peak of 283 m/z that accounted for the loss of a C18 2hydroxy fatty acyl group (with transfer of one H atom to the sphingosine). These fragments confirmed that the sphingosine base contained sevent een carbons as was postulated from the NMR data and that th e 2-hydroxy chain consisted of eighteen carbons. A search of the chemical literature reveal ed that compound 113a was a previously isolated glucosphingolipid from the ascidian Phallusia fumigata called phalluside 2.84 O H HO H HO H H OH H O OH OH NH O OH 591 283 Figure 26. The most characteristic fragment ions in the APCI -MS data of glycosphingolipid 113a 74

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The other three gl ycosphingolipids ( 113b-d ) were determined to be homologous to 113a based on a comparison of their 1H NMR data and their similar chromatographic properties. Compounds 113b-d were all found to contain a glucose sugar residue, and a ceramide composed of the same 4,8,10triuns aturated, 9-methyl branched sphingoid base and 2-hydroxy fatty acid. The only portion of the molecules that remained unassigned was the length of the alkyl chain of the fatty acid. Mass spectral an alysis using APCI as the ionization method revealed that the alkyl chains of the three glucosphingolipids differed from each other by only a single methylene. Compound 113b showed an [M + Na]+ of 790 m/z indicating that it had a molecular weight of 767 g/mol and formula of C44H81NO9 Fragmentation of the sugar from the ceramide portion resulted in a lo ss of 162, resulting in an [M + Na 162]+ of 628 m/z This peak revealed that the fatty acid portion of the ceramide contained one more methylene than compound 113a A fragmentation peak of moderate intensity was observed at 610 m/z accounting for the cleavage of the sugar unit as well as the dehydration of one of the alcohols of the ceramide ([M + Na 180]+). Compound 113c demonstrated an [M + Na]+ of 804 m/z indicating a molecular weight of 781 g/mol and molecular formula of C45H83NO9. An [M + Na 162]+ of 642 m/z was observed for the loss of the sugar unit and an [M + Na 180]+ for the loss of the sugar unit and dehydration of a hydroxyl group. An alysis of the mass spectrometric data revealed that the fatty acid chain of 113c contained one more methylene than 113b Compound 113d had a [M + Na]+ pseudomolecular ion peak at 818 m/z and an [M + Na 162]+ of 656 m/z for the cleavage of the glucose residue. The usual [M + Na 180]+ was observed at 612 m/z for the dehydration of a hydroxyl from the precursor ion 75

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[M + Na 162]+. The molecular formula was established as C46H85NO9, indicating that the alkyl chain of the fatty acid consisted of one more methylene than 113c A comparison of the 1H NMR spectra of compounds 114a and 114b (Figure 25) to those of 113ad showed that they were clearly two different series of glycosphingolipids. It was evident from the 1H NMR spectrum that 114a and 114b had fewer olefins, with the absence of the H-10 ( 6.00) proton being particularly appa rent. Further analysis revealed the absence of the C-19 olefinic methyl of the C-8/C-9 trisubstituted olefin as well based on the disappearance of the intense singlet at 1.64 ppm. The proton NMR spectrum suggested that the C-8/C-9 tr isubstituted olefin was not present either because of the disappearance of the H-7 ( 2.13) proton, which was originally shifted further downfield than the other methylenes because it was between to unsaturated systems. A full NMR data set of the inseparable mixture of 114a and 114b confirmed that the C-4/C-5 disubstituted alkene was the only unsaturation present in the molecule. Performing a cursory evaluation of the chemical shifts and general splitting pattern of the sugar protons to those of compounds 113ad strongly suggested that the saccharide residue was a glucopyranoside. Unfortunately, due to the lim ited amount of sample isolated an optimal proton NMR spectrum could not be obtained in order to extract all of the coupling constants. 76

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8 7 6 5 4 3 2 1 PPM Figure 27. 1H NMR spectrum of glycosphingolipid 114a-b in d6-DMSO, 500 MHz O H HO H HO H H OH H O OH (CH2)y O H NH O (CH2)x OH 1 2 3 4 5 6 CH3 1" 2" 1' 2' 3' 4' 5' 6' CH3 With the general structure of the gluc osphingolipid backbone established the only remaining structural features to be assigned were the numbe r of carbons in both the fatty acid chain and sphingosine base. Unlike the firs t series of compounds isolated, all of the carbons of the sphingosine unit could not be identified by NMR spectroscopy. Mass spectrometric data was utilized in order to determined the number of carbon atoms in the ceramide portions of both 114a and 114b however, it provided no insight into how they were distributed amongst the sphingosine and fatty acid. Compound 114a showed an [M + Na]+ pseudomolecular ion of 738 m/z, indicating a mass of 715 g/mol and a molecular formula of C40H77NO9. Dehydration of the saccharide resulted in an [M + Na 162]+ of 576 m/z meaning that the ceramide contained 34 carbon atoms. A de hydration of one of the alcohols was observed with an [M + Na 180]+ at 558 m/z. Compound 114b exhibited an [M + Na]+ of 752 m/z and an [M + Na 162]+ of 590 m/z signifying that the ce ramide moiety contained 35 carbon 77

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atoms, which was one more methylene than 114a An [M + Na 180]+ of 572 m/z arising from the loss of an alcohol from the ceramide was observed as it had been for all of the other glucosphingolipids isolated from S.adareanum Although the mass spectrometric data was helpful in discerni ng the number of carbon atoms present in the ceramide, more advanced measures will have to be taken in order to determine their distribution between the two co mponents of the ceramide. Li terature precedence suggests that either hydrolysis of both the sacchari de and the amide or tandem mass spectrometry can be used to achieve this. A search of the chemical literature fo r compounds similar to those discussed above resulted in quite a few compounds with the same general stru cture, many of them originating from marine organism s, but suggests that compounds 113b d as well as 114ab are new glucosphingolipids. Future isolati on of these compounds in higher yields is necessary in order to obtain the necessary physical data for publication and biological evaluation. 78

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CHAPTER FOUR: Anti-leishmanial Sterols from the Orange, Encrusting Antarctic Sponge Artemisina plumosa 4.1 Introduction to Leishmania Leishmaniasis is a group of neglected tropi cal diseases caused by twenty species of protozoan parasites of the genus Leishmania and are transmitted by thirty species of sandfly.94 These diseases afflict eighty-eight co untries, all in tropical or temperate regions, with an infection rate of appr oximately two million men, women, and children each year.95 The species of Leishmania are divided according to their geographic location of endemic species and are designated as ei ther Old World (Eastern Hemisphere) or New World (Western Hemisphere). The primary reservoir hosts for all Leismania spp. are vertebrates such as rodents, canids, marsupi als, and humans, however, the parasite is carried by the female phlebotamine sandfly of the genus Phlebotamus in the Old World and Lutzomyia in the New World.96 Infection by Leishmania spp. results in three clearl y distinguishable clinical manifestations regarded as visc eral, mucocutaneous, and cutaneous.97 In general, the visceral form is the most deadly and a ffects some of the internal organs, the mucocutaneous form affects the skin and mu cous membranes, and the cutaneous form affects the skin. Approximately ninety percent of vis ceral infections have been found to 79

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occur in Brazil, Bangladesh, India, Nepal and Sudan.96 Mucocutaneous infections are prevalent in Brazil, Peru, and Bolivia; wher eas cutaneous infections are prevalent in Afghanistan Brazil Iran Peru Saudi Arabia and Syria .96 There is no vaccine to prevent infection by Leishmania spp. instead there are only a few conventional drugs that have b een used to treat the infections.96 These drugs are not only expensive but the parasites are quickly developing a resistance to them and they have several side effects.98 For these reasons, the search fo r new anti-leishmanial drugs is of paramount interest to medicinal chemists Natural products have recently been found to be potential sources for the treatment of a number of tropical diseases caused by protozoans and other parasites.96,99,100 Many anti-leishmanial natu ral products have been discovered from the marine realm such as renieramycin A ( 115) from the Japanese marine sponge Neopetrosia sp.,101 euplotin C ( 116) from the marine ciliate protist Euplotes crassus,102 and isoakaterpin (117) from the sponge Callyspongia sp.103 These compounds have been of particular interest to our labor atory because of the implications they have for our research. Literature precedence that validated marine secondary metabolites as potential anti-leishmanial agents prompted a new collaboration to be made between our natural products laboratory and one with th e ability to perform high-throughput screening against Leishmania spp. parasites. 80

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N O O MeO H O O N O O OMe O O H OCOCH3 H H H H O O SO3Na SO3Na 1 15 116 117 In an effort to contribute to the search for new and more cost effective treatments for leishmaniasis our laboratory has screen ed over 800 extracts of Antarctic marine organisms against Leishmania donovani Only 13 of these crude extracts demonstrated inhibitory activity against the parasite. For many of the extracts, anti-leishmanial activity was lost upon separation, indicating a syne rgistic relationship amongst its components. However, the organic extract of the orange, encrusting sponge Artemisina plumosa demonstrated a significant increas e in inhibition upon separation. 81

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4.2 Bioassay-guided fractionation of Artemisina plumosa Freeze-dried sponge was extracted three times for a period of 24 hours in a 1:1 solution of dichloromethane:methanol, after which the organic extracts were combined and concentrated. The crude organic extract was subjected to sepa ration via MPLC using a normal phase silica gel pre-packed column a nd a tertiary gradient from 100% hexanes to 100% ethyl acetate then to 100% methanol. Bioassay of these initi al fractions allowed for the identification of the fraction respons ible for the originally observed activity. A proton NMR spectrum of this fraction indicated that it consisted mainly of a sterol(s). The sterol-containing fraction was separated a number of times using HPLC, alternating between normal and reversed phase to separate sterols by differences in both their rings and side chains. When it appeared th at a pure compound was obtained based on 1H NMR data and TLC, a full NMR data set was acqui red only to reveal that the sample was comprised of more than one sterol with very closely related structures. Of these inseparable mixtures, three general steroida l skeletons were identified all of which contained an exomethylene in the side chain. Th e least polar of the steroids were a series of steroidal ketones with the general structure shown in Figure 26, containing a single unsaturation independent of the side chai n exomethylene. Two dimensional NMR data suggested that this unsaturati on was located on the side chain as well. The second series was a group of sterols with what appeared to be an unsaturation in the B-ring in addition to the exomethylene (Figure 27). And the third series were sterols with unsaturations in both the B-ring and the side chain as well as the exomethylene (Figure 28). 82

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7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Ch i l Shift ( ) O A B C D O A B C D Figure 28. 1H NMR spectrum of steroid series 1 in CDCl3, 600 MHz 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 05 Ch i l Shift ( ) HO HO Figure 29. 1H NMR spectrum of steroid series 2 in CDCl3, 500 MHz 83

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7.0 65 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 15 1.0 0.5 Ch i l Shift ( ) HO H O Fi g ure 30. 1H NMR s p ectrum of steroid se r ies 3 in CDCl3 500 MHz Each of the steroid skeletons were tested against the Leishmania donovani parasites in order to determin e which of the three was most responsible for the observed bioactivity as well as to prio ritize the order in which to m ove forward with the fractions. The results revealed that all three steroidal sk eletons were active against the parasites but there was only a slight difference observed in their activity. The keto -steroid (Figure 26) demonstrated an IC50 of 5 g/mL, the doubly unsaturated sterol (Figure 27) demonstrated an IC50 of 4 g/mL, and the sterol containing thre e unsaturations (Figure 28) was the most active demonstrating an IC50 of 3.5 g/mL. A literature search for other sterols possessing anti-leishmanial activity showed sterols that also contain an exom ethylene group in the side chain ( 118123 ). Clerosterol ( 118)104 was isolated from the fruits of Cassia fistula and the norselic acids ( 119123) 84

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from the Antarctic sponge Crella sp.105 A superficial comparison of these sterols indicates that the basic four ring steroid sk eleton and the exomethylene group in the side chain are the only structurally features that all of them have in common. Since not all steroids possess anti-leishmanial activity it can be postulate d that the exomethylene plays an important role in the steroids bioactivity. H H O HOOC RO NorselicacidA( 119 ,R=H) NorselicacidE( 123 ,R=Ac) H H O HOOC HO H H NorselicAcidB( 120 ) H H O HOOC H NorselicAcidC( 121 ) H H O HOOC HO H NorselicAcidD( 122 ) HO 118 A search of the chemical literature for other sponges of the Artemisina genus surprisingly revealed that the chemical inve stigation of only one other sponge from the 85

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genus had been reported. The Antarctic sponge Artemisina apollinis was found to elaborate twenty-eight C25-C30 sterols as well as a few ster oidal ketones, all of which were separated using argentation chro matography and characterized by GC-MS.104 The complex mixture was able to be broken down into four steroidal nuclei and twelve side chains (Figure 29).106 O H R H R HO R HO H R HO A B CD Figure 31. Steroid nuclei and side chains found in Artemisina apollonis The variety and distribu tion of steroids found in A. apollonis are very similar to those observed in the collection of Artemisina plumosa currently being studied in our laboratory and suggests that an unusually high number of sterols may be present in this fellow Antarctic sponge as well. At least two other sponges in th e literature al so report a large number of sterols elaborated with the sa me general variation in steroidal nuclei and side chains.107,108 These closely related metabolites were also separated using more advanced methods of chromatography and characterized by GC-MS. 86

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It appears that furthe r separation of the sterol -containing fractions of A. plumosa is plausible using the methodol ogies reported in other papers for sponges containing such a large number of closely related sterols. Howe ver, it should be noted that the authors of these papers were only able to isolate a fe w pure sterols if any while the others were characterized strictly by GC-M S. In many instances, sterols were derivatized as their sterol acetates and separated by argentation chromatography to aid in further separation than that achieved using the usual normal and reversed phase conditions.104-107 The sterol acetates were then deacetylated and clean ed up using reversed phase HPLC. Although this method has the ability to provide addi tional separation of the sterols based on the number and location of the unsaturations the difficulty remains in separating those that only differ in alkyl substitution. There is still a great deal of research to be done on this Antarctic sponge and it should be continued for many reasons. Further chemical investigati on will hopefully lead to a detailed account of its many substituents which is important since only one other sponge of the Artemisina genus has been chemically investigated to date. In addition, the search for new treatments of leishmaniasis re mains a pressing issue and natural products still appear to be an underestimated and untapped source of anti-infective agents. 87

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CHAPTER FIVE: Conclusion The chemical investigation of the Antarctic tunicate Synoicum adareanum has led to the isolation of a number of secondary meta bolites that have yet to be fully evaluated for their biological activities. The palmerolides have proven to be potent inhibitors of vATPase as well as selectively cytotoxi c against UACC-62 melanoma cells, and are anticipated to operate by a polymodal mechanism of action. Preliminary structure activity relationship studies have provide d insight into the structural features necessary to retain cytotoxicity and v-ATPase inhi bition but have failed to de couple the bioactivities from one another. It is anticipate d that further studies into their mechanism of action will reveal that they bind to multiple receptors. Determining their binding site(s) will be the next step towards making the palmerolides pl ausible drug candidates. The stereochemical assignment of the palmerolides described in this dissertation is significant because complete characterization is necessary in order to move them into the next stage of drug discovery and it also allows fo r the total synthesis of thes e compounds in order to access them from another route than the natural source. S. adareanum was also found to elaborate a seri es of glucosphingolipids but they were isolated in such small quantities that bioassay and complete characterization was not possible. Extracting a larger amount of the tuni cate will lead to the isolation of these 88

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compounds in greater qu antitites which would allow for the acquisition of the necessary physical data as well as to provide enough samp le to submit to a number of assays that similar compounds have shown activity in. The Antarctic orange, encrusting sponge Artemisina plumosa was separated by bioassay-guided fractionation against Leishmania donovani which revealed that it produces a number of anti-leishmanial sterol and steroidal ketone constituents. Continued fractionation will lead to pure compounds that can be further evaluated as potential antileishmanial treatments. The search for treatments for leishmaniasis that are cheap and easy to administer is still underway and mari ne natural products are recently proving to be a potential source. The chemical investigation of both of th ese marine invertebrates has led to the discovery of a number of bioactive metabolite s that warrant further investigation into their activity. The research discussed in th e following chapters describes our efforts to contribute to both the field of natural products chemistry and drug discovery through the chemical investigation of Antarctic organisms. Although there is still a great deal of work to be done on these projects, significant cont ributions have been made and described in this dissertation. 89

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CHAPTER SIX: Experimental 6.1 General Experimental Procedures Optical rotations were measured on a Rudolph Research Analytical AUTOPOL IV digital polarimeter. IR and UV spectra were measured on a Nicolete Avatar 320FT infrared and a Hewlett-Packard 8452A diode array spectrophotometer, respectively. NMR spectra were recorded on Varian Inova 500 MHz and 600 MHz instruments. Chemical shifts are give as (ppm) with TMS as internal standard. The low resonance mass spectra were recorded on an Agilent Technologies LC/MSD VL electrospray ionization mass spectrometer. The high res onance mass spectra were recorded on an Agilent Technologies LC/MSD TOF electrospra y ionization spectrometer. Flash column chromatography was carried out on EM Science silica gel 60 of 230-400 mesh. High performance liquid chromatography was carri ed out on preparative YMC-Pack ODS-AQ reverse phase columns (250 x 20 mm) and analytical columns (250 X 10 mm) using an LC-8A Shimadzu multi-solvent delivery system, an SCL-10A Shimadzu system controller, and an SPD-10A Shimadzu UV-Vis detector. 90

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6.2 Biological Material The tunicate Synoicum adareanum and sponge Artemisina plumosa was collected by hand using SCUBA near Palmer Stati on on the Antarctic Peninsula between 20002008. The specimens were immediately frozen and kept frozen until extraction. A voucher specimen of the tunicate was identi fied by Dr. Linda Cole at the Smithsonian Institution, Washington, D.C. and the sponge identified by Dr. Rob van Soest at the Instituut voor Taxonomische Zoologie, Amsterdam, The Netherlands. 6.3 Extraction of S. adareanum and Isolation Secondary Metabolites Freeze dried S. adareanum was extracted with CH2Cl2/MeOH. The combined extract was concentrated and the residue was partitioned between EtOAc and H2O. Subsequently, the EtOAc la yer was dried with MgSO4 and concentrated in vacuo. The crude organic extract was subjected to fl ash column chromatography using a binary gradient of EtOAc/MeOH to give ten frac tions. The less polar (1 %-6% MeOH/EtOAc) of the fractions were further separated using 40% H2O/MeCN, and the palmerolidecontaining fractions obtained from the se paration were double purified using 20-30% H2O/MeOH to afford pure palmerolides A, C, D, E, F, G and K. The more polar fractions (20%-50% MeOH/EtOAc) were fu rther purified using 50% H2O/MeCN, and the palmerolide-containing fractions obtained from the separation were double purified using 50% H2O/MeOH to afford pure palmerolides B and H. The glycoshingolipids eluted toward the middle of the gradient around 8-10% MeOH/EtOAc. These fractions were separated by repeated high pressure/perfo rmance liquid chromatography (HPLC) using 91

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repeated normal phase silica gel and revers ed phase C-18 columns to afford a crude glycolipid mixture. Palmerolide B (96) : white solid; [ ]D 25= + 1.6 ( c = 0.1, MeOH); UV/Vis (MeOH): max ( )= 216 (1756), 240 (603); IR (thin film): 3514, 3433, 1648, 1633, 1510, 1392, 1275, 1190 cm-1; LR ESIMS (-) m/ z 663.3[M H]-, LR ESIMS (+) m/z 567.3 [M + H H2SO4]+, HR ESIMS (-) m/z 663.29417 (C33H47N2O10S requires. 663.29569), HR ESIMS (+) m/z 567.3430 (C33H46N2O6 requires 567.3429); 1H and 13C NMR, see Appendices A-3 and B-33. Palmerolide C (97): white solid; [ ]D 25= 27.1 (c = 0.1, MeOH); UV/Vis (MeOH): max ( ): 216 (1002), 248 (635); IR (thin film): 3364 (br), 2933, 1697, 1637, 1446, 1387, 1274, 1182, 1018, 978 cm-1; LR ESIMS (+) m/z 608.3 [M + H + Na]+, HR ESIMS (+) m/z 585.3534 (C33H49N2O7 requires 585.3540); 1H and 13C NMR, see Appendices A-4 and B-58. Palmerolide D (98): colorless solid; [ ]D 25= +67 ( c = 0.5, MeOH); UV/Vis (MeOH): max ( ): 216 (1742), 248 (528); IR (thin film): 3327, 2939, 2829, 2061, 1716, 1558. 1455, 1261, 1025, 975 cm-1; LR ESIMS (+) m/z 625.6, HR ESIMS (+) m/z 625.3864 (C36H53N2O7 requires 625.3853); 1H and 13C NMR, see Appendices A-1 and B-1. Palmerolide E (99): colorless solid, [ ]D 25= +17 ( c= 0.1, MeOH); UV/Vis (MeOH): max ( ): 216 (1295), 248 (645);IR (thin film): 3635, 2940, 2830,1715, 1637, 1540, 1387, 92

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1276, 1194, 1079, 938 cm-1; LR ESIMS (+) m/z 601.5, HR ESIMS (+) m/z 512.2634 (C27H39NO7Na requires 512.2624); 1H and 13C NMR, see Appendices A-1 and B-9. Palmerolide F (100): yellow solid; [ ]D 22= -67.1 ( c= 0.5, MeOH); UV/Vis (MeOH): max ( )= 213 (413), 262 (229); IR (thin film) 3340, 3013, 1705, 1642, 1524, 1318, 1216, 1187, 1018, 976 cm-1; LR ESIMS (+) m/z 607 [M + Na]+; HR ESIMS (+) m/z 607.3359 (C33H48N2O7Na requires 607.3359); 1H and 13C NMR data, see Appendices A-2 and B17. Palmerolide G (101) : yellow solid; [ ]D 22= -27.1 ( c= 0.1, MeOH); UV/Vis (MeOH): max ( )= 216 (719) 262 (403); IR (thin film) 3383, 2927, 1705, 1638, 1522, 1457, 1377, 1279, 1216, 1194, 1024 cm-1; HR ESIMS [M + Na]+ m/z 607.3350 (C33H48N2O7Na requires 607.3359) 1H and 13C NMR data, see Appendices A-2 and B-26. Palmerolide H (102): colorless solid, [ ]D 25= -27 ( c= 0.1, MeOH); UV/Vis (MeOH): max( ) = 217 (1232), 248 (712); IR (thin film) 3515, 3400 (br), 2925, 2856, 1653, 1633, 1517, 1208, 1040 cm-1; LR ESIMS (-) m/z 703.3 [M H]-, LR ESIMS (+) m/z 607.3 [M + H H2SO4]+, HR ESIMS m/z 703.3258 (C36H52N2O10S requires 703.3270); 1H and 13C NMR, see Appendices A-3 and B-49. Palmerolide K (104): yellow solid, [ ]D 22= -30.1 ( c= 0.1, MeOH); UV/Vis (MeOH): max ( ) = 216 (1492), 262 (705); IR (thin film): 3383, 2927, 1707,1379, 1215, 1025 cm-1; 93

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LR ESIMS (+) m/z 534 [M + HCOO]-, HR ESIMS (+) m/z 534.2704 (C28H40NO9 requires 534.6190); 1H and 13C NMR, see Appendices A-4 and B-69. 6.4 Acetylation of the Glycosphingolipids from S. adareanum The crude glycolipid-contain ing fraction was acetylated with Ac2O in pyridine at 25 C for 18 h. The reaction mixture was separated by repeated HPLC on silica columns using n-hexane/ethyl acetate and n-hexane/isopropanol. Two fractions, each of which contained a mixture of peracetylated glycosphingolipids, were obtained and subsequently deacetylated by dissolving each fraction in MeOH and adding a solution of MeONa in MeOH (0.4 M). The reaction was al lowed to proceed for 18 h at 25 C after which the crude reaction mixtures were concentrated and partitioned between chloroform and water. The organic layers were concentrated and purified by reversed phase HPLC using C-18 and a gradient from 20% H2O/MeOH to 100% MeOH. The first fraction injected contained glycosphingolipids 113ad which eluted towards the end of the gradient in the order of their lipophilicity as was to be expected (113a 113d). The longer the fatty acid side chain the higher the c oncentration of MeOH necessary for it to elute from the column. The second fraction inject ed contained glycosphingolipids 114ab and resulted in an inseparable mixture of the two. Glycosphingolipids 113a-d were purified on a normal phase HPLC column using 20% MeOH/CH2Cl2. 94

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6.5 Preparation of Palmerolide A Analogs 6.5.1 Preparation of 107. Palmerolide A was dissolved in methanol and subsequently transferred into a round bottom flask to be concentrated in vacuo It was then dissolved in a minimal am ount of methanol (1-2 drops) a nd then diluted to half of the volume of the reaction flask with ethanol. A magnetic stir bar and activated Pd/C was added to the flask followed by the attachment of an H2 (g) balloon to the mouth of the flask. The reaction was allowed to stir for 24 hour s at rt and quenched with ethyl acetate. The crude reaction mixture was filtered over Ce lite. The filtrate was concentrated then purified on an analytical reverse phase C18 HPLC column using 100% MeOH. Compound 107: Clear oil, [ ]D 30= -3.5 ( c = 0.4, CDCl3); UV/Vis (MeOH): max= 211 (341), 300 (86). 1H NMR (500 MHz, d6-DMSO) 7.71 (1H, m, 24-N H ), 6.31 (1H, br s, OCON H 2), 4.85 (1H, m, H-19), 4.49 (1H, m, H11), 3.43 (1H, m, H-10), 3.38 (1H, m, H7), 3.07 (2H, m, H2-24), 2.27 (2H, m, H2-2), 2.17 (2H, m, H2-2), 1.92 (1H, s, H-3), 1.64 (1H, m, H-20), 1.52 (2H, m, H2-23), 1.51 (1H, m, H-12a), 1.47 (2H, m, H2-9), 1.47 (1H, m, H-18a) 1.38 (1H, m, H-12b), 1.36 (2H, m, H2-8), 1.36 (1H, m, H-22), 1.32 (1H, m, H18b), 1.30 (1H, m, H-17), 1.26 (2H, H2-3, H2-4, H2-5, H2-6, H2-13, H2-14, H2-15, H2-16), 1.07 (2H, m, H2-21), 0.84 (3H,s, H3-4), 0.84 (3H, s, H3-5), 0.80 (3H, s, H3-27), 0.80 (3H, d, H3-26), 0.80 (3H, s, H3-25); 13C NMR (125 MHz, d6-DMSO) 173.4 (C-1), 171.9 (C-1), 157.7 (O C ONH2), 75.78 (C-11), 74.7 (C-19), 71.8 (C-7), 70.5 (C-10), 45.6 (C-3), 40.0 (C-24), 37.7 (C-22), 36.9 (C-23), 34.8 (C-2), 34.3 (C-2), 28.5-30.0 (C-3,C-4, C-5, C-6, C-8, C-9, C-12, C-13, C-14, C-15, C-16, C-17, C-18, C-20, C-21), 22.9 (C-4) 22.9 (C-5), 20.0 (C-26), 19.4 (C-27), 15.0 (C-25) LR ESIMS (+) m/z 599.4 [M+1]+, HR 95

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ESIMS (+) m/z 599.4678 [M+H]+ (C33H62N2O7 requires 599.4630), m/z 621.4463 [M+Na]+ (C33H62N2O7Na requires 621.4449), see Appendices B-75 and C-2. 6.5.2 Preparation of 108, 109, and 110. Palmerolide A was dissolved and transferred into a round bottom flask and concentrated in vacuo. One granule of DMAP and a magnetic stir bar was added to the reaction flask before placing it under nitrogen. Dry dichloromethane was added followed by five equivalents of both Hunigs base and 4bromo-benzoyl chloride. The reaction was allowed to stir overnight at rt before being quenched with methanol. The crude reaction mixture was separated on a reverse phase C18 analytical HPLC column equilibrated with 30% H2O:MeOH and run with 100% MeOH. Compound 108: white solid, [ ]D 22.7= -24.3 ( c = 0.2, CDCl3); UV/Vis (MeOH): max= 248 (634). 1H NMR (500 MHz, d6-DMSO) 9.86 (1H, d, 10.4, 24-N H ), 7.88 (2H, d, 8.5, Bz), 7.75 (2H, d, 8.5, Bz), 6.87 (1H, dd, 10.1, 14.2, H-24), 6.76 (1H, ddd, 5, 10, 15, H-3), 6.54 (2H, br, OCON H 2 ), 6.09 (1H, dd, 11.1, 14.6, H-15), 5.86 (1H, d, 14.2, H-23), 5.85 (1H, m, H-8), 5.82 (1H, d, 15, H-2), 5.71 (1H, br s, H-2), 5.70 (1H, m, H-9), 5.6 (1H, d, 11, H-16), 5.45 (1H, ddd, 4.5, 10.2, 14.7, H-14), 5.35 (1H, m, 10-O H ), 5.35 (1H, m, H-7), 5.15 (1H, d, 9.7, H-21), 4.86 (1H, m, H-19), 4.53 (1H, m, 5, 10.5, H-11), 4.21 (1H, m, H10), 2.70 (1H, qdd, 6.5, 7.5, 9.7, H-20), 2.17 (2H, m, H-4), 2.16 (1H, m, H-18a), 2.13 (3H, s, H3-5), 2.00 (1H, m, H-18b), 1.99 (2H, m, H2-13), 1.84 (3H, s, H3-4), 1.74 (1H, m, H-6a), 1.72 (3H, s, H3-25), 1.65 (1H, m, H-6b), 1.64 (3H, s, H3-27), 1.64 (1H, m, H12a), 1.41 (1H, m, H-5a), 1.17 (1H, m, H-5b), 0.95 (1H, m, H-12b), 0.91 (3H, d, 6.6, H326); 13C NMR (125 MHz, d6-DMSO) 165.3 (C-1), 164.1 (O C (O)pBzBr), 163.1 (C-1), 96

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156.5 ( C (O)NH2), 151.8 (C-3), 148.9 (C-3), 134.9 ( C 1-pBz), 132.6 (C-22), 131.9 (C-14), 131.9 ( C 2-pBz), 131.9 ( C 6-pBz), 131.7 (C-17), 131.1 ( C 3-pBz), 131.1 ( C 5-pBz) 130.2 (C21), 129.8 ( C 4-Br), 129.7 (C-16), 128.2 (C-8), 127.8 (C-9), 126.7 (C-15), 122.2 (C-24), 120.1 (C-2), 118.1 (C-2), 116.4 (C-23), 76.5 (C-7), 74.7 (C-11), 73.8 (C-19), 68.9 (C10), 44.5 (C-18), 40.4 (C-6), 36.8 (C-20), 31.9 (C-4), 29.4 (C-12), 29.4 (C-13), 27.0 (C4), 24.5 (C-5), 19.6 (C-5), 17.1 (C-26), 16.2 (C-27), 12.7 (C-25); LR ESIMS (+) m/z 767.2 [M + 1], HR ESIMS (+) m/z 767.2921 [M+H]+ (C40H51 BrN2O8 requires 767.2902), see Appendices B-80 and C-3. Compound 109: white solid, [ ]D 22.7= -23.6 ( c = 0.3, CDCl3); UV/Vis (MeOH): max= 253 (609). 1H NMR (500 MHz, d6-DMSO) 9.86 (1H, d, 10.4, 24-N H ), 7.92 (2H, d, 8.5, Bz), 7.78 (2H, d, 8.5, Bz), 6.87 (1H, dd, 10.5, 14.5, H-24), 6.76 (1H, ddd, 5.4, 9.5, 15.2, H-3), 6.56 (2H, br, OCONH 2 ), 6.15 (1H, dd, 10.8, 14.9, H-15), 5.87 (1H, d, 14.5, H-23), 5.81 (1H, d, 15.7, H-2), 5.71 (1H, br, s, H-2), 5.65 (1H, m, H-8), 5.65 (1H, m, H-9), 5.63 (1H, m, H-16), 5.48 (1H, m, H-14), 5.47 (1H, m, 4.4, 7.4, H-10), 5.16 (1H, d, 9.8, H-21), 4.87 (1H, m, H-11), 4.81 (1H, m, 4.7, H-19), 4.81(1H, m, 7-O H ), 3.94 (1H, ddd, 5.4, H7), 2.70 (1H, qdd, 6.5, 7.5, 9.7, H-20), 2.17 (2H, m, H2-4), 2.21 (1H, m, H-18a), 2.11 (3H, s, H3-5), 2.06 (2H, m, H2-13), 2.05 (1H, m, H-18b), 1.83(3H, s, H3-4), 1.71 (3H, s, H3-25), 1.63 (3H, s, H3-27), 1.59 (1H, m, H-12a), 1.45 (1 H, m, H-6a), 1.43 (1H, m, H12b), 1.39 (1H, m, H-5a), 1.36 (1H, m, H6b), 1.18 (1H, m, H-5b), 0.90 (3H, d, 6.7, H26); 13C NMR (125 MHz, d6-DMSO) 165.8 (C-1), 164.3 (O C (O)pBzBr), 163.6 (C-1), 156.8 ( C (O)NH2), 152.3 (C-3), 149.7 (C-3), 137.8 ( C 1-pBz), 133.1 (C-22), 132.4 ( C 2pBz), 132.4 ( C 6-pBz), 131.7 ( C 3-pBz), 131.7 ( C 5-pBz), 131.6 (C-14),130.3 (C-17), 130.1 (C-21), 129.7 (C-9), 127.4 ( C 4-Br), 123.9 (C-16), 128.1 (C-8), 127.6 (C-15), 122.6 (C97

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24), 121.4 (C-2), 118.6(C-2), 116.8 (C-23), 74. 9 (C-10), 74.2 (C11), 72.3 (C-19), 71.1 (C-7), 43.5 (C-18), 37.4 (C-6), 37.2 (C-20), 32.5 (C-4), 30.7 (C-12), 29.4 (C-13), 27.6 (C4), 24.4 (C-5), 19.9 (C-5), 17.4 (C-26), 16.6 (C-27), 13.1 (C-25); LR ESIMS (+) m/z 767.2 [M + 1], HR ESIMS (+) m/z 767.2921 [M+H]+ (C40H51 BrN2O8 requires 767.2902), see Appendices B-85 and C-3. Compound 110 : white solid, [ ]D 30= -47 ( c = 0.5, CDCl3); UV/Vis (MeOH): max= 212 (1896), 348 (152). 1H NMR (500 MHz, d6-DMSO) 9.85 (1H, d, 10.7, 24-N H ), 7.90 (2H, d, 8.8, Bz), 7.84 (2H, d, 8.3, Bz), 7.75 (2H, d, 8.8, Bz), 7.71 (2H, d, 8.3, Bz), 6.87 (1H, dd, 10.3, 14.7, H-24), 6.78 (1H, ddd, 5, 10, 15, H-3), 6.57 (2H, br, OCON H 2 ), 6.16 (1H, dd, 11.2, 14.7, H-15), 5.95 (1H, d, 4.9, H-9), 5.85 (1H, d, H-23), 5.83 (1H, m, H-2), 5.81 (1H, d, H-8), 5.70 (1H, br, s, H-2), 5.65 (1H, m, H-8), 5.63 (1H, m, H-16), 5.52 (1H, m, H-14), 5.52 (1H, m, H-10), 5.43 (1 H, m, H-7), 5.16 (1H, d, 9.8, H-21), 4.88 (1H, m, H-19), 4.87 (1H, m, H-11), 2.71 (1 H, qdd, 6.5, 7.5, 9.7, H-20), 2.17 (2H, m, H2-4), 2.21 (1H, m, H-18a), 2.11 (3H, s, H3-5), 2.06 (2H, m, H2-13), 2.05 (1H, m, H-18b), 1.83(3H, s, H3-4), 1.72 (1H, m, H-6a), 1.71 (3H, s, H3-25), 1.66 (1H, m, H-6b), 1.63 (3H, s, H3-27), 1.62 (1H, m, H-12a), 1.43 (1H, m, H-12b), 1.39 (1H, m, H-5a), 1.18 (1H, m, H-5b), 0.90 (3H, d, 6.7, H3-26); 13C NMR (125 MHz, d6-DMSO) 166.0 (C-1), 164.3 (O C A(O)pBzBr), 164.3 (O C B(O)pBzBr), 163.6 (C-1), 157.1 ( C (O)NH2), 152.6 (C3), 149.9 (C-3), 137.8 ( C A1-pBz), 134.9 ( C B1-pBz), 133.1 (C-22), 132.7 ( C A2-pBz), 132.7 ( C A6-pBz), 132.7 ( C B2-pBz), 132.7 ( C B6-pBz), 131.7 ( C A3-pBz), 131.7 ( C A5-pBz), 131.7 ( C B3-pBz), 131.7 ( C B5-pBz),131.6 (C-14),130.3 (C-17) 130.1 (C-21), 129.8 ( C A4-pBz), 127.4 ( C B4-Br), 123.9 (C-16), 128.1 (C-9), 127.8 (C -8), 127.6 (C-15), 122.6 (C-24), 121.4 (C-2), 118.6(C-2), 116.8 (C-23), 74.9 (C10), 74.2 (C-11), 72.3 (C-19), 71.1 (C-7), 98

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43.5 (C-18), 37.4 (C-6), 37.2 (C-20), 32.5 (C-4 ), 30.7 (C-12), 29.4 (C-13), 27.6 (C-4), 24.4 (C-5), 19.9 (C-5), 17.4 (C-26), 16.6 (C -27), 13.1 (C-25); LR ESIMS (+) m/z 971.2 [M + Na], m/z 967.3 [M+ NH3], HR ESIMS (+) m/z 949.2257[M+H]+ (C47H54Br2N2O9 requires 949.2269) see Appendices B-90 and C-4. 6.5.3 Preparation of Compounds 111and 112. Palmerolide A was dissolved and transferred into a round bottom flask and concentrated in vacuo The flask was placed under nitrogen and approximately 1.1 eq of NaOMe dissolved in dry MeOH was added. The reaction was allowed to stir for 3 hr at room temperature before being quenched with wet MeOH. The crude reaction mixture was se parated on a reverse pha se C18 analytical HPLC column equilibrated with 30% H2O:MeOH and run with 100% MeOH. Compound 111: [ ]D 30= -27 ( c = 0.3, CDCl3) UV/Vis (MeOH): max = 211 (2164). 1H NMR (500 MHz, d6-DMSO) 9.86 (1H, d, 10.5, 24-N H ), 6.86 (1H, dd, 10.1, 14.2, H24), 6.76 (1H, ddd, 5, 10, 15, H-3), 6.06 (1H, dd, 11.1, 14.6, H-15), 5.86 (1H, d, 14.2, H23), 5.76 (1H, d, 15, H-2), 5.70 (1H, br, s, H2), 5.61 (1H, d, 11, H-16), 5.57 (1H, m, H9), 5.49 (1H, m, H-8), 5.43 (1H, ddd, 4.5, 10.2, 14.7, H-14), 5.35 (1H, m, H-7), 5.15 (1H, d, 9.7, H-21), 4.86 (1H, m, H-19), 3.97 (1H, m, H-10), 3.83 (1H, m, H-7), 3.32 (1H, m, H-11), 2.70 (1H, qdd, 6.5, 7.5, 9.7, H-20), 2.17 (2H, m, H2-4), 2.18 (1H, m, H-18a), 2.13 (3H, s, H3-5), 2.00 (1H, m, H-18b), 1.91 (2H, m, H2-13), 1.84 (3H, s, H3-4), 1.74 (1H, m, H-6a), 1.72 (3H, s, H3-25), 1.65 (1H, m, H-6b), 1.64 (3H, s, H3-27), 1.64 (1H, m, H12a), 1.41 (1H, m, H-5a), 1.17 (1H, m, H-5b), 0.95 (1H, m, H-12b), 0.91 (3H, d, 6.6, H326); 13C NMR (125 MHz, d6-DMSO) 165.3 (C-1), 163.1 (C-1), 151.8 (C-3), 148.9 (C3), 132.6 (C-22), 131.9 (C-14), 131.7 (C-17), 130.2 (C-21), 129.8 ( C -Br), 129.7 (C-16), 99

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127.8 (C-9), 126.7 (C-15), 122.2 (C-24), 120.1 (C -2), 118.1 (C-2), 116.4 (C-23), 74.4 (C-19), 74.3 (C-11), 73.4 (C-10), 73.1 (C-7), 44.5 (C-18), 40.4 (C-6), 36.8 (C-20), 31.9 (C-4), 29.4 (C-12), 29.4 (C-13), 27.0 (C-4), 24.5 (C-5), 19.6 (C-5 ), 17.1 (C-26), 16.2 (C-27), 12.7 (C-25); LR ESIMS (+) m/z 542.4 [M+1], m/z 564.3 [M+ Na], HR ESIMS (+) m/z 542.34600 [M+H]+ HR ESIMS (+) m/z 767.2921 [M+H]+ (C32H47NO6 requires 542.3476), m/z 564.3293 [M+Na]+ (C32H47NO6Na requires 564.3296), see Appendices B-95 and C-5. Compound 112: [ ]D 30= -24 ( c = 0.5, CDCl3); UV/Vis (MeOH): max = 211 (1546). 1H NMR (500 MHz, d6-DMSO) 9.86 (1H, d, 10.5, 24-N H ), 6.86 (1H, dd, 10.1, 14.2, H24), 6.11 (1H, dd, 11.1, 14.6, H-15), 5.86 (1H, d, 14.2, H-23), 5.80 (1H, m, H-9), 5.71 (1H, br, s, H-2), 5.66 (1H, d, 11, H-16) 5.55 (1H, ddd, H-14), 5.51 (1H, m, H-8), 5.15 (1H, d, 9.7, H-21), 4.82 (1H, m, H-19), 4.25 (1 H, m, H-7), 3.71 (1H, m, H-10), 3.70 (1H, m, H-3), 3.24 (1H, m, H-11), 2.70 (1H, qdd, 6.5, 7.5, 9.7, H-20), 2.45 (1H, dd, H-2a), 2.23 (1H, m, H-2b), 2.18 (1H, m, H-18a), 2.13 (3H, s, H3-5), 2.00 (1H, m, H-18b), 1.91 (2H, m, H2-13), 1.84 (3H, s, H3-4), 1.81 (H-4a), 1.65 (1H, m, H-6a), 1.72 (3H, s, H3-25), 1.64 (3H, s, H3-27), 1.64 (1H, m, H-12a), 1.57 (1H, m, H-5), 1.49 (1H, m, H-6b), 1.15 (1H, m, H-4b), 0.95 (1H, m, H-12b), 0.91 (3H, d, 6.6, H3-26); 13C NMR (125 MHz, d6DMSO) 170.4 (C-1), 163.6 (C-1), 152.1 (C-3 ), 134.8 (H-8), 133.6 (C-22), 132.0 (C17),131.9 (C-9), 131.4 (C-14), 130.5 (C-21), 128 .0 (C-16), 127.3 (C-15), 122.8 (C-24), 118.9 (C-2), 117.1 (C-23), 75.5 (C-3), 75.0 (C19), 73.6 (C-11), 72.2 (C-7), 66.9 (C-10), 44.5 (C-18), 40.4 (C-2), 36.8 (C-20), 34.0 (C12), 31.4 (C-4), 29.4 (C -13), 28.7 (C-6), 27.0 (C-4), 19.6 (C-5), 18.8 (C-5), 17.7 (C26), 16.2 (C-27), 12.8 (C-25); LR ESIMS (+) m/z 542.3 [M+1], m/z 564.3 [M+ Na], HR ESIMS (+) m/z 542.3468 [M+H]+ 100

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(C32H47NO6 requires 542.3476), m/z 564.3283 [M+Na]+ (C32H47NO6Na requires 564.3296), see Appendices B-100 and C-6. 6.7 Cytotoxicity assay. All tissue culture components were purchased from Fisher Scientific. The UACC-62 human melanoma cell line was provided by the National Cancer Institute (NCI), Division of Cancer Treatment and Diagnosis (DCTD). The cancer cells were cultured in RPMI supplemented with 10% fe tal bovine serum, 2 mM glutamine, and 50 g/ml gentamicin. The cytotoxicity of the compounds were determined using the sulphorhodamine B (SRB) assay. A 96-well plate was inoculated with 100 L of cell suspension in each well at a plating density of 5,000 cells/well. The plate was incubated at 37C with 5% CO2, 95% air and 100% relative humi dity for 24 hours prior to addition of experimental drugs. The cells were dosed by adding 100 L of media containing 5 different drug concentrations (10 M, 1 M, 0.1 M, 0.01 M, 0.001 M) of the compounds to be tested to the respective wells in triplicate. The assay plate was incubated for 48 hours following dr ug addition at 37C with 5% CO2, 95% air and 100% relative humidity. The plate was removed from the incubator and the media aspirated from the wells. The cells were fixed using 50 L of 10% cold trichloroacetic acid and kept at 4C for 1 hour, subsequently being washed five times with distilled water. The plates were air-dried at room temperatur e and the cells staine d with 100 L of SRB solution (0.4% w/v SRB in 1% v/v acetic acid) for 10 minutes The plate was washed five times with 1% acetic acid to remove excess SRB present in the wells and allowed to air dry. Protein bound stain was sol ubilized with 200 L of tris base and mixed. The 101

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absorbance was measured at 515 nm using a BioTek Synergy 2 microtitre plate reader and the IC50 values determined by plotting the optical density of SRB against the drug concentration for each well using the program Table Curve 2D v5.01. 6.8 Leishmania Assay Using a Beckman Coulter Biomek 3000 automated sampler, 120 L of Hanks Buffered Salt Solution (HBSS) was added to e ach well of a 96-well plate. Mother plates were made by adding 5 L of crude extracts that had been suspended in DMSO at a concentration of 60 mg/mL to the first column of the 96-well plate. Serial dilutions were made across the plate resulting in tw elve concentrations ranging from g ng/mL. The wells of the mother plate were aspirated to ensure a homogenous mixture of the samples then 10 L was transferred from each well into a daughter plate. The Leishmania parasites were added to each well (90 L) of the daughter plate and plate incubated for 3 days at 37C. After 3 days approximately 20 L of (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetraz olium) (MTS) is added to each well and the plate incubated for 4 hr at 37C. The MTS reacted with a dehydrogenase enzyme in the live parasite and was reduced to a colored formazan product which was quantified by reading the plate at 490 nm. The amount of formazan detected was directly proportional to the number of living parasites. 102

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List of References 1. Sarker, S.D.; Latif, Z.; Gray, A. I., Eds. Natural Products Isolation Second Edition, Humana Press: Totowa, New Jersey, 2006. 2. Bhakuni, D.S.; Rawat, D.S. Bioactive Marine Natural Products Anamaya Publishers: New Delhi, India, 2005. 3. Newman, D.J.; Cragg, G.M. Natural produc ts as sources of new drugs over the last 25 years. J. Nat. Prod. 2007, 70, 461-477. 4. Aspirin. The Columbia Encyclopedia Sixth Edition, Columbia University Press: New York, 2009. 5. Newman, D.; Cragg, G.; Snader, K. Natura l products as sources of new drugs over the period 1981-2002. J. Nat. Prod. 2003, 66, 1022-1037. 6. Lee, M.D.; Manning, J.K.; Williams, D.R.; Kuck, N.A.; Testa, R.T.; Borders, D.B. Calicheamicins, a novel family of antitumor antibiotics. III: Isolation, purification and characterization of calickeamicins B1 Br, 1 Br, 2 I, 3 I, 1 I, 1 I and 1 I. J. Antibiotics 1989, 42, 1070. 7. Yotsu-Yamashita, M.; Kim, Y.H.; Dudle y, Jr., S.C.; Choudary,G.; Pfahnl, A.; Oshima, Y.; Daly, J.W. The structure of zetekitoxin AB, a saxitoxin analog from the Panamanian golden frog Atelopus zeteki : A potent sodium-channel blocker. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 4346-4351. 8. Haefner, B. Drugs from the d eep: marine natural products as drug candidates. Drug Discov. Today 2003, 8, 536-544. 103

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32. Simone, M.; Erba, E.; Damia, G.; Vikha nskaya, F.; Di Francesco, A.; Riccardi, R.; Bailly, C.; Cuevas, C.; Fernandez Sousa-Faro, J. M.; D'Incalci, M. Variolin B and its derivate deoxy-variolin B: New marine natural compounds with cyclindependent kinase inhibitor activity Eur. J. Cancer 2005, 41, 2366-2368. 33. Zhang,H. L.; Hua, H. M.; Pei, Y. H.; Yao, X. S. Three new cytotoxic cyclic acylpeptides from marine Bacillus sp. Chem. Pharm. Bull. 2004, 52, 1029-1030. 34. Gustafson, K.; Roman, M.; Fenical, W. The macrolactins, a novel class of antiviral and cytotoxic macrolides from a deep sea marine bacterium. J. Am. Chem. Soc. 1989, 111, 7519-7524. 35. Kim, H.-H.; Kim, W.-G.; Ryoo, I.-J.; Ki m, C.-J.; Suk, J.-E.; Han, K.-H.; Hwang, S.-Y.; Yoo, I.-D. Neruonal cell protecti on activity of macrolactin A produced by Actinomadura sp. J. Microbiol. Biotechnol. 1997, 7, 429-434. 36. Ankisetty, S.; Nandiraju, S.; Win, H.; Park, Y. C.; Amsler, C. D.; McClintock, J. B.; Baker, J. A.; Diyabalanage, T. K.; Pasaribu, A.; Singh, M. P.; Maiese, W. M.; Walsh, R. D.; Zaworotko, M. J.; Baker, B. J. Chemical investigation of predator deterred macroalgae from the Antarctic peninsula. J. Nat. Prod. 2004, 67, 12951302. 37. Davyt, D.; Enz, W.; Manta, E.; Navarro, G.; Norte, M. New chromenols from the brown alga Desmarestia Menziesii. Nat. Prod. Lett. 1997, 9, 305-312. 38. Gompel, M.; Leost, M.;. De Kier Joffe, E. B.; Puricelli, L.; Franco, L. H.; Palermo, J.; Meijer, L. Meridianins, a ne w family of protein kinase inhibitors isolated from the ascidian Aplidium meridianum. Bioorg. Med. Chem. Lett. 2004, 14, 1703-1707. 107

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70. Xie, X.-S.; Padron, D.; Liao, X.; Wang, J.; Roth, M.G.; De Brabander, J. K. Salicylihalamide A inhibits the V0 sector of the V-ATPase through a mechanism distinct from bafilomycin A1. J. Bio. Chem. 2004 279, 19755-19763. 71. Nicolau, K.C.; Leung, G. Y. C.; Dattatraya, H. D.; Ramakrishna, G.; Sun, Y.-P.; Lim, C. S.; Chen, D. Y.-K. Chemical synthesis and biological evaluation of palmerolide A analogues. J. Am. Chem. Soc. 2008, 130, 10019-10023. 72. Natori, T.; Morita, M.; Akimoto, K.; Koezuka, Y. Agelasphins, novel antitumor and immunostimulatory cerebrosides from the marine sponge Agelas mauritianus Tetrahedron 1994, 50, 2771-2784. 73. Natori, T.; Koezuka, Y.; Higa, T. Novel antitumor and immunostimulatory cerebrosides from the marine sponge Agelas mauritianus Tetrahedron Lett. 1993, 34, 5591-5592. 74. Pettit, G. R.; Yuping, T.; Knight, J. C. Antineoplastic Agents. 545. Isolation and structure of turbostatins 1-4 from the Asian marine mollusk Turbo stenogyrus. J. Nat. Prod. 2005, 68, 974-978. 75. Jin, W.; Rinehart, K. L.; Jares-Erijman, E. A. Ophidiacerebrosides: Cytotoxic glycosphingolipids containing a novel sphingosine from a sea star. J. Org. Chem ., 1994 59, 144-147. 76. Shin, J.; Seo ,Y. Isolation of new ceramides from the gorgonian Acabaria undulate. J. Nat. Prod. 1995, 58, 948-953. 77. Li, H.Y.; Matsunaga, S.; Fusetani, N. Ha licylindrosides, antifungal and cytotoxic cerebrosides from the marine sponge Halichondria cylindrata Tetrahedron 1995, 51, 2273-2280. 112

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78. Carter, G.T.; Rinehart, K. L., Jr. Apli diasphingosine, an antimicrobial and anti tumor terpenoid from an Aplidium sp. (marine tunicate). J Am. Chem. Soc. 1978, 100, 7441-7442. 79. Shibuya, H.; Hawasshima, K.; Sakagami, M.; Kawanishi, H.; Shimomura, M.; Ohashi, K.; Kitagawa, I. Sphingolipids and Gl ycerolipids. I. : Chemical structures and ionophoretic activities of soyacerebrosides I and II from soybean. Chem. Pharm. Bull. 1990, 38, 2933-2938. 80. Kobayashi, J.; Ishibashi, M.; Nakamura, H.; Hirata, Y.; Yamasu, T.; Sasaki, T.; Ohizumi, Y. Experientia 1988, 44, 800-802. 81. Hirsch, S.; Kashman, Y. New glycosphi ngolipids from marine organisms. Tetrahedron 1989, 45, 3897-3906. 82. Constantino, V.; Mangoni, A.; Fattorusso, E. Glycolipids from sponges, III. Glycosyl ceramides from the marine sponge Agelas conifer. Liebigs. Ann. Chem. 1995, 12, 2133-2136. 83. Mansoor, T. A.; Shinde, P.B.; Luo, X .; Hong, J.; Lee, C.-O.; Sim, C. J.; Son, B.W.; Jung, J.H. Renierosides, cerebrosides from a marine sponge Haliclona ( Reniera ) sp. J. Nat. Prod. 2007, 70, 1481-1486. 84. Duran, R.; Zubia, E.; Ortega, M.; Naranjo, S.; Salva, J. Phallusides, new glucosphingolipids from the ascidian Phallusia fumigata. Tetrahedron 1998, 54, 14597-14602. 85. Aiello, A.; Fattorusso, E.; Mangoni, A.; Menna, M. Three new 2,3-dihydroxy fatty acid glycosphingolipids from the Mediterranean tunicate Microcosmus sulcatus. Eur. J. Org. Chem. 2003, 4, 734-739. 113

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86. Higuchi, R.; Kagoshima, M.; Komori, T.; Biologically active glycosides from asteroidea, XXII. Glycosphingol ipids from the starfish Astropecten latespinosus, I. Structures of three new cerebrosides, astrocerebroside A, B, and C and of related nearly homogeneous cerebrosides. Liebigs. Ann. Chem. 1990, 659-663. 87. Kawano, Y.; Higuchi, R.; Komori, T. Biologically active glycosides from asteroidea, XIX. Glycosphingo lipids from the starfish Acanthaster planci 4. Isolation and structure of five new gangliosides. Liebigs. Ann. Chem. 1990 4350. 88. Higuchi, R.; Natori T.; Komo ri, T. Biologically active glycosides from steroidea, XX. Glycosphingolipids from the starfish Asterina pectinifera 1. Isolation and characterization of Acanthacerebrosides B and structure elucidation of related, nearly homogeneous cerebrosides. Liebigs. Ann. Chem. 1990, 51-55. 89. Kawano, Y.; Higuchi, R.; Isobe, R.; Komori T. Biologically active glycosides from Asteroidea, XIII. Glycosphi golipids from the starfish Acanthaster planci, 2. Isolation and structure of six new cerebrosides. Liebigs. Ann. Chem 1988 19-24. 90. Constantino, V.; de Rosa, C.; Fattoruss o, E.; Imperatore, C.; Mangoni, A.; Irace, C.; Maffettone, C.; Capasso, D.; Malorni, L.; Palumbo, R.; Pedone, C. Oreacerebrosides: bioactive cerebrosides with a triunsaturat ed sphingoid base from the sea star Oreaster reticulates Eur. J. Org. Chem. 2007 31, 5277-5283. 114

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91. Chebaane, K.; Guyot, M.; Occurrence of erythro-docosasphinga-4,8-dienine, as an ester, in Anemonia sulcata Tetrahedron Lett 1986, 27, 1495-1496. 92. Chakrabarty, M.; Batabyal, A.; Barua, A. K.; Patra, A. New ceramides from the hypotensive extract of a sea anemone, Paracondylactis indicus J. Nat. Prod., 1994, 57, 393-395. 93. Sun, D.-D.; Dong, W.-W.; Li, X.; Zhang, H. -Q. Isolation, structural determination and cytotoxicactivity of two new ceramides from the root of Isatis indigotica. Sci. China Ser. B-Chem. 2009, 52, 621-625. 94. Desjeux, P. Human leishmaniases: epid emiology and public health aspects. World Health Stat. Q. 1992, 45, 267-275. 95. Killick-Kendrick, R. Phlebotomine vectors of the leishmaniases: a review. Med. Vet. Entomol ., 1990, 4, 1-24. 96. Bora, D. Epidemiology of visceral leishmaniasis in India. Natl. Med. J. India 1999, 12, 62-68. 97. Rochaa, L.G.; Almeidab, J.R.G.S.; Macedob, R.O.; Barbosa-Filhob, J.M. A review of natural products w ith antileishmanial activity. Phytomedicine 2005, 12, 514. 98. Mishra, B.; Singh, R.; Srivastav, A.; Trip athi, V.; Tiwari, V. Fighting against leishmaniasis: search of alkaloids as future true potential anti-leishmanial agents. Mini-Reviews in Medicinal Chemistry 2009, 9, 107-123. 99. Wright, C.W.; Phillipson, J.D. Natural products and the development of selective antiprotozoal drugs. Phytother. Res. 2006, 4,127-139. 100. Donia, M.; Hamann, M.T. Marine natura l products and their potential 115

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applications as anti-infective agents. Lancet-Infec. Dis. 2003 3, 338. 101. Nakao, Y.; Shiroiwa, T.; Murayama, S.; Matsunaga, S.; Goto, Y.; Matsumoto, Y.; Fusetani, N. Identification of renieramycin A as an antileishmanial substance in a marine sponge Neopetrosia sp. Mar. Drugs 2004, 2, 55-62. 102. Savoia, D.; Avanzini, C.; Allice, T.; Callone, E.; Guella, G.; Dini, F. Antimicrobial activity of euplotin C, th e sesquiterpene taxono mic marker for the marine ciliate Euplotes crassus Antimicrobial Agents and Chemotherapy 2004, 48, 3828. 103. Gray, C.; de Lira, S.; Silva, M.; Pimenta, E.; Thiemann, O.; Oliva, G.; Hajdu, E.; Andersen, R.; Berlinck, R. Sulfated meroterpenoids from the Brazilian sponge Callyspongia sp. are inhibitors of the antileishmaniasis target Adenosine phosphoribosyl transferase. J. Org. Chem. 2006, 71, 8685-8690. 104. Sartorelli, P.; Andrade, S.; Melh em, M.; Prado, F.; Tempone, A. Isolation of antileishmanial sterol from the fruits of Cassia fistula using bioguided fractionation Phytother. Res. 2007, 21, 644. 105. Ma, W.; Mutka, T.; Vesley, B.; Amsler, M.; McClintock, J.; Amsler, C.; Perman, J.; Singh, M.; Maiese W.; Zaworotko, M.; Kyle, D.; Baker, B. Norselic acids A-E, highly oxidized anti-i nfective steroids that deter mesograzer predation, from the Antarctic sponge Crella sp. J. Nat. Prod. 2009, 72, 1842 1846. 106. Selde, A.; Deluca, E.; Gros, E.; Rovirosa, J.; San Martin, A.; Da ria, J. Steroids from aquatic organisms. 19. New sterols from the Antarctic sponge Artemisina apollonis. Zeitschrift fur Naturforschung 1999, 45, 83-86. 116

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107. Arreguin-Espinosa, R.; Arreguin, B.; Hernandez-Santoyo, A.; RodriguezRomero, A. Sterol Composition a nd Biosynthesis in the Sponge Spheciospongia vesparia. J. Chem. T echnol. Biotechnol. 1998, 72, 245-248. 108. Theobald, N.; Wells, R.; Djerassi, C. Minor and trace sterols in marine invertebrates. 8. Isolation, structure elucidation, and partial synthesis of two novel sterols-stelliferasterol and isostelliferasterol. J. Am. Chem. Soc. 1978, 100, 7677-7684. 109. Morris, L. J. Separations of lip ids by silver ion chromatography. J. Lipid Res. 1966, 7, 717-732. 117

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Appendices 118

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Appendix A: NMR Data Tables A-1 1H and 13C NMR Spectral Data for Palmerolides D ( 98) and E ( 99)a Palmerolide D Palmerolide E Positionb H C HMBC H C HMBC 1 165.5 165.2 2 5.76 ( d, 15.8) 120.6 1,4 5.77( d, 15.7) 120.3 1, 4 3 6.71 ( ddd 4, 11.5, 15.7) 149.4 1,2,5 6.74 ( ddd 4.3, 11.5, 15.7) 149.7 1, 4,5 4 a 2.14 ( m ) 32.4 2, 3,5 2.15 ( m ) 32.3 2,3,5 b 2.11 ( m ) 2.00 (m ) 2,3 5 a 1.31 ( m ) 24.2 1.31 ( m ) 25.0 b 1.05 ( m ) 1.07 (m ) 6 a 1.51 ( ddd 4.4, 7.7, 11.2 )37.8 1.50 ( ddd 4.4) 37.8 4,5,7,8 b 1.33 ( m ) 1.30 (m ) 5,7,8 7 3.82 ( ddd 4.4, 6.5, 7.9) 72.6 3.83 ( ddd 4.4, 6.8, 8.2) 72.5 9 8 5.53 ( dd 7.9, 15.3) 133.6 7, 9,10 5.54 ( dd 8.2, 15.4) 133.6 7,9,10 9 5.49 ( dd 2.9, 15.3) 129.0 7,8,11,10 5.49 ( dd 2.9, 15.4) 128.9 8,7,10 10 4.15 ( s ) 69.4 11 4.13 (br s ) 69.2 8-13 11 4.48 ( ddd 1.9, 5, 10.6) 75.2 9,10,12,13,1 4.47 ( ddd 1.5, 5.1, 10.7) 75.1 9-13,1 12 a 1.60 ( m) 29.5 1.61 29.4 13 b 1.01 ( m ) 1.05 (m ) 13 13 1.94 ( m ) 29.5 1.97 ( m ) 29.4 12,14,15 14 5.41 ( ddd 5, 10, 14.6) 132.7 13,16 5.43 ( ddd 5.2, 9.8, 14.8) 132.2 12/13,16 15 6.04 ( dd 11.6, 14.6) 126.4 6.05 ( dd 10.7, 14.8) 126.3 13,14,16 16 5.59 ( d 11.6) 127.8 14,15,17,18,25 5.61( d, 10.7) 128.0 14-18,25 17 131.7 131.1 18 a 2.16 ( dd 1.4, 12.8) 43.3 16,17,25 2.16 ( dd 1.4, 12.4) 43.0 16,17,25 b 2.00 ( dd 11, 12.8) 16,17,19,25 2.07 ( dd 11, 12.4) 16,17,19,25 19 4.84 ( ddd 1.4, 7.7, 11) 73.9 1,26 5.02 ( ddd 1.4, 7.5, 11) 72.6 1,17,20,21,26 20 2.68 ( qdd 6.9, 7.7, 9.7) 36.7 19,21 2.64 ( qdd 6.8, 7.5, 9.3) 38.1 19,21,22,26 21 5.14 ( d 9.7) 130.2 19,26,27 6.55 ( dd 1.5, 9.3) 154.9 19,20,23,25,27 22 132.0 138.7 23 5.86 ( d 14.6) 117.0 21,24,27 9.40 (s ) 195.6 21,22,27 24 6.85 ( dd 10.4, 14.6) 122.2 22 25 1.60 ( s ) 16.3 16,17,18 1.63 ( s ) 16.1 16,17,18 26 0.89 ( d 6.7) 17.2 19,20,21 1.01 (d 6.8) 15.5 19,20,21 27 1.70 ( s ) 12.8 21,22,23 1.68 ( s ) 9.2 21,22,23 1 162.9 2 5.81 (s ) 119.7 1,4,8 3 152.7 4 3.34(s ) 40.3 2,3,5 5 143.0 6 4.72 (s ) 111.9 4,7 7 1.61 (s ) 22.0 4,5,6 8 1.76 (s ) 25.1 2,3,4 1 156.8 156.7 1-NH2 6.45 (br) 6.48 (br) 7-OH 4.53 (d 3.8) 4.70 ( d 4.1) 10-OH 5.19 ( m 4.5) 5.18 ( d 4.9) 24-NH 9.94 ( d 10.3) 1 a500 MHz for 1H, 125 MHz for 13C, DMSOd6. 119

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Appendix A (continued) A-2 1H and 13C NMR Spectral Data for Palmerolides F ( 100) and G ( 101)a Palmerolide F Palmerolide G Positionb H C HMBC H C HMBC 1 165.3 165.5 2 5.77 (1H, d, 16.0) 120.5 1,4 5.76 (d, 15.4) 120.5 1,4 3 6.71 (1H, ddd, 4.8, 10.3, 16.0)149.2 1,2, 4,5 6.71 (ddd, 4.8, 10, 15.4) 149.4 1,2,4,5 4 a 2.11 (1H, m) 32.3 2,3,5,6 2.10 (m) 32.6 3,5 b 1.84 (m) 1.84 (m) 5 a 1.31 (1H, m) 24.9 6,7 1.28 (m) 25.1 b 0.98 (1H, m) 7 1.06 (m) 6 a 1.48 (1H, m) 37.7 5,7,8 1.49 (ddd,4.4,7.7, 11) 38.6 5,7,8,9 b 1.30 (1H, m) 5,7,8 1.31 4,7,8 7 3.83(1H, d, 4.0) 72.5 6,9 3.83(ddd, 4.4, 6.5, 7.9) 73.4 8 5.55 (1H, dd, 7.8, 15.9) 133.5 6,7, 9,10 5.54 (dd, 7.9, 14.8) 134.5 7,10 9 5.48 (1H, dd, 2.3, 15.8) 128.8 8 5.46 (dd, 2.2, 14.8) 129.7 7,10 10 4.14 (1H, br s) 69.1 9,12/13 4.13 (s) 70.2 11 11 4.48 (1H dd, 4.6, 11.2) 75.0 9,10,12/13,1 4.48 (ddd, 1.5, 4.9, 10.8) 76.1 9-12,1 12 a 1.60 (1H, m) 29.3 11,12/13 1.13 30.1 10,13 b 0.97 (1H, m) 12/13,14 0.98 (m) 13 1.94 (2H, m) 29.3 12/13,14,15 1.98(m) 30.1 14,15 14 5.41 (1H, ddd, 4.2, 10.4, 15.0)131.9 12/13,16 5.41 (ddd, 4.4,10.1,14.8) 132.9 13-16 15 6.05 (1H, dd, 11.2, 15.0) 126.3 16,17,12/13 6.03 (dd, 11.1, 14.8) 127.3 13-16 16 5.59 (1H, d, 10.6) 127.7 14,15,18,25 5.58 (d, 11.1) 128.6 16,18 17 131.5 132.5 18 a 2.15 (1H, m, 1.7) 43.2 16,17,25 2.16 (dd, 1.5, 13.2) 37.1 16-19 b 1.99 (1H, q, 12) 16,17,19,20,25 1.98 (dd, 12, 13.2) 16,17 19 4.84 (1H, 1.7, 7.9, 12) 73.7 1,17,21,26 4.82 (ddd 1.5, 8, 12) 74.5 17,18,26 20 2.68 (qdd, 6.7,7.9,9.6) 36.5 18,19,21,22,26 2.65 (qdd, 6.6, 8, 10) 40.3 19,21,22,25 21 5.14 (1H, d, 9.6) 130.1 19,20,23,26, 27 5.01(d, 10) 129.3 18,23 22 132.3 132.1 23 5.87 (1H, d, 15.0) 116.9 21,22,24,2 7 6.19(d,14.3) 109.8 21,24 24 6.77 (1H, dd, 10.3, 15.0) 121.8 22,23,1 6.93(dd, 10.4, 14.3) 125.4 21,22,23 25 1.61 (3H, s) 16.1 16,17,18 1.60 (s) 17.1 14,16 26 0.90 (3H, d, 7.6) 17.0 19,21,20 0.89 (d, 6.6) 18.2 19,20,21 27 1.70 (3H, s) 12.6 21,22,23 1.76 (s) 21.1 21,22,23 1 167.4 164.1 2 2.91 (1H, s) 44.5 1,3,4,5 5.68 118.7 1,4,5,27 3 139.8 153.6 4 a 4.82 (1H, br s ) 113.5 2,5 2.12 20.1 1-5 b 4.79 (1H, br s) 2,3,5 5 1.71 (3H, s) 22.2 1,2,3,4 1.84 28.0 2,3,4 7-OH 4.69 (1H, br s) 4.73 (d,4.4) 10-OH 5.17 (1H, d, 3.8) 9 5.20 (d, 4,7) 24-NH 9.93 (1H, d, 10.3) 23,24,1 9.97 (d,10.2) 1 OCONH2 6.48 (2H, br) 156.6 6.48 (s) 157.6 a500 MHz for 1H, 125 MHz for 13C, DMSOd6. 120

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Appendix A (continued) A-3 1H and 13C NMR Spectral Data for Palmerolides B ( 96) and H ( 102) a Palmerolide B Palmerolide H Positionb H C HMBC H C HMBC 1 168.2 168.2 2 5.70 (1H, d, 15) 122.3 1,4 5.70 (d, 15.5) 122.3 3,4 3 6.74 (1H, ddd, 4, 11, 15) 150.7 1,4,5 6.75 (ddd, 4, 11.5, 15.5) 150.7 1,2,4 4 a 2.14 (1H, m) 34.0 2.14 (1H, m) 33.9 2,3 b 2.10 (1H, m) 2.10 (1H, m) 2,3,5 5 a 1.34 (1H, m) 26.1 1.32 (1H, m) 26.1 3,4 b 1.14 (1H, m) 1.10 (1H, m) 3,6 6 a 1.55 (1H, m) 31.8 1.56 (1H, m) 31.8 6,8,9 b 1.08 (1H, m) 1.10 (1H, m) 7 4.57 (1H, m) 77.4 6,8, O C ONH2 4.58(1H, m) 77.5 7,9,10 8 4.20 (1H, m) 72.1 9 4.20 (1H, m) 72.2 7,11,12 9 5.72 (1H, m) 133.4 8, 10 5.74 (1H, m) 132.3 8,12 10 5.64 (1H, m) 131.0 8, 11 5.63 (1H, m) 131.1 9,10,13, O C ONH2 11 4.64 (1H, m) 81.6 4.66 (1H, m) 81.9 9-12,1 12 a 1.79 (1H, m) 36.6 11 1.80 (1H, m) 36.6 13,15,16 b 1.53 (1H, m) 11 1.55 (1H, m) 13 a 1.96 (1H, m) 30.9 1.96(1H, m) 30.8 14,15,25 b 1.21 (1H, m) 1.21 (1H,m) 14,16 14 5.38 (1H, ddd, 4, 10.5, 14.5) 133.3 15 5.38 (1H, m) 133.4 5,16,18,25 15 6.01 (1H, dd, 10.5, 14.5) 128.0 14,17 6.02 (1H, dd, 10, 14.5) 128.1 16,19,25 16 5.57 (1H, d, 10.9) 129.8 15,18 5.57(1H, d, 11.5) 128.1 16,18 17 132.8 133.0 18 a 2.15 (1H, m) 45.2 2.15 (1H, m) 45.2 9,20,26,27 b 1.99 (1H, m) 45.2 20 2.00 (1H, m) 18,19,21,26 19 4.82 (1H, m) 76.6 1,20 4.84 (1H, m) 76.2 21,23,27 20 2.68 (qdd, 5,10,10) 38.7 2.68 38.7 21,24,27 21 5.08 (1H, d, 10) 132.2 19,20,22,25 5.09(1H, d, 10) 132.3 22,23,1 22 134.5 134.5 15,16,18 23 5.85 (1H, d, 15) 119.6 21,22,24,25 5.87(1H, d, 14.6) 119.7 22,24,25 24 6.86 (1H, dd, 15) 122.7 22,23,1 6.87(1H, d, 14.6) 122.8 22,23,1 25 1.58 (3H, s) 16.7 16,17,18 1.59 (3H, s) 13.2 16,18 26 0.88 (3H, d, 6.5) 17.7 19,21,20 0.89 (3H, d, 6.7) 17.7 19,20,21 27 1.71 (3H, s) 13.2 21,22,23 1.72 (3H, s) 16.6 22,23 1 166.7 166.0 2 5.64 (1H, br s) 118.9 1,3,4,5 5.76 (1H, s) 120.5 3 154.9 155.7 1,4,8 4 1.81 (3H, s) 27.7 1,2,3,5 3.44 (2H, s) 42.1 5 2.09 (3H, s) 20.4 1,2,3,4 144.6 2-7 6 4.69 (2H, s) 112.6 7 1.61 (3H, s) 22.4 4,8 8 1.76 (3H, s) 24.8 4,5,6 O C ONH2 159.8 159.9 2,3,4 a500 MHz for 1H, 125 MHz for 13C, CD3OD. 121

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Appendix A (continued) A-4 1H and 13C NMR Spectral Data for Palmerolides C ( 97) and K ( 104) a Palmerolide C Palmerolide K Positionb H C HMBC H C HMBC 1 166.9 64.5 2 5.73 (1H, d, 15.5) 122.0 1,4 5.75 (1H,d, 14.7) 120.7 1,4 3 6.77 (1H, ddd, 7.4, 7.5, 15.5) 149.8 1,2,4, 5 6.81 (1H, ddd,7.4, 7.5,14.7) 149.4 1,4,5 4 a 2.11(1H, m) 31.7 2,6 2.15 (1H, m) 30.7 2,3,5 b 2.06 (1H, m) 2,3,5,6 2.13 (1H, m) 2,3,5 5 a 1.93 (1H, m) 32.2 6,7 1.37 (1H, m) 27.9 3,4,6,7 b 1.84 (1H, m) 3,7 1.34 (1H, m) 3 6 5.54 (1H, m) 131.8 5,7 5.55 (1H, m) 130.6 2,7,8 7 5.58(1H, d, 4.0) 131.1 6,8 5.55(1H, m) 130.6 2,6,8 8 3.96 (1H, dd, 7.8, 15.9) 72.8 6,7,9 3.97 (1H, dd, 7.8, 15.9) 72.0 6,7 9 3.56 (1H, m) 75.6 10 3.57 (1H, m) 74.8 6,7,10,11 10 4.56 (1H, ddd, 2.2,7.2,10.5) 74.2 11,O C ONH2 4.56 (1H, m, 2.5,7,10.5) 73.4 9,11,12 11 a 1.49 (1H, m) 28.7 10,12 1.34 (1H, m) 29.8 12 b 1.30 (1H, m) 10,12 1.30 (1H, m) 12 12 1.95 (1H, m) 30.1 10 1.54 (1H, m) 30.1 13,14 13 a 2.01 (1H, m) 30.1 15 1.97(1H, m) 31.2 12,14 b 1.89 (1H, m) 15 1.92 (1H, m) 12,14 14 5.46 (1H, ddd, 5, 10, 15) 132.3 13,15,16 5.48 (ddd, 5, 10, 15) 132.1 15 15 6.08 (1H, dd, 11.5, 15.0) 127.2 16,17,12/13 6.10 (1H,dd, 11.8, 14.7) 126.4 13 16 5.63 (1H, d, 11.5) 128.8 14,15,25 5.67 (!H,d, 11.8) 128.3 17,18,26 17 132.5 130.1 18 a 2.14 (1H, m,10.5) 44.1 16,17,19,20,25 2.18(dd) 42.9 1620,26 b 1.58 (1H, m,) 16,17,19,25 19 4.85 (1H, ddd,2.2,7.8,10.5) 74.7 1,17,18, 20,21,26 5.04 (1H, q,6.7) 72.5 1,17,21,26 20 2.70 (1H,qdd, 6.7, 7.8,9.5) 37.4 16,19, 21,22,26 2.98 (1H, m) 37.3 19-22,25 21 5.15 (1H, d, 9.5) 130.5 19,20, 23,26,27 6.58(1H, d, 10.3) 154.5 20,23,24,25 22 133.3 138.4 23 5.85 (1H, d, 14.6) 117.2 21,22,24,27 9.41(1H, s) 194.9 22,24 24 6.85 (1H, dd, 9, 14.6) 122.8 22,23,1 1.68(3H, s) 8.62 21,22,23 25 1.69 (3H, s) 13.3 16,17,18 1.03 (3H,d,6.7) 15.6 19,20,21 26 0.90 (3H, d, 7) 17.8 19,20,21 1.61(3H,s) 15.6 16,17,18 27 1.59 (3H, s) 16.5 21,22,23 1 164.0 2 5.68 (1H, s) 118.8 1,3,4,5 3 152.5 4 1.82 (3H, s) 27.4 1,2,3,5 5 2.11 (3H, s) 20.3 1,2,3,4 8-OH 4.62 (1H, d, 5.12) 4.62 ( 1H, d, 5) 6,7,8,9 9-OH 4.72 (1H, d, 4.76) 9 4.72 (1H, d, 5) 8,9,10 24-NH 9.85 (1H, d, 10) 23,24,1 OCONH2 6.37 (2H, br) 157.6 6.36 (2H, br) 156.7 a500 MHz for 1H, 125 MHz for 13C, DMSOd6 122

PAGE 136

Appendix A (continued) A-5 1H and 13C NMR Spectral Data for glycosphingolipid 113a Position C (mult.) H (mult., J [Hz]) 1 a b 69.3 (CH2) 3.93 (dd, 10.5, 5.8) 3.53 (dd, 10.5, 3.8 2 53.5 (CH) 3.83 (m) 2-NH -7.41 (d, 9.5) 3 70.6 (CH) 3.99[a] 3-OH -4.94 (m) 4 131.1 (CH) 5.43 (d, 15.6, 6.7) 5 130.0 (CH) 5.59[a] 6 31.6 (CH2) 2.01(m) 7 27.0 (CH2) 2.13 (m) 8 129.2 (CH) 5.39 (t, 7.0) 9 133.0 (C) -10 135.4 (CH) 6.00 (d, 15.6) 11 127.1 (CH) 5.51[a] 12 32.0 (CH2) 2.02 (m) 13 25.2 (CH2) 1.35[a] 14 29.2 (CH2) 1.25[a] 15 29.2 (CH2) 1.25[a] 16 32.2 (CH2) 1.22[a] 17 22.7 (CH2) 1.26[a] 18 14.6 (CH3) 0.84 (t, 6.5) 19 12.9 (CH3) 1.64 (s) 1 104.2 (CH) 4.12 (d, 7.6) 2 73.1 (CH) 2.96 (8.5, 3.8) 2-OH -4.97[a] 3 77.9 (CH) 3.13 (dd, 9.0, 3.5) 3-OH -4.96[a] 4 69.7 (CH)v 3.04 (dd, 9.0, 4.4) 4-OH -4.91(m) 5 74.0 (CH) 3.14[a] 6 a b 61.6 (CH2) 3.66 (dd, 10, 5.9) 3.41(dt, 11.7, 5.7) 6-OH -4.52 (t, 6.0) 1 175.7 (C=O) -2 71.6 (CH) 3.81 (m) 2-OH -4.00[a] 3 a b 35.2 (CH2) 1.56 (m) 1.41 (m) 4 25.4 (CH2) 1.29[a] 6-17 29.5 (CH2) 1.24[a] 18 14.6 (CH3) 0.84 (t, 6.5) 500 MHz for 1H, 125 MHz for 13C, DMSOd6 123

PAGE 137

Appendix A (continued) A-6 1H and 13C NMR Spectral Data for glycosphingolipids 114a-b Position C (mult.) H 1 a b 69.3 (CH2) 3.93 3.53 2 53.5 (CH) 3.83 2-NH -7.41 3 70.6 (CH) 3.99 3-OH -4.94 4 131.1 (CH) 5.43 5 130.0 (CH) 5.59 6 y 29.0(CH2) 1.26 y-terminus 14.6 (CH3) 0.84 1 104.2 (CH) 4.12 2 73.1 (CH) 2.96 2-OH -4.97 3 77.9 (CH) 3.13 3-OH -4.96 4 69.7 (CH)v 3.04 4-OH -4.91 5 74.0 (CH) 3.14 6 a b 61.6 (CH2) 3.66 3.41 6-OH -4.52 1 175.7 (C=O) -2 71.6 (CH) 3.81 2-OH -4.00 3 a b 35.2 (CH2) 1.56 1.41 4 25.4 (CH2) 1.29 5 x 29.5 (CH2) 1.24 x-terminus 14.6 (CH3) 0.84 500 MHz for 1H, 125 MHz for 13C, DMSOd6 124

PAGE 138

125 Appendix B: Selected NMR Data 10 8 6 4 2 0 PP M B-1 1H NMR Spectrum of Palmerolide D ( 98 ) in DMSOd6, 500MHz O O NH2 O H N O HO O OH

PAGE 139

126 Appendix B (continued) 200 150 100 50 0 PP M B-2 13C NMR Spectrum of Palmerolide D ( 98 ) in DMSOd6, 500MHz

PAGE 140

127 Appendix B (continued) B-3 gCOSY of Palmerolide D ( 98 ) in DMSOd6, 500 MHz

PAGE 141

128 Appendix B (continued) B-4 gHMBC of Palmerolide D ( 98 ) in DMSOd6 500 MHz

PAGE 142

129 Appendix B (continued) B-5 gHMQC of Palmerolide D ( 98 ) in DMSOd6, 500 MHz

PAGE 143

130 Appendix B (continued) B-6 ROESY of Palmerolide D ( 98 ) in DMSOd6, 500 MHz

PAGE 144

131 Appendix B (continued) 10 9 8 7 6 5 4 3 2 1 0 ChilShift() B-7 1H NMR spectrum of palmerolide D (+)-MTPA diester in DMSOd6, 500 MHz H H HH H H H H O H R H O MTPA O H M T PA 5 6 7 8 9 10 11 12 1.23 1.59 1.42 5.48 5.57 5.84 5.53 4.78 1.43

PAGE 145

132 Appendix B (continued) 9 8 7 6 5 4 3 2 1 ChilShift() B-8 1H NMR spectrum of palmerolide D (-)-MTPA diester in DMSOd6, 500 MHz H H HH H H H H O H R H O MTPA O H M T PA 5 6 7 8 9 10 11 12 1.24 1.62 1.45 5.51 5.71 5.75 5.62 465 1.26

PAGE 146

133 Appendix B (continued) 8 6 4 2 PP M B-9 1H NMR Spectrum of Palmerolide E ( 99 ) in DMSOd6, 500MHz O O NH2 O HO O OH O

PAGE 147

134 Appendix B (continued) 200 150 100 50 0 PP M B-10 13C NMR Spectrum of Palmerolide E ( 99 ) in DMSOd6, 500MHz

PAGE 148

135 Appendix B (continued) B-11 gCOSY of Palmerolide E ( 99 ) in DMSOd6, 500 MHz

PAGE 149

136 Appendix B (continued) B-12 gHMBC of Palmerolide E ( 99 ) in DMSOd6, 500 MHz

PAGE 150

137 Appendix B (continued) B-13 gHMQC of Palmerolide E ( 99 ) in DMSOd6, 500 MHz

PAGE 151

138 Appendix B (continued) B-14 ROESY of Palmerolide E ( 99 ) in DMSOd6, 500MHz

PAGE 152

139 Appendix B (continued) 9 8 7 6 5 4 3 2 1 0 ChilShift() B-15 1H NMR spectrum of palmerolide E (+)-MTPA diester in DMSOd6, 500 MHz H H HH H H H H O H R H O MTPA O H M T PA 5 6 7 8 9 10 11 12 1.23 1.66 5.49 5.56 5.82 5.52 4.78 1.40

PAGE 153

140 Appendix B (continued) 9 8 7 6 5 4 3 2 1 0 ChilShift() B-16 1H NMR spectrum of palmerolide E (-)-MTPA diester in DMSOd6, 500 MHz H H HH H H H H O H R H O MTPA O H M T PA 5 6 7 8 9 10 11 12 1.24 1.69 5.51 5.73 5.75 5.60 4.64 1.25

PAGE 154

141 Appendix B (continued) 10 8 6 4 2 0PP M B-17 1H NMR Spectrum of Palmerolide F ( 100 ) in DMSOd6, 500 MHz O O NH2 O H N O HO O OH

PAGE 155

142 Appendix B (continued) 00 150 100 50 PP M B-18 13C NMR Spectrum of Palmerolide F ( 100 ) in DMSO-d6, 500MHz

PAGE 156

143 Appendix B (continued) B-19 gCOSY of Palmerolide F ( 100 ) in DMSOd6, 500 MHz

PAGE 157

144 Appendix B (continued) B-20 gHMBC of Palmerolide F ( 100 ) in DMSOd6 500 MHz

PAGE 158

145 Appendix B (continued) B-21 gHMQC of Palmerolide F ( 100 ) in DMSOd6 500 MHz

PAGE 159

146 Appendix B (continued) B-22 ROESY of Palmerolide F ( 100 ) in DMSOd6, 500 MHz

PAGE 160

147 Appendix B (continued) 10 9 8 7 6 5 4 3 2 1 0 ChilShift() B-23 1H NMR spectrum of palmerolide F (+)-MTPA diester H H HH H H H H O H R H O MTPA O H M T PA 5 6 7 8 9 10 11 12 1.23 1.59 1.39 5.48 5.56 5.83 5.52 4.76 1.37

PAGE 161

148 Appendix B (continued) 1 0 9 8 7 6 5 4 3 2 1 ChilShift() B-24 1H NMR spectrum of palmerolide F (-)-MTPA diester H H HH H H H H O H R H O MTPA O H M T PA 5 6 7 8 9 10 11 12 1.25 1.63 1.41 5.52 5.71 5.76 5.62 4.67 1.24

PAGE 162

149 Appendix B (continued) 10 8 6 4 2 0 PP M B-25 1H NMR Spectrum of Palmerolide G ( 101 ) in DMSOd6, 500MHz O O O NH2 O OH HO NH O

PAGE 163

150 Appendix B (continued) 180 160 140 120 100 80 60 40 20 0 PP M B-26 13C NMR Spectrum of Palmerolide G ( 101 ) in DMSOd6, 500MHz

PAGE 164

151 Appendix B (continued) B-27 gCOSY of Palmerolide G ( 101 ) in DMSOd6, 500 MHz

PAGE 165

152 Appendix B (continued) B-28 gHMBC of Palmerolide G ( 101 ) in DMSOd6, 500 MHz

PAGE 166

153 Appendix B (continued) B-29 gHSQC of Palmerolide G ( 101 ) in DMSOd6, 500 MHz

PAGE 167

154 Appendix B (continued) B-30 ROESY of Palmerolide G ( 101 ) in DMSOd6, 500 MHz

PAGE 168

155 Appendix B (continued) 9 8 7 6 5 4 3 2 1 0 ChemicalShift(ppm) B-31 1H NMR spectrum of palmerolide G (+)-MTPA diester H H HH H H H H O H R H O MTPA O H M T PA 5 6 7 8 9 10 11 12 1.27 1.60 4.79 1.41 1.04 1.41

PAGE 169

156 Appendix B (continued) 0 9 8 7 6 5 4 3 2 1 0 ChemicalShift(ppm) B-32 1H NMR spectrum of palmerolide G (-)-MTPA diester H H HH H H H H O H R H O MTPA O H M T PA 5 6 7 8 9 10 11 12 1.17 1.63 1.41 4.65 1.29

PAGE 170

157 Appendix B (continued) 7 6 5 4 3 2 1 PP M B-33 1H NMR Spectrum of Palmerolide B ( 96 ) in CD3OD, 500 MHz O O H N O O NH2 O HO O O S O O

PAGE 171

158 Appendix B (continued) 0 0 150 100 50 0 PP M B-34 13C Spectrum of Palmerolide B ( 96 ) in CD3OD, 500 MHz

PAGE 172

159 Appendix B (continued) B-35 gCOSY of Palmerolide B ( 96 ) in CD3OD, 500 MHz

PAGE 173

160 Appendix B (continued) B-36 gHSQC of Palmerolide B ( 96 ) in CD3OD, 500 MHz

PAGE 174

161 Appendix B (continued) B-37 gHMBC of Palmerolide B ( 96 ) in CD3OD, 500 MHz

PAGE 175

162 Appendix B (continued) B-38 ROESY of Palmerolide B ( 96 ) in CD3OD, 500 MHz

PAGE 176

163 Appendix B (continued) B-39 gHSQMBC of Palmerolide B ( 96 ) in CD3OD, 500 MHz

PAGE 177

164 Appendix B (continued) B-40 gHSQMBC of Palmerolide B ( 96 ) in CD3OD, 500 MHz 2,3JC,H H8-C6,7,9,10

PAGE 178

165 Appendix B (continued) B-41 gHSQMBC of Palmerolide B ( 96 ) in CD3OD, 500 MHz 2,3JC,H H7-C6,8

PAGE 179

166 Appendix B (continued) 7 6 5 4 3 2 1 PP M B-42 1H NMR Spectrum of Compound 103 in CD3OD, 500 MHz O O H N O OH HO

PAGE 180

167 Appendix B (continued) 160 140 120 100 80 60 40 20 0 ChilShift() B-43 13C NMR Spectrum of Compound 103 in CD3OD, 500 MHz

PAGE 181

168 Appendix B (continued) B-44 gCOSY of Compound 103 in CD3OD, 500 MHz

PAGE 182

169 Appendix B (continued) B-45 gHMBC of Compound 103 in CD3OD, 500 MHz

PAGE 183

170 Appendix B (continued) B-46 gHMQC of Compound 103 in CD3OD, 500 MHz

PAGE 184

171 Appendix B (continued) 8 7 6 5 4 3 2 1 PP M B-47 1H NMR spectrum of (+)-MTPA monoester of Compound 103 H H OH MTPA H OH H H 6 7 8 9 10 6.25 5.75 5.18 4.95 1.71

PAGE 185

172 Appendix B (continued) 7 6 5 4 3 2 1 PP M B-48 1H NMR spectrum of (-)-MTPA monoester of Compound 103 H H OH MTPA H OH H H 6 7 8 9 10 6.26 5.76 5.17 4.94 1.67

PAGE 186

173 Appendix B (continued) 7 6 5 4 3 2 1 PP M B-49 1H NMR Spectrum of Palmerolide H ( 102 ) in CD3OD, 500 MHz O O H N O O NH2 O HO O O S O O

PAGE 187

174 Appendix B (continued) 0 160 140 120 100 80 60 40 20 0 B-50 13C NMR Spectrum of Palmerolide H ( 102 ) in CD3OD, 500 MHz

PAGE 188

175 Appendix B (continued) B-51 gCOSY of Palmerolide H in ( 102 ) CD3OD, 500 MHz

PAGE 189

176 Appendix B (continued) B-52 gHSQC of Palmerolide H ( 102 ) in CD3OD, 500 MHz

PAGE 190

177 Appendix B (continued) B-53 gHMBC Spectrum of Palmerolide H ( 102 ) in CD3OD, 500 MHz

PAGE 191

178 Appendix B (continued) B-54 ROESY of Palmerolide H ( 102 ) in CD3OD, 500 MHz

PAGE 192

179 Appendix B (continued) B-55 gHSQMBC of Palmerolide H ( 102 ) in CD3OD, 500 MHz

PAGE 193

180 Appendix B (continued) B-56 gHSQMBC of Palmerolide H ( 102 ) in CD3OD, 500 MHz 2,3JC,H H8-C6,7,9,10

PAGE 194

181 Appendix B (continued) B-57 gHSQMBC of Palmerolide H ( 102 ) in CD3OD, 500 MHz 2JC,H H7-C8

PAGE 195

180 Appendix B (continued) 10 8 6 4 2 PP M B-58 1H NMR Spectrum of Palmerolide C ( 97 ) in d6-DMSO, 500 MHz O H N O O OH OH O O NH2

PAGE 196

181 Appendix B (continued) 150 100 50 0PP M B-59 13C NMR Spectrum of Palmerolide C ( 97 ) in d6-DMSO, 500 MHz

PAGE 197

182 Appendix B (continued) B-60 gCOSY Spectrum of Palmerolide C ( 97 ) in d6-DMSO, 500 MHz

PAGE 198

183 Appendix B (continued) B-61 gHSQC of Palmerolide C ( 97 ) in d6-DMSO, 500 MHz

PAGE 199

184 Appendix B (continued) B-62 gHMBC of Palmerolide C ( 97 ) in d6-DMSO, 500 MHz

PAGE 200

185 Appendix B (continued) B-63 ROESY of Palmerolide C ( 97 ) in d6-DMSO, 500 MHz

PAGE 201

186 Appendix B (continued) B-64 gHSQMBC of Palmerolide C ( 97 ) in d6-DMSO, 500 MHz

PAGE 202

187 Appendix B (continued) B-65 gHSQMBC of Palmerolide C ( 97 ) in d6-DMSO, 500 MHz 2,3 J C,H

PAGE 203

188 Appendix B (continued) B-66 gHSQMBC of Palmerolide C ( 97 ) in d6-DMSO, 500 MHz 2,3 J C,H C11 (28.7 ppm) H10 (4.56 ppm) OCONH2(156.7 ppm) C8 (72.87 ppm) C9 (7455 )

PAGE 204

189 Appendix B (continued) 9 8 7 6 5 4 3 2 1 0 ChilShift() B-67 1H NMR spectrum of palmerolide C (+)-MTPA monoester H H OH MTPA H OH H OR 6 7 8 9 10 5.70 5.79 5.53 3.92 4.58

PAGE 205

190 Appendix B (continued) 9 8 7 6 5 4 3 2 1 ChilShift() B-68 1H NMR spectrum of palmerolide C (-)-MTPA monoester H H OH MTPA H OH H OR 6 7 8 9 10 5.79 5.89 5.56 3.81 4.54

PAGE 206

191 Appendix B (continued) 10 8 6 4 2 PP M B-69 1H NMR Spectrum of Palmerolide K ( 104 ) in d6-DMSO, 500 MHz O O OH OH O O NH2 O

PAGE 207

192 Appendix B (continued) 200 50 100 50 0 PP B-70 13C Spectrum of Palmerolide K ( 104) in d6-DMSO, 500 MHz

PAGE 208

193 Appendix B (continued) B-71 gCOSY of Palmerolide K ( 104 ) in d6-DMSO, 500 MHz

PAGE 209

194 Appendix B (continued) B-72 gHSQC of Palmerolide K ( 104 ) in d6-DMSO, 500 MHz

PAGE 210

195 Appendix B (continued) B-73 gHMBC Spectrum of Palmerolide K ( 104 ) in d6-DMSO, 500 MHz

PAGE 211

196 Appendix B (continued) B-74 ROESY of Palmerolide K ( 104 ) in d6-DMSO, 500 MHz

PAGE 212

197 Appendix B (continued) 9 8 7 6 5 4 3 2 1 0 PP M B-75 1H Spectrum of Palmerolide A Hydrogenation Product ( 107 ) in DMSOd6, 500 MHz O O O NH2 O HO H N O OH

PAGE 213

198 Appendix B (continued) 180 160 140 120 100 80 60 40 20 PP M B-76 13C Spectrum of Palmerolide A Hydrogenation Product ( 107 ) in DMSOd6, 500 MHz

PAGE 214

199 Appendix B (continued) B-77 gCOSY of Palmerolide A Hydrogenation Product ( 107 ) in DMSOd6, 500 MHz

PAGE 215

200 Appendix B (continued) B-78 gHMBC of Palmerolide A Hydrogenation Product ( 107 ) in DMSOd6, 500 MHz

PAGE 216

201 Appendix B (continued) B-79 gHMQC of Palmerolide A Hydrogenation Product ( 107 ) in DMSOd6, 500 MHz

PAGE 217

202 Appendix B (continued) 10 8 6 4 2 PP M B-80 1H NMR Spectrum of Palmerolide A C-7 p -bromobenzoate ( 108 ) in DMSOd6, 500 MHz O O O NH2 O HO H N O OR 7 R= O B r

PAGE 218

203 Appendix B (continued) 160 140 120 100 80 60 40 20 ChilShift() B-81 13C Spectrum of Palmerolide A Palmerolide A C-7 p -bromobenzoate ( 108 ) in DMSOd6, 500 MHz

PAGE 219

204 Appendix B (continued) B-82 gCOSY of Palmerolide A Palmerolide A C-7 p -bromobenzoate ( 108 ) in DMSOd6, 500 MHz

PAGE 220

205 Appendix B (continued) B-83 gHMBC of Palmerolide A C-7 p -bromobenzoate ( 108 ) in DMSOd6, 500 MHz

PAGE 221

206 Appendix B (continued) B-84 gHMQC of Palmerolide A C-7 p -bromobenzoate ( 108 ) in DMSOd6, 500 MHz

PAGE 222

207 Appendix B (continued) 10 8 6 4 2 PP M B-85 1H NMR Spectrum of Palmerolide A C-10 p -bromobenzoate ( 109 ) in DMSOd6, 500 MHz O O O NH2 O RO H N O OH 8 R= O B r

PAGE 223

208 Appendix B (continued) 160 140 120 100 80 60 40 20 PP M B-86 13C NMR Spectrum of Palmerolide A C-10 p -bromobenzoate ( 109 ) in DMSOd6, 500 MHz.

PAGE 224

209 Appendix B (continued) B-87 gCOSY of Palmerolide A C-10 p -bromobenzoate ( 109 ) in DMSOd6, 500 MHz

PAGE 225

210 Appendix B (continued) B-88 gHMBC of Palmerolide A C-10 p -bromobenzoate ( 109 ) in DMSOd6, 500 MHz

PAGE 226

211 Appendix B (continued) B-89 gHMQC of Palmerolide A C-10 p -bromobenzoate ( 109 ) in DMSOd6, 500 MHz

PAGE 227

212 9 8 7 6 5 4 3 2 1 PP M Appendix B (continued) B-90 1H NMR Spectrum of Palmerolide A C-7/C-10 p -bromobenzoates ( 110 ) in DMSOd6, 500 MHz O O O NH2 O R2O H N O OR1 9 R1=R2= O B r O B r

PAGE 228

213 Appendix B (continued) 160 140 120 100 80 60 40 20 0 PP M B-91 13C NMR Spectrum of Palmerolide A C-7/C-10 p -bromobenzoates ( 110 ) in DMSOd6, 500 MHz

PAGE 229

214 Appendix B (continued) B-92 gCOSY of Palmerolide A C-7/C-10 p -bromobenzoates ( 110 ) in DMSOd6, 500 MHz

PAGE 230

215 Appendix B (continued) B-93 gHMBC of Palmerolide A C-7/C-10 p -bromobenzoates ( 110 ) in DMSOd6, 500 MHz

PAGE 231

216 Appendix B (continued) B-94 gHMQC of Palmerolide A C-7/C-10 p -bromobenzoates ( 110 ) in DMSOd6, 500 MHz

PAGE 232

217 Appendix B (continued) 10 8 6 4 2 PP M B-95 1H NMR Spectrum of Palmerolide C-11 alcohol ( 111 ) in DMSOd6, 500MHz O O H N O OH HO OH 7 10 11

PAGE 233

218 Appendix B (continued) B-96 13C NMR Spectrum of Palmerolide C-11 alcohol ( 111 ) in DMSOd6, 500MHz

PAGE 234

219 Appendix B (continued) B-97 gCOSY of Palmerolide C-11 alcohol ( 111 ) in DMSOd6, 500 MHz

PAGE 235

220 Appendix B (continued) B-98 gHMBC of Palmerolide C-11 alcohol ( 111 )in DMSOd6, 500 MHz

PAGE 236

221 Appendix B (continued) B-99 gHSQC of Palmerolide C-11 alcohol ( 111 ) in DMSOd6, 500 MHz

PAGE 237

222 Appendix B (continued) 10 9 8 7 6 5 4 3 2 1 PP M B-100 1H NMR Spectrum of Palmer olide A C-3 alcohol ( 112 ) in DMSOd6, 500MHz O O H N O OH HO OH 7 10 11 OH

PAGE 238

223 Appendix B (continued) B-101 13C NMR Spectrum of Palm erolide A C-3 alcohol ( 112 ) in DMSOd6, 500MHz. 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ChilShift()

PAGE 239

224 Appendix B (continued) B-102 gCOSY of Palmerolide A C-3 alcohol ( 112 ) in DMSOd6, 500 MHz

PAGE 240

225 Appendix B (continued) B-103 gHMBC of Palmerolide A C-3 alcohol ( 112 ) in DMSOd6, 500 MHz

PAGE 241

226 Appendix B (continued) B-104 gHMQC of Palmerolide A C-3 alcohol ( 112 ) in DMSOd6, 500 MHz

PAGE 242

227 7 6 5 4 3 2 1 PP M Appendix B (continued) O H HO H HO H H OH H O OH (CH2)6 OH NH O (CH2)15 OH 1 2 3 4 5 6 7 8 9 10 11 CH3 CH3 1" 2" 18" 18 1' 2' 3' 4' 5' 6' B-105 1H NMR Spectrum of glycosphingolipid 113a in d6-DMSO, 500 MHz C43H79NO9

PAGE 243

228 Appendix B (continued) 180 160 140 120 100 80 60 40 20 PPM B-106 13C NMR Spectrum of glycosphingolipid 113a in d6-DMSO, 125 MHz

PAGE 244

229 Appendix B (continued) B-107 gCOSY data of glycosphingolipid 113a in d6-DMSO, 500 MHz

PAGE 245

230 Appendix B (continued) B-108 gHMBC data of glycosphingolipid 113a in d6-DMSO, 500 MHz

PAGE 246

231 Appendix B (continued) B-109 gHSQC data of glycosphingolipid 113a in d6-DMSO, 500 MHz

PAGE 247

232 Appendix B (continued) B-110 ROESY data of glycosphingolipid 113a in d6-DMSO, 500 MHz

PAGE 248

233 Appendix B (continued) 8 7 6 5 4 3 2 1 0 ChilShift() B-111 1H NMR Spectrum of glycosphingolipid 113a in d5-pyridine, 500 MHz

PAGE 249

234 Appendix B (continued) 7 6 5 4 3 2 1 PP M B-112 1H NMR Spectrum of glycosphingolipid 113b in d6-DMSO, 500 MHz O H HO H HO H H OH H O OH (CH2)6 O H NH O (CH2)16 OH 1 2 3 4 5 6 7 8 9 10 11 CH3 CH3 1" 2" 18" 18 1' 2' 3' 4' 5' 6' C44H81NO9

PAGE 250

235 Appendix B (continued) 7 6 5 4 3 2 1 PP M B-113 1H NMR Spectrum of glycosphingolipid 113c in d6-DMSO, 500 MHz O H HO H HO H H OH H O OH (CH2)6 OH NH O (CH2)17 OH 1 2 3 4 5 6 7 8 9 10 11 CH3 CH3 1" 2" 18" 18 1' 2' 3 4' 5' 6' C45H83NO9

PAGE 251

236 Appendix B (continued) 7 6 5 4 3 2 1 PP M B-114 1H NMR Spectrum of glycosphingolipid 113d in d6-DMSO, 500 MHz. O H HO H HO H H OH H O OH (CH2)6 OH NH O (CH2)18 OH 1 2 3 4 5 6 7 8 9 10 11 CH3 CH3 1" 2" 18" 18 1' 2' 3 4' 5' 6' C46H85NO9

PAGE 252

237 Appendix B (continued) 7 6 5 4 3 2 1 PPM B-115 1H NMR Spectrum of glycosphingolipid 114a-b in d6-DMSO, 500 MHz. O H HO H HO H H OH H O OH (CH2)y O H NH O (CH2)x OH 1 2 3 4 5 6 CH3 1" 2" 1' 2' 3' 4' 5' 6' CH3

PAGE 253

238 Appendix B (continued) 150 100 50 PP M B-116 13C NMR Spectrum of glycosphingolipid 114a-b in d6-DMSO, 500 MHz

PAGE 254

239 Appendix B (continued) B-117 gCOSY data of glycosphingolipid 114a-b in d6-DMSO, 500 MHz

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240 Appendix B (continued) B-118 gHMBC data of glycosphingolipid 114a-b in d6-DMSO, 500 MHz

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241 Appendix B (continued) B-119 gHMQC data of glycosphingolipid 114a-b in d6-DMSO, 500 MHz

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242 Appendix C: Mass Spectral Data C-1 LR ESIMS (positive) data for 103

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243 Appendix C (continued) C-2 HR ESIMS (positive) data for 107

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244 Appendix C (continued) C-3 HR ESIMS (positive) data for 108 and 109

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245 Appendix C (continued) C-4 HR ESIMS (positive) data for 110

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246 Appendix C (continued) C-5 HR ESIMS (positive) data for 111

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247 Appendix C (continued) C-6 HR ESIMS (positive) data for 112

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248 Appendix C (continued) C-7 LR APCI-MS (positive) data for glycoshingolipid 113

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249 Appendix C (continued) C-8 LR APCI-MS (positive) data for glycoshingolipid 113a

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250 Appendix C (continued) C-9 LR APCI-MS (positive) data for glycoshingolipid 113b

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251 Appendix C (continued) C-10 LR APCI-MS (positive) data for glycoshingolipid 113c

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252 Appendix C (continued) C-11 LR APCI-MS (positive) data for glycoshingolipid 113d

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253 Appendix C (continued) C-12 LR APCI-MS (positive) data for glycoshingolipids 114a-b

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254 Appendix D: Bioassay Data D-1 Palmerolide A UACC-62 data plot Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.95461361 DF Adj r^2=0.93645905 FitStdErr=0.085229247 Fstat=77.121135 a=0.090975965 b=0.9548449 c=0.011263561 d=0.91447148 0.001 0.01 0.1 1 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.97622158 DF Adj r^2=0.96671021 FitStdErr=0.065711532 Fstat=150.53475 a=0.080745796 b=0.78674703 c=0.035593224 d=2.080209 0.001 0.01 0.1 1 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0.002 M (v-ATPase) 0.0 24 M (U A CC 6 2 ) co m pa r ed to NC I 0.0 1 8 MO O O NH2 O HO H N O OH

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255 D-2 Palmerolide B UACC-62 data plot Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=099927187 DF Adj r^2=0.99898061 FitStdErr=0.013250897 Fstat=50320399 a=0.0068052017 b=1.0029625 c=0.23506557 d=1.2374706 0.001 0.01 0.1 1 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.99999089 DF Adj r^2=0.99996357 FitStdErr=0.0027772691 Fstat=36603.506 a=0.070727065 b=0.93127039 c=0.26880172 d=1.6539878 0.001 0.01 0.1 1 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 O O H N O O NH2 O HO O O S O O 0.023 M (v-ATPase) 0.25 M (UACC-62)

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256 D-3 Palmerolide C UACC-62 data plot Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.96957639 DF Adj r^2=0.96146343 FitStdErr=0.036818162 Fstat=169.96913 a=0.059800515 b=0.41034065 c=0.12257149 d=3.7661815 0.001 0.01 0.1 1 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0 0.1 0.2 0.3 0.4 0.5 0.6 Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.9987207 DF Adj r^2=0.99837956 FitStdErr=0.016245663 Fstat=4163.6209 a=0.086230565 b=092315348 c=0.089321713 d=1.7605982 0.001 0.01 0.1 1 10 0 0.1 02 03 0.4 05 0.6 0.7 08 09 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 O H N O O O O NH2 OH OH 0.150M (v-ATPase) 0.11M (UACC-62)

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257 0.025 M (v-ATPase) 0.002 M ( UACC-62 ) D-4 Palmerolide D UACC-62 data plot Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.98799544 DF Adj r^2=0.98399392 FitStdErr=0.036349087 Fstat=356.6406 a=0.059483839 b=1.5312387 c=0.0010091719 d=1.4206199 0.001 0.01 0.1 1 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.98577193 DF Adj r^2=0.94308771 FitStdErr=0.087756255 Fstat=23.09453 a=0.032591955 b=1.3640241 c=0.00285681 d=0.58449391 0.001 0.01 0.1 1 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 O O NH2 O H N O HO O OH

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258 D-5 Palmerolide E UACC-62 data plot Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.99410493 DF Adj r^2=0.99073631 FitStdErr=0.032194718 Fstat=449.6885 a=0.13716696 b=0.84116638 c=5.5371635 d=4.5722235 0.001 0.01 0.1 1 10 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.99903732 DF Adj r^2=0.99848721 FitStdErr=0.015597364 Fstat=2767.3643 a=0.040817254 b=0.95296052 c=4.1467107 d=5.8459316 0.001 0.01 0.1 1 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 O O NH2 O HO O OH O 10 M (v-ATPase) 5 M (UACC-62)

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259 D-6 Palmerolide F UACC-62 data plot Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.99596443 DF Adj r^2=0.9943502 FitStdErr=0.028279405 Fstat=904.91999 a=0.14335109 b=0.83890449 c=0.69987786 d=4.6438677 0.001 0.01 0.1 1 10 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.99933685 DF Adj r^2=0.99904212 FitStdErr=0.012903565 Fstat=5023.216 a=0.064133334 b=0.93586667 c=0.81461891 d=7.0785315 0.001 0.01 0.1 1 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 O O NH2 O H N O HO O OH 0.063 M (v-ATPase) 0.76 M ( UACC-62 )

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260 D-7 Palmerolide G UACC-62 data plot Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.99908376 DF Adj r^2=0.99867654 FitStdErr=0.014004236 Fstat=3634.7294 a=0.003431838 b=0.99678018 c=1.4786218 d=2.6186998 0.001 0.01 0.1 1 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.99985018 DF Adj r^2=0.99979025 FitStdErr=0.0058273255 Fstat=24469.833 a=0.010000012 b=0.98999999 c=0.93405357 d=5.8896305 0.001 0.01 0.1 1 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 O O O NH2 O OH HO NH O 0.0065 M (v-ATPase) 1.2 M (UACC-62)

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261 D-8 Palmerolide H UACC-62 data plot Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.99274514 DF Adj r^2=0.9898432 FitStdErr=0.040066471 Fstat=501.74198 a=0.10207459 b=0.89969451 c=0.023309733 d=2.0054029 0.001 0.01 0.1 1 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.99690293 DF Adj r^2=0.9956641 FitStdErr=0.026816115 Fstat=1180.2478 a=0.11086954 b=0.88912924 c=0.014991273 d=5.0628148 0.001 0.01 0.1 1 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 O O H N O O NH2 O O O S O O HO 0.021 M (v-ATPase) 0.019 M (UACC-62)

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262 D-9 Palmerolide A pBrBz C-7 monoester UACC-62 data plot Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.99706517 DF Adj r^2=0.99589124 FitStdErr=0.024832382 Fstat=1245.6958 a=0.089945228 b=0.87790476 c=0.34690528 d=1.8393336 0.001 0.01 0.1 1 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.9956321 DF Adj r^2=0.99388493 FitStdErr=0.025497327 Fstat=835.79007 a=0.11634907 b=0.73110503 c=0.73598201 d=5.1082043 0.001 0.01 0.1 1 10 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.99657083 DF Adj r^2=0.99519917 FitStdErr=0.02180372 Fstat=1065.592 a=0.12240146 b=0.73659195 c=0.71567909 d=3.0065856 0.001 0.01 0.1 1 10 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 O O O NH2 O HO H N O OR R= O B r 0.002 M (v-ATPase) 0.600 M (UACC-62)

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263 D-10 Palmerolide A pBrBz C-10 mo noester UACC-62 data plot Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.9894007 DF Adj r^2=0.98516098 FitStdErr=0.040023251 Fstat=342.26822 a=0.12068255 b=0.74684554 c=0.3185954 d=1.729595 0.001 0.01 0.1 1 10 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.98326172 DF Adj r^2=0.97656641 FitStdErr=0.049913722 Fstat=215.39209 a=0.12202144 b=0.75375364 c=0.39619193 d=1.6592696 0.001 0.01 0.1 1 10 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.99666403 DF Adj r^2=0.99532964 FitStdErr=0.026692665 Fstat=1095.4629 a=0.099949409 b=0.88954677 c=0.21192074 d=1.699866 0.001 0.01 0.1 1 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0. 2 0. 3 0. 4 0. 5 0. 6 0. 7 0. 8 0. 9 1 O O O NH2 O RO H N O OH R= O B r 0.014 M (v-ATPase) 0.309 M (UACC-62)

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264 D-11 Palmerolide A carbamate hydrolysis product UACC-62 data plot Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.99605295 DF Adj r^2=0.99447413 FitStdErr=0.028713103 Fstat=925.29735 a=0.10741706 b=0.88007182 c=0.58094259 d=2.5713388 0.001 0.01 0.1 1 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.95737659 DF Adj r^2=0.94032723 FitStdErr=0.091480651 Fstat=82.358056 a=-0.27791647 b=1.4402454 c=0.39690655 d=0.40115028 0.001 0.01 0.1 1 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 O O OH HO H N O OH 0.009 M (v-ATPase) 0.489 M (UACC-62)

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265 D-12 Palmerolide A NaOH conjugate addition/carb amate dehydration product UACC-62 data plot Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.99999938 DF Adj r^2=0.99999752 FitStdErr=0.00074214912 Fstat=537716.26 a=0.018000001 b=0.982 c=0.87529052 d=6.7979591 0.001 0.01 0.1 1 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Eqn 8013 LgstcDoseRsp(a,b,c,d)r^2=0.9999993 DF Adj r^2=0.99999722 FitStdErr=0.00081109102 Fstat=479485.05 a=0.021000001 b=0979 c=0.82414906 d=6.971688 0.001 0.01 0.1 1 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 O O HO H N O OH OH 0.026 M (v-ATPase) 0.85 0 M(UACC 62)

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266 About the Author Jaime Heimbegner Noguez received her B achelor of Science with a major in chemistry in May 2004 from Sweet Briar Colleg e. She then moved to Tampa, Florida to pursue a Doctorate in organic chemistry, more specifically natural products chemistry, from the University of South Florida under the instruction of Dr Bill Baker. Jaime’s research is based on the isolation, structur e elucidation, and biol ogical evaluation of secondary metabolites from Antarctic marine invertebrates. She has presented her research at a number of nationa l meetings within her field and was chosen to be a Thrust Scholar by the Florida Center of Excellence for Biomolecular Identification and Targeted Therapeutics for her multidisciplinary research. Jaime intends to pursue a career in drug discovery as a natural products chemist.